Enzymes, Industrial (overview)

Enzymes, Industrial (overview) B C Saha and D B Jordan, US Department of Agriculture-Agricultural Research Service, Peoria, IL, USA R J Bothast, South...
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Enzymes, Industrial (overview) B C Saha and D B Jordan, US Department of Agriculture-Agricultural Research Service, Peoria, IL, USA R J Bothast, Southern Illinois University, Edwardsville, IL, USA Published by Elsevier Inc.

Defining Statement Introduction Classification Starch Conversion Lignocellulosic Biomass Conversion Enzymes in the Production of Functional Oligosaccharides and Other Neutraceuticals Enzymes in the Modification of Fats and Oils Enzymes in the Animal Feed Industry Enzymes in the Pulp and Paper Industry

Glossary biotechnology Collection of techniques involving living organisms or their derivatives (e.g., enzymes, genes) that can be used to make or modify products, frequently for industrial and commercial purposes. chiral Geometrical attribute of an object (e.g., molecule), which cannot be superimposed on its mirror image (its enantiomer). dextrose equivalent (DE) Quantification of total reducing sugars as glucose percentage. Unhydrolyzed starch has a DE of zero and glucose has a DE of 100. enantioselective reaction A chemical reaction that produces the two enantiomers of a chiral molecule in unequal amounts. liquefaction Process in which starch granules are dispersed or gelatinized in aqueous solution and then partially hydrolyzed by a thermostable -amylase.

Abbreviations CDs CGTase DE DP

cyclodextrins cyclomaltodextrin glucanotransferase dextrose equivalent degree of polymerization

Defining Statement ‘Enzymes, Industrial (Overview)’ surveys applications of enzymes in the manufacture and modification of foods,

Enzymes in the Fruit Juice Processing Industry Enzymes in the Meat and Fish Processing Industry Enzymes in the Dairy Industry Enzymes in Detergents Enzymes in the Leather Industry Enzymes in the Production of Bulk and Fine Chemicals Analytical Applications of Enzymes Enzyme-Replacement Therapy Further Reading

pitch Mixture of highly viscous liquids comprising terpenes, oils, and other hydrocarbons occurring in wood. prebiotics A nondigestive food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already in the gastrointestinal tract. racemate An equimolar mixture of two enantiomers. recombinant DNA DNA formed by joining pieces of DNA from two or more organisms. saccharification Process in which polysaccharides are hydrolyzed to their component monosaccharides. tannin Water-soluble phenolic compounds (molecular weights of 500–3000) that can combine with protein, cellulose, and other biopolymers to form cross-linked complexes.

DS HFCS SSF

dry substance basis high-fructose corn syrups simultaneous saccharification and fermentation

materials, nutraceuticals, pharmaceuticals, monitoring devices, bulk chemicals, and fine chemicals. Most industrial enzymes are currently produced by recombinant microorganisms. A number of enzymatic processes have

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already replaced conventional chemical processes. The application of enzymes in some processes has helped to develop processes unavailable to conventional chemistry.

Introduction

been identified in nature for mediating biological processes. As such, although enzymes are often designated by using short trivial names, they also have been assigned longer systematic names and four-part classification numbers (EC number, ‘enzyme commission number’) by the enzyme nomenclature system maintained by the International Union of Biochemistry and Molecular Biology and the International Union of Pure and Applied Chemistry. Thus, depending on the type of reaction catalyzed, enzymes are divided into six main classes:

Enzymes play key roles in numerous biotechnology products and processes that are commonly encountered in the form of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. At present, the most frequently used enzymes in biotechnology are hydrolases, which catalyze the breakdown of molecules. Enzymes can display regioand stereospecificity, properties that have been exploited for asymmetric synthesis and racemic resolution. Chiral selectivity of enzymes has been employed to prepare enantiomerically pure pharmaceuticals, agrochemicals, chemical feedstocks, and food additives. Enzymatic methods have replaced numerous conventional chemical processes, and they have afforded practical routes to products that were unprecedented by conventional chemistry. At present, hundreds of enzymes are used industrially. As the industrial enzyme market has expanded at a rate of about 10% annually, microbial enzymes have largely replaced the traditional plant and animal enzymes, and most of them are produced recombinantly. DNA technology has been used to modify substrate specificity and improve stability properties of enzymes for increasing yields of enzyme-catalyzed reactions. Also, it has been used in metabolic engineering of cellular metabolism to increase yields of fermentation products. Enzymes are environmentally friendly; they work under moderate conditions of temperature, pH, and pressure, their catalyzed reactions rarely form wasteful side products, and the proteins themselves are biodegradable and generally pose no threat to the environment. This article reviews applications of enzyme biotechnology in industry.

The major classes are further subdivided into subclasses. For example, the nickel-containing enzyme that catalyzes hydrolysis of urea to ammonia and carbon dioxide has the commonly accepted trivial name urease, the systematic name urea amidohydrolase, and the enzyme commission number EC 3.3.1.5. Whereas the hydrolases are the most frequently used enzymes in industry, the ligases stand as the only main class of enzymes that are not currently used as catalysts in industrial-scale production. Table 1 lists some microbial enzymes and their uses in industry.

Classification

Starch Conversion

Enzymes, produced by living systems, are protein catalysts that can accelerate chemical reactions, the transfer of electrons, atoms, or functional groups, by several orders of magnitude while maintaining high fidelity of reaction trajectories and producing low levels of side-reaction products. They catalyze nearly all the reaction types known in organic chemistry. For catalytic activity, enzymes may require organic (e.g., coenzymes such as flavin mononucleotide and thiamine pyrophosphate) or inorganic (e.g., Mg2þ) cofactors that, like the enzyme itself, are not consumed in the catalyzed reactions. Over 4000 enzymes, each catalyzing different reactions, have

Feedstocks containing starch include most of the cereal grains (corn, sorghum, barley) and tuberous crops such as potatoes. Starch contains about 15–30% amylose and 70–85% amylopectin. Amylose (MW about 300 000) is a long linear polymer of -1,4-linked glucose residues, whereas, amylopectin (MW about 1.5–3  109) is a branched polymer having both -1,4 and -1,6 linkages. The branched chains may contain from 20 to 30 glucose units. Enzymes have largely replaced the use of strong acid and high temperature to break down starchy materials. Three types of enzymes are involved in starch bioconversion: endo-amylase ( -amylase, EC 3.2.1.1), exo-amylases

1. Oxidoreductases. Transfer of electrons from one substrate molecule to another (e.g., dehydrogenases, reductases, oxidases). 2. Transferases. Transfer of functional group from one substrate molecule to another (e.g., glycosyl transferases, acetyl transferases, and aminotransferases). 3. Hydrolases. Transfer of functional group from substrate to water (e.g., glycoside hydrolases, peptidases, esterases). 4. Lyases. Elimination of functional group from substrate with the formation of double bonds. Thus, bonds are cleaved using a different principle than hydrolysis (e.g., pectate lyases break glycosidic linkages by -elimination). 5. Isomerases. Transfer of groups from one position to another in the same molecule (e.g., glucose isomerase). 6. Ligases. Addition of function group to substrate usually coupled with ATP hydrolysis (e.g., glycine– tRNA ligase).

