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3.5. Medical and Industrial Utilization of Enzymes

Enzymes have been significant industrial products for more than a hundred years. However, the range of potential applications is increasing rapidly. With the advent of recombinant DNA technology, it has become possible to make formerly rare enzymes in large quantities and hence to reduce cost. Also, in pharmaceutical manufacturing, the desire to make chirally pure compounds is leading to new opportunities. Chirality is important in a product; in a racemic mixture, one enantiomer is often therapeutically useful while the other may cause side effects and add no therapeutic value. The ability of enzymes to recognize chiral isomers and react with only one of them can be a key component in pharmaceutical synthesis. Processes that depend on a mixture of chemical and enzymatic synthesis are being developed for a new generation of pharmaceuticals.

Technological advances have facilitated the use of enzymes over an increasingly broad range of process conditions. Enzymes from organisms that grow in unusual environments (e.g., deep ocean, salt lakes, and hot springs) are increasingly available for study and potential use. New enzymes and better control of reaction conditions allow the use of enzymes in the presence of high concentrations of organics, in high-salt aqueous environments, or at extreme temperatures, pH, or pressures. As we couple new insights into the relationship of enzyme structure to biological function with recombinant DNA technology, we are able to produce enzymes that are human designed or manipulated (see Chapter 14, “Utilizing Genetically Engineered Organisms,” Section 14.9, on protein engineering). We no longer need to depend solely on natural sources for enzymes.

Another emerging technology is the use of cell-free systems. This approach uses cell lysates; such crude mixtures have been used historically for industrial purposes for nonenergy-requiring single-step reactions and are relatively inexpensive. Recent advances allow the use of cell-free production for complex, multistep synthesis of high-value products. Such approaches potentially allow intensification of bioprocesses for complex reaction networks and show potential for industrial applications for products that would normally be intracellular. Such systems have been scaled up to over 100 l.

In 2014, U.S. sales of industrial enzymes were about $4.5 billion, and sales are projected to grow to $6.5 billion by 2020. The products made by enzymatic processes are worth many more billions of dollars. Food and beverage account for about one-third of the market. The detergent market is next in size with animal feed, biofuels, paper and pulp, and textiles being significant. Table 3.5 lists some industrially important enzymes.

Table 3.5. Some Industrially Important Enzymes

Name

Example of Source

Application

Amylase

Bacillus subtilis, Aspergillus niger

Starch hydrolysis, glucose production

Glucoamylase

A. niger, Rhizopus niveus, Endomycopsis

Saccharification of starch, glucose production

Trypsin

Animal pancreas

Meat tenderizer, beer haze removal

Papain

Papaya

Digestive aid, meat tenderizer, medical applications

Pepsin

Animal stomach

Digestive aid, meat tenderizer

Rennet

Calf stomach/recombinant E. coli

Cheese manufacturing

Glucose isomerase

Flavobacterium arborescens, B. coagulans, Lactobacillus brevis

Isomerization of glucose to fructose

Penicillinase

B. subtilis

Degradation of penicillin

Glucose oxidase

A. niger

Glucose → gluconic acid, dried-egg manufacture

Lignases

Fungal

Biopulping of wood for paper manufacture

Lipases

Rhizopus, pancreas

Hydrolysis of lipids, flavoring and digestive aid

Invertase

S. cerevisiae

Hydrolysis of sucrose for further fermentation

Pectinase

A. oryzae, A. niger, A. flavus

Clarification of fruit juices, hydrolysis of pectin

Cellulase

Trichoderma viride

Cellulose hydrolysis

Proteases hydrolyze proteins into smaller peptide units and constitute a large and industrially important group of enzymes. Proteases constitute about 60% of the total enzyme market. Industrial proteases are obtained from bacteria (Bacillus), molds (Aspergillus, Rhizopus, and Mucor), animal pancreas, and plants. Most of the industrial proteases are endoproteases. Proteases are used in food processing, such as cheese making (rennet), baking, meat tenderization (papain, trypsin), and brewing (trypsin, pepsin); in detergents for the hydrolysis of protein stains (subtilisin); and in tanning and the medical treatment of wounds.

Pectinases are produced mainly by A. niger. The major components in pectinases are pectin esterase, polygalacturonase, and polymethylgalacturonatelyase. Pectinases are used in fruit juice processing and wine making to increase juice yield, reduce viscosity, and clear the juice.

Lipases hydrolyze lipids into fatty acids and glycerol and are produced from animal pancreas, some molds, and yeasts. Lipases may be used to hydrolyze oils for soap manufacture and to hydrolyze the lipid-fat compounds present in wastewater streams. Interesterification of oils and fats may be catalyzed by lipases. Lipases may also be used in the cheese and butter industry to impart flavor as a result of the hydrolysis of fats. Lipase-containing detergents are an important application of lipases.

