Applications of Microbial Enzymes in Food Systems and in Biotechnology

Applications of Microbial Enzymes in Food Systems and in Biotechnology MATTHEWJ. TAYLORAND TOMRICHARDSON Department of Food Science, University of Wisconsin, Madison, Wisconsin I. Introduction .............................. 11. Conventional AppIications of Microbial Enzymes In Systems ..................... .................

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

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

C. Dental Hygiene .

V. Future Uses of Microbial Enzymes in Food Systems . . . . . . . . A. Introduction .......................... B. Modification of Protein Functionality . . . . . . . . . . . . . . . . . . C. Plastein Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Antioxidants . . . . . . ........... VI. Future Uses of Mic VII. Conclusion . . . . . . . ............... References . . . . . . . ..........

7

8 8 10 18 18 19 19

22 22 22 23 25 25 25 26 27 28 29 31 31

1. Introduction The biochemical diversity of microorganisms makes them logical sources of a wide variety of enzymes for use in food and other biotechnological systems. Genetic manipulation of microorganisms increases their potential for the production of enzymes, possibly including enzymes from mammalian sources. In addition, improved techniques for enzyme production and purification-such as af3nity chromatography-are making microbial enzymes increasingly competitive. Thus, the possibilities of producing microbial enzymes to catalyze virtually any desired reaction are nearly endless. However, of the 2000 enzymes known, fewer than 20 are now used with any commercial significance. All enzymes currently used in food processing and biotechnology represent only a $60 million industry (estimate from Skinner, 1975) in the United States. 7 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 25 . Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0026252

8

MATTHEW J. TAYLOR AND TOM RICHARDSON

Additional and different microbial enzymes are needed for several purposes. Food enzymologists are using various enzymes to improve current food technology and to utilize new food sources. Organic chemists are beginning to use enzymes to catalyze specific stereochemical reactions, such as in the synthesis of pharmaceuticals. There are increasing attempts to use enzymes as therapeutic agents in medicine. Microorganisms will probably supply most of these needed enzymes. Applications of enzymes benefit from new technologies, such as enzyme immobilization, which may increase processing efficiency and decrease costs. Immobilization of enzymes onto specific electrodes permits rapid, specific, and sensitive analyses for natural products, such as glucose and urea. Enzyme immobilization in uiuo or ex uivo may be useful in the treatment of certain diseases. The virtually limitless array of microbial enzymes coupled with recent innovations in biotechnology lead to predictions of a bright future for enzyme technology. This review summarizes applications of enzymes in food and biotechnology and suggests future trends and applications.

II. Conventional Applications of Microbial Enzymes in Food Systems A. SOURCESOF MICROBIALENZYMES Selected strains of molds, bacteria, and yeasts are currently used as sources of enzymes for food processing. Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis are the three most useful, well-known, and safe microbial sources for enzymes. Modern fermentation techniques allow production of unlimited quantities of microbial enzymes in a reproducible and controlled manner. Large-scale production of microbial enzymes has been discussed by Erickson (personal communication). Five microorganisms (two fungi, two yeasts, and one bacterium) yield enzyme preparations which are generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) (Table I). Enzymes that degrade starch or proteins are the types of enzymes most used, and the two aspergilli species supply most of the required enzymes. There are several other microorganisms which serve as sources for enzyme preparations approved by the FDA for food use (Table 11). These enzyme preparations are useful because of a special application, an exceptional yield, or a desirable stability. The glucose isomerase of Streptomyces sp. and the fungal rennets have been particularly successful commercially. Glucose isomerase is used in the manufacture of high-fructose syrup from cornstarch,

9

APPLICATIONS OF MICROBIAL ENZYMES

TABLE I SOURCESOF MICROBIAL ENZYMES FOR FOODUSE: "GRAS" ORGANISMS~ Microorganism

Enzyme(s)

Bacillus subtilis

Amylase (high temperature), neutral protease, alkaline protease

Aspergillus wyzae, Aspergillus niger

Amylase, glucoamylase, protease, lactase, acid protease, catalase, glucose oxidase, lipase, anthocyanase, naringinase, cellulase, hemicellulase, pentosanase, pectinase

Saccharomyces cereoisiae

Invertase

Kluyoeromyces fragilis

Lactase

"Reprinted from Haas (1971, p. 122), by courtesy of Arlington Publ. Co.

and the fungal rennets are quickly supplanting the dwindling supply of calf rennet in cheese making. Perhaps, one impedance to wider application of microbial enzymes in food systems is the necessity to demonstrate the safety and wholesomeness of enzymes from microorganisms not generally recognized as safe (GHAS). Petitioning the FDA for approval for the intended use is costly and time consuming. TABLE I1 SOURCESOF MICROBIALENZYMESFOR FOODUSE: NON-GRASORGANISMS Microorganism Mucw miehei, M . Pusillus var. Lindt, Endothia parasitica Trichoderma viride Micrococcus lysodeikticus Bacillus lichenifmiis Penicillium funiculosum Rhizopus spp. Streptomyces sp. Mottierella uinocea var. raffinose-utilizer Trametes sanguranea Escherichia coli, Saccharomyces lactis

Enzyme(s)

References

Rennets

Sternberg (1976)

Cellulase, pectinase, hemicellulase Catalase Carbohydrase, protease Dextranase Amylase, glucoamylase, pectinase, lipase, protease Glucose isomerase a-Galactosidase

Kulp (1975)

Unspecified mixture to soften fruit Lactase

Scott (1975a)

Scott (1975~) Scott (1975a) Scott (1975a) Underkofler (1976) Mermelstein (1975) Scott (1975a)

Shukla (1975)

10

MATTHEW J. TAYLOR AND TOM RICHARDSON

B. APPLICATIONS Available space does not permit even a cursory discussion of the many existing or proposed uses of microbial enzymes in food systems. Therefore, the significant commercial applications of microbial enzymes have been listed in Table 111, and an extensive compilation of other applications is presented in Tables IV-VIII. One example (bread making) of the application of microbial enzymes to food is discussed below, but for a more detailed analysis of the various uses of microbial enzymes in foods the reader is referred to several excellent reviews (Ory and St. Angelo, 1977; Reed, 1975a; Underkofler, 1976; Whitaker, 1972, 1974). Enzymes have great utility in food processing, where they perform a particular purpose. The processing of such common foods as bread, beer, cheese, and soft drinks requires microbial enzymes as an integral part of their manufacture. The advantages of enzymes in food processing over alternative physical or chemical manipulations are several. First, enzymes catalyze a specific action,. avoiding potentially undesirable side reactions resulting from less specific processing methods. Second, the extremes of p H or temperature usually necessary for chemical or physical treatments are avoided by using enzymes-and this minimizes side reactions as well. In addition, less processing energy is required. Third, removal of enzymes after use is usually not necessary since they are present in low concentration and, in many circumstances, are inactivated by subsequent processing. A commercially successful enzyme must have the following characteristics (Beck and Scott, 1974). Foremost, the cost of using the e'nzyme must be less than the increased value of the food. The enzyme must be sufficiently active at the pH, temperature, and substrate concentration normally in the food. It is not usually practical to manipulate a food to attain optimum conditions for enzyme activity. Instead, one searches for an enzyme that has an optimum activity closely matching the conditions in the food. The enzyme preparation must be safe-i.e., free from toxins, carcinogens, and pathogenicity. Employing enzymes already approved for food use is more desirable than submitting to the costly procedure for FDA approval of a new enzyme. Last, the commercially successful enzyme must be available in required purity, stability, assayability, and controllable activity. In many cases, microbial enzymes conform more closely to these characteristics than do alternative plant or animal enzymes. The use of microbial enzymes in bread illustrates the application of microbial enzymes in a food system. Enzyme usage in baking results from the deficiency of some naturally occurring enzymes in present-day wheat flour. Deficiencies in natural enzymes is the result of mechanical harvesting (beginning in the 1920s) which prevents incipient germination of the wheat.