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Table 1 Microbial enzymes and their uses in industry Enzyme

Producer

Use

Acetolactate decarboxylase Aminoacylase Amine oxidase -Amylase

Lactobacillius sp. Bacillus brevis Aspergillus oryzae Aspergillus niger Bacillus amyloliquefaciens Bacillus licheniformis Bacillus stearothermophilus Bacillus polymyxa Lactococcus lactis

Acceleration of maturation process in beer production

-Amylase Aminopeptidase Anthocyanase Arabinofuranosidase L-Aspartate

ammonia lyase 4decarboxylase Catalase L-Aspartate

Cellulases

Chitinase Cyclomaltodextrin glucanotransferase Fumarase -Galactosidase (raffinase)

-Galactosidase (lactase)

-Glucanase Glucoamylase

Glucose isomerase

Glucose oxidase Invertase Isoamylase Laccase Lipase

Naringinase Nitrile hydratase Pectinases Penicillin acylase Phytase

A. niger A. niger Aureobasidium pullulans Escherichia coli Pseudomonas dacunhae A. niger Trichoderma reesei Penicillium purpurogenum Clostridium thermocellum Trichoderma harzianum Streptomyces sp. Bacillus sp. Brevibacterium flavum A. niger Aspergillus pheonics Saccharomyces cerevisiae A. niger Kluyveromyces fragilis Candida pseudotropicalis A. niger Bacillus subtilis A. niger Endomycopsis fibuligera Rhizopus niveus Bacillus coagulans Actinoplanes missouriensis Streptomyces olivochromogenes A. niger Penicillium amagasakiense Aspergillus sp. Saccharomyces sp. Pseudomonas amylodermosa Coriolus versicolor A. niger Candida rugosa Geotrichum candidum Humicola lanuginosa Rhizomucor miehei A. niger Rhodococcus rhodochrous A. niger A. oryzae Bacillus megaterium Aspergillus ficuum A. niger

Optical resolution of amino acids Optical resolution of racemic amines Starch liquefaction, viscosity reduction, brewing, baking, confectionery, textile, detergents Starch saccharification to maltose, brewing, baking Protein break down to peptides, cheese, soy sauce, removes bitter taste, tenderize meat, removes haze from beer Decolorizing fruit juices and wines Liberates arabinose, animal feed, wine making, lignocellulosic biomass conversion, delignification of pulp Makes aspartic acid from fumarate and ammonia Makes L-alanine from L-aspartic acid Molecular oxygen removal, lemonades, egg whites, contact lens care Fruit and vegetable processing, juice clarification, oil extraction, biomass conversion, textile industries, detergents, deinking, animal feeds, digestive aid Antimicrobial activity Cyclodextrin production Conversion of fumaric acid into malic acid Raffinose hydrolysis to sucrose and galactose in sugar beet, flatulence reduction, processed legume-based foods Milk sugar hydrolysis to glucose and galactose; cheese whey hydrolysis Improved feed utilization, brewing, baked goods Starch saccharification to glucose, brewing, baking, alcohol production Conversion of glucose into fructose, High-fructose corn syrups (HFCS) Oxygen scavenger, beverages, eggs, fruit juices, wine Invert sugar production, baking, confectionery, artificial honey manufacture Starch debranching, brewing, maltose production Oxygen removal, lignin degredation Cheese ripening, flavor development, fat and oil modification, transesterification of fatty acids, stereoselective transformation, pitch control in pulp and paper industry, detergents, organic synthesis Citrus fruit juice debittering Acrylamide production from acrylonitrile, nicotinamide production Fruit juice processing, coffee processing, wine Antibiotic production Liberation of phosphate groups, animal feed (Continued )

284 Applied Microbiology: Industrial | Enzymes, Industrial (overview) Table 1 (Continued) Enzyme

Producer

Use

Proteinases (serine proteinase, carboxylproteinase, metalloproteinase) Pullulanase

B. subtilis Aspergillus sp.

Protein hydrolysis, cheese, meat, fish, cereal, fruit, beverage, baking, leather, laundry detergents, gelatin hydrolysis peptide synthesis

Aerobacter aerogenes Bacillus acidopullulyticus Mucor sp. A. oryzae

Starch debranching; glucose and maltose production, brewing Milk coagulation, dairy industry Improved solubility of instant tea, reduces chill haze formation in beer, wine making, animal feed additive Synthesis of aspartame Pulp and paper making, fruit and vegetable processing, juice clarification, extraction processes, biomass conversion, textile industries, detergents, deinking, animal feeds, digestive aid

Rennet Tannase Thermolysin Xylanases

Bacillus thermoproteolyticus Trichoderma sp. Aspergillus sp.

(glucoamylase or glucan 1,4- -glucosidase, EC 3.2.1.3; -amylase, EC 3.2.1.2), and debranching enzymes (pullulanase, EC 3.2.1.41; isoamylase, EC 3.2.1.68). -Amylase hydrolyzes internal -1,4-glycosidic bonds of starch at random in an endo-fashion producing malto-oligosaccharides of varying chain lengths. It cannot act on -1,6 linkages. The enzyme is produced by bacteria such as Bacillus lichiniformis, Bacillus subtilis, and Bacillus amyloliquefaciens and fungi such as Aspergillus oryzae. Glucoamylase cleaves glucose units from the nonreducing end of starch and it can hydrolyze both -1,4 and -1,6 linkages of starch. It is, however, slower in hydrolyzing -1,6 linkages. Glucoamylase is produced by various genera of fungi such as Endomycopsis, Aspergillus, Penicillium, Rhizopus, and Mucor. -Amylase hydrolyzes the -1,4-glycosidic bonds in starch from the nonreducing ends, generating maltose. The enzyme is unable to bypass the -1,6 linkages and leaves dextrins, known as -limit dextrins. -Amylase is produced by a number of microorganisms including Bacillus megaterium, Bacillus cereus, Bacillus polymyxa, Thermoanaerobacter thermosulfurogenes, and Pseudomonas sp. Pullulanase (pullulan -1,6-glucanohydrolase) or isoamylase (glycogen -1,6-glucanohydrolase) cleaves the -1,6linked branch points of starch and produces linear amylosaccharides of varying lengths. Pullulanase is produced by Aerobacter aerogenes and isoamylase is produced by Pseudomonas amyloderamosa. The dual-function enzyme, amylopullulanase, hydrolyzes both -1,4 and -1,6 linkages of starch and generates DP2–DP4 (DP, degree of polymerization) as products. The enzyme has potential for use in both liquefaction and saccharification of starch. Amylopullulanase is produced by various anaerobic as well as aerobic bacterial species.

Production of Glucose Syrup D-Glucose (dextrose) can readily be produced from starch by acid hydrolysis. However, this process has disadvantages such as a low yield of glucose (85%), formation of

undesirable bitter sugar (gentiobiose), and the inevitable formation of salt (from subsequent neutralization with alkali) and coloring materials. With the discovery and development of thermostable -amylase from Bacillus licheniformis, an enzymatic process has replaced the acid hydrolysis process. Enzymatic production of glucose from starch usually involves two essential and distinct steps: liquefaction and saccharification. First, an aqueous slurry of corn starch (30– 35%, dry substance basis, DS) is gelatinized (105  C, 5 min) and partially hydrolyzed (95  C, 2 h) by a highly thermostable -amylase to a dextrose equivalent (DE) of 10–15. The optimal pH for the reaction is 6.0–6.5 and Ca2þ (1 mmol l1) is required. Ca2þ is a structural factor needed by -amylase for maintaining protein stability and it does not participate in catalysis as an enzyme cofactor. DNA technology has been used to modify the Ca2þ binding site to improve binding affinity and lower Ca2þ levels needed for stabilization. During liquefaction, -amylase hydrolyzes -1,4 linkages at random, lowering the viscosity of the gelatinized starch. Liquefied and partially hydrolyzed starches are known as maltodextrins and are widely used in the food industry as thickeners. In the saccharification step, temperature of the reaction is brought to 55–60  C, pH is lowered to 4.0–5.0, and glucoamylase is added. Long reaction times of 24–72 h are required depending on the enzyme dose and the percent of glucose desired in the product. Efficiency of saccharification with glucoamylase can be improved by adding pullulanase or isoamylase. This supplemental enzyme addition increases the glucose yield (about 2%), lowers the saccharification time from 72 to 48 h, allows for increased substrate concentrations (to 40%, DS), and lowers the use of glucoamylase by up to 50%. Thus, a glucose yield of 95–97.5% is achieved by including a starch-debranching enzyme with glucoamylase. At high glucose concentrations, glucose molecules can polymerize in a reaction called reversion, forming unwanted byproducts such as maltose, isomaltose, and higher saccharides that decrease the glucose yield and

Applied Microbiology: Industrial | Enzymes, Industrial (overview)

purity. The polymerization reaction can be catalyzed by glucoamylase itself or by another enzyme called transglucosidase (EC 2.4.1.24), which is often present in crude glucoamylase preparations. Typically, glucose syrups (DE 97–98) having 96% glucose contain 2–3% disaccharides (maltose and isomaltose) and 1–2% higher saccharides.

