Advances in Applied Microbiology, Volume 40

ADVANCES IN

Applied Microbiology VOLUME 40

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ADVANCES IN

Applied Microbiology Edited by

SAUL NEIDLEMAN Vacaville, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 40

Academic Press San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX International Standard Serial Number: 0065-2 164 International Standard Book Number: 0-12-002640-6 PRINTED IN THE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 0 0 B B 9 8 7 6

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CONTENTS

Microbial Cellulases: Protein Architecture. Molecular Properties. and Biosynthesis

AJAYSINGH AND KIYOSHIHAYASHI I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Microbial Sources of Cellulolytic Enzymes ........................... I11. Protein Architecture and Molecular Properties ........................

IV. Structural and Catalytic Properties .................................. V Cellulase Biosynthesis ............................................. VI . Future Prospects .................................................. References., ......................................................

.

1 2 6 15 26 34 35

Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective

D . K . O’TOOLE I . Introduction ...................................................... I1. Early Developments ............................................... 111. Lactenin .......................................... ........... IV. Studies with Lactic Acid Bacteria ................................... V. Effect of Cow Ration on Milk as a Growth Medium for Bacteria . . . . . . . . . VI . Role of Inhibitors in Milk Quality ................................... VII. Stimulators of the Growth of Lactic Acid Bacteria ..................... VIII . Growth Inhibitors of Other Bacteria .................................. IX. Applications of the Antimicrobial Systems in Milk .................... X . Conclusion .......................... Appendix A ......................... Appendix B ......................... ........................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 47 48 53 69 71 72 77 81 82 83 84 85

Challenges in Commercial Biotechnology . Part I. Product. Process. and Market Discovery

ALE^

PROKOP

I . Introduction

...................................................... .................................. I11. Primary Market Classification ....................................... I1. Biocatalysts: Sources for Discovery

V

95 108 113

vi

CONTENTS

IV. Pathway to Commercialization: Discovery and Development (D & D) Cycle ..................................................... V. Product Discovery ................................................. VI . Process Discovery ................................................. VII. Market Discovery ................................................. VIII . Interactions of Discovery Aspects within the D & D Cycle . . . . . . . . . . . . . . References........................................................

114 115 141 148 149 152

Challenges in Commercial Biotechnology. Part II. Product. Process. and Market Development ALE; PROKOP

I . Introduction

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

I1. Product Development ..............................................

111. Process Development and Scale-Up .................................. IV. Market Development ............................................... V. Outlook .......................................................... References........................................................

155 156 173 209 227 233

Effects of Genetically Engineered Microorganisms on Microbial Populations and Processes in Natural Habitats JACK

D. DOYLE.GUENTHER STOTZKY. GWENDOLYN MCCLUNG. AND CHARLES W . HENDRICKS

I . Introduction ...................................................... I1 Aquatic Environments ............................................. I11 Activated Sludge .................................................. IV Soil V. Plants ........................................................... VI Discussion ....................................................... References ........................................................

. . . .

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

237 245 248 249 261 267 280

Detection. Isolation. and Stability of Megaplasmid-Encoded Chloroaromatic Herbicide-Degrading Genes within Pseudomonas Species

DOUGLASJ . CORK AND AMJADKHALIL I . Introduction ...................................................... I1. Selected Model Haloaromatic and Aromatic Compounds of Agricultural and Industrial Importance ............................... I11. Detection and Isolation of Large Plasmids from Pseudomonos spp . . . . . . .

289 290 301

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IV. Effect of Alternative Carbon Sources on the Stability and Curing of a Large Molecular Weight Plasmid. pDKl .......................... V . Summary ........................................................ References........................................................

308

INDEX.................................................................

323

CONTENTSOFPREVIOUSVOLUMES .........................................

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Microbial CelI ulases: Protein Arch itect ure, Molecular Properties, and Biosynthesis AJAYSINGH AND KIYOSHIHAYASHI Biomaterials Conversion Laboratory National Food Research lnstitute Tsukuba, Ibaraki 305, Japan

I. Introduction 11. Microbial Sources of Cellulolytic Enzymes

111.

IV.

V.

V1.

A. Fungi B. Bacteria Protein Architecture and Molecular Properties A. Cellobiohydrolases B. Endoglucanases C. P-Glucosidases Structural and Catalytic Properties A. Structural Organization B. Glycosylation C. Synergistic Mechanism D. Catalytic Mechanism Cellulase Biosynthesis A. Induction and Regulation B. Localization C. Biosynthesis in Recombinant Cells Future Prospects References

I. Introduction

Cellulose is the most abundant carbohydrate polymer in the biosphere. An estimated synthesis rate of cellulose is approximately 4 X lo7tons per year. For a long-range solution to our resource problems of energy, chemicals, and food, cellulose is the most promising renewable carbon source that is available in large quantities. Today, the need for oil substitutes appears to be acute mainly because of an increase in oil prices and a higher risk of shortage. Enzymatic hydrolysis of cellulose has been widely studied during the last 20 years as a way to provide fermentable sugars, which, in turn, could be converted into various value-added chemicals and fuels. However, because of the physical nature of the cellulose molecule and the fact that its natural occurrence is always accompanied by associated materials, hemicellulose and lignin, the efficiency of cellulolytic enzymes deserves improvement in most cases. 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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AJAY SINGH AND KIYOSHI HAYASHI

Enzymatic hydrolysis of cellulose, although not yet commercially attractive, is expected to be feasible in the long run, as has been the case with starch hydrolysis (Bisaria and Mishra, 1989). The inherent complexity of cellulolytic enzyme mixtures, in terms of the number of enzyme components and their specificity, has caused difficulty in our understanding of the cellulase enzyme system. Considerable progress in our understanding of the role of various enzyme components in cellulose degradation has recently been provided by studies involving the powerful tools of molecular biology, genetic engineering techniques, monoclonal antibodies, and protein chemistry (BBguin, 1990; Kubicek, 1992). In recent years, recombinant DNA technology has provided a means for isolating, characterizing, and manipulating the genes for a large number of different proteins (Glick and Pasternak, 1989). Therefore, the use of r-DNA technology applied to the cellulase system will facilitate not only a better understanding of catalytic functioning, cooperative interactions between different enzyme components, and regulation of these enzymes, but also the development of practical systems for the utilization of native cellulose. The cellulase-cellulose system and the importance of cellulose bioconversion research have already been subjects of detailed studies including cellulose structure and pretreatment (Chang et al., 1981; Young and Rowell, 1986; Fan et al., 1982; Lipinsky, 1979; Ghosh and Singh, 1993),cellulose bioconversion (Mandels, 1982; Bisaria and Ghose, 1980; Klyosov, 1986; Singh and Kumar, 1991; Singh et al., 1992a; Mishra and Singh, 1993), cellulase purification and properties (Ghose, 1987; Wood, 1985; Enari and Niku-Paavola, 1987; Merivuori et al., 1985a; Gong et al., 1979), cellulase production and applications (Lee et al., 1980; Fan et al., 1987; Ryu and Mandels, 1980; Ladisch et al., 1983; Mandels, 1985; Lachke and Deshpande, 1988; Srinivasan and Seeta Laxman, 1988; Whitaker, 1990; Vallander and Eriksson, 1990), and the genetics of cellulolytic microorganisms (Durand et al., 1988; Orpin, 1988; Shoemaker et al., 1981; Knowles et al., 1988; Pentilla et a]., 1988; Pasternak and Glick, 1987; Kuhad and Singh, 1993a). Recent advances in the nature, protein architecture, and regulation of biosynthesis and secretion of the cellulase enzyme system will be discussed in the present article. II. Microbial Sources of Cellulolytic Enzymes

Cellulases are synthesized by a large number of microorganisms (Table I). These organisms have in common the production of extracellular hydrolytic enzymes that attack the cellulose polymer. However, rela-

MICROBIAL CELLULASES

3

TABLE I WITH RESPECT TO MOSTWIDELY STUDIED MICROORGANISMS CELLULOLYTIC POTENTIAL

Fungi

Bacteria

White-rot Phanerochaete chrysosporiurn Coriolus versicolor Brown-rot Poria placenta Lanzitus trabeurn Soft-rot Trichoderma reesei Penicillium funiculosum Fusariurn oxysporum Aspergillus niger Anaerobic Neocalimastix frontalis

Aerobic Cellulomonas fimi Pseudomonas fluorescens Bacilus subtilis Cellvibrio gilvus Cytophaga hutchinsonii Anaerobic Clostridiurn thermocellurn Acetovibrio cellulolyticus Ruminococcus albus Actinomycetes Therrnoactinomyces curvata Streptornyces flavogriseus

tively few microorganisms are able to produce the necessary group of enzymes for degradation of crystalline cellulose (Mandels and Weber, 1969).

A. FUNGI Fungi are the most studied organisms with respect to cellulose degradation and cellulase production. Fungi producing the necessary enzymes for the cell-free degradation of crystalline cellulose generally belong to the Ascomycetes and Deuteromycetes groups or to the whiterot Basidiomycetes (Eriksson and Johnsrud, 1982). Brown-rot Basidiomycetes also degrade cellulose but they seem to lack exo-1,4-Pglucanases (Ljungdahl and Eriksson, 1985). The white-rot group of fungi is rather heterogenous; however, they have in common the capability to degrade lignin as well as other lignocellulose components (Eriksson, 1981).The most studied white-rot fungus was first isolated from wood chip piles and was given the name Chrysosporium lignorum (Bergman and Nilsson, 1966). The name was later changed to Sporotrichum pulverulentum for its imperfect stage (von Hofsten and von Hofsten, 1974) and then to Phanerochaete chrysosporium (Burdsall and Eslyn, 1974) for its perfect stage. The fungus is characterized as thermotolerant and has an optimum growth rate around 38" C (Eriksson and Pettersson, 1975a). It produces five

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AJAY SINGH AND KIYOSHI HAYASHI

endo-l,4-/3-glucanases (EC 3.2.1.4), one exo-1,4-/3-glucanase (EC 3.2.1.91), and two 1.4-/3-glucosidases (EC 3.2.1.21) (Eriksson and Pettersson, 1975b; Deshpande et al., 1978). Brown-rot fungi, which rapidly depolymerize cellulose, seem to utilize a mechanism of cellulose degradation different from that operating in P. chrysosporium. Brown-rot fungi apparently lack the synergistic endo-exo-glucanase cooperation to degrade crystalline cellulose because they lack exo-1,4-/3-glucanase activity (Highley, 1975, 1980, 1987a,b; Highley and Murmanis, 1985). Among brown-rot fungi, Poria placenta, Tyromyces palustris, and Lenzites trabeum are the best known examples of cellulolytic organisms (Eriksson et a]., 1990). Soft-rot fungi have the capability to deplete both polysaccharides and lignin; however, the polysaccharides are the main targets for this group. The ability of soft-rot fungi to produce all the necessary extracellular enzymes for degradation of crystalline cellulose varies. The best known of those producing a complete enzyme system is Trichoderma reesei (Mandels and Reese, 1964; Mandels, 1975; Gritzali and Brown, 1979; Fagerstam and Pettersson, 1980; Bhikhabhai et al., 1984). Trichoderma koningii (Wood and Phillips, 1969; Wood and McCrae, 1972, 1975, 1986a; Fan et a]., 1987), Penicillium funiculosum (Wood et al., 1980; Wood and McCrae, 1982, 1986b,c), Penicillium citrinum (Kuhad and Singh, 1993b), Fusarium solani (Wood, 1969, 1971, 1975; Wood and McCrae, 1977), Fusarium oxysporum (Kumar et al., 1991, 1992; Singh et al., 1992a,b; Christakopoulos et al., 1989, 1990), and Aspergillus niger (Singh et al., 1988a,b, 1989a,b, 1990a,b,c, 1991a,b) are other well known examples of cellulolytic soft-rot fungi. Trichoderma reesei secretes large amounts of different cellulases that solubilize crystalline cellulose efficiently by their combined action. It produces at least three endo-1,4-p-glucanases (EG I-111), two exo-l,4-/3-glucanases (cellobiohydrolases, CBH-I and CBH-11), and 1,4-P-glucosidases (Stahlberg et al., 1988; Teeri et al., 1987; Enari and Niku-Paavola, 1987; Enari et al., 1981).

The anaerobic rumen-inhabiting fungi also have the capability to degrade cellulose to glucose. The fermentation of cellulose by a rumen anaerobic fungus results in the formation of acetate. CO,, formate, acetate, lactate, and hydrogen (Orpin, 1977; Bauchop, 1981). The cellulolytic enzyme system in one of the rumen anaerobic fungi, Neocalimastix frontalis, has been studied by several groups (Pearce and Bauchop, 1985; Wood et al., 1986). The major digestion product by the action of N. frontalis cellulase on Avicel (microcrystalline cellulose) is glucose and not cellobiose (Pearce and Bauchop, 1985).This suggests that excess P-glucosidase is produced relative to cellulase by N. frontalis. This

MICROBIAL CELLULASES

5

contrasts with the situation in T. reesei, which produces only small quantities of P-glucosidase (Mandels, 1982). Wood et al. (1986) isolated an extracellular enzyme system from N. frontalis that was several fold more active in solubilizing cotton fibres per unit of endo-l,4-P-glucanase than the cellulase of T. reesei mutant strain Rut C-30, one of the most active cellulases.

B. BACTERIA

Degradation of cellulose by bacterial systems occurs both aerobically and anaerobically. The cellulase systems of bacteria are not directly comparable to those of fungi. Bacterial degradation of cellulose seems to take place by a cluster of enzymes rather than by individual endoand exoglucanases as in the case of fungi (Ljungdahl and Eriksson, 1985). Many bacteria lack 1,4-P-glucosidases and employ phosphorylases for the degradation of cellobiose. Cellulases of aerobic bacteria, such as the Cellulomonas species, appear to be tightly bound to their cellulose substrates and appear in the culture medium only in the stationary phase (B6guin et al., 1977). Langsford et al. (1984) reported up to 10 components with carboxymethyl cellase (CMCase)activity as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE). Various cellulolytic species of Bacillus, such as B. subtilis, B. polymyxa, B. brevis, B. licheniformis, and B. cereus, have been described (Priest, 1977; Knosel, 1971; Robson and Chambliss, 1984, 1986, 1987; Thayer, 1978). All of these species are able to hydrolyze carboxymethyl cellulose with cellobiose as the main product. The cellulolytic Pseudomonas species has been studied by Ueda et al. (1952) and the best-studied strain was Pseudomonas fluorescens var. cellulosa (Yamane et al., 1970; Gilbert et al., 1987). Cellvibrio gilvus, which grows well on cellulose, cellobiose, and other cellodextrins, has a complex extracellular cellulase system similar to P. fluorescens. Degradation products are chiefly cellobiose and cellotriose (Storvick et a]., 1963; Alaexander, 1968; Sasaki et al., 1983; Blackall et al., 1985; Wynn and Pemberton, 1986). Actinomycetes form an important part of the microbial community responsible for lignocellulose degradation. Generally, actinomycetes cellulases are inducible extracellular enzymes, which appear to attack cellulose in a way similar to fungal cellulases (Stutzenberger and Kahler, 1986; Stutzenberger and Lupo, 1986; Hagerdal et a]., 1978; Calza et a]., 1985; Bartley et al., 1984; MacKenzie et al., 1984, 1987; MacKenzie, 1986). The thermophillic species of Thermomonspora and Thermoacti-

6

AJAY SINGH AND KIYOSHI HAYASHI

nomyces and the mesophilic species of Streptomyces are the most studied organisms. The cellulolytic system of anaerobic bacteria has gained considerable interest mainly because of the possibility of use in industrial processes. Clostridium thermocellum produces a multicomponent cellulolytic complex termed “cellulosome” (Lamed et al., 1983a,b; Lamed and Bayer, 1988).In young culture of C. thermocellum, cellulosome is tightly bound to the substrate. After the consumption of cellulose, the bound enzymes are released in to the culture medium (Lamed et al., 1985; Cornet et al., 1983). Several endo-l,4-/3-glucanases with M, values ranging from 56 to 125 kDa have been purified from this organism (Wu and Demain, 1988; Ng and Zeikus, 1981). Several other anaerobic bacteria including Acetovibrio cellulolyticus (MacKenzie et al., 1987; Saddler and Khan, 1981), Ruminococcus flavefaciens (Pettipher and Latham, 1979; Latham et al., 1978), Ruminococcus albus (Wood and Wilson, 1984; Stack and Cotta, 1986), and Bacteriodes succinogens (Groleau and Forsberg, 1983) have also been studied with respect to cellulose degradation and cellulase production. Ill. Protein Architecture and Molecular Properties

Cellulose is a homopolymer of about 8000-12,000 glucose units linked linearly by /3-1,4-glucosidic linkages, forming a crystalline unit held together by hydrogen bonding (Brown, 1982). Based on this structure, Reese et al. (1950) postulated the involvement of two different types of enzymes in the degradation of natural cellulose. According to this hypothesis, a C, enzyme renders the crystalline cellulose accessible for hydrolytic attack and a C, enzyme then hydrolyzes the modified cellulose. However, evidence for the existence of the C, enzyme is still obscure. The cellulolytic enzymes have been defined as the enzymes hydrolyzing cellulose, thereby yielding soluble sugars small enough to pass through microbial cell walls (Eriksson, 1967). While considerable progress has been made in the understanding of the nature and function of cellulolytic enzymes, a modification of this definition seems necessary today. In addition to the hydrolytic enzymes, oxidative and phosphorolytic enzymes also participate in cellulose degradation in some microorganisms. There are at least three major types of hydrolytic enzymes involved in the degradation of native cellulose to glucose (Mandels and Weber, 1969). 1. Endoglucanases (endo-l,4-/3-~-glucan-4-glucanohydrolase, EC 3.2.1.4) that randomly attack the cellulose chain and split B-1,4glucosidic linkages.

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MICROBIAL CELLULASES

2. Exoglucanases, present as cellobiohydrolases (exo-l,4-/3-~-glucan 4-cellobiohydrolase, EC 3.2.1.91) that release either cellobiose or glucose from the nonreducing end of cellulose. 3. P-Glucosidase or cellobiase (EC 3.2.1.21) that hydrolyzes cellobiose and other water soluble cellodextrins to glucose.

Phanerochaete chrysosporium produces two types of oxidative enzymes, cellobiose : quinone oxidoreducatase (cellobiose dehydrogenase, EC 1.1.5.1) and cellobiose oxidase for oxidation of cellobiose (Westermark and Eriksson, 1974b; Ayers et a]., 1978; Dekker, 1980; Coudray et al., 1982; Morpeth and Jones, 1986). Some aerobic and anaerobic bacteria produce phosphorylases that are also involved in cellulose degradation. A wide variety of substrates (Wood, 1988) have been used to study the specificity of cellulolytic enzymes (Table 11). The most commonly used substrates have been cotton fiber, Avicel (microcrystalline cellulose), phosphoric acid-swollen cellulose, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), cellobiose, p-nitro-phenyl-P-Dglucoside, and cellodextrins. Properties of individual enzymes are discussed in the following sections. A. CELLOBIOHYDROLASES Cellobiohydrolases degrade cellulose by hydrolyzing cellobiose units from the nonreducing end of the chain and have very limited action on substituted celluloses such as CMC and HEC (Bisaria and Mishra, 1989). They are very active on swollen, partially degraded, amorphous TABLE I1 ASSAYOF CELLULOLYTIC ENZYMESINVOLVEDIN CELLULOSE DEGRADATION Enzyme Endo-l,4-P-glucanase Exo-l,4-P-glucanase

1,4-p-Glucosidase Cellobiose: quinone oxidoreductase Cellobiose oxidase

Substrate Cellulose derivatives (CMC, HEC), cellodextrins Amorphous cellulose, cellodextrins, pnitrophenyl-P-cellobioside, p-nitrophenyl-P-lactoside p-Nitrophenyl-P-D-glucoside, cellobiose, cellodextrins 2,6-Dichlorophenol indophenol (DCIP), cellobiose Cellobiose, cellodextrins

Product Reducing sugars Reducing sugars,cellobiose pnitrophenol p-Nitrophenol, glucose reducing sugars Decrease in absorbance at specific wave length Onic acid

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AJAY SINGH AND KIYOSHI HAYASHI

cellulose; however, oh soluble cello-oligosaccharides, cellotriose to cellohexose, their activity decreases with small chain length. They are not very active against crystalline cellulose but exhibit highly cooperative synergistic action in the presence of endoglucanases (Enari and NikuPaavola, 1987; Bisaria and Mishra, 1989).Avicel, with a degree of polymerization (DP) of about 200 has more chain ends accessible to the enzyme and is degraded more rapidly than the cotton fiber [DP 10,000). Cellulases are characterized by a multiplicity of enzyme components whose exact number varies from one organism to another. Heterogeneity of the cellobiohydrolase component from T. reesei and Penicillium pinophilum has been studied in detail [Wood et al., 1988; Wood and McCrae, 1986a). Two immunologically distinct cellobiohydrolases, CBH I and CBH 11, have been detected in the extracellular medium of T. reesei using polyclonal antibodies (Fagerstam and Pettersson, 1979). Both are glycoproteins differing in the amount of carbohydrate attached to the protein and also in amino acid composition (Enari and NikuPaavola, 1987; Wood et al., 1988). The nature and origin of these isoenzymic forms are not yet completely understood. However, these differences could be due to genetic factors, partial proteolysis, or differential glycosylation (Coughlan, 1985). CBH I composes the major part [about 60%) of the cellulolytic enzyme mixture synthesized by T. reesei. Its gene has been cloned and sequenced [Teeri et al., 1983; Chen et al., 1990). It codes for a polypeptide with a corresponding M, of 58 kDa. When two different mutants, T. reesei L27 and T. reesei QM 9414, were compared, the data for gene sequence were consistent with the primary structure of CBH I protein [Fagerstam et al., 1984). Purification of CBH I has been attempted by several researchers (Nummi et al., 1983; Beldman et al., 1985; Okada eta]., 1986; Hostomska and Mikos, 1984; Odegaard et al., 1984).Purified CBH I has been found to exhibit pronounced heterogeneity (Gum and Brown, 1977), which has been specified as 0- and N-linked glycosylation (Salovuori et al., 1987; Perera et al., 1990). Purification data also indicate considerable differences in the M, of the purified proteins. However, relative proportions of amino acids present [Table 111) in these proteins are quite comparable (Gum and Brown, 1977; Shoemaker et al., 1983; Odegaard et al., 1984). Molecular weight and carbohydrate content of cellulases and P-glucosidase from T. reesei are presented in Table IV. The second cellobiohydrolase, CBH 11, which splits cellobiose from cellulose, has also been purified and characterized (Fagerstam and Pettersson, 1979). Its physicochemical properties, such as the isoelectric point and M,, are similar to those of EG I, a form of endoglucanase.

9

MICROBIAL CELLULASES TABLE 111 AMINOACIDCOMPOSITION OF CELLULASE COMPONENTS FROM Trichoderma reesei CBH I Amino acid Ala Arg Asx CYS Glx GlY His Ile Leu LYS Met Phe Pro Ser Thr TYr TrP Val

EG I

EG 111

A

B

C

D

E

D

5.8 1.8 11.3 4.8 8.2 12.5 1.0 2.4 5.6 2.6 1.2 3.0 5.6 11.3 11.5 4.8 1.8 4.6

7.1 2.1 12.4 4.7 10.0 15.1 1.0 2.1 6.1 2.9 1.5 3.4 5.0 10.8 12.7 5.0 2.4 5.3

5.9 1.8 12.7 4.7 7.0 11.0 2.0 2.9 5.4 2.1 2.2 2.2 5.6 13.5 10.3 5.3 1.6 4.5

7.3 1.4 12.7 3.3 6.6 10.7 1.2 3.8 5.7 1.6 1.2 2.1 7.6 11.8 11.1 4.0 2.6 5.2

7.0 2.5 12.3 3.0 8.0 10.6 1.2 5.3 5.8 1.5 1.0 3.2 4.7 10.5 11.0 3.5 2.8 5.5

8.0 2.8 12.3 3.0 8.5 11.8 1.5 4.8 5.2 1.7 0.7 3.0 5.8 9.6 10.0 3.3 2.5 5.3

Note. Values of amino acids are given as percentage of total residues. Reference A (Bhikhabhai et al., 1984),B (Odegaardet al., 1984),C (Shoemakeret al., 1983),D (Shoemakerand Brown, 1978),E (Saloheimo et al., 1988).