TABLE 111 SIGNIFICANTCOMMERCIAL FOODUSESOF MICROBIAL ENZYMES Application Baking

Purpose

Brewing

Starch degradation Wheat gluten degradation Saccharification (mashing)

Cheese making

Chillproofing Coagulation

Corn syrups

Saccharification

Eggs Fruit juices Soft drinks Wine

High-fructose syrup Preventing browning when dried Clarification, filtration Oxygen removal Pressing, clarification, filtration

Enzyme(s)

Source

References

Amylase Protease Amylase, glumamylase Protease Rennet

Fungal Fungal Bacterial, fungal

Barrett (1975) Barrett (1975) Bass and Cayle (1975)

Fungal, bacterial Fungal

Amylase, glucoamykse Glucose isomerase Glucose oxidase Pectinases Glucose oxidase Pectinases

Fungal

Bass and Cayle (1975) Sternberg (1976); Nelson (1975); Green (1977);Sardinas(1976) MacAllister et al. (1975)

Bacterial Fungal Fungal Fungal Fungal

MacAllister et al. (1975) Scott (1975b,c) Neubeck (1975) Beck and Scott (1974) Neubeck (1975)

PROPOSED OR EMERGING APPLICATIONS Application

Purpose

OF

TABLE IV MICROBIALENZYMES AFFECTINGSTRUCTURAL ELEMENTS OF FOODS Enzyme(s)

Source

Plant cell separation

Filtration and lautering aid Chillproofing Increasing porosity Facilitating concentration Fermentation aid Liquid center manufacture Peeling, softening, etc.

Shellfish

Cleaning, peeling

Soft drinks Tea (instant)

Clouding agent Solubilizingsolids

Cellulase, pectinase, hemicellulase Carbohydrases, cellulases Pectinases Tannase

Increasing yield

Cellulase

Fungal

Reducing foaming

Pectinase

Fungal

Beer Beer Candied fNit Coffee Coffee Confections

References

p-Glucanase

Bacillus subtilis

Bass and Cayle (1975)

Tannase Complex mixture Pectinase, hemicellulase Pectinase Pectinase

Aspergillus niger Trametes sanguranea, Aspergillus niger Fungal

Beck and Scott (1974) Scott (1975a)

Fungal Fungal

Underkofler (1976) Underkofler (1976)

T r i c h o h a viride

Beck and Scott (1974)

Aspergillus niger

Scott (1975a)

Unspecified Aspergillus niger

Scott (1975a) Sanderson and Coggon (1977); Scott (1975a) Sanderson and Coggon (1977). Sanderson and Coggon (1977)

Underkofler (1976)

TABLE V PROPOSED OR EMERGING APPLICATIONS OF MICROBIAL CARBOHYDRASES I N FOODS ~~

Purpose

Bean products

Removal of flatulence factors Hydrolyzing rathose

a-Galactosidase

Aspergillus niger

Liener (1977)

a-Galactosidase

Scott (1975a)

Increasing fermentable sugar Juice thinning

Lactase

Mwtierella oiwcea var. raffinoseutilizer Yeast

Scott (1975a)

Syrup manufacture

Amylase

Penicillium funiculosum Bacterial, fungal

Corn syrup

Candy scrap salvage Liquid center formation High-maltose syrup

Amylase Invert ase Pullulanase

Bacterial, fungal Yeast Bacterial

Honey

Artificial SYNP

Invertase

Yeast

Starch

Saccharification at high temperature Synthesis from starch

Amylase

Bacillus 1icheni;fonis

Starch phosphorylase, sucrose phosphorylase, amylase, glucoamylase, glucose isomerase

Yeast, fungal, and bacterial

Underkofler (1976) Beck and Scott (1974) Beckand Scott (1974); MacAllister et al. (1975) Beck and Scott (1974); Whitaker (1972) Beck and Scott (1974); Scott (1975a) Butler et al. (1976)

Beet sugar Bread Cane sugar I-

W

~~

Application

Chocolate, cocoa Confections

Sucrose

Enzyme

Dextranase

Source

References

Barrett (1975)

Reed (1975b)

TABLE VI APPLICATIONS OF MICROBIAL ENZYMES I N DAIRYSYSTEMS PROPOSED OR EMERGING Application

Purpose

Enzyme

Source

Cheese making

Flavor development

Lipase

Fungal

Concentrated milk Dried milk

Increasing stability Oxygen removal

Yeast Fungal, bacterial

Ice milk Milk Milk fat

Preventing sandiness Use by consumers intolerant tolactose Pasteurization Flavor modification

Lactase Glucose oxidase, catalase Lactase Lactase Catalase Lipase

Fungal, bacterial Fungal

Whey

Increasing use

Lactase

Yeast

Yeast Yeast

References Huang and Dooley (1976); Shahani (1975); Brockerhoff and Jensen (1974) Richardson (1975) Scott (1975b) Richardson (1975) Kulp (1975); Shukla (1975) Scott (1975~) Shahani et al. (1976); Arnold et al. (1975) Richardson (1975)

TABLE VII

PROPOSED OR EMERGING APPLICATIONS OF MICROBIAL ENZYMES I N FOODPROTEIN SYSTEMS

Application

Purpose

Enzyme

Egg albumen Fish protein Gelatin

Pasteurization Solubilization Degreasing bones

Catalase Protease Lipase

Meat

Tenderization

Protease

Protein hydrolysate Single-cell protein

Hydrolysis

Protease

Yeast cellwalllysis

P-Glucanase

Source Fungal Bacillus subtilis Rhizopus arrhizus var. delemar Aspergillus oryzae, Bacillus subtilis Bacterial Arthrobacter spp., Bacillus circukzns

References Scott (1975~) Scott (1975a) Scott (1975a) Bernholdt (1975) Bemholdt (1975); Yamamoto (1975) PhatT (1977)

TABLE VIII PROPOSED OR EMERGING MISCELLANEOUS APPLICATIONS OF MICROBIALENZYMES IN FOODS Application

Purpose

Enzyme(s)

Beer

Removing off-flavor

Diacetyl reductase

Beer

Oxygen removal

Digestion

Aids in digestion

Feed supplement Flavor enhancers Fruit juice

Increasing conversion RNA hydrolysis

Glucose oxidase, catalase Amylase, cellulase, lipase, protease Protease, amylase, hemicellulase Ribonucleases

Debittering

Naringinase

Penicillum citrinum, Streptomyces griseus Fungal

Fruit

Low-methoxyl pectin Antinutritional factor removal Fermentation aid

Pectinesterase

Fungal

Neubeck (1975); Chandler and Nicol (1975) Neubeck (1975)

Phytase

Fungal

Liener (1977)

Polyphenol oxidase

Fungal

Wheat Tea (instant)

Source

References Scott (1975a)

Aerobacter aerogenes, immobilized whole yeast cells Fungal, bacterial

Scott (1975b)

Fungal, bacterial

Sizer (1972)

Fungal, bacterial

Scott (1975a); Unno et al. (1977) Underkofler (1976)

-

Sanderson and Coggon (1977)

