Immobilized Enzymes and Cells

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Author: Nathan P. Colowick | Nathan P. Kaplan | Klaus Mosbach

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Contributors to V o l u m e 136 Article numbersare in parenthesesfollowingthe names of contributors. Affiliationslisted are current. OBSIDIANA ABRIL (25), Research and De-

PETER S. J. CHEETHAM (40), PPF Internavelopment, Angenics, Inc., Cambridge, tional, Ashford, Kent TN24 OLT, England Massachusetts 02139 ICHmO CHIBATA (41, 43), Research andDeL. ANDERSSON (I 1), Ferring AB, S-200 62 velopment Headquarters, Tanabe SeiyMalm6, Sweden aku Co., Ltd., Yodogawa-ku, Osaka 532, Japan RICHARD ARMENTA (9), Molecular Devices Corporation, Palo Alto, California 94304 DEBBIE C. CRANS (25), Department of Chemistry, Colorado State University, D. AURIOL (23), BioEurope, F-31400 TouFort Collins, Colorado 80523 louse, France PIERO CREMONESl (14), ltalfarmaco, MASAKI AZUMA (36), Technical Research S.p.A., Milan, Italy Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamagachi M. J. DANIELS (34), Genencor, Inc., South Prefecture 747, Japan San Francisco, California 94080 JOHANN BADER (28), Deutsches Patentamt, M. DELUCA (8), Department of Chemistry, D-8000 Miinchen 2, Federal Republic of University of California, San Diego, La Germany Jolla, California 92093 J. L. BARET (38), Novo lndustrie Enzymes ROBERT K. DINELLO (9), Chiron CorporaS.A., F-75017 Paris, France tion, Emeryville, California 94608 L. A. BEHIE (30), Department of Chemical G. FLEMINGER (17), Department of Biotechand Petroleum Engineering, The Univernology, The George S. Wise Faculty of sity of Calgary, Calgary, Alberta, Canada Life Sciences, TeI-Aviv University, RaT2N 1N4 mat-Aviv, 69 978 TeI-Aviv, Israel LARRY G. BUTLER (22), Department of BioSABURO FUKUI (27), Department of Induschemistry, Purdue University, West Latrial Chemistry, Faculty of Engineering, fayette, Indiana 47907 Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan G. J. CALTON (45), RhOne.Poulenc Research Center, Savage, Maryland 20763 MURRAY C. FUSEE (42), Research Division, W. R. Grace & Co., Washington ReBERNARD CAMBOU (12), Laboratory of Apsearch Center, Columbia, Maryland plied Biochemistry, Department of Ap21044 plied Biological Sciences, Massachusetts Institute of Technology, Cambridge, G. M. GAUCHER (30), Division of BiochemMassachusetts 02139 istry, Department of Biological Sciences, Faculty of Science, The University of CalM. L. CAMPBELL (45), RhOne.Poulenc Regary, Calgary, Alberta, Canada T2N 1N4 search Center, Savage, Maryland 20763 S. GESTRELIUS (32), Ferring AB, S-200 62 GIACOMO CARREA (14), Istituto di Chimica Malm6, Sweden degli Ormoni, Consiglio Nazionale delle Ricerche (C.N.R.), 20131 Milan, Italy IAN GIBBONS (9), Biotrack, Inc., Sunnyvale, California 94086 Z. M. S. CHANG (7), Artificial Cells and Organs Research Centre, Faculty of Medi- HELMUT G/3NTHER (28), Lehrstuhl fiir Orcine, McGill University, Montreal, Queganische Chemie und Biochemie im Orbec, Canada H3G 1 Y6 ganisch-Chemischen lnstitut der Technisix

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CONTRIBUTORS TO VOLUME 136

chen Universitgit Miinchen, D-8046 REINHOLD KELLER (47), Hoechst AktiengeGarching, Federal Republic of Germany sellschaft, D-6230 Frankfurt am Main 80, Federal Republic of Germany NORIO HAGI (46), Biotechnology Research Center, Toyo Soda Manufacturing Co., ALEXANDER M. KLISANOV (12), LaboraLtd., Yamaguchi-ken 746, Japan tory of Applied Biochemistry, Department of Applied Biological Sciences, MassaS. HARBRON (13), Department of Biochemchusetts Institute of Technology, Camistry, University College London, London bridge, Massachusetts 02139 WCIE 7JE, England ANDREAS KONNECKE (18), Department of URSULA HAUFLER (26), Fachbereich BioloBiochemistry, Biosciences Division, Karlgic, Universitgit Bremen, D-2800 Bremen Marx-University, DDR-7010 Leipzig, 33, Federal Republic of Germany German Democratic Republic LARS HEDSYS (21), Pure and Applied BioY. KONNO (49), Dowa Engineering Co., chemistry, Chemical Center, University Ltd., Tokyo 100, Japan of Lund, S-221 O0 Lund, Sweden RIET HILHORST (20), Department of Bio- BETTINA Koep (29), Henkel KGaA, D-4000 Diisseldorf 1, Federal Republic of Gerchemistry, Agricultural University Wamany geningen, 6703 BC Wageningen, The FRIEDRICH KRAUSS (28), Lehrstuhlfar OrNetherlands ganische Chemie und Biochemie im OrBERNARD L. HIRSCHBEIN (25), Imaging Reganisch-Chemischen lnstitut der Technissearch, Polaroid Corporation, Camchen Universitdt Miinchen, D-8046 bridge, Massachusetts 02139 Garching, Federal Republic of Germany T. IMAIZUMI (49), The University of Tokyo, L. J. KRICKA (8), Department of Clinical Shibuya-ku, Tokyo 113, Japan Chemistry, Wolfson Research LaboratoKEIICHI INUZUKA (36), Kyowa Hakko ries, University of Birmingham, BirmingKogyo Co., Ltd., Ube Plant, Ube-shi, ham B15 2TH, England Yamaguchi Prefecture 755, Japan MARIA-REGINA KULA (2), lnstitut far EnSHIGEAKI IRINO (46), Chemical Research zymtechnologie der Universitgit DiisselCenter, Toyo Soda Manufacturing Co., dorf in der Kernforschungsanlage Jiilich Ltd., Yamaguchi-ken 746, Japan GmbH, D-5170Jiilich 1, Federal Republic HANs-DIETER JAKUBKE (18), Department of of Germany Biochemistry, Biosciences Division, KarlCOLJA LAANE (20), Unilever Research LaMarx-University, DDR-7010 Leipzig, boratorium, 3130 AC Vlaardingen, The German Democratic Republic Netherlands VIOLETA G. JANOLINO (39), Department of Food Science, North Carolina State Uni- KEITH J. LAIDLER (6), Department of Chemistry, Faculty of Science, University versity at Raleigh, Raleigh, North Caroof Ottawa, Ottawa, Ontario, Canada lina 27695 KIN 6N5 VILLY J. JENSEN (33), Molecular Biology, PER-OLOE LARSSON (21), Pure and Applied Novo Industri A/S, DK-2880 Bagsvaerd, Biochemistry, Chemical Center, UniverDenmark sity of Lund, S-221 O0 Lund, Sweden VOLKER KASCHE (26), Arbeitsbereich M. D. LILLY (13), Department of Chemical Biotechnologie H, Biotransformation and Biochemical Engineering, University und -Sensorik, Technische Universitiit College London, London WC1E 7JE, EnHamburg-Harburg, D-2100 Hamburg 90, gland Federal Republic of Germany A. LOPEZ (23), Departamento de Alimentos ROMAS J. KAZLAUSKAS (25), Research and DEPg, Facultad de Quimica, Mexico Development Center, General Electric D.F., Mexico Company, Schenectady, New York 12301

CONTRIBUTORS TO VOLUME 136

xi

P. L. LuIsI (19), lnstitut fiir Polymere,

SHOGO NOJIMA (35), Catalysts and Chemi-

Swiss Federal Institute of Technology, EidgenOssische Technische Hochschule (ETH)-Zentrum, CH-8092 Zurich, Switzerland HIDEKATSU MAEDA (3), Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Ibaraki-ken 305, Japan MATS-OLLE MXNSSON (1, 10, 11), Pure and Applied Biochemistry, Chemical Center, University of Lund, S-221 O0 Lund, Sweden M. ABDUL MAZID (6), Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 P. MONSAN (23), BioEurope, F-31400 Toulouse, France KAZUYUKI MORIHAgA (16), Kyoto Research Laboratories, Toho Pharmaceutical Industry Company, Limited, Kyoto Prefecture 617, Japan KLAUS MOSBACH (1, 10, 11, 21, 32), Pure and Applied Biochemistry, Chemical Center, University of Lund, S-221 O0 Lund, Sweden RYONOSUKE MUNEYUKI (16), Research Division, S. T. Company, Limited, Shimoochiai, Shinjuku-ku, Tokyo 161, Japan T. MURAYAMA(49), Oga Operation Office, Akita Oil Storage Co., Ltd., Funagawa, Funagawa-ko, Oga City, Akita Prefecture 010-05, Japan MINORU NAGASHIMA (36), Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamaguchi Prefecture 747, Japan T. J. NAI~NDgANATHAN (13), Wellcome Biotechnology Ltd., South Eden Park, Beckenham, Kent BR3 3BS, England STEFAN NEUMANN (28), Consortium far Elektrochemische, Industrie GmbH, D8000 Miinchen, Federal Republic of Germany SADAO NOGUCHI (36), Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamaguchi Prefecture 747, Japan

cals, Industry Co., Ltd., Chiyoda-ku, Tokyo I00, Japan TATSUSHI OKA (16), Shionogi Research Laboratories, Shionogi and Company, Limited, Osaka 553, Japan HIROSUKE OKADA (4), Department ofFermentation Technology, Faculty of Engineering, Osaka University, Osaka 565, Japan KIYOTAKAOYAMA(46), Chemical Research Center, Toyo Soda Manufacturing Co., Ltd., Yamaguchi-ken 746, Japan F. PAUL (23), BioEurope, F-31400 Toulouse, France URSULA PFITZNER (31), Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 LuTz RIECHMANN(26), Fachbereich Biologie, Universitgit Bremen, D-2800 Bremen 33, Federal Republic of Germany G. P. ROYER (15), Biotechnology Division, Amoco Corporation, Amoco Research Center, Naperville, Illinois 60566 J. DAVID ROZZELL (44), Genetics Institute, Cambridge, Massachusetts 02140 SUSANNERUG8 (33), Process Development, Novo Industri A/S, DK-2880 Bagsvaerd, Denmark T. SAKATA (49), Metal Mining Agency of Japan, Minato-ku, Tokyo 105, Japan HmOTOSHX SAMEJIMA (36), Kyowa Medex Co., Ltd., Chiyoda-ku, Tokyo 100, Japan MERTEN SCHLINGMANN (47), Hoechst Aktiengesellschaft, D-6230 Frankfurt am Main 80, Federal Republic of Germany NILS SIEGBAHN(10), Perstorp Biolytica AB, S-223 70 Lund, Sweden HELMVT SIMON (28), Lehrstuhlfiir Organische Chemie und Biochemie im OrganischChemischen Institut der Technischen Universitiit Miinchen, D-8046 Garching, Federal Republic of Germany PAUL J. SKUDDER (39), Process Development Department, APV International Ltd., West Sussex RHIO 2QB, England B. STEINMANN-HOFMANN(19), Institutfiir Polymere, Swiss Federal Institute of

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CONTRIBUTORS TO VOLUME 136

Technology, EidgenOssische Technische CEES VEEGER (20), Department of BiochemHochschule (ETH)-Zentrum, CH-8092 istry, Agricultural University WaZurich, Switzerland geningen, 6703 BC Wageningen, The Netherlands" HIDEO SUZUKI (5), Fermentation Research Institute, Agency of Industrial Science CHRISTIAN WANDREY (2), Institut fiir and Technology, Ministry of International Biotechnologie 2 in der KernforsTrade and Industry, Ibaraki-ken 305, chungsanlage Jiilich GmbH, D-5170 JiiJapan lich 1, Federal Republic of Germany SIGFRID SVENSSON (21), Carbohydrate ICHIRO WATANABE (48), Central Research Chemistry, Chemical Center, University Laboratory, Nitto Chemical Industry Co., of Lund, S-221 O0 Lund, Sweden Ltd., Tsurumi-ku, Yokohama 230, Japan HAROLD E. SWAISGOOD (39), Department GEORGE M. WmTESIDES (25), Department of Food Science, North Carolina State of Chemistry, Harvard University, CamUniversity at Raleigh, Raleigh, North bridge, Massachusetts 02138 Carolina 27695 G. WIENHAUSEN (8), Department of BiolSATORU TAKAMATSU (43), Research Laboogy, University of California, San Diego, ratory of Applied Biochemistry, Tanabe La JoUa, California 92093 Seiyaku Co., Ltd., Yodogawa-ku, Osaka ROLF WOERNLE (47), Hoechst Aktienge532, Japan seUschaft, D-6230 Frankfurt am Main 80, ISAO TAKATA (41), Research Laboratory of Federal Republic of Germany Applied Biochemistry, Tanabe Seiyaku Cm-HuEY WONG (25), Department of Co., Ltd., Yodogawa-ku, Osaka 532, Chemistry, Texas A&M University, ColJapan lege Station, Texas 77843 ATSUO TANAKA (27), Department oflndusL. L. WOOD (45), RhOne.Poulenc Research trial Chemistry, Faculty of Engineering, Center, Savage, Maryland 20763 Kyoto University, Yoshida, Sakyo-ku, TOMIAKI YAMADA (35), Biotechnology ReKyoto 606, Japan search Department, Research and DevelTAKASHI TANAKA (37), Central Research opment Division, JGC Corporation, Laboratories, Ajinomoto Co., Inc., KaMinami-ku, Yokohama 232, Japan wasaki-ku, Kawasaki 210, Japan IORDANIS THANOS (28), Lehrstuhl fiir Or- K o z o YAMAMOTO (41), Research Laboratory of Applied Biochemistry, Tanabe ganische Chemie und Biochemie im OrSeiyaku Co., Ltd., Yodogawa-ku, Osaka ganisch-Chemischen Institut der Technis532, Japan chen Universitiit Miinchen, D-8046 SHIGERU YAMANAKA (37), Central ReGarching, Federal Republic of Germany search Laboratories, Ajinomoto Co., TETSUYA TOSA (41, 43), Research LaboraInc., Kawasaki-ku, Kawasaki 210, Japan tory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka YOSHIMITSU YAMAZAKI (3, 5), Fermenta532, Japan tion Research Institute, Agency of Industrial Science and Technology, Ministry of JOHANNES TRAMPER (24), Department of International T r a d e and Industry, Food Science, Food and Bioengineering lbaraki-ken 305, Japan Group, Agricultural University Wageningen, 6703 BC Wageningen, The A. YARON (17), Department of Biophysics, Netherlands The Weizmann Institute of Science, 76 100 Rehovot, Israel EDWIN F. ULLMAN (9), Syva Company, Palo Alto, California 94304 MEINHART H. ZENK (31), Lehrstuhl PharITARU URAaE (4), Department of Fermentamazeutische Biologie, Universitgit Mantion Technology, Faculty of Engineering, chen, D-8000 Miinchen 2, Federal RepubOsaka University, Osaka 565, Japan lic of Germany

Preface Volumes 135 through 137 of Methods in Enzymology, Immobilized Enzymes and Cells, Parts B through D, include the following sections: (1) Immobilization Techniques for Enzymes; (2) Immobilization Techniques for Cells/Organelles; (3) Application of Immobilized Enzymes/Cells to Fundamental Studies; (4) Multistep Enzyme Systems and Coenzymes; (5) Immobilized Enzymes/Cells in Organic Synthesis; (6) Enzyme Engineering (Enzyme Technology); (7) Analytical Applications with Emphasis on Biosensors; (8) Medical Applications; and (9) Novel Techniques for and Aspects of Immobilized Enzymes and Cells. The first three sections appear in Volume 135, the next three in Volume 136, and the last three in Volume 137. Immobilization techniques for enzymes, Section (1), has already been treated in Volume XLIV of this series. Immobilization techniques for cells/organelles, Section (2), an area which seems to have great potential, especially for the application of immobilized yeast and plant and animal cells, is covered for the first time in these volumes. Sections (3) and (4) have been dealt with previously. Section (5), the use of immobilized enzymes/cells in organic synthesis, has probably not been covered before. It is my firm opinion that in the not too distant future we will see a number of processes employed which are based, in part, on the examples given in this section. Section (6) on industrial uses updates the material presented in Volume XLIV. The examples given are, to the best of my knowledge, in operational use today or, at least, on a pilot plant level. Section (7), analytical applications with emphasis on biosensors, is the subject of a great deal of research at present, and it may very well be that in the not too distant future we will witness a breakthrough, i.e., many applications of a number of such devices. The medical area, covered in Section (8), seems promising, but certainly more research is required to fully exploit any underlying potential. Finally, in Section (9), I have collected a number of contributions that did not seem to fit in any of the other sections, but do address important and novel developments. I would like to note that although major emphasis in these volumes has been placed on immobilization in its strictest sense, preferentially, covalent attachment of enzymes or entrapment of cells, one should not view immobilizedsystems in too limited a manner. In fact, bioreactors confined by ultrafilter membranes or hollow fiber systems belong in this category, and the various systems appear to overlap. Immobilization techniques as applied to affinity chromatography or immunoassays such as ELISA are not included to any extent in these volumes since they have xiii

xiv

PREFACE

been adequately covered in other volumes of this series (e.g., Volumes XXXIV and 104 on affinity techniques). An area that was originally scheduled for inclusion is synzymes or artificial enzymes. These include attempts to create catalysts mimicking enzymes by coupling of functional groups to, for instance, cyclodextrin [e.g., D'Souza et al. (Biochem. Biophys. Res. Commun. 129, 727-732, 1985) and Breslow et al. (J. Am. Chem. Soc. 108, 1969, 1986)], to crown ethers [Cram et al. (J. Am. Chem. Soc. 107, 3645, 1985)], or to solid matrices [Nilsson and Mosbach (J. Solid-Phase Biochem. 4, 271, 1979) and Leonhardt and Mosbach (Reactive Polymers, in press)]. Related to these studies are attempts to create cavities in polymers with substrate-binding properties ]notably by Wulff et al. (e.g., Reactive Polymers 3, 261, 1985; and previous publications by these authors) and Arshady and Mosbach (Makromol. Chem. 182, 687, 1981)]. This exciting area is presently in a rapid state of development, and the methodology involved should soon be made available in a more comprehensive context. Mention should be made of the developments in the utilization of recombinant DNA technology for the immobilization (and affinity purification) of biomolecules. I refer to the reported fusion of "affinity tails" as polyarginine (Smith et al., Gene 32, 321, 1984), of polycysteine [B01ow and Mosbach, Proceedings of the VIII International Conference on Enzyme Engineering, Annals of the New York Academy of Sciences, in press (presented 1985)], or of protein A (Nilsson et al., EMBO J. 4, 1075, 1985) to enzymes facilitating their purification and immobilization. These preparations can be obtained by fusion of the respective groups as "tail" to the NH2 or COOH termini of the enzyme or by site-directed mutagenesis leading to substitution on the enzyme structure. DNA technology can also be usefully employed to create new multienzyme complexes, fusing enzymes acting in sequence to one another (B01ow et al., Bio/Technology 3, 821, 1985) as an alternative to their co-immobilization on supports; similarly, attachment of "tails" allowing reversible coenzyme binding may be accomplished. The same technology has also been used recently in attempts to prepare esterase mimics from the ground up (Biilow and Mosbach, FEBS Lett. 210, 147, 1987). Since this is such a rapidly moving area, I advise the reader, apart from the usual standard books in this area, to read the proceedings of the Enzyme Engineering Conferences 1-8 (Wiley, first conference; Plenum Press, second-sixth conferences; and Annals of the New York Academy of Sciences, seventh and eighth conferences); Biochemical Engineering, Volumes I-III and subsequent volumes; Annals of the New York Academy of Sciences, 1983; the patent book "Enzyme Technology, Recent Advances" (S. Torrey, ed.), Noyes Data Corporation, Park Ridge, New

PREFACE

XV

Jersey, 1983; and Biotechnology Review no. 2. In addition, in the following journals many articles relating to immobilized enzyme and cell research can be found: Biotechnology and Bioengineering (John Wiley & Sons); Trends in Biotechnology (Elsevier, The Netherlands); Bio/Technology (Nature Publishing Co., U.S.); Applied Biochemistry and Biotechnology (The Humana Press, Inc., U.S.); Applied Biochemistry with Special Emphasis on Biotechnology; Biotechnology Letters (Science and Technology Letters, England); Applied Microbiology and Biotechnology (Springer-Verlag, Germany); Enzyme and Microbial Technology (Butterworth Scientific Limited, England); Biosensors (Elsevier Applied Science Publishing Ltd., England). In studies with immobilized systems, sometimes useful, not immediately obvious "by-products" may be obtained. I refer to the finding that immobilized Escherichia coli cells, when kept in media without selection pressure, show improved plasmid stability (de Taxis du PoEt, P., Dhulster, P., Barbotin, J.-N., and Thomas, D., J. Bact. 165, 871, 1986). An additional example would be the improved regeneration of plants using immobilized protoplasts discussed in Section (2). I would like to express the hope that these volumes present an overview of the various areas in which immobilized enzymes and cells are used, act as a stimulus for further research, and provide methodological "know-how." The proper choice of support and/or immobilization technique for a particular application may not always be easily accomplished, but I hope that guidance to do so is found in these volumes. Putting these volumes together has been a time-consuming and, at times, frustrating undertaking. Without the coeditors, Drs. Lars Andersson, Peter Brodelius, Bengt Danielsson, Stina Gestrelius, and Mats-Olle M~nsson, the volumes would not have materialized. Because of the number of coeditors, some heterogeneity in the editing has resulted. Contributors to the various sections are from substantially different disciplines, and again this has contributed to the heterogeneity that can be found. Part of the editing of the three volumes was carried out in Ziirich, where I held a chair in biotechnology at the Swiss Federal Institute of Technology. Without the enormous efforts and skills of the staff of Academic Press, these volumes would never have reached production. I also owe much gratitude to my secretaries, notably lngrid Nilsson, for their highly qualified help. Finally, I would like to thank the contributors for their efforts. These volumes are dedicated to the memory of the late Professors N. O. Kaplan and S. P. Colowick, with whom I had highly fruitful discussions, especially at the beginning of this undertaking.

K L A U S MOSBACH

METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF

Sidney P. Colowick and Nathan O. Kaplan

VOLUME VIII. Complex Carbohydrates

Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism

Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation

Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XlI. Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XlII. Citric Acid Cycle

Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids

Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids

Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions

Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR xix

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M E T H O D S IN E N Z Y M O L O G Y

VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes

Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques

Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

METHODS IN ENZYMOLOGY

xxi

VOLUME XXXII. Biomembranes (Part B)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I - X X X

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B)

Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)

Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)

Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes

Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND

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METHODS IN ENZYMOLOGY

VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEEF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C)

Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIII. Biomembranes (Part D" Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LV. Biomembranes (Part F: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence

Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture

Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

M E T H O D S IN E N Z Y M O L O G Y

xxiii

VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVlE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA

Edited by RAY Wu VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)

Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME 71. Lipids (Part C)

Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D)

Edited by JOHN M. LOWENSTEIN

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METHODS IN ENZYMOLOGY

VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONEAND HELEN VAN VUNAK1S VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX Edited by EDWARD A. DENNIS AND MARTHAG. DENNIS VOLUME 76. Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE

VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A)

Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B)

Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C)

Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays)

Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS

M E T H O D S IN E N Z Y M O L O G Y

XXV

VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNN1NGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites

Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D)

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Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

xxvi

METHODS IN ENZYMOLOGY

VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Edited by

SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B)

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Edited by P. MICHAEL CONN VOLUME 104. Enzyme Purification and Related Techniques (Part C)

Edited by WILLIAM B. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems

Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS

METHODSIN ENZYMOLOGY

xxvii

VOLUME 109. Hormone Action (Part I: Peptide Hormones)

Edited by LUTZ BIRNBAUMERAND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME I 11. Steroids and Isoprenoids (Part B)

Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A)

Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Corn-

pounds

Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)

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VOLUME 115. Diffraction Methods for Biological Macromolecules (Part

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VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS

VOLUME 117. Enzyme Structure (Part J)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology

Edited by ARTHUR WEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C)

Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101

XXVIn

METHODS IN ENZYMOLOGY

VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G)

Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 123. Vitamins and Coenzymes (Part H)

Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P. MICHAEL CONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K)

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METHODS IN ENZYMOLOGY

xxix

VOLUME 133. Bioluminescence and Chemiluminescence (Part B)

Edited by MARLENE DELUCA AND WILLIAM D. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B)

Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C)

Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) (in preparation)

Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E)

Edited by VICTOR GINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and CalmodulinBinding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes I02-119, 121-134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids)

Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines

Edited by SEYMOUR KAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids

Edited by WILLIAM B. JAKOBY AND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM

XXX

M E T H O D S IN E N Z Y M O L O G Y

VOLUME 146. Peptide Growth Factors (Part A) (in preparation)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B) (in preparation)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes (in preparation)

Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B) (in preparation)

Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunocheraical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) (in preparation) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells (in preparation)

Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques

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Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E) (in preparation)

Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) (in preparation)

Edited by RAY Wu

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IMMOBILIZED

ACTIVE COENZYMES

3

[1] I m m o b i l i z e d A c t i v e C o e n z y m e s By MATS-OLLE MANSSON and K t n u s MOSBACH This section includes a number of contributions concerning immobilized multienzyme systems and coenzymes. Other contributions addressing these systems in a different context are also found in Vols. 135 and 136 (articles by Rozzell, Luisi and Steinmann-Hofmann, Scheller et al., Carrea and Cremonesi, Murachi and Tabata, Laane and Veeger, and Chibata et al.). Furthermore, in a previous volume in this series I similar questions and methodologies have been presented. In the following we wish to discuss briefly general aspects especially related to immobilized coenzymes and also to provide a number of additional references. Coenzyme-dependent enzymes are of great potential interest for the synthesis of interesting stereospecific compounds and for analytical applications. Paralleling attempts to use immobilized coenzymes or coenzyme analogs in affinity chromatography 2 the question of their coenzymatic activity in the immobilized state has been addressed. The first immobilization, e.g., of NAD(H), showing coenzymatic activity was reported in 1971.3 Later, synthesis of chemically better defined analogs, several of which can be seen in Table I, was carried out. The main interest has focused on the redox coenzyme NAD, but NADP, ATP, and CoA have also been immobilized as active coenzymes. Preparation of Immobilized Active Coenzymes Coenzymes usually have to be modified to allow proper immobilization and regeneration. This modification of a coenzyme can be accomplished in basically two ways. One is the preassembly approach and the other has been called the solid-phase modular approach. In the preassembly approach the coenzyme is first modified and then assembled with a spacer molecule to increase the steric availability for the coenzyme of the enzyme and subsequently coupled to a support. In the solid-phase modular approach the coenzyme or a modified coenzyme is coupled to a matrix previously substituted with spacer molecules carrying reactive functional groups. 1 K. Mosbach (ed.), this series, Vol. 44. 2 K. Mosbach, in "Advances in Enzymology" (A. Meister, ed.), Vol. 46, p. 205. Wiley, New York, 1978. 3 p. O. Larsson and K. Mosbach, Biotechnol. Bioeng. 13, 393 (1971).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

4

[1]

M U L T I S T E P E N Z Y M E SYSTEMS A N D C O E N Z Y M E S

TABLE I SUBST1TUENTS USED FOR MODIFICATION OF N A D AND A T P IN THE

Compound NAD

Substituent

N-6 POSITION Reference

CH2COOH CH2~NH(CH2)6NH2

a

O CH2~HCH2COOH OH (CH2)2NH2 (CH2)6NH2 (CH2)2~NHCH(CH2)4NH~CH~-~-CH2 O

C~O

c,d e f

O

I

OCH 3 CH2CHCH2OCCH~CH2

I

II

II

II

OH O CH2CNHCH2CNHCH2CHCH2NHCC~CH2

I

III

O O OH CH2CNH(CH2)6NHCC~CH2

II

OCH3

III

O OCHs (CH2)2CNH(CH2)2CNH(CH2)2NHCC~---CH2

II

II

III

O O OCH3 CH2CNH(CH2)2CNH(CH2)2NHCC---~CH2

II

II

O O (CH2)2COOH (CH2)2~NH(CH2)2NH2 ATP

O CH2COOH CH2~NH(CH2)6NH2

III

OCH3

k k

O CNH(CH2)6NH2

II

O CHzCNH(CH2)6NHCC~CH2

II

O (CH2)6NH2

III

OCH3

M. Lindberg, P. O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973). b p. Zappelli, A. Rossodivita, and L. Re, Eur. J. Biochem. 54, 475 (1975). c H. L. Schmidt and G. Grenner, Eur. J. Biochem. 67, 295 (1976). a A. F. Buckmann, M.-R. Kula, R. Wichmann, and C. Wandrey, J. Appl. Biochem. 3, 301 (1981). e D. B. Craven, M. J. Harvey, and P, D. G. Dean, FEBS Lett. 38, 320 (1974). a

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IMMOBILIZED ACTIVE COENZYMES

5

In the authors' opinion the preassembly approach has significant advantages. First, chemically well-defined preparations can be obtained and second, the preassembled coenzyme-spacer molecule can be characterized prior to coupling and studied in homogeneous solution, yielding useful information in advance concerning kinetic constants such as Kdi~s and Km. The different variations of the solid-phase modular approach all suffer from the drawback that heterogeneous preparations containing excess spacers on the matrix may be obtained which can give rise to unwanted nonspecific interactions with the enzyme. The syntheses of NAD(P) and ATP analogs have been described in a previous volume of this series.l The probably most widely applied synthetic route involves alkylation of the N-1 nitrogen of the adenine nucleus [a part of both NAD(P) and ATP] by reagents such as iodoacetic acid, 4 3,4-epoxybutanoic acid, 5 or ethyleneimine. 6,7 After reduction of the alkaline-labile NAD to the stable NADH, a Dimroth rearrangement is carried out to convert the N-1 derivative to the corresponding desired N-6 analog. This N-6 analog is then used as a starting material for the synthesis of analogs with different spacers carrying different reactive groups (Table I). For an immobilized coenzyme to be used as an active coenzyme in an enzyme reactor or as a component of a biosensor, special requirements have to be fulfilled. Water-soluble polymers should be used as they do not cause as much diffusional hindrance as a water-insoluble matrix. In addition the degree of substitution is of importance since it has been reported that a high degree of substitution will lead to inhibition of the enzyme. 8 Widely used water-soluble polymers are dextran and polyethylene glycol 4 M. Lindberg, P. O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973). 5 p. Zappelli, A. Rossodivita, and L. Re, Eur. J. Biochem. 54, 475 (1975). 6 H. L. Schmidt and G. Grenner, Eur. J. Biochem. 67, 295 (1976). 7 A. F. Btickmann, M.-R. Kula, R. Wichmann, and C. Wandrey, J. Appl. Biochem. 3, 301 (1981). s S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochem. 114, 101 (1981).

I M . Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). g F. LeGoffic, S. Sicsic, and C. Vincent, in "Enzyme Engineering" (W. H. Weetall and G. P. Royer, eds.), Vol. 5, p. 127. Plenum, New York, 1980. h y. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2277 (1981). i y. Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, Biochem. J. 95, 109 (1984). J S. Adachi, M. Ogata, H. Tobita, and K. Hasimoto, Enzyme Microb. TechnoL 6, 259 (1984). , M. Lindberg and K. Mosbach, Eur. J. Biochem. 53, 481 (1975). I H. Suzuki and Y. Yamazaki, this volume [5]. my. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2091 (1981). " W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N.Y. Acad. Sci. 437, 257 (1984).

6

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[1]

(PEG), but polymers such as polyethyleneimine (PEI) have also been used. Polymerizable coenzymes such as the acrylic derivatives listed in Table I are immobilized in a polymerization reaction together with other monomers, e.g., acrylamide. The coenzyme content of the final polymer will then depend on the ratio of coenzyme monomer to the other monomers added. The polymeric derivatives obtained in this way are heterogeneous mixtures of various polymer chains. The "coenzymatic activity" of such a coenzyme moiety placed on a polymer chain may depend on its position in the polymer and on the size of the polymer. 9 Properties of Immobilized Active Coenzymes A coenzyme analog synthesized according to the preassembly method has, as mentioned before, the advantage that it can be thoroughly studied, both chemically and enzymatically prior to immobilization. One aspect of great importance is whether the modification has influenced on the interactions between enzyme and coenzyme. Generally, the N-6 position of NAD is the point of choice for substitution since it has been shown that for many dehydrogenases the exocyclic amino group of the parent NAD molecule points out of the active site. 1° Thus, spacers attached at this position also will project out from the active site of the enzyme, and interference with coenzyme binding will be minimized or nonexistent. However in some instances the C-8 position has also proved useful, at least in affinity chromatography (for an overview, see Ref. 11). The easiest way to determine the amount of immobilized coenzyme on a water-soluble polymer is to record the UV spectrum for the coenzyme and, based on the absorption peak, determine its concentration. 1 It is important that the reference cell during recording of the UV spectrum carries the equivalent amount of nonsubstituted polymer. The degree to which an immobilized coenzyme can be enzymatically reduced or oxidized is also of great interest. The kinetic properties of immobilized NAD derivatives vary depending on both what enzyme and what coenzyme derivative are being used. In general the Km values are higher and the Vmaxvalues are lower for the immobilized NAD than for the free NAD. In a comparison of coenzymatic activity between free NAD, N-6-substituted NAD, dextran-NAD,

9 S. Adachi, M. Ogata, H. Tobita, and K. Hashimoto, Enzyme Microb. Technol. 6, 259 (1984). J0 C. I. Br~inden, H. JOrnvall, H. Eklund, and H. Furugren, in "The Enzymes" (P. D. Boyer, ed.), Vol. 11, 3rd Ed., p. 103. Academic Press, New York, 1975. " M. O. M~nsson and K. Mosbach, in "Coenzymes and Cofactors" (D. Dolphin, R. Poulson, and O. Avramovic, eds.). Wiley, New York, in press.

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IMMOBILIZED ACTIVE COENZYMES

7

and agarose-bound NAD, the relative initial rates toward alcohol dehydrogenase were found to be 100, 61, 16, and 0.7, respectively. ~2 Apart from the different number of interactions possible between coenzyme and support that are dependent on the size of the latter, there are two factors which influence these initial reaction rates when compared to the free coenzyme. First, the modification of the coenzyme can alter its binding to the active site. Also, the polymer to which the coenzyme is anchored may influence the binding of the coenzyme to the active site, e.g., because of its hydrophilic/hydrophobic character. In different reports, the effect of polymer molecular weight, 9 NAD density on the polymer, 8 and length of the spacer ~3 on the enzymatic reaction have been discussed. Applications of Immobilized Active Coenzymes Immobilized active coenzymes such as NAD have found use in analytical systems as well as in enzyme reactors. The reactor of choice is the membrane reactor in which a semipermeable membrane retains the high molecular weight immobilized coenzyme together with the enzymes. 14 The design of the reactor will of course be different if the enzyme-coenzyme system is of a totally "self-sustained" form where both the enzyme and the coenzyme are integrated parts of the same polymer. ~5Alternative systems that allow continuous processes to be carried out have also been studied. Following one approach, an NAD analog has been coupled directly onto an enzyme, allowing it to act as a prosthetic group~6,~7; alternatively the enzyme is coimmobilized with an NAD analog to a support in a predetermined configuration. 16,~8 An important issue both in the reactor systems and for analytical systems is the requirement for regeneration of the coenzyme because of its high cost and the fact that it is needed in stoichiometric amounts relative to the product formed. Regeneration can be accomplished chemically, electrochemically, or enzymatically. At present, enzymatic regeneration has the most advantages, especially because of its high specificity in regeneration. However, electrochemical procedures are gaining in importance (in this context see Ref. 18a). Table II summarizes several appli12 p. O. Larsson and K. Mosbach, FEBS Lett. 46, 119 (1974). J3 y . Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, Biochem. J. 95, 109 (1984). ~4 M.-R. Kula and C. Wandrey, this volume [2]. ~5Y. Yamazaki and H. Maeda, this volume [3]. 16 M. O. Mhnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457. x7 C. Woenckhaus, R. Koob, A. Burkhard, and H. G. Schaefer, Bioorg. Chem. 12, 45 (1983). ~s K. J. Laidler and M. A. Mazid, this volume [6]. ~8~I. Thanos, J. Bader, H. Giinther, S. Neumann, F. Krauss, and H. Simon, this volume [28].

8

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[1]

TABLE II APPLICATIONS OF IMMOBILIZED N A D ANALOGSa

Product or compound analyzed Enzyme reactors with Alanine Alanine Leucine Benzaldehyde Lactate Lactate Lactate Lactate Lactate Alanine Malate Malate Propanediol Propanediol Lactate Phenylalanine Analytical systems Glutamate Pyruvate Alcohol Alcohol Lactate Alcohol Alcohol Lactate Glutamate Lactate Glutamate

Coenzyme

Enzymesb

coupled enzyme regeneration Dextran-NAD Dextran-NAD PEG-NAD Polyacrylate-NAD PEG-NAD Dextran-NAD PEI-NAD Polylysine-NAD Dextran-NAD PEI-NAD Polyacrylate-NAD Dextran-NAD Coimmobilized NAD Enzyme-bound NAD Enzyme-bound NAD PEG-NAD

GalDH + AlaDH AIaDH + LDH FDH + LeuDH ADH (*) ADH + LDH ADH + LDH ADH + LDH ADH + LDH ADH + LDH AIaDH + LDH FDH + MDH ADH + MDH ADH (*) ADH (*) ADH + LDH FDH + PheDH

Dextran-NAD Dextran-NAD Polyacrylate-NAD Coimmobilized NAD Electrode-bound NAD Electrode-bound NAD Dextran-NAD Dextran-NAD Dextran-NAD Dextran-NAD Dextran-NAD

GDH + LDH GDH + LDH ADH + diaphorase ADH LDH ADH ADH LDH GDH LDH GDH

Reference

c e : e h J k m

n.o n P q q r s s t t ' "

GalDH, Galactose dehydrogenase; AIaDH, alanine dehydrogenase; FDH, formate dehydrogenase; LeuDH, leucine dehydrogenase; ADH, alcohol dehydrogenase, LDH, lactate dehydrogenase; MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; and PheDH, phenylalanine dehydrogenase. b (,) Indicates coupled-substrate enzymatic regeneration. c p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). a M.-R. Kula and C. Wandrey, this volume [2]. e C. W. Fuller, J. R. Rubin, and H. J. Bright, Eur. J. Biochem. 103, 421 (1980). z H. Okada and I. Urabe, this volume [4]. g P. O. Larsson and K. Mosbach, FEBS Lett. 46, 119 (1974). h j. R. Wykes, P. Dunill, and M. D. Lilly, Biotechnol. Bioeng. 17, 151 (1975). i y . Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). J Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). k W. Marconi, G. Prosperi, S. Giovenco, and F. Morisi, J. Mol. Catal. 1, 111 (19751976).

[2]

CONTINUOUS ENZYMATIC TRANSFORMATION

9

cations of immobilized active N A D in analysis and as a component in enzyme reactors. In addition a polyethylene glycol-bound ATP ~9 and N A D P 2° have recently been used in enzyme reactor studies. It should be kept in mind that native coenzymes for a number of applications remain an alternative (see, e.g., the contribution on ATP recycling). 2~ In this context a recent publication on "an affinity chromatographic reactor for highly efficient turnover of dissociable cofactors" applying free NAD in an ultrafiltration hollow fiber tube should be mentioned. 22 19 W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N. Y. Acad. Sci. 437, 257 (1984). 20 K. Okuda, I. Urabe, and H. Okada, Eur. J. Biochem. 151, 33 (1985). 2~ D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C.-H. Wong, O. Abril, and G. M. Whitesides, this volume [25]. 22 O. Miyawaki, N. Osato, and T. Yano, Agric. Biol. Chem. 49, 2063 (1985).

I y . Yamazaki and H. Maeda, this volume [3]. m j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). n M. O. MAnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457, o S. Gestrelius, M. O. MAnsson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). p E. Schmidt, E. Fiolitakis, and C. Wandrey, Enzyme Eng. 8 (in press). q H. Suzuki and Y. Yamazaki, this volume [5]. • K. J. Laidler and M. A. Mazid, this volume [6]. s T. Yao and S. Musha, Anal. Chim. Acta 110, 203 (1979). t A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). " Y. Sakaguchi, M. Sugahara, J. Endo, and T. Murachi, J. Appl. Biochem. 3, 32 (1981).

[2] C o n t i n u o u s E n z y m a t i c T r a n s f o r m a t i o n in an Enzyme-Membrane Reactor with Simultaneous NADH Regeneration B y M A R I A - R E G I N A K U L A a n d CHRISTIAN WANDREY

Enzymes as catalysts are especially noted for their high stereo- and regiospecificity. To accomplish the synthesis of chiral compounds often not only an enzyme is needed as catalyst but in addition a low molecular weight coenzyme which participates in the reaction. For a large number of redox processes NAD(H) or NADP(H) serves as coenzymes for dehydrogenases and is utilized in stoichiometric amounts in the course of the reaction. These coenzymes are readily dissociable from the enzyme and require a separate second reaction for regeneration. I S. S. Wang and C. K. King, Adv. Biochem. Eng. 12, 119 (1979). METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.

[2]

CONTINUOUS ENZYMATIC TRANSFORMATION

9

cations of immobilized active N A D in analysis and as a component in enzyme reactors. In addition a polyethylene glycol-bound ATP ~9 and N A D P 2° have recently been used in enzyme reactor studies. It should be kept in mind that native coenzymes for a number of applications remain an alternative (see, e.g., the contribution on ATP recycling). 2~ In this context a recent publication on "an affinity chromatographic reactor for highly efficient turnover of dissociable cofactors" applying free NAD in an ultrafiltration hollow fiber tube should be mentioned. 22 19 W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N. Y. Acad. Sci. 437, 257 (1984). 20 K. Okuda, I. Urabe, and H. Okada, Eur. J. Biochem. 151, 33 (1985). 2~ D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C.-H. Wong, O. Abril, and G. M. Whitesides, this volume [25]. 22 O. Miyawaki, N. Osato, and T. Yano, Agric. Biol. Chem. 49, 2063 (1985).

I y . Yamazaki and H. Maeda, this volume [3]. m j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). n M. O. MAnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457, o S. Gestrelius, M. O. MAnsson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). p E. Schmidt, E. Fiolitakis, and C. Wandrey, Enzyme Eng. 8 (in press). q H. Suzuki and Y. Yamazaki, this volume [5]. • K. J. Laidler and M. A. Mazid, this volume [6]. s T. Yao and S. Musha, Anal. Chim. Acta 110, 203 (1979). t A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). " Y. Sakaguchi, M. Sugahara, J. Endo, and T. Murachi, J. Appl. Biochem. 3, 32 (1981).

[2] C o n t i n u o u s E n z y m a t i c T r a n s f o r m a t i o n in an Enzyme-Membrane Reactor with Simultaneous NADH Regeneration B y M A R I A - R E G I N A K U L A a n d CHRISTIAN WANDREY

Enzymes as catalysts are especially noted for their high stereo- and regiospecificity. To accomplish the synthesis of chiral compounds often not only an enzyme is needed as catalyst but in addition a low molecular weight coenzyme which participates in the reaction. For a large number of redox processes NAD(H) or NADP(H) serves as coenzymes for dehydrogenases and is utilized in stoichiometric amounts in the course of the reaction. These coenzymes are readily dissociable from the enzyme and require a separate second reaction for regeneration. I S. S. Wang and C. K. King, Adv. Biochem. Eng. 12, 119 (1979). METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.

l0

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

Since NADH and NADPH are complex and rather labile organic chemicals they are quite expensive. For the application of dehydrogenases in the enzyme-catalyzed synthesis of chiral compounds an efficient coenzyme regeneration is therefore needed in order to make such a process economically viable. A simple comparison of cost of I mol of NADH (665 g/mol, value -$1000) with 1 mol of chiral a-hydroxy or a-amino acid (-100 g/mol, value -$2) shows that separation, external regeneration, and recycling of the coenzyme would require an overall yield of 99.98% per pass to keep the coenzyme cost at 10% of the product value. Simultaneous regeneration offers a chance to overcome the stoichiometric relation between product and coenzyme by an efficient internal recycling. In principle several experimental approaches are possible. ~-6 Here we concentrate on an enzymatic regeneration of NADH by a second enzyme-catalyzed reaction. Our approach follows the strategy developed in cellular metabolism in which NADH is produced and consumed by a series of balanced reactions. 6-~° Considering ultimately a technical application it should be noted that besides the anticipated conversion, a second substrate is now needed in a stoichiometric amount and two products are formed in equimolar concentration. Therefore the cosubstrate should be cheap enough and neither cosubstrate or coproduct should be obnoxious to the enzymes involved. In the authors' laboratories formate dehydrogenase is preferred as a general NADH-regenerating enzyme for several reasons: the enzyme is readily available in large amounts from Candida boidinii grown on methanolH,~2; the equilibrium of the reaction catalyzed lies far on the side of CO2 and therefore NADH formationS1; and the 2 H. Simon, J. Bader, H. Giinther, S. Neumann, and J. Thanos, Ann. N. Y. Acad. Sci. 434, 171 (1984). 3 Z. Shaked, J. J. Barber, and G. M. Whitesides, J. Org. Chem. 46, 4100 (1981). 4 M. O. M~nsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457. M. Aizawa, R. W. Coughlin, and M. Charles, Biotechnol. Bioeng. 18, 209 (1976). 6 R. Wichmann, C. Wandrey, A. F. Biickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 7 C. Wandrey, R. Wichmann, W. Leuchtenberger, A. B/ickmann, and M.-R. Kula, U.S. Patent 4,326,031, European Patent 80,104,346.4; U.S. Patent 4,304,858, European Patent 80,104,345.6. s W. Leuchtenberger, C. Wandrey, and M.-R. Kula, DOS 3,307,094.6, Spanish Patent 530,155. 9 A. F. Bfickmann, U.S. Patent 4,443,594, European Patent 79,102,954.9. ~ C- Wandrey and R. Wichmann, in "Application of Isolated Enzymes and Immobilized Cells to Biotechnology" (A. Laskin, ed.), p. 177. 1985. H H. Schtitte, J. Flossdorf, H. Sahm, and M.-R. Kula, Eur. J. Bioehem. 62, 151 (1976). ~2 K. H. Kroner, H. Schiitte, W. Stach, and M,-R. Kula, J. Chem. Technol. Bioteehnol., Biotechnol. 32B, 130 (1982).

[2]

CONTINUOUS ENZYMATIC TRANSFORMATION

HCO0-

reduced subsfrate

u.~

PEG2oooo-NAD"

/z~

•

PE52oooo-NAOH k0H

~A

C0z

1l

oxidized subsfrafe

FIG. 1. Reaction scheme. FDH, Formate dehydrogenase; E, enzyme; PEG 20000-NAD + NADH, coenzyme derivatives.

coproduct CO2 is easily separable. The overall reaction scheme is outlined in Fig. 1. It is apparent that for fast NADH recycling, ready access to both enzymes becomes crucial and reactor systems with inherent mass transfer resistances should be avoided. Therefore an enzyme-membrane reactor was chosen. Here the reaction will proceed in homogenous solution. 6-1° The enzymes are retained in the reactor by virtue of their high molecular weight, utilizing an ultrafiltration membrane as a selective barrier. However, the difference in molecular weight between product and coenzyme is not sufficient to achieve satisfactory results by a membrane separation. Therefore we attempted to increase the molecular weight of the coenzyme by attaching it covalently to a water-soluble polymer such as polyethylene glycol (PEG). 9 This way the retention of the coenzyme by an ultrafiltration membrane could be accomplished together with separation of the coenzyme from the product stream. Provided the coenzyme activity is not lost upon modification, the basic design of the reactor should allow continuous enzymatic conversion and regeneration of NADH to proceed. Coenzyme D e r i v a t i v e s The synthesis of the PEG-NADH is outlined in Fig. 2 and has been described in detail by Bfickmann et al. 9,13 The modified coenzyme is readily soluble in water, and its concentration can be determined from absorbance at 340 or 259 nm, respectively. Assuming that the molar absorption coefficients are identical with the native coenzyme the following values have been used for calculation: 6220 M -~ cm -I for NADH at 340 nm, and 18,000 M -~ cm -~ for NAD at 259 nm. A number of dehydrogenases has been tested with regard to their ability to accept the modified coenzyme. u A. F. Biickmann, M.-R. Kula, R. Wichmann, and C, Wandrey, J. Appl. Biochem. 3, 301 (1981).

12

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

NH2 ~NH~ "~N/~"~ N

LRib-- p--p

\N /

"k

2H*

--.,bJ

PEG--COO-

L.ib-- p-- p--Rin /

CQrbodiimide NH2

reduction

h R i b - - p - - P--Rib ~

Dimroth

H

PEG--

C - - N --CH~--CH~~'- NH

H

H

in reorrQngement

O

o

N

N

NH2

LRib-- p--p--Rib

-]

FIG. 2. General synthesis route to modified coenzymes. Rib, Ribose; P, phosphate; PEG, polyethylene glycol.

TABLE I KINETIC PARAMETERS OF DEYHYDROGENASES FOR NATIVE NADH AND P E G - N A D H a

Conditions

Native NADH

PEG-NADH

Temperature

Vmax

Km

Vmax

Krn

Enzyme b

Substrate

pH

(°C)

(U/rag)

(mM)

(U/mg)

(raM)

AIaDH LeuDH PheDH L-LDH D-LDH L-HicDH b-HicHD FDH

Pyruvate Ketoleucine Phenylpyruvate Pyruvate Pyruvate Ketoleucine Ketomethionine Formate

9 8 8.5 9 9 8.5 8 8

25 25 25 25 25 25 25 40

20.9 13.2 37.2 308.0 37.0 511.0 259.0 1.2

0.022 0.033 0.047 0.001 0.289 0.044 0.170 0.300

10.3 11.5 17.7 150.0 4.3 214.0 240.0 2.6

1.290 0.029 0.099 0.080 0.060 0.144 0.240 0.330

a Activity per milligram protein in the enzyme preparation. b AlaDH, Alanine dehydrogenase; LeuDH, leucine dehydrogenase; PheDH, phenylalanine dehydrogenase; L-LDH, L-lactate dehydrogenase; D-LDH, D-lactate dehydrogenase; L-HicDH, L-hydroxyisocaproate dehydrogenase; D-HicDH, D-hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase.

[2]

13

CONTINUOUS ENZYMATIC TRANSFORMATION

The results are listed in Table I. Often the Vmaxvalue obtained with the derivative is in the range 50-100% in comparison with the native coenzyme, while the Km value may change by an order of magnitude. It should be noted that formate dehydrogenase exhibits an even higher initial reaction velocity with the modified coenzyme than with the native. 6 Besides the examples listed in Table I the following enzymes are known to accept P E G - N A D ( H ) as coenzyme: alcohol dehydrogenase, isopropanol dehydrogenase, malate dehydrogenase, glutamate dehydrogenase. So far, glucose dehydrogenase from Bacillus megaterium is the only dehydrogenase detected that does not accept P E G - N A D H as a substrate. Retention

The retention of the coenzyme in the reactor becomes very important for continuous operation. In Fig. 3 the loss of coenzyme by incomplete retention in the reactor is plotted as a function of the operating time. The data show that retentions better than 99.9% are necessary to avoid excessive loss of coenzyme at a typical mean residence time of 1 hr. The high

\

" " " ........ \

8.8

............

~

O

\ e. 4

\

.........

PEG_20000_NAD(H) R = 9 9 93 kELU = 1 7 X/d

.........

PEG-1000g-NADCH)~""~ R

\\

= gg82

kELU =

\,

\,

4 3

X

X/d

N ""

0.2 R

~'". 2 4 . 0 ~/cl "'"--.....,...

= 99.00

kELU I 8

2

I 4

I I~

I 8

I 18

I 12

I 14

1 10

I 18

I

I

2a

22

24

TIME l D Flo. 3. Elution of P E G - N A D ( H ) across a YM5 (Amicon) membrane in a continuously operated membrane reactor; residence time Z = l hr. R, Retention (R = 99.00% for comparison); ke~u, elution loss (due to incomplete retention); PEG 10000 and PEG 20000, molecular weight 10,000 and 20,000.

14

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

retention is a very stringent requirement when choosing the right membrane and depends on the average molecular weight of the coenzyme derivative and also on the pore size distribution determining the actual cutoff behavior of a given membrane. The performance of a membrane selected should be checked by experiments lasting for several days in order to allow assessment of the data with sufficient confidence. Retention R is defined by Eq. (1): R = (Cr-

Cf)/Cr

(1)

where Cr and Cf are the concentration of the compound of interest in the retentate and filtrate, respectively. The washout can be described as a function of time by Eq. (2): Cr/Cro = e -[~1 - R)/~-]t

(2)

Cro is the retentate concentration at time zero, t is the elapsed time, and r the mean residence time. The apparent retention R can be calculated according to Eq. (2) from a plot of l n ( C r / C r o ) versus operating time. The elution loss Kelu is then defined by Eq. (3): Kelu = (1 - R ) / r

(3)

Even for rather high retention, washout becomes critical as residence times go down, since it depends on residence times as well as retention. In Fig. 4 the retention of PEG, carboxylated PEG, and the final preparation of the P E G - N A D H are represented. Experiments were performed within the membrane reactor as described below. Concentration of PEG was measured by refractive index; P E G - N A D H was determined by UV absorbance at 260 nm employing a cuvette with a 1-mm light path for the retentate and a 10-mm light path for the filtrate to compensate partly for the large difference in concentration of the different streams. It should be noted, however, that the apparent retention depends on the hydrodynamics of the system employed. Considering its importance for coenzymedependent processes the apparent retention should be reexamined if other reactor configurations are evaluated or hydrodynamic conditions are drastically changed. The absolute loss of coenzyme is also a function of the coenzyme concentration, which should therefore be as small as possible in the reactor. The lower limits of coenzyme concentration are defined by the necessity to saturate the enzymes involved in order to operate with their maximal catalytic efficiency. Table I shows that P E G - N A D H derivatives exhibit comparatively low Kmvalues, so that in many cases stationary coenzyme concentrations below 0.5 mM are sufficient for operation. Since the enzymes employed in the membrane reactor are much larger

[2]

15

CONTINUOUS ENZYMATIC TRANSFORMATION t

8.6

8.4

A D ¢

8.2 o

8

PE8-28888 PEG-28088-COOH PEG-28888-NADH

-

0

I

I

I

I

I

I

I

2

3

4

5

6

TIME

7

/ D

FIG. 4. Elution of P E G and P E G derivatives across a YM5 (Amicon) m e m b r a n e ; residence time ~" = 1 hr.

than the coenzyme derivative, elution losses of the catalyst itself are negligible. Experimental Setup of an Enzyme-Membrane Reactor The ultimate goal of an experiment determines the degree of sophistication the experimental setup of an enzyme-membrane reactor has to meet. A simple homemade version utilizing commercially available equipment is illustrated in Fig. 5. It has been successfully used for the determination of the stereospecificity of enzymes in crude extracts during a screening procedure.~4 A similar version could be employed if the preparation of small amounts (1-100 mmol) of a certain chiral compound is of prime interest. However, if the enzyme-membrane reactor is operated to evaluate coenzyme utilization and to use the data for feasibility studies or the design of a large-scale process several, safety and control features as well as precise measuring devices have to be installed. 14 W. H u m m e l , H. Schiitte, and M.-R. Kula, Ann. N . Y. Acad. Sci. 434, 194 (1984).

16

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

p-

i

i . . . . . . . . . . . . . . .

FIG. 5. Simple version of a laboratory enzyme-membrane reactor. The glass vessel (1) contains the substrate solution in 0.3 M ammonium formate or sodium formate, pH 7.0-7.5. In the thermostated (25 ° water bath) reactor (2), 0.5 ~mol/ml PEG-NADH, 3 U/ml formate dehydrogenase, and 3-5 U/ml of the second dehydrogenase are placed in 10 ml substrate solution containing 0.3 M formate. The reaction mixture is circulated by means of a peristaltic pump (4) passing through an ultrafiltration device (3). A CEC unit from Amicon or a similar device fitted with a YM5 membrane (Amicon) can be conveniently employed. With a CEC unit the circulating rate is set to approximately 30 ml/hr. This will create a small pressure differential and lead to a filtrate flux of approximately 3 ml/hr and a residence time of 3 hr. Filtrate flux is controlled and readjusted manually about twice a day if necessary. Filtrate is collected in bulk or with a fraction collector. The reactor (2) is closed airtight and the liquid level kept constant by the pneumatic overhead arrangement (1). The reactor is stirred using a magnetic stirrer to ensure proper mixing.

Figure 6 gives a flow diagram of a continuous process including all measuring points. Figure 7 shows a laboratory reactor which has been assembled in the mechanics workshop of the authors. The guiding principles in the design were to achieve a high ratio of membrane surface to internal retentate volume, to incorporate temperature control, and to allow easy access to the membrane for setup and changes between experiments. Polarization control is effected by operating the membrane reactor

~

enzyme ~~c ° e no z y mI e ~ ~

~J I product

go substrate I 2 S

METERINfi PUMP STERILE FILTER ENZYME MEMBRANE REACTOR

4 5 6

PHOTOMETER POLARIMETER RECORDER

F

FIG. 6. Flow diagram of a laboratory-scale membrane reactor with magnetic stirrer and thermostating jacket.

[2]

17

CONTINUOUS ENZYMATIC TRANSFORMATION

lid

se mc se sti

bc )strate sa

FIG. 7. Details of a laboratory membrane reactor (the magnetic bar is inserted in a plastic disk to minimize the retentate volume).

on a magnetic stirrer and inserting a magnetic bar underneath the membrane. In general the speed of the stirrer is set to 200 rpm. For long-time operations special care has to be taken to achieve and maintain sterile conditions in the reactor. After complete assembly a solution containing a suitable disinfectant such as 0.1% peroxyacetic acid is pumped through the reactor, and at the same time all tubing and filters are flushed. The disinfectant is replaced after several hours, at least 5, by sterile water followed by substrate solution. At this point the integrity of all seals is also tested. Finally the selected enzymes and PEG-NADH are added aseptically or pumped into the reactor and concentrated on the retentate side. Reaction starts at zero conversion and the time course until steady state is obtained can be followed and utilized to test the kinetic model based on measurement of initial rates, which has previously been developed to describe the reactor performance. 6,1° Because chiral compounds are produced, polarimetry is the method of choice to follow conversion in the reactor effluent. A port to the retentate side, closed by a septum, serves to draw periodically samples from the reactor using sterile syringes and to measure coenzyme content and enzyme activities. If desired, on-line control of enzyme activity and coenzyme levels is possible, but this requires for each single case a detailed

18

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

100

80 g,g 80

z cl H OC bJ Z 0 (J

40

SPACE-TIME-YIEL = D214 G/(LND) 100 MMOL/L KETDISOCAPROATE 400 MMOL/L NH4-FORMATE pH = 8 , 0 , T = 2S * • = 7S MIN

20

0

0

|

I

I

I

}

1

I

I

10

20

3B

48

SO

50

70

80

TIME /

90

D

FIG. 8. L-Leucine production with F D H , L e u O H , and PEG 2 0 0 0 0 - N A D H .

298 U/KG PRODUCT

i~ rl D -I i~r~

O

(3 b.

FDH I

I

I

0

20

30

I

40 TIME

/

ADDITION

1

I

I

i

50

80

70

80

D

FIG. 9. L-Leucine production and deactivation of F D H .

90

[2]

19

CONTINUOUS ENZYMATIC TRANSFORMATION

261U/KGPRODUCT1 ._1 \

I w 1

t LEUDH

o 0

-

I

ADDITION

I

I

1

I

1

I

I

I

10

20

30

40

SO

60

70

80

90

TIME / D FIG. 10. L-Leucine production and deactivation of LeuDH.

kinetic model in order to differentiate the activity of the enzymes involved. 15 The two enzymes and the coenzymes utilized in the coupled reaction will have different rates of decomposition. The component limiting the performance of the reactor can be identified and added with the substrate stream in order to restore conversion and performance. After sufficiently long operating times the consumption of enzyme activity and coenzyme loss per unit weight of product can be determined with confidence and put into an economic model of the reaction in order to optimize the process studied. For calculation and design it is important to maintain a constant flow. A pulse-free piston pump (Reichelt Chemietechnik, Heidelberg) was found satisfactory for prolonged operation with the low flow rates required for the laboratory reactor. Performance of Reactor Figures 8-11 demonstrate 3 months' performance of a laboratory enzyme-membrane reactor for the production of L-leucine. A cycle number of 80,000 was obtained for the coenzyme. The cycle number is defined 15 R. Wichmann and C. Wandrey,

Enzyme Eng. 6,

311 (1982).

20

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[2]

I

100000

O.B

00000

86

60808

0.4

48000 ~ \

0.2

20000

d

0

0 0

10

20

38

40 TZME /

50

60

70

80

90

D

FIG. 11. L-Leucine production, deactivation of PEG 20000-NADH, and development of cycle number.

here as moles of product obtained per moles of coenzyme lost in the process (for whatever reasons). Figure 11 shows that the cycle number increases from the start of the experiment and levels off after - 2 0 days. Figures 9 and 10 demonstrate that the reactor was operated at rather low stationary enzyme concentrations. Increasing the enzyme content in the reactor would give higher space-time yields. Productivities up to 1 kg/ liter-day have been realized, which is much higher than reported values for fixed bed reactors. The optimal ratio of the enzyme activities depends on the kinetic constants, and concentrations are not necessary equal. It is one of the advantages of the enzyme-membrane reactor that the concentration of the enzymes involved in the process can be chosen and maintained at will. The system is amenable for modeling based on kinetic data and can therefore be optimized on rational grounds. Enzymes isolated from different organisms can be mixed and applied together in homogeneous phase. For this reason the enzyme-membrane reactor appears to have advantages compared to the utilization of whole cells, as long as the number of single reactions taking place remains fairly small and the enzymes employed are sufficiently stable in solution during operation. Results shown in Figs. 9 and 10 indicate that these conditions can be fulfilled. The average enzyme

[3]

COIMMOBILIZATION OF

NAD

AND DEHYDROGENASES

21

activity consumption with respect to both enzymes was less than 300 U/ kg product over a period of 3 months of continuous operation. Acknowledgment The coenzyme preparation was from A. F. Biickmann, and the enzymes were isolated by H. Schiitte and K. H. Kroner. The chemical engineering measurements at continuous operation were carried out by Mrs. U. Mackfeld. The engagement of all these co-workers is gratefully acknowledged.

[3] C o i m m o b i l i z e d S y s t e m o f N A D w i t h D e h y d r o g e n a s e s B y YOSHIMITSU YAMAZAKI a n d HIDEKATSU M A E D A

Recycling of NAD is a prerequisite for the industrial application of dehydrogenases. The most effective way of realizing this is to maintain both NAD and the coupled dehydrogenases in one reactor system (bioreactor) and recycle NAD in situ. Several methods have been developed for this purpose: NAD bound to a water-soluble polymer and the dehydrogenases are either placed all together in an ultrafiltration apparatusf1-3 enclosed with a semipermeable membrane, 4,5 microencapsulated, 6 or immobilized in a collagen membrane. 7 Solid-phase coimmobilization of NAD and a dehydrogenase ~,9 and the covalent binding of an NAD derivative in or near the active site of dehydrogenases ~°,~ have also been reported.

l y . Yamazaki and H. Maeda, Agric. Biol. Chem. 50, 3213 (1986). S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. Btickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). 5 A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). 6 j. Grunwald and T. M. S. Chang, J. Appl, Biochem. 1, 104 (1979). 7 Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). 8 S. Gestrelius, M.-O. M~nsson, and K. Mosbach, Fur. J. Biochem. 57, 529 (1975). 9 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982). 10 M.-O. Mhnsson, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 86, 455 (1978). I~ M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[3]

COIMMOBILIZATION OF

NAD

AND DEHYDROGENASES

21

activity consumption with respect to both enzymes was less than 300 U/ kg product over a period of 3 months of continuous operation. Acknowledgment The coenzyme preparation was from A. F. Biickmann, and the enzymes were isolated by H. Schiitte and K. H. Kroner. The chemical engineering measurements at continuous operation were carried out by Mrs. U. Mackfeld. The engagement of all these co-workers is gratefully acknowledged.

[3] C o i m m o b i l i z e d S y s t e m o f N A D w i t h D e h y d r o g e n a s e s B y YOSHIMITSU YAMAZAKI a n d HIDEKATSU M A E D A

Recycling of NAD is a prerequisite for the industrial application of dehydrogenases. The most effective way of realizing this is to maintain both NAD and the coupled dehydrogenases in one reactor system (bioreactor) and recycle NAD in situ. Several methods have been developed for this purpose: NAD bound to a water-soluble polymer and the dehydrogenases are either placed all together in an ultrafiltration apparatusf1-3 enclosed with a semipermeable membrane, 4,5 microencapsulated, 6 or immobilized in a collagen membrane. 7 Solid-phase coimmobilization of NAD and a dehydrogenase ~,9 and the covalent binding of an NAD derivative in or near the active site of dehydrogenases ~°,~ have also been reported.

l y . Yamazaki and H. Maeda, Agric. Biol. Chem. 50, 3213 (1986). S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. Btickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). 5 A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). 6 j. Grunwald and T. M. S. Chang, J. Appl, Biochem. 1, 104 (1979). 7 Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). 8 S. Gestrelius, M.-O. M~nsson, and K. Mosbach, Fur. J. Biochem. 57, 529 (1975). 9 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982). 10 M.-O. Mhnsson, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 86, 455 (1978). I~ M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

22

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[3]

We report still another method in which the dehydrogenases and a polymerizable NAD derivative are coentrapped in a polyacrylamide gel. The gel is prepared by radical copolymerization, and consequently NAD is bound on the matrix which encloses the enzymes. The purpose of this article is to outline the preparation of the coimmobilized gels of NAD and dehydrogenases and to discuss their application to bioreactors for synthesis and analysis. Synthetic Procedures Preparation o f Polymerizable N A D Derivatives

The principle used is the carbodiimide-mediated coupling of N6-carboxymethyl-NAD (1) with amines containing a methacryloyl group. The amines 2 and 3 (see Fig. 1) were synthesized as described below. Muramatsu et al., ~2 Le Goffic et al., ~3 and Yamazaki et al. 14 also reported methods for synthesis of polymerizable NAD derivatives. 6-(Tritylamino)hexylamine dihydrochloride (4). ~5 To a solution containing 30.6 g of 1,6-diaminohexane and 39.2 g of triethylamine in 200 ml of isopropanol is slowly added a solution of trityl chloride (88 g in 100 ml of isopropanol plus 150 ml of chloroform). The reaction mixture is stirred at room temperature for 3 hr and then the solvents and the remaining triethylamine are removed under reduced pressure. The residual solid is mixed with benzene (1.2 liters) and water (1.2 liters) and vigorously shaken in a separatory funnel. After separation of the two phases, the aqueous layer is discarded and the organic layer is washed successively with water (1 liter) and water containing 2.5% KH2PO4 (0.7 liters x 2). Centrifugation (1800 rpm for 10 min) is necessary to promote the phase separation. As part of the product is contained in the aqueous layers, it is recovered by extraction with benzene (0.5 liters each time). All organic layers are combined and evaporated under reduced pressure. The oily ~2M. Muramatsu, 1. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 13 F. Le Goffic, S. Sicsic, and C. Vincent, Eur. J. Biochem. 108, 143 (1980). 14 y . Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, J. Biochem. (Tokyo) 95, 109 (1984). In this work, stopped-flow reaction kinetics was investigated on the binding of several species of polymer-bound NAD derivatives to yeast alcohol dehydrogenase in the presence of an inhibitor pyrazole. The result showed that the binding of the polymer derivatives of NAD to the enzyme was not essentially weaker and slower than that of native NAD, but was even faster in some cases in spite of an expected large steric hindrance between the polymer derivatives and the enzyme. ~5This section is reprinted with permission from Y. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2091 (1981).

COIMMOBILIZATION OF NAD AND DEHYDROGENASES

[3] H2N 7 ~ f ~ . N H

2 ~ 3 c'CI

.~,~'cooet*

)

~ ) 3 c _ ~ N H

~-4

> ¢, ~C-N~COOEt

CI-

3 DCC

H 3 N ~

~'-7;"°"

>

~'-

5

N

2

__ ,~c-N.'ff"'J'-/""=

23

o

~coo,.occ

6 N

N

~/A¢OH

T,.o,

+ ,c ,.~;o

%~ _"~ TosO-

)

N,~L,,./N

7

3

....--.(R 2or3 NAD-> "> N6-carboxymethyI-NAO ~ I

tk~ ~

o

'%-'%-'

~

: r-o+o-~-o-7 .,o~ I I~Ho~ o- o.

8

oH OH u

9

R

=

o

H

I0 R

OH H /

o H/

: N~7-~.~N~

0

Fro. I. Synthetic scheme of polymerizable NAD derivatives. (b3C-, Trityl group; DCC, dicyclohexylcarbodiimide; TosOH, p-toluenesulfonic acid; AcOH, acetic acid; EDC, ethyldimethylaminopropylcarbodiimide hydrochloride.

residue is dissolved in 1 liter of chloroform. This solution is divided into 4 portions and each is extracted 3 times with 0.5 liters of ice-cold 0.1 M HCI. Phase separation is promoted by centrifugation. The aqueous layers are combined, adjusted to a pH of about 13 with 2 M NaOH, and saturated with NaCI. The aqueous solutions (l.5 liters × 4) are respectively extracted with chloroform (0.5 and 0.3 liters). The chloroform layers are combined, dried over Na2SO4, and evaporated under reduced pressure. The residue is dissolved in 400 ml of ether. Into the solution is bubbled dry HCI gas at - 2 0 ° to give a white precipitate. The gas bubbling is stopped when no more precipitate appears. The precipitate is collected by suction filtration, dried in vacuum, and crystallized from ethanol-ether to give 28 g (25% yield) of 4 as prisms, dec. 207 °. 6-Methacrylamidohexylammonium chloride (2).15 Preparation 4, 27.6 g, is dissolved in a mixture of 40 ml of dimethylformamide, 150 ml of chloroform, and 13 ml of triethylamine. To the ice-cold solution is slowly added a chloroform solution (30 ml) containing 22.4 g of methacrylic acid and a small amount of hydroquinone, and then another chloroform solution (40 ml) containing 28.2 g of dicyclohexylcarbodiimide is added under

24

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[3]

stirring. The reaction mixture is stirred at 4 ° for 15 min and then at room temperature for 1 hr. The formed dicyclohexylurea is filtered off and the filtrate is concentrated to about 50 ml in an evaporator at 40°. To the concentrate is added 800 ml of benzene. The insoluble materials are filtered off and the filtrate is washed successively with alkaline water (pH 13 with NaOH, 300 ml × 2), water (200 ml), and 2.5% KH2PO4 (200 ml x 2). After drying over Na2SO4, the benzene layer is evaporated under reduced pressure. The oily residue, N-[6-(tritylamino)hexyl]methacrylamide, shows a substantially single spot in TLC [Rf = 0.66 on silica gel F254 developed with benzene/methanol (9 : 1), detected under UV light and by the ninhydrin spray method], but crystallization does not occur. So, the total amount of the residue is dissolved in a mixture of acetic acid (70 ml) and water (30 ml) to remove the trityl group. The solution is maintained at 100° for 10 min and then cooled. To the ice-cold solution is added I00 ml of water. The precipitate of triphenylcarbinol is filtered off. A small amount of hydroquinone is added to the filtrate, which is concentrated to about 10 ml in an evaporator at 30°. The addition of water (100 ml), filtration, and concentration are performed again. Then, 100 ml of toluene is added to the concentrate to remove the remaining acetic acid by azeotropic distillation. After concentration under reduced pressure, 70 ml of 1 M HC1 is added to the oily residue. This mixture is concentrated in an evaporator and then the remaining water and acetic acid are removed under reduced pressure as azeotropic mixtures with ethanol and toluene, respectively. The final residue is crystallized from ethanol-ether to give 10 g (69% yield) of 2 as needles, mp 113-114 °. Ethyl 2-(tritylamino)ethanoate (5). Commercially available glycine ethyl ester hydrochloride, 15 g, is dissolved in 160 ml of chloroform. To this solution was added 24 g of triethylamine and 32 g of trityl chloride. The mixture is stirred at room temperature for 6 hr and then 160 ml of benzene is added. The insoluble salt is filtered off and the filtrate concentrated to give a syrupy residue, which is crystallized from methanol. Thus 36 g (98% yield) of 5 is obtained as prisms, mp 116°. N-(3-Amino-2-hydroxypropyl)-2-(tritylamino)acetamide (6). 15 A solution containing 12.5 g of 5, 70 g of 1,3-diamino-2-propanol (Tokyo Chemical Industries Ltd., Tokyo), and 20 g of ethylene glycol in 40 ml of isopropanol is refluxed for 4 hr. The solvents and the unreacted diamine are removed in vacuum, and the resultant glassy residue is dissolved in 200 ml of chloroform. After washing with water (200 ml), the solution is dried over NaESO4 and concentrated under reduced pressure. The residue is finally precipitated from ethanol-ether-hexane to give 6 as a crystalline solid (13.5 g, 96% yield, dec. 138-141°).

[3]

COIMMOBILIZAT1ON OF NAD AND DEHYDROGENASES

25

N - [ 2 - Hydroxy - 3 - [ 2 - ( tritylamino ) acetamido ] propyl ] methacrylamide (7). 15 Preparation 6, 14.3 g, is treated with methacrylic acid and dicyclohexylcarbodiimide in a similar manner to that of 4, but dichloromethane is used instead of chloroform and triethylamine is not used. The reaction mixture is similarly worked up. Then, the washed, benzene layer is divided into 4 portions. Each portion (200 ml) is extracted three times with 0.05 M HCI (ice-cold solutions of 800,500, and 500 ml). The aqueous layers are combined, adjusted to a pH of about 13 with 2 M NaOH, saturated with NaCl, and extracted three times with chloroform [600,400, and 200 ml for each combined solution (1.8 liters)]. All the chloroform layers (1.2 liters x 4) are combined, dried over Na2SO4, and evaporated under reduced pressure. The viscous residue is crystallized from chloroform-benzene to give 7 as fine needles (11.14 g, 66% yield, dec. 155157°). [N-(2-Hydroxy-3-methacrylamidopropyl)carbamoyl ]methylammonium p-toluene sulfonate (3). 15 The trityl group of 7 (11.1 g) is removed with acetic acid in the same way as described for 2. In the purification stage, p-toluenesulfonic acid (4.62 g, monohydrate, dissolved in 10 ml of ethanol) is used instead of HCI since the corresponding hydrochloride is very hygroscopic. The product is precipitated from ethanol-ether as a crystalline solid (7.7 g, 82% yield, dec. 110-115°). N6-[ N-[ N - ( 2 - H y d r o x y - 3 - m e t h a c r y l a m i d o p r o p y l ) c a r b a m o y l m e t h y l ] c a r b a m o y l m e t h y l ] - N A D (9) and N6-[N-(6-methacrylamidohexyl)carba m o y l m e t h y l ] - N A D (10). 16 N6-Carboxymethyl-NAD (1) is prepared according to the method of Mosbach et al. 17 Then 1.2 g o f l , 2.7 g of 3, and 50 mg of hydroquinone are dissolved in 14 ml of water and the pH is adjusted to 4.8 with 2 M LiOH. To the solution is added 0.53 g of ethyldimethylaminopropylcarbodiimide hydrochloride (8, Fluka) with continuous stirring at room temperature. The pH is maintained at 4.8 with 2 M LiOH and 1 M HC1. After l hr, another portion of 8 (0.28 g) is added and the stirring is continued. After completion of the coupling [within 3 hr from the initial addition of 8; monitored by TLC on PEI-ceUulose F (Merck) developed with 0.1 M LiCI], the reaction mixture is applied to a gel permeation column (BioGel P-2, 2 cm x 1.5 m; elution with water). The nucleotides are eluted in effluent 130-220 ml separately from other compounds (the unreacted amines, etc.). The main fractions are pooled and concentrated to about 10 ml in an evaporator at 30°. The concentrate is applied to a Dowex l-X2 column (1.5 x 50 cm, HCOO- form) and the elution is performed by a linear gradient made of 500 ml of 0.2 M formic i6 y . Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2277 (1981). 17 K. Mosbach, P.-O. Larsson, and C. Lowe, this series, Vol. 44, p. 859.

26

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[3]

acid and 500 ml of 2 M formic acid. Fractions of the main peak (effluent 480-600 ml) are combined and concentrated in an evaporator at 30°. An excess amount of chilled acetone is added to the concentrate. The white precipitate is collected by centrifugation, washed with acetone, and dried in vacuum to give 1.05 g of 9 as white powder; 68% yield from 1, based on UV absorption measurement ('~266 = 23.0 × 103 M 1 c m - 1 ) 16 Similarly, 10 is prepared by the coupling of 1 with 2 in 27% yield. These NAD derivatives show a single spot in various TLC systems with the following Rf values: 0.53 (9), 0.74 (10), and 0.46 (NAD) on cellulose F (Merck) developed with isobutyric acid/1 M aqueous NH3 (5/3, v/v) saturated with EDTA; 0.08 (9), 0.03 (10), and 0.16 (NAD) on cellulose F developed with 0.1 M phosphate buffer (pH 6.8)/ammonium sulfate/n-propanol (100/60/2, v/w/v); and 0.57 (9), 0.57 (10), and 0.28 (NAD) on PEI-cellulose F developed with 20 mM LiC1. The spots are located by viewing under UV light (254 nm).

Preparation of Coimmobilized Gel of NAD and Dehydrogenases Enzymes. Yeast formate dehydrogenase (FDH; lyophilized powder), horse liver alcohol dehydrogenase (LADH; suspension in 20 mM phosphate buffer, pH 7.0, containing 10% ethanol), and pig heart diaphorase (dihydrolipoamide dehydrogenase; suspension in 3.2 M ammonium sulfate) were purchased from Boehringer-Mannheim. Thermus thermophilus malate dehydrogenase (MDH; solution in 50% glycerol) was given to us by Dr. N. Miwa of Mitsubishi Petrochemical Co., Ltd. (Ibaraki), whom we wish to thank for his generosity. Standard Assay Procedures. 18 A Hitachi 124 spectrophotometer with a thermostated cell holder is used to follow the absorbance change at 340 nm in the dehydrogenase assays. The following substrate solutions are used: LADH--0.56 M ethanol, 2 mM NAD, 75 mM semicarbazide-HC1, 9.8 mM glutathione, and 62 mM sodium pyrophosphate-18 mM glycine buffer (pH 8.7) in a total volume of 3.00 ml; FDH--35 mM sodium formate, 1 mM NAD, and 87 mM potassium phosphate buffer (pH 7.5) in a total volume of 3.00 ml; MDH--0.52 mM potassium oxaloacetate, 0.2 mM NADH, 98 mM potassium phosphate buffer (pH 7.5) in a total volume of 2.98 ml; and diaphorase--5 mM sodium olAipoate, 0.2 mM NADH, 0.3 mM NAD, 0.94 mM EDTA, 0.69 mg/ml bovine serum albumin (Miles), and 80 mM potassium phosphate buffer (pH 5.9) in a total volume of 2.88 ml. The reaction is started by addition of the enzyme solution (20 p.l) and is This section is reprinted with permission from Y. Yamazaki and H. Maeda, Agric. Biol. Chem. 46, 1571 (1982).

[3]

COIMMOB|LIZATION OF

NAD

AND DEHYDROGENASES

27

the temperature is maintained at 30° in all cases. Proteins are determined by Lowry's method with bovine serum albumin as standard. ~9 F D H - M D H - N A D Gel. 2° The gel containing FDH, MDH, and NAD is prepared as follows: 15 mg (14.7/xmol) of the polymerizable NAD derivative 9 is dissolved in 0.15 ml of 0.05 M sodium pyrophosphate buffer (pH 8.5) and neutralized with 0.1 M NaOH. To the solution are added 0.69 g of acrylamide (recrystallized from benzene), 36.3 mg of methylenebisacrylamide, and 1.1 ml of the pyrophosphate buffer. Nitrogen gas is bubbled into the solution for 5 min under cooling with ice. Then, the following solutions are added successively to the above solution: 0.25 ml of MDH solution (in 50% glycerol; containing 342 U and 3.08 mg protein), 0.3 ml of FDH solution (in 0.1 M phosphate buffer, pH 7.5; containing 7.02 U and 2.93 mg protein), and two 0.15-ml solutions in the pyrophosphate buffer containing 4.5 mg of K2S208 and 11.25 mg of dimethylaminopropionitrile. The final solution is removed from the ice bath and N2 gas is bubbled into it at room temperature for about 5 min until viscosity increases. Then the solution is kept in the ice bath for 20 min. The formed gel is crushed in a blender with 50 ml of ice-cold 0.1 M phosphate buffer, pH 7.5, and thoroughly washed with the buffer on a Btichner funnel. The total weight of the gel (in a wet state) was 3.51 g. The particle size (average of the major and minor diameters) of the crushed gel was in the range of about 0.01-0.8 mm. The protein content, enzyme activity, and NAD content of the gel and their immobilization yield or recovery were as follows: protein content, 1.38 mg/g (81%); FDH, 0.16 U/g (8%); MDH, 31 U/g (32%); NAD, 3.6 /zmol/g (86%). Assay procedures for the gel are described below. L A D H - d i a p h o r a s e - N A D Gel. ~8 This gel is prepared as described above, but using the following amounts of reagents: 60 mg (56.4/zmol) of the polymerizable NAD derivative 10 in 0.2 ml of the pyrophosphate buffer (pH 8.5), 1.2 g of acrylamide, 66 mg of methylenebisacrylamide, 6 mg of bovine serum albumin, 1.5 ml of the buffer, 0.6 ml of LADH suspension (20.1 U and 6.60 mg protein), 0.6 ml of diaphorase suspension (137 U and 6.42 mg protein), 3 mg K2S208 in 0.15 ml of the buffer, and 7.5 mg dimethylaminopropionitrile in 0.15 ml of the buffer. This gel showed the following properties: protein, 2.43 mg/g (59% recovery); LADH, 0.23 U/g (5%); diaphorase, 3.7 U/g (12%); NAD, 7.5/~mol/g (61%). 19 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 2o This section is reprinted with permission from Y. Yamazaki, H. Maeda, and K. Kamibayashi, Biotechnol. Bioeng. 24, 1915 (1982).

28

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[3]

Estimation o f Immobilized Enzyme Activity, Protein, and N A D 18 The activities of the immobilized dehydrogenases were assayed by the flow method using a substrate solution of the same composition as in the standard assay procedure. The apparatus consisted of a test tube held in a water bath (30°) and a flow system with a flow cuvette in the spectrophotometer and a peristaltic pump. A net was attached at the inlet of the flow system to filter the gels. At first, the flow system was filled with the substrate solution (total volume of 1.3 ml) and then another substrate solution (three times the volume in the standard procedure) was added to the test tube. After equilibration for 3 min at 30°, the reaction was started by addition of the gel to the test tube with a constant circulation of the solution through the whole system at a flow rate of 2.3 ml/min. The reaction mixture in the test tube was also stirred by a magnetic stirrer. The absorbance change at 340 nm was continuously monitored. The experiment was repeated with different amounts of the gel and the free enzyme. The reaction rate (A340/min) was proportional to the number of milligrams of the wet gel as well as the number of units of the free enzyme determined by the standard method. From these data the enzyme activity (U/g wet gel) was calculated. The immobilized proteins were estimated by amino acid analysis (with a Hitachi 835 amino acid analyzer with an autosampler) after acid hydrolysis. A sample of 100-400 mg of the gel was mixed with 15 ml of 6 M HC1. The mixture (in a sealed vial) was kept at 120° for 30 hr and then the insoluble materials were removed by filtration. A 13-ml portion of the filtrate was evaporated to dryness at 60°. The residue was redissolved in 1.5 ml of 0.06 M HC1 and applied to the analyzer. The protein content (mg/g wet gel) was calculated from the combined peak area of 11 species of amino acids (Asp, Thr, Set, Glu, Pro, Ala, Val, Ile, Leu, Phe, and Lys; other amino acids had decomposed or overlapped with unknown compounds on the chromatograms) using the corresponding calibration curves which were prepared with the hydrolyzates of the free proteins (FDH + MDH and LADH + diaphorase + albumin, mixed in the same ratios as in the immobilization). The combined peak area was proportional to the used amount of the gels as well as that of the free proteins. The coexistence of polyacrylamide gels had no effect on this protein analysis as shown by a comparative experiment with albumin plus protein-free gels and albumin only. The NAD content was estimated from phosphate determinationsz~ after exhaustive washing of the gel with 1 M NaCl. 21 G. R. Bartlett, J. Biol. Chem. 234, 466 (1959).

[3]

COIMMOBILIZATION OF NAD AND DEHYDROGENASES

29

Applications

Continuous Production of L-Malate by the Coimmobilized F D H - M D H - N A D Gel 2° The principle is the coupled reaction: formate

matrix-bound FDH /

CO2

~ matrix-bound ) NADH

t.-malate MDH ~ oxaloacetate

The gel (1.4 g) was packed in a column tube and a solution containing 0.2 M sodium formate and 2 mM sodium oxaloacetate in 0.1 M phosphate buffer (pH 7.5) was passed upward through the column at a flow rate of 2.4 ml/hr. The temperature was kept at 30°. The substrate solution was saturated with chloroform before use for preventing bacterial growth in either the solution or the column. It was further kept in an ice bath and changed to a freshly prepared one, usually every 12 hr. At the points indicated by arrows A and E in Fig. 2, the substrate solution was changed to a solution lacking only formate. It was again changed to the original substrate solution as indicated by arrows B and F. A similar experiment was run for removal and addition of oxaloacetate at the points C and D. The eluent from the column was divided into fractions of 1-hr duration and then held at 100° for 5 min to decompose the unreacted oxaloacetate. The L-malate in the fractions was determined by the method using hydrazine, NAD, and pig heart malate dehydrogenase (Boehringer-Mannheim). 22 The result is shown in Fig. 2. The L-malate production by the F D H - M D H - N A D gel was continued for 3 weeks. When formate was removed from the substrate solution (as indicated by arrow A), the L-malate production ceased. It was, however, restored to the original level by the addition of formate to the solution. This result clearly shows that L-malate was produced with recycling of the matrix-bound NAD. The L-malate production was, of course, stopped by the removal of oxaloacetate from the substrate solution (as indicated by arrow C) and resumed by the addition of the substrate. The concentration of the produced L-malate gradually decreased with time, but even at the final stage of the operation about 60% the initial value was observed and 22O. H. Lowry and J. V. Passonneau, in "A Flexible System of Enzymatic Analysis," p. 203. Academic Press, New York, 1972.

30

MULTISTEP ENZYME SYSTEMS AND COENZYMES

3E E

[3]

LO

O

0

J

I I00

~ i

L

I

r

,

3

,

~

5

-~'B , , 7

200 .--

400

300

_ 1 ~ * _ F r o c t l o n NO. h (.,,/U, , , , 9 II 13

,

,

15

L

, 17

500 ,E ~" 19

'~,'F ,

i 21

Days

FI6.2. Continuous production of L-malate in a column reactor packed with the F D H M D H - N A D gel. 2° The arrows indicate the removal of formate (A and E) and oxaloacetate (C) from the substrate solution and the addition of the removed substrate to the solutions (B, D, and F).

the recycling of the matrix-bound NAD was again confirmed by the second experiment concerning the removal and addition of formate at the points E and F. The total amount of L-malate produced during the operation was about 1300/zmol. Therefore the turnover number for each NAD molecule was 260 cycles, assuming that the total number of bound NAD molecules were available to the enzymes. The gel was recovered from the column and the contents of protein, the enzymes, and NAD were determined. The data and the residual percentages were as follows: protein, 0.77 mg/g (56%); FDH, 0.16 U/g (100%); MDH, 1.4 U/g (5%); NAD, 3.6 /xmol/g (100%). The value of NAD was obtained from phosphate analysis and the residual percentage of enzymatically active form could not be determined. In spite of this ambiguity, it can be concluded that long-term production of L-malate was achieved by the stable supply of the matrix-bound NADH. This is because the rate-limiting step of the coupled reaction is considered to be the reaction of FDH since the content of FDH was remarkably less than that of MDH, and moreover, no loss of FDH was found for the recovered gel. Therefore, the gradual decrease in the L-malate production by the F D H M D H - N A D gel must be due to the loss of MDH. This enzyme is very

[3]

COIMMOBILIZATION OF NAD AND DEHYDROGENASES

31

Sample solution

r~x ,.~,~jlnjector I

~

~

.~-" ~._r~ '~ J

.~

~

Gels of coimmobilized

/

[~I I

~a[~]l I

LADH-Diaphorase-NAD spectrophotometel E x 560nm

i~ ~i;_~ ~'~ Em580nm I~---~ ;--'L~L____~ FlOwcell ~,

(+~-Lipoatesolution j / ~

Resazur n so ut on

FIG. 3. Illustration of the ethanol analyzer using the coimmobilized LADH-diaphoraseNAD gel. ~sThe solutions in the two reservoirs are mixed at an equal flow rate by a peristaltic pump and sent to the column via the injector (Rheodyne, Model 7125). The column is packed with the L A D H - d i a p h o r a s e - N A D gel (5 x 110 mm). The eluent is led to the flow cell (14 p.l) in the fluorescence spectrophotometer (Hitachi 204), which is operated at an excitation wavelength of 560 nm and an emission wavelength of 580 nm. When a solution containing ethanol is added by the injector, resorufin is formed in the column by the coupled reaction of LADH and diaphorase, and the amount thereof (detected as fluorescence intensity) is continuously monitored.

stable as it maintains the whole activity in the incubation at 70° for 3 hr. 23 The reduced protein content of the recovered gel suggests a leakage of MDH from the gel. This was supported by the following experiment. A gel containing only MDH was similarly prepared, packed in a column, and continuously washed with the above substrate solution for 3 weeks in the same way as in the operation of the reactor column. The residual percentages of enzyme activity and protein content of the recovered gel were 4 and 15%, respectively. It was therefore concluded that MDH was removed with time from the reactor column by leakage, but that since its content was large compared with the content of FDH, the loss of MDH was not sharply reflected in the rate of L-malate production.

Ethanol Analyzer Using the Coimmobilized LADH-Diaphorase-NAD Gel 18 The L A D H - d i a p h o r a s e - N A D gel was applied to an ethanol analyzer composed of a column reactor and a fluorescence spectrophotometer (Fig. 3). The system is based on the enzymatic oxidation of NADH with 23 N. Miwa, personal communication (1981).

32

MULTISTEP ENZYMESYSTEMSAND COENZYMES

[3]

"G

i

_=

\

B

1T

[ EtOH] I0 (rnM)

25

50

I0

25

50

I0

25

5O

FIG. 4. Reproducibility of the ethanol analyzer/8 To the system shown in Fig. 3 were applied 0.12-ml solutions of ethanol in 0.05 M phosphate buffer (pH 7.5). The ethanol concentrations are given in the figure. This figure shows the trace of the fluorescence intensity. The operational conditions were as follows: buffer, the same as above; DLlipoate, 5 mM in the reservoir; resazurin, 32 tzM in another reservoir; temperature, 25°; flow rate, 0.95 ml/min; column size, 5 x 110 mm; sensitivity of the photometer, 6 x 10; recorder range, 5 mV. Injection points are indicated by arrows. resazurin, 24 but practically, lipoate (a natural substrate of diaphorase) was

also used to promote the oxidation. The response of this analyzer (the ethanol ~

/matriN-bDUnd ~x~

LADH

/

~N/~"~/"O~o~~

O

~hor~ (fluorescent)

acetaldehyde

0~.~.~

matrix-bound NADH

0 ....~jO

-

N O resazurin sum of fluorescences represented by peak areas) was reproducible with regard to the applied amount of ethanol as shown in Fig. 4. The response increased with increasing ethanol concentration but seemed to saturate at high concentration. The fluorescence intensity reached maximum at 4.5 min after the sample injection at a flow rate of 0.95 ml/min and then gradually returned to its original level. The time required for the return and the response intensity were affected by many factors: pH, temperature, concentrations of lipoate and resazurin, column size, and flow rate. From an investigation on these factors, :8 the optimal conditions were chosen as given in the legend to Fig. 4. The long-term use of the ethanol 24G. G. Guilbault and D. N. Kramer, Anal.

Chem.

37, 1219 (1965).

[3]

COIMMOBILIZATION OF N A D AND DEHYDROGENASES

33

analyzer was tested in the same way as in Fig. 4, but the temperature was maintained at 15° during the operation. Between 9 and 12 samples were applied each day, and then the column was washed with the lipoate solution only and stored in a refrigerator until the next operation. Thus, this analyzer was used 84 times in 1 week for a total operation time of 41 hr. The responses gradually decreased with time, but about 50% of the initial value was still obtained at the final stage. The residual contents (percentage relative to the initial value) of the enzymes and NAD in the recovered gel were as follows: protein, 87%; LADH activity, 41%; diaphorase activity, 21%; and NAD, 100% as phosphate recovery. In a stability experiment it was found that both the free enzymes maintained 100% of the initial activity for 43 hr at 15° in a solution of the same composition as in the analyzer column. The decrease of the analyzer response is due to the loss of the enzymes and it must have been caused by leakage from the gel. Repeated use of NAD as an analytical reagent has also been studied by Davies and Mosbach 4 and Malinauskas and Kulys 5 based on a potentiometric or amperometric procedure. Their systems use a semipermeable membrane to retain a water-soluble polymer-bound NAD along with enzymes. Concluding Remarks As this article shows, the entrapment of dehydrogenases in polyacrylamide gels prepared with the usual monomers plus a radically polymerizable NAD derivative is a useful technique for the immobilization of coupled enzyme systems including the coenzyme. The advantages of the present coimmobilization method are (1) the simplicity, in that NAD and the enzymes are simultaneously immobilized in a single step, and (2) the applicability of the resultant gel to simple column-type reactors, without any need for an additional material such as ultrafiltration membrane. The principal disadvantage is that the reaction mixture cannot be supplemented with the enzyme even when it has been removed from the gel and the bound NAD still remains intact. The ultrafiltration method has an advantage in this connection. Thus, it is essential in the present method to use stable enzymes. The second important problem of this method is the leakage of enzymes from the gels. The molecular weight of Thermus MDH has been found to be relatively low (6 × 104).23 A cross-linking pretreatment to increase its molecular size or preparation of the gel at higher monomer concentrations (to reduce the pore size of the gel 25,z6) z5 C. J. O. R. Morris, in "Protides of the Biological Fluids" (H. Peeters, ed.), p. 543. Elsevier, Amsterdam, 1%6. 26 j. S. Fawcett and C. J. O. R. Morris, Sep. Sci. 1, 9 (•966).

34

MULTISTEP

ENZYME

SYSTEMS AND COENZYMES

[4]

would be effective to prevent the leakage. In contrast, FDH was well retained in the gel even though this enzyme is also not such a large protein (MW = 7.4 x I04, information from the maker). These facts suggest that the degree of leakage of proteins from the gel is dependent not only on the molecular size of the proteins but also on other factors, for instance, their shape and affinity to the matrix. It may be possible to increase the effect on enzyme retention of the affinity factor by selecting more suitable gels and/or chemically modifying the surface of the proteins. Since the present work has shown that long-term recycling of matrix-bound NAD by the simultaneously immobilized enzymes is possible, this method can be expected to find wide use once the above problem of the leakage has been solved.

[4] P o l y m e r i z a b l e N A D D e r i v a t i v e a n d M o d e l E n z y m e Reactor with Recycling of Polyethylene Glycol-Bound NAD By HIROSUKE OKADA and ITARU URABE

Many enzymes require the participation of readily dissociable cofactors such as NAD for their catalytic activities. The continuous utilization of these enzymes requires the retention and regeneration of the coenzymes. For this purpose, several methods of covalently attaching NAD to water-soluble polymers have been reported. 1,2These NAD derivatives are also useful for the study of the interaction between enzymes and NAD. In this chapter, methods for preparing a polymerizable NAD derivative, N A D - N 6-[N-( N-acrylo yl- l -metho x ycarbon yl- 5-aminopentyl )propio amide], and a polyethylene glycol-bound NAD derivative (PEG-NAD) are described (Fig. 1). The polymerizable NAD is a unique derivative, which easily copolymerizes with other vinyl monomers such as acrylamide to give macromolecular NAD derivatives (polymeric NAD). 3 Furthermore, various kinds of polymeric NAD can be obtained by copolymerization with different vinyl monomers in different molar ratios: .~ From investigations into the coenzymatic properties of the polymeric NAD, it has been suggested that NAD derivatives of smaller molecular 1 K. Mosbach, P.-O. Larsson, and C, Lowe, this series, Vol. 44, p. 859. 2 C. R. Lowe, in "Topics in Enzyme and Fermentation Biotechnology" (A. Wiseman, ed.), Vol. 5, p. 13. Horwood, Chichester, England, 1981. 3 M. Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 4 S. Furukawa, Y. Sugimoto, I. Urabe, and H. Okada, Biochimie 62, 629 (1980). 5 S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochern. 114, 101 (1981).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

34

MULTISTEP

ENZYME

SYSTEMS AND COENZYMES

[4]

would be effective to prevent the leakage. In contrast, FDH was well retained in the gel even though this enzyme is also not such a large protein (MW = 7.4 x I04, information from the maker). These facts suggest that the degree of leakage of proteins from the gel is dependent not only on the molecular size of the proteins but also on other factors, for instance, their shape and affinity to the matrix. It may be possible to increase the effect on enzyme retention of the affinity factor by selecting more suitable gels and/or chemically modifying the surface of the proteins. Since the present work has shown that long-term recycling of matrix-bound NAD by the simultaneously immobilized enzymes is possible, this method can be expected to find wide use once the above problem of the leakage has been solved.

[4] P o l y m e r i z a b l e N A D D e r i v a t i v e a n d M o d e l E n z y m e Reactor with Recycling of Polyethylene Glycol-Bound NAD By HIROSUKE OKADA and ITARU URABE

Many enzymes require the participation of readily dissociable cofactors such as NAD for their catalytic activities. The continuous utilization of these enzymes requires the retention and regeneration of the coenzymes. For this purpose, several methods of covalently attaching NAD to water-soluble polymers have been reported. 1,2These NAD derivatives are also useful for the study of the interaction between enzymes and NAD. In this chapter, methods for preparing a polymerizable NAD derivative, N A D - N 6-[N-( N-acrylo yl- l -metho x ycarbon yl- 5-aminopentyl )propio amide], and a polyethylene glycol-bound NAD derivative (PEG-NAD) are described (Fig. 1). The polymerizable NAD is a unique derivative, which easily copolymerizes with other vinyl monomers such as acrylamide to give macromolecular NAD derivatives (polymeric NAD). 3 Furthermore, various kinds of polymeric NAD can be obtained by copolymerization with different vinyl monomers in different molar ratios: .~ From investigations into the coenzymatic properties of the polymeric NAD, it has been suggested that NAD derivatives of smaller molecular 1 K. Mosbach, P.-O. Larsson, and C, Lowe, this series, Vol. 44, p. 859. 2 C. R. Lowe, in "Topics in Enzyme and Fermentation Biotechnology" (A. Wiseman, ed.), Vol. 5, p. 13. Horwood, Chichester, England, 1981. 3 M. Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 4 S. Furukawa, Y. Sugimoto, I. Urabe, and H. Okada, Biochimie 62, 629 (1980). 5 S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochern. 114, 101 (1981).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

['fir]

POLYMERIZABLE N A D DERIVATIVE AND P E G - N A D

L- Lysine 1) CHz=CHCOCI 2) HCIICH3OH C.OOCH3 H2NCH(CH2)4NHCOCH=CH 2 i

F

I

;oocm

35

NAD Polyethylene glycol 1) Propiolactone I SOCt2 ~v 2) Rearrangement CI(CHzCHtO)nCHtCHzCI NHCH2CH2COOH I NH~ J. ~CONH 2 ~' N" j'~I---N ~ ~t" H2N(CH2CH20)nCH2CH2N H2 /

I,"-C2<..bo.yot,yl)-N,ol

NHCHiCHiCONHCH(CHi)4NHCOCH=CH2 L~N~t~1 N N ~ CONH2

R--P--P--I~

IPolymerizable NADI

I Copotymerization

I C.rbod,,r.,de

I

J ~ NHCHtCH2CONH(CHzCHzO)nCH2CH2N Hz L~N I ~ N iI N N ~'~cONH2 !

i

R--P--P--R

I Polyethylene glycol -bound NADI

IPotym.eric NAD] FIG. 1. Synthesis of a polymerizable NAD derivative and a polyethylene glycol-bound NAD derivative. R, Ribose; P, phosphate. size and lower NAD content in the polymer chain have higher cofactor activity. Therefore, we have prepared P E G - N A D , which has only one N A D moiety located at a terminal o f the linear, flexible, and hydrophilic chain o f polyethylene glycol. P E G - N A D has good cofactor activity and is applicable in a continuous e n z y m e reactor. 6,7 To use these macromolecular NAD derivatives in an e n z y m e reactor, it is necessary to understand the behavior o f the system in which the reactions of dehydrogenases are coupled by the recycling of the NAD derivative. Methods for a theoretical analysis and the experimental operation of a model e n z y m e reactor containing lactate and alcohol dehydrogenases and P E G - N A D are also described. 7 Preparation and Polymerization of Polymerizable NAD Derivative N6-(2-Carboxyethyl ) - N A D

N A D (4.0 g, 5.6 mmol) is dissolved in a small volume of water (about 10 ml), and the pH of the solution is adjusted to 6 with 2 M LiOH. 6 s. Furukawa, N. Katayama, T. Iizuka, 1. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 7 N. Katayama, I. Urabe, and H. Okada, Eur. J. Biochem. 132, 403 (1983).

36

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[4l

3-Propiolactone (1 ml, 16 mmol) is added to the solution, and the reaction mixture is stirred in the dark at room temperature, its pH being kept at 6 with a pH-stat (Hiranuma, PS-11) charged with 2 M LiOH. A substantial amount of propiolactone is hydrolyzed to hydroxypropionic acid and consumes LiOH. When the rate of LiOH addition slows down, propiolactone (1 ml) is further added. The alkylation of NAD is monitored by thin-layer chromatography on silica gel (solvent system A: isobutyric acid/water/ 28% aqueous NH3 = 66/33/1.7, by volume), and about 80% of NAD is alkylated after 2 days with the addition of a total of 8 ml of propiolactone. The rate of the alkylation is almost constant within the pH range of 5-7, but the rate of the hydrolysis of propiolactone increases with the increase in pH. The reaction mixture is then acidified to pH 3 with 6 M HCI and poured slowly into 20 volumes of vigorously stirred cold acetone/ethanol (1/1). The precipitate is collected by centrifugation, washed with acetone/ ethanol (1/1), then acetone, and dried in a vacuum. The crude 1-(2-carboxyethyl)-NAD (4.8 g) is dissolved in 200 ml of 1.3% NaHCO3 (pH 8.5). The solution is deaerated with N2 gas, kept at 45 °, and Na2S204 (2 g) is added. The vigorous bubbling of N2 gas is continued for 5 min, and the ratio of the absorbances at 340/260 nm reaches a value between 4 and 5. The pH of the solution is readjusted to 8.5, the reduction is terminated by bubbling air through the solution for 10 min at room temperature, and the pH is then raised to 11 with 2 M LiOH. The solution is incubated for 2 hr at 70° with the pH kept at 11, and 1-(2carboxyethyl)-NADH is rearranged to N6-(2-carboxyethyl)-NADH. The rearrangement is monitored by the change in the ultraviolet spectrum, of which the absorption maximum shifts from 260 to 266 nm and the shoulder at 300 nm disappears. The solution is cooled to room temperature, pH is adjusted to 7.3 with 2 M HCI, and 80% acetaldehyde (2 ml) and yeast alcohol dehydrogenase (1000 U) are added. The pH of the solution is kept at 7.3 with the pH-stat charged with 2 M HCI. The enzymatic oxidation is followed spectrophotometrically until the absorbance at 340 nm almost disappears (overnight). The solution is acidified to pH 3 with 2 M HCI, concentrated at 30° under reduced pressure, and poured into 20 volumes of vigorously stirred cold acetone/ethanol (1/1). The precipitate is collected by centrifugation, washed with acetone/ethanol (1/1) and acetone, and dried. The crude N6-(2-carboxyethyl)-NAD is dissolved in water and chromatographed over a DEAE-Sephadex A-25 (CI-) column (2.5 x 30 cm) with a linear gradient of 0-250 mM LiCI. The ultraviolet spectra in water and in 1 M KCN 8 of the effluent are monitored, and the fractions contains S. P. Colowick, N. O. Kaplan, and M. M. Ciotti, J. Biol. Chem. 191, 447 (1951).

[4]

POLYMERIZABLE NAD DERIVATIVEAND PEG-NAD

37

ing the desired product (eluted at about 130 mM LiC1) are combined and concentrated. Precipitation with acetone/ethanol (1/1) as above gives N 6(2-carboxyethyl)-NAD in a yield of about 30% (1.3 g). N6-(2-Carboxyethyl)-NAD shows an ultraviolet spectrum in water with an absorption maximum at 266 nm (e = 18,600 M -j cm -1) and a nuclear magnetic resonance spectrum in 2H20 with aliphatic side-chain proton signals at 8 = 2.56 (2H, t, CH2COO) and 3.65 ppm (2H, t, N6-CH2) in addition to the NAD proton pattern. The Rr values of thin-layer chromatography on silica gel in solvent system A of NAD, 1-(2-carboxyethyl)NAD, and N6-(2-carboxyethyl)-NAD are 0.40, 0.14, and 0.37, respectively.

N~-Acryloyl-t-lysine Methyl Ester Acryloyl chloride is prepared according to the procedure of Stempel et L-Lysine monohydrochloride (27.6 g, 0.15 mol) is dissolved in hot water (70 ml), and basic cupric carbonate (15 g) is added to the solution. After addition of 4 M NaOH (37.5 ml) and 2,6-bis(tert-butyl)-p-cresol (50 mg), the solution is chilled to - 5 °, and both acryloyl chloride (13.7 g, 0.15 mol) and 4 M NaOH (37.5 ml) are added dropwise to the solution at - 5 ° over 1.5 hr with vigorous stirring. The pH of the reaction mixture is adjusted to 6 with 5 M HC1, and the precipitate formed is collected by filtration and washed with water. The precipitate is suspended in water (150 ml), dissolved by lowering the pH of the solution to 1.5 with 5 M HCI, and reprecipitated by raising the pH to 6 with Na2CO3. The precipitate is collected by filtration and washed with water to yield cupric N ~acryloyl-L-lysinate. The product is suspended in water (150 ml) and dissolved by acidification with 5 M HCI. Hydrogen sulfide is introduced into the solution, and cupric sulfide formed is filtered off. The filtrate is evaporated at 30 ° under reduced pressure. To the residue ethanol is added and evaporated to remove water. N~-Acryloyl-L-lysine hydrochloride is obtained in crystalline form. The product is converted to its methyl ester by treatment with 21% methanolic hydrogen chloride (200 ml) for 48 hr at room temperature. N~-Acryloyl-L-lysine methyl ester hydrochloride is obtained as brown syrup in a yield of about 50% (20.5 g). The nuclear magnetic resonance spectrum of the product in ZHEO shows these proton signals: 6 = 1.3-1.7 (4H, m, "y,~-CH2), 1.92 (2H, m, flCH2), 3.26 (2H, m, e-CH2), 3.77 (3H, s, C O O C H 3 ) , 4.09 (1H, t, a-CH), 5.66 (IH, dd, COC--~-~CH), 6.07-6.23 ppm (2H, m, C O C H ~ C H ) . The Rf values of thin-layer chromatography on silica gel in propanol/ethyl aceal. 9

9 G. H. Stempel, R. P. Cross, and R. P. Mariella, J. Am. Chem. Soc. 72, 2299 (1950).

38

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[4]

tate/water/28% aqueous NH3 (6/1/3/1, by volume, solvent system B) of L-lysine. HC1, cupric N~-acryloyl-L-lysinate, N~-acryloyl-L-lysine • HCI, and N~-acryloyl-L-lysine methyl ester. HC1 are 0.03, 0.82, 0.65, and 0.84, respectively. NAD-Nr-[N-(N-Acryloyl-l-methoxycarbonyl-5aminopentyl )propionamide]

N~-Acryloyl-L-lysine methyl ester hydrochloride (2.5 g, 10 mmol) is dissolved in water (10 ml), and the pH is adjusted to 4.5 with 2 M LiOH. To the solution are added N6-(2-carboxyethyl)-NAD (0.5 g, 0.7 mmol), hydroquinone (10 mg), and aqueous solution (1 ml) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.34 g, 7 mmol). The reaction mixture is stirred for 20 hr at room temperature, and its pH is kept at 4.5. NAD-N6-[N-(N-Acryloyl-l-methoxycarbonyl-5-aminopentyl)pro pionamide] (polymerizable NAD derivative) is obtained by the precipitation with acetone/ethanol (1/1) and by the chromatography on DEAESephadex A-25 column as described above. The yield is about 90% (0.4 g), the overall yield from NAD being approximately 25%. The polymerizable NAD derivative shows an Rr value of 0.56 on thinlayer chromatography on silica gel in solvent system A, an ultraviolet spectrum in water with an absorption maximum at 266 nm (e = 22700 M-1 cm-1), and a nuclear magnetic resonance spectrum in 2HzO with characteristic side-chain proton signals at 8 = 1.1-1.5 (4H, m, 3,4-CH2), 1.6-1.8 (2H, m, 2-CH2), 3.11 (2H, m, 5-CH2), and 3.62 ppm (3H, s, COOCH3) for the 1-methoxycarbonyl-5-aminopentyl group, 8 = 2.75 ppm (2H, m, CH2CO) for the propionamide group, and 8 = 5.63 (1H, dd, C O C ~ C H ) and 6.0-6.2 ppm (4H, m, C O C H = C H combined with two anomeric protons of the ribose) for the vinyl group. The NAD derivative has high cofactor activities (70-90%) relative to native NAD for alcohol (horse liver and yeast), lactate (rabbit muscle), and malate (pig heart) dehydrogenases. Polymeric NAD Derivative

Copolymerization is carried out in an aqueous reaction mixture containing 1 mM NAD-N6-[N-(N-acryloyl-l-methoxycarbonyl-5-aminopen tyl)propionamide], 300 mM acrylamide, 0.8% 3-(dimethylamino)propionitrile (pH 7.5), and 0.2% potassium persulfate. N2 gas is bubbled through the solution for 3 min, and the solution is kept in an ice bath for 2 hr. The reaction mixture is then dialyzed thoroughly against water, and a solution of polymeric NAD derivative is obtained in a yield of more than 90%. The polymeric NAD derivative shows an identical ultraviolet spectrum to the polymerizable NAD derivative, and is reduced completely

[4]

POLYMERIZABLE N A D DERIVATIVE AND P E G - N A D

39

with yeast alcohol dehydrogenase. The relative molecular mass is about 5 x l05. The relative cofactor activities (NAD -- 100) for alcohol and malate dehydrogenases are 15-35%, but for lactate dehydrogenase, the NAD derivative is a competitive inhibitor with respect to NAD. The conditions of the polymerization described above are very flexible. For example, the molar ratio of the polymerizable NAD derivative and acrylamide can freely be changed to give polymeric NAD derivatives with different NAD densities. The comonomer, acrylamide, can also be replaced with other compounds having an acryloyl group. It should be pointed out that the polymeric NAD derivatives thus obtained are heterogeneous mixtures of various polymer chains especially with respect to their molecular size. The cofactor activity of an NAD moiety on a polymer chain may depend on its position in the polymer chain and on the size of the polymer. These heterogeneities should be borne in mind when interpreting the properties of macromolecular NAD derivatives. Preparation of Polyethylene Glycol-Bound NAD(H)

a,co-Dichloropoly(ethylene glycol) Polyethylene glycol (Mr 3000-3700, 30 g) is dissolved in CHCI3 (55 ml), and dry pyridine (0.2 ml) is added. Thionyl chloride (30 ml) is added dropwise to the solution at 30° with stirring. After refluxing for 8 hr, the reaction mixture is chilled in an ice bath, and water (100 ml) is added to decompose excess thionyl chloride. The water layer is extracted with CH2C12, and the organic layers are combined and dried over anhydrous Na2SO4, and the solvent is evaporated. The product is precipitated from the residue ( - l 0 ml) by addition of ether (200 ml), harvested by filtration, and dried. Yield: 29 g; content of Cl: 2.6%.

a,to-Diaminopoly(ethylene glycol) a,to-Dichloropoly(ethylene glycol) (30 g) is converted into a,codiaminopoly(ethylene glycol) by heating in ethanol saturated with ammonia as described by Biickmann et al.l° After evaporation, ether is added and the precipitate is harvested by filtration and dried. The precipitate is dissolved in 5% NaHCO3 (-50 ml), extracted with CH2C12,concentrated, and precipitated with ether as described above. The crude c~,co-diaminopoly(ethylene glycol) is dissolved in water and chromatographed over a SP-Sephadex C-25 (H +) column (4 x 80 cm) with a linear gradient of 0-40 mM NaCl. The concentration of amino group in the effluent is measured 10 A. F. Biickmann, M. Morr, and G. Johansson, Makromol. Chem. 182, 1379 (1981).

40

M U L T I S T E P E N Z Y M E SYSTEMS AND COENZYMES

[4]

using 2,4,6-trinitrobenzenesulfonic acid," and the fractions containing the desired product are combined, concentrated, and extracted with CH2CI2. The organic layer is dried over anhydrous NaESO4, evaporated to remove the solvent, and tx,o-diaminopoly(ethylene glycol) is obtained in a yield of about 70% (22 g).

Polyethylene Glycol-Bound NAD a,to-Diaminopoly(ethylene glycol) (20 g) is dissolved in water (30 ml), the pH is adjusted to 4.5 with 2 M HCI, and N6-(2-carboxyethyl)-NAD (1 g) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide.HCI (4.6 g) are added to the solution. The reaction mixture is stirred for 24 hr at room temperature with the pH kept at 4.5, dialyzed against water by use of a Spectrapor 6 dialysis membrane (Mr cutoff 1000, Spectrum Medical Industries Inc.), and passed through a SP-Sephadex C-25 (H +) column (4 × 60 cm), a DEAE-Sephadex A-25 (C1-) column (1.5 × 20 cm), and then the SP-Sephadex C-25 column. The eluted fractions are combined, concentrated, and lyophilized to yield polyethylene glycol-bound NAD (PEGNAD, 2.4 g). The preparation of PEG-NAD contains about 0.2 g of a,todiaminopoly(ethylene glycol), which is removed by repeated passage through the SP-Sephadex C-25 column. P E G - N A D shows an ultraviolet spectrum in water with an absorption maximum at 266 nm (e = 23,500 M 1 cm-l), and good cofactor activity for various kinds of dehydrogenases.

Polyethylene Glycol-Bound NADH P E G - N A D is reduced to PEG-NADH with yeast alcohol dehydrogenase in a reaction mixture (10 ml) containing 50 mM Tris-HCl buffer (pH 9.0), 100 mM ethanol, 50 mM semicarbazide • HC1, 5 mM PEG-NAD, and the enzyme (200 U). After 3 hr at 37° the reaction mixture is heated in a boiling-water bath for 2 min to inactivate the enzyme, and dialyzed against 5 mM NazCO3 (pH 10.0) by using Spectrapor 6 dialysis membrane. Steady-State Analysis of Coupled Two-Enzyme Reactor

Theoretical Analysis Consider the following two reactions catalyzed by dehydrogenases, E1 and E2: El " PI + C,,~

(1)

S., + Co~ - " p,_ + C~

(2)

Si + Crd E,

11 R. F i e l d s , Biochem. J. 124, 581 (1971).

[4]

POLYMERIZABLE N A D DERIVATIVE AND P E G - N A D

41

where Crd and Cox are the reduced and oxidized forms of an NAD derivative, respectively. When the two reactions are coupled by the recycling of the coenzyme, the overall reaction is SI + $2 -

El, E, -" Pi + P_,

(3)

In a continuous reaction system, the enzymes and the coenzyme are kept in a reactor, the substrates (S~ and $2) are fed continuously at a fixed flow rate, and the products (P~ and P2) and nonconverted substrates leave the reactor at the same flow rate. The material balances for the reaction system at steady state can be written as follows if it is assumed that the reaction mixture in the reactor is completely mixed. v=

v= [Sl]0 = [$2]0 = [Pt] =

-vl

= vz

[Pl]/r [S1] + [P1] [$2] + [P2] [P2]

(4) (5) (6) (7) (8)

where v is the rate of P1 production, v~ and v2 are the rates of reduction of the coenzyme by E~ and Ez, respectively, r is mean residence time, and [ ]0 and [ ] show the concentration in the eluent and in the reactor, respectively. The reaction rates of the enzymes (vl and v2) are expressed by the following equations: Vf,l Vra[E1]([Cox][Pl] - [Crd][Sl]/Keq,l) Ol = Or,lKicox,lKpl + Vr,lKpl[Cox] + Vr,zKcox,l[P1]

(9) + Vr, l[Cox][P1] + Vf, l(gcrd,l[S1] + g s l [ C r d ]

+ [Crd][S~])/Keq,~ + Vf,~Kcrd,~[Cox][S~]/Ko.~ Kicox,~ + Vr, l Kcox,~[Crd][P~]/Ki.d,~ Vf,2 Vr,2[E2]([Cox][S2] - [Crd][Pz]/Keq,2) V2 = Vr,2Kicox,2Ks2 + Vr,2Ksz[Cox] + Vr,2Kcox,2[S2]

(10) + Vr,2[Cox][S2] + Vf,2(Kcrd,2[P2] + Kp2[Crd] + [Crd][P2])/Keq,2 + Vf,2Kcrd,2[Cox][P2]/Keq,2Kicox,Z

+ Vr,2Kcox,2[Crd][S2]/Kicrd,2 where Vf,l, Vr,1, Vf,2, and Vr,2 are maximal reaction velocities (V) of the reduction (f) and oxidation (r) of the coenzyme by the enzymes (El and E2); K~oxa, K~rd,l, K~ox,2, and Kerd,2 are the limiting Michaelis constants for the coenzymes; Kp~, Ks~, Kp2, and Ks2 are the limiting Michaelis constants for the substrates; Ki~ox,l, Ki~rd,~, Kicox,2, and Kicrd,Z are the dissociation constants for the enzyme-coenzyme complex. These equations are de-

42

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[4]

rived from an ordered Bi-Bi mechanism with negligible steady-state concentration of ternary complex (Theorell-Chance mechanism). ~2 The kinetic constants are obtained from initial rate studies of each enzyme reaction. Keq,~ and Keq,2 are the equilibrium constants of these enzyme reactions and are calculated by the Haldane equations:

Vf, f gicrd,l Ksl

(11)

Keq,l = Vr, l Kicox,l Kpl

Vf'2 Kicrd'2gP2 Ke,,2 =

(12)

Vr,2Kicox,2Ks2

The yield of P~ from Sl, Y, the total enzyme concentration, [E]t, the mole fraction of El, Ef, the total coenzyme concentration, [C]t, and the mole fraction of Crd, Cf, are defined as follows, by assuming that the fraction of the coenzyme bound to the enzymes is negligible: Y= [E]t-Ef = [C]t = Cf--

[PI]/[S1]0 × 100 [El] + [EL] [E1]/[E]t [Cox] + [Cr0] [Crd]/[C]t

(13) (14) (15) (16) (17)

At one fixed set of values of [E]t, El, [C]t, [Sl]0, [$2]0, and Y, values of Cf, v, and ~" are calculated from Eqs. (4)-(17). By changing the values of Y, the relationship between ~" and Y is obtained. The steady-state values of the enzyme reactor can thus be calculated for any operational conditions.

Example For a model enzyme reactor, the following system is used: El, lactate dehydrogenase (rabbit muscle); EL, alcohol dehydrogenase (horse liver); Cox, P E G - N A D ; Crd, P E G - N A D H ; S~, pyruvate; P~, L-lactate; $2, ethanol; P2, acetaldehyde. Table I shows the kinetic constants of the enzymes. These values were obtained from initial rate studies using P E G NAD(H) as coenzyme, and were used for the calculation of steady-state values. Figure 2 shows the theoretical relationship between ~- and Y obtained as described above. With different sets of operational conditions (in Fig. 2, [ethanol]0 is changed), different curves are obtained. The method of continuous operation of the model enzyme reactor is as follows. The enzymes and P E G - N A D were kept in a model 8MC ultrafiltration apparatus (Amicon Corp., Lexington, MA) fitted with a UM2 i2 I. H. Segel,

in " E n z y m e

Kinetics," p. 593. Wiley, New York, 1975.

[4]

POLYMERIZABLE N A D DERIVATIVE AND P E G - N A D

43

TABLE I KINETIC CONSTANTS OF LACTATE DEHYDROGENASE (RABBIT MUSCLE) AND ALCOHOL DEHYDROGENASE (HORSE LIVER)a Lactate dehydrogenase Vfj K~oxj

22.6 50,6 2330 542 413 21.2 501 2.79

Kpl Kicox.i Vrj K,,~j Ks~ K~¢rdj

--- 0.7 sec -j ± 6,5 ~tM 4- 5 9 0 / t M ± 166/zM ± 12 sec 1 ± 1.1 /xM -+ 27/.tM ± 0,50/zM

Alcohol dehydrogenase Vf,2 Kcox,2 Ks2 Kicox.2 Vr.2 Kcrd.2 Kp2 K~rd,2

0.591 18.7 1950 22.0 10.8 33.1 192 8.74

- 0.023 sec -~ ± 1.5/xM ± 210/xM -+ 3.4/.tM ± 0.2 sec -~ ---- 0.9/zM ± 9 ~M ± 1.05 IzM

Initial rate studies were carried out at 30° in 30 mM sodium phosphate buffer, pH 7.5. Reaction mixture contained the following components. For coenzyme reduction by lactate dehydrogenase: 5-100 mM DL-lactate, 50-400 /.tM PEG-NAD, 30 mM hydrazine, and 3.95 nM enzyme; for coenzyme oxidation by lactate dehydrogenase: 0.57-5.7 mM pyruvate, 5 - 5 0 / z M P E G NADH, and 2.36 nM enzyme; for coenzyme reduction by alcohol dehydrogenase: 1-10 mM ethanol, 6.25-50/zM PEG-NAD, 10 mM semicarbazide' HC1, and 0.584/xM enzyme; for coenzyme oxidation by alcohol dehydrogenase: 0.15-1.5 mM acetaldehyde, 6.25-50/.tM PEG-NADH, and 0.117/zM enzyme. The enzyme concentrations are expressed as subunit concentration. Initial rates were measured spectrophotometrically at 340 nm. Results are expressed -+ standard error.

r Et hanoi ]o 100 I

lO0 mM

f

80

60 rnM

60

30 mM

>" 40 /

10 mM

20

0

I

I

I

1

2

3

-c(h) FIG. 2. Theoretical relationship between residence time (r) and the yield of L-lactate (Y) at steady state with different ethanol concentrations in the eluent ([ethanol]0). Conditions for calculation are [lactate dehydrogenase] = 0.12/xM, [alcohol dehydrogenase] = 2.88/zM, [C]~ = lmM, [pyruvate]0 = 5 mM.

44

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[4]

100

8O

60

> 40

20 I

0

I

I

50 100 150 [ E t h a n o L ] o (raM)

200

FIG. 3. Effect of ethanol concentration in the eluent ([ethanol]0) on the yield of L-lactate (Y) at steady state. (0) Y determined experimentally; error bars indicate fluctuation range; (O) Y calculated for each set of experimental conditions; ( ) Y calculated with the average conditions of these experiments. The average conditions: [lactate dehydrogenase] = 0.11/,~M, [alcohol dehydrogenase] = 2.64/zM, [C]t = 0.749 mM, [pyruvate]0 = 5.0 mM, ~- = 2.61 hr.

ultrafiltration membrane, and the apparatus was placed in a temperaturecontrolled chamber (Tabai LN-110). The continuous enzyme reaction was carried out at 30° in 30 mM sodium phosphate buffer, pH 7.5, containing 0.1% streptomycin and 0.02% NaN3 by continuous ultrafiltration with substrate solution containing pyruvate and ethanol in the buffer as eluent under N2 pressure, and the volume of the reaction mixture was kept at 6.5 ml. The filtrate was collected in fractions at 1-hr intervals, and the Llactate concentration and volume of each fraction were measured. LLactate was assayed with lactate dehydrogenase and NAD.13 The concentration of L-lactate became constant 10-15 hr after the start of the reaction. The steady-state values of L-lactate concentration and residence time 0") were obtained as average values during the steady state over 10 hr (10 measurements). Their fluctuation range was less than 10% of the average value. The concentration of PEG-NAD and the activities of the enzymes in the reactor decreased gradually during the continuous operation. The quasi-steady-state values were estimated from initial and final values by assuming a first-order rate of decrease. The difference between the initial and final values of a steady state was less than 20% of the average value. Figure 3 shows the effects of ethanol concentration in the eluent at a t3 I. Gutman and A. W. Wahlefeld, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd Ed., p. 1464. Academic Press, New York, 1974.

[5]

E N Z Y M E REACTOR BASED ON a q T W O - P H A S E SYSTEMS

45

fixed value of z. The solid and open circles show experimental and theoretical values, respectively. The solid line was calculated with the average conditions of fixed variables in the figure. Deviation of an open circle from the solid line is due to the deviation of the fixed variables of the experiment from their average values. Generally, the steady-state values obtained by continuous operation of the enzyme reactor are lower than those obtained by calculation based on the kinetic model. These differences are thought to be due to the simplifications made for the kinetic model, in which we assumed a simplified ordered Bi-Bi mechanism and neglected the effects of substrate inhibition and so on. However, the differences are not large in the region of Ef -> 0.03 and [C]t -> 0.7 raM, and the steady-state behavior of the enzyme reactor can be explained and predicted semiquantitatively by the simple kinetic model.

[5] A d e n o s i n e 5 ' - T r i p h o s p h a t e R e c y c l i n g in a n E n z y m e Reactor Based on Aqueous Two-Phase Systems B y HIDEO SUZUKI a n d YOSHIMITSU YAMAZAKI

The retention and in situ regeneration of coenzymes (e.g., NAD or ATP) in a reactor are prerequisites for the industrial use of dehydrogenases, kinases, or ligases. One possible approach to this is to bind the coenzymes to water-soluble polymers and use them with the enzymes in an ultrafiltration apparatus. J-3 However, this method has the disadvantage that enzymes as well as the polymers carrying coenzymes tend to be adsorbed on the ultrafiltration membrane and thus lost from the reaction mixtures. J The need for developing a better method for separating polymers from low molecules reminded us of countercurrent partitioning using aqueous two-phase systems. These systems were developed by Albertsson 4 and l y. Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). 2 S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. B0ckmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd Ed. Wiley, New York, 1971.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[5]

E N Z Y M E REACTOR BASED ON a q T W O - P H A S E SYSTEMS

45

fixed value of z. The solid and open circles show experimental and theoretical values, respectively. The solid line was calculated with the average conditions of fixed variables in the figure. Deviation of an open circle from the solid line is due to the deviation of the fixed variables of the experiment from their average values. Generally, the steady-state values obtained by continuous operation of the enzyme reactor are lower than those obtained by calculation based on the kinetic model. These differences are thought to be due to the simplifications made for the kinetic model, in which we assumed a simplified ordered Bi-Bi mechanism and neglected the effects of substrate inhibition and so on. However, the differences are not large in the region of Ef -> 0.03 and [C]t -> 0.7 raM, and the steady-state behavior of the enzyme reactor can be explained and predicted semiquantitatively by the simple kinetic model.

[5] A d e n o s i n e 5 ' - T r i p h o s p h a t e R e c y c l i n g in a n E n z y m e Reactor Based on Aqueous Two-Phase Systems B y HIDEO SUZUKI a n d YOSHIMITSU YAMAZAKI

The retention and in situ regeneration of coenzymes (e.g., NAD or ATP) in a reactor are prerequisites for the industrial use of dehydrogenases, kinases, or ligases. One possible approach to this is to bind the coenzymes to water-soluble polymers and use them with the enzymes in an ultrafiltration apparatus. J-3 However, this method has the disadvantage that enzymes as well as the polymers carrying coenzymes tend to be adsorbed on the ultrafiltration membrane and thus lost from the reaction mixtures. J The need for developing a better method for separating polymers from low molecules reminded us of countercurrent partitioning using aqueous two-phase systems. These systems were developed by Albertsson 4 and l y. Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). 2 S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. B0ckmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd Ed. Wiley, New York, 1971.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

46

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[5]

are useful in collecting cell particles 5 and biopolymers. 6'7 They are prepared by mixing aqueous solutions of different polymers. For instance, when a solution of dextran in water is mixed with a solution of polyethylene glycol (PEG) in water, the mixture becomes turbid and after a while it separates into two phases. If proteins or other polymers are added to the mixture, they are often asymmetrically partitioned to the upper and the lower phases, depending on the experimental conditions. Low molecular weight molecules are usually almost symmetrically partitioned into both phases. Thus one can see the possibility of an enzyme reactor based on the principle that enzymes and the polymer-bound coenzymes are retained in the lower phase while the low molecular weight products are extracted to the upper phase. The purpose of this article 8 is to present an outline of such a reactor in the case of kinases, especially for the coupled system of hexokinase and acetate kinase. Design of the System In constructing a reactor of this type, the first step is to find an aqueous two-phase system in which all catalysts (here, acetate kinase, hexokinase, and polymer-bound ATP) are mostly partitioned to either the upper or the lower phase, and in which phase the enzyme reaction can actually proceed. There are many factors affecting aqueous two-phase systems: (1) the species of polymers and their ratios, (2) the species of buffers, their pH, and concentrations, (3) the species of salts in the media and their concentrations, (4) temperature, and so on. An additional factor in the present case is the structure of the ATP carrier, which must be carefully selected with consideration given to what polymers are used for the aqueous two-phase system. We chose dextran as the ATP carrier and a combination of dextran and PEG for the basic phase system) In this system, the upper phase is rich in PEG and the lower phase is rich in dextran; therefore dextran-bound ATP goes to the lower phase by the tendency of similar materials to meet. In contrast, Johansson ~° reported 5 A.-L. Smeds, A. Veide, and S.-O. Enfors, Enzyme Microb. Technol. 5, 33 (1983). 6 K. H. Kroner, H. Hustedt, and M.-R. Kula, Biotechnol. Bioeng. 24, 1015 (1982). 7 A. Veide, A.-L. Smeds, and S.-O. Enfors, Biotechnol. Bioeng. 25, 1789 (1983). 8 The original publication is Y. Yamazaki and H. Suzuki, Biseibutsu Kogyo Gijutsu Kenkyusho Kenkyu Hokoku 52, 33 (1979). 9 A recent review of the syntheses of polymer-bound ATP is by Y. Yamazaki and H. Maeda, Yuki Gosei Kagaku Kyokaishi 41, 1088 (1983). to G. Johansson, Biochim. Biophys. Acta 222, 381 (1970).

[5]

ENZYME REACTOR BASED ON aq TWO-PHASE SYSTEMS

47

that acidic proteins were mostly partitioned in the lower phase when a charged PEG derivative, PEG sulfonate, was used in preparing the system of PEG and dextran. This can possibly be accounted for by electric repulsion. Dextran-bound ATP is negatively charged in neutral solutions owing to the phosphate groups. Thus, a more favorable partition of dextran-bound ATP to the lower phase can be expected in a phase system supplemented with a negatively charged PEG. Hexokinase (pI 4.5-4.81~) and serum albumin (pI 4.7-4.9, j2 used as enzyme stabilizer) are also expected from their isoelectric point to show a higher affinity to the lower phase in such a system. Johansson used PEG sulfonate ( - O 3 S - C H z C H z O (CH2CHzO)n-CHzCH2-SO3-), but we used a more easily preparable polymer, PEG sulfate (-03S-O-CH2CH20-(CH2CH20),-CH2CHz-O-S03-). Synthetic procedures of dextran-bound ATP and PEG sulfate are described below. Preparation of Dextran-Bound A TP 13

The principle used is the introduction of a spacer chain having a terminal amino group to the N-6 site of adenine nucleus and the succeeding coupling to BrCN-activated dextran using the amino group. In the reaction shown in Fig. 1 ADP is used as the starting material, but the ATP derivative, N6-[N-(6-aminohexyl)carbamoyl]-ATP, which is formed probably from the disproportionation (2ADP = ATP + AMP), is purified by column chromatography. One gram of ADP (free acid, Boehringer-Mannheim) is dissolved in 50 ml of hexamethylphosphoramide with stirring at 70°, and then 20 ml of freshly distilled hexamethylene diisocyanate is added. After incubation at 75 ° for 2 hr, the reaction mixture is cooled to 0 ° and slowly poured into an ice-cold two-phase mixture of chloroform (750 ml) and acidic water (750 ml, pH 1 with HC1), under vigorous stirring. The resultant emulsion is transferred into a separatory funnel and allowed to stand overnight at room temperature. The aqueous layer becomes clear and is separated from the chloroform layer, which is reextracted with 750 ml of acidic water (pH 1). The combined aqueous layer is neutralized with 1 M LiOH and concentrated on a rotary evaporator at 40 ° to approximately 100 ml. To the concentrate is added 1 liter of an ice-cold mixture made of ethanol u M. Kunitz and M. R. McDonald, J. Gen. Physiol. 29, 393 (1946). t2 E. G. Young, in "Comprehensive Biochemistry" (M. Florkin and E. H. Stotz, eds.), Vol. 7, p. 22. Elsevier, New York, 1963. u This section is reprinted with permission from Y. Yamazaki, H. Maeda, and H. Suzuki, Eur. J. Biochem. 77, 511 (1977),

48

MULTISTEP ENZYME SYSTEMS AND COENZYMES

H N . , ~ N . " % / " k ~ NC0 ADP

OCNo (CHI) Q- NCO •

t~j ~ - - ~ "~N'~'N /

[5]

HN . - " ~ N ~ / ~ I )

N.~..,,~ N ~'~NI~"N "~

OH OH

H3 CB-

OH OH

Chromatographlc Separation I Dextran-bound ATP

<

I

Br CN-activated dextran FIG. 1. Synthetic scheme of dextran-bound ATP (HMPA, hexamethylphosphoramide; (~),, mono-, di-, or triphosphate).

and acetone (1/I). The precipitate is collected by centrifugation and dried in vacuum. The resultant white powder is dissolved in water, adjusted to pH 7, and adsorbed on a Dowex I-X2 (C1-) column (1.4 × 55 cm). The column is washed with 150 ml of 50 mM LiCI (pH 5.0). A linear gradient of LiCI is then applied. The mixing vessel contains 50 mM LiCI (1.2 liters, pH 5.0) and the reservoir 350 mM LiC1 (1.2 liters, pH 2.0). The effluent is collected as 15-ml fractions. The nucleotides are eluted into three major peaks (fractions 400-600, 900-1100, and 1400-1600 ml). These peaks correspond to the AMP derivative, ADP derivative, and ATP derivative, respectively. The fractions in the latter major peak are pooled, adjusted to pH 7 with 1 M LiOH, and concentrated on a rotary evaporator at 30° to approximately 30 ml. To the concentrate is added 300 ml of an ice-cold mixture of ethanol-acetone (I/1). The precipitate is collected by centrifugation and dried in vacuum. This crude product is again purified by Dowex l-X2 column chromatography in a similar manner as described above. The precipitation procedure is repeated twice and the final precipitates are dried for 24 hr over P205 in vacuum. Thus, 193 mg of N6-[N-(6 aminohexyl)carbamoyl]-ATP is obtained as white powder (12% yield as tetralithium salt). Coupling of the ATP derivative to soluble dextran is carried out essentially according to the method by Mosbach e t al. 14 Dextran T40 (2 g, Pharmacia) is dissolved in 20 ml of distilled water and activated at pH 11.0 by addition of 100 mg cyanogen bromide in 2 ml of water and continuous 14 K. Mosbach, P.-O. Larsson, and C. Lowe, this series, Vol. 44, p. 859.

[5]

ENZYME REACTOR BASED ON a q TWO-PHASE SYSTEMS

49

titration with I M NaOH at room temperature. After 5 min the consumption of NaOH ceases, and then the pH is lowered to 9.5 with 0.1 M HCI. To the activated dextran solution is added 600 mg of the above ATP derivative dissolved in 5 ml of 0.1 M NaHCO3, pH 8.5. After the reaction mixture has been stirred overnight at room temperature, 4 ml of 0.8 M ethanolamine-HCl buffer, pH 8.0, is added to quench any residual active groups. The reaction mixture is allowed to stand for an hour at room temperature and then applied to a Sephadex G-50 column (2.5 x 112 cm) equilibrated with 0.1 M LiCI. Elution is performed with 0.1 M LiCI, and the effluent is collected as 15-ml fractions. Dextran-bound ATP is eluted in fractions 12-20 and the uncoupled derivative in fractions 21-40. The fractions containing the polymer are pooled, concentrated 5-fold by ultrafiltration (Amicon PM10 membrane) and then dialyzed against 0.1 M triethanolamine-HCl buffer, pH 7.6, for 2 days. The buffered solution of dextran-bound ATP is used in all subsequent studies. Determination of phosphate ~5 and total hexoses ~6 has indicated a nucleotide content of 43 /xmol/g dry dextran and a coupling yield of 15%.

Preparation of PEG Sulfate 8 PEG 4000 (10 g, Wako Pure Chemical Industries Ltd., Osaka) is dissolved in 20 ml of freshly distilled chloroform. To this ice-cold solution is added 0.84 ml of chlorosulfonic acid. The mixture is gently shaken for 10 min under cooling with ice and then stirred at room temperature for 1 hr. The reaction mixture is again cooled with ice and mixed with about 20 g of ice and 20 ml of water containing 1 g of NaOH. This mixture is vigorously stirred at room temperature for 10 min. The emulsion is evaporated under reduced pressure at 35° to remove chloroform. The residual syrup is mixed with 100 ml of water and 100 ml of ethanol. The solution (pH 2) is neutralized with 2 M NaOH and then concentrated in vacuum at 65°. The remaining water is removed as the ethanol azeotrope. The residue is dissolved in 50 ml of hot ethanol. Insoluble salts are filtered off and washed with ethanol (I0 ml, 2x). The combined filtrate and washings are allowed to stand at - 2 0 ° overnight. The precipitate is collected by filtration and then dissolved in 100 ml of chloroform. After addition of ether (400 ml), the solution is cooled to -20 ° for 2 hr. The precipitate is collected by suction filtration and dried in vacuum at 80° for 1 hr. Thus 7.83 g of PEG sulfate was obtained as white solids, mp 48 °. Analysis: Found: S, 1.2%. Calcd. for NaSO4(CH2CHzO)70SO3Na: S, 1.9%; the degree of polymerization 70 is an assumption based on the ~5 G. R. Bartlett, J. Biol. Chem. 234, 466 (1959).

16 j. H. Roe, J. Biol. Chem. 212, 335 (1955).

50

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[5]

average molecular weight (3000, information from the manufacturer) of PEG 4000. PEG sulfate migrated faster (5.7 cm/hr) to the anode than PEG (4.2 cm/hr) in zone electrophoresis [on a cellulose-coated glass plate (Avicel SF, 5 x l0 cm, 0.2-0.25 mm thick) using 10 mM phosphate buffer, pH 6.8, and applying a potential of 600 V for 30 min; spots were located in 12 vapor]. Investigation of Optimal Partition The reagents used for making the system are (1) enzymes and polymers to be partitioned: hexokinase, acetate kinase, bovine serum albumin, and dextran-bound ATP; (2) polymers for the preparation of twophase systems: dextran T500, PEG 4000, and PEG sulfate; (3) substrates for the enzymes: glucose and acetyl phosphate. Other factors to be selected are species of buffer and additional salts, pH, and temperature. However, the ranges within which these can be selected is narrowly limited for the enzyme reaction. We used triethanolamine-HC1 buffer (pH 7.6). The optimal conditions for partitioning all of the proteins and dextran-bound ATP into the lower phase were investigated by changing the concentrations of dextran, PEG, PEG sulfate, and the buffer. Determination o f Partition Coefficients ~7

A dextran solution (20%, w/w) is prepared by dissolving l0 g of dextran T500 (Pharmacia) into 40 ml of water. PEG + PEG sulfate solutions (20%, w/w; ratio of PEG sulfate/PEG = 0/1, 1/3, or I/l) are prepared by dissolving l0 g of PEG 4000 or mixtures of PEG 4000 and PEG sulfate into 40 ml of water. Phase systems containing 8% (w/w) PEG + PEG sulfate and 8% (w/w) dextran are prepared by mixing the 20% solutions (0.8 g each) and 0.4 ml of triethanolamine-HCl buffer (10, 50, or 150 mM), pH 7.6, containing the materials to be tested. The amounts of the materials are given in the legends to Figs. 2 and 3. Yeast hexokinase (BoehringerMannheim) and E. coli acetate kinase (Boehringer-Mannheim) are dialyzed against 2 mM triethanolamine-HCl buffer, pH 7.6, overnight before use. The systems are mixed by gentle shaking and allowed to separate by centrifugation (3000 rpm, for l0 min) at about 20°. Samples (0.2 ml each) are removed from the upper phase and then from the lower phase using hole pipettes. The partition coefficient (K, concentration in the upper phase divided by concentration in the lower phase) for each material is ~v R e p r o d u c e d from Y a m a z a k i and Suzuki 8 with permission.

[5]

51

ENZYME REACTOR BASED ON aq TWO-PHASE SYSTEMS

calculated from the following values of the samples for each phase: absorbance at 269 and 280 nm for dextran-bound ATP and albumin, respectively, and activity for enzymes. The absorbances are measured, following adequate dilution of the samples with water, using a blank prepared from the corresponding system without the materials. The enzymes are assayed from the initial velocities of absorbance change (A340/min) in the following reaction mixtures at 25°: for hexokinase, in a total volume of 3.03 or 3.10 ml containing 242/zmol triethanolamine-HC1 buffer (pH 7.6), 20/zmol MgSO4, 667 ~mol glucose, 8.2 /zmol ATP, 2.2/zmol NADP, 1.4 U yeast glucose-6-phosphate dehydrogenase (Boehringer-Mannheim), and hexokinase; and for acetate kinase, in a total volume of 3.00 or 3.19 ml containing 204/~mol triethanolamineHCI buffer (pH 7.6), 4/zmol MgSO4, 1000/zmol acetate, 3.3/zmol phosphoenolpyruvate, 16.2/zmol ATP, 0.96/zmol NADH, 15 U rabbit muscle pyruvate kinase (Boehringer-Mannheim), 40 U beef heart lactate dehydrogenase (Miles), and acetate kinase. Figure 2 shows that the proteins and dextran-bound ATP were all partitioned more than 90% in the lower phase when PEG contained 25% PEG sulfate. This fact confirms the validity of our prediction regarding the design for a two-phase system. In addition, there was a tendency for the lower buffer concentration to give a higher affinity to the lower phase. The system, however, did not contain the substrates for the enzymes. The partition coefficients of the three catalysts were thus also determined in the same aqueous two-phase system as the above except that all the necessary substances for the coupled reaction of hexokinase and acetate kinase were added (Fig. 3). Dextran-bound ATP %PEG sulfate 0

25

50

Hexoklnase %PEG sulfate 0

25

50

o£

oT

oT

Acetate kinase %PEG sulfate 0

o

:}5

o

50

oT

Albumin %PEG sulfate 2S 50

o-

oT

-- ~

"It

-2 -

-2

-

FI6. 2. Partition of dextran-bound ATP, hexokinase, acetate kinase, and albumin in aqueous two-phase systems containing dextran T500 and PEG plus PEG sulfate, t7 Quantities in the 0.4 ml of triethanolamine-HC1 buffer: dextran-bound ATP, 7 mg; hexokinase, 10 U; acetate kinase, 1 U; bovine serum albumin (Miles), 2 mg; the buffer: 2 mM (O), 10 mM (A), or 30 mM ([~). The buffer concentrations were calculated by assuming homogeneous dispersion and a total volume of 2 ml. % PEG sulfate = [PEG sulfate/(PEG sulfate + PEG)] x 100. K is the partition coefficient.

52

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[5]

%PEG sulfate 0 0

25

-

0

)

- -

m

-

1.6

- 1.6

FIG. 3. Partition of dextran-bound ATP, hexokinase, and acetate kinase in aqueous twophase systems containing dextran T500, PEG plus PEG sulfate, and the substrates for the enzymes.17Quantities in the 0.4 ml of triethanolamine-HCl buffer (10 mM): dextran-bound ATP, 3.5 rag; hexokinase, 10 U; acetate kinase, 1 U; albumin, 2 mg; MgSO4, 14/xmol;acetyl phosphate, 15/xmol;and glucose, 180 tzmol. (O) Dextran-bound ATP; (A) hexokinase; (V1) acetate kinase. When the substrates and Mg 2+ ion were present in quantity in the system, the partition coefficients of all three catalysts were found to increase as compared to those obtained in the former system lacking the substances. The reduced affinity of the catalysts to the lower phase can possibly be interpreted as an electric repulsion between them and the acetyl phosphate present in the lower phase, or as the formation of ionic bonds between them and the PEG sulfate through the Mg 2+ ion. The results in Fig. 3 are somewhat undesirable for our purpose. Nevertheless, the three catalysts were still partitioned 6-20 times more in the lower phase when P E G sulfate was added to the system. We surmised that the catalysts could be retained in a reactor using the aqueous two-phase system by carrying out countercurrent partitioning several times. Application to Repeated E n z y m e Reaction

Repeated Production of Glucose 6-Phosphate 17 The principle is the coupled reaction: glucose

/-

dextran-bound ATP

/"

acetate

hexokinase acetate kinase d/ ~ dextran-boundJ ~,. glucose 6-phosphate ADP - - acetyl phosphate

F o u r test tubes are used (see Fig. 4). A phase system is prepared by mixing 0.2 g of 20% (w/w) (75% P E G + 25% P E G sulfate) solution, 0.2 g

[5]

ENZYME REACTOR BASED ON aq TWO-PHASE SYSTEMS

tube

A B substrate/'~'~ Ition~ t

C

53

D ~'~ IO0*C,5rain t ~)determi natioofn

glucose 6-phosphate

,

|Dextran

[ ~

~PEG+PEG sulfate [Substrates, Buffer

Oextran PEG+PEG sulfate Enzymes Dextran-bound ATP Substrates,

[Dextran

~PEG +PEGsulfate tBuffer

Buffer

FIG. 4. Illustration of the procedure for repeatedly producing glucose 6-phosphate in an aqueous two-phase system) 7

of 20% (w/w) dextran T500 solution, and 0.1 ml of 10 mM triethanolamine-HCl buffer, pH 7.6, containing 1 mg of dextran-bound ATP, 3.5 /.~mol MgSO4, 3.8 /~mol acetyl phosphate (Boehringer-Mannheim), 45 /~mol glucose, 0.5 mg albumin, 2.8 U hexokinase, and 0.08 U acetate kinase. This mixture is placed in test tube A. In tubes B, C, and D are placed the lower phases (0.25 ml each; the same volume as that of the lower phase in tube A) from the corresponding system minus all of dextran-bound ATP, the enzymes, albumin, and the substrates. In addition, a substrate solution is prepared which is the upper phase of the system having the same composition as the above, but without the three catalysts and albumin. The initial content in tube A is incubated at 25 ° for 30 rain with shaking and then allowed to separate by centrifugation for 10 rain. The upper phase is transferred to tube B with a pipette and a new substrate solution (0.25 ml; the same volume as that of the upper phase) is added to tube A. The tubes A and B are incubated at 25 ° for 30 min with shaking. After centrifugation, the upper phase in tube B is transferred to tube C and that in tube A to tube B. Then, a new substrate solution (0.25 ml) is added to tube A. In this manner, the upper phases in tube A are extracted four times. Immediately after the final extraction, the upper phases are heated to 100° for 5 min and then subjected to the enzymatic determination of glucose 6-phosphate. The cooled sample is mixed with 2 ml of 0.1 M triethanolamine-HCl buffer (pH 7.6) and centrifuged to remove the denatured proteins. One volume of the supernatant (1 ml) is mixed with 1.5 ml of the buffer, 0.1 ml of 0.1 M MgSO4, and 0.2 ml of NADP solution (10 mg/ml). Absorbance increment at 340 nm after addition of yeast glucose6-phosphate dehydrogenase (10/xl, 3.5 U) and incubation at 28 ° for 30 min

54

M U L T I S T E P E N Z Y M E SYSTEMS A N D C O E N Z Y M E S

[5]

is read, from which the concentration of glucose 6-phosphate is calculated using the value of ~340 = 6.2 × 10 3 M -1 cm 1. The four times repeated extraction procedure was carried out to reextract some part of the catalysts partitioned in the upper phases into the lower phases. In this way, glucose 6-phosphate was repeatedly produced in the final upper phases (Fig. 5) even though the catalysts were added only one time in tube A. The maximum amount of glucose 6-phosphate production was 1.9/xmol (in phase 9). This value was comparable to that obtained at the equilibrium of the coupled reaction in one batch experiment (see the original workS), and therefore the time (at phase 9) was considered to be the start of a steady state for this production. The total operation was repeated nine times and stopped, since the substrate solution was used up. Thus, the final upper phases 1-9 had passed the four times extracting procedure before the heating treatment, but the final upper phases I0, 11, and 12 were heated when withdrawn from tubes C, B, and A, respectively. In other words, the final upper phases 11 and 12 had not contacted with the lower phases in tubes C and B, respectively. The fact that the quantities of glucose 6-phosphate in the final upper phases 11 and 12 were less than those of the preceding final upper phases (8-10) suggests that some of the enzymes and dextran-bound ATP, which had been supplied in tube A, was displaced into tubes B and C by moving the upper phases. Conversely, for retaining most of the catalysts as long as possible in the whole system, the upper phases should be reextracted more times by the lower phases. Concluding Remarks The present work provides a prospect for using aqueous two-phase systems to retain enzymes, especially coupled enzyme systems including @

I

2

3

4

5

15

7

8

9

I0

II

12

Number of the Upper Phase

FIG. 5. Repeated enzymatic production of glucose 6-phosphate in an aqueous two-phase system containing dextran T500 and PEG plus PEG sulfate.t7 Quantities of glucose 6-phosphate in the upper phases finally withdrawn out of the whole system are plotted against the numbers of these phases.

[5]

ENZYME REACTOR BASED ON aq TWO-PHASE SYSTEMS

55

coenzymes, in bioreactors. In another experiment, we also found that native ATP was partitioned more in the lower phase (K = 0.6) in a system containing dextran T500, PEG, and PEG sulfate. 8 Pollak and Whitesides 18 found that another important coenzyme, NADP, was partitioned more in the lower phase (K = 0.44) in an aqueous two-phase system composed of Ficoll (polysucrose) and UCON (copolymer of propylene glycol and ethylene glycol). These facts suggest the possibility that the coenzymes might be retained, without being bound to polymers, in the reactor if countercurrent partitioning is repeated several times. Of course, a small portion of the catalysts actually passes into the upper phase and is finally drawn out of the whole system. This loss is intrinsic and cannot be avoided. However, it can be minimized if the partition coefficients are exceedingly high or low. A possible way to realize this is to find a more desirable phase system by changing the materials and/or the conditions for the systems or by chemically modifying the catalysts. In fact, when carboxymethyl dextran was used as the ATP carrier, this ATP derivative showed a remarkably lower partition coefficient (<0.01) than the usual dextran-bound ATP in the systems for Fig. 2 which contained PEG sulfate. 8 The present method will be useful if the required enzymes are too unstable to be immobilized on solid supports or to be employed in usual solutions, although stable in polymer solutions. It is highly possible that such a situation will be encountered, since several enzymes were reported to be stabilized by polymers.19 Furthermore, if the substrates and/or the products are of relatively high molecular weight and thus the usual techniques of enzyme immobilization cannot be employed, the aqueous twophase partition method will be useful. Bioreactors based on aqueous two-phase systems have also been reported for ethanol production 2°,2~and aminoacylase reaction. 22

18 A. Pollak and G. M. Whitesides, J. Am. Chem. Soc. 98, 289 (1976). z9 R. D. Schmid, in "Advances in Biochemical Engineering" (T. K. Ghose, A. Fiechter, and N. Blakebrough, eds.), Vol. 12, p. 64. Springer-Verlag, Berlin, 1979. z0 I. Kfihn, Biotechnol. Bioeng. 22, 2393 (1980). 2~ B. Hahn-H~igerdal, E. Andersson, M. Lrpez-Levia, and B. Mattiasson, Biotechnol. Bioeng. Symp. 11, 651 (1981). 22 W. Kuhlmann, W. Halwachs, and K. SchiJged, Chern.-lng.-Tech. 52, 607 (1980).

56

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[6]

[6] A l c o h o l D e h y d r o g e n a s e C o i m m o b i l i z e d w i t h Its C o e n z y m e

By KEITH J. LAIDLER and M. ABDUL MAZID Whereas much work has been done on the separate immobilization of enzymes and coenzymes, little has as yet been done on their coimmobilization. Many enzymes, such as the dehydrogenases, require the participation of easily dissociable coenzymes, and for obvious practical and economic reasons it is useful to be able to immobilize them side by side. Coimmobilization is, however, only successful if certain conditions are satisfied. It is important for the enzyme and coenzyme to be held in a suitable spatial arrangement and for the coenzyme to be easily regenerated during the course of reaction. The problem of spatial arrangement may often be controlled by the introduction of suitable spacing groups; these can be placed between the supporting matrix and the coenzyme, which is thus more flexible. The matter of regeneration of the coenzyme also has to be dealt with by positioning it suitably with respect to the enzyme. Because of these factors the coimmobilization of enzymes and coenzymes has not always been met with complete success. In the case of alcohol dehydrogenase and its coenzyme nicotine adenine dinucleotide (NAD), the recycling of the latter has been achieved for many years, I-9 and derivatives of the coenzyme with flexible side arms have been synthesized~°-~2; there are, however, few reports on their coimmobilization. Immobilization of alcohol dehydrogenase itself has been l N. K. Gupta and W. G. Robinson, Biochirn. Biophys. Acta 118, 429 (1%6). 2 C. L. Woodley and N. K. Gupta, Anal. Biochem. 43, 341 (1971). 3 M. P. Schulman, N. K. Gupta, A. Omachi, G. Hoffman, and W. E. Marshall, Anal. Biochem. 60, 302 (1974). 4 G. E. Glock and P. McLean, Biochem. J. 61, 381 (1955). 5 C. A. Villee, Biochem. J. 83, 191 (1962). 6 T. F. Slater and B. Sawyer, Nature (London) 193, 454 (1962). 7 T. F. Slater, B. Sawyer, and U. Strauli, Arch. Int. Physiol. Biochim. 72, 427 (1964). s j. S. Nisselbaum and S. Green, Anal. Biochem. 27, 212 (1969). 9 C. Bernofsky and M. Swan, Anal. Biochem. 53, 452 (1973). io C. R. Lowe and K. Mosbach, Eur. J. Biochem. 49, 511 (1974). 11 M. Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 12 H. L. Schmidt and G. Grenner, Eur. J. Biochem. 67, 295 (1976).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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reported by a number of workers, 13-18 and the preparation and uses of NAD analogs for affinity adsorbents are well documented in this series of volumes) 9,2° However, it was only in 1975 that Gestrelius et al. 21 first reported the preparation of an alcohol dehydrogenase-NAD(H)Sepharose complex which did not require for its activity the addition of any soluble coenzymes. It involved coimmobilization of horse liver alcohol dehydrogenase and an NAD(H) analog, N6-[(6-aminohexyl)carbamoylmethyl]-NAD(H), to Sepharose 4B under conditions which permitted binary complex formation between the enzyme and the coenzyme derivative prior to their immobilization into the gel. A somewhat similar method for the coimmobilization of alcohol dehydrogenase and NAD on a water-dispersible high molecular weight polymer containing methyl methacrylate and acrylic acid has been reported in a Japanese patent. 22In another approach, Grunwald and Chang23-25described the microencapsulation of a multienzyme system involving yeast alcohol dehydrogenase and malate dehydrogenase with soluble dextran-NAD, for the continuous regeneration of the latter within a collodion-hemoglobin24 and a nylonpolyethyleneimine microcapsule. 25 We have more recently reported the covalent attachment of NAD and yeast alcohol dehydrogenase to the interior surface of a partially hydrolyzed nylon tube. 26 Materials for Immobilization Alcohol dehydrogenase occurs in a variety of plant and animal organisms. The most widely studied alcohol dehydrogenase, a cytoplasmic protein, is usually obtained from bakers' yeast which is also the source of the coenzyme NAD. The other commonly studied alcohol dehydrogenase is that obtained from mammalian liver, particularly equine liver. Both 13 W. E. Hornby, D. J. Inman, and A. McDonald, FEBS Lett. 23, 114 (1972). 14 A. C. Hohansson and K. Mosbach, Biochim. Biophys. Acta 370, 348 (1974). 15 N. Kelly, A. Flynn, and D. B. Johnson, Biotechnol. Bioeng. 19, 1211 (1977). l~ M. J. Brougham and D. B. Johnson, Int. J. Biochem. 9, 283 (1978). 17 S, Barry, T. Griffin, and D. B. Johnson, Int. J. Biochem. 9, 289 (1978). is M. A. Mazid, Ph.D. Thesis, University of Ottawa, Ottawa, Ontario, Canada, 1981. 19 M. J. Harvey, D. B. Craven, C. R. Lowe, and P. D. G. Dean, this series, Vol. 34, p. 242. 20 K. Mosbach, P.-O. Larsson, and C. Lowe, this series, Vol. 44, p. 859. 21 S. Gestrelius, M.-O. Mansson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). 22 Nitto Electric Industrial Co. Ltd., Japanese Patent 57,150,387 [82,150,387] (CI. C12NI 1/ 08), September 17, 1982. 23 j. Grunwald and T. M. S. Chang, Biochem. Biophys. Res. Commun. 81, 565 (1978). 24 j. Grunwald and T. M. S. Chang, J. Appl. Biochem. 1, 104 (1979). 25 j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). 26 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982).

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

[6]

alcohol dehydrogenase and its coenzyme are commercially available in different grades from a number of suppliers, such as Sigma Chemical Company in the United States, Boehringer-Mannheim in Germany, and several others. The supporting materials such as Sepharose and dextran are available from Sigma and Pharmacia Fine Chemicals (Uppsala, Sweden), while Bio-Rad Laboratories (Richmond, VA) sells them under the trade name of BioGel A. Nylons can be obtained in different forms from a number of sources. All other materials including various reagents and chemicals, notably 1-cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMC), a water-soluble condensation agent, are also available commercially. Coimmobilization Methods The various methods of coimmobilization involve somewhat different principles. They all basically use a derivative of the coenzyme such as N 6[(6-aminohexyl)carbamoylmethyl]-NAD which provides some degree of flexibility in binding with the enzyme under restrictions imposed by its immobilization, except in the case of microencapsulation in which the coenzyme would not otherwise be retained. The preparation and uses of NAD derivatives have been reviewed earlier in this series.19,2° Our own method of coimmobilization will be described as an example of covalent attachment to the inner surface of a nylon tube. Gestrelius e t al. 21 coimmobilized horse liver alcohol dehydrogenase and the NAD(H) analog, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, to Sepharose 4B following the commonly used method of cyanogen bromide activation under alkaline conditions. 27 The enzyme and the NADH analog were mixed prior to immobilization to permit initial formation of the binary enzyme-coenzyme complex, thereby allowing them to interact more efficiently after the immobilization. Both thermal and storage stability of the enzyme was increased to some extent, allowing a high recycling rate which resulted from protection of the active site by the coimmobilized coenzyme, the latter being fixed in or near the active site of the enzyme. The coenzyme was used in its reduced form (NADH) because of its lower dissociation constant than that for NAD, 28 which thus favored binary complex formation, while the analog was chosen to provide a "spacer arm" as well as a functional group suitable for covalent binding to the CNBr-activated Sepharose matrix. This method may be compared 27 R. Axen, J. Porath, and S. Ernbach, Nature (London) 214, 1302 (1967). 28 H. Theorell and A. D. Winer, Arch. Biochem. Biophys. 83, 291 (1959).

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ALCOHOL DEHYDROGENASE COIMMOBILIZED WITH N A D

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with that reported by the Japanese workers, 22 in which the supporting gel consisted of a copolymer of methylmethacrylate and acrylic acid, and in which the polymerization was initiated by potassium sulfate. The coupling of the mixed enzyme and NAD was carried out in a suspension of gel particles in the presence of the water-soluble carbodiimide 1-cyclohexyl3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMC) at a temperature of 5°. Grunwald and Chang 24 described the preparation of immobilized NAD-N6-[N-(6-aminohexyl)acetamide] with soluble dextran T70 (Pharmacia) activated with cyanogen bromide, following the method of Larsson and Mosbach. 29Their procedure for coimmobilization involves preparation of semipermeable microcapsules 23'25 by interfacial polymerization in which an aqueous phase containing yeast alcohol dehydrogenase, malate dehydrogenase, dextran-NAD analog, 1,6-diaminohexane, and polyethyleneimine (used as a microcapsule filler) is emulsified in an organic phase of I : 4 (v/v) chloroform : cyclohexane solution with Span 85, followed by the addition of terephthaloyl chloride. The polymerization reaction is terminated by the addition of extra organic solvent, and the resulting microcapsule is sedimented by centrifugation. Finally, the covalent attachment procedure used by ourselves with nylon tubing 26 involves activation of the inner surface of nylon tubing by partial acidic hydrolysis, followed by condensation of the amino group of N6-[(6-aminohexyl)carbamoylmethyl]-NAD with the liberated carboxyl group, and then coupling of the enzyme to the amino groups via glutaraldehyde. This method leads to a stable enzyme-coenzyme tube and one that is ideally suitable as an open tubular heterogeneous enzyme reactor (OTHER), in which diffusional limitations are unimportant since the enzyme and the coenzyme are both suspended in solution from the interior surface of the tube. However, the activity of such a system is limited by the extent to which the coenzyme molecules are properly oriented with respect to the active site of the enzyme. This method will now be described in some detail. A t t a c h m e n t Procedure Inside a N y l o n Tube

The coimmobilization of the enzyme, yeast alcohol dehydrogenase (233 U/mg protein), and the coenzyme analog, N6-[(6-aminohexyl)carba moylmethyl]-NAD, synthesized by the method of Lindberg et al., 3° is .,9 P.-O. Larsson and K. Mosbach, F E B S Lett. 46, 119 (1974). 3o M. Lindberg, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973).