Thus, a methodological problem has long hindered the purification and characterization of this enzyme. This problem has been solved by using affinity chromatography on thiocellobiose coupled to Affigel (van TilTABLE IV MOLECULAR WEIGHT AND CARBOHYDRATE CONTENT OF CELLULASES AND p-GLUCOSIDASES FROM Trichoderma Reesei Enzyme CBH I EG I EG 111 p-Glucosidase

Mr ( m a 1

Carbohydrate (%]

64.0 50.3 55.0 44.7 34.5 42.2 76.6 79.7

6.0 7.2 11.0 12.0 15.0 18.0 0 1.3

Reference Bhikhabhai et al. (1984) Odegaard et al. (1984) Shoemaker et 01. (1983) Hakansson et al. (1979) Odegaard et al. (1984) Salovuori et al. (1987) Odegaard et al. (1984) Chirico and Brown (1987)

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AJAY SINGH AND KIYOSHI HAYASHI

beurgh et al., 1984) or immunoadsorption (Niku-Paavola et al., 1986). Its gene, cbh2, has also been cloned and sequenced and codes for a 471-amino acid protein whose composition is identical to that of purified CBH I1 (Bhikhabhai et al., 1984; Teeri et al., 1987; Chen et al., 1987). CBH I and CBH I1 lack any apparent homology in their amino acid sequences (Chanzy et al., 1983; Teeri et al., 1987), and while CBH I binds preferentially to crystalline regions, CBH I1 binds to both the crystalline and amorphous regions of cellulose. This reflects the differences in the active surfaces of the enzymes. Although CBH I and CBH I1 from T. reesei QM9414 show some common features, such as binding to the same affinity column and high activity toward microcrystalline cellulose (van Tilbeurgh et a]., 1984; Fagerstam and Pettersson, 1980), the modes of action of the two enzymes seem to differ considerably. The reaction patterns toward 4-methylumbellyferyl-~-~-glucoside, MeUmb(Glc),, and MeUmB(Glc),, are totally different from the two enzymes. With MeUmb(Glc), as substrate no MeUmb(Glc), could be observed as a reaction product from CBH I hydrolysis (van Tilbeurgh et al., 1982, 1986),while it is the main product from CBH I1 hydrolysis. Further studies are needed to reveal the nature and functions of CBH I and CBH 11. Characterization studies require pure enzymes because of strong synergistic interactions between different types of cellulases, and even traces of activities other than those expected can obscure the results.

B. ENDOGLUCANASES Endoglucanases (CMCases)hydrolyze CMC or swollen-cellulose in a random fashion; as a result, there is a rapid decrease in chain length and a slow increase in reducing groups. They also act on cellodextrins; their action on cellodextrins decreases as the chain length decreases (Wood, 1985; Bisaria and Mishra, 1989). Three different endoglucanases, i.e., EG I, EG 11, and EG 111, are produced by T. reesei, which also occur in multiple forms (Biely et al., 1985; Kammel and Kubicek, 1985; Labudova and Farkas, 1983; Messner et al., 1988; Hrmova et al., 1986). However, there are different views regarding the classification, nature, and functions of various isoenzymic forms of endoglucanases because of controversies in the values of M,, and carbohydrate contents of different enzymes present in any one cellulase system (Bbguin, 1990). The major endoglucanase component, EG I (previously known as Endo-11; Hakansson et al., 1979), represents approximately 5-10% of the secreted proteins in T. reesei QM9414 cultures. The enzyme has

MICROBIAL CELLULASES

11

been isolated and characterized in several laboratories (Henrissat et al., 1985; Bhikhabhai et al., 1984; Niku-Paavola et al., 1985; Penttila et al., 1986; Saloheimo et al., 1988; Nieves et al., 1990; Luderer et al., 1991). The M, of EG I glycoprotein has been reported to be from 43 to 55 kDa by different researchers (Henrissat et al., 1985; Niku-Paavola et al., 1985). The nature of carbohydrate residue is still unclear. According to the amino acid (aa)sequence, six putative N-glycosylation sites would be present (at aa 78,164,204,208,281,and 394). The size of glycosylated fragments was not compatible with the usual (Man) ,(GlcNAc), or (Man),(GlcNAc), structure (Salovuori et al., 1987). It has also been reported that 70% of EG I carbohydrate was 0-glycosidically linked (Salovuori, 1987), which indicates less occupation of the resulting Nglycosylation sites in EG I. The N-terminal sequence of EG I indicates considerable sequence homology between EG I and CBH I in spite of the clear differences in substrate specificity (Bhikhabhai and Pettersson, 1984). Since the enzyme is comparatively resistant to proteolysis (Luderer et al., 1991) and enzymatic deglycosylation (Kubicek et al., 1987), postsecretional modification appears to contribute little to the high number of multiple forms of endoglucanases (Sheir-Neiss and Montenecourt, 1984; Farkas et al., 1982; Biely and Markovic, 1988; Hayn and Esterbauer, 1985).However, Hakansson et al. (1979) found no detectable glycosylation of purified 50-kDa EG I, thus pointing to a possible postsecretional modication. A second endoglucanase, EG 111, has also been characterized by several researchers (Saloheimo et al., 1988; Beldman et al., 1985; Bhikhabhai et al., 1984; Stahlberg et al., 1988). Multiple forms of EG I11 have been purified exhibiting M, values of 48, 48, and 37 kDa with corresponding PIS of 5.4, 5.7, and 4.8 (Stahlberg et al., 1988; Saloheimo et al., 1988).Two other proteins exhibited amino acid composition closely resembling those reported for EG I1 and EG IV (Shoemaker and Brown, 1978). The nature of third EG 111 (M, 48 kDa, PI 5.1) has not yet been identified. However, a fingerprint very similar to that of EG I11 (M, 48 kDa, PI 5.7) has been obtained using CNBr-peptide mapping (Kubicek, 1992). This indicates that it is a very closely related subspecies. A number of other endoglucanases have also been isolated from T. reesei cultures that could not be identified as either EG I or EG 111. Bhikhabhai et al. (1984) purified a 48-kDalpI 4.5 endoglucanase which differs from EG I11 with respect to its amino acid composition and Nterminal sequence. Its carbohydrate content and amino acid composition are sufficiently unique to suggest that this protein is not related to either EG I11 or EG I. It could be the product of an additional EG gene

12

AJAY SINGH AND KIYOSHI HAYASHI

whose isolation is required to characterize this protein. Similarly, NikuPaavola et al. (1985) purified a 43-kDa/pI 4.0 endoglucanase with low (2-7%) carbohydrate content and postulated that it constitutes a proteolytic fragment. Other researchers (Gong et al., 1979; Hakansson et al., 1978; Hong et al., 1986; Ulker and Sprey, 1990) have also purified lowmolecular-weight endoglucanases (20-25 kDa) which lack glycosylation. Their formation appeared to depend strictly on the presence of cellulose in the medium, unlike other cellulases [Messner et al., 1988; Hrmova et al., 1986). However, the nature of these proteins is still unclear, These enzymes are rapidly degraded by fungal extracellular proteases and constitute only a very small portion of the secreted proteins (Kubicek, 1992). Further studies are needed in order to explore the nature of these enzymes. The cellulolytic enzyme system of bacteria seems to be quite complex. Yamane et al. [1970) identified two extracellular (A and B) and one cell-bound (C) endoglucanase components of bacteria. P. fluorescens var. cellulosa, which were purified and characterized. All three of these endoglucanases hydrolyzed a variety of substances, including crystalline and amorphous cellulose and cellodextrins. The mode of action toward CMC appears to be similar to that shown by fungal endoglucanases. Yoshikawa et al. (1974) later reported that components A and B were not homogeneous. In subsequent work by Gilbert et al. (1987) on the molecular biology of the cellulase complex from P. fluorescens, it was shown that four distinct isolated endoglucanase genes exhibited no homology with one another. On the other hand, Wolff et al. (1986) could isolate only one endoglucanse gene from this organism, suggesting that the multiplicity of endoglucanases from this species obtained in earlier studies is a result of post-translational modification of a single gene product. In another widely studied bacterial species, Cellulomonas fimi, Langsford et al. (1984) noted up to 10 components with CMCase (endoglucase) activity as determined by nondenaturing polyacrylamide gel electrophoresis. Some of the active components were glycosylated. The glycosylated enzymes bind strongly to Avicel and are stabilized by this substrate, whereas enzymes free in solution are unstable and are altered by proteolysis and deglycosylation. Studies were focused on the two tightly substrate-bound enzymes designated CB 1 and CB 2 (Gilkes et al., 1984). Both components were glycoproteins with mannose as the sole glycosyl component (BBguin et al., 1987). However, the mode of action and substrate specificities were found to be different. Both hydrolyze CMC but with different specific activities (370 and 85 units/ mg protein for CB 1 and CB 2, respectively). This finding led researchers

MICROBIAL CELLULASES

13

to characterize CB 1 as an endoglucanse and CB 2 as an exoglucanase (Gilkes et al., 1984; Warren et aJ., 1987). There are relatively few reports (Sashihara et al., 1984) of Bacillus sp. producing endoglucanses. The best characterized example is B. subtilis DLG (Robson and Chambliss, 1987). The endoglucanse was confirmed by measuring the ability of the enzyme to reduce the viscosity of a CMC solution, which was similar in fashion to T. reesei EG I. However, the B. subtilis enzyme exhibited almost twice the specific activity of T. reesei EG I when trinitrophenyl CM was used as the substrate. The DNA sequence of B. subtilis DLG endoglucanase gene indicates that the enzyme is coded for a single sequence at the Nterminal end (Robson and Chambliss, 1987). Such a signal is believed to be necessary for an efficient export of protein in most organisms, and is usually composed of a short sequence of charged amino acids followed by a longer stretch of strongly hydrophobic amino acids (Perlman and Halvorson, 1983). The DNA sequence also revealed that the endoglucanase gene of B. subtilis has a coding capacity for an approximately 55-kDa protein. However, the isolated extracellular endoglucanase was only 35.2 kDa. The apparent differnece wasmost likely due to post-translational modification (Robson and Chambliss, 1986). C. P-GLUCOSIDASES The least studied of the enzymes involved in cellulose hydrolysis are P-glucosidases. They represent only a very small portion (about 1%) of the total extracellular proteins secreted by T. reesei. p-Glucosidases hydrolyze degradation products of cellulose to glucose. They have maximum activity toward cellobiose and their action on cellodextrins decreases as chain length increases. p-Glucosidases are characterized by their transferase activity, retention of the P-configuration of glucose on hydrolysis, inhibition by low concentrations of gluconolactone, and high activity toward cellotriose and less activity toward larger cellodextrins (Bisaria and Mishra, 1989). The purification of p-glucosidases from extracellular culture filtrates, intracellular extracts, and plasma membranes of organisms has been reported by several groups (Hofer et al., 1991; Bergham and Pettersson, 1974; Enari et al., 1981; Gong et al., 1977; Chirico and Brown, 1987; Schmid and Wandrey, 1987; Jackson and Talburt, 1988; Wilhelm and Sahm, 1986; Singh et al., 1990d; Umile and Kubicek, 1986). A comparison of the amino acid composition of P-glucosidases from T. reesei is given in Table V. Two immunologically distinct forms (I,Mr 50 m a , pl 6.1; II,Mr 35 kDa, pl 5.7) of extracellular P-glucosidases have been re-

AJAY SINGH AND KIYOSHI HAYASHI

14

TABLE V OF @-GLUCOSIDASES FROM Trichoderma reesei AMINOACIDCOMPOSITION

Amino acid Ala Asx CYS Glx GlY His Ile Leu LYS

Met Phe Pro Ser Thr TYr TrP Val

Bergham and Pettersson (1974) 10.1 3.4 12.5 1.3 6.7 10.8 1.2 4.8 7.1 3.0 1.3 2.6 5.6 8.2 7.1 3.5 2.1 8.2

Odegaard et al. (1984) 10.3 3.3 13.4 0.6 7.2 11.4 1.4 4.3 6.6 2.9 1.0 3.9 7.0 9.5 7.8 3.7

ND 5.1

Chirico and Brown (1987) 10.0 3.0 12.5 0.8 6.9 10.5 1.2 4.3 7.1 3.3 1.0 2.5 5.0 9.6 6.8 3.4 3.0 9.0

Note. Values of amino acids given as percentage of total residues.

ported by Hofer et al. (1991).They provided evidence that partial proteolysis of the 80-kDa P-glucosidase results in the formation of two different protein fragments. It is also very likely that some of the pglucosidases described earlier (Bergham and Pettersson 1974; and Enari et al., 1981) are proteolytic fragments of the 80-kDa P-glucosidase. Intracellular and plasma membrane P-glucosidases have also been identified. However, the exact genetic and biochemical relationship among these different P-glucosidases is not yet clear. It has been suggested that extracellular enzyme may result from the release of intracellular or cell wall-bound enzyme to the outside medium upon autolysis. Although the role and intracellular location of this enzyme are still unclear, it has been proposed to function in controlling the accumulation of cellulase inducers (Inglin et a]., 1980; Loewenberg, 1984). Enari et al. (1981) purified the P-glucosidase of T. reesei that showed an apparent molecular weight of 35 kDa on gel chromatography. The M, calculated from SDS electrophoresis of the purified P-glucosidase was 73 kDa. The discrepancy was most likely due to the glycoprotein nature of the enzyme (0.1 mg sugar/mg protein). Glycoproteins are known to behave abnormally in SDS electrophoresis. Another p-

MICROBIAL CELLULASES

15

glucosidase component of 130 kDa was also observed, but this component represented only 2% of the total p-glucosidase activity. Similar results have been reported for T. viride p-glucosidases (Bergham and Pettersson, 1974; Gong et al., 1977). Species of Aspergillus and Sporotrichum seem to produce pglucosidases of higher molecular weight than does Trichoderma. Purified p-glucosidase from A. niger has a molecular weight of 150 kDa and consists of two polypeptide chains that can be separated by SDS-PAGE (Enari et al., 1981). Aspergillus oryzae has been reported to form an enzyme with M, 218 kDa, which also consists of two polypeptide chains (Mega and Matsushima, 1979). Meier and Canevascini (1981) isolated two different p-glucosidases from S. thermophile, one with M, 440 kDa, having only aryl-P-glucosidase activity] while the other, with M, 40 kDa, having cellobiase activity with only low activity toward arylP-glucosides. Kashiwagi et al. (1991) reported the isolation of a p-glucosidase gene from C. gilvus and the purification and properties of the cloned pglucosidase. The purified enzyme has M, 82 kDa and PI 6.0. It attacked cellobiose very slowly, compared to its activity toward higher cellooligosaccharides. The same research group recently reported the nucleotide sequence of the p-glucosidase gene from C. gilvus (Kashiwagi et al., 1993). The P-glucosidase from C. gilvus was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 base pairs encoding 854 amino acid residues with a characteristic signal peptide. The p-glucosidase of C. thermocellum was shown to be very heat stable (Ait et al., 1979). The purified enzyme has M, 50 kDa and PI 4.6. However, the affinity of the enzyme for cellobiose and p-nitrophenylP-D-glucoside (PNPG) was very low with a K, value of 2 X lop3M for PNPG and 8.3 x low2M for cellobiose. It was postulated that the pglucosidase has an unspecific action and may participate in hydrolyzing saccharides having other types of p-structure linkages. The cellobiose and cellodextrins produced may instead enter the cell where they are metabolized by the specific cellodextrin and cellobiose phosphorylases to yield glucose-1-phosphate and free glucose as has been demonstrated by Alaexander (1968). IV. Structural and Catalytic Properties

A. STRUCTURAL ORGANIZATION A comparison of the sequence of about 50 fungal and bacterial cellulase genes and of some other polysaccharide hydrolases has offered evidence that enzyme proteins acting on insoluble substrates like cellu-

AJAY SINGH AND KIYOSHI HAYASHI

16

lose are composed of separate domains, which allow a spacial separation of the sites involved in substrate recognition and enzyme activity (Beguin, 1990; Kubicek, 1992). A remarkable sequence homology (Fig. I) can be detected between the N-terminus of EG I11 and the N- or Ctermini of three other T. reesei cellulases (Teeri et al., 1987). Perhaps the most interesting feature emerging from the sequence analysis of four cellulase genes in T. reesei (Knowles et al., 1988) is the presence of a short conserved region at either the N-terminal (CBH I, EG 111) or C-terminal (CBH I, EG I) end of the corresponding protein (blocks A and B, Fig. 2). The finding that partial proteolysis by papain can cleave CBH I and CBH I1 into an enzymatic active “core,” and a cellulose adsorbing “tail” domain, gave evidence for the domain organization of the T. ressei cellulases (van Tilbeurgh et a)., 1986; Tomme et al., 1988). Small-angle X-ray scattering studies revealed a rather unusual tadpolelike shape of both CBH I and CBH 11, with a large isomorphic head and a flexible terminal extension (Schmuck et al., 1986; Abuja et al., 1988a,b). Block A (about 30 aa) is rich in glycine residues, which are often found in turns in protein structures and cysteine residues, the positions of which are strictly conserved in all four cellulases (Knowles et al., 1988). These features indicate that Block A might fold into a small separate domain stabilized by disulfide bridges. On the other hand, the block B sequence, which always joins the A block to the main body of the enzyme, is rich in hydroxyl amino acids and proline (Lehtovaara et al., 1986; Knowles et al., 1987). In CBH I, this region has been shown to be heavily glycosylated (Allen, 1983). There is also evidence that in CBH I1 most of the glycans are located in this particular region (Fagerstam et al., 1984). Crystallization of either of these enzymes has not yet been possible. However, CBH I1 was crystallized after proteolytic removal of the AB region (Bergfors et a]., 1989). The removal of the AB region from CBH I and CBH I1 has been shown to decrease significantly

8

1

7

n

n

n

n -

CBH I ( C ) H Y l G Q C G G I 1 G Y l S G l Q T V l C l A S l G l T T l C l Q V L l N l P l Y Y l S I

I

I

I

I

I

I

I

I

I

I

l

l

I

C B H I I ( N ) V W I G Q C G G I 0 N WIS G l P T C l C l A S l G l S T l C l V Y S l N l D l Y Y l S I

EG I

I

I

I

I I

I I

I I

I

l

l

I

( C ) H WIG Q C G G I 1 G Y l S G I C K T I C I T S l G l T T l C l Q Y S l N l D l Y Y l S I

I

I

I

I

I

I

I

I

I

I

l

l

I

EG I I I C N ) V W I G Q C G G I 1 G WIS G l P T N I C I A P I G I S A l C l S T L l N l p l y Y l A I

u

u

u

u -

FIG. 1. Comparison of amino acid sequence homology among four cellulase components of Trichoderma reesei. The residues conserved in all four proteins are blocked.

17

MICROBIAL CELLULASES Trichoderma reesei

CBH

I

lntron

ss H2N I I 1

CBHll

EG 1

1

-I+

H

m?j+:l H

I

B A

ism

1

H

E G 111

1

Clostridi um thcrmocell u m

EG A EG 0 EG

COOH

Ser pro

-,-I Thrid; ... ...

r [

D

Cellulomonas fimi

50% homologous Pro-Thr rich

-

EN G EX G

I

*. ......

Aspergillus nigpr ( Glucoamylase)

Ser -Thr -rich G1

[

62

1

.

Starch binding domain

I ,.,...:.'i FH C...

..... ::: r:.

FIG.2. Structural organization of the cellulase genes from Trichoderma reesei, Clostridiurn thermocellum, and Cellulornonas fimi and glucoamylase genes from Aspergillus niger. The terminal domains and the putative hinge regions rich in hydroxyl amino acids and proline are indicated. The catalytic domains are represented by open boxes and the intron positions are shown by solid bars.

their affinity to microcrystalline cellulose, Avicel, while the activity on soluble substrates is retained (van Tilbeurgh et a]., 1986).These results suggest that the terminal regions are at least in part responsible for the binding of the enzyme to insoluble, crystalline cellulose and that the hydrolytic site is located in the larger domain (Knowles et a]., 1988). Thus, the AB region forms a functional domain wherein the A part interacts with cellulose and the B part provides a flexible arm connect-

18

AJAY SINGH AND KIYOSHI HAYASHI

ing the catalytic and adsorptive regions of the enzyme (Tomme et al., 1988;Johansson et al., 1989). The results obtained by Stahlberg et al. (1988)show that this also was the case with EG 111. While the binding domain is composed of two clearly distinguishable regions, A and B, the main part of the enzyme is the core protein containing the active site. According to van Tilbeurgh et al. (1985),the specificity of the EG I11 shows some similarity in the fact that both EG I11 and CBH I1 have their homologous domains at the N-termini, in contrast to CBH I and EG I, which have similar sequences at the C-termini. In contrast to EG I, no transferase activity was observed for EG 111.Differences in transferase activities among the five endoglucanases isolated from S. pulverulentum were also demonstrated by Streamer et al. (1975). Detailed knowledge of the three-dimensional structure of cellulases is not available, mainly because of the failure to crystallize these proteins. However, the crystallization of the catalytic core protein of CBH I1 of T. reesei (Bergfors et al., 1989)has revealed that it consists of a sevenstrand, singly wound parallel alp barrel. Extended loops from the barrel produce a large channel for cellulose binding. The active site is located at the C-terminus of the p-sheet and could be identified clearly using data collected with an inhibitor diffused into the crystal. It is present in the tunnel, through which the cellulose threads and two aspartic acid residues form the catalytic residue. Comparable investigations for other cellulase components are still lacking. It has been proposed that the various cellulases secreted by T. reesei may differ in the number of surface loops forming the active site (Kraulis et al., 1989).The tunnellike structure may be typical of exoglucanases (cellobiohydrolases) because it allows an attack only from the end of the cellulose chain. The primary structure of cellulases from various bacteria and fungi, deduced from the nucleotide sequences, is diverse. However, the structural organization of the Cellulomonas fimi cellulases closely resembles that of the T. reesei cellulases (Fig. 2). A region of about 100 aa at the N-terminus of the endoglucanase (ENG) and at the C-terminus of exoglucanase (EXG) is 50% conserved in the two enzymes and is linked to the rest of the protein by a strictly conserved stretch of alternating Pro and Thr residues (O’Neill et al., 1986;Wong et al., 1986).Extensive amino acid homologies among C. thermocellum endoglucanases EG A, EG B, and EG D are also found in the terminal region. A direct repeat of 24 aa is found in the C-termini in all three enzymes. The proline-hydroxy amino acid sequences have also been shown to be conserved in the ENG gene of Bacillus sp. (Fukumori et al., 1986a,b)and CEL I

MICROBIAL CELLULASES

19

of Microbispora bispora (Yablonsky et al., 1988). The function of the terminal repeats in these C. thermocellum endoglucanases is not clear; however, they may be related to the binding of enzymes to the large glycosylated subunits of cellulosome (S, or SL),which may also have a role similar to that of the T. reesei block A tail (Bbguin et al., 1985; Grepinet and Bbguin, 1986; Joliff et al., 1986a,b). Fungal glucoamylases have been shown to bear functional similarity to cellulases in terms of cleavage of glucosidic linkages (Ueda, 1981; Svensson et al., 1983). Glucoamylase G 1 from A. niger (Fig. 2) has a region of approximately 100 aa at its C-terminus that is responsible for binding to insoluble starch but apparently is not required for the hydrolysis of small soluble substrates (Svensson et al., 1986). This terminal domain is linked to the rest of the protein by a glycosylated region rich in hydroxyl amino acids and proline similar to the putative hinge region in the T. reesei cellulases. The small G 2 form of this glucoamylase is generated by the proteolytic removal of the starch binding domain from G 1. It seems that the proteolytic removal of the substrate binding domain from both cellulase and glucoamylase may be an important mechanism for the modification of their substrate specificities during the course of hydrolysis of complex insoluble substrates (Knowles et al., 1988).Once the insoluble substrate has been degraded to short, soluble oligosaccharides, enzymes with higher specific activities toward the more soluble substrates would be available. In this way the microorganisms can increase the diversity of enzymes encoded by a limited number of genes. B. GLYCOSYLATION

Most of the fungal and a number of bacterial cellulases are glycoproteins with sugars attached at asparagine (N-linked)or serine and threonine (0-linked) residues. Most of the 0-glycosylation occurs in the hinge region of the protein, whereas N-glycosylation is restricted to the core domain (Kubicek, 1992). The carbohydrate content of cellulases varies from 1 to 10% (Enari and Niku-Paavola, 1987) and the principal sugar in all the cases studied is mannose, but other sugars such as glucose, galactose, xylose, N-acetyl glucosamine, and galactosamine have also been detected (Salovuori et al., 1987). The presence of carbohydrate moieties in microbial cellulases suggests their functional significance in cellulolysis. Because of the many hydroxyl groups available, the carbohydrates can hydrogen bond with the substrate and may also increase the solubility of the protein (Darnel1et al., 1986). Affinity

20

AJAY SINGH AND KIYOSHI HAYASHI

of mannan polymer to cellulose crystals has been demonstrated, which suggests the functional role of mannose-rich carbohydrates of cellulases in binding of crystalline cellulose (Chanzy et al., 1979, 1984). When produced in E. coli in nonglycosylated form, the CBH I of T. reesei (Knowles et al., 1988) and the two cellulases of C. fimi (Gilkes et a]., 1984) retained their activities on soluble substrates. The core proteins of CBH I and CBH I1 lacking the heavily 0-glycosylated terminal tail were found still active on the cello-oligosaccharides (van Tilbeurgh et al., 1986). These results demonstrate that glycosylation is not required for the hydrolysis of small soluble substrates. The heavily glycosylated region is localized near the C-terminus of CBH I and N-terminus of CBH I1 (Tomme et al., 1988; McHale and Coughlan, 1981). EG I and EG I1 also contain N- and 0-linked carbohydrates (Bhikhabhai and Pettersson, 1984; Pentilla et al., 1986). In CBH I, three N-glycosylation sites are occupied (Gum and Brown, 1977; Salovuori et al., 1987) and the N-linked carbohydrate resembles the highly conserved (Man),(ManNAc), structure found in other organisms (Salovuori et al., 1987). 0-Glycosylation lacks defined target sequence. The presence of mono-, di-, tri-, and tetrasaccharide chains has been revealed by several researchers (Kruszewska et al., 1989).In microsomes of T. reesei, attachment of these oligosaccharides to protein has also been observed. The function of 0-glycosylation has been discussed with respect to protection against proteolysis or adsorption to cellulose; however, it may also be required for efficient secretion of cellulases (Messner and Kubicek, 1988; Kubicek et al., 1987). Glycosylation has also been reported to play an important role in the stability of extracellular cellulases in T. reesei. N-Linked glycosylation appears to stabilize the structure of the cellulases and thereby protect them from enzymatic attack during secretion (Merivuori et al., 1985b; Sanchez et al., 1982; Onishi et al., 1979; Speake et al., 1981). An optimum level of N-linked glycosylation is required for efficient binding to and subsequent hydrolysis of crystalline cellulose (Pentilla et al., 1988). It has also been shown by these authors that native CBH I1 of T. reesei bound more effectively onto crystalline cellulose than the highly N-glycosylated enzyme produced by a recombinant yeast (Pentilla et al., 1988). While native CBH I and CBH I1 hydrolyzed highly crystalline cellulose, the recombinant enzymes were able to degrade less crystalline cellulose with a reduced efficiency. While N-linked glycosylation appears to be involved in imparting a specific conformation to the cellulases for binding to and hydrolysis of crystalline cellulose, 0-linked glycosylation seems to be required for secretion of the active cellulase.