APPLICATIONS OF MICROBIAL ENZYMES

17

The missing enzymes are important in providing machining and other functional properties to the dough. Amylases from malted cereal, fungal, or bacterial sources are now added to flour at the mill or bakery to replace the amylases that once originated in the grain. The addition of fungal proteases to flour has also gained wide usage. Traditionally, and still in Europe, enzyme-active soy flour was added to bread flour (Rackis, 1977). The lipoxygenase present in the soy flour generated lipohydroperoxides that bleached wheat pigments, oxidized gluten for improved dough properties, reduced staling, and decreased binding of added shortening. Soybean lipoxygenase exerts its beneficial effect on both traditional fermented or mechanically developed bread. Although the lipoxygenase used is currently not of microbial origin, Satoh et al. (1976) have recently isolated a lipoxygenase-like enzyme from Fusarium oxysporum. Perhaps the source of lipoxygenase for use in bread making will at some future time be microbial. Other microbial enzymes suggested for use in bread making include lactase, to increase the fermentable sugars, and pentosanase, to allow use of the tailings fraction of flour (Barrett, 1975). Either malt or fungal amylases are added to flour for use in bread making. In bread, amylases ensure continued formation of fermentable sugars, improve dough properties, and improve the structure and keeping quality of the bread. Flour does not naturally contain enough fermentable sugar to sustain the vigorous yeast fermentation needed for lively doughs and large loaf volumes. The application of amylases also results in lower dough viscosity, increased loaf volume, improved crumb score (grain and texture), and a softer, more compressible crumb. a-Amylase, which rapidly degrades amylose to maltose and maltotriose and more slowly degrades it to maltose and glucose, is used. The @amylase naturally present in the flour aids in completing the conversion of starch to maltose. Fungal amylases (mostly from Aspergillus oryzae), since they have a lower temperature for inactivation than do the cereal or bacterial amylases, may be more useful in bread making. Fungal amylases do not appreciably decrease dough viscosity as the starch gelatinizes, resulting in much stronger loaves than those made with bacterial amylase (Barrett, 1975). Bacterial a-amylase, noted for its high thermal stability, is used in baked products needing an exceptionally soft or sticky crumb. Natural levels of protease in wheat flour are too low to have a significant effect during bread making. Fungal proteases are added to modify the wheat gluten and any milk proteins present. The limited proteolysis results in a softer, more extensible dough that requires less mixing time. Loaf volumes are substantially increased. Newer methods of bread production (mass production, automatic equipment, tight time schedules, etc.) require strict control of mixing times and the production of doughs with optimum handling

18

MATTHEW J. TAYLOR AND TOM RICHARDSON

properties. Approximately two-thirds of the white bread made in the United States is treated with proteolytic enzymes derived from A. oryzae (Barrett, 1975). Fungal proteases are necessary additives in modem bakeries. Another microbial enzyme suggested for bakery use is phytase from Aspergillus ficcum (Liener, 1977). This enzyme would hydrolyze phytate, an antinutritional factor present in wheat. Phytate is a natural chelator of di- and trivalent cations, such as calcium, magnesium, zinc, and iron. Metal ions and phytate form complexes which are poorly absorbed from the intestines. However, only when the diet consists predominantly of high-phytate cereals is mineral metabolism sufficiently disturbed to result in a real nutritional problem (Bitar and Reinhold, 1972; Reinhold et al., 1973). In addition to the direct use of microbial enzymes in baking, microbial enzymes are used in the manufacture of several bread ingredients, such as corn sweeteners and hydrolyzed dairy products. Certainly the use of microorganisms in bread making extends well beyond the yeast needed for leavening. The foregoing example of employing microbial enzymes illustrates how they can be used to alter the physical, chemical, and biochemical properties of a food as exemplified by improved rheology, texture, color, aroma, flavor, and nutrition. 111. Conventional and Proposed Uses of Microbial Enzymes in Biotechnology

A. INTRODUCTION Enzymes used in biotechnology other than in foods represent a sizeable dollar market-about two-thirds the size of the market for food enzymes (Beck and Scott, 1974). Their use could be greater because of the wider diversity of potential applications. Many of the enzymes used in biotechnology are derived from microbial sources. Microbial enzymes are often used in analysis, such as the clinical use of hngal glucose oxidase and bacterial catalase to measure blood glucose to monitor the diabetic condition (Underkofler, 1976). In the past they were widely used in detergents, although concern for consumer safety greatly decreased the large market for microbial enzymes in laundry detergents (Kelly and Fogarty, 1976; Langguth and Liss, 1971). However, the addition of enzymes (particularly proteases) to detergents is apparently staging a comeback (Christensen et al., 1978). Microbial enzymes are also used in medicine (Sizer, 1972); in the utilization of waste cellulose from food, paper, and municipal refuse (Kulp, 1975; Hajny and Reese, 1969; Andren and Mandels, 1976); and in other biotechnological applications. Again, limited space does not allow a discussion of the various applications; instead, representative uses of microbial enzymes in

APPLICATIONS OF MICROBIAL ENZYMES

19

biotechnology have been listed in Table IX. Several examples suffice to illustrate the potential for microbial enzymes in biotechnology.

B. ORGANICSYNTHESIS The use of microbial enzymes in organic synthesis has great potential, but

so far the applications have been limited (Jones et al., 1976; Whitesides, 1976; Sih et al., 1977; Abbott, 1976). However, the catalysis of difficult

stereospecific reactions by enzymes will probably increase-particularly in the synthesis of pharmaceuticals. As the need for more sophisticated drugs, hormones, and antibiotics increases, specific enzymes may be used to catalyze desired conversions. Intact microorganisms have been used in most organic syntheses but wholly or partially purified enzymes are rapidly gaining application. The manufacture of L-dihydroxyphenylalanine (L-dopa), a useful agent in the treatment of Parkinson’s disease, was made much easier by a microbial tyrosine hydroxylase which catalyzes the conversion of L-tyrosine to L-dopa (Whitesides, 1976). Esterases from Saccharomyces cerevisiae and A. niger have been used in the simultaneous resolution and asymmetric synthesis of compounds useful as precursors in the synthesis of naturally occurring prostaglandins (Muira et al., 1976). In a more ambitious synthesis, Archer et al. (1975) have accomplished the total enzymatic synthesis of a cyclic decapeptide antibiotic, gramicidin S (GS). Gramicidin S is used as a growth factor in animal feed, increasing conversion of feed grain to animal protein. The overall synthesis of GS, shown below, required the use of enzymes from three microorganisms. 2 Leu

+ 2 Pro + 2 Phe + 2 Om + 2 Val + 10 ATP A> GS + 10 AMP + 10 PPi El1

Enzyme fractions EI and EIIare from Bacillus brevis and catalyze the synthesis of GS from its constituent amino acids. Adenylate kinase from S. cerevisiae and acetate kinase from Escherichia coli are used to regenerate ATP in situ. This enzymatic regeneration of ATP was a major factor in the successful synthesis of GS.

C. DENTALHYGIENE The potential use of microbial enzymes in dental hygiene illustrates a novel application in biotechnology. The etiology of dental caries is admittedly complex; nonetheless, dental caries are associated with the presence in the mouth of Streptococcus mutans, which produces a viscous plaque that tightly adheres to teeth. This plaque is necessary for caries formation. The enzymatic degradation of this plaque has been studied by several re-

TABLE IX CONVENTIONAL APPLICATIONS OF MICROBIAL ENZYMES IN BIOTECHNOLOGY

0 E3

Application

Purpose

Cellulose conversion

Utilization of waste cellulose from food, paper, and municipal refuse General, enzyme electrodes, clinical

Analysis Detergents

Enzyme

Source

References

Cellulase

Trichoderma uiride, Aspergillus niger, others

Kulp (1975); Hajny and Reese (1969); Srinivasan (1975)

Glucose oxidase, urease, etc.

Various microbial

Laundry additive

Protease/amylase

Bacillus suhtilis

Cold-soluble laundry starch Dry cleaning spot removal Drain cleaner

Amylase

Bacterial

Guilhault (1976); Gray et al. (1977); Bergmeyer (1974) Langguth and Liss (1971); Damhmann et al. (1971); Wang and Humphrey (1969) Underkofler (1976)

Proteaselamylase

Fungal

Underkofler (1976)

Lipase

Bacterial, fungal

Underkofler (1976)

Leather Medical

Bating, dehairing Blood clot dissolution Inflammation treatment

Bacterial, fungal Streptococcus sp. Streptococcus sp.