60

M U L T I S T E P E N Z Y M E SYSTEMS AND COENZYMES

[6]

summarized in the following scheme: Nylon,'-,~,,,CONH,,,,,,,,~

nylon partial hydrolysis H+/H20

) ~COO-

H3H +,,',,,,,,~

H2N(CH2)6NHCOCH2 + NAD

) " , " , ~ C O H N ( C H 2 ) 6 N H C O C H 2 - N A D + H3N +,'.~,~ CMC, pH 5, 21° glutaraldehyde ' """~COHN(CHz)6NHCOCH2-NAD + OCH(CH2)3CH=N"~"" pH 8.5, 0-5 o H2N-enzyme "~'~COHN(CHz)6NHCOCHz-NAD + enzyme--N=CH(CH2)3CH=N,,,,,,,,~ pH 7.5, 0-5 °

This procedure is essentially the same as that described in a previous paper 26and is a modification of our earlier method for the covalent attachment of the enzyme only31; in that procedure the carboxyl groups of the partially hydrolyzed nylon tubing is blocked by reaction with benzidine. Nylon tubing (type 6, from John Tullis, Scotland) 1 m in length and of 0.068 cm internal radius is used as a tightly wound coil. The tube is first filled with a mixture of 18% (w/v) CaCI2 in a solution of 18% (v/v) water in methanol, and incubated for 30 min at 40 °, followed by rinsing with water at a flow rate of 5 cm3/min for 20 min, to increase the available surface area. Partial hydrolysis of the inner surface is then accomplished at 40 ° by pumping 4 M HCI at a flow rate of 5 cm3/min. This is stopped after 20 min by washing the tube, at the same flow rate, with ice-cold distilled water for 30 min. In the next step, 100 mg of the NAD derivative is dissolved in 10 cm 3 water to which 500 mg of the carbodiimide CMC has been added, and the pH is adjusted to 5. This solution is circulated for 24 hr at a flow rate of 0.5 cm3/min through the tube kept in a water bath at room temperature (-21°). The tube is then rinsed with water at a flow of 5 cm3/min for 1 hr. The enzyme is coupled to the free amino groups, liberated during the acid hydrolysis of the tube, through the bifunctional reagent glutaraldehyde. A solution of 12.5% (v/v) glutaraldehyde in 0.2 M borate buffer, pH 8.5, is passed through the tube in which the NAD analog is already attached, immersed in crushed ice, at a flow rate of 1 cm3/min for 2 hr. Any unreacted glutaraldehyde is removed by washing the tube with 0.1 M phosphate buffer, at pH 7.5, at a flow rate of 5 cm3/min for some time. The last step involving the coupling of the enzyme to the glutaraldehyde residues is carried out by circulating overnight a solution of 10 mg enzyme in 10 cm 3 buffer (0.1 M phosphate, pH 7.5) at a flow rate of 0.5 cma/min, the tube being maintained in a bath of crushed ice. Finally, any unbound enzyme is removed by perfusing the tube with 0.1 M phosphate buffer, pH 7.0, containing 0.5 M NaC1, at a flow rate of 5 cm3/min for 3 hr. 31 M. A. M a z i d a n d K. J. L a i d l e r , Biochim. Biophys. Acta 614, 225 (1980).

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ALCOHOL DEHYDROGENASE COIMMOBILIZED WITH NAD

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The tube is next filled with 0.1 M phosphate buffer, at pH 7.5, containing 1 mM EDTA and 0.1 mM 2-mercaptoethanol and, when not in use, is stored below 5° in a water bath kept in a refrigerator. Kinetic Studies The reaction usually studied involves the oxidation of ethanol by NAD with the production of acetaldehyde and NADH, the reduced form of the immobilized coenzyme. Unless the latter is recycled efficiently, very little reaction occurs and it is not practical to measure the rate. A number of methods, both enzymatic and nonenzymatic, are available for this purpose. ~-9 We found that the rapid nonenzymatic reduction of 2,6-dichlorophenolindophenol (DCPIP) dye by NADH in the presence of phenazine ethosulfate (PES) is suitable in our tubular flow reactor system. Gestrelius et al. 21 also studied different methods, and following the method of Schulman et al. 3 found that the most successful regeneration was accomplished by using the coupled oxidoreduction between the two alternative substrates, ethanol and lactaldehyde. Our method is similar to that described by Slater and co-workers6.7; the entire reaction scheme is as follows: -reduced PES --. 2,6-dichlorophenol ethanol ](NAD ~(phenazine ethosul-~( indophenol (DCPIP) acetaldehyded~NADH J\fate (PES) */~reduced DCPIP

In this method, PES acts as a mediator for electron transport during the oxidation of NADH, and reduces DCPIP. Phenazine methosulfate has also been used, but PES is chemically more stable. It is to be noted that PES and DCPIP can oxidize 2-mercaptoethanoP 2 which was used for the storage of the tube, and any traces of the latter must therefore be removed before a kinetic run is carried out. Reagents and Methods

Both PES and DCPIP were obtained from the Sigma Chemical Company. They were used in fixed concentrations of 0.25 and 70 mM, respectively, which were sufficiently high to permit measurement of the N A D ethanol reaction rate. The concentration of the substrate ethanol was varied from 5 to 50 mM in the reaction mixture with PES and DCPIP. All solutions were freshly prepared in 0.1 M phosphate buffer, pH 7.5 - 0.05, made up with doubly distilled deionized water. They were protected from 32 C. Bernofsky and K. M. Royal, Biochim. Biophys. Acta 215, 210 (1970).

62

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[6]

exposure to air and light, which reoxidizes DCPIP. The flow-kinetic runs were carried out as described previously, 3~ using a Pye Unicam SP 1800 UV spectrophotometer. Rates were calculated from the decrease in absorbance of DCPIP at 600 nm, under steady-state conditions, in which the absorption coefficient was taken to be 20,000 M -~ cm -~.33 Also, the activity of the tube was monitored periodically by assays at a linear flow rate of 0.69 cm/sec with 20 mM ethanol, 0.25 mM PES, and 70 mM DCPIP, at 25.5 ° and pH 7.5. The tube showed no measurable loss of enzymatic activity over a period of 1 month.

Analysis of Data The methods of analysis of flow-kinetic data are based on the Kobayashi-Laidler theory 34 developed originally for one-substrate reactions catalyzed by immobilized enzymes; the procedure has been described in a number of previous papers 34-4° and recently reviewed. 41 The methods have been found to be equally applicable to two-substrate reactions under the limiting conditions of one substrate in excess while the other is varied) 1,42-44However, the present system is essentially a one-substrate one, since the coenzyme is immobilized and immediately recycled so that its concentration is practically constant. The equations used in the analyses were summarized in a previous paper 26 and are given here very briefly. The apparent Michaelis constant Km,app is related to the inherent Michaelis constant K~ through the mass transfer coefficient kL as follows: Km,app =

Vm

t

K~n + ~ L = Km +

~ 8 (\D2/ rt ~1/3 O f 1/3

(1)

where Vm is the limiting rate for the immobilized enzyme at the flow rate vf, r is the radius and L the length of the tube, and D is the diffusion coefficient for the substrate in solution. 33 D. S. Coffey and L. Hellerman, Biochemistry 3, 394 (1964). 34 T. Kobayashi and K. J. Laidler, Biotechnol. Bioeng. 16, 99 (1974). 35 p. S. Bunting and K. J. Laidler, Biotechnol. Bioeng. 16, 119 (1974). 36 T. T. Ngo and K. J. Laidler, Biochim. Biophys. Acta 377, 317 (1975). 37 D. Narinesingh, T. T. Ngo, and K. J. Laidler, Can. J. Biochem. 53, 1061 (1975). 38 T. T. Ngo, D. Narinesingh, and K. J. Laidler, Biotechnol, Bioeng. 18, 119 (1976). 39 T. T. Ngo and K. J. Laidler, Biochirn. Biophys. Acta 525, 93 (1978). 4o T. T. Ngo, K. J. Laidler, and C. F. Yam, Can. J. Biochem. 57, 1200 (1979). 4~ K. J. Laidler and P. S. Bunting, this series, Vol. 64, p. 227. 42 M. A. Mazid and K. J. Laidler, Biochim. Biophys. Acta 614, 237 (1980). 43 N. J. Daka and K. J. Laidler, Can. J. Biochem. 56, 774 (1978). 44 N. J. Daka and K. J. Laidler, Biochim. Biophys. Acta 612, 305 (1980).

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ALCOHOL DEHYDROGENASE COIMMOBILIZED WITH N A D

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The mass transfer coefficient kL is a measure of the rate at which the substrate passes through a diffusion boundary layer established at the wall of the tube. There can be full diffusion control at low substrate concentrations and low flow rates, and under these conditions the concentration of the product at the exit of the tube is given by

(DL ] 2/3 [P]eD = 2.56 \r-Tvf/ [S]

(2)

At the other extreme, the exit concentration is given by 2L [p]o

VM = -rof

(3)

where VMis the inherent enzymatic reaction rate as given by an equation of the Michaelis-Menten form. The extent of diffusion control can also be evaluated on the basis of double-logarithmic plot of the dimensionless parameters qb and 19 defined as follows:

( ufr2~2/3 \~--~: p = Km,app/[S]

(4) (5)

Results and Discussion Table I summarizes results obtained from Lineweaver-Burk plots at different flow rates; the plots were linear over the concentration range of 5-50 mM ethanol. The Michaelis parameters, Km.avpand Vm, were obtained from the best fit, using the method of least squares. Included in the table are the extrapolated values of Km and V~ at infinite flow rates [Eq. (1)], i.e., under conditions when there are no diffusional effects. The value of 1.5 mM for the inherent Michaelis constant Km is significantly lower than that of 4.6 raM, found previously3~ with only the enzyme attached to the inner surface of a nylon tube. This means the present system is a more efficient one, which in turn indicates that there are fewer steric restrictions to reaction, and that the coenzyme has been suitably positioned on immobilization. Similar results to ours were obtained by Gestrelius et al. 2~ The activity of our preparation was rather low compared to that for the free enzyme and coenzyme in solution, as reported earlier by Larsson and Mosbach. 45 Our results also show that, on 45 P.-O. Larsson and K. Mosbach,

Biotechnol. Bioeng. 13, 393 (1971).

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

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TABLE I VALUES OF Kin,apt, (ETHANOL) AND Vm AT VARIOUS FLOW RATES a

Flow rate, of (cm/sec)

Km,app(ethanol) (raM)

V" (10 -9 M sec ~)

Reference

2.14 2.16 2.20 1.99 1.85 1.5 (Kin)

74.6 77.3 82.3 84.5 87.8 - 100

26 26 26 26 26 26

Enzyme and NAD coimmobilized 0.25 0.40 0.69 1.27 2.00 (extrapolated)

Enzyme immobilized; saturation with N A D o¢ (extrapolated)

4.6

~ 1000

31

a At 25.5 ° at pH 7.5.

an absolute basis, the tube with the coimmobilized enzyme and the coenzyme is less active than that used in our previous work, 31 in which only the enzyme was immobilized and the NAD was present in saturating concentrations. This is evident from the V" value of 10 -7 M sec -I which is an order of magnitude lower than that of 10 -6 M sec -1, obtained in our previous work. 3~ Such a result may be hard to improve upon, since it is obviously difficult to immobilize as much coenzyme as can be made available in a saturation experiment. We studied other methods to immobilize the coenzyme 4s-5° but none of them led to a preparation as active or stable as the one we have described. Our procedure follows that of Lindberg e t al. 3° for the synthesis of the coenzyme analog, the attachment being at the N-6 group of the adenine moiety, which in conjunction with the method of attaching the enzyme appears to be quite satisfactory. The success of the method is attributable to the fact that both enzyme and coenzyme are attached covalently to the surface of the tube, and that there are neutralizations of surface charges; the latter have a detrimental effect on enzymatic activity. 43,44,5~ Figure 1 shows a double-logarithmic plot of product concentration at 46 J. R. W y k e s , P. Dunhill, a n d M. D. Lilly, Biochim. Biophys. Acta 286, 260 (1972). 47 M. Guilford, P.-O. Larsson, and K. Mosbach, Chem. Scr. 2, 165 (1972). S. Barry and P. O ' C a r r a , FEBS Left. 37, 134 (1973). 49 R. L a m e d , Y. Levin, and M. Wilcheck, Biochim. Biophys. Acta 304, 231 (1973). 50 M. J. H a r v e y , C. R. L o w e , D. B. Craven, and D. G. Dean, Eur. J. Biochem. 41, 335 (1974). 51 p. S. Bunting and K. J. Laidler, Biochemistry 11, 4477 (1972).

[6]

65

ALCOHOL DEHYDROGENASE COIMMOBILIZED WITH N A D

-4.4

-4.6

-48

-5.0

-% oo ~ - 5 . 2

-54 20raM 10 mM 7OmM 5.0mM - 5"6 j~ _ L _ - 0,60

-045

-0.30 -0.15 IOglo(V f / c r n $'1)

0

0.15

0.30

FIG. I. Double-logarithmic plots of product concentrations at the exit against flow rates for the l-m tube, at various ethanol concentrations as indicated. From Mazid and Laidler -'6 by permission of John Wiley and Sons, Inc.

the exit against flow rate with different concentrations of the substrate ethanol. The slopes of the lines are from - 0 . 9 0 to -0.92. A slope of - 2 / 3 is expected when there is full diffusion control [Eq. (2)] and of - 1 when there is no diffusion control [Eq. (3)]. The fact that the slopes are not far from unity is thus consistent with little diffusion control. This is confirmed by the calculation of the dimensionless parameters [Eqs. (4) and (5)] as shown in Table II, the values of • being obtained using a diffusion coefficient of 4 × 10 -6 c m 2 s e c -1 for ethanol. 3],37 Inspection shows that the points lie well inside the diffusion-free region of the plot of qb against p (compare, for example, Fig. 2 of Ref. 31), since the qb values are very low as a result of the low product concentrations compared to the concentrations of the substrate [Eq. (4)]. Our previous work with the immobilized

66

MULTISTEP ENZYME SYSTEMS AND COENZYMES

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T A B L E II DIMENSIONLESS PARAMETERS qb AND p AT DIFFERENT ETHANOL CONCENTRATIONS AND FLOW RATES Parameter values at three flow rates, uf (cm sec -~) 0.25

0.69

2.00

[C2HsOH] (mM)

10 3 (])

p

10 3 qb

to

10 3 qb

p

5.0 10.0 50.0

8.5 5.0 1.2

0.43 0.21 0.04

6.6 3.9 0.9

0.44 0.22 0.04

5.3 3.0 0.7

0.37 0.19 0.04

enzyme only 31 also showed that there is little diffusion control with respect to ethanol when NAD is present at saturating concentrations, but that the slopes of the double-logarithmic plots (see Fig. l) had anomalously low negative values of -0.57 to -0.60 and -0.40 to -0.45 with different concentrations of saturating NAD. This result was accounted for by the product inhibition, which was more pronounced at lower flow rates when the products remain attached to the surface, as a result of longer residence times, and block further reaction. However, direct tests showed that there was no product inhibition in the coimmobilized system either by NADH or by acetaldehyde, the former being immediately reoxidized in the recycling mechanism and the latter being produced in the reaction in sufficiently low concentrations. Conclusions Alcohol dehydrogenase has been coimmobilized with its coenzyme, NAD. Our procedure for covalent attachment of both enzyme and a coenzyme analog inside a nylon tube showed higher efficiency compared to a similar system with only the enzyme immobilized, indicating that on coimmobilization the coenzyme has been suitably positioned with respect to the active center of the enzyme. The higher efficiency also suggests that the spacer arm through which the coenzyme is attached to the tube is of an appropriate length. On an absolute basis, however, the activity was low, and this was also observed in the coimmobilization by other methods. The activity may be improved by introducing a longer and more flexible spacer arm; any coenzyme immobilized at a distance away from the active center could then be available for enzymatic activity. Our method showed no measurable loss of enzymatic activity over a period of 1 month, and the success of the method was attributed to covalent attach-

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RECYCLINGov NAD(P) By MULTIENZYMESYSTEMS

67

ment as well as to the neutralization of surface charges in the nylon tube. Apart from low activity, however, a system of the kind described would be ideally suitable as an open tubular heterogeneous enzyme reactor (OTHER) in applications such as automated analysis, with no need to add soluble coenzyme.

[7] R e c y c l i n g o f N A D ( P ) b y M u l t i e n z y m e S y s t e m s I m m o b i l i z e d b y M i c r o e n c a p s u l a t i o n in Artificial Cells

By T. M. S. CHANG Semipermeable microcapsules have been used as artificial cells encapsulating enzymes, cells, cell extracts, adsorbents, immunogens, and other biologically active materials. ~-5A number of artificial cell systems are now being applied in the treatment of patients or in biotechnology. 5 The possible applications of microencapsulated enzymes were proposed and demonstrated in animal experiments some time ago using mostly single enzyme systems. TM For more widespread applications, it will be important to have microencapsulated multienzyme systems. There is no problem in enclosing high concentrations of any number of enzyme systems within the artificial cells, since this can be done by dissolving the enzymes in the solution before microencapsulation.l-4 Enzymes immobilized within semipermeable microcapsules are in close proximity and in free solution, and a large number of different enzyme systems can be enclosed at high concentrations. In this way, cofactor in the microcapsules can interact much more freely with the multienzyme systems. Furthermore, the large surface area to volume relationship and the ultrathin membrane are such that substrates and products of the enzyme reaction can equilibrate across the membrane at a rate which is at least 100 times that of standard dialysis membrane systems. This paper is an updated discussion of our studies on the recycling of NAD(P) by multienzyme systems immobilized by microencapsulation within artificial cells. i T. M. S. Chang, Science 146, 524 (1964). 2 T. M. S. Chang, F. C. Macintosh, and S. G. Mason, Can. J. Physiol. Pharmacol. 44, 115 (1966). 3 T. M. S. Chang, "Artificial Cells." Thomas, Springfield, Illinois, 1972. 4 T. M. S. Chang, "Biomedical Applications of Immobilized Enzymes and Proteins," Vols. 1 and 2. Plenum, New York, 1977. T. M. S. Chang, "Microencapsulation including Artificial Cells." Humana, New York, 1984.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[7]

RECYCLINGov NAD(P) By MULTIENZYMESYSTEMS

67

ment as well as to the neutralization of surface charges in the nylon tube. Apart from low activity, however, a system of the kind described would be ideally suitable as an open tubular heterogeneous enzyme reactor (OTHER) in applications such as automated analysis, with no need to add soluble coenzyme.

[7] R e c y c l i n g o f N A D ( P ) b y M u l t i e n z y m e S y s t e m s I m m o b i l i z e d b y M i c r o e n c a p s u l a t i o n in Artificial Cells

By T. M. S. CHANG Semipermeable microcapsules have been used as artificial cells encapsulating enzymes, cells, cell extracts, adsorbents, immunogens, and other biologically active materials. ~-5A number of artificial cell systems are now being applied in the treatment of patients or in biotechnology. 5 The possible applications of microencapsulated enzymes were proposed and demonstrated in animal experiments some time ago using mostly single enzyme systems. TM For more widespread applications, it will be important to have microencapsulated multienzyme systems. There is no problem in enclosing high concentrations of any number of enzyme systems within the artificial cells, since this can be done by dissolving the enzymes in the solution before microencapsulation.l-4 Enzymes immobilized within semipermeable microcapsules are in close proximity and in free solution, and a large number of different enzyme systems can be enclosed at high concentrations. In this way, cofactor in the microcapsules can interact much more freely with the multienzyme systems. Furthermore, the large surface area to volume relationship and the ultrathin membrane are such that substrates and products of the enzyme reaction can equilibrate across the membrane at a rate which is at least 100 times that of standard dialysis membrane systems. This paper is an updated discussion of our studies on the recycling of NAD(P) by multienzyme systems immobilized by microencapsulation within artificial cells. i T. M. S. Chang, Science 146, 524 (1964). 2 T. M. S. Chang, F. C. Macintosh, and S. G. Mason, Can. J. Physiol. Pharmacol. 44, 115 (1966). 3 T. M. S. Chang, "Artificial Cells." Thomas, Springfield, Illinois, 1972. 4 T. M. S. Chang, "Biomedical Applications of Immobilized Enzymes and Proteins," Vols. 1 and 2. Plenum, New York, 1977. T. M. S. Chang, "Microencapsulation including Artificial Cells." Humana, New York, 1984.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

68

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[7]

Basic Methods for Immobilization of Multienzymes by Microencapsulation

Preparation of Cellulose Nitrate Membrane Microcapsules Cellulose nitrate membrane microcapsules are prepared using an updated procedure based on earlier publications. 1-7 Hemoglobin (bovine hemoglobin Type 1, 2× crystallized and dialyzed, and lyophilized, Sigma Co.), 15 g, is dissolved in 100 ml of distilled water and filtered (Whatman No. 42). Multienzymes and other materials to be microencapsulated are dissolved or suspended in 2.5 ml of this hemoglobin solution. The final pH is adjusted to pH 8.5 with Tris buffer and the hemoglobin concentration adjusted to 10 g/dl. This solution, 2.5 ml, is added to a 150-ml glass beaker. Water-saturated ether (analytical-grade ether shaken with distilled water in a separating funnel and water discarded), 25 ml, is added. The mixture is immediately stirred with a Fisher Jambo magnetic stirrer at 1200 rpm (setting of 5) for 5 sec. While stirring is continued, 25 ml of a cellulose nitrate solution is added (this solution is prepared earlier by evaporating 100 ml of USP collodion to a thin sheet and redissolving in 100 ml of a mixture of 82.5 ml ether and 17.5 ml absolute alcohol). Stirring is continued for another 60 sec. The beaker is covered and allowed to stand unstirred at 4° for 45 min. The supernatant is decanted and 30 ml of n-butyl benzoate added. The mixture is stirred for 30 sec at the same magnetic stirrer setting. The beaker is allowed to stand uncovered and unstirred at 4° for 30 rain. Then the butyl benzoate is removed completely after centrifugation at 350 g for 5 min. A Tween 20 (Atlas Powder Co.) solution, 25 ml, at 50% (v/v) concentration, pH 7.0, is added. Stirring is started at a setting of 10 for 30 sec. Water, 25 ml, is added and stirring continued at a setting of 5 for 30 sec; then 200 ml of water is added. The supernatant is removed and the microcapsules are washed 3 more times with 200 ml of a 1% Tween 20 solution, pH 7. The microcapsules are then suspended in a suitable buffer. In properly prepared microcapsules, there should not be leakage of hemoglobin after the preparation. Hemoglobin at a concentration of 10 g/dl is necessary for successful preparation. Furthermore, this high concentration of protein stabilizes the enzymes during the preparation, reaction, and storage. 8 However, crude hemoglobin contains enzymes which inactivate NADH and unless highly purified hemoglobin is used, purification using affinity chromatography on 6 T. M. S. Chang, this series, Vol. 44, p. 201. 7 y . T. Yu and T. M. S. Chang, Int. J. Biomat. Med. Devices Artif. Organs 8, 273 (1980). 8 T. M. S. Chang, Biochem. Biophys. Res. Commun. 44, 1531 (1971).

[7]

RECYCLING OF N A D ( P ) BY MULTIENZYME SYSTEMS

69

an NAD+-Sepharose column is required. 9 The long-term stability of microencapsulated multienzyme activity can be greatly increased by crosslinking with glutaraldehyde, 8 but this is done at the expense of greatly reduced initial enzyme activity.

Preparation of Polyamide Membrane Microcapsules by lnterfacial Polymerization Polyamide membrane microcapsules of 100 /zm mean diameter are prepared using an updated method based on earlier methods. 1-6:0-14 Just before use the following two solutions are prepared. (l) Terephthaloyl chloride (ICN K & K Inc.), 100 mg, is added to 30 ml of an organic solution (chloroform : cyclohexane, 1 : 4) kept in an ice bath. This is covered and stirred with a magnetic stirrer for 4 hr and then filtered with Whatman No. 7 paper. (2) A diamine-polyethyleneimine solution is prepared by dissolving 0.378 g NaHCO3 and 0.46 g 1,6-hexadiamine (J. T. Baker Chemical Co.) in 5 ml distilled water, and adjusting the pH to 9 with 6 N HCI. Then 2 ml 50% polyethyleneimine (ICN K & K Inc.) is added to the diamine solution, the pH readjusted to 9, and the final volume made up to I0 ml with distilled water. The hemoglobin solution, 10 g/dl, is prepared as described above for cellulose nitrate microcapsules. Multienzymes and other materials are added to the hemoglobin solution. Two and one-half milliliters of this hemoglobin solution and 2.5 ml of the diamine-polyethyleneimine solution are mixed for 10 sec in a 150-ml beaker placed in an ice bath. A 0.5% (v/v) Span 85 (Atlas Powder Co.) organic solution (chloroform : cyclohexane, 1 : 4), 25 ml, is added and stirred in the Fisher Jambo magnetic stirrer at speed setting of 2.5 for 60 sec. The terephthaloyl chloride solution prepared earlier is added (25 ml) and the reaction is allowed to proceed for 3 rain with the same stirring speed. The supernatant is discarded and another 25 ml of the terephthaloyl chloride solution is added. The reaction is carried out with stirring for another 3 rain. The supernatant is discarded. Then 50 ml of the 0.5% Span 85 chloroform-cyclohexane solution is added and stirred for 30 sec. The supernatant is discarded. After this the procedure with Tween 20 as describd for cellulose nitrate micro9 j. Grunwald and T. M. S. Chang, J. Appl. Biochem. 1, 104 (1979). 10j. C. W. Ostergaard and S. C. Marting, Biotechnol. Bioeng. 15, 561 (1973). " R. B. Aisina, N. F. Kazanskafa, E. V. Lukasheva, and V. Berezin, Biokhimiya 41, 1656 (1976). 12j. Grunwald and T. M. S. Chang, Biochem. Biophys. Res. Commun. 81, 565 (1978). 13 j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). ]4 y. T. Yu and T. M. S. Chang, Enzyme Microb. TechnoL 4, 327 (1982).

70

MULTISTEP ENZYME SYSTEMS AND COENZYMES

/

\

/

ALCOHOL

x

ethanoI-"'-/-',-DEHYDROCENASE

+,

/+

NAD ~'T'~NAD

malate

[7]

\

,.

- acetaldehyde

NAD H "q------'~NADH

~ -.~ DEHYDROGENAS E y

oxaloacetate

%

FIG. 1. High concentrations of yeast alcohol dehydrogenase and malate dehydrogenase immobilized in solution within semipermeable microcapsules. NAD + and NADH can equilibrate rapidly across the membrane to be recycled in the microcapsules.

capsules is used here for the transfer of the microcapsules into the buffer solution. Failure in preparing good microcapsules is frequently due to diamine or diacids which have been stored after they have been opened. A new, unopened bottle will usually solve the problems. Unlike the cellulose nitrate microcapsules, in interfacial polymerization the hemoglobin solution can be replaced by a 10% polyethyleneimine solution adjusted to pH 9.u However, the microcapsules prepared without hemoglobin may not be as sturdy. Cross-linking with glutaraldehyde could also be carried out. 8 As stated before, this markedly decreases the initial enzyme activity, though the long-term stability of the remaining enzyme activity after cross-linking with glutaraldehyde is greatly increased.

Recycling of Free NADH and NADPH Recycling of Free N A D + and N A D H by Microencapsulated Yeast Alcohol Dehydrogenase and Malate Dehydrogenase us'n6 (Fig. I) Yeast alcohol dehydrogenase, 2.8 rag, and 0.22 mg of malate dehydrogenase are dissolved in 2.5 ml of the hemoglobin solution prepared as described under the basic methods, and then immobilized within cellulose nitrate microcapsules by the procedure described above. The final preparation is suspended in 0.1 M phosphate buffer, pH 8.0 at 4 °. Recycling of free NAD + is studied by suspending the microcapsules in the presence of 0.0196 mM NAD +, 0.217 M ethanol, 0.981 mM oxaloacetic acid, and 0.1 M phosphate, pH 8.0. Recycling of free NAD + is determined as the production of malic acid as follows. Reactions are ~5 j. Campbell and T. M. S. Chang, Biochim. Biophys. Acta 397, 101 (1975). t6 j. Campbell and T. M. S. Chang, Biochem. Biophys. Res. Commun. 69, 562 (1976).

[7]

RECYCLING OF

urea

/ /

+/

glucose '

BY MULTIENZYME SYSTEMS

~ UREASE .~

I

i glutamates------

NAD(P)

x. NH3

7

~

GLUTAMATE DEHYDROCENASE 9

/;+

71

NH 3

~ ketoglutarate

,,

t

~ ,-~1 GLUCOSE / ~ DEHYDROCENASE

-\ ,~ / pyruvate~CLUTAMATE-PYRUVAT~ TRANSANINASE ~

\

',glucuronate /

) /

/ ketoglutarate

--

+ alantne

FIG. 2. High concentrations of four enzymes (urease, glutamate dehydrogenase, glucose dehydrogenase, and glutamate pyruvate transaminase) immobilized in solution within semipermeable microcapsules. Substrates and NAD ÷ can equilibrate rapidly across the membrane for the multistep enzyme reaction in the conversion of urea to ammonia to glutamic acid to alanine.

allowed to be carried out and then terminated after preset time intervals by heating the reaction mixture at 100° for 5 min. The reaction mixture is centrifuged, and 0.5 ml of the supernatant is used in the following assay for malic acid. 17 The supernatant, 0.5 ml, is added to 1.5 ml NAD ÷ (2 raM), 0.5 ml 30 mM glutamic acid, and 1.0 ml 0.1 M glycine/NaOH, pH 10.0. The change in absorbance at 340 nm after the addition of 5 ttl glutamate oxaloacetate transaminase (aspartate aminotransferase) and 25 /zl malate dehydrogenase is noted. This procedure is standardized by subjecting known amounts of malic acid to the same assay. Recycling of free NADH is studied in a similar fashion to NAD + except that 0.0196 mM NADH is used instead of NAD +. Recycling of N A D H in the Conversion of Urea and Ammonia into Amino Acid w'-2° (Fig. 2) The hemoglobin solution (3 ml of 10 g/100 ml solution) is prepared as described under the basic method, but in addition containing 1.0 mg of urease (Worthington Biochemical Corp., 60 U/mg), 16 mg of L-glutamate dehydrogenase (Sigma Chemical Co., 50 U/mg, 10 mg/ml), 0.16 mg of glucose dehydrogenase (Boehringer-Mannheim, 150 U/mg), 4.0 mg of glutamate pyruvate transaminase (aspartate aminotransferase) (Sigma 17 T. t8 T. ,9 T. 2o K.

Kato, S. J. Berger, J. A. Carter, and O. H. Lowry, Anal. Biochem. 53, 83 (1973). M. S. Chang and C. Malouf, Artif. Organs 3, 38 (1979). M. S. Chang, C. Malouf. and E. Resurreccion. Art([] Organs (Suppl.) 3, 284 (1979). F. Gu and T. M. S. Chang, Biomat. Artif. Cells Artif. Organs 15, 289 (1987).

72

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[7]

Chemical Co. 91 U/mg), and 20 mg of NADH (Sigma Chemical Co.). NADH is used to stabilize glucose dehydrogenase during preparation and is dialyzed out of the microcapsules after preparation. The remaining steps for the microencapsulation of hemoglobin-enzyme solution are the same as described under the basic method for cellulose nitrate membrane microcapsules. Ammonia substrate solution is made up of glucose (1 g/dl), KCI (5 mM/liter), MgC12 (5 mM/liter), ammonium acetate (20 mM/liter), a-ketoglutarate (20 mM/liter), pyruvic acid (20 mM/liter), NADH (0.5 mM/ liter), and ADP (0.1 mM/liter). For urea assay, the same solution as above is used except that ammonium acetate is replaced by urea (50 mg/dl). Our results show that this multienzyme system is effective in the sequential conversion of urea into ammonia, then ammonia into glutamic acid, and finally glutamic acid into another amino acid, alanine (Fig. 2). The conversion of urea into ammonia takes place extremely rapidly, nearly all the urea being converted into ammonia within 10 min. However, the rate of conversion of ammonia sequentially into glutamic acid and then into alanine was 0.3 ixM/ml microcapsules per minute. This is not likely due to the delay in the recycling of NADH in the formation of glutamic acid, since when microcapsules were used to convert ammonia into glutamic acid only, the rate of formation of glutamic acid was 2.4 tzM/ ml microcapsule per minute. When the microcapsules were used to convert glutamic acid into alanine only, the rate was only 0.8 tzM/ml microcapsules per minute even with a 20 mM/liter glutamic acid substrate solution. Further analysis showed that by increasing the amount of transaminase the rate of conversion can be increased to 1.80 tzM/ml microcapsules per minute. Thus by optimizing the enzyme concentrations and the other reaction rate, a much more efficient system is possible. The high rate of recycling of NADH was made possible only by the use of a very high specific-activity glucose dehydrogenase (150 U/mg). Microencapsulated multienzyme systems with NAD-dextran can convert ammonia and urea into essential amino acids. 2°

Recycling of NADPH in the Sequential Conversion of Urea and Ammonia into Amino Acid 2j (Fig. 3) Urease (10 rag), glutamate dehydrogenase (8 mg), and glucose-6-phosphate dehydrogenase (2 mg) are dissolved in 1.5 ml of hemoglobin solution. This is then microencapsulated using the procedure of interfacial polymerization described under the basic methods. The rate of formation of 6-phosphogluconate is used to measure the 21 j. C o u s i n e a u and T. M. S. Chang, Biochem. Biophys. Res. Commun. 79, 24 (1977).

[7]

RECYCLINGOF NAD(P) BY MULTIENZYMESYSTEMS / .

urea

' I

/

u~ 3

GLUTAMATE DEHYDROCENASE :

+l

/

NADP ~ - - ~ N A D P

~ \

"--

\

glutamate-I

glucose-6-P

"% ~ U R E A S E ~~H ~3N~

N

+

~ ketoglutarate

t

NADPH ~

GLUCOSE-6-P / DEIIYDROGENASE

73

I

NADPH

/ -~ 6 - p h o s p h o g l u c o n a t e /

/

FIG. 3. High concentrations of three enzymes (urease, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase) for the multistep enzyme reaction of conversion of urea into the amino acid glutamic acid.

cyclic regeneration ofNADP + : NADPH. The microcapsules, 100/~1, containing the muitienzyme system are suspended in 3.0 ml of 0. I M phosphate, pH 7.5, containing 10 mM of urea, 4 mM o f a-ketoglutarate, 0. I mM of ADP, and 5 mM of glucose 6-phosphate, followed I rain later by the addition of the cofactor NADPH at 0.21 mM to initiate the cyclic process. Aliquots of the Suspension are withdrawn at various intervals and heated at 100° for 2 min, and the filtrate analyzed for 6-phosphoglucohate as follows. The filtrate (0.5 ml) is added to 1.0 ml of 0.1 M phosphate, pH 7..5, containing 6-phosphogluconate dehydrogenase (0.15 units) and 2 mM NADP + at 37°. After 30 min of incubation, the observed increase of the absorbance at 340 nm is noted and read on a standard curve. The rate of conversion of urea into glutamate and the recycling of NADPH could be followed by the rate of formation of 6-phosphogluconate, the decrease in urea level, or the formation of glutamic acid. Ammonium acetate could be used in this reaction instead of urea. Glutamate is formed at the same rate irrespective of whether urea or ammonium acetate is used as substrate. This suggests that the rate-limiting step in this sequential reaction is in the conversion of ammonia into glutamic acid. Recycling of NAD + in Stereospecific Steroid Oxidation 22 (Fig. 4) Another application is demonstrated in Fig. 4. Here microcapsules containing 3cz-hydroxysteroid dehydrogenase and the bacteria Leuconostoc mesenteroides with NADH oxidase have been used in stereospecific 22 F. Ergan, D. Thomas, and T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 61 (1984).

74

MULTISTEP ENZYME SYSTEMS AND COENZYMES

/

andros

t erone

\

t3a-HYDROXYSTEROID~ ----~ DEHYDROCENASE

i

,,0++ d o ÷ , water

~

[7]

\ ~

NADH

,!

/ OXIDASE

~androstanedione

•

NADH oxygen

I

j

FIG. 4. Recycling of NAD ÷ in stereospecific steroid oxidation. Here the bacteria Leuconostoc mesenteroides as the source of NADH oxidase is microencapsulated with 3a-

hydroxysteroid dehydrogenase. steroid oxidation. 22 The substrate required to recycle N A D + is oxygen, which is c o n v e r t e d into water. Recycling of D e x t r a n - N A D H Retained within Microcapsules 9A2A3 (Fig. 5) The a b o v e studies show that microencapsulated multienzyme s y s t e m s can carry sequential e n z y m e reactions and can recycle the required cofactors. F o r in vitro studies, cofactors in the external substrate solution can freely equilibrate across the microcapsule m e m b r a n e to be recycled in the microcapsules. H o w e v e r in applications for medicine or in industrial reactors, it will be important to retain the cofactor inside the microcapsules for recycling. This w a y , it will not be necessary to have cofactors supplied f r o m outside the microcapsules. Studies were therefore carried out here to covalently link cofactor to macromolecules, and retain these inside microcapsules for recycling 9a2a3 (Fig. 5).

/ ethanol

\

¢ ALCOHOL " ~ DEHYDROCENASE DEXTRAN

,\ malate

. ~

SAD+

~ ~ acetaldehyde

DEXTRAN

I

SADH )!

/,

MALATE DEHYDROCENASE~"

I oxaloacetate

FIG. 5. Recycling of dextran-NADH retained within semipermeable microcapsules.

[7]

RECYCLING OF N A D ( P ) BY MULTIENZYME SYSTEMS

75

Preparation of Dextran-NAD + NAD+-N6-[N-(6-aminohexyl)acetamide] is coupled to dextran T7023 with slight modifications. 12 NAD + could also be coupled to polyethyleneimine, 16 albumin, 24 or hemoglobin 24 and immobilized in the microcapsules. 16,24

Preparation of Cellulose Nitrate Membrane Microcapsules Containing Dextran-NAD and Multienzyme Systems 9 Dextran-NAD + solution (1.5 ml) containing 3-5 mg of NAD + coupled to 150 mg of dextran TT0 is mixed with 1.5 ml of hemoglobin solution (15 g/dl prepared as described under the procedure of cellulose nitrate membrane microcapsules). The pH is adjusted to 8.5 and 30 mg of yeast alcohol dehydrogenase (300 U/mg) and 100/zl of malate dehydrogenase (1870 U/100/xl) are added to the hemoglobin-dextran-NAD + solution. The pH is again adjusted to 8.5. The microcapsules are then prepared by the method described in the earlier section for cellulose nitrate membrane microcapsules. The microcapsules prepared are stored at 0° in phosphate buffer (0.1 M, pH 8.0).

Preparation of Polyamide Membrane Microcapsules Containing Dextran-NAD ÷ and Multienzyme Systems 13 The microcapsules are prepared as described under basic procedure of preparation using interfacial polymerization. Dextran-NAD + (200 mg), containing 5 mg of immobilized NAD ÷, is dissolved in 1.5 ml of the specially prepared hemoglobin solution. Yeast alcohol dehydrogenase (10 mg of 305 U/mg) and malate dehydrogenase (1.65 mg of 1130 U/mg) are added and dissolved in this solution. The resulting solution is mixed with 1.5 ml of the diamine-polyethyleneimine solution in a 150-ml beaker kept in an ice bath. The rest of the procedure is as described under the section on basic procedure. The resultant microcapsules are suspended in 0.1 M phosphate buffer and stored at 4°.

Measurements of the Recycling of Dextran-NAD + within the Microcapsules This could be carried out in two ways. One way is to carry out stirbatch studies as described for the recycling of free NAD + in the earlier section. Alternatively, recycling activity measurements are made by us23 K. Mosbach, P. O. Larsson, and C. R. Lowe, this series, Vol. 44, p. 859. 24 Ho P. Wahl and T. M. S. Chang, J. Mol. Catal. 39, 147 (1987).

76

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[7]

ing a continuous flow shunt chamber prepared from a microcolumn (0.5 × 3 cm) containing 0.5 ml of microcapsules. 9,13Glass wool is placed at both ends of the column to retain the microcapsules and to allow the passage of fluid. A solution of Tris buffer (0.1 M, pH 8.0) containing the substrates (200 mM ethanol and 5 mM oxaloacetate) is passed through the column at a flow rate of 0.1 ml/min and the effluent is collected in 0.5-ml fractions, which are then boiled at 100° for 5 min to destroy excess oxaloacetate and cooled in an ice bath. The concentration of malate in the column effluent is measured. Results The recycling activity as followed by the production of malate is shown in Table I. The microcapsules contained about 1 mg of immobilized NAD ÷ per 1 ml of microcapsules. Thus, in the continuous flow reactor, no cofactor is required in the substrate solution perfusing the column. In the microcapsules, prepared using purified hemoglobin as described, both the recycling activity and the storage stability are severalfold higher than in the case of the microcapsules prepared using crude hemoglobin. The recycling activity of the shunt is high, 65% of the oxaloacetic acid being converted into malic acid by one passage through the shunt. The dextranNAD ÷ present within the microcapsules is regenerated two times each minute during the reaction. The shunt showed good stability, the reaction rate remaining constant for the first hour and 83% of the original activity TABLE I RECYCLING OF DEXTRAN-NAD + WITHIN CELLULOSE NITRATE MEMBRANE MICROCAPSULES N A D ÷ regenerated

Sample Crude Hb

Purified Hb

Glutaraldehyde-Hb

Storage time (days)

No. of cycles per hour

Quantity in microcapsules (/zmol/hr/ml)

0 1 2 5 0 I 4 7 0 7

7.0 4.2 2.8 1.05 120.0 117.5 84.0 37.3 4.9 4.0

9.7 6.0 4.0 1.5 150.0 147.0 105.0 46.5 6.0 4.8

[7]

RECYCLING OF

NAD(P)

BY MULTIENZYME SYSTEMS

77

being retained even after 3 hr of continuous reaction. The same shunt could be stored and reused several times. The cross-linked microcapsules showed complete stability during 3 hr of continuous reaction although their initial activity was only about 10% of that of the untreated microcapsules. In the case of the polyamide membrane microcapsules, the dextranNAD ÷ within the microcapsules was regenerated twice each minute during the continuous reaction of the shunt. The stability of the recycling activity was very good. After the recycling activity reached its maximal value, it remained stable at this value and no significant decrease in activity was observed during the 3 hr of reaction. After this reaction, each shunt was stored at 4 ° . Sixty-three percent of the original activity was retained after 7 days and 41% was still retained after 12 days. The microcapsules could be further stabilized by cross-linking with glutaraldehyde but this cross-linking caused a sharp decrease in the initial recycling activity within the microcapsules. Both yeast alcohol dehydrogenase and malate dehydrogenase are much more stable in the microencapsulated form when compared with the same enzymes in solutions. Stability of microencapsulated malate dehydrogenase is much higher than that of yeast alcohol dehydrogenase. Furthermore, there is a correlation between the decrease in the microencapsulated dextran-NAD + recycling activity and the decrease in the activity of the microencapsulated yeast alcohol dehydrogenase. The stability of the recycling activity of the microcapsules is therefore related to the stability of yeast alcohol dehydrogenase. Recycling of Free NADH Retained within Lipid-Polymer Membrane Microcapsules j4'25-28 (Fig. 6) Cofactors covalently linked to macromolecules such as dextran or polyethyleneimine can be retained within semipermeable microcapsules to be recycled enzymatically. However, linkage of cofactors to macromolecules increases steric hindrance and reduces their rate of reactions with enzymes. In biological cells such as erythrocytes, free cofactors and multienzymes systems are all retained within the cells in free solution. Thus studies were carded out here to immobilize free cofactors inside microcapsules with membranes impermeable to cofactors but permeable to the initial substrates. In this way, the free cofactor can function without steric 25 y . 26 y . 27 y . 2s E.

T. Yu and T. M. S. Chang, FEBS Lett. 125, 94 (1981). T. Yu and T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 27, 535 (1981). T. Yu and T. M. S. Chang, Enzyme Eng. 6, 163 (1982). Ilan and T. M. S. Chang, Appl. Biochem. Biotechnol. 13, 221 (1986).

78

MULTISTEP ENZYME SYSTEMS AND COENZYMES

urea

[7]

NH 3

Itpld-polymer membrane

alcohol

acetaldehyde

FIG. 6. Recycling of free NADH retained within microcapsules having a lipid-polymer membrane which is impermeable to NADH. The permeability of the lipid-polymer membrane can be adjusted.

hindrance in close proximity to the enzymes. Furthermore, all enzymes and cofactors inside the microcapsules are in free solution. Lipidpolyamide membrane microcapsules have been prepared. 3,4 These are permeable to lipophilic molecules but have negligible permeability to hydrophilic molecules as small as K + and Na+. 29 Preparation ~4

Lipid-polyamide microcapsules of 100 tzm mean diameter containing multienzyme systems, cofactors, and o~-ketoglutarate are prepared as described. 14The first part is similar to the procedure described earlier in this paper under basic procedure for the preparation of polyamide microcapsules. To 2.0 ml of the hemoglobin solution prepared as described earlier in this chapter is added 12.5 mg glutamate dehydrogenase (bovine liver, type III, 40 U per mg, Sigma Co.), 6.25 mg alcohol dehydrogenase (yeast, 330 U per mg, Sigma Co.), 0.5 mg urease (51 U/mg, Millipore Co.), 1.18 mg ADP, and either NAD + (0.52, 105, 2.11, or 21.13 mg) or NADH (21.13 mg) dissolved in 0.25 ml of water. Finally, 0.25 ml of a solution containing ot-ketoglutarate (56.5 rag), MgClz (2.5 mg), KCI (0.93 rag) is added. Two and one-half milliliters of the hemoglobin-enzyme solution so prepared is added to 2.5 ml of the diamine-polyethyleneimine solution prepared as described in the basic method section. The remaining steps are the same as described except that the Tween 20 steps are omitted 29 A. M. Rosenthal and T. M. S. Chang, J. Membr. Sci. 6, 329 (1980).

[7]

RECYCLINGOF NAD(P) av MULTIENZYMESYSTEMS

79

here. Instead, after a wash with the 0.5% Span 85 organic solution as described, the following steps are carried out to apply the lipids to the polyamide membranes. The microcapsules are rinsed twice with 10 ml of a lipid-organic liquid. (This is prepared earlier as follows: 1.4 g lecithin and 0.86 g cholesterol are added to 100 ml tetradecane and stirred for 4 hr at room temperature. If a more permeable lipid membrane is required to allow urea to diffuse across, then the lipid compositions should be 0.43 g cholesterol and 0.7 g lecithin.) Then another 10 ml of the lipid-organic liquid is added and the suspension is slowly rotated for 1 hr at 4° on a multipurpose rotator. After this, the supernatant is decanted, and the lipid-polyamide membrane microcapsules are recovered and left in this form at 4 ° without being suspended in aqueous solution until it is added to the substrate solution just before the reaction. The procedure should be practiced and the microcapsules prepared should be tested for absence of leakage of enzymes or cofactors before being used in experimental studies.

Assay Method The microcapsules so prepared can act on either ammonia or urea. For assaying using ammonia, a substrate solution is made up to contain Tris (100 mM), ammonium acetate (20 mM), and alcohol (200 raM) with a final pH adjusted to 9. At pH 9, about 38% of ammonia is present in the nonionized form (NH3), which can cross the lipid-polyamide membrane. At pH 7.4, most of the ammonia is present in the ionized form (NH4+), which cannot cross the lipid-polyamide membrane. For assaying using urea, urea (50 mg/dl) replaces ammonium acetate in the above substrate solution. In each experiment, 2 g lipid-polyamide microcapsules is added to 4 ml of the substrate solution in a stoppered flask. The reaction is carried out in a rotary shaker at 30° and 160 rpm. At present intervals, 0.4-ml aliquots of the supernatant are taken and 0.04 ml 0.5 N HC1 is added to each. These are then heated in boiling water for I min and the ammonia and/or urea concentrations measured using a Technicon autoanalyzer. Since glutamate formed inside the microcapsules is retained inside, the microcapsules are disrupted and the glutamate measured by HPLC.

Results With cofactor recycling, 0.25 #mol NAD ÷ per 2 g of microcapsules was sufficient to convert 10/~mol of ammonia into glutamate. The number of recyclings was 40 in 3 hr (Table II). By increasing the amount of NAD + in the microcapsules from 0.25 to 0.5, 1.0, and 10/.Lmol, the reaction rates

80

[7]

MULTISTEP ENZYME SYSTEMS AND COENZYMES

TABLE II COFACTOR RECYCLING WITH DIFFERENT AMOUNTS OF NAD÷ RETAINED WITHIN LIPID-POLYAMIDE MEMBRANE MICROCAPSULES

NAD÷ (/~mol) in 2 ml microcapsules 0.25 0.50 1.00 10.00

Number of cycles after 45 min 10 16 8 0.8

-+ 10 -+ 3.2 -+ 1.6 -+ 0.2

90 min 26 -+ 19 -+ 9.6-+ 1.2 ---

11 4 2 0.2

135 min 32 21 12 1.3

-+9 _+ 4 -+ 1.2 -+ 0.2

180 min 40 22 13 1.5

-+9 _+ 4 -+ 1.2 -+ 0.2

were only increased slightly. With recycling of 0.5/xmol NAD ÷ per 2 g of microcapsules, the rate of conversion of ammonia into glutamate is comparable to the use of 10/xmol NADH per 2 g of microcapsules with no recycling. When 10 ~mol of NADH per 2 g microcapsules was used without recycling, the reaction stopped as soon as 10 ~mol ammonia was converted into glutamate. However, when 2 g microcapsules containing 0.25, 0.5, 1.0, or 10/~mol NAD ÷ with recycling were used, the conversion continued even after 10/xmol ammonia was converted into glutamate by each 2 g of microcapsules. By decreasing the proportion of cholesterol in the lecithin-cholesterol lipid solution used for complexing, it was possible to obtain a membrane with good permeability to urea but with no leakage of NAD ÷ and otketoglutarate. When microcapsules containing urease and the multienzyme system with NAD ÷ recycling were used, ammonia formed from urea was converted into glutamate. However, since the rate of conversion of urea into ammonia was faster than the rate of conversion of ammonia into glutamate, the ammonia level increased before it fell as ammonia was converted more slowly into glutamate. The glutamate formed is retained within the microcapsules, which can be disrupted and the glutamate measured by HPLC as shown in Table III. The membrane of the standard lipid-polyamide membrane microcapsules was not permeable to urea, so that no glutamate was detectable inside the microcapsules. When the permeability of the lipid-polyamide membranes was increased by the methods described above, the rate of conversion of urea into ammonia and then into glutamate increased with increasing permeability (Table III). The lipid-polyamide membrane retains enzymes, unmodified cofactors, and a-ketoglutarate in solution within the microcapsules (Fig. 5). Ammonia and urea equilibrating into the microcapsules are converted

[7]

RECYCLINGOF NAD(P) BY MULTIENZYMESYSTEMS

81

TABLE Ill CONVERSION OF UREA INTO GLUTAMIC ACID WITH NADH RECYCLING IN LIPID-POLYAMIDE MEMBRANE MICROCAPSULES

Permeability to urea

Glutamic acid (#tool/liter in 3 hr)

Low Medium High

0 2.42 6.93

into glutamate. Alcohol entering the microcapsules acts as substrate for the conversion and recycling of NAD + to NADH. Thus cofactors can be immobilized without modification. Even some substrates, e.g., t~-ketoglutarate, can also be immobilized. Since none of the cofactors or enzymes is immobilized directly to solid polymers, there is no diffusion restriction or steric hindrance within the "intracellular" confines of the microcapsules. A very small amount of NAD + is required when the cofactor is recycled. A larger amount of NADH can be microencapsulated for the reaction if recycling of cofactor cannot occur, so that the enzymes and cofactors may all be included in the microcapsules. The required substrate, aketoglutarate, can also be retained within the microcapsules. Thus, when used in a microcapsule column for perfusion, cofactors or a-ketoglutarate need not be added to the perfusate. The product, glutamate, can be used as the initial material for another enzymatic reaction. Microcapsules containing more complex multienzyme systems can convert ammonia into glutamate which is then converted into other amino acids (e.g., alanine) by transaminases inside the same microcapsule.19,28 The a-ketoglutarate formed during the transamination reaction can be recycled in the reaction. 19,28 Summary Multistep enzyme systems can be immobilized in solution within semipermeable microcapsules. With the ability to recycle cofactors, a number of potentially useful systems have been made possible. Furthermore NAD + can be retained inside the microcapsules by two approaches. (l) NAD + can be linked to macromolecules such as dextran or polyethyleneime. However, in this form, there are significant increases in steric hindrance and diffusion restrictions. (2) "Artificial cells" consisting of

82

M U L T I S T E P E N Z Y M E SYSTEMS A N D C O E N Z Y M E S

[8]

lipid-polyamide membrane microcapsules containing multienzyme systems, cofactors, and substrates can retain NAD + in the free form. Analogous to the intracellular environments of red blood cells, free NAD + in solution inside the microcapsules is effectively recycled by the multistep enzyme systems which are also in solution. Enzymes in the microcapsules are in high concentrations and in close proximity to one another. Any number and any concentration of different enzyme systems can be microencapsulated all within one artificial cell, within the limit of solubility of the total amount of enzymes. Products of sequential reactions inside the microcapsules are at much higher concentrations than outside. All these factors result in an optimal intracellular environment for multistep enzyme reactions. External substrates in the form of lipophilic or small hydrophilic molecules can equilibrate across the membrane to participate as initial substrates in the multistep reactions in the microcapsules. A number of potential applications are possible using this approach. The lipid-polyamide membrane artificial cell can also be used in basic research as a biochemical cell model for the simpler types of biological cells such as erythrocytes.

[8] Bioluminescent Assays U s i n g C o i m m o b i l i z e d E n z y m e s By G. WIENHAUSEN, L. J. KRICKA, and M. DELUCA The present article will discuss the uses of luciferases coimmobilized with other enzymes for various analytical purposes. The quantitative determination of metabolites and enzymes is coupled to the luciferase reactions which are measured as light production. The assays are sensitive, rapid, and specific. Two different luciferases have been used: the firefly (Photinus pyralis) luciferase (EC 1.13.12.7, Photinus-luciferin 4-monooxygenase) and the bacterial (Vibrio harveyi) luciferase (EC 1.14.14.3, alkanal, reduced-FMN : oxygen oxidoreductase). Firefly luciferase catalyzes the ATP-dependent oxidative decarboxylation of luciferin (LH2) with the emission of light [reaction (1)]. LH2 + ATP + 02

luciferase, L ~ O

+ A M P + PP~ + CO2 + hv

(1)

Any enzyme or substrate which is coupled to ATP production or disappearance can be assayed with firefly luciferase, as shown in reaction (2). X-P + ADP

METHODS IN ENZYMOLOGY, VOL. 136

E

, ATP + X

(2)

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

82

M U L T I S T E P E N Z Y M E SYSTEMS A N D C O E N Z Y M E S

[8]

lipid-polyamide membrane microcapsules containing multienzyme systems, cofactors, and substrates can retain NAD ÷ in the free form. Analogous to the intraceUular environments of red blood cells, free NAD ÷ in solution inside the microcapsules is effectively recycled by the multistep enzyme systems which are also in solution. Enzymes in the microcapsules are in high concentrations and in close proximity to one another. Any number and any concentration of different enzyme systems can be microencapsulated all within one artificial cell, within the limit of solubility of the total amount of enzymes. Products of sequential reactions inside the microcapsules are at much higher concentrations than outside. All these factors result in an optimal intracellular environment for multistep enzyme reactions. External substrates in the form of lipophilic or small hydrophilic molecules can equilibrate across the membrane to participate as initial substrates in the multistep reactions in the microcapsules. A number of potential applications are possible using this approach. The lipid-polyamide membrane artificial cell can also be used in basic research as a biochemical cell model for the simpler types of biological cells such as erythrocytes.

[8] Bioluminescent Assays U s i n g C o i m m o b i l i z e d E n z y m e s By G. WIENHAUSEN, L. J. KRICKA,and M. DELUCA The present article will discuss the uses of luciferases coimmobilized with other enzymes for various analytical purposes. The quantitative determination of metabolites and enzymes is coupled to the luciferase reactions which are measured as light production. The assays are sensitive, rapid, and specific. Two different luciferases have been used: the firefly (Photinus pyralis) luciferase (EC 1.13.12.7, Photinus-luciferin 4-monooxygenase) and the bacterial (Vibrio harveyi) luciferase (EC 1.14.14.3, alkanal, reduced-FMN : oxygen oxidoreductase). Firefly luciferase catalyzes the ATP-dependent oxidative decarboxylation of luciferin (LH2) with the emission of light [reaction (1)]. LH2 + ATP + 02

luciferase

~ L=O

+ A M P + P P t + CO2 + hv

(1)

Any enzyme or substrate which is coupled to ATP production or disappearance can be assayed with firefly luciferase, as shown in reaction (2). X-P + ADP

METHODS IN ENZYMOLOGY, VOL. 136

E

, ATP + X

(2)

Copyright © 1987by Academic Press, Inc. All rights of reproduction in arty form reserved.

[8]

B I O L U M I N E S C E N T ASSAYS

83

Either the concentration of the phosphorylated compound X-P or the specific kinase (E) can be determined. Bacterial luciferase catalyzes the oxidation of FMNH2 and a longchain aldehyde, a reaction which also results in light emission. F M N H z + R C H O + O2

luciferase

> F M N + R C O O H + H20 + hv

(3)

This reaction by itself is of limited analytical usefulness unless it is coupled to the production of FMNH2, a reaction which is catalyzed by an NADH- or NADPH-specific oxidoreductase [Eqs. (4) and (5)]. NADH + H + + FMN NADPH + H + + FMN

oxidoreductase oxidoreductase

~ N A D + + FMNH2

(4)

~ N A D P ÷ + FMNH2

(5)

By coupling the luciferase with the oxidoreductase, any dehydrogenase which produces N A D H or NADPH can be assayed, as shown in reaction (6). XH~ + NAD(P) +

E

, X + NAD(P)H + H +

(6)

Either XH2, the reduced substrate, or the enzyme catalyzing the oxidation of XH2 can be measured. What are the advantages of using coimmobilized enzymes for these assays? In general the enzymes are more stable than the soluble forms. The sensitivity of the assays is greatly increased due to the fact that the immobilized luciferase is present in a microenvironment with locally high concentrations of ATP or FMNH2. For example, coimmobilized N A D H : FMN oxidoreductase and bacterial luciferase produce about 100 times more light per picomole of NADH than comparable amounts of soluble enzymes. Another important aspect is that immobilized enzymes can be incorporated into flow cells where they can be used for multiple assays. Source

of the Enzymes

The experiments reported here have all been performed with enzymes purified in our laboratory. Firefly luciferase is purified as described in Vol. LVII of this series.~ Bacterial luciferase is purified from Vibrio harveyi according to Hastings et al. z The oxidoreductases are purified also from V. harveyiJ There is a diaphorase from microorganisms available 1 M. D e L u c a and W. D. McElroy, this series, Vol. 42, p. 3. 2 j. W. Hastings, T. O. Baldwin, and M. Z. Nicoli, this series, Vol. 42, p. 135. 3 E. Jablonski and M. D e L u c a , Biochemistry 16, 2932 (1977).

84

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[8]

from Boehringer-Mannheim which can be used instead of the oxidoreductases. This enzyme will use either NADH or NADPH as a substrate. We do not have any experience in immobilizing commercially available enzymes; however, there is one report in which the commercial bacterial oxidoreductase and luciferase have been coimmobilized on nylon tubing. 4 Other experiments have indicated that the commercial enzymes can be successfully immobilized onto Sepharose using the same procedures described here. 5 A combined luciferase/oxidoreductase preparation partially purified from Photobacterium fischeri can be purchased from the following companies: Boehringer-Mannheim, Lumac/3M, Calbiochem-Behring/ Hoechst, LKB-Wallac, Analytical Luminescent Laboratory, and Packard Instruments. Sigma Chemical Co. Ltd. sells the enzymes either from P. fischeri or as a preparation from V. harveyi. Firefly luciferase and luciferin are commercially available from the following companies: Boehringer-Mannheim, Calbiochem-Behring/ Hoechst, Sigma Chemical Co. Ltd., LKB-WalIac, Lumac/3M, Turner Design, Packard Instruments, and Analytical Luminescent Laboratory. Instrumentation

The instruments used for measuring light emission can be a very simple design with a photomultiplier tube, power supply, and a light-tight reaction chamber. It is also possible to buy more sophisticated automated luminometers with microprocessor-controlled automatic injection systems. The various commercially available instruments were reviewed by Picciolo et al. in 1978. 6 A more recent discussion of the then currently available instruments can be found in an article by Stanley. 7 A different approach to measuring the light emission from luminescent assays is the use of a high-speed (ASA 20000) instant photographic film, as described by Green et al. ~ Assays

Firefly Luciferase. This enzyme is assayed by injecting 100 ~1 of 0.02 M ATP into 500/.d of 0.025 M glycylglycine buffer, pH 7.8, containing 1 × 10 -4 M D-luciferin, 8 x 10 -3 M MgC12, and an appropriate amount of 4 A Roda, S. Girotti, S. Ghini, B. Gricolo, G. Carrea, and R. Bovara, Clin. Chem. 30, 206 (1984). 5 K. Green, L. J. Kricka, G. H. G. Thorpe, and T. P. Whitehead, Talanta 31, 173 (1984). 6 G. L. Picciolo, J. W. Deming, D. A. Nibley, and E. Chappelle, this series, Vol. 42, p. 550. 7 p. E. Stanley, in "Clinical and Biochemical Luminescence" (L. J. Kricka and T. J. N. Carter, eds.), p. 219. Dekker, New York, 1982.

[8]

BIOLUMINESCENT ASSAYS

85

either soluble or immobilized luciferase. The amount of enzyme to be used is dependent on the sensitivity of the luminometer. The peak light intensity is proportional to the concentration of enzyme present and is expressed in relative light units. If the amount of enzyme is kept constant and limiting amounts of ATP are injected, then light intensity is proportional to the ATP concentration. Bacterial Luciferase. Either the soluble or immobilized enzyme is assayed by the injection of 100 Izl 1.5 x 10 -4 M FMNH2 into 500 tzl of 0.1 M potassium phosphate buffer, pH 7.0, containing 0.0005% (v/v) decanal and luciferase. The FMNH2 is prepared by photoreducing FMN in the presence of 5 × 10-3 M EDTA. 8 The buffer-decanal mixture is made from a 0.05% decanal-water suspension which should be prepared freshly every 4 hr. Oxidoreductases. These enzymes are assayed spectrophotometrically by measuring the initial rate of oxidation of NADH or NADPH as the decrease in absorbance at 340 nm. The reaction is initiated by adding I00 /A of 2 mM N A D H or NADPH to 0.9 ml of potassium phosphate buffer, pH 7.0, containing the enzyme and 0.13 mM FMN. The immobilized oxidoreductases are assayed in the same way except the reaction mixture in the cuvette is stirred continuously to avoid settling of the Sepharose. Diaphorase. The conditions for the determination of the activity of the diaphorase are the same as those used in the oxidoreductase assays. The oxidation of N A D H or NADPH in the absence of added FMN is subtracted from that in the presence of FMN.

Coupled Reaction of Coimmobilized Oxidoreductase and Luciferase. Maximum initial light intensity is measured upon the addition of 10-20/~1 of the Sepharose-bound enzyme suspension into 0.5 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 0.04 mM NADH or NADPH, respectively, 0.003 mM FMN, and 0.0005% decanal. Other Enzymes. All of the other enzymes which are coimmobilized with the oxidoreductase and luciferase are assayed spectrophotometrically according to published procedures. 9 Immobilization Procedure The mixture of enzymes to be immobilized contain 5 mg of bacterial luciferase and 2-3 IU of either the NADH or NADPH : FMN oxidoreduc8 j. Lee, Biochemistry 11, 3350 (1972). 9 H. U. Bergmeyer, "Methods of Enzymatic Analysis." Verlag Chemie Weinheim/Academic Press, New York, 1971.

86

MULTISTEP

c.,

P

r~

ENZYME

c,l

t"q

SYSTEMS

AND

COENZYMES

e,l

[8]

i-,,I

~

t'q

?

?

?

e~ i

×

E

?

X

T T T x

~

b

T

x

? ~-

x

T

x

T

x

~

x

-×××~-×~-×-m M d ~

md

md----

,t"q e,~

0 .<

t',,~ t-.2 t'q

~"-.I ~"~

e'q

[."~ ,r'~ eq

i.

Z

e

Z

e Z 0 ¢,.) >. ,<

0) "~

< ~Z.~

~ _

~Z~

,~ Z.~ ~ Ez~~' ~

~ Z . ~~ .. ~~

~ ~Z

~Zm E

~2

2

z

f~

[8]

BIOLUMINESCENT

O,

~

t~ ~

~

87

ASSAYS

t~ ~

t~

o.

,

~.

.~

-

"~

X

X

~

X

--~'~x-

-

X

~

X

X

z

-~.~ _~.~ "" ~

0

~-

~

~

.~

.~ ~,~.~ =-~ ~

'-

~

~

-~

~

z~

,t~ e~ ~

t~

~n

n~

-~

~.x× ~x××

r....~ X

t~

×

~-~ e,,.h vh

z~z~

X

X

X

~,~ X

x X

X

~

=~

~

.~ ~.~,~'~ . . . ~ o _ ~

~'~'~

~

X

.-

n~

<

X

X

~

88

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[8]

tase or diaphorase. Additional enzymes are added usually in the amount of 2-8 IU. If the final protein concentration of the mixture is less than 8 mg/ml, BSA is added to reach this concentration. The volume of the coupling mixture is 2.5-3.0 ml. The enzymes are dialyzed overnight in the cold against 4 liters of 0.1 M sodium pyrophosphate, pH 8.0. One gram of Sepharose was activated according to the method of March et al.~° and this is added to the enzyme mixture. The suspension is stirred gently at 4 ° for 16 or 2 hr at room temperature. The Sepharose is then washed extensively with 0.1 M potassium phosphate buffer, pH 7.0, and 1 M NaC1 in 0.1 M potassium phosphate, pH 7.0.~1 The Sepharose-enzymes are suspended in 6 ml of 0.1 M potassium phosphate, pH 7.0, containing 0.02% sodium azide and 2 mM dithiothreitol (DTT). Additional DTT, 20/zl of 0.1 M, is added each week. If diaphorase or the NADH : FMN oxidoreductase is used instead of the NADH-dependent oxidoreductases, the storage buffer contains 0.5 mM glutathione rather than DTT. This is essential because DTT is a substrate for these enzymes. The efficiency of the immobilization is determined by measuring the enzyme activities recovered on the Sepharose. The activities recovered on the Sepharose are expressed as a percentage of the initial activities added to the Sepharose. Storage and Stability of Immobilized Enzymes The immobilized enzymes can be stored at 0-4 ° in buffer containing either DTT or glutathione and azide for several months. If more DTT is added weekly, as discussed above, the enzymes remain fully active. For longer storage glycerol is added to the enzyme-Sepharose suspension to give a final concentration of 15%. The immobilized enzymes are then rapidly frozen in liquid nitrogen and kept at - 2 0 ° indefinitely with no loss of activity. Repeated slow freezing and thawing result in a considerable loss of activity. Metabolites Assayed In general, the immobilized enzymes are added to the buffer mixture containing everything except the compound to be measured and the background light is determined. Then the specific compound is added, the contents of the tube are mixed, and either the peak light or the rate of increase of light is measured. When diaphorase is used instead of the ~0 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974L tl j. Ford and M. DeLuca, Anal. Biochem. 110, 43 (1981).

[8]

BIOLUMINESCENT ASSAYS

89

TABLE II RECOVERY OF ENZYME ACTIVITIES IMMOBILIZED ON SEPHAROSE Activity recovered on Sepharose Enzyme coupled

(%)

NADH : FMN oxidoreductase Creatine kinase Firefly luciferase Hexokinase Malate dehydrogenase Lactate dehydrogenase 7a-Hydroxysteroid dehydrogenase Bacterial luciferase

90 84 80 60 45 25 20 15

oxidoreductase, the most reproducible results are obtained when the reaction is initiated by the injection of NAD. Table I contains the data 1~-15for the various metabolites which have been measured using coimmobilized enzymes. The final volume for all of the assays listed is 0.5 ml. The buffers and substrate concentrations are different for the various assays. While it is very possible that other assay conditions could be substituted we have found these concentrations to give the best results. In general the various compounds can be assayed in the range of 1010,000 pmol. The lower limit of detection will depend upon the amount of active enzymes that are immobilized as well as on the equilibrium constants for the various enzymes. In addition, the sensitivity of the assay is limited by the amount of background light, which is determined by the purity of the substrates and enzymes used. Many of the commercially available dehydrogenases contain an aldehyde dehydrogenase that gives light in the presence of decanal. These enzymes must be further purified before use in the luminescent assays as described previously. ~1It is necessary to determine the range of linearity of light vs concentration of substrate for each new preparation of immobilized enzymes. Standards should be included every day with unknown samples. Table II shows some representative enzyme recoveries obtained after 12 G. Wienhausen and M. DeLuca, Anal. Biochem. 127, 380 (1982). 13 j. Schoelmerich, G. P. van Berge Henegouwen, A. F. Hofmann, and M. DeLuca, Clin. Chim. Acta 137, 21 (1984). ~4j. Schoelmerich, J. E. Hinkley, I. A. Macdonald, A. F. Hofmann, and M. DeLuca, Anal. Biochem. 133, 244 (1983). l~ A. Roda, L. J. Kricka, M. DeLuca, and A. F. Hofmann, J. Lipid Res. 23, 1354 (1982).

90

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[8]

a

_3,----'-"-'-"

./

03 I-Z I.--r"

t.u

"~

m ee"

/

TIME (min) FIG. 1. Time course o f light output in the assay of D-glucose: (a) background, (b) 2, (c) 5, and (d) 10 pmol of glucose.

immobilization on Sepharose. These recoveries range from almost 100% to a low of 15%. Kinetics The kinetics of light emission are also variable with the different combinations of enzymes. ~2 For example, in the assay for glucose with the four-enzyme system--hexokinase, glucose-6-phosphate dehydrogenase,

bf ,

0'3

r..D

._,.~_ L.I.I

c,,s

0

1

0

t

i

1

2

TIME (min) FIG. 2. Time course of light output in the assay of NADP+: (a) background, (b) 2 pmol of N A D P ÷.

~3.1, ~I31]dLLq/1/N O.l,OHd

t~

=

~.IF

oqoi

°

I I I I

I

L

92

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[8]

100

0 0

10

20

30

40

50 60 70 TIME (rain)

80

90

lOO 110

FIG. 4. Typical analytical traces obtained for repeated analysis of 10 pmol/ml of NADH; 2-min samples, 4-min wash.

N A D H : FMN oxidoreductase, and bacterial luciferase--the slope of increasing light is measured as shown in Fig. 1. With other assays, such as shown for NADP +, the peak light intensity is measured (Fig. 2).12 Each combination of enzymes must be evaluated independently and the best method for measuring the compound of interest is then used. Automation One of the advantages of the coimmobilized enzymes is the possibility of using them in an automated system. The Sepharose-enzymes can be packed into small flow cells and the cells placed in front of the phototube in a standard luminometer. Such a system was developed by Kricka et al. 16The flow cells could be reused in some cases for 500-700 consecutive assays. The reproducibility was good and the lower limit of detection of various metabolites was approximately 1 pmol. Recently the automated system has been modified. ~7A schematic of the modified flow manifold is shown in Fig. 3A, and Fig. 3B shows the design of the immobilized flow 16 L. J. Kricka, G. K. Wienhausen, J. E. Hinkley, and M. DeLuca, Anal. Biochem. 129, 392 (1983). 17 D. Vellom, M. DeLuca, J. Hinkley, A. Loucks, and H. Egghart, in "Analytical Applications of Bioluminescence and Chemiluminescence" (L. J. Kricka, P. E. Stanley, G. H. P. Thorpe, and T. P. Whitehead, eds.). Academic Press, New York, 1984.

[9]

E N Z Y M E C H A N N E L I N G I M M U N O M E T R I C ASSAYS

93

cell and its orientation in the luminometer. By reducing the total volume in the system and positioning the immobilized enzymes very close to the phototube the sensitivity of the assays has been increased significantly. It is possible to measure as little as 6 fmol of NADH in a 60-/zl sample. A typical analytical trace for repeated analysis of NADH is shown in Fig. 4. The reproducibility is good and 10 samples per hour can be measured. We believe that with some further modifications we will be able to increase the sensitivity and the speed of these automated assays.

[9] N o n s e p a r a t i o n E n z y m e C h a n n e l i n g Immunometric Assays

By IAN GIBBONS, RICHARD ARMENTA, ROBERT K. DINELLO, and EDWIN

F.

ULLMAN

Enzymes are often used as labels in immunoassays because of their stability and ease of detection. In many enzyme immunoassays, the enzyme-labeled reagent binds to a surface and the immobilized enzyme is subsequently measured. For example, in the enzyme-linked immunosorbent assay (ELISA), l antigen binds to antibody which has previously been adsorbed to a solid surface. Usually, unbound antigen is removed. Enzyme-labeled antibody is then added and binds to antigen on the solid surface. After carefully washing away unbound reagent, measurement of the bound enzyme gives a direct indication of the quantity of antigen originally present. "Homogeneous" enzyme immunoassays 2 were devised as a way to eliminate the tedious separation steps required by conventional techniques. In homogeneous methods, the binding of antigen to antibody modulates the activity of the bound label. The original homogeneous methods were adapted to measure both drugs 3 and proteins 4 and rely on competition between enzyme-labeled antigen and sample antigen for a limited quantity of antibody. When measuring small amounts of antigen, low concentrations of antibody must be used and the binding reactions J A. H. W. M. Schuurs and B. K. Van Weeman, Clin. Chim. Acta 81, 1 (1977). 2 K. E. Rubenstein, R. S. Schneider, and E. F. Ullman, Biochem. Biophys. Res. Commun. 47, 846 (1972). 3 G. L. Rowley, K. E. Rubenstein, J. Huisjen, and E. F. Ullman, J. Biol. Chem. 250, 3759 (1975). 4 I. Gibbons, C. Skold, G. L. Rowley, and E. F. Ullman, Anal. Biochem. 102, 167 0980).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[9]

E N Z Y M E C H A N N E L I N G I M M U N O M E T R I C ASSAYS

93

cell and its orientation in the luminometer. By reducing the total volume in the system and positioning the immobilized enzymes very close to the phototube the sensitivity of the assays has been increased significantly. It is possible to measure as little as 6 fmol of NADH in a 60-/zl sample. A typical analytical trace for repeated analysis of NADH is shown in Fig. 4. The reproducibility is good and 10 samples per hour can be measured. We believe that with some further modifications we will be able to increase the sensitivity and the speed of these automated assays.

[9] N o n s e p a r a t i o n E n z y m e C h a n n e l i n g Immunometric Assays

By IAN GIBBONS, RICHARD ARMENTA, ROBERT K. DINELLO, and EDWIN

F.

ULLMAN

Enzymes are often used as labels in immunoassays because of their stability and ease of detection. In many enzyme immunoassays, the enzyme-labeled reagent binds to a surface and the immobilized enzyme is subsequently measured. For example, in the enzyme-linked immunosorbent assay (ELISA), l antigen binds to antibody which has previously been adsorbed to a solid surface. Usually, unbound antigen is removed. Enzyme-labeled antibody is then added and binds to antigen on the solid surface. After carefully washing away unbound reagent, measurement of the bound enzyme gives a direct indication of the quantity of antigen originally present. "Homogeneous" enzyme immunoassays 2 were devised as a way to eliminate the tedious separation steps required by conventional techniques. In homogeneous methods, the binding of antigen to antibody modulates the activity of the bound label. The original homogeneous methods were adapted to measure both drugs 3 and proteins 4 and rely on competition between enzyme-labeled antigen and sample antigen for a limited quantity of antibody. When measuring small amounts of antigen, low concentrations of antibody must be used and the binding reactions J A. H. W. M. Schuurs and B. K. Van Weeman, Clin. Chim. Acta 81, 1 (1977). 2 K. E. Rubenstein, R. S. Schneider, and E. F. Ullman, Biochem. Biophys. Res. Commun. 47, 846 (1972). 3 G. L. Rowley, K. E. Rubenstein, J. Huisjen, and E. F. Ullman, J. Biol. Chem. 250, 3759 (1975). 4 I. Gibbons, C. Skold, G. L. Rowley, and E. F. Ullman, Anal. Biochem. 102, 167 0980).

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become inconveniently slow. This problem can be overcome by adopting a noncompetitive method and driving the binding reactions with excess reagents. However, the use of excess labeled reagents frequently leads to high unmodulatable background signals which limit sensitivity. The present article describes a set of nonseparation immunoassays in which the reagents are enzyme-labeled antibodies. These methods were designed to measure low concentrations of macromolecular antigens with excess reagents while limiting the background due to the unbound reagents. The assays provide sensitivity comparable to that of ELISA while greatly simplifying the assay protocol. The principle on which these assays are based is called enzyme channeling immunoassay. 5 Channeling occurs when two enzymes catalyzing sequential reactions [reaction (1)] are brought together at a surface or other microenvironment where diffusion-controlled exchange with bulk solution is limited. Substrates

enzyme 1 enzyme2 ~ Product 1 ~ Product 2

(1)

Under these conditions, the reduced rate of diffusion of Product 1 away from the microenvironment permits the local concentration of the product to exceed that of the bulk phase. Product 2 is therefore produced more rapidly than when the enzymes are free in solution or when only one enzyme is on the surface. Several immunoassay methods have been demonstrated in which antigen-antibody binding reactions bring together enzyme-labeled reagents at a surface? '6 For the methods reported here, glucose oxidase (GO) was selected as the first enzyme and horseradish peroxidase (HRP) as the second. Together they catalyze the reaction shown in reaction (2). Glucose + O~ -

H202 OO~ + Gluconolactone )

Colored (oxidized) product

HRP , f ~. Chromogen

+

(2)

H 20

These enzymes were chosen for their high turnover numbers and stability. A variety of chromagens are substrates for HRP. 2,2'-Azinodi-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS), which produces an easily 5 D. J. Litman, T. M. Hanlon, and E. F. Ullman, Anal. Biochem. 106, 223 (1980). 6 E. F. Ullman, I. Gibbons, L. Weng, R. DiNello, S. N. Stiso, and D. J. Litman, in "Diagnostic Immunology: Technology Assessment and Quality Assurance" (R. M. Nakamura and J. H. Rippey, eds.), pp. 31-46. College of American Pathologists, Skokie, Illinois, 1983.

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ENZYME CHANNELING IMMUNOMETRIC ASSAYS

95

measured green product, is useful for this purpose. In order to reduce background signal and provide linear enzyme rates, a scavenging agent is employed to destroy hydrogen peroxide in the bulk solution and suppress color due to unbound reagent. Catalase, which converts hydrogen peroxide to water and oxygen, was found to be very effective. The theory of these assays has been described elsewhere 6 and will not be further discussed here. The capsular antigen of Haemophilus influenzae known as polyribose phosphate (PRP) was used in our work as typical of many antigens of clinical interest. PRP is a repeating heterogeneous polymer with many similar antigenic sites. 7 Three enzyme-channeling assays for PRP will be described. The advantages and limitations of each will be discussed. The materials and reagents employed for the assays are largely the same and are specified below. Materials Used and Reagent Preparation

Enzymes. HRP (type VI), glucose oxidase (type V from Aspergillus niger), and catalase (bovine liver) were the products of the Sigma Chemical Co., St. Louis, MO 63128. Antigen. A pure preparation of PRP, the capsular antigen of Haemophilus influenzae, type b, was the kind gift of Dr. Porter Anderson. The antigen is a polyribosylribitol phosphate 7 which had an average molecular weight of 500,000. Antibodies. Rabbit anti-PRP was obtained from the New York State Department of Health. The immunoglobulin fraction was purified from the antiserum by precipitation with ammonium sulfate. The stock solution had a concentration of 80 mg/ml. Anti-GO was raised in sheep by repeated immunization with 100 /zg of the amine derivative of GO (described later) in incomplete Freund's adjuvant. The immunoglobulin fraction of the antiserum was prepared by precipitation with 50% ammonium sulfate. Chemicals. ABTS, Tween 20, ovalbumin, and glucose: Sigma Chemical Co., St. Louis, MO 63128. Sodium metaperiodate: Fisher Chemical Co., Pittsburgh, PA 15219. Sodium borohydride: J. T. Baker Co., Phillipsburg, NJ 08865. 1-Fluoro-2,4-dinitrobenzene: Eastman Kodak, Rochester, NY 14650. Equipment. Microtiter plates and covers: "Serocluster" Costar, Cambridge, MA 02139. Absorbances are measured either with a Gilford Stasar 7 R. M. Crisel, R. S. Baker, and D. E. Dorman, J. Biol. Chem. 250, 4926 (1975).

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

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III spectrophotometer, Gilford Instrument Labs, Oberlin, OH 44074, or with a Model 210 vertical beam reader, Artek Systems Corp., Farmingdale, NY 11735. Assay incubations are generally done with shaking using a Bellco (Vineland, NJ) miniorbital shaker set at "4."

Measurement of Protein Concentrations Proteins are quantitated spectrophotometrically. HRP is measured at 403 nm assuming a molar extinction of 1.12 x 105 cm -~ M -~ and a molecular weight of 40,000. Native GO is measured at 450 nm assuming a molar extinction of 2.95 x 104 cm -~ M -~ and a molecular weight of 160,000. After derivatization with 1-fluoro-2,4-dinitrobenzene, the extinction of GO at 450 nm increases to 6.76 x 10 4 c m -1 M -1. IgG is assumed to have a molar extinction of 2.4 x 105 cm -l M -t at 280 nm and a molecular weight of 160,000. The composition of conjugates of GO and HRP is deduced from the appropriate spectral ratios correcting the absorbance at 280 nm for the contribution of the enzymes as follows: A4o3/A28o = 2.0 (HRP) and A45o/A28o = 0 . 1 7 (fluorodinitrobenzene-modified GO).

HRP-Labeled Antibody Conjugation of HRP to anti-PRP is according to Wilson and Nakane. s HRP is dissolved in distilled water at 4.3 mg/ml. Four milliliters of freshly prepared sodium periodate (0.1 M) is added to 20 ml of the HRP solution to oxidize the enzyme carbohydrate. After the reaction mixture is stirred for 20 min at room temperature, 2.4 ml of a 1 M glycerol solution is added to terminate the reaction. Stirring is continued for 30 min, followed by overnight dialysis against three changes of 500 ml 2 mM sodium acetate, pH 4.5, to remove the reagents. Anti-PRP diluted to 25 mg/ml in water is dialyzed against 10 mM sodium carbonate, pH 9.6. Coupling to HRP is achieved by mixing 70 mg oxidized HRP (22 ml) with 110 mg antibody (5 ml). The pH of the mixture is adjusted to 9.6 with NaOH (0.1 M) and the reaction allowed to proceed for 2 hr at room temperature. After the mixture is cooled to 0°, the Schiff base complex is reduced by addition of 1.5 ml of a freshly prepared solution of NaBH4 (4 mg/ml of water). This mixture is incubated at 0° for 2 hr, dialyzed overnight against PBS to remove reagents (PBS, 10 mM sodium phosphate, pH 7.2, with 150 mM NaCI), and then concentrated to 5 ml by vacuum ultrafiltration through a collodion bag. The conjugates are s M. B. Wilson and P. Nakane, in "Immunofluorescence and Related Staining Techniques Proc. Int. Conf., 6th" (W. Knapp, K. Holubar, and G. Wick, eds.), p. 215. Elsevier/North Holland, Amsterdam, (1978).

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ENZYME CHANNELING IMMUNOMETRIC ASSAYS

97

separated from unconjugated enzyme and antibody by chromatography on a column (2.5 × 90 cm) of BioGel A-5m eluted with PBS. Fractions of 5 ml are collected. For us, fractions 39 to 57 represented the bulk of the conjugates. These fractions were pooled and concentrated 10-fold by ultrafiltration. A conjugate molar ratio of 1.4 HRP/IgG was determined spectroscopically. The conjugates were heterogeneous in molecular weight ranging from about 1 to 3 × 106 as judged by their elution position. Conjugated enzyme retained 80% of its activity, and complete activity was retained by conjugated antibody, as shown by the ability to compete with native antibody for PRP. Glucose Oxidase-Labeled Antibody

The method of Johnson et al. 9 is used. Glucose oxidase is first allowed to react with 1-fluoro-2,4-dinitrobenzene to block amino groups. GO (200 mg) is dissolved at 5 mg/ml in 0.1 M NaHCO3, pH 8.2. 1-Fluoro-2,4-dinitrobenzene (3.5 ml of a 1%, w/v, ethanol solution) is then added and the reaction allowed to proceed for 1 hr at room temperature. The reagent is removed by dialysis against 0.3 M NaHCO3, and the product concentrated by ultrafiltration to 20 ml. Oxidation of the enzyme carbohydrate is achieved by addition of 2 ml NalO4 (0.4 M). After reaction for 20 min at room temperature, 1 ml glycerol (1 M) is added. One hour later the product is dialyzed against 10 mM sodium carbonate, pH 9.6. A solution of 90 mg of anti-PRP in 3 ml of 10 mM NaHCO3 is mixed with the solution of oxidized GO (200 mg) giving a final volume of 25 ml. After reaction for 14 hr at room temperature, the solution is cooled to 0° and the Schiffbase complexes are reduced by addition of 1.6 ml NaBH4 (4 mg/ml). The mixture is allowed to incubate for 2 hr at 0° before dialysis against PBS. The product is then concentrated to 5 ml and chromatographed as described for the HRP conjugate. In our studies, fractions 5156 were pooled. The conjugate pool had a molar ratio of 0.8 GO per IgG. Conjugates ranged in molecular weight from about 1 to 2 × 106. The GO activity was 30% compared with starting material. Derivatization o f Glucose Oxidase with Amino Groups

A solution of GO (40 mg/ml) in 2 mM sodium acetate (pH 4.5) is combined with an equal volume of freshly prepared NaIO4 (0.2 M in the same solvent). Oxidation is allowed to proceed for 20 rain at room temperature and is followed by purification on a Sephadex G-50 column by elution with 2 mM sodium acetate buffer, pH 4.5. Ethylenediamine (3 M 9 R. B. Johnson, R. M. Libby, and R. M. Nakamura, J. lmmunoassay 1, 27 (1980).

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

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in 0.5 M Na2CO3, pH 9.5) is added to a final concentration of 0.2 M. After incubation for 1.5 hr at room temperature, the solution is cooled to 0° and the Schiff base complex is reduced by addition of 0.1 volume freshly prepared NaBH4 (4 mg/ml). Following a 2-hr incubation at 0°, aminated GO is recovered by gel filtration over Sephadex G-50.

Microtiter Plates Coated with Glucose Oxidase and Anti-PRP Aliquots (200/A) of a mixture of the amino derivative of GO (40/zg/ml) and anti-PRP (40/zg/ml) dissolved in 60 mM Na2CO3 (pH 9.6) are placed in the wells of microtiter plates. After incubation at 37° for 4 hr unbound material is removed by washing with PBS containing 0.05% Tween 20. Glucose oxidase activity equivalent to about 50 ng of free GO is bound to each well. Coated plates are stored at 4° with PBS-Tween in the wells.

Assay Reagents The HRP and GO conjugates of anti-PRP are stable when stored at 4° in PBS containing 0.01% NaN3 as a preservative. Working dilutions are made up immediately before use. NaN3 is an inhibitor of HRP, but the concentration in the assays is so low as to be negligible. ABTS solutions when freshly made are strongly colored due to traces of oxidized material. Since the color would interfere with measurements of HRP activity, the ABTS solutions are bleached by stirring at room temperature overnight. Stock concentrated solutions of catalase are made by diluting the enzyme suspension to 4 mg/ml in PBS and dialysing against the same solvent to remove the thymol preservative. Concentrations are measured spectrophotometrically assuming a molar extinction coefficient at 450 nm of 548,000 cm -I M -1 (MW = 250,000). The solutions can be stored at 4° and used for up to 30 days.

Enzyme Channeling Immunoassays

Coated-Surface Enzyme Channeling lmmunoassay This assay resembles ELISA except that no separation steps are required. Usually, microtiter plates are used as the solid support. GO is coimmobilized on the solid phase together with antibody. An aminated derivative of GO is used because a much greater activity can be adsorbed to the microtiter plate surface than is possible with native GO. The assay principle is illustrated in reaction (3).

[9]

H20 BULK SOLUTION

99

E N Z Y M E C H A N N E L I N G I M M U N O M E T R I C ASSAYS

G1

I ~TS

~TS

~

02 ~

Cat

ii

/I /

H202

Colored P duct

GIu

Ag

UNSTIRRED LAYER GO

Ab

/11

[Catalase]>[HRP] in Bulk Solution

GO

i

AboAg-(Ab-HRP)

f

T~ I ) / I I

(3)

[ I I

[HRP]>[Catalase] at Surface

Reagents are prepared using PBS containing 0.02% ovalbumin as buffer. Reagent A contains 4.5 ~g HRP conjugated to anti-PRP per milliliter. Reagent B contains 25 mM ABTS, 625 mM glucose, and 500 /zg catalase/ml. Microtiter plates to which GO and anti-PRP have been adsorbed are used as the coated surface. Samples (I00/xl) of PRP dissolved in PBS are added to the wells of the microtiter plate. The plate is covered and incubated for 3 hr at 37°. Then reagent A (50/xl) is added and incubation continued for a further 2 hr at 37°. Finally, reagent B (100/xl) is added to generate color. A405is read using an Artek vertical beam reader after incubation for 30 min at 37 °. The assay response obtained (Fig. 1) resembles that of an ELISA assay using similar reagents. Absorbance increased with added antigen over the range 0.1-3 ng. In the absence of antigen, the enzyme channeling assay gave a higher absorbance and higher background rate than did the corresponding ELISA. This was due to absorbance by catalase and to a background rate of color production not suppressed by catalase. At high antigen concentrations (>3 ng per assay), the response diminished because the reagents were no longer in excess over antigen. This is typical of immunometric assays with no separation. This assay has a more convenient protocol than ELISA in that the need for the usual separation and washing steps has been eliminated. However, like ELISA it is slow because of the time required for macromolecules to bind to the solid surface, and the solid-phase reagent is inconvenient to store and to prepare reproducibly.

Soluble Reagent Enzyme Channeling Immunoassay: Microtiter Plate Protocol To solve some of the problems inherent in the coated-surface assay, an assay was constructed that only required reagents in solution. Since

100

[9]

M U L T I S T E P E N Z Y M E SYSTEMS AND COENZYMES

2.2

1.8

g

,~

1.4

1.o~0.6~

II II

//

t

t

0.01

t

0.1 PRP (ng)

t

1

10

FIG. 1. Coated-surface enzyme channeling immunoassay. Experimental details are given

in the text.

enzyme channeling requires an immobilized phase, an appropriate phase was caused to form during the assay by precipitating one of the reagents [see reaction (4)]. Ab-GO

+

Ab-HRP

I o~-GO

Ag

>

(Ab-GO).Ag. (Ab-HRP)

l

a-GO Ag,(Ab-HRP)

Ab Ib /~b Abt Abl

OyOvyyO

Ab Ab Ib Ab Ab

Precipitate forms

Precipitate forms with channeling

without channeling

(4)

do

YYYY

In this assay, GO-labeled antibody replaces GO and antibody adsorbed to the microtiter plate. Since the antigen is polyepitopic, it can simultaneously bind both GO-labeled antibody and HRP-labeled antibody in solution. The use of labeled antibodies at high concentrations ensures that the reaction is almost instantaneous. Anti-GO is then added to pre-

[9]

ENZYME CHANNELING IMMUNOMETRIC ASSAYS

101

cipitate all the GO-labeled antibody in a process that can be accelerated by shaking. The precipitate forms as small particles. Coprecipitation of the HRP-antibody conjugate with the GO immune complex serves to signal the presence of antigen. Reagents are made up as follows in PBS containing 0.05% Tween 20 and ovalbumin at 1% (reagents A and B) or 0.1% (reagent C). Reagent A contains 120/.Lg HRP conjugated to anti-PRP and 200/.Lg GO conjugated to anti-PRP per milliliter. Reagent B contains anti-GO diluted to 0.44 mg/ ml. Reagent C contains 12 mM ABTS, 284 mM glucose, and 450 /zg catalase/ml. In the assay protocol, 10-~1 samples containing PRP are mixed with 10 /zl of reagent A and 10/xl of reagent B in the wells of a microtiter plate. The plate is covered and gently shaken for 90 min at 37°. Reagent C (220 /zl) is then added and the initial absorbance of the wells at 415 nm is recorded using an Artek vertical beam reader. The increase in absorbance over 30 min at 37° is measured, the plate being shaken between readings. Results obtained with this method are given in Fig. 2. A linear response from 0 to 500 pg per tube (microtiter plate well) was seen. As little as I0 pg of PRP was detected. This represents a considerable improvement over the coated-surface assay. Two factors contribute to the improvement: (1) All the antigen is bound to reagent in the soluble reagent

0.4

.__q 0.3 0

E

t" LF)

"~ o.2

<

0.1

0

I

I

I

I

I

100

200

300

400

500

Polyribose Phosphate (PRP) per Tube (pg) FIG. 2. Soluble reagent enzyme channeling immunoassay: microtiter plate protocol. Experimental details are given in the text. Reproduced by permission from Ullman e t al. 6

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

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method, whereas only a small fraction of antigen becomes bound to the surface in the coated-surface method. (2) There is a lower background rate in the soluble reagent method. Although much more sensitive, the soluble reagent method is still quite slow because of the time required to form the GO precipitate and the separate incubation needed to generate color.

Soluble Reagent Enzyme Channeling lmmunoassay: Rapid Protocol A much more convenient method resulted from two changes. First, polyethylene glycol is added to speed up particle formation. Second, the assay response is measured over only I rain in a temperature-regulated flow cell. Not only is this protocol faster, but it provides somewhat better sensitivity than the microtiter plate protocol. Reagents are made up as follows in PBS containing 0.1% ovalbumin. Reagent A contains 55 /zg HRP conjugated to anti-PRP and 20 Izg GO 0.08

0,06 0 O3 0 if3 ,T, ,< <1

0.04 .m

(~

E UJ

0.02

o

I 50

I 1oo

1,50

PRP (PO)

FIG. 3. Solublereagent enzyme channeling immunoassay: rapid protocol. Experimental details are given in the text.

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103

conjugated to anti-PRP per ml. Reagent B contains anti-GO diluted to 0.44 mg/ml. Reagent C contains 10% polyethylene glycol 6000, plus 0.2% Tween 20. Reagent D contains 10 mM ABTS, 250 mM glucose, and 500 ~g catalase/ml. The PRP samples are dissolved in human serum. In the assay protocol, a 10-/A serum sample is mixed with 10/A of reagent A, 10 ~1 of reagent B, and 10/~1 of reagent C. After incubation at room temperature with orbital shaking for 15 min, 960/zl of reagent D is added. The mixture is subjected to brief vortex mixing prior to aspiration into the flow cell of a Stasar spectrophotometer thermostated to 37°. The change in A415is measured between 10 and 60 sec after the preset temperature is reached. As seen in Fig. 3, the assay has detected as little as 5 pg of PRP. This assay is suitable for automation in a flow analyzer since the time required to read the signal is short (<1 min). The overall time of the assay (16 min) is much less than a typical ELISA and comparable sensitivity is achieved. The protocol can be further simplified by combining reagents A and C. Conclusions The homogeneous methods discussed here demonstrate the potential of nonseparation techniques to match ELISA in performance. The assay protocols represent a significant advance over ELISA in convenience and flexibility.

[10] I m m o b i l i z e d a n d S o l u b l e S i t e - t o - S i t e D i r e c t e d E n z y m e Complexes Composed of Alcohol Dehydrogenase and Lactate Dehydrogenase

By NILS SIEGBAHN, MATS-OLLE M,~NSSON, and KLAUS MOSBACH Coimmobilization of enzymes on solid supports leads to heterogeneous preparations in which the active sites of the enzymes are found "at random" in relation to one another. A bifunctional NAD analog, bisNAD, has been reported j as a useful reagent for affecting affinity precipitation of enzymes. The bis-NAD can also be used to obtain both an immobilized and a soluble two-enzyme system in which the two different P. O. Larsson and K. Mosbach, FEBS Left. 98, 333 (1979).

METHODS IN ENZYMOLOGY, VOL. 136

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[10]

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conjugated to anti-PRP per ml. Reagent B contains anti-GO diluted to 0.44 mg/ml. Reagent C contains 10% polyethylene glycol 6000, plus 0.2% Tween 20. Reagent D contains 10 mM ABTS, 250 mM glucose, and 500 ~g catalase/ml. The PRP samples are dissolved in human serum. In the assay protocol, a 10-/A serum sample is mixed with 10/A of reagent A, 10 ~1 of reagent B, and 10/~1 of reagent C. After incubation at room temperature with orbital shaking for 15 min, 960/zl of reagent D is added. The mixture is subjected to brief vortex mixing prior to aspiration into the flow cell of a Stasar spectrophotometer thermostated to 37°. The change in A415is measured between 10 and 60 sec after the preset temperature is reached. As seen in Fig. 3, the assay has detected as little as 5 pg of PRP. This assay is suitable for automation in a flow analyzer since the time required to read the signal is short (<1 min). The overall time of the assay (16 min) is much less than a typical ELISA and comparable sensitivity is achieved. The protocol can be further simplified by combining reagents A and C. Conclusions The homogeneous methods discussed here demonstrate the potential of nonseparation techniques to match ELISA in performance. The assay protocols represent a significant advance over ELISA in convenience and flexibility.

[10] I m m o b i l i z e d a n d S o l u b l e S i t e - t o - S i t e D i r e c t e d E n z y m e Complexes Composed of Alcohol Dehydrogenase and Lactate Dehydrogenase

By NILS SIEGBAHN, MATS-OLLE M,~NSSON, and KLAUS MOSBACH Coimmobilization of enzymes on solid supports leads to heterogeneous preparations in which the active sites of the enzymes are found "at random" in relation to one another. A bifunctional NAD analog, bisNAD, has been reported j as a useful reagent for affecting affinity precipitation of enzymes. The bis-NAD can also be used to obtain both an immobilized and a soluble two-enzyme system in which the two different P. O. Larsson and K. Mosbach, FEBS Left. 98, 333 (1979).

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active sites of two different enzymes are facing one another. 2,3 The bisNAD is used as a directing aid for the orientation of the active sites. After cross-linking of the two enzymes with glutaraldehyde, the bis-NAD template is removed, leaving the active sites still positioned against one another. By such an active site arrangement of the two enzymes it can be expected that the diffusion of the product of the first enzyme to the juxtaposed active site of the second enzyme would be facilitated when compared with "at random" immobilized enzyme systems. Such site-tosite enzyme systems might also serve as models for enzyme complexes of consecutively operating enzymes, which are believed to be of importance in the channeling of labile intermediates and in the regulation of metabolism. 4 Site-to-Site Orientation

General Procedure Buffer A: 0.1 M sodium phosphate, pH 7.5, 0.1 M NaC1. Buffer B: 0.1 M sodium phosphate, pH 7.5, 0.5 M NaC1. Buffer C: 0.1 M sodium phosphate, 0.1 M NaCI, 0.05 M oxalate, and 10 mM pyrazole. All washing steps of the gels are performed on a glass filter funnel. Incubation of the gels is normally carried out in 25 ml Erlenmeyer flasks under gentle stirring.

Immobilized System Moist Sepharose 4B, 2 ml, is activated with 25 /zl tresyl chloride (Fluka Buchs, Switzerland) as described elsewhere in this series) The activated gel is subsequently washed with several volumes of buffer A. Horse liver alcohol dehydrogenase (ADH, 2.7 U/mg) (Boehringer-Mannheim, FRG), 12 mg, is dissolved in about 2 ml buffer A and added to the activated gel. The coupling is allowed to proceed for 2 hr at room temperature and the immobilized enzyme preparation is then washed with several volumes of buffer A. The remaining active tresyl groups on the 2 M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983). 3 N. Siegbahn, M.-O. MAnsson, and K. Mosbach, Appl. Biochem. Biotechnol. 12, 91 (1986). 4 G. R. Welch (ed.), "Organized Multienzyme Systems." Academic Press, New York, 1985. 5 K. Nilsson and K. Mosbach, this series, Vol. 104, p. 56, and Vol. 135 [3].

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Sepharose are quenched with 0.25 M Tris-HCl, pH 8.0, for 2 hr at room temperature. The gel is then washed with several volumes of buffer B and finally with buffer C. About 200 nmol bis-NAD (Sigma Chem. Co., St. Louis, MO) in 2 ml buffer C is added and during 10 min at 0° allowed to form a ternary complex with the active site of ADH in the presence of pyrazole (Fig. 1, step 1). In order to remove excess of bis-NAD and to maintain the formed ternary complex the gel is washed 5 times with 5 volumes of buffer C. The amount of bis-NAD that remains affinity bound is calculated by subtracting the bis-NAD removed during washing from the amount initially added. Lactate dehydrogenase (LDH) (from beef heart, 490 U/mg, Sigma Chem. Co., St. Louis, MO), two times the molar amount of affinity-bound bis-NAD, dissolved in a minimal volume of buffer C, is added to the gel, for affinity coupling of LDH to the NAD entity of the affinity-bound bisNAD pointing out from the immobilized ADH (Fig. 1, step 2). After 5 min of incubation the gel is washed 5 times with 2 ml of cold buffer C. The amount of LDH that remains affinity bound to the gel is determined in the same way as for the determination of remaining bis-NAD described above. The gel is subsequently suspended in 2 ml cold buffer C and glutaraldehyde is added to a final concentration of 0.06% (Fig. 1, step 3). The cross-linking is allowed to proceed for 2.5 hr at room temperature. After the cross-linking step the gel is first washed and then suspended in 0.2 M Tris-HCl, pH 8.0, in order to quench the remaining unreacted aldehyde groups and kept at 4° overnight. After quenching, the gel is washed with several volumes each of buffer A, then buffer B, and finally buffer A containing 0.1 M isobutyramide, 0.05 M oxamate, and 1 mM

ADH

LDH

FIG. 1. Schematic presentation of preparation of immobilized site-to-site directed twoenzyme complex. See text for further explanation. The oligomeric nature of the enzymes has not been taken into account. ('~) Pyrazole; (O) Oxalate; ([] []) bis-NAD.

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MULTISTEP ENZYME SYSTEMS AND COENZYMES

ADH

([]

~

[10]

ADH

FIG. 2. Preparation of reversibly immobilized alcohol dehydrogenase. ( 4 ) Pyrazole; []) bis-NAD.

NADH. This final treatment is carried out because it is expected that NADH, together with isobutyramide and oxamate, will form new ternary complexes with ADH and LDH, respectively. The NADH will then compete with remaining enzyme-bound bis-NAD and thereby displacing it from the active sites of the two enzymes (Fig. 1, step 4). The gel is finally washed with several volumes of buffer B and buffer A in order to remove the NADH.

Soluble System A soluble site-to-site oriented enzyme complex is obtained by immobilizing ADH on solid phase with a cleavable disulfide bond (Fig. 2, steps 14) prior to the active site orientation of LDH. After the cross-linking of the two enzymes the enzyme complex is split off from the solid phase by cleaving the disulfide bond between the ADH part of the complex and the matrix. An amino-group-containing gel is obtained by coupling diaminoethane to cyanogen bromide-activated Sepharose 4B.6 In order to introduce thiol groups onto the gel, 2 ml 20 mM N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Pharmacia Fine Chem. Co., Uppsala, Sweden) in ethanol is added to 5 ml amino-gel suspended in 5 ml buffer A. After coupling for 2 hr at 25° the SPDP-activated gel is washed with several volumes of buffer A. The 2-pyridyl disulfide-Sepharose obtained is then easily converted into the free thiol form by treating 5 ml of moist gel with 5 ml 100 mM 1,4dithioerythritol (DTE) (Boehringer-Mannheim, FRG), dissolved in buffer A, for 30 rain at room temperature (Fig. 2, step 2). The gel is subsequently washed with several volumes of buffers B, A, and C. The amount of thiol groups on the gel is determined by cleavage of the disulfide bond and then spectrophotometric measurement of the amount 6 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochern. 60, 149 (1974).

[10]

ENZYME COMPLEXES COMPOSED OF A D H AND L D H

107

of released pyridine-2-thione (extinction coefficient at 343 nm of 8080 cm-~ M-~ 7). Since the free thiol groups can take part in unwanted side reactions, the thiol-Sepharose is used immediately after the cleavage. SPDP-activated ADH is prepared as follows 7 (Fig. 2, step 3). ADH (312 nmol), 25 mg, is dissolved in 6 ml of buffer C and in order to protect the active sites of ADH during the SDDP activation step, about 1/zmol of bis-NAD, dissolved in 100/xl distilled water, is added. A ternary complex between the active sites of ADH, pyrazole, and bis-NAD is thereby formed. A five molar excess of SPDP (20 mM in ethanol) is dropwise added to the solution of protected ADH and the reaction is allowed to proceed 30 min at 25°. The unreacted SPDP is removed by dialysis of the ADH solution against three changes of 1 liter of buffer C. The amount of SPDP coupled to ADH can then be determined as described above for the solid-phase approach. The SPDP-activated and dialyzed ADH is mixed with the thiolSepharose immediately after the above-mentioned reduction of the 2pyridyl disulfide-Sepharose, and the coupling is allowed to proceed 16 hr at 4°. The immobilized ADH is then carefully washed with cold buffers A, B, and C. The affinity immobilization of LDH and the subsequent cross-linking with glutaraldehyde are performed as described above (see Fig. 1). Quenching of unreacted aldehyde groups is accomplished by incubation for 24 hr at 4 ° with 20 ml 0.2 M Tris-HC1, pH 8.0, containing 33 mM NaCNBH3 and 10 mM AMP. This gentle treatment with NaCNBH3 reduces the Schiff bases that are formed by the reaction of aldehyde and Tris. 8 AMP is added to the quenching solution to remove possible remaining affinity-bound bis-NAD by competition. AMP may also protect the active sites of ADH and LDH during the quenching procedure. After quenching, the gel is washed with buffers B and A to remove all AMP. Finally 5 ml of buffer A, containing 100 mM DTE, is added to the gel in order to cleave the disulfide bonds between ADH and the matrix. After 25 min of incubation at 25° the gel is washed 4 times with 5 ml of 50 mM DTE in buffer A. After this treatment about 40% of immobilized ADH activity is recovered in the effluent. The effluent is made 25 mM with respect to oxalate in order to carry out affinity purification of the A D H LDH complex. The oxalate-containing solution (50 ml) is subsequently applied to a 10-ml column of weakly substituted NAD-Sepharose. The NAD-Sepharose is obtained by coupling 0.55 /.tmol N6-[N-(6-amino 7 T. Stuchburg, M. Shipton, R. Norris, J. P. G. Malthouse, K. Brocklehurst, J. A. L. Herbert, and H. Susckitzky, Biochem. J. 151, 417 (1975). 8 R. Borch, M. Bernstein, and D. Durst, J. Am. Chem. Soc. 93, 2897 (1971).

108

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[10]

hexyl)carbamoylmethyl]-NAD, 9 available from Sigma Chem. Co., to 10 ml Sepharose 4B activated with CNBr (50 mg CNBr/ml gel). 6 The NAD-Sepharose is packed in a column and equilibrated with 25 mM oxalate in 50 mM sodium phosphate, pH 7.5, prior to the affinity purification of the A D H - L D H complex. The complex is, in the presence of oxalate, affinity bound to the NAD column since a ternary complex is formed with NAD, oxalate, and the active sites of LDH of the complex. After application of the sample, the NAD-gel is washed with 25 mM oxalate in 50 mM sodium phosphate, pH 7.5, until all non-affinity-bound enzymes have been removed. The L D H - A D H complex is then eluted from the affinity column by changing the elution buffer to buffer A, thereby excluding the oxalate in the elution buffer. The protein fractions that contain the A D H - L D H complex are collected and used for further experiments. Enzyme Assays All assays with immobilized enzymes (5-100 mg of moist gel) are carried out in a stirred 25-ml vessel, containing the immobilized enzyme suspended in 15 ml of assay solution which is continuously pumped with a peristaltic pump through a filter, allowed to pass through a flow cuvette in a spectrophotometer (Hitachi 181), and recycled back to the reaction vessel. All assays with soluble enzymes are carried out in I- or 3-ml cuvettes. Lactate Dehydrogenase (LDH) Activity. LDH activity is determined (in buffer A) by measuring the decrease in absorbance at 340 nm with 0.3 mM and 5 mM pyruvate as substrates. Lipoamide Dehydrogenase (LiDH) Activity. LiDH activity is determined in buffer A by the method described by Massey. 10The reduction of 0.7 mM ferricyanide (e420 = 1040 cm -1 M -1) is followed with 0.2 mM N A D H as second substrate. Alcohol Dehydrogenase (ADH) Activity. ADH activity is measured in buffer A as the increase in absorbance at 340 nm with 1 mM NAD and 50 mM ethanol as substrates, unless a coupled substrate assay is used. In the coupled substrate assay the ADH activity is measured in buffer A containing 50 mM oxalate using 5 mM benzyl alcohol and 5 mM acetaldehyde as substrates (Fig. 3). The coenzyme NAD is regenerated in the active site of ADH after addition of the substrates. The required coenzyme is added to the immobilized enzyme system in the following order. First the immo9 M. Lindberg, P. O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973). 10 V. Massey, this series, Vol. 9, p. 272.

[10]

ENZYME COMPLEXESCOMPOSEDOF ADH AND LDH

ADH ~

109

LDH

ethanol benzylaleohol FIG. 3. Site-to-site directed complex with readded Dis-NADas coenzyme in a coupled substrate assay. The oligomeric nature of the enzymes has not been taken into account. (Q) Oxalate; ( t ~ [ ] ) bis-NAD.

bilized A D H - L D H complex is washed with several volumes of buffer A containing 50 mM oxalate. Bis-NAD, 50 nmol, is dissolved in 1 ml of the same buffer and added to the gel, allowing its affinity binding to the active sites of LDH. After 30 min at room temperature excess of bis-NAD is removed by washing the gel with a few volumes (2-3) of buffer A containing 50 mM oxalate. One of the two NAD entities of the affinity-bound bisNAD is accordingly pointing outward from the active site of L D H (Fig. 3). This NAD entity is then able to reach the active site of ADH and can there act as a coenzyme for the coupled reaction. A molar extinction value at 279 nm for the product, benzaldehyde, of 1400 cm -1 M -I ~1 is used. In order to determine the maximum activity the solution is subsequently made 50/xM with respect to NAD. In the corresponding coupled substrate assay for the soluble system, bis-NAD is added to the enzyme complex to a final concentration of 50 nM in the cuvette and incubated for 10 min at room temperature in order to saturate all active sites of L D H with bis-NAD. Benzaldehyde formation is measured as before. In order to obtain maximum coupled substrate activity, 5/xl 10 mM NAD is added to the assay solution.

Scavenger Enzyme Assay In order to investigate the effects of the orientation and general proximity of the active sites of L D H and ADH, a third enzyme, lipomide dehydrogenase (LiDH), was introduced as a scavenger enzyme (Fig. 4). For the immobilized enzyme system, ADH (2 mg) and LiDH (3 mg, 136 U/mg) are coimmobilized to tresyl chloride-activated Sepharose 4B (2.5 g of moist gel). L D H is subsequently site-to-site immobilized to the immobilized ADH as described above. In the control experiment all three en11C. W. Fuller, J. R. Rubin, and H. J. Bright, Eur. J. Biochern. 103, 421 (1980).

I I0

[10]

MULTISTEP ENZYME SYSTEMS AND COENZYMES

A

B

/

NA

\

\

NAD+ ~ /

NAD+ NADH,/

NAD+/

NAD+

FIG. 4. Scavenger assay with lipoamide dehydrogenase (LiDH) as an enzyme competing with LDH for NADH formed by ADH.

zymes (alcohol, lactate, and lipoamide dehydrogenases) are immobilized randomly : 1.2, 0.6, and 2.0 mg, respectively, are used per 2.5 g of tresyl chloride-activated moist gel. The scavenger enzyme assay is performed as follows, The gel is suspended in 13.5 ml buffer A containing 1 mM NAD and 0.75 mM ferricyanide. Ethanol (final concentration 50 mM) is added and the reduction of ferricyanide is measured as the decrease in absorbance at 420 nm. Pyruvate is subsequently added to a final concentration of 5 mM in order to measure the competition between LiDH and LDH for the NADH formed by ADH. The amount of NADH oxidized by LDH is thus measured indirectly as the decrease in LiDH activity. The soluble site-to-site enzyme complex was also assayed with the scavenger enzyme assay using the same system as for the immobilized enzymes. Characterization of Site-to-Site Enzyme Complexes

ADH Activity In order to characterize both the immobilized and soluble enzyme complexes the ADH activity was determined by a coupled substrate assay with bis-NAD affinity bound to LDH acting as coenzyme. As is shown in Table I, as much as 33% o f the ADH activity obtained in the presence of excess of NAD (50/~M) was obtained upon readdition of 50 nM bis-NAD to the immobilized A D H - L D H complex. In a reference experiment, ADH and LDH were randomly coimmobilized and for this preparation much less activity (4%) was observed upon readdition of bis-NAD. The soluble enzyme complex was characterized in a similar way. For the first measurement no coenzyme was added to the assay solution, However, some ADH activity was observed. The same effect was also

[10]

ENZYME COMPLEXES COMPOSED OF A D H AND L D H

111

TABLE I RELATIVE A D H ACTIVITY OF SOLUBLE AND IMMOBILIZED COMPLEXES OF A D H AND L D H AT DIFFERENT N A D CONCENTRATIONS

Relative ADH activitya

System Immobilized Site-to-site oriented enzymes Randomly coupled Soluble Site-to-site oriented enzymes Separate enzymes

0

50 nM bis-NADb

50 ~tM NAD

11 0

33 4

100 100

20 0

55 <1

100 100

a ADH activity was determined with a coupled substrate assay using three different coenzyme concentrations. Relative ADH activity = (ADH activity at different coenzyme concentration/activity with 50/zM NAD) × 100. b The bis-NAD affinity bound to LDH in the immobilized system corresponds to a nominal concentration of approximately 50 nM.

noted for the immobilized site-to-site directed enzyme complex as shown in Table I. This was probably due to some remaining bis-NAD that had not been removed by washing. As the washing procedure and the purification of both the immobilized and the soluble site-to-site directed enzyme complexes were rather extensive, it indicates that the bis-NAD template was physically entrapped between the active sites of the two enzymes. This was probably due to the cross-linking treatment since adding more glutaraldehyde during the cross-linking procedure caused an even higher coupled substrate activity prior to adding any coenzyme to the assay solution. The coupled substrate activity for the soluble enzyme complex increased when the bis-NAD concentration increased to 50 nM in the presence of oxalate. A 50 nM concentration of bis-NAD corresponds to the nominal concentration of bis-NAD in the corresponding immobilized system. The soluble enzyme complex reached, at 50 nM bis-NAD, about 55% of the ADH activity obtained with an excess of NAD (50/zM) NAD. The corresponding system of separate soluble enzymes showed, in the presence of oxalate, no measurable activity (< 1%) at 50 nM bis-NAD compared to the maximum activity obtained with 50/zM NAD. From these experiments it becomes apparent that one moiety of the bis-NAD can interact with ADH while the other half of the molecule is still affinity bound to LDH. This indicates that the overall geometry of the two enzymes relative to one another, obtained upon cross-linking, is re-

112

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[10]

tained also after the majority of the bis-NAD templates had been washed away. The results from the two reference experiments, i.e., randomly immobilized and soluble separate enzymes (Table I), show that the bisNAD analog added does not interact to any large extent with ADH under the conditions used. This is most likely due to the fact that the two enzymes are too far apart or are not properly oriented toward one another.

Scavenger Enzyme Assay To further investigate the effects of the orientation of the active sites of ADH and LDH, a third enzyme, lipoamide dehydrogenase (LiDH), was introduced in an assay as a scavenger enzyme (Fig. 4). When NAD and ethanol were added to such an enzyme system, NADH and acetaldehyde were formed. The N A D H formed was subsequently oxidized by LiDH when ferricyanide was added. When pyruvate then was added, LDH competed with LiDH for the NADH formed by ADH. This competing reaction was measured as the decrease in LiDH activity. Only ADH operated under substrate saturation conditions and, since LDH and LiDH were in excess, it was ensured that all NADH molecules produced by ADH were rapidly oxidized. The amount of N A D H oxidized by LDH and LiDH, respectively, would be expected to be determined by the relative total numbers of TABLE II RELATIVE NADH-OxIDIZING ACTIVITIES OF THE THREE-ENZYME SYSTEM COMPRISING ADH, LDH, Ar~r~LiDH NADH oxidized in "scavenger" assay (%p By LiDH System Immobilized Site-to-site oriented enzymes Random coupling Soluble Site-to-site oriented enzymes Separate enzymes

Theoretical

By LDH

Found

Theoretical

19

Found

81

50

95.5

99.5

37

17

63

83

37

39

63

61

4.5

50 0.5

The theoretical percentage of NADH oxidized by either LDH or LiDH was calculated from the obtained separate enzyme activities as the ratio of found Vma~activities of LDH and LiDH, LDH/(LHD + LiDH), and LiDH/(LDH + LiDH), respectively.

[10]

ENZYME COMPLEXES COMPOSED OF A D H AND L D H

113

enzyme units of the two enzymes since the Kn~values for NADH of LiDH and LDH are approximately the same under the conditions used. In a randomly coupled three-enzyme system the amount of NADH oxidized by LiDH was that to be expected on these grounds. As is shown in Table II the values were 99.5% found and 95.5% expected. For the system with LDH juxtaposed to ADH, it was predicted that 81% of formed NADH would be oxidized by LiDH. However, it was found that only 50% of the formed NADH was oxidized by LiDH, showing the channeling effect. The soluble site-to-site enzyme complex was also studied with the scavenger enzyme assay and in principle the same kind of results were obtained as for the immobilized system (Table II). The results obtained for both the immobilized system and the soluble system indicate that, given two alternative routes, the one to LDH is preferred over the one to LiDH when LDH and ADH are site-to-site oriented.

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PRODUCTION

OF OPTICALLY

ACTIVE COMPOUNDS

117

[11] O v e r v i e w

By L. ANDERSSON,M.-O. M/~NSSON,and K. MOSBACH In this section we have collected a relatively large number of contributions reflecting the growing interest in the use of immobilized enzymes/ cells for organic syntheses. Striking is the fact that many reactions are carried out in organic solvents. We would like to draw the reader's attention to a recent publication and references cited therein related to this topic describing enzyme modification with polyethylene glycol resulting in enzymes formidably stable in organic solvents [K. Takahashi, Y. Kodera, T. Yoshimoto, A. Ajima, A. Matsushima, and Y. Inada Biochem. Biophys. Res. Commun. 131, 532 (1985)]. Also in an article by Nilsson and Mosbach in Vol. 135 [3] of this series an example of enzyme application in organic solvents, more specifically on peptide synthesis, is given. Other aspects not treated here but of potential importance include the increased use of thermostable enzymes in connection with coenzyme recycling [see, for instance, Enzyme Eng. 8, in press (1987)]. Surely it can be anticipated that in the future we will see increasing use of immobilized systems containing genetically engineered enzymes, such as those obtained by site-directed mutagenesis.

[12] E n z y m a t i c P r o d u c t i o n o f O p t i c a l l y A c t i v e C o m p o u n d s in B i p h a s i c A q u e o u s - O r g a n i c S y s t e m s

By

ALEXANDER M. KLIBANOV a n d BERNARD CAMBOU

Production of optically active compounds is one of the most challenging problems in modern organic chemistry. 1.2 In addition to its fundamental interest, this area is also of great practical significance. As new generations of specialty chemicals (pharmaceuticals, agricultural chemicals, flavors, food additives, etc.) become increasingly complex, a growing number of them contain chiral carbon atoms. Since the ultimate function of most of the aforementioned substances is to interact with biological i E. L. Eliel and S. Otsuka (eds.), "Asymmetric Reactions and Processes in Chemistry." American Chemical Society, Washington, D.C., 1982. 2 j. D. Morrison (ed.), "Asymmetric Synthesis." Academic Press, New York, 1983.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc, All rights of reproduction in any form reserved,

[12]

PRODUCTION

OF OPTICALLY

ACTIVE COMPOUNDS

117

[11] O v e r v i e w

By L. ANDERSSON,M.-O. M/~NSSON,and K. MOSBACH In this section we have collected a relatively large number of contributions reflecting the growing interest in the use of immobilized enzymes/ cells for organic syntheses. Striking is the fact that many reactions are carried out in organic solvents. We would like to draw the reader's attention to a recent publication and references cited therein related to this topic describing enzyme modification with polyethylene glycol resulting in enzymes formidably stable in organic solvents [K. Takahashi, Y. Kodera, T. Yoshimoto, A. Ajima, A. Matsushima, and Y. Inada Biochem. Biophys. Res. Commun. 131, 532 (1985)]. Also in an article by Nilsson and Mosbach in Vol. 135 [3] of this series an example of enzyme application in organic solvents, more specifically on peptide synthesis, is given. Other aspects not treated here but of potential importance include the increased use of thermostable enzymes in connection with coenzyme recycling [see, for instance, Enzyme Eng. 8, in press (1987)]. Surely it can be anticipated that in the future we will see increasing use of immobilized systems containing genetically engineered enzymes, such as those obtained by site-directed mutagenesis.

[12] E n z y m a t i c P r o d u c t i o n o f O p t i c a l l y A c t i v e C o m p o u n d s in B i p h a s i c A q u e o u s - O r g a n i c S y s t e m s

By

ALEXANDER M. KLIBANOV a n d BERNARD CAMBOU

Production of optically active compounds is one of the most challenging problems in modern organic chemistry. 1.2 In addition to its fundamental interest, this area is also of great practical significance. As new generations of specialty chemicals (pharmaceuticals, agricultural chemicals, flavors, food additives, etc.) become increasingly complex, a growing number of them contain chiral carbon atoms. Since the ultimate function of most of the aforementioned substances is to interact with biological i E. L. Eliel and S. Otsuka (eds.), "Asymmetric Reactions and Processes in Chemistry." American Chemical Society, Washington, D.C., 1982. 2 j. D. Morrison (ed.), "Asymmetric Synthesis." Academic Press, New York, 1983.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc, All rights of reproduction in any form reserved,

118

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[12]

receptors (consisting of asymmetric building blocks), it turns out that very frequently only one out of two or more isomers of chiral molecules is biologically active. This means that one has to be able either to readily resolve racemic mixtures of enantiomers or to asymmetrically synthesize optical isomers. Conventional chemical methods for the former, although often used, are laborious, time-consuming, and inefficient.3 Chiral chemical syntheses, on the other hand, despite some spectacular recent developments, 4,5 are still not generally applicable to large-scale preparations. Enzymes are uniquely suitable for asymmetric transformations. ~ This is because they are built of only L-amino acids and hence their active centers constitute asymmetric environments which are likely to react differently with different enantiomers. Despite this and other virtues of enzymes as catalysts in organic syntheses, 6-9 there are also some major obstacles. 9 One of the most serious of them stems from the fact that enzymes are supposed to function in water and therefore have traditionally been used in aqueous solutions. That represents a severe limitation with respect to organic syntheses, because (1) a majority of compounds on interest to organic chemists are poorly soluble in water, and (2) in a number of instances the position of equilibrium for a desired reaction is unfavorable in aqueous solutions or water participates in a side reaction which competes with the desired one. In 1977, Klibanov e t al. 1° proposed an approach to solve both of the above problems. The gist of the method was to employ biphasic aqueousorganic mixtures. The enzyme is located in the aqueous phase, while the substrates are dissolved in a water-immiscible organic solvent constituting the organic phase. Substrates and products can freely diffuse between the two phases and yet the solubility problem does not come up. This approach was illustrated using chymotrypsin-catalyzed esterification reaction between N-acetyl-L-tryptophan and ethanol: whereas in water the yield of N-acetyl-L-tryptophan ethyl ester was only about 0.01%, in a biphasic system consisting of approximately 99% chloroform and 1% wa-

3 j. Jacques, A. Collet, and S. H. Wilen, "Enantiomers, Racemates and Resolutions." Wiley, New York, 1981. 4 C. H. Behrens and K. B. Sharpless, Aldrichimica Acta 16, 67 (1983). H. S. Mosher and J. D. Morrison, Science 221, 1013 (1983). 6 j. B. Jones and J. F. Beck, in "Applications of Bochemical Systems in Organic Chemistry" (J. B. Jones, C. J. Sih, and D. Perlman, eds.), Part I, p. 107. Wiley, New York, 1976. 7 C.-H. Wong and G. M. Whitesides, Aldrichimica Acta 16, 27 (1983). 8 M. A. Findeis and G. M. Whitesides, Annu. Rep. Med. Chem. 19, 263 (1984). 9 A. M. Klibanov, Science 219, 722 (1983). 10 A. M. Klibanov, G. P. Samokhin, K. Martinek, and I. V. Berezin, Biotechnol. Bioeng. 19, 1351 (1977).

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

119

ter it was nearly 100%. 10 Since the first paper, ~° the theory of the enzymatic esterifications in biphasic aqueous-organic mixtures has been refined and applied to the production of other esters. 11-~s Although the approach described above has not been used for asymmetric transformations (either already optically pure N-acetyl-L-amino acids or nonchiral acids served as substrates), it is clear that the methodology, in principle, may have a potential in that regard. However, detailed analysis TMof enzymatic esterifications in biphasic systems shows that this approach is not generally applicable for preparative conversions. The latter require high (preferably molar/liter) concentrations of substrates to boost productivity. Most acids (one of the substrates) will partition from organic to the aqueous phase where they will dissociate. Hence a high concentration of the acid in the organic phase will result in its high concentration in the aqueous phase. This, in turn, will drastically reduce the pH in the aqueous phase (a simple calculation shows that there is no way even a concentrated buffer could prevent that) and, consequently, cessation of the enzymatic reaction (unless the enzyme is an acid hydrolase, and those are rare). It occurred to us ~5that in order to avoid the above complications, one could use enzymatic transesterifications [Eq. (1)] instead of esterifications. Since all reactants are esters and alcohols, there is no pH change during the reaction. In the present paper, we describe the use of this technology for the preparative production of a number of optically active compounds. Rationale Most hydro-lyases, 16 in particular carboxylesterases, 17 can catalyze not only their "natural" reaction of hydrolysis, but also that of transesterification: RICOOR2 + RsOH --~ RtCOOR3 + R2OH

(1)

In the absence of water or in a biphasic system with a very low overall H K. Martinek, A. N. Semenov, and I. V. Berezin, Biochim. Biophys. Acta 658, 76 (1981). 12 K. Martinek and A. N. Semenov, Biochim. Biophys. Acta 658, 90 (1981). ~3j. L. Vidaluc, M. Baboulene, V. Speziale, A. Lattes, and P. Monsan, Tetrahedron 39, 269 (1983). ~4 B. Cambou and A. M. Klibanov, Biotechnol. Bioeng. 27, 1449 (1985). 15 B. Cambou and A. M. Klibanov, J. Am. Chem. Soc. 106, 2687 (1984). ~6 L. B. Spector, "Covalent Catalysis by Enzymes," Chap. 4. Springer-Verlag, New York, 1982. 17 p. Greenzaid and W. P. Jencks, Biochemistry 10, 1210 (1971).

120

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[12]

fraction of water, the reaction of hydrolysis may be almost completely suppressed. Suppose that either Rl or R2 or R3 contains a chiral carbon. Furthermore, let us assume that the carboxylesterase catalyzing reaction (1) is stereospecific, i.e., it accepts only one enantiomer (of either the ester or the alcohol) as a substrate. Then enzymatic transesterification shown in Eq. (1) can be used for preparative resolution of racemic ester R1COOR2 or racemic alcohol R3OH provided that the second, nonchiral substrate is present in a molar excess. The reactive enantiomer will be completely enzymatically converted to a new chemical entity, thereby making it easy to separate it from the unreacted one. A solution of a stereospecific carboxylesterase in an appropriate aqueous buffer serves as the aqueous phase. A mixture of the substrates (RICOOR2 and R3OH) constitutes the organic phase; that is, to enhance the productivity, no additional solvent for the substrate is used. Using pig liver carboxylesterase and lipase from Candida cylindracea as stereospecific catalysts, we have implemented transesterifications in biphasic systems to preparatively resolve both racemic alcohols (R3 contains a chiral carbon) and racemic esters (R1 contains a chiral carbon). ~4,~5 Experimental Design Conceptually, the system described above represents an emulsion of an aqueous solution of an enzyme in a mixture of substrates. However, from the experimental standpoint it is advantageous instead to use porous supports whose pores are filled with an aqueous solution of an enzyme. First, such beads are more mechanically robust than water droplets; second, they can be readily separated from products at the end of the enzymatic reaction; third, they can be used repeatedly. The organic (substrate) phase should be presaturated with an aqueous buffer (of the pH optimal for the enzyme action) to avoid loss of water from the beads due to its partitioning. Three different porous supports are used for entrapment of stereospecific esterases: Sepharose, Chromosorb, and titania. A racemic alcohol or ester is dissolved in a molar excess of the second substrate, the mixture is saturated with an aqueous buffer solution, and then the enzyme confined to the pores of a solid support is added. The system is vigorously shaken at room temperature. Periodically, the organic phase is analyzed by gas chromatography. After the reaction is completed, the enzyme-containing beads are recovered by filtration, and the organic phase obtained is separated by aqueous extractions and/or fractional distillation.

[12]

PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

CH3CH2COCH3 + HOCH2CH2R

121

-CH3CH2COCH2CH2R÷ CH30H

R = CH(0CH3)CH3 (3-meth0xy-l-buton0l) R = CH(CH3)CH2CH3 (3-methyl-l-pentonol)

R = CH(CH3)CH2CH2CH2CH(CH3) 2 (3,7-dlmethYl-l-octanol) R = CH(CH3)CH2CH2CH=C(CH3) 2

(cltronello])

FIG. 1. Hog liver carboxylesterase-catalyzed resolution of racemic primary alcohols (1-4) via transestefification reactions.

Preparative Resolution of Racemic Primary Alcohols Catalyzed by Pig Liver Carboxylesterase

Materials Pig liver carboxylesterase (EC 3.1.1.1) was purchased from Sigma Chemical Co. in the form of a suspension in 3.2 M ammonium sulfate, and had a specific activity of 160 units/mg protein. Porous supports used for "immobilization" of the enzyme, Sepharose 4B and Chromosorb 101, were also obtained from Sigma. The former was an aqueous suspension of 40- to 190-~m beads (agarose concentration 4%). Chromosorb 101 (commonly used as a carrier in gas-liquid chromatography) consisted of 125- to 150-/xm particles with pore diameter of 0.30.4/xm. All primary alcohols used in this work, both in the racemic and in the optically pure form (when available), were obtained commercially. Propionic esters of 1-4 (see Fig. 1) were synthesized from propionyl chloride and the corresponding alcohol following the general procedure of Morris and Green. 18The boiling points of the propionic esters thus prepared were as follows (760 mm Hg): of 1, 181°; of 2, 174°; of 3, 342°; and of 4, 250°. Methyl propionate employed in this study as a "matrix" ester was obtained from Aldrich and was used without further purification.

Methods Assays. All alcohols and esters in this work are determined gas chromatographically using a 6-foot glass column packed with Analab's Super Pak 20 M (N2 cartier gas, 10 ml/min; detector and injector port temperatures 250°). (1) In the case of 1, the temperature of the column is increased 18 j. F. Morris and E. H. Green, J. Am. Chem. Soc. 26, 293 (1901).

122

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[12]

from 51 ° to 240 ° at 16°/min. The retention times observed are 1.6 min for methyl propionate and methanol, 6.4 min for 1, 7.1 min for 3-methoxy-1butyl propionate, and 5.2 min for propionic acid. (2) In the case of 2, the temperature of the column is 90 °. The retention times observed are 1.1 min for methyl propionate and methanol, 2.0 min for propionic acid, 2.8 min for 2, and 3.6 min for 3-methyl-l-pentyl propionate. (3) In the case of 3 and 4, the temperature of the column is 135°. The retention times observed are 1.0 min for methyl propionate and methanol, 1.5 min for propionic acid, 3.0 min for 3, 4.1 min for 3,7-dimethyl-l-octyl propionate, 3.5 min for 4, and 4.5 min for citronellyl propionate. All optical rotations are measured at 589 nm (sodium line) and 25 ° using a Perkin-Elmer 243B polarimeter. Preparation of the Enzymatic Catalyst. Solutions of hog liver carboxylesterase are entrapped either in Sepharose or in Chromosorb as described below. (1) Swollen Sepharose 4B, 2ml, is washed with water and with 0.1 M phosphate buffer (pH 8.0). The resin is thoroughly dried on a glass filter, and then the resultant gel is cut into small (approximately 2 x 2 mm) pieces with a razor blade. The beads produced are added to 4.6 ml of an enzyme suspension, 10.9 mg/ml, and allowed to absorb it (there was no liquid left afterward). (2) Chromosorb 101 (100-120 mesh), 1 g, is washed with water and with 0.1 M phosphate buffer, and then dried on a glass filter. After that, the beads are added to 1.1 ml of an enzyme suspension, 10.9 mg/ml, and allowed to completely absorb it. We have found that (for reasons not presently clear) Sepharose is a better support in the transesterification with 1 as a nucleophile, while Chromosorb is superior with 2, 3, and 4 (in terms of the half-life of the reaction at a given amount of the enzyme present in the system). Both enzymatic preparations are stored at 4 ° for several days with no appreciable loss of the catalytic activity. Alkaline Hydrolysis of Esters. Enzymatically prepared optically active esters of propionic acid can be converted to the corresponding optically active alcohols by alkaline hydrolysis. Propionic esters of 1, 2, 3, and 4 are hydrolyzed at pH 12 and 37 ° in a pH-stat's cuvette. Saturated solutions of the esters in 0.1 M aqueous solution of KCI with 10% of acetone are placed in a 100 ml water-jacketed cuvette and are hydrolyzed until completion (several hours). Then the solutions are partly evaporated under vacuum and the alcohols are extracted with ether, followed by its evaporation.

Results Hogs liver carboxylesterase entrapped in Sepharose or Chromosorb was used to asymmetrically catalyze transesterifications shown in Fig. 1:

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

123

methyl propionate (an inexpensive and readily available ester) was employed as a matrix in which the methyl moiety was stereoselectively replaced with alcohols 1-4. (-)-3-Methoxy-l-butanol (1) was dissolved in a molar excess of methyl propionate and the mixture was saturated with phosphate buffer, followed by addition of carboxylesterase entrappedsin Sepharose beads. The system was shaken at room temperature; periodically, the organic phase was analyzed gas chromatographically. Figure 2 shows the time course of the enzymatic transesterification. One can see that the concentration of 1 gradually decreases, while the concentration of the transesterification product, 3-methoxy-l-butyl propionate, concomitantly increases (no propionic acid is formed, indicating that hydrolysis was completely suppressed); after 20 hr no further reaction takes place. The enzyme-containing beads were then recovered by filtration. The organic phase obtained was separated by aqueous extractions and evaporation under vacuum. As a result, optically active (+)-1 and (-)-3-methoxy-1butyl propionate were collected. We have found that the former was

c o n c e nt r a t ion, M 2

I

fI

14 hours

I

Ii

28

FIG. 2. The time course of the reaction between methyl propionate and (-)-1 catalyzed by hog liver carboxylesterase entrapped in Sepharose beads. (O) Disappearance of methyl propionate; (C]) accumulation of (-)-3-methoxy-1-butyl propionate. The organic phase consisted of methyl propionate containing 2 M (-+)-1 (and was saturated with 0.1 M phosphate buffer, pH 8.0). The aqueous phase contained 9600 units of the enzyme entrapped in 6 g of Sepharose beads swollen with 0.1 M phosphate buffer (pH 8.0). The mixture was shaken at room temperature.

124

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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totally unreactive in the esterase-catalyzed reaction with methyl propionate. On the other hand, (-)-1 produced from the (-)-ester by an alkaline hydrolysis was nearly fully acylated in the enzymatic transesterification. These results suggest that hog liver carboxylesterase has essentially absolute stereoselectivity in the reaction of 1 with methyl propionate, and that the transesterification stops at a 50% conversion (Fig. 2) because the reactive (-)-isomer is exhausted. The enzymatic catalyst recovered after the preparative synthesis was rinsed with ether presaturated with phosphate buffer (to remove accumulated methanol), and repeatedly employed for the preparative transesterification. After each use, no more than a quarter of the enzymatic activity was lost (as judged on the basis of the half-life of the enzymatic reaction). Sepharose is by no means a unique support for the enzyme entrapment; e.g., we have successfully used porcine liver carboxylesterase confined to Chromosorb beads for preparative production of three other couples of optically active esters and alcohols. Using the same approach as described above for 1, we have found that only S- (but not R-) isomers of 2 and 3 can act as nucleophiles in the transesterification reaction shown in Fig. 1 catalyzed by the carboxylesterase. As a result, gram quantities of R(+)-2 and -3 [and their S(-)-propionic esters] were prepared with the enantiomeric excess of 94 and 97%, respectively. Both ( - ) - and (+)-fl-citronellols (4) are natural products which are widely used in perfumery. 19,20Although methods for the synthesis of the optical isomers of 4 have been developed, they are rather inefficient and usually afford low optical purity, zl-24 (---)-4 can be easily made by reduction of synthetic citronellal or geranio119,2° but its resolution, as well as that of most racemic alcohols, 3 is very cumbersome. We have attempted to employ our enzymatic approach for the resolution of (+-)-4. Starting with 20 g of racemic 4, 6.9 g of R(+)-4 [ee (enantiomeric excess) = 96%] and 4.8 g of S(-)-4 (ee -- 92%) were obtained. As in the case of the other alcohols tested, only S(-)-4 was reactive toward the enzyme. Hence hog liver carboxylesterase exhibits absolute S-stereoselectivity in the transesterification reaction. Remarkably, the enzyme recognizes configuration of 19 A. B. Booth, in "Kirck-Othmer Encyclopedia of Chemical Technology," Vol. 19, 2nd Ed., p. 803. Wiley (Interscience), New York, 1969. 2o "The Merck Index," 9th Ed., p. 310. Merck, Rahway, New Jersey, 1976. 21 R. von Rienacker and G. Ohroff, Agnew. Chem. 73, 240 (1961). 22 R. von Rienacker, Chimia 27, 97 (1973). 23 T. Shono, Y. Matsmara, K. Hibino, and S. Miyawaki, Tetrahedron Lett. 15, 1295 (1974). 24 M. Hidai, H. Ishiwatari, H. Yagi, E. Tanaka, K. Onozawa, and Y. Uchida, Chem. Commun. 170 (1975).

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125

the chiral carbon which is two methylene groups away from the alcohol's reactive hydroxyl. It is significant that there is a close parallelism between the stereospecificity of the enzyme in the transesterification reaction (Fig. 1) and that in the reaction of hydrolysis. We have found that in water (pH 8.0) hog liver carboxylesterase catalyzes the hydrolysis of only the (-)-isomer of propionic esters of 1, 2, 3, and 4 (the reactions were carried out to completion in a pH-stat's cuvette; then the products were extracted with ether and characterized). An ideal enzyme to be used as a catalyst in organic chemistry has to meet the following two requirements: (1) it should have a very broad substrate specificity, and (2) it should display a marked selectivity (e.g., stereo) in the conversion of its substrates. Porcine liver carboxylesterase clearly satisfies the latter requirement: it is stereospecific with respect to the nucleophile in reaction (1). However, it does not meet the former requirement. We found that the carboxylesterase has a rather narrow specificity with regard to the alcohols it accepts as nucleophiles in the transesterification reaction; only primary alcohols having no substituents in the first two methylene groups adjacent to the hydroxyl react at appreciable rates. Since a number of chiral secondary alcohols are of practical significance, it was necessary to extend our methodology to them, i.e., to identify an estei-ase that will be both stereospecific and able to accept secondary alcohols as nucleophiles in the transesterification reaction. This work is described in the next section.

Procedure Enzymatic Production of (-)- and (+)-3-Methoxy-l-butanol. A mixture containing 92 ml of methyl propionate and 28 ml of racemic 1 is saturated with 0.1 M aqueous sodium phosphate (pH 8.0). Following removal of the aqueous phase on a separation funnel, 6 g of Sepharose beads containing hog liver carboxylesterase is added. The mixture is transferred to a 200-ml screw-cap bottle and shaken on an orbit shaker at 250 rpm and room temperature for 20 hrs. Then the enzymatic catalyst is recovered by filtration, and the liquid phase obtained is washed with 3 volumes of distilled water. The washings are combined and evaporated at 15 mm Hg; at 50 ° 11.7 g of (+)-1 (93% yield, 97% purity as judged by gas chromatography) with [a]D = 15.1° (neat) was collected. The organic phase remaining after aqueous extraction is evaporated at 15 mm Hg; at 40 ° 16.7 g of (-)-3-methoxy-l-butyl propionate (80% yield, 98% purity by GC) with [a]o = -16.3 ° (neat) was obtained. The ester is hydrolyzed as described above, resulting in 6.1 g of (-)-1 (48% overall yield, 95% purity

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by GC) with [ a ] D = - - 14.3 ° (c = 9, methanol). To our knowledge, optically active 1 has not been synthesized before. Therefore, we could not determine the enantiomeric excess of the enzymatically prepared (+)- and ( - ) 1 (or the (-)-ester).

Enzymatic Production of R(+)-2 and S(-)-Methyl-l-pentyl Propionate. A mixture containing 22.3 ml of methyl propionate and 7.7 ml of racemic 2 is saturated with the phosphate buffer as described above and then 2 g of Chromosorb beads with hog liver carboxylesterase is added. The mixture is placed in a 50-ml screw-cap bottle and shaken for 53 hr. Following r e c o v e r y of the enzymatic catalyst, methanol and methyl propionate from the organic phase are removed by rotary evaporation and the remainder is distilled at 10 mm Hg. At 54 ° 2.6 g of R(+)-2 (75% yield, 96% purity by GC) with [a]D = 9.8 ° (C = 10, methanol), which corresponds to ee = 94% [lit. 25 [a]D = 10.4 ° (C = 10, methanol)], was obtained. At 71 ° 2.5 g of S ( - ) - 3 - m e t h y l - l - p e n t y l propionate (44% yield, 94% purity by gas chromatography) with [odd = - 8 . 6 ° (c = 10, methanol) was collected.

Enzymatic Production of R( +)-3 and S(-)-3, 7-Dimethyl-l-octyl Propionate. The procedure is the same as for 2 except the reaction time is 96 hrs. Upon distillation at 10 mm Hg, at 105 ° 3.3 g of R(+)-3 (61% yield, 96% purity by GC) with [~]D = 4.9 ° (C = 5, ether), which corresponds to ee = 97% [lit. 26 [C~]D = 5.1 ° (C = 5.03, ether)], was obtained. At 121 ° 3.0 g of S(-)-3,7-dimethyl-l-octyl propionate (40% yield, 95% purity by GC) with [aiD = --5.1 ° (C = 5, ether) was collected. Enzymatic Production of R(+)- and S(-)-4. A mixture containing 39.7 ml of methyl propionate and 24.8 ml of (-+)-4 is saturated with the buffer as described above and 4.9 g of Chromosorb beads with hog liver carboxylesterase is added. The mixture is placed in a 100-ml screw-cap bottle and shaken for 96 hr. Then the beads are filtered out, and the organic phase is distilled at 10 mm Hg. At 108 ° 6.9 g of R(+)-4 (69 ° yield, 96% purity by GC) with [a]D = 5.0 ° (neat), which corresponds to ee = 96% [lit. 26 [a]D = 5.2 ° (neat)], was obtained. At 123° 8.0 g of S(-)-citronellyl propionate (58% yield, 97% purity by GC) with [a]D = --3.8 ° (neat) was collected. The ester is hydrolyzed as described in Methods, resulting in 4.9 g of S ( - ) - 4 (48% overall yield, 94% purity by GC) with [a]D = --4.4 ° (C = 20, methanol), which corresponds to ee -- 92% [as compared to [a]D = --4.8 ° (C = 20, methanol) for the authentic samples of S ( - ) - 4 purchased from Pfaltz and Bauer]. 25 K. Mori, T. Suguro, and M. Uchida, Tetrahedron 34, 3119 (1978). 26 N. Cohen, C. Scott, C. Neukom, R. J. Lopresti, G. Weber, and G. Saucy, Helv. Chim. Acta 64, 1158 (1981).

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

127

Preparative Resolution of Racemic Secondary Alcohols Catalyzed by Yeast Lipase

Materials Lipase from Candida cylindracea (EC 3.1.1.3, triacylglycerol lipase) was purchased from Sigma Chemical Co.; it was a powder with a specific activity of 2415 units/mg protein. Chromosorb I01, described in the preceding section was used as a support for immobilization. All secondary alcohols used in this work, both in the racemic and in the optically pure form (when available), were obtained commercially. Butyric esters of 5-10 (see Fig. 3) were synthesized from butyryl chloride and the corresponding alcohol following the general procedure of Morris and Green. ~8The boiling points of the butyric esters thus prepared were as follows (760 mm Hg): of 5, 151°; of 6,210°; of 7, 250°; of 8, 223°; of 9, 170°; and of 10, 220 °. Tributyrin, employed as a matrix ester, was obtained from Sigma and was used without further purification.

Methods Assays. All alcohols and esters in this work are determined gas chromatographically using a 6-foot glass column packed with Analab's "Super Pak 20 M " (N2 carrier gas, 10 ml/min; detector and injector port temperatures 250°). The temperature of the column is increased from 51 ° to 240° at 16°/min. The retention times observed are 11-13 min for tributyrin and

0 TRIBUTYRIN + H O C H R I R 2 ~ CH3CH2CH2~0CHRi R2 + DIBUTYRIN

Z

Ri = CH3

R2 = CH2CH3

RI = CH3

R2 = (CH2)5CH3

(2-BUTANOL)

R! = CH3

R2 = C6H5

(2-OCTANOL)

(SEC....N-PHENETHYLALCOHOL)

RI = CH3 R2 = CH2CH2CH=C(CH3)2 RI = CH3

R2 = CH2Ct

RI = H

R2 = CH(CL)CH20

I_I RI = H

R2 = CH(0H)CH2CH3

10

(6-METHYL-S-HEPTEN-2-OL)

(I-CHLORO-2-PROPANOL) (2,3-DICHLOROPROPANOL) (I,2-BUTANEDIOL)

FIG. 3. Yeast lipase-catalyzed resolution of racemic alcohols (5-11) via transesterification reactions.

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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dibutyrin (a broad shapeless peak), 6.0 min for butyric acid, 1.8 min for 5, 3.2 min for 2-butyl butyrate, 5.0 min for 6, 6.6 min for 2-octyl butyrate, 6.6 min for 7, 7.8 min for sec-phenethyl butyrate, 5.0 rain for 8, 6.7 min for 6methyl-5-hepten-2-yl butyrate, 3.0 min for 9, 4.9 min for 1-chloro-2-propyl butyrate, 6. I min for 10, 7.4 min for 2,3-dichloroprop-l-yl butyrate, 6.1 min for 11, and 7.1 min for 2-hydroxybut-l-yl butyrate. All optical rotations are measured at 589 nm (sodium line) and 25 ° using a Perkin-Elmer 243 B polarimeter. Preparation of the Enzymatic Catalyst. Lipase from C. cylindracea is entrapped in Chromosorb beads. One gram of the beads is impregnated with 0.1 M phosphate buffer (pH 8.0), and they are dried on a filter paper. Then 67 mg of the enzyme powder is added to the beads and mixed thoroughly, and the mixture is allowed to equilibrate at room temperature for 5 hr. Alkaline Hydrolysis of Esters. Enzymatically prepared optically active butyric esters can be converted to the corresponding optically active alcohols by alkaline hydrolysis. (1) Butyric esters of 5 and 11 are hydrolyzed at pH 12 and 37° in a pH-stat's cuvette. Saturated solutions of the esters in 0.1 M aqueous solution of KCl with 10% of acetone are placed in a 100-ml water-jacketed cuvette and hydrolyzed until completion (several hours). Then the solutions are partly evaporated under vacuum and the alcohols are extracted with ether, followed by its evaporation. (2) Butyric esters of 6, 7, and 8 (about 10 g) are dissolved in 50 ml of dry methanol saturated with KOH. The esters are completely solvolyzed within 1 hr (as judged by gas chromatography). Then methanol is evaporated under vacuum and the precipitated KOH is removed by filtration; the resultant solution is washed with ether and dried with anhydrous MgSO4. (3) In the case of butyric esters of 9 and 10, the alkaline treatment results both in hydrolysis and in epoxidation 27 of the alcohols produced. Butyric ester of 9, 11.9 g, and butyric ester of 10, 10.5 g, are dissolved in I 1 ml of dry nbutanol and 12 ml of dry methanol, respectively. Then the resultant solutions are added dropwise to 50 ml of dry n-butanol or methanol, respectively, saturated with KOH, at 4 °. The mixtures are stirred at room temperature for 1 hr during which time the epoxidation is completed as judged by gas chromatography. (Under condition 1 of the first Assays section, the retention times of both authentic, commercially obtained and enzymatically prepared propylene oxide and epichlorohydrin were 1.3 and 2.5 min, respectively.) Then the mixtures are stored at 4 ° overnight to precipitate KCI, and filtered; the solvents are evaporated under vacuum; the solutions are filtered again, washed with ether, and dried with anhydrous MgSO4. 27 N. F. Blau, J. W. Johnson, and C. G. Stuckwisch, J. A m . Chem. Soc. 76, 5106 (1954).

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

129

Epoxidation of 9 and 10. The procedure used is a modification of the method of Blau et al. 27 and is identical to that described in (3) of the preceding section except that 6.3 g of 9 and 8.2 g of 10 are used instead of their butyric esters.

Results

As mentioned earlier, the major drawback of hog liver carboxylesterase as a practical catalyst is its narrow nucleophile specificity. To overcome this obstacle, we have endeavored to search for another commercially available esterase which will be both stereoselective and nonspecific in the transesterification process. Such an esterase was found among lipases. Since the natural function of lipases is to hydrolyze triglycerides, 28-3° one of them, tributyrin (glyceryl tributyrate), was employed by us as a matrix ester. This ester is inexpensive and readily available. (Another likely candidate, triacetin, is too soluble in water and also is less reactive with most lipases. 29 Triglycerides of fatty acids are inferior because of their high molecular weight and cost per mole.) We have discovered that lipase from the yeast C. cylindracea, when placed in a biphasic system, where the aqueous phase is a solution of the enzyme in water confined to the pores of Chromosorb and the organic phase is a solution of an alcohol in tributyrin, (1) accepts a wide variety of alcohols (both secondary and primary) in the transesterification reaction (Fig. 3), and (2) if the alcohol is chiral, displays a marked stereoselectivity in accepting only one isomer as a nucleophile. In the case of all chiral alcohols tested, the time course of lipase-catalyzed transesterifications resembled that shown in Fig. 2. In all cases the process was carried out to apparent completion; no significant amount of butyric acid was detected (i.e., enzymatic transesterification nearly totally suppressed hydrolysis). Table I shows some examples of preparative resolution of secondary alcohols catalyzed by yeast lipase. The general scheme of this process was conceptually the same as in hog liver carboxylesterase-catalyzed resolutions and is illustrated in Fig. 4. To test the reusability of lipase entrapped in the Chromosorb beads, the enzymatic catalyst was used for four consecutive transesterifications 28 p. Desnuelle, in "The Enzymes" (P. D. Boyer, ed.), Vol. 7, 3rd Ed., p. 575. Academic Press, New York, 1972. 29 H. Brockerhoff and R. G. Jensen, "Lipolytic Enzymes." Academic Press, New York, 1974. 30 K. M. Shahani, in "Enzymes in Food Processing" (G. Reed, ed.), 2nd Ed., Chap. 8. Academic Press, New York, 1975.

130

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TABLE I PREPARATIVE RESOLUTION OF RACEMIC SECONDARY ALCOHOLS VIA TRANSESTERIFICATION IN BIPHASIC SYSTEMS CATALYZED BY YEAST LIPASEa Production of Unreactive isomer

Alcohol

Configuration of the reactive isomer

Reaction time (days)

2-Butanol 2-Octanol sec-Phenethyl alcohol 6-Methyl-5-hepten-2-olc

R R R S

1.6 5.8 6.2 5.3

Yield (g) 4.9 7.7 7.9 8.3

(70%) (77%) (79%) (83%)

ee (%) 89 95 88 91

Reactive isomer b Yield (g) 4.6 6.0 6.1 5.8

(65%) (60%) (61%) (58%)

ee (%) 93 92 85

In all cases the organic phase consisted of 2 M solution of the racemic alcohol in tributyrin; the aqueous phase consisted of a solution of lipase from Candida cylindracea in 0.1 M phosphate (pH 8.0). The mixtures were shaken at room temperature, followed by removal of the catalyst by filtration and separation of the products by distillation. b Obtained from the enzymatically produced butyric esters by alkaline hydrolysis, followed by ether extraction. ' Resolution of optical isomers of this alcohol (also called sulcatol) is particularly interesting because their nonequimolar mixture serves as an aggregation pheromone of a scolytid beetle [K. J. Byrne, A. A. Swigar, R. M. Silverstein, J. H. Borden, and E. Stokkink, J. Insect. Physiol. 20, 1895 (1974)].

with 5 as a nucleophile. After each resolution, about 75% of the initial enzymatic activity was retained. Enzymatically prepared optically active alcohols and esters can then be chemically converted to other optically active compounds. We have illustrated this in the lipase-catalyzed transesterification (Fig. 3) using 9 and 10 as nucleophiles. In each case only the R-isomer was reactive. Both the remaining S-alcohols and butyric esters of the R-alcohols were converted to epoxides using KOH treatment, z2 All four optically active epoxides produced are useful as chiral building blocks, 31 for example: R(+)propylene oxide in the synthesis of R-reciferolide 3z or chiral a_methylene-y-lactones33; S(-)-propylene oxide for the production of Ssulcatop4; both R ( - ) - and S(+)-epichlorohydrin for the synthesis of a variety of pharmaceuticals. 31'35 3~ A. S. Rao, S. K. Paknikar, and J. G. Kirtane, Tetrahedron 39, 2323 (1983). 32 K. Utimoto, K. Uchida, M. Yamaga, and H. Nozaki, Tetrahedron Lett. 18, 3641 (1977). 33 L. D. Martin and J. K. Stille, J. Org. Chem. 47, 3630 (1982). 34 B. D. Johnston and K. N. Slesser, Can. J. Chem. 57, 233 (1979). 35 j. j. Baldwin, A. W. Raab, K. Mensler, B. H. Alison, and D. E. McClure, J. Org. Chem. 43, 4876 (1978).

[12] matrix ester

PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

esterase

racemic

+

alcohol

>

filtration

• o o• •

new, optically active ester

washing_

+

131

remaining optically active alcohol

reuse

I organic phase I aq ueousd~Xtrr oc t ion n under vacuum

optically active alcohol (Ist enantiomer)

new, optically active ester I ~= alkaline hydrolysis optically active

alcohol (2nd enantiomer) FIG. 4. Schematic illustration of the resolution of racemic alcohols using esterase-catalyzed transesterifications.

All the alcohols enzymatically resolved thus far were monohydric. It was of obvious interest to explore whether the lipase-catalyzed transesterifications can be used for the preparation of optically active dials; 1,2butanediol was chosen as a model. To avoid formation of both 1- and 2esters, the enzyme, in addition to stereoselectivity, must also exhibit positional specificity. To examine that we studied the lipase-catalyzed transesterification with 2 M 1-butanol (mimicking the primary hydroxyl in 11) and 2 M 5 (mimicking the secondary hydroxyl in 11) as nucleophiles. It was determined that the half-life of the former reaction exceeds that of the latter by the factor of 12; that is, the enzyme is indeed regiospecific. This led to the facile enzymatic resolution according to the scheme in Fig. 4: from 20 g to racemic 11, 7.2 g o f R ( + ) - l l (ee = 88%) and 7.0 g of S ( - ) - l l (ee = 90%) (the unreactive and reactive isomers, respectively) were prepared.

Procedure Enzymatic Production of S(+)- and R(-)-5. A mixture containing 77.7 ml of tributyrin and 17.3 ml of racemic 5 is equilibrated with the phosphate buffer, and 3.8 g of Chromosorb beads with yeast lipase is added. The mixture is placed in a 200-ml screw-cap bottle and shaken at 250 rpm and room temperature for 40 hr. Then the enzymatic catalyst is recovered

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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by filtration, and the organic phase is distilled at 18 mm Hg. At 28 ° 4.8 g of S(+)-5 (69% yield, 97% purity by GC) with [a]D = 12.0 ° (neat), which corresponds to ee = 89% [lit. 36 [a]D = 13.5 ° (neat)], was obtained. At 50 ° 10.2 g o f R ( - ) - 2 - b u t y l butyrate (75% yield, 98% purity by GC) with laID = --11.3 ° (neat) was collected. The ester was hydrolyzed as described above, resulting in 4.6 g of R ( - ) - 5 (65% overall yield, 95% purity by GC) with [a]D = --12.8 ° (C = 5, ether), which corresponds to ee = 93% [as compared to [a]D = --13.8 ° (C = 5, ether) for the authentic sample of R ( - ) - 5 purchased from Fluka]. Enzymatic Production of S(+)- and R(-)-6. The procedure employed is the same as for 5 except the mixture contains 52 ml of tributyrin, 25 ml of racemic 6, and 3.1 g of the catalytic beads, and it is shaken for 140 hr; distillation is done at 4 mm Hg. At 33 ° 7.7 g of S(+)-6 (77% yield, 95% purity by GC) with [a]D = 9.4 ° (neat), which corresponds to ee = 95% [lit. 37 [OdD = 9.9 ° (neat)], was obtained. At 50 ° 12.6 g of R ( - ) - 2 - o c t y l butyrate (82% yield 97% purity by GC) with [~]D = --11.8 ° (neat) was collected. Alkaline hydrolysis of the ester resulted in 6.0 g of R ( - ) - 6 (60% overall yield, 96% purity by GC) with [OdD = - 9 . 1 ° (neat), which corresponds to ee = 92% [lit. 37 [a]D = --9.9 ° (neat)]. Enzymatic Production of S(-)- and R( +)-7. The procedure employed is the same as for 6 except the mixture consists of 63.3 ml of tributyrin, 19.8 ml of racemic 7, and 3.3 g of the catalytic beads, and is shaken for 150 hr. Upon vacuum distillation, at 44 ° 7.9 g of S ( - ) - 7 (79% yield, 94% purity by GC) with [a]D = --36.4 ° (neat), which corresponds to ee = 88% [lit. 38 [et]D = --41.3 ° (neat)], was obtained. At 62 ° 11.8 g of R(+)-sec-phenethyl butyrate (82% yield, 93% purity by GC) with [a]D = 42.3 ° (neat) was collected. Alkaline hydrolysis of the ester resulted in 6.1 g of R(+)-7 (61% overall yield, 97% purity by GC) with [a]D = 33.6 ° (neat), which corresponds to ee = 85% [lit. 38 [a]D = 39.5 ° (neat)]. Enzymatic Production of R(-)- and S(+)-8. The procedure employed is the same as for 6 except the mixture consists of 53.5 ml of tributyrin and 23.8 ml of racemic 8, and it is shaken for 128 hr. Upon vacuum distillation, at 30 ° 8.3 g of R ( - ) - 8 (93% yield, 97% purity by GC) with [a]D = --16.9 ° (neat), which corresponds to ee = 91% [lit. 34 [C~]D = --18.5 ° (neat)], was obtained. At 44 ° 12.2 g of S(+)-6-methyl-5-hepten-2-yl butyrate (79% yield, 96% purity by GC) with [a]D ---- 14.8 ° (neat) was collected. Alkaline hydrolysis of the ester resulted in 5.8 g of S(+)-8 (58% overall yield, 95% 36 "The Merck Index," 9th Ed., p. 197. Merck, Rahway, New Jersey, 1976. 37 y . y . Lin, D. N. Palmer, and J. B. Jones, Can. J. Chem. 52, 469 (1973). 38 "Aldrich Catalog/Handbook of Fine Chemicals," p. 918. Aldrich, Milwaukee, Wisconsin, 1982-1983.