21

MICROBIAL CELLULASES

C. SYNERGISTIC MECHANISM

It is now a well-established fact that degradation of crystalline cellulose by microorganisms is carried out by a multicomponent enzyme complex wherein the individual components interact in a synergistic way to convert the insoluble substrate into glucose. Individual enzyme components have been considered unable to break down the crystalline cellulose to any substantial extent. However, when the components are recombined they act in a synergistic manner to render the cellulose soluble (Coughlan, 1985; Finch and Roberts, 1985). Eriksson and Wood (1985) have shown that the individual exoglucanases, endoglucanases, and P-glucosidases degrade practically none of the crystalline cellulose, while a mixture of the three enzymes causes extensive hydrolysis. Wood and McCrae (1979) have demonstrated the synergistic effect beween cellobiohydrolase and endoglucanases and also between cellobiohydrolase and P-glucosidase (Table VI). A similar synergism has been demonstrated between exo- and endoglucanses from F. solani (Wood and McCrae, 1979; Wood, 1980), S. pulverulentum (Streamer et al., 1975), Trichoderma viride and P. funiculosum (Selby, 1969), Taleromyces emersonii (McHale and Coughlan, 1980),and Sclerotium rolfsii (Sadana and Patil, 1985). Cross-synergism between cellobiohydrolase from one fungus with endoglucanse to another has also been demonstrated (Wood, 1975). However, the extent of cooperation seems to vary considerably (Table VII). Thus endo- and exoglucanses from the fungi F. solani, P. funiculosum, and T. koningii can be exchanged and give rise to extensive solubi-

TABLE VI COTTON-SOLUBILIZING ACTIVITY OF CELLOBIOHYDROLASE, AND p-GLUCOSIDASE OF ENDOGLUCANASE, Trichoderma reesei Cotton solubilization Enzyme

(%I

Culture filtrate Cellobiohydrolase (CBH) Endoglucanase (EG) p-Glucosidase (BG) CBH + EG CBH + BG CBH + EG + BG

71 1 1 0 53 20 72

22

AJAY SINGH AND KIYOSHI HAYASHI TABLE VII SYNERGISTICEFFECTSOF CELLOBIOHYDROLASEAND ENDOGLUCANASE FROM DIFFERENT SOURCES IN SOLUBILIZING COTTON Source of cellobiohydrolase

Source of endoglucanase

Solubilization of cotton (oh]

F. solani F. solani T. koningii T. koningii P. funiculosum T. koningii P. funiculosum

F. solani T. koningii T. koningii F. solani T. koningii M. verrucario M.verrucaria

59 51 54 79 51 20 11

lization of cellulose. However, when exoglucanase from these fungi acted together with the endoglucanases of rumen anaerobic bacteria Ruminococcus albus and B. succinogenes, no synergistic effect was obtained (Wood, 1983).Wood (1988)demonstrated a synergistic action between cellulase components from the anaerobic thermophillic bacterium C. thermocellum and T. koningii (Table VIII). Clostridium thermocellum cellulase cannot extensively break down cotton cellulose in the absence Ca2+or dithiothreitol (DTT). However, a combination of C. thermocellum cellulase and T. koningii cellobiohydrolase can break down cellulose even in the absence of Ca2+or DTT. Apart from endo/exo-type synergism, two unusual types of synergism have also been reported. Combined action of Irpex lacteus endoglucanases causes considerable hydrolysis of crystalline cellulose (Kanda et al., 1976).The two endoglucanases differ in the way they attack soluble cellulose. Another type of synergistic action exo/exo-type was reported TABLE VIII HYDROLYSIS OF COTTON BY CELLULASES OF Clostridium thermocellum AND Trichoderma koningii C. thermocellum cellulase Addition

C. thermocellum cellulase

None DTTa CaCI, DTT + CaCl, DTT,dithiothreitol.

8 0 20

35

+

T. koningii cellulase 40 35 55 68

MICROBIAL CELLULASES

23

by Fagerstam and Pettersson (1980).They found that the hydrolysis of crystalline cellulose by the combined action of CBH I and CBH I1 of T. reesei was more than twice that calculated from the extent of hydrolysis by each of the separate enzymes. Brown and Gritzali (1984)also found that the exo/exo-synergism is important in the intact cellulase system as it degrades crystalline cellulose. The synergistic action of cellulases has been found to depend on the ratio of the individual enzymes, the degree of substrate saturation, and the type of substrate (Henrissat et al., 1985;Woodward et al., 1988). The action of CBH I and CBH I1 and the endoglucanases EG I and EG I1 purified from T. reesei was evaluated against different substrates (Henrissat et al., 1985).Synergism between the endoglucanases and CBH I1 follows the normal patterns for endo/exo type synergism. CBH I degrades the P-D-glucan from barley in a typical endo-patter. With cellulose as substrate, the synergism between EG I or EG I1 and CBH I depends on the structural and ultrastructural features of the substrate. CBH I demonstrates an unusually strong and irreversible binding to cellulose (Reese, 1982;Chanzy et al., 1983).Sprey and Lambert (1983) even suggest that there is a lectin-type binding between cellulases and cellulose substrate. The synergistic action exhibited by CBH I and CBH I1 seems to be due to the mixture of both endo- and exo-type character of the functions of both of these enzymes. A scheme for the hydrolysis of cellulose was suggested by Enari and Niku-Paavola (1987).Accordingly, hydrolysis of insoluble native cellulose is accomplished by the synergistic action of the two enzymes CBH I and CBH I1 (Fig. 3). The endoglucanases participate in the hydrolysis only at a later stage when soluble cellodextrins are hydrolyzed. The synergistic action of purified cellulases from T. reesei in the hydrolysis of cellulose was found to decrease with increasing substrate concentration, depending strongly on the type of cellulose used, and was maximum on crystalline cellulose (Nidetzky et al., 1993).However, the activity of individual enzymes was greatest on amorphous cellulose. The binary combinations CBH I/EG I11 and CBH I/CBH I1 exhibited a greater degree of synergism on crystalline cellulose, Synergistic action between two immunologically distinct cellobiohydrolases (I and 11) from P. pinophilum has also been demonstrated (Wood and McCrae, 1986a).The maximum rate of attack on Avicel was exhibited when the two enzymes were mixed in the ratio 1 :1. It was suggested that they may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of nonreducing groups that would exist in cellulose. Only CBH I1 from P. pinophilum acts synergistically with cellobiohydrolases of T. koningii and F. solani

24

AJAY SINGH AND KIYOSHI HAYASHI

CBH I

+ CBH I 1

b GLUCOSE

FIG.3. Suggested scheme for the enzymatic hydrolysis of cellulose.

to solubilize Avicel. CBH I1 showed no synergistic action with the endoglucanases of T. koningii or F. solani, but CBH I did.

D. CATALYTIC MECHANISM Cellulases catalyze the breakdown of the p-1,4-glucosidic linkage. It has been proposed that cellulose hydrolysis occurs by an acid catalysis mechanism at the active site (Knowles et al., 1987).Attempts to identify the active sites of the cellulases have been carried out by chemical modification and inhibition studies as well as sequence homologies among related enzymes (Paice et al., 1984; Paice and Jurasek, 1979; Dinur et a]., 1986). The active sites of many p-glucosidases have been found to involve aspartic acid residues. The results of Clarke and Yaguchi (1985) on the effects of chemical modifications of Schizophyllum commune EG I supported the hypothesis that carboxyl groups are involved in the hydrolysis of cellulose. Limited homologies in the cellulase primary structures to the active sites of hen egg white lysozyme (HEWL) have been noted (Paice et al., 1984; Teeri et a]., 1987). Figure 4 shows the weak homologies that have been proposed between lysozymes and cellulases. A comparison of amino acid sequences between T. reesei cellulase and HEWL indicates little sequence homology; how-

25

MICROBIAL CELLULASES

HEWL S.C.

EG I

C. f . EXG C. f . ENG

I35 I FI E S NIF N T Q A n 133 rJ NI E SIC A ElFlG N 136 SI E I F l N l L v V ~ A ~ E + I 13181 P I E lY A V G I F A I L

+

- -

N T D G S I T D Yl I I P G V K N I T D Yl I +

M K

W D AlTlE P I

I

S N Y Q TlTlA E U

-

HEWL S.C.

EG I

T.r.

CBH I1

135 I n FI E SIN F N T Q A T - 1 N I R N T D G S T - I D Yl 133 1 n 1 I - n I I N I E S I C A E F G N l Q l - I N I I l P l Q l V l K N T -ID Yl I 244 + I I I I I I I I I + LI E I C I N Y A V T l Q l L l N l L l P l N l V l A M Y L l D l A

-

u u u u

U

FIG.4. Comparison of amino acid homologies between hen egg white lysozyme (HEWL) and cellulases. S.C., Schizopyllum commune; C.f., Cellulomonas fimi; T.r., Trichoderma reesei.

ever, the EG I of S. commune has been found to bear a considerable sequence homology with the HEWL and it has been suggested that the catalytic mechanisms are likely to be similar in the two cases (Yaguchi et a]., 1983). Lysozyme is, however, the smallest of the carbohydrate-binding proteins and only one-third of the size of a typical cellulase. Thus it cannot be the only possible model for cellulases. Sequence analysis of different cellulases has revealed that the primary structures of S. commune EG I and T. reesei EG I11 are similar, although the aa sequence at their Ntermini are totally different (Saloheimo et ~ l . 1988). , Currently, in the absence of any definitive evidence amylases provide at least as good a model for the active site of cellulases. The localization of the active site of cellulases is a difficult task in the absence of solved three-dimensional structures. Crystallization of only two cellulases EG D of C. thermocellum (Joliff et a]., 1986b) and the catalytic domain of CBH 11 of T. reesei (Knowles et a]., 1987) have so far been successful. Some recent techniques such as computer-aided modeling and site-directed mutagenesis would also be useful in predicting the location of the active site (van Custem et al., 1987).Availability of such information is expected to lead to the design of newer enzymes with superior rates of catalysis.

26

AJAY SINGH AND KIYOSHI HAYASHI

Although considerably detailed information is now available on the structure and molecular properties of the cellulase components of T. reesei and other organisms, our understanding about the degradation of crystalline cellulose is less complete. Firm knowledge of the kinetics and substrate specificities of individual cellulase components is required to postulate a model for cellulose degradation. The introduction of a low-molecular-weight chromogenic 4-methylumbelliferyl-~-glycoside has eliminated the problem of cross-contamination of enzymes purified to homogeneity (van Arsdell et al., 1987;Pentilla et a]., 1987). There are pronounced differences in the kinetics of glycoside hydrolysis even between CBH I and CBH 11, and EG I and EG 111.In the case of CBHs, few differences are apparent between two enzymes, but differences do occur with higher homologues. CBH I attacks at sites other than the nonreducing end in the higher homologues and hydrolyzes both cellobiosides and lactosides. On the other hand, CBH I1 shows strict substrate specificity, three to four contiguous p-1&linked glycosyl residues being required (Claeyssens et al., 1989;van Tilbeurgh et al., 1989). It has also been deduced that CBH I acts with overall retention of configuration, whereas CBH I1 acts with overall reversion (Claeyssens et a]., 1990).

Endoglucanases act via rentention of their configuration (Claeyssens et a]., 1990).EG I has been classified as a nonspecific endoglucanase because it hydrolyzes not only cellulose but also xylan (Biely and Markovic, 1988).Its hydrolytic reaction proceeds via transfer reactions and the apparent hydrolytic parameters (Kcat,&/Km) increase steadily as a function of the number of glucose residues (Claeyssens et al., 1990).It probably possesses several subunits, each capable of binding a glucose molecule, and the glycosyl intermediates formed during hydrolytic reactions are then transferred to acceptor molecules. V. Cellulase Biosynthesis

A. INDUCTION AND REGULATION

Biosynthesis of cellulases is generally assumed to be subject to biochemical and genetic regulation. Cellulases are inducible enzymes, the best inducers being cellulose, cellobiose, lactose, and sophorose (2-0P-D-glycopyranosyl-D-glucose).Synthetic compounds such as palmitate and acetate esters of disaccharides and thiocellobiose have also been shown to function as inducers of cellulases (Reese et al., 1969;Rho et a]., 1982).In virtually all the microorganisms examined, the synthesis of cellulases is induced by the presence of cellulose and repressed in the presence of glucose. The fact that cellulose is insoluble in water and

MICROBIAL CELLULASES

27

cannot enter the microbial cell suggests that it cannot be the inducing component. This appears to be logical in view of the differences in structures of different natural cellulosic substrates. The most generally accepted view of the induction process is that the organisms produce a basic level or a constitutive amount of cellulase. This gives rise to soluble hydrolysis products of cellulose, which enter the microbial cells and function as inducers (Eriksson et al., 1990). Trichoderma species contain cellulases located on the surface of conidia, which appear to be involved in the initial attack on cellulose (Kubicek, 1987; Kubicek et al., 1987). CBH I1 has been found to be the major conidial bound cellulase in some hyperproducing strains (Messner et al., 1991). A putative cyclic AMP-dependent transcription activator binding region is about 400 base pairs upstream of the start point of the cbh2 gene, and a correlation between cAMP levels and sporulation in T. viride has been reported (Chen et al., 1987; Farkas et al., 1990). The induction by cellobiose appears to be concentration dependent, with high concentration having an inhibitory effect due to the build up of intracellular glucose (Coughlan, 1985; Hrmova et al., 1986). Cellobiose has been found to be an inducer of cellulases in S. pulverulentum, S. thermophile, and Neurospora crassa (Eriksson and Hamp, 1978; Canevascini et al., 1979; Eberhart et al., 1977). However, it does not seem to be an inducer of cellulases in T. reesei, at least not with washed mycelium (Eriksson and Hamp, 1978; Sternberg and Mandels, 1979). A considerable difference between T. viride and S. pulverulentum in regulating the production of cellulase was observed by Eriksson and Hamp (1978). In S. pulverulentum, endoglucanase activity was induced by cellobiose at low concentrations such as 1 mg/liter, while cellobiose at the same or even higher concentrations did not induce endoglucanase activity in T. viride QM6a. Sophorose is the most potent inducer of both exo- and endocellulase synthesis in T. reesei and its mutants (Nisizawa et al., 1971; Mandels et al., 1962; Sternberg and Mandels, 1979). However, the enzyme activity was about half that of the cellulose-induced enzyme. While sophorose is a very efficient inducer in low concentrations, in high concentration it inhibits production of cellulases. This has been explained by assuming that sophorose is enzymatically hydrolyzed to glucose, which inhibits cellulase synthesis (Nisizawa et al., 1972). The de novo induction of cellulase synthesis by sophorose has been proven by the inhibitory effect of the translational inhibitor puromycin on enzyme synthesis (Nisizawa et al., 1971, 1972). Montenecourt (1983) found no particular differences in intracellular cAMP levels between induced and noninduced cultures following exposure to sophorose and the induction of endoglucanase .

28

AJAY SINGH AND KIYOSHI HAYASHI

The intracellular level of some key metabolites at the time of cellulase induction in a T. reesei mutant was investigated by Farkas et al. (1987). The period of cellulase induction was preceded by a transient peak of ATP concentration and accompanied by an increased cAMP level. Addition of substrate, lactose, briefly stopped endoglucanase synthesis, increased ATP and glucose-6-phosphate concentration, and decreased the cAMP levels. Endoglucanase secretion again resumed after 2 hr. The increased levels of cAMP at the time of cellulase synthesis indicate that it may play a role in modulating catabolite repression (Cochet et al., 1984). Interestingly, T. reesei strains synthesizing higher cellulase activity in the medium display higher steady-state levels of CBH I- and CBH 11-mRNA in the mycelia, suggesting that transcription may limit cellulase production in the lower producer strain (Messner and Kubicek, 1991; El-Gogary et al., 1989).The mechanism of activation of cellulase gene transcription is a challenge in our understanding of how cellulase biosynthesis is regulated. Cellulase synthesis is generally considered to be subject to carbon catabolite repression (Nisizawa et al., 1971; Canevascini et al., 1979). Almost no cellulase is formed during growth on glucose, glycerol, and other carbon sources related to glycolytic metabolism (Nisizawa et al., 1972). The derepression of enzyme synthesis occurs on the exhaustion of glucose in Myrothecium verrucaria cultures (Hulme and Stranks, 1970). The catabolite repression of cellulase synthesis has also been shown in T. reesei and other fungi and in some bacteria (Steward and Leatherwood, 1976; Eriksson and Hamp, 1978; Canevascini et al., 1979). No correlation was found between cellulase repression and intracellular cAMP levels in fungi (Montenecourt et al., 1981);however, cAMP regulates the biosynthesis of cellulases in bacteria (W. E. Wood et al., 1984). It was recently shown that the addition of glucose to T. reesei cultures producing cellulases only decreased the rate of synthesis of CBH I by 40% and the fungus continued to produce cellulase up to 40 hr (Messner and Kubicek, 1991). The results were similar when a number of other glucose analogues or inhibitors of glucose uptake or metabolism were used. There was no evidence that glucose or a catabolite thereof in fact controls the transcription of the cellulase gene (Kubicek, 1992). Alterations detected at the level of the endoplasmic reticulum in some T. reesei mutants suggest that glucose may affect the development of the secretory pathway or some of its components (Ghosh et a]., 1982; Glenn et al., 1985). The decreased level of a key enzyme (dolicholphosphate-mannose synthase) in the 0-glycosylation pathway in T. reesei has been found during growth in glucose (Kruszewska et al., 1989). The above observations clearly indicate that the role of glucose in cellulase synthesis requires further investigations.

MICROBIAL CELLULASES

29

The role of L-sorbose in the induction and regulation of cellulase and P-glucosidase biosynthesis and secretion in fungi is well documented (Nanda et al., 1982, 1986; Bisaria et al., 1986; Kubicek, 1983; Sahoo et al., 1986; Duff et al., 1985; Kawamori et al., 1986). L-Sorbose inhibits P-1,3-glucan synthetase, with the result that P-glucan content decreases in the cell wall (Kubicek, 1983) and enhanced levels of P-glucosidase, which are closely associated with P-1,3-glucan, are released into the culture medium. Kawamori et al. (1986) suggested sorbose as an inducer of cellulase synthesis in a mutant strain, T. reesei PC-3-7,because endoglucanse synthesis on sorbose was inhibited in the presence of cycloheximide. The addition of sorbose to cellulose/cellobiose-grown cultures of T. reesei QM 9414 resulted in a sevenfold increase in extracellular cellulase activity (Nanda et al., 1986). Such activity could not be explained by sorbose-induced changes in cell wall composition since cellnlases are predominently extracellular (Vaheri et al., 1979; Nanda et al., 1986). The specific consumption rate of cellobiose in strain QM 9414 was also reduced in the presence of sorbose. A molecular model based on the above mentioned rate of sorbose was proposed by Bisaria and Mishra (1989). The effect of sorbose on synthesis of P-glucosidase and cellulase is given in Table IX. The synthesis of cellulases in fungi can be hampered by factors other than catabolite repression. A wide variety of phenols can repress the synthesis of both cellulases and xylanases in Chaetomium globosum and S. commune (Varadi, 1972). The enzyme synthesis was considerably repressed even at concentrations less than 1mM of vanillin, vanillic acid, and vanillyl alcohol. Production of endoglucanase was drastically repressed in a phenol oxidase-less mutant (Phe 3) of S. pulverulentum in the presence of kraft lignin and phenols even at a concentration of 1 mM. However, Miiller et al. (1988) demonstrated that cellulase production in white-rot fungi is stimulated by lignin-

TABLE IX EFFECTOF L-SORFJOSE ON CELLULASE BIOSYNTHESIS IN Trichodermo reesei

Extensive branching, septation, and increase in the number of hyphal tips. Inhibition of p-1,3-glucan synthetase. Decrease in the level of cell-wall p-1,3-glucan. Reduced level of intracellular p-glucosidase. 5. Release of cell-wall-bound p-glucosidase. 6. Increase in extracellular enzyme secretion. 7. Reduction of the cellobiose uptake into the cell. 8. Reduction of intracellular breakdown of an active inducer, sophorose. 9. Minimized catabolite repression.

1. 2. 3. 4.

30

AJAY SINGH AND KIYOSHI HAYASHI

related phenolic compounds. They suggested that stimulation of cellulase synthesis was due to an indirect rather than a direct induction by the phenols themselves. Phenol oxidases produced by the white-rot fungi oxidize phenolic compounds to their corresponding quinones. These quinones are further reduced to phenols by cellobiose : quinone oxidoreductase (Westermark and Eriksson, 1974a). Cellobiose is oxidized to cellobionolactone in this reaction, which is a powerful inducer of cellulases (Bruchmann, 1978). The extracellular cellulase activity is dependent not only on mechanisms regulating their biosynthesis but also on the presence of specific inhibitors regulating the activity of enzymes themselves. The role of one such inhibitor, gluconolactone, has been studied for the regulation of P-glucosidase of S. pulverulentum. Gluconolactone is produced either by oxidation of glucose by glucose oxidase or by hydrolysis of cellobionolactone. The K, values for gluconolactone as a competitive inhibitor of the two extracellular P-glucosidases from S. pulverulentum were 3.5 X and 15 X lo-’ M (Deshpande et al., 1978). B. LOCALIZATION Generally, microbial cellulases are regarded as extracellular enzymes because of their appearance in the culture medium during growth. However, some of the cellulase components have been demonstrated to be cell-wall or membrane bound. In T. reesei QM 9414 large amounts of cellulases are secreted into the medium when grown at a low concentration of cellobiose. Under induced conditions, P-glucosidase was mainly detected in cell debris and was not released unless cell autolysis took place (Vaheri et al., 1979). However, Kubicek (1981) noticed an appreciable association of endoglucanase with the cell wall fraction of T. reesei QM 9414 during its growth on cellulose and cellobiose. The cell-bound endoglucanse was bound to the mannoproteins located at the outer region of cell walls and could be released effectively by treatment with a-mannanase or trypsin (Gander, 1974). Although membrane-bound P-glucosidases have been reported in T. reesei and M. bispora they are predominently cell wall associated in most of the fungi (Kubicek, 1982, 1983; Dekker, 1981; Sprey, 1986; Umile and Kubicek, 1986; Yablonsky et al., 1988). Cell-wall-bound Pglucosidase is released into the medium after the completion of exponential phase. T. reesei is known to contain a most efficeint cellulase system but it is deficient in P-glucosidase activity. Since the major portion of P-glucosidase activity in T. reesei is cell-wall associated, various methods to release it from cell walls have been investigated by

MICROBIAL CELLULASES

31

several research groups (Kubicek, 1981; Nanda et al., 1982; Kubicek and Pitt, 1982; Bisaria et al., 1986). Lytic enzymes such as chitinase, p-1, 3-glucanse, a-1, 3-glucanase, and p-1, 6-glucanase have been observed in the culture filtrate of T. viride grown on S. commune cell walls (de Vries and Wessels, 1972, 1973). Cultures supplemented with chitin resulted in a higher release of P-glucosidase (Nanda et al., 1982). Chitin has also been found to co-induce p-1, 3-glucanase along with chitinase in T. reesei (Kubicek, 1981; Dekker, 1981).P-Glucosidase has been suggested to be located within the intramural space (Kubicek, 1981), and it can be released by treatment of cell walls with chitinase and p-1, 3-glucanase. On the other hand endoglucanase is released by treatment with a-mannanase or trypsin. The p-1, 3-glucan of the cell wall plays an important role in the release of P-glucosidase (Kubicek, 1983). Mutants of T. pseudokoningii, having low levels of wall-bound p-1, 3 -glucanase, released almost no wall-bound P-glucosidase. LSorbose, known to cause paramorphogenesis in fungi, inhibits pl, 3-glucanase synthetase and increases extracellular 6-glucosidase activity (Mishra and Tatum, 1972; Bisaria et al., 1986). In sorbosesupplemented cultures of T. reesei, peripheral hyphae of the mycelial clumps were highly branched with stubby tips and short internodal distances (Bisaria et al., 1986).On the other hand, in cellobiose medium, the mycelium grew as long, tapering hyphae with sparse branching. Similar results on localization and secretion of p-glucosidase from T. pseudokoningii have been reported. Sprey (1986) used ferritinconjugated antibodies in electron microscopic studies to show that p-glucosidase was localized mainly in the outermost exopolysaccharide layer, in the plasma membrane, and, to a lesser extent, in the carbohydrate portions of the cell wall. A cellulose degradation mechanism was proposed by Sprey (1986) in which a sequential degradation of cellooligomers is accomplished by @-glucosidaseattached to an exopolysaccharide layer, followed by glucan and plasma membrane-located pglucosidase action on cello-oligomers of successively lower degrees of polymerization. However, it is still not certain if the same or different forms of p-glucosidases are present in the plasma membrane and the cell walls.