Leukemia therapy

Protease Streptokinase Streptokinase, protease Streptokinase, streptodornase L-Asparaginase

Skin graft aid

Collagenase

Organic chemistry

Pharmaceutical synthesis

Various

Clostridium histolyticum Various microbial

Paper

Starch modification for paper coating Wallpaper removal Silver recovery from spent film Desizing

Amylase

Bacterial

Amylase Protease

Bacterial Bacterial

Sih etal. (1977); Jones et al. (1976); Whitesides (1976) MacAllister et al. (1975); Kulp (1975) Underkofler (1976) Underkofler (1976)

Amylase, protease

Bacterial, fungal

Underkofler (1976)

Wound debridement

Photography Textiles

Streptococcus sp. Erwinia carotoowa, Escherichia coli

Underkofler (1976) Sizer (1972) Sizer (1972); Underkofler (1976) Sizer (1972); Underkofler (1976) Sizer (1972); Cooney and Handschumacher (1970) Sizer (1972)

22

MATTHEW J. TAYLOR AND TOM RICHARDSON

searchers. For instance, Woodruf et al. (1976) used a dextranase &om Penicillium funiculosum and a cariogenanase from Bacillus sp. A significant reduction in plaque and in dental caries resulted when these two enzymes were used in animals and in man. Toothpaste fortified with a mixture of enzymes from A. niger and A. oryzae reduced calculus and soft accretions more than did an unfortified toothpaste (Sizer, 1972). Budtz-Joergensen and Kelstrup (1977) used a mutanase-protease mixture to reduce dental plaque and improve the clinical condition of palatal mucosa (denture stomatititis). It is ironic that certain microbial enzymes are involved in the production of cariogenic sugars and that other microbial enzymes can be used, in part, to prevent the destructive effects of these sugars.

IV. Use of Immobilized Microbial Enzymes in Food Systems and in Biotechnology A. INTRODUCTION The immobilization of enzymes is an emerging technology that involves the fixing of an enzyme within or onto an insoluble matrix. The preparation of an immobilized enzyme involves an enzyme, a support material and a method of immobilization. Many microbial enzymes have been immobilized. Support materials for enzyme immobilization are quite diverse as are their physical forms. Although many immobilization methods have been described, they may be categorized as adsorption, covalent attachment of the enzyme to the matrix, or physical entrapment within membrane systems. Immobilized enzymes have been applied in a few commercial situations, but many potential uses remain to be exploited. However, the lack of longterm stability of many immobilized enzyme catalysts, particularly those used in treating such complex foods as milk, precludes their commercialization at present. Several excellent reviews describe this technology in great detail (Zaborsky, 1973; Wingard et al., 1976; Olson and Cooney, 1974; Skinner, 1975; Messing, 1975; Pye and Wingard, 1974; Mosbach, 1976).

B. COMMERCIAL USESIN

THE

FOODINDUSTRY

Immobilized enzymes are used in at least two significant commercial food-processing operations (Olson and Richardson, 1974a; Weetall, 1975, 1977). The first large-scale commercial application of immobilized enzymes in the world utilizes glucose isomerase from Streptomyces sp. to produce high-fructose syrups from cornstarch (Bucke, 1977; MacAllister et al., 1975; Aschengreen, 1975). The discovery of the glucose isomerase was a key factor in commercial development. The overall process involves liquefying raw

APPLICATIONS OF MICROBIAL ENZYMES

23

cornstarch, saccharifying the liquid starch with fungal amylases and glucoamylases to glucose, isomerization of the resulting glucose to fructose by the glucose isomerase, and refining. Clinton Corn Products, of Clinton, Iowa, immobilizes this enzyme by ion exchange onto DEAE-cellulose which is then used in a packed-bed reactor in a continuous flow system (Mermelstein, 1975). Isomerization is carried out at 60-70°C under slightly alkaline conditions. The glucose isomerase requires Mn2+, Co2+, or Mg2+ as a cofactor, which is removed during refining. The substrate is a high-dextrose equivalent (DE) corn syrup of 3 0 5 0 % solids of which 93% are glucose. The product is 42% fructose-further conversion of feedstream to fructose is limited by the equilibrium of the enzyme. The half-life of the immobilized glucose isomerase is approximately 15-25 days. Other companies are also using immobilized glucose isomerase in the manufacture of high-fructose syrup, and other immobilization techniques for glucose isomerase have been described (Mermelstein, 1975; Bucke, 1977; Mosbach, 1976). The other significant commercial application of immobilized enzymes in food technology is the use of immobilized microbial enzymes or whole cells to produce desired biochemicals. A well-known example is the resolution of racemic mixtures of amino acids resulting from chemical synthesis by immobilized L-amino acid acylase (Chibata et al., 1976). This is a commercial process in Japan. The enzyme is from A . oryzae and is ionically bound to DEAE-Sephadex. The immobilized enzyme selectively removes the acetyl group from the L isomer of a racemic mixture of the acetylated amino acid. The resulting free L form is easily separated by crystallization from the acetylated D form, which is then racemized chemically to regenerate more acylated L-amino acid. L-Methionine is the most important amino acid prepared by this technique. The resolution of chemically synthesized amino acids is quite useful since the biologically active L isomers are preferred over the racemic mixtures in food and feed applications. Chibata et al. have also developed several syntheses of various biochemicals using whole microbial cells entrapped within polyacrylamide gels (Weetall, 1977; Chibata and Tosa, 1976; Chibata et al., 1974) (Table 10). Other researchers have also employed this process but have used different methods of immobilization (Fukui and Ikeda, 1975; Vieth et al., 1975; Lagerlof et al., 1976). The immobilized microbial cells have long half-lives (greater than 100 days in several cases), produce high yields of product, and decrease processing costs as much as 40%. C. PROPOSEDUSESIN

THE

FOODINDUSTRY

Many other microbial enzymes have been immobilized for potential use in food systems and in biotechnology. Immobilized microbial lactase has been extensively studied in the treatment of whey, milk, and other dairy products

24

MATTHEW J. TAYLOR AND TOM RICHARDSON

(Ford, 1975; Wondolowski, 1976; Woychik and Holsinger, 1977). For instance, lactase with an optimum pH near 4.5 is available from A. niger for the treatment of acid whey (from cottage cheese), whereas lactases with pH optima near neutrality are available from Kluyveromyces fiagilis, Saccharomyces lactis, and E . coli for the treatment of sweet whey (from Cheddar and most other cheeses). Dairy products treated with lactase could be consumed more readily by people who are intolerant to lactose. Also, for technical reasons, hydrolysis of the lactose would allow greater usage of whey in such foods as ice cream. Oligosaccharides, such as rattinose, stachyose, and verbascose, are largely responsible for the gas, nausea, cramps, and diarrhea sometimes experienced after the ingestion of legumes. Fungal a-galactosidase (particularly from A. niger) has been used to hydrolyze these flatulence-producingfactors in soy milk (Liener, 1977). The use of immobilized a-galactosidase has potential in the treatment of soy milk, but there are no reports of such. Proteases from Mucor miehei have been covalently attached to porous glass beads for the continuous coagulation of milk, but porcine pepsin was the most suitable enzyme of the several investigated (Cheryan et al., 1975). However, the choice of an immobilized protease in a continuous cheesemaking process remains open. Catalase has been immobilized to remove H202remaining after the cold pasteurization of milk (Richardson and Olson, 1974;Olson and Richardson, 1974b).Although the source of catalase has been beef liver, catalase is also available from fungal and bacterial sources. Various microbial enzymes have been immobilized for the industrial-scaledetoxification of pesticides in production waste waters, pesticide containers, and spray tank rinse waters (Munnecke, 1978). Glucoamylase has been immobilized by TABLE X PRODUCTION OF BIOCHEMICALS USING IMMOBILIZED MICRbBlAL CELLSa’b Microorganism

Achromobacter liquidum

Brevibacterium ammoniagenes Escherichia coli Pseudomonas putida

Substrate L-Histidine

Enzyme

L-Histidine ammonialyase Fumaric acid Fumarase Ammonium fumarate L-Aspartase Penicillin Penicillin amidase L-Arginine Arginine deiminase

Product Urocanic acid

L-Malic acid a as par tic acid 6-Aminopenicillanic acid L-Citrulline

“Source: Chibata et al. (1974); Chibata and Tosa (1976); Weetall (1977). *The microbial cells were entrapped within a polyacrylamide gel.

