Enzymes are biocatalysts produced by living cells to make specific biochemical reactions of the metabolic processes of the cells.

Enzymes are highly specific in their action on substrates. All enzymes which have been purified are protein in nature, and may or may not possess a nonprotein prosthetic group. Boidin and Effront (1917) in France pioneered in the production of bacterial enzymes. Microbial cultivated enzymes have replaced the animal or plant enzymes. Microbial enzymes are enzymes derived from micro-organisms and are produced through fermentation of these organisms. Microbial enzymes include the fungal and bacterial amylases, diastases, and others. For many uses, microbial enzyme has replaced plant or animal enzyme. Microbial enzyme has well known application as biocatalyst in several areas of industry such as biotechnology, agriculture and pharmaceutical.

Variety of Enzymes
Yeast Enzyme Invertase (Sucrase) Saccharomyces cerevisiae o Digest sucrose into fructose and glucose. o Used to create candies with a soft center and chocolate covered cherries. Lactase Saccharomyces fragilis o Used to degrade lactose into glucose and galactose. o Given to lactose intolerant. o Used in the production of ice cream and frozen desserts.

Bacteria Enzyme Amylase Bacillus subtilis o Found in a variety of organisms, including the saliva of humans. o Sugar digesting enzyme. o Used to create various syrups.

Fungal Enzyme Pectinase Aspergillus niger o Commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice, including apple and sapota.

Methods of Enzyme Production

Production of enzymes takes place in the following steps :

Isolation of Microorganisms, Strain Development and Preparation of Inoculum Microorganisms are isolated on culture media following the microbiological techniques. Aim for isolating a suitable microorganism lies in: (a) production of enzyme in high amount and other metabolites in low amount (b) completion of fermentation process in short time (c) utilization by the microorganisms of low cost culture medium Once a suitable microorganism is obtained its enzyme producing ability is optimized by improving strains and formulating culture medium (pH and temperature). Strains of microorganisms are developed by using mutagens i.e. mutagenic chemicals and ultraviolet light. Inoculum of enzyme producing strains developed after treatment of mutagens is prepared by multiplying its spores and mycelia on liquid broth. Medium Formulation and Preparation Culture medium is formulated in such a way that should provide all nutrients supporting for enzyme production in high amount but not for good microbial growth. For this purpose, an ideal medium must have a cheap source of carbon, nitrogen, amino acids, growth promoters, trace elements and little amount of salts. Care must be taken to maintain pH during fermentation. The typical constituents of media for enzyme fermentation: Carbohydrates: Molasses, barley, corn, wheat and starch hydrolysate Proteins: Meals of soybean, cotton seed, peanut and whey, corn steep liquor and yeast hydrolysate. Sterilization and Inoculation of Medium, Maintenance of Culture and Fluid Filteration Medium is sterilized batch-wise in a large size fermenter. For this purpose, continuous sterilization method is now becoming popular. After medium is sterilized, inoculation with sufficient amount of inoculum is done to start fermentation process. Fermentation process is the same as described for antibiotic production. Traditional method of enzyme production has been the surface culture technique where inoculum remains on upper surface of broth. Nowadays submerged culture method is most widely practiced because of less chances for infection and possibility for more yield of enzymes.