IN RECOMBINANTCELLS C. BIOSYNTHESIS

The number of structural genes for each cellulase type (except CBH 1/11 and EG I/III) and the regulatory genes have not been completely identified because of the lack of a well defined genetic cycle in T. reesei.

32

AJAY SINGH AND KIYOSHI HAYASHI

Although a sexual stage has been reported in Trichoderma, mating types analogous to S. cerevisiae and Neurospora crassa are not available (Webster, 1964;Mortimer and Hawthorne, 1966).Because traditional methods of mapping the structural and regulatory genes are not available the mechanism of regulation at the molecular level has been studied by isolating mutants with altered physiology (Gallo et al., 1978;Kawamori et al., 1985;Nevalainen et al., 1980;Ghosh et al., 1982).In a number of hyperproducing mutants there has been a coordinated increase in all the components of cellulases, indicating involvement of a major regulatory gene (Sheir-Neiss and Monenecourt, 1984; Ghosh et al., 1982). Furthermore, the concomitant loss of crystalline and soluble substrate-hydrolyzing activity, and mannase and xylanase activities in some cellulase-negative mutants, also indicated that the biosynthesis of these enzymes may be regulated by a single gene (Nevalainen and Palva, 1978).Advances in genetic engineering and molecular biological techniques and successful cloning (Table X) of some fungal and bacterial cellulase genes have made it possible to identify and understand the role of structural and regulatory genes involved in cellulase biosynthesis. Escherichia coli has been the most common host for cloning of different cellulase genes. However, cellulase genes have also been efficiently cloned and expressed in Zyrnomonas mobilis (Misawa et al., 1988; Lejeune et al., 1988),B. subtilis, B. megatarium (Lee and Pack, 1987), and S.cerevisiae (Skipper et al., 1985;Shoemaker et a]., 1983;Pentilla et al., 1987). The expression of cellulase genes in heterologous hosts must ensure transcription of the cloned gene through cloned natural promotors or the control of the promotors of the vector system, efficient translation of the gene, and subsequent export of the cellulase protein to the extracellular medium (Bisaria and Mishra, 1989).The expressed gene product has been identified either by CMCase plate assay (Teather and Wood, 1982) or by immunoprecipitation of enzymes (Whittle et al., 1982;Gilkes et a]., 1984;Bdguin et al., 1985;Teeri et al., 1987; Chen et al., 1987).The genomic library constructed in the phage lambda vector has been screened for cellulase gene by hybridizing with two distinct cDNA probes: one synthesized from mRNA formed during growth of T. reesei on glucose, and the other from mRNA induced on cellulose (Skipper et al., 1985;Pentilla et al., 1987;Shoemaker et al., 1983;Teeri et al., 1983).The relevant cDNA sequences were identified by hybrid selection to the corresponding mRNAs and immunoprecipitation of proteins synthesized in vitro. The secretion of cloned gene product in bacteria and yeast appears to be governed by the same molecular mechanisms as in the native

MICROBIAL CELLULASES

33

TABLE X MOLECULAR CLONING OF CELLULASE GENE Donor cell Bacillus subtilis Cellulomonas fimi C. fimi

C. uda Clostridium thermocellum

EIulinia chrysanthemi Microbispora bispora Pseudomonas fluorescens Ruminococcus flavefaciens Thermomonospora sp. YX Aspergillus niger Candida pelliculosa Schizophyllum commune Trichoderma reesei

Host cell Escherichia coli (PBC51 E. coli (pBR322) E. coli (pBR322)

Cellulase gene type

Reference Nakamura et al. (1986)

Exoglucanase (ExGI Endoglucanase (ENGI Endoglucanase

O’Neill et al. (1986) Wong et al. (1986)

Zymomonas rnobilis (pZA22) E. coli (pBR322) (PHC791

Endoglucanase cel A cel B

(hLIC7) (h1059) (pBR325) E. coli (pMMB34)

cel C cel D P-Glucosidase Endoglucanase

Romaneic et al. (1987) GrBpinet and BBguin (1986) Joliff et al. (1986a) Joliff et al. (1986b) Kadam et al. (1988) Barras et al. (1984)

E. coli (pBR322)

Endoglucanase

Yablonsky et al. (1988)

E. coli (pBR322)

Endoglucanase

Wolff et al. (1986)

E. coli (pMEB2OO)

Cellulase

E. coli (pBR322)

Endoglucanase

E. coli ( ~ 3 0 3 0 )

P-Glucosidase

Barros and Thomson (1987) Collmer and Wilson (1983) Pentilla et al. (1984)

E. coli (YEp13)

p-Glucosidase

Kohchi and Toh (1985)

E. coli (hgtll, pBR328) E. coli (h1059) (h1059) (A1059) (h1059)

Endoglucanase

Willick et al. (1987)

Cellulases CBH I CBH I1 EG I EG 111

Teeri et al. (1983) Teeri et al. (1983) Pentilla et a]. (1986) Saloheimo et al. (1988)

Misawa et al. (1988)

host system. The N-terminal nucleotide sequences of both the cloned bacterial and fungal cellulases predict an initial sequence of 25 to 34 amino acids with a charged amino acid at the N-terminus and lengthy hydrophobic sequences (Watson, 1984; Wickner and Lodish, 1985; Pugsley and Schwartz, 1985). A complete secretion of enzymes to the

34

AJAY SINGH AND KIYOSHI HAYASHI

extracellular medium occurs when the cellulase genes are cloned in Bacillus sp. or S. cerevisiae (Robson and Chambliss, 1986; Lee and Pack, 1987; van Arsdell et al., 1987; Shoemaker et al., 1983). On the other hand, the presence of the N-terminal sequence directs its export to the periplasm when E. coli is the host (Sharma et al., 1987).Trichoderma reesei cellulases are generally overglycosylated in yeast, which indicates that post-translational modifications in yeast and filamentous fungi are different. VI. Future Prospects

Trichoderma reesei is one of the most efficient producers of extracellular enzymes; more than 50% of all proteins produced by this organism are secreted into the extracellular medium. Although our understanding of the molecular biology and enzymology of cellulase biosynthesis has advanced rapidly in recent years, more detailed knowledge in this area may be necessary in order to increase the production of cellulolytic enzymes by this fungus. The most,exciting aspect of cellulase biochemistry currently being examined is the discovery of how the threedimensional structure of cellulases brings about the degradation of cellulose. These studies are required in order to improve cellulases by modifying their active center or the structures involved, and also to explain the need for different ratios of individual cellulase components for the optimal hydrolysis of cellulose. From an application point of view, the desirable properties of cellulases are high catalytic efficiency, high thermal stability, and low end-product inhibition (Bissetand Sternberg, 1978; Reese and Mandels, 1980;Ryu and Mandels, 1980).Adsorption of cellulases on cellulose is another critical factor governing hydrolysis of amorphous and crystalline cellulose (Klyosov et al., 1982,1986; Singh et al., 1 9 9 2 ~ )The . role of glycosylation in the binding efficiency of cellulases onto crystalline cellulose needs to be further investigated so that the cellulases with high binding efficiency can be developed. The advent of recombinant DNA technology has dramatically accelerated research in the field of cellulase enzyme systems. Efforts have been directed to clone genes from cellulolytic organisms with the desired molecular properties. In the past few years a large number of cellulase genes have been isolated, characterized, and expressed in a variety of hosts. Cloning of genes for individual components of the cellulase system would allow the biosynthesis of pure components and also provide information on their relative importance in the enzymatic hydrolysis of cellulose. Understanding the enzymology of cellulose hydrolysis appears to be more difficult than anticipated earlier. This is mainly

MICROBIAL CELLULASES

35

because of the very complex and variable nature of the natural substrate cellulose. The development of an efficient transformation system opens the possibility of constructing improved strains producing cellulases with higher specific activities on particular substrates. An equally important aspect would be improvement in the thermoresistance of these enzymes. Although protein engineering offers the necessary technology, the isolation and characterization of thermostable cellulase and their genes are required. With this work as a foundation, the development of new cellulolytic organisms that can be used to exploit the enormous amount of waste cellulose may be feasible. ACKNOWLEDGMENT STA Fellowship provided to AS. by JRDC and JISTEC, Japan, is gratefully acknowledged.

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Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective D. K. O’TOOLE Department of Biology and Chemistry City University of Hong Kong Kowloon, Hong Kong

I. 11. 111. IV.

V. VI.

VII.

VIII.

IX. X.

Introduction Early Developments Lactenin Studies with Lactic Acid Bacteria A. Agglutinin for Lactic Acid Bacteria B. The Lactoperoxidase System C. Autoinhibition by Hydrogen Peroxide D. Thiocyanate in Milk E. Lactoferrin F. Other Possible Inhibitors for Lactic Acid Bacteria in Milk G. Seasonal Variation in Bacterial Activity in Milk H. Heated Milk as a Growth Medium for Lactic Acid Bacteria I. Effect of Culture Manipulations J. Effect of Milk on Morphology of Starter Cultures Effect of Cow Ration on Milk as a Growth Medium for Bacteria Role of Inhibitors in Milk Quality A. Raw Milk Quality Stimulators of the Growth of Lactic Acid Bacteria A. Investigations of Milk and Milk Components B. Stimulation by Minerals C. Complex Substances as Stimulators D. Commercial Stimulators Growth Inhibitors of Other Bacteria A. Inhibitors for Enterobacteria B. Inhibitors for Bacilli C. Inhibitory Factor for Propionibacteria D. Inhibition of the Staphylococci E. Inhibition of Leptospiras F. Inhibition of Mycoplasmas Applications of the Antimicrobial Systems in Milk Conclusion Appendix A Appendix B References

I. Introduction

Milk is a complex biological fluid which is secreted by female mammals from their mammae and which contains liberal quantities of fat, 45 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 8 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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carbohydrate, protein, minerals, and vitamins, which are ideal for the growth of the mammal’s offspring but which also supply many of the nutrient requirements of bacteria. Consequently, many types of bacteria grow rapidly in milk and milk products. This fact motivates the technologically advanced industry practices developed by the dairy industry to protect milk and dairy products from deterioration during processing, storage, and distribution. Thus, the growth-supporting properties of milk are well known and appreciated; not as well appreciated and understood are the growth inhibiting properties of milk. In addition, some suggest that the growth of bacteria in milk is further controlled by stimulating factors which promote bacterial growth, as suggested for cheese starter bacteria (Czulak and Meanwell, 1951). Thus the growth we see may be the net response of the bacteria to two different forces, an inhibiting force which retards the growth of or destroys bacterial cells and a stimulating force which enhances bacterial growth under the conditions of the test. The source of these inhibiting and stimulating factors is inherent; that is, they are part of the natural protective systems in the milk, but they can be influenced by the female’s diet which can be deficient in essential constituents that would normally be transmitted to milk. Cow milk has been most extensively studied, but the inhibiting properties have been noted in milk from buffaloes (Natarajan and Dudani, 1961; Patel, 1969), camels (El Agamy et a]., 1992), goats (Hammer and Babel, 1957), and humans. Milk from all mammals probably shows these properties. Most of the work done in this area has been concerned with the effect of milk on particular organisms with few attempts, with the exception of the lactoperoxidase-thiocyanate-hydrogen peroxide system, to link the observations with those found with other organisms. The main concern of this review is the lactic acid bacteria and the effect of milk on their growth, but propionibacteria, coliforms, bacilli, and others are also dealt with because it cannot be assumed that these separate agents bear no relationship to those factors inhibiting the lactic acid bacteria. The growth of many other organisms is known to be inhibited in milk (see Appendix A). Reviews include those of Jones and Little (1927), Reiter and Moller-Madsen (1963), Reiter and Oram (1967), and Reiter (1978, 1985). By far the most prolific worker in this field within the last 30 years has been Dr. Bruno Reiter. Interest in these substances has waxed and waned over the decades. There seem to be four main reasons for interest in their presence. The first important studies celntered on the possibility of using the inhibitory properties to control mastitis and began before the antibiotic era. The

BACTERIAL GROWTH IN MILK

47

second and most fruitful studies began when their effects on lactic acid bacteria, the cheese-making bacteria as well as mastitis bacteria, were studied. The third series of studies began to investigate their role in controlling and influencing milk quality. The fourth strand of study was concerned with the effect of milk on the growth of organisms pathogenic to young mammals, distinct from the mastitis organisms which infected the mammary glands, and includes the protective properties of milk for infants. II. Early Developments

Fokker’s research in 1890 is thought to be the first to note the germicidal properties of milk; he found that raw goat milk soured more slowly than heated goat milk that was similarly inoculated (Hammer and Babel, 1957). Jones and Little (1927) reported Heinemann’s conclusion that milk contains a bactericidal substance, specific for certain organisms, which was destroyed by heating at 60°C for 30 min. They added that Rosenan and McCoy, after noting that bacterial numbers declined during the first 8-10 hr postmilking, suggested that agglutination and phagocytosis caused the decline. However, Chambers found no relationship between agglutination and growth inhibitory properties except for their equal susceptibility to heat, 80°Cfor 2 min, and reported specificity with regard to the cow and bacterial species employed. Jones and Little (1927), on the contrary, reported that Sherman and Curran found that young cultures of Streptococcus lacticus inoculated into fresh milk had a 30-min lag period compared to controls. (Note that many early names are now invalid; readers are directed to Index Bergyana (Buchanan et al., 1966) and the latest edition of Bergey’s Manual of Systematic Bacteriology to check the fate of these taxons; the lactic acid streptococci are now placed in the genus Lactococcus). Hanssen (1924) noted that in fresh milk the maximum decline in numbers of Salmonella typhi and Salmonella paratyphi B took place within 30 min. The milk retained this activity for 24 hr during storage in ice; it seemed that when the cow was receiving, among other things, fresh grass, turnip, and cabbage, the bactericidal activity was greatest, and that bactericidal activity was the least when receiving no fresh grass but only ensilage (Hanssen, 1924). He thought that the bactericidal property of milk was closely related to oxidases and peroxidases, as both had a similar temperature limitation of 75°C for 15 min. He proposed that the enzymes came from the feed (Hanssen, 1924). The first systematic studies were those of Jones and associates, who found that milk and whey had bacteriostatic activity for a nonhemolytic

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mastitis streptococcus; it failed to grow 4-6 hr postinoculation (Jones and Little, 1927), and it did not use the substance in the lag period; rather, the organism adapted itself to the inhibitory principle (Jones and Simms, 1929). The activity was natural, inherent in cow milk, and specific in that Bacterium coli and Bacterium bovisepticus cultures were not inhibited by milk inhibitory for streptococcus but B. bovisepticus could absorb some of the activity for the streptococcus from milk (Jones and Little, 1927);on the contrary, cells of a scarlet fever streptococcus, live or dead, did not absorb the principle (Jones and Simms, 1929) even though the substance killed the organism (Jones, 1929). Colostrum contained less “principle” than normal milk, and charcoal removed some, and Kieselguhr none, of the principle which was destroyed by heat (80°C for 20 min) but not by drying (Jones, 1928). The substance was not concentrated in the rennet whey as blood proteins are, and it had a different heat inactivation pattern from that of the bactericidal or bacteriostatic property of cow serum. Their technique ruled out the agglutination theory and they concluded that the substance was not “alexin” (complement) (Jones and Little, 1927). Jones and Simms (1930) proposed the name “lactenin” for this substance(s), a name applied for many years to any unknown substance that inhibited bacterial growth in milk, and suggested that it was a protein even though unaffected by trypsin. It existed as a salt of whatever cation was present, it had a low isoelectric point, it contained only C, H, 0, and N and no reducing sugars, and its natural advantages and possible uses were unknown (Jones and Simms, 1930). Ill. Lactenin

Further investigations began in the 1950s, the first studies being those of Wilson and Rosenblum. They tested streptococci for their sensitivity to lactenin and obtained the results shown in Table I. Wilson and Rosenblum (1952a) used a group A streptococcus (327W) and found that milk retained a bactericidal effect for up to 24 hr irrespective of inoculum size. Growth improved but no inactivation of lactenin occurred when 5% normal human, rabbit, sheep, or horse serum was added to milk. Lactenin was partially inactivated in milk when defibrinated human, rabbit, or sheep blood was added. Their most important observation (Wilson and Rosenblum, 1952b) was that rigid exclusion of oxygen prevented the activity of lactenin. They examined substances for their ability to inactivate lactenin (Wilson and Rosenblum, 1952b), the results of which are in Appendix B, and found that cysteine (100 ppm), sodium thioglycollate (100 ppm),

49

BACTERIAL GROWTH IN MILK TABLE I SEROLOGICAL GROUPSOF STREPTOCOCCI SENSITIVE TO LACTENIN IN RAW MILK Group A B C, D,E, H,

F G K

L

Number of strains

Strains sensitive

477

477 1

25 31, 25, 4, 4,10 3 25 3

0 1

5 2

Note. From Wilson and Rosenblum (1952a).

glutathione (100 ppm), and 2,3-dithiopropanol(lo ppm) were the most effective. Auclair began similar investigations (Auclair and Hirsch, 1953) with the conception that he was dealing with a very complex phenomenon. He confirmed an earlier observation (Wilson and Rosenblum, 1952b) that the antistreptococcal properties of milk were not due to lysozyme. He quantified the bactericidal properties of milk using dilution assays which were prepared in sterile skim milk adjusted to pH 7.0 to which was added an equal volume of glucose-peptone-broth inoculated with 5% v/v of an 18-hr serum-broth culture of Streptococcus pyogenes, strain “Richards.” After 6 hr at 37”C, distilled water (5x test volume) was added and the pH determined. The response curve obtained (Auclair and Hirsch, 1953) (Fig. 1)was explained as follows: milk contains a small quantity of stimulator and at least two separate inhibitors which they called “lactenin 1” and “lactenin 2” (Fig. 2). Lactenin 1 (Ll) occurs mainly in colostrum, is most stable at pH 6.0-6.5, and is 90% destroyed at 68°C. Lactenin 2 (L2) occurs mainly in milk, is most stable at pH 7.0, and is 95% destroyed at 74°C. L1 and L2 act synergistically, each having low potency in the absence of the other (Auclair and Berridge, 1953). Anaerobiosis and cysteine abolish the inhibitory activity of both lactenins (Auclair and Hirsch, 1953) but do not destroy the lactenins, but L2 is destroyed by H,O, and L1 remains relatively unaffected (Auclair, 1954b). Occurrence of the lactenins was not correlated with udder infection nor with stage of lactation, age of cow, or the season of the year (Auclair, 1954b). Auclair (1954b) found that Streptococcus lactis was more seriously inhibited by L1 and L2 together than L2 alone but Streptococcus agalactiae was more sensitive to L2 than L1 and L2 together, and that Staphylo-

D. K. O'TOOLE

50

7.0

6.5

PH 6.0

5.5

If2

1/4

1/8

1/16

1032

1/64

1/128 If256 1/512 1/1024 If2048 1/4096 1/8192

Control

Dilution of sample FIG.1. The dose-response curve showing the pH change in midlactation milk and colostrum inoculated with S. pyogenes strain 'Richards'. ( X ) Standard, (0)milk from cow Pandora in midlactation, (A)colostrum from cow Rosalie. From Auclair and Hirsch (1953).Reprinted with the permission of Cambridge University Press.

coccus aureus was only slightly inhibited by L1 and L2. L1 and L2 could be partially separated using acetone fractionation in small steps (Auclair and Berridge, 1953), whereas on electrophoresis, which gave good separation of L2, the activity of neither lactenin could be linked with a corresponding protein brand. Auclair (1954b) concluded that variation in the activity of lactic acid bacteria in milk was due to variations in natural stimulators in milk. At that time Jag0 (1954)noted that the growth of lactic starter cultures in pasteurized milk varied. He concluded there was an inhibitor associated with the fat globule because buttermilk was inhibitory but butter granules were not, and as the concentration of cream added to raw milk increased, so did inhibition of the cultures. Heated separator slime (primarily white blood cells and manure) neutralized the inhibitor. He suggested (Jago,1954) the inhibitor was an enzyme and that it interfered with the utilization of growth-promoting substances normally present in milk. Jago's studies were instigated because of variations in the activity of lactic cultures in cheese making, as reflected in activity tests done by

51

BACTERIAL GROWTH IN MILK

7.0

6.5

PH 6.0

5.5

If2

1/4

1/77

1/16

1/32

1/64

Ill28

1,256 1412 1/1024 1,2048

Control

Dilution of milk FIG.2. The hypothetical effects of decreasing concentrations of two inhibitory substances and one stimulatory substance on the acid production of S.pyogenes as it relates to the standard response curve of the organism to milk. (0) Dose-response of the test organism to raw milk, ( x ) inhibitory substance I,, (0)inhibitory substance I,, [A) stimulatory substance S. From Auclair and Hirsch (1953). Reprinted with the permission of Cambridge University Press.

inoculating 9 ml of whole milk in a test tube and measuring acidity after 6 hr at 30°C. By such tests lactic cultures are classed as either fast, producing about 0.6% acid in this time, or slow, producing less acid in this time. Wright and Tramer (1957)pointed out, however, that fast cultures produced acid throughout the tube but slow cultures (e.g., Streptococcus cremoris 60, 803) often produced acid only in the cream layer. Prolific growth of slow strains occurred in the cream layer, as observed microscopically, but not in the bulk of the milk (Wright and Tramer, 1957). Slow cultures also showed improved acid production when tubes were inverted every half-hour but only if air was excluded by placing wax on top of the milk, as air reduced acid production by fast and slow cultures (Wright and Tramer, 1957). Elimination of creaming by homogenization or heating (82°C) or addition of rennet or agar improved acid production by slow starters, and their growth was better in skim milk than in whole milk. They showed that if milk was separated, the milk and cream were separately heated for 30 min at either 63 or 76"C, and the milk was reconstituted from portions sub-

52

D. K. O’TOOLE

jected to different heat treatments, the activity of the cultures and the cream-rising properties of the milk depended on the heat treatment of the skim milk. In skim milk heated at 63°C for 30 min, slow starters formed clusters that deposited on the bottom of tubes; in whey from such milk the organisms formed floccules of massed agglutinated cells. Growth was normal in whey from milk heated to 76°C for 30 min. Activity and chain length of the organisms were not related as even slow cultures formed shorter chains in milk heated to 76°C for 30 min. Wright and Tramer (1957) named the substance responsible for this activity “agglutinin.” Wright and Tramer (1958) noted a second type of slow starter, of which S. cremoris 972 was one; even when creaming was prevented, this type showed increased activity only when the milk used was heated to a high temperature. Streptococcus cremoris 803 was affected by the agglutination effect and its maximum activity occurred in whole milk heated to 80°C for 24 sec, but 90°C for 24 sec was necessary for the maximum activity of 972. The addition of 2-10% milk to autoclaved or steamed milk, and to wheys from these milks, inhibited the 972 culture, but dilutions of milk heated to 63°C in milk heated to 85°C gave a different dose-response curve. This difference was due to the presence of reducing compounds in milk heated to 85-88”C, measurable with the sodium nitroprusside test; these compounds could be destroyed by H,O, treatment (30-100 ppm) followed by catalase treatment. Wright and Tramer (1958) found a relationship between milk peroxidase and the inhibitory properties of milk as Hanssen (1924) had suggested. Culture activity and peroxidase activity behaved similarly in whey heated to various temperatures. In addition, whey was not affected by exposure to pH 2.82, but below this pH culture activity increased and peroxidase activity decreased. Milk heated to 82°C contained residual peroxidase of about 1%of that originally present in the milk. There was an interaction between the inhibitor in whey and H,O,, as whey treated with 10 ppm H,O, showed little adverse effect on peroxidase but at 20 ppm peroxidase was destroyed and culture activity improved. The inhibitor in a mixture of homogenized and commercially sterilized milk was overcome with cysteine but excess of cysteine was inhibitory, i.e., there was a critical level of cysteine needed to neutralize the inhibitor in high-temperature short-time pasteurized milk. Sodium hydrosulfite (Na,S2O,.2H,O) also counteracted the inhibition caused by peroxidase, but not that caused by agglutinin, and overcame a partial bacteriostatis of 972 in homogenized, pasteurized milk. Sodium azide, which inactivates catalase (critical concentration about 1 mM), also inactivated peroxidase and the inhibitor. A similarity was shown between the lactenin of Auclair which af-

BACTERIAL GROWTH IN MILK

53

fected S. lactis M1 and that affecting strain 972 (Wright and Tramer, 1958). They found colostrum to be low in peroxidase compared to milk; Auclair found colostrum poor in lactenin 2. They tested colostrum for agglutinin and found correspondence between the creaming properties of milk induced by colostrum and the activity of agglutinin-sensitive organisms; they suggested that colostrum contains 10 times as much agglutinin as normal milk. They supported the concept that the removal of inhibition can be achieved by either inactivation of peroxidase or reduction of a postulated inhibitory oxidation product. They also noted that although many studies of slow cheese starters have found that the inhibitory effect of milk disappears when it is heated to 71°C for 30 min, this treatment was also just sufficient to inactivate peroxidase in milk. Wright and Tramer (1958) concluded that L1 was equivalent to agglutinin and L2 was equivalent to peroxidase, i.e., lactoperoxidase. Auclair and Portmann (1959) reexamined the question and found that the substance inhibiting 760 was different from L l ; they named it L3 and reported that it was different from the substance causing agglutination of fat globules. Since about 1959, investigations into the inhibitory properties of milk are confounded by separate studies of peroxidase and agglutinin and further studies of the general growth-supporting properties of milk for various bacteria, I shall deal with the agglutinin and peroxidase investigations separately as far as possible. Most studies deal with single strains of streptococci, but mixed cultures are also inhibited by milk (Randolph and Gould, 1966). IV. Studies with Lactic Acid Bacteria

A. AGGLUTININ FOR LACTICACIDBACTERIA

The observations of Wright and Tramer were not only quickly confirmed but also extended, in that it was shown that 803 not only rose with the fat globules but also deposited to the bottom of the culture (Stadhouders, 1963). Keogh (1958) showed that there was generally a relationship between inhibition in raw whole milk and a positive ring test; she also noted excessive numbers of organisms in the cream layer when compared with the skim milk layer and prevented inhibition by skimming and by adding agar. She further showed (Keogh, 1958) that some cultures were consistently faster in milk heated to 98°C for 15 min than in raw milk (strains K, DR7, R6, US3, R1, DRC1, DRC2, DRC3) but others were as slow or slower in heated milk as in raw milk (strains C1, C2, C3, Clo). The capability of skimming to remove the inhibitory effect of raw milk depended on milk supply for some cultures (strains DR7, K, US3, DRC3); the removal of inhibition by agar also

54

D. K. O’TOOLE

depended on milk supply, but a slightly different group of strains showed the variation (strains DR7, HP, R1, DRC1). The acetoneextracted and -dialyzed precipitate produced by Auclair and Berridge (1953) which contained L1 and L2 also contained agglutinin, as it produced a positive ring test and caused microscopic agglutination of bacteria and contained peroxidase (Keogh, 1958). When added to heated milk the extract also stimulated some bacteria in a manner similar to that found in raw milk. Gillies (1961a) showed that inhibition of HP and R6 was prevented in whole, pasteurized milk constantly rotated in a bottle from which air was excluded. HP was agglutinated around fat globules. As the percentage cream added to sterile reconstituted skim milk increased, the susceptible strain HP showed a rapid decrease in acid production followed by a gradual increase and then a final decrease (Fig. 3). This final decrease as the percentage cream approached 1OOO/” was shown also by a resistant strain C13. The inhibitory property attached to the fat globule was destroyed by heating at 76°C for 30 min (Gillies, 196lb). The inhibitory factor for R6 could be washed out of cream after two 50

Suscepuble strain HP Resistant strain C13

0

- - - -.