APPLICATIONS OF MICROBIAL ENZYMES

2s

TABLE XI APPLICATIONSOF ENZYMEELECTRODES" REPRESENTATIVE Substrate measured Amygdalin Cholesterol Glucose L-Amino acids L-Lysine Urea

Enzyme P-Glucosidase Cholesterol oxidase, cholesterol esterase Glucose oxidase L-Amino acid oxidase Lysine decarboxylase Urease

Membrane electrode type CN- ion selective 0, selective

0, selective 0,selective, cation selective CO, selective Ammonia selective, C02 selective

"For more examples and greater detail, see Guilbault (1976) or Bowers and Carr (1976).

many workers to convert starch to glucose (Zaborsky, 1973; Weetall et al., 1974; Weetall and Havewala, 1972). These few examples are representative of the wide variety of microbial enzymes immobilized for use in food systems and in biotechnology.

D. ENZYMEELECTRODES Enzyme electrodes offer a novel but an increasingly useful analytical technique. Enzyme electrodes allow rapid, sensitive, and specific analyses for various components in a complex mixture, such as a food. Many microbial enzymes have been used in enzyme electrodes. The enzyme is immobilized onto the sensing portion of a specific ion or gas electrode. A product from the reaction of the enzyme with the measured substrate is detected by the electrode. An example is glucose oxidase fued onto an oxygen electrode for the measurement of glucose (Nilsson et al., 1973). Applications are numerous (Table XI) and are discussed in more detail elsewhere (Kessler et al., 1976; Guilbault, 1976; Bowers and Carr, 1976; Sternberg et al., 1976).

V. Future Uses of Microbial Enzymes in Food Systems A. INTRODUCTION Enzymes currently used in foods are largely hydrolytic, degrading enzymes. Enzymes of potential future use include those that catalyze other than hydrolytic reactions. In this section, a microbial source for an enzyme is not stressed, since these suggested future uses are not so well defined that the source of the enzyme is of concern. Furthermore, the potential for the implantation of the genetic information for a nonmicrobial enzyme into a

26

MATTHEW J. TAYLOR AND TOM RICHARDSON

microorganism and subsequent microbial production of that enzyme suggests the possibility for producing plant and animal enzymes from microorganisms. For example, calf rennet is the preferred milk coagulant in the cheese industry but demand exceeds supply. Instead of using fungal rennets in cheese-making, it may eventually be possible to produce calf rennet from a calf rennet gene transplanted to a microorganism. However, since genetic engineering has as yet an undefined role in our society, and since this technology is just now emerging, this suggestion remains speculative.

B. MODIFICATION OF PROTEINFUNCTIONALITY A largely unexplored area for the use of enzymes is in the modification of proteins to improve their functionality (Richardson, 1977). Protein functionality includes such characteristics as solubility, emulsifying capacity, water and fat binding, whippability, and elasticity. This may become even more important as more processed foods are produced, since processed foods generally require proteins with a high degree of functionality. However, the manufacture of the processed food may destroy much of the protein functionality present. Enzyme treatment may increase protein functionality before processing or restore functionality after processing. For instance, many useful protein concentrates are rendered insoluble (at neutral pH values) by denaturing process treatments (Fujimaki et al., 1977). Limited proteolytic hydrolysis can restore solubility, but this treatment degrades the protein, adversely affecting other functional properties. Instead, one might enzymatically glycosylate, phosphorylate, or hydroxylate proteins before processing to provide needed solubility, at the same time keeping the protein intact. Enzymes involved in the postribosomal modification of proteins might prove useful in this regard (Whitaker, 1977). It is desirable that enzymes should find application in the improvement of protein functionality. Enzymatic modification of food proteins would probably be more acceptable than chemical modification since enzymatic treatment is more “natural,” more specific, and less prone to undesirable side reactions. Since they are essentially denatured in their native forms, likely proteins with which to study enzymatic modification are the caseins, a major group of food proteins presently in surplus. Thus, denaturation during modification would not be of concern and the random coil structure of the caseins would allow good access to reactive sites. A potential example of enzymatic modification of protein functionality is the use of thiol-disulfide isomerase or sulfhydryl oxidase along with disulfide reductase to yield disulfide interchange reactions in a food protein (Whitaker, 1977). A thiol-disulfide isomerase was first purified from beef

APPLICATIONS OF MICROBIAL ENZYMES

27

liver but has been recently purified from Candida claussenii (Kurane and Minoda, 1975). Bread is a food system that relies upon disulfide interchange in the development of the dough and which may benefit from these enzymes. The use of these enzymes may reduce or eliminate the need for chemical flour oxidizers, which are routinely added to improve flour for baking. Besides, the machining of whole wheat doughs may be eased with use of these enzymes, allowing mass production of a product increasingly in demand. Another potential enzymatic modification of protein functionality might involve lysyl oxidase or transaminases to increase crosslinking and structure formation in a protein-based food system (Whitaker, 1977). Lysyl oxidase can catalyze the oxidation of lysine (to a-amino adipic acid-&semialdehyde) and hydroxylysine (to a-hydroxy-a-amino adipic acid-S-semialdehyde) in oitro leading to the crosslinking of collagen. The linkages arise from the nonenzymatic interaction of the oxidized amino acids with another oxidized residue, a lysine, a hydroxylysine, or a histidine, located elsewhere in the collagen polypeptide chain. Transaminases catalyze the covalent attachment of amino-containing substrates to the y-carboxyl group of glutamic acid residues in proteins. Factor XI11 of the fibrin system slowly forms EN-(y-glutamy1)-lysine crosslinkages in fibrinogen and in other proteins. Perhaps some of these crosslinking enzymes will help provide structure to a formulated food of the future, such as reformed vegetable puree.

C. PLASTEINREACTION The plastein reaction provides another possible future use of enzymes in food technology. The plastein reaction takes advantage of the reversible nature of protease activity. Upon adjustment of the reaction conditions, net synthesis of new protein-like materials, rather than proteolysis, can be achieved. Most plastein research has employed one enzyme to partially hydrolyze a protein; then a change of conditions and enzyme is used to effect a resynthesis of peptide bonds but in a different sequence. In the past pepsin, chymotrypsin, or papain have largely been used but there is apparently nothing preventing the use of microbial proteases. In fact, the different specificities of the various microbial proteases should encourage their study. There are many possible uses of the plastein reaction (Fujimaki et al., 1977). Since the plastein reaction can be used to covalently bind amino acid esters into proteins, food proteins could be nutritionally improved by the incorporation of selected essential amino acids. Soybean protein fortified with methionine had a protein efficiency ratio (PER) greater than casein (Arai et al., 1975).

28

MATTHEW J. TAYLOR AND TOM RICHARDSON

The functionality of food proteins can be modified, such as by increasing the

solubility of denatured soy protein upon incorporation of glutamic acid (Yamashita et al., 1975). Proteins for special diets can be prepared such as a low-phenylalanine plastein for consumption by phenylketonurics (Yamashita et al., 1976). One could also remove unwanted or toxic constituents too tightly bound to the protein for removal by normal processing, by limited hydrolysis of the protein, extraction of the impurities, and resynthesis into a new food protein (Fujimaki et al., 1977). Since the cost of a plastein is primarily dependent on the cost of the protease, the recovery of the enzyme becomes paramount for practical purposes. The use of immobilization technology, affinity chromatography, or less expensive microbial proteases is worthy of consideration.