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Corn starch slurry (30–35% DS, pH6.0–6.5, Ca2+ 50 ppm) Liquefaction Thermostable α-amylase Gelatinization (105 °C, 5 min) Dextrinization (95 °C, 2 h)

Liquefied starch DE 10–15

Production of High-Fructose Corn Syrups Glucose isomerase (also known as xylose isomerase, EC 5.3.1.5) is an example of the highly successful application of enzyme biotechnology to an industrial process that has no commercially viable route through conventional chemistry. Chemical isomerization of glucose to fructose at high pH and high temperature leads to undesirable side products, some of which are colored and have off flavors. Enzymecatalyzed isomerization (at moderate pH and temperature) does not form undesired side products. Produced intracellularly by Streptomyces, Bacillus, Arthobacter, and Actinoplanes species, glucose isomerase is used to convert glucose into fructose to exploit the greater sweetness of fructose over glucose and sucrose. High-fructose corn syrups (HFCSs) are prepared by enzymatic isomerization of glucose syrups (DE 95–98; 40–50%, DS) in column reactors containing immobilized enzyme. Reactors are run at pH 7.5–8.0 and 55–60  C. Mg2þ (5 mmol l1) is usually added as a cofactor and stabilizer of the isomerase, and it also alleviates inhibition of the enzyme by Ca2þ (present due to some carryover from its use in starch liquefaction with amylase). Immobilized glucose isomerase has a stability half-life of around 200 days in industrial practice. At equilibrium, the interconversion reaction of glucose and fructose modestly favors glucose over fructose at reactor temperatures (55–60  C). Therefore, at most, glucose isomerase can produce HFCS containing 42% fructose from glucose syrups (95% glucose). Two grades of HFCS are available in the market – HFCS-42 and HFCS-55 which contain 42% and 55% fructose, respectively, based on dry substance. HFCS-42 syrup is fractionated in a chromatographic column to yield 90– 95% fructose syrup, which can be blended back with HFCS-42 to make HFCS-55 (which matches the sweetness of sucrose on an equivalent mass basis) for use in soft drinks. Figure 1 summarizes the steps involved in the conversion of starch into HFCSs using glucose isomerase, the largest biocatalytic process in current practice with millions of tons of HFCS produced annually. DNA technology has been used to introduce amino acid substitutions in glucose isomerase that enhance its thermal stability. Further increases in the thermal stability of glucose isomerase are desirable because the equilibrium constant (fructose/glucose) increases with temperature and if the enzyme could withstand 90  C, then it could produce HFCS-55 without necessitating a chromatographic enrichment step.

Saccharification Glucoamylase (60 °C, pH4.0–4.5, 24–72 h)

Glucose syrups DE 95–96 Isomerization Glucose isomerase (pH7.5–8.0, 55–60 °C, 5 mM Mg2+)

High-fructose corn syrups (42% fructose) Figure 1 Production of high-fructose corn syrups (HFCS) from starch.

Production of High-Maltose Conversion Syrups Various maltose-containing syrups are used in the brewing, baking, soft drink, canning, confectionery, and other food industries. There are three types of maltose-containing syrups: high-maltose syrup (DE 35–50, 45–60% maltose, 10–25% maltotriose, 0.5–3% glucose), extra high-maltose syrup (DE 45–60, 70–85% maltose, 8–21% maltotriose, 1.5–2% glucose), and high conversion syrup (DE 60–70, 30–47% maltose, 35–43% glucose, 8–15% maltotriose). Production of these syrups from starch generally involves liquefaction and saccharification, as in the production of glucose. However, in this process, the liquefaction reaction is terminated when the DE reaches about 5–10 since a low DE value increases the potential for attaining high maltose content. Depending on the maltose content of the syrup desired, saccharification is generally performed by using a maltogenic amylase such as -amylase, -amylase with pullulanase or isoamylase, or a fungal -amylase at pH 5.0–5.5 and 50–55  C. High conversion syrups are produced from liquefied starch (DE, generally 40) by saccharification with a carefully balanced mixture of -amylase or fungal amylase and glucoamylase. After partial saccharification, the syrup is heated to destroy the enzyme action and prevent further glucose formation.

Production of Cyclodextrins Cyclodextrins (CDs) are cyclic oligosaccharides composed of six or more -1,4-linked glucose units. CDs of 6, 7, and 8 -D-glucose residues are called -, -, and

-CD, respectively. CDs possess a hydrophilic exterior and a hydrophobic interior. The latter affords the ability to form inclusion complexes with hydrophobic chemicals making CDs useful in the food, cosmetic, pharmaceutical,

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and plastic industries as emulsifiers, antioxidants, stabilizing agents, and traps for volatiles. CDs are produced industrially from starch or dextrins by cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) by intramolecular transglycosylation (cyclization reaction). The ratio of -, -, and -CDs produced from soluble starch depends on the source of CGTase (B. megaterium, Bacillus macerans, Bacillus circulans, Bacillus ohbensis, Klebsiella pneumoniae). Unmodified CDs have low aqueous solubility. Branched CDs such as maltosyl CDs can be prepared by using the reverse reaction of pullulanase with maltose and CDs as substrates. Glucosyl CDs can be prepared by treatment of maltosyl CDs with glucoamylase. Such branched CDs are highly soluble in water and organic solvents.

Following liquefaction, enzymatic saccharification using glucoamylase and fermentation using the conventional yeast are carried out simultaneously. The addition of protein-splitting enzymes (proteases) releases soluble nitrogen compounds from the fermentation mash and promotes growth of the yeast, decreasing fermentation time. The residue left after fermenting the sugars is known as distiller’s grains, which is used as animal feed. Typically, large-scale industrial fermentation processes provide 12–15% (v/v) ethanol with an ethanol yield as high as 95% of theoretical, on the basis of starch feedstock. At present, ethanol is the most widely used renewable fuel in the transportation sector, with about 5 billion gallons produced in the United States in 2006 and with another 6 billion gallons of annual capacity under construction.

Production of Ethanol The process of making ethanol from starch involves three basic steps: (1) preparation of the glucose feedstock, (2) fermentation of glucose to ethanol, and (3) recovery of ethanol. Enzymes have an important role in preparing the feedstock, that is, in converting starch into the fermentable sugar, glucose. Corn kernels contain 60–70% starch, and it is the dominant source (97%) of starch feedstock used for ethanol production in the United States. Two methods are used industrially to process corn for making starch accessible to enzymes in subsequent treatment. In the wet-milling process, corn is steeped in acidic water solutions and the oil, protein, and fiber fractions are successively removed as products leaving the starch fraction. Enzymatic liquefaction and saccharification of the starch fraction are then carried out for the production of glucose, as described above. In the beverage–alcohol industry, -amylase is also used. Microbial enzymes have replaced the traditional hydrolytic enzymes formerly supplied by adding malt. Glucose is fermented by the traditional yeast Saccharomyces cerevisiae to ethanol, which can be recovered by distillation. In beverage ethanol processes, the beer may be treated with acetolactate decarboxylase (EC 4.1.1.5) from Bacillus brevis or Lactobacillus sp. to convert acetolactate into acetoin via nonoxidative decarboxylation – this prevents spontaneous oxidative decarboxylation of acetolactate that yields diacetyl and its undesirable aroma. Saccharification and fermentation steps can also be carried out concurrently in a process known as simultaneous saccharification and fermentation (SSF). In SSF, the fermenting yeast consumes glucose as it is produced rather than allowing it to accumulate. In the United States, most ethanol (over 80% in 2006) from corn is produced from corn processed through dry grind facilities because of the lower capital investment required in comparison to that of wet mills. In the typical dry grind process, corn is mechanically milled to a coarse flour. Oil, protein, and fiber fractions are not isolated.