I

I

I

I

20

40

60

80

100

Percentage cream added FIG.3. The effect of increasing concentration of cream in milk on the acid production of lactic cultures resistant and susceptible to inhibitors in milk. From Gillies (1961bj. This figure provided by the Department of Primary Industries, Queensland, from their Journal QJAAS published by the DPI Queensland.

BACTERIAL GROWTH IN MILK

55

washings in sterile tap water, but the cream was still inhibitory after one washing and agglutinated organisms were present in the fat layer. When the water from the second washing was added to sterile skim milk, R6 was inhibited. There was no agglutination of the organism. The addition to pasteurized whole milk of homologous rabbit antisera to resistant strains caused those strains to act like susceptible strains (Gillies, 196lb). By the acriflavine test all resistant strains were smooth and all susceptible strains were rough (Gillies, 196lb). Gillies (196lb) concluded there was no firm proof that the inhibition of susceptible strains in milk was due to specific antibodies, but Randolph (1963) found that R1 was inhibited by a factor associated with the immune globulin fraction in milk, which suggested an antibody as the cause of inhibition. Specific antibodies in milk inhibiting lactic streptococci (strains ML1, KH, DR7) had previously been demonstrated by McPhillips (1958). Portmann and Auclair (1959) found that of 15 strains of lactic streptococci, 11were sensitive to both inhibition and agglutination, and could be placed in five groups according to how they behaved in raw skimmed milk absorbed with suspensions of the organisms. These groups were: 760, DR7, K; E8,972; 803, R1, US3; KH, ML1; and HP. Strains C3, C10, T7, and DRC3 were unaffected. However, a sensitive strain of ClO was derived from a resistant culture of ClO and shown to be in a group different from these five groups (Auclair and Vassal, 1963). Portmann and Auclair (1959) suggested that inhibition and agglutination may be due to the same substance. Stadhouders (1963),in addition to confirming the prevention of inhibition by rennet, showed that agglutination of the bacteria before removal into the cream layer was not necessary. The agglutinins were inactivated in homogenized skim milk and were present in whey at the same concentration as that in milk, i.e., they were whey proteins. The euglobulin fraction of milk promoted agglutination and inhibited acid production by 803, whereas the pseudoglobulin fraction was without effect (Stadhouders, 1963). The distribution of the agglutinins between the cream and skim milk fractions of milk were dependent on temperature (Stadhouders, 1963). Stadhouders and Hup (1970) distinguished within the euglobulin antibodies a group called cryoglobulins which not only agglutinated bacteria and fat globules separately but also attached bacteria to fat globules. The antibodies which attach bacteria to fat globules are very specific, and the binding sites on the antibodies for bacteria are the same as those which agglutinate fat globules and bacteria. Goat milk fat globules removed the antibodies which agglutinate fat globules in cow milk. They thought that L1 and L3 were equivalent (Stadhouders and Hup, 1970).

56

D. K. O’TOOLE

An investigation of the agglutination phenomenon (Emmons et al., 1966a) using a sensitive test (Emmons et a]., 1965) for agglutinating antibodies to streptococci was prompted by problems associated with cottage cheese manufacture (Emmons et al., 1966b, 1967). Instead of rising on fat globules into the cream layer (Emmons et al., 1966a) the organisms agglutinated and settled to the bottom of the vat (Emmons et al., 1966b)(cottagecheese is made from skimmed milk). This produced a bottom layer 0.5-1 cm thick with a very low pH and generally slow acid development in the rest of the vat; the curd tended to shatter easily and was “mealy.” Absorption of the milk with saline-washed heatkilled cells reduced the sediment, but even so there was no absolute relationship between agglutination titres in whey and the extent of settling in skim milk (Emmons et a]., 1966b). Old milk and excessive Ca ions may be linked with the problem (Emmons et al., 1966b) but pasteurization at 71-74OC for 30 min virtually eliminated it (Emmons et al., 1967). Using two strains sensitive (Rl, DR7) and one resistant (C2) to agglutination, Emmons et al. (1966a)showed that for R 1 and DR7 colostrum was high in agglutinating antibodies whose concentration rapidly decreased to a relatively constant level within 2 weeks of parturition, and that antibodies to C2 were virtually nonexistent. The concentration of antibodies in milk was independent of breed and age of cow and of antibody titre in blood serum in which concentrations were higher than in milk. The agglutinating antibodies for C2, R1, and DR7 showed similar results for milk and blood from Short Horn beef cattle, swine, and Shropshire sheep, and similar results for blood from Clydesdale horses, Holstein bulls, dairy calves, unbred dairy heifers, and rabbits (Emmons et al., 1966a).Blood serum from hens also showed agglutination of lactic streptococci (McPhillips, 1960). Heat-killed cells of R1 removed agglutination and inhibitory principles for R1, but not for KH and HP, from skim milk and whey (Randolph and Gould, 1968). The inhibitor was bound firmly to R1 cells but some could be removed by 0.8% NaCl at 45°C for 2 hr and 15% NaCl at 37°C for 1 2 hr (Randolph and Gould, 1968). The inhibitory principle for R1 was associated with the immunoglobulins in milk (Randolph and Gould, 1968). It had been concluded earlier (Portmann et al., 1960) that lactenins were very similar to antibodies because vaccination of cows with sensitive and resistant lactic acid bacteria had resulted in increased inhibition of the organisms by the milk. B. THELACTOPEROXIDASE SYSTEM

The identification of L2 with lactoperoxidase (LP) was further strengthened when it was shown that both were destroyed by similar

BACTERIAL GROWTH IN MILK

57

processes and at a similar rate (Portmann et al., 1959). LP occurs in saliva, uterine fluid, and white blood cells (Reiter, 1973), and has since been found in goat milk (Zapico et al., 1990), ewe milk (Medina et al., 1989),and guinea pig milk and saliva (Stephens et al., 1979). It appears to exert a greater inhibitory effect in winter milk than in milk produced at other times of the year (Stadhouders, 1961) but more than LP is involved in the inhibition of microorganisms. Hydrogen peroxide (H202),the second component in the system, was discovered when it was shown that LP, but not horseradish peroxidase, inhibited S. cremoris 972 under aerobic conditions and catalase reversed this inhibition (Jago and Morrison, 1962). In a study (Pickering et al., 1962) involving S . lactis (C6, C10, 712, ML3) and S. crernoris (C7, 927, 972, 803), a wide range of hydrogen donors were oxidized by LP in the presence of a limited range of H,02 concentrations. H202 generating systems such as ascorbic acid, glucose plus glucose oxidase, and the hypoxanthine plus xanthine oxidase system added to the LP system inhibited lactic acid bacteria (Klebanoff et al., 1966). In milk, xanthine oxidase and ascorbic acid (Reiter and Oram, 1967) as well as H202produced by the organisms during aerobic growth (Jago and Morrison, 1962; Klebanoff et a]., 1966) could well be the source of H202 needed for the LP system to function. The microbial H202is generated by NADH oxidase, and if the organism produces insufficient NADH peroxidase, then H202builds up in the medium (Hogg and Jago, 1970a,d). Thiocyanate, the third component in the system, was discovered during studies on the antilactobacillus system in saliva (Klebanoff and Luebke, 1965).LP could replace a heat-labile, nondialysable component of the antilactobacillus system in which thiocyanate was already known to be a component, Iodide (Hogg and Jago, 1970c; Klebanoff and Luebke, 1965; Reiter and Oram, 1967), indican (Reiter and Oram, 1967), and bromide (Hogg and Jago, 1970c) can replace the thiocyanate in the system. Strain 972 but not 803 was inhibited by 2 p M KSCN in the presence of other components of the LP system and both strains were inhibited by 10 p M KSCN alone (Oram and Reiter, 1966a; Reiter and Oram, 1967). When thiocyanate was removed from milk with ion exchange resins 972 could grow normally in the cheese-making process (Reiter, 1973). With the recognition of H202as a component of the system, H202generating systems have been used to activate the system to control microbial growth. These H202-generatingsystems include urea peroxide (Earnshaw et al., 1989, 1990), the glucose oxidase system (Dionysius et al., 1992; Earnshaw et a]., 1989),and sodium carbonate peroxyhydrate (Dionysius et al., 1992) as well as pure H202(Siragusa and Johnson, 1989).

58

D. K. O'TOOLE

What is the inhibitor? It was suggested (Jago and Morrison, 1962) that an oxidized product of a substrate present in milk was the cause. Attempts (Oram and Reiter, 1966b) to isolate an intermediate failed but there were similarities between the activities of the product causing inhibition and sulfur dicyanide (S(CN),).However, the inhibitor (Hogg and Jago, 1970b) was pH dependent (the system is inactivated in milk by raising pH to 7.5), was unstable to heating at 60°C or above for 20 min, was stable for several days at a low temperature, had a pK, of about 5.2, and was influenced by excess thiocyanate in the medium. From a study (Maguire and Dunford, 1971) of the kinetics of formation of the primary compound from the LP-catalyzed oxidation of thiocyanate by H,Oz, it was found that the second-order rate constant, k,,was pH independent (range 3.0-10.8) and had a value of (9.2 k 0.9) x lo6 M-' sec-'. Either cyanosulfurous or cyanosulfuric acid was suggested (Hogg and Jago, 1970b) as the likely inhibitor, but hypothiocyanite (OSCN)was subsequently suggested (Aune and Thomas, 1977) and confirmed as the inhibitor (Pruitt and Tenovuo, 1982).Jones and Simms (1930) were proved wrong in that S was also involved in the inhibitor. The LP system was neutralized by catalase (Mickelson, 1966) when the organism was grown under aerobic conditions (Jago and Morrison, 1962) and was inhibited by thiosulfate, CSH, thiourea, methimazole, cysteine, ergothioneine, azide, ascorbic acid (Klebanoff et al., 1966), fluoride, cyanide (Pickering et al., 1962),and thioglycollic acid (Mickelson, 1966). Some thought (Stadhouders and Veringa, 1962) that the inhibition of lactic acid bacteria by LP and the prevention of that inhibition by cysteine were related. Cysteine was proposed to act as an H donor in a mixture with H,O, and LP, whereas in the absence of H,Oz it formed an irreversible compound with LP. Sensitive organisms may have an absolute requirement for free cysteine (Stadhouders and Veringa, 1962). The sensitivity of lactic streptococci to LP in milk was related to their capacity to inactivate LP (Stadhouders and Veringa, 1962). Pickering et al. (1962)found that lactic streptococci modified LP, and that all of the cultures of S. lactis and S . cremoris tested for sensitivity to LP showed a response similar to 803. Natural and modified LP differed in that natural LP showed a peak of activity at 0.1 mM H,O,, while modified LP showed activity over a broad range of H,Oz concentrations with peak activity at 30 mM H,O,; KF and KCN inactivated both forms but each form showed a different percentage inactivation by a given concentration of the inactivators; natural LP was more resistant to heat inactivation of 64°C for 20 min at pH 6.0 than modified LP, and velocity constants and activation energies of heat denaturation

BACTERIAL GROWTH IN MILK

59

differed for the two forms. Further studies showed that with strain C10 inactivation of LP was due to a buildup of excessive H,O, in the medium during aerobic growth; up to 0.11 mM H,O, was measured in the culture (Hogg and Jago, 1970a). Preincubation of 972 with LP and subsequent transfer of the cells to an LP-free medium containing the other constituents of the LP system resulted in inhibition of the organisms, which was counteracted by adding catalase to the LP-free medium (Steele and Morrison, 1969). In addition to modifying LP and producing excessive quantities of H,O,, the lactic streptococci produce an NADH and NADPH oxidizing enzyme which catalyzes the reduction of the inhibitor to a noninhibiting product (Hogg and Jago, 1970d; Oram and Reiter, 1966a). This enzyme was present in extracts of 803 and could neutralize the inhibitor for 972 (Oram and Reiter, 1966a). A schematic representation (Hogg and Jago, 1970d) of the action of LP on lactic streptococci is shown in Fig. 4.

FIG.4 . The postulated sites of activity of the lactoperoxidase-thiocyanate-hydrogen peroxide-generated inhibitor in lactic cultures. From Hogg and Jago (1970d).

60

D. K. O’TOOLE

How does the inhibitor influence the streptococci? Because the LP system prevented lactic acid formation and oxygen uptake in 972, it was suggested (Oram and Reiter, 1966a) that the system acts on the energy-generating process, i.e., the Embden-Meyerhof-Parnas pathway. The activities of enzymes in inhibited cells were examined and compared with those in uninhibited cells of two strains, 972 and 803 (Oram and Reiter, 1966a). Inhibited cocci of both strains had no hexokinase activity. Hexokinase in cell-free extracts was inhibited by the system and the inhibition was not reduced by dialysis or the addition of reducing substances (Oram and Reiter, 1966a). Thiocyanate alone did not inhibit hexokinase in extracts. Whole inhibited cells washed in Ringer phosphate showed normal hexokinase activity (Oram and Reiter, 1966a). Lactic dehydrogenase, NADH oxidase, and NADH peroxidase had normal activity in cell-free extracts of inhibited cocci, but some inhibited cocci (Oram and Reiter, 1966a) had higher phosphohexose isomerase activity [972), phosphohexokinase activity [803), and glyceraldehyde-%phosphate dehydrogenase activity (972, 803). Inhibited cocci also have partially inhibited fructose diphosphate aldolase activity (Hogg and Jago, 1970d). In resting cells of Lactobacillus acidophilus, though, the LP system inhibited the accumulation of glutamic acid and certain other amino acids (Clem and Klebanoff, 1966). In addition to the lactic starter organisms S. cremoris and S. lactis, and L. acidophilus, other organisms inhibited by the LP system are Streptococcus faecalis, Staphylococcus albus, Escherichia coli, Bacillus megatherium (Klebanoff et al., 1966),S. pyogenes, and S. agalactiae, which showed a delay of up to 6 hr before growth initiation that could be abolished with catalase (Mickelson, 1966). Recent reviews of the LP system include those of Pruitt and Reiter (1985) and Wolfson and Sumner (1993).

c. AUTOINHIBITION BY HYDROGEN PEROXIDE Another explanation offered for inhibition of lactic acid bacteria in milk is that they produce autoinhibitory levels of H,O, (Gilliland and Speck, 1969). Cultures were grown in reconstituted skim milk containing 0.01 mg/ml of catalase and H,O, levels were measured. Cultures containing catalase reduced the pH to 5.0 some 2 1 to 54 min faster than controls in which H,O, accumulated (Gilliland and Speck, 1969).There was no accumulation of H,O, in tubes containing catalase, whereas the addition of pyruvate reduced H,O, accumulation with no effect on acid production (Gilliland and Speck, 1969). Reduced glutathione was a

BACTERIAL GROWTH IN MILK

61

little more effective at preventing H,O, production and stimulated acid production slightly, but 0.5 mM FeSO, prevented H,O, production and greatly increased acid production, even more than catalase boosted acid production (Gilliland and Speck, 1969). Autoclaved catalase had no effect. A micrococcus isolate, F4, and a preparation of its capsular material increased growth and acid production of S. lactis, S. cremoris, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus casei var. casei (Nath and Wagner, 1973). Some of this stimulation was due to catalase but some other factor was also involved, as pure catalase could not restore the maximum stimulation obtained with the F4 associated material. The maximum accumulation of H,O, occurred during the most rapid decrease in pH (Nath and Wagner, 1973). The autoinhibitory levels of H,O, in milk could be modified by the natural levels of catalase in milk, which vary with the severity of subclinical mastitis in the cows producing the milk.

D.THIOCYANATE IN MILK In view of the importance of thiocyanate in the lactoperoxidase system, the concentrations that occur in milk and how they come to be there are of interest. Two precursors of this substance in milk isolated from plants of the Brassica family are glucobrassicin (Virtanen, 1961) and neoglucobrassicin (Reiter and Moller-Madsen, 1963), the former being a thioglucoside containing an indole group (Virtanen, 1961).Thiocyanate can be formed by enzymatic processes from mustard oil glucosides (Virtanen and Cmelin, 1960). Seasonal variations in the thiocyanate content of milk have been noted. In France a mean maximum of about 10 ppm NaSCN was obtained in summer, with a mean minimum of about 4 ppm NaSCN in winter (Boulange, 1959). Results from Victoria were similar; there was a yearly average of 4.9 ppm (range 1.4 to 10.8 ppm), the minimum values being obtained in August (Lawrence, 1971). In feeding experiments (Virtanen and Cmelin, 1960) a mixture of ammonium and potassium thiocyanate was fed to individual cows. This caused a jump in the thiocyanate content of milk from 0.24 to 1.4 mg Yo within 24 hr in one case, and in another case a jump to the same level from 0.12 mg Yo was observed. There was a lag period of 10 to 28 days before the thiocyanate content of the milk dropped to the pretreatment level (Virtanen and Cmelin, 1960). Clearly female diet can influence the thiocyanate content of milk.

62

D. K. O'TOOLE

E. LACTOFERRIN A red protein isolated from milk in 1960 (Groves,1960)is now known as lactoferrin. Studies showed that this protein had iron-binding properties (Gordon et al., 1962)and that bovine milk contained low levels of Fe (Oram and Reiter, 1968).Many bacteria require relatively high levels of Fe in their growth medium, such as species of Bacillus (Oram and Reiter, 1968) and E. coli (Reiter, 1978),and when they are inoculated into milk containing high levels of lactoferrin their growth is retarded or death occurs. Thus the activity of lactoferrin is antinutritional in character. However, the antibacterial effect of lactoferrin is neutralized by citrate in the milk (Bishop et al., 1976). Levels of lactoferrin in bovine milk vary from 0.1 to 0.35 mg/ml (Gaunt et al., 1980),but in human milk levels are very high (greater than 2 mg/ml) (Reiter, 1978). In bovine milk the level of lactoferrin is inversely related to the milk production levels of individual cows (Thomas and Fell, 1985). F. OTHER POSSIBLE INHIBITORS FOR LACTIC ACIDBACTERIAIN MILK Other inhibitors for bacterial growth in milk are possible but have not yet been recognized. The fatty acids caprylic, capric, lauric, and myristic showed some inhibition for S. cremoris, S. lactis and Streptococcus diacetilactis, whereas oleic acid was more selective, mainly affecting S.cremoris C13 (Anders and Jago, 1964).The oleic acid altered the metabolism of pyruvate by C13 by inhibiting the enzyme involved in the formation of acetate (Anders and Jago, 1970). Butyric, caproic, palmitic, stearic, arachidic, linoleic, and linolenic acids were ineffective (Anders and Jago, 1964). Various milk-associated volatile compounds (Table 11) were found by the disc assay method to be inhibitory for strains of S. lactis, S . cremoris, S . diacetilactis, Leuconostoc citrovorum, and S. aureus (Kulshrestha and Marth, 1970). The influence of fatty acids in milk may be complicated by an interaction with Ca2+ions (Galbraith et al., 1971). Streptococcus faecium is inhibited by 1 mM sodium lauryl sulfate, 0.35 mM linoleic acid, and lauric acid; however, the activity of the latter compound was counteracted by 1 mM CaZ+and 1 mM Mgz+,and that of linoleic by 2.0 mM Ca2+but not by Mg2+(Galbraith et al., 1971). Of 42 compounds tested for their inhibitory activity against S . agalactiae (see Appendix B) 8 were inhibitory at 200 pg/ml with serine, tyrosine, and glycyl-1-glutamic acid being least inhibitory (Brown,

63

BACTERIAL GROWTH IN MILK TABLE I1

SOME MILKASSOCIATEDVOLATILECOMPOUNDSTHATCANCAUSEINHIBITIONOF BACTERIAL GROWTH Substance (100 ppm)

S. lactis

S . cremoris

S . diacetilactis

L. citrovorum

Formaldehyde Glyoxal Decanoic acid Butyric acid Formic acid Hexanoic acid Diacetyl

+ + +"

+ + (1 strain] +" +a +a +a

+ + + ( 1 strain]

S. aureus _____

~~

+ + +

+ +

+

+

When tested at 100 ppm these substances caused no inhibition. Note. From Kulshrestha and Marth (1970).

1974).The most inhibitory were L-asparagine, L-isoleucine, L-leucine, L-methionine, and L-valine. The D-isomers of isoleucine and leucine were inactive up to 100 pg/ml. Only D-a-keto-p-methyl-N-valeric acid, of the precursors of isoleucine, leucine, and valine, was active at 1 mM or less (Brown, 1974).L-Cystine, which could neutralize to some extent the natural inhibitors of milk, could not counteract the effect of Lisoleucine (Brown, 1974). It is possible that some of the above free amino acids may be the cause of inhibition of lactic acid bacteria in milk under natural conditions. However, of these substances, it is more likely that the fatty acids are a cause of inhibition because of the phenomenon of lipolysis in raw milk. Under natural conditions and in some circumstances the natural lipase in some milk supplies can be activated and a reasonable amount of free fatty acid produced before the milk is processed. These residual fatty acids can cause defects in milk and may also interfere with the activity of bacteria in milk.

G. SEASONAL VARIATIONIN BACTERIALACTIVITY IN MILK

Bacterial activity in milk apparently varies with the seasons (Czulak and Meanwell, 1951;Hunter, 1949).This seasonal variation could be due to either the practice of seasonal calving, which is accompanied by physiological changes in the milk associated with the lactation of the cow, or the availability of pastures for the animals. No studies have set out to separate these two factors. Brown and McDonald (1982)used acid production and plate counts to measure the effect of lactation of individual cows on the growth of

64

D. K. O’TOOLE

S. agalactiae. They found that over the lactation as the yield of milk dropped the suitability of milk for the growth of the culture increased. In the milk from one cow, counts after growth in milk from early versus late lactation varied from 0.4 to 1.6 x lo9 CFU per milliliter. Bactericidal and bacteriostatic activity of milk for S. aureus var. aviarius was less apparent in February (Northern Hemisphere) than at other times of the year (Bergaminiand Solinas, 1950).The most notable effect is on starter activity. A low period of activity was noted in England from December to March (Czulak and Meanwell, 1951) and a similar activity pattern (Fig. 5) was noticed in Australia (Jago, 1954). In New Zealand when seasonal calving was practiced, one fast strain, C13, showed decreasing activity as the season progressed but a slow strain, AM1, showed an increase in activity (Lawrence and Pearce, 1972). Season, particularly spring, had a significant effect on gas, acid production, and multiplication of S. diacetilactis and Streptococcus paracitrovorus, the latter being quite sensitive (Gibahman, 1960). Inhibition seemed greatest when pastures were in a state of decline (Jago and Swinbourne, 1959). Others have noted no seasonal effects (McEwen

0.8

0.7 U .-

m U

U .-”

m

0

; 0.6 ” 2 8

‘ 2

0.5

0.4

I 1 Sept.

I

I

4

I

I

4

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

I

I

I

I

I

Apr.

May

June

July

Aug.

Months of the year (1952-3) FIG.5. Variation over 1 year (Southern hemisphere) in lactic acid production by lactic cultures resistant and suseptible to the natural inhibitors in raw milk when grown in boiled and raw milk. Curves A and C show results from susceptible strains in boiled (0) and raw [O)milk, and curves B and D are results from resistant strains in boiled [A)and raw (A) milk. There were eight susceptible and two resistant strains. From Jag0 (1954). Reprinted with permission of Cambridge University Press.