D . ANTIOXIDANTS Selected enzymes may find a useful role as antioxidants in certain food systems. Lipid oxidation is a continuing problem in foods, contributing to loss in nutritional value, deterioration of organoleptic quality, and formation of toxic substances. The compartmentalization responsible, in part, for the oxidative stability of living organisms is lost in most foods postharvest or postmortem. As a result, chemical antioxidants are used to inhibit oxidation. However, enzyme antioxidants may be more acceptable because of their “natural” quality. Three enzymes suggested as food antioxidants are superoxide dismutase (SOD), catalase, and glutathione peroxidase (Table XII). Presumably they would function as antioxidants by removing highly reactive oxidative species from the food. Catalase and SOD are available from microbial sources (Fridovich, 1975, 1976). Superoxide dismutase derived from certain marine bacterial strains has been used in the protection of oxidizable systems from autoxidation (Michelson, 1977). T A B L E XI1 ENZYMES PROPOSED AS ANTIOXIDANTS 1. 2 0;

+ 2 Hf

2. 2 H,O,

3. ROOH

Superoxide dismutase

Catalase

> H202 + O2

> 2 H,O

Glutathione peroxida e

n 2GSH GSSG

?=-

+ 0, ROH

+ H20

APPLICATIONS OF MICROBIAL ENZYMES

29

VI. Future Uses of Microbial Enzymes in Biotechnology Microbial enzymes have additional potential in other areas of biotechnology that is only now being recognized. Two potential uses for microbial enzymes in medicine and pharmacology are discussed here. L-Asparaginase from Erwinia carotouora or Escherichia coli has been used in the treatment of acute lymphocytic leukemia (Mauer and Simone, 1976). Leukemic cells require L-asparagine but normal cells do not (Kidd and Sobin, 1966). Starving leukemic cells by injection of microbial L-asparaginase to deplete circulating L-asparagine is a useful therapy (Cooney and Handschumacher, 1970). However, the rapid removal of the injected enzyme from the blood stream, along with immunogenicity and toxicity of the enzyme, are limitations to this treatment (Mashburn and Landia, 1970). As a result, there has been much research designed to increase the biological half-life and decrease the antigenicity of microbial L-asparaginase. L-ASparaginase has been modified chemically, immobilized within extracorporeal shunts, and entrapped within semipermeable microcapsules. For instance, glycosylated L-asparaginase was cleared from the circulation of mice at only half the rate of the underivatized enzyme (Marsh et al., 1977). Research in our laboratory has focused on the immobilization of L-asparaginase in uiuo so as to increase its biological half-life. Generally, the technique involves attaching one-half of a bifunctional electrophilic reagent to the therapeutic enzyme, leaving the other half free to further react with tissue nucleophiles. When the enzyme-reagent conjugate is introduced into living tissue, the remaining free reactive group is attacked by reactive groups available in the tissue. As a result, the therapeutic enzyme is immobilized in viuo, which may increase the half-life and mod+ the antigenicity of the enzyme in the body (Fig. 1).In our laboratory, L-asparaginase and parabenzoquinone were reacted together to form an enzyme-quinone conjugate (parabenzoquinone reacts with free amino groups). Upon incubation with human red blood cells, an enzyme-red blood cell adduct was formed, illustrating the binding of the conjugate to human tissues (Mattarella, 1978). It remains to be seen whether the enzyme covalently bound to red blood cells would have an increased circulating lifetime over free L-asparaginase. This example illustrates a possibility in enzyme therapy for an increased therapeutic effect resulting from enzyme immobilization in uiuo. Another potential future use of microbial enzymes is in the treatment of various lysosomal storage diseases. Lysosomes are essentially intracellular particles containing digestive enzymes that are presumably involved in turnover of tissue components. In lysosomal storage diseases, the deficiency of a particular lysosomal enzyme, such as P-glucuronidase or sphingomyelinase,

30

MATTHEW J. TAYLOR AND TOM RICHARDSON

1

+

-R

R-A '

CBIFUNCTDNAL REAGENT)

IN VITRO --

@=*A

I

CENZYMEREAGENT CONJUGATE )

CENZYME IMMOBILIZED, FIG. 1. Enzyme immobilization i n uioo for therapy. R, R', reactive groups specific for each other; A, electrophilic group; :Z, nucleophilic group.

results in incomplete degradation of these materials in the lysosome, creating various clinical symptoms (Neufeld et al., 1975). The lysosomes of the patient become enlarged and swollen with ingested material. A reasonable therapy would be to replace the missing enzyme in the lysosome. Current research is concentrating on the use of human placental enzymes for replacement because of their lower antigenicity in humans. However, many of the replacement enzymes could come from microorganisms, possibIy as the result of transferring the genetic information in the human placenta to the microorganism. In any event, a likely delivery system for the enzyme to the lysosome is by entrapment within phospholipid vesicles, termed liposomes (Marx, 1978), which upon injection would be internalized by leukocytes and delivered to the lysosomes for degradation. Once within the lysosome, the enzyme would be liberated as the liposome was degraded, thereby freeing the enzyme to exert its therapeutic effect (Pagan0 and Weinstein, 1978).

APPLICATIONS OF MICROBIAL ENZYMES

31

VII. Conclusion Present applications of microbial enzymes in food systems and in biotechnology are substantial but their potential is even greater. Enzymes provide a specific method relatively free of side reactions to catalyze a desired effect. For technical and economic reasons, microorganisms are becoming increasingly the best source for enzymes. Modern fermentation techniques, improved purification schemes, immobilized enzyme methods, etc., are making the use of microbial enzymes increasingly attractive. There are many technically feasible applications for microbial enzymes and the task is to pursue those that may significantly improve the social welfare.

ACKNOWLEDGMENTS This review was made possible by support from the College of Agriculturd and Life Sciences, University of Wisconsin at Madison, Madison, Wisconsin 53706. We are also grateful to Dr. Gerald Reed (Amber Laboratories, Milwaukee, WI) for his helpful comments.

REFERENCES Abbott, B. J. (1976). Ado. Appl. Mimobiol. 20, 203-257. Andren, R. K., and Mandels, M. (1976). Process Biochem. 11(8),2 4 , 6-11. Arai, S., Yamashita, M., and Fujimaki, M. (1975). Cereal Foods World 20, 107-112. Archer, M. C., Colton, C. K., Cooney, C. L., Demain, A. L., Wang, D. I. C., and Whitesides, G. M. (1975). In “Enzyme Technology: Grantees-Users Conference” (E. K. Pye, ed.), pp. 1-8. Natl. Sci. Found., Washington, D.C. Arnold, R. G., Shahani, K. M., and Dwivedi, B. K. (1975). /. Dairy S c i . 58, 1127-1143. Aschengreen, N. H. (1975). Process Biochem. 10(4), 17-19, Barrett, F. F. (1975). In “Enzymes in Food Processing” (6.Reed, ed.), pp. 301-330. Academic Press, New York. Bass, E. J., and Cayle, T. (1975). In “Enzymes in Food Processing’ (G. Reed, ed.), 2nd Ed., pp. 455-471. Academic Press, New York. Beck. C. I., and Scott, D. (1974). In “Food Related Enzymes” 0. R. Whitaker, ed.), Advances in Chemistry Series No. 136, pp. 1 3 0 . Am. Chem. SOC.,Washington, D.C. Bergmeyer, H. U., ed. (1974).“Methods of Enzymatic Analysis,” 2nd Ed., Vols. 1 4 . Academic Press, New York. Bernholdt, H. F. (1975). In “Enzymes in Food Processing” (G. Reed, ed.), 2nd Ed., pp. 473492. Academic Press, New York. Bitar, K., and Reinhold, J. G. (1972). Biochim. Biophys. Acta 268, 442-447. Bowers, L. D., and Carr, P. W. (1976). Anal. Chem. 48, 544-558. Brockerhoff, H., and Jensen, R. G. (1974). “Lipolytic Enzymes.” Academic Press, New York. Bucke, C. (1977). In “Topics in Enzyme and Fermentation Biotechnology (B)” (A. Wiseman, ed.), pp. 147-171. Halsted Press, Chichester, England. Budtz-Joergensen, E . , and Kelstrup, J. (1977). Scand. J . Dent. Res. 85, 209-215.