Lignocellulosic Biomass Conversion Lignocellulosic biomass includes various agricultural residues (straws, hulls, stems, cobs, stalks), deciduous and coniferous woods, municipal solid wastes (paper, cardboard, yard debris, wood products), waste from the pulp and paper industry, and energy crops (switchgrass, miscanthus). These materials are structurally diverse and compositions vary widely (cellulose, 35–50%; hemicellulose, 20–35%; lignin, 10–25%; proteins, oils, and ash, 3–15%). Native lignocellulosic biomass is resistant to enzymatic hydrolysis and effective pretreatment of the materials is crucial for improving access of enzymes to substrates for enabling efficient enzymatic action. Various pretreatment options are available to fractionate, solubilize, hydrolyze, and separate cellulose, hemicellulose, and lignin components. These include steam explosion, dilute acid, concentrated acid, alkali, SO2, alkaline peroxide, ammonia fiber expansion, and organic solvents. Dilute acid pretreatment at high temperature can hydrolyze hemicellulose to simple sugars (xylose, arabinose, and other sugars) and acids (acetic, glucuronic), which are water-soluble. The insoluble residue contains cellulose and lignin. The lignin can be extracted with solvents such as ethanol, butanol, or formic acid. Alternatively, enzymatic hydrolysis of cellulose with lignin present produces glucose, and the residues are lignin plus any unreacted materials. Cellulose Conversion Cellulose is a water-insoluble, linear polymer of 8000– 14 000 glucose units linked by 1,4- -D-glucosidic bonds. The enzyme system for the conversion of cellulose into glucose comprises endo-1,4- -glucanase (cellulase, EC 3.2.1.4), exo-1,4- -glucanase (cellulose 1,4- -cellobiosidase; EC 3.2.1.91), and -glucosidase (EC 3.2.1.21). These cellulolytic enzymes with -glucosidase act sequentially

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and cooperatively to degrade crystalline cellulose to glucose. Endoglucanase acts in random fashion on the regions of low crystallinity of the cellulosic fiber, whereas exoglucanase removes cellobiose ( -1,4-glucose dimer) units from the nonreducing ends of cellulose chains. Synergism between these two enzymes is attributed to the endo–exo form of cooperativity and has been studied extensively in the degradation of cellulose by Trichoderma reesei. Glucosidase hydrolyzes cellobiose and, in some cases, larger cellooligosaccharides to glucose. The enzyme can be rate limiting to the enzymatic hydrolysis of cellulose, as both endoglucanase and cellobiohydrolase activities are inhibited by cellobiose. Thus, -glucosidase not only produces glucose from cellobiose but also lowers cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently. However, like -glucanases, most -glucosidases are subject to product (glucose) inhibition. Many microorganisms are cellulolytic. However, cellulases from only two fungi (Trichoderma and Aspergillus) have been used extensively. Saccharification of cellulosic materials at commercial scale has been hindered by the cost of the cellulase enzymes and also by the cost of pretreatment of the biomass substrate.

Hemicellulose Conversion Hemicelluloses are heterogeneous polymers of pentoses (xylose and arabinose), hexoses (mannose), sugar acid (glucuronic acid), and organic acids. Xylans, which are the major hemicelluloses of many plant materials, are heteropolysaccharides comprising a homopolymeric backbone chain of 1,4-linked -D-xylopyranose units and side chains stemming from the xylose backbone that may contain arabinose, galactose, glucuronic acid (or its 4-Omethyl ether), and acetic, p-coumaric, and ferulic acids. Complete enzymatic hydrolysis of xylan requires endo -1,4-xylanase (EC 3.2.1.8), -xylosidase (EC 3.2.1.37), and several accessory enzyme activities which are necessary for hydrolyzing xylose substitutions (side chains). The accessory enzymes include -L-arabinofuranosidase (EC 3.2.1.55), -glucuronidase (EC 3.2.1.139), acetyl xylan esterase (EC 3.1.1.72), feruloyl esterase (EC 3.1.1.73), and p-coumaroyl esterase. Endo-xylanase randomly attacks the main chain of xylan, and the -xylosidase hydrolyzes xylobiose and xylooligosaccharides to xylose. -L-Arabinofuranosidase and -glucuronidase remove the arabinose and 4-O-methyl glucuronic acid substituents, respectively, from the xylan backbone. The esterases hydrolyze the ester linkages between xylose units of the xylan and acetic acid (acetyl xylan esterase) or between arabinose side chain residues and phenolic acids such as ferulic acid (feruloyl esterase) and p-coumaric acid (p-coumaroyl esterase). Depolymerizing and side-group cleaving enzymes act synergistically to break down hemicellulose to simple

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sugars. Some xylanases do not hydrolyze glycosidic bonds between xylose units which are substituted at positions C2 or C3; so the side chains must be cleaved before the xylan backbone can be completely hydrolyzed. On the other hand, several accessory enzymes only remove side chains from xylooligosaccharides. These enzymes require xylanases to partially hydrolyze hemicellulose before side chains can be cleaved. Although the structure of xylan is more complex than cellulose and requires several different enzymes with different substrate specificities for complete hydrolysis, the polysaccharide does not form tightly packed crystalline structures (owing to the varied substitutions at C2 and C3 positions of xylose of the xylose chain) and is thus more accessible to enzymatic hydrolysis. Xylanolytic enzymes are produced by numerous microorganisms such as Trichoderma sp., Aspergillus sp., Fusarium sp., and Bacillus sp. Currently, the high cost of hemicellulosic hydrolases prohibits their use in a fully enzymatic saccharification process. It is anticipated that cellulosic bioethanol facilities will initially use high temperature and acid pretreatments of lignocellulose materials to achieve complete saccharification of the hemicellulose fraction, and subsequent saccharification of the remaining cellulose fraction would be carried out by cellulosic hydrolases. As the hemicellulosic enzymes become more cost competitive, they could be incorporated into processes that have milder pretreatments of lignocellulose. Milder pretreatments are desirable because they produce lower amounts of by-products from the five-carbon sugars, some of which (e.g., fufurals) are inhibitory to fermenting organisms. For example, alkaline pretreatments may be used to saponify ester groups from hemicellulose, and remove much of the lignin so that three enzyme activities (endo-xylanase, -xylosidase, and -arabinofuranosidase) would be required for hydrolysis of hemicellulose. The traditional strains of S. cerevisiae, used for fermenting glucose to ethanol, do not metabolize the five-carbon sugars (mainly xylose) of hemicellulose. Two approaches have been taken to improve bioethanol production from the pentoses: (1) incorporation of genes that code for enzymes that metabolize the pentoses (e.g., xylose reductase, xylitol dehydrogenase, xylose isomerase, and xylulokinase) into S. cerevisiae and other ethanologens and (2) metabolic engineering of other ethanologens, that already have the ability to ferment both hexoses and pentoses, to improve ethanol tolerance and yields. Some progress has been made in both approaches.

Lignin Conversion Lignin is a water-insoluble, long-chain heterogeneous polymer composed largely of phenylpropane units which are most commonly linked by ether bonds. The

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conversion of cellulose and hemicellulose into fuels and chemicals leaves lignin as a byproduct. In recent years, removal of lignin from lignin–carbohydrate complex has received much attention because of potential application in the pulp and paper industry. The lignin barrier can be disrupted by a variety of pretreatments rendering the cellulose and hemicellulose more susceptible to enzymatic attack. The basidiomycete, Phanerochaete chrysosporium, is able to degrade lignin in a H2O2-dependent process catalyzed by extracellular peroxidases (lignin peroxidase and manganese peroxidase). Due to extreme complexity of the problem, a great deal of research remains to reveal the essential factors involved in lignin biodegradation.

