|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, and (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. Procedure for development of antibiotic producing strains is given in Selection of culture of penicillium.
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. For a specific microbe pH, temperature and formulation of culture medium is optimized prior to inoculation. Production of enzymes increases with the concentration of culture medium. Aunstrup et al, (1979) have given the following 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.
Central Food Technological Research Institute (CFTRI), Mysore, has developed technology for conversion of tapioca starch to glucose by using fungal enzymes. Enzymes have also been isolated from bacterial cultures which convert glucose to fructose in starch hydrolysate. At this institute, about 15 tonnes of high fructose syrup (dry weight basis) could be obtained from tapioca starch from one hectare land as against only 5 tonnes of the same from sugarcane.
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 (see Fermentation process
Traditional method of enzyme production has been the surface culture technique where inoculum remains on upper surface of broth. Now-a-days 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.). Use of continuous culture technique for cellulase production by Trichoderma
has been suggested by Mitra and Wilke (1975).
Growth conditions e.g.
pH, temperature and oxygen are maintained in fermenter at optimum level. These factors differ microbe to microbe and even in the same species of a microbe. 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 5°C 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. For detailed description readers are advised to study elsewhere (see
Wisemen, 1975; Trevan, 1987). The main steps of purification are: (i)
preparation of concentrated solution by vacuum evaporation at low temperature or by ultrafiltration, (ii)
clarification of concentrated enzyme by a polishing filtration to remove other microbe, (iii)
addition of preservatives or stabilizers, for example, calcium salts, proteins, starch, sugar, alcohols, sodium chloride (18-20 per cent), sodium benzoate, etc. (iv)
precipitation of enzymes with acetone, alcohols or organic salts, e.g.
ammonium sulfate or sodium sulfate, (v)
drying the precipitate by free drying, vacuum drying or spray drying, and (vi)
packaging for commercial supply (Aunstrup et al,