32

MATI’HEW J. TAYLOR AND TOM RICHARDSON

Butler, L. G., Squires, R. G., and Kelly, S. J. (1976). In “Enzyme Technology and Renewable Resources,”Proceedings of a Grantees-Users Conference 0. L. Gainer, ed.), pp. 119-122. Natl. Sci. Found., Washington, D.C. Chandler, V. V., and Nicol, K. J. (1975). CSZRO Food Res. Q . 35,(4), 79-88. Cheryan, M., van Wyk, P. J., Olson, N. F., and Richardson, T. (1975). Biotechnol. Bioeng. 17, 585-98. Chibata, I., and Tosa, T. (1976). In “Immobilized Enzyme Principles” (L. B. Wingard, Jr., E. Katchalski-Katzir, and L. Goldstein, eds.), Applied Biochemistry and Bioengineering, Vol. 1, pp. 329357. Academic Press, New York. Chibata, I., Tosa, T., Sato, T., Mori, T., and Yamamoto, K. (1974). In “Enzyme Engineering’ (E. K. Pye and L. B. Wingard, Jr., eds.), Vol. 2, pp. 303-313. Plenum, New York. Chibata, I., Tosa, T., Sato, T., and Mori, T. (1976). In “Immobilized Enzymes” (K. Mosbach, ed.),pp. 746-759. Academic Press, New York. Christensen, P. N., Holm, P., and Sonder, B. (1978). J. Am. Oil Chem. SOC. 55, 109-113. Cooney, D. A., and Handschumacher, R. E. (1970). Annu. Reu. P h a m c o l . 10, 421450. Dambmann, D., Holm, P., Jensen, V., and Nielsen, M. H. (1971). Deu. Znd. Mirrobiol. 12, 11-23. Ford, J. R. (1975). Enzyme Technol. Dig. 4, 23-29. Fridovich, I. (1975). Annu. Reo. Biochem. 44, 147-159. Fridovich, I. (1976). In “Free Radicals in Biology” (W. A. Pryor, ed.), Vol. 1, pp. 238-271. Academic Press, New York. Fujimaki, M., Arai, S., and Yamashita, M. (1977). In “Food Proteins: Improvement through Chemical and Enzymatic Modification” (R. E. Feeney and J. R. Whitaker, eds.), Advances in Chemistry Series, No. 160, pp. 156-la. Am. Chem. Soc., Washington, D.C. Fukui, S., and Ikeda, S.-I. (1975). Process Biochem. 10(5),3-8. Gray, D. N., Keyes, M. H., and Watson, B. (1977). Anal. Chem. 49, 1067-1078. Green, M. L. (1977). J. Dairy Res. 44, 159-188. Guilbault, G. C. (1976). “Handbook of Enzymic Methods of Analysis.” Dekker, New York. Haii, G. J. (1971). Food Prod. Deu. 5(6), 120. Hajny, G. J., and Reese, E. T., eds. (1969). “Cellulases and Their Applications,” Advances in Chemistry Series, No. 95. Am. Chem. Soc., Washington, D.C. Huang, H. T., and Dooley, J. G. (1976). BiotechnoL Bioeng. 18, 909-919. Jones, J. B., Sih, C. J., and Perlman, D., eds. (1976). “Applications of Biochemical Systems in Organic Chemistry.” Wiley, New York. Kelly, C. T., and Fogarty, W. M. (1976). Process Biochem. 11(6), 3-9. Kessler, M., Clark, L. C., Jr., Luebbers, D. W., Silver, I. A., and Simon, A,, eds. (1976). “Ion and Enzyme Electrodes in Biology and Medicine.” Univ. Park Press, Baltimore, Maryland. Kidd, J. G., and Sobin, L. H. (1966). Cancer Res. 26, 208-211. Kulp, K. (1975). In “Enzymes in Food Processing” (G. Reed, ed.), 2nd Ed., pp. 53-122. Academic Press, New York. Kurane, R., and Minoda, Y. (1975). Agric. B i d . Chem. 39, 1417-1422. Lagerlof, E., Nathorst-Westfelt, L., Ekstrom, B., and Sjoberg, B. (1976). In “Immobilized Enzymes” (K. Mosbach, ed.), pp. 759-768. Academic Press, New York. Langguth, R. P., and Liss, R. L. (1971). In “Kirk-Othmer Encyclopedia of Chemical Technology” (A. Standen, ed.), 2nd Ed., Suppl., pp. 294309. Wiley (Interscience),New York. Liener, I. E. (1977). In “Food Proteins: Improvement Through Chemical and Enzymatic Modification” (R. E. Feeney and J. R. Whitaker, eds.), Advances in Chemistry Series, No. 160, pp. 283300.Am. Chem. Soc., Washington, D.C.

APPLICATIONS OF MICROBIAL ENZYMES

33

MacAllister, R. V., Wardrip, E. K., and Schnyder, B. J. (1975). In “Enzymes in Food Processing (G. Reed, ed.), 2nd Ed., pp. 331359. Academic Press, New York. Marsh, J. W. Denis, J., and Wriston, J. C., Jr. (1977). J . Biol. chern. 252, 7678-7684. Man, J. L. (1978). Science 199, 1056-1057, 1128. Mashburn, L. T., and Landia, L. M. (1970). In “Recent Results in Cancer Research: Experimental and Clinical Effects of L-Asparaginase” (E. Grundmann and H. F. Oettgen, eds.), pp. 48-57. Springer-Verlag, Berlin and New York. Mattarella, N. L. (1978). M.S. Thesis, Univ. of Wisconsin, Madison. Mauer, A. M., and Simone, J. (1976). Cancer Treat. Rev. 3, 17-41. Mermelstein, N. H. (1975). Food Technol. 29(6), 20-26. Messing, R. A., ed. (1975). “Immobilized Enzymes for Industrial Reactors.” Academic Press, New York. Michelson, A. M. (1977). U.S. Patent 4,029,819. Mosbach, K., ed. (1976). “Immobilized Enzymes,” Methods in Enzymology, Vol. 44. Academic Press, New York. Muira, S., Kurozumi, S., Toru, T., Tanaka, T., Kobayashi, M., Matsubara, S., and Ishimoto, S. (1976). Tetrahedron 32, 1893-98. Munnecke, D. M. (1978). Process Biochern. 13(2), 14-16, 31. Nelson, J. H. (1975). J. Dairy Sci. 58, 1739-50. Neubeck, C. E. (1975). In “Enzymes in Food Processing (G. Reed, ed.), 2nd Ed., pp. 397442. Academic Press, New York. Neufeld, E. F., Lim, T. W., and Shapiro, L. J. (1975). Annu. Reu. Biochem. 44, 357376. Nilsson, H., Akerlund, A. C. and Mosbach, K. (1973). Biochirn. Biophys. Acta 320, 529534. Olson, A. C., and Cooney, C. L. (1974). “Immobilized Enzymes in Food and Microbial Processes.” Plenum, New York. Olson, N. F., and Richardson, T. (1974a). In “Enzyme Engineering (E. K. Pye and L. B. Wingard, Jr., eds.), Vol. 2, pp. 329336. Plenum, New York. Olson, N. F., and Richardson, T. (1974b). J . Food Sci. 39, 653-59. Ory, R. L., and St. Angelo, A. J., eds. (1977). “Enzymes in Food and Beverage Processing,” ACS Symposium Series, No. 4 7 , pp. 244-282. Am. Chem. Soc., Washington, D.C. Pagano, R. E., and Weinstein, J. N . (1978). Annu. Reu. Biophys. Bioeng. 7, 435. Phaff, H. J. (1977). In “Food Proteins: Improvement Through Chemical and Enzymatic Modification’’ (R. E. Feeney and J. R. Whitaker, eds.), Advances in Chemistry Series, No. 160, pp. 244-282. Am. Chem. Soc., Washington, D.C. Pye, E. K., and Wingard, L. B., Jr., eds. (1974). “Enzyme Engineering,” Vol. 2. Plenum, New York. Rackis, J. J. (1977). In “Enzymes in Food and Beverage Processing (R. L. Ory and A. J. St. Angelo, eds.), ACS Symposium Series, No. 47, pp. 244-265. Am. Chem. SOC.,Washington, D.C. Reed, G., ed. (1975a). “Enzymes in Food Processing,” 2nd Ed. Academic Press, New York. Reed, G. (1975b). In “Enzymes in Food Processing” (6.Reed, ed.), 2nd Ed., pp. 549-545. Academic Press, New York. Reinhold, J. G., Nedayati, H., Lahimgarzadeh, A., and Nasr, K. (1973). Ecol. Food Nutr. 2, 157-162. Richardson, G . H. (1975). In “Enzymes in Food Processing” (G. Reed, ed.), 2nd Ed., pp. 361395. Academic Press, New York. Richardson, T. (1977). In “Food Proteins: Improvement Through Chemical and Enzymatic Modification” (R. E. Feeney and J. R. Whitaker, eds.), Advances in Chemistry Series, No. 160, pp. 185-243. Am. Chem. Soc.,Washington, D.C.