Enzymes in the Production of Functional Oligosaccharides and Other Neutraceuticals Nutraceuticals are food supplements thought to benefit human health. Various oligosaccharides (molecules containing 2–10 monosaccharides linked by glycosidic bonds), prepared by using enzymes, are relatively new functional food ingredients used to promote the growth of Bifidobacterium species in the human colon. This activity categorizes them as prebiotics, nondigestible food ingredients that may beneficially affect the host by selectively stimulating the growth and/or the activity of a limited number of bacteria in the colon. These should not be confused with probiotics, which consist of bacteria (Bifidobacterium and Lactobacillus species) added directly to food and drink. Additional functional attributes of oligosaccharides include inhibition of dental plaque formation, improved mineral absorption, enhanced antitumor immune response, and improved control of blood glucose. Oligosaccharides are generally many times sweeter than glucose and they are often used as food additives (breads, candies). The annual manufacture of thousands of tons of prebiotic oligosaccharides provides a commercial example of successful use of reverse reactions of glycosidases and transglycosylation reactions catalyzed by glycosyltransferases. Isomaltooligosaccharides are prepared from maltose by the transglucosidase activity of -glucosidase (EC 3.2.1.20). These oligosaccharides contain glucose residues linked by -1,6-glycosidic bonds and induce a bifidogenic response. Glycosyl sucrose (coupling sugar) is prepared from starch and sucrose by using CGTase. In this case, CGTase catalyzes a transglycosylation reaction whereby glycosyl moieties are transferred from starch to glucose residues of sucrose by -1,4 linkages. Coupling sugar is used as an anticariogenic sweetening agent; it is not a substrate for formation of insoluble glucan and it inhibits formation

of insoluble glucan from sucrose. Gentio-oligosaccharides are prepared from glucose by enzymatic transglucosylation. They contain glucose residues linked by -1,6glycosidic bonds and promote the growth of bifidobacteria. Xylo-oligosaccharides are produced from xylan substrates by the action of endo-1,4- -xylanase. These xylo-oligosaccharides promote the growth of bifidobacteria and are used in prebiotic drinks. Fructooligosaccharides (neosugars, mixtures of Glu–Fru2, Glu–Fru3, and Glu–Fru4) are produced from sucrose using the transfructosylation activity of the enzyme -fructofuranosidase (EC 3.2.1.26) from Aspergillus niger. They are also produced from inulin, a linear, -2,1-linked fructose polymer initiated by a glucose unit, through controlled enzymatic hydrolysis using inulinase. The nonreducing disaccharide trehalose ( -D-glucospyranosyl- -D-glucopyranoside) is prepared on the industrial scale from liquefied starch in reactions mediated by malto-oligosyltrehalose synthase (EC 5.4.99.15) and malto-oligosyltrehalose trehalohydrolase (EC 3.2.1.141). Trehalose is used as a low-impact sweetener (low-insulin response and low-cariogenic profile) and stabilizer (cryoprotection for frozen foods). Acariogenic disaccharides palatinose (isomaltulose, 6- -D-glucopyranosyl-D-fructofuranose) and trehalulose (1- -D-glucopyranosyl-Dfructose) are prepared from sucrose in reactions mediated by palatinose synthase (isomaltulose isomerase, EC 5.4.99.11) from Protaminobacter rubrum and Pseudomonas acidophilia. Enzyme from the two different sources produces different ratios of palatinose to trehalulose from sucrose: 9 for the P. rubrum enzyme and 0.11 for the P. acidophilia enzyme. Larger oligosaccharides of the two disaccharides are prepared by using glucosyltransferase reactions. Lactosucrose is prepared from lactose and sucrose by transfructosylation with -fructofuranosidase. It consists of a lactose molecule to which a fructose moiety is joined at the glucose residue by a -2,1-glycosidic bond. Galacto-oligosaccharides are produced from lactose using the galactosyltransferase activity of -galactosidase from Kluyveromyces fragilis, Kluyveromyces lactis, B. circulans, and A. oryzae. Other nutraceuticals include digestive aids, vitamins, and other biochemicals. Lactase, raffinase, and amylase are the most commonly used digestive-aid supplements for humans. The one -galactosidase that act on lactose (present in dairy products) and the -amylase that acts on starch (starchy foods) are taken in tablet or liquid form during meals. They promote digestion of the sugars before they reach the colon where they could, otherwise, be digested by bacteria and cause flatulence and other ill effects. Optimized fermentation processes are used for production of many of the vitamins and biochemicals used as dietary supplements such as riboflavin, coenzyme Q , and L-carnitine.

Applied Microbiology: Industrial | Enzymes, Industrial (overview)

Enzymes in the Modification of Fats and Oils Lipases (EC 3.1.1.3, glycerol ester hydrolases) are a ubiquitous class of enzymes which catalyze hydrolysis, esterification (synthesis), and transesterification (group exchange of esters). These enzymes are used for diverse purposes such as fat hydrolysis, flavor development in dairy products, ester synthesis, transesterification of fats and oils, production of chiral organic compounds, washing and cleaning products, and treatment of domestic and industrial products. Lipolytic reactions occur at the lipid– water interface. There are two broad types of lipases based on their positional specificity. Nonspecific lipase releases fatty acids from all three positions of the glycerol moiety and is used to hydrolyze fats and oils completely to free fatty acids and glycerol. These are produced by Candida sp., Staphylococcus sp., and Geotrichum sp. The other type of the enzyme is 1,3-specific lipase which releases fatty acids from 1,3 positions and preferentially free fatty acids and di- and monoglycerides as the reaction products. This type of lipase is produced by Aspergillus, Mucor, Rhizopus, and Pseudomonas sp. Conventional fat hydrolysis by high-temperature steam splitting (250–260  C at 50 bar) consumes large amounts of energy and creates environmental concerns. Lipases can be used to hydrolyze fats and oils with excellent yields, but the high cost of the enzymes, exacerbated by enzyme stability problems, currently makes the process uneconomical. Similarly, lipase-catalyzed transesterification of plant glycerides to make alkyl esters (plus glycerol byproduct) for use as biodiesel and replace base-catalyzed processes would be a huge market for enzyme biotechnology; the need to overcome severe cost restraints to become economically competitive is an ongoing research effort. In the meantime, higher value and lower bulk products such as edible and nonedible fats and oils with specialized, improved, or new properties are produced by the action of regio-specific lipases. For example, the 1,3-regio-specific lipase from Rhizomucor miehei is used on the industrial scale to replace palmitic acid moities of palm oil with stearic acid yielding stearic–palmitic–stearic triacylglycerol, which is a cocoa butter substitute. Similarly, enrichment of oils with highly unsaturated fatty acids can be made by lipase-mediated transesterification reactions to produce nutraceuticals. Lipases are also used to assist in the extraction of fats and oils and for the development of cheese flavor. Owing to their stability in organic solvents, lack of cofactor requirement, and range of substrate selectivities, lipases are the most utilized enzymes in mediating reactions in organic synthesis with hundreds of research papers on the subject. There is an increasing adaptation of the versatile microbial lipases in the largescale synthesis of fine chemicals, in various organic

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synthesis processes including the preparation of chiral compounds in enantiomerically pure form.