BACTERIAL GROWTH IN MILK

65

and White, 1950), and Auclair found no correlation between season and lactenin content of bulk milk (Berridge, 1955). But the ability of certain strains of S. Iactis to ferment normally in spring season milk was linked with the organism’s greater proteolytic activity, its ability to synthesize nicotinic acid, and its requirement for more biotin but less riboflavin (Speck, 1962). An attempt (Reiter, 1973) to correlate seasonal variations in free and protein bound amino acids with the occurrence of difficulties in cheese and butter manufacture, associated with slow starter growth, ended in failure despite a sophisticated approach. In an earlier attempt (Anderson et al., 1955) to correlate the peptide, peptone, and proteose peptone content of milk with activity of lactic acid bacteria, a correlation between increased peptide content and lactic acid production was reported. The evidence, however, was not very strong. This slowness of starter activity, particularly in winter months, was thought to be linked with availability of nitrogen in the milk (Garvie and Mabbitt, 1956). Czulak and Meanwell (1951) originally postulated that there was a constant concentration of inhibitor which was counteracted by a growth factor in milk which varied with the season, being lowest in winter. They thought that uninhibited cultures could synthesize the growth factor. Stadhouders (1961) noted higher inhibitory activity due to peroxidase in winter. He suggested that a thermolabile stimulator inactivated peroxidase partially but irreversibly in summer milk, and that either thermostable substances or stimulative substances formed on heating were also involved. Despite this he did not detect a particularly noticeable seasonal variation in starter activity, although he did note a seasonal variation in agglutinins (Stadhouders, 1963). LP system-associated susceptibility of Streptococcus uberis, another mastitis-causing organism, to late lactation milk, secretions obtained after drying off and up to 21 days postcalving, was positively related to the susceptibility of cows to challenge from the organism (Marshall et al., 1986).

H. HEATED MILKAS A GROWTHMEDIUM FOR LACTICACIDBACTERIA In many of the studies of the inhibitors in milk, heated milk of some description has been used as a diluent. It is assumed that heating to kill extraneous organisms has no effect on the growth-supporting or -inhibiting properties of milk, but this assumption is not valid as Brown (1967) has shown (Fig. 6). Whether the diluent is steamed or autoclaved can have a significant influence on results. A summary of studies of

D. K. O'TOOLE

66 7.0 .8

.6 .4 .2

6.0 .8

pH

.6 .4

.2 5.0

,..

.8

.6 .4

4.2

*

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

t I

I

I

I

I

I

I

0

2

4

8

16

32

64

'

I

'

128

256

512

Dilutions FIG.6. The effect of different heat treatments of the diluent milk on the inhibitory activity of pasteurized milk for S . agalactiae 660. Diluent milks were heated at (1) 80°C for 30 min, (2) 90°C for 30 min, (3) 100°C for 30 min, (4) 121OC for 15 min. From Brown (1967).

the effect of heating on the growth-supporting properties of milk are tabulated in the brief review of Feldstein and Westhoff (1979). More specifically, the ability of lactic acid bacteria to grow in milk heated at various temperatures and times has been studied. Acid production by s. lactis and s. cremoris was more rapid in milk heated to 76.7"C momentarily than in milk heated at 62.8"Cfor 30 min (Rice, 1942).A culture of S. lactis grew better in autoclaved skim milk (115°C for 15 min) than in low heat milk (80°Cfor 10 min) although autoclaved milk showed greater variability in its growth-supporting properties; with increased time of heating at 80 and 100°C there was increased stimulation of starter growth (Foster, 1952). The beneficial effect of heating milk strongly was lost when the casein was removed (Foster,

BACTERIAL GROWTH IN MILK

67

1952). Autoclaved milk stimulated acid production in low heat milk; one part to two parts low heat milk as well as autoclaved milk alone supported growth, and peptonized milk (100 pg/ml) stimulated acid production in low heat milk in a similar manner (Foster, 1952). Cycles of stimulation and inhibition of lactic starter cultures, linked with the presence of denatured serum protein cysteine, were observed in milk heated to various temperatures (Greene and Jezeski, 1956). In milk heated for 30 min there was a primary period of stimulation up to 72°C inhibition at 80 and 9O"C, and further stimulation at 95 and 120°C. Inhibition caused by heating at a temperature below 100°C was a unique function of the available cysteine concentration in milk. Volatile sulfides arising from heated sulfydryl compounds were also implicated (Greene and Jezeski, 1956), evidence for which is provided by Henningson and Kosikowski (1957) who found that if S. pyogenes grew in whey heated to 80°C for 5 or 10 min they could demonstrate the presence of free sulfydryl groups. As the maximum growth of the organism in whey heated at 100°C for various periods decreased, there was a corresponding decrease in the concentration of sulfydryl groups as shown by a decrease in the reaction intensity of the nitroprusside test (Henningson and Kosikowski, 1957). Heated, i.e., denatured, lactalbumin had a stimulating effect similar to that of heated milk (Henningson and Kosikowski, 1957). A study (Lyster, 1964) of the effect of heating skim milk at various temperatures for 15 min on the liberation of sulfydryl groups has shown (Fig. 7) that as milk is heated above 70°C there is a rapid increase in the concentration of free sulfydryl groups in the milk. A similar response pattern was obtained when the milk was heated for 1 min at similar temperatures (Kirchmeier et al., 1984). These observations can explain some of the variation in the ability of heated milk to support the growth of lactic acid bacteria.

I. EFFECTOF CULTURE MANIPULATIONS The sensitivity of some strains to the inhibitors in milk is affected by the maintenance procedures adopted for the culture before testing. Some strains, e.g., S. lactis ClO, although insusceptible to inhibitors in raw and pasteurized milk, developed susceptibility on prolonged daily subculture in autoclaved milk (Jago and Swinbourne, 1959). This was due to the presence in the ClO culture of cells resistant and sensitive to both lactoperoxidase (LP) and agglutinin (Auclair and Vassal, 1963). Those sensitive to agglutinin were also sensitive to LP, and those sensitive to LP were not necessarily sensitive to agglutinin. Streptococcus

D. K. O'TOOLE

68 0.18

1

0.16 0.14 0.12

5z-

0.10

Y 0.08 0.06 0.04 0.02

40

50

60

70

80

90

100

Temperature,'C FIG. 7. The -SH content of skim milk heat for 15 min at various temperatures. (0) Free -SH,(0)total -SH.From Lyster (1964).Reprinted with permission of Cambridge University Press.

cremoris 972 contained cells sensitive to both inhibitors but the agglutinin sensitive 760 contained cells (100%) sensitive to agglutinin and cells (48%) sensitive to LP (Auclair and Vassal, 1963). Auclair and Vassal (1963) suggested that subculturing in autoclaved milk unmasked a specific antigen on the surface of the cells for which a homologous antibody exists normally in cow milk. More recently, growth conditions for a Listeria monocytogenes Scott A culture, a nonlactic organism, were shown to influence the length of lag periods induced by the lactoperoxidase system. Culture inocula prepared from shaken cultures began growth sooner than statically grown cells (Siragusa and Johnson, 1989). Apparently aerobic growth prepared the organisms better for growth in the presence of the LP system than anaerobically grown cells.

J. EFFECTOF MILKON MORPHOLOGY OF STARTER CULTURES In a comparison (Morris and Edwards, 1949) of growth rates of S. Iactis, as measured by the number of colony forming units (CFU) per milliliter and methylene blue reduction times, it was found that CFU per milliliter was lower in raw milk than in heated milk (70°C for 1hr)

BACTERIAL GROWTH IN MILK

69

even though methylene blue reduction times were similar. Cultures could be placed in three groups: (a) those forming long chains in raw milk and short chains and diplococci in heated milk; (b) those forming clumps in raw milk; and (c) those forming short chains in raw and heated milk. The ratio of the average numbers of cocci per chain in raw and heated milk for six strains were 21.25 : 1.7; 2.5 : 1.4; 6.9 : 1.0; 3.6 : 1.0; 11.0 : 1.75; and 1.0 : 1.2. A protein was thought to cause the phenomenon, but the clumping was not due to agglutination and the chain inducing ability of milk was destroyed when milk was heated at 68 and 70°C for 1 hr (Morris and Edwards, 1949). Peptone added to milk (Garvie and Mabbitt, 1956) changed the morphology of lactic streptococci; in its presence the long axis of the organism was along the chain, and in its absence the long axis of the cocci was across the chain. V. Effect of Cow Ration on Milk as a Growth Medium for Bacteria

Because the thiocyanate concentration in milk can be increased by altering the cow’s ration, one could expect a cow’s ration to influence bacterial growth in her milk as cows graze different pastures and consume different rations. Pounden and associates investigated aspects of the effect of cow rations on the growth of S. agalactiae, an important lactic acid bacterium that causes mastitis. They wanted to measure the relative resistance of animals to S. agalactiae without actually exposing the animals to the organism, so they measured acid production by the organism in the milk-the less acid produced the more resistant the milk, and perhaps the cow, to the organism (Pounden, 1952). Among factors examined were the feeding of vitamin A, citrates, and phosphates, intravenous injections of vitamin E, phosphates, and ascorbic acid, and the roughage fed (Pounden et al., 1952). Only roughage seemed to have any effect, but other factors could not be ruled out (Pounden, 1952).

Milk from a group of Holstein-Friesian and Jersey cows receiving high-quality roughage (alfalfa hay and silage) was consistently more inhibitory for S . agalactiae than milk from a group fed low-quality timothy hay (Pounden et a ] . ,1952). In another experiment three groups of cows were selected and put on blue grass pasture, on a legume and brome grass pasture, and on a low-quality mixed hay, mostly timothy, which had an average protein content of 7.16%; alfalfa hay had an average protein content of 17.51% (Pounden, 1952). During three seasons milk from the group fed low-quality hay was significantly (P < 0.01) more inhibitory for the microorganism than milk from cows on higher-quality pasture (Pounden et a]., 1956a). Introduction to pas-

70

D. K. O’TOOLE

ture after feeding stored food during winter was correlated with increased incidence of streptococcal mastitis (Pounden et al., 1956a). The addition of corn silage to a timothy hay and grain ration resulted in greater acid production in milk (Pounden et al., 1956b). When changing from pasture (alfalfa, blue grass) to a forage crop silage there was a slight increase in the inhibition of organisms in the milk, but a transfer from pasture to a hay, grain, and silage (corn or forage crop) ration seemed to have no effect on acid production by the organism (Pounden et al., 1956b). Cows on a ration of fresh-cut pasture of alfalfa, Ladino clover, and grass (soilage) or a ration of silage made from that pasture (plus a grain mixture in both cases) showed a difference in resistance to streptococcal mastitis; the silage-fed group had a lower incidence of mastitis (Pounden et al., 1958) and their milk had greater inhibitory activity for S. agalactiae (Pounden et al., 1960).The activity of a hemolytic staphylococcus was also lower in milk from silage-fed cows, as the organism took longer to reduce methylene blue in such milk than to reduce methylene blue in milk from silage-fed cows. Pounden’s main concern was mastitis; he presents evidence of changes in the growth-supporting properties of milk as cow ration changes, and he notes flare-ups of mastitis which occur coincidentally with these ration changes (Pounden and Frank, 1961; Pounden et al., 1958). Pounden et al. (1958) noted, for example, one herd in which 5% of cows had mastitis in the month before going on to pasture and 28% had mastitis in the month following the ration change. Changes in the growth-supporting properties of milk tended to be inversely related to level of milk production, i.e., low milk production was related to increased bacterial inhibition (Pounden et al., 1957), which is contrary to the results of Brown and McDonald (1982). When cows were placed on pasture there was an initial decrease in bacterial inhibition which was followed by a partial recovery of the inhibitory properties of milk (Schalm et al., 1971). One explanation (Schalm et al., 1971) offered for Pounden’s observations is that pasture increased milk secretion, thus diluting leucocyte numbers and humoral factors which normally act as bactericidal agents in milk. Schalm et al. (1971) suggested that the partial increase in inhibitory factors is due to subsequent reestablishment of the disturbed equilibrium of the defence mechanisms. No inhibitory effecton yoghurt cultures, S . lactis or S. thermophilus, was noted in milk from cows fed a weed, Lepidium bonariense (Luck et al., 1966). The activity of a lactic streptococcus, ML, was greatly reduced during a drought and improved markedly immediately after rain (Hunter, 1949). The addition of 0.01% tryptone to cheese milk counteracted the adverse effect of drought on milk (Hunter, 1949).Speck

BACTERIAL GROWTH IN MILK

71

(1962) noted variations in milk composition, including proteose-peptone content, due to pasture and dry feeding but found no correlations with culture activity. Differences in starter growth were more dependent on culture characteristics than cow ration (Speck, 1962).

VI. Role of Inhibitors

in Milk Quality

Inhibitors in milk have played a number of roles. First, they have been found to influence tests for coliforms and for antibiotic content of milk. Second, they have been used to manipulate the keeping quality of milk, as outlined later in this review. A. RAW MILKQUALITY Milk quality depends on its microbiological status which is shown by various traditional tests. Trout et al. (1951) found that the inhibitory properties of milk were instrumental in maintaining the bacteriological and organoleptic properties of raw milk. Although they were thought to improve the keeping quality of milk from tropical countries, no proof of this was found in lactenin levels (Auclair, 1968). The inhibitors may also mask the true bacteriological condition of raw milk (Morris, 1945). Milk containing large numbers of coliforms but having long methylene blue reduction times, that is, apparently of good quality, contained substances which were specifically bactericidal and bacteriostatic for coliform organisms (Morris, 1943).In addition, Clegg et al. (1956) found that they retard the growth of thermoduric bacteria in raw milk but do not improve the keeping quality of pasteurized milk, a view contradicted by recent studies which showed that activation of the LP system in milk improved the keeping quality of pasteurized milk (Kamau et al., 1991). When testing milk for the presence of Campylobacter jejuni the rate of isolation was improved by inactivating the LP system by raising the pH to 7.5 (Beumer et al., 1988). During one test for antibiotic residues in milk a culture of Bacillus stearothermophilus var calidolactis is used (other cultures are also used) and false positives are obtained, sometimes due to the natural inhibitors in milk. Lysozyme, lactoferrin, and the LP system inhibit the organism at levels found in milk, and there was a synergistic effect between lysozyme and lactotransferrin (Carlsson and Bjorck, 1987). It was suggested that with elucidation of the LP system in milk, the system could be activated to improve the keeping quality of refrigerated milk in view of its activity against pseudomonad bacteria (Bjorck, 1978);

72

D. K. O’TOOLE

this idea was extended to short time keeping quality of milk in tropical countries with low-technology dairy farming environments (Bjorck et al., 1979a). By adding 10 ppm thiocyanate and 8.5 ppm hydrogen peroxide, the level of bacteria in milk at 30°C remained static for 7-8 hr (Bjorck et al., 1979b). Tests in Sri Lanka (Harnulv and Kandasamy, 1982) and Kenya (Ridley and Shalo, 1990) showed that the method could be used to extend the keeping quality of water-cooled evening milk. The treatment did not affect the quality of the milk for cheddar and cottage cheese manufacture (Zall et al., 1983), an important point in view of the effect of natural inhibitors on the growth of lactic acid bacteria. However, acid production and cell growth by yoghurt cultures were delayed in milk in which the LP system was activated (Basaga and Dik, 1994). VII. Stimulators of the Growth of Lactic Acid Bacteria

Although a stimulator in milk is postulated, its identity is not known. The most common approach to the problem has been to test compounds at random for their stimulative properties in milk. In addition, many compounds have been found which are essential or stimulate the growth of lactic acid bacteria under certain conditions. A. INVESTIGATIONS OF MILKAND MILKCOMPONENTS Auclair (1954a) noted the stimulation of S. agalactiae by colostrum, an observation made earlier with S. lactis and S. cremoris for which the addition of 10-25% colostrum induced more rapid acid production (Rice, 1942). Among the substances tested by Auclair which might replace the activity of colostrum, it was found (Auclair, 1954a) that only sulfur-containing compounds such as NaS and thioglycollic acid (1 mM) and cysteine and cystine (0.01 mM) were effective. Auclair (1954a) suggested that the concentration of certain sulfur compounds normally present in milk was responsible for marked differences in the growth rate of some organisms. The rate of acid production by lactic streptococci varied in different milk fractions (Gilliland and Olson, 1963), whole milk, skim milk, buttermilk, whey, whey from buttermilk, and buttermilk from washed cream. Acid production was more rapid (Gilliland and Olson, 1963) in whole milk and buttermilk than in skim milk, and even more rapid in a 1: 1 mixture of the skim milk and buttermilk. Brown (1974) studied 110 low-molecular-weight compounds (see Appendix B) for possible stimulators of S. agalactiae with the aim of

BACTERIAL GROWTH IN MILK

73

finding what may be the natural stimulators of milk. All compounds were tested in a 1 : 3 2 dilution of pasteurized skim milk (60°C for 30 min) in steamed skim milk (100°C for 45 min) supplemented with 300 pg/ml of NH,SCN. The only stimulators (at an initial concentration of 1pg/ml) found were hydrolyzed cephalin, cytidine-5'-triphosphate, and sulfur-containing substances which, in descending order of stimulative activity, were L-cystine, L-cysteine, DL-homocystine, reduced glutathione, DL-homocysteine, dithiothreitol, NaS, and sodium thioglycollate; DL + allo-cystathionine was initially inhibitory but became stimulative on dilution.

B. STIMULATION BY MINERALS Mineral requirements and their interaction with other requirements of the organisms have been studied. Streptococcus cremoris HP and S. lactis C10 show a requirement for Fe and V, the latter metal replacing the requirement for the former, but Mo was without effect (Reiter and Oram, 1968). The quantities for maximum growth of the organisms were 0.002 ppm (ClO),0.001 ppm (HP) of Fe, and 0.01 ppm (ClO), 0.04 ppm (HP) of V (Reiter and Oram, 1968). Fe activated aldolase in cell-free extracts of lactic starters, and the sequestration of Fe in milk reduced lactic acid production by S. thermophilus (Reiter and Moller-Madsen, 1963). Strains of S. cremoris and S. lactis were stimulated by the addition of 0.4% sea salt or 0.4 ml seaweed (kelp) extract per 10 ml of pasteurized skim milk (Olsen and Qutub, 1970). Of 1 2 trace elements present in seaweed, Fe, Mg, and Mo, each at 2 ppm, Se at 1 ppm, 2-4 ppm Zn, and 2 ppm Co stimulated acid production; B, Pb, and Li (at 8 ppm) had no effect or were slightly inhibitory, but Cu, I, and Hg at >4 ppm were inhibitory. A mixture of Fe (2 ppm) + Mg (4 ppm) + Se (0.5 ppm) significantly (P < 0.01) increased the rate of acid production in milk (Olsen and Qutub, 1970). A stimulative component of tomato juice was replaceable with Mn and 63 of 71 strains of lactic acid bacteria required this mineral (Stamer et al., 1964). Lactobacillus acidophilus and Lactobacillus bifidus were stimulated when 0.1-0.3% hexametaphosphate was added to a modified milk, presumably due to the removal of Ca2+ions (Sasaki et al., 1959). The amino acid requirements of s. thermophilus were partially dependent on and affected by the presence of Ca2+in the medium (Reiter and Moller-Madsen, 1963). This organism requires K+ and Mg2+;the latter is not replaceable with Ca2+ (Reiter and Moller-Madsen, 1963). Streptococcus lactis and S. cremoris responded to acetate and citrate, a reaction that may be related to Ca2+and Mg2+requirements of the organisms (Reiter and MollerMadsen, 1963).

74

D. K. O’TOOLE

C. COMPLEX SUBSTANCES AS STIMULATORS

In addition to simple organic compounds there are some complex substances which stimulate lactic acid bacteria and which have been studied to determine their active constituents. Some of these stimulators are pancreas extract (Koburger et al., 1963; Sandine et al., 1956),tomato juice (Stamer et al., 1963),corn steep liquor (Johnson et al., 1971; Sasaki et al., 1959), a liver extract (Garvie and Mabbitt, 1956; Sasaki et aJ., 1959), an aqueous extract from thermophilic methane fermentations (Khandak et al., 1972), chitin hydrolysate (Sasaki et al., 1959), peptone (Wilson and Rosenblum, 1952a), and tryptone (Hunter, 1949). Tryptone stimulated the growth in milk of the slow strain S. cremoris ML, and cystine, cysteine, and glutathione, but not tryptophan, produced stimulation similar to that produced by tryptone (Hunter, 1949). However, tryptophan and threonine were stimulative for strains of S. cremoris in another study (Reiter, 1973). Generally, strains of S. lactis grow adequately using sodium caseinate as the sole nitrogen source; S. cremoris is more fastidious and requires alanine and proline at concentrations similar to those found in milk, and some strains require tyrosine and lysine (Reiter, 1973). Pancreas extract (Dahiya and Speck, 1963) improved acid production by lactic streptococci in fresh milk so that it compared favorably to reconstituted skim milk. This extract could be replaced by inosine, the best of the purines and purine ribosides tested, but the availability of various nitrogen sources, such as amino acids and peptides, also seemed important (Dahiya and Speck, 1963). In an earlier study (Sandine et aJ., 1956) two stimulants for L. casei and S. lactis were found in pancreas extract. They were active in the presence of “Streptogenin” (a term coined by Woolley to describe peptides and crude extracts containing peptides that stimulate bacteria of the family Lactobacteriaceae). One appeared to be a peptide containing lysine, aspartic acid, serine, glycine, glutamic acid, threonine, alanine, proline, valine, and probably leucine and isoleucine. A peptide (MW ~ 4 7 0 0 produced ) by S. Jactis which stimulated L. casei had similar composition but contained, in addition to the above, arginine, histidine, ornithine, phenylalanine, and tyrosine; none of these are sulfur amino acids (Branen and Keenan, 1970). Johnson et al. (1971) implicated inosine (1 pg/ml) and hypoxanthine adenine in pancreas extract as the stimulants for S. Jactis. Two strains of S. cremoris (KH and HC) produced a substance stimulating acid production by R6 which was not a peptide, amino acid, or an amine; by UV spectral analysis, it appeared to be guanine which, like adenine and guanosine, stimulated growth

BACTERIAL GROWTH IN MILK

75

when added to milk (Kothari and Nambudripad, 1973). Adenine and adenosine were the active constituents in tomato juice stimulating L. bulgaricus (Cogan et al., 1968a) but an inhibitor also present for this organism was a nucleotide containing xylose and adenine (Cogan et al., 1968b). For L. citrovorum tomato juice contained the stimulants fructose and CO,. Corn steep liquor (Johnson et al., 1971) contained four constituents that stimulated S . lactis and S . cremoris, one of which was a peptide containing an aromatic amino acid; the others were not nucleotides or nucleosides, but could have been purine or pyrimidine bases. When the growth of streptococcal starter cultures were compared in fresh skim milk and reconstituted skim milk, growth in the fresh skim milk was reduced but could be enhanced by the addition of inosine; however, purines and purine ribosides were not as effective (Dahiya and Speck, 1964). Hydrolyzed casein and yeast extract stimulated L. casei (Hutchings et al., 1941). There were two separate substances, one of which was biotin. The other was unknown but contained an acid and probably an amino group, and was not a nucleotide (Hutchings et al., 1941). This substance stimulated Lactobacillus helvecticus, Lactobacillus delbruckii, S. lactis, and Propionibacterium pentosaceum (Hutchings et al., 1941).Under defined conditions, p-aminobenzoic acid, methionine, adenine, guanine, hypoxanthine, and xanthine were growth factors for Lactobacillus arabinosus, and Lactobacillus pentosus, S . lactis, and Leuconostoc mesenteroides responded to purine bases but not to the other substances (except that adenine did not stimulate L. mesenteroides) (Snell and Mitchell, 1942). A L. bulgaricus strain requiring lactose, but not growing on glucose or galactose, was stimulated by pyruvate (Snell et al,, 1948). When added to media containing all known growth essentials, peptone, and plant and animal tissue extracts, increased early growth of L. casei. The growth factor (Pollack and Lindner, 1943) neither was a peptide nor was it glutamic acid which did cause a strong growth response. The vitamins required by S. lactis, S . cremoris, S. diacetilactis, and S . thermophilus are nicotinic acid, pantothenate, and biotin, but pyridoxal stimulates growth (Reiter and Moller-Madsen, 1963). Menadione (2-methyl-1&naphthoquinone), related to vitamin K, has been suggested (Berridge, 1955) as the inhibitor associated with cream in milk ( Jago, 1954). When menadione and menadione diphosphate (tetrasodium 2-methyl-l,4-naphthohydroquinone diphosphate acid ester) were fed to cows it had no effect on starter culture activity even though menadione added to milk was inhibitory (Wilkowske et al., 1955).