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

133

purity by GC) with [O/]D = 16.5 ° (neat), which corresponds to ee = 95% [lit. 34 [Ct]D = 18.6 ° (neat)]. Enzymatic Production of S(-)- and R( + )-Propylene Oxide. A mixture consisting of 79.1 ml of tributyrin and 16.2 ml of racemic 9 is saturated with the buffer, and 3.8 g of the lipase-containing Chromosorb beads is added. The mixture is placed in a 200-ml screw-cap bottle and shaken for 52 hr. Following separation of the catalyst, the organic phase is distilled at 4 mm Hg. At 18° 6.3 g of S(-)-9 (70% yield, 95% purity by GC) with [a]D = -- 5.7 ° (neat) were obtained; this alcohol was converted to 2.4 g of S ( - ) propylene oxide (43% overall yield, 93% purity by GC) with [a]D = --4.9 ° (C = 1, chloroform) which corresponds to ee = 68% [lit. 39 laiD = --7.2 ° (C ---- 1, chloroform)]. At 28 ° 11.9 g of R(+)-l-chloro-2-propyl butyrate (76% yield, 94% purity by GC) with [a]D = 6.3 ° (neat) was collected; the ester was converted (see above) to 2.7 g of R(+)-propylene oxide (50% overall yield, 94% purity by GC) with [a]D = 4.8 ° (C = 1, chloroform) which corresponds to ee = 67% [lit. 39 [ a ] D = 7.2 ° (c = 1, chloroform)]. Enzyma tic Production of S (+)- a nd R (-) -Epichlorohydrin. The procedure employed is the same as for propylene oxide except the mixture consists of 62 ml of tributyrin, 15 ml of racemic 10, and 3.1 g of the enzymatic catalyst, and is shaken for 50 hr. At 28 ° 8.2 g of S(+)-10 (82% yield, 94% purity by GC) with [aid = 5.6 ° (neat) was obtained; this alcohol was converted to 3.6 g of S(+)-epichlorohydrin (51% overall yield, 95% purity by GC) with [a]D = 23.5 ° (C = 1.2, methanol) which corresponds to ee = 71% [lit. 35 [aid -- 33.0 ° (C = 1.2, methanol)]. At 52 ° 10.5 g of R(-)-2,3-dichloro-l-propyl butyrate (70% yield, 95% purity by GC) with [aid = --8.9 ° (neat) was collected; the ester was converted (see above) to 3.0 g of R(-)-epichlorohydrin (43% overall yield, 95% purity by GC) with [a]D = --23.0 ° (C = 1.5, methanol) which corresponds to ee -67% [lit. 35 [a]D = --34.3 ° (c = 1,5, methanol)]. It is worth noting that the most likely reason for a relatively low enantiomeric excess obtained is temperature-induced racemization of optically active 9 and 10 (and their butyric esters). In agreement with this hypothesis, when the enzymatically produced 9 was distilled at a lower vacuum (20 mm Hg) and higher temperature (38 °) only 10% ee was obtained for the resultant propylene oxide. Enzymatic Production of R(+)- and S(-)-ll. A mixture consisting of 92 ml of tributyrin and 20 ml of racemic 11 is saturated with the buffer, and 4.4 g of the lipase-containing Chromosorb beads is added, followed by shaking for 36 hr. The beads are removed by filtration and the resultant 39 "Aldrich Catalog/Handbook of Fine Chemicals," p. 997. Aldrich, Milwaukee, Wisconsin, 1982-1983.

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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liquid is washed with 3 volumes of water. The washings are combined and evaporated in a rotary evaporator; the remainder is washed with ether and dried with anhydrous MgSO4. As a result, 7.2 g of R ( + ) - l l (72% yield, 96% purity by GC) with [a]D = 11.0 ° (C = 16, ethanol), which corresponds to ee = 89% [lit. 4° [aiD = 12.4 ° (C = 16, ethanol)], was obtained. The organic phase left after aqueous extraction is distilled at 5 mm Hg. At 50 ° 13.9 g of (-)-1,2-butyl butyrate (79% yield, 95% purity by GC) with [a]D = --14.6 ° (neat) was collected; this ester was converted to 7.0 g of S ( - ) - l l (70% overall yield, 94% purity by GC) with [a]D = --11.6 ° (C = 2.5, ethanol) which corresponds to ee = 90% [lit. 41 [a]D = --12.9 ° (C = 2.5, ethanol)]. Preparative Resolution of Racemic Esters Catalyzed by Yeast Lipase

Materials Lipase from Candida cylindracea (EC 3.1.1.3) was purchased from Sigma Chemical Co.; it was a powder with a specific activity of 2415 units/ mg protein. The enzyme was confined to the pores of titania. Titania ST6A was a generous gift of Dr. N. R. Clark of Sterling Organics and was in the form of 400- to 700-/zm particles with pore diameter of 2000-10,000 ,~. Racemic 2-(p-chlorophenoxy)propionic acid and n-butanol were obtained from Aldrich. Methyl 2-(p-chlorophenoxy)propionate and butyl 2(p-chlorophenoxy)propionate were synthesized by us using Fischer esterification. 42

Methods Assays. All esters and alcohols are determined by gas chromatography as described in the previous section. The retention times observed are 1.5 min for methanol, 1.6 min for butanol, 9.1 min for 12, and 11.0 min for butyl 2-(p-chlorophenoxy)propionate. Optical rotations are measured at 589 nm (sodium line) and 25 ° using a Perkin-Elmer 243B polarimeter. Preparation of the Enzymatic Catalyst. Candida cylindracea lipase is entrapped in the pores of titania beads. One gram of the beads is impregnated with 0.1 M phosphate buffer (pH 8.0), followed by drying on a filter 4o K. Mori, M. Sasaki, S. Tamada, T. Suguro, and S. Masuda, Heterocycles 10, 111 (1978). 4~ K. Mori, M. Sasaki, S. Tamada, T. Suguro, and S. Masuda, Tetrahedron 35, 1601 (1979). 4: B. A. E. MacClement, R. G. Carrier, D. J. Phelps, and P. R. Carey, Biochemistry 20, 3438 (1981).

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PRODUCTION OF OPTICALLY ACTIVE COMPOUNDS

C,~/~O~HCOOCH3 CH3

+ HOCH2CH2CH2CH3~C,~/-~O~HCOOCH2CH2CH2CH3 ~ CH3

+ HOCH3

~2 FIG. 5. Yeast lipase-catalyzed resolution of racemic ester 12 via a transesterification reaction.

paper. Then 300 mg of the enzyme powder is added to the beads, mixed thoroughly, and the mixture allowed to equilibrate for 5 hr at room temperature. Alkaline Hydrolysis of Esters. Compound 12 and the corresponding butyl ester are hydrolyzed at pH 12.0 in 0.1 M aqueous solution of KCI containing 20% (v/v) of acetone at 37°. The reaction (followed potentiometrically) is complete after 4 hr. Then the pH is adjusted to pH 1.0 with concentrated HC1, followed by extraction of 1 with ether. The etheral solution is dried with anhydrous MgSO4 and evaporated under vacuum.

Results Thus far, we have used esterase-catalyzed asymmetric transesterifications in biphasic systems for preparative resolution of racemic alcohols (Fig. 4). The technology we have developed, 15 however, is not limited to that particular class of compounds. To illustrate this point, we have applied our method for the resolution of a racemic ester. Derivatives of 2-(p-chlorophenoxy)propionic acid are widely used as herbicides 43 with only one enantiomer being biologically active. 44 We have attempted to resolve methyl ester of racemic 2-(p-chlorophenoxy)propionic acid (12) using the reaction shown in Fig. 5. In this case nbutanol (the smallest water-immiscible alcohol) was used both as the "matrix" nucleophile and the solvent for 12. (---)-12 is dissolved in a molar excess of n-butanol, and the mixture is saturated with phosphate buffer, followed by addition of lipase entrapped in titania beads. The mixture is shaken at room temperature and the extent of the enzyme-catalyzed transesterification is followed gas chromatographically. After 30 hr the degree of conversion reaches 50% and the transesterification stops. Subsequent analysis of the products (see below) has shown that this is due to absolute stereospecificity of yeast lipase: only the R-isomer of the ester is reactive toward the enzyme and could be transesterified. This affords a facile preparative resolution of 43 R. Cremlin, "Pesticides: Preparation and Mode of Action," p. 143. Wiley, Chichester, England, 1978. 44 K. H. Buchel (ed)., "Chemistry of Pesticides," p. 357. Wiley, New York, 1983.

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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racemic 12 via enzymatic transesterification in the biphasic system described above. Procedure

Twenty milliliters of 2 M solution of 12 in n-butanol is saturated with 0.1 M phosphate buffer (pH 8.0). Then 3 g of the titania beads containing 0.7 g of lipase is added. The mixture is placed in a 100-ml screw-cap bottle and shaken on an orbit shaker at 250 rpm and 25 °. Periodically, 1-/xl aliquots are withdrawn and analyzed gas chromatographically as described above. When the reaction stops, the enzyme-containing beads are separated from the liquid phase by filtration, followed by its vacuum (4-5 mm Hg) distillation. At 85 ° 3.5 g of (S)-12 (81% yield, 98% purity by GC) which has a specific optical rotation [a]D = --41.1 ° (C ---- 50, ethanol) is obtained; the latter corresponds to an enantiomeric excess of 96%. 45 At 95 ° 4.1 g of (R)-butyl 2-(p-chlorophenoxy)propionate (80% yield, 97% purity by GC) with [a]D = +43.5 ° (C = 50, ethanol) (which corresponds to ee of 97% 45) is obtained. In both cases the ee is determined on the basis of the optical purity of the acid produced from the ester by alkaline hydrolysis. The enzymatically produced (S)-12 can be racemized and subjected to another enzyme-catalyzed transesterification, thereby leading to a continuous process for the preparation of the biologically active R-isomer 44from the racemic mixture. We have found that the racemization can be conveniently based on the Claisen condensation: incubation of a methanol solution of the S-ester at 60° in the presence of catalytic amounts of sodium methoxide (prepared by dissolution of metallic sodium in methanol in the 1 : 10, w/w, ratio) for 16 hr affords complete racemization of the ester. Concluding Remarks As reported in this paper, our approach, enzymatic stereoselective transesterification in biphasic systems, has been successfully tested with two different esterases, hog liver carboxylesterase and yeast lipase. As a result, a number of optically active alcohols and esters were produced with high yield and enantiomeric purity. It appears that our work represents a significant advance in the use of enzymes for preparative production of chiral compounds 6-8 as the methodology developed (1) is very simple, efficient, and readily amenable to a scaleup; (2) is applicable to a wide variety of water-insoluble substances; (3) employs commercially available enzymes; (4) does not require expensive cofactors; and (5) is 45 D. T. Witiak, T. C.-L. Ho, R. E. Hakney, and W. E. Connor, J. Med. Chem. 11, 1086 (1968).

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137

carried out in the presence of only about 1% of water, which greatly simplifies product recovery. A natural extension of the biphasic system with a low water content would be a monophasic, purely organic medium. We have recently found 46 that at least one esterase, porcine pancreatic lipase, can vigorously function in nearly anhydrous organic solvents. Under such conditions the enzyme acquires remarkable new properties; for example, it becomes extremely thermostable and more specific. 46 We believe that the use of enzymes in aqueous-organic or organic media represents one of the most promising directions in enzyme technology. Addendum In addressing the basic question of how much water in the reaction medium enzymes actually require, we discovered that that amount in many cases can be close to z e r o . 47 This phenomenon has led to a new area of enzyme technology, biocatalysis in organic s o l v e n t s . 48'49 When placed in nonaqueous solvents instead of water, enzymes become extremely thermostable, 46 exhibit a new substrate specificity, 5° and can catalyze reactions impossible in aqueous solutions. 47 The last feature has been utilized by us to carry out novel efficient bioconversions including lipasecatalyzed regioselective acylation of glycols 5~ and sugars 52 and stereoselective conversions 53 in dry organic solvents, polyphenol oxidase-catalyzed regiospecific oxidations of phenols in organic media, 54 alcohol dehydrogenase-catalyzed asymmetric oxidoreductions in nonaqueous milieu, 55 and peroxidase-catalyzed depolymerization of lignin in organic media (but not in water)) 6 Biocatalysis in organic solvents indeed promises to have a profound impact on both basic and applied enzymology.

46 A Zaks and A. M. Klibanov, Science 224, 1249 (1984). 47 A. Zaks and A. M. Klibanov, Proc. Natl. Acad. Sci. U.S.A. 82, 3192 (1985). 48 A. M. Klibanov, CHEMTECH 16, 354 (1986). 49 M. Waks, Proteins 1, 4 (1986). 50 A. Zaks and A. M. Klibanov, J. Am. Chem. Soc. 108, 2767 (1986). 5t p. Cesti, A. Zaks, and A. M. Klibanov, Appl. Biochem. Biotechnol. 11, 401 (1985). 52 M. Therisod and A. M. Klibanov, J. Am. Chem. Soc. 108, 5638 (1986). 53 G. Kirchner, M. P. Scollar, and A. M. Klibanov, J. Am. Chem. Soc. 107, 7072 (1985). 54 R. Z. Kazandjian and A. M. Klibanov, J. Am. Chem. Soc. 107, 5448 (1985). 55 j. Grunwald, B. Wirz, M. P. Scollar, and A. M. Klibanov, J. Am. Chem. Soc. 108, 6732 (1986). 56 j. S. Dordick, M. A. Marietta, and A. M. Klibanov, Proc. Natl. Acad. Sci. U.S.A. 83, 6255 (1986).

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I M M O B I L I Z E D E N Z Y M E S / C E L L S IN O R G A N I C S Y N T H E S I S

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[13] T w o - L i q u i d P h a s e B i o c a t a l y t i c R e a c t o r s

By M. D. LILLY, S. HARBRON,and T. J. NARENDRANATHAN Two liquid phase biocatalytic reactors have been developed over the last I0 years to meet the growing need for biological catalysis of selective and stereospecific conversions of a range of water-immiscible and waterinsoluble organic compounds of interest to the chemical and biological industries. In these systems the organic phase consists of either the reactant alone or the reactant dissolved in a water-immiscible solvent. Advantages and disadvantages of two-liquid phase systems have been described elsewhere.l,2 One particular advantage of this approach is the possibility of operating the reactor with a large proportion of the volume occupied by organic phase which can be readily separated from the aqueous phase at the end of the reaction. This overcomes the usual need to work in dilute aqueous solutions with all the associated problems of product recovery. The development of biocatalytic reactors for these conversions involving a water-immiscible organic phase requires an understanding of both the kinetic properties of the biocatalyst and the diffusion and mass transfer characteristics of the reactants and products. A knowledge of the influence that the organic phase has on the biocatalyst and the factors likely to contribute to the long-term stability of the biocatalyst is also needed. Types of Reactions It is worth distinguishing in a simple manner between the possible configurations of two-liquid phase biocatalytic reactions so that a rational approach can be made to a better understanding of their kinetic behavior. Such a scheme is given in Table I in which the reaction types are classified according to four criteria. It is important to distinguish first between those reactions which take place in the aqueous phase and those which occur at the aqueous-organic interface. Representative examples of systems where the reaction site is in the aqueous phase or at the aqueous-organic interface are given in Table II and Table III, respectively. The second major distinction depends on how much aqueous component is added: if a discrete aqueous phase is present then it may be either the continuous (type 1) or the discontinuous phase (type 2). For this i M . D . L i l l y , J. Chem. Technol. Biotechnol. 32, 162 (1982). 2 M . D . L i l l y , Philos. Trans. R. Soc. London, Ser. B 300, 391 (1983).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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139

TABLE I CLASSIFICATIONOF Two-LIQUID PHASE BIOCATALYTICREACTIONS Criterion Reaction site Aqueous phase

Biocatalyst Organic phase

Possible configuration Aqueous phase Aqueous-organic interface Discrete aqueous continuous phase (type 1) Discrete aqueous discontinuous phase (type 2) No discrete aqueous phase (type 3) Free Immobilized No added solvent Added solvent

purpose "discrete" indicates the presence of a definite aqueous phase external to the biocatalyst. This is illustrated in Fig. la, which relates to reactor types 1 and 2, and shows the substrate concentration profile arising from mass transfer across the aqueous-organic interface to the surface of the biocatalyst (immobilized enzyme, free or immobilized cell). For reactions involving enzymes dissolved in the aqueous phase, the corresponding concentration profiles are shown in Fig. lb. For simplicity TABLE II TYPES OF Two-LIQUID PHASE BIOCATALYTICREACTIONS:AQUEOUSPHASE REACTION Catalyst Type 1. Discrete aqueous continuous phase Free biocatalyst No added solvent Bacillus subtilis Nocardia corallina Pseudomonas oleovorans Pseudomonas putida Various microorganisms Added solvent Steroid 20/3-dehydrogenase

Nocardia rhodochrous Pseudomonas oleovorans

Reaction

Substrate (phase ratio)

Ester hydrolysis Epoxidation Epoxidation Epoxidation Ester hydrolysis

Menthyl acetate (0.05-1.0) a Tetradecene (0.05) b Octene (0.02-1.0F 1,7-Octadiene (0.02-1.00) a Menthyl acetate (0.04) e

20/3-Reduction

Prednisolone (1.0Y Cortisol (1.0) f Cortisone (1.0)Y Deoxycorticosterone (1.0y Prednisone (1.0) r Cholesterol (0.8)g 1,7-Octadiene (0.02-1.0) h

3r-Dehydrogenation Epoxidation

(continued)

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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TABLE II (continued) Catalyst Immobilized biocatalyst No added solvent Pseudomonas putida Added solvent Steroid 3a-dehydrogenase Steroid fl-dehydrogenase Steroid 20fl-dehydrogenase Type 2. Discrete aqueous discontinuous phase Free biocatalyst No added solvent Bacillus subtilis Pseudomonas oleovorans Added solvent Chymotrypsin Nocardia erythropolis Nocardia rhodochrous Pseudomonas oleovorans Rhodotorula minuta Immobilized biocatalyst Added solvent Chymotrypsin Nocardia erythropolis Type 3. No discrete aqueous phase Free biocatalyst No added solvent No examples Added solvent Nocardia rhodochrous

Nocardia rhodochrous Nocardia rhodochrous Rhodotorula minuta Immobilized biocatalyst No added solvent Bacillus subtilis

Reaction

Substrate (phase ratio)

Epoxidation

1,7-Octadiene (0.02-1.0) h

3a-Dehydrogenation fl-Dehydrogenation 20/3-Reduction

Androsterone (0.5) ~ Testosterone (0.5) ~ Cortisone (0.5) /

Ester hydrolysis Epoxidation

Menthyl acetate (1.0-2.3) ° Octene (1.0-4.0y

Ester formation

N-Acetyltryptophan and leucineamide (5-50) / Cholesterol (1.5) k Cholesterol (1.1-2.9F 1,7-Octadiene (1.0-1.63) h Menthyl succinate (3.33) I

3/3-Dehydrogenation 3/3-Dehydrogenation Epoxidation Ester hydrolysis

Ester formation 3fl-Dehydrogenation

N-benzoylphenylalanine and ethanol (15)" Cholesterol (0.53-0.67) k

Ester hydrolysis

Cholesterol (3.7-10F," Dehydroepiandrosterone (10).,o ¢i-Sitosterol (10)" Stigmasterol (10)" Pregnenalone (10)° Testosterone (10)P Androstenedione (15-20)q Testosterone (10)P Menthyl succinate (10)~

Ester hydrolysis

Menthyl acetate (7)~

3/3-Dehydrogenation

17/3-Dehydrogenation A~-Dehydrogenation

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TWO-LIQUID PHASE BIOCATALYTIC REACTORS

141

TABLE II (continued) Catalyst Added solvent Chymotrypsin

Reaction

Ester hydrolysis Ester formation

Lipoxygenase Nocardia erythropolis Nocardia rhodochrous

Fat oxidation 3/3-Dehydrogenation 3/3-Dehydrogenation

Nocardia rhodochrous Nocardia rhodochrous

AI-Dehydrogenation

Rhodotorula minuta

Ester hydrolysis

17/3-Dehydrogenation

Substrate (phase ratio)

N-Acetyltyrosine ethyl ester (100) r N-Acetyltryptophan and ethanol (100)r Linoleic acid (4)s Cholesterol (0.78-1.13) k Cholesterol (2.5-10) n,° Dehydroepiandrosterone (2.5-10)~, o Pregnenalone (2.5-10) ° fl-Sitosterol (2.5-10y Stigmasterol (2.5-10) n Testosterone (2.5-10)P Androstenedione (15-20)q Testosterone (2.5-10)p Menthyl succinate (2-2.5) t

"I. K. Brookes, Ph.D. Thesis, University of London, 1984. b K. Furuhashi, A. Taoka, S. Uchida, I. Karube, and S. Suzuki, Eur. J. Appl. Microbiol. Biotechnol. 12, 39 (1981). ¢ M. J. de Smet, H. Wynberg, and B. Witholt, Appl. Environ. Microbiol. 42, 811 (1981). d S. Harbron, T. J. Narendranathan, and M. D. Lilly, Proc. 3rd Eur. Congr. Biotechnol. 4, 369 (1985). e y. Yamaguchi, T. Oritami, N. Tajima, A. Komatsu, and T. Moroe, Nippon Nogei Kagaku Kaishi 50, 475 (1976). s p. Cremonesi, G, Carrea, F. L. Ferrara, and E. Antonini, Biotechnol. Bioeng, 17, 1101 (1975). g B. C. Buckland, P. Dunnill, and M. D. Lilly, Biotechnol. Bioeng. 17, 815 (1975). h R. D. Schwartz and C. J. McCoy, Appl. Environ. Microbiol. 34, 47 (1977). i G. Carrea, F. Colombi, G. Mazzola, P. Cremonesi, and E. Antonini, Biotechnol. Bioeng. 21, 39 (1979). J A. N. Semenov, I. V. Berezin, and K. Martinek, Biotechnol. Bioeng. 23, 355 (1981). k p. Atrat, E. Huller, and C. Horhold, Z. Allg. Mikrobiol. 20, 7 (1980). l T. Omata, N. Iwamoto, T. Kimura, A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. U , 199 (1981). " K. Martinek, A. N. Semenov, and I. V. Berezin, Biotechnol. Bioeng. 23, 1115 (1981). n T. Omata, T. Iida, A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol, Biotechnol. 8, 143 (1979). o T. Omata, A. Tanaka, and S. Fukui, J. Ferment. Technol. 58, 339 (1980). P S. Fukui, S. A. Ahmed, T. Omata, and A. Tanaka, Eur. J. Appl. Microbiol. Biotechnol. 10, 289 (1980). q T. Yamane, H. Nakatani, E. Sada, T. Omata, A. Tanaka, and S. Fukui, Biotechnol. Bioeng. 21, 2133 (1979). • A. M. Klibanov, G. P. Samokhin, K. Martinek, and I. V. Berezin, Biotechnol. Bioeng. 19, 1351 (1977). s T. Yamane, Enzyme Eng. 6, 141 (1982).

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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T A B L ~ III TYPES OF Two-LIQUID PHASE BIOCATALYTIC REACTIONS: REACTION AT INTERFACE

Catalyst

Reaction

Substrate (phase ratio)

Free biocatalyst

Rhizopus arrhizus Immobilized biocatalyst Lipase

Triglyceride hydrolysis

Olive oil (50) a

Interesterification

Olive oil and stearic

Interesterification

Myristic acid and palm midfraction (35-85) c

acid (22.5) b

Lipase

G. Bell, J. R. Todd, J. A. Blain, J. D. E. Patterson, and C. E. Shaw, Biotechnol. Bioeng. 23, 1703 (1981). b K. Yokezeki, S. Yamanaki, K. Takinami, Y. Hirose, A. Tanaka, K. Sonomoto, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 14, 1 (1982). c R. Wisdom, P. Dunnill, M. D. Lilly, and A. Macrae, Enzyme Microbiol. Technol. 7, 567 (1985). a

it has been assumed that the aqueous phase is well mixed (i.e., C 4 = C [ ) . With microbial cells, as the biocatalyst concentration is increased, mixing in the aqueous phase is reduced, and eventually a cell paste is obtained with no discrete aqueous phase outside the biocatalyst as shown in Fig. lc (reactor type 3). A similar situation can occur when immobilized cells or enzymes are used, for instance, in a packed bed. The substrate concentra(a) BIOCATALYST

AOUEOUS PHASE

OROANICPHASE

:film lfitm ',

I i C~

( b} BIOCATALYST

ORGANIC PHASE

(d,~ved in a~leous phase)

(c) BIOCATALYST

ORGANIC PHASE

:f+tmIf+t~_

FIG. 1. Concentration profiles of reactant at steady state in a two-liquid phase systems. Mass transfer from the organic phase to the biocatalyst. (a) Discrete aqueous phase, separate biocatalyst; (b) discrete aqueous phase, biocatalyst dissolved in aqueous phase; (c) increased biocatalyst concentration such that there is no aqueous phase.

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143

TWO-LIQUID PHASE BIOCATALYTIC REACTORS

tion profile within the biocatalyst (Fig. la, b, c) will depend on the nature, form, and intrinsic activity of the biocatalyst. For an immobilized biocatalyst reactor with a discrete aqueous phase, it has been assumed in Tables II and III that the immobilized particles remain associated with the discrete aqueous phase, and reactor types 1 and 2 are possible. However, if the particles are found predominantly in the organic phase, then a type 3 configuration will result. Further subdivisions in Table I relate to whether the biocatalyst is free or immobilized and whether the organic phase contains added solvent. In Tables II and III the phase ratio is defined as the volume of organic phase per volume of aqueous phase including biocatalyst. In our own experiments with either octadiene or menthyl acetate in the absence of biocatalyst, phase inversion occurred between 1.2 and 1.6. 3 In many of the twoliquid phase systems described in the literature, no information is given on the phase ratio at which inversion occurs. We have therefore assumed that at phase ratios below one, the aqueous phase is continuous and at phase ratios above unity the organic phase is continuous. Kinetic Considerations Once a system has been classified in this manner it is possible to develop equations to describe its performance. For a reaction catalyzed by cells or immobilized biocatalyst with discrete aqueous phase (Fig. la), the following relationships 4 for steady-state conditions can be obtained: ds/dt = v, the aqueous phase volumetric reaction rate = k2A2(C1

--

---- k 3 A 3 ( C 3

--

C2) C4)

(1) (2) (3)

(4)

= ksAs(C~ - C5) C3 = K32C2 C5 -- K56C6

(5) (6)

where k, A, C, and K are mass transfer coefficients (liter • T-l), areas (liter 2 • liter-3), substrate concentrations (mol • liter-3), and partition coefficients, respectively. Equations (1) to (6) give C6 \K32 ~

= 1

K32C, \k2Az + ~

+~

3 T. J. Narendranathan, S. Harbron, and M. D. Lilly, unpublished results. 4 S. Harbron, T. J. Narendranathan, and M. D. Lilly, P r o c . 3 r d Eur. C o n g r . 369 (1985).

(7)

B i o t e c h n o l . 4,

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[13]

3= zoo-

100

0

0.0

a0z

oh6 o.b8 o.io

o.i2

0i4

g DCW/ml aqueousphaseor g dry foam

FIG. 2. The conversion of 1,7-octadiene to 7,8-epoxy-l-octene by free (filled symbols) and entrapped (open symbols) Pseudomonas putida in small tubes agitated on a reciprocating arm shaker at 1300 strokes per minute. Phase ratios: (©) 0.11; (A) 0.43; ([~) 1.00.

The right-hand side of Eq. (7) is a measure of the effectiveness of the overall mass transfer, and ranges between 0 (high mass transfer resistance) and 1 (low mass transfer resistance). The relationship between v and C6 will depend on the nature and form of the biocatalyst, and if the biocatalyst is immobilized, will need to take into account the diffusion of the substrate into and product out of the support. From a knowledge of the kinetic parameters involved it is therefore possible to predict the behavior of the reactor. An illustration of the influence of phase ratio and biocatalyst concentration on the performance of two-liquid phase reactors is shown in Fig. 2. 5 As the concentration of free cells in the aqueous phase is increased, the system becomes progressively limited by the rate of transfer of the substrate, resulting in the observed fall in specific activity. The effect is less pronounced at higher phase ratios, since an increase in the phase ratio toward the point of inversion gives an increased interracial area per unit volume of aqueous phase, 3 and the mass transfer limitation is thereby reduced. The ability to reduce mass transfer limitations in this way is offset by the increased volume of the organic phase. When this is taken into account the overall volumetric rate for the reactor is reduced at higher phase ratios. With immobilized biocatalyst, diffusion into the catalyst may be the rate-limiting step, in which case changes in phase ratio will have little or no effect. This diffusion limitation will become more pronounced as the s S. Harbron and M. D. Lilly, unpublished results.

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145

cell loading in the biocatalyst is increased, resulting in an observed fall in specific activity. Such a situation with immobilized Pseudomonas putida is illustrated in Fig. 2. Agitation is the other major factor likely to influence the interfacial area. For example, in the conversion of cholesterol to cholestenone by Nocardia rhodochrous 6 in a stirred tank reactor, an increase in agitation from 9 to 15 rev/sec gave a corresponding increase in reaction rate. This increase was attributed to an increased interfacial area between the organic phase and the aqueous cell suspension. For menthyl ester hydrolysis by Bacillus subtilis the specific reaction rate in a 50-ml working volume reactor was independent of agitation above 13 rev/sec. Under these conditions the specific reaction rate decreased above and below an organic phase ratio of 0.3 to 0.35. At a lower speed of 6.5 rev/sec, the optimum phase ratio shifted to about 1.0. 7 Effect of Organic Solvent

The organic solvent can have two general effects on two-liquid phase biocatalytic reactions. First, it can affect the partitioning of reactants and products between the aqueous and organic phase. This can be used to advantage if the product has an inhibitory effect on the biocatalyst, as for example with the epoxidation activity ofPseudomonas oleovorans 8 which is inhibited by the accumulation of epoxide product in the aqueous phase. Addition of relatively large volumes of cyclohexane resulted in the favorable partitioning of the epoxide into the organic phase and over 90% conversion of the reactant, octadiene, was achieved as a result. In another case N. rhodochrous entrapped in a hydrophilic resin had activity that was closely related to the partition coefficient of the reactant cholesterol, between the resin and different organic solvents used. 9 A similar relationship was obtained when the same solvent was used, but cells were entrapped in a range of different resins. 9,1° Second, the solvent may have a deleterious effect on the structure of the enzyme and the integrity of the cell. Several workers have observed large differences in catalytic activities with different solvents. Various organic extraction reagents have been examined for their toxic and inhibi-

6 B. C. Buckland, P. Dunnill, and M. D. Lilly, Biotechnol. Bioeng. 17, 815 (1975). 7 I. K. Brookes, Ph.D. Thesis, University of London, England, 1984. 8 R. D. Schwartz and C. J. McCoy, Appl. Environ. Microbiol. 34, 47 (1977). 9 T. Omata, A. Tanaka, and S. Fukui, J. Ferment. Technol. 58, 339 (1980). i0 T. Omata, T. Iida, A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 8, 143 (1979).

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tory properties on anaerobic bacteria. 11 At concentrations above those required to saturate the aqueous phase, alcohols (C5-C12), ketones (C5C8), benzene derivatives, isoamyl acetate, and diisopropyl ether were toxic, whereas alkanes (C6-Clz) and diisoamyl ether were not. For the conversion of cholesterol to cholestenone by N. rhodochrous, properties of the solvent which appear to minimize any damage have been reported to be low polarity, 6 low solubility in water, lz and a low value for the dielectric constant. 9 Similar observations have been made with 20fl-hydroxysteroid dehydrogenase. 13 Immobilization of the cell or enzyme can reduce the magnitude of any deleterious effects of organic solvents, fl-Hydroxysteroid dehydrogenase immobilized on Sepharose gave a preparation that retained about 60% of its activity after 2 months of continuous use in the presence of ethyl acetate, whereas the free cell preparation under similar conditions lost 80-90% of its activity after 4-5 days.14 Long-term stability of Rhodotorula minuta, which catalyzes the conversion of menthyl succinate to menthol, could similarly be achieved by immobilization. 15Free cells had a half-life of about 50 hr whereas some immobilized cell preparations had a half-life of 1520 hr. Immobilization of N. rhodochrous in polyacrylamide was found to protect the cholesterol degradation pathway from the organic solvent in which the cholesterol was dissolved. 2 Choice of Reactors For a reaction which is not limited by diffusion or mass transfer, the degree of conversion achieved in a packed bed reactor will be higher than in a continuous stirred tank reactor, except at very high substrate concentrations and low conversion rates. If the reaction is diffusion limited then a packed bed reactor becomes kinetically even more favorable than the well-mixed continuous reactor. The kinetic behavior of a batch reactor is similar to that of a packed bed reactor but the overall productivity is less because of reactor downtime. For the above and other reasons 16 packed beds of immobilized enzymes and cells have been widely used for reactions involving water-soluble reactants and products. H M. J. Playne and B. R. Smith, Biotechnol. Bioeng. 25, 1251 (1983). 12j. M. C. Duarte and M. D. Lilly, Enzyme Eng. 5, 363 (1980). ~3 p. Cremonesi, G. Carrea, L. Ferrara, and E. Antonini, Biotechnol. Bioeng. 27, 1101 (1975). 14 G. Carrea, F. Colombi, G. Mazzola, P. Cremonesi, and E. Antonini, Biotechnol. Bioeng. l l , 39 (1979). 15 T. Omata, N. Iwamoto, T. Kimura, A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 11, 199 (1981).

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For two-liquid phase reactions it is essential to create sufficient interfacial area to allow adequate rates of transfer of the reactant and product between the phases. Much of the published experimental work has been done in shaken tubes and there are only a few reports on the use of reactors, either stirred tank or packed bed. Lipase-catalyzed interesterification reactions have been carried out in both batch stirred tank reactors and continuous packed bed reactors.17 Since dried catalyst particles have virtually no activity, up to 10% (v/v) water was added to hydrate the catalyst particles even though this resulted in some hydrolysis of the substrate. These catalyst particles were used for 600 hr in the packed bed and for 10 successive batches each of about 16 hr duration in the stirred tank reactor without appreciable loss of activity. A packed bed reactor gave better yields than a stirred tank reactor for the continuous hydrolysis of triglyceride in organic solvent systems using immobilized lipase from Rhizopus arrhizus. 18 The conversion of cholesterol to cholestenone by Nocardia 6 was studied in a 500-ml stirred tank reactor and the reaction rate found to be independent of agitation above a stirrer speed of 15 rev/sec. The cells retained 52% of their activity after seven batches lasting a total of 69 hr. A large stirred tank is also being used in the United States for a two-liquid phase steroid conversion. Experimental Methods Immobilization Techniques Only a limited number of immobilization techniques have been used so far in two-liquid phase systems. Although in some cases support materials already used for completely aqueous reaction systems have been used (porous glass, 19Sepharose,14 polyacrylamide, and alginate12), several new techniques have been developed which are particularly suitable for use in two-liquid phase systems. Several examples of methods are given below. Using hydrophobic photo-cross-linkable resin prepolymers. 2° One gram thawed cells suspended in a 2 ml of water-saturated solvent under ~6M. D. Lilly and P. Dunnill, this series, Vol. 44, p. 717. 17 A. R. Macrae, J. Am. Oil Chem. Soc. 60, 243A (1983). 18 G. Bell, J. R. Todd, J. A. Blain, J. D. E. Patterson, and C. E. Shaw, Biotechnol. Bioeng. 23, 1703 (1981). 19 A. M. Klibanov, G. P. Samokhin, K. Martinek, and I. V. Berezin, Biotechnol. Bioeng. 19, 1351 (1977). 2o S. Fukui, S. A. Ahmed, T. Omata, and A. Tanaka, Eur. J. Appl. Microbiol. Biotechnol. 10, 289 (1980).

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mild sonication is mixed with 1 ml of same solvent containing 1 g hydrophobic photo-cross-linkable resin polymer (ENTP-2000, 2~ ENTB-1000, or mixtures of ENTP-2000 and ENT-4000) and 10 mg photosensitizer (benzoin ethyl ether). The mixture is placed on a sheet of transparent polyester and illuminated with near-UV light for 3 min. The resin gel formed is cut into 1.5-mm-square pieces for use. Using hydrophilic photo-cross-linkable resin prepolymers. 2° The procedure for the immobilization of hydrophilic photo-cross-linkable resin prepolymers (ENT 4000 or mixtures of ENT-4000 and ENTP-2000) is the same as above, except potassium phosphate buffer (pH 7.0, 20 mM) is substituted for the organic solvent. Using urethane prepolymers. 4,z° One gram thawed cells is suspended under mild sonication in 2 ml of potassium phosphate buffer (pH 7.0, 20 mM) and mixed with previously melted 1 g urethane prepolymer (PU3, PU6, or mixtures of PU3 and PU6) at 40 °. The gel formed is cut into 3-mm cubes for use. Polyurethane beads 4 are prepared by pouring the above mixture into 50 ml liquid paraffin being agitated at 1250 rpm. After 20 min the liquid paraffin is decanted, and the beads formed are washed with buffer, dried between filter paper, and stored on ice till used. Immobilization on Celite. 22 Two parts of the lipase from Rhizopus delemar (7200 U/g) are dissolved in 20 parts of distilled water and 5 parts Celite added with stirring at 0°. Then 30 parts acetone are added over 5 min and stirring is continued for 30 min more. The solid product formed is filtered off and dried at 20° under reduced pressure. Reactor Systems Stirred tank reactors. 6 A glass stirred tank (0.5 liter working volume, 75-mm diameter) with four stainless-steel baffles (7 mm width) is used for the oxidation of cholesterol to cholestenone by N. rhodochorous. Agitation is carried out with a four-blade fiat plate turbine impeller (22 mm diameter) located 22 mm above the reactor bottom and driven by a variable speed (0-6000 rpm) motor. Either air is supplied by a small pump, or oxygen is fed directly from a pressurized cylinder to the gas inlet which is a capillary glass tube ending directly beneath the impeller. A reflux condenser is fitted to the air outlet of the reactor. 2t Abbreviations: ENTP-2000, a derivative of polypropylene glycol 2000; ENTB-1000, a derivative of polybutadiene; ENT-4000, a derivative of polyethylene glycol 4000; PU3, a polyester diol of urethane containing 57% polyethylene glycol; and PU6, a polyester diol of urethane containing 91% polyethylene glycol. 22 M. H. Coleman and A. R. Macrae, United Kingdom Patent 1,577,933 (1980).

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One hundred grams of thawed Nocardia cells suspended in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol (at 20°C, oxygen flow rate 100 ml/min and stirrer speed 2200 rpm) converted all the cholesterol to cholestenone in 5.5 hr. The reaction rate was constant at 35 g/liter-hr for the first 4 hr. Shaken f l a s k reactor. 2° One gram of thawed Nocardia cells were washed with water-saturated solvent and mixed with a given amount of crystalline electron acceptor (if required) by mild sonication. This mixture is then added to a solution of steroid (e.g., testosterone) in 10 ml solvent in a 30-ml glass-stoppered conical flask and incubated in a thermostated bath at 30° with shaking at 120 strokes/min. A reaction mixture containing 1 g thawed cells and 173/zmol of testosterone and 16/zmol of phenazine methosulfate (electron acceptor) in 10 ml water-saturated benzene-n-heptane solvent (4 : 1 v/v) mixture at 30° converted 167/zmol of testosterone in 2 hr. P a c k e d bed reactor. 22 A mixture of palm midfraction (1 part) and myristic acid (0.4 parts) dissolved in 100-120C petroleum ether (3.2 parts) is saturated with water and then continuously pumped at a flow rate of 22 ml/hr through a bed of hydrated catalyst (5.0 g) prepared from Rhizopus niveus lipase and kieselguhr. The reactor temperature is 40 °. Almost complete (greater than 95%) interesterification was obtained during a 400-hr reactor operation. Future Prospects The examples listed in Tables II and III indicate the increasing interest in conversions involving water-insoluble or water-immiscible reactants and products. Because of their complex nature much more work needs to be done before our understanding of the kinetic behavior of these reactions reaches the same level as that for reactions involving water-soluble reactants and products. Nevertheless the operational stabilities that are being reported for some systems are encouraging, and it seems likely that several commercial processes will be introduced in the next few years.

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IN ORGANIC SYNTHESIS

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[14] E n z y m e - C a t a l y z e d S t e r o i d T r a n s f o r m a t i o n s in Water-Organic Solvent Two-Phase Systems

By

GIACOMO CARREA a n d PIERO CREMONESI

The potential of enzyme-catalyzed reactions in organic chemistry is now remarkable. This is due partly to the increased number of available enzymes, to their use in the immobilized form, and to the existence of efficient systems of coenzyme regeneration. However, substrates poorly soluble in water such as steroids present special difficulties in this area since the enzyme-catalyzed transformations must be carried out using large reaction volumes and consequently large amounts of biological catalysts and cofactors as required. Also, the use of large reaction volumes makes the recovery of products more difficult. Attempts to increase steroid solubility by adding water-miscible organic solvents to the reaction medium have given unsatisfactory results since high solvent concentrations progressively give rise to inhibition, decreased specificity, and denaturation of the enzymes.l In contrast, when organic solvents which are practically immiscible or poorly miscible with water are used, the situation becomes substantially improved. In this case, a two-phase system consisting of water and an organic solvent is established. The water phase contains enzymes and hydrophilic cofactors, and the organic phase high concentrations of hydrophobic substrates. On shaking, substrates diffuse into the aqueous phase where they undergo the enzyme-catalyzed transformation and then the formed products return to the organic phase. In this system, the concentration of solvents in water is low and not dependent on the ratio of the two phases, even if the partial volume of the organic phase is much greater than that of the aqueous phase, and the inhibitory and denaturing effects are much lower than those induced by comparable concentrations of miscible solvents. 2-8 L. G. Butler, Enzyme Microb. Technol. 1, 253 (1979). 2 p. Cremonesi, G. Carrea, G. Sportoletti, and E. Antonini, Arch. Biochem. Biophys. 159, 7 (1973). 3 G. Lugaro, G. Carrea, P. Cremonesi, M. M. Casellato, and E. Antonini, Arch. Biochem. Biophys. 159, I (1973). 4 p. Cremonesi, G. Carrea, L. Ferrara, and E. Antonini, Eur. J. Biochem. 44, 401 (1974). s G. Carrea, P. Cremonesi, M. M. Casellato, and E. Antonini, Steroids Lipids Res. 5, 162 (1974). 6 p. Cremonesi, G. Carrea, L. Ferrara, and E. Antonini, Biotechnol. Bioeng. 17, 1101 (1975).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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In the present article, the specific oxidation-reduction of hydroxylketo groups of steroids, catalyzed by NAD(P)÷-dependent hydroxysteroid dehydrogenases, is described and discussed. The reactions are carried out in two-phase systems and are coupled to the enzymatic regeneration of the coenzymes. Materials

20fl-Hydroxysteroid dehydrogenase (17,20/3,21-trihydroxysteroid: NAD ÷ oxidoreductase, EC 1.1.1.53) (specific activity, l0 U/mg protein), yeast alcohol dehydrogenase (alcohol:NAD ÷ oxidoreductase, EC 1.1.1.1) (specific activity, 200 U/mg protein), formate dehydrogenase (formate : NAD ÷ oxidoreductase, EC 1.2.1.2) (specific activity, 3 U/mg protein), glutamate dehydrogenase (L-glutamate : NAD(P) ÷ oxidoreductase, EC 1.4.1.3) (specific activity, 90 U/mg protein), L-lactate dehydrogenase (L-lactate : NAD ÷ oxidoreductase, EC 1.1.1.27) (specific activity, 220 U/ mg protein), NAD ÷, NADP ÷, NADH, and NADPH are obtained from Boehringer-Mannheim. 3 (or 17)fl-Hydroxysteroid dehydrogenase (fl-hydroxysteroid : NAD(P) ÷ oxidoreductase, EC 1.1.1.51) (specific activity, 18 U/mg protein) and steroids are purchased from Sigma. 12a-Hydroxysteroid dehydrogenase (specific activity 2 U/mg protein) is extracted from Clostridium group P as described by Macdonald et al. 9 Sepharose 4B and Sepharose CL-4B are obtained from Pharmacia and CNBr and tresyl chloride from Fluka. All other reagents and compounds are of analytical grade. Assays

Enzymatic activity is measured in a 3-ml cuvette by spectrophotometric monitoring at 340 nm of the formation or consumption of NAD(P)H. The conditions for the various assays are as follows: 20fl-hydroxysteroid dehydrogenase in 0.05 M potassium phosphate buffer, pH 7, containing 0.17 mM NADH and 0.18 mM cortisone; fl-hydroxysteroid dehydrogenase in 0.1 M sodium phosphate buffer, pH 8.5, containing 0.3 mM NAD ÷ and 0.05 mM testosterone; 12a-hydroxysteroid dehydrogenase in 0.1 M potassium phosphate buffer, pH 8, containing 0.3 mM NADP ÷ and 1 mM cholic acid; alcohol dehydrogenase in 0.1 M sodium phosphate 7 G. Carrea, S. Colombi, G. Mazzola, P. Cremonesi, and E. Antonini, Biotechnol. Bioeng. 21, 39 (1979). 8 E. Antonini, G. Carrea, and P. Cremonesi, Enzyme Microb. Technol. 3, 291 (1981). 9 j. A. Macdonald, J. F. Jellet, and D. E. Mahony, J. Lipid Res. 20, 234 (1979).

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buffer, pH 9, containing 4 mM NAD + and 0.3 M ethanol; formate dehydrogenase in 0. I M potassium phosphate buffer, pH 7, containing 0.5 mM NAD + and 50 mM formate; glutamate dehydrogenase in 0.1 M potassium phosphate buffer, pH 8, containing 0.1 M ammonium acetate, 20 mM o~ketoglutarate, 1 mM ADP, and 0.15 mM NADPH; lactate dehydrogenase in 0.1 M sodium phosphate buffer, pH 8.5, containing 1 mM pyruvate and 0.15 mM NADH. The activity of immobilized enzymes is likewise determined, and during activity measurements the gel is suspended in the test solution by continuously stirring the mixture with a magnetic cylinder (9 mm diameter and 3 mm height) placed in the cuvette.

Enzyme Immobilization 20fl-Hydroxysteroid dehydrogenase and alcohol dehydrogenase are immobilized separately onto CNBr-activated Sepharose 4B, whereas 12ahydroxysteroid dehydrogenase and glutamate dehydrogenase are coimmobilized onto Sepharose CL-4B activated with tresyl chloride. Sepharose 4B is activated at pH 10.5 using 65 mg CNBr/ml of settled gel, following the method described by Axen et al. lo The coupling of 20/3hydroxysteroid dehydrogenase (34 U, previously dialyzed at 4° against 0.1 M potassium phosphate buffer, pH 8) or alcohol dehydrogenase (640 U) to CNBr-activated Sepharose 4B (10 ml) is carried out in 0.1 M potassium phosphate buffer, pH 8, containing 1 mM NAD ÷, under gentle stirring of the coupling mixture (20 ml) at 4° overnight. The unreacted groups on the matrix are blocked by treating the gel with 0.1 M ethanolamine, pH 8, for 3 hr. Then, the supernatants are withdrawn and the immobilized enzymes thoroughly washed with 0. I M potassium phosphate buffer, pH 7, before testing the enzymatic activity. Sepharose CL-4B is activated with tresyl chloride following the method described by Nilsson and Mosbach. 11 The matrix (10 ml of settled gel) is washed stepwise on a glass-filter funnel with 150 ml of each of the following solutions: distilled water, 30 : 70, 60 : 40, 80 : 20 of acetone:water (v/v), acetone, and finally acetone dried over a molecular sieve. The gel is then added to 3 ml of dry acetone and 0.5 ml of anhydrous pyridine and reacted with 0.25 ml of tresyl chloride under vigorous stirring. After activation for 10 rain the gel is washed with 150 ml of each of the following solutions: acetone, 80 : 20, 60 : 40, 30 : 70, 15 : 85 of acetone : 1 mM HCI (v/v), and finally 1 mM HCI. The coupling of 12ct-hydroxysteroid dehydrogenase (32 U) and glutamate dehydrogenase (115 U) to R. Axen, J. Porath, and S. Ernback, Nature (London) 214, 1302 (1967). i1 K. Nilsson and K. Mosbach, Biochern. Biophys. Res. Commun. 102, 449 (1981).

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TABLE I RECOVERY OF ACTIVITY OF IMMOBILIZED ENZYMES

Added activity a

Immobilized activity

Activity in the supernatant

Enzyme

(U)

U

%

U

%

20fl-Hydroxysteroid d e h y d r o g e n a s e

34 640 32 l 15

20 300 11 56

59 47 34 49

Undetectable 5 3 4

0 l l0 3

Alcohol dehydrogenase 12a-Hydroxysteroid d e h y d r o g e n a s e

Glutamate dehydrogenase

a The enzymes were coupled to 10 ml of settled, activated gel.

to the activated matrix (10 ml) is carried out in 0.1 M potassium phosphate buffer, pH 8, under gentle stirring of the coupling mixture (18 ml) at 4 ° overnight. The sample is then processed as described in the case of Sepharose 4B-immobilized enzymes. The recovery of activity of the immobilized enzymes is shown in Table I. Activities of the immobilized enzymes were found to be 34-59% of those of the added soluble enzymes, depending on the enzyme preparations used (Table I); the enzyme activity left in the supernatant was, in all cases, a small fraction of the added one. The Michaelis constants of the immobilized enzymes were generally higher than those of the free enzymes (Table II), except for immobilized glutamate dehydrogenase whose Km.appvalue for tx-ketoglutarate was significantly lower than that of the free enzyme. T A B L E II MICHAELIS CONSTANTS OF FREE AND IMMOBILIZED ENZYMES

Enzyme 20fl-Hydroxysteroid dehydrogenase

Substrate or cofactor NADH

Cortisone Alcohol dehydrogenase

NAD +

12a-Hydroxysteroid dehydrogenase

NADP +

Ethanol Cholic acid methyl ester Glutamate dehydrogenase

NADPH

tx-Ketoglutarate

Free enzyme Km

Immobilized enzyme Km,app

(/zM)

(/~M)

7 42 74 12,500 15 110

26 98 360 16,000 45 130

35 680

230 240

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Steroid Transformations

Reduction of 20-Ketosteroids to 20fl-Hydroxysteroids The stereospecific reduction of progesterone and cortisone is described next. The reduction of progesterone is based on the following reactions: Progesterone (pregn-4-ene-3,20-dione) + N A D H + H + pregn-4-ene-20fl-hydroxy-3-one + N A D ÷ H C O O H + N A D + ~ CO2 + N A D H + H ÷

(1) (2)

where reaction (1) is catalyzed by 20fl-hydroxysteroid dehydrogenase and reaction (2), which serves to regenerate NADH, by formate dehydrogenase. The reaction system is made up as follows. Aqueous phase: 15 ml of 0.1 M potassium phosphate buffer, pH 7, containing 0.2 M formate, 1 mM dithiothreitol, 15 mg of serum albumin, 3 /zmol of NAD ÷, 21 U of 20flhydroxysteroid dehydrogenase, and 24 U of formate dehydrogenase. Organic phase: 15 ml of butyl acetate containing 1.2 mmol (375 mg) of progesterone. The reaction is initiated by shaking the system, contained in a bottle (50 ml) placed on a mechanical oscillating device (oscillating angle approx. 40°, 100 oscillations per minute). The reaction is followed by thin-layer chromatography (of the organic phase) on silica gel GF254 plates (Merck) in the system benzene-methanol (9 : 1, v/v). The complete reduction of progesterone to the 20fl-hydroxyderivative was obtained in 30 hr. The completeness of transformation was also due to the high value (about 10iS) of the overall equilibrium constant for the coupled reactions. The turnover number (moles of product generated per mole of coenzyme added) for the coenzyme was 400. At the end of the run, the residual activity of 20fl-hydroxysteroid dehydrogenase and formate dehydrogenase was 60 and 65%, respectively. The reduction of cortisone is based on the following reactions: Cortisone (pregn-4-ene-17a,21-dihydroxy-3,11,20-trione) + N A D H + H ÷ ~ pregn-4-ene-17a,20fl,21-trihydroxy-3,11-dione + N A D ÷ CH3CH2OH + N A D + ~---CH3CHO + N A D H + H ÷ O CH3CHO + N H 2 - - N H - - C O - - N H 2

II

~ CH3CH=N--NH--C--NH2

(3) (4)

+ H20

(5)

where reaction (3) is catalyzed by 20fl-hydroxysteroid dehydrogenase and reaction (4) by alcohol dehydrogenase. Semicarbazide, reacting with acetaldehyde, shifts the equilibrium to the right and allows for the complete conversion of cortisone.

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155

The reaction system is made up as follows. 7 Aqueous phase: 50 ml of 0.05 M potassium phosphate buffer, pH 7, containing 1.5 ml of ethanol, 0.8 mmol of semicarbazide, 0.04 mmol NAD ÷, 12 U of Sepharose 4Bimmobilized 20/3-hydroxysteroid dehydrogenase (6 ml), and 150 U of Sepharose 4B-immobilized alcohol dehydrogenase (5 ml). Organic phase: 100 ml of ethyl acetate containing 0.72 mmol (260 mg) of cortisone. After 3 hr of shaking (100 oscillations/min) approximately 95% of cortisone was converted into the 20/3-hydroxy derivative. Thin-layer chromatography on silica gel GF254plates in the system water-methanoldiethyl ether-dichloromethane (1.2:8:15:77, v/v) revealed only trace amounts of steroid by-products. The turnover number for the coenzyme was about 20. This low value is due, at least in part, to the increased gm values of the immobilized enzymes for NAD(H) (Table II), and therefore high concentrations of coenzymes in the medium were needed to assure high reaction rates. Also, the NADH regenerating system based on alcohol dehydrogenase is less suitable than that based on formate dehydrogenase since it needs the presence of semicarbazide which also can react with the keto groups present in the steroids and yield by-products. 6 After 8 days of use, the residual activity of 20/3-hydroxysteroid dehydrogenase was approximately 35%. Oxidation of Testosterone to Androstenedione The process is based on the following reactions: Testosterone (androst-4-ene-17/3-hydroxy-3-one) + N A D ÷ androstenedione (androst-4-ene-3,17-dione + N A D H + H + Pyruvate + N A D H + H + ~ lactate + NAD +

(6) (7)

where reaction (6) is catalyzed by fl-hydroxysteroid dehydrogenase and reaction (7) by lactate dehydrogenase. The overall equilibrium constant value for the coupled reactions is 9.4 × 103. 4 The reaction system is made up as follows. Aqueous phase: 10 ml of 0.1 M potassium phosphate buffer, pH 8.5, containing 0.2 M sodium pyruvate, 1 mM dithiothreitol, 15 mg of serum albumin, 3/zmol of NAD +, 20 U of/3-hydroxysteroid dehydrogenase, and 100 U of lactate dehydrogenase. Organic phase: 20 ml of butyl acetate containing 1.04 mmol (300 mg) of testosterone. The reaction is initiated by shaking (100 oscillations/ min) and then followed by thin-layer chromatography on silica gel GF254 plates in the system ethyl acetate-benzene-hexane (100:80:60, v/v). The complete oxidation of testosterone to androstenedione was obtained in about 25 hr. The turnover number for NAD ÷ was 350. At the end of the run, the residual activity of/3-hydroxysteroid dehydrogenase and lactate dehydrogenase was 54 and 70%, respectively.

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Oxidation of Cholic Acid Methyl Ester to 12-Ketochenodeoxycholic Acid Methyl Ester The process is based on the following reactionsJ2: Cholic acid methyl ester (3a,7a, 12t~-trihydroxy-5fl-cholan-24-oic acid methyl ester) + NADP + 12-ketochenodeoxycholic acid methyl ester (3a,7a-dihydroxy-12-oxo-5fl-cholan-24-oic acid methyl ester) + NADPH + H ÷ a-Ketoglutarate ÷ NADPH + NH3 + H + ~- L-glutamate + NADP + + H20

(8) (9)

where reaction (8) is catalyzed by 12a-hydroxysteroid dehydrogenase and reaction (9) by glutamate dehydrogenase. The overall equilibrium constant value for the coupled reactions is about 3.5 x 106.12 The reaction system is made up as follows. Aqueous phase: 10 ml of 0.1 M potassium phosphate buffer, pH 8, containing 0.2 M ammonium acetate, 0.1 M a-ketoglutarate, 0.75 mM ADP, 1 mM dithiothreitol, 1 /zmol of NADP ÷, and 5.5 U of 12a-hydroxysteroid dehydrogenase coimmobilized onto Sepharose CL-4B (5 ml) with 28 U of glutamate dehydrogenase. Organic phase: l0 ml of butyl acetate containing 0.47 mmol (200 mg) of cholic acid methyl ester. The reaction is initiated by shaking (100 oscillations/min), and then followed by thin-layer chromatography on silica gel plates in the system chloroform-methanol-acetic acid (20:2: 1, v/v); the plates are sprayed with Komarowsky's reagent. 13 An almost complete oxidation of cholic acid methyl ester to 12-ketochenodeoxycholic acid methyl ester was achieved in 3 days. The turnover number for NADP ÷ was 470. After 3 months of continuous use, the residual activity of 12a-hydroxysteroid dehydrogenase and glutamate dehydrogenase was 48 and 60%, respectively. General Considerations The described steroid transformations exemplify the feasibility of carrying out enzymatic reactions in two-phase systems. Many other steroids can be converted, under similar conditions, using the examined enzymes2'4-6 (20fl-hydroxysteroid dehydrogenase, 3 (or 17)/3-hydroxysteroid dehydrogenase, and 12a-hydroxysteroid dehydrogenase), or other enzymes such as 3a-hydroxysteroid dehydrogenase, laccase, 3 peroxidase, and steroid isomerase. 14In all cases, the use of two-phase systems drastically reduced reaction volumes and allowed an easy separation of ~2G. Carrea, R. Bovara, P. Cremonesi, and R. Lodi, Biotechnol. Bioeng. 26, 560 (1984). 13 I. A. Macdonald, J. Chromatogr. 136, 348 (1977). i4 p. Cremonesi, G. Mazzola, and L. Cremonesi, Ann. Chim. 67, 415 (1977).

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157

the products, which are predominantly present in the organic phase, from the enzymes and cofactors, which are present in the aqueous phase. The parameters that influence the effectiveness of enzymatic reactions in two-phase systems may be summarized as follows: effect of organic solvents on the stability and activity of enzymes, solubility and partition of reagents and products between the two phases, ratio between the volumes of the organic and the aqueous phases, rate of transfer of reagents and products between the phases. A detailed examination of such parameters was made in previous articles, s,15 Regarding the choice of the organic solvents, the ones most suitable for our purposes were butyl acetate and ethyl acetate, 2-s,lz since they have high solubilizing capacity for steroids and weak inhibiting and denaturing effects on enzymes. The regeneration of the costly coenzymes markedly reduces the cost of the process and should make it possible to fully exploit the great specificity of hydroxysteroid dehydrogenases for preparative-scale transformations of steroids in two-phase systems. Also, the use of coupled enzymatic reactions highly improves transformation yields when overall equilibrium constant values are favorable. The immobilization of the enzymes made it possible to use them repeatedly and increased their stability, particularly in the case of tresyl chloride activation of the matrix. It should also be emphasized that, contrary to what happened with separately immobilized enzymes, coimmobilized enzymes regenerated the coenzymes very effectively. is G. Carrea, Trends Biotechnol. 2, 102 (1984).

[15] D e b l o c k i n g in P e p t i d e S y n t h e s i s with Immobilized Carboxypeptidase Y

By G. P. ROYER Introduction The selectivity of enzymes and the mild conditions under which they operate suggest their use in the synthesis of complex, unstable biomolecules and drugs. One application is in deblocking reactions. In sequential synthesis many reactions are run one after the other. Accumulation of impurities generated by harsh deblocking reagents results in a complex mixture of closely related products. Sequential synthesis of biopolymers METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[15]

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the products, which are predominantly present in the organic phase, from the enzymes and cofactors, which are present in the aqueous phase. The parameters that influence the effectiveness of enzymatic reactions in two-phase systems may be summarized as follows: effect of organic solvents on the stability and activity of enzymes, solubility and partition of reagents and products between the two phases, ratio between the volumes of the organic and the aqueous phases, rate of transfer of reagents and products between the phases. A detailed examination of such parameters was made in previous articles, s,15 Regarding the choice of the organic solvents, the ones most suitable for our purposes were butyl acetate and ethyl acetate, 2-s,lz since they have high solubilizing capacity for steroids and weak inhibiting and denaturing effects on enzymes. The regeneration of the costly coenzymes markedly reduces the cost of the process and should make it possible to fully exploit the great specificity of hydroxysteroid dehydrogenases for preparative-scale transformations of steroids in two-phase systems. Also, the use of coupled enzymatic reactions highly improves transformation yields when overall equilibrium constant values are favorable. The immobilization of the enzymes made it possible to use them repeatedly and increased their stability, particularly in the case of tresyl chloride activation of the matrix. It should also be emphasized that, contrary to what happened with separately immobilized enzymes, coimmobilized enzymes regenerated the coenzymes very effectively. is G. Carrea, Trends Biotechnol. 2, 102 (1984).

[15] D e b l o c k i n g in P e p t i d e S y n t h e s i s with Immobilized Carboxypeptidase Y

By G. P. ROYER Introduction The selectivity of enzymes and the mild conditions under which they operate suggest their use in the synthesis of complex, unstable biomolecules and drugs. One application is in deblocking reactions. In sequential synthesis many reactions are run one after the other. Accumulation of impurities generated by harsh deblocking reagents results in a complex mixture of closely related products. Sequential synthesis of biopolymers METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

158

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS HOAv'O("~O)N~OH

[15]

PEG

Carboxymethylation

Attachment of glycylmethionine

PEG'-Met-OH

FIG. 1. Preparation of the PEG handle.

(peptides and nucleic acids) appears to be an area where enzyme catalysis would prove useful. Carboxypeptidase Y (CPY; EC 3.4.16.1, serine carboxypeptidase) is an exopeptidase found in yeast. The enzyme is unusual in that it has a broad specificity. It catalyzes the release of L-amino acids, including proline, from the C terminus of a polypeptide chain. Rates of amino acid release vary but not to the extent exhibited by other exopeptidases such as pancreatic enzymes. CPY also acts as an esterase. It rapidly cleaves the C-terminal alkyl ester group of large and small peptides. The pH optima of these two activities are widely separated. At pH 6 the enzyme acts as a peptidase, while at pH 8.5 the enzyme acts as an esterase with little or no peptidase activity. This behavior suggested to us that the enzyme would be of use in the synthesis of peptides. Our first thought was that one could cleave the C-terminal ester and leave the side-chain ester groups of aspartic acid and glutamic acid intact. Thus, the condensation of fragments would be facilitated by this procedure of selective saponification. Also, we demonstrated a method to resolve racemic mixtures of blocked peptide and amino acid esters; the enzyme will not hydrolyze the ester group of a terminal D-amino acid.l Regiospecific cleavage of the aester of blocked diester derivatives of glutamic acid and aspartic acid can be accomplished with the immobilized enzyme. Our main interest in CPY has involved the concept of a method for the synthesis of peptides in aqueous systems using very mild conditions for deblocking. 2 Small protected peptides can be deblocked in alcohol/water mixtures. However, as the size of the peptide increases the solubility decreases. To use the immobilized CPY in the presence of high levels of organic solvent is not possible. This problem was solved by growing the peptide chain on a water-soluble "handle," namely polyethylene glycol (PEG). The derivatization of the PEG is shown in Fig. 1. The terminal i G. P. Royer, H. Y. Hsiao, and G. M. Anantharamaiah, Enzyme Eng. 5, 461 (1980). 2 G. P. Royer and G. M. Anantharamaiah, J. A m Chem. Soc. 101, 3394 (1979).

[15]

D E B L O C K I NIN G PEPTIDE SYNTHESIS WITH CPY

159

PEG'-Met-OH ~ H-AA1-OR PEG'-Met-AA1-OR Deblockwith I-CPY PEG'-Met-AA1-OH I Extension PEG'-Met [AAI....AAN] -OH I CNBr Freepeptide FIG. 2. Chain elongation and release.

hydroxyl groups of PEG were carboxymethylated; glycylmethionine was then added. The methionine was incorporated in order to provide a cleavable (CNBr) linkage between the peptide and the PEG carrier. Chain elongation and release are shown schematically in Fig. 2. The first amino acid of the target sequence is added as the ester, using watersoluble carbodiimide. Following removal of excess reagents, the ester protecting group is hydrolyzed by treatment with immobilized CPY. Coupling/deblocking cycles are repeated until the sequence is complete. The finished peptide is then removed by cleavage with cyanogen bromide. In the event that methionine is part of the target sequence, it can be added as methionine sulfoxide which prevents reaction with cyanogen bromide. Reduction of the released peptide yields the desired sequence. Procedures Enzyme. Carboxypeptidase Y can be prepared from yeast according to the procedure of Kuhn et al.3 Final purification can be accomplished using the affinity column of Johansen et al. 4 The enzyme is commercially available from Pierce Chemical Co. and Sigma Chemical Co. An immobilized form of the enzyme is also available from Pierce. Assay. CPY-catalyzed ester hydrolysis can be followed titrimetrically or spectrophotometrically with N-a-acetyl-L-tyrosine ethyl ester (ATEE). 5 The titrimetric assay is convenient for the immobilized enzyme. An automatic titration system (pH-stat) equipped with a strip chart recorder is needed. When reactions are run at alkaline pH, a stream of 3 R. W. Kuhn, K. A. Walsh, and H. Neurath, Biochemistry 13, 3871 (1974). 4 j. I. Johansen, K. Breddam, and M. Ottesen, Carlsburg Res. Commun. 41, 1 (1976). 5 R. Hayashi, this series, Vol. 45, p. 568.

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[15]

nitrogen is directed at the surface of the liquid in the reaction vessel. The buffer is phosphate (5 mM, pH 8) which contains 0.1 M KC1. An ATEE stock solution (0.2 M) can be prepared with ethanol as a solvent. The titrant is 10 mM NaOH, standardized and protected from the atmosphere with an ascarite (NaOH) trap. The temperature of the reaction is maintained at 25 ° with a jacketed reaction vessel which is connected to a circulating water bath. The final substrate concentration is 10 mM. Enzyme concentrations are approximately 1 /xg/ml for the soluble enzyme and 1 mg/ml for immobilized enzyme. Enzyme Immobilization. There are several methods in the literature for immobilization of CPY. 6'7 The enzyme can be attached directly to aminohexylamino-agarose using water-soluble carbodiimide. It can be adsorbed to immobilized concanavalin A. The carbohydrate can be oxidized and reacted with diamines to give a species which can be immobilized on supports containing active acylating groups or aldehydes, The preferred procedure (the latter) appears in this series. 8 CP Y-Catalyzing Deblocking of Peptide Esters. Conditions and results for the deblocking of peptide esters are shown in Table I. The deblocking reaction is carried out at pH 8.5 with the aid of a pH-stat. The reactant is dissolved in methanol or DMF and kept as a stock solution (about 100 mg/ ml). The concentration of the organic solvent in the final reaction mixture should not exceed 25%. The reaction is started by addition of the immobilized enzyme (20-80 ATEE units, standard). When base uptake ceases, the enzyme is filtered, the reaction mixture is acidified (pH 2-3), and the solution is concentrated with a rotary evaporator at a temperature of 40 ° until precipitation occurs. The crystallization of the peptide derivatives is accomplished with ethanol/water. Water is added to a 20% ethanolic solution of the peptide until turbidity appears. The solution is cooled to complete crystallization. If available, seed crystals should be added before cooling. Preparation of CM-PEG. PEG (14 g, MW 6000-7000) and potassium tert-butoxide (10 g) are dissolved in tert-butanol (150 ml) by warming. To this solution ethyl bromoacetate (5 ml) is added dropwise over a period of 10 rain. After 2 hr of stirring, the solvent is removed by rotary evaporation. The residue is taken up in 100 ml of 1 N NaOH. After 2 hr at room temperature, the pH of the mixture is adjusted to 2 with HCI. The C M PEG is recovered by partitioning into chloroform (two 200-ml portions). The chloroform layer is washed with water and then dried with anhydrous 6 F. A. Liberatori, J. E. Mclsaac, Jr., and G. P. Royer, FEBS Lett. 68, 45 (1976). 7 H. Y. Hsiao and G. P, Royer, Arch. Biochern. Biophys. 198, 379 (1979). 8 G. P. Royer, this series, Vol. 135 [10].

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161

TABLE I DEBLOCKING OF PEPTIDE ESTERS

Peptide ester Z-Asn-Phe-OEt Boc-Asp(O-Bzl)-Phe-OEt Z-Asp(OMe)-OMe Z-Phe-D-Ala-OEt 1 : 1 mixture of Z-Phe-D-Ala-OEt and Z-Phe-L-AIa-OEt

Yield (%)

Concentration/solvent

Product

20 mg in 160 ml of 25% MeOH 10 mg in 15 ml of 20% MeOH 20 mg in 20 ml of 25% DMF 10 mg in 50 ml of 25% DMF 10 mg of each in I00 ml of 25% DMF

Z-Asn-Phe-OH

95

Boc-Asp(O-Bzl)-Phe-OH

84

Z-Asp(OMe)-OH

98

No reaction

--

Z-Phe-L-AIa-OH

95

NazSO4. After rotary evaporation of the solvent, 12 g of derivatized P E G remains.

CP Y-Catalyzed Deblocking of Peptides Attached to the PEG Handle. The deblocking o f P E G / M e t - O E t is accomplished by treatment with immobilized CPY at p H 8.5 at room temperature. The pH-stat is used to maintain the pH and to follow the course of the reaction. P E G ' - M e t - O E t (3 g, P E G ' is P E G - g l y c i n e ) is dissolved in 40 ml of water. Immobilized e n z y m e (30 A T E E units, standard) is added and the pH is quickly adjusted to 8.5. The reaction mixture is stirred with an overhead stirrer at a speed sufficient to maintain the enzyme beads in suspension. The titrant used is 0.1 M N a O H . The time required for hydrolysis of P E G ' - M e t - O E t under these conditions is 5 hr. The hydrolysis rate goes up dramatically as the chain is extended, probably because of subsite binding of neighboring amino acids. When the reaction is complete (cessation of base uptake), the reaction mixture is filtered to remove the immobilized enzyme. The P E G - p e p t i d e solution can be used directly for coupling of the next amino acid. It is essential to remove all of the e n z y m e from the solution. If e n z y m e remains behind and the p H is adjusted to 6, peptidase activity will result in the degradation of the chain. Comments In related work on the use of proteases in peptide synthesis, John Glass and co-workers have shown that trypsin can be used to deprotect the a-amino group. 9 The coupling segments were Cbz-arginyl peptides. 9 C. Myers and J. D. Glass, Proc. Natl. Acad. Sci. U.S.A. 72, 2193 (1975).

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[16]

The Cbz-arginyl group is cleaved by trypsin. To avoid endopeptidase activity, temporary blocking groups for arginyl and lysyl side chains in the target sequence were employed. In another clever application of proteases, Glass and Pelzig have demonstrated enzyme-cleavable side-chain protection. Arginine methyl ester was attached to side-chain carboxyl groups using water-soluble carbodiimide. Trypsin was used to remove the methyl ester of the arginine moiety and then carboxypeptidase B was used to remove the arginyl group.l° In conclusion, immobilized CPY is a versatile tool for the protein chemistry lab. It can be used for deblocking in peptide synthesis,2 resolution of racemic mixtures of blocked amino acid esters or peptides, 1 sequencing of peptides, 6 total hydrolysis of peptides for analysis, 11 and condensation reactions in peptide synthesis. ~2 The attachment of C-terminal amino acid amides can be accomplished using CPY to catalyze the peptide bond formation starting with the peptide ester and the amide. Also, the enzyme should be of interest in peptide modification (semisynthesis) and fragment condensation of peptides. 10 j. D. Glass and M. Pelzig, Proc. Natl. Acad. Sci. U.S.A. 74, 2739 (1977). ~1 G. P. Royer, W. E. Schwartz, and F. A. Liberatore, this series, Vol. 47, p. 40. 12 K. Breddam, F. Widmer, and J. T. Johansen, Carlsburg Res. Commun. 45, 361 (1980).

[16] U s e o f I m m o b i l i z e d Achrornobacter P r o t e a s e I for Semisynthesis of Human Insulin

By

K A Z U Y U K I MORIHARA, RYONOSUKE M U N E Y U K I ,

and TATSUSHI OKA Porcine insulin can be enzymatically converted into human insulin by two different methods. In condensation reactions catalyzed by trypsin (EC 3.4.21.4) I and protease I from Achromobacter lyticus (EC 3.4.21.50), 2 L-threonine tert-butyl ester can be coupled to 30B-dealanine porcine insulin (DAI) to obtain an ester derivative of human insulin. In the K. Morihara, T. Oka, and H. Tsuzuki, Nature (London) 280, 412 (1979). 2 K. Morihara, T. Oka, H. Tsuzuki, Y. Tochino, and T. Kanaya, Biochem. Biophys. Res. Commun. 92, 396 (1980).

METHODS IN ENZYMOLOGY, VOL. 136

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IMMOBILIZED

ENZYMES/CELLS

IN ORGANIC

SYNTHESIS

[16]

The Cbz-arginyl group is cleaved by trypsin. To avoid endopeptidase activity, temporary blocking groups for arginyl and lysyl side chains in the target sequence were employed. In another clever application of proteases, Glass and Pelzig have demonstrated enzyme-cleavable side-chain protection. Arginine methyl ester was attached to side-chain carboxyl groups using water-soluble carbodiimide. Trypsin was used to remove the methyl ester of the arginine moiety and then carboxypeptidase B was used to remove the arginyl group.l° In conclusion, immobilized CPY is a versatile tool for the protein chemistry lab. It can be used for deblocking in peptide synthesis,2 resolution of racemic mixtures of blocked amino acid esters or peptides, 1 sequencing of peptides, 6 total hydrolysis of peptides for analysis, 11 and condensation reactions in peptide synthesis. ~2 The attachment of C-terminal amino acid amides can be accomplished using CPY to catalyze the peptide bond formation starting with the peptide ester and the amide. Also, the enzyme should be of interest in peptide modification (semisynthesis) and fragment condensation of peptides. 10 j. D. Glass and M. Pelzig, Proc. Natl. Acad. Sci. U.S.A. 74, 2739 (1977). ~1 G. P. Royer, W. E. Schwartz, and F. A. Liberatore, this series, Vol. 47, p. 40. 12 K. Breddam, F. Widmer, and J. T. Johansen, Carlsburg Res. Commun. 45, 361 (1980).

[16] U s e o f I m m o b i l i z e d Achrornobacter P r o t e a s e I for Semisynthesis of Human Insulin

By

K A Z U Y U K I MORIHARA, RYONOSUKE M U N E Y U K I ,

and TATSUSHI OKA Porcine insulin can be enzymatically converted into human insulin by two different methods. In condensation reactions catalyzed by trypsin (EC 3.4.21.4) I and protease I from Achromobacter lyticus (EC 3.4.21.50), 2 L-threonine tert-butyl ester can be coupled to 30B-dealanine porcine insulin (DAI) to obtain an ester derivative of human insulin. In the K. Morihara, T. Oka, and H. Tsuzuki, Nature (London) 280, 412 (1979). 2 K. Morihara, T. Oka, H. Tsuzuki, Y. Tochino, and T. Kanaya, Biochem. Biophys. Res. Commun. 92, 396 (1980).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[16]

SEMISYNTHESIS OF HUMAN INSULIN

163

other method, the terminal alanine residue of the B chain in porcine insulin is exchanged for amides and esters of L-threonine in a single transpeptidation step catalyzed by either trypsin, 3 carboxypeptidase Y (EC 3.4.16.1, serine carboxypeptidase) 4 or Achromobacter protease 1.5 The protected insulin derivative obtained from the enzyme-catalyzed reactions is subsequently deblocked in a one-step chemical procedure which yields human insulin. For large-scale production, Achromobacter protease I seems to be the ideal enzyme to use in enzyme-assisted semisynthesis of human insulin. The enzyme exhibits several characteristics that make it suitable to use for synthetic purposes. For example, the rate of condensation of DAI to L-threonine tert-butyl ester catalyzed by Achromobacter protease I is at least 10 times higher than that observed for the trypsin-catalyzed condensation reaction. Also, the enzyme specifically cleaves peptide bonds containing an L-lysine residue at the carboxyl side of the splitting point in protein and peptide substrates, 6 preventing the formation of by-products. Furthermore, the amount of enzyme required for the enzymatic coupling reaction is 50-100 times less than that required in transpeptidation reactions catalyzed by other proteases, and the same product yields are afforded by the two approaches) It should finally be added that Achromobacter protease I may be useful for direct preparation of DAI which cannot be prepared in a single step using other proteolytic enzymes. For efficient use of enzymes in industry, continuous operation of the enzyme-catalyzed process is desirable. This can be achieved by using immobilized enzymes, which are known to be stable, reusable, and also easily recoverable. This article explains the immobilization of Achromobacter protease I as well as the use of the immobilized enzyme in the enzyme-assisted semisynthesis of human insulin, which is schematically depicted in Fig. 1. Assay Procedure of the Immobilized Enzyme

Principle. Ester and amide derivatives of N~-acylated L-lysine are used as substrates for enzyme activity determinations of both soluble 6 and immobilized enzymes. 3 A. Jonczyk and H.-G. Gattner, Hoppe-Seyler's Z. Physiol. Chem. 362, 1591 (1981); J. Markussen, United Kingdom Patent 2,069,501A (August 26, 1981). 4 K. Breddam, F. Widmer, and J. T. Johansen, Carlsburg Res. Commun. 46, 361 (1981). 5 K. Morihara and T. Oka, "Peptide Chemistry, 1982," p. 231, Protein Research Foundation, Osaka, Japan, 1983. 6 T. Masaki, T. Fujihashi, K. Nakamura, and M. Soejima, Biochim. Biophys. Acta 660, 51 (1981).

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IMMOBILIZED ENZYMES/CELLSIN ORGANICSYNTHESIS

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Gly

~ - ~ / Phe

Asn22

29 30

Arg

Lys-Ala

Porcine insulin

~ Immobilized enzyme Gly 29

Phe - Lys Des-alanyl(B30)-insulin (p-DAI) t ~--.- Thr-OBu t Immobilized enzyme Gly ~ - - - ~ /

Asn

Phe Human insulin tertiary butyl

Lys-Thr-O But ester

1 VT---1

.~-- CF3COOH

Gly

/

/

Phe / / Human insulin

29

30

Lys-Thr

FIG. 1. Scheme for enzyme-assistedsemisynthesisof human insulin using immobilized protease I.

Achromobacter

Reagents and Apparatus N~-Tosyl-L-lysine methyl ester (TLME), 20 mM in 2 M KCI Sodium hydroxide, 0.01 or 0.025 N Immobilized Achromobacter protease I (lysine endopeptidase) Automatic titration apparatus Procedure. To a small glass tube (12 × 60 mm) containing immobilized enzyme (10 mg wet gel), 1 ml of water is added. The mixture is vigorously stirred and 1 ml of TLME is added to the homogeneous suspension. The pH of the reaction mixture is maintained at 7.0 by automatic titration with 0.01 or 0.025 N NaOH at 20 - 2°. The activity of the immobilized enzyme is expressed as micromoles of substrate hydrolyzed by the enzyme per minute and gram (wet weight) of immobilized enzyme added to the assay mixture at 20 ± 2°.

[16]

SEMISYNTHESIS OF HUMAN INSULIN

165

Enzyme Immobilization Preliminary studies7 indicate that at least 6/zM enzyme is required to obtain practical product yields in the enzyme-catalyzed coupling reactions using immobilized Achromobacter protease I. Therefore, different methods of enzyme immobilization were investigated to obtain preparations containing immobilized enzymes showing sufficiently high specific activities. The best results were obtained after coupling the enzyme to poly(t-glutamic acid) (poly-Glu-OH) or silica gel containing covalently bound poly(L-glutamic acid). The enzyme immobilized on other commonly used supports such as CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals), Affi-Gel 15 (Bio-Rad), and agar (Difco Bacto) showed low specific activities.

Covalent Coupling to Poly(L-Glutamic Acid) Reagents Sodium poly(L-glutamate) (MW 50,000), Protein Research Foundation, Minoh, Osaka Amberlite IR-120 1- Ethyl- 3 - (3- dimethylaminopropyl)carbodiimide hydrochloride (WSCD), Protein Research Foundation, Minoh, Osaka Achromobacter protease 18 (487/zmol TLME/min-mg at pH 7.0, 761 /zmol TLME/min-mg at pH 8.0, 20 -+ 2°), Wako Pure Chemical Industries, Osaka) Procedure. Amberlite IR-120 (4 ml of settled beads, acid form) is added to a solution containing sodium poly(e-glutamate) (411 mg) in water (10 ml). After several minutes of reaction, the precipitate which forms is removed from the beads by filtering the slurry through a thin cotton layer and by washing the beads retained on the cotton layer with several small volumes of water. To the poly-Glu-OH (acid form) suspended in the combined washing solutions (30 ml), Achromobacter protease I (11.3 mg) in water (2 ml) and WSCD (340 mg) in water (4 ml) are added successively, and the mixture is stirred gently for 35 rain at room temperature. After centrifugation (1000 g × 10 rain) of the reaction mixture, the supernatant, which shows no enzyme activity, is removed and the precipitate is suspended in 0.1 M acetate buffer (30 ml), pH 4.0. The mixture is centrifuged 7 R. Muneyuki, T. Oka, and K. Morihara, "Peptide Chemistry, 1981," p. 113, Protein Research Foundation, Osaka, Japan, 1982. 8 T. Masaki, M. Tanabe, K. Nakamura, and M. Soejima, Biochim. Biophys. Acta 660, 44 (1981).

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(I000 g x 10 min), and after additional washing with 1 M NaCI (30 ml), the precipitate is transferred to a filter paper. The filter paper containing the precipitate is squeezed to remove excess water, and after drying 0.317 g of immobilized enzyme is obtained. This dry weight of the immobilized enzyme corresponded to 1.63 g wet weight of the same preparation. The activity of the immobilized enzyme was 907 p.M/min-g wet gel when the preparation was assayed at pH 7.0. This activity corresponded to 27% of the original activity of the soluble enzyme used in the immobilization step. Immobilized Achromobacter protease I showed considerable solubility at pH 6.5 (the pH of the enzymatic condensation reaction). Moreover, the solubility caused some difficulties because the human insulin ester derivative that was produced was difficult to separate from the immobilized enzyme; for example, it could not be separated by filtration on glass filter. The enzyme was therefore immobilized on an insoluble silica gel premodifled by substitution with poly(L-glutamic acid).

Covalent Coupling to Silica Gel Containing Immobilized Poly(L-Glutamic Acid) Reagents Sodium poly(L-glutamate) (MW 50,000) Lichrosorb-NH2 (3-aminopropyl silica gel; 10/zm mean particle size) for liquid chromatography (Merck) Achromobacter protease I WSCD Procedure. Step I. An aqueous solution (5 ml) containing sodium poly-L-glutamate (1.0 g) is added to 1 N HC1 (30 ml). The solution is left standing for 2 hr in a thermostated water bath kept at 0°. After centrifugation of the reaction mixture (450 g x 15 min), the precipitate is suspended in 0.1 N HC1 (30 ml). The mixture is left standing for another hour at room temperature and was then centrifuged (450 g x 15 min). The obtained precipitate is dried under vacuum at around 60°, yielding a white solid for poly-Glu-OH (0.778 g). Step 2. Lichrosorb-NH2 (5.0 g) is suspended in water (20 ml) and the resulting slurry is poured into a glass cylinder filled with water. The height of the liquid in the cylinder is 30 cm after addition of the silica gel. The portion precipitation after 20 min is collected, and the gel is washed successively on a sintered glass filter with 0.002 N HC1 (800 ml), water (300 ml), 0.003 N NaOH (500 ml), water (300 ml), ethanol-water (1/1, v/v) (100 ml), and ethanol (50 ml). After drying overnight at room temperature

[16]

SEMISYNTHESIS OF HUMAN INSULIN

167

the obtained silica gel (3.33 g) contains 1.21% nitrogen (determined after elemental analysis). Step 3. Poly-Glu-OH (381 mg of dry weight) and the silica gel (680 mg of dry weight), both obtained as described above, are added to N,N-dimethylformamide (10 ml). The slurry is added to N,N-dimethylformamide (5 ml) containing WSCD (331 mg). After stirring at 60° for 4 hr, the reaction mixture is poured into 0.1 N HCI (40 ml) and the slurry centrifuged (1000 g × 10 min). The precipitate obtained is washed successively by repeated centrifugation with 0.05 N HC1 (60 ml), 0.005 N NaOH (60 ml), 0.2 M NaHCO3 (60 ml), and water (60 ml). The solid material is then transferred to a glass filter and washed with 0.002 N HC1 (200 ml) and water (200 ml). The modified silica gel is dried under vacuum at 50°, giving 783 mg of dry silica-bound poly-Glu-OH. The nitrogen concentration of the modified silica gel is 3.31% (determined after elemental analysis). Step 4. Silica-bound poly-Glu-OH (87.5 mg) is added to a water solution (0.5 ml) containing WSCD (16.2 mg), and the mixture is stirred for 2.5 min at room temperature. To this suspension, Achromobacter protease I (0.65 mg) in water (0.5 ml) is added, followed by addition of 0.1 M sodium phosphate buffer (pH 1!.8, 0.5 ml). After continuous stirring for 2.5 hr at room temperature, the reaction mixture is filtered on a Bfichner funnel and the precipitate containing immobilized enzyme is washed successively with 1 M acetate buffer (pH 7.6, 50 ml), water (50 ml), 1 M acetate buffer (pH 5.0, 50 ml), and water (50 ml). The immobilized enzyme is transferred to an Erlenmeyer flask containing 2 M NaC1 (50 ml). After stirring the slurry for 30 min, the immobilized enzyme is washed with water on a BiJchner funnel until no enzyme activity can be detected in the washing solution. The wet gel (176 mg) showed an enzyme activity of 615/zM/min-g wet gel when assayed at pH 8.0. It is worth noting that the activity of the silica-bound enzyme varied with the pH of the sodium phosphate buffer used in the coupling reaction. Thus, preparations assayed at pH 8.0 showed activities of 124,263,346,510, and 615/zM/min-g of wet gel when the pH of the added phosphate buffer was 7.5, 8.8, 9.8, 10.8, and 11.8, respectively. When 0.2 M sodium phosphate buffer (pH 11.8) was added in the immobilization step, the activity of the immobilized enzyme dropped to 134/.tM/min-g wet gel at pH 8.0. Preparation of DAI Using Immobilized Enzyme

Principle. Achromobacter protease I can be used to hydrolyze porcine insulin by specifically releasing the C-terminal alanine residue of the B chain in insulin. 6

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[16]

Reagents and Apparatus Porcine insulin (lot 1FJ91, 26.2 U/mg), Eli Lilly Silica-bound Achromobacter protease I, 615/zM/min-g wet gel (prepared as described above) Ammonium carbonate, 0.1 M, pH 8.3 Amino acid analyzer, Hitachi KLA Model 5 Procedure. Silica-bound enzyme is packed in a small glass column (6.5 × 12 mm, containing 310 mg of wet gel) and was equilibrated with 0.1 M ammonium carbonate, pH 8.3 (equilibration buffer). Porcine insulin (167 mg, 1 mM) dissolved in 29 ml of the equilibration buffer is applied to the column and the solution is pumped through the column at a flow rate of 0.5 ml/hr at room temperature. Since the hydrolysis is not completed after one passage through the column, the substrate solution must be recirculated. Three, six, and nine passages lead to 34, 69 and 100% hydrolysis of the porcine insulin. The rate of hydrolysis is determined from the content of analine in the elution buffer (for alanine determinations, amino acid analyses of the eluates are carried out). After 10 passages, the column is washed with equilibration buffer until the buffer contains no DAI. The combined eluates are lyophilized and the formed DAI is ultimately purified by gel chromatography on Sephadex G-50 (4.2 × 134 cm, 1.85 liter gel) using 0.5 M acetic acid as the eluant. 2 The yield of DAI was 153 mg (92%). Preparation of Human Insulin Using Immobilized Enzyme

Principle. The enzymatic condensation of DAI to Thr-OBu t has been catalyzed successfully by soluble Achromobacter protease I in organic aqueous cosolvents under controlled pH. 2 Similar reaction conditions are employed for the enzymatic condensation using immobilized enzyme. Reagents and Apparatus DAI is prepared as described above. DAI is also prepared after digesting porcine insulin in the presence of soluble carboxypeptidase A 9 (EC 3.4.17.1). L-Threonine-tert-butyl ester (Thr-OBut), bp 85-88°/3 mm Hg, is prepared as described elsewhere, i° Silica-bound Achromobacter protease I, 615 ~M/min-g wet gel (prepared as described above) 9 E. W. Schmitt and H.-G. Gattner, Hoppe-Seyler's Z. Physiol. Chem. 359, 799 (1978). 10 E. Vowinkel, Chem. Ber. 11~, 16 (1967).

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169

Acetate buffer, 5 M, pH 5.7

1,4-Butanediol Apparatus for high-performance liquid chromatography: Waters Assoc. Model 6000A pump, Rheodyne Model 7120 injector, and Japan Spectrooptics UVIDEC-100 variable-wavelength UV detector Assay. The enzyme-catalyzed coupling of DAI to Thr-OBu t is followed by spectrophotometric measurement of the decrease in DAI absorbance at 220 nm. The analyses are performed using reversed-phase liquid chromatography ~ (column, Nucleosil 5C~8, 0.4 x 20 cm; eluant, 30.5% CH3CN in 5 mM phosphate buffer, pH 3.0, containing 5 mM sodium 1-butane sulfonate and 50 mM Na2SO4). The product yield of the enzymatic reaction is calculated from the ratio of the determined peak areas of [30B-Thr-OBut]insulin (retention time, 14.2 min) and the remaining DAI (retention time 5.3 min). Procedure. DAI (162 mg), Thr-OBu t (400 mg), and water are added to a mixture containing 1,4-butanediol (1.12 ml) and acetate buffer (5 M, pH 5.7, 1.07 ml). The final volume of the reaction mixture is 7.81 ml and the pH of the mixture is 6.5. The solution is applied to a small column (6.5 × 12 mm) packed with silica-bound Achromobacter protease I (310 mg of wet gel). The column has been preequilibrated with the reaction mixture containing no DAI and Thr-OBu t. The flow rate is adjusted to 85/zl/hr by applying nitrogen pressure (0.2 atm) to the top of the column. The reaction mixture is recirculated through the column at room temperature to complete the enzymatic condensation reaction. In the first eluate, 45% of DAI is converted into [30B-Thr-OBut]insulin. The yield increases to 63 and 80% after the second and third runs, respectively. The combined eluates and the solution obtained after washing the column with 2 M acetate buffer (pH 6.5) are pooled and applied to a column packed with Sephadex G-50 (4.2 x 134 cm, 1.85 liter gel). Column chromatography is conducted using 0.5 M acetic acid as the eluant as described, z The insulin fraction obtained after the gel chromatography is lyophilized and the residue is then applied to a column packed with DEAE-Sephadex A-25 (4 x 34 cm, 427 ml gel) which has been preequilibrated with 0.01 M Tris buffer, pH 7.4, containing 7 M urea (equilibration buffer). On application of a linear NaC1 gradient (0-0.3 M, total volume of 1.5 liter) in the presence of equilibration buffer, [30B-Thr-OBut]insulin is eluted first and then DAI. 2 After removal of the urea by gel filtration, fractions containing [30B-Thr-OBut]insulin are lyophilized. The protected insulin derivative is deprotected by treatment with trifluoroacetic acid (TFA) in the presence of anisole. 2 The yield of human insulin is 59% (95 mg).

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The experiments discussed in this article have been helpful in solving some of the principal problems in the semisynthesis of human insulin from porcine insulin. Although they were done on a semipreparative scale, the obtained results indicate that application of immobilized Achromobacter protease I in the semisynthesis of human insulin has great potential as a tool for the large-scale production of human insulin.

[ 17] Application of I m m o b i l i z e d A m i n o p e p t i d a s e s to t h e S e q u e n t i a l H y d r o l y s i s of P r o l i n e - C o n t a i n i n g P e p t i d e s

By G. FLEMINGERand A. YARON Sequence-ordered hydrolysis of peptide chains by exopeptidases followed by quantitative amino acid analysis is a mild method for determination of the amino acid sequence, which is sensitive to the amino acid configuration and structure. ~ The most convenient procedure uses enzymes in the immobilized form, 2,3 permitting removal of enzyme-free samples for analysis as well as the use in sequence of several enzymes in the same reaction mixture. Peptide bonds associated with proline residues--present in a polypeptide chain--are unique in their susceptibility to hydrolysis by aminopeptidases. 4 Clostridial aminopeptidase (CAP; EC 3.4.11.13) 5-7 isolated from Clostridium histolyticurn is able to cleave N-terminal proline residues from polypeptide chains at rates comparable to cleavage rates of other N-terminal amino acid residues. The enzyme does not cleave Nterminal residues if followed by proline. The Xaa-Pro bond is resistant also to other aminopeptidases, except to aminopeptidase P (AP-P; EC 3.4.11.9) which cleaves this bond exclusively,s-12In the present article we describe application of glass-bound CAP and AP-P to the sequential hydrolysis of proline-containing polypeptides, t3 I A. N. Glazer, R. J. Delange, and D. S. Sigman, in "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work, eds.), Vol. 4, Part 1, p. 38. Elsevier/North Holland, Amsterdam, 1976. 2 G. P. Royer and J. P. Andrews, J. Biol. Chem. 248, 1807 (1973). 3 K. D. Vosbeck, B. D. Greenberg, and W. M. Award, Jr., J. Biol. Chem. 250, 3981 (1975). 4 R. Waiter, W. H. Simmons, and T. Yoshimoto, Mot. Cell. Biochem. 30, 111 (1980). 5 E. Kessler and A. Yaron, Eur. J. Biochem. 63, 271 (1976). 6 E. Kessler and A. Yaron, this series, Vol. 45, p. 544. 7 G. Fleminger, Z. Bohak, and A. Yaron, in preparation (1987). 8 A. Yaron and A. Berger, this series, Vol. 19, p. 521.

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The experiments discussed in this article have been helpful in solving some of the principal problems in the semisynthesis of human insulin from porcine insulin. Although they were done on a semipreparative scale, the obtained results indicate that application of immobilized Achromobacter protease I in the semisynthesis of human insulin has great potential as a tool for the large-scale production of human insulin.

[ 17] Application of I m m o b i l i z e d A m i n o p e p t i d a s e s to t h e S e q u e n t i a l H y d r o l y s i s of P r o l i n e - C o n t a i n i n g P e p t i d e s

By G. FLEMINGERand A. YARON Sequence-ordered hydrolysis of peptide chains by exopeptidases followed by quantitative amino acid analysis is a mild method for determination of the amino acid sequence, which is sensitive to the amino acid configuration and structure. ~ The most convenient procedure uses enzymes in the immobilized form, 2,3 permitting removal of enzyme-free samples for analysis as well as the use in sequence of several enzymes in the same reaction mixture. Peptide bonds associated with proline residues--present in a polypeptide chain--are unique in their susceptibility to hydrolysis by aminopeptidases. 4 Clostridial aminopeptidase (CAP; EC 3.4.11.13) 5-7 isolated from Clostridium histolyticurn is able to cleave N-terminal proline residues from polypeptide chains at rates comparable to cleavage rates of other N-terminal amino acid residues. The enzyme does not cleave Nterminal residues if followed by proline. The Xaa-Pro bond is resistant also to other aminopeptidases, except to aminopeptidase P (AP-P; EC 3.4.11.9) which cleaves this bond exclusively,s-12In the present article we describe application of glass-bound CAP and AP-P to the sequential hydrolysis of proline-containing polypeptides, t3 I A. N. Glazer, R. J. Delange, and D. S. Sigman, in "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work, eds.), Vol. 4, Part 1, p. 38. Elsevier/North Holland, Amsterdam, 1976. 2 G. P. Royer and J. P. Andrews, J. Biol. Chem. 248, 1807 (1973). 3 K. D. Vosbeck, B. D. Greenberg, and W. M. Award, Jr., J. Biol. Chem. 250, 3981 (1975). 4 R. Waiter, W. H. Simmons, and T. Yoshimoto, Mot. Cell. Biochem. 30, 111 (1980). 5 E. Kessler and A. Yaron, Eur. J. Biochem. 63, 271 (1976). 6 E. Kessler and A. Yaron, this series, Vol. 45, p. 544. 7 G. Fleminger, Z. Bohak, and A. Yaron, in preparation (1987). 8 A. Yaron and A. Berger, this series, Vol. 19, p. 521.

METHODS IN ENZYMOLOGY, VOL. 136

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Assay Method

Principle. The assay of CAP is based on measurement by the acid ninhydrin colorimetric method 14 of proline released from the tripeptide Pro-Gly-Pro. In order to ascertain that only this enzyme activity is measured in preparations of various degree of purity, they were occasionally tested also with the tetrapeptide Pro-Gly-Pro-Pro by the same method. In the assay of AP-P the amount of proline released from poly(L-proline) is determined analogously. Reagents Assay buffer. Sodium barbital buffer (50 raM) is purified by passage through a Chelex 100 column (analytical-grad~ chelating resin, mesh 50-100, BioRad Laboratories, Richmond, CA) and mixed with sodium citrate and COSO4 to final concentration of 50 and 10 mM, respectively. The pH is adjusted to 8.2. Pro-Gly-Pro solution, 0.5 mM in the assay buffer. Poly-L-proline solution (4.17 × 10-5 M) is prepared by dissolving the polymer (MW 6000) in cold assay buffer. The number average molecular weight of poly-L-proline is determined by potentiometric titration of the amino end groups (pKa 9.0). 8 Ninhydrin reagent. Ninhydrin (1.5 g) is dissolved in a mixture of acetic acid (60 ml) and 6 M phosphoric acid (40 ml) by warming at 70° . Prior to use the reagent is diluted with an equal volume of acetic acid. Enzymes. Frozen solutions of soluble C/~P (80 units/ml) in 50 mM sodium phosphate buffer, pH 7.4, containing 1.0 mM EDTA ("storage buffer") and of AP-P (120 units/ml) in 50 mM sodium acetate buffer, pH 5.6, containing 2.0 mM sodium citrate are stored at - 2 0 ° . The glass-bound enzymes are stored in storage buffer at 4 °. The isolation and purification of CAP 5,6 and of AP-P 8 have been described. The procedures for CAP were recently improved 7 mainly due to the finding that most of this extracellular enzyme is cell bound and can be released by mild sonication without causing any apparent damage to the

9 G. Fleminger, A. Carmel, D. Goidenberg, and A. Yaron, Eur. J. Biochem. 125, 609 (1982). i0 E. Holtzman, G. Pillay, T. Rosenthal, and A. Yaron, Anal. Biochem. 162, in press (1987). 11 p. Dehm and A. Nordwig, Eur. J. Biochem. 17, 364 (1970). 12 W. Sidrowicz, J. Szechinski, P. C. Canizaro, and F. J. Behal, Proc. Soc. Exp. Biol. Med. 175, 503 (1984). 13 G. Fleminger and A. Yaron, Biochim. Biophys. Acta 743, 437 (1983). 14 W. Troll and J. Lindsley, J. Biol. Chem. 215, 655 (1955).

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cell. The collected enzyme sonicate had to be purified only 15-fold to achieve homogeneity. Enzyme Immobilization and Properties of the Immobilized Enzyme Preparations Amino-glass beads (100 rag, Coming MAO-3930) are mixed with a fresh solution of glutaraldehyde in water (2.5%, 2.0 ml) for 60 min in vacuo at room temperature in a rotating syringe. The beads are washed with water and the storage buffer (100 ml). CAP in the same buffer (0.1 mg/ml, 80 units/mg, 2.0 ml) or AP-P in 0.1 M sodium acetate buffer, pH 5.6, containing 2.0 mM sodium citrate (0.1 mg/ml, 120 units/mg, 2.0 ml) is added to the activated glass beads and slowly mixed in the rotating syringe partially immersed in an ice bath for 3 hr. The beads are thoroughly washed with the storage buffer and stored in the same buffer at 4 ° . To inactivate any potential serine protease activity the immobilized preparations are treated once with 0.5 ~M phenylmethane sulfonylfluoride in the storage buffer for 20 min at room temperature. The enzyme activity of the beads is 18 and 8% of the original activity of the soluble CAP and AP-P used for immobilization, respectively. No enzymatic activity is released from the beads into the supernatant when the beads are kept in buffers at pH 7.4-8.6 at 4 ° for several days, as judged by the inability of the supernatant to hydrolyze the respective substrate under assay conditions. Stability. Both glass-bound enzymes are stable for at least 5-7 months when kept in the storage buffer at 4° even when frequently used for peptide hydrolysis. Activity of some less used preparations are stable for 8-10 months. The glass-bound enzymes loses activity irreversibly in solutions containing urea at concentrations of 0.5 M or more. Full activity is preserved at 0.2 M NaCI. At higher concentrations, activities are reversibly inhibited. In 50 mM sodium barbital (pH 8.2), 50 mM sodium citrate, 10 mM COSO4 glass-bound CAP is fully active in presence of 10% dimethyl sulfoxide; glass-bound AP-P is fully active up to 5% and at 10% dimethylsulfoxide the activity is 30% lower. The inhibition is reversible. In contrast to soluble CAP which loses activity partially at SDS concentrations exceeding 1 Izg/ml and has no activity at 100 ~g/ml, glass-bound CAP is fully active in presence of SDS concentrations up to 10/xg/ml, and at 100 tzg/ml still has 80% of its original activity. Adsorption. No adsorption to the glass-bound enzymes under the assay conditions is observed with any of the substrates used. Only cytochrome c and substance P are slightly adsorbed in absence of NaCI.

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Absence of Endopeptidase Activity. Denatured cytochrome c which has an acetylated N terminus is treated with the immobilized aminopeptidases under the assay conditions. No ninhydrin-positive material is formed as would be expected if endopeptidases were present in the enzyme preparations. Dependence on pH and Temperature. The pH range of maximal activity of glass-bound CAP and AP-P is 7.8-8.2 and 8.1-8.4, respectively. The optimal temperature is at 40°; at 55° the enzymes are irreversibly inactivated within 1 min. Metal Ion Requirements. 15 The dependence of enzymatic activity on COSO4 of both glass-bound enzymes is bell shaped. For CAP the inhibition at high Co 2+ concentrations is abolished in presence of cobalt citrate which acts as a metallobuffer. For AP-P the inhibition is partially reduced. In the presence of the metallobuffer (50 mM citrate) maximal activity for CAP and AP-P is reached at a total Co z+ concentration of 1.0 mM (1 /xM free Co 2÷) and 5-10 mM (4-9/~M free Co2+), respectively. Dissociation of the enzyme-Co 2+ complex is very slow for glass-bound CAP, and when once exposed to Co 2+ ions, the beads can be washed with Co2+-free buffer and used repeatedly for hydrolysis of peptides in absence of Co z+ ions. This stabilization effect caused by immobilization is not observed for cellulose-bound CAP and glass-bound AP-P. The association constants derived for the interaction of Co 2+ with glass-bound CAP and AP-P are KE-co = 4.5 X 106 M -1 and KE-co = 8.2 x 105 M -1, respectively. 13The immobilization of CAP to glass beads has no effect on the Km found with leucine p-nitro anilide as the substrate. The value (0.5 mM) is the same for the soluble and the glass-bound enzyme and is not influenced by flow rate of the substrate through the enzyme-coated beads packed into a column, indicating that no major diffusion barrier interferes with the enzyme activity. Enzyme Activity Measurements

Soluble Enzymes. CAP or AP-P (10-50/zl, 0.01-0.2 units) is incubated with Pro-Gly-Pro or poly-L-proline solution (1.0 ml), respectively, for 30 min at 40°. Ninhydrin reagent (5 ml) is added to terminate the reaction and the solution is heated for 30 rain in a boiling water bath. After cooling, the absorption at 520 nm is measured. The proline concentration is calculated with the aid of a calibration curve constructed with known concentrations of proline (10-200 nmol). 15 G. Fleminger and A. Yaron, Biochim. Biophys. Acta 789, 245 (1984).

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Immobilized Enzymes. Glass-bound CAP or AP-P (100 mg dry weight) with the Pro-Gly-Pro or poly-L-proline solution, respectively, is rotated at 80 rpm in a 5-ml plastic syringe equipped with a sintered plate and partially immersed in a 40 ° water bath. The needle outlet is plugged by sticking it into a rubber stopcock. After 5 min the proline concentration in the supernatant is determined as described for the soluble enzymes. Definition of Unit. One unit of activity is defined as the amount of soluble or glass-bound CAP or AP-P which catalyzes the formation of 1.0 /zmol of proline in 30 min under the assay conditions. In addition to the above described assays, colorimetric and fluorimetric methods have been developed. CAP can be assayed spectrophotometrically with leucine p-nitroanilide as the substrate. ~6 For aminopeptidase P the intramolecularly quenched fluorogenic substrate Phe(p-NOE)-Pro-Pro-HN-CHz-CH2-NH-ABz, where Phe(p-NO2) is the pnitrophenylalanyl quenching group and ABz is the 2-aminobenzoyl fluorophore, was developed. 9 An improvement in sensitivity is achieved ~° by replacing the Phe(p-NO2) group with N~-(2,4-dinitrophenyl)-L-lysine. A colorimetric method with Gly-Pro-Hyp is used in the isolation of the hog kidney enzyme. ~ Procedure for Sequential Hydrolysis of Polypeptides with Immobilized Aminopeptidases Glass beads (100 mg dry weight) containing immobilized aminopeptidase and the solution of the peptide (0.2-0.5 raM, 1 ml) in the assay buffer containing 0.1 M NaCI and norleucine (0.2-0.5/zmol) are incubated at 40 ° as described under the assay procedure, and aliquots of 10-100/zl of the supernatant are withdrawn for amino acid analysis at different time intervals. For continuation of the hydrolysis with the second enzyme the entire supernatant is pressed through a sintered disk into another syringe containing the other enzyme beads. The enzyme-coated glass beads are washed with the assay buffer prior to each hydrolysis step. Samples (10100/zl) of the initial peptide solution, of enzymatic hydrolyzates, and of the same after total acid hydrolysis (6 N HCI, 110% 22 hr, in vacuo) and drying are diluted with 0.2 M citrate buffer, pH 2.2, and subjected to amino acid analysis using a Beckman Model 121 Automated Amino Acid Analyzer. The total hydrolysis of aliquots is required as a control for potential loss of peptides or their fragments by adsorption to the glass beads. Norleucine is used as an internal standard. 16 A. Carmel, E. Kessler, and A. Yaron, Eur. J. Biochem. 73, 617 (1977).

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Sequential Hydrolysis o f Peptides with Immobilized Aminopeptidases Two kinds of peptides serve as model substrates for the immobilized aminopeptidases: (1) peptides devoid of proline, or those in which a proline residue is present at the amino end, which are hydrolyzed by CAP alone; and (2) peptides containing proline inside the polypeptide chain, requiring for their hydrolysis both CAP and AP-P. Hydrolysis of [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu, 0.2 mM) by immobilized CAP results in a rapid liberation of the N-terminal tyrosine (/1/2 = 3 min), which is followed by the fast appearance of glycine (tvz = 5 min) and a slower hydrolysis of the remaining dipeptide, Phe-Leu. Complete hydrolysis is achieved within 2 hr. The time course of the sequential hydrolysis of substance P octapeptide (Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, 0.5 mM) is shown in Table I. All of the N-terminal proline is released within 75 min, followed by the successive release of glutamine, phenylalanine, glycine, and leucine, as expected. After 135 min the hydrolysis is still not complete, apparently due to the slow cleavage of the Gly-Leu peptide bond and of the methionine amide. The octapeptide angiotensin II (Asn-Arg-Val-Tyr-Val-His-Pro-Phe, 0.5 mM), which contains a proline residue at position 7, is completely hydrolyzed in three steps, as shown in Table II. First, CAP is used to remove the five residues beginning with the N-terminal asparagine. Complete release of these amino acids is achieved after 1 hr. In the second step, AP-P is applied for 30 min to release histidine completely from the remaining tripeptide His-Pro-Phe. Further incubation with CAP for 30 min results in complete hydrolysis of the remaining dipeptide Pro-Phe. TABLE I ENZYMATIC HYDROLYSIS OF SUBSTANCE P OCTAPEPTIDEa Percentage of amino acid residues released Incubation time (min) 16 30 75 135

Pro xGln ~,.Gl%- Phe ~ Ph%- Gly - Leu - Met- NH2

55 80 98 105

23 50 90 100

7 15 76 90

0 3 27 75

0 0 22 68

0 0 0 15

a Concentration, 0.5 mM, in the assay buffer containing 5% dimethyl sulfoxide, using immobilized clostridial aminopeptidase (CAP) (100 mg dry weight).

176

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

TABLE II ENZYMATIC HYDROLYSIS OF

Step I

II III

Enzyme

Incubation time (min)

CAP CAP CAP CAP CAP AP-P CAP

3 5 10 20 60 30 30

[Asn ~, ValS]ANGIOTENSIN

II a

Cumulative percentage of amino acid residues released A s n - Arg 40 70 95 100 100 100 102

N.D. N.D. N.D. N.D. 95 95 N.D.

Val

- Tyr-

0 40-60 57-95 88-100 100 97 105

0 38 57 88 98 95 92

Val - His - Pro - Phe 0 0-20 15-53 80-92 100 97 105

N.D. N.D. N.D. N.D. 0 108 N.D.

0 0 0 0 0 0 100

0 0 0 0 0 0 90

a The peptide (0.5 raM) was incubated alternately with immobilized clostridial aminopeptidase (CAP) and aminopeptidase (AP-P) (100 mg dry weight each) and samples were withdrawn for amino acid analysis at various time intervals. The amounts of Val 3 and VaP released were estimated from the total amount of valine, asparagine, and tyrosine release. N.D., Not determined.

The applicability of the two immobilized aminopeptidases in achieving complete hydrolysis of polypeptide chains is further demonstrated with tuftsin (Th~-Ly~-P~-Arg where ~ CAP,--~ AP-P). The two enzymes are applied alternately as indicated by arrows above the sequence. In this case, the expected amino acids appear within 20 min at each step. The nonapeptide bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, 0.5 raM), containing three proline residues in position 2, 3, and 7, is completely hydrolyzed in four consecutive steps by alternately applying the immobilized AP-P and CAP as shown in Table III. The synthetic sequential polymer (Pro-Gly-Pro)r6 is consecutively hydrolyzed by alternately exposing the polymer solution to the two immobilized enzymes as shown in the following scheme (the numbers above the arrows represent the consecutive steps of hydrolysis):

112

P3r

4

I

Pro-Gly-Pro-Pro-Gly-Pro ....

In this experiment 10 hydrolysis steps were performed. In each step one equivalent of proline was found to be released as determined by colorimetric analysis. Altogether 50% of the total proline present in the substrate was released by the enzymes.

[17]

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AMINOPEPTIDASES FOR PROLYL PEPTIDES TABLE III ENZYMATIC HYDROLYSIS OF BRADYKININa

Step

Enzyme

Incubation time (rain)

I II III IV

AP-P CAP AP-P CAP

10 20 20 20

Cumulative number of equivalents of amino acid residues released Arg

Pro

Gly

Phe

Ser

0.9 N.D. N.D. 1.9

1.1 2.1 2.2 3.3

0 1.0 0.95 1.0

0 0.8 0.85 1.80

0 0 0.83 0.87

a Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, 0.5 mM) was hydrolyzed using immobilized aminopeptidase P (AP-P) and clostridial aminopeptidase (CAP) (100 mg dry weight each) alternatively.

Discussion Useful enzymatic preparations are obtained by coupling AP-P and CAP to amino-glass beads by the glutaraldehyde method. Resistance of cytochrome c to hydrolysis and the ordered release of amino acids during the hydrolysis, demonstrated for several peptides, shows that the preparations are free of interfering enzyme activities. The resistance of X-Pro bonds to hydrolysis by CAP and the absolute specificity of AP-P for Nterminal amino acids which are followed by a proline residue make it possible to degrade polypeptides up to any proline residue present in the peptide chain, resulting thereby in peptide fragments with Pro or X-Pro at the amino end. Substrates in the range from tripeptides to dodecapeptides were readily degraded. The stepwise degradation of the sequence-ordered polymer (Pro-Gly-Pro)~, consisting of an average of 30 amino acid residues, demonstrates that the enzymatic hydrolysis is applicable to quite high molecular weight substrates and that at least 10 successive transfers between the enzyme beads can be carried out with the release of a full equivalent of the expected amino acids in each step. Longer polypeptide chains such as reduced and carboxymethylated trypsin inhibitor from lima bean (Mr 9000) and soybean (Mr 21,500), reduced and carboxymethylated RNase (Mr 13,600), and apomyoglobin (Mr 17,900) are not hydrolyzed by either soluble or glass-bound CAP. Sequential degradation with the immobilized aminopeptidases may find application in the analysis and modification of natural and synthetic

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proline-containing oligopeptides. Immobilized CAP and AP-P may also be helpful in the sequencing of proline-containing peptides using dipeptidyl aminopeptidase I (DAP I; EC 3.4.14.1) and dipeptidyl aminopeptidase IV (DAP IV; EC 3.4.14.5). 17 Since these enzymes cannot hydrolyze the X-Pro bond, degradation stops two residues before proline. These two residues can be cleaved off by the combination of CAP and AP-P, thereby permitting continuation of the degradation by DAP I or DAP IV. Hydrolysis of polypeptides, using a mixture of various immobilized proteolytic enzymes, that proceeds almost to completion has been achieved. ~,18 The residual peptides, resisting further hydrolysis, have been shown to contain proline residues. The immobilized CAP and AP-P should therefore be helpful in completing their hydrolysis. ~7 H. C. K r u t z s c h and J. J. Pisano, this series, Vol. 47, p. 391. ~s j. L a s c h , R. Koelsch, P. Roth, A. Gabert, J. Marguardt, and H. H a n s o n , Acta Biol. Med. Ger. 35, 735 (1976).

[18] P e p t i d e S y n t h e s i s U s i n g I m m o b i l i z e d P r o t e a s e s

By HANS-DIETER JAKUBKE and ANDREAS Kt)NNECKE Although the well-established techniques of chemical peptide synthesis have been successful in making peptides and small proteins of biological and industrial interest (see Ref. I for several reviews), synthetic methodology is still in need of innovations. The use of enzymes should provide a biological alternative to chemical peptide synthesis. There has been a revival of interest in the ability of proteolytic enzymes to catalyze the formation of peptide bonds for the synthesis of biologically active peptides and the semisynthesis of proteins (see Ref. 2 for several reviews). The main attraction of proteases lies in their capacity to effect peptide bond formation stereospecifically without the need of side-chain protection. In protease-mediated peptide synthesis, the enzymatic specificity prevents the formation of undesired by-products often formed in the course of conventional chemical synthesis. E. Gross and J. Meienhofer, " T h e Peptides: Analysis, Synthesis, Biology," Vols. 1-9. A c a d e m i c Press, N e w York, 1979-1987. z H.-D. Jakubke, P. Kuhl, and A. K r n n e c k e , Angew. Chem. Int. Ed. Engl. 24, 85 (1985).

METHODS 1N ENZYMOLOGY, VOL. 136

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proline-containing oligopeptides. Immobilized CAP and AP-P may also be helpful in the sequencing of proline-containing peptides using dipeptidyl aminopeptidase I (DAP I; EC 3.4.14.1) and dipeptidyl aminopeptidase IV (DAP IV; EC 3.4.14.5). 17 Since these enzymes cannot hydrolyze the X-Pro bond, degradation stops two residues before proline. These two residues can be cleaved off by the combination of CAP and AP-P, thereby permitting continuation of the degradation by DAP I or DAP IV. Hydrolysis of polypeptides, using a mixture of various immobilized proteolytic enzymes, that proceeds almost to completion has been achieved. ~,18 The residual peptides, resisting further hydrolysis, have been shown to contain proline residues. The immobilized CAP and AP-P should therefore be helpful in completing their hydrolysis. ~7 H. C. K r u t z s c h and J. J. Pisano, this series, Vol. 47, p. 391. ~s j. L a s c h , R. Koelsch, P. Roth, A. Gabert, J. Marguardt, and H. H a n s o n , Acta Biol. Med. Ger. 35, 735 (1976).

[18] P e p t i d e S y n t h e s i s U s i n g I m m o b i l i z e d P r o t e a s e s

By HANS-DIETER JAKUBKE and ANDREAS Kt)NNECKE Although the well-established techniques of chemical peptide synthesis have been successful in making peptides and small proteins of biological and industrial interest (see Ref. I for several reviews), synthetic methodology is still in need of innovations. The use of enzymes should provide a biological alternative to chemical peptide synthesis. There has been a revival of interest in the ability of proteolytic enzymes to catalyze the formation of peptide bonds for the synthesis of biologically active peptides and the semisynthesis of proteins (see Ref. 2 for several reviews). The main attraction of proteases lies in their capacity to effect peptide bond formation stereospecifically without the need of side-chain protection. In protease-mediated peptide synthesis, the enzymatic specificity prevents the formation of undesired by-products often formed in the course of conventional chemical synthesis. E. Gross and J. Meienhofer, " T h e Peptides: Analysis, Synthesis, Biology," Vols. 1-9. A c a d e m i c Press, N e w York, 1979-1987. z H.-D. Jakubke, P. Kuhl, and A. K r n n e c k e , Angew. Chem. Int. Ed. Engl. 24, 85 (1985).

METHODS 1N ENZYMOLOGY, VOL. 136

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The application of enzymes for organic synthesis is increasing rapidly in importance 3 and has been stimulated to a considerable degree by the availability of immobilized enzymes. Proteases can be immobilized without loss of function, and the potential of immobilized proteolytic enzymes for peptide synthesis has been demonstratedY The purpose of this contribution is to draw attention to peptide coupling reactions on a preparative scale in which immobilized proteases can be used with the advantage of avoiding reaction conditions which are normally required for chemical condensations. The simplified work-up procedure that becomes possible when immobilized proteases are used, the long-term stability of the immobilized enzyme preparations, and the successful reutilization experiments are among the advantages to such an approach. Approaches to Enzymatic Peptide Synthesis In general, the methods of protease-mediated peptide bond formation can be classified into two basic strategies according to the type of carboxyl component used. In the first strategy this component has a free carboxyl terminus, and the formation of the peptide bond occurs with thermodynamic control as the reverse of peptide hydrolysis. In the second strategy the carboxyl component is employed in an activated form, mainly as an alkyl ester, and the synthesis occurs with kinetic control by competitive partitioning of a rapidly formed acyl-enzyme intermediate between the nucleophile and water. These two strategies are fundamentally different due to the energy required for the conversion of the substrates to the peptide products. In the following two subsections, some kinetic and thermodynamic aspects of protease-catalyzed peptide synthesis are briefly discussed and illustrated by representative experimental procedures. The discussion is valid for both native and immobilized proteases and provides a theoretical basis for understanding the strategy and the problems associated with the use of proteases as biocatalysts for peptide bond formation. The Kinetic Approach Serine and cysteine proteases are known to catalyze acyl transfer from specific substrates to various nucleophiles via an acyl-enzyme intermedi3 j. B. Jones, this series, Vol. 44, p. 831. 4 A. KOnnecke, R. Bullerjahn, and H.-D. Jakubke, Monatsh. Chem. 112, 469 (1981). 5 A. Kfnnecke, M. H~insler, V. Schellenberger, and H.-D. Jakubke, Monatsh. Chem. 114, 433 (1983).

180

[18]

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ate. In reactions of this type, the protease reacts rapidly with an amino acid or peptide ester R]COOR 3 to form an acyl-enzyme intermediate R~CO-E that reacts, in competition with water, with the amino acid- or peptide-derived nucleophile H2NR 2 to form a new peptide bond6,7: RIco-NHR2

I<s R1COOR3

÷

E

+

E

k2

RICO-E

k3[H20] RIcooH + E

(1) The partitioning of the acyl-enzyme intermediate between water and the added nucleophile is the rate-limiting step. Under kinetic control the peptide product RICO-NHR z should accumulate, provided k4[HzNR 2] ~> k3[H20]. However, with increasing reaction time, a soluble product will be degraded in a secondary hydrolysis step. Therefore, the reaction conditions must be carefully considered in order to obtain the optimal product yield. Peptide bond formation by partitioning of acyl-enzyme intermediates may also be controlled by the specificity of the enzyme. Structural factors in the nucleophile which increase the extent to which it is bound productively at the S'-subsite of the enzyme promote synthesis. The type and concentration of the nucleophile influence the partitioning of the acylenzyme intermediate, and influence therefore the success of peptide synthesis. Since only the free-base form of the amino component reacts with the acyl-enzyme intermediate, and the pK of this reactant is about 8, the pH of the reaction mixture should preferably be higher than this. Examples

Materials and Immobilization Procedures Enzymes are all from commercial sources: o~-chymotrypsin (EC 3.4.21.1.), Le~iva (Prague, Czechoslovakia); papain (EC 3.4.22.2.), J. Fastrez and A. R. Fersht, Biochemistry 12, 2025 (1973). 7 A. Kfnnecke, V. Schellenberger, H.-J. Hofmann, and H.-D. Jakubke, Pharmazie 39, 785 (1984).

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Merck (Darmstadt, FRG); trypsin (EC 3.4.21.4) and thermolysin (EC 3.4.24.4.), Boehringer-Mannheim (FRG). Carriers for enzyme immobilization include Enzacryl AA and AH from Koch Light (now distributed by Aldrich), and carboxymethyl cellulose powder from Reanal (Budapest, Hungary). The utility of these materials for covalent binding of enzymes has been described in previous volumes of this series. Macroporous silica with 20 nm pore diameter is silanized, succinylated, and subsequently transformed into activated N-hydroxysuccinimide ester, 8 and the carboxymethylcellulose powder is used as hydrazide. 9 Immobilization of chymotrypsin ~° and thermolysin II to Enzacryl AH and trypsin 9 to carboxymethylcellulose azide is accomplished by standard procedures. Activated silica is coupled with chymotrypsin (1 g carrier and 100 mg enzyme) in 8 ml 0.05 M borate buffer (pH 8.4) for 2 hr at room temperature. Crude papain (3 g) is suspended in 15 ml 0.1 M phosphate buffer (pH 7). After the solution is stirred for 1 hr, insoluble material is separated by centrifugation. The supernatant (60 units/ml) is incubated with 1 g activated Enzacryl AA for 24 hr at 4 °. Trypsin (100 mg in 8 ml 0.1 M phosphate buffer, pH 7) is coupled to 1 g activated Enzacryl AA for 2 hr at room temperature. Residual reactive groups of the carriers are blocked with aqueous 0.5 M ammonium hydroxide/1 M ammonium chloride (acyl azide or ester carriers) or 0.01% aqueous ot-naphthol solution (Enzacryl AA). Adsorbed protein is removed by exhaustive washings of the gels. Immobilized enzymes are stored in water at 4 °, except immobilized papain, which is washed with methanol and dried at 70° . A s s a y 12 of immobilized chymotrypsin preparations is spectrophotometrically performed with N-glutaryl-Leu-Phe-Nan ~2a as substrate. ~3 Trypsin and papain are assayed titrimetrically with Bz-Arg-OEt as substrate using the Radiometer pH-stat TTT ld assembly at pH 7.9 and 6.2, respectively. Titrimetric assay procedures are more convenient for immobilized proteases, 12 and the measure of activity per gram of support is 8 H.-H. Weetall, this series, Vol. 44, p. 134. 9 E. M. Crook, K. Brocklehurst, and C. W. Warthon, this series, Vol. 19, p. 963. 10j. K. Inman and H. M. Dintzis, Biochemistry 8, 4074 (1969). i1 R. Epton, B. L. Hippert, and T. H. Thomas, this series, Vol. 44, p. 84. 12 B. Mattiason and K. Mosbach, this series, Vol. 44, p. 335. 12a Amino acid symbols except Gly denote L configuration. Other abbreviations: Ac, acetyl; Bz, benzoyl; Z, benzyloxycarbonyl; OMe, methyl ester; OBu t, tert-butyl ester; Nan, 4nitroanilide. Symbols and abbreviations conform to the IUPAC-IUB Commission recommendations. t3 H.-D. Jakubke, H. D~iumer, A. K6nnecke, P, Kuhl, and J. Fischer, Experientia 36, 1039 (1980).

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sufficient for practical use. The protein content is determined from the difference in absorbance of the enzyme solution prior to and after immobilization. Substrates and reagents are from Serva (Heidelberg, FRG) and Reanal (Budapest, Hungary), respectively. All chemicals and solvents are reagent grade. The amino acid and peptide derivatives used as carboxyl and amino components in the enzyme-catalyzed condensations are drawn from our collection or are synthesized by standard procedures. Prior to use, their identity and purity are confirmed by thin-layer chromatography, melting point determination, elementary analysis, and HPLC. Porcine des-Ala (B30) insulin and Thr-OBu t are kindly supplied by Dr. K.-D. Kaufmann, Institute of Drug Research, Academy of Sciences of GDR. High-performance liquid chromatography (HPLC) is carried out with Liquochrom 307 equipment of Labor MIM (Budapest, Hungary), using isocratic elution and variable-wavelength UV detection. Separations are performed with a Hewlett-Packard prepacked 200 × 4.6 mm column containing LiChrosorb RP-18, 10 ~m, and a homemade 150 × 4 mm column packed with high-temperature silanized RP-6 material 14based on LiChrosorb Si-100, 10 /~m. Solvents are degassed separately by sonication in vacuo, if necessary. It should be stressed that immobilization, assays, and also the enzymatic synthesis (see below) do not require special equipment, and, therefore, can be performed in any laboratory.

Chymotrypsin-Catalyzed Synthesis of Ac-Phe-Ala-NH215 Chymotrypsin bound to macroporous silica (250 mg wet gel containing about 2.5 mg of immobilized protein) is placed in a reaction vessel commonly used for solid-phase peptide synthesis with a G 4 frit at the bottom (10 mm in diameter), fitted with an overhead stirrer. A solution of AlaNH2, obtained by neutralization of 49.4 mg (0.4 mmol) Ala-NH2 • HCI with 0.1 ml cold 4 N NaOH and dilution with 1.6 ml 0.2 M carbonate buffer (pH 10), is added, and the reaction mixture is stirred efficiently (avoiding any contact of the stirrer with the glass wall of the vessel), The coupling reaction is initiated by addition of 0.3 ml of a stock solution of Ac-PheOMe in dimethylformamide containing 44.2 mg (0.2 mmol of the ester). After 12 min the solution is removed by suction filtration, and the immobilized enzyme is washed successively with three 3-ml portions of water. The next coupling cycle can be started by adding the substrate solutions in the order given above. The combined filtrates are worked up by addition 14 T. Welsch and H. Frank, J. Chromatogr. 267, 39 (1983). 15 A. K6nnecke and H.-D. Jakubke, unpublished results.

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of 1 ml 1 N HCI and rotary evaporation to dryness. The residue is dried in vacuo, and inorganic salts are removed by extraction of solid with three 30-ml portions of boiling ethyl acetate. The ethyl acetate extract is subsequently rotary evaporated to dryness. The residue is dissolved in a minimum amount of methanol, and the peptide is precipitated by addition of two volumes of diethyl ether. Yield was 34.4 mg (62%); mp 241-242 ° (see Ref. 6; mp 242-244 °) unchanged after recrystallization from methanol.

Papain-Catalyzed Formation of Z-Arg-Ala-NH216 Phosphate buffer (2 ml, 0.1 M, pH 8; with 0.3 M NaCl, 1 mM EDTA) containing 0.1 M Z-Arg-OMe • HCI and 0.2 M AIa-NHz • HCI as reactants is placed in the titration beaker of the autotitration device TTT ld thermostated at 35°. The solution is rapidly stirred, and the pH adjusted to 8 with ! N NaOH. The condensation is initiated by addition of 20 mg dry Enzacryl AA-papain (6.9 BAEE units). A slow stream of argon is bubbled through the solution and the reaction is allowed to proceed for 23 min with automatic readjustment of the pH to 8. The extent of synthesis may roughly be estimated from the amount of base consumed by the formation of Z-Arg-OH. More precisely, the reaction is monitored by HPLC using RP-6 column and methanol/0.05 M phosphate buffer, pH 7 (30/70, v/v) as eluent; capacity factors used were k' 2.21 for Z-Arg-OH, 3.28 for Z-ArgAla-NH2, and 8.86 for Z-Arg-OMe with methanol as to marker. After 23 min Z-Arg-OMe was completely consumed and 78% Z-Arg-Ala-NH2 along with 22% Z-Arg-OH were formed. After the immobilized papain was washed with water, it was used in a repeat of the experiment, which provided the same result after 26 rain.

Trypsin-Catalyzed Synthesis of Z-Lys-Leu-NHe 5 Enzacryl AA-trypsin (350 mg wet gel containing about 2.5 mg of immobilized protein or 70 units) is placed in a reaction vessel similar to the one used for the synthesis of Ac-Phe-Ala-NH2. A solution of 79.4 mg (0.24 mmol) Z-Lys-OMe. HCI and 60 mg (0.36 mmol) Leu-NHE. HC1 in 2 ml t M carbonate buffer (pH 10) containing 12.5% (v/v) dimethylformamide is added to the catalyst, and the mixture stirred vigorously. After 45 min the liquid phase of the reaction mixture is removed by suction, and the precipitated product removed from the immobilized trypsin by extraction with three 2-ml portions of precooled (4°) methanol followed by 3 ml ~6 V. Schellenberger, A. K6nnecke, and H.-D. Jakubke, in "Peptides, 1984" (U. Ragnarsson, ed.), p. 201. Almqvist & Wiksell, Stockholm, 1984.

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water. The immobilized enzyme is washed additionally with five 3-ml portions of water, and the next coupling cycle is started by adding new solutions of substrates. After rotary evaporation of the methanol from filtrate, the residue is dissolved in about 50 ml water, applied directly to an Amberlite IRA-400 ion-exchange column (2 x 6 cm), and subsequently eluted with water until no change in optical density occurs. The eluate is rotary evaporated and the residue dried in vacuo. The yield was 51.8 mg (66%) pure (TLC and HPLC) Z-Lys-Leu-NH2; mp 140-145 ° (dec.); [a]~3 - 10.2 ° (c 1; MeOH). After five cycles the yield still amounted to 67%, and the activity of the immobilized trypsin was 71% of the initial acdespite repeated treatment with methanol. Chymotrypsin-Catalyzed Synthesis o f Z-Gly-Phe-Leu-NHe 4

Wet Enzacryl AH-chymotrypsin (350 mg; about 3 mg of immobilized protein) is placed in a small tube equipped with a magnetic stirrer. Stock solutions of 1.6 ml 0.2 M carbonate buffer (pH 10) containing 23.4 mg (0.2 mmol) Leu-NH2. HCI and 400/zl carbon tetrachloride containing 73.5 mg (0.2 mmol) Z-Gly-Phe-OMe are added. After vigorous stirring for 2 hr, the solution is removed by suction filtration and the enzyme is treated with three 5-ml portions of dimethylformamide for removing the precipitated product from the immobilized biocatalyst. The immobilized enzyme is washed with 5 ml water, and the next coupling cycle is started by adding reactants. The dimethylformamide extract is diluted with about 40 ml water and stored overnight at 4 °. The precipitated product is isolated by filtration and dried in vacuo. Yield was 60 mg (64%); mp 209-211°; [a] 22 - 20.1 ° (c 1; MeOH); after the third use of the immobilized enzyme preparation, the yield still amounted to 57% (53.4 mg). The Thermodynamic Approach The thermodynamic barrier to peptide bond synthesis is predominantly due to the energy required for the transfer of a proton from the positively charged a-amino group to the negatively charged a-carboxyl group. The equilibrium constant Kco, of the conversion of the nonionized reactants into the product is determined by the concentration of the nonionized forms in Kion : K,on RICOO - + Ha+NR 2 .

K¢on ~ RICOOH

+ H2NR:

.

• R1CO--NHR:

+ H20

(2)

Because of the unfavorable equilibrium position, relatively high concentrations of the reactants are required. In order to achieve an appreciable

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185

degree of synthesis, one needs to resort to some expedients and unusual techniques. 2 Furthermore, the reaction can be shifted toward synthesis by the insolubility of the newly formed peptide when the solubility of the product is below the equilibrium concentration. The main reason for promoting synthesis is to increase the equilibrium constant K~o, for proton transfer from the H3N + to C O 0 - end groups of reactants. This is accomplished by addition of high concentrations of water-miscible organic cosolvents.17 A similar favorable influence on this equilibrium constant can also be achieved by working in biphasic organic-aqueous mixtures. 18Furthermore, in special cases water-immiscible cosolvents promote synthesis by continuous removal of product from equilibrium if the peptide formed is much more soluble in the organic solvent.19 The only function of the protease is to accelerate the attainment of the equilibrium Kcon. Examples

Thermolysin-Catalyzed Synthesis of Z-Phe-Leu-NH25 To a small tube fitted for magnetic stirring, 29.9 mg (0.1 mmol) Z-PheOH, 21 mg (0.16 mmol) Leu-NH2, and 2 ml 0.2 M Tris-maleate buffer (pH 7) are added. The condensation is initiated by adding 300 mg wet Enzacryl AH-thermolysin (containing about 0.6 mg protein). The coupling reaction is allowed to proceed for 20 hr at room temperature with constant stirring. The precipitated product is separated from the immobilized thermolysin by treatment with three 3-ml portions of cold dimethylformamide followed by addition of 5 ml water. The extract was diluted with 35 ml water and stored at 4° overnight. The precipitated product is filtered and dried in vacuo, yielding 34.5 mg (84%) pure (TLC and HPLC) Z-Phe-Leu-NH2, mp 191-192 ° (see Ref. 19; mp 190-192°). After four uses of the immobilized thermolysin, the yield still amounted to 70%.

Trypsin-Catalyzed Formation of Bz-Gly-Lys-Leu-OMe 2° A plastic vial (2 ml) is filled with 7.3 mg (0.025 mmol) Bz-Gly-Lys-OH and 90.8 mg (0.5 mmol) Leu-OMe. HCI. The substrates are dissolved by adding 400/xl dimethyl sulfoxide and 50/zl 0.2 M Tris-HC1 buffer (pH 7, 17 G. 18 K. 19 p. 2o A.

A. Homandberg, J. A. Matfis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). Martinek and A. N. Semenov, J. Appl. Biochem. 3, 93 (1981). Kuhl and H.-D. Jakubke, Z. Chem. 22, 407 (1982). K6nnecke, M. Hansler, and H.-D. Jakubke, Z. Chem. 26, 292 (1986).

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[18]

10 mM CaC12). The apparent pH is adjusted to 7 with 8 N sodium hydroxide, and buffer is added to a total volume of 500/zl. The condensation is initiated by adding 150 mg wet cellulose-bound trypsin (66 units), and the reaction allowed to proceed for 2 hr at room temperature with constant shaking. HPLC analysis (RP-6 column eluted with methanol/0.1% phosphoric acid (1 : 1, v/v)) indicated the formation of 93% Bz-Gly-Lys-Leu-OMe. After five uses of the immobilized enzyme preparation, no loss in yield or activity was observed.

Trypsin-Catalyzed Formation of Human Insulin tert-Butyl Ester from Porcine 30&deAla-insulin and Threonine tert-Butyl Ester 30B-deAla-porcine insulin (30 mg, 5/xmol) and 52.5 mg (0.3 mmol) ThrOBu t are dissolved in 300/zl dimethylformamide and 150/zl 0.25 M TrisHCI buffer (pH 6.6, 10 mM CaCI2) in a plastic vial (2 ml). The apparent pH is adjusted to 6.6 with 6 N HCI, and buffer added to a final volume of 500 /A. Carboxymethylcellulose-bound trypsin (150 mg wet gel containing 66 units of enzyme) is added, and the coupling reaction is allowed to proceed at room temperature with constant shaking. As indicated by HPLC, equilibrium is reached within 90 min. Yield was 70% human insulin tert-butyl ester; the amount of 23B-30Bde-octapeptide insulin was below 2%. The analytical separations were performed using RP-18 column eluted with A + B21 (70:30, v/v) [A is 0.1 M monobasic sodium phosphate in water/2-methoxyethanol (95 : 5, v/v) adjusted to pH 2 with 85% phosphoric acid; B is acetonitrile/2-methoxyethanol (95 : 5, v/v)]. Due to the sensitivity of the retention of insulins to small changes of eluent composition, nearly reproducible results can only be obtained by mixing the eluent by weight; k' was 3.6 for 30B-deAla insulin and 10.8 for human insulin ester. Isolation of the product could be accomplished by conventional chromatographic procedures as described in literature. 22 Discussion As already discussed by Jones 3 in a previous volume of this series, the use of soluble and immobilized enzymes in synthetic organic chemistry can be of advantage if the reactions to be catalyzed require high regioand/or stereospecificity. A very important problem of classical peptide synthesis is to maintain chiral integrity during peptide bond formation. 2J W. M6nch and W. Dehnen, J. Chromatogr. 147, 415 (1978). 22 A. Jonczyk and H.-G. Gattner, Hoppe-Seyler's Z. Physiol. Chem. 362, 1591 (1981).

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This is guaranteed using proteases as stereospecific biocatalysts. The experimental procedures given in this contribution demonstrate that covalently bound proteases catalyze peptide bound formation as effectively as the native enzymes, since the substrate specificity and stereospecificity appear not to be altered by immobilization. Although the use of immobilized enzymes offers some important advantages, several requirements must be fulfilled: (1) The immobilization procedure should be as simple as possible and provide high and reproducible activity yields. Covalent coupling procedures should be preferred to other immobilization techniques to minimize leakage. (2) Especially for thermodynamically controlled synthesis, the need of rapid attainment of the equilibrium requires high enzyme concentrations. (3) The support material should be cheap, easily derivatized, and stable under the reaction conditions, and should possess no significant swelling capacity. The essential advantages in the application of covalently bound proteases can be summarized as follows: (1) The immobilized protease can easily be recovered from the reaction mixture. (2) The peptides synthesized are free of contamination by proteolytic activities and denaturated protein. (3) Due to the increased stability in the presence of organic solvents, higher concentrations of such solvents can be used to influence the position of the thermodynamic equilibrium. For example, in the presence of 80% (v/v) dimethyl sulfoxide, the equilibrium concentration of Bz-GlyLys-Leu-OMe (see procedure) is 93%. 20 In contrast, native trypsin is already inactivated by concentrations higher than 65% (v/v) of this cosolvent. (4) The simple recovery and enhanced stability of the immobilized enzyme preparations guarantee the multiple reuse of the biocatalysts. Kinetically controlled syntheses promise favorable results for use with immobilized proteases, because only low concentrations of enzymes and organic solvents are needed merely to dissolve the reactants. On the other hand, thermodynamically controlled reactions need high enzyme concentrations for rapid attainment of equilibrium. A favorable position of the equilibrium can be obtained only in the presence of high concentrations of organic cosolvents (see procedures for human insulin ester or Bz-GlyLys-Leu-OMe) which, unfortunately, reduce the activity of the enzyme dramatically. Prognosis Due to the high degree of specificity of proteases, enzymatic peptide synthesis does not have the versatility of chemical coupling methods and therefore suffers from some limitations. It is evident from the foregoing discussion that some of the known deficiencies (e.g., coprecipitation of

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soluble proteases with products, inactivation of native enzymes by organic solvents, costs of enzymes) can be overcome by employing immobilized instead of native proteases. For continuous processing, the introduction of suitable solubilizing protective groups may be helpful in overcoming solubility problems with oligopeptides. A main goal of research in this field is to find an appropriate set of proteolytic enzymes which will widen the scope of enzymatic peptide bond formation. It may be expected that the methodology of protease-catalyzed peptide synthesis and semisynthesis of proteins using the approach suggested here will be considerably improved in the near future. Acknowledgments We are grateful to Dr. T. Welsch for generous help with analytical HPLC. We are much obliged to Dr. John H. Jones for reading and commenting on the manuscript.

[19] A c t i v i t y a n d C o n f o r m a t i o n of E n z y m e s in R e v e r s e M i c e l l a r Solutions

By P. L. LuIsI and B. STEINMANN-HOFMANN In this paper some of the basic concepts and latest results concerning enzymes in reverse micelles are reviewed. In a first part we consider the general experimental aspects, and in the second part a review of enzyme activity and conformation is presented. Of necessity, some important aspects of the field are not adequately covered, for example, the thermodynamic and kinetic problems connected with the formation and stabilization of reverse micelles. Also the biological aspects are not treated in this review, but the reader is referred to other reviews or books that have appeared in the last few years ~-5 as well as to the series of articles by Eicke, Robinson, Magid, Hauser, Montal, El Seoud, Laane, de Schryver, and their co-workers in Ref. 6. J. H. Fendler, "Membrane Mimetic Chemistry," Chap. 3. Wiley (Interscience), New York, 1982. 2 H. F. Eicke, Top. Curr. Chem. 87, 85 (1980). 3 K. Martinek, Eur. J. Biochem. 155, 453 (1985).

METHODS IN ENZYMOLOGY, VOL. 136

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[19]

soluble proteases with products, inactivation of native enzymes by organic solvents, costs of enzymes) can be overcome by employing immobilized instead of native proteases. For continuous processing, the introduction of suitable solubilizing protective groups may be helpful in overcoming solubility problems with oligopeptides. A main goal of research in this field is to find an appropriate set of proteolytic enzymes which will widen the scope of enzymatic peptide bond formation. It may be expected that the methodology of protease-catalyzed peptide synthesis and semisynthesis of proteins using the approach suggested here will be considerably improved in the near future. Acknowledgments We are grateful to Dr. T. Welsch for generous help with analytical HPLC. We are much obliged to Dr. John H. Jones for reading and commenting on the manuscript.

[19] A c t i v i t y a n d C o n f o r m a t i o n of E n z y m e s in R e v e r s e M i c e l l a r Solutions

By P. L. LuIsI and B. STEINMANN-HOFMANN In this paper some of the basic concepts and latest results concerning enzymes in reverse micelles are reviewed. In a first part we consider the general experimental aspects, and in the second part a review of enzyme activity and conformation is presented. Of necessity, some important aspects of the field are not adequately covered, for example, the thermodynamic and kinetic problems connected with the formation and stabilization of reverse micelles. Also the biological aspects are not treated in this review, but the reader is referred to other reviews or books that have appeared in the last few years ~-5 as well as to the series of articles by Eicke, Robinson, Magid, Hauser, Montal, El Seoud, Laane, de Schryver, and their co-workers in Ref. 6. J. H. Fendler, "Membrane Mimetic Chemistry," Chap. 3. Wiley (Interscience), New York, 1982. 2 H. F. Eicke, Top. Curr. Chem. 87, 85 (1980). 3 K. Martinek, Eur. J. Biochem. 155, 453 (1985).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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189

Preparative Aspects

Purification of Aerosol OT The system which has mostly been used in connection with enzymes solubilized in hydrocarbon micellar solutions is isooctane/water AOT, where AOT stands for Aerosol OT, namely bis(2-ethylhexyl)sodium sulfosuccinate. In turn, the reason for the use of this system for enzymes CH

yH~-- CO0- CH2-- CH- CH2-- CH2--CH2--CH3 NO® @03 S-- CH-CO0- C H ~- ?H- CH2--CH ~- CH~- CH ~

#H2 CH 3 Bis(2-ethylhexyl)sodium sulfosuccinate, "Aerosol OT" (AOT)

stems simply from the fact that it is the most studied system per se due to the work of Eicke in Basel} ,7 Reverse micellar solutions are simply prepared by dissolving AOT in a hydrocarbon, up to a concentration ranging between 25 and 300 mM, then adding the desired amount of water (typically in the range of 0.1-5%), and shaking by hand (usually no sonication is needed), until a clear solution is obtained. There is of course only a limited region of values of AOT-water concentration and temperature where the reverse micellar aggregates are stable. This is shown in Fig. 1, which is a typical phase diagram 8 representing the different equilibria existing in this ternary system. AOT can be obtained from different commercial sources. For most technical applications, its purification and a check of chemical purity are not essential, but in fine chemical work (e.g., spectroscopy and enzymatic activity) a series of artifacts can originate from impurities in the surfactant. The most commonly employed commercial sources of AOT are Fluka (product 86140), Cyanamid (24967-01 Complenix-100), Serva (20540), and Sigma (D0885). In all these samples one can detect UVabsorbing impurities and acid impurities, as welt as salts.

4 p. L. Luisi and L. S. Magid, Crit. Rev. Biochem. 20, 409 (1986). 5 p. L. Luisi, Angew. Chem. 97, 446 (1985). 6 p. L. Luisi and B. Straub (eds.), "Reverse Micelles." Plenum, New York, 1984. 7 M. Zulauf and H. F. Eicke, J. Phys. Chem. 83, 480 (1979). 8 B. Tamamushi and N. Watanabe, Colloid Polymer Sci. 258, 174 (1980).

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J-C8H 18

H20

50

AOT

FIG. I. Phase diagram of aerosol OT/water/isooctane system at 25°. L~, Aqueous micellar solution; 2L, emulsion; L2, reverse miceUar solution; 2C, liquid crystals of lamellar (D) and hexagonal (F) types; L + LC, liquid crystals in equilibrium with solution (mainly L2). From Tamamushi and Watanabe. 8

For our purposes (spectroscopic work and enzyme activity determinations) we found that the purification methods described in the literature were not entirely satisfactory. Some of our first experiments, in which we simply purified AOT according to literature, reflect this situation, for example our first paper on a-chymotrypsin. 9 The extreme pH sensitivity of the activity data was due in part to acid impurities, as later documented. 6 We now rely entirely on a HPLC purification procedure, which is satisfactory for eliminating UV-absorbing and acid impurities from the commercial samples. The procedure is as follows. A preparative reversed-phase HPLC column (~Bondapak C~8, Perkin-Elmer, 25 × 2.5 cm) is used. The elution mixture is methanol-water 75:25 (v/v) with a flow rate of 10 ml/min. AOT from Serva (300 mg/ml) is dissolved in the same mixture, and 5 ml of this solution is injected at each run. Both UV and RI (refractive index) detection give the same elution profile (Fig. 2a). The fractions of the central peak are collected. Typically, about 5.5 g of purified AOT can be obtained after 10 injections ( - 1 day of work), which corresponds to an overall yield of 52%. Figure 2, b and c ~° show the UV spectra and the titration curve for our current AOT preparation method compared to other methods. The UV absorbance between 250 and 340 nm is clearly the lowest of all AOT samples. The pH titration curve shows 9 S. Barbaric and P. L. Luisi, J. Am. Chem. Soc. 103, 4239 (1981). l0 p. L. Luisi, P. Meier, V. E. Imre, and A. Pande, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds.), p. 332. Plenum, New York, 1984; see also B. Steinmann, H. J/ickle, and P. L. Luisi, Biopolymers 25, 1133 (1986).

[19]

ENZYMES IN REVERSE MICELLES

191

od 220

I

0.5

0.1

1 J

O-

:

I

0

4

~ 3 o

1

250

270

290 nrn

310

330

1

2 prnoles

3

4

5

NaOH

FIG. 2. (a) HPLC chromatography ofAOT. A reverse a-phase C~8 column (25 x 2.5 cm) has been used; flow rate 10 ml/min, elution mixture methanol/water 75 : 25 (v : v). (The arrow indicates the beginning of sample collection.) (b, c) UV absorption (b) and potentiometric titration profiles (c) of various AOT samples. The spectra were obtained using 50 mM AOT solutions in UV-grade isooctane, and titrations were performed in 10 ml of 1 : 1 (v : v) watermethanol solution of - 2 . 2 mmol of AOT, using 0.1 N NaOH as the titrating agent. AOT samples: ( n ) Obtained from Serva; (11) purified as in ref. 10a; (Q) purified as in ref. 10b; (A) purified as in ref. 11; (A) purified by HPLC. Luisi et al? °

that the obtained preparation is neutral; i.e., the beginning of the curve lies at pH 7. t0a C. A. Martin and L. J. Magid, J. Phys. Chem. 85, 3938 (1981). ~0bp. D. I. Fletcher, N. F. Pen-ins, B. H. Robinson, and C. Toprakcioglu. 6 U M. Wong, J. K. Thomas, and T. Novak, J. Am. Chem. Soc. 99, 4730 (1977).

192

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[19]

The AOT samples, once carefully dried under vacuum from the solvents used for the purification steps, can be kept over periods of months without apparent deterioration. The water content in the AOT powder can be checked by NMR or Karl Fischer titration, and is usually lower than 0.1 mol water per mole AOT. The physical characterization of reverse micelles before the uptake of enzymes is an important endeavor, both to study the micelles per se and also for understanding the mechanism of the solubilization of biopolymers. As already mentioned, structure and composition of the micelles will not be considered in detail here. It should simply be mentioned that on the basis of different physical methods, reverse micelles appear to be spherical, and mostly monodisperse. 7,11-14Also, the dimensions are determined by the ratio w0 = [water]/[AOT]. 7'11 Reverse micelles swell by increasing w0; their diameter generally ranges between 40 and 200 ,~. It is relevant for the discussion of enzyme activity and conformation that up to w0 = 6-8 the water of the water pool is assumed to be firmly bound to the micellar internal wall. Only above such a w0 limit can one speak of free water 11,15 (actually beyond this point the term microemulsion would be more correct than the term micelle).

Solubilization of Enzymes in Reverse Micellar Solutions Three methods have been proposed and utilized for the solubilization of enzymes in reverse micellar solutions. They are schematically illustrated in Figs. 3-5. The "injection method" (Fig. 3a) is the simplest: first one prepares the hydrocarbon solution of the surfactant, for example 50 mM AOT; and to this solution a small amount of a concentrated aqueous protein solution is added. For example, to 1 ml of the micellar hydrocarbon solution, 10/xl of a 100 mM lysozyme solution in 50 mM phosphate buffer, pH 7, is added. The enzyme solution must be added gently and in tiny droplets. After a short period of turbidity, gentle hand shaking produces the clear solution of lysozyme in the hydrocarbon micellar solution. It is useful to build "stability diagrams" like those represented in Fig. 3b, which give the wo/T region where the micellar solution is clear and stable, with or without the enzyme. The problem with the injection technique is that one does not know a priori the amount of protein that a given reverse micellar solution can take, especially when one carries out spectroscopic investigations for which a large amount of enzyme in solution is needed. There is then the 12j. Sunamoto, T. Hamada, T. Seto, and S. Yamamoto, Bull, Chem. Soc. Jpn. 53, 583 (1980). ~3j. Tabony, A. Llor, and M. Drifford, Colloid Polymer Sci. 261, 938 (1983). 14 F. M. Menger, J. A. Donohue, and R. F. Williams, J. Am. Chem. Soc. 95, 286 (1973). i5 p. D. Fletcher and B. H. Robinson, Ber. Bunsenges. Phys. Chem. 85, 863 (1981).

[19] a

ENZYMES IN REVERSE MICELLES

Wo 70

protein in water

193

j .;:.,\. i ¢.

•

~ ' ~,D

60 50

I i I !

\

". --

. . . . . . . . . . .

40 cloodv

,

.

li

30

/ jl/

clear solution

20 10

-30

-20

-10

0

10

20

30

40

50

60

70 t

°C

FIG. 3. Solubilization by the injection technique. (a) Injection procedure. (b) Stability curve. A, 50 mM AOT/isooctane/50 mM borate buffer, pH 8.5. B, system as in A + lipoxygenase, Coy = 1.6 × 10 -7 M. C, system as in A + ribonuclease, coy = 1.75 × 10 -4 M. D, system as in A + ribonuclease, Cov = 1.75 × 10-5 M. Coy is the overall concentration, i.e., referred to the total volume (hydrocarbon and water).

danger of preparing supersaturated solutions. In this case the excess protein may tend to precipitate out of the micellar solution, giving rise to a decrease in the optical density during time and to artifacts during the physicochemical characterization. In principle, one should first construct stability diagrams of the type seen in Fig. 3b, in which the concentration of protein is varied as an independent parameter for the micellar stabilization. This is, however, very lengthy and complicated. The second method to solubilize proteins in a hydrocarbon micellar solution is the phase-transfer m e t h o d 16A7 shown diagrammatically in Fig. 4a. In this method the protein is in the water solution and the supernatant is the hydrocarbon micellar solution. With gentle shaking, and with a rate and efficiency which depend on buffer, pH, and concentration, the protein initially present in the water phase is transferred into the micellar solution. An example is given in Fig. 4b. The process of transfer is very slow, as it requires a few hours. A new study of this process, from which these data have been taken, will soon be published by our group. The protein solubilized in the hydrocarbon phase can be transferred again into the aqueous solution with an inverse treatment (the "backward transfer"), by changing the nature of the salt in the water phase. The two ~6 p. L. Luisi, F. J. Bonner, A. Pellegrini, P. Wiget, and R. Wolf, Heir. Chim. Acta 62, 740 (1979); see also Meier. 5° 17 M. E. Leser, G. Wei, P. L. Luisi, and M. Maestro, Biochem. Biophys. Res. Comrnun. 135, 629 (1986).

194

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[19]

surfactant in hydrocarbon

protein in water

FIG. 4. Solubilization by phase transfer. (a) Transfer from the aqueous solution. (b) "Forward transfer" of trypsin from an aqueous solution into AOT/isooctane as a function of salt concentration. From M. Leser et al.t7

processes can be combined together to give the so-called "double transfer"lG,17: a micellar hydrocarbon solution is now bridging two water phases in an H tube. The protein is initially present in the first water phase, and is vectorially transferred into the second water phase by crossing the hydrocarbon bridge. The reverse micelles are the agents of such transport, and the driving force is provided by the difference in the salt concentration between the two water phases. The advantage of the phase-transfer solubilization method with respect to the previous one lies mostly in the fact that the obtained micellar solution is thermodynamically stable; no supersaturation effect is possible. Also, the amount of water which is transferred into the hydrocarbon phase is the one which corresponds to the thermodynamic equilibrium of the system. The advantages are often overweighed by the disadvantages: the procedure is slow, the water and protein concentrations so obtained cannot be varied, and they must be determined at the end of the transfer. Because of the lengthy time required the activity of the enzymes also decreases, although not drastically.17 Let use consider finally transfer from the solid state of proteins, which is of course particularly suited for water-insoluble proteins (Fig. 5). In fact, by this method Delahodde et al.18 have been able to solubilize the water-insoluble lipophilin, a constituent of the myelin structure which has never been investigated in solution (see Fig. 5b). An example of this procedure, the case of lysozyme, which is very soluble in water, is shown in Fig. 5b.19 However, in this case the procem A. Delahodde, M. Vacher, C. Nicot, and M. Waks, F E B S Lett. 172, 1603 (1984); see also, M. Waks, Proteins 1, 4 (1986). t9 C. Grandi, R. E. Smith, and P. L. Luisi, J. Biol. Chem. 256, 837 (1981).

[19]

ENZYMES IN REVERSE MICELLES

195

% Transfer Trypsin 90

pH = lO

20 m M B o r a t e 50 m M A O T CaC12

8O

----- N a C I

70

,

6O

50

\

4O

% %. % k

%. x

%, %

30

20

10

|

0,001

,.

I

0.005

i

I

!

O,01

0.05

O I

M

Fro. 4b.

dure of solubilization from the solid state may offer some advantages.

From a practical point of view, one can reach higher concentrations of solubilized protein, since one is not limited (as in the injection method) by the maximal concentration which can be reached in the aqueous stock solution. Furthermore, thermodynamically stable solutions are obtained, where w0 can be varied independently, in contrast to the previous phasetransfer method.

196

IMMOBILIZED

ENZYMES/CELLS

b

IN ORGANIC

~ 2/-,O

#

: {/{/t'

200

;1-1

0 C~

{

~20

> 0

[19]

SYNTHESIS

40

{

! I

=~ 11L1

¢o

22!2 33'3 ~Ez s%--%

21o 3'o to

5'o oo

% H20

2t,~

protein powder

2O T ~1~

9A~6 ~,o u n ~,~2

1'

O

~cZ.

/

_J

111

o

2~2 3~3

Wo ~.'o z'o 3.'0

%H~O

J

4M.

555

d.o

s'o

60

FIG. 5. Solubilization by phase transfer from the solid state. (a) Extraction from the solid state. (b) Solubility of lysozyme during extraction from the solid states, expressed in overall concentration (A) and concentration in the water pool (B). To 50 mM AOT/isooctane containing various amounts of water (0,©) or aqueous buffer (A, A) solid lysozyme was added, followed by a short shaking, as described in the text. From Grandi e t a l . 19

It is interesting to observe both from Fig. 5 and from the literature 18 that the maximal solubilization does not take place at the largest w0 values, but instead at rather small ones. In the case of lipophilin discussed above, the French group found the maximal solubility at w0 = 5.6, and the same value was obtained from the myelin basic protein, another component of the myeline structure that is however soluble in water. This sur-

[19]

ENZYMES IN REVERSE MICELLES

197

prising feature is a clear indication that the water inside the water pool is not "normal" water, but acquires novel properties, including a novel solubilization power.

Characterization of Protein-Containing Reverse Micelles The experimental determination of the size and structure of the micellar aggregates which host the biopolymers is more complicated than that of the "unfilled" micelles, mostly because the system under study will generally be heterogeneous, i.e., composed by "filled" (containing the biopolymer) and "unfilled" micelles. Actually, it is rather difficult (with the exception of a few cases) to obtain a high degree of occupancy of the micelles, and most of the experimental methods for the determination of the molecular weight or size (light scattering, osmometry, photon correlation, fluorescence, etc.) will reach an average value. Since also the occupancy degree is generally not known a priori (this depend on the model for the protein uptake), the. knowledge of the molecular weight of the enzyme-containing micelles is indeed not straightforward. One needs an experimental method that is able to discriminate a small amount of filled micelles over a larger background of unfilled ones. Ultracentrifugation with a UV-monitoring device seems to be the best method, since measuring at around 280 nm, where only the protein absorbs, makes the unfilled micelles "invisible." Furthermore, by adding an appropriate chromophoric small molecule to the system, which is present in both filled and unfilled micelles, as suggested by Martinek's group, 2° one may be able to demonstrate both filled and unfilled micelles. One such sedimentation run is shown in Fig. 6A. However, it is not easy to proceed from these data to molecular weight evaluation. Sedimentation and diffusion coefficients must be extrapolated to infinite dilution (as shown in Fig. 6B) of micelles in order to avoid artifacts due to intermolecular association (as is always the case in macromolecular systems having finite virial coefficients). But this is difficult because micelles dissociate at very low surfactant concentration, and there are some conceptual problems in the definition of the partial specific volume of the sedimenting particle. Equilibrium sedimentation runs are probably best suited for determining the molecular weight, but in order to determine the dimensions (Stokes radius) one needs again diffusion coefficients, which have to be measured in independent runs, and extrapolated to c = 0. Finally the molecular weight should be determined as a function of w0 and other micellar parameters. 20 E. Martinek, A. V. Levashov, Y. L. Khmel'nitskii, N. L. Klyachko, V. Y. Chernyak, and, V. I. Berezin, Dokl. Akad. Nauk SSSR 258, 1488 (1981).

198

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

°i/-

[19]

A

OD

S, S'

18 ¸

16

/

DNP ÷ LYS

LYS

1,

/ ,

2'5

go

w6o

c A O T (mM)

FIG. 6. Ultracentrifugation experiments with AOT reverse micelles. (A) UV scanner traces of the time-dependent sedimentation profile (concentration in terms of optical density at 350 or 296 nm versus radial position) of three different micellar solutions (w0 = 7, 7, 100 m M AOT in isooctane) at 48,000 rpm and 25 °. DNP, 2,4-Dinitrophenol; Lys, lysozyme. (B) Extrapolation of the determined sedimentation coefficients (S) to infinite dilution (AOT in isooctane, w0 = 6.9) for reverse micelles with and without entrapped lysozyme: - - , without lysozyme; - . - , with lysozyme ([lysozyme]/[AOT] = 10 3). From Zampieri et al. 21a

Table I collects some typical experimental data on the molecular weight determination of filled and unfilled micelles. One notices in all cases an enlargement of molecular mass, which is larger than the sum of the two initial components (unfilled micelles and lysozyme, which has a molecular mass of 14,500 Da). The simplest way to interpret the data is shown in Fig. 7. This water shell model, based mainly on geometrical considerations and mass balance, has been elaborated into a method which permits the calculation of the dimensions of the filled micelle as a function of w0 once the molecular weight and dimensions of the protein and the unfilled micelle are known. 2~ However, on the basis of recent measurements, it appears that this first-approximation model may need s o m e r e v i s i o n . 21a 2i F. J. Bonner, R. Wolf, and P. L. Luisi, J. Solid-Phase Biochem. 5, 255 (1980). 21a G. G. Zampieri, H. J~ickle, and P. L. Luisi, J. Phys. Chem. 90, 1849 (1986).

[19]

ENZYMES IN REVERSE MICELLES

199

TABLE I MOLECULAR WEIGHTS OF LYSOZYME-CONTAINING REVERSE MICELLES a

W0

AOT (mM)

Molecular weight b

6.9 7.8 10. 11.7

100 100 50 100

60,000-168,000 c 40,000-96,000 d 91,000-122,000 d 81,000-303,000 c

o The aqueous solution is 20 m M borate buffer, pH 8.5. The overall e n z y m e concentration is in the range 50-100 /~M. All measurements are performed at room temperature. From G. Zampieri e t al. 2Ia w0 = [H20]/[AOT]. b The first figure is related to "unfilled," the • second to "filled" micelles. c F r o m equilibrium measurements. d F r o m sedimentation and diffusion measurements.

Conformation and Activity of Enzymes in Reverse Micellar Solutions

Some General Experimental Considerations The concentration of protein in the hydrocarbon micellar solution can be easily checked by spectroscopic methods, for example, by measurement of the optical density at 280 nm. Once the enzyme is solubilized in the hy~irocarbon micellar solution, its activity needs to be assayed. This can be done in the same way as for an aqueous solution of the enzyme, namely by addition of the substrate to the enzyme solution, or vice versa. Several possibilities of this operation protein

® reverse micelle

FIG, 7. E n z y m e s in reverse micelles. The water shell model.

200

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[19]

can be used in a micellar solution. (1) The two reagents (enzyme and substrate) can be added to each other as two hydrocarbon miceUar solutions; (2) one of the two reagents can be added to the hydrocarbon micellar solution in small amounts (microliter quantities) as an aqueous buffered solution; (3) finally the substrate, when directly soluble in hydrocarbon, can be added in the pure liquid form (e.g., linoleic acid to lipoxygenase). 22 Which of the assay methods is best depends on the particular system under study. Perhaps a word of warning is necessary concerning the order of mixing. When the enzyme is not very stable in the micellar solution, it is better to add the enzyme as the last component from the aqueous stock solution, to minimize the time that the enzyme stays in the micellar solution. One such case is lysozyme which we will discuss later. The study of the enzyme properties in the micellar solution, from this point on, can be carried out exactly following the procedures used for aqueous solutions. The micellar enzyme solutions are in fact clear, and spectroscopic enzyme assays to measure activity as well as circular dichroism, absorption, fluorescence, or NMR studies can be readily performed. However, there is one conceptual difficulty that sets these systems apart from normal aqueous solutions: the definition of concentration (and therefore of the Michaelis-Menten constant Kin) for water-soluble reagents, such as hydrophilic enzymes or their water-soluble substrates, is not unambiguous. One can refer the concentration to the overall volume (ov) of the solution (hydrocarbon plus water) or to the volume of the water alone. For a micellar solution containing 1% water (v : v), and for the simple case where the guest substance is only present in the water pools, the two numbers differ by a factor of 100. Km,ovis a phenomenological parameter, i.e., it refers to the analytical concentration in the whole system4'23; Km,wpassumes a structural model, i.e., one in which water is segregated in the water pools (wp), which very rapidly exchange materials with each other. There is no reason a priori to prefer to express the concentration as overall or as water pool, but misconceptions may arise. A numerically larger Kr~ value is in fact associated with a lower affinity of the enzyme for the substrate. One should be careful at this point not to confuse a trivial mathematical transformation with physical reality. In our case, enzyme saturation (or half-saturation at Km) is obtained with the same amount of added substrate regardless of whether one considers 22 p. L. Luisi, P. L~lthi, I. Tomka, J. Prenosil, and A. Pande, Proc. 7th Enz. Eng. Conf., Ann. N.Y. Acad. Sci. 434, 549 (1984). 23 p. D. I. Fletcher, N. F. Perrins, B. H. Robinson, and C. Toprakcioglu, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds.). Plenum, New York, 1984.