Enzymes in the Animal Feed Industry Animal feed is composed mainly of plant materials such as cereals, agricultural and grain milling byproducts, and agricultural waste residues. These contain nonstarch polysaccharides (such as -glucan, cellulose, hemicellulose, and lignin), protein, and phytic acid. Monogastric animals (those with one stomach) generally cannot fully digest and utilize the fiber-rich feedstuffs. Due to the complex nature of the feed materials, starch sequestered by -glucans and pentosans is also not digestible. -Glucan, arabinoxylan, and phytic acid, present in many feedstuffs, act as antinutritional factors as these materials interfere with the digestibility, absorption, and utilization of nutrients, adversely affecting animal production. -Glucan contains glucose residues linked via -1,3and -1,4-glycosidic linkages. The use of -glucanases in poultry feed allows the incorporation of large quantities of barley in the diet. Arabinoxylans (pentosans) are heteropolymers containing mainly xylose and arabinose. Arabinose side chains in arabinoxylans are often associated with ferulic acids which form linkages between the arabinoxylans and lignin molecules in the plant cell walls. The addition of arabinofuranosidase in wheatbased diets can facilitate the removal of antinutritional characteristics of these diets. Phytic acid (myo-inositol hexaphosphate) represents the major storage form of phosphates in plants. Up to 80% of the grain phosphorus is bound in the form of phytic acid. Phytic acid forms complexes with metal ions such as calcium, magnesium, iron, and zinc, thus preventing their assimilation by the animal. Addition of suitable microbial phytase (EC 3.1.3.16, phytate-6-phosphatase) liberates part of the bound phosphorus and makes it possible to reduce the amount of supplements (phosphorus, calcium, and other nutrients) added to the animal diet. Using phytase, which is now commercially available, in animal feed can alleviate environmental pollution from bound phosphorous in animal waste and development of dietary deficiencies in animals. The most common source of microbial phytase is Aspergillus ficuum. Protein utilization from vegetables can be enhanced by using microbial proteases. Thus, feed utilization and digestion by animals can be enhanced by adding enzymes to the feed. Also, doing so reduces overall waste by enhancing the efficiency of the digestion process. Various microbial enzymes are now used as feed enhancers and hold the prospect of serving larger roles in animal and poultry production.

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Enzymes in the Pulp and Paper Industry In recent years, tremendous research efforts have been made to reduce the amount of chlorine used for bleaching of kraft pulp after the pulping processes. Chlorine-based (chlorine, chlorine dioxide, and hypochlorite) bleaching can result in the discharge of chloroorganics such as chlorinated phenols and chlorinated dioxins into the environment. Environmental regulatory pressures have prompted the pulp and paper industry to adapt new technology to reduce or eliminate the presence of various contaminants in the bleaching plant effluents. The main constituents of wood are cellulose, hemicellulose, and lignin. Research in the use of enzymes in pulp manufacture involves the degradation or modification of hemicellulose and lignin without disturbing the cellulose fibers. Pretreatment of kraft pulp with xylanase promotes a decrease in lignin content (kappa number) and a brightness increase of the treated pulp. Xylanase facilitates lignin removal with high specificity from lignin–carbohydrate complexes. Such enzymatic pretreatment has reduced the amount of chlorine needed to reach a target brightness in pulp in chemical bleaching by 20–30%. The use of laccase (EC 1.10.3.2) to promote degradation of lignin and bleaching of pulp has attracted considerable interest as a costeffective replacement for chlorine bleaches. Research into improving recombinant expression levels and pH and thermal stabilities of laccase is ongoing. For high-quality pulps, extensive mechanical debarking is essential, but the process consumes substantial amounts of energy and wastes large amounts of cellulosic raw material. The border between wood and bark is the cambium which consists of a single layer of cells. This living cell layer produces xylem cells toward the inside of the stem and phloem cells toward the outside. Both cambium and phloem layers have high pectin content. Enzymes (mainly polygalacturonase) specific for the hydrolysis of the cambium and phloem layers facilitate bark removal. This new and attractive approach for enzymatic debarking shows great potential for saving both energy and raw material. Pitch is the sticky resinous material in wood. The removal of pitch by chemical pulping and bleaching is not particularly efficient. Treatment with lipases has been found to be useful in reducing pitch deposits since lipases hydrolyze the triglycerides in the wood resin to fatty acids and glycerol making the material less viscous. The enzyme does not affect the cellulose quality. Removal of ink is an important part of waste paper processing. Conventional deinking involves pulping of the paper in highly alkaline solution. It has been reported that cellulase enzymes can increase the efficiency of the deinking process. But there is concern that treatment of the secondary fibers by cellulases may decrease the fiber

strength. Currently, cellulases are used to partially hydrolyze pulp to decrease its water retention capacity, which lowers time and energy input of the paper drying process.

Enzymes in the Fruit Juice Processing Industry Enzymes can play important roles in preparing and processing various fruit and vegetable juices such as apple, orange, grapefruit, cranberry, pineapple, grape, carrot, and lemon. Fruits and vegetables are particularly rich in pectic substances. Pectin, a hydrocolloid, has a great affinity for water and can form gels under certain conditions. The addition of exogenous enzymes, such as pectinases, pectin lyase, pectin esterase, and polygalacturanase, reduces viscosity and improves pressability as the pectin gel collapses. For complete liquefaction of fruits and vegetables, hemicellulases, cellulases, and amylases can be used in addition to pectinases. Arabinan, a linear polymer of arabinofuranose units with 1,5 linkages, is an important component of fruit cell walls and may cause haze in fruit juice concentrate. Haze formation can be prevented by using endo-arabinanases (arabinan endo1,5- -L-arabinosidase; EC 3.2.1.99) to break down arabinan into soluble, low molecular weight degradation products (arabinofurano-oligosaccharides). Various commercial enzyme preparations are currently available for such use in the fruit juice industry. Enzymatic de-bittering of grapefruit juice can be achieved through the application of fungal naringinase preparations. The enzyme preparation contains both -rhamnosidase (EC 3.2.1.40) and -glucosidase activities. -Rhamnosidase first breaks down naringin, an extremely bitter flavanoid, to rhamnose and prunin and then -glucosidase hydrolyzes prunin to glucose and naringenin. Prunin bitterness is less than one-third of that of naringin. However, -rhamnosidase is competitively inhibited by rhamnose and -glucosidase is inhibited by glucose. Immobilized enzymes are used in flow-through reactors to solve the inhibition problems. Naringenin is reported to provide antioxidant and other beneficial effects to human health. Another enzyme, the flavoprotein glucose oxidase (EC 1.1.3.4), is used to scavenge oxygen in fruit juice and beverages to prevent color and taste changes upon storage. Glucose oxidase is produced by various fungi such as A. niger and Penicillium purpurogenum.

Enzymes in the Meat and Fish Processing Industry Proteinases, either indigenous (cathepsin) or those obtained from plants and microorganisms, are used in the meat and fish industries to tenderize meat and solubilize fish

Applied Microbiology: Industrial | Enzymes, Industrial (overview)

products. Plant proteinases include papain (EC 3.4.4.12) from papaya, ficin (EC 3.4.4.24) from figs, and bromelain (EC 3.4.4.24) from pineapples. Microbial enzymes include fungal (A. oryzae, A. niger) and bacterial (B. subtilis, B. lichiniformis) proteases. Tenderization of meat can be achieved by keeping the rapidly chilled meat at 1–2  C to allow proteolysis by indigenous enzymes (cathepsins and the Ca2þdependent neutral protease). Current practice in the United States favors immersion of meat cuts in concentrated enzyme solutions, followed by vacuum packaging and refrigeration for up to 3 weeks. Enzymes are also used to facilitate separation of hemoglobin from blood proteins and removal of meat from bones. For the preparation of pet food, minced meat or meat by-products are hydrolyzed by proteases to produce a liquid meat digest or a slurry with a much lower viscosity. Pets like the savory flavor generated by peptides and amino acids are produced from the enzymatic hydrolysis of meat. Fish protein concentrates are generally prepared by treating ground fish parts with a protease. After hydrolysis, the bones and scales are removed by screening, and the mixture of solubles and undigested fish solids is dried or separated by centrifugation. The yield of solubles, mainly amino acids and peptides, is generally 60–70% of the initial fish solids. Enzymatic treatment of fish stick water (press water obtained in fish meal manufacture) is performed with a microbial protease (from B. lichiniformis) before concentration by evaporation. This reduces its viscosity to 20–50% which helps to increase the final solid contents by 55–73% under industrial conditions. Thus, the use of enzymes substantially saves in drying costs. Enzymes (xanthin oxidase, catalase, nucleoside phosphorylase, and nucleotidase) can also be used for testing the quality and freshness of fish.