76

D. K. O’TOOLE

In synthetic media glutamine and glutamic acid stimulated the growth of S. lactis, L. pentosus, L. arabinosus, and L. casei (Pollackand Lindner, 1942). Another growth factor for S. Jactis was not a known vitamin, nor could it be replaced with a combination of 23 amino acids (Smith, 1943). Folic acid, however, was suggested as a growth factor for this species (Keresstesy et a]., 1943).A substance in yeast extract stimulating the oxidation of pyruvate by S. faecalis was not leucine, phenylalanine, methionine, cysteine, glutathione, folic acid, or vitamins (O’Kane and Gunsalus, 1948), but it was suggested (Snell and Brequist, 1949) that it may be the same as a factor replacing acetate which stimulated L. casei. Acetate is also essential for lactic acid streptococci, but in S. faecalis it was replaceable with lipoic acid or mevalonic acid (Reiter and Moller-Madsen, 1963). Sodium inocinate was sufficiently stimulative for L. bulgaricus to reduce the manufacturing time for yoghurt by 1 hr. This organism and S. crernoris were also slightly stimulated by lacticol monostearate (Tsugo and Tanigucki, 1962). N-Acetyl-glucosomine, 1-amino lactose, lactulose, and N-acetyl lactosamine had no effect on various lactic acid bacteria (Tsugo and Tanigucki, 1962).Formic acid, up to 50 ppm, stimulates the growth of L. bulgaricus when growing with S. therrnophilus (Bottazzi et al., 1971). The growth of lactic streptococci was stimulated in milk to which pgalactosidase was added (Gilliland et al., 1972). This was apparently due to the organisms ability to grow faster on the glucose released by the enzyme than on lactose (Gilliland et al., 1972). D. COMMERCIAL STIMULATORS

Several commercial stimulators have been developed, particularly to overcome periods of low culture activity in milk such as spring and autumn (Hylmar et al., 1969).There was “stimulac” from East Germany, and “starter” composed of (Hylmar et al., 1969) yeast extract (10 g), casein acid hydrolysate (15 g), casein enzyme hydrolysate (15 g), peptone (15 g), calf heart infusion (150 ml), pancreas infusion (150 ml), calf muscle infusion (150 ml), 85% lactic acid (20 ml), distilled water (500 ml); an inoculum of 0.1%in milk was recommended. Yeast autolysate or yeast hydrolysate at 0.05-0.02% was recommended (Gibahman, 1960). Another commercial agent, “Lac-Val” (essentially a soluble yeast extract), stimulated (Weber, 1971) S. lactis, S. therrnophilus, S. diacetilactis, and L. helveticus at concentrations of 0.3-0.67%. The agent is thought to stimulate through reducing the lag phase (Weber, 1971).

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VIII. Growth Inhibitors of Other Bacteria

A. INHIBITORS FOR ENTEROBACTERIA The enterobacteria comprise a wide range of organisms including the coliforms. Milk samples containing large numbers of coliforms but exhibiting long methylene blue reduction times led to the observation that milk contained substances which were specifically bactericidal and bacteriostatic for coliform organisms [Morris, 1943). When inoculated into raw milk at 37°C the coliform count dropped from about 400,000 to 80 per ml within 6 hr [Morris, 1943). The substance is present in milk and whey in equal concentrations; in milk serum, 0.25% gave 99% kill of the organisms in sterile milk [Morris and Edwards, 1950). The concentration varied with the source of cow milk, just as it did in goat milk, and it was destroyed after 30 min at 53°C but was not destroyed within a similar time at 52°C [Morris and Edwards, 1950). Suspensions of heat-killed coliform organisms could absorb the factor but the effectiveness of the absorption depends on the temperature and time combination used to kill the organisms. Dead cells of S. lactis were also effective as absorbents of the factor [Morris and Edwards, 1950). Agglutination of coliform organisms by raw milk has been demonstrated [Portmann et al., 1962). Milk taken 1 2 hr after the induction of the inflammatory response in a cow udder by the injection of distilled water caused a sharp decline in numbers of Aerobacter aerogenes [ Jain et a]., 1967). Heating at 56°C for 30 min removed the effect, and 1% guinea pig serum added to the heated milk and to normal milk with no bacteriostatic properties resulted in reduced A. aerogenes counts [ Jain et al., 1967). This same organism added to normal milk and mastitic milk [somatic cell counts of >500,00O/ml) showed a range of responses. Some normal milks supported growth but others, and mastitic milks, either inhibited the growth of the organism or killed it [Carrol and Jain, 1969). Colostrum had low bactericidal activity which was enhanced in some cases by guinea pig serum. Heat-killed A. aerogenes removed the activity from milk and serum [Carrol and Jain, 1969). These observations can be explained by the presence of complement in milk which has been detected using guinea pig erythrocytes and a conglutin test (Reiter and Oram, 1967). It is regularly found in bulk milk, in colostrum, and toward the end of lactation but is irregularly present in normal cow milk [Reiter and Oram, 1967). Reiter and Oram (1967) suggest that complement and the formation of antibody-antigen complexes with gram-negative organisms are the cause, in many in-

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stances, of the bactericidal activity of milk against gram-negative organisms. Species of Yersinia (Farrag and Marth, 1992) are also killed by milk and can be controlled by the activation of the LP system. B. INHIBITORS FOR BACILLI An inhibitor which specifically inhibits Bacillus species was reported (Cheeseman and Jayne-Williams, 1964). First found affecting B. stearothermophilus, 23 of 60 spored thermophiles were inhibited by the substance. Other species inhibited are Bacillus coagulans (Ashton and Busta, 1968),Bacillus subtilis (Oram and Reiter, 1968; Pugh, 1970),and Bacillus calidolactis (Pugh, 1970), a variety of B. stearothermophilus used in tests to detect antibiotics in milk. The inhibitor is present in raw, pasteurized, and dried milk and is associated with the 30% (NH,),SO, precipitate from milk serum at pH 6.5 (Cheeseman and Jayne-Williams, 1964). It is stable on heating to 80-90°C for 60 min but is destroyed by 121°C for 15 min. The factor is not dialysable, separates with milk protein when filtered on Sephadex C-25, and is destroyed by trypsin and chymotrypsin (Cheeseman and Jayne-Williams, 1964). About 20 mM FeSO, counteracts it (Ashton and Busta, 1968; Cheeseman and Jayne-Williams, 1964; Pugh, 1970),as does MgSO,, but the latter is not as effective as FeSO, (Ashton and Busta, 1968; Cheeseman and Jayne-Williams, 1964). It is now thought to be lactotransferrin (Reiter and Oram, 1967), but whether there is more than one inhibitor for bacilli was disputed (Ashton and Busta, 1968).The concentration of a substance in milk inhibiting B. stearothermophilus increased as the protein content of the milk increased (Ashton and Busta, 1968). It was associated with the casein fraction of the milk of which it was a constant property. It was similar to lactotransferrin in that iron and magnesium counteracted its effect but dissimilar in that it showed greater heat stability (Ashton and Busta, 1968). The evidence for a second substance is that the secretion from nonlactating cows contained a substance not suppressed by Fe ions, with greater heat stability than lactotransferrin (Oram and Reiter, 1968), which was not inactivated by antiserum. Another inhibitor for bacilli, neutralized by Fe ions and probably lactotransferrin, was found whose concentration varied inversely with milk yield, maximum titres being present in the dry period secretion (Pugh, 1970). The mechanism by which this substance acts on the organism is unknown but Ashton and Busta (1968)suggested that the inhibitor may bind iron essential for the growth of the organism. Hence the addition

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of excess Fe ions saturates the sites on the inhibitor and the essential Fe ions become available for microbial growth (Ashton and Busta, 1968). It is known that lactotransferrin from bovine, human, and goat sources, when not fully saturated with Fe, has no effect on the activation of spores of B. stearothermophilus but it inhibits their germination (Oram and Reiter, 1968; Reiter and Oram, 1967). The bacteriostatic activity of lactotransferrin is also enhanced by Zn2+,Co2+,Mn2+,Ni2+,and Cuz+ (Oram and Reiter, 1968). c.

INHIBITORY

FACTOR FOR PROPIONIBACTERIA

The inability of Propionibacterium shermanii to grow in whey from a cheese factory was noted during a search for bacteriophage to these organisms (Vedamuthu et al., 1968).Similar activity was noted in whey from Cheddar and Colby cheese manufacturing plants and in whey obtained by direct acidification and by renneting. The factor also inhibited Propionibacterium arabinosum, Propionibacterium fruedenreichii, Propionibacterium jensenii, Propionibacterium pentosaccum, Propionibacterium peterssonii, E. coli, Micrococcus varians, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putrefaciens, and S. faecalis (Vedamuthu et al., 1968). It occurred at all times of the year, stored well at 5°C for many months, was stable in the range pH 4-7, was destroyed by boiling (Vedamuthu et al., 1968, 1971) and by 82°C for 16 sec, and was nondialysable (Vedamuthu et al., 1968).The activity was associated with the immunoglobulin portion of milk whey, presumably the pseudoglobulin fraction (Vedamuthu et al., 1971). There was some correlation between hemolytic activity of the organisms (eight species of propionibacteria tested) and susceptibility to bactericidal activity (Vedamuthu et al., 1971). Heating the whey for 10 min destroyed a band on disc gel electrophoresis associated with the activity (Vedamuthu et a]., 1971).

D. INHIBITIONOF THE STAPHYLOCOCCI Some (Baumgartner et al., 1965; Meeder, 1964a,b) have denied that raw milk is lethal for bacteria and for staphylococci in particular. The coagulation times for 31 strains of S. aureus var. aviarius were longer for 84% of the organisms in milk heated to 100°C than in milk heated to 65°C (Bergamini and Solinas, 1950).This property of milk was stable at 4°C.In Russian work (Mutevin, 1963),milk was shown to have strong antistaphylococcal activity. After some 10,000 tests on milk and udder secretions, four lysozymes or antibacterial substances were proposed

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as the cause. They were called lysozyme of the udder (U), lysozyme of milk (M), thermostatic lysozyme (T), and basic lysozyme (B). The characteristics of these substances, adapted from two tables in the paper of Mutevin (1963),are shown in Table 111.

E. INHIBITION OF LEPTOSPIRAS In addition to type-specific antibodies, milk contains a heat-stable factor which inactivates and lyses leptospiras (Kaplan et al., 1962).This factor has been demonstrated in milk from cows, goats, and humans, and in human colostrum. Virulent and avirulent strains of Leptospira hyos, Leptospira icterohaemorrhagiae, and Leptospira pomona are dissolved by the factor. The factor persists in milk stored at 4OC for 2 months and survives pasteurization at 80°C for 5 min, but is destroyed by boiling (Kaplan et al., 1962).