[19]

ENZYMES IN REVERSE MICELLES

201

overall or local concentration. Or in other words, if Km is numerically larger when expressed in water pool units, the substrate concentration is larger by the same factor, so that the enzyme fractional saturation is the same. Another controversial conceptual point, which is very important for the experimentalist, is the determination of the pH of the miceUar solutions. The argument has been much debated, z4,25 and no general agreement has been reached. We believe that the situation can be summarized as follows. In principle, there is no simple direct way either to define or to determine the pH in the water pool, mainly because the water of the water pool is a novel solvent, for which no calibration has been as yet offered. However, it can be accepted with fair reliability that the pH of the water pool is close to that of the stock buffered aqueous solution used to prepare the water pool of the reverse micelles, particularly at w0 values larger than 10-15. A useful experimental check is the determination of the apparent acidity by using the 3tp NMR of phosphate buffers in the water pool. 25,1° After this discussion of the main operational problems encountered in the field, we can now present an analysis of the conformation and activity of enzymes solubilized in apolar solvent via reverse micelles. We will analyze the various classes of enzymes, trying to emphasize in each case the major changes in activity and conformation brought about by the micellar environment with respect to bulk water. Most of the data presently available in the literature about proteins in reverse micellar systems are presented in Tables II-V. In particular, Table II reports data about proteases, Table III about hydrolases other than proteases, Table IV about oxidoreductases and related enzymes, and Table V about nonenzymatic proteins. A collection of kinetic parameters (Km and kcat) in reverse miceUes, compared with aqueous solutions, is offered in Table VI. Proteases

As apparent from Table 1I, 1'9'16'27-33 the most widely studied enzyme in hydrocarbon micellar solutions is a-chymotrypsin. The first report de24 O. A. El Seoud, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds,), p. 81. Plenum, New York, 1984. 25 R. E. Smith and P. L. Luisi, Heir. Chim. Acta 63, 2302 (1980). z6 p. L. Luisi and C, Laane, Trends Biotech. 4, 153 (1986). 27 p. L. Luisi, F. Henninger, M. Joppich, A. Dossena, and G. Casmati, Biochem. Biophys. Res. Commun. 74, 1384 (1977). 18 K. Martinek, A. V. Levashov, N. L. Klyachko, V. I. Pantin, and I. V, Berezin, Biochim. Biophys. Acta 657, 277 (1981). 29 F. M. Menger and K. Yamada, J. Am. Chem. Soc. 101, 6731 (1979).

202

IMMOBILIZED

ENZYMES/CELLS

o

t-

IN ORGANIC

•- ¢ , , ~

~

•. ~

~

,~

'~.~

:~

.~

~

:~

~

o

.~ ,-.,

[19]

SYNTHESIS

•

~ .'~

~ . ~ . ~ :~

:~

~

'~ ~,

~

~

z

g~ o

o

~-

¢h

~,

~a

<

>

z ua 0

E 0

o

¢11

.~.

4~

• t " ,.~ ~'~ P"-. O

<

o

r.~

[19]

ENZYMES IN REVERSE MICELLES

eq

•

~.£oo

e,I

203

.=.

~

b

~='~

o

N

~

z

;~

z

,.a

~'~

<<

~

~

0

Z

0

°~

0 o

0

b~ ~>:'&

o;~oo

,:5

0 ..=

2

'S

k~ a=l

e-

E

•~

'a 0

E

I=

.-.=

0

.z

204

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[19]

scribed the protein dissolved in cyclohexane via the cationic surfactant methyltrioctylammonium chloride.16 The CD spectrum is very similar to that in water, which was taken as evidence that the conformation of the main chain was not too different in the two systems. The activity of the enzyme, however, had decreased to zero. It was not established whether enzyme-entrapping reverse micelles are formed in this case. Two other proteases, trypsin and pepsin, were investigated in methyltrioctylammonium chloride. 16As in the case of a-chymotrypsin, the UV spectra show a small red shift, the fluorescence spectra are blue-shifted, and the quantum yields are 2-5 times higher than in water. The CD spectrum of trypsin in the far-UV shows only slightly reduced ellipticities compared to water, while the near-UV CD has almost disappeared. Again, no activity for either enzyme could be determined. The authors state that only slight conformational changes take place in the micellar solution because the differences in the far-UV CD spectra are only small compared to water. The changed fluorescence and near-UV CD spectra would only be due to small perturbations of the aromatic chromophores. The case is different for the AOT/hydrocarbon/water system. Four research groups that have studied a-chymotrypsin in this type of reverse micelles report that the enzyme remains active. 9,28-3°At a pH corresponding to the optimal pH in water, Menger and Yamada found initially almost no activity in reverse micelles. 29 It became clear, however, that this apparent decrease in enzyme activity was due to a shift of the pH profile of 1.5 pH units to more alkaline values in the AOT micelles. In fact, at the optimum pH and w0 = 25, kca t w a s tWO times higher than in water for the substrate N-acetyl-t-tryptophan methyl ester. The enhancement of activity was confirmed by other workers 9 in the low w0 region, using a different substrate. Interestingly, at low w0 the helix content seems to be higher than in bulk water solution (Fig. 8). (This conclusion is inferred from CD data on the basis of the calculation procedure of Chen e t al. 34) Since in aqueous solution, a-chymotrypsin exists in equilibrium between two forms with different activities and different CD properties, 35 it has been proposed 30 C. Kumar and D. Balasubramanian, in "Solution Behavior of Surfactants" (K. L. Mittal and E. J. Fendler, eds.), Vol. 2, p. 1207. Plenum, New York, 1982. 31 p. D. I. Fletscher, R. B. Freedman, J. Mead, C. Oldfield, and B. H. Robinson, Colloids Surface 10, 193 (1984). 32 p. Douzou, E. Keh, and C. Balny, Proc. Natl. Acad. Sci. U.S.A. 76, 681 (1979). 33 p. L. Luisi and R. Wolf, in "Solution Behavior of Surfactants" (K. L. Mittal and E, J. Fendler, eds.), Vol. 2, p. 887. Plenum, New York, 1982. 34 Y.-H. Chen, J. T. Yang, and H. M. Martinez, Biochemistry 11, 4120 (1972). 35 A. R. Fersht, J. Mol. Biol. 64, 497 (1972).

[19]

205

ENZYMES IN REVERSE MICELLES ~

m

40 :~

A

/

.,. ~; . . f-/\.\

'.

/z.'

/'/ I: /

_ ¢/~/....."...,., ~'/./.,."

-4

,,,f:(.. . . . .

-40

.',

]-

,:2'

..: ,.,. :

-80

/b"

-6

~;4 //:

-8 210

230

250 ~(nm)

270

290

310

FIG. 8. CD s p e c t r a of ~ - c h y m o t r y p s i n in the far (a) and near (b) UV region (overall c o n c e n t r a t i o n 11/~M) in w a t e r and A O T / i s o o c t a n e at different w0: , water, pH 7.9; --water, pH 9.8; - . - w0 = 22.5; . . . . w0 = 18; ... w0 -- 13.5. F r o m Barbaric and Luisi. 9

that in the miceUar environment the same holds, but instead the conformational equilibrium is shifted toward the more active form. 9 Kumar and Balasubramanian investigated a-chymotrypsin in several other micellar systems 3° at pH 7.4. They found the best activity in reverse micelles of Brij 56 and sodium dodecyl sulfate (85-87% of activity in water). The CD spectra obtained in these solutions were almost identical to those in water. In Triton X-100 the enzyme was only half as active as in the aqueous solution, and in CTAB and sodium laurate, it had lost its activity. Martinek et al. studied trypsin in the cationic surfactant CTAB in chloroform/octane solution. 2s In this system, the enzyme remains active. In AOT, however, at w0 = 25, they found a much lower activity for the enzyme, by using the same substrate. The authors explain the difference by electrostatic interactions between the micelle wall and the substrate. Spectroscopic data are not presented. While the interpretation offered by Martinek is plausible, it is also important to recall the results of Kumar and Balasubramanian 30 who found that a-chymotrypsin shows a modified conformation in CTAB and, correspondingly, no activity. This would mean that not the nature of the substrate, but a change in the enzyme, may cause the loss of activity. In contrast to the results obtained by Martinek et al. 28 Douzou and co-workers found that trypsin retains its

206

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[19]

activity in AOT/heptane. 32 A broad temperature range (-23 ° to +30 °) was investigated, thus using the micellar system for cryoenzymology. In fact, reverse micelles appear to be useful for low-temperature studies because under certain conditions the entrapped water does not freeze well below 0°. Other Hydrolases As shown in Table I I I , 26'28'36-39 ribonuclease was first investigated in AOT/octane. 36 The UV absorption spectrum is practically identical to that of the aqueous solution. Also the CD spectra both in the far- and near-UV range of the aqueous and the micellar solutions are almost superimposable, indicating that no large conformational change takes place (w0 was - 18). A few data exist for pyrophosphatase in reverse micelles of Brij 56 in cyclohexanefl8,4° The rate of hydrolysis of pyrophosphate was studied as function of w0 and it was found that at the optimum w0 = I0 the enzyme had the same activity as in water. Another interesting aspect of enzymatic catalysis in reverse micelles is the use of water-insoluble substrates as in the case of lipids. Misiorowski and Wells 38used phospholipase in ether/methanol solutions of phosphatidylserine. Malakhova et al. studied pancreatic lipase in AOT reverse micelles, 37 thus avoiding the problem of having a fixed substrate concentration, as in the case of the phosphatidylcholine micelles. 38 The optimum rate for the hydrolysis of triolein was found at w0 = 12. A short-chain substrate, tributyrin, was cleaved by an order of magnitude less effectively than the long-chain substrate triolein, The use of reverse micelles in this case made it possible for the first time to compare the substrate specificity of pancreatic lipase under conditions in which the adsorption step of the enzyme on the surface of the substrate agglomerates is absent. Finally, let us consider the case of lysozyme in reverse micelles. A first investigation was made by Grandi et al.19 The far-UV CD spectra of this enzyme in AOT/isooctane showed higher ellipticities than in water. In the near-UV range, the spectra changed from positive values to more 36 R. Wolf and P. L. Luisi, Biochem. Biophys. Res. Commun. 89, 209 (1979). 37 E. A. Malakhova, B. I. Kurganov, A. V. Levashov, I. V. Berezin, and K. Martinek, DokL Akad. Nauk SSSR 270, 474 (1983). 37a D. Han and J. S. Rhee, Biotech. Bioeng. 27, 1250 (1986). 3s R. L. Misiorowski and M. W. Wells, Biochemistry 13, 4921 (1974). 39 A. Oshima, H. Narita, and M. Kito, J. Biochem. (Tokyo) 93, 1421 (1983). 4o A. V. Levashov, N. L. Klyachko, V. I. Pantin, Y. L. Khmel'nitskii, and K. Martinek, Bioorg. Khim. 6, 929 (1980).

[19]

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or less negative ellipticities, depending on the w0 value. Since the UV absorption spectra in the near-UV range were the same in water and in the micellar solution, these CD changes could be safely attributed to conformational changes. Despite this, the enzyme's activity was found to be comparable to that in water. The situation was then interesting: how can an enzyme function with a markedly changed conformation? This question has recently been clarified ~° by carrying out CD spectra in the presence of the inhibitors NAG (N-acetylglucosamine) and tri-NAG (Fig. 9) from which it became clear that the CD changes from water to micelles corresponded to enzyme denaturation. The reason why the original investigation of Grandi et al. ~9failed to recognize this is that chitins were used as substrate, which are not readily soluble in the micellar hydrocarbon system, and the assay was then performed by adding the enzyme last to the micellar solution. In this way, it had no time to denature. However, the final conclusion ever is that when the enzyme works, it does so with the right conformation. In CTAB and the neutral surfactant tetraethylene glycol dodecyl ether, lysozyme exhibits almost water-like CD spectra. The activity is comparable to that in water, and a denaturation process like in AOT does not occur.l° Oxidoreductases

Spectroscopic and activity data are available for horse liver alcohol dehydrogenase (LADH) in the system isooctane/AOT/water 41m (see Table IV16'4°-51). The UV and CD spectra of the aqueous and micelle systems are very similar/~ The CD spectra show some changes with respect to water, but the main conformational features seem to remain similar in the two systems. The fluorescence spectra show a decrease in quantum yield

41 p. Meier and P. L. Luisi, J. Solid-Phase Biochem. 5, 269 (1980). 42 K. Martinek, L. Y. Khmel'nitskii, A. V. Levashov, and I. V. Berezin, Dokl. Akad. Nauk S S S R 263, 737 (1982). 43 K. Martinek, A. V. Levashov, Y. L. Khmel'nitskii, N. L. Klyachko, and I. V. Berezin, Science 218, 889 0982). " A. N. Erjomin, S. A. Usanov, and D. J. Metelitza, Vestsi Akad. Navuk. BSSR, Set. Khim. Navuk. 3, 65 0982), 4~ A. N. Erjomin and D. J. Metelitza, Biochim. Biophys. Acta 732, 377 (1983). 46 p. Douzou, P. Debey, and F. Franks, Biochim. Biophys. Acta 523, I (1978). 47 M. P. Pileni, Chem. Phys. Left. 81, 603 (1981). 4s R. Hilhorst, C. Laane, and C. Veeger, Proc. Natl. Acad. Sci. U.S.A. 79, 3927 (1982). 49 R. Hilhorst, C. Laane, and C. Veeger, FEBS Lett. 159, 31 (1983). 5o p. Meier, Ph.D. Dissertation No. 7222, ETH (Swiss Federal Institute of Technology), Zurich (1983). 51 C. Gitler and M. Montal, FEBS Lett. 28, 329 (1972).

[19]

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and a blue shift of several nanometers. This may be due to a more pronounced quenching of the exposed Trp-15 by water molecules in the miceUar system. The activity of the enzyme in water and in micelles is not very different for the respective optimum conditions, despite some changes in the spectroscopic properties. Martinek et al. showed that LADH in AOT/octane changes its substrate specificity. 42 They investigated the oxidation of alcohols with the coenzyme N A D + and studied the dependence of the number of carbon atoms of the alcohol molecule on the enzyme activity. In water, the activity of LADH is highest at n = 8, i.e., octanol is the best substrate. In

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AOT/octane, the maximum was found at n = 4 regardless of Wo. The authors explain this changed substrate specificity in terms of the changed local concentration of the alcohols in the micelle. A 20-fold velocity enhancement for the oxidation of pyrogallol with peroxidase was found in AOT/octane with respect to the water solution. 43 The optimum water content was at Wo = 10. No substrate inhibition was found in the micellar system, whereas in aqueous solution peroxidase is inhibited by excess of substrate. 28 The reaction catalyzed by lipoxygenase represents another case of reactivity with a water-insoluble substrate.16,40,50 The hydroperoxidation of linoleic acid could be followed directly by UV spectroscopy because clear solutions were obtained in reverse AOT micellar solutions. The reaction proceeded only half as fast as in the aqueous solution and the enzyme-substrate affinity was smaller than in water, as shown by the higher Km value (see Table VI). As Luisi et al. explain, 16 it might be possible that the optimum pH was not reached in the micellar solution because of the instability of aqueous enzyme stock solutions with pH values higher than 10. A highly organized system for an efficient coupling between hydrogenase and a photochemical system that produces reducing equivalents and protons necessary for hydrogenase action was obtained by entrapping hydrogenase in reverse CTAB micelles. 26,48The stability of hydrogenase was higher in reverse micelles than in the aqueous system. Furthermore, the same group has described a multienzyme system for the site-specific enzymatic reduction of apolar, poorly water-soluble ketosteroids that could also be enclosed in CTAB reverse micelles, z6:9 Table V 18,3°,52-55 summarizes reverse micellar systems using miscellaneous nonenzymatic proteins. The kinetic parameters k~at and Km for several enzymes in reverse micellar systems are presented in Table WI. 9'25'26'28'29'32'36'41'43'50'56'57 Concluding Remarks The use of reverse micelles is a general method to solubilize proteins in aprotic media. Although with all probability proteins are not really in 52 A. Darszon, R. Strasser, and M. Montal, Biochemistry 18, 5205 (1979). s3 A. Darszon, L. Blair, and M. Montal, FEBS Lett. 107, 213 (1979). 54 M. Sch6nfeld, M. Montal, and G. Feher, Biochemistry 19, 1535 (1980). 55 j. Wirz and J. P. Rosenbusch, in "Reverse Micelles" (P. L. Luisi and B. Stranb, eds.), p. 231. Plenum, New York, 1984. 56 p. D. I. Fletcher, A. M. Howe, B. H. Robinson, and D. C. Steytler, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds.), p. 69. Plenum, New York, 1984. 57 O. A. E1 Seoud, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds.), p. 81. Plenum, New York, 1984.

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the organic solvent, but rather in water microdroplets (at least for hydrophilic enzymes), the micellar system may contain only 0.3% water, and the system shows properties which are typical of a bulk apolar solution: for example, hydrocarbon-soluble substrates can be directly added to the bulk micellar solution and are accepted by the enzyme, as shown by the examples of lipoxygenase 16,4°and LDH 41 and by the complex reduction of the ketosteroid accomplished by Laane and co-workers. 49 Actually, the hydrocarbon micellar system of the enzyme is a delicate balance of properties both of the aqueous protein solutions and of hydrocarbon solutions, and in the exploitation of this balance resides the whole interest in the field, both from the biotechnological and the academic point of view. Another point of interest, which we have not dwelled on in this paper, is the fact that nucleic acids, large plasmids, and bacterial cells can be solubilized in hydrocarbon micellar solutions) 8,59Applications to biotechnological and basic research can also be explored. Although the reverse micellar system, and particularly the hydrocarbon/AOT/water system, is transparent in the far UV, only very few groups have reported spectroscopic/conformation studies. This is unfortunate; in addition to the importance of the conformational aspects per se, the lack of information about conformation makes the comparison between the results of different research groups rather difficult. For example, the discrepancy in the activity of proteases (a-chymotrypsin in particular) may well be due to partial unfolding under certain conditions. The discrepancy of results from different laboratories may be partly due to differences in the surfactant purity. This is particularly so for AOT. It would be useful for future work in the field if each group would characterize AOT samples by at least the following three parameters: absorption at 280 nm, a titration profile as in Fig. 2, and a conductibility measurement as a criterium for the salt content. These three figures would at least permit the comparison of AOT preparations from different laboratories, which at present is still rather difficult. In addition to purification procedures, there are several other areas to be explored. One is the structure of the micellar aggregate; the "water shell" model 21 is only one of the possible models and its generality is open to debate. Data about the nature and properties of water in the water pool indicate that the relationship between water of the water pool and the guest enzyme is, to say at least, surprising. Maximal activity is generally not present at the largest water content, but at w0 values around 10-13. Also, the optimal solubility of proteins in reverse micelles occurs at relas8 V. E. Imre and P. L. Luisi, Biochem. Biophys. Res. Commun. 107, S.38 (1982). 59 G. Hating, F. MensdOrfer, and P. L. Luisi, Biochem. Biophys. Res. Commun. 127, 911 (1985).

216

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[20]

tively low w0 values. Finally, the phenomenon of "superactivity" of certain hydrolases, such as a-chymotrypsin, ribonuclease, and lysozyme, is present only at very small water content. Under this condition the conformational perturbations are the largest, and the micelle should be the smallest, i.e., the least hospitable. This is an area which needs clarification, and which can have relevance for enzymology as a whole.

[20] D e s i g n o f R e v e r s e d M i c e U a r M e d i a for t h e E n z y m a t i c Synthesis of Apolar Compounds

By

COLJA L A A N E , R I E T HILHORST,

and

CEES VEEGER

Reversed micelles are tiny oases of water stabilized in an organic solvent with the aid of surfactants. It has been shown that enzymes entrapped in these pools are capable of synthesizing apolar compounds such as steroids,l-3 oxidized polyunsaturated fatty a c i d s / a n d peptides. 4 In this paper we will focus on the reversed micellar enzymology of 20fl-hydroxysteroid dehydrogenase (cortisone reductase; 17,20/3,21-trihydroxysteroid: NAD ÷ oxidoreductase; EC 1.1.1.53), which catalyzes the reduction of various ketosteroids to the corresponding 20fl-hydroxysteroids at the expense of NADH. Preparation of Cetyltrimethylammonium Bromide (CTAB) Reversed Micellar Media

Principle. A reversed micellar medium consists of at least three components: an aqueous solution, a water-immiscible organic solvent, and a surfactant. The mechanism leading to the formation of reversed micelles is the tendency to extend the interfacial area until the concentrations of surfactants are sufficiently low to achieve a nonnegative interfacial tension. 5 By far the best studied three-component reversed micellar medium contains AOT [bis(2-ethylhexyl)sodium sulfosuccinate] as surfactant and R. Hilhorst, C. Laane, and C. Veeger, FEBS Lett. 159, 225 (1983). 2 R. Hilhorst, R. Spruijt, C. Laane, and C. Veeger, Eur. J. Biochem. 14,t, 459 (1984). 3 R. Hilhorst, Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands (1984). 4 p. L. Luisi, P. Liithi, I. Tomka, J. Prenosil, and A. Pande, Enzyme Eng., in press (1987). 5 j. Th. G. Overbeek, Faraday Discuss. Chem. Soc. 65, 7 (1978).

METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

216

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[20]

tively low w0 values. Finally, the phenomenon of "superactivity" of certain hydrolases, such as a-chymotrypsin, ribonuclease, and lysozyme, is present only at very small water content. Under this condition the conformational perturbations are the largest, and the micelle should be the smallest, i.e., the least hospitable. This is an area which needs clarification, and which can have relevance for enzymology as a whole.

[20] D e s i g n o f R e v e r s e d M i c e U a r M e d i a for t h e E n z y m a t i c Synthesis of Apolar Compounds

By

COLJA L A A N E , R I E T HILHORST,

and

CEES VEEGER

Reversed micelles are tiny oases of water stabilized in an organic solvent with the aid of surfactants. It has been shown that enzymes entrapped in these pools are capable of synthesizing apolar compounds such as steroids,l-3 oxidized polyunsaturated fatty a c i d s / a n d peptides. 4 In this paper we will focus on the reversed micellar enzymology of 20fl-hydroxysteroid dehydrogenase (cortisone reductase; 17,20/3,21-trihydroxysteroid: NAD ÷ oxidoreductase; EC 1.1.1.53), which catalyzes the reduction of various ketosteroids to the corresponding 20fl-hydroxysteroids at the expense of NADH. Preparation of Cetyltrimethylammonium Bromide (CTAB) Reversed Micellar Media

Principle. A reversed micellar medium consists of at least three components: an aqueous solution, a water-immiscible organic solvent, and a surfactant. The mechanism leading to the formation of reversed micelles is the tendency to extend the interfacial area until the concentrations of surfactants are sufficiently low to achieve a nonnegative interfacial tension. 5 By far the best studied three-component reversed micellar medium contains AOT [bis(2-ethylhexyl)sodium sulfosuccinate] as surfactant and R. Hilhorst, C. Laane, and C. Veeger, FEBS Lett. 159, 225 (1983). 2 R. Hilhorst, R. Spruijt, C. Laane, and C. Veeger, Eur. J. Biochem. 14,t, 459 (1984). 3 R. Hilhorst, Ph.D. Thesis, Agricultural University, Wageningen, The Netherlands (1984). 4 p. L. Luisi, P. Liithi, I. Tomka, J. Prenosil, and A. Pande, Enzyme Eng., in press (1987). 5 j. Th. G. Overbeek, Faraday Discuss. Chem. Soc. 65, 7 (1978).

METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES

217

isooctane or hexane as oil phase. For preparation and characterization, see Luisi and Wolf 6 and Eicke. 7 The disadvantage of a three-component system is that the properties of the interphase are determined mainly by the surfactant and are therefore difficult to vary. However, by adding a fourth component, usually a cosurfactant that partitions between the interphase and the continuous phase, it is possible to vary the properties of both phases with respect to each other. Suitable cosurfactants are aliphatic alcohols ranging from butanol up to decanol. To our knowledge any organic solvent or mixtures of solvents can be used provided it is immiscible with water. Our standard four-component system consists of an aqueous solution, the cationic surfactant cetyltrimethylammonium bromide (CTAB), hexanol as cosurfactant, and octane as organic solvent. The concentration of CTAB is fixed at 0.2 M in octane. The concentration of water and hexanol are expressed as weight percentage with respect to the CTAB-octane solution. Another often used expression for the water content in a reversed micellar medium is w0, which is defined as the molar ratio of water to CTAB. In general, an optically clear reversed micellar solution is formed within an hour after mixing at concentrations of 9-30% alcohol and 435% of an aqueous solution both with respect to 0.2 M CTAB in organic solvent. Procedure. A reversed micellar medium consisting of an aqueous solution of HEPES (N-2-hydroxyethylpiperazine N'-ethanosulfonic acid), 10% hexanol, and 0.2 M CTAB in octane at a w0 = l0 contains 1.46 g CTAB; 20 ml octane; 1.8 ml hexanol; 0.72 m150 mM HEPES, pH 7.6. The brand of CTAB is important. Without purification enzyme activity with 20fl-hydroxysteroid dehydrogenase is highest with CTAB purchased from Serva or Merck. The sequence in which the components are mixed is not important. Furthermore, to obtain equilibrium as rapid as possible it is advisable to stir the solution vigorously after each addition. Determination of a HEPES-Hexanol-CTAB-Octane Phase Diagram

Principle. To gain insight into the way the composition of reversed micelles influences the activity of enzymes in their water pool, a physical picture of the system such as a phase diagram is desired. Since the CTAB 6 p. L. Luisi and R. Wolf, in "Solution Behaviour of Surfactants: Theoretical and Applied Aspects" (E. J. Fendler and K. L. Mittal, eds.), pp. 887. Plenum, New York, 1981. 7 H.-F. Eicke, Top. Curr. Chem. 87, 85 (1980).

218

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[20]

to octane ratio is kept constant, the quaternary phase diagram becomes a pseudo-ternary phase diagram. This implies that two components can be varied freely, whereas the ratio of the other two is kept constant (see Fig. I). Procedure. A series of 2 ml solutions of 0.2 M CTAB in octane containing varying amounts of 50 mM HEPES (pH 7.6) are prepared in Bausch and Lomb 0.5-in. colorimeter test tubes having a rubber stopper. Hexanol is titrated into these tubes with a microsyringe under vigorous mixing with a Vortex mixer at 25 °, until just clear. The titration is contin-

HEXANOL 0,,100

CONTINUOU

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100 / 0

~ o 5'0

WATER ~

~*%100 ETAB/OETANE

FIG. 1. Pseudo-ternary phase diagram for the system CTAB, octane, hexanol, and 50 mM HEPES, pH 7.6. The phase diagram was determined as described in the text. All quantities are expressed as percentage of total weight. The concentration of CTAB was 9.4% (w/w) with respect to octane (0.2 M). Keeping this amount constant allowed determination of 20fl-hydroxysteroid dehydrogenase activities in this plane of the phase diagram while varying the water and hexanol content. The shaded area represents the region where equilibrium in the presence of prednisone or progesterone, NADH, and enzyme was obtained rapidly, i.e. within mixing time. In this area initial enzyme activities were measured according to procedure 1 (see text). Furthermore the lines 4.4% (w/w) H20 (w0 = 10) and 10% (w/w) hexanol with respect to CTAB-octane are drawn. Variation of the concentration along these lines allows the largest change of the water and hexanol concentration in the shaded area where enzyme activity was measured. (©), Area where reversed micelles are formed within 4 hr.

[20l

ENZYMATIC REACTIONS IN REVERSED MICELLES

219

ued until the solution becomes turbid again. At relatively low water and hexanol contents (see right corner of the triangle in Fig. 1) the transition from a clear to a turbid solution is very sharp and quick, but at higher water and hexanol contents the time needed to establish equilibrium can be several hours. Furthermore, gradual transitions occur due to the fact that opalescent solutions are obtained at higher water content. In case that no clear-cut transition is observed by eye, a refractometer or any kind of fluorometer can be very helpful in determining phase transitions. Similarly, 2 ml solutions of 0.2 M CTAB in octane containing varying amounts of hexanol are titrated with 50 mM HEPES (pH 7.6) until just clear and subsequently until turbid. When all transition points thus obtained are expressed in weight percentages of total weight the phase diagram shown in Fig. 1 is obtained. In the bootlike area, at relatively low water and hexanol content, reversed micelles are formed within 4 hr after mixing. Criteria for the presence of reversed micelles are that the solution is optically transparent and that the ohmic resistance of the solution is high (> 106 fl/cm). Entrapment of Enzymes in Reversed Micelles According to Luisi et al., 6 Martinek et al., 8 and Menger and Yamada, 9 there are essentially three techniques that can be used to solubilize enzymes into reversed micellar media (Fig. 2). Procedure.

1. A small amount (few % v/v) of a concentrated aqueous stock solution of enzyme is added by a microsyringe under vigorous stirring to the reversed micellar medium (Fig. 2A). In general the final overall enzyme concentration is within the range 1-20/zM, which is well below the actual micelle concentration in most reversed micellar media studied so far. For a 0.1 M AOT in heptane solution the micelle concentration is approximately 0.5 mM at w0 = 20, l° and for a 0.2 M CTAB in octane medium the concentration is within the range 1-4 mM (Laane, unpublished results). Hence most reversed micelles are empty. This technique is the most frequently used since it is rapid and extremely simple. Instead of mechanical agitation the medium can also be sonicated) ~ We prefer, however, mechanical agitation with a Vortex mixer. 8 K. Martinek, A. V. Levashov, N. L. Klyachko, and I. V. Berezin, Dokl. Acad. Nauk. USSR 236, 920 (1977); Dokl. Biochem. (Engl. Transl.) 236, 951 (1977). 9 F. M, Menger and K. Yamada, J. Am. Chem. Soc. 101, 6731 (1979). 10 p. D. I. Fletcher and B. H. Robinson, Ber. Bunsenges. Phys. Chem. 85, 863 (1981). H p. Douzou, E. Keh, and C. Balny, Proc. Natl. Acad. Sci. U.S.A. 76, 681 (1979).

220

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

A

B

[20]

c

Flo. 2. The three techniques to solubilize a protein in a reversed micellar medium. (A) Injection technique, by which a few microliters of the buffered stock solution of the protein are added to the surfactant solution in hydrocarbon or to a solution that already contains water. (B) Extraction from the solid state, in which the protein powder is added to a solution of hydrocarbon containing the surfactant and water. (C) Phase transfer technique in which the hydrocarbon solution of the surfactant is in contact with an aqueous solution containing the protein. From Lnisi and Wolf. 6

A few minor disadvantages of this technique are that it is difficult to encapsulate high concentrations of enzyme in the reversed micellar solution and that the enzyme may be inactivated slightly due to a "concentration shock" during the injection. Furthermore, one has to be aware of the possibility that even a few hours after injection the solution may be thermodynamically unstable due to microscopic structural rearrangements. These problems are generally overcome by the second technique. 2. The enzyme is added in the solid state as a fine powder to a complete reversed micellar solution (Fig. 2B). Subsequently the protein is dissolved by vigorous shaking or sonication. 9 This procedure takes much more time than the first one, and is reserved mainly for relatively stable and hydrophobic proteins. When it is necessary to obtain highly concentrated solutions of protein, the dry protein is added in excess and the portion remaining undissolved after a few hours is removed by centrifugation or filtration. As was shown by Martinek and co-workers ~2the solubility of a-chymotrypsin in 0. l M AOT micelles reached a maximum of - 1 mM which is probably more than the micellar concentration in this medium. Hence all reversed micelles will contain at least one protein. 3. The enzyme is solubilized in the water pools of the micellar medium by a phase transfer from a bulk aqueous solution to the reversed ~2A. V. Levashov, Yu. Khmelnitski, N. L. Klyachko, and K. Martinek, in "Surfactants in Solution" (K. L. Mittal and B. Lindman, eds.), Vol. 2, p. 1069 (1984).

[20]

ENZYMATIC REACTIONS IN REVERSED M1CELLES

221

micellar solution (Fig. 2C). This technique may be useful for studying the kinetics and mechanism of solubilization. 6 At present it is not possible to give general guidelines for the solubilization of enzymes in reversed micelles. Neither is it possible to predict whether an enzyme will be active in such media. Too many unpredictable factors such as the local pH around the enzyme and electrostatic and hydrophobic interaction between the enzyme and its microenvironment determine the solubility and the activity of a given protein. Likewise, the temperature is a factor that should be considered. For example the solubility of liver alcohol dehydrogenase in AOT-isooctane reversed micelles increases substantially when the temperature is raised from 22 to 300. 6 Furthermore, one should be aware of the fact that some proteins containing a noncovalently bound cofactor can loose this cofactor upon solubilization. For example, flavodoxin from Megasphaera elsdenii loses its FMN upon solubilization in CTAB-hexanol-octane reversed micelles, but not in an AOT-isooctane medium (A. J. W. G. Visser, unpublished results). To date, the solubilization of proteins in reversed micellar media is in the "trial and error" stage. Yet, the general consensus seems to be that for every enzyme it is possible to design a reversed micellar medium in which the enzyme is active.

Procedure for 20fl-Hydroxysteroid Dehydrogenase Encapsulation. Prior to use 20fl-hydroxysteroid dehydrogenase from Boehringer-Mannheim is diluted to a concentration of - 2 mg protein/ml and dialyzed against 7 mM HEPES (pH 7.6) to remove ammonium sulfate. According to technique 1 the enzyme is then added by microsyringe to the standard CTAB reversed micellar medium.

Determination of Enzyme Activity in Reversed Micelles

Principle. Since reversed micellar solutions are optically transparent, enzymatic reactions can be followed using conventional spectroscopic techniques. Alternatively, enzyme activities can be followed under these conditions with a common 02 electrode, standardized by adding known amounts of Oa to the micellar system, in the case of oxidases, monooxygenases, and dioxygenases (Laane, unpublished results). Another powerful technique is HPLC, in particular for apolar compounds such as steroids. 1-3 Procedures for 20fl-Hydroxysteroid Dehydrogenase. 20fl-Hydroxysteroid dehydrogenase requires both NADH and a 20-ketosteroid for catalysis. Hence, its activity can be followed spectroscopically by measur-

222

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[20]

ing the rate of NADH consumption at 340 nm or by following the rate of steroid conversion by HPLC. Procedure 1. A dialyzed stock solution of 20fl-hydroxysteroid dehydrogenase is prepared as described above. Freshly prepared 50 mM solution of steroid in chloroform, 30/zl, is pipetted into a Bausch and Lomb 0.5-in. test tube. After evaporation of the chloroform 1.5 ml of a H E P E S hexanol-CTAB-octane micellar solution is added together with 5 ttl 5 mM N A D H in 50 mM HEPES (pH 7.6). While mixing the reaction is started by the addition of 5/zl stock solution of 20fl-hydroxysteroid dehydrogenase. Subsequently the reaction is monitored at 340 vs 380 nm using any kind of spectrophotometer. Procedure 2. At time intervals 25-/xl aliquots from a complete reaction mixture (see procedure 1) is added to 100/zl acetonitrile in a 1-ml Eppendorf centrifuge tube. This step is required to stop the reaction and to precipitate CTAB specifically, because CTAB interferes with the detection procedure. After freezing for about 20 min at - 2 0 ° the samples are centrifugated for 2 min at 12,000 g in an Eppendorf centrifuge. Under these circumstances most steroids remain in solution and can be analyzed by HPLC equipped with a Phase Sep Spherisorb 10/~ ODS column. The steroids can be detected in most cases between 200 and 280 nm and can be eluted from the column using a mixture of acetonitrile and water. For progesterone this ratio is 80 : 20 (v/v) and for prednisone 25 : 75 (v/v). For the determination of initial enzyme activity procedure I is preferred, since it is the most rapid and the simplest. In Fig. 1 the area is shown in which initial 20fl-hydroxysteroid dehydrogenase activities could be measured. Procedure 2 is preferred when long-term experiments are performed (see Fig. 6). Determination of the Intrinsic Rate Parameters of Enzymes in Reversed Micellar Media

Principle. The methods employed to determine the intrinsic rate parameters kcat and Km in aqueous media are also applicable in reversed micellar media. The only difference with an aqueous system is that in a reversed micellar medium one can relate the Km of a water-soluble substrate either to the overall (K °v) or to the water pool concentration (KWp). This in contrast to the Km for apolar compounds, which of course is always related to the overall concentration. In most studies so far the observation is that the K wp is much larger than in water, whereas the K °v is about the same as in water. 6,j3 We disagree with these authors that the 13 S. Barbaric and P. L. Luisi, J. A m . Chem. Soc. 103, 4239 (1981).

[20]

E N Z Y M A T I C REACTIONS IN REVERSED M1CELLES

223

close resemblance between the K~nv and the K m in water is accidental. In our opinion the only physically meaningful expression for Km in reversed micellar media is K~v . Theory. When an enzymatic reaction is considered that obeys Michaelis-Menten kinetics the following reactions take place: kl E + S .

k2 " ES

, E + P

k-i

(1)

where E is the enzyme, S the substrate, and P the product, kl, k_ ~, and kz are rate constants. It is clear that the velocity (v) of such a reaction does not depend on the way the concentrations are expressed. Hence Vmax

Vmax

v = 1 + (K°V/S °v) = 1 + (KWp/S wp)

(2)

When 0 is the volume fraction of the water phase, the water pool and overall concentrations are related as follows S °v = OS wp

(3)

As a result Eqs. (4) and (5) can be written K ° v / o s wp = K w p / S wp

K °v = OK wp

(4) (5)

Since Km is defined as K m = (k-1 + kz)/kl

(6)

and k_~ and k2 are first-order rate constants, k~ v=

(1/O)k~ p

(7)

the correction factor (1/0) indicates that the substrate is dispersed over the whole volume of the solution and in fact reflects the frequency factor of collision and exchange between micelles. Hence K °v is relevant and not Kwp.

Deviations of K°mv from Km values found in aqueous media may originate either from microenvironmental effects on the enzyme itself or from interface-substrate interactions. Km a n d keat V a l u e s f o r 20fl-Hydroxysteroid D e h y d r o g e n a s e . For catalysis 20fl-hydroxysteroid dehydrogenase requires the water-soluble substrate NADH and in case of a reversed micellar system an apolar steroid, for example progesterone. The K °v values for NADH and for progester-

224

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[20]

TABLE I MICHAELIS-MENTEN PARAMETERSFOR 20/~-HYDROXYSTEROID DEHYDROGENASE IN AQUEOUS AND REVERSED MICELEAR MEDIAa K~nv (~M) Medium Aqueous Reversed micellar

NaC1 (mM)

NADH

Progesterone

kcat (sec -~)

0 500 0 500

3.6 3.4 11.0 3.7

10 n.d. 400 n.d.

10.9 6.2 14.4 7.6

a Experiments were performed in 50 mM Bistris, pH 6.3, or in reversed micellar solutions of 0.2 M CTAB in octane, 10% hexanol, w0 = 10, containing 50 mM Bistris, pH 6.3, in the water pool. Similar solutions were prepared containing 500 mM NaCI. For the determination of K °v for NADH the progesterone concentration was kept constant at 5 mM and for the determination of K °v for progesterone the overall NADH concentration was fixed at 50 ~M. n.d., Not determined. (Taken from ref. 3.)

one as well as the kcat values are listed in Table I both for an aqueous and for a CTAB reversed micellar medium. Compared to an aqueous solution, kcat increases in CTAB reversed micelles. This increase is not caused by a shift in pH of the water pool, since in aqueous solutions of pH 7.0 and 6.3 similar values for kcat a r e obtained. Upon enclosure in reversed micelles, K °v for NADH increases. Considering that NADH is negatively charged and CTAB positively, it can be envisaged that NADH is associated with the interface. Hence, the effective concentration available to 20/3-hydroxysteroid dehydrogenase could be lower, resulting in an increase in K°mv. In a reversed micellar medium the differences in K °v for NADH and kcat decrease at high ionic strength, indicating that the changes in k~atand K °v for NADH are caused mainly by electrostatic effects imposed by the charge of the head groups of the surfactant. In contrast to the K °v for NADH, that for progesterone increases markedly upon enclosure in reversed micelles, which could be due to a relatively low concentration of progesterone in the microenvironment of the enzyme compared to the bulk concentration. In conclusion it can be said that the enzyme experiences an essentially aqueous environment in reversed micelles, since kcat and K °v for NADH hardly change upon enclosure in the presence of salt, whereas the change in K°mv for progesterone is not caused by an effect on the enzyme itself, but by changes in the microenvironment.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES

225

Determination of the Parameters That Regulate the Enzymatic Conversion of Apolar Substrates in Reversed MiceUes

Principle. When an enzyme is encapsulated in a reversed micelle and the substrate is present in the oil phase, the substrate has to cross barriers such as the interphase and, if present, the aqueous layer surrounding the enzyme before catalysis takes place. It can therefore be expected that for apolar substrates the enzyme activity will be influenced by (1) the thickness of the water layer around the protein, and (2) the concentration of substrate in the interphase. Consequently, a reversed micellar medium should be designed in such a way that the water content is as low as possible and that the solubility of the substrate in the interphase is high with respect to the continuous phase. The water layer around the enzyme can easily be varied by adjusting w0. The radius of the water pool increases when w0 increases. TM As a measure for the solubility of a substrate in the interphase the hydrophobicity value of the interphase, log P i , c a n be used. 2,3 Since the interphase contains only cosurfactant and CTAB the semiempirical formula log Pi -

ao

a0+ 1

log Pcosurfactant +

1

log PCTAB

(8)

can be defined, where a0 is the molar ratio of the cosurfactant to CTAB in the interphase. Similarly, a log P for the continuous phase, log Pcph, can be defined according to Eq. (9), log Pcph = Co log ecosurfactant -1- (1 - c0) log Porganic solvent

(9)

where Co is the mole fraction of the cosurfactant in the continuous phase and thus equal to E q . (10). In this formula Ccph, p, M °, and My are the amount of cosurfactant in the continuous phase (/zl/ml), the density (kg/liter), and the molecular weight of the organic solvent (o) and the cosurfactant (c), respectively. CcphpC/MCr CO = CcphpC/MCr "k (1000 -- Ccph)p°/M °

(10)

By determining the log P values of the different components of the system, Ccphand a0 (see below), log Pcph and log Pi c a n be calculated for any given composition of the reversed micellar medium. Hence a quantitative measure for the hydrophobicity of the different phases in a reversed mi14 R. A. Day, B. H. Robinson, J. H. R. Clarke, and J. V. Doherty, J. Chem. Soc., Faraday Trans. 1 75, 132 (1979).

226

IMMOBILIZED

ENZYMES/CELLS

IN ORGANIC

SYNTHESIS

[20]

S-

F 'T

E % E

>, >,

ru -I-

C3 tf~

0

01 02 03 04 }logPi- [ogPs I

\\0%

FIG. 3. The influence of ling P~ - log Psi and Ilog Pc~h -- log Psi on the 20/3-hydroxysteroid dehydrogenase activity with prednisone as substrate. For explanation, see text; for details, see Hilhorst e t al. 2.3

cellar medium can be obtained, which can be compared to the hydrophobicity of the substrate (log Ps) and related to the enzyme activity. Since small differences between log P values indicate good mutual solubilities, high substrate concentrations in the interphase and thus high enzyme activities are expected when Ilog P i - log Psi is minimal and when [log Pcph - log Psi is as large as possible. With these rules for any given apolar compound a medium can be composed that assures optimal enzymatic conversion. Figure 3 shows the results obtained with 20fl-hydroxysteroid dehydrogenase in H E P E S - C T A B - h e x a n o l - o c t a n e reversed micelles with prednisone as substrate. The results clearly show the effects of Ilog P~ - log Psi and ]log Pcph -- log Psi on the enzyme activity and that these effects are additive and thus amplify each other. Determination of log P. Log P is defined as the logarithm of the partition coefficient in an octanol water two-phase system. J5 For simple molecules log P values are known or can be calculated from hydrophobic fragmental constants according to Rekker. 15 For more complicated structures such as steroids log P can be determined easily as follows. One micromole of apolar compound is shaken overnight in sealed vials containing 1 ml octanol and 1 ml of an aqueous solution. The composition of the aqueous solution and the temperature should be the same as those used in the enzymatic assay. The concentration of the apolar com~5 R. F. Rekker, " T h e Hydrophobic Fragmental Constant." Elsevier, Amsterdam, 1977.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES I

I

I

227

I

300

3 -5

200

x

~oo-~vl I

I

I

I

1

2

3

4

V ocfane (m[)

Fi6. 4. Typical example of a titration curve for the HEPES-hexanol-CTAB-octane medium. For explanation see text. Ccphis the amount of hexanol in the continuous phase (in ~l/ml); V~ is the amount of hexanol (in p.1) in the interphase, and V denotes total volume.

pound in both phases can be determined afterward using the appropriate detection technique. Determination ofccph and ao. The mole fraction of cosurfactant in the interphase (a0) and its amount in the continuous phase (Ccph) are determined by phase boundary titrations according to Bowcott and Schulman. 16 For the HEPES-hexanol-CTAB-octane system the procedure is as follows. A 2 ml solution containing 0,2 M CTAB in octane and 50 mM HEPES (pH 7.6) at the desired water content is prepared in a Bausch and Lomb 0.5-in. test tube. This solution is titrated using a microsyringe with hexanol to clarity under vigorous shaking. Upon dilution with octane the solution becomes turbid again because of extraction of the hexanol from the interphase into the continuous phase. This process of dilution and titration is repeated 5 times in the same tube. Each titration is carried out in duplicate or triplicate at the temperature used in the enzyme assay. A plot of the minimal amount of hexanol required for the formation of a clear solution vs the total volume of octane added should yield a straight line (Fig. 4). The slope represents the amount of hexanol in the continuous phase in microliters hexanol per milliliter continuous phase (ccph), whereas the intercept on the Yaxis (V~) represents the amount of hexanol in microliters present in both the interphase and the water pool. Since 16 j. E. Bowcott and J. H. Schulman, Z. Elektrochem. 59, 283 (1955).

228

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS (c0)surfattant~.~

2~'' '~÷

N'~'+

2 - ~ / f - MV ~

i

~H2ase

,

.

.

.

'+

[20]

.

~/~

APOLAR

AUM t - I ~ K E T O S T E R O I O

.~L,pOH

",..,2My++2H+J ~ N A D

..........

j% HSOH~

+'

J~APOLAR

A~

~ofl-HYDROXYSTER0ll]

FIG. 5. Scheme for the Hz-driven regeneration of NADH and the subsequent reduction of an apolar steroid in a reversed micellar medium. For clarity all the water-soluble components of the system are drawn in one micelle. A more realistic view is that all components are distributed among all micelles that are in rapid exchange with each other. H2ase, Hydrogenase; LipDH, dihydrolipoamide dehydrogenase; HSDH, 20/3-hydroxysteroid dehydrogenase; and MV, methyl viologen. From Hilhorst et al. ]

hexanol is very poorly soluble in water, the latter amount is negligible. V~ times the density of the cosurfactant and divided by its molecular weight yields the amount of hexanol in the interphase (ci) in micromoles. Since all surfactant molecules are localized in the interphase, a0 can be calculated according to Eq. (11), ao

= ciMS/S

(11)

where S is the amount of surfactant in the assay in milligrams and M s the molecular weight of the surfactant. Multienzymatic Reactions in Reversed Micellar Media Principle. An important property of reversed micelles is that they are in rapid exchange with each other. Hence, enzymes distributed among these microdroplets can communicate with each other either directly or through low molecular weight mediators. Figure 5 shows an example of a carrier-mediated multienzymatic system in a reversed micellar medium. ~ Cytochrome-c3 hydrogenase (EC 1.12.2.1) and dihydrolipoamide dehydrogenase (EC 1.8.1.4) are used for the in situ regeneration of NADH which is consumed by 20fl-hydroxysteroid dehydrogenase during the site- and stereospecific reduction of an apolar ketosteroid to its corresponding 20fl-hydroxyform. Procedure. An aqueous solution (70 tzl) is injected into 1.5 ml of a Vortex-stirred 0.2 M solution of CTAB in hexanol-octane 1 : 4 (v/v) containing 1.0 mM steroid unless stated otherwise. Stirring is continued until the solution becomes clear. Any assay vial can be used, provided it contains a gastight rubber septum that allows the withdrawal of aliquots from the reaction mixture.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES I

i

229

i

~E E

o

i

co i

~i lC

x o

o

=~ 0 ~.

0

~l

I

6

I

9

Time (hrs)

FIG. 6. Time course of 20fl-hydroxypregn-4-en-3-one formation at different concentrations of progesterone in a HEPES-hexanol-CTAB-octane reversed micellar medium. See text for procedure. Progesterone concentrations: 0.2 mM (IlL 1.0 mM (A), and 5.0 mM (N). From Hilhorst et al. 1

The aqueous solution contains pig heart dihydrolipoamide dehydrogenase (2.5 /.tM), 20fl-hydroxysteroid dehydrogenase (3.0/zM), methyl viologen (2.5 mM), and NAD ÷ (1.0 mM) in 50 mM HEPES (pH 7.6). After six cycles of 30 sec evacuation, 15 sec flushing with O2-free argon, and 5 min of bubbling with O2-free H2, 10/xl hydrogenase isolated from Desulfovibrio vulgaris 17 (final concentration of 0.6/xM in the water pool) is added to initiate the reaction. During the reaction the incubation is shaken mildly at 25 ° . Figure 6 shows the time course of progesterone reduction. The results clearly demonstrate that the reaction rate declines when the conversion is almost complete. Furthermore, at higher concentrations the reaction rate increases and the reaction proceeds steadily for >10 hr, indicating that the multienzyme system is stable for a considerable length of time. Acknowledgments We wish to thank Mrs. J. C. Toppenberg-Fang for typing the manuscript, Mr. M. M. Bouwmans for drawing the figures, and the Pharmaceutical Industry Duphar BV, Weesp, and the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) for financial support. 17 H. M. Van der Westen, S. G. Mayhew, and C. Veeger, F E B S L e t t . 86, 122 (1978).

230

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[21]

[21] D i s a c c h a r i d e S y n t h e s i s w i t h Immobilized fl-Galactosidase B y P E R - O L O F L A R S S O N , L A R S H E D B Y S , S I G F R I D S V E N S S O N , and

KLAUS MOSBACH Many carbohydrates are known to interfere in cellular recognition mechanisms in a very efficient way. j This fact forms the basis for the design of new drugs that would, for example, prevent the adhesion of bacteria on mucosal surfaces. The carbohydrate drug could be envisaged to block the bacterial receptors, making them unable to recognize or bind to the host cell, a process known to be a crucial initial step of the pathogenesis. The interest in such biologically active carbohydrates has prompted the development of new methods for carbohydrate synthesis. Normal organic chemical methods for di- and oligosaccharide synthesis are usually laborious and time consuming, due to a number of protection and deprotection steps. The use of enzymes for carbohydrate synthesis is conceptually very attractive, as the procedure often is a single-step reaction. However, several isomers may be formed. When only a few isomers are formed, as is the case here, the workup is also straightforward, making the overall procedure very convenient. Under the proper conditions, the enzyme fl-galactosidase will catalyze a transgalactosylation reaction (Fig. 1). 2 The fl-galactoside, lactose, combines initially with the enzyme to form a galactosylated enzyme with concomitant release of glucose. The galactosylated enzyme may experience two fates. Either it is hydrolyzed with water to give free enzyme and galactose, or it may transfer its galactosyl moiety to a second sugar, in this case N-acetylgalactosamine. This transgalactosylation reaction leads to the formation of galactosyl-N-acetylgalactosamine. Under normal aqueous conditions the final equilibrium is shifted far toward hydrolysis. However, the transfer reaction is comparatively fast, and during an intermediate phase a substantial yield from the transfer reaction is obtained. A third route is also evident from the scheme, namely the reversal of the hydrolysis reaction. For any substantial synthesis to occur, the water activity must be kept low. The procedures given below describe the synthesis of the disaccharide galactosyl-N-acetylgalactosamine [fl-D-Gaip-(I-6)-D-GalNAc] in good 1 N. Sharon and H. Lis, Chem. Eng. News March 30, p. 21 (1981). 2 K. Wallenfells and R. Well, in " T h e E n z y m e s " (P. Boyer, ed.), Vol. 7, 3rd Ed., p. 617. A c a d e m i c Press, N e w York, 1972.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

[21]

DISACCHARIDESYNTHESIS

231

Galactosyl transfer

"GIc E + GaI-GIc

+ GalNAc E-Gal

ll

+H20

GaI-GalNAc + E

Hydrolysis

E+Gal

FIG. 1. Simplified scheme of proposed mechanism of/3-galactosidase, showing the transfer and the hydrolysis reactions. E,/3-Galactosidase; Gal-Glc, lactose; GalNAc, N-acetylgalactosamine; Gal-GalNAc, galactosyl-N-acetylgalactosamine, Reproduced from Hedbys et al. 4 with permission.

yield from lactose and N-acetylgalactosamine and in low yield from galactose and N-acetylgalactosamine via reversal of the hydrolysis reaction. The enzyme was used in the immobilized form to facilitate its reuse and its easy removal from the reaction mixture when the yield was at its maximum. In addition, and of relevance for future studies, immobilization may lead to more stable enzyme preparations of importance when carrying out enzymatic synthesis at elevated temperatures and in organic solvents and/or solvents with low water activity. Methods

Immobilization of fl-Galactosidase to Sepharose CL-4B The immobilization is carried out by the tresyl chloride m e t h o d ) : Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden), 5 g of moist gel, is washed with water and then transferred stepwise to organic solvent by washing with a series of solvents: 30 : 70, 50 : 50, and 70 : 30 acetone:water, followed by acetone, and finally acetone dried with a molecular sieve) The gel is filtered off, carefully avoiding any moist air to be sucked through the gel bed, and then transferred to a 10-ml beaker containing 1.13 ml dry acetone and 95/xl pyridine. Tresyl chloride, 6 125 3 K. Nilsson and K. Mosbach, this series, Vol. 104, p. 56. 4 L. Hedbys, P.-O. Larsson, K. Mosbach, and S. Svensson, Bi oc he m . Biophys. Res. Commun. 123, 8 (1984). 5 One hundred grams of Linde molecular sieve type 4 A is added to 1 liter of acetone. Drying time is 24 hr. 6 Tresyl chloride (2,2,2-trimethylsulfonyl chloride) was obtained from Fluka, Buchs, Switzerland. Tresyl chloride-activated Sepharose gel is available from Pharmacia Fine Chemicals, Uppsala, Sweden.

232

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[21]

/zl, is added under vigorous magnetic stirring to rapidly disperse the activating agent. The mixture is then stirred at a slow rate for 15 min at room temperature. The activated gel is washed with acetone and then transferred to an aqueous solvent by washing with 70 : 30, 50 : 50, and 30 : 70 acetone : 5 mM HC1 in water, followed by 5 mM HCI. The activated gel is stored at 4° until used. The activated gel, about 5 g wet weight, is washed with 0.20 M sodium phosphate buffer, pH 8.0, and mixed with 6 mg of Escherichia coli [3galactosidase 7 in 6 ml of the same buffer. The suspension is shaken gently for 2 hr at room temperature. The [3-galactosidase-carrying gel is washed with the above buffer, followed by 0.2 M Tris-HCl, pH 8.0, in which it is kept for 30 min to block any remaining tresyl groups. The gel is then washed with 0.5 M NaC1, water, and finally 0.10 M sodium phosphate buffer, pH 7.0, containing 2 mM MgCIz. The activity of the gel is routinely determined from its ability to hydrolyze o-nitrophenyi-[3-o-galactoside. The gel typically has an activity of 48 U/g of gel, which corresponded to about 50% of added activity.

Enzyme Assays [3-Galactosidase activity is determined from initial rate measurements of o-nitrophenyl-[3-D-galactoside hydrolysis at 24°. The assay mixture contains 1.0 mM o-nitrophenyl-[3-D-galactoside, 2 mM MgC12, and 0.10 M sodium phosphate buffer, pH 7.0. The hydrolysis reaction is followed at 420 nm, where the product has a molar absorption coefficient of 2050 Mcm -~ (pH 7). 8 When immobilized enzyme is assayed, a spectrophotometric cell equipped with a magnetic stirring device is used to keep the particles suspended. Normally 2-5 mg of gel is used per assay (2 ml assay volume).

Preparation of Galactosyl-N-acetylgalactosamine [[3-D-Galp-(1-6)-D-GalNAc] via Transglycosylation Immobilized enzyme, 1.4 g (70 U), is suspended in 17 ml 0.10 M potassium phosphate buffer, pH 6.5, containing 0.22 M N-acetylgalactosamine and 0.45 M lactose. The mixture is shaken gently at room temperature, and samples taken intermittently for HPLC analysis to determine the correct time for breaking the reaction, i.e., when the yield was at its 7 Grade VI was obtained from Sigma Chemical Co., St. Louis, MO. The preparation used had a specific activity of 80 U/mg with the assay conditions given under "Enzyme Assays." 8 The molar absorption coefficient is very pH sensitive around pH 7 (pK, for o-nitrophenol = 7.2). To avoid this problem, the assay may be run at pH 8.

[21]

DISACCHARIDESYNTHESIS

233

maximum. Samples are diluted with 9 volumes acetonitrile : water 3 : 1, and injected on a LiChrosorb-NH2 column (Merck; 5-/~m particles, 25 cm length). The mobile phase consists of acetonitrile : water 3 : I. The effluent is monitored at 235 nm, at which wavelength acetamido sugars are detected. The k' value of N-acetylgalactosamine was determined to be 1.6 and the k' value for the product galactosyl-N-acetylgalactosamine was 3.6. After 30 hr incubation, the reaction is stopped by spinning down the gel. As an extra measure of precaution, the supernatant is boiled for 3 min to denature any traces of enzyme, and freeze-dried. The yield, according to HPLC measurements, is at this stage about 25%. The product /3-DGalp-(1-6)-D-GalNAc is isolated in pure form by using a combination of chromatographic techniques. 4

Preparation of Galactosyl-N-acetylgalactosamine [/3-D-Galp-(1-6)-D-GalNAc] via Reversal of the Hydrolysis Reaction Immobilized enzyme, 5 U, is suspended in 0.50 ml of 0.10 M potassium phosphate buffer, pH 6.5, containing 0.31 M galactose and 0.31 M 2acetamido-2-deoxy-D-galactose. The mixture is gently shaken at room temperature for 24 hr before the reaction is stopped and the mixture treated as above. Comme~s The described transferase-type synthesis gave a very good yield of galactosyl-N-acetylgalactosamine [fl-D-Galp-(1-6)-D-GalNAc]. Analysis of the reaction mixture showed that small amounts of the disaccharides/3D-Galp-(1-6)-D-Gal and/3-D-Galp-(1-6)-D-GIc also had been formed. Remarkably, only (1-6)-linked GalGalNAc could be f o u n d . 4 Preliminary results with enzymes from other sources indicate that other linkages are possible to obtain. The product galactosyl-N-acetyl-galactosamine had not been described earlier and was therefore identified/characterized by sugar and methylation analyses using gas chromatography-mass spectrometry as analytical tools. Proton NMR data and the observed optical r o t a t i o n (o~D = 26°) were also in agreement with the proposed structure? The reversed hydrolysis reaction, on the other hand, resulted in only low yield under the conditions given here. To make the latter reaction suitable for preparative applications, the conditions must be altered substantially. However, recent preliminary studies with several glycosidases, including/3-galactosidase, suggest that satisfactory yields (10-50%) are possible, provided that a combination of measures are taken such as very high substrate concentration and high temperature (to increase substrate solubility and decrease water activity).

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[22] S y n t h e s i s o f U r e a w i t h U r e a s e in W a t e r - O r g a n i c Solvent Mixtures B y LARRY G . B U T L E R

The synthesis of urea in animals involves several enzymes and is driven by hydrolysis of 3 mol of ATP per mole of urea synthesized. 1 Urea can be readily synthesized nonenzymatically from the inorganic salt ammonium cyanate; this original synthesis by W6hler in 1828 was a landmark in the history of chemistry. 2 It has now become possible to demonstrate the synthesis of urea from an inorganic salt in a reaction catalyzed by a single enzyme, independent of hydrolysis of ATP or any other high energy compound. The inorganic salt is ammonium carbonate; the enzyme is urease, which catalyzes urea synthesis by reversal of urea hydrolysis2 Reversal of hydrolytic reactions was originally considered as a biosynthetic route; the first claim of an enzyme-catalyzed synthesis reaction was by reversal of the hydrolytic reaction catalyzed by maltase (a-glucosidase). 4 Because water is not only the solvent but is a reactant at high concentration, the equilibrium for hydrolytic reactions lies far toward hydrolysis when carried out in aqueous media. Methods for overcoming this thermodynamic constraint have been developed so that reversal of peptide bond hydrolysis has become a useful method for semisynthesis of proteins 5 and is being extended to large-scale synthesis of specialized materials such as aspartame. 6 Hydrolysis of urea is equivalent to hydrolysis of two peptide bonds, so the equilibrium lies even farther toward hydrolysis than for comparable reactions such as peptide bond hydrolysis. 7 Nevertheless, synthesis of urea from ammonium carbonate can readily be detected and quantitated. Replacement of part of the solvent water by water-miscible organic solvents dramatically increases the yield of urea. 3 This increase was presumed to be due to the corresponding decrease in water activity, shifting the equilibrium toward urea synthesis, 3 but an effect of the organic solJ H. 2 F. 3 L. 4 C. 5 R. 6 K. 7 A.

A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). W#hler, Poggendorff's Annalen 12, 253 (1828). G. Butler and F. J. Reithel, Arch. Biochem. Biophys. 178, 43 (1977). Hill, J. Chem. Soc. 73, 634 (1898). E. Offord, "Semisynthetic Proteins." Wiley, New York, 1980. Oyama and K. Kihara, CHEMTECH 14, 100 (1984). Kapune and V. Kasche, Biochem. Biophys. Res. Commun, 80, 955 (1978).

METHODS IN ENZYMOLOGY, VOL. 136

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UREASE-CATALYZED UREASVNTHEStS

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vent on ionization a may also be involved. Several problems associated with synthesis by reversal of enzymatic hydrolysis reactions are illustrated by investigation of the effects of reaction conditions on the rate and extent of urea synthesis as catalyzed by urease. Source of Enzyme. Jack bean urease extracted and purified by the standard acetone crystallization procedure 9 with minor modifications ~° was used in demonstrating urease-catalyzed urea synthesis) Utilization of urease from other sources should be possible, although conditions for good reaction rates would have to be established for the particular enzyme used. Conditions which determine the position of equilibrium of the urea hydrolysis reaction (see below) are of course independent of the source of the enzyme. Assays

Urea Synthesis. In order to obtain readily measurable amounts of urea it is necessary to use relatively high concentrations of substrate. Typical conditions are 0.75 M (NH4)zCO3, 5 mM EDTA, and 5 mM 2-mercaptoethanol (no additional buffer is necessary). The urease concentration should be about I Sumner Unit 11 per milliliter. Under these conditions Jack bean urease catalyzes urea synthesis at about 2% of the rate at which it hydrolyzes urea (conditions for urea hydrolysis specified below). Urea concentration is determined spectrophotometrically by a modification ~2of the Fearon reaction.13 Incubated samples, zero time blanks, or urea standards containing up to 1/zmol urea in a total volume of 0.2-1.0 ml in screw-capped tubes are quenched by addition of 5 ml of 52% H3PO4 (600 ml of 85% H3PO4 diluted with water to 1 liter). Samples can be kept at room temperature at this stage for up to an hour without significant loss of urea, so that several samples can be run together through the remainder of the procedure. To each quenched sample is added 1.0 ml of an aqueous solution containing 6 g of 2,3-butanedione monoxime and 0.3 g thiosemicarbazide (both from Aldrich Chem. Co.) per liter. After heating for 30 min on a boiling water bath, samples are cooled to room temperature and their absorbance at 530 nm is determined immediately. The assay is not affected by high concentrations of ( N H 4 ) 2 C O 3 . At smaller sample volumes (0.2-0.5 ml) the apparent extinction coefficient for urea (approxi8 G. A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). 9 G. Mamiya and G. Gorin, Biochim. Biophys. Acta 105, 382 (1965). ~0C. M. Kaneshiro, Ph.D. Dissertation, University of Oregon, Eugene (1975). N j. B. Sumner, this series, Vol. 2, p. 378. ~2 M. K. Schwarz, this series, Vol. 17, p. 857. 13 W. R. Fearon, Biochem. J. 33, 902 (1939).

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mately 2 × l06 M -~ c m - 0 is relatively insensitive to the presence of organic solvents in the sample. For samples in which the equilibrium concentration of urea is being determined, it is advisable, after measuring the urea concentration, to add fresh urease and incubate again until the urea concentration is constant. Urea Hydrolysis. For comparison to urea synthesis, urea hydrolysis can be measured by an adaptation of the same assay using 2 mM urea as substrate, in the presence of 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 20 mM imidazole acetate, pH 7.0. This urea concentration is below the Km for urea 14 so the rate decreases continually throughout the incubation. Data points at four different reaction times are plotted semilogarithmically; the first-order rate constant is determined from the slope. Effect of Organic Solvents on Enzyme Stability The use of organic solvents to enhance the yield of urea is possible only if urease activity can be maintained in the presence of the solvents. Fortunately, Jack bean urease is known to be rather stable in organic solvents such as glycerol.~5 Moreover, it is extracted and crystallized in 32% acetone. 9 In connection with these experiments it was observed that urease in 50% aqueous dimethylformamide, ethanol, or pyridine irreversibly lost most of its activity in a period of a few hours at room temperature. However, activity was recovered in high yield from urease dissolved in 50% aqueous solutions of acetone or polyhydroxylic alcohols such as glycerol and 1,2-propanediol. In an attempt to stabilize Jack bean urease against denaturation and loss of catalytic activity by high concentrations of organic solvents, the enzyme was immobilized and cross-linked by two independent methods. In neither case, however, did immobilized cross-linked urease have improved stability in the presence of organic solvents) Subsequent work was done only with soluble urease. Effect of Organic Solvents on Reaction Rate Organic solvents affect urea hydrolysis in much the same way that they affect urea synthesis .3 The effect of organic solvents on reaction rate can thus be conveniently determined by measuring urea hydrolysis, which requires less enzyme than urea synthesis. The effects are strongly dependent on the nature of the organic solvent, as well as its concentra~4 K. R. L y n n , Biochim. Biophys. Acta 146, 205 (1967). ~5 C. C. Contaxis and F. J. Reithel, J. Biol. Chem. 246, 677 (1971).

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U R E A S E - C A T A L Y Z E D UREA SYNTHESIS

237

tion. The solvents were reagent grade but not redistilled; further purification of the solvents might be warranted. When compared at the same concentration (50%), the rate of urea hydrolysis ranged from undetectable (in ethanolamine) up to equal activity in acetone to that observed in strictly aqueous solution. 3 Rates in 50% aqueous glycerol, dimethylformamide, ethanol, and pyridine were 15, 49, 56, and 65% of the rate in water. In acetone and glycerol, in which urease is stable indefinitely, the activity as a function of organic solvent concentration shows rather different patterns, with most of the activity retained up to 75% acetone and much less activity observed at relatively low glycerol concentrations) Solvent selection for proposed urea synthesis processes wouldrequire optimization of the particular solvent for enzyme stability, reaction rate, and yield of urea (see later section). Effect of Organic Solvent, Substrate, and Hydrogen Ion Concentration on Yield of Urea The equilibrium concentration of urea, determined in solvents in which urease is stable so that achievement of equilibrium can be assured, increases as the water activity (related to concentration) diminishes. The yield of urea at equilibrium is approximately a second-order function of water concentration (activity) as predicted by the equilibrium constant. 3 The yield of urea as a function of (NH4)2CO3 concentration, at constant solvent composition, strongly increases as the substrate concentration increases. 3 In 50% glycerol, the observed urea yield was greater than the theoretical yield (which assumes concentration and activity are equivalent) at low substrate concentrations and lower than the theoretical yield at high substrate concentrations. The yield of urea is greatly enhanced in alkaline conditions) H + is a by-product of urea synthesis from HCO3-, according to Eq. (1). 2 NH4 + + H C O 3 - --0 u r e a + 2 H 2 0 + H +

(1)

Effect of the Nature of the Organic Solvent on Yield of Urea The equilibrium concentration of urea differs greatly depending upon which organic solvents are utilized to replace part of the water. Compared at a concentration of 50%, the equilibrium concentration of urea in aqueous acetone is over 12-fold higher than in aqueous glycerol. 3 Other organic solvents tested gave yields between these values, with hydroxylic solvents generally showing the lowest yields) As determined by measurement of the solubility of urea and ammonium carbonate, the primary effect of organic solvents is on the activity of the salt, rather than urea.

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Use of Organic Solvents to Study the Reaction Mechanism It has been reported that the products of urease-catalyzed urea hydrolysis are ammonium and carbamate ions. 16 Measuring urea synthesis rather than hydrolysis, it is possible to show that maximum initial rates are obtained only if the substrate ammonium bicarbonate has been dissolved for an hour before urease is added; freshly dissolved substrate gives much slower initial rates of urea synthesis s This difference is likely due to a requirement for synthesis from carbamate, which forms nonenzymatically at a slow rate after ammonium bicarbonate is dissolved. In order to prove this point, carbamate concentration should be measured in both fresh and aged substrate solutions, and the rate of synthesis from ammonium carbamate should be directly compared with the rate of urea synthesis from ammonium carbonate. Significance The small amounts of urea synthesized (approximately O. 1% yield) by reversal of the urease reaction are not likely to warrant consideration of this process for large-scale urea production. Urease does, however, provide an interesting model for investigation of the reversal of hydrolytic enzyme reactions, both because of the historical significance of urea synthesis, and because urease illuminates several factors which limit utilization of the technology of reversal of enzymatic hydrolysis. Perhaps most important is the realization that no single set of conditions is optimal for all the parameters affecting urea synthesis. For example, conditions which give high urea yields also give slow rates of synthesis, and conditions in which synthesis is rapid give poor yields. Enzyme efficiency and stability and substrate solubility likewise may be affected differently by organic solvents. Some of these problems may be partially overcome by using mixtures of different organic solvents.

~6R. L. Blakely, J. A. Hinds, H. E. Kunze, E. C. Webb, and B. Zerner, 1991 (1969).

Biochemistry 8,

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IMMOBILIZEDDEXTRANSUCRASE

239

[23] D e x t r a n S y n t h e s i s U s i n g I m m o b i l i z e d Leuconostoc rnesenteroides D e x t r a n s u c r a s e

By P. MONSAN, F. PAUL, D. AURIOL, and A. LOPEZ Dextransucrase [ a ( 1 ~ 6)-glucan:o-fructose-2-glucosyltransferase E.C.2.4.1.5] catalyzes the synthesis of a glucose polymer, dextran, according to the reaction: n Sucrose ~ (glucose)n + n fructose dextran

Dextran is a branched polymer in which the linear parts result from linear condensation of o-glucopyranosyl units by a ( 1 --~ 6)-osidic bonds, whereas branching points result from a(1 ~ 3), a(1 ~ 2), or a(1 ~ 4) bonds. The degree, nature, and length of branching are related to the strain of bacteria which produced the enzyme and the conditions of polymer synthesis.~ Dextransucrase from Leuconostoc mesenteroides NRRL B512 F, a nonpathogenic lactic bacterium, produces a low-branched dextran polymer containing 95% a ( 1 ~ 6) bonds and 5% o~(1~ 3) bonds, and which is highly soluble in water. This explains the exclusive use of this strain for industrial production of dextran. Produced in the same manner since 1950, dextran is mainly used as a blood plasma substitute (clinical dextran) and chromatography support. 2 Dextran is also involved in the process of dental caries: pathogenic bacteria of the genus Streptococcus produce insoluble dextran, which favors the adhesion of bacteria on the teeth and causes the formation of dental plaque. 3 Leuconostoc mesenteroides dextransucrase is an exocellular enzyme induced by its substrate, sucrose, the only inductor known at present. 4 Dextran synthesis is thus performed outside the cell, without the intervention of activated precursors or cofactors, as is the case for the synthesis of the majority of polysaccharides.5 In vitro dextran synthesis is thus A. Jeanes, W. C. Haynes, C. A. Wilham, J. C. Ranki, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist, J. Am. Chem. Soc. 76, 5041 (1954). 2 A. Jeanes, in "Extracellular Microbial Polysaccharides" (P. A. Sandford and A. Laskin, eds.), Symp. Ser. Vol. 45, p. 284. American Cancer Society, Washington, D.C., 1977. 3 R. L. Sidebotham, in "Advances in Carbohydrate Chemistry and Biochemistry" (R. S. Tipson and D. Horton, eds.), Vol. 30, p. 371. Academic Press, New York, 1974. 4 W. B. Neely, Arch. Biochem. Biophys. 79, 154 (1959). 5 R. C. W. Berkeley, G. W. Gooday, and D. C. EUwood, "Microbial Polysaccharides and Polysaccharases." Academic Press, New York, 1979.

METHODS IN ENZYMOLOGY, VOL. 136

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possible using a cell-free purified enzyme preparation. The energy required for the condensation of o-glucopyranosyl residues is provided by hydrolysis of the glucose-fructose osidic bond of the sucrose molecule. This energy is conserved by the formation of o-glucosyl-enzyme covalent complexes. The action mechanism of dextransucrase described by Robyt e t al. 6"7 involves two identical fixation sites of D-glucosyl residues, from which the latter are transferred to the reducing end of the growing dextran chain. This chain remains covalently bonded to the enzyme. During polymer synthesis, the enzyme forms an enzymatically active covalent complex with both glucose and dextran. Dextran release occurs when the fructose produced during the reaction is sufficiently concentrated to act as acceptor of the dextran chain. As fructose is a weak acceptor, the molecular weight of the polymer is extremely high, greater than 106.3 Fructose may also act as acceptor for D-glucosyl residues, resulting in the formation of leucrose, o~-o-glucopyranosido-(1 ~ 5)-fructopyranose. 8 However, other molecules, not involved in the reaction, may also act as acceptors for D-glucosyl and dextranosyl residues. Maltose is a particularly efficient acceptor causing the release of small oligodextrans containing a maltose residue at the reducing end. 9,1° These oligodextrans are themselves excellent acceptors.11,12 Control of dextran molecular weight during synthesis with the resulting direct production of polymers of sizes required for pharmaceutical and analytical applications may thus be envisaged. Along with a deeper understanding of the transfer mechanism of oglucopyranosyl residues and the possibilities of molecular weight control of dextran, dextransucrase production and purification have also been improved, in particular using fed-batch culture and ultrafiltration.13.14 We have recently developed an original purification process involving liquidliquid extraction of dextransucrase directly from culture broth. 15The enzymatic preparation thus obtained has a very high specific activity, 6 j. F. Robyt, B. K. Kimble, and T. F. Walseth, Arch. Biochem. Biophys. 165, 634 (1974). 7 j. F. Robyt and H. Taniguchi, Arch. Biochem. Biophys. 174, 129 (1976). F. H. Stodda, E. S. Sharpe, and H. J. Koepsell, J. Am. Chem. Soc. 78, 2514 (1956). 9 H. M. Tsuchiya, N. N. Hellman, H. J. Koepsell, J. Corman, C. S. Stringer, S. P. Rogovin, M. O. Bogard, G. Bryant, V. H. Feger, C. A. Hoffman, F. R. Senti, and R. W. Jackson, J. Am. Chem. Soc. 77, 2412 (1954). ~0j. F. Robyt and T. F. Walseth, Carbohydr. Res. 61, 433 (1978). n F. Paul, E. Oriol, D. Auriol, R. M. Willemot, and P. Monsan, Eur. Congr. Biotechnol. 3, (1984). 12 j. F. Robyt and S. H. Eklund, Carbohydr. Res. 121, 279 (1983). 13 p. Monsan and A. Lopez, Biotechnol. Bioeng. 23, 2027 (1981). 14 j. F. Robyt and T. F. Walseth, Carbohydr. Res. 68, 95 (1979). 15 F. Paul, P. Monsan, and D. Auriol, French Patent 8,307,650 (1983).

[23]

IMMOBILIZEDDEXTRANSUCRASE

241

greater than 170 U/mg protein and is free from contaminating saccharolytic activity. ~5,~6 Little research has been made into immobilization of L. mesenteroides dextransucrase. Robyt et al. 6,7 immobilized dextransucrase on BioGel P2 activated by glutaraldehyde in order to show the synthesis mechanism of dextran, and, in particular, the synthesis of a(1 ---* 3) branching points on the dextran linear chain. Recently, Reilly's group immobilized the enzyme by covalent bonding on various supports. ~7 But the most active derivatives were obtained by dextransucrase adsorption on phenoxyacetyl cellulose powder. 18 In our case, dextransucrase was immobilized on porous silica activated by glutaraldehyde in order to compare the kinetic behavior of the enzyme in free and immobilized form, and the nature of the dextran thus obtained. 13,19,2° Assay Methods Dextransucrase A s s a y . Enzyme activity is determined by measuring the initial production rate of reducing sugars in a solution containing 2 ml 3.3 M sucrose solution (Merck, Germany) in water, 0.24 ml 1 M sodium acetate buffer (pH 5.2) containing 2.5 g/liter calcium chloride, a volume of enzymatic solution with an activity between 2 and 20 U, and a volume of distilled water to obtain a final volume of 12 ml. Reaction takes place at a controlled temperature of 30° with stirring by magnetic rod. The amount of reducing sugars is determined using the reagent 3,5-dinitrosalicylic acid, 2~ with aliquots taken at 5-rain intervals over a period of 20 min, diluted if necessary to maintain reducing sugar concentration between 0.2 and 2 g/liter. Evolution of optical density as a function of time allows the initial production rate of reducing sugars to be determined. Dextransucrase activity (U) is defined as the amount of enzyme releasing 1/~mol of reducing sugar per minute. Protein Determination. Protein concentration is determined by the method of Lowry et al. 22 using bovine serum albumin as reference. 16 F. Paul, D. Auriol, E. Oriol, and P. Monsan, Enzyme Eng. 7, in press (1986). 17 n . Kaboli and P. J. Reilly, Biotechnol. Bioeng. 22, 1055 (1980). ~8 H. N. Chang, Y. S. Ghim, Y. R. Cho, D. A. Landis, and P. J. Reilly, Biotechnol. Bioeng. 23, 2647 (1981). ~9A. Lopez and P. Monsan, Biochimie 62, 323 (1980). 2o p. Monsan and A. Lopez, in "Advances in Biotechnology" (M. Moo-Young, C. W. Robinson, and C. Vezina, eds.), Vol. 1, p. 679. Pergamon, New York, 1981. 2~ j. B. Sumner and S. F. Howell, J. Biol. Chem. 108, 51 (1935). 52 O. H. Lowry, N. H. Rosebrough, A. L. Farr, and R. H. Randall, J. Biol. Chem. 193, 265 (1951).

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Dextran Determination. Dextran concentration is determined by the anthrone method using glucose as reference. 23

Production of Dextransucrase by Fed-Batch Culture of Leuconostoc mesenteroides Dextransucrase is produced by cultivation of the strain L. mesenteroides N R R L B512 F in a nutritive medium at 27° containing 2,4 yeast extract (Biokar, France), 20 g/liter; dipotassium hydrogenophosphate, 20 g/liter; sucrose, 20 g/liter; sodium chloride, 0.01 g/liter; magnesium sulfate (MgSO4"7H20), 0.2 g/liter; manganese sulfate (MnSO4. H20), calcium chloride (CaCI2" 2H20), and iron sulfate (FeSO4" 7H20), 0.01 g/liter. The pH of the medium is adjusted to 6.9 by pure orthophosphoric acid addition before sterilization. After the addition of a volume of preculture corresponding to 10% of total culture volume, cell growth is measured by following optical density at 650 nm of the culture medium diluted 10 times with distilled water. At the beginning of the exponential growth phase, a concentrated sucrose solution (800 g/liter) is added at a rate of 20 g/hrliter of culture medium. Sucrose must remain present in the medium to fulfill its role as growth carbon source, inducer, and substrate for dextransucrase, pH is maintained at 6.7, corresponding to optimum enzyme production. 24Cell growth and dextransucrase production are shown in Fig. 1. The addition of sucrose allows both the cell growth phase and dextransucrase production phase to be prolonged. Under these conditions, final culture activity was 8.8 U/ml, corresponding to a culture productivity of 1.2 U/ml-hr. 19 On completion of the growth phase, the culture medium is recuperated and centrifuged (20 rain, 4°, 10,000 g). The supernatant is immediately adjusted to pH 5.2 by the addition of pure orthophosphoric acid, thus constituting crude enzymatic extract. Purification of Dextransucrase Ultrafiltration and Gel Permeation. Crude enzymatic extract, 250 ml, is subjected to ultrafiltration in a hollow fiber cartridge (HIP 100-20, Amicon) with a nominal molecular weight cutoff of 100,000. The solution is diafiltered with an amount of 20 mM acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter and corresponding to 10 times the volume of the solution to be purified. The experiment is carried out at 4 °. Because of 23 T. A, Scott and E. H. Helvin, Anal. Chem. 25, 1956 (1953). 24 H. M. Tsuchiya, H. J. Koepsell, G. Bryant, M. O. Bogard, V. H. Feger, and R. W. Jackson, J. Bacteriol. 64, 521 (1952).

[23]

IMMOBILIZEDDEXTRANSUCRASE I

I

I

243 I

s

2

2

a

6

g

TIME (hr)

FIG. 1. Production of dextransucrase during fed-batch culture of L. mesenteroides NRRL B512(F). Absorbance (El); dextransucrase activity (A).

its very high apparent molecular weight the dextransucrase remains inside the fibers (Table I). This retentate is directly subjected to gel permeation: 40 ml of enzymatic preparation is placed on a 4 x 100 cm column containing 1 liter of Ultrogel AcA 34 (IBF, France), of separation range between 20,000 and 350,000, and eluted with a 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter. Chromatography is performed at 4°. Figure 2 shows the separation obtained19: it may be seen that dextransucrase elutes at the void volume and clearly separates from the greater part of the proteins present in the extract. Specific activity for a typical experiment as described in Table I attained 103 U/mg protein after the two successive steps of ultrafiltration and gel permeation. Final yield was 51%. Phase Partition. We have developed an original process for the purification of dextransucrase which is based on the fact that the latter is of dextranosyl-enzyme form and which takes into account the incompatibility of dextran with polyethers, e.g., polyethylene glycol (PEG). 15 The addition of a PEG aqueous solution to a dextran aqueous solution, when both are at suitable concentrations in the medium, leads to the appearance of two phases, each containing between 80 and 95% water. The top phase is rich in PEG and the bottom phase is rich in dextran. This phenomenon

244

[23]

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

TABLE I DEXTRANSUCRASE PURIFICATION FROM L. mesenteroides BY ULTRAFILTRATION AND GEL CHROMATOGRAPHY

Property Initial volume (ml) Final volume (ml) Protein concentration (mg/ml) Dextransucrase activity (U/ml) Dextransucrase total yield (%) Specific activity (U/mg protein)

Diafiltration'~

Gel permeation chromatography b

--5.12

250 291 0.075

40 100 0.013

7.63

4.25

1.34

Crude extract

100 1.5

65 56.7

51 103

a Experimental conditions: crude enzymatic extract was diafiltered with 10 times its volume of 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/ liter, using a HIP 100-20 hollow fiber cartridge (Amicon) with a nominal molecular weight cutoff of 100,000. b Experimental conditions: diafiltered enzyme solution was chromatographied on a 4 x 100 cm column containing 1 liter of Ultrogel AcA 34 (IBF, France) and eluted with a 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter at 4°.

is called phase partition. 25 Such two-phase systems are used for the partition of media containing cells or organelles, 25 cell debris, and enzymes. 26 In our case, the presence of dextran in the solution to be purified only requires the addition of PEG. The covalent association of enzyme and dextran, in particular, results in the presence of dextransucrase in the dextran-rich bottom phase. A 50% (w/v) PEG solution (Merck, molecular weight 1500) in water is slowly added to the solution to be purified (150 ml) in a glass beaker cooled in a 4 ° water bath and continually stirred. Phase partition occurs when the solution, at first translucid, becomes turbid. The system thus obtained is centrifuged at 8000 g for l0 min at 4 °. After centrifugation, the top phase is discarded. The volume of the bottom phase is then adjusted to the initial volume of the solution to be purified (150 ml) using 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter. This enzymatic preparation may be sub25 p. A. Albertsson, "Partition of Cell Particles and Macromolecules." Wiley, New York, 1971. 26 M. R. Kula, in "Applied Biochemistry and Bioengineering" (L. B. Wingard, E. Katchalski-Katzir, and L. Goldstein, eds.), Vol. 2, p. 71. Academic Press, New York, 1979.

[23]

IMMOBILIZEDDEXTRANSUCRASE I

I

I

I

245 I

E r-

--

4

v

C) o0

>-

c~

t

-3

LU !

\

(I) cr

~ t.d (/3

_2~

0 u') Cn

X ttt

20 40 60 B0 FRACTION NUMBER (1.5mr/tube)

100

FIG. 2. Gel filtration of dextransucrase on an Ultrogel AcA 34 column. Experimental conditions: column, 4 x 100 cm; eluent, 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter, 4 °. Absorbance (---); dextransucrase activity (A).

jected to further phase partition as described above. Thus, in a typical experiment, five successive phase partition steps were carried out on crude extract, resulting in a final specific activity of 171 U/mg protein and 1.35 U/mg dextran with a final yield of 95%. The amount of protein represented 0.8% of the total amount of the purified preparation. Comparison of the two methods described shows that repeated phase partition leads to a degree of purity much greater than that obtained by ultrafiltration and chromatography. Phase partition is quick and easy and allows the rapid concentration of crude enzymatic extract which has a positive effect on enzyme stability. Dextransucrase Immobilization Immobilization Procedure Porous Silica. A porous silica (Spherosil, Rh6ne-Poulenc, France) available as beads of various diameters and porosities is used. The support is aminated by y-aminopropyltrimethoxysilane coupling in boiling

246

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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T A B L E II CHARACTERISTICS OF SPHEROSIL a POROUS SILICA SUPPORTS Specific area (m2/g)

Pore size (nm)

6 37 120 180 445

550 80 25 15 7

a The diameter o f Spherosil (Rh6ne-Poulenc, France) beads ranges from 100 to 200 ~ m .

xylene for 24 hr. The diameter of the Spherosil particles ranges from 100 to 200/xm and the specific area from 6 to 445 m2/g. The characteristics of these supports are given in Table II. Glutaraldehyde Activation. A 100 mg amino-silica sample is activated with 20 ml of 2% glutaraldehyde solution in 0,05 M pyrophosphate buffer, pH 8.6. The glutaraldehyde solution is prepared from a commercial 25% glutaraldehyde solution in water (Merck) and its pH is adjusted before use. The samples are agitated by roller-mixing (Denley Mixer A 257) for 3 hr at 20 °. The supernatant is sucked up after the silica beads settled. Excess glutaraldehyde is washed off by repeated contact of the support with 20 ml of the above pyrophosphate buffer over a period of 1 hr at 20°. The activated support is washed until the supernatant no longer shows any reaction with Schiff's reagent for aldehyde detection. The support is then washed three times with 20 ml of the buffer solution in which immobilization is then to take place (if not pyrophosphate buffer). This method constitutes the optimization of the parameters influencing support activation and enzyme immobilization on porous silica as previously described.27, 28 Enzyme Immobilization. Dextransucrase immobilization is performed in 10 mM acetate buffer (pH 5.2). Dextransucrase solution, 15 ml, with a specific activity of 65.3 U/mg protein is brought into contact with 100 mg activated silica for 12 hr at 4 ° in 20-ml tubes and roller-mixed as during activation. After removal of the supernatant, the support is first thoroughly washed with 10 mM sodium acetate buffer (pH 5.2), then washed with 1 M sodium chloride solution to desorb the noncovalently grafted enzyme fraction (2 hr at 4°), and finally washed with acetate buffer. A blank is carried out by bringing a nonactivated support into contact with :7 p. M o n s a n , J. Mol. Catal. 3, 371 (1977-1978). 28 p. M o n s a n , Eur. J. Appl. Microbiol. Biotechnol. 5, 1 (1978).

[23]

IMMOBILIZEDDEXTRANSUCRASE l >.k-.-

t

247 I

30

I.(_.)

<(

mtm

u3

20 U') Z rr'

.,.-. X LI.I t~ ILl N _.l C)

~r

30

A A

)--

I

I 400 SPECIFIC AREA ( m 2 / g )

200

I 600

FIG. 3. Influence of specific area of silica support on immobilized dextransucrase activity. Experimental conditions: see Table III.

the enzymatic solution under the same conditions as for immobilization. The blank should show that there is no dextransucrase activity on the support, and that there is therefore no measurable irreversible adsorption of the enzyme. The immobilized enzyme is stored in 10 mM sodium acetate buffer (pH 5.2). Immobilized Enzyme Assay. Enzymatic activity of dextransucrase derivatives is measured directly in the immobilization tube using the same assay conditions as for the free enzyme. Periodic samples allow the production of reducing sugars in the reaction mixture to be determined.

Effect of Support Specific Area on Dextransucrase Immobilization Enzymatic solution, 15 ml, with an activity of 6.3 U/ml was reacted with samples of 100 mg of activated silica of varying specific areas. Support specific activity, expressed as U/g support, very rapidly decreased as the specific area of the silica samples increased (Fig. 3). The curve obtained thus differs noticeably from that generally obtained for enzyme immobilization on porous silica of varying specific area; an optimum range is most often observed for specific areas between 50 and 120 m2/g. 27'29In the case of dextransucrase, the greatest activity was obtained 29 R. A. Messing, Biotechnol. Bioeng. 16, 897 (1974).

248

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[23]

for the support with the lowest specific area, 6 m2/g, corresponding to an average pore diameter of 550 nm (Table II). This may be attributed to the macromolecular size of the dextranosyl-enzyme complex which penetrates the porous support with ever-increasing difficulty as the average diameter of the pores decreases. The relatively constant activity obtained from 122 m2/g on to higher specific areas corresponds to immobilization on the support surface. Further immobilization experiments were carried out with silica of specific area 6 and 24 mE/g. The influence of the amount of enzyme reacted with activated silica on the specific activity of the support was also determined. The experiment described in Table III shows that immobilization activity depends directly on the total amount of enzyme reacted with the support. Effect o f Maltose Addition on Dextransucrase Immobilization As seen above, dextransucrase exists as a high apparent molecular weight dextranosyl-enzyme complex (Fig. 2). The acceptor effect of an appropriate molecule such as maltose may allow the release of the dextran chain and thus a decrease in size of the active enzymatic complex. The effect of maltose addition to the immobilization medium on the activity grafted onto the support was determined. Dextransucrase 95 U was reacted with 100 mg silica of specific area 6 and 24 m2/g in the presence of 40 mM maltose. The immobilized specific activity was much greater in the presence of maltose (Table IV). This may be attributed to the fact that the enzyme partially freed from the dextran chains in the presence of maltose is of smaller apparent size, and may therefore more easily penetrate to the immobilization sites within the support. This interpretation is confirmed T A B L E III EFFECT OF AMOUNT OF ENZYME ON DEXTRANSUCRASE IMMOBILIZATION EFFICIENCY a Total activity (U)

Specific area (m2/g)

Activity (U/g support)

9.75 95 9.75 95

6 6 24 24

4.4 29.5 8.3 14

a Experimental conditions: 100 mg amino-Spherosil activated with glutaraldehyde was brought into contact with 15 ml d e x t r a n s u c r a s e solution in 10 m M s o d i u m acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter for 12 hr at 4 °.

[23]

IMMOBILIZEDDEXTRANSUCRASE

249

TABLE IV EFFECT OF MALTOSE ADDITION ON DEXTRANSUCRASE IMMOBILIZATIONa Maltose (raM)

Specific area (m2/g)

Activity (U/g support)

40 0 40 0

6 6 24 24

40.4 29.5 30.9 14.0

a Experimental conditions: see Table III. by the fact that the effect of maltose addition is even greater for the 24 m2/g specific area support, i.e., as average silica pore diameter decreases (Table II). The above experiments have allowed the preparation of an immobilized derivative of L, mesenteroides dextransucrase with an activity of up to 40.4 U/g silica.

Kinetic Characterization of Immobilized Dextransucrase The production rate of reducing sugars was measured as function of time for immobilized enzyme samples of differing specific area and activity (Fig. 4). In the case of the sample of specific area 6 m2/g, the production of reducing sugars remained constant for the first 50 min of reaction, corresponding to a conversion rate of 3.4% and a specific activity of 40.4 U/g. However, in the case of a sample of specific area 24 m2/g, a decrease in the reducing sugar production rates was observed as from 30 min of reaction, corresponding to a conversion rate of only 1.7% and a lower specific activity of 31 U/g. This experiment shows the existence of internal diffusional limitations which become all the more rapidly apparent as the average pore diameter decreases, The action mechanism of the enzyme in the absence of acceptors would imply that dextran is only released in the medium after it has attained a very high molecular weight (over several millions), regardless of the sucrose conversion rate. 9 It follows that dextran of very high molecular weight produced by the enzyme causes plugging of the support pores. The resulting increased viscosity induces high resistance to intraparticular mass transfer, and, in particular, fructose diffusion to the exterior of the support (Fig. 4). Substrate internal diffusional limitations have also been shown by studying immobilized dextransucrase activity in function of initial sucrose concentration. Lineweaver-Burk reciprocal plots, 1/V versus 1/S, show widely differing behavior for free and immobilized dextransucrase (Fig. 5). In the case of the

250

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS I

~.1.5

I

I

I

[23]

I

_

_

E v U..I U 0 m,W m 0

n

o.s

_

n~

LL

I0

20 TIME

30 40 ( rain )

50

FIG. 4. Kinetics of fructose production catalyzed by immobilized dextransucrase derivatives with different activities and specific areas: 40.4 U/g support and 6 m2/g (V); 31 U/g support and 24 m2/g ([]); 14 U/g support and 24 m~/g (~).

2

I

I

2

1

D ~g o

E1.5

1.5 ).

laJ O3

1

rv {-9 Z Cr

X

~

0.s

-

0.5 r~ BA N -A

I 0.05

I 0.10 (ram "1 )

I 0.15

m O

1/S FIO. 5. Effect of initial sucrose concentration on free (A) and immobilized (A) dextransucrase initial reaction rate; reciprocal plot.

[23]

IMMOBILIZEDDEXTRANSUCRASE

251

free enzyme, behavior is of the classic Michaelis-Menten type, and the apparent Michaelis constant is 27 mM. However, in the case of the immobilized enzyme, there is a marked deviation in linearity on the reciprocal plot, thus rendering impossible calculation of the apparent Km of the enzyme for sucrose. This behavior implies as above the existence of diffusional limitations on mass transfer within the support which are rapidly aggravated by the fact that the reaction product, dextran, brings about an increase in viscosity (plugging) in the immobilized enzyme's microenvironment, thus limiting even more substrate accessibility. Diffusional resistance is thus affected not only by the support itself, which induces a substrate concentration gradient within the pores (internal diffusion), but also by the reaction product, dextran, accumulated in the vicinity of the enzyme (plugging).

Characterization of Dextran Produced by Immobilized Dextransucrase Synthesis of High Molecular Weight Dextran. The question may be asked whether the use of immobilized dextransucrase preparations for the synthesis of high molecular weight dextran allows a structural modification of the dextran produced, in particular with regard to apparent molecular weight. To answer this question, two syntheses were carried out under identical conditions, one using the free enzyme and the other using the enzyme immobilized on silica of specific area of 6 m2/g, with sucrose concentration 100 g/liter, 20 mM sodium acetate buffer (pH 5.2), at 30°. Both synthesis products were chromatographed on a column of Ultrogel A2 (IBF, France) and compared with the chromatogram of a commercial solution of dextran blue of molecular weight 2,000,000 (Fig. 6). Both products were eluted long before the dextran blue, and the dextran obtained using the immobilized enzyme showed the highest apparent molecular weight. This difference in apparent molecular weight must be due to microenvironmental conditions, in particular increased viscosity within the pores resulting in high diffusional resistance. In the light of these results, it is difficult to envisage the use of immobilized dextransucrase for the production of high molecular weight dextran, bearing in mind the sharp increase in intraparticular diffusional resistance during polymer synthesis. However, it may be possible to use such immobilized enzymatic preparations for controlled molecular weight dextran synthesis in the presence of acceptors since the reaction products do not increase resistance to intraparticular transfer due to their low molecular weight. Dextran Synthesis in the Presence of Acceptors. Syntheses using the free and immobilized enzyme were carried out in the presence of three

252

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS I

E

2

I

[23]

I

/'i

_

E z o

r\

tow h-

/

/

//i

Z L~ ~.)

Z 0

I

Z I-X LI.I

/ / \ /

\

\ \ I0 ELUTION

20 VOLUME

30 (rot)

FIG. 6. Gel filtrationof dextran solutions on a Ultrogel A2 column: dextran blue (-.-); dextran synthesisby free (--) and immobilized ( ) dextransucrase. good glucosyl and dextranosyl residues acceptors: maltose, dextran of average molecular weight MW 9400 (Sigma), and oligodextrans (see production conditions in Table V). Reaction conditions were as follows: Free enzyme: 3.17 U/ml in sodium acetate buffer (pH 5.2) containing calcium chloride 0.05 g/liter, sucrose concentration 200 g/liter, acceptor concentration 20 g/liter at 30°. Immobilized enzyme: 500 mg immobilized dextransucrase (7.3 U) in the same buffer as for the free enzyme, sucrose concentration 200 g/liter, acceptor concentration 100 g/liter for a final volume of 6 ml. The synthesis products were analyzed on a column of Ultrogel AcA 34 as described in Table V. When maltose was used as acceptor it was observed that, in the case of the free enzyme, an acceptor concentration of 20 g/liter, i.e., a sucrose/maltose molecular ratio of 10, was enough to orient the reaction mechanism totally toward oligodextran production. These oligodextrans appeared in the total volume of Ultrogel AcA 34 column (molecular weight <20,000). However, to achieve the same result in the case of the immobilized enzyme, the maltose concentration had to be increased to 100 g/liter (Table V). Behavioral differences between immobilized and free enzyme were once again underlined, related to the presence of a concentration gradient for both substrate and acceptor.

[23]

IMMOBILIZED DEXTRANSUCRASE

253

TABLE V DEXTRAN SYNTHESISBY IMMOBILIZED DEXTRANSUCRASEIN PRESENCEOF ACCEPTORSa Dextran apparent molecular weight Nature of acceptor

>350,000 b

<20,000 c

Maltose Dextran (MW 9400)d Oligodextrans e

0.3 0.5 1.2

99.7 99.5 98.8

Experimental conditions: 500 mg immobilized dextransucrase (7.3 U) was reacted at 30° with 6 ml of a sucrose solution of 200 g/liter in 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter and one of the three acceptors at a concentration of 100 g/liter. b Product characterized by elution at the void volume of a gel permeation column using Ultrogel AcA 34 (IBF, France) corresponding to the exclusion of a globular protein, molecular weight >350,000. Molecular weight values are given for comparison and do not correspond to the actual dextran molecular weights, which have a much different hydrodynamic behavior in aqueous solution than that of a globular protein. Product characterized by elution at the total volume of a column using Ultrogel AcA 34, corresponding to a globular protein. d MW: Weight-averaged molecular weight determined by the manufacturer (Sigma). e Oligodextrans produced by free dextransucrase under the following conditions: sucrose 200 g/liter; maltose 20 g/liter; enzymatic activity 3.2 U/ ml at 30°. After total conversion of sucrose, the oligodextran solution was purified using a column of Ultrogel AcA 34 (eluted at the total volume) and concentrated under reduced pressure.

W i t h r e g a r d to t h e a c c e p t o r e f f e c t o f o l i g o d e x t r a n s , b e h a v i o r w a s differe n t in t h e c a s e o f t h e f r e e e n z y m e . T h e c o n c e n t r a t i o n o f 20 g / l i t e r u s e d w a s n o t e n o u g h to o r i e n t t h e r e a c t i o n t o t a l l y t o w a r d t h e s y n t h e s i s o f l o w m o l e c u l a r w e i g h t d e x t r a n ; 30% o f t h e p r o d u c t o b t a i n e d a p p e a r e d at t h e v o i d v o l u m e o f t h e U l t r o g e l A c A 34 c o l u m n ( T a b l e V). T h i s is d u e to t h e f a c t t h a t t h e s u c r o s e / a c c e p t o r m o l e c u l a r ratio is l o w e r t h a n w i t h m a l t o s e .

254

I M M O B I L I Z E D E N Z Y M E S / C E L L S IN O R G A N I C SYNTHESIS

[24]

In the case of the immobilized enzyme the oligodextran concentration of 100 g/liter used, was, as with maltose, enough to orientate the reaction toward low molecular weight dextran synthesis (Table V) (molecular weight <20,000). Throughout the various synthesis conditions, no significant production of intermediate molecular weight dextran, corresponding, for example, to the synthesis of clinical dextran (MW 70,000), was observed. However, such a type of dextran may be obtained by performing two consecutive synthesis steps using the free enzyme; the oligodextrans produced during the first step are used as acceptors during the second step. At 4° with a sucrose concentration of 200 g/liter and oligodextran concentration of 20 g/liter in the presence of 3.2 U free enzyme/ ml, 40% of the synthesis product corresponds to a polymer of molecular weight, estimated as in Table V, similar to that of clinical dextran. ~3 In conclusion, improved L. mesenteroides dextransucrase production and purification conditions make available highly purified enzyme preparations. Such preparations may be used for the controlled in vitro synthesis of high purity dextran. In particular, the direct synthesis of intermediate molecular weight dextran fractions, at present industrially produced by acid hydrolysis of high molecular weight dextran, may also be envisaged. If the production of high molecular weight dextran using immobilized dextransucrase is hampered by problems of increased medium viscosity, such preparations may however be used in the presence of acceptors such as maltose for the synthesis of oligodextrans, which constitutes the first step in the synthesis of intermediate molecular weight dextran.

[24] P r e p a r a t i o n of I m m o b i l i z e d M i l k X a n t h i n e Oxidase a n d Application in Organic S y n t h e s e s

By

JOHANNES

TRAMPER

Nearly all chemical reactions occurring in living cells are catalyzed by enzymes. Many of these reactions yield interesting products which are otherwise difficult or impossible to synthesize. That alone makes many enzymes potentially useful as catalysts in organic syntheses. All enzymes are specific with respect to the reaction they catalyze; however, almost all enzymes are not specific with respect to substrate. In other words, they METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

254

I M M O B I L I Z E D E N Z Y M E S / C E L L S IN O R G A N I C SYNTHESIS

[24]

In the case of the immobilized enzyme the oligodextran concentration of 100 g/liter used, was, as with maltose, enough to orientate the reaction toward low molecular weight dextran synthesis (Table V) (molecular weight <20,000). Throughout the various synthesis conditions, no significant production of intermediate molecular weight dextran, corresponding, for example, to the synthesis of clinical dextran (MW 70,000), was observed. However, such a type of dextran may be obtained by performing two consecutive synthesis steps using the free enzyme; the oligodextrans produced during the first step are used as acceptors during the second step. At 4° with a sucrose concentration of 200 g/liter and oligodextran concentration of 20 g/liter in the presence of 3.2 U free enzyme/ ml, 40% of the synthesis product corresponds to a polymer of molecular weight, estimated as in Table V, similar to that of clinical dextran. ~3 In conclusion, improved L. mesenteroides dextransucrase production and purification conditions make available highly purified enzyme preparations. Such preparations may be used for the controlled in vitro synthesis of high purity dextran. In particular, the direct synthesis of intermediate molecular weight dextran fractions, at present industrially produced by acid hydrolysis of high molecular weight dextran, may also be envisaged. If the production of high molecular weight dextran using immobilized dextransucrase is hampered by problems of increased medium viscosity, such preparations may however be used in the presence of acceptors such as maltose for the synthesis of oligodextrans, which constitutes the first step in the synthesis of intermediate molecular weight dextran.

[24] P r e p a r a t i o n of I m m o b i l i z e d M i l k X a n t h i n e Oxidase a n d Application in Organic S y n t h e s e s

By

JOHANNES

TRAMPER

Nearly all chemical reactions occurring in living cells are catalyzed by enzymes. Many of these reactions yield interesting products which are otherwise difficult or impossible to synthesize. That alone makes many enzymes potentially useful as catalysts in organic syntheses. All enzymes are specific with respect to the reaction they catalyze; however, almost all enzymes are not specific with respect to substrate. In other words, they METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

[24]

IMMOBILIZED MILK XANTHINE OXIDASE PREPARATION

255

TABLE 1 REASONS TO CONSIDER THE APPLICATION OF ENZYMES IN ORGANIC SYNTHESES Many enzymatic reactions yield products which are otherwise difficult or impossible to synthesize Enzymes are reaction specific with no undesired side reactions Enzymes are generally not substrate specific, thus the range of applications is not confined to natural substrates Enzymes demand mild reaction conditions Enzymatic reaction rates are generally high, at least when natural substrates are involved

are able to convert compounds other than their natural substrate by that particular type of reaction. Compared to most ordinary chemical catalysts, enzymes perform their task under very mild conditions, i.e., in aqueous solutions at ambient conditions or at least close to it. Enough reasons thus exist to seriously consider the application of enzymes in organic syntheses (Table I). Obviously, there must also be reasons why so few enzymes have actually been applied as catalysts in syntheses until now.

When an enzyme is to be used for synthetic purposes, isolation from its natural environment is often desirable and in many cases even mandatory. For instance, it is not practically feasible to use milk as such in the case of bovine milk xanthine oxidase, the enzyme of interest in this paper. The stability of isolated enzymes is usually low, and their solubility in water makes repeated or continuous use difficult. Consequently, effective application of most isolated enzymes as such is rather limited. Immobilization of enzymes is a technique which can make possible a more effective use of enzymes, and several immobilized enzymes are presently applied as catalysts in industrial processes. 1 In the laboratory, however, both free and immobilized enzymes are rarely used as catalysts in syntheses. To a large extent this is due to the fact that only a very limited number of enzymes meet the desired requirements mentioned in Table II. 2 The aim of my studies 3 on milk xanthine oxidase was to develop a simple procedure to make a xanthine oxidase preparation that can be conveniently and profitably used in the organic chemistry laboratory, in other words, a preparation that meets these requirements. 1 p. B. Poulsen, in "Biotechnology and Genetic Engineering Reviews." Intercept, in preparation. 2 j. B. Jones, this series, Vol. 44, p. 831. 3 j. Tramper, "Oxidation of Azaheterocycles by Free and Immobilized Xanthine Oxidase and Xanthine Dehydrogenase." Pudoc, Wageningen, The Netherlands, 1979.

256

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[24]

TABLE II CRITERIA AN (IMMOBILIZED) ENZYME SHOULD MEET TO BECOME GENERALLY ACCEPTED AS A ROUTINE CATALYST IN THE ORGANIC CHEMISTRY LABORATORYa The enzyme must catalyze a reaction of general preparative interest Sufficient specificity data should be available to enable reliable predictions to be made The enzyme should be commercially available The enzyme must be fairly cheap The enzyme should be sufficiently stable A convenient experimental procedure must be available From ref. 2.

Xanthine Oxidase The enzyme xanthine oxidase (EC 1.1.3.22) from cow's milk efficiently catalyzes the oxidation of many azaheterocycles (Fig. 1), 4 which by other means are often less reactive to controlled oxidation. Azaheterocyclic chemistry is an interesting and important area, because many of the compounds involved possess important biochemical and pharmacological properties. A large number of purines containing hydroxyl, amino, methyl, mercapto, and halogeno groups and purine N-oxides are oxidized by xanthine oxidase, although at greatly different rates. Also, 2or 8-azapurines are good substrates. Replacement of the imidazole (C4C5N7CSN 9) of the purines by a pyrazole ring (C4C5C7NSN9) gives a series of compounds that can serve as substrates, although they are more remarkable with respect to the strong inhibition they show in the oxidation of xanthine to uric acid by xanthine oxidase. 5 A wide variety of pyrimidines, pteridines, and other heterocyclic compounds are oxidized as well, some of them quite rapidly. We have studied homologous series of new potential substrates. One series consisted of 7-R-pteridin-4-ones with R = methyl, ethyl, n-propyl, isopropyl, and tert-butyl (Fig. 2). With these compounds, the influence of the size of the alkyl substituent on the rate of oxidation and on the affinity of the enzyme for the substrate was determined. 6 With the exception of the tert-butyl derivative, which is very slowly oxidized, the 7-alkylpteridin-4-ones have maximum oxidation rates comparable to that of the natural substrate xanthine. By means of another series consisting of 7-(4-X-phenyl)pteridin-4-ones (X = H, OMe, Br, CN, and NO2) the effect of substituent X was investigated (Fig. 3). 7 It 4 C. J. Suckling and K. E. Suckling, Chem. Soc. Rev. 3, 387 (1974). 5 R. C. Bray, in "The Enzymes" (P. D. Boyer, ed.), Vol. 12, p. 299. Academic Press, New York, 1975. 6 j. Tramper, W. E. Hennink, and H. C. van der Plas, J. Appl. Biochem. 4, 263 (1982). 7 j. Tramper, A. Nagel, H. C. van der Plas, and F. MOiler, Recl. Tray. Chim. Pays-Bas 98, 224 (1979).

[24l

257

IMMOBILIZED MILK XANTHINE OXIDASE PREPARATION

H

H

H ~ l ~J-~H

H

H

pyrimidine

H/~I~I~H

purine

pferidine

FIG. 1. Examples of azaheterocyclic compounds.

0 H N ' ~ N~--'-'l/H

0 HN" ~ N v H

immobilized

H FIG. 2. Oxidation of 7-alkylpteridin-4-ones to 7-alkyllumazines by immobilized milk xanthine oxidase.

0

0

HN"~''~N~'/H

imrn0biLized

./y

"x

H

-..-

H N ~ ' I N~---~H~ ." y

"x

H

FIG. 3. Oxidation of 7-(4-X-phenyl)pteridin-4-ones to 7-(4-X-phenyl)lumazines by immobilized milk xanthine oxidase.

was found that the more electron attracting is X, by either resonance or induction, the lower the rate of reaction. Summarizing, the huge amount of data concerning substrate specificity of milk xanthine oxidase allows rather reliable predictions to be made about substrate properties of untested potential substrates. Milk xanthine oxidase is commercially available. The enzyme (Boehringer-Mannheim) has a specific activity of 0.4 U/mg (25°; hypoxanthine as substrate). The storage stability of this preparation at 4° is good, but the operational stability of both the free and immobilized enzyme is very poor. Considerable stabilization is achieved by coimmobilization of xanthine oxidase with superoxide dismutase and catalase, and especially by entrapment of the enzyme in glutaraldehyde cross-linked gelatin. 8 However, considering the ultimate stability and the price of the enzyme, application in syntheses is generally not justified. Therefore, we have developed simple immobilization procedures that have milk as starting 8 j. Tramper, F. MOiler, and H. C. van der Plas, Biotechnol. Bioeng. 20, 1507 (1978).

258

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[24]

material and that do not involve enzyme isolation procedures. The resulting preparations of immobilized xanthine oxidase are very cheap, highly active, relatively stable, and well suited for organic syntheses. 9 These procedures are described below, and the applicability of the resulting immobilized enzyme preparation is illustrated by means of the oxidation of easily accessible substrates. In addition, the immobilization of commercial xanthine oxidase onto n-octyl-Sepharose 4B is described, not only because the adsorbed enzyme can be used for syntheses as well, but also because of its suitability for determination of kinetic properties of (new) substrates. Experimental

Materials. Xanthine oxidase (EC 1.I.3.22) from cow's milk (Boehringer-Mannheim); freeze-dried CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals); n-octylamine (Aldrich); gelatin powder (Merck) or ordinary cooking gelatin; glutaraldehyde, 25% in water (Fluka); xanthine, puriss (Fluka); l-methylxanthine, puriss (Fluka); local flesh whole milk (unpasteurized, nonsterilized). Skimmed milk is obtained by overnight storage of fresh whole milk at 4 ° (to accomplish dissociation of xanthine oxidase from the fat globule membranes) and the fat layer is removed by centrifugation at low speed. Glass-distilled water is used throughout and trishydroxymethylaminomethane/HCl buffer, pH 8.1 (I = 0.01) containing 0.1 mM EDTA is also used. All other materials are of analytical or biochemical grade. Adsorption of Xanthine Oxidase onto n-Oetyl-Sepharose 4B. Immobilization of xanthine oxidase by adsorption onto n-octyl-substituted agarose has been originally described by Hofstee and OtilliC ° and slightly adapted by us 8 by the following procedure. Commercial xanthine oxidase is diluted to a concentration of 0.2 mg/ml with buffer and dialyzed against Tris buffer at 4 ° (3 ×). The required amount of CNBr-activated Sepharose 4B is swollen for 15 rain in 1 mM HCI and washed on a sintered glass filter (porosity G3) with the same solution. A total of approximately 200 cm 3 per gram dry gel is added in several aliquots, the supernatant being sucked off between successive additions. The swollen Sepharose (! g --3.5 ml) is suspended in a mixture of n-octylamine, dimethylformamide (DMF), and water brought to pH 9 with hydrochloric acid. The volume ratio used is about 1 : 1 : 50 : 50. The mixture is rotated or shaken (not magnetically stirred) for 2 hr at room temperature and then thoroughly 9 j. T r a m p e r , H. C. van der Plas, and F. MOiler, Biotechnol. Lett. 1, 133 (1979). ~0 B. J. Hofstee and N. F. Otillio, Biochem. Biophys. Res. Commun. 53, 1137 (1973).

[24]

IMMOBILIZED MILK XANTHINE OXIDASE PREPARATION

259

washed on a sintered glass filter with DMF/water (1 : 1, v/v) containing I M NaCI, water, ethene glycol/water (1 : 1, v/v) containing 1 M NaC1, and then water, with subsequent repetition of this sequence. The n-octylSepharose 4B is stored overnight in Tris buffer to inactivate any reactive groups left. Dialyzed xanthine oxidase solution is rotated or shaken (not magnetically stirred) with n-octyl-Sepharose 4B (10: l, v/v) for 2 hr at room temperature. The resulting immobilized enzyme preparation is washed and stored in Tris buffer at 4° until used. Oxidation of Xanthine to Uric Acid by Adsorbed Xanthine Oxidase (Fig. 4). An amount of adsorbed enzyme corresponding to 10 mg protein is suspended in 50 cm 3 Tris buffer and rotated in a round-bottomed flask. Concentrated xanthine solution (2 mM; dissolve by means of alkali, as little as possible) is added at such a speed that no substrate accumulates. Check by measuring extinction at 296 n m (~uricacid = I0.1 mM -1 cm-1; ~xanthine = 0.6 mM -1 cm-~). When about 0.1 mmol is added (in 8 hr), the addition is stopped and the reaction continued for about 1 hr to oxidize most of the xanthine. The immobilized enzyme is removed by filtration and the filtrate acidified to pH 4-5 and evaporated in vacuo to about 10 ml. The precipitate formed upon cooling is filtered, washed with ethanol and ether, and dried. Yields of more than 80% are possible. Preparation of Immobilized Xanthine Oxidase Using Milk as Starting Material. Gelatin is dissolved in fresh whole milk (1 : 20, w/w) at 60 °. The solution is rapidly frozen in liquid nitrogen and lyophilized, and the freeze-dried material thoroughly grounded in a mortar or in a Gulatti analysis mill. The powder is added to a vigorously stirred 0.5% glutaraldehyde solution of water and acetone (1 : 1, v/v) and the stirring continued for 30 min at room temperature. About 25 ml of glutaraldehyde solution per gram of powder is used. Finally, the cross-linked preparation is exhaustively washed with Tris buffer and stored in buffer at 4 ° until used. An alternative for this procedure is the spray drying of fresh whole milk. For this we have used a Biichi 190-minispray dryer with an air inlet temperature of 55 ° and a liquid flow of 1I0 cm3/hr (unpublished results). The spray-dried milk powder is pushed through a sieve to obtain small agglomerates, whose size can be varied by the grit size. The agglomerates are immediately added via a funnel to glutaraldehyde (0.5%) in acetone/ 0

0

0AN H

H H

imm00,ized

xanthine 0xidase"-

0 H

H

FIG. 4. Oxidation of xanthine (R = H) and 1-methylxanthine (R = CH3) to uric acid and 1-methyluric acid by immobilized milk xanthine oxidase.

260

I M M O B I L I Z E D E N Z Y M E S / C E L L S IN ORGANIC SYNTHESIS

[24]

water (1 : 1, v/v). The cross-linking is continued for 30 min at room temperature. By means of sieves fine and large clumps are removed. The preparation is thoroughly washed with Tris buffer on these sieves and stored in buffer at 4° until usage. This spray-drying method is only suitable for preparing larger quantities, as initially a substantial amount of material is lost in the spraying chamber. The procedures described above yield the most suitable preparations, but whole milk with or without gelatin, skimmed milk with or without gelatin, and commercial xanthine oxidase in gelatin solution can all be used as starting material in both procedures. In addition to lyophilization and spray-drying the following methods can also be used. Gelatin is dissolved in milk (1: 10, w/w) at 60° and poured onto a polyethylene surface such that a thin film forms. The stiff gel which forms at 4° is cut into small strips which are immersed in a solution of 0.25% glutaraldehyde in 0.1 M citrate buffer, pH 6 (1 : 4, v/v) at 4 ° for 15 hr. The strips are then cut into small cubes, which are treated with fresh glutaraldehyde solution in a similar way as the strips. The cubes are finally washed with Tris buffer and stored in buffer at 4° until use. Instead of cubes, flat disks can be made by extruding the solution dropwise through a Pasteur pipet onto parafilm using, if available, a fraction collector which moves synchronously to drop speed. After drying at room conditions the disks are hardened with a glutaraldehyde solution as above, or with a 0.5% glutaraldehyde solution at room temperature for 30 min. By changing the initial gelatin concentration the diameter and thickness of the disks can be varied. A lower concentration gives larger and thinner disks (unpublished results). Variation in the ratio of materials is possible and skimmed milk or commercial xanthine oxidase can be used as well. Oxidation of 1-Methylxanthine to 1-Methyluric Acid (Fig. 4). An amount of immobilized xanthine oxidase, corresponding to 100 g freezedried material before cross-linking, is exhaustively washed with Tris buffer at 4° until A280nm in the filtrate is about zero. Subsequently the material is poured in a column and washed with 1 dm 3 Tris buffer followed by 1 dm 3 water (pH 8). One hundred milligrams of 1-methylxanthine is dissolved in a minimal amount of NaOH solution (4 M) and diluted to 600 ml with water. The pH is brought to 8.1 with Tris buffer or HC1 and the solution pumped through the column at 4° (50-100 cm3/hr). The solution is recycled once or twice if not all the substrate is converted at once. This can be determined by measuring the difference in A290nm ( A ~ = 10.3 mM -1 cm -1) of influent and effluent, as well as by adding to a sample (1.8 c m 3) of the effluent 0.2 cm 3 buffer and 0.5 cm 3 diluted commercial xanthine oxidase solution (0.4 mg/ml) and measuring the increase in A290n m . This

[9-4]

IMMOBILIZED MILK XANTHINE OXIDASE PREPARATION

261 E f:g oq

/

c~

.T

0

c¢

rq

o z~..T Z

Z

0

r~

c¢

"I-

E

_= >,

o -.,

£h >.,

e, Q

z I

//

©

0

-,

t t ~ "r~

~ . oo • au e q J o s q V

o~

262

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[24]

increase is compared with the increase obtained when a sample of the influent is treated in the same way. When the former is negligible and A290 nm maximal, both indicating complete conversion, the effluent is acidified to pH 5 with 2 M HCI. The solution is evaporated in v a c u o to a small volume, cooled (0°), and filtered and the residue extensively washed with water and dried. Yields over 60% are possible. Conclusion The procedures described in this paper are simple and yield immobilized xanthine oxidase preparations which meet the desired requirements (Table II) to a large extent. The rather low operational stability is more than compensated for by the very low price when milk is used as starting material. All the procedures can in practice be conveniently used for oxidation of many compounds on a preparative scale and, as a result of the reaction specificity, products of high purity can be collected. Figure 5 illustrates the reaction specificity of enzymes. The occurrence of several sharp isosbestic points strongly indicates the absence of side reactions. Tramper" provides an extensive overview of the work on xanthine oxidase and on the application of immobilized enzymes to syntheses in general.

11 j. Tramper, in "Solid-Phase Biochemistry: Analytical Synthetic Aspects" (W. H. Scouten, ed.), p. 393. Wiley, New York, 1983.

[25]

ENZYMATICREGENERATIONOF ATP

263

[25] E n z y m a t i c R e g e n e r a t i o n of A d e n o s i n e 5 ' - T r i p h o s p h a t e : Acetyl Phosphate, Phosphoenolpyruvate, Methoxycarbonyl Phosphate, Dihydroxyacetone Phosphate, 5-Phospho-a-D-ribosyl Pyrophosphate, Uridine-5'-diphosphoglucose 1 By D E B B I E C . CRANS, ROMAS J. KAZLAUSKAS, BERNARD L . HIRSCHBEIN, C H I - H U E Y W O N G , OBSIDIANA ABRIL, and GEORGE M . WHITESIDES

ATP is a key cofactor in enzyme-catalyzed synthesis. It must be used in catalytic amounts and regenerated in situ in enzyme-catalyzed reactions in order to minimize expense and simplify isolation of products. 2 Here we describe the best methods currently available for regeneration of ATP from ADP and AMP and detail several applications of these methods in enzyme-catalyzed syntheses. In particular, we give procedures for synthesis of three phosphate donors--acetyl phosphate, 3 phosphoenolpyruvate, 4 methoxycarbonyl phosphateS--and apply these reagents to syntheses of sn-glycerol 3-phosphate,6 dihydroxyacetone phosphate, 7 glucose 6-phosphate, 5-phospho-a-D-ribosyl pyrophosphate,8 and uridine-5'diphosphoglu¢ose.9 Three procedures for the enzymatic regeneration of ATP are presently available which are useful in practical-scale organic synthesis (Fig. I). One is based on acetyl phosphate (AcP) as the phosphorylating agent and acetate kinase as the catalyst; the second uses phosphoenolpyruvate (PEP) and pyruvate kinase; the third uses methoxycarbonyl phosphate [CH3OC(O)OPO3 2-, MCP] and acetate kinase. The advantages and disadvantages of each method are summarized in Table I. Acetyl phosphate is Research was supported by National Institutes of Health (NIH) Grants GM 26543 and GM 30367. R.J.K. gratefully acknowledges support as a NIH postdoctoral fellow 1983-1984, GM 09339. 2 G. M. Whitesides, C.-H. Wong, and A. Pollak, Adv. Chem. Ser. 185, 205 (1982); G. M. Whitesides and C.-H. Wong, Aldrichimica Acta 16, 27 (1983). 3 D. C. Crans and G. M. Whitesides, J. Org. Chem. 48, 3130 (1983). 4 B. L. Hirschbein, F. P. Mazenod, and G. M. Whitesides, J. Org. Chem. 47, 3765 (1982). 5 R. J. Kazlauskas and G. M. Whitesides, J. Org. Chem. 50, 1069 (1985). 6 V. M. Rios-Mercadillo and G. M. Whitesides, J. Am. Chem. Soc. 101, 5828 (1979). 7 C.-H. Wong and G. M. Whitesides, J. Org. Chem. 48, 249 (1983). 8 A. Gross, O. Abril, J. M. Lewis, S. Geresh, and G. M. Whitesides, J. Am. Chem. Soc. 105, 7428 (1983). 9 C.-H. Wong, S. L. Haynie, and G. M. Whitesides, J. Am. Chem. Soc. 105, 115 (1983).

METHODS IN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.

264

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

Reactant

Product

ATP

ADP

[25]

chromoS-1 CH3OH4"C02

ofl-co~

CH30~OPO~_

-~o~Po)Lco2-

Acefete/r/nose

,~,,-,,,,~ek , ~

FIG. 1. Enzyme-catalyzed regeneration of ATP.

very easily prepared; it is a phosphoryl donor of intermediate strength; it is moderately stable in solution. Acetate k/nose is subject to modest inhibition by acetate ion but this product inhibition is practically important only for reactions carried out in solutions containing acetate concentrations greater than 1 M. Phosphoenolpyruvate has excellent stability in solution, and is a very strong phosphoryl donor. Its synthesis is, however, more complex than that of AcP. Moreover, pyruvate k/nose is subject to inhibition by pyruvate. To minimize the effects of this inhibition, the reaction must be carried out in dilute solution to keep the pyruvate concentration low, pyruvate must be removed from the reaction mixture as it is formed, 1° or high concentrations of enzymes and PEP must be used. Methoxycarbonyl phosphate is comparable to PEP in its high phosphoryl donor strength, but resembles acetyl phosphate in its ease of synthesis. The product remaining after phosphoryl transfer from MCP to A D P - methyl carbonate--hydrolyzes rapidly in solution to methanol and carbon dioxide. This decomposition minimizes problems arising during isolation of products and resulting from product inhibition (bicarbonate, which can also inhibit acetate k/nose, is easily removed by purging). The principal disadvantage of methoxycarbonyl phosphate is that it decomposes in solution inconveniently rapidly (tl/2 = 20 min, pH 7, 25°) under conditions used for enzymatic synthesis. Both pyruvate kinase and acetate kinase have high specific activity and show excellent stability in immobilized form. Pyruvate kinase is currently the less expensive enzyme. Further, it is effective for regeneration of ATP from ADP at lower concentrations of ADP than is acetate kinase since the Michaelis constant for pyruvate kinase [Km(MgADP) = 0.1 10 Enzymatic assay of pyruvate in reactors using PEP often shows lower (~×2) than expected concentrations of pyruvate. The low pyruvate concentrations are likely due to polymerization of pyruvate and serve to reduce the effects of product inhibition.

[2S]

ENZYMATIC REGENERATION OF A T P

265

TABLE I PROPERTIES OF PHOSPHORYLATING REAGENTS USED IN ATP REGENERATION

O CHsCOPO32AcP

Property Ease of preparation AG~d (kcal/mol) ~ Half-life for hydrolysis (hr) pH 7, 25° 0° Product inhibition Ki (mM)

OP032CO2PEP

O CH30 OPO32MCP

+++ - 10.1

+ - 12.8b

++ - 12.4

21 c 960 Acetate 400, NC e

~103d -105 Pyruvate 10, C e

0.3 15 HCO 3 500, NE e

W. P. Jencks, in "Handbook of Biochemistry" (H. A. Sober, ed.), 2nd Ed., p. J-185. Chemical Rubber Company, Cleveland (1970). Standard free energy of hydrolysis at pH 7, based on a standard state of 1 M total stoichiometric concentration of reactants and products, except hydrogen ion, and on an activity of pure water of 1.0. b Other authors have used - 14.8 kcal/mol [H. G. Wood, J. J. Davis, and J. Lochmiiller, J. Biol. Chem. 241, 5692 (1966)]. This value, however, is based on a AG~,d for ATP of -9.1 kcal/mol instead of -7.3 kcal/mol which was used to calculate the Ag~d for acetyl phosphate and methoxycarbonyl phosphate. Measurement of the equilibrium constant for the ATP-mediated equilibrium: acetate + PEP ~- acetyl phosphate + pyruvate showed that the phosphoryl donor potential of PEP is - 2 . 5 kcal/mol greater than that for acetyl phosphate at pH 7.6. This value is consistent only with - 12.8 kcal/mol as the value of AG~yd of PEP. c Half-life for the dianion in phosphate buffer, pH 6.9 from G. Di Sabato and W. P. Jencks, J. Am. Chem. Soc. 83, 4400 (1961). d Half-life for dianion calculated using activation parameters determined at 75° from S. J. Benkovic and K. J, Schray, Biochemistry 7, 4090 (1968). NC, Noncompetitive; C, competitive.

mM] 11 is lower than that for acetate kinase [Km(MgADP) = 0.4 raM].12 In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase is used for regeneration of ATP. The former is preferable for large-scale work in which economy is an important consideration; the latter is used in instances in which the requirement for a strong phosphorylating reagent outweighs the relative inconvenience of the preparation of phosphoenolpyruvate, or in which a slow rate of enzyme-catalyzed reaction dictates the use of a hydrolytically

stable phosphorylating agent. The syntheses of each phosphoryl donor i1 A. S. Mildvan and M. Cohn, J. Biol. Chem. 241, 1178 (1965). 12 C. A. Janson and W. W. Cleland, J. Biol. Chem. 249, 2567 (1974).

266

[25]

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

l,O H3PO4

+

f, Ethyl acetate, O'C, 6 hr 2. H20,NaHC03, oH 3

0 2.0 (CH5~)20

=

0 2 CH3~;OPO 3- NO+

:5.Extract, O°C 4, H20 , NaOH

ioO K2HPO4

¢

5,0CH50~CI

CHa~OH LOBrz, CHzCl2 4.5 hr

'

95%

7"5hr'-5°C, KOH m= H20, pH 8 CH30 OPOa2-K~"÷ 2CHsOH ~" 2KHCO3'I" 3KCI 75%

~'~'o.

,.P(OC.~.

,. ~.,Shr.20"C.

Br

Et20 reflux 4.5 hr

2. 1.0 KOH, EtOH, 0 ° C

90% yield 97% purity

.~ C.~=CCO~,.?PO~'K+ 50% yield 95% purity

FIG. 2. Syntheses of phosphoryl donors.

(Fig. 2) are detailed below. Syntheses which produce AMP can also be coupled to these systems for ATP regeneration using adenylate kinase, a phosphotransferase which catalyzes formation of 2 ADP from ATP and AMP. 13 The principles of ATP regeneration are also applicable to the regeneration of other nucleoside triphosphates since acetate kinase and pyruvate kinase accept other nucleoside phosphates as substrates.14 Dihydroxyacetone phosphate and sn-glycerol 3-phosphate are synthesized on 1 M scales via phosphorylation catalyzed by glycerol kinase using acetyl phosphate as the ultimate phosphoryl donor. These represent the best currently available syntheses for these synthons. Dihydroxyacetone phosphate is useful for sugar phosphate syntheses via aldolase-catalyzed reactionsT; sn-glycerol 3-phosphate can be used for synthesis of enantiomerically pure phospholipids. 15Synthesis of 5-phospho-a-o-ribosyl pyrophosphate (PRPP), an important intermediate in nucleoside and nucleotide biosynthesis, 16 can be effected starting from either ribose or ribose 5-phosphate and using phosphoenolpyruvate as the ultimate phosphoryl ~3 R. L. Baughn, O. Adalsteinsson, and G. M. Whitesides, J. Am. Chem. Sac. 100, 304 (1978). J4 Acetate kinase accepts ADP, CDP, UDP, GDP, IDP, dADP, dGDP, and dCDP [P. I. Bauer and G. Varady, Anal. Biochem. 91, 613 (1978)]. Pyruvate kinase accepts ADP, CDP, UDP, GDP, IDP, and dADP [K. M. Plowman and A. R. Krall, Biochemistry 4, 2809 (1965)]. ~5R. Radhakrishnan, R. J. Robson, Y. Takagaki, and H. G. Khorana, this series, Vol. 72, p. 408, ~6 S. C. Hartman and J. M. Buchanan, J. Biol. Chem. 233, 451 (1958); I. Lieberman, A. Kornberg, and E. S. Simms, J. Biol. Chem. 215, 403 (1955).

[25]

ENZYMATIC REGENERATION OF A T P

267

AMP H20 H+ "203P0~.~0

H

HOOpRP ~p AmP ~

~203PO..~

~".---b.AMP~-'

0 ,

•

0

A

."~CO~

~

J~,.CO 2-

O-?-O-?-O-

HO OH O- OFIC. 3. Enzymatic synthesis of PRPP from AMP using in situ ATP regeneration.

donor. 8 Ribose 5-phosphate is readily accessible from AMP via acidcatalyzed hydrolysis (Fig. 3). Both acetyl phosphate and phosphoenolpyruvate are used in the preparation of a mixture of nucleoside triphosphates from a hydrolyzate of yeast RNA and the further conversion of the UTP in this mixture to uridine-5'-diphosphoglucose, an intermediate in oligosaccharide biosynthesis 9 (Fig. 4). Phosphoryl Group Donors

Disodium Acetyl Phosphate) Reaction of acetic anhydride with phosphoric acid in ethyl acetate, followed by extraction of the acetyl phosphate into water, extraction of acetic acid from the aqueous solution, and neutralization of the reaction mixture yields acetyl phosphate as an aqueous solution. This solution may be used for ATP regeneration without further manipulation. This procedure was designed to avoid the isolation of acetyl phosphate as the diammonium salt 17since ammonium ion causes the precipitation from solution of the magnesium ion required for activity of the kinase as NH4MgPO4 during ATP regeneration. Several other procedures which yield aqueous solutions of acetyl phosphate have been described, 3,5 but the procedure described here is generally the most convenient. Phosphoric acid (85%, 2.0 mol, 135 ml) is dissolved in 1.2 liters of ethyl acetate in a 2-liter flask. The solution is cooled to 0°, and precooled 17 j. M. Lewis, S. L. Haynie, and G. M. Whitesides, J. Org. Chem. 44, 864 (1979).

268

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

RNA

Nuc/eosePl Oligonucleotides

~ P/VPose,Pi ADP+ GDP +

"Aco

÷

UDP

.

M/~P~

o.

HK,XIcK,AcP

PPose 2Pi x

o

j

o o?

UDPGP

PPi

O-

UDP--GIc - HO~Z)H

FIO. 4. Enzymatic synthesis of UDP-Glc from glucose and RNA.

(0°) acetic anhydride (4.0 mol, 376 ml) is added over 40 min. The mixture is stirred for 6 hr at 0 ° and added to a suspension of - 1 liter of water, 500 g of ice, and 168 g of sodium bicarbonate in a 5-liter flask. The resulting mixture is stirred at 0 ° until no more carbon dioxide evolves (approximately 30 min). The organic layer is separated and discarded. The resulting solution (pH -3.0) is washed with one 1.8-liter portion and one 1.0liter portion of ethyl acetate to remove most of the acetic acid. After neutralization of the aqueous solution of acetyl phosphate to pH 7 by addition of 10 M sodium hydroxide (-200 ml), approximately 40 ml of ethyl acetate separates as a second phase. The ethyl acetate layer is separated and discarded. Using this procedure, the concentration of acetyl phosphate in the final solution (1.68 liters) was 1.10 M as determined by enzymatic assay~S; the yield was 1.86 mol (93%). The acetate concentration was 0.4 M as determined by ~H NMR spectroscopy. Monopotassium Phosphoenolpyruvate? This procedure is an improvement over an earlier procedure of Clark and Kirby ~9 in that the is G. M. Whitesides, M. Siegel, and P. Garrett, J. Org. Chem. 40, 2516 (1975). 19 V. M. Clark and A. J. Kirby, Biochem. Prep. 11, 101 (1966).

[25]

ENZYMATICREGENERATIONOF ATP

269

hydrolysis of the dimethyl ester of PEP occurs more rapidly using the conditions described here and the isolation of K÷PEP - is faster and more convenient. Pyruvic acid (480 g of 95% pure material, 5.17 mol), 20 drops of concentrated H2SO4, and 450 ml of CH2C12 are added to a 3-liter, threenecked flask equipped with an overhead stirrer, an addition funnel, and a reflux condenser connected to a bubbler. Bromine (265 ml, 5.17 mol) is added dropwise over a 3.5-hr period to the stirred solution. A white precipitate forms when the addition of bromine is nearly complete. The suspension is stirred for one additional hour and diluted with 40 ml of cyclohexene and 200 ml of ligroin (bp 35-60°). The reaction mixture was cooled in an ice bath. The bromopyruvic acid is collected by filtration, washed with 300 ml of ligroin, and dried at 0.1 torr for 12 hr. Yield: 804 g [mp 64-67 ° (lit. 19 mp 70°), 97% pure, 2° 4.65 mol, 90% yield based on pyruvic acid]. Bromopyruvic acid is converted to the dimethyl ester of PEP (2-hydroxyacrylic acid dimethyl phosphate) in a 12-liter, threenecked flask equipped with a reflux condenser connected to a bubbler, an additional funnel, and a magnetic stirrer. A solution of 752 g (4.37 mol) of bromopyruvic acid (97% pure, used without further purification) in 1.25 liters of dry ether is added dropwise at a rate sufficient to maintain the ether at reflux (3.5 hr) to a stirred solution of 557 mol (4.72 tool) of trimethyl phosphite in 3.85 liters of dry ether. The reaction mixture is stirred for 1 hr at ambient temperature, and the ether removed by rotary evaporation. Crude dimethyl PEP (1000 g) is obtained as a brown viscous oil. This oil is dissolved in 1.67 liters of water, and the solution is stirred at 20° for 15 hr. The spontaneous hydrolysis reaction proceeds to completion in this time and produces 2.64 mol (60%) of PEP and 0.2 tool (5%) of pyruvate (by enzymatic assay). The solution is cooled in an ice bath, and 267 g of solid K O H (85% pure, 4.0 mol) is added (to produce a solution with pH 2.8) followed by 2.7 liters of absolute ethanol. The white precipitate which forms is collected by filtration, washed with 800 ml of cold absolute ethanol, and dried at 0.1 torr, yielding 531 g of K÷PEP - (95% pure by enzymatic assay, 2.45 mol, 50% yield based on crude pyruvic acid). The 31p NMR spectrum of K÷PEP - (0.5 M, D20) consisted of a single peak at -4.51 ppm (85% H 3 P O 4 external reference; resonances downfield o f H3PO4 are reported as positive). The 1H NMR spectrum of K÷PEP - (in D20) consisted of a multiplet at 5.88 ppm (1 H) and a multiplet at 5.54 ppm (1 H) downfield from an internal 2,2-dimethyl-2-silapentane-5-sulfonic acid reference. 20 The purity of bromopyruvic acid was determined by enzymatic assay using an assay for pyruvic acid 22 and from its IH NMR spectrum.

270

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

Methoxycarbonyl Phosphate. 5 A 2-liter Erlenmeyer flask containing 0.40 liters of 1.0 M K2HPO4 (70 g, 0.40 mol) is cooled to a temperature between 0 and - 5 ° in an ice-saltwater bath. Methyl chloroformate (93 ml, 1.20 mol) is added over 6 hr with stirring. The major part of the methyl chloroformate remains as a separate liquid phase during the reaction. The pH is maintained at 8.0 --+ 0.5 by adding 13 N KOH using a pH controller and peristaltic pump; approximately 200 ml is required. The reaction is complete after 7.5 hr as evidenced by the disappearance of the methyl chloroformate phase. Enzymatic assay carried out analogously to that for acetyl phosphate 18showed that the resulting solution was 0.42 M in MCP. Integration of 31p NMR signals showed conversion of 75% of the phosphate to MCP. The NMR spectrum was obtained using a 90 ° pulse angle and a relaxation delay of --5Tl (12 sec). The solution was used directly in synthesis or stored at - 8 0 °. The dilithium salt of MCP is isolated by a method analogous to that used for the isolation of the trilithium salt of succinyl phosphate. 2~ Cold (0°) aqueous solution (2.0 M, 0.30 liter) is added to 0.15 liter of a solution containing 0.30 M MCP (45 mmol) and 0.38 M phosphate (prepared as described above, using 1 equiv of methyl chloroformate). Cold (0°) absolute ethanol (0.50 liter) is added and the resulting precipitate (mostly inorganic phosphate) removed by filtration and discarded. Additional cold ethanol (0.70 liter) is added to the filtrate to precipitate methoxycarbonyl phosphate. The precipitate is collected by filtration, washed with absolute ethanol (1 × 100 ml), ethyl ether (2 × 100 ml), dried under vacuum at 0 ° for 2 hr, and stored at - 8 0 °. The 3Jp NMR showed 85% of the phosphorus as MCP, the remainder as inorganic phosphate. 31p NMR (H20 pH 7) -0.98 (s); J3C NMR (DzO, pD 7) 8 54.9 (q IJc.H = 149 Hz) 153.1 (s); 1H NMR ( D 2 0 , pD 7) 8 2.9 (s). Solid dilithium MCP and frozen aqueous solutions of dipotassium MCP (pH 7-8) are stable for months at - 8 0 °. MCP hydrolyzes rapidly in aqueous solution at ambient temperatures (tv2 = 20 min, pH 7, 25°; tl/2 = 15 hr, pH 7, 0°). Enzymatic Assays General. Standard methods 22 are used to assay enzyme activities and purity of reagents unless otherwise noted. Enzymes and biochemicals are obtained from Sigma Chemical Co., St. Louis, MO. PRPP synthetase (EC 2.7.6.1) is isolated from strain (Su 422) of the nonpathogenic Salmonella 21 S. Kaufman, Arch. Biochem. Biophys. 50, 506 0954). 22 H. U. Bergmeyer, "Modern Methods of Enzymatic Analysis," 2nd Ed. Verlag Chemie, Weinheim, Federal Republic of Germany, 1974.

[25]

ENZYMATIC REGENERATION OF A T P

271

typhimurium supplied by Switzer who has previously purified the enzyme to homogeneity,z3 Acetate kinase activity is measured in the direction of ATP synthesis by the reaction sequence 18 AcK Acetyl p h o s p h a t e + A D P ~ acetate + A T P HK A T P + glucose ~ glucose-6-phosphate + A D P G-6-PDH Glucose 6-phosphate + NAD(P) ~ 6-phosphogluconate acid + NAD(P)H

The assay solution (total volume 3.0 ml) contains: Tris buffer (0.15 M, pH 7.6), glucose (5 mM), ADP (10 raM), acetyl phosphate (5 mM), NAD(P) (0.6 mM), hexokinase (HK, 9 U), glucose-6-phosphate dehydrogenase (G-6-PDH, 9 U) and acetate kinase (AcK, -0.03 U). The formation of NAD(P)H is monitored spectrophotometrically at 340 nm (e = 6220 M -1 cm -1) as a function of time at 25°. Acetyl phosphate and methoxycarbonyl phosphate concentrations are measured using the same assay system by using acetate kinase (9 U), omitting acetyl phosphate but instead adding an aliquot of the solution containing acyl phosphate. Glucose 6-phosphate (G-6-P) is measured in the same assay system using NAD(P) ÷ (0.6 mM) and glucose-6-phosphate dehydrogenase (9 U) and then adding an aliquot of the solution containing glucose 6-phosphate. PRPP Synthetase. A modification of the Ferrari method 24 is employed: PRPP synthetase , PRPP + A M P AdK AMP + ATP ~ 2 ADP PK 2 ADP + 2 PEP ~ 2 A T P + 2 pyruvate LDH 2 pyruvate + 2 N A D H ~ 2 lactate + 2 N A D

A T P + ribose 5-phosphate

The assay mixture (total volume 1.0 ml) contains triethanolamine buffer (100 mM, pH 7.6), K2HPO4 (100 raM), ribose 5-phosphate (r-5-P, 5 mM), ATP (3 mM), PEP (0.8 mM), MgSO4 (10 mM), KC1 (142 mM), NADH (0.2 mM), lactate dehydrogenase (LDH, 2 U), pyruvate kinase (PK, 2 U) and adenylate kinase (AdK, 2 U). PRPP. The determination of PRPP concentrations is based on the decrease in optical density at 295 nm upon the conversion of 5-fluoroorotate to 5-fluoro-UMP.z5 23 R. L. Switzer and K. J. Gibson, this series, Vol. 51, p. 3. 24 M. Ferrari, A. Giacomello, C. Salermo, and E. Messina, Anal. Biochem. 89, 355 (1978). 25 j. G. Flaks, this series, Vol. 6, p. 473.

272

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

orotidine-5-pyrophosphorylase , fluoroorotidine-5-P + PP~ orotidine-5-P-decarboxylase 5-fluoroorotidine-5-P ,5-fluoro-UMP

PRPP + 5-fluoroorotate

The assay solution (final volume ! ml) contains Tris-HC1 buffer, pH 8.5 (20 mM), MgCI2 (2 mM), 5-fluoroorotic acid (0.2 raM), orotidine-5-Ppyrophosphorylase (0.5 U), and orotidine-5-P-decarboxylase (0.5 U). Nuclease P~. The phosphodiesterase activity of nuclease PI using yeast RNA (Boehringer-Mannheim) as substrate is used to assay activity 26 (one unit of activity liberates 1/xmol of acid soluble nucleotide from RNA in 1 min at 37°). The procedure involves incubation of nuclease Pt with yeast RNA, separation of the mononucleotide products from polynucleotides by precipitation of these polynucleotides with uranyl acetate, and measurement of the mononucleotide concentration in the supernatant spectrophotometrically at 260 nm. A 1.10-ml aliquot of a dilute solution of nuclease P~ (containing - 5 U) initiates the reaction in a mixture containing 0.18 ml of 30 mM sodium acetate buffer, pH 5.33; 0.20 ml of a solution of yeast RNA (5 mg/ml) in the acetate buffer; and 20 ~1 of 10 mM ZnSO4. Distilled water (0. I0 ml) instead of enzyme is added to the blank reaction sample. The test tubes are incubated for 15 rain at 37°. The reaction is quenched by the addition of 1.0 ml of a solution containing 0.25% (w/v) uranyl acetate in 10% (v/v) perchloric acid. The tube is rapidly vortexed, then placed in an ice-water bath for 20 rain before sedimenting (5000 g) the undigested RNA. The supernatant is diluted by a factor of 10 and the absorbance of the sample is measured against the blank at 260 nm. An average extinction coefficient for nucleoside monophosphates at 260 nm of 10,600 M -1 cm -1 is used for calculations. Polynucleotide Phosphorylase (PNPase). Activity is measured by monitoring the disappearance of NADH at 340 nm due to the following reactions. 27 PNPase Poly(A) + Pi ~ ADP PK ADP + PEP , A T P + pyruvate LDH Pyruvate + N A D H ~ L-lactate + N A D +

To assay the PAN-immobilized PNPase, poly(A) is treated with nuclease P~ before the addition of PNPase. To a 3-ml plastic cuvette containing 1.5 26 K. R o k u g a w a , T. Fujishima, A. K a n i n a k a , and H. Yoshino, J. Ferment. Technol. 58, 509 (1980); B o e h r i n g e r - M a n n h e i m specification sheet for nuclease P~ (cat. no. 236 225). 27 S. O c h o a a n d S. Mii, J. Biol. Chem. 236, 3303 (1961).

[25]

ENZYMATICREGENERATIONOF ATP

273

ml of HEO is added 25/zl of poly(A) (1% w/v, pH 6.0), 10 tzl of MnCI2 (0.05 M), and 10/zl (50 U) of nuclease PI (1 mg suspended in 0.1 ml of 3 M ammonium sulfate). The mixture is incubated at 25 ° for 10 min; then the following reagents are added: 0.3 ml of potassium phosphate (0.1 M, pH 7.4), 30/xl of MgCI2 (0.5 M), 0.3 ml of glycylglycine (20 mM, pH 7.4), 0.12 ml of PEP (50 mM), 45/zl of N A D H (25 mM), 20 U each of PK and L D H (10/zl each), and aliquots of immobilized PNPase. The rate of NADH oxidation is monitored at 340 nm (25 °) to determine the activity of PNPase. UDPglucose Pyrophosphorylase (UDPGP). Activity was assayed using the following reactions: UDP-GIc + PPi PGM

G-1-P.

NAD

• G-6-P

UDPGP

~ G-1-P + UDP

~ NADH + 6-phosphogluconate

G-6-PDH

The following reagents are added to a 1.0-ml cuvette: 830/~1 of 50 mM Tris-chloride, pH 8.2; 35/zl of 45 mM sodium pyrophosphate; 35 ~1 of 29 mM UDPglucose; 35 tzl of 0.2 M MgCI2; 35/zl of 10 mM NAD ÷ ; 20/zl of 1 mM glucose 1,6-diphosphate; 3/zl of a suspension of G-6-PDH in ammonium sulfate solution ( - 3 U); and 20/zl of a suspension of phosphoglucose mutase (PGM) in ammonium sulfate solution ( - 8 U). The rate of absorbance change at 340 nm is measured until the rate is constant. A 20-tzl aliquot of a solution of UDPGP is added to start the reaction. The difference in rates is proportional to the UDPGP activity. UDPglucose (UDP-GIc). Activity is assayed by measuring the UDPGlc dehydrogenase (UDPGDH)-catalyzed oxidation of UDPglucose to UDPglucuronate. :8 UDP-GIc

UDPGDH

~ UDPglucuronate + 2 NADH

2NAD

The assay solution contains 0.86 ml of distilled water, 0.1 ml of 1.0 M glycine buffer, pH 8.6, 20/zl of 50 mM NAD, and 10 tzl of UDPGDH dissolved in water (-0.1 U). The initial absorbance of the assay solution is recorded before adding the sample (20/zl) to initiate the reaction. Enzyme-Catalyzed Syntheses

General. The enzymatic reactions were carried out in three-necked, round-bottomed flasks, which were modified to accommodate a pH elec2sj. L. Strominger, E. S. Maxwell, and H, M. Kalckan, this series, Vol. 3, p. 975.

274

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

trode. The pH of the reaction mixtures was controlled with a Weston Model 7561 pH controller which regulated the addition of acid or base using an LKB 10200 peristaltic pump. Prior to the addition of the immobilized enzymes, all solutions were deoxygenated by purging with argon using a gas dispersion tube. The enzymatic reactions were conducted at room temperature under an argon atmosphere unless otherwise specified. Immobilization o f Enzymes Enzymes. Enzymes obtained as suspensions in ammonium sulfate solutions were dialyzed twice against 150 volumes of 50 mM HEPES buffer (pH 7.5) at 4° before use; small quantities of ammonium ion left in solution compete with the a,to-diamine (TET) for active ester groups on the PAN. N-Acryloxysuccinimide. N-Hydroxysuccinimide (115 g, 1.0 mol) and triethylamine (110 g) were dissolved in 1500 ml of chloroform at 0 °. Acryloyl chloride (Aldrich, 100 g, I. 1 mol) was added dropwise over a 20min period to the stirred reaction mixture. After being stirred an additional 20 min at 0 °, the solution was washed with 800-ml portions of icecold water and saturated brine, dried with MgSO4, and filtered; 50 mg of 2,6-di-tert-butyl-4-methylphenol (polymerization inhibitor) was added to the chloroform solution which was concentrated to a volume of 300 ml in vacuo using a rotary evaporator and filtered. Ethyl acetate (30 ml) and 200 ml of n-hexane were added slowly with stirring to the chloroform solution which was left to stand at 0 ° for several hours. The precipitated, colorless crystals were separated by filtration and washed with an ice-cold 100-ml portion of a mixture of n-hexane and ethyl acetate (4 : 1), then with another 100-ml portion of n-hexane and ethyl acetate (9 : 1), and finally with two 100-ml portions of n-hexane. The crystals were dried in vacuo at ambient temperature to constant weight; 118 g (70%) was obtained at this stage, mp 69.5-71.0 ° (lit. mp 67°). This material is pure enough for the PAN preparation. A slightly purer product (mp 70.5-71.5 °) could be contained by the recrystallization from a mixture of n-hexane and ethyl acetate. The product has the expected spectral characteristics: NMR (CDC13) 2.85 (s, 4 H), 6.0-7.0 (muir, 3 H); IR (Nujol mull) 1800, 1775, 1735, 1260, 995, 870 cm -~. Poly (acrylamide - co- N - acryloxysuccinimide) (PAN). A 5000-ml, round-bottomed flask, equipped with a Teflon-coated magnetic stirring bar (0.5 x 2.0 in.) and a reflux condenser, was charged with acrylamide (275 g, 3.85 mol), N-acryloxysuccinimide (30 g, 178 mmol), AIBN (1.75 g, 11 mmol), and 2500 ml of THF (AR grade, distilled from CaH2). The reflux condenser was capped with a serum stopper and the flask degassed

[25]

ENZYMATIC REGENERATION OF A T P

275

with nitrogen for 30 min with vigorous stirring to remove dioxygen. The flask was maintained in a constant-temperature water bath at 50° under slight positive pressure of nitrogen for 24 hr. After 24 hr, 1000 ml of THF was added to the flask and the contents were stirred for 10 min. The precipitated white polymer was washed on the funnel four times with 1000-ml aliquots of dry THF, transferred to a vacuum desiccator, and dried under vacuum (0.02 torr) for 24 hr at room temperature; 325 g (106%) of a white, very fluffy produce was obtained. An assay of this polymer showed that it contained 215 /zmol of active ester groups per gram: IR (Nujol mull) 3340, 3200, 1730, 1660, 1210, 1070 cm -I. Assay for the Active Ester Content of PAN. PAN (-50 mg, - 5 0 tool of active ester groups, dried under vacuum at 0.01 torr and 45° for 24 hr) was dissolved and made up to volume in distilled water in a 5-ml volumetric flask. A 50-/xl aliquot of this solution was added into a 5-ml quarts cuvette containing 3000 /~1 of HEPES buffer (0.1 M, pH 7.5), 50 liters of 1 M ethylamine solution, and 10 txl of a 1 M solution of mercaptoethanol; the rate of the appearance of N-hydroxysuccinimide was followed spectrophotometrically at 259 nm at 25°; after the reaction was completed (-90 min) and the increase of the absorbance leveled off, the active ester concentration was calculated. When required, the total concentration of neutral (NHSH) and deprotonated N-hydroxysuccinimide (NSH-) was estimated from the observed concentration of the anion using Eq. (1). At [NHS-] = 1 + 10pKaN"sH-pH-' [NHSH] + [NHS-]

(1)

pH 7.5, for pKaNHsrt = 6.0, this correction is approximately 3% ([NHS-]/ [NHSH] + [NHS-] = 0.969), and was ignored. Procedure for Immobilization of Enzymes. PAN (3.0 g, 450/xmol/g, 1350/zmol of active ester groups) was placed in a 50-ml beaker and 12.0 ml of 0.3 M HEPES buffer (pH 7.5, containing the appropriate substrates of this enzyme to be immobilized; see below) was added. The polymer was quickly (within 1 rain) dissolved by mechanical grinding with a glass rod against the glass wall of the beaker and a 1-in. magnetic stirring bar was dropped into the polymer solution. The solution was stirred magnetically for 30 sec, and 150/xl of 0.5 M dithiothreitol solution and 1.275 ml of triethylene tetramine (1275 tzmol of primary amino groups) were added with vigorous stirring; 30 sec later, 1000/zl of a solution of the enzyme was added. In less than 2 min, the solution set to a transparent, resilient gel. The gel was allowed to stand for 1 hr at ambient temperature and then was transferred to a blender containing 185 ml of HEPES buffer (50 mM,

276

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

pH 7.5, containing 50 mM ammonium sulfate). Blending at low speed for 3 min followed by 30 sec at high speed reduced the gel to a suspension of particles having -100 ~m diameter. The suspension was separated by centrifugation and the gel washed twice with buffer containing no ammonium sulfate. The final suspension was diluted to 150 ml using the same HEPES buffer. Immobilizations were carried out in the presence of substrates or products intended to occupy the active site and protect it against modification during immobilization. 29 The concentration of substrates used to protect the enzyme active site during immobilization and the immobilization yields were glycerol kinase (GK) (glycerol 20 mM, ADP 12 mM, ATP 4 mM, 82%), AcK (AcP 12.5 mM, ADP 20 mM, 40%), PK (PEP 6.7 mM, ADP 25 mM, 46%), PRPP synthetase (PRPP 3 mM, r-5P 5 mM, AMP 4 mM, ATP 3 mM, 80%), HK (glucose 25 mM, ADP 10 mM, 62%), PNPase (inorganic phosphate 50 mM, MgCI2 5 mM, 28%), PGM (40%), UDPGP (UDP-GIc 0.2 mM, MgCI2 15 mM, 38%), inorganic pyrophosphatase (PPase) (no dithiothreitol was added, MgC12 10 mM, inorganic pyrophosphate 2 mM, 51%). s n - G l y c e r o l 3 - P h o s p h a t e . 6 A 2-liter aqueous solution of glycerol (1 mol), ATP (8 mmol), MgCI2" 6H20 (30 mmol), and 2-mercaptoethanol (17 mmol) was adjusted to pH 7.0 and deoxygenated. A suspension of immobilized glycerol (600 U) and acetate kinase (800 U) was added and stirred magnetically at ambient temperatures under argon. Disodium acetyl phosphate (I. 1 mol in 1.2 liters of solution) kept at 0° was added via a peristaltic pump over 5 days. The pH was kept at 7.0 (+0.5) using a pH controller and the automatic addition of 4 M sodium hydroxide solution. The reactor was left for 2 days after the end of acetyl phosphate addition at which time enzymatic assay showed 97% conversion of glycerol to snglycerol 3-phosphate. The solution was separated from the enzyme-containing gel by decantation followed by centrifugation. The enzymes were washed with 200 ml of deoxygenated water and centrifuged, and the supernatant added to the main reaction fraction. The solution was passed through charcoal (-50 g) to remove ATP, and concentrated in v a c u o to 0.5-1 liters. A saturated solution of barium chloride (0.2 mol) was added, and the precipitate, consisting primarily of barium phosphate, was separated by filtration. Additional barium chloride (1 mol) and ethanol (3 liters) was added to precipitate the barium salt of sn-glycerol 3-phosphate. The salt was allowed to precipitate and settle for 2 days to facilitate filtration. After drying in v a c u o , a total of 0.92 mol (92%) of sn-glycerol 3z9 A. Pollak, H. Blumenfeld, M. Wax, R. L. Baughn, and G. M. Whitesides, J. A m . Chem. Soc. 102, 6324 (1980).

[25]

ENZYMATIC REGENERATIONOF ATP

277

phosphate was obtained (314 g of solid containing 90% barium sn-glycerol 3-phosphate as determined by enzymatic assay). The turnover number for ATP during the synthesis was 115, and the activities of enzymes recovered in the gel were GK 95%; AcK 83%. D i h y d r o x y a c e t o n e P h o s p h a t e . 7 The enzyme reaction was repeated for three consecutive runs each of 0.32 mol generating a total of 0.95 mol of DHAP. T o a l-liter deoxygenated solution containing dihydroxyacetone (36.0 g, 0.4 mol), ATP (2.20 g, 4 mmol), MgCI2" 6H20 (1.63 g, 8 mmol), and 2-mercaptoethanol (4 mmol) was added PAN-immobilized glycerol kinase (1500 U, determined with glycerol as substrate) and PAN-immobilized acetate kinase (1700 U). To this mixture a deoxygenated solution of acetyl phosphate (0.45 mol, 410 ml) was added using a peristaltic pump over 16 hr. The mixture was stirred at room temperature under argon, and the pH was automatically controlled at 6.7-7.0 by addition of aqueous NaOH (4 M) through a peristaltic pump. The reaction was stopped when enzymatic assay indicated 98% conversion (16 hr). After separation of the reaction solution from the enzyme-containing gel, the solution was filtered through charcoal (-25 g). The solution was adjusted to pH 4.0 and stored at 4°; the concentration of DHAP retained 90% of its original value after 1 month. A solution of 0.4 mol of dihydroxyacetone phosphate (1.6 liters, 0.25 M) was prepared as above using the recovered enzymes. Quantitative 31p NMR was used to determine inorganic phosphate content and a solution of barium chloride (14.6 g, 60 mmol) was added to precipitate it at pH 6. After the precipitate was removed by filtration, the dihydroxyacetone phosphate was precipitated by addition of barium chloride (0.4 mol, 97.6 g) and three volumes of ethanol. Drying in oacuo resulted in 116 g (0.33 mol, 87% pure calculated as Ba. DHAP by enzymatic assay, overall yield 82.5%). A solution of 0.4 mol of dihydroxyacetone phosphate was prepared as above (1.6 liters, 0.25 M) using the recovered enzymes. After removal of the inorganic phosphate by precipitation as barium phosphate at pH 6.0, and reducing the pH to 3.0 with Dowex 50-X8 (hydrogen form), the solution was passed through charcoal (-25 g). Acetic acid was removed by extraction with two l-liter portions of ethyl acetate, and the pH was adjusted to 4.8 by addition of NaOH (2 M). The solution was concentrated in vacuo to -500 ml and lyophilized. The isolated monosodium dihydroxyacetone phosphate (119.0 g) was 82% pure by enzymatic assay (0.32 mol, 119 g, 79.7% overall yield). The turnover number for ATP was 83 and the recovered activities for the enzymes after three runs were GK 62%, AcK 30%.