34

MATTHEW J. TAYLOR AND T O M RICHARDSON

Richardson, T., and Olson, N. F. (1974). In “Immobilized Enzymes in Food and Microbial Processes” (A. C. Olson and C. L. Cooney, eds.), pp. 19-40. Plenum, New York. Sanderson, G. W., and Coggon, P. (1977). In “Enzymes in Food and Beverage Processing’ (R. L. Ory and A. J. St. Angelo, eds.), ACS Symposium Series, No. 47, pp. 12-26. Am. Chem SOC.,Washington, D.C. Sardinas, J. L. (1976). Process Biochem. 11(4), 10-17. Satoh, T., Masuda, Y., Takashio, M., Satoh, K., Beppu, T., and Kei, A. (1976). Agric. B i d . Chem. 40, 953-961. Scott, D. (1975a). In “Enzymes in Food Processing” (C. Reed, ed.), 2nd Ed., pp. 493517. Academic Press, New York. Scott, D. (1975b). I n “Enzymes in Food Processing” (G. Reed, ed.), 2nd Ed., pp. 519-547. Academic Press, New York. Scott, D. (1975~).In “Enzymes in Food Processing (G. Reed, ed.), 2nd Ed., pp. 219-254. Academic Press, New York. Shahani, K. M. (1975). In “Enzymes in Food Processing (G. Reed, ed.), 2nd Ed., pp. 181217. Academic Press, New York. Shahani, K. M., Arnold, R. G., Kilara, A., and Dwivedi, B. K. (1976). Biotechnol. Bioeng. 18, 891-907. Shukla, T. P. (1975). Crit. Reo. Food Technol. 5, 325356. Sih, C. J., Abushanab, E., and Jones, J. B. (1977). Annu. Rep. Med. Chem. 12, 298308. Sizer, I. W. (1972). Ado. Appl. Microbiol. 15, 1-11. Skinner, K. J. (1975). Chem. Eng. News. 53(33), 22-41. Srinivasan, V. R. (1975). In “Enzyme Technology: Grantees-Users Conference” (E. K. Pye, ed.), pp. 100-106. Natl. Sci. Found., Washington, D.C. Sternberg, J. C., Updike, S. J., and Lehane, D. L. (1976). In “Microtechniques for the Clinical Laboratory” (M. Werner, ed.), pp. 129-151. Wiley, New York. Sternberg, M. (1976). Ado. AppZ. Microbiol. 20, 135-157. Underkofler, L. A. (1976). In “Industrial Microbiology” (B. M. Miller and W. Litsky, eds.), pp. 128-164. McGraw-Hill, New York. Unno, N., Yoshino, M., and Murata, M. (1977). Ger. Pat. 2,705,878. Vieth, W. R., Venkatasubramanian, K., Constantinides, A., and Bernath, F. R. (1975). In “Enzyme Technology: Grantees-Users Conference” (E. K. Pye, ed.), pp, 64-72. Natl. Sci. Found., Washington, D.C. Wang, D. I. C., and Humphrey, A. E. (1969). Chem. Eng. (N.Y.) 86(27), 108-120. Weetall, H. H. (1975). Process Biochem. 10(6), 3-11, 22, 30. Weetall, H. H. (1977). In “BiotechnologicalApplications of Proteins and Enzymes” (Z. Bohak and N. Sharon, eds.), pp. 103-126. Academic Press, New York. Weetall, H. H., and Havewala, N. B. (1972). BiotechnoZ. Bioeng. Symp. 3, 241-266. Weetall, H. H., Havewala, N. B., Garfinkel, H. M., Buehl, W. M., and Baum, G . (1974). Biotechnol. Bioeng. 16, 169-179. Whitaker, J . R. (1972). “Principles of Enzymology for the Food Sciences.” Dekker, New York. Whitaker, J. R., ed. (1974). “Food Related Enzymes,” Advances in Chemistry Series, No. 136. Am. Chem. Soc., Washington, D.C. Whitaker J. R. (1977) In “Food Proteins: Improvement Through Chemical and Enzymatic Modification” (R. E. Feeney and J. R. Whitaker, eds.), Advances in Chemistry Series, No. 160,pp. 95-155. Am. Chem. Soc.,Washington, D.C. Whitesides, G. M. (1976). Tech. Chern. (N.Y.) 10, Part 2, 901-927. Wingard, L. B., Jr., Katchalski-Katzir, E., and Goldstein, L., eds. (1976). “Immobilized Enzyme Principles,” Applied Biochemistry and Bioengineering, Vol. 1. Academic Press, New York.

APPLICATIONS OF MICROBIAL ENZYMES

35

Wondolowski, M. V. (1976). Proc. Int. Biodegrud. Symp., 3rd 0. M . Sharpleyand A. M.Kaplan, eds.), pp. 1033-1040. Appl. Sci. Publ., Barking, England. Woodruf, H. B., Nollstadt, K. H., Wdton, R. B., and Stoudt, T. H. (1976). Deo. 1nd.MicrobioL 17, 405416. Woychik, J. H., and Holsinger, V. H. (1977). In “Enzymes in Food and Beverage Processing” (R. L. Ory and A. J. St. Angelo, eds.), ACS Symposium Series, No. 47, pp. 67-79. Am. Chem. Soc., Washington, D.C. Yamamoto, A. (1975). In “Enzymes in Food Processing” (G. Reed, ed.), 2nd Ed., pp. 123-179. Academic Press, New York. Yamashita, M., Arai, S., Kokubo, S., Aso, K., and Fujimaki, M. (1975). J. Agric. Food Chem. 23, 2730. Yamashita, M., Arai, S., and Fujimaki, M. (1976). 1. Food Sci. 41, 1029-1032. Zaborsky, 0. R. (1973). “Immobilized Enzymes.” CRC Press, Cleveland, Ohio.