F. INHIBITION OF MYCOPLASMAS Experimental mastitis could be induced in cows with T-mycoplasmas (Gourlay et a]., 1972) but strains varied in their virulence and cows showed differences in susceptibility. Endotoxin-stimulated mastitic milk had lower mycoplasmacidal activity, but activity varied from cow to cow. This contrasted with pyrogen-stimulated mastitis, which gave TABLE 111 CHARACTERISTICS OF FOURANTISTAPHYLOCOCCAL SUBSTANCES IN MILK Group Characteristic ~

~~~

Diffusion in agar Delay growth of “lachrymalmicrococcus” Delay growth of staphylococcus Presence in normal milk Presence in colostrum Resists heating to 60°C Resists 65°C for 30 min Resists 70°C for 30 min Resists 75°C for 30 min Resists 100°C Udder lysozyme, U; milk lysozyme, M; thermostatic lysozyme, T; basic lysozyme, B. Note. Adapted from Mutevin (1963).

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milk with higher bactericidal activity. The activity was present in the whey from the milk; when the whey was fractionated it was associated with the immunoglobulin fraction but complement was not involved. Activity was lost on dialysis (Brownlie et al., 1974).The active principle could be absorbed by heterologous and homologous mycoplasmas and erythrocytes (Howard et al., 1975). IX. Applications of the Antimicrobial Systems in Milk

Jones and Simms (1930) concluded that the possible uses of lactenin were unknown but in the last 15 years a number of applications have been tested. First, Bjorck et al., (1979a,b) demonstrated the use of the LP system for short-term preservation of raw milk before processing. Activation of the LP system retarded the growth of gram-negative bacteria and allowed storage at relatively elevated temperatures without the milk deteriorating in quality, When stored at 5°C the growth of gramnegative psychrotrophic organisms was suppressed so that it took 4 days for LP-activated raw milk to reach the same count as that of the control milk at 1 day (Swart et al., 19901. This application was further tested in Kenya (Ridley and Shalo, 1990) and Sri Lanka (Harnulv and Kandasamy, 1982), and was shown to work with buffalo milk in India (Chakraborty et al., 1986). Kamau et al. (1990) showed that the growth of L. monocytogenes in raw milk could be controlled for 5 days at 10°C and for 8 hr at 35"C, and for S. aureus growth at both temperatures. In addition, Zapico et al. (1993) showed that the effect of the LP activated system in goat's milk depended on the strain of L. monocytogenes so that growth by 2 of 3 strains was prevented over 10 days at 4°C. The shelf life of pasteurized milk at 10°C was increased by 20 days after activation of the LP system by addition of 2.4 M SCN and 0.6 M H,O, followed immediately by holder pasteurization of 63°C for 30 min (Kamau et al., 1991). Second, L. monocytogenes was eliminated from French style fresh cheese made from milk contaminated with the organism by spreading the LP system on the surface of the cheese (Denis and Ramet, 1989). Similar experiments were done with cottage cheese during which it was shown that E. coli and S. typhimurium were killed by the LP system (Earnshaw et al., 1989). The LP system could provide a further safety feature for these cheeses to eliminate important pathogens as well as by extending shelf life. Third, the rearing of calves from dairy cows requires the feeding of milk replacers and consequently calves are often susceptible to infection with enterotoxigenic coli. Through activation of the LP system in the

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calf replacers and addition of lactoferrin, infections were reduced in severity and duration, even in those animals already infected, when fed LP activated milk replacer (Still et al., 1990),and calf weight gains were greater (Reiter et al., 1981).A dose of 4000 units of lactoperoxidase per liter of milk replacer prevented development of diarrhea in calves due to enterotoxigenic E. coli and was even sufficient to treat the disease (Prieels et aI., 1989). The addition of acetic or butyric acid to milk fat substitutes in milk replacer contributed to a reduction in the enterobacteria (gram negative) flora in the abomasum of calves (Glas and Schaafsma, 1988). X. Conclusion

The growth of bacteria in milk, because of its complex nature, is not as simple as it may at first appear. It seems that the net bacterial growth measured is the result of the organisms resisting inhibitory systems present in the milk and being stimulated by some unknown substances also present. Interest in this phenomenon stems from three important aspects of milk. First, there is the industrial use of milk. Milks from cows, goats, buffaloes, and camels are important industrial commodities. Anything which enhances or detracts from the usefulness of milk for this purpose is important. Second, when farmers are harvesting milk from their farm animals the animals are prone to mastitis or infection of the udder. Mastitis lowers productivity, reduces milk quality, and decreases the income of the farmer. Third, milk is the food of young mammals, human babies in particular, and anything which will enhance the growth and health of a human body, as well as that of other young mammals, is considered desirable. The relationship of the inhibitory systems to what was originally called lactenin does not appear to have been established. The lactoperoxidase-thiocynate-hydrogen peroxide system has been most thoroughly studied and could easily be what Auclair called L2, except for the fact that L1 and L2 act synergistically (Auclair and Berridge, 1953) and L1 appears to be an antibody (Portmann et al., 1960). So far there seems to have been no attempt to investigate how an antibody could influence the LP system. Although L1 may be an antibody, no explanation as to how an antibody could be inactivated by anaerobiosis has been offered. Further, lactenin is not inactivated by cyanide, yet the LP/SCN-/H,O, system is. Growth inhibition in milk of lactic cultures could not be explained by the LP system or by the H,O, concentration (Roginski et al., 1984).

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The role of antibodies in the inhibition of lactic acid bacteria is not clear. I suggest, however, that the role that they play in milk is similar to that played by antibodies to groups A, B, C, and D streptococci in rabbit serum. Larson et al. (1974) showed that human serum, as it was diluted, had the effect of exhibiting bactericidal activity when in concentrated solution but was bacteriostatic for the organism as the serum was diluted. It may be that a complement-like reaction is involved with the inhibition of milk. That is, once the specific antibodies attach to the organism other substances in the milk then attach to the antigen-antibody complex. Another possible mechanism causing the inhibition of lactic acid bacteria involves a lack of Fez+.Lactic cultures, in the absence of sufficient Fez+ may not be able to produce sufficient NADH peroxidase, or other enzymes necessary to decompose hydrogen peroxide. Hence, sufficient hydrogen peroxide could build up in the milk to allow the oxidation of thiocyanate by LP. The depletion of the Fez+could be due to lactotransferrin, the inhibitor of bacilli. One further inhibitor of bacteria in milk is the presence of free fatty acids in the milk due to the activation of the lipase in milk. Activation of lipase is variable and depends on the machinery being used to handle the milk, as well as the diet of the cow. The natural stimulator(s) in milk for lactic acid bacteria is unknown. An essential nitrogen source has been suggested as the stimulator, as have sulfydryl-containing substances. Whatever its identity, there appears to be a stimulator associated with the fat globule membrane. This is indicated by the work of Jag0 and Gillies, which shows that the growth-supporting properties of cream varied as the cream was diluted in skim milk. Further work is required to determine what the nature of the stimulator is in the cream fraction in milk. A fully integrated understanding of the growth of bacteria in milk awaits elucidation. Appendix A Species of bacteria inhibited by milk include: A. aerogenes, B. subtilis, B. coagulans, B. stearothermophilus,E. coli, species of Flavobacterium, L. lactis, Lactobacillus jugurti, L. helveticus, L. bulgaricus, L. hyos, L. icterohaernorrhagiae, L. pomona, M. varians, Mycoplasma bovigenitalium, M. agalactiae, M. bovoculi, P. arabinosum, P. fruedenreichii, P. jensenii, P. pentosaceum, P. peterssonii, P. aeruginosa, P. fhorescens, P. putrefaciens, S. typhi, S. paratyphi B, Shigella dysenteriae, Shigella paradysenteriae, S. aureus, S. albus, S. lactis, S. cremoris, S. diacetilactis, S. dysagalactiae, S. faecalis, S. citrophilus, S. liquefaciens, S. uberis, S. pyogenes, S. thermophilus, and S. agalactiae.

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Appendix B The following substances were tested for their interaction with lactenin by Wilson and Rosenblum (1952b). Inactivated lactenin: sodium thioglycollate (100 ppm), cysteine (100 ppm), glutathione (100 pprn), 2,3-dithiopropanol (1 ppm). Did not inactivate lactenin but did inhibit lactenin-sensitive organisms: hydroxylamine, sodium fluoride, sodium azide, sodium pyrophosphate, sodium selenite. Did not inactivate lactenin but inhibited sensitive and resistant organism: urethane, 2,4dinitrophenol, methionine, carbon monoxide. Did not inactivate lactenine: sodium cyanide. Did not interfere with lactenin: glutamine, riboflavin, pyridoxine, biotin, folic acid, calcium pantothenate, nicotinic acid, guanine, xanthine, p-aminobenzoic acid. Sodium ascorbate had no effect. Substances tested for inhibitory and stimulative activity in milk by Brown (1974) were as follows. Compounds tested for inhibitory activity: those indicated by an asterisk below and DLtryptophan. Compounds tested for stimulative activity: Vitamins: Ascorbic acid, biotin, choline, cyanocobalamin, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, thiamine monophosphate chloride, thiamine pyrophosphate chloride. Amino acids and related compounds: L-alanine*, L-arginiine*, L-asparagine*, L-aspartic acid*, L-citrulline*, DL + allo-cystahionine*, L-cysteine, L-cystine, L-glutamic acid*, L-glutamine*, glycine*, L-histidine*, DL-homocysteine, m-homocystine, m-allo-dhydroxylysine* , L-hydroxyproline*, L-isoleucine*, L-leucine*, L-lysine*, L-methionine*, L-ornithine*, L-phenylalanine*, L-proline*, L-serine*, L-threonine*, L-tyrosine*, L-valine*, p-alanine*, P-alanyl-histidine (L-carnosine), P-alanyl-L-methyl histidine (Lanserine), 3-anthraniloylalanine (m-kynurenine)* , m-a-amino-N-butyric acid*, yamino-butyric acid*, 2-aminoethanesulfonic acid (Taurine)*, L-cysteic acid*, glycylL-glutamic acid*, glutathionine (reduced and oxidized), m-methionine-m.-sulfoxide*, phosphoserine*. N-methyl-glycine (Sarcosine)*, N-a-phenylacetyl-L-glutamine*, Phospholipids, fatty acids and related compounds: glycerol, P-glycero-phosphate*, DLa-glycerophosphate*, glycerophosphoryl-ethanolamine*,DL-3-phospho-glyceraldehyde,3-phospho-~-glyceric acid, 2-phosphoglyceric acid, 2-phosphoenol-pyruvic acid, 0-phospho-ethanolamine*, potassium isobutyrate, potassium isovalerate, potassium N-valerate, potassium 2-methylbutyrate, a-keto-isocaproic acid*, a-keto-isovaleric acid*. acid*, DL-a-keto-p-methyl-N-valeric Cephalin, lecithin, lysophosphatidyl ethanolamine, phosphatidyl ethanolamine, and phosphatidyl serine were hydrolyzed by the method of DawsonlSGO.After evaporation to dryness, the water-soluble material was tested. Nucleosides and nucleotides: Adenosine-5’-mono- and diphosphate, cytidine, cytidine5‘-monophosphate, cytidine-5’-diphosphate, cytidine-5’-triphosphate, guanosine, guanosine-5’-monophosphate,guanosine-5’-diphosphate, guanosine-5’-triphosphate, inosine, inosine-5’-monophosphate, inosine-5’-diphosphate, inosine-5‘-triphosphate, uridine, uridine-5‘-monophosphate,uridine-5‘-diphosphate, uridine-5’-triphosphate, guanosine-5’-diphosphomannose,uridine-5‘-diphosphoglucose,uridine-5’-diphosphogalactose.

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Other compounds: Sodium citrate, creatinine*, dithiothreitol, ethanolamine (Seramine)*, D-glucosamine, hippuric acid, N-acetyl-neuraminic acid, neuramine-lactose, orotic acid, sodium sulfide, sodium thioglycollate, urea*, uric acid.

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Anders, R. F., and Jago, G. R. (1970). The effect of fatty acids on the metabolism of pyruvate in lactic acid streptococci. J. Dairy Res. 37, 445-456. Anderson, A. W., Parker, R. B., and Elliker, P. R. (1955). The nutritional requirements of lactic streptococci isolated from starter cultures. 111. Variation in growth-promoting properties of fresh whole milk. J. Dairy Sci. 38,1083-1088. Ashton, D. H., and Busta, F. F. (1968).Milk components inhibitory to Bacillus stearothermophilus. J. Dairy Sci. 51, 842-847. Auclair, J. E. (1954a). Stimulation of certain bacteria by raw milk. Nature (London] 173, 491.

Auclair, J. E. (1954b). The inhibition of micro-organisms by raw milk. 111. Distribution and properties of two inhibitory substances, lactenin 1 and lactenine 2.1. Dairy Aes. 21, 323-336.

Auclair, J. E. (1968). Bacteriostatic-properties of milk from tropical countries. Ann. Bull.-Int. Dairy Fed. 4, 7-44. Auclair, J. E., and Berridge, N. J. (1953). The inhibition of micro-organisms by raw milk. 11. The separation and electrophoretic examination of two different inhibitory fractions. J. Dairy Aes. 20, 370-374. Auclair, J. E., and Hirsch, A. (1953). The inhibition of micro-organisms by raw milk. I. The occurrence of inhibitory and stimulatory phenomena. Methods of estimation. J. Dairy Res. 20, 45-59. Auclair, J.E., and Portmann, A. (1959). Inhibitory action of the lactenins on the lactic streptococci starters of cheese. Int. Dairy Cong. Proc., 15th, London, 1959, Vol. 2, pp. 580-586. Auclair, J. E., and Vassal, Y. (1963). Occurrence of variants sensitive to agglutinins and to lactoperoxidase in a lactenin-resistant strain of Streptococcus lactis. J. Dairy Res. 30, 345-349.

Aune, T. M., and Thomas, E. L. (1977). Accumulation of hypothiocyanate ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80, 209-214. Basaga, H., and Dik, T. (1994). Effect of the lactoperoxidase system on the activity of starter cultures for yogurt production. Milchwissenschaft 49, 144-146. Baumgartner, H., Kastli, P., Walser, V., and Needer, K. (1965).Experiments on the bactericidal properties of milk. Pathol. Microbiol. 28, 3-11; cited in Dairy Sci. Abstr. 27, 2857.

Bergamini, L., and Solinas, J. (1950). Research on some properties of lactenin and the sensitivity to it of some strains of Staphylococcus aureus type aviarius. Boll. SOC. Ital. Biol. Sper. 35, 805-808; cited in Dairy Sci. Abstr. 22, 745. Berridge, N. J, (1955). Inhibitory substances of bacterial and other origins in milk and milk products. J. Sci. Food Agric. 6, 65-72. Beumer, R. R., Cruysen, J. J. M., and Birtantie, I. R. K. (1988). The occurrence of Campylobacter jejuni in raw cows’ milk. J. Appl. Bacteriol. 65, 93-96.

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Bishop, J. G., Schanbacher, F. L., Ferguson, L. C., and Smith, K. L. (1976). In vitro growth inhibition of mastitis-causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo-lactoferrin. Infect. Immun. 14, 911-918. Bjorck, L. (1978). Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118. Bjorck, L., Claesson, O., and Schulthes, W. (1979a). Antibacterial effect of the lactoperoxide system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118. Bjorck, L., Claesson, O., and Schulthes, W. (1979b). The lactoperoxidase/thiocyanate/ hydrogen peroxide system as a temporary preservative for raw milk in developing countries. Milchwissenschaft 34, 726-729. Bottazzi, V., Battistotti, B., and Vescove, M. (1971). Continuous production of yoghurt cultures and stimulation of Lactobacillus bulgaricus by formic acid. Milchwissenschaft 26,214-219. Boulange, M. (1959). Fluctuation saisonniere du taux des thiocyanates dans le lait frais de vache. C. R. Seances SOC.Biol. Ses Fil. 153, 2019-2020. Branen, A. L., and Keenan, T. W. (1970). Identification of a stimulant for Lactobacillus casei produced by Streptococcus lactis. Appl. Microbiol. 20, 757-760. Brown, R. W. (1967). Factors affecting growth of Streptococcus agalactiae in milk. J. Dairy Sci. 50, 1572-1584. Brown, R. W. (1974). Compounds affecting Streptococcus agalactiae growth in milk. J. Dairy Sci. 57, 797-802. Brown, R. W., and McDonald, T. J. (1982). Effect of time of sample collection, milk composition, and stage of lactation on the growth-promoting properties of milk for Streptococcus agalactiae. Am. J. Vet. Res. 43, 973-978. Brownlie, J., Howard, C. J., and Gourlay, R. N. (1974). Mycoplasmacidal activity of bovine milk for T-mycoplasmas. J. Hyg. 73, 415-423. Buchanan, R. E., Holt, J. G., and Lessel, Jr., E. F. (1966). “Index Bergeyana: An Annotated Alphabetic Listing of Names of the Taxa of Bacteria.” Williams and Wilkins, Baltimore, MI). Carlsson, A., and Bjorck, L. (1987). The effect of some indigenous antibacterial factors in milk on the growth of Bacillus stearothermophilus var calidolactis. Milchwissenschaft 42,282-285. Carrol, E. J., and Jain, N. C. (1969). Bactericidal a-‘ivity of normal milk, mastitic milk, and colstrum against Aerobacter aerogenes. Am. J. Vet. Res. 30, 1123-1132. Chakraborty, B. K., Chaudry, S. S., Alex, K. A., Jacob, G., and Soni, G. J. (1986).Application of the lactoperoxidase system for preserving buffalo milk produced in Indian villages. Milchwissenschaft 41, 16-19. Cheeseman, C. C., and Jayne-Williams, D. J. (1964). An inhibitory substance present in milk. Nature (London) 204, 688-689. Clegg, L. F. L., Sergeant, V., Franklin, J. C., and Auclair, J. E. (1956). The effect of lactenin on the growth of thermoduric bacteria in milk, with some observations on “bitty cream.” Dairy Ind. 21, 128-133. Clem, W. H., and Klebanoff, S. J. (1966). Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidasethiocyanate system. J. Bacteriol. 91, 1848-1853; cited in Dairy Sci. Abstr. 28, 2471. Cogan, T. M., Gilliland, S. E., and Speck, M. L. (1968a). Identification of stimulants for Lactobacillus bulgaricus in tomato juice. Appl. Microbiol. 16, 1215-1219. Cogan, T. M., Gilliland, S. E., and Speck, M. L. (1968b). Characterization of a inhibitor for Lactobacillus bulgaricus in tomato juice. Appl. Microbiol. 16, 1220-1224.

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Wilson, A. T., and Rosenblum, H. (1952a). The antistreptococcal property of milk. I. Some characteristics of the activity of lactenin in vitro. The effect of lactenin on the hemolytic streptococci of the several serological groups. J. Exp. Med. 95, 25-38. Wilson, A. T., and Rosenblum, H. (1952b). The antistreptococcal property of milk. 11. The effects of anaerobiosis, reducing agents, thiamine, and other chemicals on lactenin action. J. Exp. Med. 95, 39-50. Wolfson, L. M. and Sumner, S. S. (1993). Antibacterial activity of the lactoperoxidase system: a review. J. Food Prot. 56, 887-892. Wright, R. C., and Tramer, J. (1957). The influence of cream rising upon the activity of bacteria in heat-treated milk. J. Dairy Res. 24, 174-183. Wright, R. C., and Tramer, J. (1958). Factors influencing the activity of cheese starters. The role of milk peroxidase. J. Dairy Res. 25, 104-118. Zall, R. R., Chen, J. H., and Dzurec, D. J. (1983). Effect of thiocynate-lactoperoxidasehydrogen peroxide system and farm heat treatment on the manufacturing of cottage cheese and cheddar cheese. Milchwissenschaft, 38, 203-206. Zapico, P., Gaya, P., Nuiiez, M., and Medina, M. (1990). Lactoperoxidase and thiocyanate contents of goats’ milk during lactation. Lett. Appl. Microbiol. 11,90-92. Zapico, P., Gaya, P., Nubez, M., and Medina, M. (1990). Goats’ milk lactoperoxidase system against Listeria monocytogenes. J. Food Prot. 56, 988-990.

Challenges in Commercial Biotechnology. Part 1. Product, Process, and Market Discovery ALESPROKOP Department of Chemical Engineering Vanderbilt University Nashville, Tennessee 37235

I. Introduction A. Biotechnology, Process, and Process Systems B. Interdisciplinary Nature of Biotechnology C. Industrial Aspects of Biotechnology D. Systems Approach 11. Biocatalysts: Sources for Discovery A. Definitions and Classification B. Organismic Biocatalysts C. Enzyme-Based Biocatalysts D. Biocatalyst Processing and Reactors III. Primary Market Classification IV. Pathway to Commercialization: Discovery and Development (D&D)Cycle V. Product Discovery A. Classification of Products and Screens B. Examples of Screens C. Route of Discovery vs Route of Manufacturing D. Example of Discovery of a New Chemical Class (and of Product Development) E. Example of Discovery from Plant Biotechnology F. Special Considerations for Discovering (and Developing) Products of Different Industrial Sectors G. Approaches to Discovery H. Role of Serendipity in Product Discovery I. How the Top 20 Drugs Were Discovered VI. Process Discovery A. Approaches to Process Discovery B. Biotransformations C. Examples of Bioprocess Discovery VII. Market Discovery WI. Interactions of Discovery Aspects within the D&D Cycle A. Product Discovery vs Product Development B. Product Discovery vs Process DiscoverylDevelopment C. Market Discovery vs Development References

1. Introduction

A. BIOTECHNOLOGY, PROCESS, AND PROCESS SYSTEMS The word biotechnology has various meanings. In the United States (and in some western European countries) it has frequently become 95 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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restricted to, and almost synonymous with, the application of genetic engineering. In the rest of the world biotechnology has retained its original definition, so that it comprises all the technical aspects of cultivating cells or tissues and the products and processes derived from such cultures, including enzyme technology (Finn, 1988).A definition by the Office of Technology Assessment (OTA) of the U.S.Congress states that “biotechnology, broadly defined, includes any technique that uses living organisms (or parts of organisms) to make or modify products, to improve plants or animals, or to develop microorganisms for specific use” (Office of Technology Assessment, 1984). Mimicking of naturally occurring materials should be added to the above definition. Implicit in the above definitions is a processing aspect of handling biological material (process engineering) and the real object of our investigation-process. A process is a sequence of operations or steps resulting in the production of a certain product or in a particular treatment or raw materials. A technical field dealing with bioprocessing is called bioprocess engineering or bioprocessing. Most biotechnological processes consist of a chemical reaction process section (fermentation, or bioreaction section) and of a subsequent product recovery section. Sometimes, operations preceeding and including fermentation are called upstream processing (e.g., media preparation, sterilization) and those of product recovery are called downstream processing. A process system can be defined by addressing the interrelated and interdependent nature of elements of a process. It can be arbitrarily defined as consisting of the upstream and downstream segments, including those of fermentation, cell recovery, and product recovery processing. Each process system interacts with its environment by means of mass, energy, and information flows. A set of elements and flows determines the internal structure of the process system. Such elements are then described as structural elements. Time and space conglomerates of elements, having a certain common similarity, are called subsystems.

B. INTERDISCIPLINARY NATURE OF BIOTECHNOLOGY Bioprocesses are systems in which complete living cells, tissues, or their components (e.g.,organelles) are used to effect desired physical or chemical changes. Bioprocess systems (biocatalysts) could potentially replace some current chemical catalysts; they include enzymes, microbial cells, and plant/animal tissue cells, and they draw from many disciplines of science and engineering. Table I summarizes some major

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TABLE I INTERACI'ION OF DISCIPLINE-SYSTEM-PROCESS IN BIOTECHNOLOGY Discipline Microbiology, ecology, taxonomy Chemistry, enzymology Genetics, molecular biology, genetic engineering Physiology, cell biology, plant sciences Process engineering physical sciences Economics, law, social sciences Systems engineering

Biocatalyst system"

Process aspects

M, P/A

Culture choice

E, M, P/A E, M, P/A

E, M, P/A

Reactions, product recovery Product discovery Process discovery Process operation: response to environment Enzyme reactors, process operation optimization, control and design Business and regulatory aspects

M, P/A

Systems analysis and synthesis

M, P/A E, M, P/A

E,enzyme; M, microbial; PIA, plantlanimal.

interactions between systems, processes, and disciplines in biotechnology and provides a foundation for industrial applications of biotechnol-

ogy in several sectors: 1. Pharmaceuticals, including medical diagnostics and biosensing 2. Animal and plant agriculture 3. Specialty chemicals and food additives 4. Commodity chemicals and energy production 5 . Services within the environmental area.

From these it is thus clear that biotechnology is a multidisciplinary field and has multiindustry applicability. Two categories of biotechnology are sometimes distinguished: 1. Traditional (before the invention of genetic engineering) 2. New (after the invention of genetic engineering).

It is predicted that new biotechnology, related to genetic engineering and hybridoma technologies, will account for a large fraction of the total biotechnology industry in the future, although both the traditional and the new will coexist for a long time.

c . INDUSTRIAL ASPECTSOF BIOTECHNOLOGY The central problem here is a definition of research and developmental aspects (R&D)that must be completed to bring a product to market.

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Other terms often used are discovery and development. Definitions are given in Table 11, which also includes regulatory, legal, and social constraints imposed on a product (“soft constraints”; they are denoted as such because they are hard to quantify and solutions are not obvious). The general scheme of R&Dactivity in an industrial setting is depicted in Fig. 1,indicating the long path to market and the tremendous cost and time involved. Besides soft constraints, economic constraints also play a decisive role. The process must be designed to give the private sector an advantage in the marketplace. The optimal process must be defined within the context of competing goals or means of competitive advantage [Wheelwright, 1991): 1. First to market [strong proprietary position, safe, and efficacious product) 2. High quality 3. Low cost. TABLE I1

INDUSTRIAL (RBrD) ASPECTSOF BIOTECHNOLOGY Term Discovery Product discovery Process discovery Market discovery Development Product development Process development and scale-up Market development Regulatory aspects Health, safety, and environment Industrial Legal aspects Social and ethical

Description Product screen via genetic, biological, chemical, and molecular means Process discovery via catalyst selection, metabolic pathway optimization and statistical improvement Market forecasting, strategic planning and market analysis Product improvement via product formulation and delivery, via improved processing and manufacturing Inoculum development, process scale-up, control, optimization, and process and plant design Product uniqueness, market orientation, and technical factors FDA, USDA, EPA, NIH, etc. GLP, GMP, QC, validation Trade secrets, patents, licensing Policy, technology transfer, public perception, ethical issues

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

I

Processdiscovery

Forecasting

1

Optimization

Economics I

I

'-I I

I I

I I

99

Process Development and Scale-up

i-

-hi

11-4 I

I 4 I

Manufacturing

Product in Market Market Development I

t - _ _ - _ _ _ - - _ - , - - L - _ - - FIG.1. R&D in perspective (---, information transfer; -product transfer].

Each of these offer advantages that can be realized only at the expense of the others. In choosing between them, the best market performance must be sought. The first to market typically requires a strong patent (license) position, granted approval (safety and efficacy), and short process development times. High quality requires high purity and activity, as well as consistent production (process validation). The cost is related to the complexity of processing, the efficiency of each step, the scale of operation, and the degree of manufacturing and equipment flexibility. The product uniqueness and its life cycle expectancy are also important. Once the competitive advantage is identified it must be maintained because of product specifications and available resources.

D. SYSTEMS APPROACH The differentiation of a system into subsystems allows for an investigation of an organization of a system at different levels of hierarchy. The general system is then composed of elements, subsystems, and levels of hierarchy, bound by the corresponding relations within each system. Structural elements, also called elementary events (processes), then play certain functions. The hierarchy of living systems and of a

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hypothetical biotechnological process is depicted in Table 111, presenting a relationship between hierarchy, structure, and function (elementary events). Table IV lists examples of individual subsystems with corresponding elementary events. Such decomposition of a system via breakdown into its components involves not only an analytical approach but also a synthetic view, both germane to a systems approach (general systems approach). Such an approach attempts to point out related properties of the investigated phenomena by selecting relevant (systemic) properties, relations, and functions. The process of synthesis is based on a reduction of information at each level of hierarchy by means of purposeful selection of important systemic phenomena and by identification of interactions among them. Note that the complex metabolic network presented for L-lysine biosynthesis is already highly simplified, featuring only important elements and interactions. The population level is treated as a quasi-homogeneous system composed of identical cells (another oversimplification) and the reactor level (not shown) is featured by perfect reactor mixedness and oxygen balances. The synthesis or transition to the higher levels of hierarchy (also denoted as scale-up) can be thus carried out by lumping of lower specific levels into higher (systemic) levels of hierarchy, since it may be difficult to incorporate all properties of specific levels. The selection of systemic properties and reactants is not governed by strict rules and is a matter

TABLE 111 HIERARCHY OF A BIOTECHNOLOGICAL PROCESS Hierarchy level

Structural element

Molecular molecules Organelles

Atoms, macromolecules Macromolecules

Cell

Organelles

Population

Cells

Reactor

Elementary zones

Biotechnological process

Unit equipment

Note. Adapted from Table VII of Prokop (1982).

Function (elementary event) Synthesis/decay of epigenetic control Duplication of organelles, metabolic manifestations of cells Translocation of substances, complex biochemical reactions, morphological differentiation, cell cycle control Growth and multiplication, interaction between cells Reactions, mass transfer, hydrodynamics, heat exchange, diffusion, etc. Unit operations: upstream, reactor, downstream

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TABLE IV LISTOF SUBSYSTEMS AND ELEMENTARY EVENTS OF L-LYSINEBIOSYNTHESIS Subsystems 1. Maintenance 2. Glycolysis/energy

3. Lysine

4. Protein 5. Threonine/homoserine

Elementary events Oxygen and substrate uptake, maintenance of cellular functions Control of glycolysis, energy generation and control, provision of building blocks for biosynthesis Lysine biosynthesis pathway, positive glycolytic flux control, negative feedback control, metabolic block at homoserine dehydrogenase, negative feedback by oxygen excess Protein synthesis, growth, negative feedback by oxygen excess Threonine/homoserine pathway, two metabolic blocks (homoserine dehydrogenase, homoserine kinase),two auxotrophic mutations (homoserine, threonine)

Note. Adapted from Fig. 6 of Prokop (1982).

of art exercised by scientists and engineers. At a cellular level, typically, systemic events and reactants are usually those at the branching points of metabolism, undergoing feedback and feedforward control. At the level of reactor, typically, mass transfer (oxygen)may control the general reaction scheme and is thus typically chosen as an elementary event (for economic reasons). A major postulate of the systems approach is a recognition that the performance of the whole is not the sum of individual parts, but a consequence of the relationship of the performance between the parts (levels of hierarchy). For example, oxygen uptake of cells (in a population) is coupled with oxygen transfer and oxygen balance at the reactor level, giving rise to a reactor model. A further step in the systems approach is separation of conditions leading to a limitation of either biological or physical conditions (regime analysis, Part I1 of this series). The last step is usually construction of a mathematical model of the system and simulation by means of a model. The model is conceived as a formal description of a real process and reflects a relationship between input, state, and output variables of the system. Simulation is then an investigation of a system by means of mathematical manipulation of a model. The systems approach thus embodies two faceted view: analytical and synthetic. Tools of analysis are most often discussed in chemical reaction engineering textbooks; methods of synthesis are typical for

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discovery and developmental aspects. An effective biotechnology R&D thus uses two components of investigative study (Fig. 2): 1, Specific knowledge of disciplines as a structural basis for development of different application fields 2. General problem-solving strategies represented in the integrated systems approach, as a basis of “knowledge pathway.”

As such, the first component represents a specialized view, the second one a generalists view (Table V). In fact, in real life, the problem-solving

(7 Tools of analysis

Specific knowledge: Microbiology Biochemistry Chemistry Physics Genetics, etc.

(r> Methods of synthesis

General problem strategies: speculative thinking personalizedthinking data-bound thinking relational thinking

Product discovery Product development and scale-up Plant design Market development Societal effects

a FIG.2. Components of effective R&D activity.

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TABLE V CONCEPTUAL DIFFERENCES BETWEEN SPECIALISTSAND GENERALISTS AND TYPEOF SOLUTIONS AND METHODS Specialists 1. A whole to be divided into

components 2. The dimension of depth 3. Exclusive postures and intentions 4. Deduced principles 5. An accurate view 6. A sole expert 7. Narrow skill 8. Solution seeker close-ended problems 9. Application-oriented 10. Well-defined or definable limits 11. Usually one correct answer 12. Rigorous techniques available

Generalists Components associated into a whole The dimension of breadth Mutual concerns/interactions Induced concepts A representative view (scheme, analogy) An interdisciplinary team Broad views Problem definition solutions are openended Systems/concepts awaiting applications Ill-defined or nonexistent limits Multiplicity of correct answer Generahague techniques available

Note. Adapted from Gilbreath (1987,p. 89).

approaches do not always fall into only two such categories. According to Lefton et al. (1980) each analytical and synthetic view can be of two types (Tl-T2 and T3-T4)(Fig. 3). From an individual perspective, it may be useful to identify and be aware of what kind of thinking mode one is using. An example of Tl-T4 types drawn from biotechnological R&D is given in Table VI. More examples will be presented while discussing scale-up methods (Part I1 of this series). A general systems researcher starts with the laws of different disciplines (analysis), searches for similarities among them, and then announces to the world a new “law about laws” using generalization (synthesis)(Weinberg,1975,1982).The power of generalization through induction is that we can use the general laws to draw conclusions about cases not yet observed. Yet such a situation may not be always right. This leaping from discipline to discipline does not always work. Most of the time errors are due to either improper use of assumptions of oversimplifications of the real world at the problem formulation stage. The question is, then, why not to wait for more evidence (data) before generalization? The answer lies in the explosive growth of knowledge. We just cannot afford to wait for more specifics to come. The world is pressing us to contribute and to solve problems. The general systems researcher is constantly jumping to conclusions on insufficient evidence; this is a prerequisite for rapid learning. There is a danger in-

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intuitive

Conceptual

Speculative thinking: generalization based on theoly and intuition rules of thumb

Relational thinking: logical analysis of data via relationships and generalizations

Personalized specific and personal decisions based on past experience and

Data-bound thinking: statistical and pattern analysis of data

Systematic

Concrete

Information evaluation Information gathering FIG.3. Types of problem-solving approaches (adapted with permission from Lefton et al. (19801, Fig. 42, p. 445, copyright 1980 by Ballinger-Harper Business). TABLE VI EXAMPLES OF DIFFERENTMETHODS OF THINKING IN R&D Method of thinking TI. Relational T2. Data-bound T3. Personalized

T4. Speculative

Example Specific rate analysis, regime analysis, morphological analysis Statistical analysis, pattern analysis Origins of scale-up problems, personnel experience with facilities/equipment,microbiologist’s knowledge of culture characteristics, chemist’s knowledge of product Rule of thumb in engineering analysis, “gut” feelings of specialists on biologicalkhemical characteristics

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volved, in that some generalizations may not be correct. Why do we need such general systems thinking in biotechnology? The answer is related to the nature of the inherent complexity of industrial R&D problems. Solutions to industrial biotechnology need to come from the contributions of multidisciplinary specialists. Therefore, one needs to provide a conceptual framework by which the multidisciplinary specialists can have a common representation of their latest understanding of the biotechnical systems. In general, types of systems with respect to thinking and their complexity are of three kinds (Fig. 4). Examples taken from biotechnology are listed in Table VII. Obviously, a systems approach is useful for systems of Type 111, since the problem is too diverse for analysis and too structural for a statistical treatment (typically in the development area). It represents an attempt to organize “pockets of knowledge” of the system and subsystems. The position of Randomness (diversity)

Most complex

111 Organized complexity

Complexity (number of hierarchies, subsystems, elementary processes)

FIG.4. Types of systems with respect to methods of thinking (adaptedwith permission from “Introduction to General Systems Thinking,” Weinberg (1975), Fig. 1.9, p. 18, copyright 1975 by John Wiley, reprinted with permission of John Wiley & Sons).

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EXAh.lF'LES OF COMPLEXITY OF BIOTECHNOLOGICAL SYSTEMS AND INVESTIGATIVE TOOLS

Type of system

I. Organized simplicity

11. Unorganized complexity

111. Organized complexity

Tools used

Example

Analytical/deterministic reaction mechanisms, Michaelis-Menten equation, enzyme inhibitiodactivation Statistical/stochastic, stochastic: factorial experimental design screening

Single enzyme reacting system Microbial biotransformations (e.g., steroids]

Systems approachanalysis and synthesis: specific rate analysis, pattern analysis checklist

Secondary metabolite production in complex media (e.g., antibiotics) Bioremediation Cell culture in serum media Biocorrosion/biofouling Microbial and cell culture in chemically defined media Microbial growth on single limiting substrate Sterility problems in biotechnology and food processing

specific examples in Table VII will move as our understanding and knowledge increase and more synthetic tools become available. Figure 5 depicts a multiplicity in pathways of the discovery (product or process) procedure. This is the most significant finding of this series of two articles: the multiplicity of solutions in practical biotechnology, requiring a general systems (global,holistic) view. There are many ways a product can be manufactured, and different alternatives are to be evaluated using industrial constraints: 1. Total discovery and development time 2. Cost (process economy, market position) 3. Quality (product quality, consistency, stability, etc.).

As a rule, many industrial (biotechnology) problems are open-ended, and multiple solutions are available on both the chemical and biological processing sides. These include, naturally, nonprofitable tracks, fallible tracks, and back-tracking. Different questions may be posed regarding how to produce a given product: 1. What to use-Chemical vs enzymatic vs fermentation route? 2. What analoghariant molecule?

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Pathway number of R&D

Multiple solutions to industrial problems

Time, cost & quality dependent

FIG.5. Multiple pathways to open-ended problem solutions.

3. What is the best therapeutic formulation/delivery? 4. What are the process variants available once a route is fixed?

The systems approach provides the best available methodology to guide us in finding an effective solution of complex biotechnological problems. A summary of strategies involved in R&D in part at discovery, mostly at product and process development and scale-up, is shown in Table VIII. All of the above material presented in the Introduction merely defines the status of biotechnology and, most importantly, explains the importance of the systems approach. Specific examples will be presented below. TABLE VIII STRATEGIES INVOLVEDIN R&D Strategy Empirical SystemsIEngineering Principles Simplification Interactions (feedback) Separation Model and simulation

Example Trial and error, statistically bound approach Systematic (rational) process development Reduction of information Identification of relationships between subsystems Regime analysis [biological vs. physical phenomena) Structural model of metabolism coupled with a reactor model

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II. Biocatalysts: Sources for Discovery A. DEFINITIONS AND CLASSIFICATION A biocatalyst in biotechnology represents a laboratory observation on an organism or an isolated enzyme activity having the potential to be used in developing a marketable product. For product definition and its interrelationship to biocatalysts see Section IV. Compared to chemical processing, the use of biocatalysts in biotechnological processing offers many advantages (Table IX). The same table also lists disadvantages. Biocatalysis enables one to carry single- or multistep reactions with high stereo- and regiospecificity under mild conditions. The chirality effects in drugs and agricultural chemicals present new challenges and the need to isolate a single isomer from a racemic mixture. Novel chemistries via enzymatic catalysis lead to new routes for manufacturing of novel molecules, to novel routes for existing chemicals, and to new technologies adding value to feedstocks and by-products. The emergence of biotechnology-based technologies is due to:

The explosive increase in understanding of biological systems The development of large-scale bioprocessing techniques The maturity of the petrochemical-based chemical industry The increasing environmental pressures and restrictions on conventional chemical technology. 1. 2. 3. 4.

TABLE IX COMPARISON OF BIOTECHNOLOGICAL AND CONVENTIONAL CHEMICAL PROCESSES Advantages

Disadvantages

Multistep enzymatic conversions in one processing step

Dilute reactant conditions resulting in low productivity

Structural and stereochemical specificity

Large equipment and capital costs

Mild conditions of temperature, pressure, and pH

Complex purification because of complex reaction conditions

High conversion efficiency when optimized

Complexity of multiphase reaction conditions

New routes to novel molecules

Protein denaturation and cell lysis

Use of renewable resources

Low health and environmental risk

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Besides the use of different intact natural organisms as biocatalysts, recent developments in biotechnology allow for advanced tools to be applied in biocatalysis. 1. Advanced breeding and genetic engineering tools (Protoplast fusion, Plasmid transfer, Recombinant DNA technology), leading to the creation of new traits at the cellular level in plants, animals, microorganisms, and man 2. Hybridoma technology, leading to the production of pure and specific antibodies (Liddell and Cryer, 1991) 3. Plant tissue culturing, providing for manipulation and propagation of plants and of their genetic traits (Stafford and Warren, 1991) 4. Whole-cell and organelle immobilization, providing for practical exploitation of catalytic properties in specially designed reactors (Hartmeier, 1986) 5. Enzyme immobilization, providing for their more extended practical use (in enzyme reactors)(Hartmeier, 1986) 6. Protein engineering, allowing the modification of protein (enzyme) structure and activity (Moody and Wilkinson, 1990)