278

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[25]

Ribose 5-Phosphate (r-5-P) by Acid-Catalyzed Hydrolysis of AMP. 8 Dowex 50W-X8 (910 g, H + form) was placed in a 4-liter Erlenmeyer flask and distilled water was added to a total volume of 2.2 liters. The flask was heated to boiling and stirred with an overhead stirrer. Disodium AMP (Sigma, 131 g, 262 mmol) was rapidly added to the flask (<2 min). The mixture was kept at boiling with stirring for 8.5 min and then immediately cooled in an ice-water bath. When the temperature of the mixture had dropped to 35°, the Dowex was removed by filtration. The filtrate was further cooled to 0° and the pH was adjusted to 7.5 with 10 N NaOH. The solution was concentrated on a rotary evaporator to - 1 liter. Lyophilization of the concentrated solution yielded 79.0 g of pale yellow hygroscopic powder, which contained 246 mmol of disodium r-5-P (94% yield, 83% purity). 5-Phospho-a-o-ribosyl Pyrophosphate from r-5-P.8 To a solution (1 liter) containing disodium r-5-P (33 g, 100 mmol, 83% purity), disodium ATP (2.8 g, 5.6 mmol), MgCI2 • 6H20 (3.2 g, 16 mmol), K+PEP - (46 g, 200 mmol, 90% pure), K2HPO4 (4.1 g, 30 mmol), and disodium EDTA (336 mg, 1 mmol) were added separately immobilized PRPP synthetase (90 U), PK (180 U), and AdK (250 U). The reaction was run at 30°, pH 7.4. After 4 days 75 mmol of PRPP had been formed. The solution was separated from the gel by centrifugation and stirred at 4 ° with activated charcoal (40 g). The resulting solution contained 70 mmol of PRPP and was stored at - 8 0 °. The recovered enzymes activities were PRPP synthetase, 82%; PK, 72%; AdK, 60%. Conversion of RNA to a Mixture of Nucleoside Triphosphates. 9 Yeast RNA (15 g, 95% pure) and nuclease Pl (1 mg) in 20 ml of water containing 0.1 mM MnCI: (pH 6.0) was stirred at 50° for 1 hr. The mixture was cooled to room temperature and neutralized to pH 8.0 by addition of cold aqueous NaOH (5 N), then transferred to a l-liter solution containing potassium phosphate (0.2 M), MgClz (2 mM), PEP monopotassium salt (80 mM), and PAN-immobilized PNPase (8 U in 20 ml of gel) and PK (50 U in 1 ml of gel). The reaction mixture was stirred at room temperature under argon for 4 days with the pH controlled in the range 8.0-8.5. Enzymatic analysis 3° indicated the mixture contained 30 mmol of nucleoside triphosphates (NTP) (68% yield based on RNA with a monomer equivalent MW of 360). No further increase in the yields of NTP was observed on further digestion. After separation of the gel by decantation, the solution was adjusted to pH 3.0 at 4° by addition of cold concentrated HC1 with stir30 W. Grfiber, H. Mrllering, and H. U. Bergmeyer, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), p. 2080. Verlag Chemie, Weinheim, Federal Republic of Germany, 1974.

[25]

ENZYMATIC REGENERATIONOF ATP

279

ring, and the precipitated material was removed by filtration. The filtrate was adjusted to pH 8.0 at 0 ° by addition of 5.0 N NaOH, and the solution concentrated under reduced pressure at 35° to a volume of 50 ml. The resulting solution contained a mixture of NTP (27 mmol as determined by enzymatic assay, 3° 62% overall yield based on RNA). HPLC analysis of the mixture showed the relative composition to be ATP, 24%; UTP, 28%; GTP, 30%; CTP, 18%. This mixture showed 5% decomposition after storage for 2 months at - 2 0 ° (pH 8.0). It can be used without further purification for enzymatic syntheses (see below). The enzymatic activities recovered from this reaction were PNPase, 75%; PK, 92%. Glucose 6-Phosphate. 9 This preparation illustrates the use of the crude nucleoside triphosphate mixture prepared in the preceding procedure as an ATP source for generating the glucose 6-phosphate necessary for UDP-glucose synthesis. To a 2-liter solution containing 5 ml of the NTP mixture (2.7 mmol), glucose (1.0 mol), H K (330 U), AcK (310 U), and mercaptoethanol (0.75 g, 5 raM) was added (NH4)2 AcP 17 (1.1 mol, 86% pure) over a period of 4 days. (The ammonium salt of AcP was used only because this procedure was developed before the procedure for synthesis of ammonium-free AcP described above.) The solution was separated from the enzyme-containing gels by decantation. Barium chloride (0.2 mol) was added to precipitate inorganic phosphate, and the precipitate was removed by filtration. Another portion of barium chloride (1.0 mol) was added followed by addition of 6 liters of ethanol. The precipitated solid (412 g) contained 0.68 mol of barium glucose 6-phosphate as determined by enzymatic assay (84% purity, 63% yield based on glucose). The turnover numbers and recovered activities were NTP, 185 (82%); HK, 92%; AcK, 81%. UDPglucose (UDP-GIc). 9 A portion of the NTP solution (40 ml) containing 6.2 mmol of UTP was diluted to 200 ml, followed by addition of PAN-immobilized UDPGP (50), PPase (60 U), PGM (52 U), and glucose 6-phosphate (6.2 mmol). The reaction mixture was stirred under argon for 20 hr with the pH controlled at 7.5 (-+0.5). Enzymatic analysis of the solution indicated that it contained 6 mmol of UDP-Glc (97% reaction yield) and 14 mmol of a mixture of nucleoside triphosphates. The solution was concentrated under reduced pressure at a temperature below 40° to a volume of 30 ml. A portion (3 ml) of the above solution containing 0.6 mmol of UDP-GIc was applied to a column (2.7 × 42 cm) of Bio-Rad P-2 equilibrated with water, and eluted with water at a rate of 0.6 ml/min. Fractions (6 ml) were collected and the absorbance at 260 nm was measured. The fractions with elution volumes between 70 and 90 ml were combined and lyophilized. The resulting amorphous powder (0.4 g) contained 92% of dipotassium UDP-GIc (0.57 mmol) as determined enzymati-

280

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

cally. The fractions with elution volumes 90-130 ml contained ATP, GTP and CTP, and were combined and lyophilized (0.6 g). This quantity is equivalent to a yield of dipotassium UDP-GIc of 92% based on glucose 6phosphate. Acknowledgments Our work with PRPP s y n t h e t a s e depended on generous help and advice from Professor R. L. Switzer and co-workers. Drs. Akiva Gross, Jerome Lewis, and Alfred Pollak contributed significantly to the d e v e l o p m e n t o f various of the procedures described.

[26] E q u i l i b r i u m a n d K i n e t i c a l l y C o n t r o l l e d S y n t h e s i s w i t h E n z y m e s : Semisynthesis of Penicillins and Peptides l

By

VOLKER KASCHE, URSULA HAUFLER,

and LUTZ R I E C H M A N N

The increased availability of hydrolases and their potential biotechnical application as catalysts in the synthesis of condensation products have led to an increased interest in studying these enzymes. Some products that have been synthesized using hydrolases as biocatalysts are given in Table I. The enzyme-catalyzed synthesis of the condensation product AN can be carried out either as an equilibrium-controlled process: EH AOH + NH.

" AN + H20

(1)

where the enzyme only accelerates the rate with which the equilibrium is obtained, or as a kinetically controlled process:

AB + N H

EH

, AN + HB

H20 EH

* A O H + N H + HB

(2)

where an activated substrate AB (ester or amide) is used. In the latter process the enzyme acts as a transferase transferring the group A from AB to a nucleophile NH. Generally the biosynthesis of condensation ~This study has been supported by Deutsche Forschungsgemeinschaft (Ka 505/3-1), Bundesminister for Forschung und Technologie (PTB 8477), and Dr. O. R0hm Ged~ichtnisstiftung.

METHODS IN ENZYMOLOGY. VOL. 136

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

280

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

cally. The fractions with elution volumes 90-130 ml contained ATP, GTP and CTP, and were combined and lyophilized (0.6 g). This quantity is equivalent to a yield of dipotassium UDP-GIc of 92% based on glucose 6phosphate. Acknowledgments Our work with PRPP s y n t h e t a s e depended on generous help and advice from Professor R. L. Switzer and co-workers. Drs. Akiva Gross, Jerome Lewis, and Alfred Pollak contributed significantly to the d e v e l o p m e n t o f various of the procedures described.

[26] E q u i l i b r i u m a n d K i n e t i c a l l y C o n t r o l l e d S y n t h e s i s w i t h E n z y m e s : Semisynthesis of Penicillins and Peptides l

By

VOLKER KASCHE, URSULA HAUFLER,

and LUTZ R I E C H M A N N

The increased availability of hydrolases and their potential biotechnical application as catalysts in the synthesis of condensation products have led to an increased interest in studying these enzymes. Some products that have been synthesized using hydrolases as biocatalysts are given in Table I. The enzyme-catalyzed synthesis of the condensation product AN can be carried out either as an equilibrium-controlled process: EH AOH + NH.

" AN + H20

(1)

where the enzyme only accelerates the rate with which the equilibrium is obtained, or as a kinetically controlled process:

AB + N H

EH

, AN + HB

H20 EH

* A O H + N H + HB

(2)

where an activated substrate AB (ester or amide) is used. In the latter process the enzyme acts as a transferase transferring the group A from AB to a nucleophile NH. Generally the biosynthesis of condensation ~This study has been supported by Deutsche Forschungsgemeinschaft (Ka 505/3-1), Bundesminister for Forschung und Technologie (PTB 8477), and Dr. O. R0hm Ged~ichtnisstiftung.

METHODS IN ENZYMOLOGY. VOL. 136

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

[26]

KINETICALLY CONTROLLED SYNTHESIS WITH ENZYMES

NH

HB EH + AB..

"EH ,, AB .

7_~E -

k~

~

A

281

~ EH + AN

NH

NH

EH

EH + AOH

NH

HB EH + AB.

'EH ', AB.

"~ ' E

A \

_k~

EH

~E

H20

AOH

A.

k~ ,N ~ ..

"EH + AN

~3,N

EH

AOH

NH

FIG. 1. Possible mechanisms for semisynthesis catalyzed by penicillin amidase and

serine proteases. Symbols, EH, enzyme; AB, donor ester; HB, leaving group, alcohol; E - A , acyl-enzyme; NH, nucleophile; AOH, hydrolysis product; AN, condensation product; dotted lines indicate a noncovalent binding.

products is kinetically controlled [Eq. (2)]. 2 Whether this kinetically controlled process can compete with the equilibrium-controlled process in the biotechnological synthesis of condensation products must be answered by studies on the mechanism and the yield-controlling factors. Some of these factors will be analyzed here in connection with the enzyme-catalyzed semisynthesis of/3-1actam antibiotics and peptides. Mechanism Two different mechanisms have been proposed for hydrolase-catalyzed condensation reactions. One (Fig. 1A) involves no specific nucleophile binding to the reactive enzyme-substrate (acyl-enzyme or noncovalent complex) intermediate. This mechanism is most frequently used to explain condensation reactions catalyzed by proteases. 3,4 In the other 2 B. Alberts, D. Bray, J. Lewis, M. Raft, K. Roberts, and J. D. Watson, "Molecular Biology of the Cell," p. 81. Garland, New York, 1983. 3 I. S. Fruton, Ado. Enzymol. Relat. Areas Mol. Biol. 53, 239 (1982). 4 D.-D. Jakubke and P. Kuhl, Pharmazie 37, 89a (1982).

282

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KINETICALLY CONTROLLED SYNTHESIS WITH ENZYMES

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~ ,-~

283

284

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

mechanism (Fig. 1B) the nucleophile must be bound to the reactive enzyme-substrate (acyl-enzyme or noncovalent complex) intermediate. 5,6 The desired condensation product can only be formed from this ternary complex. This mechanism involves two reactive enzyme-substrate intermediates that both can react with H20. To distinguish between the two mechanisms, the initial rates of formation of AN (VAN) and AOH (VA) can be measured as function of the nucleophile content [NH]. From this, the apparent ratio of the deacylation rate constants 7,8 (Fig. 1) can be determined: k~ __ VAN[ U 2 0 ] k3/app vA[NH]

(3)

(k~/k3)app = k~/k3

(4)

This ratio is

for the mechanism without nucleophile binding (Fig. 1A) and k~

k3,N

k3/app = k3KN + k3,N[NH]

(5)

for the mechanism with nucleophile binding (Fig. 1B). Thus the nucleophile concentration influences (k~/ka)app for homogeneous enzyme preparations catalyzing reactions in accordance with the mechanism in Fig. lB. This relation has been used to show that the mechanism in Fig. 1B applies for the synthesis of semisynthetic penicillins and peptides catalyzed by penicillin amidase from Escherichia coli and mammalian serine proteases, respectively. 7-9 Normally the nucleophile is a charged species that contributes to the ionic strength. Because the rate constants and (k~/k3)app depend on the ionic strength, it is essential to keep it constant in such studies. Experimental Procedures Enzymes. Penicillin amidase from E. coli (EC 3.5.1.1 l) was a gift from Dr. K. Sauber (Hoechst AG). It was purified to homogeneity by hydrophobic chromatography on a TSK SW 3000 G column (7 × 600 mm, 5 A. L. Fink and M. L. Bender, Biochemistry 8, 5109 (1969). 6 K. Wallenfels and O. P. Malhorta, Adv. Carbohydr. Chem. 16, 239 (1961). 7 L. Riechmann and V. Kasche, Biochem. Biophys. Res. Commun. 120, 686 0984). a V. Kasche, U. Haufler, and R. Z611ner, Hoppe-Seyler's Z. Physiol. Chem. 365, 1435 (1984). 9 A. g . Klesov, A. L. Margolin, and V.-Y. K. Shvyadas, Bioorg, Khim. 3, 654 (1977).

[26]

KINETICALLY CONTROLLED SYNTHESIS WITH ENZYMES

285

LKB). The enzyme was eluted with 0.005 M KH2PO4, pH 7.4, and was homogeneous as judged from analysis by isoelectric focusing: Bovine o~chymotrypsin (EC 3.4.21.1, Worthington DCI) and trypsin (EC 3.4.21.4, Merck 24579) were purified by affinity chromatography using soybean trypsin inhibitor-Sepharose as a biospecific adsorbent.l°,u The isolated/3trypsin was used for synthesis the same day as it was prepared. Penicillin amidase from E. coli was immobilized in Eupergit C and was a gift from Dr. D. Kramer (R6hm GmbH). The unreacted oxirane groups were inactivated with mercaptoethanol. Activity of immobilized enzyme was determined using 10 mM benzylpenicillin in phosphate buffer (I = 0.2 M), pH 7.8, at 37°, and the activity was found to be 150 units/g dry gel. The average particle diameter in solution was 85/zm. Substrates. Cephalexin and phenylacetic acid were obtained from Sigma. Phenylacetylglycine was prepared as described in Ref. 12. D-Phenylglycine methylester was provided by Dr. D. Kr~imer (R6hm); 6-aminopenicillanic acid (6-APA), ampicillin, benzylpenicillin, and 7-aminodeacetoxycephalosporanic acid (7-ADCA) were provided by Dr. Sauber (Hoechst). N~-Benzoyl-L-arginine ethyl ester (Bz-Arg-O-Et) was obtained from Merck (Darmstadt, West Germany), N~-benzoyl-Larginine (Bz-Arg) and L-valineamide (VaI-NH2) from Sigma. All substrates eluted as single peaks in high-performance liquid chromatography (HPLC). Synthesis of Condensation Products. The substrates are dissolved in buffer or H20. Trishydroxymethylaminomethane (Tris) should not be used as a buffer in these experiments as it is a good nucleophile at pH - 8 and can react with the acyl-enzyme)3 The pH of the solution is adjusted to the desired pH, and then the biocatalyst is added. The solution is kept in a thermostated vessel that is stirred or rotated to keep the particles in suspension. The pH is kept constant during the reaction. At different times samples are withdrawn, diluted, filtered, and analyzed by HPLC using a Spectra Physics solvent delivery system (SP 8700) and a Spectra Physics detector (SP 8400). The following conditions are used to separate the products and reactants: Benzylpenicillin synthesis: RP-18 (10 /xm) column (4.6 × 250 mm from Knauer); isocratic elution (2 ml/min) with 35% (v/v) methanol in 0.067 M KH2PO4, pH 4.7, at 56°; detection at 225 nm. Ampicillin and cephalexin synthesis: RP-8 (10/zm) column (4.6 × 250 mm from Knauer); elution (2 ml/min) for 2 min with 5% (v/v) methanol in 10 V. 11 V. ~2V. ~3V.

Kasche, Biochem. Biophys. Res. Commun. 38, 875 (1970). Kasche, Arch. Biochem. Biophys. 173, 269 (1976). Kasche and B. Galunsky, Biochem. Biophys. Res, Commun. 104, 1215 (1982). Kasche and R. Z611ner, Hoppe-Seyler's Z. Physiol. Chem. 365, 631 (1982).

286

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

0.067 M KH2PO4, pH 6.0, and then with 25% (v/v) MeOH in the same buffer for 5 min at 56°; detection at 225 nm. Peptide synthesis: RP-18 (10 /xm) column (4.6 × 250 mm from Knauer); isocratic elution (1.2 ml/min) with 70% (v/v) methanol in 0.03 M KH2PO4, pH 4.6, at room temperature; detection at 250 nm. The concentrations are determined from calibration curves using stock solutions of the different compounds. Factors Influencing the Yield of Condensation Product Enzyme Concentration For the kinetically controlled processes in Table I a kinetically controlled maximum j2 in the desired condensation product AN is often observed. In these cases the yield of AN in the kinetically controlled process is much larger than the yield observed in the equilibrium-controlled process (Fig. 2). The kinetically controlled maximum is also obtained much earlier than the final equilibrium. The nonactivated substrate AOH is generally a poorer substrate for the enzyme than the activated substrate. Thus higher concentrations of acyl-enzyme and rates of formation of the condensation product AN can be obtained in the kinetically controlled process than in the equilibrium-controlled process. As long as the rate of formation of AN in the kinetically controlled process exceeds the rate of hydrolysis of AN, higher than equilibrium yields of AN will be observed. When the product AN is soluble and formed according to the mechanism in Fig. 1B, its concentration at the kineticaUy controlled maximum in a batch reactor is given by the relation 14 k~,N[NH] [AN]max = k3[H20]KN + (k3,N[H20] + k~,N)[NH]

(kcat/Km)AB,NH × (kcat/Km)AN,NH [AB]

(6)

where the subscript N denotes the properties of the acyl-enzyme with bound nucleophile. This relation shows that the maximum yield is not dependent on the enzyme concentration. This is demonstrated experimentally in the synthesis of benzylpenicillin and N~-benzoyl-L-arginyl-L valine amide (Bz-Arg-VaI-NH2) (Fig. 2). Contrary to the equilibrium yield the maximum yield does depend on properties of the enzyme [deacylation rates, binding constant of the nucleophile NH (KN), turnover numbers z4 V. Kasche, Enzyme Microb. Technol. 8, 4 (1986).

[26]

287

K I N E T I C A L L Y C O N T R O L L E D SYNTHESIS W I T H ENZYMES

A 0,37

0,19

}

0.093

4037 O/ml

550

Z80

lt, O

n~

°

~ ,o

,0

,'0

, bo

rIME, rain

2

....

.......

....

I00

?'II~E, rain

FIG. 2. The influence of the enzyme content on the kineticaUyand equilibrium-controlled synthesis. (A) Penicillin amidase-catalyzed synthesis of benzylpenicillin at pH 6.0, 25° and total ionic strength 0.2 M. Initial conditions: ( ) kinetically controlled synthesis; 10 mM phenylacetylglycine and 10 mM 6-aminopenicillanic acid. (.... ) equilibrium-controlled synthesis; 10 mM phenylacetic acid and 10 mM 6-aminopenicillanic acid. (B) Trypsin (unpurifled preparation, Merck 25479)-catalyzed synthesis of Bz-Arg-Val-NH2at pH 9, 25° and total ionic strength 0.2 M. Initial conditions: ( ) kinetically controlled synthesis; 7 mM BzArg-O-Et and 83 mM VaI-NH2. (---) equilibrium-controlled synthesis; 7 mM Bz-Arg and 83 mM Val-NHz. The enzyme concentrations used are given above the maxima of each curve. (kcat), and M i c h a e l i s - M e n t e n constants (gm) for the activated substrate and product]. Equation (6) can be used for a rational analysis of the factors that influence the yield to obtain optimal reaction conditions. For soluble products larger yields and product formation rates can be obtained in the kinetically controlled [Eq. (2)] than in the equilibrium-controlled process. In this case the use of immobilized e n z y m e s is advantageous. The reaction can be easily terminated at the m a x i m u m of product formation by filtering off the biocatalyst, and residual e n z y m e must not be r e m o v e d in d o w n s t r e a m processing. F o r an insoluble product AN, practically quantitative yields can be obtained in b o t h the kinetically and equilibrium process. E v e n then the rate of p r o d u c t formation is m u c h larger for the former process. In this case immobilized e n z y m e s are of limited use.

Enzyme Properties Equation (6) shows that the maximal yield in the kinetically controlled process depends on the properties of the enzyme. Penicillin G amidases f r o m different species have b e e n shown to differ markedly in molecular

I/,40

288

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

~J

rr [Z

~u

,.J

<

~J

Z

~o~

.1

z~ ~z

°.~±

<

e~

O p~

~.r)

"<'~

o

< b. z ~2

¢)

O

~.~, , ~ Eo

o~

.O

,~

~ o ~

,.

z

L) '~

°~

r~

e~ tD

.

[26]

[26]

KINETICALLY

CONTROLLED

SYNTHESIS

WITH

ENZYMES

e% ,,.g,

.-,..i

,-.¢ ~

<<<<<<<<< g ~ g g ~ g ~ g ~

,,..:, eq

~S r',-

~

.,..:

,~ ~ ' ~

~

N ~ N N N N ~ N .~

e

>r....6s = ~, >~,

0

L)

.

a~ -'~

.~ ¢icfi ai

289

290

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

weight, r5 while the enzyme from E. coli has been shown to exist as several enzymatically active forms with different isoelectric points. 8 The heterogeneity of the enzyme from one species, and different properties of the enzyme from different species, may account for the different yields obtained using similar conditions (Table II). At the large substrate concentrations (> 100 mM) that are desirable in biotechnological applications the difference in yields for free and immobilized enzyme is marginal. At lower substrate contents the yields for the immobilized and cell-bound enzyme are lower than that found for the free enzyme. Then mass transfer effects cannot be neglected. ~6Thus to find the enzyme that gives maximal yields both enzymes from different species and different enzyme forms from one species must be studied. For serine proteases data on their hydrolytic specificity can be used to predict yields when they are used for peptide synthesis. The PI specificity in kinetically controlled synthesis correlates well with the corresponding specificity for hydrolysis. 7 The enzyme properties depend on pH and temperature. Since the nucleophilic groups used in the kinetically controlled synthesis must be uncharged, the reaction has to be carried out at a pH > the pK value for the nucleophilic groups. For 6-APA and 7-ACA (7-ADCA) the pK values are 4.5-5 and for the a-amino groups involved in the peptide synthesis they are - 8 . This limits the pH range in which high yields can be obtained in the kinetically controlled process to 6-8 for fl-lactams and 8-10 for peptides. The maximal yield has been shown to have a temperature optimum about 20 ° in/3-1actam and peptide synthesis. 14,17 Such temperature optima have not been observed for the equilibrium-controlled synthesis.18 Water Content

The equilibrium yield and the maximum yield in the kinetically controlled synthesis [Eq. (6)] depend on the water content. The latter can be reduced by adding organic (inert) solvents. This does, however, also change the properties and stability of the enzyme that may counteract the effect due to the reduction in water content (Table III). To evaluate the effect of organic solvents on the yields in equilibrium and kinetically controlled synthesis of condensation products more data on enzyme properties and stabilities in H20/organic solvent mixtures are required. This 15 E. J. Vandamme, in "Economic Microbiology" (A. H. Rose, ed.), Vol. 5, p. 467. Academic Press, New York, 1980. 16 V. Kasche, Enzyme Microb. TechnoL 5, 2 (1983). 17 K. Kato, K. Kawahara, T. Takahashi, and S. Igarasi, Agric. Biol. Chem. 44, 821 (1980). Js B. McDougall, P. Dunnill, and M. D. Lilly, Enzyme Microb. Technol, 4, 114 (1982).

[26]

KINETICALLY CONTROLLED SYNTHESIS WITH ENZYMES

291

TABLE III INFLUENCE OF ORGANIC SOLVENT ON THE YIELDS IN THE EQUILIBRIUM AND KINETICALLY CONTROLLED SYNTHESIS OF BENZYLPENICILLINa Yield (% of 6-APA)

Enzyme and organic solvent

Equilibriumcontrolled synthesis

Kinetically controlled synthesis (at maximum)

Free enzyme None 7 29 20% acetone 18 33 20% DMSO 21 27 Enzyme immobilized in Lichrospher-NH2 (d = 10/xm) None 6 30 20% acetone 11 25 20% DMSO 14 25 ° At pH 6.0, 25° and ionic strength 0.2 M catalyzed by penicillin amidase from E. coli. Initial conditions: 10 mM phenylacetylglycine, 10 mM 6-aminopenicillanic acid (kinetic approach); 10 mM phenylacetic acid, 10 mM 6-aminopenicillanic acid (equilibrium approach).

may explain the observation that in equilibrium-controlled synthesis the yield was found to depend on the enzyme content. 19 In this case the yield cannot be influenced by the enzyme content or its properties. Enzyme inactivation in the H20/organic solvent z° that was not studied in Ref. 19 may explain the observed findings. The influence on equilibria of organic solvents miscible with water can be predicted as analyzed in several studies.2~,22

Substrate Properties In both the equilibrium-controlled and the kinetically controlled process the nature of the substrate may influence both the product yields and the reaction rates. In the former case the yield is mainly affected when the equilibrium depends on the charge of the substrates. This is observed as a 19 K. 20 H. 21 G. 22 K.

Morihara and T. Oka, J. Biochem. 89, 385 (1981). H. Weetall and W. P. Vann, Biotechnol. Bioeng. 18, 105 (1976). A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). Martinek, A. N. Semenov, and I. V. Berezin, Biochem. Biophys. Acta 58, 76 (1981).

292

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[26]

pH dependence in the yield of peptide formed in the condensation of free amino and carboxyl groups. 21,~2 However, charged groups that are not involved in the condensation reaction may also influence both the product yields and reaction rates. For example, low yields and reaction rates are observed when L-aspartate reacts with e-phenylalanine methyl ester to form aspartame in the condensation reaction catalyzed by thermolysin. It is only when the amino group in L-aspartate is protected that the condensation rate is increased and an insoluble addition product between protected aspartate and L-phenylalanine methyl ester can be formed. 23 Then almost quantitative yields of protected aspartame are obtained. Endoproteases frequently contain a negatively charged residue in or near the binding site for the P~-amino acid. 24 When they are used as catalysts in peptide synthesis the carboxyl group of the amino acid nucleophile NH must be protected. An unprotected amino acid is repelled from the negatively charged binding sites for P~ amino acids in endoproteases as chymotrypsin and trypsin. This influences both the yields and rates of kinetically controlled peptide semisynthesis. 25 The nature of substrate activation influences the ratio (kcat/Km)AB in Eq. (6). To obtain larger yields more specific substrates are advantageous. For biotechnological applications substrate concentrations larger than 100 mM are desirable. The higher concentrations may contribute to the solvent properties (ionic strength, polarity), and their influence on the properties of the enzyme must also be considered. Conclusions Higher yields are obtained in the kinetically controlled synthesis [Eq. (2)] of condensation products catalyzed by enzymes than in the corresponding equilibrium-controlled synthesis [Eq. (1)]. Contrary to that of the latter process the yield of the former process depends on the properties of the used enzyme. In addition, large product yields can be obtained at the kinetically controlled maximum even when the (final) yield of the corresponding equilibrium-controlled process is negligible. Thus the kinetically controlled maximum is a suitable end point in the synthesis of condensation products catalyzed by immobilized hydrolases. 23 y. Isowa, M. Ohmori, T. Ichikawa, K. Mori, Y. Novaka, K. Kihara, K. Oyama, M. Satoh, and S. Nishimura, Tetrahedron Lett. 28, 2611 (1979). 24 H. Dugas and C. Penney, "Bioorganic Chemistry," Chap. 4. Springer-Verlag, Berlin, 1981. 25 V. Kasche, B. Galunsky, U. Haufler, and R. Z611ner, in "Enzyme Technology" (R. M. Lafferty, ed.), p. 297. Springer-Verlag, Berlin, 1983.

[27]

OPTICAL RESOLUTION OF dI-MENTHOL

293

[27] Optical R e s o l u t i o n of d l - M e n t h o l b y E n t r a p p e d Biocatalysts

By SABURO FUKUI and ATSUO TANAKA /-Menthol is widely used in the cosmetic, food, and pharmaceutical industries. /-Menthol can be isolated from peppermint oil or other mint oils; it can also be produced by optical resolution of dl-menthol mixtures obtained chemically by hydrogenation of thymol. Chemically synthesized d/-menthol, however, contains four isomers: d/-menthol, dl-isomenthol, dl-neomenthol, and dl-isoneomenthol. Thus, the separation of/-menthol from its isomers and racemates is industrially important. Resolution of menthyl ester racemates by microbial esterases in aqueous systems has been reported.l,2 However, neither menthyl ester nor menthol is soluble in water. Hence, it would be advantageous to employ an appropriate organic solvent for the stereoselective enzymatic hydrolysis of menthyl esters. In addition, racemates of menthol and other terpenoids can be resolved optically by stereoselective esterification of these racemates with lipase. In this case, it is essential to choose appropriate organic solvents both to dissolve the reactants and to shift the equilibrium toward esterification. The reaction will only succeed, however, if the used biocatalysts are not inactivated by organic solvents. As immobilization could render the biocatalysts more stable in organic solvents, we have entrapped both yeast ceils and lipase in polymer gels. This article deals with optically selective hydrolysis of dl-menthyl succinate by gel-entrapped yeast cells (Fig. l) and stereoselective esterification of d/-menthol by gel-entrapped lipase (Fig. 2). Stereoselective Hydrolysis of dl-Menthyl Succinate3

Materials and Methods Prepolyrners. Two kinds of water-miscible urethane prepolymers (PU3 and PU-6) are prepared by Toyo Tire & Rubber Co., Japan. 4-7 PU-3 T. Oritani and K. Y a m a s h i t a , Agric. Biol. Chem. 37, 1695 (1973). 2 y . Y a m a g u c h i , A. K o m a t s u , and T. Moroe, J. Agric. Chem. Soc. Jpn. 51, 411 (1977). 3 T. O m a t a , N. I w a m o t o , T. K i m u r a , A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 11, 199 (1981). 4 S. F u k u s h i m a , T. Nagai, K. Fujita, A. Tanaka, and S. Fukui, Biotechnol. Bioeng. 20, 1465 (1978).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

294

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

CH3 0-C-CH, (( ~:,CH.~"~-C-0H0 011 dl-Menthyl succlnate

~

,,,OH l_-Menthol

[27]

CH~) +

0T-C-CH'~CH~'':C'0':#'(01P /~L II d_-Henthylsuccinate

FIG. 1. Stereoselective hydrolysis of dl-menthyl succinate catalyzed by R. minuta var. texensis cells.

gives a hydrophobic gel and PU-6 a hydrophilic gel. A photo-cross-linkable resin prepolymer, ENT-4000, is synthesized by Kansai Paint Co., Japan. 7,8 Cultivation of Yeast. Various microorganisms, especially those belonging to the genera Rhodotorula and Bacillus, show high hydrolytic activity using menthyl succinate as a substrate. Among the strains examined Rhodotorula minuta var. texensis IFO 1102 was selected because it showed both high hydrolytic activity and satisfactory stereoselectivity in the catalyzed process. Rhodotorula minuta var. texensis is cultivated with shaking (220 rpm) at 30° in 500-ml shaking flasks each containing 100 ml of a medium of the following composition (per liter): treacle, 50 g; corn steep liquor, 50 g; ammonium sulfate, 5 g; mineral mixture, 10 ml. The mineral mixture was (g per liter): MgSO4.7H20, 20; FeSO4-7H20, 5; CaCI2, 2; MnC12"4H20, 0.2; NaMoO4.2H20, 0.1 and NaCI, 0.1. The pH of the medium is adjusted to 7.0 with 1 N NaOH before sterilization. Cells are harvested by centrifugation after 70 hr of cultivation, washed twice with 20 mM potassium phosphate buffer (pH 7.0), mixed thoroughly, and stored in sealed sample bottles at - 2 0 °. The frozen cells maintain their original hydrolytic activity for at least one month. Immobilization of Cells. Thawed cells (1 g wet cells) suspended in 3 ml of water are entrapped wih I g of ENT-40006.7; 1 g wet cells suspended in 2 ml of water is immobilized with I g of PU-3, PU-6, or mixtures of these prepolymers. 6,7 The gels so prepared are cut into small pieces ( - 1 x 1 mm for ENT-4000, thickness, - 0 . 5 mm; - 1 x 1 × 1 mm for PU-3 and PU-6) and used for the reaction.

s K. Sonomoto, I.-N. Jin, A. Tanaka, and S. Fukui, Agric. Biol. Chem. 44, 1119 (1980). 6 T. Omata, T. Iida, A. Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 8, 143 (1979). 7 S. Fukui, K. Sonomoto, and A. Tanaka, this series, Vol. 135 [20]. 8 A. Tanaka, S. Yasuhara, M. Osumi, and S. Fukui, Eur. J. Biochem, 80, 193 (1977).

[27]

OPTICAL RESOLUTION OF dl-MENTHOL

+

(CH2)4COOH

)

0 O-C-(CH2)4

OH dl-Menthol

5-Phenylvaleric acid

295

+ OH

Z-Menthyl-5-phenylvalerate

d-Menthol

FIG. 2. Stereoselective esterification of dl-menthol catalyzed by C. cylindracea lipase.

Reaction Conditions. Free yeast cells (1 g wet cells) are suspended in 10 ml of 20 mM potassium phosphate buffer (pH 7.0) containing 39 mM dlmenthyl succinate ammonium salt. The reaction is carried out at 30 ° with shaking (180 strokes/min) for the indicated period. Most of/-menthol formed does not dissolve in the aqueous buffer and accumulates on the surface of the cells, thereby decreasing the apparent activity of the cells. As shown in Table I, the hydrolytic activity of the cells is reduced significantly in the presence of organic cosolvents, except for 10% methanol, probably due to denaturation of the enzyme (esterase) in the yeast cells. TABLE I EFFECT OF SOLVENTS ON HYDROLYSIS OF d/-MENTHYL SUCCINATE BY FREE CELLS OF Rhodotorula minuta VAR. texensis" Substrate d/-Menthyl succinate ammonium salt

d/-Menthyl succinate

Solvent KPB b

KPB-Methanol (75 : 25) KPB-Methanol (90: 10) KPB-Dimethylformamide (50 : 50) KPB-CH3CN (50 : 50) KPB-CH3CN (65 : 35) KPB-CH3CN (80 : 20) Water-saturated benzene Water-saturated chloroform Two-phase system (KPB-solvent, 1 : 5) t-Butyl acetate Benzene Benzene-n-heptane (50 : 50) Benzene-n-heptane (40 : 60) Benzene-n-heptane (30 : 70) Benzene-n-heptane (20 : 80) Benzene-n-heptane (10:90) n-Heptane

Conversion ratio (%) 71 48 66 14 5 14 30 1 0 0 22 25 33 38 45 53 73

a From Omata et al. 3 b 20 mM Potassium phosphate buffer (pH 7.0). The cells (1 g wet cells) were incubated for 48 hr in 10 ml of KPB, KPB-water-miscible solvents, or water-saturated organic solvents (39 mM substrate), or in two-phase systems composed of 10 ml of waterimmiscible organic solvent (39 mM substrate) and 2 ml of KPB.

296

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[27]

Hydrolysis of menthyl succinate in water-immiscible organic solvents catalyzed by the free cells was not successful, because the cells could not be suspended well in such solvents. Therefore, two-phase systems composed of the phosphate buffer and organic solvents containing 39 mM substrate (1:5, v/v) were tested for the optically selective hydrolysis of dl-menthyl succinate by the free cells (Table I). After 48 hr of incubation in a two-phase system composed of 20 mM potassium phosphate buffer (pH 7.0) and n-heptane (1 : 5, v/v), the conversion ratio was almost the same as in the phosphate buffer containing d/-menthyl succinate ammonium salt. Hence, n-heptane was selected as the reaction solvent using the phosphate buffer-n-heptane two-phase system for the free cells and water-saturated n-heptane for the gel-entrapped cells. In this solvent system both substrate and products show satisfactory solubility, and under these conditions there is minimal inactivation of the hydrolytic enzyme in the cells. Concentration of dl-menthyl succinate in the organic solvent is 39 mM. The reaction is carried out at 30° with shaking (180 strokes/min). Analytical Methods./-Menthol produced from dl-menthyl succinate is assayed by gas chromatography with a JEOL-20 KFL gas chromatograph equipped with a hydrogen flame ionization detector. The steel column (2 m × 2 mm I.D.) is packed with LAC-2R-446 (20%, w/w) on Celite 545, 6080 mesh (Gasukuro Kogyo, Japan). The temperatures at the injector, in the column, and at the detector are 175, 140, and 240 °, respectively. The flow rate of the carrier gas, helium, is 44 ml/min, y-Valerolactone is used as the internal standard for the determination of/-menthol. /-Menthol is isolated from the reaction mixture as follows. The reaction mixture is washed with an equal volume of 0.1 N NaOH and dried on calcium chloride, and the solvent is removed in vacuo to yield white crystals which are recrystallized from ethanol by adding water. The melting point of/-menthol so obtained is 40-41 ° (mp in literature, 41-43°). Optical rotation of the product is measured in ethanol with a DIP-SL automatic polarimeter (Japan Spectroscopic Co.). The specific rotation [a]~5 is estimated using the following equation: [a]ZD5 = (a/lc)lO0 where a is the observed rotation; l, light path (dm); and c, concentration of/-menthol (g/100 ml). Synthesis ofMenthyl Succinate. Succinyl anhydride is used as the acyl donor for the ester synthesis because succinate produced by the enzymatic reaction can be easily recovered as succinic anhydride in high yield and because menthyl succinate can be solubilized in water as the ammonium salt. Finely powdered succinic anhydride (15.7 g) is added to a

[27]

OPTICAL RESOLUTION OF dI-MENTHOL

297

solution of dl-menthol (24.5 g) in o-xylene (30 ml). The reaction is carried out at 80 ° for 3 hr, after which the solvent is removed. The white crystals thus obtained are recrystallized from benzene (yield, 86%). The product is identified by spectrophotometric analyses (NMR, IR, and mass-spec.). The results show that the compound is indeed dl-menthyl succinate without contamination by esters of dl-isomenthol, dl-neomenthol, and dl-isoneomenthol. The water-soluble ammonium salt of dl-menthyl succinate is prepared as follows, dl-Menthyl succinate (8.3 g) is neutralized with 60 ml of 2.2 M ammonium hydroxide, and the pH is adjusted to 7.0 with 5.1 N HCI. The final concentration of dl-menthyl succinate ammonium salt is estimated to be 390 mM. This solution is diluted to 39 mM with 20 mM potassium phosphate buffer (pH 7.0).

Properties of Gel-Entrapped Cells Activities of Cells. The rate of/-menthol formation by the hydrophobic gel (PU-3)-entrapped cells was 2.5/~mol/hr-g wet cells, that of the hydrophilic gel (PU-6)-entrapped cells was 2.0/zmol/hr-g wet cells, while that of the free cells was 6.1 /~mol/hr-g wet cells. Effect of Gel Hydrophobicity. When the hydrophobicity of the gels was changed by mixing PU-3 and PU-6 in different ratios, the activity increased as the content of the hydrophobic PU-3 increased. Effect of Reaction Temperature. The optimal temperature for the free and entrapped cells was 40 ° . In most cases, however, the cells were used at 30° to minimize the thermal inactivation of the enzyme during long periods of operation; the activity of the cells at 30° was about 75% of that at 40 °. Stability of Hydrolytic Activity. When menthyl succinate ammonium salt was hydrolyzed by the free cells, about 80% of/-menthol produced by the cells was accumulated on the cell surface. Extraction of/-menthol by an organic solvent, such as benzene, significantly damaged the hydrolytic activity of the cells, thus rendering repeated use of the cells difficult. Addition of 10% methanol to the aqueous reaction mixture improved the productivity of/-menthol by the free cells, and their activity was stable for at least 10 reaction batches (total operational period, 240 hr). Figure 3 illustrates the repeated use of the free cells in the buffer-n-heptane system and of PU-3-, PU-6-, and ENT-4000-entrapped cells in water-saturated n-heptane for the hydrolysis of menthyl succinate. The free cells were recovered from the reaction mixture by centrifugation, and the immobilized cells by filtration. The half-life of the activity of the free cells was 50 hr, while that of the ENT-4000-, PU-6-, and PU-3-entrapped cells was estimated to be 560, 1315, and 1520 hr, respectively. Thus, immobili-

298

[27]

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS I

I

i

!

I

1

1

~

J

1

i

I

10o

zoo

30o

400

5oo

600

Incubation t i m

(hr)

100

A 75

25

FIG. 3. Repeated use of free and gel-entrapped R. minuta var. texensis cells in hydrolysis of dl-menthyl succinate. Each reaction was carried out at 30° by shaking the reaction mixtures (180 strokes/min) for 24 hr as described in the text. (/-1) Free cells; (O) PU-3-entrapped cells; (A) PU-6-entrapped cells; (V) ENT-4000-entrapped cells. From Omata et al?

zation greatly improved the operational stability of the hydrolytic enzyme in the yeast cells. The specific rotation of/-menthol ([a] 25 = -51 °) produced from dlmenthyl succinate by the free and entrapped cells showed that the product was pure /-menthol. The initial optical yield were maintained even after 10 reaction batches in every case. Production o f l-Menthol The acids used to form the dl-menthyl esters have to be carefully selected because they influence the optical purity of the hydrolysis product. 9 Recovery of the corresponding acids from the reaction mixture should also be high. We employed the succinate ester as the substrate because of the 100% optical purity of/-menthol produced by the enzyme of R. minuta var. texensis and because of the high recovery (75%) of 9 y . Yamaguchi, T. Oritani, N. Tajima, A. Komatsu, and T. Moroe, J. Agric. Chem. Soc. Jpn. 50, 475 (1976).

[27]

OPTICAL RESOLUTION OF d/-MENTHOL

dl-Mentho1 (2.5 kg) in xylene

~

Succinlc anhydride 80°, 3 h

299

( (1.6 kg) (,

[

dl-Menthyl succinate (3.8 kg), Y = 94% Dehydration

•

Immobilized /?. rrc/mut.a (1,3 kg dry cell) in water-saturated n-heptane 40 °, 15 h ) Succinlc acid (0.6 kg)

L _l-Menthol (I kg), Y = 86%

Racemlzat lon

(Y = 92%)

(Recovery,75%7 d_-Menthyl succinate HTdr°1}'sls) Succinic + d-Menthol acid (1.3 kg)

(1.3 kg)

FIG. 4. Flow diagram of/-menthol production by gel-entrapped R. minuta var. texensis cells. From Omata et al)

succinic acid after transformation to succinic anhydride. The remaining dmenthyl succinate was hydrolyzed with KOH. The d-menthol thus obtained was racemized by heating at 260-280 ° for 4.5 hr under hydrogen pressure of 65-72 kg/cm2 in the presence of a catalyst such as copper chromite. 1° The yield of dl-menthol was -46%. Based on the results mentioned above we have tried to produce/-menthol on a large scale. The flow diagram for the large scale production of/-menthol is shown in Fig. 4. Stereoselective Esterification of dl-Menthol"

Materials and Methods Prepolymer. Water-miscible urethane prepolymer (PU-3)7 prepared by Toyo Tire & Rubber Co., Japan, is used. PU-3 gives a hydrophobic gel, Enzyme. Lipase from Candida cylindracea (MY and OF 360; Meito Sangyo Co., Japan) and porcine pancreas (Sigma, USA) shows a high synthetic activity in the production of l-menthyl 5-phenylvalerate with a high stereoselectivity. On the other hand, enzymes from Aspergillus niger (Nagase Seikagaku Co, Japan) and Rhizopus delemar (Seikagaku Kogyo Co., Japan) are almost inactive in catalyzing the menthyl ester formation. When lipase preparations from hog pancreas (Sigma) and porcine pancreas (Wako Pure Chemicals Co., Japan) are entrapped with PU-3, rigid gels could not be obtained using these enzyme preparations. Pancreas lipase (Sigma; 19 units/mg powder) and lipase MY (30 units/mg powder) have relatively low activity, and it is rather difficult to prepare immobi10T. Yoshida, A. Komatsu, and M. Indo, Agric. Biol, Chem. 29, 824 (1965). " S. Koshiro, K. Sonomoto, A. Tanaka, and S. Fukui, J. Biotechnol. 2, 47 (1985).

300

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[27]

lized enzyme preparations showing high enzyme activity. Therefore, lipase OF 360 was selected as the enzyme for the esterification. Immobilization of Enzyme. Lipase OF 360 (100 mg; 36,000 units) dissolved in 0.2 ml of deionized water is entrapped with 0.5 g of PU-3. 6,7 The prepared gel is cut into small pieces ( - 3 x 3 x 3 mm) and is ready to be used in the esterification experiments. In some cases, 100 mg of lipase in 0.2 ml of water is adsorbed on 0.25 g of Celite 535. Celite-adsorbed lipase is also immobilized with 0.5 g of PU-3 in the absence of water. Reaction Conditions. For the esterification of menthol, choice of an acyl donor influences significantly both the reactivity and stereoselectivity of the enzyme. Longer chain fatty acids, such as stearic and oleic acids, were good substrates for the esterification reaction. Use of middle chain fatty acids, such as n-valeric, n-heptanoic, and n-nonanoic acids, resulted in a low product yield and shorter chain fatty acids, such as acetic acid, showed no reactivity. In contrast, stereoselective esterification was hardly observed using longer chain fatty acids, while/-menthol was selectively esterified using middle chain fatty acids. 5-Phenylvaleric acid was appreciably reactive, yielding optically active product. Therefore, this acid was employed as the acyl donor. Addition of an organic solvent to the reaction system was essential in order to obtain a homogeneous reaction system and to shift the reaction equilibrium favoring ester formation. Both water-miscible and water-immiscible organic solvents were examined. It was found that enzyme activity in nonpolar solvents, such as cyclohexane and isooctane, was high. The activity of the entrapped enzyme was, however, moderate in n-hexane, and low in benzene and carbon tetrachloride. No activity was observed in polar solvents, such as acetone, chloroform, dioxane, methanol, and methyl isobutyl ketone (Table II). Based on these results, lipase preparations (36,000 units) are suspended in I0 ml of water-saturated isooctane or cyclohexane containing 130 mM dl-menthol and 100 mM 5-phenylvaleric acid. Enzymatic esterification is carried out at 30° by shaking the reaction mixtures (220 strokes/ min using test tubes or 120 strokes/min using 100-ml flasks). Analytical Methods. An aliquot of the reaction mixture (50 /xl) is added to 50/~1 of methanol containing a known amount of progesterone as an internal standard. After removal of the solvent by evaporation at 4 °, the residues are dissolved in 0.2 ml of a mixture of methanol and acetic acid (100:0.5, v/v), and the solution is filtered through a Teflon membrane filter (pore size, 0.22 /.tm). Amounts of the product and of the substrates are determined by high-performance liquid chromatography using an ALC/GPC 204 instrument (Waters Co., USA) equipped with a data module (type 730; Waters) and a differential refractometer R401

[27]

OPTICAL RESOLUTION OF all-MENTHOL

301

TABLE II EFFECT OF ORGANIC SOLVENTS ON ESTERIFICATION OF d/-MENTHOL BY Candida cylindracea LIPASEa,b Organic solvent

Conversion (%)

Acetone Benzene Carbon tetrachloride Chloroform Cyclohexane Dioxane n-Hexane Isooctane Methanol Methyl isobutyl ketone "

nil l 8 nil 56 nil 18 43 nil nil

From Koshiro et al." The enzymatic reaction was carried out for 62-69 hr with PU-3-entrapped lipase as described in Fig. 5.

model (Waters). Separations are carded out on a Radial-Pak liquid chromatography cartridge C~s column (8 mm I.D. × I0 cm; d = 10/xm) using methanol-acetic acid (100 : 0.5, v/v) as the mobile phase (flow rate, 2 ml/ min) at a pressure of 70 kg/cm 2. 100

--

I

I

I

7 5 -

0

--

so

zs

o

25 Reaction

50 time

75

lO0

(h)

FIG. 5. Stereoselective esterification of menthol by entrapped lipase. Lipase OF 360 (36,000 units) was entrapped with 0.5 g of PU-3 and the immobilized enzyme was incubated at 30° in 10 ml of water-saturated isooctane containing 130 mM menthol and 100 mM 5phenylvaleric acid. (O)/-Menthol; (A) dl-menthol; (rT) d-menthol. From Koshiro et al. ~1

302

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

Properties of Entrapped Lipase Stereoselectivity of the reaction using 5-phenylvaleric acid as an acyl donor was tested by comparing different alcohol substrates, such as d-, l-, and dl-menthol, in the esterification reaction (Fig. 5). Stereoselectivity of the reaction was also analyzed by measuring optical rotation of the menthyl ester isolated from the reaction mixture containing dl-menthol. The purification was performed on a column packed with Silica gel 60 (70-230 mesh; solvent, n-hexane-diethyl ether-acetic acid, 80:20: 1, v/v). The optical purity of the product was about 100%. Based on the obtained results, we have found that about 80% of the used racemic alcohol substrate was transformed into/-menthyl ester after 70-80 hr of incubation under the conditions employed. The entrapped enzyme was more stable at 30 ° but rather unstable above 35 ° in repeated batch reactions. The decrease in enzyme activity of Celite-adsorbed lipase was more rapid than the loss of activity of PU-3-entrapped Celiteadsorbed enzyme. In addition, the production of l-menthyl 5-phenylvalerate by the entrapped enzyme after 10 batches of reaction (operational period, 930 hr) was 1.5 times greater than that of the Celite-adsorbed enzyme under identical conditions. It is noteworthy to add that other terpenoids, such as d/-borneol, could also be resolved optically by using essentially the same procedure as the one described here for lipase OF 360 from C. cylindracea.

[28] E l e c t r o e n z y m a t i c a n d E l e c t r o m i c r o b i a l R e d u c t i o n : Preparation of Chiral Compounds B y IORDANIS THANOS, JOHANN BADER, H E L M U T GONTHER, STEFAN N E U M A N N , FRIEDRICH KRAUSS,

and H E L M U T SIMON

Principles Reductases partially or completely purified or in the form of whole microbial cells are often used in the preparation of chiral compounds via stereoselective reduction of suitably substituted unsaturated substrates. Whole cells are usually applied together with sugars as the electron source. So far, the use of isolated enzymes has been restricted to pyridine nucleotide-dependent reductases. However, reductions on a preparative scale cannot be performed using stoichiometric amounts of reduced pyridine nucleotides because of their high cost. At present only enzymatic METHODS IN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc.

All rightsof reproductionin any formreserved.

302

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

Properties of Entrapped Lipase Stereoselectivity of the reaction using 5-phenylvaleric acid as an acyl donor was tested by comparing different alcohol substrates, such as d-, l-, and dl-menthol, in the esterification reaction (Fig. 5). Stereoselectivity of the reaction was also analyzed by measuring optical rotation of the menthyl ester isolated from the reaction mixture containing dl-menthol. The purification was performed on a column packed with Silica gel 60 (70-230 mesh; solvent, n-hexane-diethyl ether-acetic acid, 80:20: 1, v/v). The optical purity of the product was about 100%. Based on the obtained results, we have found that about 80% of the used racemic alcohol substrate was transformed into/-menthyl ester after 70-80 hr of incubation under the conditions employed. The entrapped enzyme was more stable at 30 ° but rather unstable above 35 ° in repeated batch reactions. The decrease in enzyme activity of Celite-adsorbed lipase was more rapid than the loss of activity of PU-3-entrapped Celiteadsorbed enzyme. In addition, the production of l-menthyl 5-phenylvalerate by the entrapped enzyme after 10 batches of reaction (operational period, 930 hr) was 1.5 times greater than that of the Celite-adsorbed enzyme under identical conditions. It is noteworthy to add that other terpenoids, such as d/-borneol, could also be resolved optically by using essentially the same procedure as the one described here for lipase OF 360 from C. cylindracea.

[28] E l e c t r o e n z y m a t i c a n d E l e c t r o m i c r o b i a l R e d u c t i o n : Preparation of Chiral Compounds B y IORDANIS THANOS, JOHANN BADER, H E L M U T GONTHER, STEFAN N E U M A N N , FRIEDRICH KRAUSS,

and H E L M U T SIMON

Principles Reductases partially or completely purified or in the form of whole microbial cells are often used in the preparation of chiral compounds via stereoselective reduction of suitably substituted unsaturated substrates. Whole cells are usually applied together with sugars as the electron source. So far, the use of isolated enzymes has been restricted to pyridine nucleotide-dependent reductases. However, reductions on a preparative scale cannot be performed using stoichiometric amounts of reduced pyridine nucleotides because of their high cost. At present only enzymatic METHODS IN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc.

All rightsof reproductionin any formreserved.

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

303

methods are available for the regeneration of NAD(P)H. Although many methods have been published l,z such systems have some intrinsic problems and are complicated because two enzymes, two substrates, two products, and the rather labile coenzymes NAD(P)+/NAD(P)H are needed for the recycling reaction. Recently two reductases 3 which reduce a broad spectrum of substrates to chiral products without using pyridine nucleotides have been described. However, methods for NAD(P)H regeneration are still very important and in this article an alternative approach using electrochemical methodology for both electroenzymatic NAD(P)H regeneration and electromicrobial reductions often exploiting NAD(P)H regeneration is discussed. Enzymes possessing prosthetic groups which can be reduced by means of direct electron transfer from a cathode are known. 4 However, a direct electrochemical reduction of a protein applied in concentrations lower than 1/xM usually is not fast enough due to limited diffusion of the reactant. Reduction rates of suitable enzymes faster by orders of magnitude can be expected by use of a low molecular weight mediator applied in concentrations ranging from 1 to 4 mM. Derivatives of 4,4'-bipyridinium dications substituted at the 1 and 1' position are useful carriers of electrons from the surface of the cathode of an electrochemical cell to a reductase reducing the substrate. The best known mediators are methyl viologen (MV 2+) and benzyl viologen (BV2+). In electromicrobial or electroenzymatic reductions we may have the following sequence of reactionsS: 2 MV 2÷ + 2 e ~ 2 MV +.

2 MV+. + ~ X

+ 2 H+ --* 2 MV2~ + H->-X--H

(1) (2)

Two enzymes capable of catalyzing reaction (2) are 2-enoate reductase (EC 1.3.1.31) from a Clostridium tyrobutyricum strain (Clostriclium La 1) and 2-oxocarboxylate reductase (so far no EC number has been given) from Proteus vulgaris or Proteus mirabilis. The latter enzyme is now called (2R)-hydroxycarboxylate-viologen oxidoreductase since the later

C. H. Wong and G. M. Whitesides, J. Am. Chem. Soc. 105, 5012 (1983) and literature cited therein. 2 G. M. Whitesides and C. H. Wong, Angew. Chem. 97, 617 (1985). 3 H. Simon, J. Bader, H. Giinther, S. Neumann, and J. Thanos, Angew. Chem. 97, 541 (1985) [Angew. Chem. Int. Ed. Engl. 24, 539 (1985)]. 4 H. L. Schmidt and H. Giinther, Phil. Trans. R. Sac. Lond. (1987), Discussion Meeting of Biosensors, London, May 1986, in press and literature cited therein. 5 H. Simon, J. Bader, H. Giinther, S. Neumann, and I. Thanos, Ann. N. Y. Acad. Sci. 434, 171 (1984), in "Enzyme Engineering 7" (A. I. Laskin, G. T. Tsao, L. B. Wingard, Jr., eds.).

304

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

described reaction (7) is reversible if carbamoylmethylviologen is used. 6 Both enzymes show an astonishingly broad substrate specificity, 3,5,7-13 high stereoselectivity, 3,5,J4 and satisfactory kinetic parameters, 3,~4 and they do not 3,5,7-9require reduced pyridine nucleotides as cosubstrates. An important group of enzymes which can be used for electroenzymatic NAD(P)H regeneration are certain methyl viologen-dependent NAD(P) ÷ reductases3&7-9: 2 MV +. + NAD(P) + + H + --* 2 MV 2÷ + NAD(P)H

(3)

Many organisms produce enzymes that catalyze this reaction, and the enzymes show specific activities of the order of 0.01-40 units/mg protein. 8 Some of the enzymes have been studied, while others have not been characterized in detail s o f a r . 3,8,9 On the basis of reaction (3) and the fact that many pyridine nucleotidedependent reductases are present in cells, electromicrobial or electroenzymatic reductions of the following type are possible: H+

(4) H+

The electrons delivered from the cathode of an electrochemical cell reduce the mediator, i.e., methyl viologen which in turn is enzymatically oxidized by NAD ÷ or NADP + with the aid of enzyme El. The reduced pyridine nucleotides can be used as cosubstrates of reductase E2 for stereospecific reduction of unsaturated substrates. Often E1 and E2 are present in the same cell species. Another possibility is the combination of E1 and E2 from two different cell sources, for example the combination of 6 H. Skopan, H. GOnther, and H. Simon, Angew. Chem., Int. Ed. Engl. 26, 128 (1987). 7 M. Biihler and H. Simon, Hoppe-Seyler's Z. Physiol. Chem. 363, 609 (1982). s H. Simon, H. Giinther, and J. Thanos, in "Enzymes as Catalysts in Organic Synthesis" (M. P. Schneider, ed.), Vol. 35. Reidel Publishing, Dordrecht, 1986. 9 j. Bader, H. Gtinther, S. Nagata, H. J. Schuetz, M. L. Link, and H. Simon, J. Biotechnol. 1, 95 (1984). ~0 H. G0nther, C. Frank, H. J. Schuetz, J. Bader, and H. Simon, Angew. Chem. Int. Ed. Engl. 229 322 (1983). u W. Tischer, W. Tiemeyer, and H. Simon, Biochimie 62, 331 (1980). ~2H. Sedimaier, W. Tischer, P. Rauschenbach, and H. Simon, FEBS Lett. 100, 129 (1979). 13 S. Neumann and H. Simon, FEBS Lett. 167, 29 (1984). ~ H. Gfinther, S. Neumann, and H. Simon, J. Biotechnol, 7, 53 (1987).

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

305

two different cell species or their crude extracts or the two purified enzymes from different sources. Reactions (4) and (2) can be performed by using cells made partially permeable, for instance by freezing and thawing, or by using pure or partially purified e n z y m e s . 3'5'8-1°'14 If methyl viologen is used in combination with whole cells of CIostridium La 1 or P. vulgar& hydrogen may be formed in a side reaction according to reaction (5): 2 MV +. + 2 H +

hydrogenase~ 2 MV 2+ + H2

(5)

In such cases benzyl viologen substituting for methyl viologen as a mediator can be used instead. However, benzyl viologen is less stable if experiments are carried out for long periods of time. The different possibilities are discussed in the examples given below.

Substrate Specificity and Some Kinetic Data of Enoate Reductase Enoate reductase from Clostridium La 1 (DSM 1460) catalyzes the following reactionS,8: 3R

2My +.+2Ia++

'-R ~

X

~ 2R/-~tR

,

H

+2~v~*

(6)

H

X = COOR I = H, CH3, C2H5, F, CI, Br, NHCHO, OCH3 R 2 = many alkyl groups, COOCH3, substituted phenyl or pyridine tings, but also furyl and thioenyl groups R 3 = residues not too bulky and rigid

The rates of different substrates have been published. 3,5,7,8In the reduced form the enzyme is especially oxygen sensitive. The specific activity of purified enoate reductase determined for the reduction of (E)-2-methyl-2butenoate is about 20 units/mg protein (1 unit protein converts 1/zmol of enoate per minute). This value is about 1.5-fold the reduction rate observed using N A D H as the electron donor. Therefore enoate reductase can also be used in combination with an NADH regenerating system such as formate dehydrogenase from Candida boidinii.11 The following rules have emerged regarding the enzyme's substrate specificity: ~R should not be too bulky; NHCOCH3 and OC2H5 cannot replace N H C H O or OCH3 if 2R is a substituted phenyl ring. The halogens F, C1, or Br are tolerated only in the a position; in contrast, halogens in the/3 position are eliminated. 12The least restrictions apply to functionalities in position 2R. Branching in the 13 position leads to diminished activi-

306

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

ties of the enzyme (2-4 units/mg). If 2R and 3R are interchanged, i.e., if E and Z isomers jaa of substrates 3R2RC=CHX are used, the products found are the corresponding enantiomers. This was shown for E- and Z-gerania t e ) The former led to the R and the latter to the S enantiomer of citronellate, respectively• Therefore only pure E and Z isomers should be used if an enantiomeric product containing a chiral center in the/3 position is desired. In substrates such a s / C = C = C R X (i.e., allene carboxylates) ~. or jC---~C--C---~CRX only the a,/3-double bond will be reduced. It can be assumed from reductions of substrates similar to 1 R H C = C R X that the E and the Z isomers will yield identical enantiomeric products• So far, all the products analyzed show an enantiomeric excess greater than 96%. 3,5 If the reductions are carried out in buffered 2H20, stereospecifically deuterated compounds can be obtained. 3,5,9 Using MV.+, enoate reductase shows a pH maximum at 5.2. 5 At pH 4.5 and 6.2 the rate is two-thirds that at pH 5.2. A pH value less than 5.2 should not be used and pH values up to 8.0 do not harm the enzyme• A pH value of 6.0-6.2 is recommended as a compromise considering the reaction rate and the stability of both reduced methyl viologen and enoate reductase. In addition, the reverse direction of reaction (6) has not been observed so far. The kinetic data of enoate reductase are favorable as can be seen in Table I. Substrate Specificity and Some Kinetic Data of (2R)-Hydroxycarboxylate-Viologen Oxidoreductase This oxidoreductase from P. mirabilis (DSM 30115) or P. vulgaris (DSM 30118) catalyzes the following reaction3,5,8,J3,4: O )~ R CO0

OH H +2H++2BV'*°r2MV'*'---+

>~C +2BV2÷or2MV2+ R OO-

(7)

R = CH3, CH2F, CTHls, C6HsCH2, C6H5, (CH3)2CHCH2, C2HsCH(CH3), HOCH2C(CH3)2, -OOC(CH2)~ (n = 1, 2, 3), -OIPI(Me)CH2CH2, 3-indolyl, 5-benzyloxy-3-indolyl II

O

The rates of reduction of different substrates have been published. 5,8,13,14 Except glyoxylate, all tested derivatives of 2-oxocarboxylates were found to be good substrates. The specific activity of purified (2R)-hydroxy_carboxylate-viologen oxidoreductase is about 1000 units/mg protein, using phenylpyruvate as the substrate (100%). Beside 2-oxomonocarboxylates, J4~According to the rules of Cahn, Ingold, and Prelog the notation is Z if the substituents of higher priority are on the same side of the C = C double bond; otherwise, it is E.

[28]

307

PREPARATIVE ELECTROENZYMATIC REDUCTIONS TABLE I KINETIC PARAMETERS DETERMINED FOR REACTIONS CATALYZED BY ENOATE REDUCTASE Substrate

Km (raM) °

Inhibitor

Ki (mM) °

Reduced methyl viologen NADH b Cinnamate (Z)-2-Bromocinnamate (E)-2-Methyl-2-butenoate (E)-Geraniate (Z)-Geraniate

0.4 0.012 0.013 0.50 1.5 0.014 0.10

Reduced methyl viologen Methyl viologen NAD ÷ Phenyl propionate Aliphatic carboxylates

10 20 0.84 30 500

Michaelis-Menten constants have been determined by analysis of initial rate studies using variable substrate concentrations and saturating concentration of NADH or reduced methyl viologen, respectively. For details of inhibitor studies, see Biihler and Simon. 7 b Instead of NADH reduced methyl viologen can be used as an electron donor.

2-oxodicarboxylates are also reduced at rates amounting to 50-75% of the rate observed for phenylpyruvate reduction. Branching in the 3-position of a substrate results in diminished reaction rates. For a quaternary carbon atom in the 3-position only about 5-7% of the reduction rate has been observed. The maximal reduction rate is observed at pH 6.75, and at pH 6.0 and 7.5 half of the maximum rate is seen. Neither NADH nor NADPH are electron donors. Typical Km and K~ values are shown in Table II. The use of this enzyme activity for the preparation of different (2S)-hydroxyTABLE II KINETIC PARAMETERS DETERMINED FOR REACTIONS CATALYZED BY 2-HYDROXYCARBOXYLATE-VIOLOGEN OXIDOREDUCTASE Substrate

Km (mM)"

Inhibitor

Reduced methyl viologen Reduced benzyl viologen Phenylpyruvate 2-Oxo-4-methylpentanoate 2-Oxo-3,3-dimethylbutanoate 2-Oxoglutarate

<0.1 0.1 0.15 2.1 7.5 2.5

(R)-Phenyl lactate (R)-2-Hydroxy-4-methylpentanoate 2-Oxo-3,3-dimethyl4-hydroxybutanoate Reduced benzyl viologen Benzyl viologen Phenylpyruvate 2-Oxo-4-methylpentanoate

a

Ki (mM)" 100 100 50 0.7 10 12 70

This reductase shows complicated enzyme kinetics. Therefore K m and K~ values are used to indicate the lower and higher concentrations of the substrates, respectively, at which the activity reaches half of its maximal value. All measurements have been conducted in 0.1 M potassium phosphate buffer, pH 7, at 34°. Oxidized methyl viologen is a positive inhibitor. From Simon e t a l . 3

308

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

carboxylates by the selective dehydrogenation of (2R)-hydroxycarboxylates from an enantiomeric mixture of an (R,S)-hydroxycarboxylate is described in ref. 6. Factors Determining the Design of the Electrochemical Cell For electrochemical reductions in water a cathode of high hydrogen overpotential is desirable but conditions favoring direct reduction of the mediator should be used. The substrate must only be enzymatically reduced. Suitable cathode materials for operation near physiological pH values and potentials set lower or equal than -460 mV versus the saturated calomel electrode [saturated calomel electrode (SCE): E0 = +244 mV] include Ag, C, and Hg while for reductions at more negative potentials (-640 mV) Pb, Bi, Sb, and Sn are electrochemically also acceptable. The potential is set by a standard three-electrode arrangement (Fig. 1). A low-power potentiostat is adequate.

/10

6

/4

l; ' \\\\ \

2

\ \ \

\\

il\ \

5

i

\\

7

I ?

J4

/

lI l ~\ \\\\,\

\\ \\ '

~ ll~\\ \ l

\\

\

/10

?

/ ~L',',ql ~\', 1

a

b

c

FZG. 1. Different types of electrochemical cells used for electroenzymatic and electromicrobial reductions. (a) The catholyte volume of the cell is 15-40 ml. 1, Glass flask; 2, mercury pool cathode; 3, Pt contact; 4, Pt anode; 5, anode compartment separated by Vycor tip (glass fiber diaphragm); 6, reference electrode; 7, 8, rubber caps; 9, stopcock; 10, stirring bar. (b) The catholyte volume of the cell is 40-60 ml. 1, cathode compartment; 2, carbon cathode foil or high surface area carbon fibers; 3, anode compartment; 4, Luggin-Haber capillary for reference electrode; 5, magnetic bar; 6, cylindrical holder for immobilized catalyst on filter paper; 7, tubes for gas bubbling and sampling. (c) The catholyte volume is 90-120 ml. 1, Glass vessel; 2, insulated platinum wire through the poly(vinyl chloride) cell cover is contacted via a mercury pool to the rotating perforated lead cathode; 3, Hg contact to cathode; 4, Pt anode; 5, Luggin-Haber capillary; 6, cation exchanging membrane; 7, rotor with magnetic bar; 8, perforated polyethylene sheet, and 9, insulated wires used to enhance turbulence of the electrolyte on both sides of the electrode; 10, tubes for gas bubbling and sampling; 11, thermostating coat.

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

309

For the diaphragm 0.5-mm foils of Nation (Du Pont) or 0.15-mm Selemion (Asahi Glass) are suitable materials. For cell type a and b (Fig. 1) also Vycor tips (supplied from Princeton Applied Research) can be used. These diaphragms fulfill the following requirements: easy proton transfer from the anolyte to the catholyte where protons are consumed [reactions (2)-(7)], sufficient conductivity, and practically no oxygen transfer from the anolyte to the catholyte. The symmetry of the cathode should be chosen in such a way that potential variations higher than -+30 mV compared with the potential value in the vicinity of the reference electrode are avoided. The cells should operate under predominantly diffusion-controlled conditions. The diffusion layer is decreased by stirring the solution by means of a magnetic stirring bar or rotating electrodes. Electrochemical cells equipped with mercury, carbon, and lead cathodes are shown in Fig. la and c. A simple, conventional construction using carbon or lead cathodes designed for small-scale experiments is shown for comparison in Fig. lb. Taking advantage of the compatibility of utilizing mercury in combination with substrates and enzymes, Hg contacts were employed in cell c. This cell has a free catholyte volume of 100 ml. The electrode area is about 150 cm 2 and offers a higher symmetry than the electrode areas of cells a and b. Compact rotating cylindrical electrodes have been tested and applied successfully. 15,~6 We have used hollow perforated cylindrical electrodes in a flow with Reynold numbers of 5000 to 15000.

Purification of Enoate Reductase Cells of Clostridium La 1 are grown in 300-liter tanks containing a crotonate medium as described for the production at maximal enoate reductase activity. 17Cells are harvested by centrifugation and then stored at - 18°. All procedures are carried out under an atmosphere of nitrogen at room temperature. All buffers used for column chromatography contain 0.02% sodium azide, 11.4 mM mercaptoethanol, 1 mM (E)-2-butenoate, 0.1 mM EDTA, and 250 mM sucrose. Columns are extensively equilibrated with respective elution buffers prior to use.

t5 D. J. Pickett, "Electrochemical Reactor Design," pp. 158-160, 231. Elsevier, Amsterdam, 1977. 16 V. G. Levich, "Physicochemical Hydrodynamics," pp. 30, 149, 304. Prentice Hall, Englewood Cliffs, New Jersey, 1962. t7 j. Bader and H. Simon, Arch. Microbiol. 127, 279 (1980).

310

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

TABLE III PURIFICATION OF ENOATE REDUCTASE FROM Clostridium La I

Purification step Crude extract Combined chromatography on DEAE-Sepharose and spherical hydroxylapatite

Volume (ml) 162 36 b

Protein concentration (mg/ml)

Activity (units)"

Specific activity (units/mg)

Yield (%)

52.1 1.5

1275 1135

0.15 20"

100 89

a One unit corresponds to the formation of 1/xmol of (R)-2-methylbutanoate/min in the presence of MV.+ (methyl viologen radical). b After ultrafiltration. c This material is often 5% contaminated as judged from analysis with analytical ultracentrifugation.

Wet packed cells (55 g, equivalent to 11 g dry weight) are suspended in 138 ml of 20 mM potassium phosphate buffer (pH 7.0), containing 10 mM (E)-2-methyl-2-butenoate, 1 mM EDTA, 40 mM hydroxylamine hydrochloride, 4.8 × l 0 6 units lysozyme, and 4 x 104 units DNase. After homogenizing the cells for 3 min employing a Vortex homogenizer, the obtained cell suspension is shaken (150 strokes/min) for half an hour at 35 ° and then centrifuged (27,000 g, 20 min at 4°). The supernatant (161 ml) is applied to a column (5.0 x 265 cm) packed with 130 ml DEAESepharose CL-6B, and the applied material is eluted with 20 mM potassium phosphate buffer (pH 7.5) at a flow rate of 160 ml/hr at room temperature. On the column a clear brown band migrates in front of a yellow band. Before the latter reaches the outlet of the column (after about 220 ml) the column is connected to a hydroxylapatite column (2.5 x 18 cm), containing 45 ml spherical hydroxylapatite (mesh size 0.075-0.18 mm, Merck, Darmstadt), preequilibrated with 700 ml of 50 mM potassium phosphate buffer (pH 7.0). After additional washings of the column with 120 ml of the phosphate buffer its concentration is increased to 0.3 M and finally to 0.7 M. Fractions containing enzyme are pooled, and the solution is concentrated by ultrafiltration (Table III). All buffers used for column chromatography contain 1 mM EDTA, 10 mM (E)-2-methyl-2-butenoate, and 40 mM hydroxylamine hydrochloride. (For further information about the purification and structural features of enoate reductase, see Kuno et al. 18). ~8 S. Kuno, A. Bacher, and H. Simon, Biol. Chem., Hoppe Seyler 366, 463 (1985).

[28]

311

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

Growth of Proteus vulgaris and Enrichment of (2 R)-Hydroxycarboxylate-Viologen Oxidoreductase The catalytic activity of (2R)-hydroxycarboxylate-viologen oxidoreductase in crude extracts depends heavily on the composition of the growth medium. The following procedure is a compromise considering both the costs of production of the enzyme and its activity in the crude extract of the cells. Growth medium: 1 liter of deionized water containing 5 g glucose, 2.5 g yeast extract, 5 g peptone, 5.1 g K2HPO4, I g sodium formate, 0.15 g (NH4)2PO4, 33 mg MgCI2" 6H20, 6.6 mg MgSO4" 7H20, 40 mg CaCI2"2H20, 0.4 mg MnSO4"H20, 0.4 mg FeSO4"TH20, 50 mg NH4C1, 10 mg (NH4)6Mo7024" 4H20, 0.02 mg biotin, I × 10 -6 M Na2SeO4, and 0.4 mg p-aminobenzoic acid. The cells are grown in a volume of 200 liters under anaerobic conditions at 37 °. After 15 hr of growth the specific activity of the reductase in the crude extract is found to be 5-10 units/rag protein. Enrichment procedure (Table IV): If not stated otherwise all procedures are carried out at 0-4 ° under anaerobic conditions. A suspension of wet packed cells (100 g) in 300 ml 0.01 M Tris-HCl buffer (pH 7.5) containing 1 mM dithioerythritol and 2 mM dithionite is broken in a French press-cell (138,000 kPa). Cell wall fragments are removed by centrifugation (10,000 g, 10 rain) and the membrane fraction in the supernatant is centrifuged (160,000 g, 180 rain). The sediment is resuspended in TABLE IV PURIFICATION OF (2R)-HYDROXYCARBOXYLATE-VIOLOGEN OXIDOREDUCTASE FROM

Proteus vulgaris

Purification step Crude extract Solubilized enzyme DEAE-cellulose Hydroxylapatite Sepharose CL-6B I and ultrafiltration Sepharose CL-6B II and ultrafiltration

Volume (ml)

Protein concentration (mg/ml)

Activity (units)"

Specific activity (units/mg)

Enrichment (n-fold)

273 195 244 126 60

20.0 10.0 1.1 0.4 0.3

22,700 14,573 14,136 9,945 4,500

4.2 b 7.5 49.0 197.3 261.1

-1.8 13.1 46.8 61.8

100 65 c 54 44 20

236.4

19

283

0.02

4,312

994.1

Yield (%)

One unit corresponds to the formation of 1/.tmol of (R)-phenyllactetate per minute in the presence of BV +. (benzyl viologen radical). b Later growth experiments resulted in 7-10 units/rag protein in crude extract. c Most of the hydrogenase is eliminated.

312

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

240 ml 0.01 M Tris-HCl (pH 7.5) containing 1 mM dithioerythritol, 4 mM dithionite, and 1.5% (v/v) polyoxyethylene 9-1auryl ether (detergent). After incubation for 10 min at room temperature the slurry is stirred in an ice bath at 150 rpm for 30 min. The supernatant obtained after centrifugation at 160,000 g for 90 rain is chromatographed on a DEAE-cellulose column (40 × 3.9 cm) containing 478 ml packed cellulose preequilibrated with 0.01 M Tris-HCl (pH 7.5) containing I mM dithioerythritol, 4 mM dithionite, and 0.8% detergent. After application of 273 ml of this solution to the column the reductase is eluted with a linear gradient of 0.1-0.7 M KC1 (total volume 2 liters) in the aforementioned buffer solution containing detergent. The enzyme is eluted at about 0.25 M KCI in 120 ml buffer. This solution is applied to a hydroxylapatite (HPT)-BioGel column (40 x 3.9 cm) preequilibrated with 10 mM Tris-acetate pH 6.5 buffer, supplemented with 1 mM dithioerythritol, 4 mM dithionite, and 0.4% detergent. The loaded column is treated with 580 ml of the equilibration buffer followed by a linear gradient of 0-0.15 M potassium phosphate (total volume 2 liters) in equilibration buffer. The enzyme elutes at about 0.06 M potassium phosphate. Two further gel filtration steps carried out on Sepharose CL-6B columns (70 × 3.9 cm and 75 × 2.5 cm) equilibrated with l0 mM Tris-HCl (pH 7.5) containing 1 mM dithioerythritol, 4 mM dithionite, 50 mM KCI, and 0.4% detergent are carried out lead to an enzyme with less than 5% impurities as judged by electrophoretical analysis. For preparative purposes the enzyme activity can be used in form of whole or broken cells. For details see Refs. 3, 14. Enzyme Assays in Cuvettes

Preparation of Reduced Mediator. Reduced viologens react extremely fast with oxygen; even trace amounts of oxygen (on a micromolar scale) must therefore be removed prior to reduction. In an electrochemical cell (i.e., cell type a or b in Fig. 1) under anaerobic conditions a solution of 0.02 M methyl- or benzyl viologen in 0.1 M Tris-acetate or potassium phosphate is reduced at potentials of -790 and -650 mV versus SCE, respectively. The reduced solutions are stored under nitrogen in suitable containers. Enoate Reductase. The enzyme can be tested using NADH 7 as well as MV -+ as substrates. Oxygen-free buffers and reagents are transferred to cuvettes under a stream of nitrogen. The cuvettes are closed with silicon rubber stoppers and alternately evacuated through a hypodermic needle and flushed with nitrogen. After this procedure substrate or enzyme is injected through the stopper using a nitrogen-flushed syringe. The test mixture (final volume 2.3 ml) contains 0.1 M potassium phosphate buffer and 0.2 mM N A D H or 1.0 mM MVt as electron donors at pH 6.0 and 5.5,

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

313

respectively. Enzyme activity determined by measuring changes in absorbance at 334 nm (NADH) or 730 nm (MV+.); one unit of enzyme consumes 1 /zmol N A D H or 2/zmol MV+./min at 25 °. For reduced methyl viologen the extinction coefficient is expressed by the relation e730 = 1.644 + 1.945/([MV+.] + 1.567) cm2/mmol. This is an empirical equation (unpublished data) determined by least square fitting of the absorbance of MV +. as a function of its concentration in 0.1 M potassium phosphate, pH 7.0. The concentration [MV.+] is expressed in millimolar. (2R)-Hydroxycarboxylate-Viologen Oxidoreductase. This enzyme does not oxidize NAD(P)H. The enzyme should be tested using BV +. instead of MV +. as mediator since the former shows little interaction with hydrogenase from Proteus and because of the unusual kinetic properties of MV -+ and MV z+ 3,14 For BV +. the relation e578 = 12 cm2/mmol is used. The test mixture (2.3 ml total volume) contains 100 mM potassium phosphate buffer (pH 7.0), 0.2 mM BV +., 2 mM phenylpyruvate (or another substrate). One unit of enzyme consumes 2/zmol of viologen per minute at 34°. Immobilization of Enoate Reductase in Calcium Alginate Gels Ionotropic gels, described by Klein and Wagner, 19 are suitable for the immobilization of purified enoate reductase, whole cells or crude extracts of Clostridium La 1, or partially purified 2-oxocarboxylate reductase. Oxygen-flee solutions are used in all steps. A solution of alginic acid in 100 mM Tris-acetate (pH 7.0) is mixed with the biocatalyst at room temperature in a stoppered vial under nitrogen to form a 5% (w/v) alginate solution at pH 7.0. Medium flow rate chromatography paper (Whatman 0.38 mm) is soaked with this solution and the ionotropic gel is allowed to harden in 0.62 M calcium acetate in 100 mM Tris-acetate (pH 7.0) for 1530 min. The filter paper is partially dried in a stream of nitrogen, transferred into the electrochemical cell in the form of a cylinder, and covered with the reaction solution in the absence of substrate (see example below). Exposure of the filter paper to air for about 0.5 min does not decrease the enzyme activity. In an electrochemical cell of type b (Fig. 1), free enoate reductase has a half-life of 6-7 hr. The half-life increases to 50 hr if 2.5 units of enoate reductase are immobilized on 50 cm 2 filter paper and used in an electrolyte containing 40 mM calcium acetate. The apparent activity is about 35% of the original activity of the free enzyme before immobilization. After the

t9 j. Klein and F. Wagner, DECHEMA-Monogr. 82, 142 (1978). ~0 S. Nagata, H. Giinther, J. Bader, and H. Simon, FEBS Lett. 210, 66 (1987).

314

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[28]

first 10 hr about 3-4% of the total initial enoate reductase activity is found in the catholyte due to leakage. In the absence of calcium acetate in the reaction medium the half-life is about 22 hr and the leakage is 16% in the first 10 hr. Immobilization of 6-7 units per 50 cm 2 filter paper, i.e., a higher package density of the enzyme, results in a preparation showing a half-life of about 140 hr and an apparent activity of 18%. About three times more product can be obtained using immobilized enoate reductase compared to the situation utilizing free enzyme. In the electrochemical cell free (2R)-hydroxycarboxylateviologen oxidoreductase is more stable than enoate reductase (half-life of free reductase was found to be 80 hr). Therefore the advantage of using immobilized enzyme in electrochemical reduction systems is not apparent so far, apart from the reusability of the immobilized enzyme. Practical Aspects In order to supply protons to the catholyte during reduction processes the anolyte is made up of 50 mM sulfuric acid, and the volume of the anolyte should be less than one-tenth of the catholyte. Systems designed for NAD(P)H regeneration are much more effective • and stable in buffers containing Tris-acetate than in potassium phosphate. Even though Tris-acetate is a weak buffer at pH 6.2 the pH stays rather constant if the anolyte is made up of sulfuric acid. All electroenzymatic reductions using pure enzyme proceed with almost 100% current efficiency. If crude cell extracts, partially purified enoate reductase (2R)-hydroxycarboxylate-viologen oxidoreductase or whole cells of Clostridium La 1 are used, most of the electrons flow to the unsaturated substrate if the maximal rate of the catalyzed reaction is high. This can clearly be seen by comparing the electromicrobial reduction of (E)-2-methyl-4-hydroxybutenoate and (E)-3-methyl-4-hydroxybutenoate. The latter is reduced at a rate that is about one-tenth of the Vmaxof the reduction rate of the former. A current efficiency of about 10% was found for the 3-methyl substrate and 50-75% for the 2-methyl substrate, when the reduction was catalyzed by a crude extract of Clostridium La I. The product of milliamps times minutes = 3.2 (i.e., 0.192 Coulomb) corresponds to the reduction of 1 ~mol substrate by two electrons. The rate of product formation in an electrochemical cell may be limited by the catalytic efficiency of the applied biocatalyst or the formation of the reduced mediator. In a stirred cell of type a or b (Fig. 1) or in the cell equipped with a rotating cathode (about 400 rpm) 0.3-0.5 mA per cm 2 cathode can be obtained in the presence of 1 mM oxidized and reduced methyl viologen at room temperature.

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

315

1.2

E

u

",la 1 , 2 " ~ ' ~ - ~ " ' ~ 9

o

'

time

-~'

a

1T ' 19 " " - " " - " ~ - -

gT"il

'

[ hours)

FIG. 2. Repeated electrochemical use of immobilized enoate reductase, (a) Reduction of methyl viologen; (b) addition of 2-methylcinnamate; (c) replacement of electrolyte and reduction of additional methyl viologen; (d, e) addition of 0.7 and 1.4 mmol (E)-2-methylbutenoate, respectively.

Repeated Use of Immobilized Enoate Reductase Enoate reductase (2.8 units) is immobilized on a filter paper (23 cm 2) at room temperature. This catalyst is transferred to cell b (Fig. 1) containing 55 ml of 0.1 M Tris-acetate (pH 6), 38 mM calcium acetate, and 1.5 mM methyl viologen. After reduction (at -760 mV) of most of the methyl viologen present in the cell (Fig. 2), 0.5 mmol of (E)-2-methylcinnamate is added. As shown in Fig. 2, after reaction for about 19 hr the current decreases significantly down to a constant background level. More than 99.2% of the substrate is converted into product according to high-performance liquid chromatography analysis. The solution containing product is removed, and the catalyst is used once more to reduce another 0.7 mmol of (E)-2-methylbutenoate. After further 22 hr of time of reaction another 1.4 mmol of the same substrate is added. As judged from analysis with gas-liquid chromatography less than 1% of substrate is left in the cell. Taking into account the experimental accuracy, about 2.1 mmol of (R)-2methylbutanoate has been formed. The immobilized catalyst, used in operation for 2 weeks, shows a half-life of about 200 hr. Use of Whole Cells of Clostridium La 1 or Partially Purified (2R)-Hydroxycarboxylate-Viologen Oxidoreductase Reduction o f an Enoate. Figure 3 shows the time course of the reduction of 10 mmol of (E)-2-methylbutenoate using as catalyst wet packed cells of C. La 1 that had been stored for 5 weeks at - 18°. An electrochemical cell of type c (Fig. 1) containing 95 ml 0.1 M potassium phosphate buffer (pH 6.4) was used at room temperature. (Additional ingredients of the reduction experiment are given in the legend of Fig. 3.)

316

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

80F

L

e

,0I

8

[28]

.- ....... ,8

,,

o

15 time

21

2T

[hours)

FIG. 3. Electromicrobial reduction of 10.0 mmol of (E)-2-methylbutenoate. (a) Methyl viologen (2 mM) is reduced; (b) whole cells of CIostridium La 1 (90 mg dry weight) are added; (c) the concentration of methyl viologen is increased to 3 mM; (d) 90% of the substrate is reduced; (e) more cells of Clostridium La 1 (55 mg) are added. Inset: Time dependence (abscissa in hours) of total charge (in milliequivalents, right-hand ordinate, ---) and production of (R)-methylbutanoate (millimoles, left-hand ordinate, ). Analysis by means of gas-liquid chromatography indicated that 90% of (E)-2-methylbutenoate had been converted into 2-methylbutanoate after 14 hr o f reaction, The conversion was higher than 99% after a total reaction time of 30 hr. In the inset in Fig. 3, it is shown that most of the available electrons (70%) were used for the reduction of the enoate substrate. After formation of the product hydrogen gas was formed at a higher reaction rate. Reduction of a 2-Oxocarboxylate. To the electrochemical cell type a (Fig. l) a solution (25 ml) containing potassium phosphate 0.1 M (pH 7.0), 3.3 m M methyl viologen, and 80 m M 2-oxo-4-methylpentanoate was added. After the initial reduction of methyl viologen at - 6 3 0 mV almost hydrogenase-free (2R)-hydroxycarboxylate-viologen oxidoreductase was added and the working potential increased to - 7 0 0 inV. The starting current of 5.5 mA lasted for 7 hr and decreased slowly to 5.1 mA after reaction for l0 hr, and finally the current went down to reach the background level. It was found that the substrate had been completely converted into product. The yield of the recrystallized product was 75%, and it showed the correct optical rotation of [a]Rs~ = + 11.3 ° (C = 1.00; H20). (R)-Propanediol Preparation by the Combination of Candida utilis and Alcaligenes eutrophus To an electrochemical cell of type a (Fig. l) a solution (40 ml) containing 0.1 M T r i s - a c e t a t e (pH 7.0) and 3.3 m M methyl viologen was added.

[29]

LONG-TERM ALKALOID PRODUCTION BY

C. purpurea

317

After reduction of methyl viologen at -700 mV at room temperature 1.4 mg (dry weight) of disrupted cells of Candida utilis, 32/zmol NAD +, 400 /~mol hydroxyacetone, and 0.65 mg (dry weight) of disrupted cells of A. eutrophus were added to the electrochemical cell. The following was observed. After addition of NAD ÷ to the cell the current increased from 0.05 to 0.35 mA as C. utilis showed some NAD + reductase activity. 3,~°,2° The addition of hydroxyacetone during the reduction of NAD + hardly increased the reaction rate because of the limiting supply of NADH in the cell. Addition ofA. eutrophus however, caused a current of 1.65 mA due to the increased NAD ÷ reductase activity. Within 16 hr of reaction 335 p.mol of (R)-propanediol was formed. It was shown by assaying enzyme activities that the applied amount of C. utilis contained 0.6 units of hydroxyacetone reductase (probably a glycerol dehydrogenase, EC 1.1.1.6) and that ofA. eutrophus 0.6 units of a methyl viologen-dependent NAD + reductase.

[29] L o n g - T e r m A l k a l o i d P r o d u c t i o n b y I m m o b i l i z e d Cells o f Claviceps purpurea 1 By BETTINA K O P P

Immobilization of biocatalysts such as enzymes, microbial cells, and plant and animal cells has attracted worldwide interest in the past few years. The high potential of cofactor regeneration, prolonged metabolic activity, catalysis of biochemical reactions under stabilized conditions as well as the mechanical stability of the matrix beads make immobilization processes attractive for research investigations as well as for commercial applications. Fungal secondary metabolites of considerable pharmaceutical interest are the ergot alkaloids produced by the pyrenomycete Claviceps. Living parasitically on grain Claviceps cells form sclerotia with plectenchymatic structures of high alkaloid content. Saprophytically cultured Claviceps mycelia form sclerotia-like cells at the end of the growth phase, and the alkaloid production is strongly related to this morphological differentiation and a change in metabolism. Mycelia of Claviceps are sensitive to mechanical stress and local oxygen deficits and show strong tendency to degeneration. Thus the saprophytic culturing in scaled-up fermenters with conventional microbiological methods presents difficulties. Application of Dedicated to Professor Dr. H. J. Rehm, Mtinster, on the occasion of his sixtieth birthday.

METHODS IN ENZYMOLOGY, VOL, 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[29]

LONG-TERM ALKALOID PRODUCTION BY

C. purpurea

317

After reduction of methyl viologen at -700 mV at room temperature 1.4 mg (dry weight) of disrupted cells of Candida utilis, 32/zmol NAD +, 400 /~mol hydroxyacetone, and 0.65 mg (dry weight) of disrupted cells of A. eutrophus were added to the electrochemical cell. The following was observed. After addition of NAD ÷ to the cell the current increased from 0.05 to 0.35 mA as C. utilis showed some NAD + reductase activity. 3,~°,2° The addition of hydroxyacetone during the reduction of NAD + hardly increased the reaction rate because of the limiting supply of NADH in the cell. Addition ofA. eutrophus however, caused a current of 1.65 mA due to the increased NAD ÷ reductase activity. Within 16 hr of reaction 335 p.mol of (R)-propanediol was formed. It was shown by assaying enzyme activities that the applied amount of C. utilis contained 0.6 units of hydroxyacetone reductase (probably a glycerol dehydrogenase, EC 1.1.1.6) and that ofA. eutrophus 0.6 units of a methyl viologen-dependent NAD + reductase.

[29] L o n g - T e r m A l k a l o i d P r o d u c t i o n b y I m m o b i l i z e d Cells o f Claviceps purpurea 1 By BETTINA K O P P

Immobilization of biocatalysts such as enzymes, microbial cells, and plant and animal cells has attracted worldwide interest in the past few years. The high potential of cofactor regeneration, prolonged metabolic activity, catalysis of biochemical reactions under stabilized conditions as well as the mechanical stability of the matrix beads make immobilization processes attractive for research investigations as well as for commercial applications. Fungal secondary metabolites of considerable pharmaceutical interest are the ergot alkaloids produced by the pyrenomycete Claviceps. Living parasitically on grain Claviceps cells form sclerotia with plectenchymatic structures of high alkaloid content. Saprophytically cultured Claviceps mycelia form sclerotia-like cells at the end of the growth phase, and the alkaloid production is strongly related to this morphological differentiation and a change in metabolism. Mycelia of Claviceps are sensitive to mechanical stress and local oxygen deficits and show strong tendency to degeneration. Thus the saprophytic culturing in scaled-up fermenters with conventional microbiological methods presents difficulties. Application of Dedicated to Professor Dr. H. J. Rehm, Mtinster, on the occasion of his sixtieth birthday.

METHODS IN ENZYMOLOGY, VOL, 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

318

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[29]

the immobilization technique should stabilize the metabolic activity of the mycelia and thus lead to an increased and continuous alkaloid production. This paper describes the batch and semicontinuous fermentation of calcium alginate-immobilized Claviceps purpurea under various conditions. Parts of the following investigations have been described previously la-3 and are summarized in this article. The following results and morphological considerations indicate that immobilization techniques perhaps simulate the conditions of parasitically formed Claviceps sclerotia and thus lead to increased and prolonged alkaloid production. General Methodological Considerations

Culture Conditions Claviceps purpurea CBS 164.59, received from the Centraalbureau voor Schimmelcultures (P.O. Box 273, 3740 AG Baarn, Netherlands), is grown in a seed medium modified after Kelleher4: mannitol 40 g/liter; saccharose 50 g/liter; succinate 10 g/liter; KH2PO4 1 g/liter; MgSO4" 7H20 0.8 g/liter; Ca(NO3)2-4H20 0.3 g/liter; NaNO3 0.1 g/liter; FeSO4.7H20 0.05 g/liter; MnSO4.4H20 0.01 g/liter; ZnSO4.5H20 0.005 g/liter; CuSO4.5H20 0.005 g/liter. The pH is adjusted with NH4OH or KOH to 5.2. The production medium contains mannitol 50 g/liter, succinate 30 g/liter, and 1 mM tryptophan as precursor for the alkaloid synthesis, and no phosphate source) Streptomycin sulfate, 100 rag/liter, is added to avoid bacterial contamination. Seed medium, 100 ml, in baffled flasks, is inoculated with mycelia from a 5- to 10-day-old malt agar slant and incubated on a rotary shaker (90 rpm) at 21°C for 8 days. After centrifugation and washing, the synnemata-forming mycelia are homogenized with an Ultra Turrax (IKA Werke, Jancke and Kunkel). The inoculum for 100 m! of production medium in Fernbach flasks and for 1 liter of production medium in an air-lift fermenter was chosen to be 1% wet weight mycelial fragments. The interior size of the l-liter air-lift fermenter is 23 cm in height and 9 cm in diameter. Five liters/min sterile filtered moist air is forced through a ia B. Kopp and H. J. Rehm, Eur. J. Appl. Microbiol. Biotechnol. 18, 257 (1983). 2 B. Kopp and H. J. Rehm, Appl. Microbiol. Biotechnol. 19, 141 (1984). 3 B. Kopp, A. H. E1-Sayed, W. Mahmoud, and H. J. Rehm, Proc. Eur. Congr. Biotechnol. 1, 281 (1984). 4 W. J. Kelleher, Adv. Appl. Microbiol. 11, 211 (1969). 5 j. E. Robbers, W. W. Eggert, and H. G. Floss, Lloydia 41, 120 (1978).

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LONG-TERM ALKALOID PRODUCTION BY C. purpurea

319

fritted glass at the fermenter bottom and passed through a reflux cooling apparatus to avoid a diminishing of the fermentation broth volume. A lockable aperture allows sterile change of media and sampling.

Immobilization Methods The immobilization technique using gel formation from ionic crosslinking of charged polymers like alginate has provided optimum entrapment of C. purpurea.la One gram wet weight fragmented mycelia is thoroughly mixed with 20 ml of sodium alginate (Manugel DJX, Kelco Ail, Hamburg) at concentrations of 1-8%; the calcium alginate beads are of about 5 mm diameter. After immobilization the beads are kept in the seed medium for 8 days to increase the biomass, and are then transferred to the production medium. The opening in the fermenter allows all steps of the immobilization procedure to be carried out in the fermentation vessel, making it easier to avoid microbial contamination. For determination of mycelium wet weight in the calcium alginate beads the gel is stirred in a 2% sodium hexametaphosphate solution (Fluka). This solution dissolves the calcium alginate, and after washing and centrifugation the wet mycelia can be weighted.

Analytical Methods for Determination of Alkaloids Samples of 10 ml each of the fermentation broth are extracted three times with about 30 ml dichloromethane. The organic phases are combined, evaporated to dryness, and dissolved in 1 ml acetonitrile. Qualitative determinations of alkaloids are performed by thin-layer chromatography on silica gel sheets developed in benzene:chloroform:ethanol (28.5 : 57 : 14.5). Quantitative determinations are carried out by high-pressure liquid chromatography (Varian 5000 chromatograph) on a LiChrosorb RP18 column (particle size 10 /zm, 25 c m x 4 mm ready packed column, Merck). The eluent consists of 0.01 M ammonium carbonate in water and acetonitrile (1 : 1).6 The flow rate is 1.5 ml/min. The alkaloids are detected at 245 nm wavelength (variable-wavelength detector UV 50, Varian). The quantitative determination is listed by an integrator (Hewlett Packard 3390 A). Alkaloid standards as references for identification were provided by the Sandoz AG (Basel, Switzerland). Table I shows the average values of the retention times. The reproducibility of the quantitative determination is satisfactory. Although a number of unknown secondary 6 H. Bethke, B. Delz, and K. Stich, J. Chromatogr. 123, 193 (1976).

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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TABLE I AVERAGE VALUES OF THE ALKALOID RETENTION TIMES PERFORMED BY HPLC

Compound

Average retention time

Relative standard deviation (%)

Ergometrine Elymoclavine Agroclavine Chanoclavine

2.80 4.10 7.05 8.50

1.0 1.8 2.3 4.2

products in the crude extract give signals, the identification of the alkaloids is not difficult.

Morphological Considerations For light microscopy the calcium alginate beads are cut into thin sections and dissolved with 2% sodium hexametaphosphate. For scanning electron microscopy the beads are freeze-dried, cut, and shadowed with conductive graphite. Improvement of the Alkaloid Production Capacity by Application of the Immobilization Technique

Claviceps purpurea CBS 164.59 produces chanoclavine, agroclavine, elymoclavine, and ergometrine as main alkaloids in the nonphosphate production medium, la Biosynthetic correlations were conducted by Rehacek. 7 Alkaloid Production and Biomass Development in Alginate of Different Concentrations Mycelia of C. purpurea were immobilized in various alginate concentrations (1 to 8%) to investigate the effect of mechanical stress on the production capacity. The beads were fermented in Fernbach flasks; the pH of the medium was adjusted with NH4OH. Synnemata of the mycelia grew out of the low concentrated alginate beads, broke off the matrix, and continued to grow mainly as free cells in the nutrient broth. Beads with 8% alginate concentration exhibited irregular form and more permanent consistency; only a few hyphae were produced. Figure 1 shows the biomass production correlated to various alginate concentrations. Free 7 Z. Rehacek, Adv. Biochem. Eng. 14, 33 (1980).

[29]

LONG-TERM ALKALOID PRODUCTION BY C. purpurea

321

15

free cells

1%

2%

3%

4%

5%

6%

7%

8%

alginate concentration

FiG. 1. Biomass production of Claviceps purpurea immobilized in different alginate concentrations. Open bar, 8 days cultivation in the seed medium; filled bar, 15 days cultivation in the production medium.

mycelia made up the main biomass production in the seed medium. Immobilized cells, however, reached high biomass amounts after being transferred to the production medium. It is presumed that the gel builds up a mechanical barrier to mycelial growth but that nutrients might accumulate in the beads and enable a delayed but nevertheless optimum growth. With increasing alginate concentrations the maximum biomass production is reduced. The absolute and specific amounts of alkaloids, however, increased with rising gel concentrations (Fig. 2, a and b). Mycelia in beads with high alginate concentrations showed an enhanced productivity of 35% total amount (Fig. 2a) and 200% specific amount (Fig. 2b). Maximum alkaloid synthesis of free mycelia occurs when nutrients of the medium other than carbon are exhausted and cell proliferation decreases. 7 By offering the mycelia a gel skeleton cell growth is mechanically restrained and a metabolic turnover from growth to production phase is induced before substrate limitation occurs. This observation leads to the supposition that immobilization techniques simulate the conditions of parasitic Claviceps sclerotia and thus lead to higher alkaloid amounts than yielded by the fermentation of free mycelia.

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[29]

12o-

"_5

•-

7o= "0

0 -

j) .o el

5o-

30lofree 1% CellS

1.4

2% 3% 4% 5% 6% alginate concentration

7%

8%

-i

b

.Ig i

"

ii"

ii

free cells

1% 2% 3% 4% 5% 6% 7%

8%

alginate concentration

FIG. 2. (a) Absolute (mg/liter) and (b) specific (mg/g) overall alkaloid production in various alginate concentrations.

[29]

LONG-TERM ALKALOID PRODUCTION BY C. purpurea

1500 -

323

o

c 1000-

.o

~0ol

? 1

5 2

4

6

10 number of reincubations

8 10 12 14 16 18 2 0 2 2 2 4 time(weeks)

15 26283032

FIG. 3. Alkaloid production in semicontinuous culture with a change of the fermentation broth every 2 weeks. ([5]) Free cells; (O) 2% alginate; (©) 4% alginate; (&) 8% alginate.

Semicontinuous Fermentation of Immobilized Mycelia

Fermentations of free and immobilized mycelia in Fernbach flasks were carried out with change of the production medium every 14 days. Data on the total alkaloid production are shown in Fig. 3. Free cells lost their production capacity after 60 days. Immobilized cells, however, retained their activity for about 200 days. The cumulative alkaloid yields of 16 fermentation cycles using a total of 1.6 liters of nutrient broth showed that the alkaloid yields of immobilized cells could be raised about 25-fold compared to their free counterparts. Similar data for immobilized microbial catalysts with regard to both the prolongation and the increase of the yields of different fungal secondary metabolites, such as antibiotics, were described by Suzuki and Karube, 8 Veelken and Pape, 9 and Kopp et al. 3 8 S. Suzuki and I. Karube, in "Immobilized Microbial Cells" (K. Venkatasubramanian, ed.), Syrup. Ser. Vol. 106, p. 59. American Chemical Society, Washington, D.C., 1979. 9 M. Veelken and H. Pape, Eur. J. Appl. Microbiol. Biotechnol. 15, 206 (1982).

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[29]

FIG. 4. Development of mycelia immobilized in 4% alginate. Sectional area of a cut bead with mycelia growth at the edge of the bead and limited development toward the interior of the bead (electron microscopy). ×40.

Morphology of Immobilized Mycelia Immobilized mycelia grew mainly under the surface of alginate beads, after the outwardly growing hyphae were abraded. Growth was progressively reduced toward the interior of the gel beads (Fig. 4). With increasing fermentation times the cells produced violet-black pigments and showed a strong tendency to break down into arthrosporoid-like structures with vacuolated elements (Fig. 5). This morphological differentiation was regarded as an indicator for a turnover in metabolism. 1° The sclerotia-like cells resembled the plectenchymatic structures in sclerotia of parasitic Claviceps. Dickerson et al. ~1 and Rehacek 7 described these sclerotia-like cells as having the dominant role in alkaloid production. ~0 B. Berde and H. O. Schild, "Ergot Alkaloids and Related Compounds." Springer, Berlin, 1978. ~1 A. G. Dickerson, P. G. Mantle, and C. A. Sczyrbak, J. Gen. Microbiol. 60, 403 (1970).

[29]

LONG-TERM ALKALOID PRODUCTION BY C, p u r p u r e a

325

FIG. 5. Development of sclerotia-like highly vacuolated mycelia in alginate beads (light microscopy). ×600.

Free mycelia of the investigated Claviceps strain showed a limited formation of arthrosporoid cells and this phenomenon might be one reason for the considerable difference in the production capacity of free and immobilized mycelia. These morphological observations also confirm the supposition that the stable alginate gel simulates the conditions of parasitically formed sclerotia and that a mechanical skeleton is necessary for the development of highly productive cells.

Effect of the Nitrogen Concentration on Alkaloid Production There are two reasons for investigating the influence of nitrogen on the physiology of immobilized Claviceps mycelia. (1) The synthesis of ergot alkaloids requires a change in metabolism, especially a decrease in the intensity of culture growth.~2 At peak biomass production alkaloid production is poor. 13 Usually the pH of the fermentation broth is adjusted with large amounts of ammonia which is also a nitrogen source for the cells and leads to extensive biomass production. (2) Immobilized Clavi12 Z. Rehacek, Process Biochem. 18, 22 (1983). 13 A. Puc and H. Socic, Eur. J. Appl. Microbiol. Biotechnol. 4, 283 (1977).

326

129]

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

+ •10

.~

ir

-9

~

0.

o

E 3

o ¢

o

~

2

3

,~

Ca(NO3) 2 g / I

FIG. 6. Influence of nitrogen concentration on biomass production and specific alkaloid production.

c e p s cells which are supplemented by a rich medium grow out of the

beads. Synnemata are abraded and grow as free cells in the nutrient broth. At the same time hyphae growing out of the alginate beads stick together so that optimum flotation and nutrient supply are decreased and culture handling as well as sample taking and alkaloid extraction are difficult. A limitation in biomass production is therefore necessary for immobilization processes and can be regulated by limiting the nitrogen concentration. The pH of the medium was adjusted with KOH instead of ammonia and nitrate as Ca(NO3)2 and NaNO3 served as the sole nitrogen source. At low concentrations (up to 1.5 g/liter) extensive mycelia development was observed below the alginate surface but only few short hyphae grew out from the surface. The alginate beads remained nearly smooth and the fermentation broth contained no free cells. Figure 6 shows the development of mycelial wet weight depending on the nitrogen concentration. The specific alkaloid production (milligrams alkaloids per gram wet weight) shows a maximum at about 1 g NO3-/liter which is different from maximum biomass production under these conditions. These results confirm the observations of Rehacek, 12 Pazoutova et al., ]4 and Kopp and Rehm ~athat the specific alkaloid production increases when the intensive biomass production ceases. ~4 S. Pazoutova, L. S. Slokoska, N. Nikolova, and T. I. Angelov, Eur. J. Appl. Microbiol. Biotechnol. 16, 208 (1982).

[29]

LONG-TERM ALKALOID PRODUCTION BY C. purpurea

327

Alkaloid Production of Immobilized Claviceps purpurea in a 1-Liter Air-Lift Fermenter Air-lift fermentation with an air flow of 5 liters/min was carried out with 4% alginate immobilized mycelia. Figure 7 shows the overall alkaloid production when biomass production is limited by low nitrogen concentrations. Compared to the yields from Fernbach flask fermentations an average 3-fold increase in alkaloid production could be obtained from airlift fermentation (2.5-fold in chanoclavine, 6-fold in agroclavine, 3.5-fold in elymoclavine, and 2.5-fold in ergometrine). Optimum flotation of the smooth alginate beads and optimum nutrient and oxygen supplies are presumably the reasons for the high productivity. Investigations are presently under way to improve alkaloid yields under semicontinuous fermentation conditions in air-lift fermenters.

Increasing Alkaloid Yields by Increasing the Particle Amount in the Fermentation Broth There are two parameters in immobilization techniques that alter the physiological activity or production capacity of immobilized cells. (1) Active biomass and thereby biologically active surfaces can be increased

A

~500 ~ 400 =" 3 0 0 "5 -~ 200-

time in days Fro. 7. Alkaloid production in a l-liter air-lift fermenter compared to that in Fernbach flasks. (C)) Free cells (Fernbach flasks); (ll) 4% alginate-immobilized mycelia (Fernbach flasks); (0) 4% alginate-immobilized mycelia (air-lift fermenter).

328

IMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[29]

700

~- 600] "~500 400 'G 3001 200

lOO] 1b

timeindays

FIG. 8. Alkaloid production in an air-lift fermenter with change in particle amount (concentration of beads in the medium): ( , ) 20% and (0) 40% alginate beads in the nutrient broth.

by decreasing particle diameter. In the case of alginate-entrapped cut mycelial fragments of C. purpurea it is not possible by reducing the needle diameter of the immobilization syringe to reduce the particle size to less than 0.5 cm because mycelia fragments obstruct the needle. (2) Active biomass can be increased by decreasing the amount of fermentation medium. The described fermentations, batch as well as semicontinuous fermentations in both Fernbach flasks and air-lift fermenters, contained 20% alginate particles with a diameter of 0.5 cm. Figure 8 shows an increase of up to 35% in the overall alkaloid production when the particle concentration and thus the immobilized biomass is doubled. It is remarkable that not only the maximum production but also the velocity of production is increased. A precondition for fermentations with particle amounts doubled is a constant bead diameter. Outgrowing hyphae enlarge the particle diameter up to 1 cm, so that flotation of the beads in the fermentation broth is nearly stopped. By limiting the biomass production with low nitrogen concentrations the beads remain smooth and the particle diameter can be kept constant. Increasing the amount of alginate in the medium to more than 40% leads to impaired flotation and nutrient supply and thereby to a reduced production capacity.

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Conclusions

The immobilization technique seems to simulate the conditions of

Claoiceps sclerotia during the natural production of alkaloids on grain and thus leads to higher alkaloid amounts than yielded by fermentation of free mycelia. The gel-entrapped mycelia can be regarded as highly active resting cells; biomass production and growth are regulated by limited phosphate and nitrogen. Compared to their free counterparts the turnover in metabolism and the formation of sclerotial cells are accelerated in immobilized mycelia which leads to an intensified and prolonged alkaloid production. The moderate immobilization method of alginate entrapment presents an adequate method for improving ergot alkaloid production processes. Furthermore, compared to conventional cultivation, the described investigations on gel application give information concerning the necessity of variation and optimization of fermentation parameters as well as the changed physicochemical conditions of the fermentation processes which are responsible for the altered physiological and morphological behavior of entrapped cells.

[30] Cell I m m o b i l i z a t i o n in t h e P r o d u c t i o n of P a t u l i n a n d Penicillin b y Penicilliurn urticae a n d

Penicillium chrysogenum By G. M. GAUCHERand L. A. BEHIE Many valuable natural products are secondary metabolites: secondary in that they do not appear to be an essential prerequisite for cell growth, maintenance, and differentiation. 1 In contrast to essential primary metabolites the commercial production of secondary metabolites has special problems. The production of the antibiotics patulin and penicillin by filamentous fungi is typical. Patulin is a polyketide derived from acetyl-CoA,2 while penicillin is a polypeptide antibiotic derived from a-aminoadipate, cysteine, and valine. The net reactions for patulin and J. W. Bennett and A. Ciegler, "Secondary Metabolism and Differentiation in Fungi." Dekker, New York, 1983. 2 G. M. Gaucher, K. S. Lam, J. W. D. GrootWassink, J. Neway, and Y. M. Deo, Dev. Ind. Microbiol. 22, 219 (1981).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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329

Conclusions

The immobilization technique seems to simulate the conditions of

Claoiceps sclerotia during the natural production of alkaloids on grain and thus leads to higher alkaloid amounts than yielded by fermentation of free mycelia. The gel-entrapped mycelia can be regarded as highly active resting cells; biomass production and growth are regulated by limited phosphate and nitrogen. Compared to their free counterparts the turnover in metabolism and the formation of sclerotial cells are accelerated in immobilized mycelia which leads to an intensified and prolonged alkaloid production. The moderate immobilization method of alginate entrapment presents an adequate method for improving ergot alkaloid production processes. Furthermore, compared to conventional cultivation, the described investigations on gel application give information concerning the necessity of variation and optimization of fermentation parameters as well as the changed physicochemical conditions of the fermentation processes which are responsible for the altered physiological and morphological behavior of entrapped cells.

[30] Cell I m m o b i l i z a t i o n in t h e P r o d u c t i o n of P a t u l i n a n d Penicillin b y Penicilliurn urticae a n d

Penicillium chrysogenum By G. M. GAUCHERand L. A. BEHIE Many valuable natural products are secondary metabolites: secondary in that they do not appear to be an essential prerequisite for cell growth, maintenance, and differentiation. 1 In contrast to essential primary metabolites the commercial production of secondary metabolites has special problems. The production of the antibiotics patulin and penicillin by filamentous fungi is typical. Patulin is a polyketide derived from acetyl-CoA,2 while penicillin is a polypeptide antibiotic derived from a-aminoadipate, cysteine, and valine. The net reactions for patulin and J. W. Bennett and A. Ciegler, "Secondary Metabolism and Differentiation in Fungi." Dekker, New York, 1983. 2 G. M. Gaucher, K. S. Lam, J. W. D. GrootWassink, J. Neway, and Y. M. Deo, Dev. Ind. Microbiol. 22, 219 (1981).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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penicillin biosynthesis are Acetyl-CoA + 3 malonyl-CoA + 3 NADPH + 3 H + + 3 02 patulin + 4 CoA-SH + 4 H20 + 3 NADP ÷ + 4 CO2 L-Cys + L-Val + phenylacetyl-CoA + 2 NADPH + 2 H ÷ + 2 02 + 2 ATP --~ penicillin G + CoA-SH + 2 NADP ÷ + 3 H20 + 2 AMP + 2 PP

The sustained production of these metabolites requires that primary metabolic pathways provide an adequate supply of cofactors and precursors to the pertinent secondary pathway. These secondary routes consist of multiple steps (-> I0 for patulin, ->4 for penicillin) each catalyzed by enzymes which are substrate specific, generally labile, and synthesized de novo via mRNAs which appear in response to derepression mechanisms associated with nutrient deprivation. It is clear both from studies of pathway mutants 2 and some enzymes 3 and from the feeding of substrate analogs 4 to enzymes and cells that these enzymes are unique to secondary metabolism and are particularly short-lived in whole-cell cultures and cell-free extracts. Thus the cessation of antibiotic production in batch cultivations of Penicillium urticae and Penicillium chrysogenum after a production period of - 2 and 5 days, respectively, is most probably due to the net loss of one or more pathway enzymes (N.B. rate of enzyme loss > rate of enzyme synthesis). There is some evidence that the large multifunctional enzymes which initiate these secondary pathways tend to be the most labile. 3 The cellular content of 6-methylsalicylic acid (6-MSA) synthetase, the first enzyme of the patulin pathway, exhibits an in vivo half-maximal lifetime of 2-6 hr depending upon the strain o f P . urticae. 2'3 In crude cellfree extracts (2 mM EDTA, 15% v/v glycerol, bovine serum albumin 3 g/ liter, pH 7.6, 30 °) the same enzyme has an in vitro half-life of - 1 7 min, while the half-life of the second pathway enzyme is 32 min. 2 In vitro stabilization studies of 6-MSA and gramicidin S synthetases 2,5 suggest that the in vivo loss of these enzymes and hence the cessation of antibiotic production may be initiated by a fall in the intracellular level of key metabolites or cofactors. The expected response to such changes in metabolite pool sizes would be less enzyme-substrate interaction and more enzyme damage via oxidation and/or less repair of this oxidation. In either case the percentage of protease susceptible conformations would increase. Thus the longevity of antibiotic-producing enzymes is inher3 J. Neway and G. M. Gaucher, Can. J. Microbiol. 27, 206 (1981). 4 G. M. Gaucher, J. W. Wong, and D. G. McCaskill, in "Microbiology--1983" (D. Schlessinger, ed.), p. 208. American Society of Microbiology, Washington, D.C., 1983. K. S. Lam, J. O. Neway, and G. M. Gaucher, Can. J. MicrobioL, submitted (1987).

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PRODUCTION OF PATULIN AND PENICILLIN

331

ently poor because secondary metabolism is generally a non-growth-associated process which occurs during a period of metabolic trauma characterized by rapidly changing metabolite levels and a proliferation of intracellular proteases. 2 Given our interest in enhancing the productivity of antibiotic fermentations and our focus on the problem of enzyme longevity we were attracted to the concept of immobilized cells by the remarkable catalyst half-lives (i.e., months to years) reported by Chibata et al. 6,7 for simple one-step primary metabolic conversions. Our objectives have been to extend this technology to secondary metabolism by developing and characterizing the performance of immobilized cell bioreactors for the longterm production of two model antibiotics, patulin and penicillin. These two antibiotics were chosen because they differ in their biosynthetic origins, in the amount that is known about their pathway enzymes, in the nutritional and growth rate changes which trigger production, and in their commercial importance. In contrast to the polyketide, patulin, the polypeptide penicillin has a little known enzymology, is less strictly associated with the nongrowth phase of submerged cultures, and is an important commercial antibiotic. Immobilization Procedure and Equipment Penicillium urticae (NRRL 2159A) and Penicillium chrysogenum (ATCC 12690 and 26818 El5) are preserved as dry conidia in silica gel (4°)s and conidial inocula are prepared from uniformly grown (28°, 10-14 days; 26 °, 7-9 days, respectively) surface cultures in either 8-dram vials (15 cm 2 surface, - 10 ml agar) or 500-ml Erlenmeyer flasks ( - 100 ml agar). For P. urticae Czapek-Dox agar (49 g Difco Czapek-Dox solution agar, and 5 g Difco Bacto-agar in 1 liter deionized water) is used, while for P. chrysogenum a glycerol-peptone agar (7.5 g glycerol, 5.0 g Bacto-peptone, 7.5 g dark sugar molasses, 0.05 g M g S O 4 , 0.06 g KH2PO4, 4.0 g NaC1, 15 g Bacto-agar in 1 liter deionized water) or a sporulation agar (1 g glucose, 4 g casamino acids, 6 g Bacto-peptone, 3 g yeast extract, 1.5 g beef extract, 20 g malt extract, 20 g KHzPO4, Jarvis and Johnson trace metals, 9 40 g Bacto-agar in 1 liter deionized water) is used for low (ATCC 6 I. Chibata, T. Tosa, and T. Sato, in "Microbial Technology" (A. J. Peppier and D. Perlman, eds.), p. 433. Academic Press, New York, 1979. 7 I. Chibata, T. Tosa, and I. Takata, Trends Biotechnol. 1, 9 (1983). 8 j. W. D. GrootWassink and G. M. Gaucher, J. Bacteriol. 141, 443 (1980). 9 F. G. Jarvis and M. Johnson, Am. Chem. Soc. J. 69, 3010 (1947); in grams/liter: MgSO4-7H20, 0.25; FeSO4"7H20, 0.1; CaC12'2H20, 0.05; ZnSO4"7H20, 0.02; MnSO4-H20, 0.02; CuSO4.5H20, 0.005.

332

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[30]

12690) and high (ATCC 26818) producing strains, respectively. As outlined in Fig. 1 conidia from one slant or flask are suspended in 5.0 ml of a sterile detergent solution (450/xl Fisher Aerosol OT, 8.5 g NaC1 in 1 liter of deionized water) by mixing on a Vortex mixer or by hand. For P. chrysogenum a small Erlenmeyer flask containing this suspension is then sonicated for - 4 min in an ultrasonic water bath to break up chains of conidia. Suspensions (5 ml) o f P . urticae or P. chrysogenum conidia (0.32 × 108 or 1-6 × 10 7 conidia/ml, respectively) are added to 200 ml of a sterile, 37° solution of 4% (w/v) K-carrageenan (NJAL 724 or 798 containing 6.0% sodium, 0.98% potassium, 0.23% calcium, and 0.22% magnesium all w/v; Marine Colloids Division, FMC Corp. P.O. Box 70, Springfield, NJ, 07081, USA). In a sterile transfer hood this carrageenanspore mixture is vigorously mixed by hand and then poured into a 316

AGAR SLANT CULTURE

CONIDIAL SUSPENSION ~r VORTEXINGOR SONICATION

DISPERSED CONIDIA CARRAGEENAN SOLUTION

I,.

CARRAGEENAN + CONIDIA KCI + CaCI2 SOLUTION WASH

PRODUCTION STARVATION PHASE PHASE

GROWTH PHASE

days-months

40h at 28' 48h at 26*

6h at2B" 3h at 26" SHAKE

FLASK

DECANT

BEADS CONTAINING CONIDIA

PHOSPHATE BUFFER

FIG. I. Protocol for the immobilization of conidia and subsequent in situ growth of cells and production of antibiotics.

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PRODUCTION OF PATULIN AND PENICILLIN

333

stainless-steel cylinder (15.6 cm long, 11 cm diameter, 1.4 liter capacity) fitted with a bottom plate (1/4 in. thick) pierced by 48 stainless-steel syringe needles (24 gauge, 3/4 in. long, beveled end) and a top plate designed to be connected to a pressurized, N2 gas supply (20-40 psi) via a glass-wool packed stainless-steel filter (1/4 in. I.D., 9 cm long). As illustrated in Fig. 2 drops of uniform size are allowed to fall from a height of - 5 - 1 0 cm into a 2-liter beaker containing - 1 . 0 liter of a gently stirred (magnet) sterile, - 1 0 ° salt solution (0.3 M KCI, 0.05 M CaCI2) over a period of - 3 min. After stirring in this salt solution for 3 hr at room temperature the liquid is decanted or drained off and the ~ 10,500 beads (3.2 - 0.15 mm diameter) are washed with three -500-ml portions of sterile deionized water to remove excess calcium. Although a number of different synthetic and natural polymers have been used for cell entrapment, gelling polysaccharides such as r-carrageenan, agarose, alginic acid, and Gelrite have been particularly effective. In preliminary experiments a conidia-containing 1% solution of Gelrite (Kelco, 8225 Aero Drive, San Diego, CA) was dropped into a 2% MgCI2, 10% methanol solution to yield very clear gel beads which performed much like those of carrageenan. Once a particular matrix such as the seaweed anionic galactan, K-carrageenan, is chosen, it is important to

FIG. 2. The casting of uniform carrageenan beads with a multineedled bead-making device.

334

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

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recognize that different commercial preparations can mean that the best gelling temperature, concentration (%) of glycan, viscosity of the liquid gel, and salt composition of the bead casting solution will vary. The correct choice of these variables will determine important characteristics such as cell survival during entrapment, the choice of a bead-casting protocol and apparatus, and gel hardening, which in turn affects bead survival under physical stress and cell growth within the gel. Thus for example Sigma K-carrageenan (Type 1II, 0.5% sodium, 2.66% potassium, and 1.75% calcium), which contained more potassium and calcium, was effective when a 2.5% solution at - 5 0 ° was cast into beads in the presence of only potassium ions. ~° At 1-2% the beads obtained were physically unstable, while at 3-4% the bead hardness prevented conidia germination. 1° In contrast the FMC K-carrageenan used at 4% and 37 ° allowed germination throughout ]° while providing beads which remained intact after gyratory shaking at 280 rpm for better than 5 weeks. An "amalgam" of two polysaccharides can increase both the strength of the solid gel and the viscosity of the liquid gel. Thus the addition of 0.5% locust bean gum (Sigma) to 1% Sigma K-carrageenan yielded physically stable beads, while 0.25% of this gum increased the viscosity of 4% FMC K-carrageenan so as to trap air bubbles during the preparation of porous beads. ~0Beads which were very porous and hence buoyant were cast as described above after the addition of detergent (450/xl Fisher Aerosol OT per liter) to a mixture of conidia and these two polysaccharides was followed by 30 sec of vigorous mixing (11,000 rpm; Sorvall Omnimixer). The diffusivity of nutrients and secondary metabolites into and out of 3.2 mm 4% K-carrageenan (NJAL 724) beads without cells is very high. For example in 0.05 M KH2PO4 buffer (pH 6.5) at 28 °, glucose, patulin, and 6-MSA have diffusivities of 4.01, 3.77, and 1.75 × 10-4 cm2/min. The poorer diffusion of 6-MSA is probably due to the negatively charged character of both 6-MSA and the carrageenan matrix. The fact that these gel beads exhibit diffusion rates which are only slightly smaller than those found in water (i.e., 4.04 × 10-4 cmZ/min for glucose) is, however, misleading. In growth medium beads inoculated with conidia rapidly develop a dense peripheral population of cells which acts as a significant barrier to transport into the bead interior. Beads possessing an established 48-hr-old cell population of P. urticae which had been killed with 0.02% sodium azide exhibited diffusivities of 4.01, 3.02, and 1.17 × 10 -4 cm2/min for glucose, patulin, and 6-MSA. While this barrier of biomass may lower some diffusion rates, it is the active uptake and metabolism of live cells which eventually curtails the continued growth of cells in the bead inte10 y . M. Deo, J. W. Costerton, and G. M. Gaucher, Can. J. Microbiol. 29, 1642 (1983).

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rior. Although porous beads improve transport in the short term (<2 days) 1° they are soon subject to the same limitation. If deemed to be of value a nutrient-deprived core can be minimized by decreasing the bead diameter although the influence of bead size and density on reactor performance may dictate a minimum bead size. Beads of smaller diameter (i.e., 2.7, 1.9, 1.6 ram) were prepared by some or all of the following modifications: using finer needles (i.e., 26-27 gauge), trimming needles straight across and inwardly tapering the tip, reducing surface tension by adding silicon antifoam agent (7 ml of a 0.75% aqueous solution of SAG 471, Union Carbide, per 100 ml of carrageenan), and pneumatically vibrating the entire bead-making device during bead formation. Finally, smaller (-1000) and larger (-260,000) batches of beads have been prepared as described above by delivering 20 or 5000 ml of the conidia-carrageenan mixture through a 30-ml Luer-lok syringe with needle or in four passes through the 1.4-liter multineedled bead maker. The continuous (65 ml/min) casting of a 10-liter batch of 2.6-mm carrageenan beads directly into a fermenter has also been achieved. A commercial scale-up of bead production will undoubtedly require that beads be cast directly into a bioreactor via vibrating needles, a resonance nozzle, 11or an aerosol spray technique. Another solution to scale-up problems may be the entrapment of conidia into prefabricated porous Celite beads (JohnsManville, #560, 0.3-0.5 mm) by capillary action as described by Gbewonyo and Wang. 12 In our hands these rigid Celite beads perform well except for some sensitivity to disintegration in vigorously shaken or stirred vessels. 13 Establishment of Antibiotic-Producing Activity In an extrapolation from studies which sought to preserve primary metabolic enzyme activities a common approach has been to immobilize cells which are actively engaged in antibiotic production. Unfortunately the lability of "secondary" enzymes can result in a significant loss of antibiotic productivity (i.e., 65-90%) during the immobilization procedure. 14Our early recognition of this problem led to the development of an in situ colonization procedure in which conidia which are naturally resistant to environmental trauma (i.e., temperature) are immobilized in gel beads and then allowed to germinate and populate the entire bead during 1i j. Tramper, Trends Biotechnol. 3, 45 (1985). 12 K. Gbewonyo and D. I. C. Wang, Biotechnol. Bioeng. 25, 967, 2873 (1983). i3 A. Jones, D. N. Wood, T. Razniewska, G. M. Gaucher, and L. A. Behie, Can. J. Chem. Eng. 64, 547 (1986). ~4y . M. Deo, J. W. Costerton, and G. M. Gaucher, Dev. Ind. Microbiol. 25, 491 (1984).

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an incubation in growth medium. ~5Thus 20 or 200 ml of freshly prepared beads (i.e., -1000 or 10,500) is shaken (280 rpm, 2.5 cm stroke) in 500-ml Erlenmeyer or 2.8-liter Fernbach flasks containing 50 or 500 ml of growth medium. For P. urticae this medium is a yeast extract-glucose-buffer medium 8 adjusted to pH 6.5 with NaOH and containing (in grams per liter) yeast extract (Difco), 5; glucose, 40; KHEPO4, 13.6; citric acid, 9.8; Na2SO4, 1; and I0 ml of the trace metals solution of Yamamoto and Segel. 16 For P. chrysogenum (ATCC 12690) this medium is a glucose medium (pH 6.2) containing (in grams per liter) glucose, 25.0; ammonium lactate and acetate, 6.0 and 3.5, respectively; KH2PO4,3.0; NaESO4,0.5; and the trace metals of Jarvis and Johnson. 9 After 24 hr a further 5 g of glucose per liter is added as a sterile 50% (w/v) solution, along with 0.38 g of phenylacetic acid per liter as a sterile, neutralized 3.8% (w/v) solution. For P. chrysogenum (ATCC 26818) this medium is a glucose-lactose (pH 6.8) medium containing (in grams per liter) glucose, 10.0; lactose, 17.1; ammonium chloride and acetate, 2.7 and 3.0, respectively; sodium lactate, 3.8; KH2PO4, 3.0; Na2SO4, 0.5; and the trace metals of Jarvis and Johnson. 9 After - 4 1 hr, l0 g of glucose and 0.5 g of neutralized phenylacetic acid per liter are added to these cultures. Penicillium urticae- or P. chrysogenum-containing beads are harvested by decantation after having reached the early stage of antibiotic production (i.e., - 4 0 hr at 28 ° or 48 hr at 26°, respectively), then washed with two 500-ml portions (i.e., per 200 ml beads) of sterile phosphate buffer (0.1 M, pH 6.5 or 6.8, respectively), and finally subjected to starvation in 500 ml of the same respective buffers by shaking for 6 hr at 28 ° or 2-3 hr at 26 °, respectively. Kinetic profiles of cell protein (mg per culture or bead), glucose utilization, and patulin production have shown that cultures inoculated with free or immobilized conidia exhibit virtually identical growth and antibiotic production kinetics.15'17'18 Thus immobilization does not appear to hinder germination, vegetative growth, or the transition to the nongrowth antibiotic production phase or idiophase. After 38 hrjust prior to the removal of P. urticae-containing beads from the growth medium, a cross section of a typical bead (Fig. 3) shows that in going from the center of a bead to its surface, there is an increasing ~5y. M. Deo and G. M. Gaucher, Biotechnol. Lett. 5, 125 (1983). 16 L. A. Yamamoto and I. H. Segel, Arch. Biochem. Biophys. 114, 523 (1966); in grams/liter: Na3 citrate. 2H20, 5; MnC12"4H20, 3; ZnCI2, 2; FeC13'6H20, 2; MgC1E'6H20, 50; CuCI2'2H20, 0.2; CaCl2 '2H20, 0.75; COC12.6H20, 0.2; MoC15, 0.1; Na2B407' 10H20, 0.1. t7 A. Jones, T. Razniewska, B. H. Lesser, R. Siqueira, D. Berk, L. A. Behie, and G. M. Gaucher, Can. J. Microbiol. 30, 475 (1984). ~8D. Berk, L. A. Behie, A. Jones, B. H. Lesser, and G. M. Gaucher, Can. J. Chem. Eng. 62, 112 (1984).

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FIG. 3. Scanning electron micrograph of a cross section of a P. urticae-containing carrageenan bead after 38 hr in growth medium and just before transfer to production medium.

population gradient of cells. Thus while at first growth is not inhibited, a growing inequality of access to nutrients does eventually inhibit cell growth in the bead interior. Maintenance of Antibiotic-Producing Activity Since biomass production is a significant part of a conventional batch process, process optimization has usually been designed to support growth as well as antibiotic production. Freed of this necessity to support significant growth and given a particular choice of reactor and operating mode (i.e., semicontinuous or continuous), we have sought to optimize the efficiency and longevity of antibiotic production while minimizing cell growth. After further derepression of antibiotic production by a short period of starvation, well-colonized beads with a rapidly emerging antibiotic-producing capacity were transferred to a bioreactor containing production medium. This medium was replenished either in a semicontinuous manner by decanting and replacement every 18-24 hr 19 or 48 hr ~5 for 19 y . M. Deo and G. M. Gaucher, Biotechnol. Bioeng. 26, 285 (1984).

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penicillin and patulin, respectively, or in a continuous manner by withdrawing and feeding medium at equal rates of 7.5 ml/hr 19or 12.0-300 ml/ hr, 18,2° for penicillin and patulin, respectively. A variety of perfectly mixed three-phase bioreactors were used. Small-scale experiments were carried out in 500-ml shake flasks iS,j9 containing 20 ml of beads and 40-50 ml of medium; in 170-ml airlift reactors (height, h -- 16 cm; diameter, d = 2.9 cm) with external loop (h = 12 cm; d = 1.9 cm), aerated via a coarseporosity sintered glass plate (d = 1.5 cm; air flow = 120 ml/min) and containing 50 ml beads and 110 ml medium; in 150-mP 9 and 1.2-liter 18 fluidized bed reactors (h = 20 or 17 cm; d = 4 or 7.5 cm), aerated via sintered glass plates (d = 4 or 7.5 cm, air flow = 500-1250 or -3710 ml/ min) and containing either 50 and 100 or 400 and 700 ml beads and medium, respectively; or in a 5-liter stirred tank 2° (h = 30 cm, d = 14 cm, 500 rpm, 3 liters/min) containing 1 liter beads and 1.2 liters medium. On a larger scale, a 19-liter air-lift fermenter (Bioengineering AG, model 1523, h = 50 cm, d = 20 cm) with internal draft tube (h = 42 cm, d = 12.5 cm), aerated with a ring sparger (air flow = 20 liters/min) and containing 5 liters of (-275,000) beads and 10 liters of medium, performed well. The development of production media utilized shake cultures with periodic medium replacement. Excellent productivity was achieved during the first two to three 24- or 48-hr passages for penicillin 19 and patulin, 15'21 respectively. The objective was to arrest the slow decline which invariably followed, by examining the effect of media modifications at later stages of production (i.e., during passages 5-15; 5-15 days for penicillin and 10-30 days for patulin). Although in the short term patulin production required only a carbon source 2,2j (i,e., glucose 25 g/liter, KH2PO4 13.6 g/liter, pH 6-6.5), long-term production (i.e., 15-36 days) required limited growth 18,2°,21 achieved by the addition of 1-10% of the yeast extract and 5-10% of the NazSO4, citrate, and trace metals found in growth medium. Similarly the long-term (i.e., 12-16 days) production of penicillin G 19 was achieved using a weak growth medium containing (in grams per liter) glucose, 10; or lactose, 15, and NH4CI, 1.2; K2804, 3.95; KH2PO4, 6.8; and phenylacetic acid, 0.4. Lactose was superior to glucose and additional productivity (/~g penicillin G/mg protein-hr) was obtained by the addition of certain amino acids (e.g., sodium glutamate, 0.84 g/ liter) and metal ions (e.g., CuSO4" 5H20, 0.0078 g/liter). For penicillin and patulin, production media containing 10-15 g glucose or >0.25 g yeast extract per liter, respectively, resulted in excessive growth, an 20 A. Jones, D. Berk, B. H. Lesser, L. A. Behie, and G. M. Gaucher, Biotechnol. Lett. 5, 785 (1983). 21 y . M. Deo and G. M. Gaucher, Appl. Microbiol. Biotechnol. 21, 220 (1985).

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increase in bead volume (e.g., 36%) and free cells, and ultimately reactor defluidization. Under optimum conditions however, growth and the free cell content were minimal. The current advantages and limitations of using immobilized versus free cells for commercial antibiotic production have been discussed. 12,14,19 Sampling Procedures and Assays Samples of immobilized cells and culture medium are obtained by decanting off the medium, washing the beads thoroughly (e.g., 3 x 50 ml distilled water per 20 ml beads for - 2 min), freezing them in liquid nitrogen, and thoroughly freeze-drying for 49-72 hr. Medium was filtered through a 0.45-/zm filter. Both dry beads and culture filtrates can then be stored at - 2 0 °. Determinations of cell growth and antibiotic productivity (in micromoles or units per milligram) are based upon protein content rather than dry weight. Dry beads are crushed to a fine powder and - 2 0 mg (i.e., - 2 0 beads) is used for a Folin-Lowry based protein assay 17designed to eliminate interferences from carrageenan and phenolics. By means of the same assay the incorporation of [~4C]leucine into protein ~7provides a measure of immobilized cell viability. We have also described a less accurate but simpler protein assay.15 For microscopic studies 1° wet beads are sectioned with a razor blade for phase-contrast microscopy or frozen and cracked for scanning electron microscopy. Culture filtrates are assayed for glucose with the Glucostat or Statzyme reagents (Worthington Biochemical), for lactose by measuring the increase in glucose when fl-galactosidase was added, and for ammonia by ion-exchange 19or colorimetric assays. 22 In addition to bioassays for patulin 8 and penicillin, 19high-pressure liquid chromatography (HPLC) is used to assay these antibiotics and their associated metabolites. For patulin, filtered medium (2 ml) is acidified to pH 2 wih 4 N HCI and extracted with 3 volumes of ethyl acetate. The combined extract is concentrated in 15-ml conical tubes at 30° under vacuum (Buchler vortex evaporator 3-2200) using the addition of methanol to remove traces of water. The residue is redissolved in 2 ml of HPLC-grade methanol and 10/zl of this solution is injected into an RP-8 column (Merck or Brownlee, l0/zm, 4.6 × 200 or 250 mm). For penicillin and on occasion for patulin, culture filtrates (10 /zl) are injected directly via a guard column (RP-18, 5/zm, 70 x 2 mm). Elution with an acetonitrile-water 2° or an acetonitrile-phosphate buffer (pH 4.7 or 5.0) 19 gradient for patulin and penicillin, respectively, is per2z M. W. W e a t h e r b u r n , Anal. Chem. 39, 971 (1967).

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formed on a Hewlett-Packard HP1084B chromatograph equipped with a variable-wavelength detector (i.e., 276 nm for patulin, 254 nm for other patulin pathway metabolites, 220 nm for penicillin and its degradation products). Use of Immobilized Cells in Fundamental Studies Fundamental biochemical questions concerning secondary metabolism are often difficult to answer because of the experimental limitations imposed by culture environment, the highly viscous and shear susceptible nature of filamentous cultures, the non-growth-associated nature and limited longevity of secondary metabolism, and the preponderance of unelucidated pathways and unavailable intermediates. These limitations can often be alleviated by the use of immobilized cells. Since in their natural habitat both the differentiation (e.g., sporulation) of soil fungi and their initiation of secondary metabolism 1,23are probably controlled by nutritional and hence metabolic gradients, immobilized cell cultures (submerged or surface) rather than homogeneous free cell cultures 8 are essential to some studies. Immobilization allows fragile cells to be grown to high densities in the absence of vigorous mechanical stirring because of the significantly better mass transport (e.g., Oz) which results from minimizing the number of free cells and hence the culture viscosity.12 For example we have grown Celite bead (Johns-Manville, No. 560, 0.3-0.5 mm)-entrapped P. chrysogenum in 1.2-liter fluidized bed reactors to a cell density of about 6 mg protein/ml (free cell culture densities reach about 0.9-1.8 mg protein/ ml). 13 In another experiment it was shown that during 127 hr of the production phase (i.e., 150-277 hr) o f a 1.2-1iter fluidized bed reactor containing carrageenan-immobilized P. urticae only 1-2% of the total protein in the reactor was associated with free cells. Since the longevity of secondary metabolism is dependent upon a delicate balance between starvation-mediated derepression and nutrientmediated cell maintenance, the maintenance of a nutritional steady state via continuous culture (i.e., chemostat) techniques is invaluable to both basic and applied research and is only possible when there is no loss (i.e., washout) of the nongrowing cells) 4,zt Thus a stirred tank (5 liters, 500 rpm, 3 liters air/min) containing 1.2 liters of medium and 1 liter of carrageenan beads populated with P. urtieae was operated on a continuous ~3 I. M. Campbell, D. L. Doerfler, B. A. Bird, A. T. Remaley, L. M. Rosato, and B. N. Davis, in "Overproduction of Microbial Products" (V. Krumphanzyl, B. Sikyta, and Z. Vanek, eds.), p. 141. Academic Press, London, 1982.

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basis for 34 days at a flow rate of 200-300 ml/hr. 2° A conventional free cell culture operated at this rate would quickly wash out. Catalytic longevity appears to be a general characteristic of immobilized cells even in batch cultures. Apart from protection from physical trauma, this longevity may result from protection from physiological trauma or discontinuities. Studies of patulin pathway enzymes have shown that a rapid centrifugation (<5 min; basket centrifuge) of a f r e e cell batch culture and a resuspension of these cells into a fermenter containing fresh production medium results in significant (i.e., 15-46%) drops in the enzyme content of the ceUs. 4 In recent experiments the specific activity of three patulin pathway enzymes and the medium concentration of the end product, patulin, were determined every 12 hr during the semicontinuous cultivation (i.e., three consecutive 48-hr incubations in fresh production medium) of both free and carrageenan-immobilized P. urticae. Both free and immobilized cell cultures produced about the same amount of patulin (i.e., 0.6 mg/ml) during the first 48-hr incubation but thereafter free cell production dropped to <0.1 mg/ml while immobilized cells continued to produce at the initial level. This superior productivity appears to be reflected in the specific activity of patulin pathway enzymes. Thus although the initial specific activities of free cells were about 5- to 7-fold higher than for immobilized cells, the free cell specific activity dropped by about 93% during the first 48-hr incubation while for immobilized cells it decayed by only about 20% over the entire 6-day period. The high, localized population density of immobilized cells may provide a buffer against rapidly changing nutrient levels in the bulk culture medium and hence may account for this increased longevity. Immobilized cells are ripe for exploitation in studies which seek to isolate new pathway intermediates, z4 to determine the metabolic relationship between intermediates (i.e., construct pathways) by whole-cell feeding experiments, 4 and to create antibiotic analogs via the strategy of mutasynthesis. 4 For example, phyllostine, a scarce, transient, late intermediate of the patulin pathway, can be efficiently produced by feeding m-hydroxybenzyl alcohol, a cheap, easily purchased, early intermediate, to carrageenan-immobilized cells of P. urticae mutant P3. This mutant lacks the first and eighth enzymes of the pathway while the other pathway enzymes can be induced to 10-fold higher levels by the early intermediate, m-hydroxybenzyl alcohol. 2 Erlenmeyer flasks (500 ml) containing 20 ml of starved, 48-hr-old immobilized cells, 50 ml of medium (0.1 M KHzPO4, pH 6.5; glucose 25 g/liter), and m-hydroxybenzyl alcohol at a final concentration of 7 mM were shaken at 28° for 2 days to yield an 24 J. Sekiguchi, G. M. Gaucher, and Y. Yamada, Tetrahedron Lett. 1, 41 (1979).

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almost complete conversion to phyllostine. The immobilized catalyst could be used in this way for at least seven times. Although generally perceived to be negative, the loss of some enzymatic activities due to immobilization can actually be useful. We have found that a chemically harsh immobilization of patulin-producing cells (not spores) of the parent P. urticae NRRL 2159A resulted in cells with a truncated biosynthetic pathway. 24 Phyllostine (300 mg), a known intermediate, was added to a suspension of P. urticae-containing polyacrylamide gel blocks (30-60 mm 3) in 0.05 M phosphate buffer (pH 6.5). After a 2-hr incubation at 30° the buffer was removed, acidified to pH 2, and extracted with ethyl acetate. Preparative TLC yielded 78 mg (26% yield) of crystalline neopatulin, a normally undetectable late intermediate. Patulin, the usual end product, was undetectable. Again this immobilized catalyst could be reused repeatedly for this purpose if stored at - 4 °. The gradual increase in the accumulation of the first intermediate of the patulin pathway (i.e., 6-MSA) as immobilized cells age has been reported 18,21 as a decline in "patulin pathway efficiency" and is another example of the selective loss of certain enzyme activities. We have recently discovered that this decline can be virtually eliminated by the semicontinuous addition of small amounts of the pathway inducer, m-hydroxybenzyl alcohol. This discovery represents an important new way of significantly increasing the longevity of antibiotic biosynthesis in immobilized systems and emphasizes the important impact of fundamental studies. Acknowledgments The essential contributions of our co-workers Alan Jones, Dimitrios Berk, Yashwant Deo, Ray Lain, Teresa Razniewska, Arun Nayar, Murray Nicholson, Denise Wood, and Phil Bakes and the support of the Natural Sciences and Engineering Research Council of Canada (Grants G-0639 and G-I 118) are gratefully acknowledged.

[31] I s o l a t i o n a n d I m m o b i l i z a t i o n o f S t r i c t o s i d i n e S y n t h a s e 1

By URSULA PFITZNER and MEINHART H. ZENK Strictosidine synthase catalyzes the synthesis of the indole alkaloid strictosidine from tryptamine and the monoterpenoid glucoside secologanin in an absolute stereospecific manner (Fig. I). Of the two theoretically This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, SFB 145, and by Fonds der Chemischen Industrie.

METHODS IN ENZYMOLOGY, VOL. 136

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almost complete conversion to phyllostine. The immobilized catalyst could be used in this way for at least seven times. Although generally perceived to be negative, the loss of some enzymatic activities due to immobilization can actually be useful. We have found that a chemically harsh immobilization of patulin-producing cells (not spores) of the parent P. urticae NRRL 2159A resulted in cells with a truncated biosynthetic pathway. 24 Phyllostine (300 mg), a known intermediate, was added to a suspension of P. urticae-containing polyacrylamide gel blocks (30-60 mm 3) in 0.05 M phosphate buffer (pH 6.5). After a 2-hr incubation at 30° the buffer was removed, acidified to pH 2, and extracted with ethyl acetate. Preparative TLC yielded 78 mg (26% yield) of crystalline neopatulin, a normally undetectable late intermediate. Patulin, the usual end product, was undetectable. Again this immobilized catalyst could be reused repeatedly for this purpose if stored at - 4 °. The gradual increase in the accumulation of the first intermediate of the patulin pathway (i.e., 6-MSA) as immobilized cells age has been reported 18,21 as a decline in "patulin pathway efficiency" and is another example of the selective loss of certain enzyme activities. We have recently discovered that this decline can be virtually eliminated by the semicontinuous addition of small amounts of the pathway inducer, m-hydroxybenzyl alcohol. This discovery represents an important new way of significantly increasing the longevity of antibiotic biosynthesis in immobilized systems and emphasizes the important impact of fundamental studies. Acknowledgments The essential contributions of our co-workers Alan Jones, Dimitrios Berk, Yashwant Deo, Ray Lain, Teresa Razniewska, Arun Nayar, Murray Nicholson, Denise Wood, and Phil Bakes and the support of the Natural Sciences and Engineering Research Council of Canada (Grants G-0639 and G-I 118) are gratefully acknowledged.

[31] I s o l a t i o n a n d I m m o b i l i z a t i o n o f S t r i c t o s i d i n e S y n t h a s e 1

By URSULA PFITZNER and MEINHART H. ZENK Strictosidine synthase catalyzes the synthesis of the indole alkaloid strictosidine from tryptamine and the monoterpenoid glucoside secologanin in an absolute stereospecific manner (Fig. I). Of the two theoretically This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, SFB 145, and by Fonds der Chemischen Industrie.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[31]

STRICTOSIDINESYNTHASE OH

CliO ~ H ~,,,, {~~'11

NH2

H Tryptamine

~ ~l,

~ N H

MeO~

343

HO..F..J./~OH 0 J'~ 0"/" CH20H 0

Secologanin J

Strictosidine

Synthase

~

HO

OH

OH

.HO,

3o. (S )- Strictosidine FIG. 1. Stereospecific condensation of [2-3H]tryptamine with secologanincatalyzed by strictosidine synthase to yield 3cz(S)-strictosidine and tritiated water (HOT).

possible molecules only the glucoalkaloid with 3ct(S)-configuration is formed. 2,3 This molecule can undergo almost endless metabolic rearrangements and modifications, 4 and thus gives rise to highly diverse structures of indole alkaloids of which more than one thousand have become known in the plant kingdom mainly in the families Apocynaceae and Rubiaceae. 5 The enzyme was partially purified in the past, 6,7 and has recently been purified to homogeneity. 8 In addition, we have discovered seven isoenzymes 8 in cell suspension cultures of Catharanthus roseus serving as plant material. Strictosidine synthases from Apocynaceae cell cultures have a molecular weight of approximately 31,000 and are composed of one single polypeptide chain only. It is therefore obvious that these enzymes are suitable for immobilization. Materials. Cyanogen bromide-activated Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Ultrogel AcA 44 was from LKB (Stockholm, Sweden). Matrex Gel Red A and Matrex Gel Blue A were from Amicon (Danvers, MA). Charcoal was from Sigma (St. Louis, MO) and tryptamine from Fluka (Buchs, Switzerland); [2-3H]trypt amine (1.86 × 10 7 Bq/mmol) was synthesized according to Treimer and 2 j. St/~ckigt and M. H. Zenk, J. Chem. Soc., Chem. Commun. 646 (1977). 3 j. StOckigt, Phytochemistry 18, 965 (1979). 4 N. Nagakura, M. Rueffer, and M. H. Zenk, J. Chem. Soc. Perkins Trans. 1 2308 (1979). 5 D. Ganzinger and M. Hesse, Lloydia 39, 326 (1976). 6 j. F. Treimer and M. H. Zenk, Eur. J. Biochem. 101, 225 (1979). 7 H. Mizukami, H. Nordlrv, S.-L. Lee, and A. J. Scott, Biochemistry 18, 3760 (1979). s U. Pfitzner and M. H. Zenk, unpublished results.

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Zenk. 6 Secologanin was isolated from Lonicera tatarica dried leaves. 9 Catharanthus roseus cell suspension cultures were grown in the dark at 24 ° in l-liter Erlenmeyer flasks at 100 rpm in Linsmaier and Skoog medium, ~° containing 60 /xM 2,3-dimethylphenoxyacetic acid as sole hormone. A dry weight of 17.0 g/liter of medium was obtained after a growth period of 7 days. The tissue was then filtered off, frozen in liquid nitrogen, and subsequently stored at - 2 0 °. Assay Method. Enzyme activities are determined by measuring the amount of tritium released into the aqueous incubation mixture during the condensation of secologanin with [2-3H]tryptamine. Strictosidine synthase is assayed as follows: 90 nmol of [2-3H]tryptamine (1.7 × 103 Bq), 1.2/xmol secologanin, 10/zmol potassium phosphate buffer (pH 6.5), and the enzyme preparation are incubated in 1.5-ml Eppendorf vials in a total volume of I00/zl for 1 hr at 37°. The incubation is stopped by the addition of 300/~1 activated charcoal (2 g coal suspended in 50 ml H20) in order to adsorb residual tritium-labeled tryptamine. Tritiated water remains in the aqueous phase. The samples are vortexed for 1 min in an Eppendorf 5432 mixer and centrifuged for 5 min at 10,000 g (Eppendorf centrifuge 5412). The clear supernatant, 200/zl, is added to 5 ml of scintillation cocktail (Rotiszint 22), and the radioactivity determined in a scintillation counter. Under these conditions 1 pkat of enzyme released 4000 dpm of tritium into the ambient aqueous reaction mixture. Aliquots of the Sepharose matrix were assayed as described and the release of 3HOH quantitated. Enzyme Preparation. The enzyme of C. roseus purified to homogeneity in our laboratory 8 usually has a specific activity of 105 nkat per milligram of protein. However, the methods described below for the isolation of strictosidine synthase provide protein preparations with enzyme activities adequate enough for immobilization. These enzyme preparations have a specific activity of either 1.5 nkat per milligram of protein after the gel filtration step, or 46 nkat per milligram of protein after further purification by hydrophobic chromatography. Enzyme Isolation Frozen tissue, I kg, is stirred in 2 liters of 100 m M potassium phosphate buffer (pH 7.0), containing 20 mM mercaptoethanol. The slurry is filtered through cheesecloth and the filtrate centrifuged (20 min, 27,000 g). The supernatant is adjusted to 35% saturation of ammonium sulfate, stirred for 30 min, and centrifuged. Ammonium sulfate is again added to 9 j. Souzu and H. Mitsuhashi, Tetrahedron Lett. 2, 191 (1970). l0 E. M. Linsmaier and F. Skoog, Physiol. Plant. 18, 110 (1965).

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the clear supernatant to give 50% saturation. After 30 min the suspension is centrifuged, and the supernatant discarded. The sediment is resuspended in 86 ml 50 m M potassium phosphate buffer (pH 7.0), containing 20 m M mercaptoethanol, and dialyzed against a 100-fold excess of this buffer. After dialysis the insoluble proteins are removed by centrifugation. Glycerol is added to the supernatant to a final concentration of 5%. The solution is then subjected to gel filtration on an AcA 44 column (5 × 100 cm; flow rate 80 ml/hr). One hundred sixty fractions of 16 ml each are collected. The strictosidine synthase-containing fractions (70-92; 355 ml) are pooled and concentrated by pressure dialysis (Berghof, Tiibingen, Typ BMU) to a final volume of 8.2 ml. This procedure allowed 98% of the enzyme activity to be recovered with 17-fold enrichment (total of 165 mg protein; 255 nkat). This preparation is already free of interfering enzyme activities. Further purification of the enzyme could be achieved if necessary, by the following steps. The enzyme preparation of the AcA 44 step (8 ml) is passed through a Matrex Gel Red A column (1.5 × 16 cm, flow rate 10 ml/ hr) equilibrated with 10 mM phosphate buffer (pH 7.0). Under these conditions the synthase is not adsorbed to the matrix and recovered quantitatively in the first 80 ml. The enzyme-containing fraction is dialyzed against 25 mM citrate phosphate buffer (pH 4.5) (10 liters), overnight. The turbid solution is clarified by centrifugation (15 min; 48,000 g), and solid ammonium sulfate is added to give 25% saturation at 0°. The enzyme is now applied to a Matrex Gel Blue A column (0.8 × 4.5 cm, flow rate 5 ml/hr) equilibrated with 25 mM citrate phosphate buffer (pH 4.5) containing 25% (NH4)~SO4. Under these conditions the enzyme is adsorbed to the column. The column is then washed with equilibration buffer until no protein is detectable in the effluent. The enzyme is eluted from the column with 10 m M potassium phosphate buffer (pH 7.0). Fractions of 1.5 ml each are collected, and the enzyme is recovered in fractions 27-30. The enzyme-containing eluate (6 ml) is dialyzed against 10 mM phosphate buffer (pH 7.0) and pressure-dialyzed to a final volume of 2.2 ml. This procedure yielded a 500-fold enriched preparation containing 46 nkat enzyme activity per milligram of protein with a yield of 40%. The purification procedure is summarized in Table I. The enzyme can be stored under frozen conditions at - 2 0 ° for periods of up to 1 year without appreciable loss of catalytic activity. Preparation of Immobilized Enzyme The above protein preparation, 3.3 ml, containing about 103 nkat synthase, was dialyzed for 12 hr against an excess of 0.1 M NaHCO3 buffer

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[31]

ORGANIC SYNTHESIS

TABLE I PURIFICATION OF STRICTOSIDINE SYNTHASE FROM I KG SUSPENSION CULTURED C. r o s e u s CELLS

Purification step Crude extract Ammoniumsulfate (35-50%) Gel filtration (AcA 44) Matrex Gel Red A Matrex Gel Blue A

Volume (ml)

Total Specific PurificaProtein activity activity Yield tion (rag) (nkat) (nkat/mg) (%) factor

2430 86

2781 1466

259 258

0.09 0.18

100 100

1 2

165

255

1.5

98

17

254 107

8.4 46.4

98 41

90 499

8.2 24 2.2

30 2.3

(pH 8.5) containing 0.5 M NaCI. CNBr-activated Sepharose, 2 g, was washed with 400 ml of 1 mM HC1 and the acid was removed by suction. The gel was washed with water and suspended in the above enzyme solution. The coupling reaction was carried out for 2 hr at room temperature with slow agitation of the enzyme-Sepharose mixture. The gel was subsequently collected on a filter and washed with 3 gel volumes of 0. I M NaHCO3 buffer (pH 8.5) containing 0.5 M NaCI. The gel was then resuspended in 16 ml of 0.1 M Tris-HC1 (pH 8.0) and again stirred slowly for a period of 2 hr at room temperature. The supernatant was removed and the gel was washed in 3 gel volumes each of 0.1 M NaHCO3 buffer (pH 8.5) and then 0. I M citrate buffer (pH 3.8), each containing 0,5 M NaCI. The washing procedure was completed with 3 gel volumes of 0.1 M phosphate buffer (pH 6.5). The amount of strictosidine synthase bound to the Sepharose was determined by assaying the enzyme activity in an aliquot of the gel suspended in 0.1 M phosphate buffer. On average 40% of strictosidine synthase activity was found to be coupled to the matrix. The immobilized enzyme was stored at 4°, and 0.02% sodium azide was added to prevent microbial growth. The activity of the immobilized enzyme was determined by incubating a known amount of enzyme (usually 10 pkat) bound to the matrix with 3Hlabeled tryptamine and secologanin at pH 6.5 and measuring the release of 3HOH. The pH profile of the synthase activity is shown in Fig. 2. The immobilized synthase shows a broad pH profile with a distinct optimum at pH 6.4, which is slightly shifted to the more acidic region of the soluble enzyme's pH optimum at pH 6.8. The soluble enzyme is active at pH 4.0 only to about 4% and at pH 8.0 only to about 15% of its maximal activity.

[31]

347

STR]CTOSIDINE SYNTHASE

100 Immobilized enzyme j

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pH FIG. 2. The effect of pH on the activity of immobilized (closed symbols) and soluble (open symbols) strictosidine synthase. The assay was conducted in the presence of 0.1 M citrate phosphate (A, A), potassium phosphate ( 0 , O), and borate (ll, F-I) buffer, respectively, under standard conditions.

In contrast, the immobilized enzyme shows still about 23% of its maximal activity at pH 4.0 and 52% activity at pH 8.0. This demonstrates that the immobilized enzyme tolerates pH shifts much better than the soluble enzyme. The influence of the secologanin concentration on the activity of the immobilized strictosidine synthase was determined by double-reciprocal plots of velocity against substrate concentration. A linear relationship was observed over a range of 1.2 to 48 mM. The apparent Km value was 2.1 mM and Vmaxwas 9.1 pkat/mg. An apparent Kmof 3.4 mM and Vmaxof 55 pkat/mg for the soluble enzyme has been found previously. 6 With tryptamine as a substrate an apparent Km value of 0.9 mM and Vmax of 18.9 pkat/mg were observed, while the soluble enzyme showed an apparent Km of 2.3 mM and Vmaxof 130 pkat/mg. The comparison of the Km values indicates an equal or even slightly increased affinity for either substrate. Vmaxvalues are obviously lower with the immobilized enzyme, and this may indicate a conformational change of the protein molecule attached to the matrix. A substrate inhibition at tryptamine concentra-

348

[31]

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

tions exceeding 1 m M had previously been observed with the soluble enzyme 6 and was also found with the immobilized synthase, although this inhibition was less pronounced. While 2.5 mM tryptamine produces 46% inhibition of the soluble synthase, the inhibition of the immobilized enzyme is only 17%. As compared with the soluble enzyme, the immobilized strictosidine synthase is remarkably thermostabile. As depicted in Fig. 3 the immobilized enzyme has a half-life of about 68 days at 37 °. About 6% of its activity can still be observed after more than 1 year at that temperature. The half-lives of the partially purified soluble enzyme and protein extract (0-70% ammonium sulfate precipitation) under identical conditions are only 5 hr approximately. When the immobilized enzyme is stored at 4 ° in the presence of 0.02% NAN3, the preparation retains 100% of its initial activity after a period of more than 3 years. In all cases discussed above, strictosidine was shown by chromatographic means to be the sole reaction product formed by the immobilized enzyme from secologanin and tryptamine as substrates. The stability of the immobilized enzyme is therefore increased about 325-fold. As described below, this property

,oo

"~

I0

Time ( hours )

Time ( days )

!

b

'

1000 Time ( hours )

'

2000

FIO. 3. Stability of immobilized ( 0 ) and soluble (©) strictosidine synthase. The enzyme was kept at 37° in the presence of 0.g2% sodium s i d e . An aliquot was removed from each sample at times indicated, washed free from azide, and assayed for activity under standard conditions. The half-life of immobilized strictosidine synthase was found to be about 68 days, whereas the half-life of the soluble enzyme was 5 hr.

[31]

STRICTOSIDINESYNTHASE

349

makes the enzyme an excellent tool for the preparative synthesis of the common intermediate in the biosynthesis and biomimetic synthesis of indole alkaloids, i.e., strictosidine. Preparative Synthesis of Strictosidine A water-jacketed glass column (1 x 6 cm) was filled with 2 g of Sepharose containing about 40 nkat of bound strictosidine synthase, and the gel was washed thoroughly with water. Aqueous unbuffered 15 mM solutions of tryptamine- HCI and of secologanin were prepared in separate flasks. The substrates were adjusted with acid or alkali to pH 6.5. Both solutions were pumped separately onto the column each at a speed of 3 ml/hr and a column temperature of 37°. The solutions were mixed in the column head and subsequently passed through the catalyst. This precaution was taken in order to prevent any chemical condensation of the reactants, which would yield both stereoisomers, i.e., 3a(S)-strictosidine and 3B(R)-vincoside. The column eluate was collected in fractions of 72 ml and checked chromatographically for product formation. In addition, the condensation reaction was monitored by pulsing with 14C-labeled tryptamine. Under these conditions substrates were transformed to more than 95%. The column was continuously run at 37° for a period of 12 days without any changes in the reaction rate. The freeze-dried column eluate yielded a total of 6 g of strictosidine. The chemical identity and optical purity of strictosidine formed by the immobilized enzyme were confirmed by acetylation of the product and subsequent UV, NMR, MS, IR, and CD spectroscopy. H The compound is stereochemically pure and all physical data are in absolute agreement with the authentic compound) General Discussion Strictosidine synthase is the first enzyme involved in secondary metabolism of plants that has been immobilized successfully. The method described for the synthesis of strictosidine has several distinct advantages. Since the immobilized enzyme is quite stable, one can accumulate relatively large amounts of the glucoalkaloid strictosidine by operating the column continuously; on the other hand, one can put the column in a refrigerator after one run and use it subsequently for another preparation of either labeled or unlabeled strictosidine. This procedure has been performed in this laboratory for a period of up to 3 years with the same n U. Pfitzner and M. H. Zenk, Planta Med. 46, 10 (1982).

350

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

[31]

column and without loss of catalytic activity of the immobilized enzyme. This column offers an additional advantage in that it can be utilized for the synthesis of the versatile biogenetic precursor molecule strictosidine labeled in particular atoms with either a radioactive or a stable isotope. Furthermore, the material labeled with isotopes can be converted to the alkaloid precursor with very high concentrations of 15N or ~3C, which make these compounds amenable to modern spectroscopic studies such as nuclear magnetic resonance. Strictosidine synthesized from tryptamine and secologanin in this way can therefore be used as a synthon for known J2 or yet unknown chemical and biological transformations to give products of academic and pharmacological interest.

12 R. T. Brown, in "lndole and Biogenetically Related Alkaloids" (J. D. Phillipson and M. H. Zenk, eds.). Academic Press, London, 1980.