Enzymes in the Dairy Industry The application of enzymes (proteases, lipases, esterases, lactase, and catalase) in dairy technology is well established. Rennets (rennin, a mixture of chymosin and pepsin obtained mainly from animal and microbial sources) are used for coagulation of milk in the first stage of cheese production. Proteases of various kinds are used for acceleration of cheese ripening, for modification of functional properties and for modification of milk proteins to reduce the allergenic properties of cow milk products for infants. Lipases are used mainly in cheese ripening for development of lipolytic flavors. Lactase ( -galactosidase, EC 3.2.1.23) is used to hydrolyze lactose to glucose and galactose as a digestive aid and to improve the solubility and sweetness in various dairy products. Many people do not have sufficient lactase to digest milk sugar. Lactose hydrolysis helps these lactose-intolerant people to drink milk and eat various dairy products. -Galactosidases

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from K. fragilis, A. niger, or A. oryzae are inhibited by galactose. Immobilized enzyme systems are used to overcome this inhibition problem and to lower the cost of lactase use. The cheese manufacturing industry produces large quantities of whey as a byproduct of which lactose represents 70–75% of the whey solids. The hydrolysis of lactose by lactase converts whey into more useful food ingredients. Lactases have also been used in the processing of dairy wastes and as a digestive aid taken by humans in tablet form when consuming dairy products. Hydrogen peroxide is used as an effective chemical sterilant for the treatment of raw milk as an alternative to pasteurization with heat. Catalase (EC 1.11.1.6), which catalyzes the decomposition of hydrogen peroxide, is used at the end of the process to remove the remaining peroxide. Bitter off-flavors that develop in ripened cheese as it matures are due to the formation of bitter flavored peptides from milk proteins. Peptidases can break down the bitter peptides as they are formed and thus help maintain the traditional flavor of cheese.

Enzymes in Detergents Currently, the detergent industry occupies about 30% of the entire industrial enzyme market. Over half of the laundry detergents contain enzymes such as protease, amylase, lipase, and cellulase. In order to perform well in laundry detergent environments, these enzymes must be very efficient, work at alkaline pH conditions (pH 9–11) and high temperatures, be stable in the presence of chelating agents, perborates, and surfactants, and possess long storage stability (>1 year). The use of enzymes allows lower temperatures and shorter periods of agitation after a preliminary period of soaking. Proteases are the most widely used enzymes in the detergent industry. DNA technology has been used extensively to modify the protein catalysts, primarily for increasing stability properties. They remove protein-based stains such as blood, egg, grass, meat sauce, body secretions, and other proteinbased soils. These detergent enzymes (serine proteases) are produced by fermentation of B. lichiniformis, B. amyloliquefaciens, or Bacillus sp. Amylases remove residues of starchy foods such as mashed potato, spaghetti, and oatmeal. Lipases facilitate the removal of fatty stains such as lipsticks, frying fats, butter, salad oil, sauces, and tough stains on collars and cuffs. The lipase from Humicola lanuginosa, produced by recombinant DNA technology, is effective at pH 12 and 60  C. Recently, an alkali-stable fungal cellulase preparation has been introduced for use in washing cotton fabrics. Treatment with these cellulase enzymes removes the small fibers extending from the fabric (termed pilling), without apparently damaging the major fibers, and restores the fabric to its ‘as new’ condition by improving color brightness, enhancing softness feel, and removing particulate soiling. Cellulases are used in textile manufacture to partially

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remove dye (indigo) from denim, producing a stone-washed appearance. Bleach-stable enzymes (amylase, protease) are now available for use in automatic dishwashing detergents.

Enzymes in the Leather Industry Animal skin is composed of 60–65% water, 30–32% protein, approximately 10% fat, and 0.5–1% minerals. Skins are soaked initially to clean them and to allow rehydration. Proteolytic enzymes effectively facilitate the soaking process. In addition, lipases have also been used to dissolve and remove fat. Dehairing is then carried out using alkaline protease, such as subtilisin, in an alkaline bath. Alkaline conditions swell the hair roots, easing removal of the hair by allowing proteases to selectively attack the protein in the hair follicle. Conventional dehairing processes require harsh chemicals such as slaked lime and sodium sulfide which essentially swell the hide and loosen and damage the hair. Enzyme-based dehairing has led to much lower pollution emissions from tanneries. Tanning wastes, consisting primarily of the so-called glue stock, cannot be discarded at waste dump sites and must be completely processed. All proteins are solubilized at a high alkaline pH with bacterial proteases in order to partition the fat from the hydrolyzate. After concentration, the protein is dried and used as a nutritional supplement in animal feed.

Enzymes in the Production of Bulk and Fine Chemicals Enzymes or whole cells have been used extensively as biocatalysts in aqueous and organic solvents for production of a variety of fine chemicals and pharmaceuticals. One such chemical is indigo dye, which has a market size of $200 million. The manufacture of indigo dye used for dyeing of clothes, particularly denims, requires a harsh chemical process that generates carcinogenes and toxic wastes. A company, Amgen, has developed a biocatalytic process for the production of indigo. The pathway forming indigo involves converting tryptophan into indole by the enzyme tryptophanase, then hydroxylating indole to indoxyl by naphthalene dioxygenase, followed by a nonenzymic step to form indigo from indoxyl (spontaneous oxidation). Aspartame (L-aspartyl-L-phenylalanine methyl ester) is a dipeptide sweetener about 200 times sweeter than sucrose, and has a market of more than $1 billion annually. Aspartame is made by using an enzymatic process. The enzyme thermolysin, a zinc metaloprotease produced by Bacillus thermoproteolyticus (EC 3.4.24.4), catalyzes amide bond formation between L-aspartic acid and

L-phenylalanine methyl ester. It is enantioselective and forms the peptide bond only with L-phenylalanine methyl ester. The enzyme is also regioselective and does not form peptide bonds with the -carboxyl group of the aspartic acid, so no bitter tasting -aspartame is formed. The equilibrium reaction carried out by thermolysin has been optimized to shift the reaction toward dipeptide formation by selective precipitation and complexation of the product. The aspartame precursor, L-aspartic acid, can be prepared enzymatically from ammonium fumarate in a reaction catalyzed by L-aspartate ammonia-lyase (EC 4.3.1.1) obtained from Escherichia coli. -Lactam antibiotics, the penicillins and cephalosporins, are the most widely used antibiotics. 6-Aminopenicillanic acid (6-APA) can be produced enzymatically (pH 7.0, 35  C) from fermentation-derived penicillins (Penicillin G) by deacylation using immobilized penicillin amidase (EC 3.5.1.11), frequently called penicillin acylase, from E. coli or B. megaterium. 6-APA is an important intermediate compound for the manufacture of about 20 semisynthetic penicillins. The iron–cobalt enzyme, nitrile hydratase (EC 4.2.1.84), hydrolyzes nitriles to their corresponding carboxamides. The immobilized enzyme from Rhodococcus rhodochrous is used to convert acrylonitrile into acrylamide, an industrially important chemical feedstock, with high efficiency on the scale of tens of thousands of tons per year, replacing a much less efficient route using conventional chemistry. On the thousands of tons scale, the hydratase is used to produce nicotinamide (from 3-cyanopyridine) and 5cyanovaleramide (from adiponitrile). The latter reaction demonstrates the regioselectivity of the Pseudomonas chlororaphis hydratase in producing only a small percentage of adipamide in comparison to the larger percentage of desired product 5-cyanovaleramide, which is used as a feedstock to make a herbicide. Addition of two equivalents of water to nitriles generates carboxylic acids in reactions catalyzed by nitrilase (EC 3.5.5.1). Nitrilase from Acidovorax facilis is used on the industrial scale to produce the synthetic feedstock 4-cyanopentanoate from 2-methylglutaronitrile. Chiral amines can be isolated by oxidizing the unwanted enantiomer from a chemically synthesized racemic mixture by using stereoselective amine oxidases (EC 1.4.3.4). For primary amines, the resulting aldehyde is readily separated from the desired amine. For secondary amines, the resulting imine can be continuously reduced to regenerate the racemic amine so that after several cycles of oxidation and reduction, only the single enantiomer remains. Racemic amines can also be resolved by using lipases to catalyze acylation of one of the enantiomers. Racemic resolution of amino acids follows a strategy wherein chemically synthesized N-acyl-DL-amino acids are hydrolyzed by an L-aminoacylase (EC 3.5.1.14) to give L-amino acids and unhydrolyzed acyl-D-amino acids, allowing facile separation. The unhydrolyzed acyl-D-amino acids