The former technique is still in use for production of some of the fungal enzymes, for example, amylase (from Aspergillus sp.), protease (from Mucor sp. and Aspergillus sp.) and pectinase (from Penicillium sp. and Aspergillus sp.). Growth conditions e.g. pH, temperature and oxygen are maintained in fermenter at optimum level. A little amount of oil is added to fermenter to control foaming as it happens during fermentation. After 30-150 h incubation, extracellular enzymes are produced by the inoculated microbe in culture medium. Most of enzymes are produced when exponential phase of growth completes but in a few cases, they are produced during exponential phase. Besides extracellular enzymes, other metabolites (10-15 per cent) are also produced in the fermented broth. These metabolites are removed after enzyme purification. When fermentation is over broth is kept at 5C to avoid contamination. Recovery of enzymes from the fermented broth (fluid) of bacteria is more difficult than from that of filamentous fungi. Fungal broth is directly filtered or centrifuged after pH adjustment. Therefore, the bacterial broth is treated with calcium salts to precipitate calcium phosphate which help in separation of bacterial cells and colloids. Then the liquid is filtered and centrifuged to remove cell debris. Purification of Enzymes Enzyme purification is a complex process. The main steps of purification are: (i) (ii) (iii) preparation of concentrated solution by vacuum evaporation at low temperature or by ultrafiltration clarification of concentrated enzyme by a polishing filtration to remove other microbe addition of preservatives or stabilizers, for example, calcium salts, proteins, starch, sugar, alcohols, sodium chloride (18-20 per cent), sodium benzoate, etc precipitation of enzymes with acetone, alcohols or organic salts, e.g. ammonium sulfate or sodium sulfate drying the precipitate by free drying, vacuum drying or spray drying packaging for commercial supply (Aunstrup et al,1979).

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Application of Microbial Enzyme

Carbohydrase Carbohydrases are enzymes which hydrolyze polysaccharides or oligosaccharides. Several carbohydrases have industrial importance, but the amylases have the greatest commercial application. Bacterial amylase preparations generally remain operative at considerably higher temperature than fungal amylases, and at elevated temperatures, there is rapid liquefaction of starch. Applications: Conversion of partially acid hydrolyzed starch to sweet syrups. Supplementing the diastatic activity of flour in baking. Processing cereal products for food dextrin and sugar mixtures and for breakfast foods. for preparation of chocolate and licorice syrups to keep them from congealing for recovering sugars from scrap candy of high starch content. Microbial amylases are used for modifying starch in vegetable purees in treating vegetables for canning

Protease Industrially available proteolytic enzymes produced by microorganisms are usually mixtures of endopeptidases (proteinases) and exopeptidases. Most fungal proteases will tolerate and act effectively over a wide pH range (about 4 to 8), while with a few exceptions, bacterial proteases generally work best over a narrow range of about pH 7 to 8. Applications: production of soy sauce, tamari sauce, and miso. In baking bread and crackers, it reduces mixing time and increases extensibility of doughs, and improves grain, texture, and loaf volume. To prevent development of undesirable haze in beer and ale when these beverages are cooled. tenderizing meats, and animal casings for processed mieats. Pharmaceutical and clinical applications for fungal proteases include their use in digestive aids, and for bacterial proteases (streptokinase-streptodornase) in debridement of wounds and by injection to relieve inflammation, bruises, and blood clots.

Pectinase The pectolytic enzymes are another important group of enzymes of microbial origin. The two well recognized types of pectolytic enzymes are pectinesterase and polygalacturonase. Applications: processing fruit juices, Addition of pectic enzymes to grapes or other fruits during crushing or grinding results in increased yields of juice on pressing. For pectin removal, clearing the white haze in the fruit juice. making high density fruit juice concentrates or purees. removing the gelatinous coating from coffee beans.

Other Hydrolytic Enzyme Other useful hydrolytic enzymes include cellulase, hemicellulase, and pentosanase. Partly due to lack of commercial availability of high-potency products, particularly for cellulase, they are not yet of major industrial importance. Aapplications:

Cellulase used in tenderizing cellulosic food products and recovering cellulosic wastes. Hemicellulases used in preventing gelation in coffee concentrates, where pectic enzymes are not effective and partially degrading various natural gums to reduce the viscosity of solutions of these gums.

Nonhydrolytic Enzyme Only two nonhydrolytic enzymes at present have large-scale industrial applications, glucose oxidase and catalase. Glucose oxidase is of fungal origin, and acts in the presence of oxygen to convert glucose to gluconic acid and hydrogen peroxide. Catalase, which is also present in commercial fungal glucose oxidase preparations, acts on hydrogen peroxide to yield water and oxygen. Applications: For paper test strips for diabetic patients, which indicate the presence of glucose in the urine by a color change when the strip is dipped into the sample. Removing residual oxygen in canned foods. Hydrogen peroxide is added to the milk to sterilize it, and catalase is used to remove the residual hydrogen peroxide before further processing the milk into cheese.