[32]

OVERVIEW

353

[32] O v e r v i e w

By S. GESTRELIUS and K. MOSBACH This section will provide the reader with information on immobilized enzyme/cell systems used in industry. Some of the processes now used worldwide have already been presented in Volume 44 of Methods in Enzymology. In Table I we have listed all processes, including those presently being tested on a pilot scale, that to the best of our knowledge are in use today. Relevant references are also provided as are names of some of the companies producing the catalysts:or applying these processes. The reader is also referred to Section II on the use of enzymes/cells in organic synthesis as a number of the examples given in this section will probably be implemented on an industrial level in the near future. In addition, in Volume 137, Section III, some related examples can be found, notably on water purification using immobilized microorganisms. Worth mentioning in this general context may also be the removal of raffinose and stachyose in sugar beet processing using "immobilized" pellets of Mortierella vinacae I and the traditional vinegar process in which cells immobilize naturally to birch twigs. This "industrial" section is heterogeneous in nature. In some cases, complete detail of the methodology is not given, probably because of "proprietary" considerations. Whenever possible the aim has been to describe not only immobilization methods but also methods (and problems) of integrating immobilized enzyme/cell-catalyzed reactions into process flow schemes. Comments on the importance of sterility are found in some of the contributions. The increasingly interesting area of mammalian cell culture technology using microcarriers or other immobilized systems, including entrapment, hollow fiber systems, and ceramics, is not included in this section and only briefly treated in Section II, Volume 135. Because of its rapid development it deserves to become the subject of a special volume in this series. In spite of problems with industrial plants reluctant to reveal their methodologies (understandably though), we hope that this section will provide the reader with an overview and will act as a stimulus for other workers in the field. We would like, in this context, to refer the reader to a recent review article by R. D. Schmid on biotechnology in Japan, which appeared in Appl. Microbiol. Biotechnol. 24, 355365, 1986. This review lists immobilized enzyme/cell systems used in Japan. J. O b e r a , S. H a s h i m o t o , a n d H. S u z u k i , Sugar Technol. Rev. 4, 209 (1976/1977).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

354

[32]

E N Z Y M E E N G I N E E R I N G ( E N Z Y M E TECHNOLOGY)

e¢-

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[32]

OVERVIEW

355

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356

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

The first paper presented in this section is one by V. Jensen and S. Rugh, which summarizes 10 years of experience with industrial-scale production and the application of immobilized glucose isomerase. A more general discussion on the industrial application of immobilized enzymes by M. J. Daniels follows. Subsequently a number of contributions are presented.

[33] I n d u s t r i a l - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Glucose Isomerase By VILLY J. JENSEN a n d SUSANNE R U G H

The development of the high-fructose corn syrup process is the story of how process engineering bridged the gap between the enzyme manufacturers and the corn syrup industry in the years around 1974. Cheap soluble enzymes had been known and used by corn syrup producers for decades. However, the industry was not familiar with the application of expensive enzymes which had to be reused and protected from deactivating components in the syrup to achieve an economic process. The enzyme producers, on the other hand, were equally unfamiliar with the production of reusable enzymes for use in such large-scale manufacturing processes. The actual development of today's immobilized glucose isomerase products and application process has therefore been a complex feedback process between enzyme producers and high-fructose corn syrup (HFCS) producers. Better immobilized glucose isomerase (IGI) products allowed improvements in the isomerization process, leading to further development of more sophisticated IGI products. An outline of the different immobilization methods used for glucose isomerase products together with an evaluation of the product characteristics obtained by the various methods is given here. Also the development of the methods used in the isomerization process and the mutual influence of product and process development are described. The glucose isomerase characteristics and the kinetics of the glucose isomerization reaction are well described elsewhere 1,2 and are not dealt with in this paper. The description of products and methods is based on literature, information from the manufacturers, and the authors' experience. It is, however, not necessarily a description of the exact methods used by manufacturers of industrial products, as such information is not available from all manufacturers. W.-P. Chen, Process Biochem. June/July 30 and 36 (1980). 2 j. A. Roels and R. van Tilburg, Starch/Staerke 31, 17 (1979).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

356

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

The first paper presented in this section is one by V. Jensen and S. Rugh, which summarizes 10 years of experience with industrial-scale production and the application of immobilized glucose isomerase. A more general discussion on the industrial application of immobilized enzymes by M. J. Daniels follows. Subsequently a number of contributions are presented.

[33] I n d u s t r i a l - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Glucose Isomerase By VILLY J. JENSEN a n d SUSANNE R U G H

The development of the high-fructose corn syrup process is the story of how process engineering bridged the gap between the enzyme manufacturers and the corn syrup industry in the years around 1974. Cheap soluble enzymes had been known and used by corn syrup producers for decades. However, the industry was not familiar with the application of expensive enzymes which had to be reused and protected from deactivating components in the syrup to achieve an economic process. The enzyme producers, on the other hand, were equally unfamiliar with the production of reusable enzymes for use in such large-scale manufacturing processes. The actual development of today's immobilized glucose isomerase products and application process has therefore been a complex feedback process between enzyme producers and high-fructose corn syrup (HFCS) producers. Better immobilized glucose isomerase (IGI) products allowed improvements in the isomerization process, leading to further development of more sophisticated IGI products. An outline of the different immobilization methods used for glucose isomerase products together with an evaluation of the product characteristics obtained by the various methods is given here. Also the development of the methods used in the isomerization process and the mutual influence of product and process development are described. The glucose isomerase characteristics and the kinetics of the glucose isomerization reaction are well described elsewhere 1,2 and are not dealt with in this paper. The description of products and methods is based on literature, information from the manufacturers, and the authors' experience. It is, however, not necessarily a description of the exact methods used by manufacturers of industrial products, as such information is not available from all manufacturers. W.-P. Chen, Process Biochem. June/July 30 and 36 (1980). 2 j. A. Roels and R. van Tilburg, Starch/Staerke 31, 17 (1979).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[33]

IGI INDUSTRIAL-SCALEPRODUCTION-APPLICATION

357

The immobilization methods described are selected so that the sequence of products obtained by these methods illustrates the development of industrial IGI products during the past l0 years. It is possible to reproduce most of the products in a well-equipped biochemical laboratory. In many cases, alternative glucose isomerase-producing microorganisms can replace those mentioned in the descriptions. It is the hope of the authors that many readers will actually perform the trials. Development of Products and Processes Initial P h a s e

The first patent, "On Production of Fructose from Glucose through the Action of Xylose Isomerase," was issued to Marshall 3 in 1960, but 14 years were to elapse before a really large industrial exploitation of the idea was developed. The basic research was carried out in Japan in the 1960s by Takasaki et al. 4 who also patented the first partially reusable glucose isomerase, 5 which was produced according to the following method. A S t r e p t o m y c e s strain (e.g., S t r e p t o m y c e s w e d m o r e n s i s ATCC 21230) is inoculated in 500 ml of a medium containing 3% wheat bran, 2% corn steep liquor, and 0.024% COC12, pH 7. After shaking for 24 hr at 30°, the broth is heat treated at 65 ° for 15 min, and the "immobilized" glucose isomerase is recovered by filtration as the enzyme has become fixed within the cells by the heat treatment. The total activity is sufficient to isomerize 150 ml of 50% glucose solution (70°, pH 5.0-7.5) to a conversion of 48% fructose in 24 hr. After isomerization the cells are recovered by centrifugation and reused in flesh glucose solutions. The activity drops by one-third in about 100 hr, corresponding to an activity half-life of approximately 170 hr. This includes both physical loss of enzyme activity and enzyme inactivation. The product was used in small industrial scale by Clinton Corn Processing Company, a division of Standard Brands, United States, from 1967 until about 1970. The isomerization was initially performed in a batch reactor. Other cell paste products, for instance the flocculated A r t h r o b a c t e r cells patented by the R. J. Reynolds Tobacco Company,6 were also industrially utilized in the initial phase of the development of glucose isomerase. The batch isomerization process with heat-fixed cells was carried out 3R. O. Marshall, U.S. Patent 2,950,288 (1960). 4 y. Takasaki, Y. Kosogi, and A. Kanbayashi, Ferment. Adv. Pap. Int. Ferment. Syrup., 3rd, 561 (1969). 5Agencyof Industrial Scienceand Technology,Japanese Patent Application 27,525 (1965); British Patent 1,103,394(1968). 6R. J. ReynoldsTobacco Company, U.S. Patent 3,645,848 (1972).

358

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

at 60-70 °. The syrup concentration was 40-45% (w/w), and the normal isomerization time was about 20 hr. Because of the long residence time, the pH had to be kept at 6.5-7.0 in order to decrease by-product formation. Approximately 10 mM of Mg2+ was added as enzyme activator. Unfortunately, the low pH necessitated the presence of about 1 mM of Co 2+ in the syrup in order to stabilize the enzyme. This was undesirable from a food approval point of view, and the Co 2+ had to be removed from the syrup after isomerization. The physical appearance of this first industrial glucose isomerase was a soft cell paste. The heat fixation stabilized the cells to some extent because the proteases in the cells were destroyed, and because the solubility of the cell components was decreased. However, the loss of cells, solubilized enzyme, and solubilized cell components to the syrups was inevitable in a batch process with this product. The instability of the product and the long residence time in the isomerization process gave rise to a significant by-product formation in the syrup. These impurities had to be removed from the syrup together with the added Mgz+ and Co 2+ by tedious filtration, ion exchange, and carbon treatment. In addition, the recovery of the cells from the batch isomerization process was difficult in industrial scale.

Industrial Development The First Continuous Isomerization Process. Clinton's work with the labor-demanding and difficult batch isomerization process with heat-fixed cells was soon substituted by a continuous process. In 1970, Lloyd et al. 7 developed a continuous process with the enzyme product in a pressurized leaf filter. A slurry of the glucose isomerase cell paste was pumped into a leaf filter, and each leaf was coated with a 2.5- to 3.5-cm layer. After coating, glucose solution was pumped continuously through the filter at a flow rate which allowed a 45% conversion of glucose to fructose. The other process parameters in the leaf filter isomerization were essentially the same as in the batch process, and Co 2+ in the syrup was still necessary. The loss of active, insoluble enzyme was avoided by the leaf filter process with the heat-fixed cells, but the leakage of soluble components from the heat-fixed cell product to the syrup was still a problem. Purified Reusable Enzyme. The success of fully launching fructose syrup and the problems associated with producing it by means of the heatfixed cells soon resulted in the development of pure reusable enzyme products. In 19708 the Clinton Corn Company patented an improved prod° 7 N. E. Lloyd, L. T. Lewis, R. M. Logan, and D. N. Patel, U.S. Patent 3,694,314 (1972). 8 K. N. Thompson, R. A. Johnson, and N. E. Lloyd, U.S. Patent 3,788,945 (1974).

[33]

I G I INDUSTRIAL-SCALE PRODUCTION--APPLICATION

359

uct, using the following methodology. Streptomyces rubiginosus ATCC 21175 is grown in submerged, aerobic culture. The cells are harvested by filtration. One kilogram of filter cake is slurried in 5 liters of deionized water with 5 mM of Co 2+ and 8 g of a cationic detergent. After stirring at 60 °, pH 6.7-6.8 for approximately 4 hr the cells are disrupted, and the cell debris is removed by filtration. The glucose isomerase containing supernatant is concentrated under vacuum. The enzyme concentrate contains some inactive material which can bind to DEAE-cellulose. This inactive material would decrease the activity of the reusable enzyme complex. Therefore, the impurities are removed by adsorption to a small amount of DEAE-cellulose at conditions which will not bind the enzyme to the DEAE-cellulose. The enzyme in the purified concentrate is then adsorbed onto DEAE-cellulose by mixing further DEAE-cellulose and enzyme at proper conditions. The DEAE-cellulose is thoroughly washed with water in order to remove fine particles before using it for enzyme adsorption. An amount of 6.25 g of moist filter cake can initially isomerize 6.5 g of glucose per hour to 49.6% conversion in a small column at 45% (w/w) substrate concentration, pH 6.5, temperature 60°. The activity halflife is around 200 hr. This purified glucose isomerase replaced the heat-fixed cells in Clinton's filter press reactor. The process parameters were the same, and Co z÷ was still needed. The adsorbed, purified glucose isomerase product represented a major improvement. The enzyme loading was high, and the syrup contamination with inactive, solubilized cell material was avoided. However, the glucose isomerase product was still a very soft gel. An absorbed enzyme system is extremely susceptible to changes in process conditions, even though the enzyme in the leaf filter press can be utilized for several hundred hours. Therefore, a truly fixed enzyme was needed. Cross-Linked Glucose Isomerase for Batch Isomerization. Glutaraldehyde cross-linked glucose isomerase has been available in industrial scale since 1974, and is still widely used by HFCS manufacturers. Novo Industri A/S produces the product Sweetzyme in accordance with the method developed by Amotz et al.9: Cells of Bacillus coagulans NRRL B 5636 are cultivated in a standard fermentation medium containing xylose. A cell concentrate is produced by centrifuging the fermentation broth at 10° to give a sludge containing approximately 12% dry matter. The concentrate is left at pH 7.9, 20 ° for 3 hr with mild stirring. To 1 kg of sludge is then added 40 ml of a 50% glutaraldehyde solution. After 1 hr the mixture has gelled into a coherent mass. The gel is broken up mechanically, diluted with one volume of water, and flocculated with a cationic flocculant to give a clear water phase. The mixture is filtered, and the filter cake is 9 S. Amotz, T. K. Nielsen, and N. O. Thiesen, Belgian Patent 809,546 (1974).

360

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

ventilated with air to remove some of the free water. The filter cake is granulated on a 1-mm screen and dried in a fluid bed with an inlet air temperature of 50 ° (drying at room temperature can be used instead of fluid bed drying). The dried product is ground and sieved to 100-350/zm particles. The activity recovery from the sludge is 50-60%. An amount of 10-15 g of the obtained product can isomerize 1 kg of glucose solids to 45% conversion in 20 hr when the substrate concentration is 40% (w/w), pH 6.5-7.0, temperature 60-65 °. The physical properties of the cross-linked, dried glucose isomerase were significantly better than the physical properties of the cell paste and DEAE-cellulose products, but the pressure drop stability was still insufficient for fixed bed operation, The cross-linked enzyme particles could be recovered from a batch process simply by allowing the particles to settle and decanting the isomerized syrup. The sedimentation was carried out in the batch reactor when the desired degree of conversion had been achieved. The isomerized syrup was then drawn off. The enzyme particles were kept covered by a protective layer of syrup, as the enzyme was susceptible to oxygen at the isomerization temperature. Fresh glucose syrup was added after drawing off the isosyrup. About 5% of the initial activity was lost per isomerization. Therefore, in order to have a constant isomerization time, the activity loss was compensated for by the addition of fresh enzyme after each isomerization. After about 40 batches, the enzyme had to be discarded due to a too high proportion of particles to liquid. The addition of Co 2+ and Mg 2+ to the syrup was still necessary. Typical process cycle durations are given in Table I. The product could also have been used in the filter press continuous process, but this process never became widespread in the syrup industry. In the years of 1974-1976, most isosyrup was produced by a batch process. The standard HFCS syrup has - 7 0 % syrup dry substance (DS), 42% fructose, 54% glucose, and 4% dextrins. The global HFCS production was 285,000 tons DS in 1974 increasing to 890,000 tons in 1976. Although the process had been significantly improved by the use of cross-linked products, it was still tedious to run a batch process, and it TABLE I CYCLE DURATION FOR BATCH ISOMERIZATION Cycle step

Duration (hr)

Isomerization Settling Settling and pump out Filling with fresh syrup

20 1 8 8

[33]

IGI INDUSTRIAL-SCALEPRODUCTION-APPLICATION

361

was evident that products for very-large-scale continuous processes had to be developed. Immobilized Glucose lsomerase for Fixed Bed Operation. Several glucose isomerase products for fixed bed operations were developed by enzyme manufacturers in the period 1974-1976. Not all of these parallel developments are well documented in the literature, but can be exemplified by the development of Sweetzyme, which is produced by Novo Industry A/S. The development process was described by Hemmingsen 1° and Carasik and Carroll.11 In the early phase of this development, a product for continuous fluid bed operation was developed by McMullen and Carasik ~2in an attempt to overcome the lack of long-range physical stability of the earliest Sweetzyme product for batch isomerization. The industrial-scale experiments, however, turned out negatively. It was impossible to achieve a stable, even flow rate distribution over the reactor cross section, and the fluid bed concept was abandoned. Sweetzyme for fixed bed operations was principally produced in accordance with the method of Sweetzyme for batch operations with a few essential modifications. Before cross-linking the cells are disrupted by pumping the cell sludge through a Manton Gaulin homogenizer with a single-stage homogenizing valve assembly. The pressure drop over the valve is 300-350 kg/cm 2. After the cross-linking and filtration process, the moist, cross-linked aggregate is extruded by means of an axial extruder with a 0.8-mm screen and dried in a fluid bed dryer. The particle fractions 400-1000/xm are used for fixed bed operations. The recovery of apparent activity is approximately 50%. The activity level and physical properties are summarized in Appendix I. Recently, an improved Sweetzyme has been developed. The immobilization method is essentially the same, but the glucose isomerase is produced by a selected strain of Streptomyces murinus. The immobilized glucose isomerase products developed and marketed by Miles (Takasweet) and Godo Shusei are principally produced in accordance with the method of Sweetzyme, i.e., the particle shaping is performed after immobilization. However, the microorganisms used are different, and there are also differences in some of the unit operations used during immobilization. The product developed and marketed by Gist Brocades (Maxazyme) is produced by a different process as patented by van Velzen 13 and de10 S. H. Hemmingsen, "Applied Biochemistry and Bioengineering," Vol. 2, p. 157. Academic Press, New York, 1979. 11 W. Carasik and J. O. Carroll, Food Technol. Oct., 85 (1983). 12 W. H. McMullen and W. Carasik, U.S. Patent 4,138,290 (1976). J3 A. G. van Velzen, U.S. Patent 3,838,007 (1972).

362

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

scribed by van Tilburg14: Actinoplanes missouriensis (NRRL B-3342) cells containing glucose isomerase are produced by submerged fermentation under aerobic conditions. After achieving the desired activity, the pH is adjusted to 8.6, and the temperature is raised to 72° in order to kill the microorganisms. The cells are recovered by centrifugation. Gelatin (in solution?) is added to the cell sludge at a temperature slightly above 40 °. The final gelatin concentration is 8% (w/v). After deaeration in a vacuum vessel, the mixture at 40 ° is sprayed into butylacetate at 6°. By this procedure, small spherical gelatin/cell particles are formed. The spheres solidify because of the low temperature, but in order to obtain particles that are sufficiently rigid in the isomerization process, the particles are crosslinked with glutaraldehyde in a concentration of 2.5% (w/v) at 10-12 °. Excess glutaraldehyde and soluble impurities are removed by several water washings. The immobilized glucose isomerase particles are then transferred into a 25% propylene glycol/water solution (preservative), drained, packed, and stored. Also, the product developed by Nagase/Denki Kagaku Kogyo, (Sweetase) differs from the traditional cross-linked products in that the enzyme-containing cells are bound to an ion-exchange resin without glutaraldehyde cross-linkinglS: Streptomyces phaechromogenes is cultivated by aerobic fermentation at 30° in a medium containing xylan extract, corn steep liquor, and salts. The culture broth is then heat treated in order to avoid autolysis and enzyme leakage from the mycelium. The mycelia are collected by filtration. The enzyme-containing mycelia are then immobilized with a water-insoluble anion-exchange resin. The anion-exchange resin contains quaternized nitrogen in pyridine rings. After immobilization, dehydration, and granulation, the granulated enzyme is dried at 60 ° for 3 hr. The main characteristics of the most important glucose isomerase products for fixed bed operation are summarized in Appendix I of this chapter. The data are based on literature, brochures, and personal communications. A strictly scientific comparison of the products is impossible as data for different products refer to slightly different application conditions, and as "optimal conditions" for a given product depend on the factor optimized (see for instance Table III for different optimizations of Sweetzyme). Comparison of Batch and Fixed Bed Processes. Table II summarizes the main differences in the isomerization parameters between the batch process and the plug flow process at the time when the Sweetzyme product for fixed bed operation was introduced into the market. The advan14 R. van Tilburg, Ph.D. Thesis, Delft University of Technology, The Netherlands (1983). J5 Japanese Patent Application 49-19082, corresponding to U.S. Patent 3,915,797.

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TABLE II COMPARISON BETWEEN BATCH AND PLUG FLOW PROCESSES

Parameter

Batch process (1974-1976)

Plug flow process (1976)

Syrup concentration pH Temperature Co 2+ Mg 2+ R e s i d e n c e time Half-life of e n z y m e Productivity

40-45% w / w 6.5-7.0 65 ° 3.5 × 10 -4 M 20x (Ca 2+) 20 hr 300 hr - 9 0 0 kg a

40-45% w / w 8.4 (column inlet) 65 ° -20x (Ca 2+) 0 . 5 - 4 hr 500 hr - 1 5 0 0 kg a

Calculated as kilogram of syrup DS per kilogram of enzyme. tages of the plug flow process in comparison with the batch process are obvious. The immediate productivity increase was substantial, so the enzyme cost of conversion was nearly halved even before the strict optimization of the plug flow process. Co 2+ was no longer needed for enzyme stability, and by-product formation decreased considerably because of the short residence time in the column. A detailed description of the process design for the plug flow process and an analysis of its merits are given by Poulsen and Zittan in Vol. 44 of this series.16

Process Optimization 1976-1979 was a period of increase in IGI and isosyrup production capacity. Much effort was given to the optimization of both the IGI manufacturing process and the isomerization process. Optimization of isomerization process concentrated on feed purity as well as on isomerization parameters. The purity of the glucose substrate is a critical factor for the enzyme activity and stability. A saccharified corn starch glucose syrup contains both particulate matter and soluble impurities such as amino acids, peptides, lipids, and ions. Many of these naturally occurring impurities are very damaging to the IGI. An insufficiently purified glucose syrup can reduce the IGI productivity to 50% or less of that obtained with a purified syrup. Particulate matter in the feed syrup may cause clogging of the columns and should therefore be removed by centrifugation and filtration. Soluble impurities, such as amino acids, peptides, and certain metal ions, are enzyme poisons and inhibitors, and should be removed by carbon treat~6p. B. R. Poulsen and L. E. Zittan, this series, Vol. 44, p. 809.

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ment and ion exchange. Ca 2+ is a naturally occurring component in starch syrups and a powerful glucose isomerase inhibitor, but many other metal ions also have a negative influence on the activity. The normal purification process comprises carbon treatment with powdered or granular carbon, as described by Herves and Bailley, ~7followed by an ion-exchange process and Mg 2÷ addition. The ion-exchange process consists of a strong cation exchanger and a weak anion exchanger as described by the suppliers of ion-exchange resins. The efficiency of the carbon treatment and the ion-exchange processes is checked by measuring the UV absorption and conductivity. A small amount of Mg z- is added before isomerization because it is a powerful activator, and because it can prevent the inhibitory effect of Ca 2÷ if the Mg 2÷ is in 10- to 20-fold surplus. The work with optimization of the isomerization parameters has been concentrated on adjusting the process to capacity, economy, and syrup quality. The production capacity is usually adapted by adjusting isomerization temperature and age of the enzyme. When a high syrup production capacity is needed, the isomerization temperature is increased to about 63 °, and old low-activity enzyme is replaced by new high-activity enzyme. The lowest cost of conversion is obtained by running at a low temperature, i.e., down to 56-57 °, and by utilization of the enzyme until only 10% residual activity remains. The quality of the syrup has been improved by lowering the isomerization pH as a low pH reduces the formation of color and by-products. Today the isomerization process is run according to the "bottleneck" model, and Table III gives a comparison between Sweetzyme used in the original and the present plug flow processes. The process optimizations have allowed a productivity increase from about 1500 kg of syrup dry substance (DS) per kilogram of enzyme up to about 5000 kg DS/kg of enzyme. The IGI products supplied by the various manufacturers have different activities per kilogram of IGI product, but the IGI prices are adjusted so that the cost of conversion follows the market price for isomerization. The cost of conversion is roughly 10-20 cents per 100 lb of isosyrup dry substance. The global isosyrup production in 1977 was about 1.2 million tons dry substance and has been steadily increased to about 6.7 million tons in 1984. Second Generation Isomerization Processes and Products

55% Fructose Corn Syrup In 1978 a new development took place in the corn syrup market when 55% high-fructose corn syrup became available. This product has a sweeti7 D. V. Herves and C. Bailley, Starch 12, 422 (1977).

[33]

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ness equivalent to sucrose and soon gained a large market share, especially in the soft drinks industry. The product was made by a new fractionation technology involving chromatographic separation of fructose from glucose. Fifty-eight percent of the global isosyrup production in 1986 was 55% fructose syrup. The separation is based on fructose adsorption on either zeolites or calcium salts of a strong acid cation-exchange resin. Water is used as eluent in the industrial process. The fractionation techniques are described in detail in the literature. 18-21The fractionation is very simple, but uneconomic to run as a batch process. It is, however, very complicated to run as an economic continuous process. The process zones are constantly moving through the columns, so the columns have several inlet and outlet valves. The process has been computerized in order to achieve optimal process control.

New Glucose Isomerase Products The process optimization was followed by the development of a new generation of IGI products. The activity level was increased by enzyme purification, and a sufficient physical stability was achieved by utilization of hard, inert carriers. The glutaraldehyde cross-linking has generally been maintained. These new products have only gained a very small market share in 1984 although at least one of the products has been available since 1980. In the period of 1979-1980, UOP Inc. developed a new IGI product. The product, Ketomax GI-100, is based on the method patented by Rohrbach 2z and described in the GRAS petition submitted to the FDA. No details are available on the exact manufacturing process, but the principle of the method is as follows. Streptomyces olivochromogenes, a mutant of ATCC 21114, is inoculated into a sterile, xylose-containing fermentation medium, and glucose isomerase-containing cells are produced under submerged, aerobic conditions. The fermentation liquid will contain about 125 g of cell dry matter per liter. The cells are recovered by centrifugation, lysed by lysozyme treatment, and filtered to remove cell debris. The enzyme is precipitated in the presence of 2-butanol, recovered by centrifugation, and redissolved in water. A ceramic alumina carrier with a particle size of 180-250/xm is treated with 6 ml of 2.5% polyethyleneimine per gram of carrier for 1 hr. Excess liquid is decanted, and the carrier is dried t8 H. t9 H. 20 H. 2J H. 22 R.

J. Biezer and A. J. de Resset, Starch 29, 392 (1977). Odawara, M. Okuso, and T. Yamazaki, U.S. Patent 4,157,267 (1979). Ishikawa, H. Tanabe, and K. Usui, U.S. Patent 4,182,633 (1980). W. Keller, A. C. Reents, and J. W. Larawey, Starch 33, 55 (1981). P. Rohrbach, U.S. Patent 4,268,419 (1981).

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to a water content of about 7%. The dried carrier is treated with a 25% (w/w) glutaraldehyde solution, 18 ml of solution per gram of carrier. After 1 hr excess glutaraldehyde is removed by washing in water. The activated carrier is then shaken with purified enzyme solution for 18 hr at 4 °. Uncoupled enzyme is removed by washing in 2 M NaCI and distilled water. The preparation is stored in the NazSO3 buffered syrup. The volumetric activity of this preparation is estimated to be about 4-fold that of nonpurifled IGI preparations. The data on the product characteristics are very scarce, but the physical stability is probably sufficient to allow application in the normal industrial IGI columns, which have a diameter of about 1.5 m and a height of about 5 m. The production cost of Ketomax must be higher than that of the present large IGI products on the market, and it is expected that the product improvement will only allow a very limited increase in cost of conversion for the starch manufacturers. Miles Kali-Chemie have developed an IGI product based on purified enzyme. The product characteristics of Optisweet 22 have been described by Weidenbach, 23 but detailed data on the manufacturing method are not available. The principle of the method is the following. Streptornyces rubiginosus cells are produced by submerged, aerobic fermentation. The cells are recovered and disrupted. The solubilized enzyme is purified. Spherical SiO2 particles, 100-200/~m, are used as carriers. The enzyme is adsorbed on the carrier and cross-linked with glutaraldehyde. The activity is about six times higher than that of other products, e.g., Sweetzyme. According to the data published by the manufacturer, it is necessary to utilize the enzyme in a specially designed system comprising sterilization, and aluminosilicate precolumn for intensive syrup purification, and isomerization columns. Further, the column height is limited to 0.4 m. Finnsugar/Fermco have developed an IGI product which is a further development of the purified Clinton IGI. The data on the immobilization are only available from brochures. The principle of the method is as follows. Streptomyces rubiginosus cells containing glucose isomerase are cultivated by aerobic submerged fermentation. The enzyme is extracted from the cells and extensively purified. The purified enzyme is concentrated and shipped as a stabilized liquid concentrate. A carrier is produced according to Sutthoff et a1.24: a DEAE-ceUulose-polystyrene-TiOz composite is formed by mixing the DEAE-cellulose and TiO2 with the polymer which has been heated to a plastic state. The composite consists of approximately 30% DEAE cellulose, 20% TiO2, and 50% polystyrene. 23 G. Weidenbach, poster presented at "Biotech 83." z4 R. F. Sutthoff, R. V. MacAllister, and K. Khaleeluddin, U . S . P a t e n t 4,168,250 (1979).

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After cooling, the composite is ground and sieved. The enzyme is adsorbed on the DEAE-cellulose in the composite carrier. The carrier can be reused after removing the old enzyme and regenerating the D E A E cellulose with sodium hydroxide. The initial activity of this product is about twice that of nonpurified IGIs. The activity half-life of the product is improved substantially since Clinton started to use the DEAE-cellulose as an absorbant. The main reason for the improvement is probably the higher operating pH now used for the Finnsugar product. But this product still suffers the inherent drawback that the enzyme is adsorbed and not covalently bound, so the activity half-life is lower than for other products, and the system is susceptible to accidental changes in process conditions. Of these second generation IGI products, only the Finnsugar/Fermco product has gained a foothold in the market.

New Syrup Refining Processes The success with the classical syrup refining methods has inspired new developments in this field. In 1981 Rohrbach and Maliarik 25,26from UOP applied for patents covering the removal of glucose isomerase inhibitors from the syrup either by reduction or by oxidation of the inhibitors. Reduction typically involves treatment of the syrup with 40-200 ppm of a metal hydride such as sodium borohydride at 60°, pH 8.0. Oxidation may be carried out using 50-500 ppm hydrogen peroxide, also at 60 °, pH 8.0. When either of these processes is used, it is claimed that the half-life obtained using the treated syrup can be doubled, compared to a standard syrup. Moreover, as claimed by Weidenbach et al. 27 from Miles KaliChemie, the use of a precolumn of SiO2 or aluminosilicate may double the enzyme productivity, compared to the process without the precolumn. Whether these processes mark a real breakthrough cannot be estimated on the basis of the present knowledge. Future Developments The past I0 years have shown a tremendous increase in the production of fructose from glucose, but the existing process can only convert a fraction of the glucose to fructose. The trend in the syrup industry is a desire for a direct production of pure fructose from glucose, or at least a direct enzymatic production of 55% HFCS, without using the compli25 R. P. Rohrbach and M.J. Maliarik, U.S. Patent 4,381,345 (1983). 26 R. P. Rohrbach and M. J. Maliarik, U.S. Patent 4,382,121 (1983). 27 G. Weidenbach, D. Bonse, and B. Meyer, European Patent Application 82,111,080.8 (1982).

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cated separation system. Patents of such processes have appeared during the past years. In 1981 Neidleman 28 from the Cetus Corporation patented a method for production of substantially pure fructose from glucose. The method comprises conversion of glucose to D-glucosone by the enzyme glucose-2oxidase and chemical hydrogenation of the D-glucosone to fructose. Apparently the method has never been scaled up, and the enzyme has not become available in large quantities. Cetus seem to have abandoned the concept. The glucose isomerase pioneers Lloyd and Horwath from Clinton, now a Nabisco Brands company, have recently patented 29.3°a process for direct production of isosyrup with 55% fructose. The degree of conversion is raised from 42%, which is the normal isosyrup fructose content, to 55% by isomerization with glucose isomerase at about 95 °. A chemically stabilized, immobilized glucose isomerase product is claimed in the patent, but the data given in the patent do not prove any stabilization. The 55% fructose can be obtained at about 95 ° because the equilibrium is shifted toward the fructose when the temperature is increased. The equilibrium is 50.6% fructose at 60°. An immobilized glucose isomerase for efficient, economic isomerization at 95 ° does not exist in the market today. The syrup manufacturers will probably be very interested in such a product, provided that the syrup quality obtained from a 95 ° process is satisfactory. Concluding Remarks The development of the isosyrup industry has shown that through a close cooperation between the producer and the user of the enzyme it has been possible to build up a completely new industry based on a new technology. In the past 10 years immobilized enzyme products have been developed which makes it possible to work in a continuous plug flow system. The enzyme cost of the isomerization has been reduced 5- to 10-fold in this period, and the isosyrup market has grown tremendously to more than 5 million tons (1984). This is by far the major industrial application of immobilized enzyme technology in the world today. The market is still expanding, and the development is continuing with emphasis on the development of more efficient products and methods for the production of 55% HFCS. 28 S. L. Neidleman, U.S. Patent 4,246,347 (1981). 29 N. Lloyd and R. O. Horwath, U.S. Patent 4,410,627 (1983). 3o N. E. Lloyd, U.S. Patent 4,411,996 (1983).

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[34] I n d u s t r i a l O p e r a t i o n o f I m m o b i l i z e d E n z y m e s

By M. J. DANIELS The immobilization of enzymes is relatively easy and there is a wealth of papers and patents to testify to this fact. Unfortunately, there are no criteria by which to judge these products on the basis of practical usefulness and economic process viability, but the lack of commercial exploitation suggests that most products are only of academic interest. In an attempt to rectify the situation this article will suggest some guidelines, based on experience gained from manufacturing and marketing immobilized enzymes produced at British Charcoals and MacDonalds, Greenock, Scotland. The enzymes considered will be hydrolases, which are relatively inexpensive, but are used in large quantities. While the conclusions may be more valid for low-cost large-volume enzymes, it is probable that the general picture will be the same for most immobilized enzyme systems. The comparison of immobilized and liquid enzyme economics for existing manufacturing processes is the final test, but first I would like to consider immobilized enzyme manufacturing costs and the productivity of several fixed enzyme systems. Manufacturing Cost The chemistry used for enzyme fixing must be applicable on a large scale, be safe for use and produce a safe, stable product, preferably from a range of enzymes. There are inexpensive and expensive supports, but even the cheapest would require some processing if consistent product quality is to be achieved, and the reuse of expensive ones would involve additional costs and losses. Consider two cases: (1) one utilizing inexpensive enzyme and support with simple processing and (2) the other using relatively expensive raw materials with a more complex process. In the following tabulation I would suggest the following general manufacturing costs per liter of product. It should be remembered that import duties, technical back-up, and point of operation running costs will need to be added and it is probably realistic to load these prices by 20%. Thus, it appears that immobilized enzymes produced from commercially available liquid enzymes at realistic loading efficiency would cost the user between $35 and $150 per liter to purchase and operate. METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ENZYME ENGINEERING (ENZYME TECHNOLOGY)

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Immobilized enzyme Cost factors Raw materials and waste disposal Factory overheads, services, packing, and distribution Quality control, salaries, and administration Profit Selling price

1

2

$ 8 8 8 6

$ 80 10 10 20

US$30

US$120

Activity/Productivity Liquid enzyme processes usually involve the treatment of substrates at 20 to 70% dry solids content at temperatures between 40 and 70°. The residence times are relatively long and the liquid enzyme dose would be 0.1 to 2 g enzyme per kilogram of dry solids treated. Immobilized enzymes must treat substrates at similar concentrations and temperatures to produce the same end result, albeit with a considerably shorter residence time and their value to the factory operation would be proportional to the total product solids produced. For practical reasons, the lifetime of the enzyme charge would probably not exceed three half-lives and the average productivity would be close to 50% of the day 1 productivity, so if the day 1 activity is described as bed volumes per hour of substrate, the dry weight productivity of 1 liter immobilized enzyme is simple to calculate. The total process cost of liquid enzyme treatments ranges from $6 to $120 per metric ton dry solids (DS), and for the two manufactured enzymes discussed earlier we can calculate the required lifetime productivities to provide equivalent process costs of liquid enzyme treatment.

Enzyme 1 Enzyme 2

Production cost/ton

Productivity (tons dry solids)

$6-$20 $30-$120

7-1.8 5-0.8

Thus, it appears that an immobilized enzyme formulation must produce 1-7 tons dry solids product during the factory lifetime to provide economics equivalent to liquid enzyme treatment. Assuming 20% dry solids and 55 days half-life which would provide some 4000 hr of operation, the average flow rates for this range of productivities, neglecting specific gravity, would be tabulated as shown below:

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INDUSTRIAL OPERATION OF IMMOBILIZED ENZYMES

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Tons product/liter

Tons solution at 20°, DS

Average liters/hr

Day 1 bed vol/hr

1 7

5 35

1.25 8.75

2.5 17.5

Enzyme 1 Enzyme 2

These calculated day 1 flows using 20% solids substrate of 2-14 bed vol/hr can also be considered as 3-24 min of empty column residence time, again for cost equivalence to liquid enzyme treatment. Finally, using a very simplistic empirical comparison, one can calculate very approximately the quantity of enzyme activity which must be loaded per liter immobilized enzyme and per gram of support; in this case the unit of activity is 1 g of commercially available product. Liquid case

l

2

Immobilized case

Time (hr)

Enzyme (g/kg DS product)

kg DS/hr

Enzyme (g/liter immobilized enzyme)

50 50

0.5 2.0

3.5 0.5

88 50

Assuming a bulk density for the support of 1.5 g/cm 3 this corresponds to 75-130 mg of enzyme activity per gram of support. Below is a summary of these calculated criteria for minimum commercial viability for an immobilized product.

Total user cost

Total productivity

Day 1 activity at 20% DS

Enzyme activity/g support

$35-$150/liter

1-7 tons/liter

2-18 bed vol/hr

75-130 mg

The fact that the majority of the systems described in the literature fall so far short of these targets suggests that their practical usefulness must be questioned. In the next sections, an economic comparison of liquid and immobilized treatments using specific enzyme systems will serve to demonstrate that financial advantage can be provided by an immobilized system.

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Liquid Enzyme Hydrolysis The time course of a typical hydrolysis is shown in Fig. 1. In the case of glucoamylase the maximum dextrose content is required, but with invertase or lactase, intermediate degrees of conversion may be required. Units on the x axis are enzyme hours, defined as kilograms enzyme at specific activity per ton DS substrate × time in hours. This concept is very useful, since within fairly broad limits, the enzyme dose can be reduced to increase the saccharification time quantitatively and vice versa. The total saccharification cost can vary widely even within the same factory, since an increased quantity of enzyme is required for rapid production when there is sudden or heavy demand and a reduced enzyme dose is often used during period of low throughput. The tank volume occupied by the syrup incurs fixed overhead and running costs which could be considered as the "time cost," defined as the running costs per hour for the volume occupied by 1 ton of substrate solids. The total costs for saccharification are, therefore, the sum of the enzyme cost and the time cost generated during the complete saccharification period. The enzyme dose and the saccharification time are inversely proportional, and so it follows that the minimum saccharification cost is when the enzyme cost equals the time cost.

10C -DX

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do FIG. 1. Time course of a typical hydrolysis. DX, Dextrose content (%). Liquid glucoamylase saccharification. Enzyme-thinned substrate, 55°, pH 4.5, 30% dry solids. See text for description of enzyme hours.

[34]

INDUSTRIAL OPERATION OF IMMOBILIZED ENZYMES

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Saccharifieation Tank System Some high-fructose corn syrup (HFCS) factories employ a continuous cascade system which is efficient in terms of tank utilization, the number of pumps needed, and the labor requirement, but it is more common to use batch saccharification. Each batch tank is operated independently and is fitted with pumps for filling and emptying so each tank is individually piped. The labor requirement is relatively high, and since the enzyme is added to the tank the fill and empty times are not entirely part of the saccharification time. Each tank is lagged and traced with low-temperature steam and would contain a stirrer in continuous operation. The running costs are obviously dependent on the size and the age of the plant, but could be estimated as follows (p.a. = per annum): Capital charges, repairs, m a i n t e n a n c e Labor Energy D o w n t i m e and miscellaneous Total

$350/m 3 p.a. 100/m 3 p.a. 100/m 3 p.a. 50/m 3 p.a. US$600/m 3 p.a.

The time cost would therefore be $0.069 per cubic meter of tank volume per hour for 360-day, 24-hr operation. The following terms are defined: EH, enzyme hours to achieve desired conversion; DS, dry solids content of syrup as percentage; C, enzyme cost per kilogram; T, minimum cost saccharification time; Do, optimum enzyme dose in kilograms of enzyme per ton DS. Thus, neglecting the specific gravity, EH = D o T

.'.

T = EH/Do

At the minimum cost DoC = T(0.069/DS) (enzyme cost equals time cost). Then, EH 0.069 =

The enzyme cost would be DoC =

c

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C

0.069 DS

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0.069

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C

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Which in other words is the minimum saccharification cost SC: SC =

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It is then possible to calculate the optimum enzyme dose and saccharification time: D o = SC/2C

and

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This procedure can be demonstrated using typical glucoamylase, invertase, and lactase hydrolysis processes performed commercially. Three enzymes are commonly used in saccharification. (1) The treatment of aamylase-treated starch with glucoamylase would be used to produce glucose syrups, alcohol, or high-fructose syrups. Raffinate is the glucose-rich recycle stream from the fructose-enrichment process. Treatment of raffinate with glucoamylase increases the monosaccharide content and ultimately that of the 55% fructose syrups produced. (2) Invertase treatment of sucrose syrups produces a range of table syrups or raw material for sorbitol/mannitol production. (3) Lactase treatment of whey or the permeate from ultrafiltered whey is used to produce glucose/galactose syrups by hydrolyzing the lactose. Glucoamylase Mainstream saccharification at 32% DS to 96% dextrose content Enzyme cost, $4/kg Enzyme hours required, 72 Raffinate saccharification at 18% DS to 2.4% dextrose content Enzyme cost, $4/liter Enzyme hours required, 24 Invertase 60% inversion using 65% solids Enzyme cost, $5/kg Enzyme hours required, 8 95% inversion using 50% solids Enzyme hours required, 72 Lactase Whey 6% solids--90% hydrolysis Enzyme cost, $120/kg Enzyme hours required, 30 Permeate 20% solids--70% hydrolysis Enzyme cost, $120/kg Enzyme hours required, 5 The calculated costs are minimum and would be exceeded if the enzyme doses were varied in either direction. The enzyme cost, of course, represents 50% of the total cost.

[34]

377

INDUSTRIAL OPERATION OF IMMOBILIZED ENZYMES

Operation

Enzyme dose (kg/ton DS)

Saccharification time (hr)

Total cost per ton DS

Glucoamylase mainstream Glucoamylase raffinate Invertase 60% conversion Invertase 95% conversion Lactase whole whey Lactase permeate

1.97 1.51 0.41 1.41 0.54 0.12

36.5 15.9 19.5 51.1 55.9 41.7

$ 15.8 12.1 4.1 14.1 128.7 28.8

Immobilized Enzyme Hydrolysis The concentration of enzyme fixed to the solid support is very high and the contact time of the substrate which is pumped through the bed is therefore very short. Thus the plant size required for a hydrolysis operation will be several hundred times smaller than the tankage required for liquid enzyme treatment. The capital cost of the plant will be considerably less, and in most cases the plant can be movable so that installation costs are minimal. The flow rate is measured in empty column volumes per hour (ECV/ HR) and the degree of conversion can be varied by changing the flow rate as shown in Fig. 2. Thus the activity of an immobilized enzyme is best quoted as the flow rate under operating conditions which produces the required degree of conversion. The productivity is also dependent on the rate of activity decay and the factory lifetime of each enzyme charge. The decay of enzyme activity in a factory environment occurs due to thermal denaturation which is exponential with respect to time, and also due to poisoning by trace impurities in the substrate, which is directly proportional to throughput. There are also practical limitations to the number of "half-lives" which can be utilized due to the acceptable "turn-down" ratio of pumps and ancillary equipment. For these reasons, it is a reasonable assumption that the enzyme will be used for three half-lives and that the average productivity during this period will be 50% of the initial productivity. The running expenses of an immobilized enzyme rig vary significantly with size, but for the purposes of this cost comparison I will assume overhead costs of $10,000 yearly per cubic meter of column capacity. Thus defining the terms A as initial flow in ECV/HR; H, half-life in days; DS, dry solids as percentage; C, immobilized enzyme costs (in dollars per cubic meter). The total costs are 3H C + 10,000 ==z~ = C + 83.3H 3oo

378

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

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ECV-HR

FIG. 2. Flow rate for immobilized glucoamylase saccharification. Enzyme-thinned substrate, 55°, pH 4.5, 30% dry solids. See text for description of empty column volumes per hour (ECV/HR).

and the productivity in tons DS during the enzyme lifetime is

A/23H24DS = 36AHDS C + 83.3H .'. Cost/ton DS 36A H DS The equivalent costs for the liquid hydrolysis processes considered earlier are as follows:

Glucoamylase Immobilized enzyme cost $30,000/m 3 Mainstream: initial activity, 4 ECV/HR; half-life 70 days Raffinate: initial activity, 15 ECV/HR; half-life 70 days Invertase Immobilized enzyme cost $30,000/m 3 60% conversion: 6 ECV/HR; half-life 80 days 95% conversion: 2 ECV/HR; half-life 80 days Lactase Immobilized enzyme cost $80,000/m 3 Whey, 90% conversion: 15 ECV/HR; half-life 30 days Permeate, 70% conversion: 20 ECV/HR; half-life 40 days

Operation

Initial rate

Empty column residence time (min)

Total process cost/ton DS

Glucoamylase mainstream Glucoamylase raffinate Invertase 60% conversion Invertase 95% conversion Lactase whole whey Lactase permeate

4 15 6 2 15 20

30 8 20 60 8 6

$11.11 5.3 3.3 12.7 84.9 14.5

[34]

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INDUSTRIAL OPERATION OF IMMOBILIZED ENZYMES

The following tabulation summarizes the comparative economics of immobilized and liquid enzymes, comparing three different enzymes, a range of degrees of conversion, and a range of syrup concentrations. Liquid

Immobilized

Operation

Residence time (hr)

Total cost

Residence time (mini

Total cost

Glucoamylase mainstream Glucoamylase raffinate Invertase 60% conversion lnvertase 95% conversion Lactase whole whey 90% Lactase permeate 70%

36.5 15.9 19.5 51.5 55.9 41.7

$ 15.8 12.1 4.1 14.1 128.7 28.8

30 8 20 60 8 6

$11.1 5.3 3.3 12.7 84.9 14.5

The minimum saccharification cost for liquid hydrolysis would be obtained if saccharification tanks were run at constant capacity 360 days per year, 24 hr per day. Immobilized enzyme systems can still be more cost effective if the total process costs are considered, although the actual enzyme cost component is greater. There is clearly some generalization in these cost comparisons and specific cases may show greater or smaller cost advantages than those calculated. The capital cost charges represent some 25% of the liquid enzyme treatment costs and unless the equipment can be utilized for other purposes the incentive to change to an immobilized enzyme system is considerably less. In conclusion, immobilized enzyme systems using sufficiently active products can offer cost advantages to the user on the basis of total manufacturing costs, in addition to a number of process advantages which are also provided. The cost advantages are not sufficient at the current stage of development to induce all liquid enzyme users to change, but further improvements are probable which could lead to even more favorable process economics.

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[35]

[35] L a r g e - S c a l e P r o d u c t i o n o f P h o t o - C r o s s - L i n k a b l e R e s i n - I m m o b i l i z e d Y e a s t a n d Its A p p l i c a t i o n to Industrial Ethanol Production

By

SHOGO NOJIMA and TOMIAKI YAMADA

In this paper we describe the technology for a continuous ethanol fermentation process developed by means of immobilized living cells using yeast cells and specially designed artificial resins. Yeast which has the ability to make ethanol was screened from Saccharomyces genus and mixed with prepolymers of photo-cross-linkable resin, then illuminated with an active ray (for example, chemical lamp) to change the prepolymers into three-dimensional cross-linked polymers in which yeast was entrapped. Specially shaped immobilized yeast was packed in a fermenter and a diluted molasses solution fed to the fermenter. The fermentation temperature was kept at about 30-32 ° and the pH at 4-5. As a result of a bench-scale test (10 liters of ethanol a day) and pilot plant test (250 liters of ethanol a day), the constant activity of the yeast and high ethanol yield on sugar during long-term operation were confirmed, and it was also established that the ethanol productivity was several times that of conventional suspended-state batch system fermentation. Introduction The batch fermentation process is generally used in the production of both potable and industrial alcohols. In order to produce alcohol as a power source, however, it is necessary to improve productivity and reduce manufacturing cost, in comparison with past production processes and, further, to minimize the energy requirement of the manufacturing process. In Japan, the Research Association for Petroleum Alternatives Development (RAPAD) is carrying out a study on the production of alcohol for power with the support of the Ministry of International Trade and Industry. The associated research and development work was started in 1980 under a 3-year joint research program by JGC Corp., Sanraku-Ocean Co., Ajinomoto Co., Kansai Paint Co., and Maruzen Oil Co., based on a subsidy granted to RAPAD. This paper is concerned with a continuous ethanol fermentation technology using yeast cells immobilized in a special synthetic resin. Research work on alcohol fermentation using immobilized microorganisms has reMETHODS IN ENZYMOLOGY,VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

[35]

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

381

cently been reported by a number of researchers. 1-17Table I shows ethanol production by different immobilization processes reported by these researchers, as compared with conventional process in which yeast cells are not immobilized. With the continuous ethanol fermentation process using immobilized yeast cells, the aim is to achieve high ethanol concentrations, high yield on sugar, and high alcohol productivity over long periods of time. This research therefore is being carried out to develop a continuous alcoholproducing process which satisfies these requirements, using alcohol-producing yeasts of the Saccharomycesgenus entrapped in photo-cross-linkable resin. Yeast Cell Immobilization Process Entrapping methods generally used to immobilize yeast cells can be broadly classified into two types: one entraps yeast cells in natural high molecular weight substances such as agar, alginate, K-carrageenan, animal gelatin, and collagen, and the other entraps them in high polymers such as polyacrylamide and photo-cross-linkable resins. Entrapping materials for yeast cell immobilization must have excellent water dispersibility to comingle uniformly with a yeast suspension, provide satisfactory alcohol fermentation characteristics to the immobilized yeast cells, and have sufficient mechanical strength to withstand long-term use. In addition, they must be easy to manufacture in quantity and possess excellent formability if commercial production is being considered. Studies of various entrapping materials satisfying these requirements have revealed that 1 H. Bachere, G. Durand, and M. Moll, French Patent 2,248,319 (1975). 2 I. Takata, T. Tosa, and I. Chibata, J. Solid-Phase Biochem. 2, 225 (1977). 3 j. M. Navarro and G. Durand, Eur. J. Appl. Microbiol. Biotechnol. 4, 243 (1977). 4 M. Kierstan and C. Bucke, Biotechnol. Bioeng. 19, 387 (1977). 5 F. H. White and A. D. Portino, J. Inst. Brewl 84, 228 (1978). 6 G. Cysewski and C. Wilke, Biotechnol. Bioeng. 20, 1421 (1978). 7 j. M. Navarro, SM1 Collogue (Toulouse, France) (1978). s I. Chibata, Chem. Eng. 24, 31 (1979). 9 M Wada, J. Kato, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 8, 241 (1979). 10 B. H~igerdal and K. Mosbach, Int. Congr. Eng. Food, Helsinki, Finland (1979). 11 I. Chibata, U.S. Patent 4,138,292 (1979). ~2M. Wada, T. Uchida, J. Kato, and I. Chibata, Biotechnol. Bioeng. 22, 1175 (1980). 13 M. Wada, J. Kato, and I. Chibata, J. Ferment. Technol. 58, 327 (1980). 14 F. B. Kolot, Process Biochem. Oct./Nov., 2 (1980). 15 I. B. Holcberg and P. Margalith, Eur. J. Appl. Microbiol. Biotechnol. 13, 133 (1981). i6 F. B. Kolot, Process Biochem. Aug./Sept., 2 (1981). 17 F. B. Kolot, Process Biochem. Oct./Nov., 30 (1981).

382

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

L~

e.,

(-

~G Z O

~2 O Z <

.1 < m

o o

co

e~

.£

7.---

E

~D

[35]

[35]

383

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

CHz il CH I C=O I 0

CHz li CH I O=C 0 / (CH~)z I

(CH2)z I

O CH3 L 0 = C--NH--CHz--C--CH z / X CHz

\

/

0 I]

,0 II

CH3 0 I I ?H~--C~--CHz--NH--C = 0

CH--NH-C--O--(--CH2CHz--O-)n--C--NI'I-CH

C--CH~ / \ CH3 CH 3 FIG. 1. Structure o f photo-cross-linkabl¢

\

CH=

/

CH~--C Y\,, CH3 OH 3 resin.

polyethylene glycol photo-cross-linkable resins, which are structured as illustrated in Fig. 1, are excellent in regard to these characteristics. A prominent feature of such resins is that not only yeast but also enzyme can be readily immobilized in their three-dimensional matrix, the size of which can be changed freely by adjusting the degree of polymerization of the polyethylene glycol molecules located between the two isophorone isocyanate molecules. ~8-23 Figure 2 shows a block flow sheet of the yeast cell immobilization process. One or more kinds of yeast of the Saccharomyces genus are usually selected for ethanol fermentation purposes. These yeasts have excellent activity and selectivity in alcohol fermentation. Immobilized yeast is produced by irradiating a mixture of such selected yeast cells and the abovementioned photo-cross-linkable resin with light of 300- to 400-nm wavelength from a low-pressure mercury lamp, i.e., the so-called chemical lamp. Figure 3 gives an external view of the continuous yeast cell immobilizer developed in our laboratory. This unit, which is providing the basic technical knowledge required for the commercial production of immobilized yeast cells in the future, is capable of continuously producing immobilized yeast sheets of 50 cm width at a rate of 1-5 m/min under aseptic conditions. 18 A. Tanaka, S. Yasuhara, S. Fukui, S. Iida, and E. Hasegawa, J. Ferment. Technol. 55, 71 (1977). 19 A. Tanaka, S. Yasuhara, M. Osumi, and S. Fukui, J. Biochem. (Tokyo) 81), 193 (1977). 2o T. Yamada, H. Yoshii, T. Iida, and H. Chiba, Pan-Pac. Synfuels Conf. 2, 455 (1982). 21 A. Tanaka, S. Yasuhara, G. Gellf, Osumi, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 5, 17 (1978). 22 S. Fukui, A. Tanaka, and G. GeUf, Enzyme Eng. 4, 299 (1978). 23 T. Omata, T. Iida, A, Tanaka, and S. Fukui, Eur. J. Appl. Microbiol. Biotechnol. 8, 143 (1979).

384

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[35]

~r.ehOto-cross-linkabl~ sin solution J ~ Ye~~ ~ 7 IMixing}~[F°rmingI~[ Irradiation©('~lmmobilized' ~ st FIG. 2. Block flow sheet of immobilization process.

Table II shows the properties of the immobilized yeast sheets produced by this process. It can be seen that the ENT series of immobilized yeast sheets produced under the present program possess from 30 to 50 times the compressive strength of those produced by the conventional yeast cell immobilization processes using polyacrylamide or r-carrageenan as immobilizing materials. ENT-3800 yeast sheets have been adopted for this continuous ethanol fermentation process because they possess the higher of the alcohol fermentation activities of the ENT series.

FIG. 3. Continuous immobilizer.

[35]

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

385

TABLE II PROPERTIES OF PHOTO-CROss-LINKABLE RESIN

Photo-cross-linkable resin

No. ENT-1000 ENT-2000 ENT-3400 ENTG-2000 ENTG-3800

Chain length (,~)

PEG content (%)

Water absorbability (%)

Tensile strength (kg/cm z)

100 200 340 200 310

60 75 82 58 65

130 305 540 240 350

15 10 6 10 7

Compressive strength (kg/cm 2)

Polyacrylamide r-Carrageenan

54 28 28 28 34

Relative fermentability 1.0 1.8 0.9 2.3 3.5

0.85 0.91

Study on Basic Technology As mentioned above, in order to establish a rational ethanol fermentation process, it is necessary to establish a more economical fermentation system in addition to improving fermentation characteristics such as yield on sugar and productivity. The results of study on some of the elemental characteristics of this alcohol fermentation process are reported in this section.

Fermentation Characteristics of Immobilized Yeast Cells The concentration of immobilized yeast ceils produced by the continuous yeast cell immobilizer, which is 108 cells/gram of resin at the initial stage of fermentation reaction, increases up to approximately 10 ~° cells/ gram of resin in 50-100 hours after the reaction starts. These cells are called immobilized living cells (Fig. 4). The rate of ethanol fermentation reaction is given in the form of the following differential equation by Aiba et al. 24

dP S d--7 = v0 (1 + P/Kp)(Ks + S) X

(1)

where P is the ethanol concentration (g/liter); t, time (hr); v0 specific rate at P = 0 (hr-~); S, glucose concentration (g/liter); Kp, empirical constant 24 S. Aiba and M. Shoda, J. Ferment. Technol. 47, 790 (1969).

386

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[35]

FIG. 4. Immobilized yeast cells (photomicrograph).

(g/liter); Ks, saturation constant (g/liter); and X, cell concentration (g/ liter). Generally Ks < S, so we get Eq. (2) by transforming Eq. (1). (dP/dt)(1/X)

uo

Figure 5 shows Lineweaver-Burk plots of immobilized yeast cells KH/ ENT-3800 based on Eq. (2). The specific rate u0 for the immobilized yeast cells used in this work is 0.094 g ethanol/ml resin-hr as determined by this figure. Type of Fermentation Vessels As shown in Fig. 6 various reactors such as packed bed, moving bed, suspended bed, and other types are considered to be suitable for ethanol fermentation using immobilized yeast cells. After study of such types of reactors, a fixed bed reactor of special configuration, namely a parallel flow-type reactor, was adopted for this process. The parallel flow reactor, shown in Fig. 7, is fitted with immobilized yeast sheets of 0.8 to 1.0 mm thickness placed parallel to the flow direction of the sugar solution. This type of fermentation vessel has the following features. (1) The packing rate of immobilized yeast in the vessel is adjustable within the

[35]

387

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

]

0.2t .H77=-197/ENTG-3800

,~ •

I

-BQ

-60

-40

2o 7o

-20

__ ~ r,~.

g-ethanol

L_

G'(~ 8o

P (g/I)

FIG. 5. Lineweaver-Burk plot of immobilized yeast KH/ENTG-3800.

range of 10-70%. Therefore, the ethanol production rate per unit volume of fermentation vessel can be improved merely by increasing the packing rate. (2) Carbon dioxide gas generated in the fermentation process can be discharged very easily. (3) Only very small quantities of the sludge contained in the molasses solution adhere to the immobilized yeast sheets, and any sludge which does adhere can be readily removed. (4) As mentioned below, a chemical cleaning method is adopted to decontaminate the fermentation vessel of a contaminated fermentation system. The configuration of this type of reactor is very suitable for such decontamination method.

f..~..~,

0

0 0

,-~

3%,°1ol 1°4oH ool O ] 0 I Oo°1"~o14

t Packed bed

Suspended bed

Parallel f l o w

Packed bed

Parallel f l o w

Suspended bed

< 50%

10--70%

10--30%

CO2 dischargeability

difficult (channeling)

easy

easy

Sludge removability

accumulate

readily removed

Type of vessels Pacldng ratio of immobilized cells

readily removed I

FIG. 6. Comparison of various types of fermentation vessels.

388

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[35]

FIG. 7. Internals of parallel flow-type reactor.

Sludge Removal Measures Adhesion of sludge in the molasses solution to the immobilized yeast sheets reduces ethanol productivity. Sludge treatment is divided broadly into two methods; one removes sludge from the molasses solution before it enters the fermentation vessel, and the other removes sludge adhering to the immobilized yeast sheets after the molasses solution enters the fermentation vessel without such pretreatment. From the standpoint of lower running cost, the latter method is more suitable for energy-saving

[35]

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

I / . /

40 ~

-~

~m

20 ~ / ~~ / ~

~.o~"~/'~

'''-'-w

389

-

ophosphate B ": pllosphate D o phosphate E

0.05 0.1

0.5 1.0 5.0 10.0 phosphate concentration ( w t %)

50.0

FIG. 8. Result of sludge removal test.

purposes. In our work on this fermentation process, therefore, a method for removing sludge efficiently with various phosphate solutions (as shown in Fig. 8), without adversely affecting the immobilized yeast cells, has been developed.

Preventing Contamination of Fermentation System To improve yield on sugar and to maintain stable continuous fermentation for long periods, it is necessary to prevent contamination of the fermentation system by contaminants such as bacteria. There are several methods for preventing contamination, for example, by sterilizing the molasses solution by heating it with steam or other means before introducing it into the fermentation vessel. To remove the effect of contaminants by energy-saving treatment, we have developed a method of preventing bacteria growth by holding the fermentation system within a low pH range, for example pH 4-4.5, and also a microbiocidal sterilization method; both methods have very little effect on the immobilized yeast. An example of such microbicidal solutions is given in Table III. We have succeeded in removing contamination by treating the molasses solution for a short time with very low concentrations of hypochlorite or sulfite solutions. Continuous Operation of Experimental Plants To obtain basic design data for designing a commercial plant and to accumulate operational expertise, a bench-scale plant and a pilot plant with ethanol production capacities of 10 and 250 liters/day, respectively, were constructed and the following operational test results have been obtained.

390

ENZYME ENGINEERING (ENZYME TECHNOLOGY) T A B L E III RESULT OF MICROBICIDAL S T E R I L I Z A T I O N

[35]

a

Microbiocide and concentration HC10

SO2

Solution

0

500 ppm

1000 ppm

0

500 ppm

1000 ppm

Yeast Contaminants

5.4 x 108

3 x 108

3 x 108

5.4 x 10 ~

3 x 108

1.3 x 107

<10 2 <10 2 0 0

0 0 0 0

<10 2 "<10 2 0 0

0 0 0 0

C--No. C--No. C--No. C--No. a

1 2 3 4

9.3 5 4 7

× × x x

10 7 10 7 10 7 l07

9.3 5 4 7

X × x ×

10 7 107 10 7 107

N u m b e r o f living cells is indicated in cells per gram resin.

Results of Bench-Scale Plant Test. The bench-scale plant was operated mainly to verify the results of the basic and elemental studies and to obtain guidelines for the design and operation of the pilot plant. Although the basic constitution of the bench-scale plant is the same as that of the pilot plant (see Fig. 9), the fermenter has three rectangular vessels placed in series, and the immobilized yeast sheets shown in Fig. 7 are loaded therein, This bench-scale plant has been operated for 14,000 hr in total, over seven different periods, from fiscal year 1980 to 1983, according to the purpose of each test. During this time we have collected a large amount of technical knowledge about the effects of different types of

-~-Stearn

V

v

x/

C o n t i n u o u s fermenter

Storage tank Sterilizer/Storage tank FIG. 9. Process flow diagram of continuous alcohol fermentation.

[35]

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

391

immobilized yeast cells, sludge adhesion, and contamination on the fermentation characteristics and decontamination methods. Pilot Plant Test. The pilot plant tests are presently being carried out in order to obtain engineering data, for designing a commercial plant, from long-term continuous fermentation tests and to confirm and demonstrate the results of the above-mentioned bench-scale plant test at the same time. The process flow diagram of the pilot plant is shown in Fig. 9 and its overall constitution is shown in Fig. 10. The pilot plant has been operated for about 8500 hr in total, over two different periods. Run 1 was to sterilize the molasses solution by heating it with steam (operation time 4750 hr) and run 2 was a nonsterilized fermentation (operation time 3750 hr). Figure 11 shows the record of operation run 2 and verifies that the immobilized yeast used in this process can maintain stable activity for long periods. Contamination can be almost

FIG. 10. Overall constitution of pilot plant.

392

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

135]

?

~0 10 •SO2w

750ppm

~

~1o.o ~7.5 6 5.0

1st stage

~c-2 . 5

/

T-

3rd stan~

~---SO 2,500 ppm

"'4~i-Decontamination

O

1000

2000 3000 Time (hr)

4000

FIG. 11. Results of pilot plant test (run 2 nonsterilized fermentation). Operation conditions: immobilized yeast, KH-197/ENTG-3800; residence time, 5.0 hr; packing ratio, 33.7 vol%; initial sugar concentration, 20 g/dl (molasses); initial pH, 5.3; no sterilization.

completely controlled by very low concentrations of sodium metabisulfite under nonsterilized fermentation. Evaluation of Process This fermentation process using immobilized yeast is still under development and is expected to be improved in the future. Table IV compares this process with other existing processes. Yeast Concentration. Yeast concentration is the major factor governing ethanol fermentation activity per unit volume of fermentation vessel. Of the existing processes, for instance, the Melle-Boinet process which recycles yeast for fermentation has a maximum yeast concentration of 10 g/liter, whereas the yeast concentration under the process reported here can be increased to 110-130 g/liter since yeast cultivated in resin, called "immobilized yeast," is used for fermentation. In addition, even if the fermentation vessel is charged with immobilized yeast at a rate of 3040%, the yeast concentration in the vessel amounts to - 4 0 g/liter. In other words, the use of this process results in an increase in fermentation rates, assuring high-speed fermentation (several to 10 times the fermentation rates of other processes). Alcohol Concentration. The final ethanol concentration obtained with this process is 10.6 vol%. In general, the ethanol concentration is associated with the kind and concentration of sugar solution. For instance, a high concentration is desirable in view of the reduction in equipment and operating costs of the subsequent ethanol separation and effluent treatment processes. Meanwhile, when aiming at the fermentation of high concentrations of ethanol, the effects of fermented products contain-

[35]

393

PHOTO-CROSS-LINKABLE RESIN-IMMOBILIZED YEAST

TABLE IV FEATURES OF CONTINUOUS ALCOHOL FERMENTATION BY IMMOBILIZED YEAST CELLS Process and fermentation type Immobilized yeast, continuous

Parameter Cell concentration (g/liter) Final alcohol concentration (g/dl) Yield on sugar (%) Fermentation time (hr) Life (months) Volume efficiency of the fermentation vessel (kg alcohol/m3-hr)

Conventional, batch

MelleBoinet, batch

3-5

9.6

Present result Sterilized 40

Nonsterilized 34

Target value 40-50

10-11

5.5-8.0

8.5

8.5

8-11

85-86 70-76 -1.3-1.5

85-86 13-20 -1.38

90-95 5 More than 6 11

90-95 5.7 More than 6 10.5

90 3-8 6-12 12-18

ing ethanol cannot be disregarded since the productivity per unit volume of fermentation vessel decreases. In other words, the optimum ethanol concentration must be determined with due consideration to the productivity per vessel. To this end, we consider that the economic advantages of the process, including types of raw materials, should be properly assessed. Theoretical Yield. Yield on sugar is an important factor in determining the basic unit of materials for producing ethanol. Yield on sugar, however, depends greatly on the properties of the raw sugar solution used for fermentation, i.e., the content of nonfermentative sugar. In general, yield on sugar is represented in terms of a ratio of theoretical to actual yield of ethanol per sugar consumed on the basis of fermentative sugar. The use of this process has indicated a good yield on sugar, i.e., 95% versus the target value of 90%. Reductions in the basic unit of materials can be expected. Alcohol Productioity. The ethanol production rate per fermentation vessel has an effect on the construction cost of the vessel and amount of immobilized yeast necessary for fermentation. According!y, improvements in productivity are extremely important in view of the economic advantages of the process. This process has demonstrated a high productivity rate compared with other existing processes (several to 10 times). We intend, however, to improve this process further in the future for higher productivity.

394

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[36]

Life of Immobilized Yeast. Factors controlling the life of immobilized yeast are the physical strength of the resin and continuity of yeast activation. With regard to resin, stable use can be expected for at least 1 or 2 years as observed from continuous use in the basic research covering several thousand hours. It has also been confirmed that even yeast which has been put in a high ethanol environment extending over a long period actively starts breeding again and completely recovers its activity under a favorable environment. It appears possible, therefore, that the immobilized yeast mentioned herein can be stably used for ethanol fermentation over a considerable period. Fermentation System. To reduce equipment and operating costs for the commercialization of a process, a simple process configuration, low water and electricity costs, and low steam consumption are necessary. This process requires practically no facilities for sludge treatment or for decontamination, in addition to having a high ethanol production rate. It also entails simple methods of decontamination, resulting in extremely low decontamination costs.

[36] L a r g e - S c a l e P r e p a r a t i o n o f C a l c i u m A l g i n a t e - I m m o b i l i z e d Y e a s t Cells a n d Its A p p l i c a t i o n to Industrial Ethanol Production

By M I N O R U

NAGASHIMA, MASAKI A Z U M A , SADAO NOGUCHI,

KEIICHI INUZUKA,

and HIROTOSHI SAMEJIMA

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidized-bed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and to keep cell viability for stable long-run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/liter gel-hr and prolonged life stability for more than one-half year. Maximum cell concentration in the carrier was attained at about 250 g dry cells/liter gel. A pilot plant with a total volume of 4 kl was constructed and has been in operation since 1982, while a semicommercial-scale plant consisting of two 10-kl-sized reactors has been in operation since 1983. As a result, it has been confirmed that 8-10% (v/v) ethanol-containing broth was continuously produced from nonsterilized diluted cane molasMETHODS IN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

394

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[36]

Life of Immobilized Yeast. Factors controlling the life of immobilized yeast are the physical strength of the resin and continuity of yeast activation. With regard to resin, stable use can be expected for at least 1 or 2 years as observed from continuous use in the basic research covering several thousand hours. It has also been confirmed that even yeast which has been put in a high ethanol environment extending over a long period actively starts breeding again and completely recovers its activity under a favorable environment. It appears possible, therefore, that the immobilized yeast mentioned herein can be stably used for ethanol fermentation over a considerable period. Fermentation System. To reduce equipment and operating costs for the commercialization of a process, a simple process configuration, low water and electricity costs, and low steam consumption are necessary. This process requires practically no facilities for sludge treatment or for decontamination, in addition to having a high ethanol production rate. It also entails simple methods of decontamination, resulting in extremely low decontamination costs.

[36] L a r g e - S c a l e P r e p a r a t i o n o f C a l c i u m A l g i n a t e - I m m o b i l i z e d Y e a s t Cells a n d Its A p p l i c a t i o n to Industrial Ethanol Production

By M I N O R U

NAGASHIMA, MASAKI A Z U M A , SADAO NOGUCHI,

KEIICHI INUZUKA,

and HIROTOSHI SAMEJIMA

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidized-bed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and to keep cell viability for stable long-run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/liter gel-hr and prolonged life stability for more than one-half year. Maximum cell concentration in the carrier was attained at about 250 g dry cells/liter gel. A pilot plant with a total volume of 4 kl was constructed and has been in operation since 1982, while a semicommercial-scale plant consisting of two 10-kl-sized reactors has been in operation since 1983. As a result, it has been confirmed that 8-10% (v/v) ethanol-containing broth was continuously produced from nonsterilized diluted cane molasMETHODS IN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

[36]

CALCIUM ALGINATE-IMMOBILIZED YEAST CELLS

395

ses for over one-half year. The productivity of ethanol was calculated to be 0.6 kl ethanol/kl reactor volume per day, with a 95% conversion yield versus the maximum theoretical yield for 8.5% (v/v) ethanol broth. Introduction

Following the successive oil crises of the 1970s, the Research Association for Petroleum Alternatives Development (RAPAD) was organized in Japan in May 1980 by 23 companies under the auspices of the Ministry of International Trade and Industry (MITI) of the Japanese Government. Development of biomass conversion and utilization technologies became one of the three main targets of RAPAD's research activities. As part of the project conducted by RAPAD, a new method of continuous alcohol fermentation using a system of immobilized yeast cells was developed. A number of techniques that maintain high cell density in the fermenter have been proposed for continuous ethanol fermentation. These are classified into three main types: physical immobilization by adsorptive binding of cells to inert carriers, 1 entrapment of cells in various hydrogels (e.g., Ca alginate, 2 carrageenan, 3 polyacrylamide,4~ representing the first reported entrapment of cells for production purposes and 4b involving coentrapment of magnetic particles), as well as the use of flocculent microorganisms.5,6 Other immobilization techniques were also tested. 7-13 For large-scale production, the following criteria should be fulfilled: (1) good conversion yield by prevention of contamination, (2) prolonged viability of the immobilized organisms, and (3) practical operational stability during the immobilization and fermentation steps. This article presents technological developments for continuous fermentation using the immobilized cell process, with the following considi J.-M. Navarro, G. Durand, B. Duterrtre, M. Moll, and G. Corrieu, Ind. Aliment. Agri. 93, 695 (1976). 2 M. Kierstan and C. Bucke, Biotechnol. Bioeng. 19, 387 (1977). 3 M. Wada, J. Kato, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 10, 275 (1980). 4a K. Mosbach and R. Mosbach, Acta Chem. Scand. 20, 2807 (1966). 4b p. O. Larsson and K. Mosbach, Biotechnol. Lett. 1, 501 (1979). 5 I. G. Prince and J. P, Barford, Biotechnol. Lett. 4, 261 (1982). 6 E. J. Arcuri, R. M. Worden, and S. E. Shumate, Biotechnol. Lett. 2, 499 (1980). 7 j. Klein and H. Eng, Biotechnol. Lett. 1, 171 (1979). 8 T. M. S. Chang, this series, Vol. 44, pp. 203,207 (1976). 9 y . Inaba, Japanese Patent KOKAI 72,011 (1981). 10 y . Ado, T. Kawamoto, M. Yoshino, and K. Kimura, Japanese Patent KOKAI 76,894 (1979). H y. lnaba, Japanese patent pending. 12 p. Bernfeld and J. Wan, Science 142, 678 (1963). 13 S. Enosono, S. Ushiro, Japanese Patent KOKAI 120,190 (1977).

396

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[36]

erations: (1) selection of the carrier, (2) design of the reactor and the shape of carrier, (3) selection of the strain, (4) prevention of contamination, (5) maintenance of yeast viability, and (6) scale-up examination. Selection of Carriers Various supports have been evaluated for their ethanol productivities and are listed in Table I. The entrapping methods carried out with various hydrogels showed high orders of activity. Carrageenan is comparable to calcium alginate, but displays undesirable characteristics during largescale preparation and operation because of its solidification at room temperature and lack of gel strength. Calcium alginate was finally chosen as an entrapping agent because of its good characteristics, which are shown in Table II. TABLE I PRODUCTIVITY OF VARIOUS IMMOBILIZED YEAST CELL PREPARATIONSa Alcohol-producing activity ¢

Immobilized systems b

mg alcohol/ g gel hr

mg alcohol/ ml gel hr

Porous epoxy resin 7 Nylon microcapsule (mc) s Unsaturated polyester mc 9 (Acetylbutyl)cellulose mc I° Porous polystyrene mc l~ Polyacrylamide gel ~2 Calcium alginate gel (I) Low methoxypectin gel (II) Carrageenan gel (III) Agar gel (IV) Silica sol ~3

26 2 4 14 14 36 40 35 40 40 50

15 1 2 8 7 20 22 19 22 22 28

a Cane molasses was fed continuously for 5 to 10 days through the columns to attain stable productivity. b I-III, These preparations were carried out following the general procedure given in Fig. 2: (1) alginate-CaC12 ; (II) low methoxypectin (Red Ribbon 3G, Unipectin Corp.)CaCI2; and (III) carrageenan-KCl. IV, Agar gel prepared by cooling at room temperature, me, Microcapsule. c g gel, weight basis of gel beads; ml gel, volumetric basis of gel beads.

[36]

CALCIUM ALGINATE-IMMOBILIZED YEAST CELLS

397

TABLE II REQUIREMENTS FOR SUPPORTS FOR MICROBIAL IMMOBILIZATION High carrier activity Availability in quantity Low cost of immobilization Ease of scale-up of operation Mechanical strength for long-life operation

Design of the Reactor In bench-scale studies, three prototype reactors were constructed to obtain the basic data required to design a pilot plant. The volume capacity of each reactor was 1 kl. Figure 1 shows these three prototype reactors. The first reactor is a tall tower-type reactor with adjustable length/diameter (L/D) ratios of 7-10. The second reactor is a short column-type reac-

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FIG. 1. Diagrams of prototype reactors. L, Length; D, diameter; H, height. CL-1 column reactor: L/D = 7-10, H = 6 m, capacity 1 kl. CL-2 column reactor: L/D = 1-2, H = 2.5 m, capacity 1 kl. CL-3 rectangular reactor: H = 1 m, capacity 1 kl (gas-liquid separation type).

398

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[36]

tor with adjustable L/D ratios of 1.5-2. The third reactor is a rectangular type whose interior is divided by a number of vertical plates between which the substrate solution flows laterally in a zigzag course from one end of the reactor to the other. As a result of these bench-scale experiments, tower- or column-type reactors were chosen. Preparation of calcium alginate cell beads is carried out according to the scheme shown in Fig, 2. One kiloliter of 2-3% sodium alginate solution mixed with 10 to 1000 ml of yeast cell culture broth was delivered from the top nozzle of the reactor into the 2% calcium chloride solution which filled the reactor. The drops of alginate solution solidify quickly when they come in contact with the calcium chloride solution. The preparation of cell beads was completed within several hours in the case of prototype reactors. Therefore, no special equipment was needed for the preparation of gel beads, and the preparation can be done even under aseptic conditions when the system is kept under sterilized conditions. After further studies on the reaction characteristics, it was found that at least two columns must be connected in series to obtain a higher conversion yield due to the strong turbulent effect caused by carbon dioxide gas which was evolved during fermentation, especially in the first columns. With a series connection, the immobilized cell beads can be filled to 60% of the total volume of each reactor. From the above results, a tentative process flow format was developed. Prevention of Contamination In early experiments, long-run operations sometimes suffered from contamination by certain bacteria, for example, the acid-producing Acetobacter oxydants or Lactobacillus sp. In order to obtain a good fermentation yield and make the continuous process stable for a long time, it is

Na-Alginate

Yeast

H20 7 1 solution

culture broth

I

sterilization i 1 Mixture

CaCl 2 solution ]

II

Immobilized Cells FIG. 2. Preparation of immobilized yeast cells.

[36]

CALCIUM ALGINATE-IMMOBILIZED YEAST CELLS

399

necessary to protect the process from contamination. As a result of studies on the characteristics of isolated contaminants, it was found that contamination could be effectively prevented if the initial pH of the inlet substrate solution was kept at 4.0 with sulfuric acid. The addition of some bactericidal substances such as sodium metabisulfite or antibiotics to the substrate solution was also found effective. By employing such measures, the contamination problem was virtually eliminated and, furthermore, the process became operable without sterilization of the inlet medium. Improvement of Yeast Cell Viability Because of the gradual decrease of activity in immobilized yeast cells during long-run operations, maintenance of yeast cell viability was also investigated. Yeast growth inside the carrier beads, which may be limited by substrate diffusion, was promoted by the presence of a small amount of dissolved oxygen, certain sterols, and unsaturated fatty acids which were required for yeast growth during fermentation as Andreasen e t al. 14 pointed out in their early experiments. Therefore, entrapment of these sterols and/or unsaturated fatty acids into gel beads was attempted. This effectively enhanced yeast growth and resulted in higher productivity (30-50 g ethanol/liter gel-hr). Particularly the holding capacity of yeast cells in gel beads correlatively increased, depending on the amount of sterols co-entrapped. Suitable aeration into the reactor also greatly enhanced yeast cell viability. Figure 3 shows features of a gel bead cross section created by the yeast cell entrapment procedure. Dense growth of yeast around the carrier was observed with electron microscopy. Selection and Improvement of Strains Suitable yeast strains were first selected from the culture stocks of our laboratories. These strains can produce 8-9% (v/v) of alcohol steadily for long periods of time at 32-32 ° . Strain improvement was carried out in order to get higher alcohol-tolerant strains. Improved strains can produce more than 10% (v/v) of ethanol from cane molasses. Strains which are tolerant at 35 ° are also available. Pilot Plant Operations

Figure 4 shows the improved process flow diagram. This process was operated without sterilization of the inlet medium and seed fermenter. ~4A. A. Andreasen and T. J. B. Stier, J. Cell. Comp. Physiol. 43, 271 (1954).

FIG.3. Scanningelectronmicrographof gel beadpreparedby the yeastcellentrapment method. Bar, 50/~m.

Sterol ("~Na-Alginate Y e a s ~

Broth

~~IM°lasses

Fermenter FIG.4.Processflowdiagramofthepilotoperation.

[36]

401

CALCIUM ALGINATE-IMMOBILIZED YEAST CELLS

FIG. 5. An overview of the pilot plant.

The principle of the pilot plant studies was to simplify the total process and to minimize the initial investment and operational costs. A final pilot plant was completed in March 1982. This pilot plant is composed of two reactor channels. One channel consists of two columns (each 1 kl) in series, and the other channel consists of three columns (0.8 kl for one and 0.6 kl for the other two). The total column volume is 4 kl, and the total productivity is 2.4 kl alcohol/day. Figure 5 shows the pilot plant.

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FIG. 6. Continuous alcohol fermentation by immobilized growing yeast cells in the pilot plant (run 7). Conversion yield (Q); ethanol (0); space velocity ( ).

402

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[36]

Pilot plant operation has been carried out since April 1982. Figure 6 shows the course of operation beginning in September 1982. In this case, 8.5-9.0% (v/v) alcohol was constantly produced from diluted cane molasses for over 4000 hr (~6 months). The productivity of alcohol is calculated as ~20 g/liter total volume-hr (33 g/liter gel-hr). This means that 600 liters pure alcohol is produced each day using a 1-kl column reactor. Also, this operation has been controlled by a computerized system, with automatic analysis of ethanol and residual available sugar in the fermented broth. Figure 7 shows another run of pilot plant operation. In this case, an alcohol-tolerant strain was employed, and more than 10% (v/v) ethanol was steadily produced for more than 2000 hr. Furthermore, a semicommercial plant was constructed at the Hofu Plant of Kyowa Hakko in April 1983. The plant consists of two 10-kl reactors in series (Fig. 8); operation was started in May 1983. Even in this large-scale plant, more than 8% (v/v) ethanol has been steadily produced for more than 1000 hr, as shown in Fig. 9. Conclusions Compared with the conventional batchwise process, the present process can be evaluated as follows. The productivity of alcohol is more than 20 times higher than with conventional batchwise fermentation. This process does not need any special equipment for the preparation of immobilized yeast cells. This means a significant decrease in the initial investment cost. This process can be operated continuously for more than 6 months without medium-sterilization and seed-fermentation steps. This

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FIG. 7. Continuous alcohol fermentation by immobilized growing yeast cells in the pilot plant (run 8). Conversion yield (Q); ethanol (0); space velocity ( ).

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[36]

CALCIUM ALGINATE-IMMOBILIZED YEAST CELLS

FIG. 8. Ten-kiloliter semicommercial reactors.

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[37]

INTERESTERIFICATION OF TRIGLYCERIDE

405

translates into a significant savings of energy and labor costs. This process can be operated automatically when a computer control system is applied. Also, using minimal instrumentation, fixed-rate control of the fermentation is available. Higher alcohol concentration (more than 10% v/v) can be attained if a suitable strain is used. In this case, still more than 10 times higher productivity, compared with conventional batchwise fermentation, can be obtained. Conversion yield is 90-95% versus the theoretical maximum yield. In total, this process should economize the alcohol fermentation process to a great extent. At present, a combined process using this immobilized yeast cell process and a vacuum fermentation technique is under investigation at the pilot plant in Hofu Plant as a project of RAPAD. Accordingly, we expect to obtain much higher alcohol fermentation efficiency through higher productivity of alcohol production and lesser amounts of waste liquor. Acknowledgment This work has been done as a part of the Biomass Utilization Project of the Research Association for Petroleum Alternatives in Japan.

[37] R e g i o s p e c i f i c I n t e r e s t e r i f i c a t i o n o f T r i g l y c e r i d e w i t h Celite-Adsorbed Lipase By SHIGERU YAMANAKA and TAKASHI TANAKA

Chemical interesterification has been used to modify the physical properties of triglyceride mixtures in the oils and fats industry. In the process, a chemical catalyst is used to promote acyl migration among glyceride molecules. The resulting products consist of glyceride mixtures in which the fatty acid moieties are randomly distributed among the glyceride molecules. It is also well known that during hydrolysis or synthesis of triglycerides lipases (EC 3.1.1.3) show specifities toward the carbon position of the glycerol molecule and the acyl residue with the use of such lipases products not obtainable by chemical interesterification methods may be produced. Interesterification with pancreatic lipase in an aqueous system has been reported for obtaining palmitate-enriched glyceride from glyceryl-l-palmitate 2,3-dioleate and palmitic acid. ~ In this reaction sysI R. W. Stevenson, F. E. Luddy, and H. L. Rothbart, J. Am. Oil Chem. Soc. 56, 659 (1979),

METHODS IN ENZYMOLOGY,VOL. 136

Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.

[37]

INTERESTERIFICATION OF TRIGLYCERIDE

405

translates into a significant savings of energy and labor costs. This process can be operated automatically when a computer control system is applied. Also, using minimal instrumentation, fixed-rate control of the fermentation is available. Higher alcohol concentration (more than 10% v/v) can be attained if a suitable strain is used. In this case, still more than 10 times higher productivity, compared with conventional batchwise fermentation, can be obtained. Conversion yield is 90-95% versus the theoretical maximum yield. In total, this process should economize the alcohol fermentation process to a great extent. At present, a combined process using this immobilized yeast cell process and a vacuum fermentation technique is under investigation at the pilot plant in Hofu Plant as a project of RAPAD. Accordingly, we expect to obtain much higher alcohol fermentation efficiency through higher productivity of alcohol production and lesser amounts of waste liquor. Acknowledgment This work has been done as a part of the Biomass Utilization Project of the Research Association for Petroleum Alternatives in Japan.

[37] R e g i o s p e c i f i c I n t e r e s t e r i f i c a t i o n o f T r i g l y c e r i d e w i t h Celite-Adsorbed Lipase By SHIGERU YAMANAKA and TAKASHI TANAKA

Chemical interesterification has been used to modify the physical properties of triglyceride mixtures in the oils and fats industry. In the process, a chemical catalyst is used to promote acyl migration among glyceride molecules. The resulting products consist of glyceride mixtures in which the fatty acid moieties are randomly distributed among the glyceride molecules. It is also well known that during hydrolysis or synthesis of triglycerides lipases (EC 3.1.1.3) show specifities toward the carbon position of the glycerol molecule and the acyl residue with the use of such lipases products not obtainable by chemical interesterification methods may be produced. Interesterification with pancreatic lipase in an aqueous system has been reported for obtaining palmitate-enriched glyceride from glyceryl-l-palmitate 2,3-dioleate and palmitic acid. ~ In this reaction sysI R. W. Stevenson, F. E. Luddy, and H. L. Rothbart, J. Am. Oil Chem. Soc. 56, 659 (1979),

METHODS IN ENZYMOLOGY,VOL. 136

Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.

406

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[37]

tem the yield of triglyceride is low, probably due to the large amount of buffer solution used. Recently, interesterification with carrier-adsorbed lipase was successfully attempted in organic solvent systems for the production of cacao butterlike fat from fats and oils containing triglycerides with oleic acid residues at the 2-position, fatty acids, aliphatic alcohol esters of fatty acids, or other fats and oils. 2~ In such a reaction system in the presence of a small amount of a trihydric alcohol such as glycerol (more than 0.1% of the raw material), water, or a buffer solution (less than 2 % of the total weight of the reaction mixture), lipase catalyzes the interesterification reaction when a carrier is used as a disperser of the enzyme. This paper describes a regiospecific interesterification reaction method for triglyceride in n-hexane using Celite-adsorbed lipase in the presence of a small amount of glycerol or buffer solution. A simplified reaction scheme is shown below (see also Table I). H2C--O--CO--Ra

H2C--O--CO--Rx

I

H C - - O - - C O - - R b + 2 R~--COOH --~ H C - - O - - C O - - R b + RaCOOH + RcCOOH

H2C--O--CO--Rc

H2C--O--CO--Rx

We will also describe the isolation method for reformed triglyceride from the reaction mixture, and analytical methods for total fatty acid composition and fatty acid in the 2-position of the triglycerides. (Analysis of fatty acid in the 2-position of triglyceride discloses the position of individual fatty acids within a triglyceride.) Experiment A: Preparation and Application of Lipase Adsorbed to Celite Coated with Glycerol

Reagents Olive oil (commercial product of Yoshida Pharmaceuticals Ltd., Japan) T. Tanaka, E. Ono, M. Ishihara, S. Yamanaka, and K. Takinami, Agric. Biol. Chem. 45, 2387 (1981). 3 T. Tanaka, M. Ishihara, and E. Ono, Hakko to Kogyo 41, 375 (1983). 4 T. Tanaka, E. Ono, and K. Takinami, British Patent 2,042,579 (1982). 5 M. H. Coleman and A. R. Macrae, British Patent 1,577,933 (1980). 6 T. Matsuo, N. Sawamura, Y. Hashimoto, and W. Hoshida, British Patent Application 2,035,359A (1980).

[37]

INTERESTERIFICAT1ON OF TRIGLYCERIDE

407

Stearic acid Glycerol [water content in glycerol was less than 1.5% (Karl Fischer titration)] Lipase (Seikagaku-kogyo Co., Japan, Rhizopus delemar, fine grade 200 units/mg solid) (one unit is defined as that liberating 1 /zmol equivalent of fatty acid from oil or fat per minute at 25°) Celite (Johns-Manville Sales Co., United States, No. 535) n-Hexane Procedure. Two grams of Celite is washed three times with about 20 ml of deionized water and dried in vacuo for 2 days at 50°. About 10 ml of methanol containing less than 0.1% water and 0.2 ml of glycerol is added to the dried Celite. After thoroughly mixing, methanol is evaporated at 50° in vacuo. The Celite coated with glycerol is transferred into 40 ml of n-hexane in a 100-ml flask and mixed thoroughly to obtain a suspension. Ten grams of olive oil and 10 g of stearic acid are added to the above suspension. Then finally 40 mg of lipase is added to the suspension. In this way lipase is adsorbed to the glycerol-coated Celite. This reaction mixture is shaken in a rubber-stoppered flask on a reciprocal shaker at 37° for 24 hr (120 strokes/min). Isolation of Triglyceride. When the enzyme reaction is completed, Celite is eliminated by decanting. The reaction mixture containing 0.2-0.5 g of triglyceride is loaded on a Florisil column. (Preparation of column: 30 g of 60-80 mesh Florisil containing 7% water is dispersed in 100 ml of n-hexane, and the suspension is packed in a 2 × 25 cm column. Then the column is washed with 5 column-volumes of n-hexane.) The column is then eluted with the solvent system composed of diethyl ether and n-hexane (1:4, v/v). The eluted fractions (40-80 ml) are collected, and concentrated to about 1 ml. After checking that this concentrate is free from diglycerides, monoglycerides, and fatty acids by the conventional analytical TLC method, it is subjected to the following analyses. Analysis of the Total Fatty Acid Composition. One hundred and fifty milligrams of triglyceride is hydrolyzed with 3 ml of 0.5 N NaOH in methanol at 80° for 10 min in a test tube equipped with a Liebig condenser. Then the fatty acid in the reaction mixture is subjected to methyl ester preparation for further analyses by gas chromatography. Preparation of fatty acid methyl esters is basically carried out using the American Oil Chemists Society method (Ce 2-66). To the hydrolyzate is added 3.5 ml of 7% BF3-methanol reagent and the reaction mixture is boiled for 2 min. After the addition of 2.5 ml of n-hexane the reaction mixture is boiled for an additional minute. After cooling to room temperature, 15-20 ml of saturated NaCI aqueous solution is added to the mix-

408

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[37]

ture. One milliliter of the n-hexane layer containing methyl esters of fatty acid is removed with a pipette and dehydrated on anhydrous sodium sulfate. This dehydrated n-hexane layer is used as the sample for analyses. The sample (0.5-5/.d) is injected directly in a gas chromatograph. Analysis is carried out by the conventional method. Analysis of the Fatty Acid in the 2-Position of a Triglyceride. The method is modified from that described by Usui et al. 7 One-tenth gram of triglyceride is added to 7.5 ml of 1% poly(vinyl alcohol) (average degree of polymerization 200), and the suspension is homogenized twice for 3 min using a homogenizer. To 7 ml of the homogenizate, 2 ml of 0.5 M phosphate buffer (pH 8.0) and 0.5 mg of lipase (Sigma Type IV porcine pancreas) are added. The reaction mixture is then kept at 40° for 1 hr. To stop the reaction, 2.5 ml of an acetone-ethanol mixture (1 : I, v/v) is added to the reaction mixture, which is then acidified by the addition of 2.5 ml of 1 N HCI. The acidified reaction mixture is subjected to extraction with diethyl ether 3 times. After the ether layer is washed with water twice, the washed ether layer is dehydrated with sodium sulfate. Monoglyceride isolated by conventional preparative TLC from the dehydrated ether layer is used as a sample for further analyses. Finally, the fatty acid composition of monoglyceride is analyzed by the same method used for total fatty acid composition analyses.

Experiment B: Preparation and Application of Lipase Adsorbed to Celite Coated with Buffer Solution

Reagents Oleic safflower oil (product of the United States, extracted from safflower species having oil rich in oleic acid) Palmitic acid 0.3 M TES [N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid] buffer (pH 6.5) Lipase I (Seikagaku-kogyo Co., Japan, Rhizopus delemar, fine grade 200 units/mg solid) Lipase II (Meito-sangyo Co., Japan, Candida cylindracea, Lipase MY, 42 units/mg solid) Celite (Johns-Manville Sales Co., United States, No. 535) n-Hexane Procedure. TES buffer (0.1 ml) is mixed with 1 g of Celite in a test tube and the mixture is shaken until Celite is coated with buffer solution as 7 H. Usui, H. Kuwayama, and M. Nagakura, Yukagaku 20, 284 (1971).

[37]

INTERESTERIFICATION OF TRIGLYCERIDE

409

uniformly as possible. Celite coated with buffer solution is transferred to a 100-ml flask containing 40 ml of n-hexane. The suspension is mixed thoroughly. Then 10 g of oleic safflower oil and I0 g of palmitic acid are added to this suspension. Finally 20 mg of lipase I or 100 mg of lipase II is added to this suspension, respectively. (In this way the enzyme is adsorbed onto the buffer-coated Celite.) The reaction mixture is shaken at 30° for 3 days on a reciprocal shaker (120 strokes/min). Isolation of Triglyceride. Same as in the case of Experiment A. General Considerations Hydrolysis and resynthesis of glycerides occur because lipase reactions are reversible. When the water content in the reaction system is restricted, the hydrolysis of the oil or fat is minimized, permitting lipasecatalyzed interesterification to occur. Free fatty acid exchanges with the fatty acid moieties of the triglycerides to produce novel triglycerides, incorporating the added fatty acid. When 1,3-specific lipase is used, the reaction occurs only at the 1- and 3-positions of the glycerides, whereas if nonspecific lipase is used, the reactions occur at all three positions. 2,s An interesterification reaction using olive oil and stearic acid is described as the first experiment (A). In this experiment, lipase is considered to be activated by a small amount of glycerol which coats the Celite. Activation in this case means that the lipase is put into an active form. In this way it is possible to convert olive oil to a new reformed fat where the oleic acid moieties at the 1- and 3-positions are replaced by stearic acid (25-30%). In the second experiment (B) an interesterification reaction is described in which a small amount of TES buffer (pH 6.5) is used as an activator. Such Celite-adsorbed lipase from Rhizopus delemar converted safflower oil to a reformed fat, where oleic acid moieties at the 1- and 3positions were replaced by palmitic acid moieties. The analytical data on the total fatty acid composition and the 2-position fatty acid composition of the fat are shown in Table I. The oleic safflower oil, composed of about 75% oleic acid and 20% linoleic acid, was converted to an interesterified fat composed of about 50% oleic acid and 40% palmitic acid. When the lipase from Candida cylindracea was used, palmitic acid exchanged with oleic acid at random on all three positions of the glyceride (Table I). s A. R. Macrae, J. Am. Oil Chem. Soc. 611, 291 (1983).

410

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[37]

TABLE I FATTY ACID COMPOSITION OF OLEIC SAFFLOWER OIL [NTERESTERIFIED TRIGLYCERIDEa

Interesterified triglyceride Oleic safflower oil

Lipase I

Lipase II

Composition and reaction

Total (%)

2-Position (%)

Total (%)

2-Position (%)

Total (%)

2-Position (%)

Palmitic acid Stearic acid Oleic acid Linoleic acid

6.2 2.2 75.5 17.0

0.2 0.5 77.2 23.2

40.1 0.8 48.4 11.1

2. I 0.1 75.0 23.0

47.0 2.0 42.2 9.1

34.0 0.1 54.1 13.2

Main reaction

I O,L

(+Pal)

I

O,L

t_- ,O,L

P,O,L

~,O,L

LP,O,L P,O,L

I

+ I~,O,L Lipase I, lipase of R. delemar; lipase II, lipase of C. cylindracea. O, oleoyl; L, linoleoyl; P, palmitoyl; and Pal, palmitic acid.

~F-Hexane l Mixing

r ~

Methanol evaporation I

Reaction vessel

(at 3 0 - 4 0 °)

FIG. 1. The lipase-catalyzed interesterification setup; A flowchart of a semiscale enzyme process is given. Addition of carrier, reactants, and catalyst is carried out according to the numbers designated.

[38]

PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE

411

Production of a cacao butterlike fat using 1,3-specific lipases is very interesting to the oils and fats industry. An illustration of an enzymecatalyzed reaction in a semiscale reactor is presented in Fig. 1. The enzyme reaction is carried out in a stirred tank fermenter, equipped with temperature recording and control at 30-40 °, under agitation for 24-72 hr. The reaction mixture is agitated at a speed giving a uniform dispersion. After the enzyme reaction, the Celite particles are separated from the reaction mixture, and the solvent is removed by evaporation. From this oils and fats fraction, the interesterified triglycerides (or cacao butterlike fat fraction) is concentrated and purified, using ethanol in an ordinary fractionation procedure, accompanied by differential scanning calorimetric analyses. In order to make this process practical from an industrial point of view, repeated use of adsorbed lipase as well as utilization of cheap raw material (like the midfraction of palm oil) and efficient fractionation of the products are indispensable.

[38] L a r g e - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Lactase By J. L.

BARET

Whey is a major by-product of the dairy industry. Most of it is spraydried or processed and used in a variety of applications as a food, a feed, or a fermentation substrate. Ultrafiltration techniques have also been developing for the past decade and are now becoming a well-established technology to recover whey proteins and also to process milk in cheese making. Secondary by-products from the ultrafiltration, known as whey or milk permeates, are now produced in a significant amount. A continuous effort for better utilization of wheys and permeates is being made by the dairy industry. The hydrolysis of lactose into glucose and galactose appears to be an interesting approach to widen the profitable uses of wheys and permeates. Hydrolyzed lactose is sweeter and more soluble than lactose; it also presents several additional advantages which allow producers to obtain new attractive products. This report presents some aspects of the immoMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

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411

Production of a cacao butterlike fat using 1,3-specific lipases is very interesting to the oils and fats industry. An illustration of an enzymecatalyzed reaction in a semiscale reactor is presented in Fig. 1. The enzyme reaction is carried out in a stirred tank fermenter, equipped with temperature recording and control at 30-40 °, under agitation for 24-72 hr. The reaction mixture is agitated at a speed giving a uniform dispersion. After the enzyme reaction, the Celite particles are separated from the reaction mixture, and the solvent is removed by evaporation. From this oils and fats fraction, the interesterified triglycerides (or cacao butterlike fat fraction) is concentrated and purified, using ethanol in an ordinary fractionation procedure, accompanied by differential scanning calorimetric analyses. In order to make this process practical from an industrial point of view, repeated use of adsorbed lipase as well as utilization of cheap raw material (like the midfraction of palm oil) and efficient fractionation of the products are indispensable.

[38] L a r g e - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Lactase By J. L.

BARET

Whey is a major by-product of the dairy industry. Most of it is spraydried or processed and used in a variety of applications as a food, a feed, or a fermentation substrate. Ultrafiltration techniques have also been developing for the past decade and are now becoming a well-established technology to recover whey proteins and also to process milk in cheese making. Secondary by-products from the ultrafiltration, known as whey or milk permeates, are now produced in a significant amount. A continuous effort for better utilization of wheys and permeates is being made by the dairy industry. The hydrolysis of lactose into glucose and galactose appears to be an interesting approach to widen the profitable uses of wheys and permeates. Hydrolyzed lactose is sweeter and more soluble than lactose; it also presents several additional advantages which allow producers to obtain new attractive products. This report presents some aspects of the immoMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

412

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[38]

bilized lactase processes developed by Corning for the hydrolysis of lactose in permeates and wheys. Lactose Hydrolysis Reaction and Applications Lactose in solution can be hydrolyzed. The reaction is slightly exothermic and can be catalyzed with acids, cationic resins in H ÷ form, or enzymes. It is not strictly quantitative because side products may form depending on the nature of the catalyst and on the pH and temperature of the reaction. These aspects were studied with demineralized whey permeates which are fairly purified lactose solutions.l The studies showed that the lactose hydrolyzed products which were obtained by acid or H ÷ resin hydrolysis were less pure than those obtained by enzymatic hydrolysis. With more complex lactose-containing feedstocks available on a large scale such as milk, wheys, and their permeates, the acid and H ÷ resin hydrolysis routes are not industrially attractive because of the complex side reactions occurring during such processes. Lactose hydrolysis by enzymatic routes presents a high selectivity. It can be carried out batchwise using soluble fl-galactosidase, also called lactase. This approach is justified in applications marginally sensitive to the cost of enzyme such as for dietetics purposes or for lactose intolerants. Immobilization of lactases was considered as a means to decrease the cost of enzymatic hydrolysis. The production of hydrolyzed lactose products at low costs is essential for applications as sweeteners or as intermediate food products such as protein-sweetener mixes. Immobilized lactase composites were developed using either Aspergillus niger or Aspergillus oryzae acid lactases. Immobilized lactase systems were designed and operated under industrial conditions to carry out the lactose hydrolysis reaction in lactic acid wheys, acidified sweet wheys, permeates, and demineralized permeates. Lactose hydrolysis can be considered to be a new unit operation which can be integrated in the transformation of a wide range of lactose-containing feedstocks. Starting from wheys, "lactolyzed" whey syrups are obtained which can be used as food ingredients or feed specialities. Depending on the level of demineralization, hydrolyzed permeates can be utilized as fermentation substrates or feed ingredients or elaborated into sweeteners or high DE syrup. (DE stands for "dextrose equivalent," defined as total reducing sugars in the syrup calculated as dextrose and expressed as a percentage of the total dry substance.) G. Coton, Address to the International Dairy Federation, Geneva, September (1979).

[38]

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413

Immobilized Lactase Composites

Preparation of Immobilized Lactase Composites fl-Galactosidases from A. niger and A. oryzae (also called acid fungal lactases) are immobilized on a porous silica carrier based on the procedures defined by Messing and Weetall. 2 This carrier is a controlled-pore SiO2 ceramic of 30/45 US mesh size (or about 0.50 mm mean particle size) and 350/k average pore diameter. The specific area is 45 m2/g, for a total pore volume of 0.6 cm3/g. This carrier is morphologically and chemically stable in a wide range of temperatures, it has good mechanical properties, and it is not biodegradable. Particle size and pore dimension are optimized to reduce external diffusion limitations to mass transfer, to limit pressure drops, and to maximize the enzymatic activity. The lactase is covalently bound to the controlled-pore silica cartier using the silane-glutaraldehyde immobilization procedure already described in the literature. 2 This technique can be briefly summarized as follows. The silanol groups on the silica surface react with 7-aminopropyltriethoxysilane to give an alkylamine-silica derivative. The free available amino groups are then activated by glutaraldehyde. The resulting activated carrier contacts the enzyme in order to obtain the immobilized lactase composite. The coupling efficiency decreases when higher lactase loading is used. This immobilization process was optimized and scaled up from the laboratory procedure to an industrial operation.

Properties of Immobilized Lactases Regarding kinetic behavior, the pH profiles of immobilized lactase composites are fairly similar to the soluble lactase with a shift to the acidic side in the range of 0.5-1 pH units. Immobilized A. niger lactase exhibits an activity of about 500 U/g at 50° at optimal pH in the range 3.5-3.8 (Table I). The Michaelis constant (Km) and the inhibition constant (Ki) were determined as Km= 0.053 M and Ki = 0.005 M. The activation energy calculated from the Arrhenius relationship of reaction rate to temperature is 12 kcal/mol. The deactivation energy determined from the variation of half-life (ti/2) as a function of temperature is 40 kcal/mol. 3 Immobilized A. oryzae lactase has an activity of 400 U/g at 40° at optimal pH in the range 4-4.5. Values of gm = 0.05 M and Ki in the range 0.020.05 M were reported 4 as well as an activation energy of 6 kcal/mol and a 2 R. A. Messing and H. H. Weetall, U.S. Patent 3,519,538. 3 j. R. Ford and W. H. Pitcher, Conf. Whey Prod. Chicago, Sept, (1974). 4 H. Hirohara, H. Yamamoto, E. Kawano, and T. Nagase, Int. Enzyme Eng. Conf., 6th (1981).

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TABLE I MAIN CHARACTERISTICS OF LACTASES AND IMMOBILIZED LACTASES Soluble lactases

Immobilized lactases

Source

Status a

pH optimum

pH stability

Temperature optimum

pH optimum

Activity b

A. niger A. oryzae

GRAS GRAS

3.5-4 4.5-5

3-8 3.5-8

55-60 50-55

3-3.5 4-4.5

500 U/g (50°) 400 U/g (40°)

GRAS, Generally recognized as safe. b Units are micromoles of lactose hydrolyzed per minute at optimum pH and defined temperature. thermal deactivation energy of 70 kcal/mol. The operational characteristics of A. oryzae lactase appeared to be more favorable to processing whey and permeates because of its optimum pH (4.5) and less inhibition by galactose. Engineering Considerations The development of immobilized lactase processes is basically dependent on several parameters: the operational characteristics of the immobilized lactase composite, the nature of the substrate feedstocks, the design of the reactor system, and the operating strategy. The overall performance of the system is the result of the interaction between these different parameters and determines the economy of the process. The key objective is to maximize the amount of hydrolyzed lactose which is processed per unit weight of catalyst. A long operational life is necessary to reach this objective. The deactivation of the immobilized lactase is mainly influenced by the operating temperature and pH, the nature of the feed, and the development of microbial contaminations. In industrial practice, several processes based on immobilized enzyme reactors are currently being used. The reactors have two points in common. First, a substrate feedstock of controlled purity is processed, and second, the development of microbial contaminations can be controlled because the substrate media are deficient and/or a selective environment can be used (temperature, pH, substrate concentration, microbial inhibitots). The situation using wheys and permeates is very different. These media are nutritionally rich and thus are excellent growth media for microorganisms. The possibility of controlling the development of microbial contaminations at low pH (below pH 3.5) and at temperatures above 35 °6 5 H. H. Weetall, N. B. Havewala, W. H. Pitcher, C. C. Detar, W. P. Vann, and S. Yaverbaum, Biotechnol. Biogen. 16, 295 (1974).

[38]

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415

is restricted for several reasons. Generally the addition of microbial inhibitors in the feedstock is industrially impossible because the presence of those substances must be avoided in the end product. The control of microbial contaminations in the reactor appeared as a critical technical problem to be solved in order for this technology to be developed.

Feed Substrate Different lactose-containing feedstocks have been evaluated. The main characteristics of these feeds are reported in Table II. The complexity of the feed increases as a function of the presence and concentrations of salts (defined as ashes), nitrogenous matter (total nitrogen x 6.38), true proteins (proteinaceous nitrogen × 6.38), and suspended solids. The highly demineralized permeates are for practical purpose very close to a pure lactose solution. However, their use may be limited because they are costly to produce by current ion-exchange techniques. In order to widen the applications of the technology it was important to develop processes that were adapted to the other different substrate feeds. Processing of permeates and electrodialyzed permeates showed that the presence of salts and different concentrations of cations and anions normally found in these feeds had no adverse effect on the performance and stability of the enzyme. The need for demineralization is therefore related to the application of the end product. The level of suspended solids in wheys and the presence of the colloids in sweet whey were found to be of particular importance during operations in a fixed bed reactor. Microbial contaminations in the feed also has a critical impact as the main source of contamination.

Reactor Design Reactors were designed in order to test the long-term stability performance of the immobilized lactase and the economics of the processes under industrial conditions. The design of the reactor aimed at maximizing the performances of the immobilized lactase reactor while minimizing or controlling at the same time operational problems such as microbial contaminations, pressure drops, and plugging. Three main types of reactors were considered: fixed bed, fluidized bed, and stirred tank reactors (Table III). A perfect fixed bed reactor behaves ideally as a plug flow reactor. An ideal continuous stirred tank reactor would behave as a perfect backmix reactor. Some backmixing is observed in fluidized bed reactors and the extent of bed expansion affects the performance. From a kinetic stand6 M. Harju, Nord. Med. Tidskr. 6, 155 (1977).

416

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

Z

z z

O

b, Z 0 O ,.=

k~

o~ O

m H

z

I

e~

o~

.r., ©

[38]

[38]

PRODUCTION-APPLICATION OF IMMOBILIZED LACTASE

417

TABLE III COMPARATIVE PERFORMANCES OF CONTINUOUS STIRRED TANK AND PLUG FLOW REACTORSa

Normalized residence timeb (units/ml-hr) Degree of conversion (%)

Continuous stirred tank

50 60 70 80 90

14 24.2 42.6 81.6 202.8

Plugf l o w

Relative amount: continuous stirred tank/plug flow reactor

6.4 9.8 14.8 22.6 37.6

2.2 2.5 2.9 3.6 5.4

Aspergillus niger lactase, Ki = 0.0054 M; K m = 0.0528 M; lactose

5% (w/w). b Normalized residence time (E/F). Plug flow reactor (E/F)pF = ~ I_

Ki

SoX + \ Ki

+

Continuous stirred tank reactor 1 [SoX + XKm(1 + XSo/Ki) ] (E/F)csTR = ~ 1 -- X J

where So is the initial lactose concentration (mol/liter); E, amount of enzyme (units); F, volumetric flow rate (ml/hr).

point a plug flow reactor appears to be the most efficient at minimizing the immobilized lactase requirement and the volume of the reactor. H o w e v e r , fixed bed reactors are k n o w n to be sensitive to the presence of suspended solids which m a y be present in feeds. This aspect was found to be of i m p o r t a n c e with w h e y feeds, so p r e t r e a t m e n t of w h e y feeds b e c a m e necessary to run satisfactory operations in a fixed bed mode. Microbial Contaminations

Sources of Contamination T h e r e are three main sources of microbial contamination that m a y affect the operations at industrial scale: microorganisms normally present in the feed substrate, microorganisms present in the reactor or adsorbed on the carrier, and accidental contamination occurring during the handling of the lactase c o m p o s i t e or other operations.

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It is not realistic to try to operate sterile reactors. However, the control of microbial contaminations to safe levels is an absolute necessity to monitor the performances of the reactor and also to guarantee the quality of the hydrolyzed products. In practice, the level of microbial contaminants in the feed substrate can be controlled by conventional heat treatment processes such as pasteurization which are well known in the dairy industry. The level of microbial contaminants in the product stream leaving the reactor is directly related to the level of contaminants in the feed, and it is also a function of the level of microbial contaminants which are present in the reactor. Because of the short residence time in the reactor, it was found that the increase of contaminants in the products is marginally related to the contaminants in the feed. The relative output/input ratios of microbial contaminants in the product and feed streams were good indicators in establishing the nature and trends of the microbial contaminations in the reactor. Approach to Immobilized Lactase Sanitation The development in the reactor of microbial contaminants, mainly yeasts and bacteria, can be critical. It was apparent that any commercially viable immobilized lactase process must incorporate a sanitizing or disinfecting procedure. This procedure must efficiently destroy the contaminating microorganisms without any appreciable effect on the immobilized lactase. Furthermore, when the product is intended for use in the food industry, the disinfecting agent often must meet governmental regulatory requirements. Methods for disinfecting immobilized enzyme reactors were evaluated. The different sanitizers or bacteriostatic agents were tested. Of them, acetic acid, which is commonly used as a dilute aqueous solution in laboratory studies, gives only limited results in industrial conditions. 7 Other known disinfectants, such as halogen derivatives, quaternary ammonium, and biguanidine polymers, were unsuitable because of partial or complete inactivation of the enzyme. Substituted diethylenetriamines of the following general formula were Ri

x

/

R4

/NCH2CH2NCHzCH2N \ R2 7 j. L. Baret, Brevet Franfais 2,471,192.

R3

R5

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PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE

419

found useful at laboratory scale to disinfect the immobilized lactase. Synergistic mixtures of dioctyldiethylenetriamine and trioctyldiethylenetriamine are commercially available from Th. Goldschmidt AG under the name Tego-Diocto BS. Disinfection processes based on the use of this product were scaled to industrial operations. 7 Operating Strategy

Industrial operations with immobilized enzyme reactors are dependent on two critical factors, activity and stability. The activity has to be maintained above some minimum value for an adequate period of time in order to guarantee satisfactory operations. When no particular care is taken, a rapid decrease in the activity is observed as a result of various phenomena such as the deposition of material within the bed, the formation of a coating around the particles, the development of microbial contaminations, channeling, or other problems. The thermal deactivation of the enzyme is not the controlling factor, as the apparent loss in activity was found to be reversible when cleaning-sanitation operations were operated on "dirty" or contaminated immobilized lactase (Table IV). Depending also on the nature and the quality of the feed, some pretreatment may be required to keep the activity constant during a period of time adequate for continuous production (Table V).

T A B L E IV HYDROLYSIS OF RAW WHEY AT p H 3.5 AND 50 °

Parameters

Test A"

Test B b

Flow rate (ml/hr) Immobilized lactase (g) Lactose (%) t = 2hr Glucose (g/liter) Degree of conversion (%) t = 6hr Glucose (g/liter) Degree of conversion (%)

102 4.68 4.26

103 4.68 4.26

17 78

16.5 76

11.8 54

11.4 53

" Test A was performed with freshly prepared immobi-

lized lactase. b Test B was performed subsequently to test A, after cleaning the immobilized lactase for 20 min with 1% aqueous acetic acid solution in a fluidized bed mode.

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[38]

TABLE V APPARENT DEACTIVATION DURING HYDROLYSIS OF CLARIFIED AND DEMINERALIZED WHEY WITH IMMOBILIZED LACTASEa

Whey

Operating temperature (°C)

Apparent half-life (hr)

Without heat treatment With heat treatment Without heat treatment With heat treatment

35 35 50 50

7 1980 4 124

a Whey was demineralized to 50% by electrodialysis (Ionics stack-pack) and acidified to pH 3.5 with HC1. It was then clarified by centrifugation on a Alfa Laval LAPX 202. Heat treatment was 1 hr up to 80°.

Hydrolysis and Cleaning-Sanitation Cycles Reactors were operated for long periods of time under semiindustrial conditions on a cyclic mode including a continuous production phase at constant temperature and cleaning-sanitation operations. 8 A first plant was operated at the Milk Marketing Board (MMB) technical division at Crudgington, United Kingdom, on a continuous basis 5 days a week with permeates demineralized by ion exchange. It processed about 350 liters/ hr, achieving 80% hydrolysis for 15-20 hr per day. The immobilized lactase was fluidized every day, when necessary with a dilute acetic acid solution (1%, volume basis). More than 100 operating cycles were carried out, during which the microbial contaminants in the hydrolyzed products were controlled in the range of 100-2000 total counts per milliliter. The conversion was maintained practically constant at the specified value during the operations at constant flow rate.

Impact of Cleaning-Sanitation Procedures on Stability Cleaning-sanitation procedures were developed to improve the operational stability for processing whey f e e d s . 9 In laboratory experiments, crude whey was received from a cheese factory and clarified with an Alfa Laval separator LAPX 202. The clarified whey was demineralized to 50% in an electrodialysis module (Ionics). It was then acidified to pH 3.5 with concentrated HC1 and stored at 2-4 °. The whey was heat-treated up to s L. A. Dohan, J. L. Baret, S. Pain, and P. Delalande, Int. Enzyme Eng. Conf. 5th (1979). 9 j. L. Baret and L. A. Dohan, Brevet Franfais 2,483,748.

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80 ° , centrifuged on the same separator, and cooled down to 4 ° . The whey was subsequently used as the feed for three columns, each column containing about 5 g of immobilized lactase, with the whey flow rate at 100110 ml/hr. Hydrolysis was conducted continuously for about 18 hr per day at 35 ° . At the end of the hydrolysis phase, columns were rinsed with water. In column 1, a solution of Alcalase 0.6L, at pH 7.5, 6 Anson units/ liter, from Novo was used to clean the immobilized lactase. It was then disinfected with a 0.1% solution of Tego-Diocto BS (Th. Goldschmidt AG). In column 2, only acetic acid was used at pH 3 and in column 3, only Tego-Diocto BS 0.1% in solution was used. These cleaning-sanitation procedures have a significant impact on the performance as seen from Table VI. This was confirmed during two semiindustrial projects using cottage cheese whey and acidified sweet whey, cleaning-sanitation procedures were optimized to maximize the productivity of the immobilized lactase.

Temperature Program The knowledge of both the activation energy and the thermal deactivation energy allows an estimation of the theoretical life of the enzyme as a

TABLE VI ACTIVITY AND STABILITY OF THE IMMOBILIZED LACTASE DURING HYDROLYSIS OF WHEY IN RELATION TO DIFFERENT CLEANING AND/OR DISINFECTING PROCEDURES

Column 1: protease and Day

Tego-Diocto BS

Column 2: acetic acid

Column 3: Tego-Diocto BS

258 202 163 117 36 (stop)

174 143 125 122 107 104 98 35 90

Activity (units/g) 1 5 10 15 17 20 26 Half-life(days) Stability (%)0

235 296 233 220 223 213 203 55 96

9 60

Stability (%) is the average statistical ratio of the activity after 17 hr to the activity after 2 hr hydrolysis over all the days of operations.

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ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[38]

function of the initial operating temperature. The theoretical life will increase when the initial operating temperature decreases, so initial low temperature operation is necessary to maximize the productivity. However, in practice, a pure thermal deactivation of the enzyme may not be the only controlling phenomenon at low temperature. The initial productivities of the reactors were maintained constant at specified values by raising the temperature when it was necessary. Actual gains in activities were about 5-10% per degree centigrade in the range of operating temperatures which were considered (20-45 °) with various feeds. This operating temperature approach in conjunction with an adequate cleaning-sanitation process made it possible to keep the performances of the reactors constant over periods of time sufficient to demonstrate the economic feasibility of the process. Industrial Developments These semiindustrial operations increased our confidence in the technology for hydrolyzing wheys and permeates. The technology was transferred to full-scale operations. Corning established joint ventures with major partners in the food industry: the Specialist Dairy Ingredient company (SDI) with the Milk Marketing Board in England, the Nutrisearch Company with the Kroger Company in the United States, and Corvire with Union Laiti~re Normande (ULN) in France. A plant processing 20,000 liters/day of sweet whey is operated by SDI at Aston (Cheshire). The hydrolysis reactor can process 1000 liters/hr of nondemineralized acidified sweet whey with a load of about 40 kg of lactase composite. Lactolyzed whey products are obtained, and are formulated as "sweet-protein" syrups which can be used as ingredients in different sectors of the food industries. The SDI production was used to develop the market for this new product in confectionary, ice cream, and baked products. Capacity expansion is under way. The most advanced operation is the Nutrisearch plant in Winchester, Kentucky, which combines the immobilized lactase technology of Corning and continuous fermentation technology of Kroger Co. Investments for that plant were 15 million dollars for a nominal capacity of 100,000 gallons of raw cottage cheese whey per day. Whey is processed by ultrafiltration to obtain a protein-rich retentate. This stream is then formulated as a dried whey protein concentrate. The permeate stream is pumped into two hydrolysis columns. These columns are 3-ft-diameter and 15-ft-high vessels filled with about 1500 kg of lactase composite. The lactolyzed permeate stream can then be fermented by a selected Saccharomyces cerevisae strain in a continuous fermenter to produce baker's yeast.

[39]

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423

A semiindustrial unit (10,000 1/day) is operated by Corvire in the U L N plant at Conde-sur-Vire. H o w e v e r , the industrial development was slower there because of the specific regulatory constraints in France. L a c t o s e hydrolysis with immobilized lactase is now an industrial reality. A subsequent expansion in industry is expected in the coming years.

[39] Continuous Treatment of Ultrahigh-Temperature Sterilized Milk Using Immobilized Sulfhydryl Oxidase 1 B y HAROLD E. SWAISGOOD, VIOLETA G. JANOLINO, and

PAUL J. SKUDDER Thiols are often responsible for undesirable flavors in foods because of their extremely low organoleptic threshold concentrations. The " c o o k e d " flavor of ultrahigh-temperature (UHT) 2 sterilized milk is a familiar example of such " o f f flavors." Aseptic packaging of U H T milk allows the product to be merchandized and stored at ambient temperature. If the flavor could be acceptable by a larger fraction o f the population, a potential energy saving could be realized. Consequently, discovery that an e n z y m e indigenous to unheated milk, and thus aesthetically acceptable as a processing aid for milk, could be used to eliminate the c o o k e d flavor 3,4 has important practical value. Isolation of Protein Fractions Having Increased Sulfhydryl Oxidase Activity Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane enzyme existing primarily in the membrane vesicle fraction of skim m i l k ) -~ This work is Paper No. 9349 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products. 2 Abbreviations used: UHT, ultrahigh-temperature; CPG, controlled-pore glass; SOX, sulfhydryl oxidase; DTNB, 5,5'-dithio-bis(2-nitrobenzoicacid); GSH, reduced glutathione. 3 H. E. Swaisgood, U.S. Patent 4,053,644 (1977). 4 H. E. Swaisgood, U.S. Patent 4,086,328 0978). 5 V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975). 6 M. B. Sliwkowski, M. X. Sliwkowski, H. E. Swaisgood, and H. R. Horton, Arch. Biochem. Biophys. 211, 731 (1981). 7 M. B. Sliwkowski, H. E. Swaisgood, and H. R. Horton, J. Dairy Sci. 65, 1681 (1982). 8 M. X. Sliwkowski, M. B. Sliwkowski, H. R. Horton, and H. E. Swaisgood, Biochem. J. 209, 731 (1983). METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[39]

UHT

STERILIZED MILK TREATMENT USING SOX

423

A semiindustrial unit (10,000 1/day) is operated by Corvire in the U L N plant at Conde-sur-Vire. H o w e v e r , the industrial development was slower there because of the specific regulatory constraints in France. L a c t o s e hydrolysis with immobilized lactase is now an industrial reality. A subsequent expansion in industry is expected in the coming years.

[39] Continuous Treatment of Ultrahigh-Temperature Sterilized Milk Using Immobilized Sulfhydryl Oxidase 1 B y HAROLD E. SWAISGOOD, VIOLETA G. JANOLINO, and

PAUL J. SKUDDER Thiols are often responsible for undesirable flavors in foods because of their extremely low organoleptic threshold concentrations. The " c o o k e d " flavor of ultrahigh-temperature (UHT) 2 sterilized milk is a familiar example of such " o f f flavors." Aseptic packaging of U H T milk allows the product to be merchandized and stored at ambient temperature. If the flavor could be acceptable by a larger fraction o f the population, a potential energy saving could be realized. Consequently, discovery that an e n z y m e indigenous to unheated milk, and thus aesthetically acceptable as a processing aid for milk, could be used to eliminate the c o o k e d flavor 3,4 has important practical value. Isolation of Protein Fractions Having Increased Sulfhydryl Oxidase Activity Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane enzyme existing primarily in the membrane vesicle fraction of skim m i l k ) -~ This work is Paper No. 9349 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products. 2 Abbreviations used: UHT, ultrahigh-temperature; CPG, controlled-pore glass; SOX, sulfhydryl oxidase; DTNB, 5,5'-dithio-bis(2-nitrobenzoicacid); GSH, reduced glutathione. 3 H. E. Swaisgood, U.S. Patent 4,053,644 (1977). 4 H. E. Swaisgood, U.S. Patent 4,086,328 0978). 5 V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975). 6 M. B. Sliwkowski, M. X. Sliwkowski, H. E. Swaisgood, and H. R. Horton, Arch. Biochem. Biophys. 211, 731 (1981). 7 M. B. Sliwkowski, H. E. Swaisgood, and H. R. Horton, J. Dairy Sci. 65, 1681 (1982). 8 M. X. Sliwkowski, M. B. Sliwkowski, H. R. Horton, and H. E. Swaisgood, Biochem. J. 209, 731 (1983). METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MILK .Centrifuge (4080 g, 50 min, 30") SKIM MILK

iChymosin (2 ml of Img/ml solution per liter of skim milk) 90 rain, 50 ° Centrifuge (16,300 g, 45 rain, 5 °)

WHEY • Add (NH4)2SO4 to 50*/.

saturation at 4* • Allow to stoodovernight,4* • Centrifuge (16,500 g, 60 rain, 4*)

• Filter (0.4S ~um filter] • Diafilter (100,000 Mr

membrane)against 47 ma sodiumphosphate, pH 7.0, 20-23*

• Filter (0.45 pm filter

,Molecular sieve chrorr~togmphy on 500 nm pore diameter glycerolpropyI-CPG equilibrated with 47 mM sodium phosphatepH 7.0

Pellet

Membrane retentate

Void volume fraction

(Crude skim milk

(Crude skim milk membrane

(Skim milk membrane

membrane fraction)

vesicles)

vesicles)

FlG. 1. Schematic illustration of methods for preparation of various skim m i l k membrane

vesicle fractions.

Several methods have been developed for preparation of active isolates directly from whey which are therefore commercially attractive 5,9-1~ (Fig. 1). Whey is obtained from the skim milk upon removal of the caseins by clotting with chymosin (rennin). This reaction occurs at the pH of milk (6.6-6.8) and results from the hydrolysis of a specific peptide bond in Kcasein; thus other proteins are not proteolyzed in this treatment. Because the preparation of SOX requires that the pH is maintained above 6, commercial whey which meets this criterion may also be suitable for enzyme preparation. Of the three methods listed in Fig. 1 for obtaining preparations of skim milk membrane vesicles, ultrafiltration and molecular sieve chromatography are the most attractive because of their simplicity. The chromatographic method has yielded the most active preparations, typically purified 300-fold from whey. 9 However, larger volumes are more easily processed by diafiltration than by chromatography on CPG-3000. Availability of membranes with a larger molecular weight cutoff would improve the purification obtained during diafiltration of whey. The size of 9 V. G. Janolino, D. A. Clare, and H. E. Swaisgood, Biochim. Biophys. Acta 658, 406 (1981). l0 H. E. Swaisgood, M. X. Sliwkowski, P. J. Skudder, and V. G. Janolino, in "Utilisation des Enzymes en Technologic Alimentaire" (P. Dupuy, ed.), p. 229. Technique et Documentation Lavoisier, Paris, 1982. 11 V. G. Janolino and H. E. Swaisgood, J. Dairy Sci. 67, 1161 (1984).

[39]

U H T STERILIZED MILK TREATMENT USING S O N

425

most of the vesicles appears to be between 200 and 300 nm in diameter, 12 so that methods which select for thissize range should yield the most homogeneous preparations of these membranes. An increase in SOX activity relative to y-glutamyltransferase activity can be obtained by a combination of diafiltration followed by chromatography on CPG-3000. Sulfhydryl oxidase may be further purified by solubilization of the membrane vesicles with nonionic detergent and isolation of this enzyme by transient covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. 8 At present, however, the quantities obtainable by this procedure prevent its commercial application. Immobilization of the Enzyme Several methods involving both covalent attachment and adsorption on inorganic matrices have been investigated as means for immobilization. Continuous processing and aseptic packaging of U H T milk require extremely high flow rates, and the enzyme support must be compatible with this requirement. Porous and nonporous glass or ceramics were chosen because of their mechanical strength, excellent flow characteristics, and the ease with which they can be cleaned. Unlike other enzymes which have been developed for use in immobilized forms, SOX is a membrane enzyme, and consequently, certain considerations are important to the choice of immobilization conditions. The pore size of the support matrix and the degree of enzyme solubilization are major factors in determination of the specific activity of the immobilized catalyst. 1°,11,~3 If the enzyme is not solubilized it is substantially excluded from the pore volume of matrices having pore diameters as large as 300 nmH; accordingly, the specific activity (U/g) of such catalysts is similar to that prepared on a nonporous support of equivalent particle dimensions. 13 However, the enzyme can be solubilized in the nonionic detergent polyoxyethylene 9-1auryl ether without loss of activity. 7 In I% solutions of this detergent substantial penetration of the pore volume of matrices with pore diameters as small as 100 nm has been observed. ~3 The above considerations are substantiated by the activities obtained for various immobilized forms of the enzyme prepared by covalent attachment to controUed-pore glass (CPG) beads. These results, listed in Table I, show that solubilization of the enzyme improves the specific activity of the immobilized catalyst. It is also clear from the data that chromatographically purified membrane vesicles, which had the highest specific t2 V. G. Janolino, C. S. Barnes, and H. E. Swaisgood, J. Dairy Sci. 63, 1969 (1980). 13 V. G. Janolino and H. E. Swaisgood, J. Dairy Sci. 61, 393 (1978).

426

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[39]

TABLE I ACTIVITIES OF IMMOBILIZED ENZYME PREPARATIONS OF VARIOUS ENZYME ISOLATES

Activity Preparation

Support

(U/g)

50% saturated (NH4)2SO4

SuccinamidopropylCPG-2000 Aminopropyl-CPG-2000

0.9"

Succinamidopropyl-

1.9 c

precipitate Solubilized diafiltered whey Chromatographically purified skim milk membrane vesicles Solubilized chromatographically purified skim milk membrane vesicles a

1.5 b

CPG-1400

Succinamidopropyl-

6.4 c

CPG-1400

From Janolino and Swaisgood.t4

b F r o m V. G. Janolino and H. E. Swaisgood (unpublished observa-

tions). c From Janolino and Swaisgood. H

activity for SOX, yielded the most active enzyme particles. In each case the succinamidopropyl-CPG was activated with water-soluble carbodiimide using either a simultaneous or a sequential activation-immobilization procedure.14 Solubilized enzyme was immobilized on the aminopropyl-CPG by preactivation of the matrix with 2.5% glutaraldehyde. Independent studies have shown that each of these three covalent immobilization methods yield preparations having comparable activity. Although covalent immobilization of purified membrane vesicles has resulted in more active preparations, our recent studies have resulted in a method of immobilization of SOX which may be more attractive for preparation of a commercial reactor. These results (Fig. 2) show that SOX is adsorbed directly from whey onto the cationic, silica-based matrix Spherosil QMA (P. J. Skudder and H. E. Swaisgood, unpublished results). Furthermore, it appears that SOX is adsorbed in preference to some of the other whey proteins. Thus, a 20-g reactor was loaded with enzyme simply by passing 600 ml of solubilized diafiltered whey through the column of beads. The resulting immobilized catalyst typically displayed activities similar to those obtained by covalently immobilizing the 50% (NH4)2SO4 precipitate. Furthermore, the activity could be stabilized t4 V. G. Janolino and H. E. Swaisgood, Biotechnol.

Bioeng. 24~ 1069

(1982).

[39]

427

U H T STERILIZED MILK TREATMENT USING S O X

i i ~ 4'0 protein coaceatratio~ in retentote

"10 50 t*.

0.03 activity

8

in

dll~lP reteotate

" •0.02

2.0 ~

.>_

3 1,0 ~

0.01

0

0

I

_ _ 1

200

400

0 600

Volume of the SOX isolate passed through matrix (ml) FIG. 2. Adsorption of SOX activity on Spherosil QMA upon passage of solubilized, diafiltered whey retentate through a 20-g column of the beads. Whey was diafiltered using a 100,000 Mr membrane against 47 mM sodium phosphate, pH 7.0, and solubilized with 1% polyoxyethylene 9-1auryl ether. The flow rate was 25 ml/hr at 5°. (©) SOX activity; ( 1 )

protein concentration in the emerging stream.

by cross-linking with a 0.1% (w/v) glutaraldehyde solution, yielding a reactor with an operational stability which appeared to be similar to that observed for covalently immobilized forms. Characteristics of Sulfhydryl Oxidase Reactors Laboratory-scale reactors of immobilized SOX have been operated continuously with UHT milk using both the fixed-bed and the fluidizedbed configurations. Most of the data have been obtained with a 2-liter reactor 1°,15containing SOX covalently immobilized on 170 g of succinamidopropyl-porous silica (200 nm pore diameter, 80/120 mesh) which was operated in both reactor configurations continuously at 30 °, which is near the temperature optimum for enzyme activity. The catalytic efficiency was comparable in either configuration; however, reactor plugging should not be a problem in the fluidized-bed mode. Characteristics of a number of is H. E. Swaisgood, V. G. Janolino, and M. X. Sliwkowski, "Proceedings: International Conference on UHT Processing and Aseptic Packaging of Milk and Milk Products," p. 67. Department of Food Science, North Carolina State University, Raleigh, North Carolina, 1980.

428

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[39]

PREPARATION

1

POTENTIAL FOR LONG-TERMSTORAGE BEFORE USE TOTAL POTENTIAL USEFULLIFE > 3 MO

1 IN-LINE PLACEMENT ~ 1 f l 3-6 DAY OPERATION

l REGENERATION WITH 4M UREA ~ REGENERATION ULTRAFILTRATION

RECOVER& REGENERATE SUPPORTBY HEAT TREATMENT FIG. 3. Schematic illustration of the proposed operational protocol for use of an immobilized SOX reactor.

smaller reactors containing various immobilized forms of the enzyme also have been determined. Conclusions drawn from these results suggest that typically a reactor has a half-life of about 1 week during continuous operation with U H T milk, and that the activity may be regenerated by washing the reactor with a sterilized 4 M urea solution. ~5,~6For example, the 2-liter reactor was operated in the fluidized-bed configuration over a 3-month period using six regeneration cycles with 4 M urea, and retained 80% of its original activity. 16 The immobilized enzyme is extremely stable during storage under conditions which are sterile or limiting to microbial growth, e.g., 10% ethanol. 17A suggested protocol for operation of a SOX reactor is outlined in Fig. 3. It should be noted that even for covalently immobilized forms, the support matrix can be regenerated after the enzyme becomes inactive, thus allowing fresh enzyme to be immobilized. Also, the 4 M urea solution can be maintained sterile and can be regenerated by ultrafiltration. The activity of SOX reactors was routinely assayed with 0.8 mM GSH 5 either in 47 mM sodium phosphate, pH 7.0, or in milk ultrafiltrate. The rate of thiol oxidation was measured from the decrease in concentration determined by reaction with DTNB ~ using e4~2 = 13,600 M -I cm-L Typically, 85-/.d aliquots were removed and added to 1.0 ml of 100 # M DTNB in 0.1 M sodium phosphate, pH 8.0, containing 10 mM EDTA, and 16 M. X. Sliwkowski and H. E. Swaisgood, J. Dairy Sci. 63 (Suppl. 1), 60 (Abstr, DR 56) (1980). 17 H. E. Swaisgood, V. G. Janolino, and H. R. Horton, AIChE-I. Chem. E. Syrnp. Set. 74, (No. 172) 25 (1978).

[39]

U H T STERILIZED MILK TREATMENT USING S O N

I

I

I

I

I

429

t

Z Z O

111 ~e ttl

Z

0 U Z Ill U n, uJ a.

/8'1

100 300 500 N O R M A L I Z E D RESIDENCE T I M E

I

I

700 900 E/F ( U N I T S - M I N / L )

FIG. 4. Conversion of substrate to products as a function of normalized residence time. (1) Column assays; (Is) recirculation assays; (A) batch assays. Reproduced with permission from Swaisgood e t al. 15

the absorbance at 412 nm read after 2 min. 8 One unit of enzyme activity corresponds to 1/zmol SH oxidized per minute at 35°. To facilitate scaling up of the reactors, the percent oxidation of 0.8 mM GSH solutions was determined as a function of a "normalized residence time" (Fig. 4)) °,]5 Using this curve, for example, if the desired

430

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

100

I

[

L

[39]

[ ...... -

E 80--

Q

ul

~

6 0 ~

--

_z soFU

uJ F-

LU Q

I,,-

~

3 0 - -

--

U E

L

2o

I 320 400 480 560 N O R M A L I Z E D R E S I D E N C E T I M E (GSH U N I T S - M I N / L )

FIG. 5. Relationship between the degree of cooked flavor and the extent of enzyme treatment of UHT milk.

degree of oxidation and the flow rate through the reactor are known, the amount of enzyme required to yield the desired result can be calculated. Flavor Modification of UHT Milk Both trained and untrained consumer panels have been used to organoleptically evaluate enzyme-treated UHT milk and compare it to untreated UHT milk and commercial pasteurized milk. 3'1°']5'18 UHT milk is H. E. Swaisgood, Enzyme Microb. Technol. 2, 265 (1980).

[39]

U H T STERILIZED MILK TREATMENT USING S O X

431

was treated using various immobilized SOX reactors, including the 2-liter reactor, and by injection of filter-sterilized enzyme into aseptic packages. The flavor was evaluated immediately after treatment with immobilized enzyme. Similar results have been observed using both methods of treatment; however, injection of soluble enzyme requires preparation of covalent affinity chromatographically purified enzyme to avoid the occurrence of proteolytic activity in the injected solution. In a triangular test, the untrained panel of judges could not distinguish enzyme-treated U H T milk from commercial pasteurized milk, but all of the panelists could distinguish between UHT milk and enzyme-treated U H T milk by headspace odor alone. To relate the degree of cooked flavor to the extent of enzyme treatment, a panel of experienced milk judges was used to evaluate the flavor of U H T milk which had received varying degrees of enzyme treatment, as measured by the normalized residence time. 10.15,18For this purpose, the units of enzyme activity were determined by assay with GSH; thus the normalized residence time is the same as that shown in Fig. 4. The degree of cooked flavor is expressed as the percentage of the panel which could detect the flavor. The relationship between these two parameters is shown in semilogarithmic form in Fig. 5. The linearity of this relationship suggests that the disappearance of the flavor compound(s) follows first-order kinetics, i.e., the concentration is much less than the Km, as would be expected for such flavor compound(s). The above results suggest that cooked flavor is reduced below the threshold level by a treatment corresponding to 60% oxidation of 0.8 mM GSH. Combination of the results in Figs. 4 and 5, using a normalized residence time of 510 units-min/liter,10 indicates that 8500 units of enzyme activity would be required per 1000 liter/hr processing rate. Typically, the specific activity of immobilized SOX varies between 0.1 and 1.0 units per milliliter of catalyst, which means that the reactor size for a 1000 liter/hr processing rate should be 8.5 to 85 liters. To maintain a constant production rate, one would predict that the most effective system would use multiple reactors staggered with respect to their age so that the oldest could be replaced on a schedule derived from knowledge of the immobilized enzyme's half-life.

432

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[40]

[40] P r o d u c t i o n o f I s o m a l t u l o s e U s i n g I m m o b i l i z e d M i c r o b i a l Cells B y PETER S . J .

CHEETHAM

In the last 25 years enzymes have acquired many actual and potential applications in the production of sugars and sweeteners. Undoubtedly the most significant so far is high-fructose corn syrup produced using immobilized glucose isomerase. Others include maltose syrups, Aspartame, and cyclodextrins. In this article I would like to give some reasons why we were interested in isomaltulose as a product and the attractions of using an immobilized cell process for its production. In particular I shall describe some of the methodological advances made and the experimental approaches we found useful, particularly when considering the requirements of a large-scale production process. The objective was to produce a pure, cheap product in as great a yield as is possible and compatible with easy operation of the process.

Properties of Isomaltulose Isomaltulose (alias palatinose, lylose; 6-O-a-D-glucopyranosyl-D-fructofuranose) is a natural component of honey. ~It has a number of interesting properties. First, isomaltulose has a sweetness typical of disaccharides, being only about a third as sweet as sucrose but with a sweetness profile very similar to that of sucrose. Isomaltulose is a reducing sugar and so causes browning reactions to occur in foods to a greater extent than does sucrose, and has potential applications in intermediate-moisture foods where the use of sucrose is restricted by its high sweetness. This low sweetness intensity is also useful in that the flavors of many isomaltulose-containing foods are masked less than in similar products containing sucrose. It can be readily crystallized and is metabolized in a very similar way to sucrose 2,3 and so can be used as a calorific bulking agent, for instance to completely replace the sucrose in foods and drinks. 4,5 Isomaltulose has a calorific value of 4 kcal/g but shows a slower i I. R. Siddiqui and B. Furgala, J. Apic. Res. 6, 139 (1%7). 2 I. Macdonald and J. W. Daniel, Nutr. Rep. Int. 28, 1083 (1983). 3 p. S. J. Cheetham, in "Developments in Sweeteners" (C. K. Lee and M. G. Lindley, eds.), Vol. 3, p. 105. Applied Science, Barking, Essex, U.K., 1982. 4 C. Bucke and P. S. J. Cheetham, United Kingdom Patent 2,066,539B [Tate & Lyle, PLC]

(1983). 5 K. Suzuki, New Food Ind. 26, 1 (1984).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[40]

ISOMALTULOSE PRODUCTION USING MICROBIAL CELLS

433

rate of release of monosaccharides into the blood. Therefore insulin release is correspondingly reduced compared with other simple sugars, creating the possibility of applications in diabetic and sports foods and drinks. Second, it appears to be noncariogenic; that is, it is not used by Streptornyces mutans to form the acid and/3-glucan polymers that are important factors in causing tooth decay.6-8 This property of isomaltulose has been exploited in a caries-preventing sugar composition containing Aspartame and isomaltulose. 9 Third, it is very much more resistant to acidic hydrolysis than. sucrose, making it very much less likely to invert when incorporated, into carbonated beverages, 4 for instance. Fourth, it has the property of being an excellent excipient in tablets, l° Finally whereas most enteric bacteria can metabolize most sugars, isomaltulose can only be utilized by bifidobacteria. Thus isomaltulose encourages the growth of the bifidobacteria and discourages the growth of potentially putrifactive microorganisms that have a tendency to cause diarrhea. It has therefore been tested in food, drink, and medicine formulations, especially those containing dairy products. 11 The crystalline structure of isomaltulose has been established: there is one intramolecular hydrogen bond between the 2 and 2' carbon atoms, and several intermolecular hydrogen bonds are present. IH NMR and 13C N M R spectroscopy has been carried out showing that the a and 13isomers are present in a ratio of 1 : 4 at 34°, very similar to the values measured for fructose.~2.~3 Several chemical derivatives of isomaltulose have been synthesized. 14 Production of Isomaltulose by Fermentation The chemical synthesis formation of isomaltulose time: as an intermediate in the South German Sugar

of isomaltulose is very difficult. The microbial has attracted commercial interest for some Palatinit (or Isomalt) manufacture, initially by Co. who produced it by fermentation using

6 K. R. Roberts and M. C. Hayes, Scand. J. Dent. Res. 88, 201 (1980). 7 y . j. Kadomura, Tokyo Dental College Soc. 82, 407 (1982). s T. Ooshima, A. Izumitani, S. Sobue, N. Okahashi, and S. Hamada, Infect. lmmun. 39, 43 (1983). 9 Mitsui Sugar Co., Japanese Kokai Tokyo Koho Application 8,218,925 (1983). l0 M. G. Lindley and S. Hathaway, United Kingdom Patent Application 2,066,640A (1981). H Calpis Shokuhin Kog (rika), Japanese Patent Application JS 7091-193 (1980). 12 W. von Dreissig and P. Luger, Acta Crystallogr., Sect. B B29, 514 (1973). 13 A. de Bruyn, J. van Beeumen, M. Anteunis, and G. Verhegge, Bull. Soc. Chim. Belg. 84, 799 (1975). 14 yon F. Loss, Z. Zucherind. 19, 323 (1969).

434

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[40]

Serratia plymuthica (NCIB 8285) or a strain described as Protaminobacter rubrum (CBS 574.77) 15, and more recently by Bayer AG 16 using the same strains. The cells were isolated from sugar beet refineries in the 1950s and grown on an undefined medium containing 5% sucrose and other nutrients until a high cell concentration was reached after - 2 4 hr and then used to convert a diluted refinery sucrose stream (20% w/w) which is aerated and maintained at pH 7.0 until all the sucrose is completely converted into isomaltulose (-12 hr). The cells are recovered using a jet separator and can be reused approximately six times, provided sterile conditions can be maintained. The isomaltulose solution is then deionized using a strong acidic cation exchanger in the H ÷ form, such as Amberlite 200, and a weak basic anion exchange in the OH- form, such as Amberlite IRA 93. The deionized solution is concentrated under vacuum to 65% (w/w) and cooled and the isomaltulose crystallized in two stages before separation in a basket centrifuge. In a continuous process cells are grown continuously and the cell suspension removed from the fermenter, mixed with a concentrated sucrose solution, and processed in a cascade of three to five stirred vessels so as to achieve complete conversion of the sucrose. The cells are separated and recycled, and the isomaltulose solution purified and crystallized as above. Crueger et al. 16have described the production of isomaltulose by continuous fermentations. Sucrose solutions (25% w/w) were converted at a dilution rate o f - 0 . 1 hr -I. Thus once a steady state had been reached the productivity of their fermenters was - 2 5 g isomaltulose. This value is higher than achieved in batch fermentations where productivities of - 1 7 g/liter-hr were reported in UK Pat. spec. 1,429,334. Since these cells can be reused up to six times total productivity may approach 120 g/liter per 84 hr. The comparable value for our immobilized cell system was 40 g/liter-hr, but of course because the immobilized cells are so stable their productivity aggregated over a long time period such as a year (200 kg/ liter-year) is very much higher than that of the continuous fermentation. The isomaltulose so formed is then hydrogenated at alkaline pH at 100-125 ° using Raney nickel Type B l l 3 Z to form Palatinit which comprises an equimolar mixture of 6-O-a-o-glucopyranosyl-o-sorbitol and 1-O-o~-D-glucopyranosyl-o-mannitol.15.16The uncrystallized product is not directly hydrogenated, presumably because the hydrogenated sugars derived from the trehalulose would make the product less pure and therefore 15 South Germane Sugar Co., United Kingdom Patent Spec. 1,429,334 (1976). ~6W. Crueger, L. Drath, and M. Munir, European Patent Application 78,100,803.2 (1979). t7 H. Schiweck, Alimenta 19, 5 (1980).

[40]

ISOMALTULOSE PRODUCTION USING MICROBIAL CELLS

435

less acceptable to regulatory authorities. Isomalt is a white crystalline material of about half the sweetness and calorific value of sucrose and which also exhibits low cariogenicity and good stability toward enzymatic and microbial hydrolysis over a wide pH range. It is manufactured and sold by Palatinit AG, a subsidiary of the South German Sugar Co. and Bayer AG, as a low-calorie bulking agent/sweetener. ~7They now use immobilized cells to produce the isomaltulose (see later) rather than fermentation methods. We have also found that isomaltulose could be formed by a solid-state fermentation of sugar cane. The cane was diced and crushed and inoculated with one of the organisms described in this paper. Following incubation at 30° for several days, isomaltulose could be recovered in low yield by hot-water extraction of the cane. This method depends on the isomaltulose-producing organisms also secreting pectinases to break down the cane, thus liberating sucrose for conversion to isomaltulose. Potential Advantages of the Use of Immobilized Cells Efforts have been made to produce isomaltulose using immobilized cells as they offer several potential advantages over the fermentation process. 18,19 Isomaltulose formation takes place most efficiently when nongrowing cells are used. Therefore, the cell production and product formation steps can be individually optimized since they are performed separately. Very high densities of cells can be achieved, much greater than could be achieved in conventional fermentations without washout occurring. Also high concentrations of substrate that are inimical to cell growth can be used. The high energy inputs required by the need for agitation, aeration, and the maintenance of sterility are reduced, very little substrate is diverted into cell growth, and less wastes are often produced. Last, continuous operation is possible such that the lengthy nonproductive periods that occur between batch fermentations are avoided or greatly reduced. Choice of Microorganism A number of strains of microorganism including several Enterobaceriacae strains were tested for isomaltulose production, z°-22 Since the mi~8p. 19 C. 20 C. :~ P. 22 p.

S. J. Cheetham, Top. Enzyme Ferment. Biotechnol. 4, 189 (1980). Bucke, Philos. Trans. R. Soc. London, Ser. B 300, 369 (1983). Bucke and P. S. J. Cheetham, U.S. Patent 4,359,531 (1982). S. J. Cheetharn, C. E. lmber, and J. Isherwood, Nature (London) 299, 628 (1982). S. J. Cheetharn, C. Garrett, and J. Clark, Biotechnol. Bioeng. 27, 471 (1985).

436

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[40]

croorganisms screened were intended for use in a nongrowing immobilized form it proved advantageous to screen them in this state so as to obtain an organism with a high activity, but also with a high stability when used in an immobilized form. Samples of each cell strain were grown and immobilized separately. Growth was aerobic at 30 ° in 500-ml shake flasks, containing 200-ml aliquots of medium consisting of sucrose (40 g/liter), which acts as a carbon and energy source and also induces the enzyme, peptone (10 g/liter), and beef extract (4 g/liter). Several strains with a high activity were obtained, three of which, all Erwinia rhapontici strains, had high operational stabilities when tested in columns. Evidence for a periplasmic location for the isomaltulose-synthesizing activity was provided by the observation that the addition of a nonlethal dose of the antibiotic lincomycin (10 rag/liter) to the complex medium enhanced the cells activity by 60%. Lincomycin is known to limit the synthesis of, or otherwise inhibit proteases that degrade other periplasmic proteins. 23 On a large scale, cells were grown in a defined medium developed as part of our process improvement program, and flocculated with a chemical flocculant prior to concentration by centrifugation. Use of a nonsucrose-limited medium was advantageous so that once growth had finished some sucrose remained to stabilize the enzyme activity. Due to the difficulties in recovering small bacterial cells using a Westphalia disk-stack centrifuge, flocculant was added to aggregate the cells and thus increase their rate of sedimentation. The cationic polyquaternary flocculants Cyanamid C573 and C577 proved to be most effective. The precise dose of flocculant was optimized for each fermentation in laboratory experiments immediately prior to addition to 800 liters of fermenter broth. The flocculant was added diluted 10-fold in water and mixed gently in the fermenter for 10 rain to ensure even distribution prior to recovery of the cells by centrifugation. Addition of excess flocculant was deleterious since the strength of the alginate pellets was weakened. During growth capsular polysaccharide was produced, which interfered with the recovery of the cells in a concentrated form by centrifugation. The extent of polysaccharide production was proportional not only to the amount of sucrose present, but also to the degree of agitation of the culture medium. Since Erwinia is a facultative anaerobe the polysaccharide is probably produced in response to high pO2 values, the capsular polysaccharide imposing a diffusional restriction on the rate of oxygen transfer from the bulk medium to the cells. Polysaccharide production appeared to be at the expense of isomaltulose production such that high 23 M. Levner, F. P. Wiener, and B. A. Rubin, Infect. Immun. 15, 132 (1977).

[40]

ISOMALTULOSE PRODUCTION USING MICROBIAL CELLS

437

activity cells were obtained from poorly agitated fermentations and vice versa. Once immobilized, the cells are in a relatively anaerobic environment due to the difficulty oxygen has in diffusing into the pellet, and no further polysaccharide is produced and more isomaltulose is formed. Immobilization The cells are collected by centrifuging at 23,000 g for 20 min at 30° 21 (see Fig. 1). The cells are evenly dispersed as a 20% (wet weight/v) slurry in freshly prepared 5% (dry weight/v) sodium alginate using an overhead turbine impeller. Dissolution of the alginate powder is much easier if warm deionized water is used. Alginate is supplied in a sterile form and can be dissolved and immobilized in autoclaved solutions if required. 24 Immobilization on a small scale is by extruding dropwise from a syringe. On a larger scale the cell slurry could be pumped through narrow-bore tubing. Gelation takes about 2-3 hr to complete at room temperature using a large excess of 0.1 M CaC12 solution (pH 6.5). Even-sized, smooth, spherical pellets with a diameter of 3-5 mm and consisting of a thin, highly polymerized skin and a core of highly porous gel are formed. Sodium alginate (Protanal LF10/60, BDH) extracted from Laminaria hyperborea, which has a high ratio of guluronic to mannuronic acid blocks and thus a high mechanical strength, is preferred in all the experiments. The gel is formed by calcium ions cross-linking guluronic acid residues in adjacent polysaccharide c h a i n s . 24 Many of the alginates tested were relatively unsuitable because of their low gel strength. On a large scale cells were immobilized by extrusion through a stainless-steel plate containing - 3 6 holes of very small diameter. The plate was coated with PTFE to facilitate extrusion. Several liters of pellets could be produced per hour using this method. The size of the pellets is governed by the diameter of the orofice used and the viscosity of the cell slurry. Thus the viscosity of the alginate is very important in order to achieve good results, 2.5% (w/v) Protanal LF10/60 having a viscosity of - 5 0 0 centipoise (cP) at 20°, for example. Spherical pellets were obtained when the droplets were allowed to fall from a height of about 30 cm into the CaCI2. Alginate gel pellets could be solubilized by a variety of substances such as EDTA or sodium hexametaphosphate. 22 The formation of isomaltulose from a pure sucrose solution was then monitored, so that no significant growth of the cells could take place. Several organisms produced isomaltulose; others either did not produce 24 C. Bucke, this series, Vol. 135 [15].

438

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[40]

Growth Media preparation & sterilization

l Growth of cells

Harvesting of cells ~ wastes (spent media) (centrifugation; flocculatlon)

Dissolution of alglnate ---gSlurrying of cells in alginate

l Immobilization by extruding into CaCl 2

t Draining off of CaCl 2 and packing into columns

Exhausted ( immobilized biocatalyst (waste)

L

Operation of immobilized cell ( reactors

Substrate preparation

Product (column eluate)

L

Crystallization Wastes (side products etc)

Centrlfugatlon (including washing)

l

Drying and Sieving

1

Product packaging

FIG. 1. A flow diagram showing the m~or unit operations in the immobilized cell process for producing isomaltulose. From Cheetham e t al. 22

[40]

ISOMALTULOSE PRODUCTION USING MICROBIAL CELLS

439

isomaltulose at a sufficient rate, were difficult to grow, produced pigment, or were potentially unsafe. 22 Although the Serratia and Protaminobacter strains had higher initial activities, three strains of Erwinia rhapontici, classified as Enterobacteria agglomerans by API tests, proved to be most suitable because their activity was very much more stable when the cells were used continuously in an immobilized nongrowing form. 22 All of the organisms tested produced trehalulose as well as isomaltulose. About 85% of the activity is initially retained following immobilization, comparison of the freshly immobilized pellets being made with free cells shaken in substrate with alginate pellets. The preferred strain of cells NCPPB 1578 was isolated as a secondary pathogen of rhubarb and was obtained in a yield of about 7 g wet packed cells per liter of medium ( - 2 × 109 viable cells per gram wet weight2~). No synthesis or degradation of enzyme appeared to take place following harvesting of the cells and prior to use of the immobilized cells. This is because the activity of both logarithmic and stationary phase cells, and cells stored for 5 hr prior to immobilization, was unaffected by the addition of chloramphenicol either to the growth medium, the alginate, or CaCI2 used for immobilization, or to the sucrose substrate. Characterization of the Cell-Free Isomaltulose-Forming Enzyme Isomaltulose formation is mediated by a single enzyme that is located in the cell's periplasmic space and that is solubilized most easily by osmotic shocking, typically by rapidly resuspending 1.5 g wet weight cells in 150 ml of ice-cold deionized water. 2~,25The enzyme is a previously undescribed hexosyltransferase with no ion or cofactor requirements and with four novel features. Unlike most other glycosyltransferases the enzyme is sucrose specific. Second, it has an intramolecular mechanism using only the glucose and fructose derived from sucrose. Labeled glucose and fructose were not incorporated in experiments in which unlabeled glucose and fructose (both 0.5 M) and ~4C-labeled glucose and fructose were added to 1.6 M sucrose to give a working radioactivity of 10/xCi/ml. After assay at 30° and pH 7.0, 2-tzl samples diluted to 2% (w/v) were chromatographed and the plates incubated with X-ray film (Kodak, Hemel Hempstead, Herts, United Kingdom) for several days before developing. Results were quantified by scraping off each spot and counting in a toluene-based scintillant containing 2,5-diphenyloxazole (4 g/liter), 1,4-bis(5-phenyloxazol2-/xl)benzene (0.1 g/liter), and Fisons Mix No. 1 emulsifier with a 104 cpm external standard. 25 Thus both the glucose and fructose moieties of 25 p. S. J. C h e e t h a m , Biochem. J. 220, 213 (1984).

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sucrose appear to be enzyme bound. Third, the enzyme is very unusual in that it resembles chemical catalysts rather than biocatalysts, since it displays reaction nonselectivity forming simultaneously isomaltulose and much smaller quantities of a second hitherto uncharacterized disaccharide which has been given the neologism trehalulose (1-O-a-D-glucopyranosyl-t~-D-fructopyranose). The trehalulose could be purified by chromatography on a 115 × 2.2 cm column of Dowex AGW X40 (200-400 mesh, K + form) or by preparative high-performance liquid chromatography with a Waters Prep LC/system 500A, with four Prep Pak 500/C1s columns (30 × 5.7 cm chain each) used in series by means of an auxiliary radial compression unit, both with degassed distilled water as the eluate. 25 13C NMR showed that the fructose moiety of the trehalulose exists predominantly in the pyranose rather than furanose. Trehalulose appears to be the first such oligosaccharide; previously combined fructopyranose has only been observed in oligosaccharides substituted in the C-5 position of the fructose. 26 An additional characteristic of the enzyme is that upon extended incubation a recycling mechanism causes the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum and then fall, and the concentration of trehalulose, the thermodynamically preferred product, to rise slowly. Thus after 10 days an isomaltulose to trehalulose ratio as low as 1.15 : 1 is reached. This is because although the rate of formation of trehalulose is only about 15% of that of isomaltulose, trehalulose is broken down much less rapidly than isomaltulose, such that a net accumulation of trehalulose takes place. Isomaltulose, but not trehalulose, could be converted to sucrose by the enzyme working in reverse. Therefore trehalulose appears to be the lower energy species. For a more detailed explanation of this phenomenon see Kashe et al. 27 The enzyme has been named isomaltulose synthease and should be classed in the EC 5.4.99.--group. 25 By contrast the enzyme from P. rubrum is not sucrose specific and can transfer glucose from sucrose to arabinose or xylose,28 which indicate that it does not have an exclusively intramolecular mechanism as with the E. rhapontici enzyme. Only - 5 % yields of products were obtained, but recently Hashimoto et al. 29 have shown that much higher yields are obtained using unnatural substrates, e.g., when 6-chloro-6-deoxysucrose as donor and methyl/3-D-arabinofuranoside as acceptor were used, a 72% yield of 5-O-o~-D-glucosylpyranosyl-/3-D-arabinofuranoside was obtained. z6 R. S. Shallenberger, "Advanced Sugar Chemistry," pp. 223-231. Horwood, Chichester, England, 1982. 27 V. Kashe, U. Haufler, and L. Riechmann, this volume [26]. 2s W. Mauch and S. Schmidt-Berg-Lorenz, Z. Zuckerind. 14, 375 (1984). 29 H. Hashimoto, M. Sekiguichi, and J. Yoshimura, Carbohydr. Res. 144, (1985).

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Interestingly neither P. rubrum or E. rhapontici enzymes acted on galactosucrose, the C-4 epimer of sucrose. Choice of Immobilization Method Whole cells were immobilized by various methods and the activity and stability of the freshly immobilized cell preparations tested (Table I). Several methods gave good yields of permanently immobilized enzyme activity and in one case, adsorption to DE-52 cellulose (B.D.H. Ltd) in which 2 g of wet cells was mixed with l0 ml of a thick slurry adjusted to pH 7.0 with Tris-HCl buffer, the activity was slightly higher than that obtained using alginate. However all the preparations were very significantly less stable than the cells entrapped in alginate (Table I). 22 Alginate immobilization also has the advantage of being a cheap and simple method using food-grade materials and uses a material with a relatively low solution viscosity but with a good strength when a gel is formed. On a large scale pellets were formed by extrusion under pressure through multiple holes of 0.5 mm diameter drilled in a 200 x 2 mm stainless-steel plate coated with PTFE. Alginate gels could be formed using calcium or several other periodic group 2 cations such as aluminium. Calcium was preferred because of the higher activities obtained and the greater mechanical strengths of the pellets. The concentration of CaCl2 (0. l M) used and the period allowed for immobilization (2 hr) did not significantly affect the activity of the

TABLE I COMPARISON OF THE ACTIVITY AND STABILITY OF Erwinia rhapontici CELLS IMMOBILIZED BY VARIOUSMETHODSa

Immobilization technique

Activity (g product/ g wet cells-hr)

Half-life (hr)

Conversion achieved (%)

Calcium alginate DEAE-cellulose Polyacrylamide Glutaraldehyde-aggregated ceils r-Carrageenan-locust bean gum Bone char Agar Xanthan-iocust bean gum

0.325 0.583 0.13 0.153 0.263 0.01 0.34 0.10

8,500 400 570 40 37.5 25 27 8

99 87 50 23.5 38 25 27 39.5

From Cheetham et al. 22 Note that no activity was retained following immobilization in collagen or cellulose acetate. Because of the interdependence of stability and the degree of conversion2x both parameters are quoted above.

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cells. The mechanical strength of the pellets was proportional to the concentration of alginate used. Pellets were of even size such that handling was easy and sieving prior to use was not required. The gel could also be extruded in the shape of ropes which were then wound or cut into sections, or formed as blocks which were granulated. These preparations had similar activities to those of the pellets, as did cells which had been immobilized in fibrous rather than gelatinous calcium alginate by injecting the cell-alginate slurry into a highly sheared CaC12 solution such as is formed in a domestic food blender. 22 When the gel was formed slowly by the inclusion of dicalcium phosphate and 1,5,-gluconolactone as a calcium-chelating agent, the activity was only a third of that achieved by the standard method. Mechanical Strength of Pellets Columns of alginate-immobilized cell pellets had excellent physical properties. Microbial degradation was never observed although microorganisms possessing alginate lyase did attack sodium alginate solutions stored for long periods. Substrate transfer into the pellets was rapid, no leakage of cells took place, and low-pressure drops were generated even when 55% (w/v) sucrose was pumped through columns of pellets at high flow rate (pressure measured by a pressure gauge between the pump and the column inlet). 3° Columns were relatively incompressible. The compressibility of the column is defined as the reciprocal of the bulk compressive modulus, that is, the change in column volume per unit pressure applied under defined conditions. Constant strain rate and stress relaxation tests in which high pressures were applied to test columns of pellets were used to simulate the behavior of much larger columns. Use of an Instron gel tester Model 1140 (High Wycombe, Bucks, UK) fitted with a 0.019-m-diameter probe moving at 0.83-6.7 × 10-3 m/sec and exerting pressures of up to 13158 kg/m 2 demonstrated that the columns had nonlinear viscoelastic behavior. 3] Compressibility decreased with the concentration of alginate used to form the pellets but increased as the concentration of entrapped cells was increased. Pellets were not fractured unless very high pressures were used and deformation was only partially reversible. Over long periods large creep effects were observed which decreased exponentially with time 3j (creep is the slow and continuous deformation commonly observed in ductile materials which in this case results in an exponential reduction in the void volume of the column). Compres3o p. S. J. Cheetham, K. W. Blunt, and C. Bucke, Biotechnol. Bioeng. 21, 2155 (1979). 31 p. S. J. Cheetham, Enzyme Microb. Technol. 1, 183 (1979).