Applied Microbiology: Industrial | Enzymes, Industrial (overview)

can be racemized by heating and presented again to the acylase for resolution. Recently, N-acylamino acid racemases and D-aminacylases have been discovered. So now, pure L- and D-amino acids can be afforded from a single incubation of N-acyl-DL-amino acid with the acylase and the racemase. In the amidase process, racemic amino acid amides are incubated with an amidopeptidase to generate the L-amino acid enantiomer from L-amino acid amide; the remaining D-amino acid amide is converted into the Damino acid by reacting it with benzaldehyde followed by hydrolysis. In the hydantoinase process, a racemic amino acid hydantoin is hydrolyzed by a D- or L-hydantoinase to the corresponding amino acid; the unreacted hydantoin can be racemized and resubmitted to the enzyme. In the transaminase process, an amino group is transferred from one amino acid to an accepting keto-acid to form a new amino acid by the aminotransferase and its cofactor (pyridoxal phosphate); this procedure is used for making L-phosphinothricin, a broad-spectrum herbicide and potent inhibitor of the enzyme glutamine synthetase. Transamination reactions can be driven to completion by relying on various strategies for removing the ketone produced from the amine donor. Industrial production of L-alanine from racemic aspartic acid is mediated by L-aspartate 4-decarboxylase (4.1.1.12) from Pseudomonas dacunhae. Reductive amination of keto acids is another potential route to amino acids. L-tert-Leucine is an intermediate used in the synthesis of several pharmaceuticals. It has been produced on the ton scale in reactions mediated by leucine dehydrogenase (EC 1.4.1.9). In this process, electron donor NADH is regenerated from NADþ by using formate dehydrogenase (EC 1.2.1.2) acting on formate. Departure from petroleum-based feedstocks to feedstocks from renewable resources has made considerable progress in recent years. One of the best examples is the use of metabolic engineering to enable E. coli to make 1,3-propanediol from glucose. Requisite genes from organisms, capable of fermenting glycerol to 1,3-propanediol, were introduced into an E. coli host strain (glycerol dehydratase and its reactivating factors) along with additional metabolic changes in the host organism for optimizing production has resulted in a strain capable of converting over 50% of the glucose feedstock mass into 1,3-propanediol. A production plant has been constructed for making the diol, which can be used for making a variety of industrial products including new-generation polyesters. In a related example of metabolic engineering, the genes for the two enzymes (tyrosine ammonia lyase and 4-hydroxycinnamic acid decarboxylase) needed to make 4-hydroxystyrene from tyrosine were introduced into a strain of E. coli, rendered insensitive to feedback inhibition from phenylalanine and tyrosine. The strain was demonstrated to produce 4-hydroxystyrene, but the yield was limited due to toxicity of 4-hydroxystyrene to the host. A biocatalytic alternative to the currently employed petroleum-based, industrial

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synthesis of catechol has been developed using glucose as substrate. Fermentation processes for lactic and glycolic acids are being improved by metabolic engineering to compete with the petroleum-based synthetic routes. The -hydroxy acids have growing markets for use in cosmetics and as precursors to biodegradable polymers.

Analytical Applications of Enzymes Enzymes are used in various analytical methods, both for medical and nonmedical purposes. Immobilized enzymes, for example, are used as biosensors for the analysis of organic and inorganic compounds in biological fluids. Biosensors have three major components: a biological component (e.g., enzyme, whole cell), an interface (e.g., polymeric thick or thin film), and a transducing element which converts the biochemical interaction into a quantifiable electrical or optical signal. A glucose biosensor consists of a glucose oxidase membrane and an oxygen electrode, while a biosensor for lactate consists of immobilized lactate oxidase and an oxygen electrode. The lactate sensor functions by monitoring the decrease in dissolved oxygen which results from the oxidation of lactate in the presence of lactate oxidase. The amperometric determination of pyruvate can be carried out with the pyruvate oxidase sensor which consists of a pyruvate oxidase membrane and an oxygen electrode. For the determination of ethanol, the biochemical reaction cell with an alcohol dehydrogenase (EC 1.1.1.1) membrane anode is used. A bioelectrochemical system for total cholesterol estimation was developed, based on a double-enzymatic method. In this system, an immobilized enzyme reactor containing cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6) is coupled with an amperometric detector system. An amino acid electrode for the determination of total amino acids has also been developed using the enzymes L-glutamate oxidase, L-lysine oxidase, and tyrosinase. Enzyme electrodes are used for continuous control of fermentation processes.

Enzyme-Replacement Therapy Several inborn errors of metabolism (or inherited metabolic diseases) in humans have been managed by enzyme-replacement therapy in which the defective or deficient enzyme is supplied intravenously. Thus, for example, Gaucher disease is treated with the human glucosylceramidase (EC 3.2.1.45), and Fabry disease with the human -galactosidase (EC 3.2.1.22). The enzymes are produced as glycoproteins by recombinant DNA technology using mammalian cell cultures (Chinese hamster ovary cells). Patients are injected recurrently (about every 2 weeks) for maintenance. PEGylation (conjugation with polyethylene glycol) of

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bovine adenosine deaminase (EC 3.5.4.4) for replacement therapy of severe combined immunodeficiency disease (‘Bubble Boy Disease’) is the first example where adverse immune responses and short lifetimes of the native enzyme were averted by chemical modification of protein. PEGylation of proteins for therapeutic use is now well established with numerous examples in pharmaceuticals. Disclaimer: The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. See also: Amylases; Biosensors; Biotransformations; Cellulases; Dairy Products; Ethanol; Lignin, Lignocellulose, Ligninase; Lipases; Proteases, Production

Further Reading Bairoch A (2000) The ENZYME database in 2000. Nucleic Acids Research 28: 304–305. Bommarius AS and Riebel BR (2004) Biocatalysis. Weinheim: Wiley-VCH.

Cabral JMS, Best D, Boross L, and Tramper J (eds.) (1994) Applied Biocatalysis. Switzerland: Harwood Academic. Crittenden RG and Playne MJ (1996) Production, properties, and applications of food-grade oligosaccharides. Trends in Food Science and Technology 7: 353–361. Dordick JS (ed.) (1991) Biocatalysts for Industry. New York: Plenum Press. Godfrey T and West S (eds.) (1996) Industrial Enzymology, 2nd edn. London: Macmillan Press. Laskin AI (ed.) (1985) Enzymes and Immobilized Cells in Biotechnology. Menlo Park, CA: Benjamin/Cumming. NC-IUBMB and Webb EC (eds.) (1992) Enzyme Nomenclature. San Diego: Academic Press. Olsen HS (2004) Enzymes at Work. Bagsvaerd, Denmark: Novo Nordisk. Patel RN (2000) Stereoselective Biocatalysis. Boca Raton, FL: CRC Press. Saha BC and Demirjian DC (eds.) (2000) Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals. Washington, DC: American Chemical Society. Tucker GA and Woods LFJ (eds.) (1995) Enzymes in Food Processing, 2nd edn. Bishopbriggs, Glasgow, UK: Blackie Academic. Uhlig H (1998) Industrial Enzymes and Their Applications. New York: John Wiley & Sons. Whitesides GM and Wong CH (1994) Enzymes in Synthetic Organic Chemistry. Amsterdam: Elsevier.