Amylases are used for the hydrolysis of starch and are produced by many different organisms, including A. niger and B. subtilis. Three major types of amylases are α-amylase, β-amylase, and glucoamylase. α-amylase breaks α-1,4 glycosidic bonds randomly on the amylose chain and solubilizes amylose. For this reason, α-amylase is known as the starch-liquefying enzyme. β-amylase hydrolyzes α-1,4 glycosidic bonds on the nonreducing ends of amylose and produces maltose residues. β-amylase is known as a saccharifying enzyme. α-1,6 glycosidic linkages in the amylopectin fraction of starch are hydrolyzed by glucoamylase, which is also known as a saccharifying enzyme. In the United States, on the average, nearly 1.3 × 109 lb/yr of glucose is produced by the enzymatic hydrolysis of starch. The enzyme pullulanase also hydrolyzes α-1,6 glycosidic linkages in starch selectively.

Cellulases are used in the hydrolysis of cellulose and are produced by some Trichoderma species, such as Trichoderma viride or T. reesei; by some molds, such as A. niger and Thermomonospora; and by some Clostridium species. Cellulase is an enzyme complex, and its formation is induced by cellulose. Trichoderma cellulase hydrolyzes crystalline cellulose, but Aspergillus cellulase does not. Cellulose is first hydrolyzed to cellobiose by cellulase, and cellobiose is further hydrolyzed to glucose by β-glucosidase. Both of these enzymes are inhibited by their end products, cellobiose and glucose. Cellulases are used in cereal processing, alcohol fermentation from biomass, brewing, and waste treatment.

Hemicellulases hydrolyze hemicellulose to five-carbon sugar units and are produced by some molds, such as white rot fungi and A. niger. Hemicellulases are used in combination with other enzymes in baking doughs, brewing mashes, alcohol fermentation from biomass, and waste treatment.

Lactases are used to hydrolyze lactose in whey to glucose and galactose and are produced by yeast and some Aspergillus species. Lactases are used in the fermentation of cheese whey to ethanol.

Other microbial β-1,4 glucanases produced by B. amyloliquefaciens, A. niger, and Penicillium emersonii, are used in brewing mashes containing barley or malt. These enzymes improve wort filtration and extract yield.

Penicillin acylase is used by the antibiotic industry to convert penicillin G to 6-aminopenicillanic acid (6-APA), which is a precursor for semisynthetic penicillin derivatives.

Among other important industrial application of enzymes is the conversion of fumarate to L-aspartate by aspartase. In industry, this conversion is realized in a packed column of immobilized dead E. coli cells with active aspartase enzyme. Fumarate solution is passed through the column, and aspartate is obtained in the effluent stream. Aspartate is further coupled with L-phenylalanine to produce aspartame, which is a low-calorie sweetener known as NutraSweet.

The conversion of glucose to fructose by immobilized glucose isomerase is an important industrial process. Fructose is nearly 1.7 times sweeter than glucose and is used as a sweetener in soft drinks. Glucose isomerase is an intracellular enzyme and is produced by different organisms, such as Flavobacterium arborescens, B. licheniformis, and some Streptomyces and Arthrobacter species. Immobilized inactive whole cells with glucose isomerase activity are used in a packed column for fructose formation from glucose. Cobalt (Co2+) and magnesium (Mg2+) ions (4 × 10–4 M) enhance enzyme activity. Different immobilization methods are used by different companies. One uses flocculated whole cells of F. arborescens treated with glutaraldehyde in the form of dry spherical particles. Entrapment of whole cells in gelatin treated with glutaraldehyde, the use of glutaraldehyde-treated lysed cells in the form of dry particles, and immobilization of the enzyme on inorganic support particles such as silica and alumina are methods used by other companies.

DL-Acylamino acids are converted to a mixture of L- and D-amino acids by immobilized aminoacylase. L-Amino acids are separated from D-acylamino acid, which is recycled back to the column. L-Amino acids have important applications in food technology and medicine.

Enzymes are commonly used in medicine for diagnosis, therapy, and treatment purposes. Trypsin can be used as an anti-inflammatory agent; lysozyme, which hydrolyzes the cell wall of gram-positive bacteria, is used as an antibacterial agent; streptokinase is used as an anti-inflammatory agent; urokinase is used in dissolving and preventing blood clots. Asparaginase, which catalyzes the conversion of L-asparagine to L-aspartate, is used as an anticancer agent. Cancer cells require L-asparagine and are inhibited by asparaginase. Asparaginase is produced by E. coli. Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide, which can easily be detected. Glucose oxidase is used for the determination of glucose levels in blood and urine. Penicillinases hydrolyze penicillin and are used to treat allergic reactions against penicillin. Tissue plasminogen activator and streptokinase are used in the dissolution of blood clots (particularly following a heart attack or stroke).

The development of biosensors using enzymes as integral components is proceeding rapidly. Two examples of immobilized enzyme electrodes are those used in the determination of glucose and urea by using glucose oxidase and urease immobilized on the electrode membrane. Scarce enzymes (e.g., tissue plasminogen activator) are finding increasing uses, as the techniques of genetic engineering now make it possible to produce usable quantities of such enzymes.

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