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sion was markedly reduced when the pellets were partially dried in a fluidized bed drier prior to use. Use of Immobilized Cell Reactors A column of regularly packed alginate pellets proved to be the obvious choice of reactor configuration, due to the higher density of pellets that could be achieved than is possible in a stirred or fluidized reactor, and because attrition of pellets occurred during prolonged use in agitated reactors. Moreover high degrees of conversion of substrate into products were achieved easily because of the plug-flow kinetics characteristic of columns. Up to 93% conversion was achieved in batch or continuous stirred reactors whereas greater than 99% conversion was regularly maintained in packed beds of up to 1 m 3 in size with the result that much higher yields of pure crystalline product were obtained. Isomaltulose could be detected by thin-layer chromatography for 16 hr in butanol/ethanol/water, ( 5 : 3 : 2 , v/v/v) using Merck silica gel plates, and then staining with carbazole for free and combined fructose. 32 The concentration of isomaltulose in the column eluates could be measured with - 9 5 % accuracy by assaying the reducing sugar concentration." Separation of sucrose, isomaltulose, and trehalulose by HPLC was difficult but good resolution was obtained using a Spherisorb 5-/zm silica column (Waters Assoc., Northwich, Cheshire, UK). zl Subsequently even better resolution was obtained using a Zorbax amino column (25 x 0.46 cm I.D.) (Du Pont Ltd., Hitchin, Herts., UK) which had been treated with acetonitrile/water (82 : 18, v/v) and maintained at 22°. This solvent was also used as the eluting solvent at 2 ml/min. Sugars in the eluate were detected using a Waters R401 refractive index detector. The assumption is made in calculating results that all the sugars have the same refractive index. Typical retention times obtained using a new column were for fructose 7.1 min, glucose 8.7 min, sucrose 15.1 min, and isomaltulose 17.3 min. Substrate was pumped through the immobilized cell columns using Watson-Marlow pumps (Falmouth, Cornwall). The relationship between the degree of conversion achieved and the residence time is shown in Fig. 2. The relationship was independent of the size of the reactor, provided that the hydrodynamic properties of the column remained constant. This curve is similar to the WF curve used extensively in chemical engineering. The flow rate was adjusted at frequent intervals such that the desired degree of conversion of sucrose into isomaltulose was maintained con32 S. Adachi, J. Chromatogr. 17, 225 (1965). 33 A. Astoor and E. J. King, Biochem J. 56, 44 (1954).

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100

O

80

1.6 . ~

c~

O

._o

~.---

c~ cO

0

"-~ g 4~

0"8

'0

"~

o.4~ ~ N e-

O.3

0.6

0.9

T.2

"~

e" O

e-

.E

1.5

Flow-rate (column volumes / h) FIG. 2. The effect of the degree of conversion of sucrose into products on the activity of columns of immobilized Erwinia cells. 2~ (11) Activity (g isomaltulose/g wet weight cells per hour); (A) percentage of the immobilized cells. (©) Molar concentration of isomaltulose in the column eluate. The initial flow rate used was about 0.1 empty column volumes of substrate/hr, giving a residence time of just over 3 hr (assuming the column has a void volume of 33%).

stantly high (Fig. 2). Thus the half-life of the column represents the time taken for the flow rate through the column to be reduced to one-half of its original value. The half-life value obtained has direct industrial relevance in that the quantity and quality of the eluate usually need to be maintained constant, for instance to facilitate subsequent processing of the product. A lag phase of about 24-36 hr occurred before a steady state was established, during which concentration gradients of reactants were formed in the column and a very limited amount of cell growth took place. The activity of the immobilized cells was optimal at 30° and pH 7.0, although it was most stable at the acidic pH values (pH 4.0-4.5) maintained by the small quantities of acid formed by the cells. Little color was generated during reaction and no microbial or chemical degradation of the pellets was observed. Activity was maximal with an initial productivity of about 40 g isomaltulose/liter reactor volume per hour when the cells contained 20% (wet wt/v) cells; higher concentrations resulted in excessive internal diffusional restrictions. The activity of the immobilized cells was independent of their aspect ratio (height to diameter ratios 3 to 48 : 1). 22 A contact time of about 3 hr was required to achieve 99% conversion of the substrate. No fouling problems were encountered.

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Stabilization

Immobilization in alginate stabilized the activity of both the intact cells and cell-free extracts. However, the stability of the immobilized whole cells was much greater than when a cell-free enzyme extract or mechanically disrupted cells were used (Table II), although the initial activities of these preparations were not appreciably different. Cells were mechanically disrupted by mixing 2 g wet wt cells with 2 g of dry sand and 2 ml of deionized water and shaking at maximum amplitude for 20 min at room temperature in a Mickle shaker (Laboratory Engineering Co., Gomshall, Surrey, UK) or by sonication as a 10% (wet wt/v) suspension in deionized water at 2.8 A for 340 min using a probe with a tip of diameter 2.5 cm and cooling the sample at 5-min intervals. 21 Viable cells were not essential for isomaltulose formation, because the activity of the cells decayed slowly and linearly with time, with a half-life of 1 year, whereas the viable cell count declined rapidly with a half-life of 300 hr, probably due to a lack of nitrogenous and trace nutrients. Viable cells counts were made by grindTABLE II OPERATIONAL STABILITIES OF THE ISOMALTULOSE-FORMING ACTIVITIES OF VARIOUS E. rhapontici

PREPARATIONS a

Type of enzyme preparation used and conditions of use Free cells maintained in exhausted growth medium Free cells used batchwise in 0.12 M sucrose Free cells used batchwise in 1.60 M sucrose (28% conversion) Immobilized cells used with 0.12 M sucrose (70% conversion) Immobilized cells supplied with 0.365 M sucrose in 100 mM HEPES buffer, pH 7.0 (95% conversion) Immobilized cells used with 1.60 M sucrose (70% conversion) Immobilized cells used with 1,60 M sucrose (99% conversion) Immobilized cell debris (45% conversion) A cell-free extract obtained by osmotically shocking cells used batchwise An immobilized cell-free extract (81.5% conversion) Immobilized cells supplied with affination syrup diluted to 1.6 M sucrose and adjusted to pH 7.0 (76% conversion) Immobilized cells supplied continuously with growth medium (90% conversion) Immobilized cells used in a continuous stirred reactor maintained at pH 7.0 Immobilized cells supplied with 1.02 M isomaltulose

Operational stability (half-life, hr) 335 25 41 55 295 2,265 8,625 190 26 620 386 76 341 300

From Cheetham et al. 2~ Unless otherwise stated preparations were tested in packed columns supplied with 1.60 M sucrose substrate. 2~m

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ing the pellets in phosphate-buffered saline (PBS), plating serial dilutions of the PBS extracts onto nutrient agar, incubating at 30° for 24 hr, and then counting the numbers of colonies formed. Total suspended cell measurements were calculated from A540 readings of the PBS extracts made versus a suitable blank solution. 21 A small amount of metabolic activity was retained by the nonviable but structurally intact immobilized cells as they continued to produce aketo acids throughout the useful life the columns. 21 The acids were measured as pyruvate equivalent by the method of Slonekar and Orentos. 34 No partitioning of sucrose between the bulk substrate and the immobilized cells was observed. A crucial factor in the success of this process was that the immobilized cells were both most active and maximally stable when high concentrations of sucrose were used, irrespective of the extent of conversion achieved, reaching a maximum at 46% w/v (55% w/w or 1.6 M). 2~ Only the immobilized and not the free cells were markedly stabilized by the concentrated sucrose solutions (Table II). The use of concentrated sucrose was additionally advantageous because the equilibrium between isomaltulose and sucrose was favored. Furthermore the use of concentrated sucrose reduced the formation of gaseous and acidic side products, the growth of contaminant microorganisms, and the activity of endogenous proteases, while greatly reducing the size of equipment required, the volumes of syrup that had to be manipulated, and the volume of water that had to be removed by evaporation prior to crystallization. 2~ The maximum concentration of sucrose that could be used was about 1.6 M. When high degrees of conversion of substrates containing higher concentrations of sucrose were achieved the solubility of isomaltulose at 30 ° was exceeded such that crystals of isomaltulose formed in the upper part of the column. The presence of crystals impeded the flow of substrate, increasing the pressure drop through the column and eventually causing complete blockage. The extent of conversion of sucrose into isomaltulose could be maximized ( - 1 % w/v sucrose remaining) and the generation of trehalulose minimized by controlling the flow rate of substrate through the column and thus optimizing its contact time with the cells (Fig. 2). Increases in the extent of conversion were accompanied by decreases in activity and by a marked increase in the stability of the immobilized cells (Table II). 2L22 Stabilization may be due to stabilization by the product or by both the product and substrate, or to the imposition of diffusional restrictions on the supply of substrate to the immobilized cells, since stability could be increased by raising the concentration of cells entrapped in the pellets. 34j. H. Slonekar and D. G. Orentos, Nature (London) 194, 478 (1962).

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Stability may also be connected with the low rate of endogenous metabolism of the cells or with their plasmolysis by the high sugar concentrations. The purity of the sucrose used was also important, since both activity and stability were greatly reduced when affination syrup, an impure sucrose stream produced during refining, was used. Little is known about the control of enzyme turnover in microbial cells 35but it was shown that loss of activity was not due to the action of contaminant microorganisms. No enzyme induction or turnover could have occurred in the immobilized Erwinia cells, first, because of the high purity of the substrate used and second because addition of chloramphenicol or benzylpenicillin (both 500/xg/ml) to the substrate, which inhibit protein and cell wall synthesis, respectively, did not affect stability. Stability was, however, enhanced by the use of buffered substrate (100 mM HEPES) and the use of packed columns rather than a 500-ml continuous stirred reactor containing 200 ml of pellets, maintained at pH 7.0 and supplied with fresh substrate at a rate of 0.016 liter/hr (Table II). Similarly, a fluidized bed reactor, in which the reactants were recycled through the reactor at very high flow rates and neutralized with NaOH after each cycle, was not advantageous. Detailed analysis indicated that the major factor governing the loss of activity is the presence of a very low concentration of cumulative inhibitor in the substrate. This is because the stability of the immobilized cells is best correlated with the volume of substrate passed through the columns. Thus irrespective of the stability achieved, the volume of substrate processed per half-life was virtually constant (Fig. 3). No loss of material from the beads was apparent even after long periods of continuous operation, and the beads retained full mechanical strength as measured with the Instron gel tester. Thus in order to achieve high productivities it is advantageous to allow complete conversion of substrate. Under these conditions the volume of substrate processed by the immobilized cells, the amount of inhibitor the enzyme is exposed to, and consequently the loss in enzyme activity are minimized. 22 The gene for the isomaltulose synthase is in effect "altruistic" since it results in the formation of a product that stabilizes the whole cells and so is beneficial to all the cells genes. Regeneration of the Immobilized Cell Activity After considerable decay of activity had taken place while using sucrose feedstock, regeneration could be achieved simply by pumping growth medium up the column at intervals, provided the medium did not contain an excess of calcium-chelating agents which would cause the 35 R. L. Switzer, Annu. Rev. Microbiol. 31, 135 (1977).

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(ENZYME TECHNOLOGY)

lO00

Volume of substrate processed / unit

I

f

column volume / half-life (llllt12) SO0

0

i $

i lO

Stability (hx 10 3) FIG. 3. The effect of the volume of 1.6 M sucrose substrate passed through a column of immobilized cells (liter of substrate processed/liter of column volume per half-lifeof use) and the operational stability of the isomaltulose-formingactivity of the cells (half-life).From Cheetham et al. 22 alginate to be solubilized. The extent of regeneration was slow, the cells having a doubling time of 64 hr, and was approximately proportional to the viable cell count prior to the administration of nutrients. Regeneration was limited by the space available for fresh cells inside the gel, although some cells did grow out of the alginate gel. Dissolved benzylpenicillin or chloramphenical prevented regeneration, indicating that the d e n o v o activity is entirely accounted for by the growth of fresh cells as enzyme induction, renewal, or derepression could have occurred in the presence of benzylpenicillin. 22 Crystallization of Isomaltulose Syrup eluted from the immobilized cell columns routinely contained less than 0.02 M sucrose and about 1.2 M isomaltulose plus side products: some glucose, fructose and sucrose, but chiefly trehalulose. The crystallization of isomaltulose is exothermic (A G = + 20 kcal/mol). The preferred method of crystallizing isomaltulose is to concentrate the eluate to about 2.0 M isomaltulose by rotary evaporation at no higher than 60 ° to minimize color formation, and then allow the syrup to cool. Seed crystals are next added to the agitated solution. Nucleation and crystallization then take place. The crystals are recovered by basket centrifugation and

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washed with water spray, dried in a fluidized bed drier, and sieved to give a purity of greater than 99% and an overall yield of about 65%. Thus the isomaltulose has been considerably purified and concentrated. Crystals contained 1 tool of water of crystallization per mole of isomaltulose 2Lz2 and have a melting point of 118-122 °, a solubility at 30° of - 7 0 g/100 ml water, a bulk density of 0.65-70 g/ml, an absolute density of 1.45 g/ml, and an E R H (equilibrium relative humidity) of 25-32% water at 80% and 22°. As the extent to which the column eluate was concentrated prior to crystallization was increased the yield of crystals increased, but their purity fell. Product was in the form of white, free-flowing crystals with chemical and microbiological purities similar to those of granulated sucrose; that is within twice the American Bottlers Standard (less than 200 mesophiles, 10 yeast, and 10 molds per l0 g). Following crystallization a mother liquor remains that contains sucrose, glucose, fructose, trehalulose, and uncrystallized isomaltulose. Applications for this syrup would be very useful to improve the profitability of the process. Storage Stability It is often important to be able to store immobilized cells without incurring excessive losses in enzyme activity. Such a facility would be useful if the cells need to be stored prior to use or when they are being transported from their site of production to the factory. Common methods of preservation used in the food industry include irradiation, dehydration by drying or freezing, chilling, or use of extremes of pH or osmotic pressures. The centrifuged cell pellet and cells slurried in alginate have moderate stabilities consistent with easy immobilization (Table III), but are insufficiently high to allow for storage, etc. Several procedures were found useful and practical in prolonging storage stability. 36 These included partially drying the immobilized cell pellets, typically to about 4050% of their original weight with a stream of warm air (30-40°), for instance in a fluidized bed dryer, storing the pellets soaked in glycerol (1 : 1-3, v/v) and under an inert atmosphere of nitrogen and including bacteriostatic agents such as benzoic acid esters (Nipa Laboratories, Ltd., Pontypridd, Glamorgan, United Kingdom) at concentrations of 0.10.6%, e.g., Nipasept Na ÷ at 0.06% (w/v). When these methods were employed no losses in activity could be measured after 500 hr storage in sealed containers at room temperature, whereas in the absence of these treatment substantial losses in activity and often considerable microbial 36 p. S. J. C h e e t h a m , U.S. Patent Application 4,443,538 (1984).

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TABLE III STORAGE STABILITY OF THE ISOMALTULOSE-SYNTHESIZING ACTIVITY OF VARIOUS ORGANISMS AND UNDER DIFFERENT CONDITIONS a

Organisms/conditions E. rhapontici NCPPB 1578' 1739 139 ATCC 29284 pellets plus a 2-fold P. rubrum excess of glycerol

NCIB 2878 S. marcesens NCIB 8285 Freeze-dried Centrifuged cell paste Alginate slurry NaCI (saturated) Ammonium sulfate (saturated) Glycerol : pellets, ratio 1:1 1:2 1:3 1:5 Glycerol + Nipasept Na ÷ (0.06%) + Nipacombin A (0.01%) + Nipaheptyl (0.0125%) Penicillin G (1.76 mg/ml) Chloramphenicol (1.76 mg/ml) Dried pellets in glycerol (ratio 1 : 2) plus Nipasept Na ÷ (0.06%) Nipacombin A (0.01%) Nipaheptyl (0.0125%) PMSF Dipicolinic acid

Storage stability (half-life, hr)

I100 1086 1471

1444 575 125 124 101 250 1550

2500 700 410 556 625 1563 6250

2420 33O

o From Cheetham) 6 Results were usually calculated from the loss in original activity following 500 hr in sealed storage containers at 20°. oo indicates that within the period of the storage trial there was no loss in activity. Note also that a number of compounds related to glycerol such as ethylene glycol, butanediol, hexyleneglycol, 1,3-propanediol, and polyethylene glycol all gave half-lives of less than 300 hr. In these experiments strain NCIB 1578 was used unless otherwise stated.

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contamination were observed even after comparatively short time periods (Table III). These storage methods were useful for all the isomaltuloseproducing organisms tested, including ATCC 29284 which has a relatively poor operational stability. 22,3~ Although drying denatured some enzyme activity, the volumetric activity of the pellets was actually increased due to the reductions in the weight and volume of the pellets caused by the drying. Drying also had the effect of increasing the mechanical strength of the pellets, and of making them smaller, less bulky, and more evenly flowing. Little rehydration occurred even after long periods of operational use. Other common preservation methods either caused excessive loss of activity or caused a loss of pellet strength; these methods included freeze-drying and addition of NaC1 or (NH4)2SO4 (Table II1). 36 Several effects appear to be involved. First, water activity is reduced due to drying and dehydration by the glycerol, together with the associated reduction in oxygen content due to the lower water content. For instance, after storage of dried and undried pellets in a 2-fold excess of glycerol for 500 hr the water activity of the glycerol had increased to aw values of 0.73 and 0.78, respectively. Also the solubility of oxygen was reduced by 45% in a 45% glycerol solution. Second, glycerol may specifically stabilize the enzyme and also act as a carbon source to support the maintenance requirements of the cells. Third, microbial contamination is discouraged by the low oxygen and water concentrations and the high osmotic pressure. An absence of microbial contamination would appear to be especially important, because antibacterial agents such as the Nipa agents or penicillin had such a marked stabilizing effect and because the addition of a saturated solution of the protease inhibitor phenazinemethane sulfonylfluoride (PMSF) alone had a marked stabilizing effect 36 (Table III). Addition of dipicolinic acid, which is present in high concentrations in bacterial spores, had no effect. Other Immobilization Methods Subsequent to Tate and Lyle's patent applications several other companies have also described the use of immobilized cells to produce isomaltulose. The South German Sugar Co. has disclosed a method similar to that used for immobilizing glucose isomerase, involving flocculating Protaminobacter rubrurn (CBS 574.77), such as with Primafloc C7 (1%), extrusion into ropes, followed by drying and cross-linking with 0.1% glutaraldehyde. 37 The immobilized cells are then used in a column main37 M. Munir, European Patent Application 81,105,743.9 (1982).

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tained at 45-60 ° through which a concentrated sucrose solution (45-75%, w/w) is passed. Complete conversion of the substrate is not required because a first crop of isomaltulose crystals can be obtained and the resulting mother liquor retreated with the immobilized cells to produce more isomaltulose. An overall yield of about 80% isomaltulose is obtained. The use of Serratia plymuthica, Serratia marcescens and Leuconostoc mesenteroides, flocculation with cationic flocculants and chitosan, and immobilization in cellulose acetates and calcium alginate were also claimed. In a later patent application the use o f P . rubrum cells attached to a polymer support was described? 8 The Mitsui Sugar Co. also entraps cells of S. plymuthica (NCIB 8285) in calcium alginate gels followed by cross-linking with slightly acidified 2% polyethyleneimine for 5 min and then 0.5% glutaraldehyde 39,4°at 5 ° for 30 min. The more involved immobilization procedure is necessary because of the tendency of the enzyme to leak from the cells. The cells are grown at 25° with aeration and agitation on a medium containing 5% sucrose, 3% corn steep liquor, 0.3% Na:HPO4, and 0.2% NaCI. The cell slurry is mixed with an equal volume of 4% sodium alginate and extruded through a dye with pores of 0.6 mm diameter into 0.15 M CaCI2. When used in columns to treat a concentrated sucrose solution (40% w/w) at 25 ° the half-life of the immobilized cells is about 23 days and an average activity of 0.037 g/ml column volume per hour is obtained. Similar results are obtained using Protaminobacter rubrum, for which the average activity is 0.15 g/ml-hr and the half-life is approximately 73 days. The optimum pH of the immobilized enzyme is 5.5, the Km is 0.14 M, and the K~ for isomaltulose is 0.31 M. The operating substrate concentration used is 40% (w/w) and the residual sucrose concentration in the column eluate is controlled at 0.8% (w/w), 85% of the sucrose being converted into isomaltulose. The column eluate is then treated with ion-exchange resin, evaporated, and crystallized as it cooled. Further crystals could be obtained from the mother liquor, or alternatively the column eluate could be solidified and pulverized into a crude product. The Mitsui Sugar Co. has announced that it plans to start production of isomaltulose using initially a 600 ton/year pilot plant in Okayama. The product will be initially sold for use in chewing gum and sugar confectionery. The product is to be supplied as crystals and in a liquid form only 70% as sweet as sucrose. A variant on this method has been carried out by Miles Laboratories, inJ8 W. Haase, P. Egerer, G. Schmidt-Kastner, and H. Perrey, European Patent Appfication 0,160,253 (1985). 39 j. Shimizu, K. Suzuki, and Y. Nakajima, United Kingdom Patent Application 2,082,591 (1982). 4o y. Nakajima, Seito Gijutsu Kenkyu Kaishi 33, 55 (1984).

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volving contacting the cells with tannic acid, polyethyleneimine, glutaraldehyde, and a epichlorohydrin/polyamine copolymer? 1 The enzyme from P. rubrurn responsible for converting sucrose into isomaltulose has been isolated by disrupting the cells in a Manton-Gaulin homogenizer. 42 This soluble enzyme was then employed entrapped in semipermeable hollow fibers, adsorbed onto CM-cellulose, titanium dioxide, covalently bound to Sepharose, aldehyde resin, sodium silicate, cyanogen bromide-activated starch, dextran 60, sodium alginate, carrageenan, and maleic anhydride/methylvinyl ether copolymer. Complete conversion of sucrose into isomaltulose together with some trehalulose, glucose and fructose could be obtained when used in reactors. The best half-life reported for the immobilized enzyme was 2075 hr. We have also found that cells can be immobilized in alginate that has been solidified in fibrous form, rather than the conventional gel form. Immobilization is carried out by rapidly mixing the alginate-cell slurry with CaCI2, for instance in a blender or Silversen mixer so that solid calcium alginate is formed virtually instantaneously. However, the particles are irregular and so are liable to abrasion and have poor packing properties in columns. The production of trehaluiose from sucrose or isomaltulose by isomaltulose-producing cells or enzymes has been the subject of a patent applicationfl The trehalulose is purified by ion-exchange chromatography and dried. Like isomaltulose, trehalulose is noncariogenic and of low sweetness; unlike isomaltulose it is more soluble and is only slowly and partially hydrolyzed in the small intestine. This would not appear to be a preferred procedure since the yields of trehalulose are likely to be lower than those obtained for isomaltulose. Conclusion Our immobilized cell process for producing isomaltulose (Fig. 1) is characterized by the use of a new enzyme possessing a novel mechanism and by the high process intensity achieved, that is the high concentration of substrate used, the virtually complete reaction obtained, and the very high operational stabilities reached. The immobilized cells are about 350 times more stable than the free cells, a half-life of about a year being achieved. Thus during one half-life of use, a column has a productivity of 41 O. J. Lantero, European Patent Application 82,109,404.2 (1983). 42 C. Kutzbach, G. Schmidt-Kastner, and H. Schutt, European Patent Application 81,107,714.8 (1982). 43 European Patent Application 109-009-A (1984).

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just over 200 kg of product/liter column-volume per year; that is a 10-liter column will process 5.2 m 3 of sucrose substrate in one half-life (per year) and the cells can produce about 1500 times their own weight in crystalline isomaltulose. 22 By comparison GI (glucose isomerase) produces 20003000 times its own weight of high-fructose syrup in its operational lifetime. This integrated continuous process has been successfully operated on a small pilot plant scale in which cells were grown in a 1-m 3 fermenter and using 25-liter columns. On a larger scale, several additional features had to be developed. These included a defined medium for the growth of cells that did not contain peptone and beef extract and that contained a bacteriostat to inhibit the growth of contaminated microorganisms, a method of flocculating cells so as to facilitate their recovery from the fermentation medium, and a large-scale method of extruding the cellalginate slurry to form pellets. This process illustrates the desirability of screening for novel enzyme activities from microorganisms isolated from unusual environments, of using simple, mild immobilization methods and high reactant concentrations, of maintaining a steady state in which high degrees of conversion of substrate into product is achieved, and of developing efficient methods of purifying and recovering products. Desirable improvements to the process include the suppression of trehalulose formation and an increase in the volumetric activity of the columns. The major limitation to improvement is the intrinsic properties of the enzyme. Improvements could possibly be achieved by the discovery of an improved isomaltulose-producing microorganism or possibly by site-directed mutagenisis. However since isomaltulose must be more expensive than sucrose it must be shown to exhibit a significantly improved functionality to be a good commercial product. It is hoped that others will also find some of the approaches and methods adopted in this study useful.

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[41] P r o d u c t i o n o f L - M a l i c A c i d b y I m m o b i l i z e d Microbial Cells B y ICHIRO CHIBATA, TETSUYA TOSA, KOZO YAMAMOTO, a n d ISAO TAKATA

L-Malic acid is widely used for medicines and food additives. It has been industrially produced from fumaric acid by the fermentative or enzymatic method employing fumarase (EC 4.2.1.2, fumarate hydratase) as biocatalyst. HOOCCH~---CHCOOH + H20 .

fumarase

• HOOCCH2CHCOOH [

OH

An enzymatic batch process using the enzyme of L a c t o b a c i l l u s b r e v i s was reported in 1960 by Kitahara et al. 1 However, from an industrial standpoint, a continuous enzyme reaction system using immobilized enzyme is considered to be more advantageous than a batch process using enzyme solubilized from microbial cells. 2 Marconi et al. 3 reported that fumarase can be efficiently immobilized into cellulose triacetate, and that the immobilized fumarase makes it possible to develop an economically attractive method to produce L-malic acid. Recently, we have developed an industrially very advantageous method for carrying out the above fumarase reaction? -7 That is, microbial cells having high fumarase activity are immobilized in a polymer gel matrices such as polyacrylamide and Kcarrageenan. The immobilized cells are packed into a column, and a substrate solution (sodium fumarate) is continuously passed through the immobilized cell column. An effluent containing L-malate is obtained without contamination from impurities such as microbial cells, components of cultural medium, and others.

t K. Kitahara, S. Fukui, and M. Misawa, J. Gen. Appl. Microbiol. 6, 108 (1960). z R. A. Messing, "Immobilized Enzymes for Industrial Reactors" (R. A. Messing, ed.), p. 1. Academic Press, New York, 1975. 3 W. Marconi, F. Morosi, and R. Mosti, Agric. Biol. Chem. 39, 1323 (1975). 4 K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Eur. J. Appl. Microbiol. 3, 169 (1976). 5 K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotechnol. Bioeng. 19, 1101 (1977). 6 I. Takata, K. Yamamoto, T. Tosa, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 7, 161 (1979). 7 I. Takata, K. Yamamoto, T. Tosa, and I. Chibata, Enzyme Microb. Technol. 2, 30 (1980).

METHODS IN ENZYMOLOGY, VOL. 136

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Thus, L-malic acid with high purity is obtained in a good yield by this method compared with the batchwise enzymatic method using cultural broth. The fumarase activity of immobilized cell column is fairly stable, and it can be used for a long period of operation. Therefore, we developed in 1974 an industrial process in which Brevibacterium ammoniagenes with high fumarase activity was immobilized in gel matrices of polyacrylamide. In 1977 we changed the conventional polyacrylamide method to the more economical r-carrageenan method. To increase productivity of L-malic acid in this system, improvement of the K-carrageenan method was attempted. It was found that the addition of polyethyleneimine or tannins to the immobilization medium was effective in increasing the stability of fumarase activity. 8,9 Since 1980 we have been operating this improved immobilized cell system for the industrial production of L-malic acid. These methods are described in this article. Assay Methods

Estimation o f L-Malic Acid. L-Malic acid is measured colorimetrically by the method of Goodman and Stark. l° Assay of Fumarase Activity Native Fumarase. A mixture of 0.1 ml of native fumarase and 1.9 ml of I M sodium fumarate (pH 7.0) is incubated at 37° for 10 min. After the reaction is stopped by the addition of 2 ml of 2 N HCI, the precipitates are removed by centrifugation (3000 g). The L-malic acid formed in the supernatant is determined colorimetrically by the method of Goodman and Stark. i0 Enzyme activity is expressed as micromoles of L-malic acid produced per hour. Free Cells. A mixture of 1.0 g (wet weight) of free cells and 30 ml of 1 M sodium fumarate (pH 7.0) is incubated at 37° for 10 rain. The reaction is stopped by the addition of 2 ml of concentrated HC1, and the L-malic acid formed is determined as in the case of native fumarase. Activity of the free cells is expressed as micromoles of L-malic acid produced per hour. Immobilized Cells. A reaction mixture of immobilized cells (amounts corresponding to 1 g of intact cells) and 30 ml of 1 M sodium fumarate (pH 7.0) is incubated with shaking at 37 °, and L-malic acid in the reaction mixture after 15 and 30 min is determined as in the case of native fumas I. Takata, K. Kayashima, T, Tosa, and I. Chibata, J. Ferment. Technol. 60, 431 (1982). 9 I. Takata, T. Tosa, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 19, 85 (1984). 10A. E. Goodman and J. B. Stark, Anal. Chem. 29, 283 (1957).

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rase. The fumarase activity is calculated from the increase in L-malic acid concentration during 15 min. Activity of the cell mixture is expressed as micromoles of L-malic acid produced per hour. Immobilization of Brevibacterium amrnoniagenes with Polyacrylamide

Culture of B. ammoniagenes. Breoibacterium ammoniagenes IAM 1645 is cultured under aerobic conditions at 30° for 24 hr in 100 liters of medium (pH 7.0) containing glucose (2 kg), fumaric acid (0.5 kg), urea (0.2 kg), KH2PO4 (0.2 kg), MgSO4.7H20 (50 g), and corn steep liquor (1 kg). The cells are collected by centrifugation. About 500 g (wet weight) of B. ammoniagenes cells is obtained from 100 liters of broth, and fumarase activity of the immobilized cells is approximately 200/xmol/hr per gram of wet cells in flesh state. Preparation of Immobilized B. ammoniagenes. Cells (1 kg of wet weight) are suspended in 2 liters of physiological saline, and the cell suspension is cooled to 8 °. In 2 liters of water, 750 g of acrylamide monomer and 40 g of N,N'-methylenebisacrylamide are dissolved, and the monomer solution is cooled to 8°. Both are mixed at 8° . To the mixture are added 0.5 liters of 5% (v/v) fl-dimethylaminopropionitrile as an accelerator of polymerization and 0.5 liters of 1% potassium persulfate as an initiator of polymerization, and the reaction mixture is allowed to stand at 20-25 ° for 15 min. The gel formed is cut into cubes (3 × 3 x 3 mm) with a knife, and thoroughly washed with physiological saline. Its fumarase activity is approximately 500/xmol/hr per gram of wet cells in fresh state. Suppression of Formation of Succinic Acid and Enhancement of L-Malic Acid Formation As the immobilized cells have an activity that forms succinic acid as a by-product, succinic acid and unconverted fumaric acid accumulate in the reaction mixture. Although fumaric acid can be easily precipitated by acidifying the reaction mixture with hydrochloric acid, it is very difficult to separate succinic acid from L-malic acid. The key step to a successful process, therefore, is to prevent the formation of succinic acid. As shown in Table I, detergents such as bile extract, bile acid, and deoxycholic acid reduce the amount of succinic acid formed by the immobilized cells. The detergents also remove the permeability barriers for substrate and/or product across the membrane of cells entrapped in the polyacrylamide gel, and thus the yield of L-malic acid is increased. Therefore, for industrial purpose bile extract treatment is economically most suitable.

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TABLE I EFFECT OF DETERGENT TREATMENTS ON FORMATION OF L-MALIC ACID AND SUCCINIC ACID"

Detergent

Concentration (%)

Formation of L-malic acid (pmol/hr-g of cells)

No addition CPC CPC SLS SL-10 Triton X-100 Bile acid Bile extract Deoxycholic acid

0.02 0.16 0.02 0.02 0.20 0.20 0.20 0.20

990 4570 3070 6050 1220 5360 6570 7480 7380

Treatment

Formation of succinic acid (mol% of Lmalic acid) 2.5-5.0 2.5-5.0 1.0-2.5 1.0-2.5 2.5-5.0 >5.0 <0.2 <0.2 <0.2

Immobilized cells (7.6 g) were suspended in 30 ml of 1 M sodium fumarate (pH 7.5) containing one of the detergents shown in the table and stood at 37° for 20 hr. The enzyme activity was determined after washing the gel thoroughly with physiological saline.

Optimal conditions for the treatment are as follows. The immobilized cells (3.5 kg) are incubated with 4.8 liters of 1 M sodium fumarate (pH 7.0) containing bile extract (28.8 g) at 37 ° for 20 hr. F u m a r a s e activity is 7500 /xmol/hr p e r 13 ml gel (corresponding to 1 g of wet cells). Continuous E n z y m e Reaction Using Immobilized B. ammoniagenes Cells Immobilized cells treated with bile extract are packed into a column, through which a continuous stream of 1 M fumarate (pH 7.0) is passed at a flow rate o f space velocity 0.15/hr. Since the effluent is not contaminated with cells or significant amount o f by-product or u n c o n s u m e d fumarate, relatively pure L-malic acid can be obtained with high yields in comparison to the e n z y m a t i c batch method. The half-life of the fumarase activity of the immobilized cell column at 37 ° is 53 days, and thus the system can be operated for fairly long periods. A disadvantage o f this system is that fumarase is partially denatured during the immobilization of the cells on polyacrylamide, owing to the reactive m o n o m e r and catalyst and/or the heat of polymerization. It is, therefore, desirable to increase the activity yield during immobilization and i m p r o v e the operational stability of the fumarase.

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Immobilization of Brevibacterium flavum with K-Carrageenan In order to improve L-malic acid productivity, immobilization methods and microbial cells with higher fumarase activity than B. ammoniagenes were screened. Among many synthetic and natural polymers tested as matrices for entrapping microbial cells, K-carrageenan was the best one. 1~ By screening microbial cells having higher fumarase activity and the stability, Brevibacteriumflavum was selected. To increase the concentration of fumarase and the operational stability of fumarase activity of immobilized preparations, culture conditions ofB. flaourn were investigated in detail. Culture of B. flavum. Brevibacterium flavum ATCC 14067 is cultured under aerobic conditions at 30° for 48 hr in 100 liters of medium (pH 7.0) containing malonic acid (2 kg), diammonium citrate (0.5 kg), KH:PO4 (0.2 kg), MgSO4" 7H20 (50 g) and corn steep liquor (2 kg). The cells are collected by centrifugation. About 300 g (wet weight) of B. flavum cells is obtained from 100 liters of broth, and fumarase activity is approximately 1500-2000 ~mol/hr per gram of wet cells in fresh state. Preparation of Immobilized B. flaoum. In 160 ml of 0.15 M NaC1 160 g (wet weight) ofB. flavum is suspended at 45 °, and 34 g of K-carrageenan is dissolved in 680 ml of 0.15 M NaCI. Both are mixed at 45 ° for 5 min, and the mixture is cooled to gelate at approximately 10° for 30 min. In order to increase the gel strength, the gel is soaked in 5 liters of 0.3 M KCI solution for 4 hr at 10°. After this treatment, the resulting stiff gel is cut into cubes (3 × 3 x 3 ram) with a knife. To enhance the fumarase activity and suppress succinic acid formation, the immobilized cells are incubated with 4.8 liters of 1 M sodium fumarate (pH 7.0) containing bile extract (28.8 g) at 37 ° for 20 hr. Fumarase activity is approximately 9900 ~mol/hr per 11 ml gel (corresponding to 1 gram of wet cells). Continuous Enzyme Reaction Using Immobilized B. flavum Ceils Immobilized B. flaoum (1000 liters) packed into a column and a solution of 1 M sodium fumarate (pH 7.0) is charged into the column at a flow rate of space velocity 0.3/hr (300 liters/hr). The half-life of the fumarase activity of the immobilized cell column at 37° is 160 days. As shown in Table II,~2 the productivity of B. flavum immobilized using K-carrageenan

tl I. Takata, T. Tosa, and I. Chibata, J. Solid-Phase Biochem. 2, 225 (1977). 12 N. B. Havewala and W. H. Pitcher, in "Enzyme Engineering" (E. K. Pye and L. B. Wingard, eds.), Vol. 2, p. 315. Plenum, New York, 1974.

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TABLE II COMPARISONOF PRODUCTIVITYOF IMMOBILIZEDB. ammoniagenes

AND B. flavum ~ Fumarase Operational activity stability (/xmol/hrat 37° ml g e l ) (half-life,days)

Immobilization method and microbial cells Polyacrylamide B. amrnoniagenes B. flavum Carrageenan B. ammoniagenes B. flavum

Relative productivity

345 400

53 94

100 273

530 900

75 160

142 897

a The productivity of immobilized cells for continuous production of L-malic acid was calculated from the following equation presented by Havewala and PitcherJ2: Productivity = f~ E0 exp(-Ko • t) dt where E0 is the initial enzyme activity, Ka is the decay constant, and t is the operational period. Each immobilized cell column was operated with the same fumarase activity (173/zmol/hr-ml gel) as that at the half-life of B. ammoniagenes immobilized with polyacrylamide, and the productivity of B. flavum immobilized with polyacrylamide was taken as 100%. is 9-fold higher than that of B. a m m o n i a g e n e s immobilized with polyacrylamide. I m p r o v e d Immobilization Methods Using K-Carrageenan and Its Derivatives F o r i m p r o v e m e n t of L-malic acid productivity, the following three methods are effective. Immobilization Using K-Carrageenan and Polyethyleneimine

In 160 ml of physiological saline 16 g (wet weight) of B . f l a v u m cells is suspended, and 30 g o f x-carrageenan is dissolved in 600 ml of physiological saline. The two preparations are mixed at 45 ° for 5 min. To the mixture 80 ml of 1.9% (w/v) polyethyleneimine aqueous solution is added and then mixed at 45 ° for 5 min. The mixture is cooled to gel at approximately 10° for 30 min. In order to increase the gel strength, the gel is soaked in 5 liters o f 0.3 M KCI solution for 4 hr at 10 °. After this treatment, the resulting stiff gel is cut into cubes (3 × 3 × 3 mm) with a knife. To enhance the f u m a r a s e activity and suppress succinic acid formation, the immobilized cells are treated with 1 M sodium fumarate (pH 7.0) containing 0.6% bile

[41]

461

L-MALIC ACID PRODUCTION BY MICROBIAL CELLS TABLE III COMPARISON OF L-MALIC ACID PRODUCTIVITY IN VARIOUS IMMOBILIZED PREPARATIONSa

Microbial cells and immobilization method

Operation temperature (°C)

Fumarase activity (p.mol/hrml gel)

Operational stability (half-life, days)

Relative productivity ~ (%)

37

345

53

100

37 37 37 50 37 50

400 900 980 1670 1110 1700

94 160 243 128 3 l0 104

273 897 1587 1990 2460 1626

B. ammoniagenes Polyacrylamide

B. flaoum Polyacrylamide r-Carrageenan K-Carrageenan + polyethyleneimine r-Carrageenan + Chinese gallotannin

The productivity of B. ammomagenesimmobilized with polyacrylamidewas taken at 10o%. extract at 37° for 20 hr according to the method described above. Fumarase activity and operational stability of the immobilized cells are shown in Table III.

Immobilization Using r-Carrageenan and Tannin In place of 1.9% (w/v) polyethyleneimine described in the above method, 1.25% (w/v) Chinese gallotannin aqueous solution is used for immobilization of B. flaoum cells using r-carrageenan. The other conditions are the same as the case of polyethyleneimine. Fumarase activity and operational stability of the immobilized cells are shown in Table III.

Immobilization Using r-Carrageenan Modified with Amines 13 Preparation of Modified r-Carrageenan, Amino-K-carrageenan. Thirty miUimoles of epichlorohydrin is added to 50 g (dry weight) of rcarrageenan suspended in 500 ml of 1 M KOH. The mixture is shaken at 60 ° for 30 rain. The activated r-carrageenan is washed twice with I liter of cold ethanol and resuspended in 400 ml of 1 M KOH. To the suspension 360 mmol of aqueous NH4OH is added. The mixture is shaken at 60° for 2 hr. The resulting amino-r-carrageenan is washed with 500 ml of 90% ethanol containing 0.1 N HCI, 500 ml of 90% ethanol containing 0.1 N NaOH, and then 500 ml of ethanol, respectively, five times. Monoethylamino and diethylamino-~-carrageenan. Fifty grams of rcarrageenan is activated with 20 mmol epichlorohydrin dissolved in 500 13 I. Takata, T. Tosa, and I. Chibata, J. Appl. Biochem. 4, 371 (1982).

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ml of 1 M KOH in the same manner as in the case of amino-r-carrageenan. The activated K-carrageenan is reacted with 360 mmol monoethylamine or diethylamine dissolved in 400 ml of 1 M KOH at 60° for 2 hr. Resultant monoethylamino- and diethylamino-K-carrageenan are washed in the same manner as in the case of amino-K-carrageenan. Immobilization. One hundred sixty grams (wet weight) ofB. flavum is suspended in 160 ml of 0.15 M NaCl at 45°, and 34 g of modified Kcarrageenan is dissolved in 680 ml of 0.15 M NaCl. Both are mixed at 45 ° for 5 rain, and the mixture is cooled to gelate at approximately 10° for 30 rain. In order to increase the gel strength, the gel is soaked in 5 liters of 0.3 M KCI for 4 hr at l0 °. After this treatment with resulting stiffgel is cut into cubes (3 × 3 × 3 mm) with a knife. To increase the fumarase activity and to suppress succinic acid formation, the immobilized cells are treated with 1 M sodium fumarate (pH 7.0) containing 0.6% bile extract at 37° for 20 hr according to the method described above. Fumarase activity and operational stability of the immobilized cells are shown in Table IV. I n d u s t r i a l P r o d u c t i o n o f L - M a l i c A c i d 4,8

A solution of 1 M sodium fumarate (pH 7.0) is passed through the immobilized cell column at 37° or 50° at an appropriate flow rate as described above. To 1000 liters of the effluent of the column, 200 liters of 38% HCI is added and cooled at 10° for 8 hr. The fumaric acid precipitated is recovered by centrifugation. About 70 kg of Ca(OH)2 is added to the supernatant until pH 6-7. After standing for 16 hr at 5% the resulting crystals of calcium L-malate dihydrate are collected by centrifugation and dried at 50°. Yield is 150 kg (90% based on effluent content). The calcium L-malate dihydrate obtained is suspended in 1000 liters of water, and 70 TABLE IV COMPARISON OF GEL STRENGTH, FUMARASEACTIVITY, AND THE OPERATIONAL STABILITY OF B. flal)urn IMMOBILIZEDWITH MODIFIED K-CARRAGEENAN

Kind of amine in modified r-carrageenan None NH2C2HsNH-

Gel strength (g/cA2)

Fumarase activity (/zmol/hrml gel)°

Operational stability at 37° (half-life, days)

910 1010 880

900 (50) 925 (51) 900 (50)

160 212 221

Values in parentheses show activity yields (%).

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liters of 60% (v/v) H2SO4is added. The resulting precipitate of CaSO4is removed by centrifugation and the supernatant is passed through a column of Ambedite IR-120 (H ÷ form) and then through a column of Amberlite IRA-93 (OH- form). The columns are washed with 500 liters of water. The effluent is concentrated at 60° in vacuo. To the syrup of Lmalic acid 700 liters of isopropyl alcohol is added, and then concentrated in vacuo. The resulting crystals of L-malic acid are collected by centrifugation and dried at 50°. Yield is 53 kg (55% based on calcium L-malate dihydrate); [a]~ = - 2 . 2 (c = 4.0 in water), mp 100°. The mother liquor can be used for another lot, or L-malic acid in the mother liquor can be recovered as the calcium salt in a good yield. The total yield of L-malic acid from fumaric acid is about 70%. Conclusion As described above, microbial cells having high fumarase activity can be easily immobilized and stabilized by the entrapment using K-carrageenan. The high stability of fumarase activity is resulted by Kcarrageenan in the gel state. Brevibacterium flavum immobilized with Kcarrageenan in the presence of polyethyleneimine or r-carrageenan modified with amines is considered to be stabilized by formation of polyion complex. On the other hand, high stability of B, flavum immobilized with K-carrageenan in the presence of tannin is considered to be induced by formation of hydrogen bond among K-carrageenan, tannin, and B. flavum cells. As shown in Table III, the productivity of B. flavum immobilized in the presence of Chinese gallotannin is highest, and is 3-fold higher than that in the absence of it. By using 1000-liter column packed with the immobilized B. flaoum 42.2 kg of L-malic acid/hr can be continuously produced for 6 months.

[42] I n d u s t r i a l P r o d u c t i o n o f L-Aspartic A c i d U s i n g P o l y u r e t h a n e - I m m o b i l i z e d Cells C o n t a i n i n g A s p a r t a s e

By

MURRAY

C. FUSEE

The use of immobilized Escherichia coli whole cells containing aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) activity for producing L-aspartic acid from ammonium fumarate has been practiced on an indusMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.

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liters of 60% (v/v) H2SO4is added. The resulting precipitate of CaSO4is removed by centrifugation and the supernatant is passed through a column of Ambedite IR-120 (H ÷ form) and then through a column of Amberlite IRA-93 (OH- form). The columns are washed with 500 liters of water. The effluent is concentrated at 60° in vacuo. To the syrup of Lmalic acid 700 liters of isopropyl alcohol is added, and then concentrated in vacuo. The resulting crystals of L-malic acid are collected by centrifugation and dried at 50°. Yield is 53 kg (55% based on calcium L-malate dihydrate); [a]~ = - 2 . 2 (c = 4.0 in water), mp 100°. The mother liquor can be used for another lot, or L-malic acid in the mother liquor can be recovered as the calcium salt in a good yield. The total yield of L-malic acid from fumaric acid is about 70%. Conclusion As described above, microbial cells having high fumarase activity can be easily immobilized and stabilized by the entrapment using K-carrageenan. The high stability of fumarase activity is resulted by Kcarrageenan in the gel state. Brevibacterium flavum immobilized with Kcarrageenan in the presence of polyethyleneimine or r-carrageenan modified with amines is considered to be stabilized by formation of polyion complex. On the other hand, high stability of B, flavum immobilized with K-carrageenan in the presence of tannin is considered to be induced by formation of hydrogen bond among K-carrageenan, tannin, and B. flavum cells. As shown in Table III, the productivity of B. flavum immobilized in the presence of Chinese gallotannin is highest, and is 3-fold higher than that in the absence of it. By using 1000-liter column packed with the immobilized B. flaoum 42.2 kg of L-malic acid/hr can be continuously produced for 6 months.

[42] I n d u s t r i a l P r o d u c t i o n o f L-Aspartic A c i d U s i n g P o l y u r e t h a n e - I m m o b i l i z e d Cells C o n t a i n i n g A s p a r t a s e

By

MURRAY

C. FUSEE

The use of immobilized Escherichia coli whole cells containing aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) activity for producing L-aspartic acid from ammonium fumarate has been practiced on an indusMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All fights of reproduction in any form reserved.

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ENZYME ENGINEERING (ENZYME TECHNOLOGY)

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trial scale in Japan since 1973. I Polyacrylamide gel encapsulation and, more recently, entrapment of the E. coli cells within a K-carrageenan gel ~ have been the immobilization methods. The advantages of using immobilized whole cells over intact cells in a batch reactor and of using extraction followed by immobilization of aspartase have been well documented by others. ~-3 Generally, the advantages relate to decreased costs associated with cell preparation and labor. In this section we describe a technique using polyurethane prepolymers for the immobilization of E. coli cells containing aspartase activity. 4 Preparation of Immobilized E. coli Cells Escherichia coli strain ATCC 11303 is grown under aerobic conditions at 37° in a medium containing ammonium fumarate (1.6%), MgSO4 • 7H20 (0.05%), KH2PO4 (0.2%), corn steep liquor (2.0%), and yeast extract (2.0%) (pH adjusted to 7.0). After a 24-hr growth period, the cells are harvested by centrifugation (5000 g, 30 min), and the wet cell paste (80% water) is mixed with the polyurethane prepolymer at ambient temperature (1 part cell paste plus 1 part prepolymer). The HYPOL hydrophilic prepolymer, prepared by capping a polyoxyalkylene polyol with an excess of toluene diisocyanate, is obtained from the W. R. Grace & Co. (Organic Chemicals Division, MA). The water and other isocyanate-reactive groups which reside on the cell surfaces, such as amines, react with the isocyanate-capped prepolymer. This reaction causes the formation of a polyurethane polymer which contains entrapped and presumably covalently bound E. coli cells containing aspartase activity. The foaming reaction is generally completed within 5-10 min and therefore rapid mixing is required to achieve uniformity within the polymer matrix. An additional 5-10 min is generally required for curing to its final semirigid form. The pH of the aqueous cell paste should be preferably in excess of 7 for optimum immobilization. Cell paste moisture content can vary, and with the E. coli strain it is preferable to use a cell paste with a moisture content in the 70-80% range for optimum cell immobilization. Based on the dry weight of the E. coli, the weight ratio of the polymer to cells in the foam can be about I : 10 to 10 : 1, but preferably from about 2 : 1 to 4 : 1 in this immobilized whole cell example. The resultant E. coli "foam" is ground t T. Sato, T. Mori, T, Tosa, 1. Chibata, M. Furui, K. Yamashita, and A. Sumi, Biotechnol. Bioeng. 17, 1797 (1975). T. Sato, Y. Nishida, T. Tosa, and I. Chibata, Biochim. Biophys. Acta 570, 179 (1979). 3 I. Chibata, in "Immobilized Microbial Cells" (K. Venkatsubramanian, ed.), Symp. Ser. Vol. 106, p. 187. American Chemical Society, Washington, D.C., 1979. 4 M. Fusee, W. Swarm, and G. Calton, Appl. Environ. Microbiol. 42, 672 (1981).

[42]

INDUSTRIAL PRODUCTION OF L-ASPARTIC ACID

465

to an average particle size of 0.5 cm in a Cumberland mill (this can be achieved manually with scissors if so desired) before being assayed for aspartase activity. Washing for - 1 hr with the substrate solution, 1.0 M ammonium fumarate (pH 9.0) containing 1.0 mM Mg 2÷, is recommended to wash out any nonbound cells prior to assaying. Assay of Aspartase Activity One gram of E. coli paste (obtained as above) is incubated in a stirred 100-ml solution of 1.0 M ammonium fumarate solution (pH 9.0) containing 1 mM Mg 2÷ and 0.1% (w/v) Triton X-100 at 37° for 60 min. Samples are removed at 15-, 30-, and 60-min intervals. The reaction is stopped by immersion in boiling water, and the cell debris is removed by centrifugation. The supernatant is analyzed for loss of fumaric acid and increase of L-malic acid. The difference between the decreased concentration of fumaric acid and the concentration of the L-malic acid produced represents the L-aspartic acid concentration. For immobilized cells, a weight of foam particles containing 1 g of cells (wet wt) is assayed similarly, except for the omission of surfactant from the assay solution. Two assays are recommended, since on occasion an "activation" occurs after the first assay. The activation phenomenon is presumably due to cell lysis which alleviates the substrate and product diffusion to and from the intracellular enzyme sites. Aspartase activity is defined in units per gram (wet wt) of cells, where 1 U is defined as the amount of enzyme giving 1/zmol of L-aspartic acid per hour under the conditions of the assay. Analysis of Fumaric Acid, L-Malic Acid, and L-Aspartic Acid The concentration of fumaric acid was measured either spectrophotometrically at 240 nm or enzymatically by a fumarase-malate dehydrogenase-coupled reaction, 5 where generated reduced nicotinamide adenine dinucleotide and H ÷ were measured by a thiazolyl blue tetrazolium bromide-diaphorase-coupled reaction. 6 fumarase

Fumaric acid + H20 . L-Malic acid + NAD ÷

malate dehydrogertase

NADH + H ÷ + tetrazolium dye

' L-malic acid

oxaloacetate + NADH + H ÷

diaphorase

NAD* + formizan dye

(1) (2) (3)

J. Williamson and B. Corkey, this series, Vol. 13, p. 434. 6 H. Mollering, W. Wahlefeld, and G. Michal, in "Principles of Enzymatic Analysis" (H. U. Bergmeyer and K. Gawehn, eds.), p. 88. Academic Press, New York, 1978.

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The equilibrium for reaction (2) lies to the left; therefore hydrazine was added to drive the reaction in the desired direction. The overall equation for this assay procedure appears as follows: Fumaric acid + HzO + N A D + + hydrazine + tetrazolium dye

(I) fumarase (2) malate dehydrogenase (3) diaphorase

o x a l o a c e t a t e - h y d r a z o n e + N A D + + formizan dye

Aspartic acid was analyzed by gas-liquid chromatography 7 with a 25-m Chirasil column (Applied Science Division, State College, PA). This analysis was performed occasionally to confirm that L-aspartic acid was being produced by the immobilized E. coli cells. Production of Aspartic Acid by Immobilized Cells in a Batch Reactor To assess the operational variables of polyurethane-immobilized E. coli cells with aspartase activity, we found the following bench-scale

system to be an ideal representation of an industrial-scale system. An 800ml batch reactor (9.87 × 10.16 cm) was used to investigate the optimum pH, temperature, and substrate concentration for the production of Laspartic acid from ammonium fumarate with the immobilized cells (Fig. 1). The immobilized cells particles were packed into the column, and a 2liter volume of ammonium fumarate containing 1 m M Mg 2+ was recycled through the bed at 5 cm/min (405 ml/min). The pump generated heat, which was controlled by a water-cooled coil inserted into the substrate reservoir. Small samples (0.5 ml) of substrate solution were withdrawn at timed intervals with a sampling pump and assayed for fumaric acid loss. For each run the reactions were allowed to continue until equilibrium was achieved. At this time the reactor system was drained, and the immobilized cells were left wet with the reaction product (i.e., ammonium aspartate) between runs. The total conversion of fumaric acid (as moles per liter) and the residence time required to accomplish this were calculated to give an average rate (moles per liter per minute). The residence time, in minutes, was calculated by multiplying the actual run time by 0.4 [immobilized cell bed volume (800 ml) divided by substrate volume (2000 ml)]. Malic acid was also determined spectrophotometrically. In one study, a 2-liter solution of 1.0 M ammonium fumarate (pH 9.0) containing 1 mM Mg 2+ (as MgSO4 • 7H20) was recycled through a column (9.87 x 10.16 cm) packed With immobilized cells (containing 126 g wet wt of cells) at a linear flow rate of 5 cm/min at 37° until no further change in 7 C. Gehrke a n d D. Stalling, Sep. Sci. 11, 101 (1967).

[42]

467

INDUSTRIAL PRODUCTION OF L-ASPARTIC ACID

=.

I

FLOW METER SUBSTRATE HOLDING- TANK J

POLYURETHANE IMMOBILIZED ~I

J

-i

t ~l~

(9.B7 x 10.16¢m1 GLASS/ BEADS

WATER ~-COOLED COIL

I

SAMPLING ~PUMP

i FLOW ADJUST VALVE

PUMP

~<~ DRAIN VALVE

/

FIG. 1. Batch reactor for production of L-aspartic acid from polyurethane-immobilizedE. coil cells.

fumaric acid was observed. This occurred at a residence time of 32 min. The above flow rate was chosen to approximate rates that will be used for industrial-scale reactors. The effluent was collected, adjusted to the isoelectric point of L-aspartic acid (pH 2.8) with H2SO4 at 90°, and then cooled. A white, crystalline material was collected by filtration, washed with water, and assayed. The crystalline material was determined to be >99% e-aspartic acid, and the yield was 227 g (85% of theoretical). The immobilized E. coli fumarase reaction produced L-malic acid which consistently represented 2 - 1% of the total fumaric acid lost. However, this by-product was not recovered in the crystalline material due to its higher solubility compared to L-aspartic acid.

Properties of Polyurethane-Immobilized E. coli Cells Containing Aspartase Activity Aspartase Activity of Various Immobilized E. coil Preparations Four polyurethane prepolymers of different isocyanate content were used to immobilize E. coli cells containing aspartase activity (Table I). Prepolymers with higher isocyanate content resulted in decreased immo-

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TABLE I EFFECT OF POLYURETHANE PREPOLYMER ISOCYANATE CONTENT ON IMMOBILIZED E. coli ASPARTASE ACTIVITY

Prepolymer

NCO content (meq/g)

E. coli a

Units/g

A B C D

2.16 1.63 2.48 1.79

48,966 61,035 46,035 68,017

a The aspartase activity of the free cells was determined to be 68,000 -+ 3000 units/g (wet

wt). bilized cell aspartase activity. Therefore, prepolymer D was used in Laspartic acid production by polyurethane-immobilized E. coli cells. Compared to other prepolymers, the superiority of prepolymer D appears to be related to its lower isocyanate content. This is not surprising since Chibata et al. 8 showed that cross-linking the E. coli cells with 2,4-toluene diisocyanate was detrimental to the aspartase. The remarkable retention of aspartase activity obtained with the polyurethane prepolymers (up to 100%) is presumed to be related to the porous, open-celled structure of the foam containing the cells, and to cell lysis, which allows a non-diffusion-limited environment for substrate and product. Effect o f Temperature All of the following operational parameters were studied using the batch reactor (Fig. 1) system. The observed rate of loss of fumaric acid with time was - 1 . 6 7 times faster at 37° than at 25 ° (Fig. 2) with 1.0 M ammonium fumarate (pH 9.0) solution as substrate. A substrate dilution by ammonium aspartate resulting from a previous test run was adjusted for in all subsequent data generated using the batch reactor (Fig. 1). The optimum operating temperature is dependent on the operational stability of the immobilized-cell aspartase activity. The immobilized cells showed good operational stability at 37° with either 1.0 or 1.5 M ammonium fumarate substrate at pH 8.5 to 9.0, with pH 8.5 preferred. Continuous operation over a period of weeks is possible before a fresh immobilized E. coli batch is required for repacking the reactor. I. Chibata, T. Tosa, and T. Sato, Appl. Microbiol. 27, 878 (1974).

[42]

INDUSTRIAL PRODUCTION OF L-ASPARTIC ACID

469

I.$ 1.4 1.3 1.2

Ill

I.I I.C

zw

z O

.3 .2 .I

~ C

0

.

I0

.

ZO

.

30

, .'2'

,

=

40

-"

-"

I

50

RESIDENCE TIME (MINUTES)

FIG. 2. Effect of temperature on the reaction rate of polyurethane-immobilized E. coli cells. The reactor contained 126 g (wet wt) E. coil cells. Two liters of 1 M ammonium fumarate (pH 9.0) was recycled at a linear flow rate of 5 cm/min. The average conversion rate at 37 ° was 0.028 mol/liter-min, and at 25" it was 0.017 mol/liter-min. The initial fall in substrate concentration from 1.0 to 0.89 M was due to substrate dilution by ammonium aspartate from the previous run. ( I ) 37°; (T) 25°.

Effect of pH The rate of reaction was decreased as the pH was lowered (Fig. 3). The reaction rate was approximately the same in the pH 8.5 to 9.2 range. With this particular immobilized E. coli cell preparation, an average reaction rate of 0.0206 tool/liter per minute was observed at pH values of 8.5, 9.0, and 9.2. Fumaric acid concentration determined at the equilibrium points was 0.011 and 0.010 M for the pH 8.3 and 9.0 runs, respectively. A fairly broad pH optimum (8.5 to 9.2) was observed for the polyurethane cell preparation, whereas the polyacrylamide-entrapped 8,9 and Kcarrageenan immobilized 2 preparation showed a more narrow pH profile with an optimum at 9.0. The broader pH profile is desirable since it offers increased engineering design flexibility. Below pH 8.5, the low ammo9 T. Tosa, T. Sato, T. Mori, and I. Chibata, Appl. Microbiol. 27, 886 (1974).

470

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[42]

'°I ttl r¢ u.~

.(

~

.4

O

.3 .2 .I

0

I0

20

30

40

50

RESIDENCETIME(MINUTES) FIG. 3. Effect of pH on the reaction rate of polyurethane-immobilized E. coil cells. Reaction conditions are the same as those in Fig. 2, except that the temperature was 37° and the pH varied. The average reaction rate was 0.0165 mol/liter-min at pH 8.3 and 0.0206 tool/ liter-rain at pH 9.0. (0) pH 8.3; (I') 9.0.

nium ion concentration results in a decreased reaction rate and less fumaric acid conversion to L-aspartic acid. Effect o f Ammonium Fumarate Concentration

An increased concentration of ammonium fumarate appears to affect negatively the immobilized-cell aspartase reaction rate (Fig. 4). The average rate in 1.5 M substrate was - 8 % lower than for 1.0 M. We repeated this stttdy on three separate occasions, utilizing three separate E. coilpolyurethane preparations to corroborate this observation. The negative effect on reaction rate attributable to increased ammonium fumarate concentration was also noted by Takamatsu et al. 1ofor a polyacrylamide gelentrapped E. coli preparation. Previous studies in our laboratory with intact cells did not show this rate difference under the same set of conditions. We believe that the rate change observed for our immobilized cell example is related to polyurethane polymer deformation caused by the higher ionic strength (1.5 M) of the ammonium fumarate solution. Such 10 S. Takamatsu, K. Yamashita, and A. Sumi, J. Ferment. Technol. 58, 129 (1980).

[42]

471

INDUSTRIAL PRODUCTION OF L-ASPARTIC ACID I.¢

w

tt.

t,~ .6

z

~

.3 .2 .I

0 0

I

I0

I

20

30

40

50

"~-

RESIDENCE TIME (MINUTES)

FIG. 4. Effect of substrate concentration on the reaction rate of polyurethane-immobilized E. coli cells. Reaction conditions are the s a m e as t h o s e given in Fig. 2, except that the t e m p e r a t u r e w a s 37 ° and the substrate concentration varied. The average reaction rate at 1.5 M a m m o n i u m f u m a r a t e concentration was 0.0227 mol/liter-min, and at 1.0 M a m m o n i u m f u m a r a t e concentration it was 0.0247 tool/liter-rain. The substrate dilution for the 1.5 M concentration resulted in a decrease to 1.28 M. ( 0 ) 1.5 M a m m o n i u m fumarate; (T) 1.0 M a m m o n i u m fumarate.

deformation might contribute to substrate and product diffusion barriers to and from the aspartase as theorized by Takamatsu et al. for polyacrylamide systems. However, in order to maximize the overall ammonium fumarate to L-aspartic acid productivity, considering reaction rates and enzyme use stability properties, we recommend using 1.5 M ammonium fumarate, pH 8.5, and 37°.

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[43] C o n t i n u o u s L - A l a n i n e P r o d u c t i o n U s i n g T w o D i f f e r e n t I m m o b i l i z e d M i c r o b i a l Cell P r e p a r a t i o n s on a n Industrial Scale By ICHIRO CHIBATA, TETSUYA TOSA, and SATORU TAKAMATSU

L-Alanine is used as a component of amino acid infusion and as a food additive because of its good taste. It has traditionally been produced from solid L-aspartic acid by a batch enzymatic method using the activity of Laspartate fl-decarboxylase of intact Pseudomonas dacunhae cells. I In turn, L-aspartic acid has been efficiently produced from ammonium fumarate using aspartase activity of immobilized Escherichia coli cells. Thus, in order to improve productivity of L-alanine, we investigated the immobilization of P. dacunhae cells and the method of continuous production of L-alanine from ammonium fumarate, using these two immobilized microbial cells according to the following reaction. Fumaric acid + NH3.

E. coli

• L-asparticacid

aspartase

P. dacunhae

L-aspartate fl-decarboxylase

, L-alanine+ CO2

In our results, L-alanine was produced efficiently by using these two immobilized microbial cells instead of the conventional batch method. In this article, the method of preparation of immobilized microbial cells suitable for industrial application and the bioreactors used for these sequential enzyme reactions are described.

Assay Methods Estimation o f L-Alanine. L-Alanine is measured by bioassay using Leuconostoc citrovorum ATCC 8081 according to the method of Snell. 2 Estimation o f L-Aspartic Acid. L-Aspartic acid is measured by bioassay using Leuconostoc mesenteroides P-60 according to the method of Henderson and Snell) Estimation o f Fumaric Acid. Fumaric acid is measured spectrophotometrically by the method of Bock and Alberty. 4 Estimation o f L-Malic Acid. L-Malic acid is measured colorimetrically by the method of Goodman and Stark)

I. Chibata, T. Kakimoto,and J. Kato, J. Appl. Microbiol. 13, 638 (1965). z E. E. Snell, this series, Vol. 3, p. 477. 3 L. M. Hendersonand E. E. Snell,J. Biol. Chem. 172, 15 (1948). 4R. M. Bock and R. A. Alberty,J. Am. Chem. Soc. 75, 1921 (1953). 5A. E. Goodmanand J. B. Stark, Anal. Chem. 29, 283 (1957). METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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473

Estimation of D-Alanine. D-Alanine is assayed by converting it to pyruvic acid using D-amino acid oxidase. The pyruvic acid is colorimetrically determined by the direct method of Friedmann and Haugen. 6 Assay of Enzyme Activities Aspartase of Free Cells. To 0.003 g (wet weight) of free cells, 2 ml of substrate solution (adjusted to pH 8.5 with ammonia) consisting of 1 M ammonium fumarate, 1 mM Mg 2÷, and 0.1% Triton X-100 is added. The mixture is shaken at 37° for 1 hr and heated at 100° for 5 min. The amount of L-aspartic acid formed is determined. Its activity is expressed as micromoles of L-aspartic acid produced per hour. Aspartase of Immobilized Cells. A mixture of 1 g of immobilized cells and I0 ml of the substrate solution is shaken at 37° for 30 rain. After removal of the gel by filtration, the amount of L-aspartic acid formed is determined. Activity is expressed as micromoles of L-aspartic acid produced per hour. L-Aspartate ~-Decarboxylase of Free Cells. To 0.02 g (wet weight) of free cells, 3 ml of substrate solution (adjusted to pH 5.5 with ammonia) consisting of 1 M ammonium L-aspartate, 0.1 mM pyridoxal Y-phosphate (PLP), 1 mM pyruvic acid, and 0.05% OP-10 is added. The mixture is shaken at 37° for 1 hr and heated at 100° for 5 min. The amount of Lalanine formed is determined. Activity is expressed as micromoles of Lalanine produced per hour. L-Aspartate ~-Decarboxylase oflmmobilized Cells. A mixture of 1 g of immobilized cells and 10 ml of the substrate solution is shaken at 37° for 1 hr. After removal of the gel by filtration, the amount of L-alanine produced is determined. Activity is expressed as micromoles of L-alanine produced per hour. Fumarase of Free Cells. To 0.02 g (wet weight) of free cells, 1.98 ml of substrate solution (adjusted to pH 7.0 with 3 N KOH) consisting of 1 M potassium fumarate and 0.1% Triton X-100 (for E. coli cells) or 0.5% OP10 (for P. dacunhae cells) is added and the mixture is shaken at 37° for 1-5 hr. The reaction is stopped by the addition of 2 ml of 2 N HCI, and the amount of L-malic acid formed is determined after the remaining fumaric acid is crystallized out and removed. Activity is expressed as micromoles of L-malic acid produced per hour. Fumarase of Immobilized Cells. A mixture of 1 g of immobilized cells and 10 ml of the substrate solution is shaken at 37° for 1-24 hr. After removal of the gel by filtration, the filtrate is acidified with an equal volume of 2 N HCI to remove the remaining fumaric acid, and the amount 6 T. E. Friedmann and G. E. Haugen, J. Biol. Chem. 147, 415 (1943).

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ENZYME ENGINEERING(ENZYME TECHNOLOGY)

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of L-malic acid formed is determined. Activity is expressed as micromoles of L-malic acid produced per hour. Alanine Racemase of Free Cells. To 0.02 g (wet weight) of free cells is added 2 ml of substrate solution (pH 10 for alanine racemase ofE. coli and pH 9 for that o f P . dacunhae, pH is adjusted with 3 N KOH) consisting of 1.2 M L-alanine, 1.2 M NH4HCO3, 0. l mM PLP, and 0.1% Triton X-100 (for E. coli cells) or 0.05% OP-10 (for P. dacunhae cells), and the mixture is shaken at 37 ° for 24 h. Then 0.9 ml of 0.0167 M sodium pyrophosphate solution (adjusted to pH 8.3 with 3 N KOH) is added to 0.1 ml of the reactant and the mixture is heated at 100° for 5 min. The amount of oalanine formed is determined. Activity is expressed as micromoles of oalanine produced per hour. Alanine Racemase of Immobilized Cells. A mixture of 1 g of immobilized cells and 10 ml of the substrate solution is shaken at 37° for 24 hr. After removal of the gel by filtration, 0.9 ml of the sodium pyrophosphate solution is added to 0.1 ml of the filtrate, and the amount of D-alanine formed is determined. Activity is expressed as micromoles of D-alanine produced per hour. Culture of Microbial Cells

Escherichia coli. Escherichia coli ATCC 11303 is cultured under aerobic conditions at 37 ° for 20 hr in 1 liter of medium (pH 7.0) containing ammonium fumarate (5 g), fumaric acid (11.4 g), corn steep liquor (20 g), Meast (autolysate of brewer's yeast, 20 g), K2HPO4 (2 g), and MgSO4" 7H20 (0.5 g). The cells are collected by centrifugation and subjected to immobilization. About 25 g (wet weight) of E. coli cells is obtained from 1 liter of broth, and its aspartase, fumarase, and alanine racemase activities are approximately 200,000, 20,000, and 1000/zmol/hr per gram of wet cells in the fresh state, respectively. Pseudomonas dacunhae. Pseudomonas dacunhae IAM 1152 is cultured under aerobic conditions at 30° for 24 hr in 1 liter of medium (pH 7.3) containing sodium L-glutamate (32 g), Meast (5 g), KH2PO4 (0.5 g), and MgSO4.7H20 (0.1 g). The cells are collected by centrifugation and subjected to immobilization. About 20 g (wet weight) o f P . dacunhae cells is obtained from 1 liter of broth, and its L-aspartate fl-decarboxylase, fumarase, and alanine racemase activities are approximately 20,000, 10,000, and 500 tzmol/hr per gram of wet cells in fresh state, respectively. Immobilization of Microbial Cells Among many methods of immobilization of microbial cells, the Kcarrageenan method (see this volume [41]) is suitable in this case. Immobilized preparations are easily obtained by this method.

[43]

PRODUCTION OF L-ALANINE

475

Immobilization ofE. coli Cells. The E. coli cells (20 g wet weight) are suspended in 20 ml of the supernatant of culture broth and warmed to 40°. r-Carrageenan (5.18 g) is dissolved in 113 ml of physiological saline and the solution is warmed to 45° . Both solutions are mixed, and the mixture is cooled to 5° to form a gel. To increase gel strength, the gel obtained is soaked in about 500 ml of cold 2% KC1 solution overnight. After this treatment, the rigid gel is cut into cubes of 3 × 3 × 3 mm with a knife. Its aspartase, fumarase, and alanine racemase activities are approximately 12,000, 1300, and 90/zmol/hr per gram of gels in fresh state, respectively. Immobilization of P. dacunhae Cells. The P. dacunhae cells (20 g wet weight) are suspended in 20 ml of the supernatant of culture broth and warmed to 45 °. K-Carrageenan (4.25 g) is dissolved in 85 ml of physiological saline and the solution is warmed to 45° . Both solutions are mixed, and the mixture is cooled to 5° to form a gel. To increase gel strength, the gel obtained is soaked in about 500 ml of cold 2% KCI solution for overnight. After this treatment, the rigid gel is cut into cubes of 3 × 3 x 3 mm with a knife. Its L-aspartate /3-decarboxylase, fumarase and alanine racemase activities are approximately 2000, 800, and 30/zmol/hr per gram of gels in flesh state, respectively. Elimination of Side Reactions When immobilized whole microbial cells are used as biocatalysts for the production of useful compounds, sometimes by-products are formed by the reaction of undesirable enzymes involved within cells. In the case of L-alanine production, both E. coli and P. dacunhae cells have alanine racemase and fumarase activities. Therefore, when the mixture of immobilized E. coli cells and immobilized P. dacunhae cells is used for production of L-alanine from ammonium fumarate, small amounts of D-alanine and L-malic acid are formed as by-products by alanine racemase and fumarase reactions, respectively. To eliminate alanine racemase and fumarase activities of E. eoli and P. dacunhae cells, several methods were investigated. As the results, pH treatments of the intact cells were found to be effective and to be industrially applicable. 7 The procedures are as follows. E. coli Cells. The pH of the culture broth involving the intact cells is adjusted to 5.0 by addition of acetic acid and the broth is incubated at 45 ° for 1 hr. After incubation, the pH of the broth is adjusted to 5.5 by addition of 6 N NaOH, and the cells are collected by centrifugation and immobilized with K-carrageenan as with intact cells. 7 S. Takarnatsu, I. Umemura, K. Yamamoto, T. Sato, T. Tosa, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 15, 147 (1982).

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TABLE I ENZYME ACTIVITIES OF IMMOBILIZED PREPARATIONS WITH OR WITHOUT pH TREATMENT OF MICROBIAL CELLS

Enzyme activities(/zmol/hr-gof gels) Microbial cells E. coli

P. dacunhae

pH treatment

L-Aspartate B-decarA s p a r t a s e boxylase

Alanine racemase Fumarase

-

12,250

--

89.4

+

12,200

--

1.0

-

--

1870

28.0

+

--

1690

0.3

131ff 1.4 745 0.5

Pseudomonas dacunhae Cells. The pH of the culture broth involving the intact cells is adjusted to 4.75 by addition of acetic acid in the presence of 0.5 mM PLP and the broth is incubated at 30° for 1 hr. After incubation, the pH of the broth is adjusted to 6.0 by addition of 6 N NaOH, and the cells are collected and immobilized with K-carrageenan as with intact cells. Table I shows the enzyme activities of pH-treated microbial cells immobilized with K-carrageenan. By use of a mixture of immobilized, pHtreated E. coli cells and immobilized, pH-treated P. dacunhae cells, ealanine can be efficiently produced from ammonium fumarate without formation of D-alanine and L-malic acid.

Stabilization of L-Aspartate fl-Decarboxylase Activity of P. dacunhae Cells by Glutaraldehyde Treatment For continuous production of L-alanine from ammonium fumarate on an industrial scale, aspartase and L-aspartate/3-decarboxylase activities of immobilized preparations are expected to be stable during long operation periods. Although aspartase activity of immobilized pH-treated E. coli cells is relatively stable, L-aspartate fl-decarboxylase activity of immobilized pH-treated P. dacunhae cells is unstable. Therefore, several methods were investigated to stabilize L-aspartate /3-decarboxylase activity of pH-treated P. dacunhae cells. In our results, glutaraldehyde treatment of the cells was found to be effective. 8 The procedure is as follows. After pH treatment of P. dacunhae cells, the broth is cooled to 10° and the cells are contacted with 5 mM glutaraldehyde for I0 min. After this treatment, the cells are collected by centrifugation and subjected to immobilization. 8 S. Takamatsu, T. Tosa, and 1. Chibata,]. Chem. Soc. Jpn. 9, 1369 (1983).

[43]

PRODUCTION OF L-ALANINE

2000

half-life A-.g~-------------~a~ ~ , - _ . (d a y s ) " O ~ O ---~"m--~--O-OI---W--O .... 260

0L •..~[r .•

--,------z--z__-LL_ •

"~ ~' 1000 l- f f Q o

-,"1 ~

L ~ N

500

477

.0_

~'~--.zx

.

o-oo

o__

PII

80

o

o

O

200

II

I

I

0

20

40 Operation

time

I

I

60 (day)

80

F]o. 1. Operational stability of L-aspartate fl-decarboxylase activity of various immobilized P. dacunhae cells. Immobilized cells (3 g) are packed into a column (1.2 x 9 cm), and l M ammonium L-aspartate solution (pH 5.5) containing 1 mM pyruvic acid and 0.1 mM PLP is passed continuously upward through the column at the rate of 3 ml/hr at 37°. Enzyme activity is determined from conversion by changing the flow rate to 18 ml/hr. (A) Immobilized untreated cells; (A) immobilized glutaraldehyde-treated cells; (O) immobilized pHtreated cells; and ( I ) immobilized pH- and glutaraldehyde-treated cells.

Figure 1 shows the operational stability of several immobilized preparations. Immobilized pH- and glutaraldehyde-treated cells show highest enzyme activity and operational stability. Bioreactors for L-Alanine Production Closed Column Reactor. When a conventional column reactor is employed for the L-aspartate/3-decarboxylase reaction, L-alanine is not efficiently produced due to backmixing of the substrate solution and deviation of pH value from optimum range by liberation of CO2 gas during enzyme reaction. These problems were solved by using a closed column reactor which performed the enzyme reaction at high pressure. 9 With this reactor, liberated CO2 gas is dissolved into reaction mixture, and almost complete plug flow of substrate solution is obtained and the pH of reaction mixture is not so changed. The efficiency of the reactor is 50% higher than that of conventional one. On the other hand, the operational stabilities of the enzyme activity are the same in both reactors. Sequential Reactions. After succeeding in the development of a closed column reactor, the most efficient conditions for the two-step enzyme reaction using immobilized pH-treated E. coli cells and immobilized pH9 M. Furui and K. Yamashita, J. Ferment. Technol, 61, 587 (1983).

478

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

Substrate so u

oo

took

I

I IImm°bi .1 F-

~,

liP" ~

(._ t/,~,,,,/,A ] -~ ~, ~

[43]

I [closed [ [column , /[

[Imm°bi'[ I Temperature

/F''Solfl L controlledJ l

V/f/////A < - w a t e r

t ( PLP iiiuvat e Pump

~

~/////////~

/~-~ ty

~ Plunger pump

ItL-A, lanineJl

FIG. 2. Flow diagram for continuous production of L-alanine by two immobilized microbial cells.

and glutaraldehyde-treated P. dacunhae cells were investigated on the basis of rate equations.l° A sequential reaction using two column reactors, one containing immobilized pH-treated E. coli cells and a closed column one containing immobilized pH- and glutaraldehyde-treated P. dacunhae cells, was found to be most efficient for L-alanine production from ammonium fumarate. In this sequential reactor system, the pH of effluent from immobilized E. coli cells column is adjusted to 6.0 from 8.5 and charged into immobilized P. dacunhae cells in the closed column. The optimum pH values of the aspartase of immobilized pH-treated E. coli cells and the L-aspartate fl-decarboxylase of immobilized pH- and glutaraldehyde-treated P. dacunhae cells are 8.5 and 6.0, respectively. Industrial Production of L-Alanine Flow Diagram. Figure 2 shows the flow diagram of industrial production of L-alanine using immobilized pH-treated E. coli cells and immobi1o S. Takamatsu, T. Tosa, and I. Chibata, Proc. Annu. Meet. Soc. Ferment. Technol. Jpn. 179 (1983).

[44]

IMMOBILIZEDAMINOTRANSFERASES

479

lized pH- and glutaraldehyde-treated P. dacunhae cells. The substrate solution (adjusted to pH 8.5 with ammonia) consisting of 1.5 M ammonium fumarate and 1 mM Mg 2+ is applied to the column containing immobilized E. coli cells at a flow rate of space velocity = 1.0/hr. After addition of PLP and pyruvic acid (these concentrations are 0.1 and 1 mM, respectively) into the effluent, pH of the solution is adjusted to 6.0 by addition of acetic acid. The solution is passed through the immobilized P. dacunhae closed column at a flow rate of space velocity = 0.06/hr and a pressure of about 8 kg/cm 2 achieved by plunger pump. Crystallization of L-Alanine from Column Effluent. The effluent of appropriate volume is concentrated to about one-fourth of its original volume and cooled to 15°. L-Alanine crystallized is collected by centrifugation or by filtration and washed with 80% aqueous ethanol. The yield of L-alanine from ammonium fumarate is about 90% (theoretical). [~]~ = + 14.8 (c = 10 in 6 N HCI). Conclusion In 1982, Tanabe Seiyaku Co. Ltd. successfully industrialized a continuous production system of L-alanine from ammonium fumarate, using a column reactor containing immobilized pH-treated E. coli cells and a closed column reactor containing immobilized pH- and glutaraldehydetreated P. dacunhae cells. By this system, L-alanine has been produced at low cost. This is considered to be the first industrial application of sequential enzyme reactions using two immobilized microbial cells.

[44] I m m o b i l i z e d A m i n o t r a n s f e r a s e s for Amino Acid Production

By J. DAVID ROZZELL Aminotransferases

Background Aminotransferases (more commonly called transaminases, EC 2.6.1._) are a widely distributed class of enzymes. These enzymes catalyze the synthesis and breakdown of amino acids in microorganisms, METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[44]

IMMOBILIZEDAMINOTRANSFERASES

479

lized pH- and glutaraldehyde-treated P. dacunhae cells. The substrate solution (adjusted to pH 8.5 with ammonia) consisting of 1.5 M ammonium fumarate and 1 mM Mg 2+ is applied to the column containing immobilized E. coli cells at a flow rate of space velocity = 1.0/hr. After addition of PLP and pyruvic acid (these concentrations are 0.1 and 1 mM, respectively) into the effluent, pH of the solution is adjusted to 6.0 by addition of acetic acid. The solution is passed through the immobilized P. dacunhae closed column at a flow rate of space velocity = 0.06/hr and a pressure of about 8 kg/cm 2 achieved by plunger pump. Crystallization of L-Alanine from Column Effluent. The effluent of appropriate volume is concentrated to about one-fourth of its original volume and cooled to 15°. L-Alanine crystallized is collected by centrifugation or by filtration and washed with 80% aqueous ethanol. The yield of L-alanine from ammonium fumarate is about 90% (theoretical). [~]~ = + 14.8 (c = 10 in 6 N HCI). Conclusion In 1982, Tanabe Seiyaku Co. Ltd. successfully industrialized a continuous production system of L-alanine from ammonium fumarate, using a column reactor containing immobilized pH-treated E. coli cells and a closed column reactor containing immobilized pH- and glutaraldehydetreated P. dacunhae cells. By this system, L-alanine has been produced at low cost. This is considered to be the first industrial application of sequential enzyme reactions using two immobilized microbial cells.

[44] I m m o b i l i z e d A m i n o t r a n s f e r a s e s for Amino Acid Production

By J. DAVID ROZZELL Aminotransferases

Background Aminotransferases (more commonly called transaminases, EC 2.6.1._) are a widely distributed class of enzymes. These enzymes catalyze the synthesis and breakdown of amino acids in microorganisms, METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

480

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

H2N~ ' H

+

0

0

[44]

+ H2N~'%H

SCHEME 1.

plants, and animals by the transfer of an amino group from an a-amino acid to a 2-ketoacid as shown in Scheme 1. The first evidence for aminotransferases was published by Needham j and Szent-Gy6rgyi and co-workers 2 who noticed a relationship between the L-glutamic acid, L-aspartic acid, and oxaloacetic acid levels in pigeon breast muscle. Banga and Szent-Gy6rgyi3 demonstrated the reversibility of glutamic-pyruvic transaminase (EC 2.6.1.2, alanine aminotransferase) by chemically isolating the amino acid products L-glutamate and Lalanine. Since that time, a large number of aminotransferases have been discovered and characterized. One feature of aminotransferases is the requirement for the small molecule, pyridoxal Y-phosphate, for catalytic activity, this cofactor being bound through a Schiff base linkage to the eamino group of an active-site lysine. Although the binding of pyridoxal 5'phosphate to the enzyme is reversible, most aminotransferases show maximal catalytic activity at cofactor concentrations of 100/~M or less. Such low saturating concentrations of pyridoxal phosphate are an important property of aminotransferases; at concentrations of 100/.~M or less, the cost of the cofactor in biocatalytic transamination processes is a relatively minor component of the total cost. The mechanism of transamination is well known, and has been reviewed previously.* The reaction catalyzed by aminotransferases occurs as the result of two distinct half-reactions: the first involves transfer of the amino group of the L-amino acid donor to pyridoxal 5'-phosphate to yield a 2-ketoacid product which is released from the enzyme and an enzymebound pyridoxamine Y-phosphate; the second is the binding of the 2ketoacid to be transaminated to the enzyme and the transfer of the amino group from pyridoxamine 5'-phosphate to this 2-ketoacid to produce the desired L-amino acid and regenerate the pyridoxal 5'-phosphate. As a result, aminotransferases characteristically exhibit Ping-Pong kinetics. D. M. Needham, Biochem. J. 24, 208 (1930). 2 E. Annau, I. Banga, A. Blazo, V. Bruckner, K. Laki, F. B. Staub, and A. Szent-Gy6rgyi, Z. Physiol. Chem. 224, 105 (1936). I. Banga and A. Szent-Gy6rgyi, Z. Physiol. Chem. 245, 118 (1937). A. E. Braunstein, "The Enzymes IX" (P. D, Boyer, ed.), Part B, pp. 379-481. Academic Press, New York, 1973.

[44]

IMMOBILIZEDAMINOTRANSFERASES

481

Advantages and Disadvantages for Use in Biocatalysis Although aminotransferases have been known for decades, these enzymes have seen little use as biocatalysts until now. However, since the normal function of aminotransferases is the biosynthesis and metabolism of amino acids, it is natural to look to these enzymes as potentially useful catalysts for the production of amino acids. In principle, almost any desired amino acid can be produced from the appropriate 2-ketoacid using an inexpensive amino acid as the amino donor. There are a number of advantages to the use of this kind of technology. (1) The aminotransferase enzymes catalyze the stereoselective synthesis of only L-amino acids from their corresponding 2-ketoacids. No D isomer is produced, and no resolution is required. (2) Aminotransferases have uniformly high catalytic rates, capable of converting up to 400/~mol of substrate/min per milligram of protein. (3) Many of the required 2-ketoacid precursors can be conveniently prepared by chemical synthesis at low cost. (4) The capital investment for an immobilized enzyme process using aminotransferases is much lower than for a fermentation process, and the productivity of the bioreactor is more than an order of magnitude higher. (5) The technology is generally applicable to a broad range of L-amino acids because aminotransferases exist with varying specificities. For example, there are enzymes specific for the transamination of amino acids with acidic side chains, aromatic side chains, branched alkyl side chains, etc. Such broad scope allows a number of different L-amino acids to be produced with the same equipment and often the same biocatalyst. We have demonstrated laboratory-scale processes for the production of a variety of L-amino acids including L-alanine, L-phenylalanine, L-tyrosine, L-tryptophan, and several others. As an example, we have immobilized the commercially available glutamic-pyruvic aminotransferase from porcine heart on porous glass by covalent attachment, and obtained a stable biocatalyst with an activity of 400 International Units per gram. A column packed with 500 mg of this immobilized enzyme was operated continuously for 6 months and produced 160 mg L-alanine/hr from pyruvic acid as a starting material. This example illustrates the potential of immobilized aminotransferases applied to the production of L-amino acids. There is one inherent disadvantage to the practice of this technology as described so far; as a catalyst, the aminotransferase can only accelerate the approach to equilibrium between the L-amino acid and 2-ketoacid precursors on one side of the equation and the 2-ketoacid and L-amino acid products on the other. Thus, the equilibrium constant for the generic transamination reaction as written in Scheme 1 is near unity, and the

482

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

HO2CCH2\C/C02H H2N~' %H L-Aspartic Acid

HO2CCH2~(CO2H

0 transamtnase

R C02H 2-KetoacJd

decarboxylase ~

Oxaloacetic Acid

+ 0

oxaloacetate

H3C

[44]

"~( 0

C02H

+ C02

Pyruvic Acid

+

R.

C02H

L-Amino Acid SCHEME 2.

conversion of a 2-ketoacid feedstock to a desired L-amino acid will not proceed to completion in most cases. The key to the development of a general and commercially successful transamination process for the production of L-amino acids lies in overcoming this problem of incomplete conversion of a 2-ketoacid to the desired L-amino acid. Driving the Reaction to Completion

Solving the problem of incomplete conversion of 2-ketoacid starting material to a desired L-amino acid required one important observation regarding the substrate specificity of aminotransferases. Although L-glutamic acid is generally considered to be the amino donor for aminotransferases that catalyze the transamination of a broad range of 2-ketoacids to L-amino acids, we have found that L-aspartic acid can also function competently as a general donor of an amino group with certain enzymes. We have worked extensively with an aminotransferase capable of using L-aspartic acid isolated from Escherichia coli. 5 When L-aspartic acid is used as the amino group donor for the transamination of a given 2-ketoacid, oxaloacetic acid is coproduced along with the desired L-amino acid. Oxaloacetate, unlike 2-ketoglutarate, is a /3-ketoacid, and as such can facilely be converted to pyruvic acid via an essentially irreversible decarboxylation step. This may be accomplished chemically by the use of certain metal ions or amines, thermally, or most preferably, enzymatically using the enzyme oxaloacetate decarboxylase. The coupled two-enzyme reaction is illustrated in Scheme 2. The important feature of this process is the decarboxylation of oxaloacetate to pyruvate. It is this essentially irreversible decarboxylation that drives the entire process to completion to produce L-amino acids in quantitative yields from the appropriate 2-ketoacid precursors. The 5 C. Mavrides and W. Orr, Biochim. Biophys. Acta 336, 70 (1974).

[44]

IMMOBILIZEDAMINOTRANSFERASES

483

pyruvic acid by-product is easily separated from the product mixture by crystallization of the L-amino acid or by ion-exchange methods. We have investigated several methods for decarboxylating oxaloacetate, including catalysis by primary amines and divalent metal ions such as Mg 2+, Mn z+ , and Zn z+ and the enzymatic decarboxylation by oxaloacetate decarboxylase (OAD, EC 4.1.1.3). This chapter will focus on driving the overall reaction by the OAD-catalyzed decarboxylation of oxaloacetate to produce pyruvate and the desired L-amino acid. Methodology

Sources and Production of Enzymes Aminotransferases can be isolated from virtually any microbial, plant, or animal source. The most easily obtained enzymes are from porcine heart, yeast, and E. coli. However, the usefulness of the individual aminotransferases for amino acid production varies. The glutamic-oxaloacetic aminotransferase from porcine heart (EC 2.6.1.1, aspartate aminotransferase), although very stable and commercially available, is of limited utility for the production of amino acids because of its high specificity for L-glutamic acid, L-aspartic acid, and the corresponding 2-ketoacids as substrates. 6 Other substrates are not transaminated at reasonable rates. Similarly, the commercially available porcine glutamic-pyruvic aminotransferase (EC 2.6.1.2, alanine aminotransferase) also exhibits the desirable properties of high stability, high specific activity, and lack of severe inhibition even at substrate concentrations up to 0.4 M, but the enzyme cannot use L-aspartic acid as the amino group donor. Thus, a highly productive immobilized biocatalyst can be prepared using this aminotransferase, and it can be used for the production of e-alanine from pyruvic acid and L-glutamate, but the reaction cannot be driven to completion by the coupling of oxaloacetate decarboxylase. These readily available enzymes have nonetheless been useful as model aminotransferases in the development and design of biocatalytic transamination processes, and data for the immobilization and use of these enzymes in bioreactors will be presented. The microorganism E coli. is one of the most useful sources of aminotransferases. Of the four aminotransferases from this microorganism characterized to date, v we have found the so-called glutamic-oxaloacetic 6 I. W. Sizer and W. T. Jenkins, this series, Vol. 5, p. 677. 7 j. T. Powell and J. F. Morrison, Eur. J. Biochem. 87, 391 (1978).

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ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[44]

TABLE I RELATIVE RATES OF PRODUCTION OF L-AMINO ACIDS USING ASPARTIC AMINOTRANSFERASE FROM E. coli a L-Amino acid

Precursor

Relative rate

L-Glutamic acid L-Phenylalanine L-Tyrosine L-Tryptophan L-2-Aminoadipic acid L-4-Phenyl-2-aminobutanoic acid L-Histidine

2-Ketoglutarate Phenylpyruvate p-Hydroxyphenylpyruvate lndolyl-3-pyruvate 2-Ketoadipate 4-Phenyl-2-ketobutyrate Imidazole 3-pyruvate

225 100 130 150 22 17 3

a Amino group donor in all cases: L-aspartic acid.

aminotransferase (EC 2.6.1.1) to be an extremely useful catalyst for e-amino acid production. This enzyme is the most stable of the E. coli aminotransferases, and is produced constitutively. 8,9 The enzyme is capable of catalyzing the transamination of a large number of 2-ketoacids to e-amino acids using either L-glutamate or L-aspartate as cosubstrate. Table I lists the relative rate of transamination for the production of a variety of amino acids. Interestingly, for use as a biocatalyst in the production of aromatic amino acids such as L-phenylalanine, the E. coli glutamic-oxaloacetic aminotransferase (GOA) is superior to the enzyme which carries out this function metabolically, the so-called aromatic-amino-acid aminotransferase (AA) (EC 2.6.1.5). The former enzyme is significantly more thermostable than the latter. In addition, the glutamic-oxaloacetic aminotransferase has catalytic rate constants for the transamination of phenylpyruvate to L-phenyalanine, p-hydroxypyruvate to L-tyrosine, and indole 3-pyruvate to L-tryptophan comparable to those for the aromatic aminotransferase. The K,~ for these aromatic amino acids is approximately an order of magnitude higher for the GOA enzyme, but given that high concentrations of substrates are generally used in a biocatalytic process, the enzyme is functioning at its maximal catalytic rate. The enzyme oxaloacetate decarboxylase (OAD) has been isolated from three different sources: Pseudomonas putida ATCC 950,1° Micro8 C. Mavrides and W. On', J. Biol. Chem. 250, 4128 (1975). 9 S. Chesne, N. Monnier, and J. Pelmont, Biochimie 60, 403 (1978). ~0A. A. Horton and H. L. Kornberg, Biochim. Biophys. Acta 89, 381 (1964).

[44]

IMMOBILIZEDAMINOTRANSFERASES

485

coccus luteus ATCC 4698,11 and Azotobacter vinelandii ATCC 478.12,13 We have purified all three of these enzymes, and have found the OADs isolated from P. putida and M. luteus as the most suitable for a biocatalytic process based on the criteria of specific activity, stability to operational conditions, activity after immobilization, and the ease of production and isolation. The enzymes may be obtained by modifications of published procedures, l°,ll Details of these purifications will be published elsewhere. 14 Immobilization of Aminotransferases and Assays for Activity A large number of immobilization methods have been examined for the immobilization of aminotransferases. Adsorption methods such as the binding of enzyme to ion-exchange resins failed to yield a stable immobilized enzyme preparation due to desorption of enzyme from the support. Entrapment in polymeric gels such as polyacrylamide produced active enzyme, but the activity was lower than that obtained using other methods. Also, this method suffered from the slow loss of activity due to diffusion of the enzyme out of the polymeric matrix. Covalent coupling of the enzyme to inert supports has proved to be the method of choice for the aminotransferases we have examined to this point, providing immobilized enzymes with high retention of activity, long-term operational stability, and good mechanical properties. The successful immobilization of aminotransferases is highly dependent on the chemistry of the immobilization technique. Because of the requirement for the binding of the cofactor pyridoxal 5'-phosphate to the 8-amino group of a lysine residue, reagents such as glutaraldehyde, p-nitrophenyl esters, N-hydroxysuccinimidyl esters, and the like, which react with amines on the protein, can deactivate aminotransferases. For this reason, it is absolutely essential that the active site be protected during the immobilization by including pyridoxal 5'-phosphate and a 2ketoacid (e.g., 2-ketoglutarate) in the reaction mixture. Alternatively, the coupling can be accomplished through carboxyl groups on the enzyme to amino groups on the support using a water-soluble carbodiimide. Methods for the immobilization of aminotransferases involving the covalent binding of enzyme through its carboxyl groups to primary H O. x2 S. 13 G. 14 G.

L. Krampitz and C. H. Werkman, Biochem. J. 35, 595 (1941). S. Lee, R. H. Burris, and P. W. Wilson, Proc. Soc. Exp. Biol. Med. 50, 96 (1942). W. E. Plaut and H. A. Lardy, J. Biol. Chem. 180, 13 (1949). Edwards, J. Heier, and J. D. Rozzell, in preparation.

486

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[44]

amines on the surface of an inert support gave immobilized aminotransferases of high activity. Typical supports include porous glasses, porous ceramics, and porous diatomaceous earths. Oxaloacetate decarboxylase can be immobilized using similar procedures, also with good retention of catalytic activity. The following are sample procedures for the immobilization of various preparations of aminotransferase and oxaloacetate decarboxylase and procedures for assaying the immobilized enzymes.

Immobilization of Glutamic-Oxaloacetic Aminotransferase from E. colifor the Production of L-Phenylalanine. Controlled-pore glass with an average pore size of 500 ,~ is converted to the aminopropyl form by reaction with triethoxy-3-aminopropylsilane using the aqueous procedure of Weetall. ~5Aminopropyl glass (2.0 g) is added to 10 ml of a solution of sodium borate (5 mM) containing pyridoxal 5'-phosphate (0.5 mM). The pH is adjusted to 7.0, and 50 mg of a lyophilized glutamic-oxaloacetic aminotransferase from E. coli, which has been partially purified to a specific activity of approximately 2.5 units/mg (when assayed for the transamination of phenylpyruvate to L-phenylalanine) is added. After dissolution of the enzyme, ethyl dimethylaminopropylcarbodiimide hydrochloride (100 mg) is added, and the reaction mixture is shaken at room temperature for 45 min on a rotary shaker. At the end of this time the suspension is poured into a funnel with a glass frit (coarse porosity) and suction filtered. The support is washed successively with three portions of potassium phosphate buffer (50 mM, pH 7.0), three portions of 200 mM NaCI, and three more times with phosphate buffer. The immobilized biocatalyst has an activity of 120 units (60 units/g) when assayed for the transamination of phenylpyruvate to L-phenylalanine with L-aspartic acid as the amino donor. The activity retained on immobilization is approximately 95%. Assays for the activity of free aspartic aminotransferase are carried out by a method similar to that described by Mavrides and Orr. 8 In a typical assay procedure, 0.700 ml potassium phosphate buffer (50 mM, pH 7.0), 0.100 ml L-aspartate (pH 7.0, 200 mM), 0.100 ml 2-ketoacid solution (pH 7.0, 100 mM), 0.030 ml NADH solution (5 mg/ml in H20), 0.050 ml malate dehydrogenase solution (1000 U/ml in 50 mM potassium phosphate buffer, pH 7.0), and 0.010 ml pyridoxal 5'-phosphate (pH 7.0, 10 raM) are pipetted into a cuvette. A background rate is determined by monitoring the change in the absorbance at 340 nm as a function of time, and the reaction is initiated by the addition of the aminotransferase sam15 H. H. Weetall, this series, Vol. 34, p. 59.

[44]

IMMOBILIZEDAMINOTRANSFERASES

487

pie (0.010 ml). Activity of the enzyme is calculated by the following equation: Activity (International Units/ml) = (AOD340/min)(100/6.2) To assay the immobilized enzyme for activity, a weighed amount of immobilized enzyme (approximately 20-40 mg) is added to 1.0 ml of a solution containing potassium phosphate buffer (50 mM, pH 7.0), L-aspartic acid monosodium salt (100 mM), phenylpyruvic acid sodium salt (100 mM), and pyridoxal 5'-phosphate (0.1 mM). Aliquots of 10/zl are taken at 1-min time intervals and diluted into 990/zl of 2.5% NaOH (w/v). After mixing, 100/xl of this solution is added to a cuvette containing 900/xl of 2.5% NaOH, and the absorbance at 320 nm is read. The concentration of phenylpyruvate remaining at 1-min time intervals is calculated (e320 = 17.5 mM -1 cm -1) and from this data an aminotransferase activity can be calculated using the following equation: Activity (International Units)/g

AOD320 min

1000 17.5 x grams of immobilized enzyme

An alternative assay is based on the quantitation of total oxaloacetate + pyruvate produced as a result of transamination (a small amount of pyruvate is produced by the spontaneous decarboxylation of oxaloacetate) by stoichiometric reduction with NADH using a combination of malic dehydrogenase and lactic dehydrogenase. The incubation of immobilized aminotransferase and substrates is carried out as above, and 20-/zl aliquots are removed at 2-min time intervals and diluted into a cuvette containing 850/zl of 50 mM potassium phosphate buffer (pH 7.0), 50/xl of a 1000 unit/ml solution of lactate dehydrogenase, 50/zl of a 1000 unit/ml solution of malate dehydrogenase, and 30 /~l of a 5 mg/ml solution of NADH. The net change in the absorbance at 340 nm is a measure of the consumption of NADH and therefore the total oxaloacetate + pyruvate p r o d u c e d (8340 = 6.2 m M -~ cm-~). Activity of the immobilized enzyme is calculated by the following equation: Activity (International Units)/g

AOD340

50

min

6.2 x grams of immobilized enzyme

A small background correction must be made for the reduction of phenylpyruvate, which is a poor substrate for lactate dehydrogenase.

488

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[44]

Immobilization of Glutamic-Oxaloacetic Aminotransferase from Porcine Heart. Aminopropyl controlled-pore glass prepared as described above (0.500 g) is suspended in 5 ml of sodium borate solution (5 mM) containing pyridoxal 5'-phosphate (0.5 raM) and the pH is adjusted to approximately 7. Glutamic-oxaloacetic aminotransferase (14 rag, specific activity 18.2 units/nag) is dissolved in the solution, followed by the addition of ethyl dimethylaminopropylcarbodiimide hydrochloride (50 rag). The reaction mixture is agitated at room temperature on a rotary shaker for 60 rain, after which time assays have shown negligible aminotransferase activity in the solution. The controlled-pore glass particles are transferred to a coarse frit funnel and washed repetitively with 10-ml portions of water, potassium phosphate buffer (pH 7.0), three times with 0.2 M NaCI, and finally with phosphate buffer. The combined washings contain 8.8 mg of protein. The immobilized aminotransferase activity is 50 units when assayed for the transamination of L-aspartate and 2-ketoglutarate. The enzymatic activity retained after immobilization is 53%. The assay for aminotransferase activity is carried out as described earlier for the quantitation of oxaloacetate + pyruvate produced using NADH, lactate dehydrogenase, and malate dehydrogenase. Immobilization of Glutamic-Pyruvic Aminotransferase from Porcine Heart. Aminopropyl controlled-pore glass prepared as described above (0.500 g) is suspended in 5 rnl of sodium borate solution (5 raM) containing pyridoxal 5'-phosphate (0.5 mM). The pH is adjusted to 7, and 30 mg of glutamic-pyruvic aminotransferase having a specific activity of 51 units/rag is dissolved in the solution. Ethyl dimethylaminopropylcarbodiimide hydrochloride (50 rag) is added, and the reaction mixture is shaken on a rotary shaker for 60 min at room temperature. At the end of this time, the reaction mixture is transferred to a glass frit funnel (coarse porosity) and the porous glass particles are washed with water, potassium phosphate buffer (50 mM, pH 7.0), three times with NaC1 (200 mM), and again with potassium phosphate buffer. The combined washings contain 20 mg of protein (assayed by the method of Bradford'6), indicating that 10 mg has been retained on the support. The activity of the immobilized enzyme was 200 units/0.5 g of support. The enzymatic activity retained after immobilization is 40% of the activity of the same quantity of native enzyme in solution. Activity of the immobilized enzyme can be determined by quantitating the amount of pyruvate remaining in the reaction mixture after fixed times. A weighed amount of immobilized enzyme (approximately 20 rag) is suspended in a solution containing sodium pyruvate (100 raM), mono16M. Bradford, Anal. Biochem. 72, 248 (1976).

[44]

IMMOBILIZEDAMINOTRANSFEILASES

489

sodium L-glutamate (100 mM), pyridoxal 5'-phosphate (0.1 mM), and potassium phosphate (50 mM). The pH of the solution is 7. Aliquots of 0.020 ml are removed at 1.0-min intervals and diluted into 0.180 ml of potassium phosphate buffer (50 mM). Ten microliters of this solution is added to a cuvette containing 0.900 ml potassium phosphate (pH 7.0, 50 raM), 0.050 ml of NADH solution (5 mg/ml in H20), and 0.040 ml lactate dehydrogenase (1000 U/ml in potassium phosphate buffer, pH 7.0, 50 mM). The net change in the absorbance at 340 nm is measured, and the average AOD340per minute is calculated. Activity of immobilized glutamic-pyruvic aminotransferase is calculated by the following equation: Activity (International Units)/g AOD340

500

min

6.2 × grams of immobilized enzyme

An alternative assay for the activity of immobilized glutamic-pyruvic aminotransferase is described below which measures the amount of 2ketoglutarate produced as a function of time. Approximately 10 mg of immobilized enzyme is suspended in 1.0 ml of a solution containing monosodium L-glutamate (I00 mM), sodium pyruvate (100 mM), potassium phosphate buffer (pH 7.0, 50 mM), and pyridoxal 5'-phosphate (0.1 mM). Aliquots of 10/zl are withdrawn at 1-min time intervals and diluted into a cuvette containing 800/xl of potassium phosphate buffer (pH 7.0), 50/xl of a 200 unit/ml solution of saccharopine dehydrogenase (Sigma Chemical Co., St. Louis, MO), 100/zl of a 100 mM solution of L-lysine, and 40/~1 of a 5 mg/ml solution of NADH. The net change in the absorbance at 340 nm is determined (e340 = 6.2 mM -1 cm-1).

Immobilization of Oxaloacetate Decarboxylase from Pseudomonas putida. Cells of Pseudomonas putida are grown, harvested, and the enzyme purified as described elsewhere, l: Aminopropyl glass (0.500 g) prepared as described earlier is suspended in 5 ml of 5 mM sodium borate buffer containing 10 mM MgC12, and 45 mg of a partially purified oxaloacetate decarboxylase having a specific activity of 30 units/mg is added. Ethyl dimethylaminopropylcarbodiimide hydrochloride (125 mg) is added and the reaction mixture is agitated on a rotary shaker for 60 min at room temperature. Assays have shown that by this time the decline of oxaloacetate decarboxylase activity in the supernatant (due to immobilization of the enzyme on the support) has slowed significantly. Independent controls show a negligible decrease in activity of the OAD due to chemical modification by the carbodiimide. The reaction mixture is transferred to a funnel with a glass frit (coarse porosity), and the immobilized

490

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[44]

enzyme is washed with water, three times with Tris-HCl buffer (50 mM, pH 8.0), containing MgCI2 (10 mM), three times with NaCI solution (200 mM), and finally with Tris buffer. The combined washings contain 14 mg protein. The immobilized oxaloacetate decarboxylase contain 275 units/ 500 mg support. The enzymatic activity retained after immobilization is 30%. To determine the activity of immobilized oxaloacetate decarboxylase, a weighed amount of immobilized enzyme (approximately 10 mg) is added to 2.0 ml of a solution containing oxaloacetate (100 mM), Tris-HCl buffer (50 mM) with pH adjusted to 8.0, and MgCI2 (10 mM). The mixture is shaken at 25 °, 10-/zl aliquots are withdrawn at 1- to 2-min intervals and diluted into a cuvette containing 990 tzl of water, and the absorbance at 262 nm is measured. The net change in the absorbance at 262 nm corresponds to the consumption of oxaloacetate (B262 = 0.78 mM -1 cm-~). Activity of the immobilized enzyme is calculated by the following equation: Activity (International Units)/g AOD262

100

rain

0.78 x grams of immobilized enzyme

Immobilization o f Glutamic-Pyruoic Aminotransferase on Porous Diatomaceous Earth. Porous diatomaceous earth from Johns-Manville (R 640, 50/100 mesh) is boiled for 12 hr in 5% nitric acid and washed with deionized water; the fine particles are decanted, and dried for 12 hr in an oven at 110°. The support is then converted to the aminopropyl derivative by the aqueous activation procedure of Weetall. j5 Aminopropyl support (5.0 g) is suspended in sodium borate (5 mM) containing 2-ketoglutarate (0.5 mM), pyridoxal phosphate (0.5 mM), and 110 mg glutamic-pyruvic aminotransferase (specific activity 49 units/mg, Lee Scientific). Ethyl dimethylaminopropylcarbodiimide-HC1 (250 mg) is added and the reaction mixture is placed on a rotary shaker for 2 hr at room temperature. At the end of this time, the support is transferred to a glass frit funnel (coarse porosity) and washed repeatedly with water and then 50 mM potassium phosphate buffer containing 0.1 mM pyridoxal phosphate. Assay for protein in the combined washings ~6 has indicated that 80 mg of protein is bound to the support. The support contains 850 units of activity. The activity retained after immobilization is 22%. Immobilization o f Glutamic-Oxaloacetic Aminotransferase on Activated Porous Diatomaceous Earth. Aminopropyl porous diatomaceous earth (500 rag) prepared as described above is suspended in 2 ml of potas-

[44]

IMMOBILIZED AMINOTRANSFERASES

491

sium phosphate buffer (50 mM, pH 7.0) containing 2-ketoglutarate (2 mM), pyridoxal phosphate (0.1 mM), and 26 mg partially purified glutamic-oxaloacetic aminotransferase from porcine heart (specific activity of 14 units/mg). Ethyl dimethylaminopropylcarbodiimide hydrochloride (I0 mg) is added and the reaction mixture is agitated on a rotary shaker for 1.5 hr. After washes as described above on a glass frit funnel with 50 mM phosphate buffer containing 0.5 M NaC1 and finally phosphate buffer, 3.3 mg of protein is found to be bound to the support by quantitation of the recovered protein. Assay of the bound aminotransferase as described earlier gives 19 units of activity on the support. The specific activity of the immobilized enzyme is 41%. Summary of Results. Using the carbodiimide method for immobilization, the three different aminotransferase (glutamic-oxaloacetic from porcine heart, glutamic-oxaloacetic from E. coli, and glutamic-pyruvic from porcine heart) could all be immobilized to porous supports bearing primary amine functional groups with good retention of activity. The results are summarized in Table II. The stability to operational conditions is also quite high. For GPA immobilized on porous glass, the immobilized enzyme showed little loss of activity over 6 months of operation as described below. Measurement of the Long-Term Stability of the Immobilized Enzymes. Measurements to determine the operational stability of immobilized enzyme were carried out by pumping substrate mixtures through a packed bed of biocatalyst at 25° and quantitating the amount of product in the effluent stream as outlined previously. In the case of glutamic-pyruvic aminotransferase, a solution of L-glutamate monosodium salt (200 mM), sodium pyruvate (400 mM), and pyridoxal phosphate (0.1 mM) with a pH between 7.0 and 7.5 was used as the substrate mixture. Activity of the TABLE II IMMOBILIZATION ON POROUS SUPPORTS

Enzyme

Support

Loading (mg/g)

Activity of immobilized enzyme (units/g)

GOA (E. coli) GOA (porcine) GPA OAD GPA GOA

Glass Glass Glass Glass Diatomaceous earth Diatomaceous earth

25 10 20 30 16 6

60 100 400 550 170 38

492

ENZYME ENGINEERING(ENZYMETECHNOLOGY)

[44]

IOO 80 % Activity

60 / 40 20 I I 5 I0

I 20

I 30

I 40

I 50

I 60

Time (days)

FIG. 1. Long-term operational stability of immobilized glutamic-pyruvic aminotransferase. biocatalyst was determined by quantitating both the amount of 2-ketoglutarate produced and the amount of pyruvate consumed by the methods described earlier. The column was operated intermittently over 2 months for periods varying between 1 day and 1 week at a time. When not in use, the immobilized enzyme columns were stored at room temperature in substrate solution. The results of the experiments are shown in Fig. 1. When immobilized glutamic-oxaloacetic aminotransferase from E. coli was mixed with immobilized oxaloacetate decarboxylase from P. putida, the resulting biocatalyst was shown to exhibit good stability and versatility for the production of a number of L-amino acids. As examples, L-glutamic acid, L-phenylalamine, L-tyrosine, L-tryptophan, L-2-aminoadipic acid, and L-4-phenyl-2-aminobutanoic acid could all be produced along with equimolar quantities of pyruvate using this two-enzyme system. Work to further expand the scope of this process is in progress. Retention o f the Pyridoxal 5'-Phosphate Cofactor in a Membrane Reactor One of the ways in which the economics of the process just described could be improved would be to eliminate the requirement for continuous addition of the pyridoxal 5'-phosphate cofactor during the operation of the bioreactor. Because pyridoxal 5'-phosphate is a small molecule approximately the same size as an amino acid (MW = 247), retaining this molecule in a bioreactor using a membrane presents a problem. In the case

[44]

IMMOBILIZED AMINOTRANSFERASES

493

I. Olethylazodlcarboxylate Phthanmide

Triphenylphosphine HO(CH=CH20). CH2CH20H

2. H2NNH~

)

H2N(CH2CH20)nCH2CH2NH2

~

Tosyl Chloride

TsO(CH2CH20)n CH=CH2OTs

1. Potassium PhthallmLde 2. H~.NINH~

,T

SCHEME 3. Synthesis of diaminopolyethylene glycol.

of nicotinamide cofactors, this problem of retention in a membrane reactor has been solved elegantly by Wandrey, Wichmann, Kula, Biickmann, and others 17-2~ by covalently attaching the cofactor to a soluble polymer. The macromolecularized cofactor retains the majority of its catalytic activity after attachment to the polymer, and it can be retained along with enzymes in a bioreactor using a membrane which allows the passage of small molecule substrates and products (such as amino acids) but not higher molecular weight compounds. We have successfully demonstrated a similar approach for retaining a catalytically active form of pyridoxal 5'-phosphate in a membrane reactor. Although a.number of polymers can be used, we chose a modified polyethylene glycol of molecular weight 4000 or greater as the backbone polymer. The first step is the conversion of the polyethylene glycol to an t~,to-diamino derivative. This can be accomplished by one of two methods. The first method involves reacting the polyethylene glycol with p-toluenesulfonyl chloride to produce a ditosylate derivative, displacement of the tosylates with potassium phthalimide, and hydrazinolysis.]9,2° An alternative method uses the reaction of polyethylene glycol with diethylazodicarboxylate and triphenylphosphine in the presence of phthalimide 22 to product the ct,to-diphthalimido derivative directly, followed by hydrazinolysis or hydrolysis to produce the a,to-diaminopolyethylene glycol. These two reaction schemes are illustrated in Scheme 3. 17 R. Wichmann, D. Wandrey, A. F. B0ckmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). is I. Urabe, N. Katayama, and H. Okeda, Enzyme Eng. 6, 239 (1982). ,9 A. F. BOckmann, M.-R. Kula, R. Wichmann, and C. Wandrey, J. Appl. Biochem. 3, 301 (1981). 20 S. Furukawa, N. Katayarna, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). zl C. Wandrey, R. Wichmann, W. Leuchtenberger, M.-R. Kula, and A. Biickmann, European Patent Application 040,281. 22 O. Mitsunobu, M. Wada, and T. Sano, J. Am. Chem. Soc. 94, 679 (1972).

494

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[44]

Diaminopolyethylene glycol so produced is reacted with p-nitrobenzoyl chloride in the presence of the acylation catalyst 4-dimethylaminopyridine, followed by reduction of the nitro groups to amines with sodium dithionite. Diazotization of the bis-p-aminobenzoyl derivative is accomplished with HCI and NaNO2 ,:3 generating the bisdiazonium derivative. Pyridoxal 5'-phosphate is very reactive in diazonium coupling reactions at pH values sufficiently basic to cause deprotonation of its phenolic hydroxyl group. Thus, incubation of the bisdiazonium derivative with pyridoxal phosphate at pH 8.0 produces a macromolecularized pyridoxal phosphate derivative in which the cofactor is attached to the modified polyethylene glycol polymer via a diazo linkage to the C-6 ring position. The synthesis of this derivatized, macromolecularized cofactor is outlined in Scheme 4. The modified, macromolecularized pyridoxal phosphate has a maximum absorbance at 413 nm compared with 389.5 nm for the native cofactor, and when the stability of the modified cofactor to laboratory light was measured, it was found to be 20- to 30-fold more stable to photochemical decomposition at neutral pH values relative to unmodified cofactor. 24 The modified pyridoxal phosphate exhibited 65-80% of the activity of native pyridoxal phosphate when used under saturating conditions as a coenzyme with several enzymes including apoglutamic-oxaloacetic aminotransferase from porcine heart, apoaspartate aminotransferase from E. coli, and apotryptophanase from E. coli. The modified cofactor was used with an aminotransferase from E. coli in an ultrafiltration unit for 1 week with no loss in activity, producing L-phenylalanine from phenylpyruvic acid and L-aspartic acid. Procedures for the preparation and use of the macromolecularized pyridoxal 5'-phosphate cofactor are described below. Synthesis of Diaminopolyethylene Glycol. Polyethylene glycol, MWav 8000 (1.0 g), is dissolved in 10 ml dry CH2C12, and 50 mg phthalimide, which has been dried at 110° overnight, 90 mg triphenylphosphine, and 65 /zl of diethylazodicarboxylate (Aldrich Chemical Co., Milwaukee, WI) are added. The reaction mixture is stirred at room temperature for 3 hr under a nitrogen atmosphere. At the end of this time, the contents of the reaction are poured into 150 ml anhydrous diethyl ether, and the flocculent white precipitate is collected and air dried. This product is then dissolved in 20 ml deionized water, and 1.1 ml of hydrazine hydrate is added. The resulting solution is refluxed for 18 hr. After cooling, the reaction mixture is extracted three times with CH2C12, and the extracts are combined and z3 p. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970). 24 C. Y. W. Ang, J. Assoc. Off. Anal. Chem. 62, 1170 (1979).

[44]

IMMOBILIZED AMINOTRANSFERASES

Z

495

z

tj

:12

z

Z

Z

212

&

0 o

-r 0 0

0

0

0

o~.,

:ff

-iZ

0

/

X: Z

Z

°l z

]= q.)

L)

U

0

'J .~

iii -13

,~ z

Z

~o

"~ e~

o

o

~ ~.-.~

~

e'~ e~

z

0

~

tD O CL

]1

c L

~0

u

~ m

+el Z

212

.4 L~ "I-

z

e

~

~

m

0

z o

I

"1"

¢j "1" z

+

r~

496

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[44]

dried over anhydrous Na2SO4. The diaminopolyethylene glycol is precipitated with anhydrous diethyl ether, and the precipitate is collected by suction filtration and air dried. Redissolution in CH2C12 and reprecipitation by the addition of diethyl ether yield a colorless product with a 65% conversion of free alcohols to amine end groups when assayed using trinitrobenzenesulfonic acid. z5 Synthesis of Pyridoxal 5'-Phosphate Attached to a Soluble Polyethylene Glycol Derivative. Diaminopolyethylene glycol (0.5 g), prepared as described above, is dissolved in 15 ml anhydrous CH2C12, and p-nitrobenzoyl chloride (60 mg), p-dimethylaminopyridine (10 mg), and triethylamine (50/~l) are added. The reaction mixture is stirred at room temperature overnight under a nitrogen atmosphere. After the reaction is complete, saturated NaHCO3 is added and the mixture is allowed to stand for 30 min. The CH2C12 layer is separated, washed three times with 15-ml portions of deionized water, and dried over anhydrous Na2SO4. The bisp-nitrobenzoyldiaminopolyethylene glycol was precipitated by the addition of 200 ml of anhydrous diethyl ether. Assay for primary amines using trinitrobenzenesulfonic acid z5 indicates at 99% conversion of available amines. The recovery of product is 374 mg. The bis-p-nitrobenzoyl derivative is dissolved in 20 ml of 0.5 M NaHCO3, pH 8.5, and 450 mg of 85% Na2S204 (Sigma) is added. The reaction mixture is stirred at 40 ° for 1 hr. The resulting solution is dialyzed for 36 hr against 4 liters of deionized water (three changes). Assay for primary amines as before has indicated a 61% conversion of nitro groups to aromatic amines. The bis-p-aminobenzoyldiaminopolyethylene glycol product is lyophilized and stored at 4 ° until needed. Bis-p-aminobenzoyldiaminopolyethylene glycol (150 mg) is reacted with 2 ml of 0.5 M HCI at 4 °, and 70 mg NaNO2 in 0.5 ml H20 prechilled to 4 ° is added. The reaction mixture is stirred for 7 min at 4°, and pH is immediately adjusted to 8.0 by the addition of chilled 0.5 M NaHCO3 and NaOH, and 40 mg of pyridoxal 5'-phosphate is added. The resulting orange solution is stirred at 4° for 12 hr protected from light. The reaction mixture is dialyzed against 2 liters of deionized water (three changes), and the product is isolated by lyophilization. The derivatized pyridoxal 5'phosphate has a reddish-orange color, and the absorbance maximum is 413.5 nm. Use of the Macromolecularized Pyridoxal 5'-Phosphate Cofactor Derivative in a Continuous Flow Membrane Reactor. For membrane bioreactor studies, an Amicon 8MC ultrafiltration apparatus with a 100-ml reservoir and a 2-ml reaction changer was used. The reservoir was period25R. Fields, Biochem. J. 124, 581 (1978).

[45]

PHENYLALANINE PRODUCTIONVIA E. coli

497

ically refilled with substrate solution over the course of the experiment. An Amicon YM5 membrane having a molecular weight cutoff of approximately 5000 was used in the membrane reactor. A substrate solution containing potassium phosphate buffer (50 mM, pH 7.0) sodium phenylpyruvate (50 mM), and L-aspartate (50 mM) was prepared, and 100 ml of this solution was used to fill the reservoir of the ultrafiltration unit. A solution of 0.5 ml of macromolecularized pyridoxal phosphate derivative prepared as above (concentration 8 mg/ml) containing 10 units of aspartic aminotransferase from E. coli and 1.5 ml of substrate solution were mixed in the reaction chamber. The flow through the membrane reactor was initiated by nitrogen gas pressure, and the solution in the reaction chamber was stirred continuously. The flow rate was maintained at 2 ml/hr. Durifig continuous operation over 1 week the average conversion of phenylpyruvate to L-phenylalanine was 78-80%, as determined by quantitation of both phenylpyruvate consumed and pyruvate + oxaloacetate produced. The assays were carried out as described earlier.

[45] P h e n y l a l a n i n e P r o d u c t i o n v i a P o l y a z e t i d i n e - I m m o b i l i z e d E s c h e r i c h i a coli: O p t i m i z a t i o n o f Cell L o a d i n g By G. J. CALTON, L. L. WOOD, and M. L. CAMPBELL

The use of polyazetidine-immobilized microbial cells for the production of specialty chemicals has been commercialized as a result of the excellent economic and engineering characteristics this catalyst provides. These advantages include high retention of activity, excellent enzyme stability of the immobilized cell, high flow rate, and support rigidity which allows expanded column configurations.~ Immobilization of a number of different microbes has been accomplished with increased lifetimes and/or retention of activity. 2 The potential use of phenylpyruvate as a starting material for the production of phenylalanine has been examined by a number of investigators. Ziehr et al. have used reductive amination as well as the transt L. L. Wood and G. J. Calton, Biotechnology 1, 1091 (1985). 2 G. J. Calton, L. L. Wood, M. H. Updike, L. Lantz II, and J. P. Hamman, in press (1986).

METHODS IN ENZYMOLOGY, VOL. 136

Biotechnology,

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[45]

PHENYLALANINE PRODUCTIONVIA E. coli

497

ically refilled with substrate solution over the course of the experiment. An Amicon YM5 membrane having a molecular weight cutoff of approximately 5000 was used in the membrane reactor. A substrate solution containing potassium phosphate buffer (50 mM, pH 7.0) sodium phenylpyruvate (50 mM), and L-aspartate (50 mM) was prepared, and 100 ml of this solution was used to fill the reservoir of the ultrafiltration unit. A solution of 0.5 ml of macromolecularized pyridoxal phosphate derivative prepared as above (concentration 8 mg/ml) containing 10 units of aspartic aminotransferase from E. coli and 1.5 ml of substrate solution were mixed in the reaction chamber. The flow through the membrane reactor was initiated by nitrogen gas pressure, and the solution in the reaction chamber was stirred continuously. The flow rate was maintained at 2 ml/hr. Durifig continuous operation over 1 week the average conversion of phenylpyruvate to L-phenylalanine was 78-80%, as determined by quantitation of both phenylpyruvate consumed and pyruvate + oxaloacetate produced. The assays were carried out as described earlier.

[45] P h e n y l a l a n i n e P r o d u c t i o n v i a P o l y a z e t i d i n e - I m m o b i l i z e d E s c h e r i c h i a coli: O p t i m i z a t i o n o f Cell L o a d i n g By G. J. CALTON, L. L. WOOD, and M. L. CAMPBELL

The use of polyazetidine-immobilized microbial cells for the production of specialty chemicals has been commercialized as a result of the excellent economic and engineering characteristics this catalyst provides. These advantages include high retention of activity, excellent enzyme stability of the immobilized cell, high flow rate, and support rigidity which allows expanded column configurations.~ Immobilization of a number of different microbes has been accomplished with increased lifetimes and/or retention of activity. 2 The potential use of phenylpyruvate as a starting material for the production of phenylalanine has been examined by a number of investigators. Ziehr et al. have used reductive amination as well as the transt L. L. Wood and G. J. Calton, Biotechnology 1, 1091 (1985). 2 G. J. Calton, L. L. Wood, M. H. Updike, L. Lantz II, and J. P. Hamman, in press (1986).

METHODS IN ENZYMOLOGY, VOL. 136

Biotechnology,

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

498

ENZYMEENGINEERING(ENZYMETECHNOLOGY)

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TABLE I EFFECT OF INCREASING ASPARTIC ACID CONCENTRATION ON THE CONVERSION OF PHENYLPYRUVATE TO PHENYLALANINE

Percentage conversion based on aspartic acid concentration pH

0.10 M

0.15 M

0.2 M

7.0 8.4

-77.6

87 90

-96.7

aminase of Pseudomonas putida. 3 This work was carried out in either hollow fiber or batch reactors. With reductive amination in an NADcoupled system, 100% conversions were obtained. However, in transamination, a maximum conversion of 80% was obtained. Bulot and Cooney4 also found that phenylpyruvate could be used as a substrate for the production of phenylalanine with a molar yield of 75% in a conventional fermentation using a valine-producing Corynebacterium glutamicum. In a similar effort, Fusee was able to produce phenylalanine via Escherichia coli, achieving a maximum yield of 67%. 5 It is readily apparent from these studies that a commercially feasible process requires recycling of phenylpyruvate unless a superior yield can be achieved. Free Cell Studies Free cell evaluation of phenylalanine production were carried out with E. coli 11303 (American Type Culture Collection), which has reasonably high levels of transaminase. Escherichia coli cells (2 g, wet weight) were sonicated for 10 rain at 4 ° after which they were incubated at 37° for 23 hr in 25 ml of an aqueous solution containing varying amounts of aspartic acid at the pH indicated, 0.1 mM pyridoxal 5-phosphate, and 0.1 M phenylpyruvic acid (Table I). Table I shows increased conversion yields of phenylalanine were achieved by increasing the ratio of aspartic acid to phenylpyruvate to greater than a I : 1 ratio. 2 Escherichia coli contains three transaminases which could participate in this reaction. Gelfand and Steinberg 6 developed three E. coli mutants 3 H. Ziehr, W. Hummel, H. Reichenbach, and M.-R. Kula, Eur. Congr. Biotechnol., 3rd, 1, 345 (1984). 4 E. Bulot and C. L. Cooney, Biotechnol. Lett. 7, 93 (1985). 5 M. C. Fusee, German Patent Application DE3,427,495 AI (1985). 6 D. H. Gelfand and R. A. Steinberg, J. Baeteriol. 130, 429 (1977).

[45]

PHENYLALANINE PRODUCTIONVIA E. coli

499

TABLE II CONVERSION OF PHENYLPYRUVATE TO PHENYLALANINE AS A FUNCTION OF TIME a

Conversion (%) Source E. coli mutant aspC 0.15 M glutamate (Glu) 0.15 M aspartate (Asp) trpB 0.15 M Glu 0.15 M Asp ilvE 0.15 M Glu 0.15 M Asp E. coli 11303 0.15 M Glu 0.15 M Asp

2 hr

12 hr

28 hr

55 hr

48 32

59 49

59 86

59 95

16 32

24 44

27 48

30 54

6 4

11 12

12 16

15 18

64 32

64 63

64 88

64 98

a Escherichia coli cell mutants (1 g wet wt, unless specified below), aspC, ilvE, and E. coli 11303 were placed in 25 ml of 100 mM phenylpyruvate, 0.1 mM pyridoxal 5-phosphate, and either 150 mM glutamate or 150 mM aspartate. The tyrB mutant was used in the above mixture at 2.5 g (wet wt) due to low activity.

each of which contained the gene for a single active transaminase, coded as follows: tyrosine transaminase, tyrB; aspartate transaminase, a s p C ; and branched-chain transaminase, ilvE. The a s p C mutant was found to contain the e n z y m e of choice for the production of phenylalanine (Table II). For the a s p C mutant and E. coli B 11303 the aspartate transaminase was not product inhibited and conversion greater than 90% could be achieved using aspartic acid rather than glutamate as the amine donor. 2 Equilibrium Studies An analysis of the end products of the phenylpyruvate to phenylalanine reaction showed that oxaloacetic acid was rapidly d e c o m p o s e d to form pyruvic acid. It is known that the rate of decarboxylation of oxaloacetic acid is increased by metals, e.g., Mg 2+, Zn 2÷, Cu 2+, Mo 2+ , Cd ~+, Fe 2+, Pb 2+, AP+, 7 and may also be accomplished by enzymatic action with oxaloacetate decarboxylase (EC 4.1.1.3). 8 To determine whether the 7 H. A. Krebs, Biochem. J. 36, 303 (1942). s D. J. Roussel, U.S. Patent 4,518,692 (1985).

500

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[45]

decarboxylation of oxaloacetic acid in the presence of E. coli aspC and 11303 was due to the presence of such metals or enzyme, an analysis of the decay of oxaloacetic acid under various reaction conditions was carried out (Table Ill). It was determined that in the presence of the substrate used to produce the high yields of phenylalanine obtained in Table II, oxaloacetic acid was rapidly decarboxylated. Based on the high yields obtained in the free cell reaction, the preparation of an immobilized catalyst suitable for use on a commercial scale was indicated. Immobilization of Microbe The standard method for preparation of the immobilized cell catalyst was as follows. A homogeneous dispersion of bacterial cells was mixed with polyazetidine prepolymer (Polycup 172 Hercules, Wilmington, DE) in equal quantities, with a 6.4-cm diameter impeller (Fisher Scientific, Washington, DC) attached to a variable speed drill (0-1200 rpm). The mixture was then slowly dispersed by pouring over Amberlite IRA-938 (Rohm & Haas, Philadelphia, PA) beads, previously dried to less than 10% moisture content, which were being slowly stirred in a mixing bowl by the wire wisk attachment of a home mixer (Kitchen Aid). The mixture was stirred gently until the E. coli-polyazetidine mixture was evenly dispersed, after which the catalyst was dried overnight in a gentle stream of air having 10% or less humidity. The dried coated beads were washed with 10 volumes of substrate solution and then used directly. Utilizing this standard method, catalyst of extremely high activity for the production of both aspartic acid j and phenylalanine 2 was prepared. TABLE III EFFECT OF VARIOUS COMMONLY USED SOLVENTS, pH, AND TEMPERATURE ON THE OXALOACETIC ACID DECAY RATE Oxaloacetic acid decay rate (half-life, min)

Solvent

pH

Temperature (°C)

Water Water Water Water and 0.35 M sodium sulfate Aspartic acid 0.15 M and phenylpyruvic acid 0.1 M

2.3 10.1 3.0 8.1

22 22 37 37

495 182 41 195

7.6

37

14

[45]

PHENYLALANINE PRODUCTION VIA E. coli

501

Optimization of Cell Loading for Phenylalanine Production A balance between amounts of polyazetidine, E. coli cells, and support is required to minimize the cost of the catalyst in the commercial production of phenylalanine. Even though IRA-938 resin is quite expensive from a commercial standpoint, the flow properties of the bead are exceptionally good. The beads as received are quite fragile, but, after curing the prepolymer, the catalyst preparation is rigid and does not easily fracture. The flow properties are due to the porous structure of the bead, which has an average pore diameter of 25,000-230,000 A. This large pore size also facilitates introduction of the E. coli-polyazetidine mixture to a larger surface area than the exterior surface of the bead. Excess polyazetidine may obscure the microbial cell, thus presenting an additional barrier to contact between the enzyme contained in the cell, the phenylpyruvate, and the aspartate molecules. Overloading of E. coli might also act as a barrier via entrapment of more than a monocellular layer on the surface of the resin bead. On the other hand, insufficient polyazetidine can contribute to incomplete immobilization of the E. coil, resulting in lowered stability and activity of the catalyst. Examination of these factors by varying the level of E. coil below a ratio of 1 : 1 : 1 with the polyazetidine and resin was carried out. The results are shown in Table IV. It can be seen on examination of the data that the activity levels between 0.25 and 1 g E. coil loading are linear, and that greater specific activity is seen at loading levels above 0.25 g E. coli/g resin. We then prepared higher ratios of E. coli as shown in Table V. By comparing the data in Tables IV and V, it can be seen that the maximum activity per gram of E. coli is obtained at 1 g of E. coli per gram of resin and polyazetidine. Table V also gives the relative volumes of the cell loadings. The volume of beads loaded with less than the 1 : 1 ratio of E. coli/g resin beads (Table IV) is constant at 1.9 ml/250 mg E. coll. The optimal level of cell loading for commercial production of phenylalanine is not governed solely by optimal activity. Consideration must also be given to the volume of the catalyst and the cost of the catalyst ingredients. The optimal commercial ratio of cell loading may then be obtained by the equation: Catalyst cost lb phenylalanine produced cost E. coli + cost polyazetidine + cost IRA-938 + cost catalyst-manufacturing labor X tl/2 specific activity

502

ENZYME ENGINEERING (ENZYME TECHNOLOGY) T A B L E IV ACTIVITY OF CATALYST PREPARATION AT E. coli: POLYAZETIDINE RATIOS LESS THAN 1 : 1a

Ratio of E. coli to polyazetidine

Specific activity (~mol/hr-g wet wt)

1: I 0.5 : 1 0.25 : 1 0.177 : 1 0.1 : 1

358 348 330 280 275

Batch a s s a y s o f the production of phenylalanine were carried out using 25 ml of 100 m M p h e n y l p y r u v a t e , 150 m M aspartic acid, and 0.1 m M pyridoxal 5-phosphate. The catalyst was prepared with the appropriate a m o u n t o f E . coli (wet wt) as s h o w n above and a c o n s t a n t level of polyazetidine (l g) and IRA-938 resin (1 g, 10% water content). The specific activity was taken at 4 hr and is given as moles phenylalanine produced per g r a m E. coli (80% moisture, wet wt) per hour.

TABLE V ACTIVITY OF CATALYST PREPARATION AT E. coli : POLYAZETIDINE RATIOS GREATER THAN 1 : Ia Ratio of E. coli b to polyazetidine 1: 1 1.1 : 1 1.15:1 1.2 : 1 1.3 : 1

1.5 : 1 2.0 : 1

Specific activity c

Volume (ml) d

460 422 451 424 414 425 362

1.9 1.7 1.5 1.6 1.4 1.3 0.7

a Batch a s s a y s were carried out as in Fig. 1. b G r a m s of E. coli per gram IRA-938 resin. e Determined as noted in Fig. I. a Determined on the basis of 250 mg E. coil (wet wt) after curing.

[45]

[46]

ASPARTAME PRODUCTION BY IMMOBILIZED THERMOASE

503

Of these factors, the specific activity and the half-life (tl/2) a r e affected most by the immobilization method. The specific activity may also be affected by the initial activity of the microorganism. The initial activity is not as important as the retained activity and the catalyst half-life (tl/2), however, due to the labor costs associated with the packing of columns. The support resin is the most expensive factor in the above equation. Thus ratios above l : l : l E. coli are clearly preferred for phenylalanine production and with half-lives in excess of 8 months, 2 the economic viability of this process is quite high. A 600 metric ton/year plant has been constructed. Catalyst columns of heights exceeding 10 ft and diameters as large as 4 ft have been used without restriction of flow rate or deterioration of flow distribution during operation. Other support resins have also been used with excellent success.

[46] P r o d u c t i o n o f A s p a r t a m e b y I m m o b i l i z e d T h e r m o a s e By

K I Y O T A K A O Y A M A , S H I G E A K I IRINO,

and

NORIO HAGI

Aspartame (a-L-Asp-L-Phe-OMe) is the methyl ester of the terminal dipeptide of the digestive hormone gastrin. It is about 200 times sweeter than sucrose and has a pleasant sweetness without a bitter aftertaste.l In the preparation of aspartame by conventional organic synthetic methods, it is necessary to protect the side-chain carboxylate of aspartic acid (Asp) in order to avoid the formation of the unwanted/3-peptide linkage. However, in order to reduce production cost it is better to omit protection and deprotection of the side-chain carboxylate. Therefore, all of the chemical synthetic methods with industrial potential do not involve protection of the side-chain carboxylate and thus ordinarily produce 20-40% of the/3 isomer, which has a bitter taste, along with the desired a isomer. 2-4 In recent years, the enzymatic synthesis of peptide by the reverse of the hydrolysis of the peptide bond has been receiving increasing attention. 5,6 We investigated whether the enzymatic method could be applied to the synthesis of aspartame, and found that some proteinases can cataI R. H. Mazur, J. M. Schlatter, and A. H. Goldkamp, J. A m . Chem. Soc. 91, 2684 (1969). 2 U,S. Patent 3,786,039 (1974). 3 U.S. Patent 3,833,553 (1974). 4 U.S. Patent 3,933,781 (1976). 5 j. S. Fruton, Ado. Enzymol. 53, 239 (1982). 6 K. Oyama and K. Kihara, Jpn. Chem. Rev. 35, 195 (1982); C H E M T E C H Feb., 100 (1984).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by AcademicPress, Inc. All rights of reproductionin any form reserved.

504

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

lyze the condensation reaction between N-protected Asp and phenylalanine methyl ester (Phe-OMe) in high yield. 7 The reaction is specific only on the a-carboxylate of Asp, even though the side-chain carboxylate is unprotected, and moreover, only the L-amino acids participate in the reaction when the racemic amino acids are used. In addition, the enzymatic reaction can be done under mild reaction conditions. From these findings we thought that the enzymatic method might become an economical way to produce the sweetener, and therefore have carried out research on the development of an industrial technology based on this novel method. Selection of the Enzymes Extensive screening of the commercially available proteinases revealed that some enzymes that cleave the peptide bond of proteins at the amine site of a hydrophobic amino acid residue can also catalyze the condensation between N-protected Asp and Phe-OMe. Thus the enzymes belonging to the group of metalloproteinases (EC 3.4.24._) are found to give the product in a high yield. Thiolproteinases (EC 3.4.22._) such as papain can also effect the reaction, but due to their concurrent esterase activity hydrolyses of the ester linkages of Phe-OMe in the reactant as well as in the product occur as side reactions, resulting in a lower yield of the desired product. Among the enzymes investigated, thermolysin, which is obtained from the strain found in a Japanese hot spring (Bacillus thermoproteolyticus Rokko), shows a high catalytic activity and a marked stability against heat, organic solvents, and extreme pH, and does not show esterase activity. 8 These properties are desirable in a catalyst for industrial use. A crude enzyme preparation is often contaminated with other enzymes that cause unwanted side reactions. However, it was found that thermoase, a crude preparation of thermolysin, which is commercially available and is much cheaper than thermolysin, does not show any side reactions, so it was used for the present study. Selection of the Protecting Group and the Raw Materials The enzymes that can catalyze the condensation between the two amino acids are endoproteinases, so it is necessary to protect the amino group of Asp and the carboxylic group of phenylalanine (Phe) in order for the reaction to take place. Several protecting groups that are commonly 7 y. Isowa, M. Ohmori, T. Ichikawa, K. Mori, Y. Nonaka, K. Kihara, K. Oyama, H. Satoh, and S. Nishimura, Tetrahedron Lett. 2611 (1979). 8 S. Endo, J. Ferment. Technol. 40, 346 (1962).

[46]

ASPARTAME PRODUCTION BY IMMOBILIZED THERMOASE

505

employed in organic peptide synthesis were found to be effective for the present reaction, and we selected the carbobenzoxyl group (Z) for the protection of Asp, since it is rather cheap and versatile, and gives a high reaction yield. Since the C-terminus of aspartame is the methyl ester, phenylalanine methyl ester was used directly. As raw materials, we selected L-Asp and DL-Phe for the following reasons: (1) L-Asp is available cheaply, whereas L-Phe is much more expensive than the racemic mixture; in fact the high price of L-Phe is currently the bottleneck in industrial production by chemical methods; (2) our kinetic study showed that Z - D - A s p acts as a competitive inhibitor in the enzymatic condensation, but D-Phe-OMe does not interfere with the reaction at all9; (3) the racemization of unreacted Z-D-Asp without destroying the Z protecting group, which is much more expensive protection group than the methyl ester protection group of Phe, is difficult, whereas the racemization of D-Phe to DL-Phe can be easily done. The entire process of the enzymatic production of aspartame, consisting of six reaction steps, is shown in the following scheme: N-Protection L-Asp + Z-CI ~ Z-k-Asp

(1)

DL-Phe + MeOH --~ DL-Phe-OMe

(2)

Z-L-Asp + DL-Phe-OMe --~ Z-L-Asp-L-Phe-OMe. D-Phe-OMe

(3)

Z-L-Asp-L-Phe-OMe.D-Phe-OMe ---} Z-L-Asp-L-Phe-OMe + D-Phe-OMe

(4)

Esterification Condensation Separation Hydrogenolysis Z-L-Asp-k-Phe-OMe ~ L-ASp-L-Phe-OMe

(5)

D-Phe-0Me~ DL-Phe

(6)

Racemization where Z-C1 is carbobenzoxychloride. Of the above reactions the key step is the enzymatic condensation. From the viewpoint of industrial production, the establishment of a technology for repeated use of the enzyme is of great importance. Use of Immobilized Thermoase Z-Asp and Phe-OMe

for the Condensation

between

The synthesis of Z-L-Asp-L-Phe-OMe from Z-L-Asp and DL-Phe-OMe and the hydrolysis back to the reactants catalyzed by thermoase are in 9 K. Oyama, K. Kihara, and Y. Nonaka, J. Chem. Soc. Perkin Trans., 356 (1981).

506

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

equilibrium, with the equilibrium lying in the hydrolysis direction (K = 1.5 M-l). ~0Therefore in order to obtain a high yield, it is necessary to shift the equilibrium toward the synthesis side. One approach is to make the product sparingly soluble in the reaction medium. In aqueous solution, the reaction between Z-L-Asp and oL-Phe-OMe gives Z-L-Asp-L-PheOMe, which deposits as an extremely insoluble salt with o-Phe-OMe, resulting in almost quantitative yield under reasonable concentration of the reactants ( - 1 M ) . 6 Another approach that may enhance the yield of the product is to add a water-miscible organic cosolvent, which can shift the equilibrium toward the synthesis side. j~ A third approach is the addition of a waterimmiscible organic cosolvent, resulting in a two-phase system. In a system where the reactants are soluble in the aqueous layer and the product in the organic layer, transfer of these compounds through the layers may overcome the unfavorable equilibrium. From the viewpoint of the application of an immobilized enzyme to the present system, the first approach is impractical, since separation of the catalyst from the deposited product is very difficult. Therefore only the latter two approaches may be of practical value. Selection of the Organic Cosolvent In selecting the organic cosolvent, one must consider the effect of cosolvent on the following aspects of the reaction: (1) the activity and the stability of the enzyme, (2) the equilibrium, (3) the solubility of the product, and (4) the partition of the reactants between the aqueous phase and the organic phase when a water-immiscible cosolvent is used. We investigated a number of organic cosolvents and found that, in the presence of a water-miscible cosolvent, the activity as well as the stability of the enzyme were markedly impaired, although some cosolvents could shift the equilibrium toward the synthesis side. (For example, in a 50% methanol-H20 solution, the equilibrium constant was found to be increased approximately 3-fold as compared with pure H20. ~2) On the other hand, with a water-immiscible cosolvent, the impairing effects were found to be generally smaller than with a water-miscible cosolvent, since in the former case, the enzyme could stay in the aqueous layer. Among these solvents, however, aliphatic and aromatic hydrocar10 K. Oyama, S. Irino, T. Harada, and N. Hagi, in "Enzyme Engineering VII" (A. I. Laskin, G. T. Tsao, L. B. Wingard, Jr., eds.), p. 95. The New York Academy of Science, New York, 1984. ii G. A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). ~2K. Oyama and S, Irino, unpublished results.

[46]

ASPARTAME PRODUCTION BY IMMOBILIZED THERMOASE

507

bons are unsatisfactory, since they hardly dissolve the product. Solvents such as chloroform, dichloroethane, ethyl and isopropyl acetates, and methyl isobutyl ketone can satisfy the requirements to some extent, but we selected ethyl acetate for the following reasons: (1) the impairing effects on the enzyme are smaller than with the others, especially chlorinated hydrocarbons, which adversely effect the enzyme; (2) the solubility of the product in ethyl acetate is high, and the partitions of the reactants into the organic layer are rather low (the partition constants between the organic layer and the aqueous layer for Z-Asp and Phe-OMe were found to be 0.13 and 0.30, respectivelyl2). These facts indicate that most of the reactants can stay in the aqueous layer where the enzymatic reaction takes place, and the product can transfer to the organic layer, thus shifting the equilibrium toward the less favorable synthesis side. Immobilization of Thermoase From the industrial point of view, the reaction rate needs to be sufficiently fast in order to minimize the capital investment. In the present case, however, the rate of condensation is much slower than in pure water system due to the presence of an organic cosolvent. In order to overcome such a handicap, it is preferable to immobilize the enzyme on a support with as high volumetric activity as possible. We investigated various immobilization methods, which include physical adsorption, ionic binding, and covalent binding. Physical adsorption was studied using Amberlite XAD-7 and XAD-8. The resins have a high adsorptive power to proteins owing to hydrophobic interactions, and are often used for the recovery of enzymes from a fermentation broth.13 In fact, Amberlite XAD-7 could immobilize the largest amount of thermoase per weight of the supporting material (Table I). In addition, the procedure for the immobilization is very simple as shown later, rendering the method very attractive. Ionic binding was studied using Amberlite IRA-94 and IRC-50, representative anionic and cationic ion-exchange resins, respectively. The method is as simple as physical adsorption, but only a small amount of the enzyme could be immobilized on the resins under various binding conditions. Covalent binding was studied using Toyopearl gel as a supporting material. Toyopearl is a hydrophilic polymer gel which was originally developed for use in high-performance gel filtration chromatography.~4 It t3 H. Y. Ton, R. D. Hughes, D. B. A. Silk, and R. Williams, J. Biomed. Mater. Res. 13,407 (1979). 14 j. Germeshausen and J. D. Karkas, Biochem. Biophys. Res. Commun. 99, 1020 (1981).

508

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

has good mechanical strength, even when heated or in the presence of organic solvents, and carries many hydroxyl groups available for immobilization. These properties are attractive in a support for immobilization of enzymes. Thermoase was immobilized on Toyopearl gel via several routes as shown in Fig. 1. Thermolysin is known to have a large number of tyrosine residues in the molecule, ~5and so immobilization via the diazo coupling method appeared promising. However, such attempts were not successful, since the activity of immobilized thermoase was very small, although good yields of immobilization were attained (Table I). The low activity of immobilized thermoase is presumably due to the destruction of its rigid three-dimensional structure, which derives from the hydrogen bondings of the tyrosine residues and which is known to be the key of the unusual stability of thermolysin. ~6 As noted in Table I, the binding by glutaraldehyde or cyanuric chloride via treatment with ethylenediamine appears to be advantageous, since these gave an immobilized enzyme with a large quantity of active thermoase. The immobilization via treatment with CNBr, followed with e-aminocaproic acid, and then with the water-soluble carbodiimide [N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride] was unsatisfactory because the yield was very poor. The experimental procedures for immobilization by physical adsorption on Amberlite XAD-7 and by covalent bindings to Toyopearl by glutaraldehyde and cyanuric chloride are described below. Materials

Thermoase: Daiwa Kasei Co. (Osaka, Japan), activity of 1.6 × l06 protease units per gram (determined by Anson's method ~7modified by Hagiharal8). Amberlite XAD-7, XAD-8, IRA-94, and IRC-50: Rohm & Haas Co. Toyopearl: Toyo Soda Manufacturing Co. (Tokyo, Japan). Calcium buffer: I0 mM, pH 7.5. L-Asp and DL-Phe: Nippon Rikagaku Yakuhin Co. (Tokyo, Japan), reagent grades. Carbobenzoxychloride (Z-CI): Kokusan Kagaku Co. (Tokyo, Japan), purity greater than 95%. Physical Adsorption to Amberlite XAD-7. Ten grams of a water-wet Amberlite XAD-7 is stirred in 50 ml of the calcium buffer containing 3 g of z~ y . Ohta, Y, Ogura, and A. Wada, J. Biol. Chem. 241, 5919 (1966). 16 y. Ohta, J. Biol, Chem. 242, 509 (1967). 17 M. L. Anson, J. Gen. Physiol. 22, 79 (1934). is B. Hagihara, Annu. Rep. Sci. Works, Fac. Sci. Osaka Univ. 2, 35 (1954).

! Z II Z

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0 .0

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I

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O ~

~--0 cD I O I

1 O I

l° O O

z

Z .U-.~

.~

I ,-o

II I Z .@Z

~ 0 cxl

Z

I o

m

! I O I

~

,.,., Z I

.m

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I

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~

%.z~ dI II

~

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,...,

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r..)

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r..)- - O

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0 0

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0

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0

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._

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d

510

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

TABLE I AMOUNT OF THERMOASE IMMOBILIZED ON THE SUPPORTS AND RELATIVE ACTIVITY AS COMPARED WITH NATIVE THERMOASE

Supporting material Physical adsorption Amberlite XAD-7 Amberlite XAD-8 Ionic binding Amberlite IRA-94 Amberlite IRC-50 Covalent binding TPL-EAGA C TPL-EAGA-ThA-RED

TPL-EACC e TPL-DAZ y

a

Amount of thermoase a

Relative activity b

2.31 2.10

1.0 1.0

0.53 0.25

0.8 0.8

1.52 1.38 0.76 1.79

1.1 0.9 0.7 0.3

Grams per l0 g of the wet immobilized thermoase. b The relative activity is determined on the basis of the same amount of the immobilized and the native enzymes. c Toyopearl activated with ethylenediamine-glutaraldehyde. d Immobilized thermoase on TPL-EAGA reduced with NaBH4. e Toyopearl activated with ethylenediamine-cyanuric chloride. I Toyopearl activated with phenylenediamine followed by diazo coupling.

thermoase at 10° for 3 hr. Immobilized thermoase is obtained by filtration followed by washing with 50 ml of the calcium buffer. Activation o f Toyopearl. Toyopearl gel is washed on a glass filter with distilled water, and dried in vacuo at 60°. Fifty grams of the dry gel, 200 ml of 1 N NaOH, and 11 ml of epichlorohydrin are placed in a 500-ml flask and stirred for 3 hr at 30°. The contents are filtered by a glass filter and washed with cold distilled water. The gel thus obtained, designated as TPL-EPH, is added to a 500 ml flask containing 63 ml of ethylenediamine and 35 ml of distilled water. The mixture is stirred for 1.5 hr at 80°, and then filtered by a glass filter. The gel is washed with acetone several times to remove unreacted ethylenediamine, and dried in vacuo at 60° overnight. This gel is designated as TPL-EDA. Immobilization by Glutaraldehyde. Fifty grams of dry TPL-EDA is added into a l-liter flask containing 500 ml of 5% glutaraldehyde. After the

[46]

A S P A R T A M E P R O D U C T I O N BY I M M O B I L I Z E D T H E R M O A S E

511

pH of the mixture is adjusted to 7.0 by 5 N NaOH, it is stirred overnight at 20°, and then filtered by a glass filter. The gel is washed successively with distilled water and with acetone to remove unreacted glutaraldehyde, and then dried in vacuo at 60°. This gel is designated as TPL-EAGA. The immobilization is carried out by stirring the mixture of 13.3 g of TPLEAGA and 250 ml of the calcium buffer containing 5% of thermoase in a 500-ml flask at 4° for 24 hr. Immobilized thermoase, designated as TPLEAGA-ThA, is obtained after filtration of the mixture and washing with the calcium buffer. The reduction of the C~-~-N double bonds of TPLEAGA-ThA is done as follows: 10 g of TPL-EAGA-ThA in 100 ml of the calcium buffer is treated with 0.5 g of NaBH4 at 4°. The stirring is continued at the same temperature for 24 hr, and then the immobilized thermoase, designated as TPL-EAGA-ThA-RED, is obtained after filtration followed with washing by the calcium buffer. Immobilization by Cyanuric Chloride. A mixture of 10 g of dry TPLEDA, 130 ml of acetone, and 22 g of cyanuric chloride in a 500-ml flask is stirred at - 10 to - 5 ° for 30 min, followed by the dropwise addition of 10 g NaHCO3 in 60 ml of distilled water at the same temperature. With the progress of the reaction the pH of the mixture decreases, so 5 N NaOH is added to keep pH at the constant value of 7.5. After 3 hr at 0°, the decrease of pH is no longer observed. The gel is filtered by a glass filter and washed with cold acetone to remove unreacted cyanuric chloride, and then dried in vacuo at 20° overnight. This gel is designated as TPLEACC. The immobilization is carried out by stirring the mixture of 3.3 g of TPL-EACC and 50 ml of the calcium buffer containing 5% of thermoase in a 100-ml flask at 4° for 24 hr. Immobilized thermoase, designated as TPL-EACC-ThA, is obtained after filtration of the mixture followed by washing with the calcium buffer. Determination of the Amounts of Thermoase Immobilized on the Supporting Materials The amounts of thermoase can be determined by hydrolyzing the enzyme into the constituent amino acids, followed by high-performance liquid chromatography (HPLC) analysis of the hydrolyzate, since the peak areas of the amino acids in the hydrolyzate are found to correlate very well with the amount of thermoase, as shown in Fig. 2, Thus approximately 50 mg of an accurately weighed, dry immobilized thermoase and 10 ml of 6 N HCI are taken into a test tube, which is then sealed under vacuum. The sealed tube is heated at 110° for 24 hr in an oil bath. The contents are evaporated to dryness on a rotary evaporator, and the residue is treated with 0.67 M citric acid-sodium citrate buffer (pH 2.2). After

512

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

6 O

O

O

~.

3 6 9 12 AMOUNTOF THERMOASE (mg)

FIG. 2. Correlation betweenthe amount of tbermoase and the peak area of amino acids of the hydrolysate of thermoase determined by the amino acid analyzer. the insoluble materials are removed by centrifugation, the supernatant solution is analyzed by an amino acid analyzer (TSK-HLC-805, Toyo Soda Manufacturing Co.) equipped with a 75 × 7.5 mm (I.D.) column packed with strong cation-exchange resin (TSK-GEL IEX-215SC, Toyo Soda Manufacturing Co.), a 10 m x 0.4 mm (I.D.) reaction coil, an ophthalaldehyde supply, and a fluorescence spectrofluorometer with the excitation at 360 nm and the emission at 450 nm. The amount of thermoase is determined from standard calibration by the peak area of glutamic acid. The operating conditions of the amino acid analyzer are as follows: temperature of the column and the reaction coil, 50°; eluent, a mixture of citric acid (28.0 g), sodium citrate (19.6 g),/3-thiodiglycol (2.5 ml), n-capric acid (0.1 ml), ethyl alcohol (80 ml), 20% Brij-35 (polyoxyethylene lauryl ether, 2.5 ml), in 800 ml deionized water, is adjusted to pH 3.3 by concentrated HCI, which is then made up to 1 liter by deionized water; o-phthaldehyde solution, a mixture of o-phthaldehyde (1 g) in 100 ml ethyl alcohol, 1 liter of the aqueous solution containing boric acid (49.4 g) with pH adjusted at 9.7 by 10 N KOH, a few drops of 2-mercaptoethanol, and 25% Brij-35 (2 ml); pressure and the flow rate of the eluent, 60-80 kg/cm 2 and 1.0 ml/min, respectively; pressure and the flow rate of the o-phthaldehyde solution, 15 kg/cm 2 and 1.0 ml/min, respectively.

Enzyme Assay An Erlenmyer flask containing accurately weighed wet immobilized enzyme (1-1.5 g) and 50 ml of Mcllvaine buffer (pH 6.0) is thermally preequilibrated at 40 ° for 5 min. The substrates stock solution (5 ml)

[46l

ASPARTAME PRODUCTION BY IMMOBILIZED THERMOASE

513

containing 2 mmol of Z-L-Asp and 4 mmol each of DL-Phe-OMe. HCI and NaOH is added, and then the flask is stoppered and placed in a waterbath shaker thermostated at 40 °. After 30 rain, l0 ml of 0.1 M EDTA is added to quench the reaction, and the amount of Z-L-Asp-L-Phe-OMe produced is measured by HPLC. The activity of immobilized thermoase is determined as a relative activity by comparison with the parallel control experiment using the standard sample solution of thermolysin. The HPLC analysis is done with a TSK-HLC-802 (Toyo Soda Manufacturing Co.) equipped with a 600 × 7.5 mm (I.D.) column packed with TSK-LS-170 (crosslinked polysaccharide gel, particle size of 5/~m, Toyo Soda Manufacturing Co.), and a UV detector (254 nm). The eluent is 0.5 M aqueous CHaCOOH-NaOH buffer (pH 6.0) with the flow rate of 9.2 ml/min (pressure 20 kg/cmZ).

Preparation of Substrates Z-L-Asp. Carbobenzoxylation of L-Asp is carried out by the standard Schotten-Baumann method by using carbobenzoxychloride (Z-C1) and aqueous NaOH.19 After the reaction, the reaction mixture is made acidic (pH ~2) by the addition of concentrated HCI, and then stirred overnight at 0°. The deposited crystals are filtered, dried, and then recrystallized from ethyl acetate-n-hexane to give the white crystals of Z-L-Asp in 80% yield. oL-Phe-OMe. HCI. Esterification of oL-Phe by CH3OH and HCI is done by the standard Fisher method, z° After the reaction, the reaction mixture is concentrated on a rotary evaporator to a slight turbidity, and then kept in a refrigerator overnight. The resulting deposited crystals are collected by filteration, washed with diethyl ether, and recrystallized from methanol-diethyl ether to give the white crystals of oL-Phe-OMe- HCI in a yield of 83%. Operation The enzymatic condensation of Z-L-Asp and DL-Phe-OMe was carried out in the biphasic system of water and ethyl acetate. A continuous column operation and a batchwise operation using thermoase immobilized by several methods were investigated. Continuous Column Operation. Continuous column operation was studied using thermoase immobilized on Amberlite XAD-7 by physical adsorption and on Toyopearl by the covalent binding through ethylenedit9 N. Izumiya, T. Kato, M. Ohno, and H. Aoyagi, "Peptide Synthesis," p. 25. Maruzen, Tokyo, 1975. 2o M. Bergmann and L. Zervas, Ber. 65, 1192 (1932).

514

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

FIG. 3. The apparatus of the continuous column operation: 1, reactants reservoir; 2, magnetic stirrer; 3, feed pump; 4, column reactor; 5, product reservoir.

amine-glutaraldehyde (TPL-EAGA-ThA). The apparatus is shown in Fig. 3. It consists of a column reactor (350 × 30 mm I.D.) packed with the catalyst, a feed pump, and two reservoir vessels of the reactants and the product. The reactor is thermostated at 40 ° by a water-circulating jacket. The reactants mixture solution, consisting of 0.12 M of Z-L-Asp, 0.31 M of DL-Phe-OMe, and 0.012 M of Ca(OAc)2 in ethyl acetate-water (7 : 3 in weight ratio), was continuously fed by the pump into the reactor from the top, and the product solution mixture exited via the bottom of the reactor. During the feed the mixture solution was magnetically stirred in order to maintain the homogeneity of the solution. Since the activity of the catalyst decreases with time, the space velocity was changed in order to maintain the reaction yield at a high level (80-90%). The results of the operations are illustrated in Fig. 4, which shows that, although Amberlite XAD-7 can immobilize a large quantity of thermoase (Table I), the decrease of the activity is so rapid that it is far less satisfactory than TPL-EAGA-ThA. Analysis of the product solution by the amino acid analyzer after the hydrolysis at 110° in 6 N HCI as described before showed that the decrease was attributable to the leakage of thermoase from the support, largely due to the presence of the organic cosolvent and a high ionic strength by the reactants. Treatment of the catalyst with glutaraldehyde (which is shown as Amberlite XAD-7* in Fig. 4) in the hope of preventing such leakage was unsuccessful. In contrast, although TPL-EAGA-ThA showed a rapid decrease of the activity at the beginning of the operation, it gradually settled down. After 20 days of the operation, the operation was stopped and analysis of the catalyst activity was measured. It showed that 45% of the initial activity was lost during that period. Batchwise Operation. Batchwise operation was studied using thermoase immobilized on Toyopearl by covalent binding methods, i.e., TPLEAGA-ThA, TPL-EAGA-ThA-RED, and TPL-EACC-ThA. The reactions were carried out by shaking the flask containing the reaction mixture

[46]

ASPARTAME PRODUCTION

BY

I M M O B I L I Z E D THERMOASE

515

1.5

o

05 E I/3

bertite XAD-7'~ erlit~ XAD-7 I

100

20O

3OO TIME(h)

4OO

I 5OO

FIG. 4. Time course of continuous column operation of the production of Z-L-Asp-L-PheOMe. D-Phe-OMe from Z-L-Asp and DL-Phe-OMe by immobilized thermoase on Toyopearl gel and Amberlite XAD-7. TPL-EAGA-ThA, Thermoase immobilized on Toyopearl activated with ethylenediamine-glutaraldehyde;Ambedite XAD-7*, thermoase immobilized on Amberlite XAD-7 followed by treatment with glutaraldehyde.

at 120 strokes/hr in a waterbath shaker thermostated at 40 °. The reaction mixture consisted of 2 g of Z-L-Asp, 3.4 g of DL-Phe-OMe, 13 ml of H20, 43 ml of ethyl acetate, 0. I g of Ca(OAc)2, and immobilized thermoase, the amount of which was chosen to give approximately the same enzyme activity for each catalyst. Therefore 6 g of TPL-EAGA-ThA, 8 g of TPLEAGA-ThA-RED, and 19 g of TPL-EACC-ThA were used. After 22 hr of the reaction, the contents of the flask were filtered by a glass filter, and the recovered catalyst was washed with the calcium buffer, then used for the next run. The filtrate was analyzed by HPLC as described before to determine the yield of the produced Z-L-Asp-L-Phe-OMe. The results of the repeated runs are shown in Fig. 5.

7"=

~ - - ~ ~ ~ -

J b,.l

EACC-Th# TPt:EAGA-ThA

ib

i;

!

20

REPEATNUMBER

FIG. 5. Results of batchwise operation of the production of Z-L-Asp-L-Phe-OMe. D-PheOMe from Z-L-Asp and DL-Phe-OMe by immobilized thermoase on Toyopearl gel. TPLEACC-ThA, Thermoase immobilized on Toyopearl activated with ethylenediamine-cyanuric chloride; TPL-EAGA-ThA, thermoase immobilized on Toyopearl activated with ethylenediamine-glutaraldehyde; TPL-EAGA-ThA-RED, NaBH4-reduced TPL-EAGAThA.

516

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[46]

As with the continuous column operation, a rapid decrease in activity was observed for all the catalysts studied during the early stage of the operation. This was then followed by a slower, gradual loss of activity during continued operation. After 20 repeated runs the operation was stopped and the activity of the used catalysts were measured, which showed that the activity loss of TPL-EAGA-ThA, TPL-EAGA-ThARED, and TPL-EACC-ThA were 38, 33, and 25%, respectively. The improvement of the stability by the reduction of the C ~ N double bond of TPL-EAGA-ThA indicates that the hydrolysis of the C~---N bonds is occurring during the operation. Although TPL-EACC-ThA is less active than TPL-EAGA-ThA-RED, the rate of the activity loss is much slower in the former than in the latter; thus the former appears to be advantageous.

Summary The enzymatic method for the production of aspartame has many advantages over chemical methods, e.g., cheap racemic phenylalanine can be used and no fl-aspartame is produced. The former leads to a lowering in the cost of raw materials and the latter to a simplified purification procedure. The disadvantages as compared with chemical methods are that the racemization of unreacted D-Phe-OMe is necessary and that an expensive catalyst is used. The racemization of D-Phe-OMe can be done easily as stated before, but the recovery and recycling of the delicate biocatalyst are not easy, although thermoase is a very stable enzyme. Here we have presented two possibilities for using immobilized thermoase, i.e., continuous column operation and batchwise operation. In both procedures, the use of an organic cosolvent is required, which leads to deactivation of the catalyst. Therefore it is most important to clarify the mechanism of deactivation by organic solvents and to find strategies to overcome it, perhaps by development of more sophisticated immobilization methods. In an industrial operation using the batchwise mode, deterioration of the immobilized enzyme occurs by the friction caused by stirring. Furthermore, separation of the immobilized enzyme is necessary after batchwise reaction, which is troublesome and costly. In the continuous column operation, channeling of the organic and aqueous layers was observed in the packed column, thus rendering the efficiency of the catalyst low, and in addition, deactivation tended to occur more rapidly than during batchwise operation. Nevertheless, the continuous column operation seems to be more advantageous than the batchwise operation from the viewpoint of industrial production needs.

[47]

PRODUCTION

OF 5'-RIBONUCLEOTIDES

517

[47] P r o d u c t i o n o f 5 ' - R i b o n u c l e o t i d e s U s i n g Immobilized 5'-Phosphodiesterase

By

REINHOLD KELLER, MERTEN SCHLINGMANN, and ROLF WOERNLE

A process for the preparation of 5'-ribonucleotides with immobilized 5'-phosphodiesterase starting from a mixture of crude nucleic acids such as those obtained in known manner from microorganisms has been described. ~ Such crude nucleic acids contain ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in a ratio from 10:1 to 4:1 depending on the kind of organism. 5'-Ribonucleotides are starting substances for the preparation of food additives and drugs. Their preparation by enzymatic hydrolysis of RNA is well known. 2 However, the 5'-phosphodiesterase enzyme used for this purpose simultaneously hydrolyzes RNA and DNA, so that 5'-deoxynucleotides are obtained as by-products in addition to the intended 5'ribonucleotides. These by-products can only be separated from the 5'-ribonucleotides with great difficulty. For preparing pure 5'-ribonucleotides it is therefore necessary to start from RNA which is practically free of DNA. 3 Various processes for the preparation of pure RNA are already known, for example selective precipitation of pure RNA by heating and subsequent treatment with acid, or by precipitation in the presence of bivalent cations: The disadvantage of these processes resides in the fact that the DNA is decomposed by a heat or acid treatment, whereby a considerable amount of RNA is likewise lost. To overcome these disadvantages the reactivity of the 5'-phosphodiesterase enzyme is modified by a suitable immobilization in such a highly selective manner that hydrolysis of DNA no longer occurs and a preliminary purification of RNA is no longer required) In this article, the preparation, enzymatic properties, and industrial application of immobilized 5'-phosphodiesterase are presented. U.S. Patent 4,206,243. 2 K. Ogata, S. Kinoshita, T. Tsunoda, and K. Aida, "Microbial Production of Nucleic AcidRelated Substance," Wiley, New York. 3 Japanese Patent Application 5,320,494. 4 Japanese Patent Application 5,455,791. 5 German Offenlegungsschrift 3,136,940.

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

518

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[47]

Methods for Analysis

Nucleotides. Mononucleotides produced by the hydrolysis of RNA or DNA are determined chromatographically with a Liquid Chromatograph LKB. The column used is a metal column of 250 mm length and 4 mm inside diameter, packed with Merck Lichrosorb RP18. Operational conditions: flow rate, 2 ml/min; pressure, 100 bar; mobile phase: dilute phosphoric acid, pH 2.1; 254 ram, UV detection. Activity of Native 5'-Phosphodiesterase. The standard enzyme assay of native 5'-phosphodiesterase is carried out as follows: 0.5 ml of diluted enzyme solution is added to the substrate solution containing 4% RNA and 0.1 mM ZnSO4 in 0.1 M acetate buffer (pH 5.0) and it is reacted 30 min at 60°. The units of enzyme activity are expressed as the amount of mononucleotides produced by l m g enzyme in 1 min. Activity of Immobilized Enzyme. For the assay of enzyme activity of immobilized 5'-phosphodiesterase, the substrate solution containing 4% RNA and 0.1 mM ZnSO4 in 0.1 M acetate buffer (pH 5.0) is passed through a glass column packed with the immobilized 5'-phosphodiesterase at space velocities of 15-30 at 60°. The units of enzyme activity are expressed as the amount of mononucleotides produced by I ml of the immobilized enzyme in 1 rain. Material. Crude 5'-phosphodiesterase is obtained from Amano Pharmaceutical Company, Japan. The activity is 13 × l06 U/g (pH 4.8). It is manufactured by a fermentation process of a selected strain of Penicillium. Enzyme Support. The enzyme support used consists of oxirane acrylic beads purchased from ROhm Pharma, Darmstadt, West Germany, having an average pore diameter of 350 A, pore volume of 1.74 ml/g, and an oxirane content of 800 t~mol/g dry weight. Immobilization of 5'-Phosphodiesterase. Optimum conditions for immobilization of 5'-phosphodiesterase are as follows: 20 g of 5'-phosphodiesterase is dissolved in 800 ml of 1 M acetate buffer (pH 4.5). The solution is poured onto 200 g epoxide carrier (Eupergit C; R6hm Pharma, Darmstadt), stirred slightly, and left standing for 3 days at room temperature. The polymer resin is filtered off, and subjected to a washing operation with the use of the following liquids: (1) double-distilled water; (2) 1 M NaC1; (3) 0. ! M acetate buffer (pH 5.0). The activity of the preparation is about 147 U/ml resin under standard assay conditions. Enzymatic Properties of Immobilized 5'-Phosphodiesterase. The optimum pH of Eupergit C-5'-phosphodiesterase shifts about 0.5 pH unit more to the acid side than that of the native enzyme (Fig. 1). There is no marked difference between the immobilized enzyme and the native one in

[47]

PRODUCTION OF 5'-RIBONUCLEOTIDES

100 % 80

519

im;yO~mi~ize.~/ ._d~ n a t i v e enzyme

~0

eY

2O

0

2

J/

t~

S

pH value

6

7

FIG. 1. pH and activity.

optimal temperature and the effects of metal ions. A significant difference is observed on the substrate specificity between the immobilized enzyme and the native one. Whereas the native enzyme splits RNA and DNA at about the same rate 6 the enzyme immobilized on Eupergit C hydrolyzes only RNA. A possible reason is that because of the morphological structure of Eupergit C the reactivity of the enzyme is influenced in such a way that only substrate molecules with a molecular weight lower than 200,000 are attacked. The stability of the immobilized 5'-phosphodiesterase in an industrial operation for a long period at 60° is shown in Fig. 2. It was known 7 that the native 5'-phosphodiesterase required Zn 2÷ ions as an enzyme activator. Therefore, the effect of Zn 2÷ ions during continuous operation of the immobilized 5'-phosphodiesterase was examined. If there are no Zn 2+ ions in the substrate solution, the enzyme activity is gradually lost. In contrast, when Zn 2+ ions exist in the substrate solution at a concentration of 0.1 mM, the enzyme activity is quite stable even after 500 days continuous operation. This result indicates the necessity for Zn 2+ ions in this reaction. Preparation of Substrate Solution. The nucleic acid solution used for this process is preferably obtained from microorganisms by extraction of 6 M. Fujimoto, A. Kuninaka, and H. Yoshino, Agric. Biol. Chem. 38, 1555 (1974). 7 K. Rokugawa, M. Fujimoto, A. Kuninaka, and H. Yoshino, Agric. Biol. Chem. 44, 1987 (1980).

100 %

n

O

\

RNA; 0,4.% DNA; 0,1ram ZnS0~,; pH 5,0; 60 *

\1.6%

~o ut ZnSO~

•

~,

,'

o

L

~

~

~

,

lo

~'

Av

~oo

d~y~ sbo

Time FIo. 2. Effect of Zn 2÷ ions during continuous operation using Eupergit C-immobilized 5'phosphodiesterase.

Crude nucleic acid RNA/DNA

l,o...,.c ,,,~o,,.i. I ! Chr°mat°graphy I

I

Puri,,.,,oo

I

I

Concentration J [Precipitation of DNAI

$

,9 S' - Ri b0nucle0fides

1

[

lsotation

]

I

0r,ing

I

0NA

FIG. 3. Flow diagram for the production of 5'-ribonucleotides.

[47]

PRODUCTION OF 5'-RIBONUCLEOTIDES

521

the lipids with ammonia and lower alcohols, and subsequent workup of the aqueous washing phase. ~The crude nucleic acids contain DNA having a molecular weight greater than 200,000, and RNA having a molecular weight of significantly below 100,000, especially from 10,000 to 50,000. Production of 5'-Ribonucleotides By applying the previous descriptions a process on a semitechnical scale for producing an amount of 10 tons/year 5'-ribonucleotides was developed. This process starting with the nucleic acid solution contains the following steps (see Fig. 3): preparation of the raw material, enzymatic hydrolysis, separation by chromatography, and concentration and isolation of the products.

Process Description (Fig. 4) Preparation of the Raw Material. The crude nucleic acid solution resulting from biomass disintegration and extraction is filtered to yield a clear solution which is the substrate for the subsequent process steps. Enzymatic Hydrolysis. The clear RNA/DNA solution is adjusted with ZnSO4 to 0.1 mM and then acidified with acetic acid to pH 5.0 and heated to 60°. This solution is then continuously passed through a column 10 in. in diameter containing a 25-in.-deep bed of immobilized 5'-phosphodies-

Crude --ChrQma0grahy nucleic acid [ r l ~

•.

.

.

.

.

Fdfrahon Purification

I

I

~

~

r ..... ~fic hydrolysis

F--

I

IT~IT--II ~I~

M

~

DNA ~

HCI ~

J ~c~

~'--

l

I

precipitation I

I AmmOnia

' ~

Drying

I

"

i Demineralized water

/

1

•

~

Concentration

I

~

Methanol recover,

L__ -Isolation . of nuc|eotides 5'-Ribonudec tides * DNA

Flo. 4. Process flow sheet for the production unit for 5'-ribonucleotides (lO tons/year).

522

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[47]

terase to hydrolyze the RNA. The flow rate is controlled to give a 100% conversion rate of RNA. The activity of 5'-phosphodiesterase column is equal in both upward and downward flows. In practice, downward flow is employed to prevent channeling of the column because air bubbles evolved from the warmed substrate solution (60°) can be easily separated in the top part of the column. Separation and Purification. After hydrolysis the pH of the resulting solution is adjusted with NaOH to 7.5; the solution is cooled by heat exchange to 25 ° and then passed continuously through a series of ionexchange columns consisting of a strong basic resin in C1- form. The nucleotides are adsorbed to the carrier. The unhydrolyzed polymer DNA passes through and is recovered after passage by precipitation with H2504 and centrifugation of the precipitate. The individual nucleotides are washed from the columns with dilute hydrochloric acid (pH 2.6; 0.002 N) whereby they are desorbed in the sequence CMP, AMP, UMP, GMP. The eluates are passed through four parallel charcoal beds, where each nucleotide is readsorbed individually. Desorption is brought about with aqueous methanolic ammonia solutions. Concentration and Isolation of the Products. The solutions are concentrated to 5% by weight and treated with methanol under cooling (5°). The nucleotides precipitate here quantitatively and can be filtered off and dried. Economy The economic aspects of production of 5'-ribonucleotides by 5'-phosphodiesterase immobilized in this way are many. By using an immobilized enzyme, the purification procedure of the reaction products becomes simple and the yield is high. A crude RNA/DNA solution such as is obtained in known manner from microorganisms can be used without an expansive preliminary purification of RNA. As shown in Fig. 2, the immobilized 5'phosphodiesterase is very stable. Thus, the cost of the enzyme is markedly reduced from that of native enzyme.

[48]

AN ACRYLAMIDE

PRODUCTION

METHOD

523

[48] A c r y l a m i d e P r o d u c t i o n M e t h o d U s i n g I m m o b i l i z e d N i t r i l a s e - C o n t a i n i n g M i c r o b i a l Cells By ICHIRO WATANABE

Acrylamide is in widespread use, for example as a starting material for the production of various polymers for use as flocculants, stock additives, or polymers of petroleum recovery. A method for the production of acrylamide has been known which includes reacting acrylonitrile with water by the use of a catalyst which incorporates copper in a reduced state. This method, however, suffers from various problems due to the complexity in the preparation of the catalyst, the difficulty in regenerating the used catalyst, and the complexity in separating and purifying the acrylamide formed. Furthermore, it is desirable to produce acrylamide under moderate reaction conditions, because compounds containing double bonds in the molecule, such as acrylamide, are readily polymerizable. It has therefore been considered advantageous to establish a process for producing acrylamide by hydrolyzing acrylonitrile with microorganisms under moderate conditions. CH2~CHCN

~ CH2=CHCONH2

nitrilase or nitrile hydralase

Only recently, microorganisms, such as Brevibacterium R312 and Pseudomonas chlororaphis B23, have been described with nitrilase activity effective in hydrolyzing acrylonitrile to acrylamide, j,2 At Nitto Chemical Industry Company, Ltd., we have investigated an enzymatic process for producing acrylamide using microorganisms and we have isolated some bacteria having high nitrilase and low amidase activities for hydrolyzing acrylonitrile to produce acrylamide of very high quality. A strain belonging to the genus Rhodococcus designated S-6 was found that produced a constitutive enzyme which hydrolyzed acrylonitrile to acrylamide without forming by-products. As the result, we recently developed a new bioreactor using immobilized cells with a cationic acrylamide-based polymer gel and judged that it was economically feasiS. Papaconstantinou, A. Grasmick, M. C. Habig, S. Elmaleh, and R. Ben Aim, Entropie 104, 10 (1982). 2 y. Asano, T. Yasuda, Y. Tani, and H. Yamada, Agric. Biol. Chem. 46, 1183 (1982).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

524

ENZYMEENGINEERING(ENZYMETECHNOLOGY)

[48]

ble to construct a commercial plant based on the pilot plant operation. Our results have not yet been published but patents have been issued. 3,4 The operation based on this technology is the very first use of biotechnology in the field of petrochemical industry. In this article, from the methodological standpoint, we report the preparation and properties of immobilized cells, and enzyme reactions for the production of acrylamide using immobilized cells.

Assay Methods Nitrilase (EC 3.5.5.1) and amidase (EC 3.5.1.4) activities are measured by the amount of acrylamide and acrylic acid produced in the enzyme reaction, respectively. Nitrilase Activity. Intact cells, 0.5 g (wet weight), (in the case of immobilized cells, the sample is fully ground in a mortar) are diluted with 0.1 M potassium phosphate buffer solution (pH 7.7) to prepare a suspension having a cell content of 0.1%. To 5 ml of this cell suspension is added 5 ml of a 5% (w/v) aqueous solution of acrylonitrile. The mixture is incubated at I0 ° for 10 min with shaking. After the reaction, the cells in the reaction mixture are immediately filtered off, and acrylamide in the filtrate is measured by gas chromatography. Gas chromatograph conditions: Shimadzu gas chromatograph, Model GC-4CM, equipped with a flame ionization detector. The column used is a glass column of 3 mm inside diameter, 1 m length, packed with Porapak PS (80 to I00 mesh). Operational conditions: column temperature, 210°; injector and detector temperatures, 240 °. The carrier gas is N2 at 40 cm3/min. The integration and calibration of peak area are carried out by a Shimadzu Chromatopac C-RIB. One unit of nitrilase activity is defined as the amount of bacteria which catalyzes the formation of 1 ~mol of acrylamide per minute. Amidase Activity. Intact cells (or fully ground immobilized cells), 1.0 g (wet weight) are diluted with 0.1 M potassium phosphate buffer solution (pH 7.7) to prepare a suspension having a cell content of 0.5%. To 5 ml of this cell suspension is added 5 ml of a 10% (w/v) aqueous solution of acrylamide. The mixture is incubated at 30° for 60 min with shaking. After the reaction, the cells in the reaction mixture are immediately filtered off, and the acrylic acid in the filtrate is measured by high-performance liquid chromatography (HPLC). 3 I. Watanabe, Y. Satoh, and T. Takano, U.S. Patent 4,248,968 (1981). 4 I. Watanabe, K. Sakashita, and Y. Ogawa, U.S. Patent 4,421,855 (1983).

[48]

AN ACRYLAMIDE PRODUCTION METHOD

525

HPLC conditions: Shimadzu liquid chromatograph LC-3A, equipped with a UV (at 210 nm) detector and a stainless-steel column of 2.6 mm inside diameter, 250 mm length, packed with Hitachi Gel #3011-0. Operational conditions: mobile phase, 0.2 M phosphoric acid; column temperature, 40°; flow rate, 1.3 ml/min; integrator, Shimadzu Chromatopac C-R1B. One unit of amidase activity is defined as the amount of bacteria which catalyzes the formation of 1/zmol of acrylic acid per minute. Analytical Methods Acrylonitrile and acrylamide are measured by gas chromatography and acrylic acid is measured by HPLC. The conditions for analysis are the same as those described above in Assay Methods. Culture of Strain S-6. Strain S-6 is cultured under aerobic conditions at 30° for 45-50 hr cultivation in 100 liters of medium (pH 7.2) containing 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% meat extract. The cells are harvested by centrifugation and washed with 20 liters of 0.05 M potassium phosphate buffer solution (pH 7.7). About 2.5 kg (wet weight) of cells are obtained from 100 liters of broth, and its nitrilase activity is approximately 10-20 p~mol acrylamide per minute per milligram of wet cells, and its amidase activity is below 0.01 ~zmol acrylic acid/min-mg (wet cell) in flesh state, respectively. Preparation of Immobilized Cells Strain S-6. The cell immobilization method used is that of entrapping cells in polymeric gels. In the production of acrylamide, the acrylamide-containing solution needs to be of high quality and any additives such as buffer salts, metal ions, and impurities from cells are undesirable in the reaction mixture. Carriers of alginate and carrageenan need some quantities of metal ions (e.g., Ca 2÷, K +) to keep the mechanical strength and enzyme stability of the cell-immobilizing gels. Accordingly, these carriers are not adequate. The most active immobilized cells were obtained by entrapping the cells into a cationic acrylamide-based polymer gel lattice. To prepare the most efficient immobilized cell preparation, the monomer composition and concentration and the amount of cells to be entrapped are important factors. Optimal conditions for cell immobilization of strain S-6 are as follows. Cells (5 kg, wet weight) are suspended in 2 liters of 0.1 M potassium phosphate buffer solution (pH 7.7), and the cell suspension is cooled to 4° (this is the handling temperature of all procedures described here). To this cell suspension, 2.5 kg of 40% (w/v) monomer solution [acrylamide/dimethylaminoethyl methacrylate/N,N'-methylenebisacrylamide 85.5 : 9.5 : 5.0 (w/w)] is added. The polymerization is initiated by the addi-

526

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[48]

FIG. 1. Scanning electron micrograph of immobilized cells strain S-6.

100 ¸

"eoi >6O >

~401

_=,2o @

10

20

30

Temperature

40

50

60

(*C)

FIG. 2. Effect of temperature on enzyme activity. The activity of intact cells was measured as described in Assay Methods at the temperatures in the figure.

[48]

527

AN ACRYLAMIDE PRODUCTION METHOD

tion of 250 ml 10% (v/v)/3-dimethylaminopropionitrile and 250 ml 10% (w/v) ammonium persulfate. The polymerization reaction starts after about 3-5 min. The temperature of the reaction mixture increases to about 30°, and polymerization is complete after 1 hr. The gel is separated from the reactor and made into granules about 1-2 mm in diameter for industrial use and washed with 0.05 M potassium phosphate buffer solution (pH 7.7). The nitrilase activity thus obtained is almost the same as that of the intact cells. In this process, it is very important to keep the temperature of the gel below 30°. An electron micrograph of the immobilized cells is shown in Fig. 1. Nitrilase Properties of Immobilized Cells Strain S-6

Temperature. The optimal temperature for the formation of acrylamide is 35 ° in intact and immobilized cells, but from the viewpoint of enzyme stability, the reaction temperature should be below I0 °. Figures 2 and 3 show the effect of temperature and the heat stability of the intact cells, respectively, and similar results are also obtained with immobilized cells. The activation energy of the immobilized cells is calculated to be 8.8 kcal/mol (at pH 8.0). pH. The pH activity profile of the nitrilase reaction is different for intact and immobilized cells, as shown in Fig. 4. The immobilized cells

I

I

I

i

!

!

2oo

\ '°ti I

--

" I

I

I

I

10

20

30

40

Treatment

I

50 (rain)

I

60

Fro. 3. Heat stability of the acrylamide-forming enzyme. The acrylamide-forming activity of intact cells was measured as described in Assay Methods at the temperature indicated in the figure. Treating conditions: 0.1% cell suspension incubated in 0.05 M (pH 7.7) potassium phosphate buffer solution. Temperatures: (O) 30°; (0)) 40°; (0) 45°; (®) 50°.

528

ENZYME ENGINEERING (ENZYME TECHNOLOGY) r

i

i

[48]

i

100 80 ~ 6o ~ 4O IX 20

PH FIG. 4. Effect of pH on enzyme activity. The acrylamide-forming activity was measured as described in Assay Methods at the pH values shown in the figure. (O) Intact cells; (0) immobilized cells.

I

,.,1o°

I

I

I

I

\

so

~®60,o

20[ I

0

"\ i

I

I

I

10 20 Acrylamide (,olo)

I

I

30

FIG. 5. Effect of acrylamide concentration on enzyme activity. The acrylamide-forming activity of intact cells was measured as described in Assay Methods. Acrylamide concentrations are indicated in the figure.

[48]

AN ACRYLAMIDE PRODUCTION METHOD

529

show an optimal activity at pH 8.5, whereas the optimal pH of the intact cells is 7.7. Acrylonitrile and Acrylamide Concentrations. Acrylonitrile and acrylamide possess a strong toxicity, and would inhibit the enzyme activity and stability. Especially, acrylonitrile influences severely, and so upon reaction it is preferable to keep the concentration of acrylonitrile in the reaction system to a level not exceeding 3% (w/v). Figure 5 shows the effect of acrylamide concentration on enzyme activity at 2.5% (w/v) acrylonitrile. Figure 6 shows the effects of acrylonitrile and acrylamide concentrations on enzyme activity during a semibatchwise reaction in which the acrylonitrile concentration was held almost constant by consecutive additions while acrylamide was accumulating. From these figures, it seems obvious that acrylonitrile (substrate) and acrylamide (product) greatly inhibit the nitrilase enzyme and that it is very important to control the concentrations of them in order to keep a high enzyme activity and stability. In our acrylamide-producing process, we consider that the merits of using the immobilized cells are the prevention of elution of impurities from the cells, increase in the separation of the cells from a reaction mixture, and increase in stability of enzyme toward acrylonitrile and acrylamide.

100;

~ ~

~ 8o ._>

~ 6o ~ 4O ¢Y 2O

0

1~) Acrylamide

i,

I 20 (%)

8

I 30

FIG. 6. Effect of acrylonitrile and acrylamide concentrations on enzyme activity, The acrylamide-accumulating reaction was carried out according to a semibatchwise reaction with a stirred reactor. (Acrylonitrile was added consecutively.) Reaction conditions: temperature, 0-3°; pH, 8.5; immobilized cell concentration, 1%; reaction times, 5-30 hr. Acrylonitrile concentration: (©) 1.0%; (~) 2.0%; (e) 3.0% (w/v).

530

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[49]

Acrylamide Production of Immobilized Cells Strain S-6 From these described properties of immobilized cells, we established the acrylamide process conditions at pH 8.0-8.5, temperature below 10°, and acrylonitrile concentration below 3% (w/v). By using our immobilized cells under these conditions, a reaction solution containing more than 20% (w/v) acrylamide can be obtained, i.e., a concentration previously reported to be necessary for an industrial process. I One example of the composition of the reaction solution is as follows: acrylamide, 20% (w/v); acrylonitrile, negligible; acrylic acid, below 0.1% (w/w) per acrylamide. Conclusion As described above, Rhodococcus cells of strain S-6 can be easily immobilized to produce a high nitrilase activity when stabilized by the entrapping method using cationic acrylamide-based polymer gel. We at Nitto Chemical Industry Company, Ltd. have examined the characteristics of such immobilized cells and have also recently designated a suitable bioreactor. We are now producing about 4000 tons of acrylamide per year.

[49] A p p l i c a t i o n o f I m m o b i l i z e d Thiobacillus ferrooxidans for Large-Scale T r e a t m e n t of Acid M i n e Drainage

By T. MURAYAMA, Y. KONNO, T. SAKATA, and T. IMAIZUMI A considerable number of abandoned underground mines still discharge acid mine drainage continuously. It has become necessary to treat the drainage without any complications from an environmental viewpoint. Mine acid is usually caused by oxidation of sulfide minerals, such as pyrite, chalcopyrite, galena, sphalerite, and argentite, which occur in many metalliferous ore deposits, Of these minerals, pyrite is the most important in forming mine acid and releasing toxic concentrations of heavy metals. Thiobacillus ferrooxidans, found naturally in acid mine drainage, increases the oxidation of sulfide minerals and supplies ferrous ion in the acid drainage as the microbial catalyst. Utilizing these characteristics of Thiobacillus ferrooxidans, bacterial oxidation is being adopted in the METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

530

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[49]

Acrylamide Production of Immobilized Cells Strain S-6 From these described properties of immobilized cells, we established the acrylamide process conditions at pH 8.0-8.5, temperature below 10°, and acrylonitrile concentration below 3% (w/v). By using our immobilized cells under these conditions, a reaction solution containing more than 20% (w/v) acrylamide can be obtained, i.e., a concentration previously reported to be necessary for an industrial process. I One example of the composition of the reaction solution is as follows: acrylamide, 20% (w/v); acrylonitrile, negligible; acrylic acid, below 0.1% (w/w) per acrylamide. Conclusion As described above, Rhodococcus cells of strain S-6 can be easily immobilized to produce a high nitrilase activity when stabilized by the entrapping method using cationic acrylamide-based polymer gel. We at Nitto Chemical Industry Company, Ltd. have examined the characteristics of such immobilized cells and have also recently designated a suitable bioreactor. We are now producing about 4000 tons of acrylamide per year.

[49] A p p l i c a t i o n o f I m m o b i l i z e d Thiobacillus ferrooxidans for Large-Scale T r e a t m e n t of Acid M i n e Drainage

By T. MURAYAMA, Y. KONNO, T. SAKATA, and T. IMAIZUMI A considerable number of abandoned underground mines still discharge acid mine drainage continuously. It has become necessary to treat the drainage without any complications from an environmental viewpoint. Mine acid is usually caused by oxidation of sulfide minerals, such as pyrite, chalcopyrite, galena, sphalerite, and argentite, which occur in many metalliferous ore deposits, Of these minerals, pyrite is the most important in forming mine acid and releasing toxic concentrations of heavy metals. Thiobacillus ferrooxidans, found naturally in acid mine drainage, increases the oxidation of sulfide minerals and supplies ferrous ion in the acid drainage as the microbial catalyst. Utilizing these characteristics of Thiobacillus ferrooxidans, bacterial oxidation is being adopted in the METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

[49]

APPLICATIONOF IMMOBILIZEDT. ferrooxidans

531

treatment of acid mine drainage at some mines in Japan. In this paper a large-scale treatment of acid mine drainage including a bacterial oxidation system is described as an example of an effective and practical application of the bacteria. Treatment of Acid Mine Drainage To neutralize acid mine drainage and at the same time precipitate heavy metals, lime neutralization has been used for many years, employing calcium hydroxide in many cases. In comparison, neutralization by calcium carbonate has several advantages such as lower cost, less volume, and faster settling of the resultant sludge. However, calcium carbonate simultaneously generates carbonic acid in the course of neutralization, which limits the maximum pH of the drainage to about 6.5. At this pH, the ferrous salt remains in solution and does not precipitate. If the ferrous salt can be oxidized to a ferric salt prior to neutralization, calcium carbonate is made available to neutralize acid mine drainage. Bacterial Oxidation in Treating Acid Mind Drainage To realize the oxidation of the ferrous salt, a bacterial oxidation system was developed at the Yanahara pyrite mine of Dowa Mining Co., Ltd., in 1974, under the guidance of Professor K. Imai of Okayama University, instead of using nitrogen oxide as a catalyst. Thiobacillus ferrooxidans, found naturally in the mine drainage in a concentration of about 106 cells/ml is used for the oxidation. A feature of the bacterial oxidation system developed in Yanahara mine is that the microbes are cultivated and well adsorbed on the surface of basic ferric sulfate, generated by the hydrolysis of ferric sulfate. They are precipitated together with the cell carrier in the next stage, and recycled to the oxidation circuit. In these continuous processes, it is possible to obtain a sufficient concentration of cells, i.e., about 108 cells/ml, and to oxidize 95-98% of the ferrous salt to ferric within 60 min. The mine adopted the improved system in the neutralization of mine drainage, resulting in a considerable saving of the treatment cost--about 320,000 Yen by microbial oxidation compared with 965,000 Yen by nitrogen oxide, based on a monthly treatment of 50,000 m 3 of drainage at pH 2.5 and 2100 mg/liter of ferrous iron. (Patent No. 797734 in Japan.) The bacterial oxidation system was also applied in a partially modified manner to the treatment of acid mine drainage at the abandoned Matsuo sulfur-pyrite mine.

532

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[49]

Application of Bacterial Oxidation to the Matsuo Mine 1,2

Background The Matsuo mine, an abandoned mine in the Iwate Prefecture in the northern part of Japan, was a large-scale underground and open-cut mine in which sulfur and pyrite ores had been mined for about 60 years from 1910 through 1971. Total output excavated stood at 29 million tons or 13 million cubic meters. Its mine drainage reached 30 m3/min in the final stage of operation late in the 1960s, at times with strong acidity (pH 1.3 to 1.5). Neutralization by calcium carbonate and calcium hydroxide was being carried out at the mine, but it was difficult to maintain sufficient water quality because of the large quantity and strong acidity of the drainage. This caused acidic pollution and the formation of a red suspension downstream in Kitakami river. The mine was closed in 1971 due to nonprofitability, but mine drainage still discharged continuously from the mine after its closure. The Japanese national government and the Iwate prefectural government have been responsible for the treatment of the drainage after the mine closed, and they decided to apply the bacterial oxidation system developed at the Yanahara mine to the Matsuo mine.

Feasibility Tests for the Application In applying the oxidation system to the neutralization at Matsuo mine, two difficult problems were considered at that time. First, the mine is located in the northern part of Japan in a mountainous area about 1000 m in altitude. Temperatures in winter fall as low as 20° below zero. Thus the problem of whether the cultivation and oxidizing activity of the bacteria could be continuously maintained needed to be examined. Second, basic ferric sulfate, used as the cell carrier at Yanahara mine, cannot be generated in the drainage at Matsuo mine because of its strong acidity level of pH 1.6. Therefore, the National Ministry of Construction entrusted a series of feasibility tests to Dowa Mining Co., Ltd. Under the guidance of Professor K. Imai of Okayama University, Professor J. Shimoiizaka of Tohoku University, and Professor T. Goto of Iwate University, tests were carried out in 1973 and 1974, utilizing Thiobacillus ferrooxidans living naturally in the drainage at Matsuo mine in a concentration of about 2 × 105 cells/ml. The cultivation test of the z E. Yabuuchi and Y. Imanaga, paper presented at the joint Mining and Metallurgy Institute of Japan-American Institute of Mining Engineering Meeting, Denver, Colorado, 1976. 2 T. Ishikawa, T. Murayama, I. Kawahara, and T. Imaizumi, paper presented at the International Symposium on Biohydrometallurgy, Cagliari, Italy, 1983. ("Recent Progress in Biohydrometallurgy." Associazione Mineraria Sarda, 09016 Iglesias, Italy 1983.)

[49]

APPLICATION OF IMMOBILIZEDT. ferrooxidans

533

microbes was carried out by the same method as at Yanahara, i.e., an oxidation-precipitation-recycling system, The results obtained from the tests are summarized as follows: 1. The cultivation and oxidizing activity of Thiobacillus ferrooxidans could be maintained under such severe conditions as those (1.35 °, pH 1.6) of the drainage. 2. Diatomaceous earth was selected as cell carrier because of its strong cell-adsorbing power, acid-resistant properties, and ready settling in tanks which enables it to be recycled to the oxidation circuit. Thus, the researchers succeeded in establishing a new bacterial oxidation system utilizing diatomaceous earth as the cell carrier for treating acid mine drainage at Matsuo mine, converting most ferrous salt to ferric within 60 min after the initial cultivating period of approximately 2 weeks. 3. In the neutralization of the drainage by calcium carbonate, subsequent to the bacterial oxidation, the ferric salt and arsenic compound contained in the drainage were precipitated by raising the pH value to 4. This means that most of heavy metals dissolved in the drainage can be eliminated in the form of precipitates by solid-liquid separation. Applicability of the bacterial oxidization system in the neutralization at Matsuo mine was clarified through these feasibility tests. (Patents No. 4139456 in the United States, No. 1527766 in the United Kingdom, No. 239957 in Canada, and No. 2639045 in West Germany.) Installation o f a N e w Neutralization Plant After the success of the feasibility tests, the Iwate prefectural government decided to install a new neutralization plant including the bacterial oxidation system at Matsuo mine with financial aid from the national Ministry of International Trade and Industry, the authority for the mining TABLE I SPECIFICATIONS FOR WATER TO BE TREATED AND AFTER TREATMENT

Sample

Quantity (mVmin)

Acid drainage Treated water

20 20

pH

Acidity to 8.4 a (mg/liter)

T-Fe b (mg/liter)

A1(mg/liter)

As(mg/liter)

1.6 4.0

4240 786

795 13

189 130

5.77 0.02

S.S c (mg/liter)

30 or less

a Acidity to 8.4 m e a n s an acidity value of water. This is usually indicated by concentrations of calcium carbonate (mg/liter) n e c e s s a r y to raise the pH value of water to 8.4. b T-Fe m e a n s total concentrations o f ferrous and ferric irons in water. c T h e insoluble arsenic contained in 30 mg/liter of S.S (suspended solutions) should be 0.10 mg/ liter.

534

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[49]

industry and environmental protection, under the guidance of the Metal Mining Agency of Japan. The fundamental policies for designing the new plant were set as follows and were authorized by the national and prefectural governments. 1. pH. The pH of river water should be maintained, as the target value, within the range 6 to 6.5 at the site of Funata bridge in the Kitakami downstream. 2. Tailings sludge generated by neutralization. The sludge including iron, arsenic, etc. should be stored in a restricted place near the plant, and never be discharged into any river or stream. 3. Capacity of the plant. Specifications for water to be treated and after treatment by the plant are shown in Table I. 4. Form of treating. The drainage should be treated through four lines, each having the treating capacity of 5 m3/min, to treat variable quantities of the drainage. 5. Capacity of sludge dam. The dam for storing hydroxide sludge should be constructed such that its capacity should be sufficient for 20 years' storage. 6. Location of the plant and the sludge dam. The plant should be installed near the portal discharging the drainage and the sludge dam should utilize the nearby marsh. The construction of the plant commenced in August 1977 and was completed in November in 1981. Total installation cost of the plant, including the sludge storage dam, was approximately 9300 million Yen (38 million U.S. dollars) in 1981. After test operation for about 4 months, the plant has been in full operation since April 1982. The Metal Mining Agency of Japan was entrusted with the operation and maintenance of the plant by the Iwate prefectural government. Contents of the Plant The outline of the plant is shown in Figs. I and 2.

Bacterial Oxidation System Bacterial oxidation, the key point of the neutralization system, is shown in Fig. 3. The system includes three processes: oxidation, precipitation, and recycling, as stated. Thiobacillus ferrooxidans is supplied naturally from the flowing drainage upstream, cultivated and well adsorbed on the surface of diatomaceous earth in the oxidation, precipitated and concentrated together with the cell carrier in its settling process subsequent to the oxidation, and

A P P L I C A T I OOF N IMMOBILIZED T. ferrooxidans

[49]

~

535

G 50m ,

#

I

FiG. 1. Layout of principal facilities of the plant. (A) Distribution tank; (B) oxidizing tanks; (C) diatomaceous earth settling tanks; (D) neutralizing tanks; (E) solid-liquid separation tanks; (F) blower house for aeration; (G) pilot plant for metal extraction.

recycled to the oxidation circuit. As was the case with the Yanahara mine, it is possible to maintain sufficient concentrations of cells, about 108 cells/ml, and to oxidize 95-98% of the ferrous salt to ferric within 60 min. However, the behavior of the microbes in these continuous processes is complex and not exactly understood. The oxidation rate of the ferrous salt is observed constantly by an oxidation-reduction potential meter situated at the outlet of oxidizing tank and the information relayed automatically to the control center of facilities. Specifications for the facilities and the materials for the oxidation are shown in Table II.

536

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[49]

Acid mine drainage

I Drainage Ireceiving [ tank Nutrient Diatomaceous earth

I 11 DistributiOntank[

I Oxidizing tank I. L Fe'+ ~ F e ' + [

I J

Diatomaceous earth settling tank CaC03 s l u r r y

[ Neutralizing tank

Aeration Coagulant (polymer)

I Aeration Coagulant (polymer) Treated water

Separation tank Sludge to dam

1

Discharge to river FIG. 2. Fundamental flow diagram of the plant.

TABLE II SPECIFICATIONS OF PRINCIPAL FACILITIES AND MATERIALS FOR OXIDATION

Facility and material Oxidizing tank Diatomaceous earth settling tank Diatomaceous earth Coagulant Nutrient

Specifications 580 m 3 of square concrete construction lined with resin; retention time, 60 min 16 m diameter × 5 m height; thickener type; retention time, 100 min Less than the 325 mesh screen; SiO2 83%, A1203 4.8%, Fe203 1.8%, made up in slurry; feeding capacity 21 liters/min Polymer, nonionic; feeding capacity 130 liters/min in solution Ammonium phosphate; feeding capacity 3 liters/min in solution

Number of facilities 4 4

APPLICATION OF IMMOBILIZED T. ferrooxidans

[49]

Nutrient

537

I Diatomace-

ous earth Aeration 11ne

Coagulant

-~Z

J

Distribution tank

Pum For recycling

Oxidizin 9 tank

Diatomaceous earth settling tank

FIG. 3. Flow diagram of bacterial oxidation system.

CaC03 slurry

Diatomaceous earth settling tank

Aeration line

Neutralizing tank

Coagulant

Solid-liquld separating tank

Pumping for sludge transporting

FIG. 4. Flow diagram of neutralization and solid-liquid separation.

538

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[49]

Neutralization and Solid-Liquid Separation After bacterial oxidation, the drainage is treated by neutralization and solid-liquid separation. Flow diagrams are shown in Fig. 4. The oxidized drainage is neutralized by CaCO3 slurry in a neutralizing tank to raise its pH value to 4.0, and suspended solids generated by the neutralization are precipitated in a solid-liquid separation tank subsequent to the neutralization. The pH value of water to be maintained after the neutralization is measured with a pH meter at outlet of neutralizing tank, and in the case of failure to reach the settled pH value of neutralized water, an alarm system adjusts the feeding volume of CaCO3 slurry. In solid-liquid separation, the ferric salt and arsenic compound are precipitated as the tailings sludge, extracted from the bottom of the separating tank, and transported by pumping to the sludge storage dam. Treated water, which contains eliminated suspended solids such as ferric salt and arsenic compound, is discharged from the surface of the tank to the river. Specifications for principal facilities and materials for neutralization and solid-liquid separation are shown in Table III.

Sludge Storage Dam The sludge storage dam is constructed near the neutralization plant. The dam is a rock-filled type with a slanting clay core, and is covered T A B L E III SPECIFICATIONS OF PRINCIPAL FACILITIES AND MATERIALS FOR NEUTRALIZATION AND SOLID-LIQUID SEPARATION

Facility and material

Specifications

Neutralizing tank

430 m 3 of square concrete construction lined with

Solid-liquid separation tank CaCO3 slurry Coagulant Blower for aeration

resin; retention time, 45 min 30 m diameter × 4.5 m height; thickener type; retention time, 5 hr Feeding capacity 0.5 mVmin Common to the settling diatomaceous earth Multiple-stage roots blower; 85 N mVmin, ° 1.3 kg/ cm2, b common to the oxidation

Number of facilities 4 4

4

a 85 N mVmin means delivery capacity of air volume of a blower. In this case, " N m TM

means normal m 3, conversion value of air volume under the condition of its pressure, per kg/cm 2. b 1.3 kg/cm 2 means delivery air pressure of blowers.

[49] pH

T. ferrooxidans

OF IMMOBILIZED

APPLICATION

539 Fe z+

Q

ma/min

mg/1

700

\ \

- 600

..'"..

L

•'J"- F e ~ +''' • .

2,0-

".,

i

\

C"'.

\

\

500

/

1.9-

400

•

1.8-

..-

.....

300

I 4 5 6 7 8 9 1 0 1 1 1 2 1 2 3 4 5 6 7 8 91011121 --

I

I

I

I

I

I

I

1982

I

Ill

I

"l

I

I

I

[

I

I

I

I~L-

1983

Ij I

-]4

2 3 4 I

I

I

1984

FIG. 5. Seasonal variation in quantity and quality of acid mine drainage at Matsuo mine.

partially with asphalt facing. The capacity of the sludge pond impounded by the dam is about 2 million m 3, which is sufficient to maintain 20 years' storage. Current Situation of the New Neutralization Plant

The quantity and quality of acid mine drainage have gradually improved since the neutralization plant began full operation in April 1982, although seasonal variations are observed as shown in Fig. 5. Therefore it

T A B L E IV ANNUAL OPERATING COST OF THE PLANT

Year

Annual cost

Annual quantity of drainage

Cost/m 3 of drainage

1982 1983

701 million Yen 682 million Yen

9.06 million m 3 8.74 million m 3

77 Yen (U.S. $0.32) 78 Y e n (U.S. $0.32)

540

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[49]

is now possible to maintain the rapid oxidation rate without any nutrient for the bacteria. The annual operating cost of the plant in 1982 and 1983 is shown in Table IV. Technical Development at Matsuo Mine To defray the treatment cost of acid mine drainage and reduce the tailings sludge volume generated from neutralization, the Metal Mining Agency of Japan is studying a new technical development--converting ferrous and ferric iron contained in the drainage to a marketable commodity as high-grade hematite and magnetite. The Agency commenced the development by bench-scale testing in 1981, and decided to install a pilot plant close to the neutralization plant at Matsuo mine in 1983. This plant was completed in July 1984 and is now operating in the testing stage.

AUTHOR INDEX

541

Author Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Abril, O., 263,267(9), 279(9) Adachi, S., 6, 7(9), 443 Adalsteinsson, O., 266 Ado, Y., 395 Ahmed, S. A., 147, 148(20), 149(20) Aiba, S., 385 Aida, K., 517 Aisina, R. B., 69, 70(11) Aizawa, M., 10 Alberts, B., 281 Albertsson, P.-/~., 45,244 Alberty, R. A., 472 Amotz, S., 359 Anantharamaiah, G. M., 158, 162(1, 2) Andersson, E., 55 Andreasen, A. A., 399 Andrews, J. P., 170 Ang, C. Y. W., 494 Angelov, T. I., 326 Annau, E., 480 Anson, M. L., 508 Anteunis, M., 433 Antonini, E., 146, 150, 151, 155(4, 6, 7), 157(2, 3, 4, 5, 6, 7, 8) Aoyagi, H., 513 Arcuri, E. J., 395 Arison, B. H., 130 Asano, Y., 523 Astoor, A., 443 Auriol, D., 240, 241 Austin, M. J., 239 Award, W. M., Jr., 170 Axen, R., 58, 152 B

Baboulene, M., 119 Bacher, A., 310

Bachere, H., 381 Bader, J., 10, 303,304, 305(3, 5, 10), 306(3, 5, 9, 10), 309 Bailley, C., 364 Baker, R. S., 95 Balasubramanian, D., 202(30), 203(30), 204, 205(30), 212(30), 213(30) Baldwin, J. J., 130 Baldwin, T. O., 83 Balny, C., 203(32), 204, 206(32), 212(32), 214(32), 219 Banga, I., 480 Barbaric, S., 190, 201(9), 202(9), 204(9), 205(9), 212(9), 214(9), 222 Barber, J. J., 10 Baret, J. L., 418, 419(7), 420 Barford, J. P., 395 Barnes, C. S., 425 Barry, S., 57, 64 Bartlett, G. R., 28, 49 Baughn, R. L., 266, 276 Beck, J. F., 118 Behal, F. J., 170(12), 171 Behie, L. A., 335,336, 338,339(17), 341(30) Behrens, C. H., 118 Bell, G., 147 Ben Aim, R., 523,530(1) Bender, M. L., 284 Bennett, J. W., 329, 340(1) Berde, B., 324 Berezin, I. V., 118, 119, 291,292(22) Berezin, V. I., 69, 70(11), 147, 197, 201, 202(28), 203(28), 204(28), 205(28), 206, 207(28, 37), 208, 209(42), 210(42, 43), 212(28, 43), 214(28, 43), 219 Berger, S. J., 71 Bergmann, M., 513 Bergmeyer, H. U., 85,270, 278,279(30) Berk, D., 336, 338, 339(17), 341(30) Berke, W., 9 Berkeley, R. C, W., 239

542

AUTHOR INDEX

Bernfeld, P., 395 Bernofsky, C., 56, 61 Bernstein, M., 107 Bethke, H., 319 Biezer, H. J., 366 Bird, B. A., 340 Blain, J. A., 147 Blair, L., 212, 213(53) Blakely, R. L., 238 Blau, N. F., 128, 129(27) Blazo, A., 480 Blumenfeld, H., 276 Blunt, K. W., 442 Bock, R. M., 472 Bogard, M. O., 240, 242 Bohak, Z., 170 Bonner, F. J., 193, 194(16), 198, 200(21), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16, 21) Bonse, D., 368 Booth, A. B., 124 Borch, R., 107 Bovara, R., 84, 156 Bowcott, J. E., 227 Bradford, M., 488 Brfinden, C. I., 6 Braunstein, A. E., 480 Bray, D., 281 Bray, R. C., 256 Breddam, K., 159, 162, 163 Bright, H. J., 109 Brockerhoff, H., 129 Brocklehurst, K., 107, 181 Brookes, I. K., 145 Brougham, M. J., 57 Brown, R. T., 350 Bruckner, V., 480 Bryant, G., 240, 242 Buchanan, J. M., 266 Buchel, K. H., 135 Bucke, C., 381, 395, 432, 433(4), 435, 437, 442 Buckland, B. C., 145, 146(6), 147(6), 148(6) Biickmann, A. F., 5, 10, 11, 13(6), 17(6, 10), 21, 39, 45,490, 493 Biihler, M., 304, 305(7) Bullerjahn, R., 179, 184(4) Bulot, E., 498 Bunting, P. S., 62, 64

Burkhard, A., 7 Burris, R. H., 485,489(12) Butler, L. G., 150, 234, 236(3), 237(3)

C Calton, G. J., 464,497, 498(2), 500(1, 2) Cambou, B., 119, 120(14, 15) Campbell, I. M., 340 Campbell, J., 70, 75(16) Canizaro, P. C., 170(12), 171 Carasik, W., 361 Carey, P. R., 134 Carmel, A., 170(9), 171, 174 Carrea, G., 84, 146, 147(14), 150, 151,155(4, 6, 7), 156, 157 Carrier, R. G., 134 Carroll, J. O., 361 Carter, J. A., 71 Casellato, M. M., 150, 157(3, 5) Casmati, G., 201,202(27) Cesti, P., 137 Chang, H. N., 241 Chang, T. M. S., 21, 57, 59(23, 24, 25), 67, 68, 69, 70, 71, 72, 73, 74(9, 12, 13, 22), 75(9, 12, 13, 16), 76(9, 13), 77, 78, 395 Chappelle, E., 84 Charles, M., 10 Cheetham, P. S. J., 432, 435, 437(21, 22), 438(22), 439, 440(25), 441(22), 442, 443(21), 445(21, 22), 446(21, 22), 447(22), 448(22), 449, 450(36), 451(36), 454(22) Chen, W.-P., 356 Chen, Y.-H., 204 Chernyak, V. Y., 197 Chesne, S., 484 Chiba, H., 383 Chibata, I., 331,381,382(11), 395, 455,456, 459, 464, 468, 469, 472, 475,476, 478 Cho, Y. R., 241 Ciegler, A., 329, 340(1) Ciotti, M. M., 36 Clare, D. A., 424 Clark, J., 435, 437(22), 438(22), 439(22), 441(22), 442(22), 445(22), 446(22), 447(22), 448(22), 451 (22), 454(22) Clark, V. M., 268

AUTHOR INDEX Clarke, J. H. R., 225 Cleland, W. W., 265 Cluskey, J. E., 239 Coffey, D. S., 62 Cohen, N., 126 Cohn, M., 265 Coleman, M. H., 148, 149(22), 406 Collet, A., 118 Colowick, S. P., 36 Columbi, F., 146, 147(14), 151, 155(7), 157(7) Connor, W. E., 136 Contaxis, C. C., 236 Cooney, C. L., 498 Corkey, B., 465 Corman, J., 240 Corrieu, G., 395 Costerton, J. W., 334, 335, 339(10) Coton, G., 412 Coughlin, R. W., 10 Cousineau, J., 72 Crans, D. C., 263,267(3) Craven, D. B., 57, 58(19), 64 Cremlin, R., 135 Cremonesi, P., 146, 147(14), 150,151,155(4, 6, 7), 156, 157(2, 3, 4, 5, 6, 7, 8, 12) Crisel, R. M., 95 Crook, E. M., 181 Cross, R. P., 37 Crueger, W., 434 Cuatrecasas, P., 88, 106, 108(6), 494 Cysewski, G., 381,382(6)

543

Delalande, P., 420 Delange, R. J., 170, 178(1) DeLuca, M., 83, 87(11, 12, 13, 14, 15), 88, 89, 90(12), 92 Delz, B., 319 Deming, J. W., 84 Deo, Y. M., 329, 330(2), 331(2), 334, 335, 336, 337, 338, 339(10, 15, 19), 340(21) de Resset, A. J., 366 DesnueUe, P., 129 Detar, C. C., 414 Dickerson, A. G., 324 DiNello, R., 94 Dintzis, H. M., 181 Doerfler, D. L., 340 Dohan, L. A., 420 Doherty, J. V., 225 Donohue, J. A., 192 Dordick, J. S., 137 Dorman, D. E., 95 Dossena, A., 201,202(27) Douzou, P., 203(32), 204, 206(32), 208, 210(46), 212(32), 214(32), 219 Drath, L., 434 Drifford, M., 192 Duarte, J. M. C., 146, 147(12) Dugas, H., 292 Dunhill, P., 64, 75 DunniU, P., 145, 146(6), 147, 148(6), 290 Durand, G., 381,395 Durst, D., 107 Duterrtre, B., 395

E D Daka, N. J., 62, 64(43, 44) Daniel, J. W., 432 Darszon, A., 212, 213(52, 53) D~iumer, H., 181 Davis, B. N., 340 Day, R. A., 225 Dean, P. D. G., 57, 58(19), 64 Debey, P., 208, 210(46) de Bruyn, A., 433 Dehm, P., 170(11), 171, 174(11) Dehnen, W., 186 Delahodde, A., 196, 212(19), 213(19)

Edwards, G., 485 Egerer, P., 452 Eggert, E. W., 319 Egghart, H., 92 Eicke, H. F., 188, 189, 192(7), 217, 219(7), 225(7) Eklund, S. H., 6, 240 Eiiel, E. L., 117 Ellwood, D. C., 239 Elmaleh, S., 523,530(1) EI-Sayed, A. H., 318, 323(3) E1 Seoud, O. A., 201,212 Endo, S., 504

544

AUTHOR INDEX

Enfors, S.-O., 46 Eng, H., 395 Enosono, S., 395 Epton, R., 181 Ergan, F., 73, 74(22) Erjomin, A. N., 208, 210(44) Ernbach, S., 58, 152

F Farr, A. L., 27, 241 Fastrez, J., 180 Fawcett, J. S., 33 Fearon, W. R., 235 Feger, V. H., 240, 242 Feher, G., 212, 213(54) Fendler, J. H., 188, 201(1), 203(1) Ferrara, L., 146, 150, 155(4, 6), 157(4, 6) Ferrari, M., 271 Fersht, A. R., 180, 204 Fields, R., 40, 496 Findeis, M. A., 118, 136(8) Fink, A. L., 284 Fischer, J., 181 Fisher, B. E., 239 Flaks, J. G., 271 Fleminger, G., 170, 171, 173, 174(9) Fletcher, P. D., 192, 200, 203(31), 204, 212, 219 Floss, H. G., 319 Flossdorf, J., 10 Flynn, A., 57 Ford, J., 87(11), 88,413 Frank, C., 304, 305(10) Frank, H., 182 Franks, F., 208, 210(46) Freedman, R. B., 203(31), 204 Friedmann, T. E., 473 Fruton, I. S., 281,503 Fujihashi, T., 163, 167(6) Fujimoto, M., 519 Fujishima, T., 272 Fujita, K., 293 Fukui, S., 145, 146, 147, 148(20), 149(20), 293, 294, 298(3), 299, 301(11), 383,455 Fukushima, S., 293 Fuller, C. W., 109 Furgala, B., 432

Furugren, H., 6 Furui, M., 464, 477 Furukawa, S., 5, 7(8), 21, 34, 35, 45,493 Fusee, M., 464, 498

G Gabert, A., 178 Galunsky, B., 285, 286(12), 292 Ganzinger, D., 343 Garrett, C., 435, 437(22), 438(22), 439(22), 441(22), 442(22), 445(22), 446(22), 447(22), 448(22), 451(22), 454(22) Garrett, P., 268, 271(18) Gattner, H.-G., 163, 168, 186 Gaucher, G. M., 329, 330, 331, 334, 335, 336, 337, 338, 339(10, 15, 17, 19), 340(21), 341,342(5, 24) Gbewonyo, K., 335,340(12) Gehrke, C., 466 Gelfand, D. H., 498 Gellf, G., 383 Geresh, S., 263,267(8) Germeshausen, J., 507 Gestrelius, S., 21, 57, 58(21), 61(21), 63(21) Ghim, Y. S., 241 Ghini, S., 84 Giacomello, A., 271 Gibbons, I., 93, 94 Gibson, K. J., 271 Girotta, S., 84 Gitler, C., 208, 211(51) Glass, J. D., 161, 162 Glazer, A. N., 170, 178(1) Glock, G. E., 56, 61(4) Goldenberg, D., 170(9), 171, 174(9) Goldkamp, A. H., 503 Gooday, G. W., 239 Goodman, A. E., 456, 472 Gorin, G., 235, 236(9) Grandi, C., 194, 196(18), 206(18), 208(18) Grasmick, A., 523, 530(1) Green, E. H., 121 Green, K., 84 Green, S., 56, 61(8) Greenberg, B. D., 170 Greenzaid, P., 119 Grenner, G., 5, 56

AUTHOR INDEX Gricolo, B., 84 Griffin, T., 57 GrootWassink, J. W. D., 329, 330(2), 331 Gross, A., 263,267(8) Gross, E., 178 Griiber, W., 278, 279(30) Grunwald, J., 2l, 57, 59(23, 24, 25), 69, 74(9, 12, 13), 75(9, 12, 13), 76(9, 12, 13), 77, 81(28), 137 Guilbault, G. G., 32 Guilford, M., 64 GOnther, H., I0, 303,304, 305(3, 5, 8, 9, 10, 14), 306(3, 5), 308(6) Gupta, N. K., 56, 61(1, 2, 3) Gutman, I., 44

H Haase, W., 452 Habig, M. C., 523,530(1) Hagi, N., 506 Hagihara, B., 508 Hahn-H~igerdal, B., 55, 381,382(10) Hakney, R. E., 136 Halwachs, W., 55 Hamada, S., 433 Hamada, T., 192 Hamman, J. P., 497, 498(2), 500(2) Hanlon, T. M., 94 H/insler, M., 179, 185 Hanson, H., 178 Harada, T., 506 Harbron, S., 143, 144, 148(4) H/iring, G., 215 Harju, M., 415 Hartman, S. C., 266 Harvey, M. J., 57, 58(19), 64 Hasegawa, E., 383 Hashimoto, H., 440 Hashimoto, K., 6, 7(9) Hashimoto, Y., 406 Hastings, J. W., 83 Hathaway, S., 433 Haufler, U., 284, 285, 290(8), 292, 440 Haugen, G. E., 473 Havewala, N. B., 414, 459 Hayashi, R., 159 Hayes, M. C., 433

545

Haynes, W. C., 239 Haynie, S. L., 263, 267, 279(9) Hedbys, L., 231,233(4) Heier, J., 485 Hellerman, L., 62 Hellman, N. N., 240 Helvin, E. H., 242 Hemmingsen, S. H., 361 Henderson, L. M., 472 Henninger, F., 201,202(27) Hennink, W. E., 256 Henseleit, K., 234 Herbert, J. A. L., 107 Herves, D. V., 364 Hesse, M., 343 Hibino, K., 124 Hidai, M., 124 Hilhorst, R., 208, 211(48, 49), 212(48, 49), 216, 221(1, 2, 3), 225(2, 3), 226(2, 3), 228(1), 229(1) Hill, C., 234 Hinds, J. A., 238 Hinkley, J. E., 87(14), 89, 92 Hippert, B. L., 181 Hirohara, H., 413 Hiromi, K., 7, 22 Hirschbein, B. L., 263,268(4) Hiusjen, J., 93 Ho, T. C.-L., 136 Hoffman, C. A., 240 Hoffman, G., 56, 61(3) Hofmann, A. F., 87(13, 14, 15), 89 Hofmann, H.-J., 180 Hofstee, B. J., 258 Hohansson, A. C., 57 Holcberg, I. B., 381 Holtzmann, E., 170(10), 171, 174(10) Homandberg, G. A., 185, 235, 291,506 Hornby, W. E., 57 Horton, A. A., 484,485(10) Horton, H. R., 423,425(7, 8), 428,429(8) Horwath, R. O., 369 Hoshida, W., 406 Howe, A. M., 212 Howell, S. F., 241 Hsiao, H. Y., 158, 160, 162(1) Hughes, R. D., 507 Hummel, W., 15,498 Hustedt, H., 46

546

AUTHOR INDEX

Ichikawa, T., 292, 504 Igarasi, S., 290 Iida, T., 145, 294, 300(6), 383 Iizuka, T., 21, 35, 45, 493 Imaizumi, T., 532 Imanaga, Y., 532 Imber, C. E., 435, 437(21), 439(21), 443(21), 445(21), 446(21) Imre, V. E., 190, 191(10), 193, 194(17), 200(17), 215 Inaba, Y., 395 Indo, M., 299 Inman, D. J., 57 Inman, J. K., 181 Irino, S., 506 Isherwood, J., 435, 437(21), 439(21), 443(21), 445(21), 446(21) Ishihara, M., 406 Ishikawa, H., 366 Ishikawa, T., 532 Ishiwatari, H., 124 Isowa, Y., 292, 504 Iwamoto, N., 146, 293, 298(3) Izumitani, A., 433 Izumiya, N., 513

J Jablonski, E., 83 Jiickle, H., 193, 194(17), 200(17) Jackson, R. W., 240, 242 Jacques, J., 118 Jakubke, H.-D., 178,179, 180, 181,182, 183, 185, 281 Janolino, V. G., 423,424,425,426,427,428, 429(10, 15), 430(10, 15) Janson, C. A., 265 Jarvis, F. G., 331,336(9) Jeanes, A., 239 Jellet, J. F., 151 Jencks, W. P., 119 Jenkins, W. T., 483 Jensen, R. G., 129 Jin, I.-N., 294 Johansen, J. I., 159, 162, 163 Johansson, G., 39, 46 Johnson, D. B., 57

Johnson, J. W., 128, 129(27) Johnson, M., 331,336(9) Johnson, R. A., 358 Johnson, R. B., 97 Johnston, B. D., 130 Jonczyk, A., 163, 186 Jones, A., 335, 336, 338, 339(17), 341(30) Jones, J. B., 118, 132, 179, 255 Joppich, M., 201,202(27) J6rnvell, H., 6

K Kaboli, H., 241 Kadomura, Y. J., 433 Kakimoto, T., 472 Kalckan, H.M., 273 Kanaya, T., 162, 168(2), 169(2) Kanbayashi, A., 357 Kaneshiro, C. M., 235 Kaninaka, A., 272 Kaplan, N. O., 36 Kapune, A., 234 Karkas, J. D., 507 Karube, I., 21,323 Kasche, V., 234,284, 285,286, 290, 292,440 Katayama, N., 21, 35, 45,490, 493 Kato, J., 381,395,472 Kato, K., 290 Kato, T., 71,513 Kaufman, S., 270 Kawahara, I., 532 Kawahara, K., 290 Kawamoto, T., 395 Kazandjian, R. Z., 137 Kawano, E., 413 Kayashima, K., 456 Kazanskafa, N. F., 69, 70(11) Kazlauskas, R. J., 263, 270(5) Keh, E., 203(32), 204, 206(32), 212(32), 214(32), 219 Kelleher, W. J., 318 Keller, H. W., 366 Kelly, N., 57 Kessler, E., 170, 174 Khaleeluddin, K., 367 Khmel'nitskii, Y. L., 197,206, 208,209(42), 210(42, 43), 211(40), 214(43), 215(40) Khorana, H. G., 266

AUTHOR INDEX Kierstan, M., 381,395 Kihara, K., 234, 292,503,504, 505, 506(6) Kimble, B. K., 240, 241(6) Kimura, K., 395 Kimura, T., 146,293,298(3) King, C. K., 9 King, E. J., 443 Kinoshita, S., 517 Kirby, A. J., 268 Kirchner, G., 137 Kirtane, J. G., 130 Kitahara, A., 188 Kitahara, K., 455 Kito, M., 206, 207(39) Klein, J., 313,395 Klesov, A. A., 284 Klibanov, A. M., 118, 119, 120(14, 15), 137, 147 Klyacbko, N. L., 197,201,202(28), 203(28), 204(28), 205(28), 206, 207(28), 208, 209(42), 210(43), 211(40), 212(28, 43), 214(28, 43), 215(40), 219, 220 Kobayashi, T., 62 Koelsch, R., 178 Koepsell, H. J., 240, 242 Kolot, F. B., 381 Komatsu, A., 293,298, 299 KOnnecke, A., 178, 179, 180, 181, 182, 183, 184(4), 185, 187(20) Koob, R., 7 Kopp, B., 318, 320(la), 323(3) Kornberg, H. L., 484, 485(10) Koshiro, S., 299, 301(11) Kosogi, Y., 357 Kramer, D. N., 32 Krampitz, O. L., 485 Krebs, H. A., 234,499 Kricka, L. J., 84, 87(15), 89, 92 Kroner, K. H., 10, 46 Krutzsch, H. C., 178 Kuhl, P., 178, 181, 185,281 Kuhimann, W., 55 Kiihn, I., 55 Kuhn, R. W., 159 Kula, M.-R., 5, 7, 9, 10, 11, 13(6), 15, 17(6), 21, 45, 46, 244,490, 493,498 Kulys, J., 21, 33(5) Kumar, C., 202(30), 203(30), 204, 205(30), 212(30), 213(30) Kuninaka, A., 519

547

Kunitz, M., 47 Kuno, S., 310 Kunze, H. E., 238 Kurganov, B. I., 206, 207(37) Kutzbach, C., 453 Kuwayama, H., 408

L Laane, C., 208,211(48, 49), 212(48, 49), 216, 221(1, 2), 225(2), 226(2), 228(1), 229(1) Laidler, K. J., 7, 21, 57, 59(26), 60, 62, 63(31), 64, 65(26, 31), 66(31) Laki, K., 480 Lam, K. S., 329, 330, 331(2), 342(5) Lamed, R., 64 Landis, D. A., 241 Lantero, O. J., 453 Lantz, L., 497,498(2), 500(2) Laraway, J. W., 366 Lardy, H. A., 485 Larsson, P. O., 3, 5, 7, 10, 21, 25, 34, 48, 57, 58(20), 59, 63, 64, 75, 103, 108, 231,395 Lasch, J., 178 Laskowski, M., Jr., 185, 235, 291,506 Lattes, A., 119 Lee, J., 85 Lee, S.-L., 343 Lee, S, S., 485,489(12) Le Goffic, F., 22 Lesser, B. H., 336, 338, 339(17), 341(30) Leuchtenberger, W., 10, 493 Levasbov,A. V., 197, 201,202(28), 203(28), 204(28), 205(28), 206, 207(28, 37), 208, 209(42), 210(42, 43), 211(40), 212(28), 214(28, 43), 215(40), 219, 220 Levich, V. G., 309 Levin, Y., 64 Levner, M., 436 Lewis, J. M., 263,267, 281 Lewis, L. T., 358 Libby, R. M., 97 Liberatori, F. A., 160, 162 Lilly, M. D., 64, 75, 138, 143, 144, 145, 146, 147, 148(4, 6), 290 Lin, Y. Y., 132 Lindberg, M., 5, 59, 64(30), 108 Lindley, M. G., 433 Lindman, B., 189, 192(4)

548

AUTHOR INDEX

Lindsley, J., 171 Link, M. L., 304, 305(9), 306(9) Linsmaler, E. M., 344 Lis, H., 230 Litman, D. J., 94 Llor, A., 192 Lloyd, N. E., 358, 369 Lodi, R., 156 Logan, R. M., 358 Lopez, A., 240, 241,242(19), 243(19) L6pez-Levia, M., 55 Lopresti, R. J., 126 Loss, von F., 433 Loucks, A., 92 Lowe, C., 25, 34, 48, 56, 57, 58(19, 20), 64, 75 Lowry, O. H., 27, 29, 71,241 Luddy, F. E., 405 Lugaro, G., 150, 157(3) Luger, P., 433 Luisi, P. L., 189, 190, 192(5), 193, 194, 196(18), 198,200,201,202(27), 203(33), 204, 206, 207(26, 36), 208, 210(41), 211(16), 212(16, 26, 36, 41), 214(9, 26, 36, 41), 215, 216, 217, 220(6), 221(6), 222 Lukasheva, E. V., 69, 70(11) Lfithi, P., 216 Lynn, K. R., 236

M MacAllister, R. V., 367 McCaskill, D. G., 330, 341(4) MacClement, B. A. E., 134 McClure, D. E., 130 McCoy, C. J., 145 McDonald, A., 57 Macdonald, I. A., 87(14), 89, 432 Macdonald, J. A., 151, 156 McDonald, M. R., 47 McDougall, B., 290 McElroy, W. D., 83 Maclntosh, F. C., 67, 68(1) Mclssac, J. E., Jr., 160, 162(6) McLean, P., 56, 61(4) McMullen, W. H., 361 Macrae, A. R., 147, 148, 149(22), 406, 409

Maeda, H., 7, 21, 22, 25, 26(16), 45 Magid, L. J., 200 Mahmoud, W., 318, 323(3) Mahony, D. E., 151 Malakhova, E. A., 206, 207(37) Malhorta, O. P., 284 Maliarik, M. J., 368 Malinauskas, A., 21, 33(5) Malouf, C., 71, 81(19) Malthouse, J. P. G., 107 Mamiya, G., 235, 236(9) M~nsson, M. O., 6, 7, 10, 21, 57, 58(21), 61(21), 63(21), 104 Mantle, P. G., 324 March, S. C., 88, 106, 108(6) Marconi, W., 455 Margalith, P., 381 Margolin, A. L., 284 Marguardt, J., 178 Mariella, R. P., 37 Marietta, M. A., 137 Marshall, R. O., 357 Marshall, W. E., 56, 61(3) Martin, L. D., 130 Martinek, K., 118, 119, 147, 185, 197, 201, 202(28), 203(28), 204(28), 205(28), 206, 207(37), 208, 209(42), 210(42, 43), 212(28, 43), 214(28, 43), 215(40), 219, 220, 291,292(22) Martinez, H. M., 204 Marring, S. C., 69 Masaki, T., 163, 165, 167(6) Mason, S. G., 67, 68(2) Massey, V., 108 Masuda, S., 134 Matsmara, Y., 124 Matsuo, T., 406 Mattiasson, B., 55, 181 Mattis, J. A., 185, 235, 291,506 Mauch, W., 440 Mavrides, C., 482,484, 486(8) Maxwell, E. S., 273 Mayhew, S. G., 229 Mazenod, F. P., 263,268(4) Mazid, M. A., 7, 21, 57, 59(26), 60, 62, 63(31), 64(31), 65(26, 31), 66(31) Mazzola, G., 146, 151, 155(7), 156, 157(7) Mazur, R. H., 503 Mead, J., 203(31), 204

AUTHOR INDEX Meienhofer, J., 178 Meier, P., 190, 191(10), 208, 210(41), 212(41), 214(41), 215(10, 41) Melvin, E. H., 239 Menger, F. M., 192, 201, 202(29), 204(29), 212(29), 214(29), 219 Mensd6rfer, F., 215 Mensler, K., 130 Messina, E., 271 Messing, R. A., 247,413,455 Metelitza, D. J., 208, 210(44, 45) Meyer, B., 368 Michal, G., 465 Mii, S., 272 Mildvan, A. S., 265 Misawa, M., 455 Misiorowski, R. L., 206, 207(38) Mitsuhashi, H., 344 Mitsunobu, O., 493 Mittal, K. L., 189, 192(4) Miwa, N., 31 Miyawaki, S., 124 Mizukami, H., 343 Moll, M., 381,395 M611ering, H., 278,465 M6nch, W., 186 Monnier, N., 484 Monsan, P., 119,240, 241,242(19), 243(19), 246, 247(27) Montal, M., 208, 211(51), 212, 213(52, 53, 54) Mori, K., 126, 134, 292,504 Mori, T., 464,469 Morihara, K., 162, 163, 165, 168(2), 169(1, 2), 291 Morikawa, Y., 21 Moroe,T., 293,298 Morosi, F., 455 Morr, M., 9, 39 Morris, C. J. O. R., 33 Morris, J. F., 121 Morrison, J. D., 117, 118 Morrison, J. F., 483 Mosbach, K., 3, 5, 6, 7, 10, 21, 25, 34, 48, 56, 57, 58(20, 21), 59, 61(21), 63, 64, 75, 103,104, 108, 152, 181,231,233(4), 381, 382(10), 395 Mosbach, R., 395 Mosher, H. S., 118

549

Mosti, R., 455 Miiller, F., 256, 257, 258 Muneyuki, R., 165 Munir, M., 434, 451 Muramatsu, M., 22, 34, 56 Murayama, T., 532 Myers, C., 161 N Nagai, T., 293 Nagakura, M., 408 Nagakura, N., 343 Nagase, T., 413 Nagata, S., 304, 305(9), 306(9) Nakajima, Y., 452 Nakamura, K., 163, 165, 167(6) Nakamura, R. M., 97 Nakane, P., 96 Nagel, A., 256 Narendranathan, T. J., 143, 144(3), 148(4) Narinesingh, D., 62, 65(37) Narita, H., 206, 207(39) Navarro, J. M., 381,395 Needham, D. M., 480 Neely, W. B., 239 Neidleman, S. L., 369 Neukom, C., 126 Neumann, S., 10, 303, 304, 305(3, 5, 14), 306(3, 5) Neurath, H., 159 Neway, J., 329, 330, 331(2) Ngo, T. T., 62 Nibley, D. A., 84 Nicoli, M. Z., 83 Nicot, C., 196, 212(19), 213(19) Nielsen, T. K., 359 Nikolova, N., 326 Nilsson, K., 104, 152, 231 Nishida, Y., 464 Nishimura, S., 292, 504 Nisselbaum, J. S., 56, 61(8) Nonaka, Y., 504, 505 Nordl6v, H., 343 Nordwig, A., 170(11), 171, 174(11) Norris, R., 107 Novak, T., 192 Novaka, Y., 292 Nozaki, H., 130

550

AUTHOR INDEX O

O'Carra, P., 64 Ochoa, S., 272 Odawara, H., 366 Offord, R. E., 234 Ogata, K., 517 Ogata, M., 6, 7(9) Ogawa, Y., 524 Ogura, Y., 508 Ohmori, M., 292, 504 Ohno, M., 513 Ohroff, G., 124 Ohta, Y., 508 Oka, T., 162, 163,165, 168(2), 169(1, 2), 291 Okada, H., 5, 9, 21, 22, 34, 35, 45, 56, 490, 493 Okahashi, N., 433 Okuda, K., 9 Okuso, M., 366 Oldfield, C., 203(31), 204 Omachi, A., 56, 61(3) Omata, T., 145, 146, 147, 148(20), 149(20), 293,294, 298(3), 300(6), 383 Ono, E., 406 Onozawa, K., 124 Ooshima, T., 433 Orentos, D. G., 446 Oriol, E., 240, 241 Oritani, T., 293,298 Orr, W., 482, 484,486(8) Oshima, A., 206, 207(39) Ostergaard, J. C. W., 69 Osumi, M., 294, 383 Otillio, N. F., 258 Otsuka, S., 117 Ottesen, M., 159 Overbeek, J. Th. G., 216 Oyama, K., 234, 292,503,504, 505, 506 P Pain, S., 420 Paknikar, S. K., 130 Palmer, D. N., 132 Pande, A., 190, 191(10), 193, 194(17), 200(17), 215(10), 216 Pantin, V. I., 201,202(28), 203(28), 204(28), 205(28), 206, 207(28), 208(40), 211(40), 212(28), 214(28), 215(40)

Papaconstantinou, S., 523,530(1) Pape, H., 323 Parikh, I., 88, 106, 108(6) Passonneau, J. V., 29 Patel, D. N., 358 Patterson, J. D. E., 147 Paul, F., 240, 241 Pazoutova, S., 326 Pellegrini, A., 193,194(16), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16) Pelmont, J., 484 Pelzig, M., 162 Penney, C., 292 Perrey, H., 452 Perrins, N. F., 200 Pfitzner, U., 343, 344(8), 349 Phelps, D. J., 134 Picciolo, G. L., 84 Pickett, D. J., 309 Pileni, M. P., 208, 210(47) Pillay, G., 170(10), 171, 174(10) Pisano, J. J., 178 Pitcher, W. H., 413,414, 459 Plaut, G. W. E., 485 Playne, M. J., 146 Pollak, A., 55, 263,276 Porath, J., 58, 152 Portino, A. D., 381 Poulsen, P. B., 255, 363 Powell, J. T., 483 Prenosil, J., 216 Prince, I. G., 395 Puc, A., 325

R Raab, A. W., 130 Radhakrishnan, R., 266 Raft, M., 281 Randall, R. J., 27, 241 Ranki, J. C., 239 Rao, A. S., 130 Rauschenbach, P., 304 Re, L., 5 Reents, A. C., 366 Rehecek, Z., 320, 321(7), 324(7), 325 Rehm, H. J., 318, 320(1a), 323(3) Reichenbach, H., 498

AUTHORINDEX Reilly, P. J., 241 Reithel, F. J., 234, 236, 237(3) Rekker, R. F., 226 Remaley, A. T., 340 Resurreccion, E., 71, 81(19) Riechmann, L., 284, 290(7), 440 Rios-Mercadillo, V. M., 263 Rist, C. E., 239 Robbers, J. E., 319 Roberts, K. R., 281,433 Robinson, B. H., 192,200,203(31), 204,212, 219, 225 Robinson, W. G., 56, 61(1) Robson, R. J., 266 Robyt, J. F., 240, 241(6, 7) Roda, A., 84, 87(15), 89 Roe, J. H., 49 Roels, J. A., 356 Rogovin, S. P., 240 Rohrbach, R. P., 366, 368 Rokugawa, K., 272, 519 Rosato, L. M., 340 Rosebrough, N. J., 27, 241 Rosenbusch, J. P., 212, 213(55) Rosenthal, A. M., 78 Rosenthal, T., 170(10), 171, 174(10) Rossodivita, A., 5 Roth, P., 178 Rothbart, H. L., 405 Roussel, D. J., 499 Rowley, G. L., 93 Royal, K. M., 61 Royer, G. P., 158, 160, 162, 170 Rozniewska, T., 335, 336, 339(17) Rozzell, J. D., 485 Rubenstein, K. E., 93 Rubin, B. A., 436 Rubin, J. R., 109 Rueffer, M., 343

S Sahm, H., 10 Sakashita, K., 524 Salermo, C., 271 Samokhin, G. P., 118, 119(10), 147 Sano, T., 493 Sasaki, M., 134 Sato, T., 331,464, 468,469,475

551

Satoh, A., 7, 22 Satoh, H., 504 Satoh, M., 292 Satoh, Y., 524 Saucy, G., 126 Sawamura, N., 406 Sawyer, B., 56, 61(6, 7) Schaefer, H. G., 7 Schellenberger, V., 179, 180, 183, 185(5) Schild, H. O., 324 Schiweck, H., 434 Schlatter, J. M., 503 Schmid, R. D., 55 Schmidt, H. L., 5, 56, 303 Schmidt-Berg-Lorenz, S., 440 Schmidt-Kastner, G., 452, 453 Schmitt, E. W., 168 Schneider, R. S., 93 Schoelmerich, J., 87(13, 14), 89 Sch6nfeld, M., 212, 213(54) Schuetz, H. J., 304, 305(9, 10), 306(9) Schiigerl, K., 55 Schulman, J. H., 227 Schulman, M. P., 56, 61(3) Schurs, A. H. W. M., 93 Schtitte, H., 10, 15 Schutt, H., 453 Schwartz, R. D., 145 Schwartz, W. E., 162 Schwarz, M. K., 235 Scollar, M. P., 137 Scott, A. J., 343 Scott, C., 126 Scott, T. A., 242 Sczyrbak, C. A., 324 Sedlmaier, H., 304 Segel, I. H., 42, 336 Sekiguchi, J., 341,342(24) Sekiguichi, M., 440 Semenov, A. N., 119, 185, 291,292(22) Senti, F. R., 240 Seto, T., 192 Shahani, K. M., 129 Shaked, Z., 10 Shallenberger, R. S., 440 Sharon, N., 230 Sharpe, E. S., 240 Sharpless, K. B., 118 Shaw, C. E., 147 Shimizu, J., 452

552

AUTHOR INDEX

Shipton, M., 107 Shoda, M., 385 Shono, T., 124 Shumate, S. E., 395 Shvyadas, V.-Y. K., 284 Sicsic, S., 22 Siddiqui, I. R., 432 Sidebotham, R. L., 239, 240(3) Sidrowicz, W., 170(12), 171 Siegbahn, N., 21,104 Siegel, M., 268, 271(18) Sigman, D. S., 170, 178(1) Silk, D. B. A., 507 Simmons, W. H., 170 Simon, H., 10, 303,304, 305(3, 5, 7, 8, 9, 10, 11, 14), 306(3, 5, 9), 308(6), 309, 310 Siqueira, R., 336, 339(17) Sizer, I. W., 483 Skold, C., 93 Skoog, F., 344 Skopan, H., 304, 308(6) Skudder, P. J., 424, 425(10), 427(10), 429(10), 430(10) Slater, T. F., 56, 61(6, 7) Slesser, K. N., 130 Sliwkowski, M. B., 423,425(7, 8), 429(8) Sliwkowskl, M. X., 423, 424, 425(8, 10), 427, 428, 429(8, 10, 15), 430(10, 15) Slokoska, L. S., 326 Slonekar, J. H., 446 Smeds, A.-L., 46 Smith, B. R., 146 Smith, R. E., 194, 196(18), 201, 206(18), 208(18), 212(25) Snell, E. E., 472 Sobue, S., 433 Socic, H., 325 Soejima, M., 163, 165, 167(6) Sonomoto, K., 294, 299, 301(11) Souzu, J., 344 Spector, L. B., 119 Speziale, V., 119 Sportoletti, G., 150, 157(2) Spruijt, R., 216, 221(2), 225(2), 226(2) Stach, W., 10 Stalling, D., 466 Stanley, P. E., 84 Stark, J. B., 456, 472 Staub, F. B., 480

Steinberg, R. A., 498 Steinmann, B., 201, 206(26), 207(26), 208(26), 212(26), 214(26) Stempel, G. H., 37 Stevenson, R. W., 405 Steytler, D. C., 212 Stich, K., 319 Stier, T. J. B., 399 Stille, J. K., 130 Stiso, S. N., 94 StOcklgt, J., 343, 349(3) Stodda, F. H., 240 Strasser, R., 212,213(52) Straub, B., 189 Strauli, U., 56, 61(7) Stringer, C. S., 240 Strominger, J. L., 273 Stuchburg, T., 107 Stuckwisch, C. G., 128 Suckling, C. J., 256 Suckling, K. E., 256 Sugimoto, Y., 34 Suguro, T., 126, 134 Sumi, A., 464, 470 Sumner, J. B., 235, 241 Sunamoto, J., 192 Susckitzky, H., 107 Sutthoff, R. F., 367 Suzuki, H., 21, 45 Suzuki, K., 432,452 Suzuki, S., 21,323 Svensson, S., 231 Swaisgood, H. E., 423, 424, 425, 426, 427, 428,429(8, 10, 15), 430 Swan, M., 56, 61(9) Swann, W., 464 Switzer, R. L., 271,447, 451(35) Szechinski, J., 170(12), 171 Szent-Gy6rgyi, A., 480 T Tabony, J., 192 Tajima, N., 298 Takagaki, Y., 266 Takahashi, T., 290 Takamatsu, S., 470, 475,476, 478 Takano, T., 524

AUTHORINDEX Takasaki, Y., 357 Takata, I., 331,381,456, 459 Takinami, K., 406 Tamada, S., 134 Tamamushi, B., 189, 190(8) Tanabe, H., 366 Tanabe, M,, 165 Tanaka, A., 145, 146, 147, 148(20), 149(20), 293,294, 298(3), 299, 300(6, 7), 301(11), 383 Tanaka, E., 124 Tanaka, T., 406 Tani, Y., 523 Taniguchi, H., 240, 241(7) Thanos, J., 10, 303,304, 305(3, 5, 8), 306(3, 5) Theorell, H., 58 Therisod, M., 137 Thiesen, N. O., 359 Thomas, D., 73, 74(22) Thomas, J. K., 192 Thomas, T. H., 181 Thompson, K. N., 358 Thorpe, G. H. G., 84 Tiemeyer, W., 304, 305(11) Tischer, W., 304, 305(11) Tobita, H., 6, 7(9) Tochino, Y., 162, 168(2), 169(1, 2) Todd, J. R., 147 Tomka, I., 216 Ton, H. Y., 507 Toprakcioglu, C., 200 Tosa, T., 331, 381,455, 456, 459, 464, 468, 469, 475, 476, 478 Tramper, J., 255, 256, 257, 258, 262, 335 Treimer, J. F., 343 Troll, W., 171 Tsuchiya, H. M., 239, 240, 242 Tsunoda, T., 517 Tsuzuki, H., 162, 168(2), 169(1, 2)

U Uchida, K., 130 Uchida, M., 126 Uchida, T., 381 Uchida, Y., 124 Ullman, E. F., 93, 94

553

Umemura, I., 475 Updike, M. H., 497, 498(2), 500(2) Urab¢, I., 5, 9, 21, 22, 34, 35, 45, 56, 490, 493 Usanov, S. A., 208, 210(44) Ushiro, S., 395 Usui, H., 408 Usui, K., 366 Utimoto, K., 130

V Vacher, M., 196, 212(19), 213(19) van Beeumen, J., 433 van Berge Henegouwen, G. P., 87(13), 89 Vandamme, E. J., 290 van der Plas, H. C., 256, 257, 258 Van der Westen, H. M., 229 Vann, W. P., 291,414 van Tilburg, R., 356, 361 van Velzen, A. G., 361 Van Weeman, B. K., 93 Veeger, C., 208, 211(48, 49), 212(48, 49), 216, 221(1, 2), 225(2), 226(2), 228(1), 229(1), 229 Veelken, M., 323 Veide, A., 46 Vellom, D., 92 Verhegge, G., 433 Vidaluc, J. L., 119 Villee, C. A., 56, 61(5) Vincent, C., 22 von Dreissig, W., 433 yon Rienacker, R., 124 Vosbeck, K. D., 170 Vowinkel, E., 168

W Wada, A., 508 Wada, M., 381,395, 493 Wagner, F., 313 Wahlefeld, A. W., 44, 465 Waks, M., 137, 196, 212(19), 213(19) Wallenfells, K., 230, 284 Walseth, T. F., 240, 241(6) Walsh, K. A., 159

554

AUTHOR INDEX

Walter, R., 170 Wan, J., 395 Wandrey, C., 5, 7, 9, 10, 11, 13(6), 17(6, 10), 19, 21, 45,490, 493 Wang, D. I. C., 335, 340(12) Wang, S. S., 9 Warthon, C. W., 181 Watanabe, I., 524 Watanabe, N., 189, 190(8) Watson, J. D., 281 Wax, M., 276 Weatherburn, M. W., 339 Webb, E. C., 238 Weber, G., 126 Weetall, H.-H., 181,291,413,414, 486 Weidenbach, G., 367, 368 Weii, R., 230 Welch, G. R., 104 Wells, M. W., 206,207(38) Welsch, T., 182 Weng, L., 94 Werkman, C. H., 485 White, F. H., 381 Whitehead, T. P., 84 Whitesides, G. M., 10, 55, 118, 263, 266, 267, 268, 270(5), 271(18), 276, 279(9), 303 Wichmann, R., 5, 10, 11, 13(6), 17(6, 10), 19, 21, 45,490, 493 Widmer, F., 162, 163 Wiener, F. P., 436 Wienhausen, G., 86(12), 87(12), 89, 90(12), 92 Wiget, P., 193, 194(16), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16) Wilcheck, M., 64 Wilen, S. H., 118 Wilham, C. A., 239 Wilke, C., 381,382(6) Wiilemot, R. M., 240 Williams, R. F., 192, 507 Williamson, J., 465 Wilson, M. B., 96 Wilson, P. W., 485,489(12) Winer, A. D., 58 Wirz, B., 137 Wirz, J., 212,213(55) Witiak, D. T., 136 Woenckhaus, C., 7

Wfhler, F., 234 Wolf, R., 193,194(16), 198, 200(21), 201(16), 203(16), 204, 206, 207(36), 208(16), 211(16), 212(16, 36), 214(36), 215(16, 21, 36), 217, 220(6), 221(6), 222(6) Wong, C.-H., 118, 136(7), 263, 267(9), 279(9), 303 Wong, J. W., 330, 341(4) Wong, M., 192 Wood, D. N., 335 Wood, L. L., 497,498(2), 500(1) Woodley, C. L., 56, 61(2) Worden, R. M., 395 Wykes, J. R., 64, 75

Y Yabuuchi, E., 532 Yagi, H., 124 Yam, C. F., 62 Yamada, H., 523 Yamada, K., 201,202(29), 204(29), 212(29), 214(29), 219, 220(9) Yamada, T., 383 Yamada, Y., 22, 34, 56, 341,342(24) Yamaga, M., 130 Yamaguchi, Y., 293,298 Yamamoto, H., 413 Yamamoto, K,, 455,475 Yamamoto, L. A., 336 Yamamoto, S., 192 Yamanaka, S., 406 Yamashita, K., 293,455,464, 470, 477 Yamazaki, T., 366 Yamazaki, Y., 7, 21, 22, 25, 26(16), 45 Yang, J. T., 204 Yaron, ,~., 170, 171, 173, 174 Yasuda, T., 523 Yasuhara, S., 294, 383 Yaverbaum, S., 414 Yoshida, T., 299 Yoshii, H., 383 Yoshimoto, T., 170 Yoshimura, J., 440 Yoshino, H., 272, 519 Yoshino, M., 395 Young, E. G., 47 Yu, Y. T., 68, 69, 77, 78(14)

AUTHOR INDEX

Z Zaks, A., 137 Zappelli, P., 5 Zenk, M. H., 343,344(8), 349 Zerner, B., 238

Zervas, L., 513 Ziehr, H., 498 Zittan, L. E., 363 Z611ner, R., 284, 285,290(8), 292 Zulauf, M., 189, 192(7)

555

SUBJECT INDEX

557

Subject Index

A ABTS. See 2,2'-Azinodi-(3-ethylbenzothiazoline-6-sulfonic acid) Acetate kinase assay, 271 partition, in aqueous two-phase systems containing dextran T500, PEG plus PEG sulfate and substrate, 52 specific activity, 264 stability in immobilized form, 264 Acetic acid sanitizers, 418 N-Acetylgalactosamine, 230-231 Acetyl phosphate properties, 265 synthesis, 263, 267-268 Achromobacter protease I covalent coupling to poly(L-glutamic acid), 165-166 covalent coupling to silica gel containing immobilized poly(L-glutamic acid), 166-167 in enzymatic conversion of porcine insulin to human insulin, 162163 in enzymatic semisynthesis of human insulin, 163-170 immobilization, 165-167 immobilized, assay, 163-164 Acid mine drainage. See also Matsuo Mine ferrous and ferric iron in, conversion to marketable commodities, 540 treatment, 531 bacterial oxidation in, 530-531 with immobilized T. ferrooxidans, 530-540 Ac-Phe-Ala-NH2, chymotrypsin-catalyzed synthesis, 182-183 Acrylamide

measurement, 525 production, 523 using immobilized nitrilase-containing microbial cells, 523-530 use, 523 Acryionitrile, measurement, 525 N-Acryloxysuccinimide, 274 N*-AcryloyI-L-lysinemethyl ester, 37-38 Actinoplanes missouriensis, 361 ADP, bioluminescent assay using coimmobilized enzymes, 87 Aerosol OT, 216 HPLC, 190-191 purification, 189-192 sources, 189 Aerosol OT/water/isooctane system phase diagram, 189 use for enzymes, 189 Agroclavine production, 320 retention time on HPLC, 320 D-Alanine, estimation of, 473 L-Alanine bioluminescent assay using coimmobilized enzymes, 86 continuous production using two immobilized microbial cells, 472-479 crystallization from reactor column effluent, 479 estimation of, 472 industrial production, 478-479 production from ammonium fumarate, elimination of side reactions, 475-476 bioreactors for, 477-478 in closed column reactor, 477 sequential reactions in, 477-478 Alanine aminotransferase. See Glutamicpyruvic aminotransferase

558

SUBJECT INDEX

Alanine dehydrogenase, kinetic parameters for native NADH and PEG-NADH, 12 Alanine racemase, assay, in free and immobilized microbial cells, 474 Albumin, partition, in aqueous two-phase systems containing dextran T500 and PEG plus PEG sulfate, 51 Alcohol. See also Ethanol continuous fermentation conventional batch, 393 by immobilized yeast cells, features, 393 Melle Boinet batch, 393 process flow diagram, 390 fermentation, basic technology, 385386 production, for power, 380 Alcohol:NAD÷ oxidoreductase. See Alcohol dehydrogenase Alcohol dehydrogenase. See also Site-tosite enzyme systems assay, 108-109 coimmobilization with coenzyme materials, 57-58 methods, 58-59 coimmobilization with NAD, 57 activity, 63, 66 analysis of data, 62-63 dimensionless parameters 6 and p definition, 63 at different ethanol concentrations and flow rates, 65-66 double-logarithmic plots of product concentrations at exit against flow rates with different concentrations of substrate ethanol, 64-65 efficiency, 63, 66 kinetic studies, 61-63 results, 63-66 coimmobilized with coenzyme, 56-67 coimmobilized with NAD, 56 free and immobilized, Michaelis constants for, 153 horse liver coimmobilization with NAD(H) analog, 57 to Sepharose 4B, 58-59 in coupled two-enzyme reactor, steady-state analysis, 42-45

immobilized site-to-site enzyme system with lactate dehydrogenase, 104-106 kinetic constants, 43 in reverse miceUes, 208-212 soluble site-to-site enzyme system with lactate dehydrogenase, 106108 source, 26 immobilization, 56-57 on CNBr-activated Sepharose 4B, 152 immobilized, recovery of activity, 153 and NAD analog, attachment procedure inside nylon tube, 59-61 PEG-NADH as coenzyme, 13 in reverse miceUes, kinetic parameters, 214 reversibly immobilized, preparation, 106 sources, 57-58 yeast covalent attachment, with NAD, to interior of partially hydrolyzed nylon tube, 57-67 and malate dehydrogenase, microencapsulation with soluble dextranNAD, 57 microencapsulated with malate dehydrogenase, recycling of NAD + and NADH, 70-71 source, 151 specific activity, 151 stability in microcapsules, 77 Alcohol dehydrogenase-NAD(H)Sepharose complex, 57 Alginate fibers, cell immobilization on, 453 Alginate-immobilized microbial cell pellets, mechanical strength, 442-443 Alkaline phosphatase, in reverse micelles, 207 Alkaloids. See also Strictosidine analytical methods for determination of, 319-320 production by C. purpurea, improved capacity, with application of immobilization technique, 320-329 retention on HPLC, 320 Alkanal monooxygenase. See Bacterial luciferase Amidase, assay, 524-525 Amino-acid dehydrogenase + formate

SUBJECT INDEX dehydrogenase, immobilized, application, 354 L-Amino acids, production using aspartic aminotransferase from E. coli, 484 Aminoacylase immobilized, application, 354 reaction, bioreactor for, based on aqueous two-phase systems, 55 N~-[(6-Aminohexyl)carbamoylmethyl]NADH, coimmobilization with alcohol dehydrogenase, to Sepharose 4B, 5759 N-(3-Amino-2-hydroxypropyl)-2-(tritylamino)acetamide, 24 Aminopeptidase, immobilized application, 354 sequential hydrolysis of peptides with, procedure, 174-177 Aminopeptidase P application to sequential hydrolysis of proline-containing polypeptides, 170-178 assay, 171 colorimetric assay, 174 definition of unit, 174 fluorimetric assays, 174 glass-bound absence of endopeptidase activity, 173 adsorption, 172-173 enzyme activity measurement, 174 metal ion requirement, 173 pH dependence, 173 stability, 172 temperature dependence, 173 immobilization, 172 soluble, enzyme activity measurement, 173 Aminotransferase in biocatalysts advantages, 481 disadvantages, 481-482 discovery, 480 immobilization, 485-492 immobilized application, 354 laboratory-scale processes using, 481 long-term stability, measurement, 491492

559

Ping-Pong kinetics, 480 pyridoxal 5'-phosphate cofactor, 480 attached to soluble polyethylene glycol derivative, 496 macromolecularized synthesis of, 494-495 use in continuous flow membrane reactor, 496-497 retention in membrane reactor, 492497 reaction catalyzed, 479-480 driving to completion, 482-483 half-reactions, 480 sources, 483-485 substrate specificity, 482 Amoxycillin, synthesis, in hydrolasecatalyzed condensation reaction, 282 Ampicillin, synthesis, in hydrolase-catalyzed condensation reaction, 282 a-Amylase-treated starch, treatment with glucoamylase, 376 Amyloglucosidase, immobilized, applications, 354 Androsterone, bioluminescent assay using coimmobilized enzymes, 87 Angiotensin II, sequential hydrolysis with immobilized aminopeptidase, 175-176 Antibiotic fermentations, longevity, 330331 increasing, 342 Apomyoglobin, 177 Aromatic-amino-acid aminotransferase, 484 Artificial cells recyling of NAD(P) by multienzyme systems microencapsulated within, 67-82 retention of NAD + within, 81-82 Aspartame, 432 enzymatic production, 503-504, 516 raw materials, 504-505 selection of enzymes, 504 selection of organic cosolvent, 506507 selection of protecting group, 504 and isomaltulose, combination, 433 preparation, by conventional organic synthesis, 503 production by immobilized thermoase, 503-516 batchwise operation, 514-516

560

SUBJECT INDEX

in continuous column operation, 513514 operation, 513-516 substrate preparation, 513 sweetness, 503 synthesis, 234 in hydrolase-catalyzed condensation reaction, 282 Aspartase assay in free and immobilized microbial cells, 473 in immobilized E. coli, 465 immobilized, application, 355 Aspartase + L-aspartic acid, immobilized, application, 355 Aspartate aminotransferase. See Glutamate pyruvate transaminase L-Aspartate ammonia-lyase. See Aspartase L-Aspartate/3-decarboxylase activity of P. dacunhae, stabilization by glutaraldehyde treatment, 476-477 assay, in free and immobilized microbial cells, 473 L-Aspartic acid estimation of, 465-466, 472 industrial production, using polyurethane-immobilized cells containing aspartase, 463-471 production, by immobilized E. coli cells in batch reactor, 466-467 Aspartic aminotransferase, E. coli, relative rates of production of L-amino acids, 484 Aspergillus niger, lipase, 299 ATP analogs, synthesis, 5 with carboxymethyl dextran carrier, partition coefficient in aqueous twophase system, 55 dextran-bound partition in aqueous two-phase systems containing dextran T500, PEG plus PEG sulfate, and substrate, 52 partition in aqueous two-phase systems containing dextran T500 and PEG plus PEG sulfate, 51 preparation, 47-49 enzymatic regeneration

with acetyl phosphate/acetate kinase, 263-264, 265 from ADP and AMP, 263-280 application to enzyme-catalyzed syntheses, 273-280 enzymatic assays, 270-273 with methoxycarbonyl phosphate/ acetate kinase, 263-264 with phosphoenolpyruvate/pyruvate kinase, 263-264, 265 phosphorylating agents used in, 263 properties, 265 phosphoryl group donors, synthesis, 267-270 procedures, 263-264 synthesis of phosphoryl donors, 265266 in enzyme reactor studies, 9 immobilized, as active coenzyme, 3 modification in N-6 position, substituents used for, 4 partition, in system containing dextran T500, PEG, and PEG sulfate, 55 Azaheterocyclic chemistry, 256 2,2'-Azinodi-(3-ethylbenzothiazoline.6. sulfonic acid), 94-95 Azotobacter vinelandii, oxaloacetate decarboxylase, 485

B Bacillus subtilis, menthyl ester hydrolysis, in two-liquid phase biocatalytic reaction, 139-140, 145 Backward transfer, in solubilization of enzymes in reverse micellar media, 193 Bacterial cells, solubilized in hydrocarbon miceUar solutions, 215 Bacterial luciferase activity recovered on Sepharose, 89 analytical usefulness, in bioluminescent assays, 83 assay, 85 reaction catalyzed, 83 source, 83-84 Baker's yeast, production, 422 Benzylpenicillin, penicillin amidase-catalyzed synthesis, 287

SUBJECT INDEX fl-lactam antibiotics, enzyme-catalyzed semisynthesis, 281-292 Bifidobacteria, utilization of isomaltulose, 433 Biocatalysts, in organic solvents, 137 uses, 137 Bioluminescent assay, using coimmobilized enzymes, 82-93 advantages, 83 automation, 91-93 enzyme sources, 83-84 immobilization procedure, 85-88 instrumentation, 84 kinetics, 90-92 metabolites assayed, 86-90 stability of immobilized enzymes, 88 storage of immobilized enzymes, 88 Biphasic aqueous-organic mixtures, enzymatic esterifications in, 118-119 Bis(2-ethylhexyl)sodium sulfosuccinate. See Aerosol OT bis-NAD, as directing aid for orientation of site-to-site enzyme systems, 103-104 DL-Borneol, optical resolution, 302 Bovine serum albumin, in reverse micellar media, 213 Bradykinin, sequential hydrolysis with immobilized aminopeptidase, 176-177 Breoibacteriurn immobilization using K-carrageenan and polyethyleneimine, 460-461 using K-carrageenan and tannin, 461 using K-carrageenan modified with amines, 461-462 nitrilase activity, 523 Brevibacterium ammoniagenes culture of, 457 fumarase activity, 456 immobilization with polyacrylamide, 457 immobilized cells continuous enzymatic production of Lmalic acid, 458 productivity of L-malic acid, 460 Brevibacterium flavum culture of, 459 immobilization, with K-carrageenan, 459 immobilized cells continuous enzymatic production of Lmalic acid, 459-460

561

productivity of L-malic acid, 460 immobilized with r-carrageenan, improvements, 461-463 immobilized with modified K-carrageenan, 461-462 1,2-Butanediol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-134 2-Butanol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-132 Bz-Arg-VaI-NH2, trypsin-catalyzed synthesis, 287 Bz-Gly-Lys-Leu-OMe, trypsin-catalyzed formation, 185-186

C Cacao butterlike fat, production, 411 by interesterification with carrier-adsorbed lipase, 406 Calcium alginate, as yeast entrapping agent, 396 Candida boidinii, 10 Candida cylindrica, lipase. See Lipase, C. cylindrica Carbohydrates, biologically active, 230 N6-(2-Carboxyethyl)-NAD, 35-37 Carboxylesterase pig liver preparation, 122 preparative resolution of racemic primary alcohols catalyzed by, 121-126 source, 121 stereoselective transesterification in biphasic systems, for preparation of racemic alcohols and racemic esters, 120-137 transesterification reaction, 119 Carboxypeptidase Y assay, 159-160 in conversion of porcine insulin to human insulin, 163 deblocking in peptide synthesis with, 157-162 procedures, 159-160 deblocking of peptide esters, 160

562

SUBJECT I N D E X

deblocking of peptides attached to PEG handle, 161 immobilization, methods, 160 immobilized, uses, 162 preparation, 159 properties, 158 K-Carrageenan beads casting, 333 production, 335 commercial preparations, variations in, 334 diffusivity of nutrients and secondary metabolites into and out of, 334-335 immobilization of microbial cells with, 474-475 immobilization of PeniciUium with, 332334 improved immobilization of microbial cells, 460-462 modification with amines, 461-462 as yeast entrapping agent, 396 Catharanthus roseus, strictosidine synthase, 343 Celite beads, entrapment of Penicillium on, 335 Cellulose nitrate membrane microcapsules containing dextran-NAD and multienzyme systems, preparation, 75 preparation, 68-69 Cephalexin, synthesis, in hydrolase-catalyzed condensation reaction, 282 Cetyltrimethylammonium bromide reverse micellar media, 205 preparation, 216-217 Chanoclavine production, 320 retention time on HPLC, 320 Chiral chemicals, synthesis, 117-118 dehydrogenases in, 9-10 Chiral compounds, preparation, 302-317 1-Chloro-2-propanol alkaline hydrolysis of esters, 128 epoxidation, 129 Cholic acid methyl ester, oxidation to 12ketochenodeoxyxholic acid methyl ester, in two-phase system, 156 Chymosin, milk clotting with, 424 Chymotrypsin immobilization, to Enzacryl AH, 181

immobilized, in peptide synthesis, 182184 a-Chymotrypsin immobilized, assay, 181 in reverse micelles, 201-205 CD spectra, 204-205 kinetic parameters, 214 superactivity, 216 Citronellol hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 propionic esters, alkaline hydrolysis, 122 Clauiceps, saprophytic culturing, 317 Claviceps purpurea alkaloid production effect of nitrogen concentration, 325326 in various alginate concentrations, 320-322 alkaloid production capacity, improvement, 320-329 alkaloid yields, increasing, by increasing particle amount in fermentation broth, 327-328 biomass development, in various alginate concentrations, 320-322 culture, 318 immobilization, methods, 319 immobilization technique, simulation of natural alkaloid production conditions, 329 immobilized ceils, 317-329 alkaloid production in l-liter air-lift fermenter, 327 morphological aspects, 320 preparation, 318 immobilized mycelia morphology, 324-325 semicontinuous fermentation, 323 mycelium wet weight in calcium alginate beads, determination, 319 Claoiceps sclerotia, 317 Clostridial aminopeptidase assay, 171 colorimetric assay, 174 definition of unit, 174 fluorimetric assays, 174 glass-bound absence of endopeptidase activity, 173

SUBJECT INDEX adsorption, 172-173 application to sequential hydrolysis of proline-containingpolypeptides, 170-178 enzyme activity measurement, 174 metal ion requirement, 173 pH dependence, 173 stability, 172 temperature dependence, 713 immobilization, 172 immobilized, properties, 172-173 isolation, 171-172 soluble, enzyme activity measurement, 173 Clostridium histolyticum, aminopeptidase. See Clostridial aminopeptidase CM-PEG, preparation, 160-161 CoA, immobilized, as active coenzyme, 3 Coenzyme modified, synthesis, 12 regeneration, 7 simultaneous regeneration, 10 Coenzyme-dependent enzymes, immobilized, activity, 3 Coimmobilization, 56 of NAD with dehydrogenase, 21-34 activity of immobilized dehydrogenases, assay, 28 applications, 29-33 advantages, 33 continuous production of L-malate with, 29-31 disadvantages, 33 enzyme leakage from gel, 33-34 ethanol analyzer using, 31-33 FDH-MDH-NAD gel, 27 immobilized proteins, determination, 28 LADH-diaphorase-NAD gel, 27 methods, 21 NAD content, determination, 28 preparation of gel, 26-27 spatial arrangement in, 56 Condensation products enzyme-catalyzed synthesis, equilibriumcontrolled, vs. kinetically controlled, 280-292 kinetically controlled synthesis with enzymes

563

compared to equilibrium-controlled synthesis, 285-292 effect of enzyme concentration, 286287 effect of enzyme properties, 287-290 effect of substrate properties, 291-292 effect of water content, 290-291 factors affecting yield, 286 kinetically controlled maximum, 286287 procedure, 285-286 substrates, 285 Condensation reactions, hydrolase-catalyzed, mechanisms, 281-284 Cortisone, stereospecific reduction, in twophase systems, 154-155 Cortisone reductase. See 20/3-Hydroxysteroid dehydrogenase Corvire, 422-423 Corynebacterium glutamicum, phenylalanine production using, 498 Creatine kinase, activity recovered on Sepharose, 89 Creatine phosphate, bioluminescent assay using coimmobilized enzymes, 87 Cyclodextrin, 432 synthesis, in hydrolase-catalyzed condensation reaction, 283 1-Cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, 58-59 Cytochrome-cs hydrogenase, in carriermediated multienzymatic system in reverse micellar medium, 228-229 Cytochrome-c hydrogenase, in reverse micelles, 211 Cytochrome P-450, in reverse micelles, 210 Cytochrome P-450, -b5 reductase, in reverse micelles, 210

D Deblocking reactions, 157-158 in peptide synthesis, with carboxypeptidase Y, 157-162 Dehydrogenase, 56 coimmobilization with NAD. See Coimmobilization, of NAD with dehydrogenases

564

SUBJECT I N D E X

for native NADH and PEG-NADH, 1113 Dextran branching, 239 clinical, synthesis, 254 concentration, determination, 242 in dental caries, 239 high molecular weight, synthesis, 251252, 254 intermediate molecular weight, synthesis, 254 low molecular weight, synthesis, 254 molecular weight, control of, during synthesis, 240, 251,254 produced by immobilized dextransucrase, characterization, 251-254 uses, 239 Dextran-NAD, recycling within cellulose nitrate membrane microcapsules, 76-77 within microcapsules, measurement, 7576 within polyamide membrane microcapsules, 77 Dextran-NADH preparation, 75 retained within microcapsules, recycling, 74-77 Dextransucrase action mechanism, 240 assay, 241 free and immobilized behavioral differences between, 249253 initial reaction rate, effect of initial sucrose concentration, 249- 251 immobilization, 241 effect of maltose addition on, 248-249 effect of support specific area on, 247-248 efficiency, effect of amount of enzyme on, 248 procedure, 245-247 immobilized assay, 247 dextran produced by, characterization, 251-254 kinetic characterization, 249-251 kinetics of fructose production catalyzed by, 249-250

from L. mesenteroides dextran synthesis, 239-240 immobilization, 241 inductor, 239 low-branched dextran polymer produced by, 239 production, by batch-fed L. mesenteroides culture, 242-243 protein determination, 241 purification, 240, 242-245 by ultrafiltration and gel chromatography, 242-245 reaction catalyzed, 239 Dextran synthesis by immobilized L. mesenteroides dextransucrase, 239-254 in vitro, 239-240 in presence of acceptors, 251-254 a,to-Diaminopoly(ethylene glycol), 39-40 Diaminopolyethylene glycol, synthesis, 493-496 Diaphorase assay, 85 pig heart, source, 26 source, 83-84 a,to-Dichloropoly(ethylene glycol), 39 2,3-Dichloropropanol alkaline hydrolysis of esters, 128 epoxidation, 129 Diethylenetriamines, substituted, as enzyme reactor disinfectants, 418-419 Dihydrolipoamide dehydrogenase. See also Diaphorase in carrier-mediated multienzymatic system in reverse micellar medium, 228-229 Dihydroxyacetone phosphate enzyme-catalyzed synthesis, 263,277 synthesis, 266 3,7-Dimethyl-l-octanol, propionic esters, alkaline hydrolysis, 122 3,7-Dimethyl-l-octyl propionate, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 Dipeptidyl aminopeptidase I, in sequencing of proline-containing peptides, 178 Dipeptidyl aminopeptidase IV, in sequencing of proline-containing peptides, 178

SUBJECT INDEX Dipeptidyl peptidase I. See Dipeptidyl aminopeptidase I Dipeptidyl peptidase IV. See Dipeptidyl aminopeptidase IV Disaccharide synthesis in hydrolase-catalyzed condensation reaction, 282 with immobilized fl-galactosidase, 230233 Disinfectants, for enzyme reactor, 418-419 Double transfer, in solubilization of enzymes in reverse micellar media, 193

E Electrochemical cell, design, 308-309 Eiectroenzymatic reduction reaction sequence, 303 scheme, 304 Electromicrobial reduction reaction sequence, 303 scheme, 304 ELISA. See Enzyme-linked immunosorbent assay Elymoclavine production, 320 retention time on HPLC, 320 [Leu]-Enkephalin, sequential hydrolysis with immobilized aminopeptidase, 175 2-Enoate reductase assay, 312-313 reduced mediator, preparation, 312 from C. tyrobutyricum, 303 immobilization, in calcium alginate gels, 313-314 immobilized, repeated use of, 315 kinetic parameters, 304, 306-307 purification, 309-310 reaction catalyzed, 305 stereoselectivity, 304 substrate specificity, 304, 305-306 Enterobacter agglomerans. See Erwinia rhapontici Enzymatic esterifications, in biphasic systems, 117-118 Enzymatic reaction, Michaelis-Menten kinetics, 223 Enzymatic transesterifications, for preparative production of optically active compounds, 119-137

565

experimental design, 120 rationale, 119-120 Enzyme channeling immmunoassay, 94, 98-103 coated surface, 98-100 soluble reagent microtiter plate protocol, 99-102 rapid protocol, 102-103 Enzyme immunoassay, 93 homogeneous, 93-94 Enzyme-linked immunosorbent assay, 93 Enzyme reactors, 7, 414. See also Membrane reactor; Open tubular heterogeneous enzyme reactor aqueous two-phase systems, applications, 54-55 continuous reaction system reaction rates of coenzymes, 41 theoretical analysis, 40-42 countercurrent partitioning using aqueous two-phase systems, 45-46 with coupled enzyme regeneration, 7-8 coupled two-enzyme steady-state analysis, 40-45 theoretical analysis, 40-42 fixed bed, 415-417 fluidized bed, 415-417 industrial operations with, strategy, 419422 model, steady-state analysis, 42-45 plug flow, 415 retention of coenzyme in, 13-15 stirred tank, 415-417 ultrafiltration system, 45 Enzymes, in organic syntheses, application of, 254-255 Epichlorohydrin, yeast fipase-catalyzed resolution of, via transesterification, 133 Ergometrine production, 320 retention time on HPLC, 320 Erwinia rhapontica, immobilized cells, isomaltulose production, 436-437 Erwinia rhapontici cells immobilized by various methods, activity and stability, 441 immobilized cells, isomaltulose production, 439 immobilized preparations, isomaltulose-

566

SUBJECT INDEX

forming activity, operational stabilities of, 445-448 isomaltulose-synthesizing activity, storage stability under various conditions, 450 Escherichia coli

aminotransferases, 483-484 aspartase activity effect of ammonium fumarate concentration, 470-471 effect of pH, 469-470 effect of temperature, 468-469 in various preparations, 467-468 cell immobilization, 464-465 culture, 474 immobilization with K-carrageenan, 475 in polyazetidine, 500 immobilized, in L-alanine production, 472-479 immobilized cells L-alanine production, elimination of side reactions, 475-476 aspartase activity, 465 immobilized cells containing aspartase, for L-aspartic acid production, 463471 immobilized with K-carrageenan, activities with and without pH treatment, 476 phenylalanine production, free cell studies, 498-499 polyurethane-immobilized cells containing aspartase activity, properties, 467-471 transaminases, 498-499 Esterase, immobilized, application, 355 Ethanol. See also Alcohol continuous fermentation, 394 using yeast immobilized in synthetic resin, 380-394 fermentation, using immobilized yeast cells, types of vessels for, 386-389 oxidation by NAD, with production of acetaldehyde and NADH, in tubular flow reactor system, 61-63 production bioreactor for, based on aqueous twophase systems, 55 yeast systems tested for, 382

Ethanol analyzer, using coimmobilized LADH-diaphorase-NAD gel, 31-33 Ethyl 2-(tritylamino)ethanoate, 24 F

Fatty acid esters, synthesis, in hydrolasecatalyzed condensation reaction, 283 FDH. See Formate dehydrogenase Fermentation vessel, 386-389 Finnsugar/Fermco IGI product, 367-368 Firefly luciferase activity recovered on Sepharose, 89 analytical usefulness, in bioluminescent assays, 82-83 assay, 84-85 reaction catalyzed, 82 source, 83-84 FMNH2, production, 83 Folch-Pi proteolipid, in reverse micellar media, 213 Formate:NAD ÷ oxidoreductase. See Formate dehydrogenase Formate dehydrogenase from C. boidinii, 305 as NADH-regenerating enzyme, 1011 for native NADH and PEG-NADH, kinetic parameters, 12-13 source, 10, 151 specific activity, 151 yeast, source, 26 Forward transfer, in solubilization of enzymes in reverse micellar media, 194, 195 6-O-a-D-Glucopyranosyl-D-fructofuranose. See Isomaltulose Fructose corn syrup, 55%, 364-366 Fumarase activity assays, 456-457 assay, in free and immobilized microbial cells, 473-474 immobilized application, 355 L-malic acid production, 455 Fumarate hydrase. See Fumarase Fumaric acid, estimation of, 465-466, 472 Fungi, immobilized cells, in fundamental studies, 340-341

SUBJECT INDEX

G fl-Galactosidase assay, 232 immobilization to Sepharose 4B, 231232 immobilized, disaccharide synthesis with, 230-233 transgalactosylation reaction, 230-23 l Galactosyl-N-acetylgalactosamine formation, 230-231 preparation via reversal of hydrolysis reaction, 231,233 via transferase reaction, 232-233 structure, 233 yield, 233 Glucagon, in reverse micelles, 211 Glucoamylase hydrolysis, 376 immobilized, saccharification, flow rate for, 377-378 liquid, saccharification, 374 liquid hydrolysis, costs, 378 6-P-Gluconate, bioluminescent assay using coimmobilized enzymes, 86 D-Glucose, bioluminescent assay using coimmobilized enzymes, 86 time course of light output, 90, 92 Glucose-6-phosphate dehydrogenase, microencapsulated, for multistep enzyme reaction of conversion of urea into glutamic acid, 72-73 Glucose dehydrogenase from B. megaterium, 13 immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 Glucose/galactose syrups, production, 376 Glucose isomerase batch isomerization, cycle duration, 360 cross-linked, for batch isomerization, 359-360 immobilization, methods, 356 industrial, first, 357-358 purified reusable, 358-359 Glucose isomerase, immobilized, 356-370 applications, 354

567

comparison of batch and fixed bed processes, 362-363 for fixed bed isomerization, 370 for fixed bed operation, 361-362 industrial-scale production-application, 353 development of products and processes, 356-364 effect of glucose substrate purity, 363 first, 357-358 first continuous isomerization process, 358 future developments, 368-369 impurities, 363-364 industrial development, 358-363 initial phase, 357-358 isomerization process, bottleneck model, 364-365 new products, 366-367 new syrup refining processes, 368 process optimization, 363-365 products, 364 second-generation isomerization processes and products, 364-368 products, 356-357 Glucose oxidase and anti-PRP, microtiter plates coated with, 98 derivatization with amino groups, 97-98 and horseradish peroxidase, reaction catalyzed together, 94 Glucose 6-phosphate enzyme-catalyzed synthesis, 263,279 repeated production, in aqueous twophase system, 52-54 L-Glutamate, bioluminescent assay using coimmobilized enzymes, 86 L-Glutamate:NAD(P)÷ oxidoreductase. See Glutamate dehydrogenase Glutamate dehydrogenase coimmobilization with 12a-hydroxysteroid dehydrogenase, on Sepharose CL-4B activated with tresyl chloride, 152-153 free and immobilized, Michaelis constants for, 153 immobilized, recovery of activity, 153 immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of

568

SUBJECT INDEX

urea to ammonia to glutamic acid to alanine, 71-72 microencapsulated, for multistep enzyme reaction of conversion of urea into glutamic acid, 72-73 PEG-NADH as coenzyme, 13 source, 151 specific activity, 151 Glutamate pyruvate transaminase, immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 Glutamic-oxaloacetic aminotransferase E. coli, 483-484 assays, 486-487 immobilization for L-phenylalanine production, 486 mixed with immobilized oxaloacetate decarboxylase from P. putida, 492 immobilization, on porous diatomaceous earth, 491 pig heart, 483 assay, 488 immobilization, 488 Glutamic-pyruvic aminotransferase immobilization, on porous diatomaceous earth, 490-491 immobilized, long-term operational stability, 492 pig heart assay, 488-489 immobilization, 488 porcine, 483 Glutamic-pyruvic transaminase, 480 Glycerides, 1,3-substituted, synthesis in hydrolase-catalyzed condensation reaction, 283 sn-Glycerol 3-phosphate enzyme-catalyzed synthesis, 263, 276277 synthesis, 266 Godo-AGI, 370 Gramicidin S synthetases, 330

H Haemophilus influenzae, capsular antigen, 95

Hexokinase and acetate kinase, enzyme reactor based on aqueous two-phase system, 46 activity recovered on Sepharose, 89 partition in aqueous two-phase systems conraining dextran T500, PEG plus PEG sulfate, and substrate, 52 in aqueous two-phase systems conmining dextran T500 and PEG plus PEG sulfate, 51 High dextrose equivalent syrup, 412 High-fructose corn syrup, 356, 432 High-fructose syrup, 376 Horseradish peroxidase chromagen substrates, 94 and glucose oxidase, reaction catalyzed together, 94 Hydantoinase, immobilized, application, 354, 355 Hydrogenase, in reverse micelles, 211-212 Hydrolase, products synthesized using, 280, 283-283 Hydro-lyase, transesterification reaction, 119 Hydrolytic reactions, reversal, 234, 503 in disaccharide synthesis, 233 urease as model for investigation of, 238 N-[2-Hydroxy-3-[2-(tritylamino)acetamido]propyl] methacrylamide, 25 (2R)-Hydroxycarboxylate-viologen oxidoreductase, 303-304 assay, 313 reduced mediator, preparation, 312 enrichment procedure, for P. vulgaris, 311 kinetic parameters, 304, 307-308 partially purified, use of, 315-316 purification, 311-312 reaction catalyzed, 306 reduction of 2-oxocarboxylate, 316 reduction of enoate, 315-316 stereoselectivity, 304 substrate specificity, 304, 306-307 in whole cells, 315-316 D-Hydroxyisocaproate dehydrogenase, kinetic parameters for native NADH and PEG-NADH, 12 L-Hydroxyisocaproate dehydrogenase,

SUBJECT INDEX

kinetic parameters for native NADH and PEG-NADH, 12 [N-(2-Hydroxy-3-methacrylamidopropyl)carbamoyl]methylammonium ptoluene sulfonate, 25 Nr- [N-[N-(2-Hydroxy-3-methacrylamido propyl)carbamoylmethyl]carbamoylmethyl]-NAD, 25-26 fl-Hydroxysteroid:NAD(P) ÷ oxidoreductase. See 3 (or 17)/3-Hydroxysteroid dehydrogenase 3a-Hydroxysteroid dehydrogenase microencapsulated, with L. mesenteroides with NADH oxidase, in stereospecific steroid oxidation, 7374 steroid transformation with, 156-157 7a-Hydroxysteroid dehydrogenase, activity recovered on Sepharose, 89 12ct-Hydroxysteroid dehydrogenase coimmobilization with glutamate dehydrogenase, on Sepharose CL-4B activated with tresyl chloride, 152153 free and immobilized, Michaelis constants for, 153 immobilized, recovery of activity, 153 source, 151 specific activity, 151 steroid transformation with, 156-157 20/3-Hydroxysteroid dehydrogenase on aqueous and reverse micellar media, Michaelis-Menten parameters for, 223-224 in carrier-mediated multienzymatic system in reverse micellar medium, 228-229 encapsulation in reverse micelles, method, 221 free and immobilized, Michaelis constants for, 153 immobilization on CNBr-activated Sepharose 4B, 152 immobilized, recovery of activity, 153 reverse micellar enzymology of, 216 in reverse micelles activity, determination, 221-222 parameters regulating activity, 226 source, 151 specific activity, 151

569

steroid transformation with, 156-157 3 (or 17)/3-Hydroxysteroid dehydrogenase source, 151 specific activity, 151 steroid transformation with, 156-157 3a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87 7a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87 12a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87

I Immobilized active coenzymes, 3-9 applications, 7-9 preparation, 3-6 degree of substitution of polymers, 56 preassembly approach, 3-6 solid-phase modular approach, 3-5 special requirements, 5 water-soluble polymers in, 5-6 properties, 6-7 Immobilized cell systems, industrial applications, 353 Immobilized enzyme hydrolysis, 377-379 flow rate, 377 productivity, 377-378 running expenses, 377-378 Immobilized enzymes activity/productivity, 372-373 criteria for use as catalyst in organic chemistry, 255-256 industrial applications, 255, 353 industrial operation, 371-379 manufacturing cost, 371-372 minimum commercial viability, calculated criteria, 373 for organic syntheses, 117 Immobilized microbial ceils applications, 354 r-carrageenan immobilization, 455-456, 474-475 improvement, 456 continuous production of L-alanine, 472479 isomaltulose production using, 432-454 L-malic acid production, 455-463

570

SUBJECT INDEX

polyazetidine immobilization, 497 reactor configuration, for isomaltulose production, 443-444 Indole alkaloids, 342-343 Insulin human preparation using immobilized Achromobacter protease I, 168-170 synthesis in hydrolase-catalyzed condensation reaction, 282 tert-butyl ester, trypsin-catalyzed formation from DAI and threonine tert-butyl ester, 186 porcine enzymatic conversion to human insulin, 162 hydrolysis using immobilized Achromobacter protease I, 167-168 Interesterification lipase-catalyzed, set-up for, 410-411 TES buffer used as activator, 409-410 of triglyceride, 405 using olive oil and stearic acid, 407-409 Invertase hydrolysis, 376 immobilized, applications, 354 liquid hydrolysis, costs, 378 Iron-cytochrome-c reductase, in reverse micelles, 210, 211 Isomaltose, synthesis, in hydrolase-catalyzed condensation reaction, 282 Isomaltulose as calorific bulking agent, 432 calorific value, 432 crystallization, 448-449 in food, drink, and medicine formulations, 433 noncariogenic nature, 433 physical properties, 433 production by fermentation, 433-435 by immobilized microbial cells, 432454 alginate-immobilized cell pellets used in, mechanical strength of, 442-443 ceil-free isomaltulose-forming enzyme in, 439-440 cell immobilization, 437-439 choice of immobilization method, 441-442

choice of microorganism, 435-437 desirable improvements, 454 immobilization methods, 451-453 large-scale method, 454 pilot plant, 454 potential advantages of, 435 productivity, 453-454 reactor configuration, 443-444 regeneration of immobilized cell activity, 447-448 stabilization, 445-448 storage stability of immobilized cells, 449-451 by solid-state fermentation of sugar cane, 435 properties, 432-433 resistance to acidic hydrolysis, 433 sweetness, 432 Isomaltulose synthease characterization, 439-441 from E. rhapontici, 440-441 from P. rubrum, 440-441 Isopropanol dehydrogenase, PEG-NADH as coenzyme, 13 Isosyrup, 356 with 55% fructose, 369 global production, 364 Isosyrup industry, development of, 356369

K Ketomax GI-100, 366-367, 370 20-Ketosteroids, reduction to 20/3-hydroxysteroids, in two-phase systems, 154155 Kinase, enzyme reactor based on aqueous two-phase system, 46 design, 46-47 determination of partition coefficients, 50-52 optimal partition, investigation of, 50-52 repeated production of glucose 6-phosphate, 52-54 Kobayashi-Laidler theory, 62

L Laccase, steroid transformation with, 156157 Lactase

SUBJECT INDEX A. niger, properties, 413-414 A. oryzae

pH optimum, 414 properties, 413-414 acid fungal, 413 deactivation during hydrolysis of whey, factors controlling, 419-420 hydrolysis, 376 immobilized applications, 354 engineering considerations for processes using, 414-417 hydrolysis of whey, and cleaningsanitation cycles, 420 industrial developments with, 422423 kinetic behavior, 413 lactose hydrolysis, 412-423 microbial contamination of reactor, 417-419 operational life, 414 production-application, 411-423 properties, 413-414 reactor design, 415-416 reactor using, activity, 419 reactor using, stability, 419 sanitation, 418-419 semiindustrial operations with, 421423 stability, effect of cleaning-sanitation cycles in reactor, 420-421 temperature of operations with, and operational life of enzyme, 421422 immobilized composites, 412 preparation, 413 liquid hydrolysis, costs, 378 thermal deactivation, 419 L-Lactate bioluminescent assay using coimmobilized enzymes, 86 production, in coupled two-enzyme reactor, steady-state analysis, 4245 L-Lactate:NAD + oxidoreductase. See LLactate dehydrogenase Lactate dehydrogenase. See also Site-tosite enzyme systems activity recovered on Sepharose, 89 assay, 108 beef heart

571

immobilized site-to-site enzyme system with alcohol dehydrogenase, 104-106 soluble site-to-site enzyme system with alcohol dehydrogenase, 106108 Lactate dehydrogenase kinetic parameters for native NADH and PEG-NADH, 12 rabbit muscle in coupled two-enzyme reactor, steady-state analysis, 42-45 kinetic constants, 43 source, 151 specific activity, 151 Lactobacillus brevis, enzymatic batch process for L-malic acid production using, 455 Lactose hydrolysis reaction, 412 hydrolyzed, 411-412 applications, 412 Lactose-containing feedstocks, 415-416 LADH. See Alcohol dehydrogenase, horse liver L-Leucine, production, in enzyme-membrane reactor, 18-20 Leucine dehydrogenase, kinetic parameters for native NADH and PEGNADH, 12 Leuconostoc mesenteroides dextransucrase. See Dextransucrase

immobilization, 452 as source of NADH oxidase, microencapsulated with 3a-hydroxysteroid dehydrogenase, for stereospecific steroid oxidation, 73-74 Leucrose, 240 Lima bean trypsin inhibitor, 177 Lipase from A. niger, 299 adsorbed to Celite coated with buffer solution, preparation, 408-409 adsorbed to Celite coated with glycerol, preparation, 406-407 from C. cylindracea, 299 Celite-adsorbed, interesterification reaction, 409-410 preparative resolution of racemic esters catalyzed by, 134-136 preparative resolution of racemic

572

SUBJECT INDEX

secondary alcohols catalyzed by, 125, 127-134 stereoselective transesterification in biphasic systems, for preparation of racemic alcohols and racemic esters, 120-137 Celite-adsorbed preparation, 406-407 regiospecific interesterification of triglyceride with, 405-411 immobilized applications, 354-355 properties of, 302 interesterification with, 405 from R. delemar, 299 Celite-adsorbed, interesterification reaction, 409-410 Lipid-polyamide membrane artificial cell, applications, 82 Lipid-polymer membrane microcapsules assaying using ammonia, 79 assaying using urea, 79 cofactor recycling with different amounts of NAD ÷ retained within, 79-80 conversion of urea into glutamic acid with NADH recycling in, 80-81 permeability, 78 preparation, 78-79 recycling of free NADH retained within, 77-81 results, 79-81 Lipoamide dehydrogenase. See also Siteto-site enzyme systems assay, 108 scavenger assay with, as enzyme competing with LDH for NADH formed by ADH, 109-110, 112-113 Lipophilin, solubilization in reverse micellar media, 194-197 Lipoxygenase, in reverse micelles, 211212 kinetic parameters, 214 Liquid enzyme hydrolysis costs, 378 time course, 374 total saccharification cost, 374 Liquid enzymes, vs. immobilized, comparative economics, 379 Luciferase. See also Bacterial luciferase; Firefly luciferase

coimmobilized with other enzymes, analytical usefulness, 82 coimmobilized with oxidoreductase, on nylon tubing, 84 coupled to oxidoreductase, 83 Luciferase/oxidoreductase preparation assay, coupled reaction, 85 source, 84 Luciferin, source, 84 Lyase, immobilized, application, 355 Lylose. See Isomaltulose Lysozyme in reverse micelles, 206-209 kinetic parameters, 214 solubilization in reverse micellar media, 194-197 superactivity, 216

M L-Malate bioluminescent assay using coimmobilized enzymes, 86 continuous production, by coimmobilized FDH-MDH-NAD gel, 29-31 Malate dehydrogenase activity recovered on Sepharose, 89 microencapsulated, with malate dehydrogenase, recycling of NAD + and NADH, 70-71 PEG-NADH as coenzyme, 13 stability in microcapsules, 77 Thermus

molecular weight, 33-34 source, 26 L-Malic acid estimation of, 456, 465-466, 472 industrial production, 455,462-463 production by immobilized microbial cells, 455-463 comparison of various preparations, 461,463 continuous enzyme reaction, 458 effect of detergent treatments, 457458 enhancement of, 457-458 suppression of succinic acid formation, 457-458 uses, 455 Malolactic enzyme, immobilized, applications, 354

SUBJECT INDEX Maltose, synthesis, in hydrolase-catalyzed condensation reaction, 282 Maltose syrup, 432 Mammalian cell culture, using microcartiers and immobilized systems, 353 Matsuo Mine application of bacterial oxidation to, 532 feasibility tests, 532-533 neutralization and solid-liquid separation, 538 neutralization plant bacterial oxidation system, 534-537 neutralization plant contents, 534-535 neutralization plant current situation, 539 neutralization plant installation, 533534 neutralization plant operating costs, 539 sludge storage dam, 538 background, 532 technical developments at, 540 Maxazyme, 361,370 MDH. See Malate dehydrogenase Membrane reactor, 7-9, 11 advantages of, 20 continuous enzymatic transformation in, with simultaneous NADH regeneration, 9-21 experimental setup, 15-19 flow diagram of continuous process in, 16 laboratory model, 16-17 optimal ratio of enzyme activities, 20 performance, 19-21 polarization control, 16-17 pulse-free piston pump for, 19 simple, 16 oL-Menthol isomers, 293 optical resolution of, by entrapped biocatalysts, 293-302 stereoselective esterification by gelentrapped lipase, 293, 299-302 analytical methods, 300-301 effect of organic solvents, 301 enzyme, 299 materials, 299 methods, 299 prepolymer, 299 reaction conditions, 300-301

573

L-Menthol uses, 293 DL-Menthyl succinate selective hydrolysis by gel-entrapped yeast cells, 293-299 analytical methods, 296 cultivation of yeast, 294 effect of solvents, 295 materials, 293-294 methods, 293-294 prepolymers, 293-294 production of L-menthol, 298-299 properties of gel-entrapped cells, 297298 reaction conditions, 295-296 synthesis, 296-297 Metalloproteinases, 504 Nr-[N-(6-Methacrylamidohexyl) carbamoylmethyl]-NAD, 25-26 3-Methoxy-l-butanol hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 125-126 propionic esters, alkaline hydrolysis, 122 Methoxycarbonyl phosphate properties, 265 synthesis, 263,270 6-Methylacrylamidohexylammonium chloride, 23-24 6-Methyl-5-hepten-2-ol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-133 3-Methyl-l-pentanol, propionic esters, alkaline hydrolysis, 122 Methyl-l-pentyl propionate, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 6-Methylsalicylic acid synthetase, half-life, 330 Micelles, filled and unfilled characterization, 197 molecular weight determination, 197-199 Microbial immobilization, requirements for supports, 396-397 Micrococcus luteus, oxaloacetate decarboxylase, 484-485 Microemulsion, 192 Microencapsulation, of multienzyme system, 57, 67-82

574

SUBJECT INDEX

applications, 67, 81-82 methods, 68-70 Microorganisms, immobilized, application, 355 Milk, ultrahigh-temperature sterilized. See UHT milk Milk permeates microbial contamination, 414-415 utilization, 411 Milk xanthine oxidase, 255 coimmobilization with superoxide dismutase and catalase, 257 commercial, 257 immobilization experimental procedure, 258-259 procedure, 257-258 specific activity, 257 storage stability, 257 Mine acid. See also Acid mine drainage cause, 530 Monoglyceride, fatty acid composition, analysis, 408 Myoglobin, in reverse miceUar media, 213

N NAD analogs, synthesis, 5 as analytical reagent, repeated use of, 33 bioluminescent assay using coimmobilized enzymes, 86 coimmobilization with dehydrogenases. See Coimmobilization, of NAD with dehydrogenases coupled to polyethyleneimine, 75 free recycling, by microencapsulated yeast alcohol dehydrogenase and malate dehydrogenase, 70-71 retained inside microcapsules, methods for, 81-82 immobilized, as active coenzyme, 3 immobilized active, uses, 7 immobilized derivatives, kinetic properties, 6-7 modification in N-6 position, substituents used for, 4 polymeric, 38-39 coenzymatic properties, 34-35 properties, 38-39

polymerizable derivative, 34 coimmobilization with dehydrogenases in polyacrylamide gel, 22 polymerization, 39 preparation, 22-26, 35-39 synthesis, 35 recycling, 21, 35, 56 in microcapsules, 81-82 in stereospecific steroid oxidation, 7374 sources, 57-58 NAD(H) as coenzyme for dehydrogenase, 9 immobilized, coenzyme activity, 3 polyethylene glycol-bound. See PEGNAD(H) regeneration, 10 NAD(P), recycling, by multienzyme systems microencapsulated in artificial cells, 67-82 NAD(P)H electroenzymatic regeneration, 303-304 practical aspects, 314 regeneration, 303 NAD(P)+ reductase, methyl viologendependent, 304 NAD-N6-[N-(N-acryloy1-1-methoxycarbonyl-5-aminopentyl)propionamide], 34, 38 NAD+-N6-[N-(6-aminohexyl)acetamide] coupled to dextran T70, 75 immobilized with soluble dextran T70 activated with cyanogen bromide, 59 NADH dehydrogenases for, 11-13 free, recycling by microencapsulated yeast alcohol dehydrogenase and malate dehydrogenase, 70-71 by multienzyme systems microencapsulated in artificial cells, 70-74 free, retained within lipid-polymer membrane microcapsules, recycling, 77-81 H2-driven regeneration of, and subsequent reduction of apolar steroid, in reverse micellar medium, 228-229 preparation, kinetic parameters of dehydrogenases for, 12

SUBJECT INDEX recycling, in conversion of urea and ammonia into amino acid, by multienzyme system in semipermeable microcapsules, 71-72 NADH:FMN oxidoreductase, activity recovered on Sepharose, 89 NADP analogs, synthesis, 5 bioluminescent assay using coimmobilized enzymes, 87 time course of light output, 90, 92 in enzyme reactor studies, 9 immobilized, as active coenzyme, 3 partition, in aqueous two-phase system containing Ficoll and UCON, 55 NADP(H) as coenzyme for dehydrogenase, 9 free, recycling, by multienzyme systems microencapsulated in artificial cells, 70-74 recycling, in sequential conversion of urea and ammonia to amino acid, in multistep enzyme reaction in microcapsules, 72-73 regeneration, I0 Nicotine adenine dinucleotide. See NAD Nitrilase assay, 524 immobilized, application, 355 Nocardia erythropolis, 3/3-dehydrogenation of cholesterol, in two-liquid phase biocatalytic reaction, 140-141 Nocardia rhodochrous, conversion of cholesterol to cholestenone, in twoliquid phase biocatalytic reaction, 139-141, 145-148 Nonseparation immunoassay advanatges of, 103 compared to ELISA, 94, 103 principle, 94 sensitivity, 94, 103 Nuclease P~, assay, 272 Nucleic acid, solubilized in hydrocarbon micellar solutions, 215 Nucleoside triphosphate conversion of RNA to mixture of, 278279 enzymatic regeneration, 266 Nutrisearch Company, 422 Nylon tubing

575

covalent attachment of alcohol dehydrogenase and NAD analog inside, procedure, 59-61 covalent attachment of alcohol dehydrogenase and NAD to, 59 oxidoreductase and luciferase coimmobilized on, 84

O 2-Octanol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-132 Oligonucleotides, synthesis, in hydrolasecatalyzed condensation reaction, 282 Open tubular heterogeneous enzyme reactor, 59, 67 Optically active alcohols and esters, enzymatically prepared, chemical conversion to other optically active compounds, 130 Optically active compounds enzymatic production in biphasic aqueous-organic systems, 117-137 enzymatic production in nonaqueous organic systems, 137 function, 117-118 Optisweet 22, 367, 370 OTHER. See Open tubular heterogeneous enzyme reactor Oxaloacetate, decarboxylation, 482-483 Oxaloacetate decarboxylase, 483,499 P. putida assay, 490 immobilization, 489-490 sources, 484-485 Oxaloacetic acid, decarboxylation, 499500 Oxidoreductase assay, 85 coimmobilized with luciferase, on nylon tubing, 84 coupled to luciferase, 83 in reverse micelles, 208-212 source, 83 2-Oxocarboxylate reductase, from Proteus, 303

576

SUBJECT INDEX

P Packed bed fermentation vessel, 387 Palatinose. See Isomaltulose Palmitate-enriched glyceride, interesterification with pancreatic lipase to obtain, 405 Pancreatic lipase porcine, monophasic system, in nearly anhydrous organic solvents, 137 in reverse micelles, 206 Pancreatic ribonuclease, in reverse micelles, 206-207 kinetic parameters, 214 Papain, immobilized assay, 181 in peptide synthesis, 183 Parallel flow reactor, 386-388 Patulin assay, in Penicillium culture filtrates, 339-340 biosynthetic reaction, 329-330 production by immobilized Penicilliurn, 329-342 PEG carboxylated, elution in membrane reactor, 14-15 elution in membrane reactor, 14-15 water-soluble handle, in peptide synthesis, 158-159 PEG-NAD cofactor activity, 35 preparation, 34, 40 synthesis, 35 PEG-NAD(H) preparation, 39-40 elution in membrane reactor, 13, 14-15 preparation, kinetic parameters of dehydrogenases for, 12 properties, 11 synthesis, 11-12 PEG sulfate, preparation, 49-50 Penicillin assay, in Penicillium culture filtrates, 339-340 biosynthetic reaction, 330 production by immobilized Penicillium, 329-342 semisynthesis, 280-292 Penicillin (G or V) acylase, immobilized, application, 355

Penicillin amidase from E. coli, 284 synthesis of semisynthetic penicillins and peptides catalyzed by, mechanism, 284 Penicillium chrysogenurn antibiotic-producing activity establishment, 335-337 maintenance, 337-339 antibiotic productivity, determination, 339 batch cultivations, cessation of antibiotic production, 330 cell growth, determination, 339 culture filtrates, assays, 339-340 immobilization equipment, 331-335 on K-carrageenan, 332-334 procedure, 331-335 immobilized, sampling procedures, 339 immobilized ceils, in fundamental studies of secondary metabolism, 340342 Penicillium urticae antibiotic-producing activity establishment, 335-337 maintenance, 337-339 antibiotic productivity, determination, 339 batch cultivations, cessation of antibiotic production, 330 cell growth, determination, 339 culture filtrates, assays, 339-340 immobilization equipment, 331-335 on K-carrageenan, 332-334 procedure, 331-335 immobilized, sampling procedures, 339 immobilized cells, in fundamental studies of secondary metabolism, 340342 patulin-producing, effect of harsh immobilization, 342 PEP, bioluminescent assay using coimmobilized enzymes, 87 Pepsin A, in reverse micelles, 203 Peptidase, immobilized, application, 355 Peptide semisynthesis, 162, 280-292 Peptide synthesis chain elongation and release, carboxypeptidase Y in, 159

SUBJECT INDEX deblocking, with immobilized carboxypeptidase Y, 157-162 enzymatic approaches, 179 protease-catalyzed advantages, 187 future of, 187-188 requirements, 187 thermodynamic approach, 179, 184187 protease-mediated kinetic approach, 179-184, 187 immobilization procedures, 180-182 materials, 180-182 strategies, 179 using immobilized proteases, 178-188 water-solube PEG handle in, 158-159 Peroxidase in reverse micelles, 210, 212 kinetic parameters, 214 steroid transformation with, 156-157 sec-Phenethyl alcohol, alkaline hydrolysis of esters, 128 L-Phenylalanine production, using immobilized aminotransferase, 484, 486 production via E. coli equilibrium studies, 499-500 optimization of cell loading, 501-503 production via polyazetidine-immobilized E. coli, 497-503 Phenylalanine dehydrogenase, for native NADH and PEG-NADH, kinetic parameters, 12 sec-Phenylethyl alcohol, yeast lipasecatalyzed resolution of, via transesterification, 127-132 Phenylpyruvate conversion to phenylalanine, effect of increasing aspartic acid concentration, 498 as starting material for phenylalanine production, 497-498 DL-Phe-OMe, preparation, 513 5'-Phosphodiesterase assays, 518 immobilization, 518 immobilized activity, and pH, 518-519 application, 355 enzymatic properties, 518-519 in industrial operation, stability, 519

577

Phosphoenolpyruvate properties, 265 synthesis, 263, 268-269 Phospholipase, in reverse micelles, 206207 Phospholipase A2, in reverse micelles, 207 5-Phospho-a-D-ribosyl pyrophosphate concentration, determination, 271-272 synthesis, 266-267 from ribose 5-phosphate, 278 5-Phospho-a-D-ribosyl pyrophosphate synthetase assay, 271 source, 270 Photinus-luciferin 4-monooxygenase. See Firefly luciferase Photinus pyralis, luciferase. See Firefly luciferase Photobacterium fischeri, luciferase/oxidoreductase preparation, 84 Photo-cross-linkable resin. See also Yeast, photo-cross-linkable resin-immobilized structure, 383 Phyllostine, production by Penicillium urticae mutant P3,341 Plasmid, solubilized in hydrocarbon micellar solutions, 215 Poly(acrylamide-co-N-acryloxysuccinimide), 274-275 assay for active ester content, 275 Polyamide membrane microcapsules containing dextran-NAD and multienzyme systems, preparation, 75 preparation, by interfacial polymerization, 69-70 Polyethylene glycol. See PEG Polynucleotide phosphorylase, assay, 272273 Polynucleotides, synthesis, in hydrolasecatalyzed condensation reaction, 282 Polyribose phosphate enzyme-channeling assays, 95-103 equipment, 95-96 materials used, 95-98 measurement of protein concentrations, 96 methods, 98-103 reagent preparation, 95-98 glucose oxidase-labeled, preparation, 97 HRP-labeled, preparation, 96-97 Porin, in reverse micellar media, 213

578

SUBJECT INDEX

(Pro-Gly-Pro)10, sequential hydrolysis with immobilized aminopeptidase, 176-177 Progesterone reduction, in enzymatic reactions in reverse micelles, 229 stereospecific reduction, in two-phase systems, 154-155 Proline-containing pelbtide, sequencing, 170, 178 (2R)-Propanediol, preparation, by combination of Candida utilis and AIcaligenes eutrophus, 316-317 Propylene oxide, yeast lipase-catalyzed resolution of, via transesterification, 133 Protaminobacter immobilization, 451-453 isomaltulose production, 439 isomaltulose-synthesizing activity, storage stability under various conditions, 450 Protaminobacter rubrum enzyme responsible for isomaltulose production immobilization, 453 isolation, 453 isomaltulose production, 434 Protease immobilized application, 355 peptide synthesis using, 178-188 in peptide synthesis, 161-162 in reverse micellar media, 201-206 Proteus vulgaris growth, 311 (2R)-hydroxycarboxylate-viologen oxidoreductase, enrichment procedure, 311 Pseudomonas chlororaphis, nitrilase activity, 523 Pseudomonas dacunhae L-aspartate fl-decarboxylase activity, stabilization, 476 culture, 474 immobilization with r-carrageenan, 475 immobilized, in L-alanine production, 472-479 immobilized cells, L-alanine production, elimination of side reactions, 476 immobilized with r-earrageenan, activi-

ties with and without pH treatment, 476 Pseudomonas oleovorans, epoxidation activity, in two-liquid phase biocatalytic reaction, 139-140, 145 Pseudomonas putida conversion of 1,7-octadiene to 7,8epoxy-l-octene, in two-liquid phase biocatalytic reaction, 139, 144-145 oxaloacetate decarboxylase, 484-485, 489-490 transaminase, 497-498 Pyrite, in formation of mine acid, 530 Pyrophosphatase, inorganic, in reverse micelles, 206-207 Pyruvate kinase specific activity, 264 stability in immobilized form, 264

R Racemic alcohols, resolution, using esterase-catalyzed transesterification, 131 Racemic esters, yeast lipase-catalyzed preparative resolution of, via transesterification, 134-136 Racemic primary alcohols, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121126 Racemic secondary alcohols, preparative resolution catalyzed by yeast lipase, 125, 127-134 Raffinate, treatment with glucoamylase, 376 Reaction center from R. sphaeroides, in reverse micellar media, 213 Reactors. SeeEnzyme reactors Reductase, 302 Research Association for Petroleum Alternatives Development, 380 research activities, 395 Retention, definition of, 14 Reverse micellar media concentration, expression, 200-201 enzymes in, 188-216 assay, 199-200 conformation and activity of, 199-212 intrinsic rate parameters, determination, 222-224

SUBJECT INDEX intrinsic rate parameters for, 200-201 HEPES-hexanol-CTAB-octane system, phase diagram, 217-219 hydrocarbon-soluble substrates added to, 215 multienzymatic reactions in, 228-229 pH determination, 201 solubilization of enzymes in, 192-197, 219-221 inje%tion method, 192-193,219-220 methods, 192, 219 phase-transfer method, 193-195, 220221 stability diagrams, 192-193 by transfer from solid state, 194-197, 220 Reverse micelles enzymatic conversion of apolar substrates in log P definition of, 226 determination of, 226-227 mole fraction of cosuffactant in interphase and its amount in continuous phase, determination, 227228 parameters regulating, determination of, 225-228 for enzymatic synthesis of apolar compounds, 216 enzymes in activity of, determination, 221-222 conformational aspects, 215 discrepancies of results between laboratories, 215 relationship between water of water pool and guest enzyme, 215 and suffactant purity, 215 vs. normal aqueous solutions, 200 water shell model, 198-199, 215 optimal solubility of proteins in, 215-216 physical characterization, before enzyme uptake, 192 preparation, 189-192 protein-containing characterization, 197-199 ultracentrifugation, 197-198 use to solubilize proteins in aprotic media, 212-215 Rhizopus arrhizus

579

immobilized lipase, continuous hydrolysis of triglyceride, in two-liquid phase biocatalytic reactors, 147 lipase, immobilization on Celite, 148 Rhizopus de&mar, lipase, 299 Rhizopus niveus, lipase, 149 Rhodococcus Strain 6 culture, 525 immobilized cells acrylamide production, 530 electron micrograph, 526, 527 enzyme activity effect of acrylonitrile and acrylamide concentrations, 529 effect of pH, 527-529 effect of temperature, 526-527 nitrilase properties, 527-529 preparation, 525-527 nitrilase activity, 523-524 Rhodopsin, in reverse micellar media, 213 Rhodotorula minuta, conversion of menthyl succinate to menthol, in twoliquid phase biocatalytic reaction, 140-141, 146 Rhodotorula minuta var. texensis culture, 294 gel-entrapped cells activity, 297 effect of gel hydrophobicity, 297 effect of reaction temperature, 297 properties, 297 stability of hydrolytic activity, 297298 immobilization, 294 Ribonuclease, superactivity, 216 5'-Ribonucleotides preparation, by enzymatic hydrolysis of RNA, 517 for preparation of food additives and drugs, 517 produced by immobilized 5'-phosphodiesterase concentration, 522 isolation, 522 purification, 522 separation, 522 production by-products, 517 effect of Zn2+ ions during continuous operation, 519-520

580

SUBJECT INDEX

enzyme support, 518 material, 518 methods, 518-521 nucleotides for, 518 preparation of substrate solution, 519521 process, 520-522 using immobilized 5'-phosphodiesterase, 517-522 Ribose 5-phosphate, synthesis, by acidcatalyzed hydrolysis of AMP, 278 RNase, reduced and carboxymethylated, 177

S Saccharification cost comparisons, 378-379 minumum cost for immobilized enzymes, 379 for liquid hydrolysis, 379 total cost, 374 Saccharification tank system, 375-377 running costs, 375-376 saccharification time, 375-376 Saccharomyces. See Yeast Secologanin source, 344 in strictosidine formation, 342 Secondary metabolism immobilized cells in fundamental studies of, 340-342 longevity, 340-341 Secondary metabolites, commercial production, 329 Semisynthesis catalyzed by penicillin amidase and serine protease, mechanisms, 281282 of human insulin, 162-170 of peniciUins and peptides, 280-292 of peptide, 162 of proteins, 234 Serine carboxypeptidase. See Carboxypeptidase Y Serine protease, synthesis of semisynthetic penicillins and peptides catalyzed by, mechanism, 284 Serratia isomaltulose production, 439

isomaltulose-synthesizing activity, storage stability under various conditions, 450 Serratia marcescens, immobilization, 452 Serratia plymuthica immobilization, 452 isomaltulose production, 434 Silane-glutaraldehyde immobilization, of lactase, 413 Site-to-site enzyme systems ADH activity, 110-112 characterization, 110-113 enzyme assays, 108-110 immobilized system, 104-106 orientation, 104-108 procedure, 104 scavenger enzyme assay, 109-110, 112113 soluble system, 106-108 Skim milk membrane vesicle fractions, preparation, 424 Sodium alginate, from L. hyperborea, for cell immobilization, 437 Soybean trypsin inhibitor, 177 Specialist Dairy Ingredient company, 422 Spezyme IGI, 370 Spherosil, porous silica supports, characteristics, 246 Steroid enzyme-catalyzed transformations in water-organic solvent two-phase systems, 150-157 assays, 151-152 enzyme immobilization, 152-153 materials, 151 methods, 154-157 parameters affecting, 157 two-liquid phase biocatalytic reactions for, 139-141 Steroid isomerase, steroid transformation with, 156-157 Streptomyces murinus, in glucose isomerase production, 361 Streptomyces olivochromogenes, in glucose isomerase production, 366 Streptomyces phaechromogenes, in glucose isomerase production, 362 Streptomyces rubiginosus, 367 in glucose isomerase production, 358

SUBJECT INDEX

Streptomyces wedmorensis, in glucose isomerase production, 357 Strictosidine formation, 342 preparative synthesis, 349-350 Strictosidine synthase, 342-350 activity, determination, 346 activity, effect of secologanin concentration, 347-348 activity, pH profile, 346-347 assay, 344 immobilization, materials, 343-344 immobilized preparation, 345-349 stability, 348-349 thermostability, 348 isoenzymes, 343 isolation, 344-346 preparation, 344 reaction catalyzed, 342-343 Substance P octapeptide, sequential hydrolysis with immobilized aminopeptidase, 175 Subtilisin, immobilized, application, 354 Sucrose syrup, invertase treatment of, 376 Sulfhydryl oxidase adsorbed directly onto Spherosil QMA, 426-427 covalent attachment to controlled-pore glass beads, 425-426 immobilization, 425-427 conditions, 425 pore volume of matrices for, 425 immobilized activities in various preparations, 426 applications, 354 stability, 428 storage, 428 isolation of milk proteins with increased activity of, 423--425 purification, 425 reactors activity, 428-429 characteristics, 427-430 normalized residence time, 429-430 operational protocol, 428 Suspended bed fermentation vessel, 387 Sweetase, 362, 370 Sweet-protein syrup, 422 Sweetzyme, 361-362, 370

58 1

T Takasweet, 361,370 Tego-Diocto BS, 419 Testosterone bioluminescent assay using coimmobilized enzymes, 87 oxidation to androstenedione, in twophase system, 155 Theorell-Chance mechanism, 42 Thermoase for aspartame production, selection, 504 immobilization, 507-5 ! 1 by cyanuric chloride, 511 by glutaraldehyde, 510-511 immobilized assay, 512-513 for condensation between Z-Asp and Phe-OMe, 505-506 determination of amounts on support materials, 511-512 substrate preparation, 513 Thermolysin, 504 immobilization to Enzacryl AH, 181 via diazo coupling, 508 immobilized application, 355 in peptide synthesis, 185 Thiobacillus ferrooxidans, immobilized application, 355 in treatment of acid mine drainage, 530540 Thiolproteinases, 504 Thiols, and food flavors, 423 Toyopearl activation, 510 as support material for thermoase immobilization, 507-510 Transaminase. See Aminotrarlsferase Transamination reaction, 480-481 equilibrium constant, 481-482 Trehalulose production, 453 in immobilized microbial cells, 440 properties, 453 Triacylglycerol lipase. See also Lipase, C. cylindrica in reverse micelles, 207 Triglyceride

582

SUBJECT INDEX

fatty acid in 2-position of, analysis, 408 isolation, 407 oleic safflower oil interesterified, fatty acid composition, 410 reformed, isolation from interesterification reaction mixture, 406 regiospecific interesterification, reaction scheme, 406 total fatty acid composition, analysis, 407-408 17,20fl,2l-Trihydroxysteroid:NAD+ oxidoreductase. See 20fl-Hydroxysteroid dehydrogenase Trisaccharides, synthesis, in hydrolasecatalyzed condensation reaction, 282 6-(Tritylamino)hexylamine dihydrochloride, 22-23 N-[6-(Tritylamino)hexyl]methacrylamide, 24 Trypsin in enzymatic conversion of porcine insulin to human insulin, 162-163 immobilization, to carboxymethylcellulose, 181 immobilized assay, 181 in peptide synthesis, 183-186 in reverse micelles, 203,205 kinetic parameters, 214 side-chain cleavage with, 161-162 Tuftsin, sequential hydrolysis with immobilized aminopeptidase, 176 Two-liquid phase biocatalytic reactions aqueous phase, 138-142 classification, 138-139 discrete aqueous continuous phase, 138140 discrete aqueous discontinuous phase, 138-140 discrete aqueous phase, 143 at interface, 138, 142-143 kinetic considerations, 143-144 no discrete aqueous phase, 140-142 steady-state conditions, 143-144 types of, 138-143 Two-liquid phase biocatalytic reactors, 138-149 advantages, 138 choice of, 146-147 concentration profiles of reactant at steady state in, 142-143

effect of organic solvent, 145-146 experimental methods, 147-149 future prospects, 149 immobilization technique on Celite, 148 using hydrophilic photo-cross-linkable resin prepolymers, 148 using hydrophobic photo-cross-linkable resin prepolymers, 147-148 using Urethane prepolymers, 148 packed bed, 146-147, 149 performance, effect of phase ratio and biocatalyst concentration on, 144 shaken flask, 149 stirred tank, 146-149 U UDPglucose activity, assay, 273 enzyme-catalyzed synthesis, 279-280 UDPglucose pyrophosphorylase, assay, 273 UHT milk cooked flavor of, 423 flavor modification, with immobilized sulfhydryl oxidase, 423,430-431 Urea hydrolysis, 234 effect of organic solvents on, measurement, in determination of urease reaction rate, 236-237 measurement, 236 products, 238 Urease immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 jack bean source, 235 stability, effect of organic solvents on, 236 microencapsulated, for muitistep enzyme reaction of conversion of urea into glutamic acid, 72-73 Urea synthesis from ammonium carbonate, 234 nonenzymatic, 234 urease-catalyzed

SUBJECT INDEX measurement, 235-236 reaction mechanism, use of organic solvents to study, 238 yield effect of hydrogen ion concentration, 237 effect of nature of organic solvent, 237 effect of organic solvent, 237 effect of substrate, 237 with urease in water-organic solvent mixtures, 234-238 Uridine-5'-diphosphoglucose. See also UDPglucose enzymatic synthesis from glucose and RNA, 267-268 enzyme-catalyzed synthesis, 263

V Vibrio harveyi, luciferase. See Bacterial luciferase

W Washout, 14 Whey clarified and demineralized, hydrolysis with immobilized lactase, 420 lactase treatment of, 376 microbial contamination, 414-415 obtained by clotting with chymosin, 424 raw, hydrolysis with immobilized lactase, 419 ultrafiltration, 411 utilization, 411 Whey syrups, lactolyzed, uses, 412

X Xanthine oxidase, 256-258. See also Milk xanthine oxidase commercial, immobilization onto Sepharose 4B, 258-259 immobilized oxidation of 1-methylxanthine to 1methyluric acid, 259-262 oxidation of xanthine to uric acid by, 259

583

preparation, using milk as starting material, 257-260 reaction specificity, 261-262 potential substrates, 256-257 reaction catalyzed, 256

Y Yeast calcium alginate-immobilized, 394-405 bench-scale studies, 397-398 cell viability for stable long-run operations, 394 contamination prevention, 398-399 design of reactor, 397-398 improvement of cell viability, 399 maximum cell concentration in carrier, 394 pH of inlet substrate solution, 399 pilot plant, 394 pilot plant operations, 399-402 process, 398 productivity, 395, 402-404 semicommercial plant, 402-403 continuous immobilizer, 383-384 conventional batchwise fermentation with, 405 compared to immobilized cells, 402 immobilization, 395 entrapping materials, requirements, 381-383 entrapping methods, 381 in photo-cross-linkable resin, 381-385 immobilized alcohol production, in various preparations, 396 fermentation characteristics, 385-387 fermentation system, contamination prevention, 389 life span, 394 maintenance of cell viability, 399-400 microbiocidal sterilization, 389-390 selection of carriers, 396 sludge removal measures, 388-389 immobilized cells, applications, 354-355 photo-cross-linkable resin-immobilized, 380-394 alcohol concentration, 392-393 alcohol productivity, 393 bench-scale plant, 389-391

584

SUB.IECT INDEX

continuous operation of experimental plants, 389-392 ethanol productivity, 380 fermentation system, 394 immobilization process, 383-384 pilot plant, 389, 391-392 properties, 384-385 theoretical alcohol yield, 393 yeast concentration, 392 strains, selection and improvement for ethanol production, 399 vacuum fermentation technique, 405

Z Z-Arg-AIa-NH2, papain-catalyzed formation, 183 Z-Asp, and Phe-OMe, condensation between, 505-506, 513-514 Z-L-Asp, preparation, 513 Z-GIy-Phe-Leu-NH2, chymotrypsin-catalyzed synthesis, 184 Z-Lys-Leu-NH2, trypsin-catalyzed synthesis, 183-184 Z-Phe-Leu-NH2, thermolysin-catalyzed synthesis, 185