Production Of Protease By Submerged Fermentation Using Rhizopus Microsporus Var Oligospous

摘要

Production of protease via submerged fermentation using Rhizopus microsporus var oligospous was studied using shake flask cultures. After 96 h of growth on a BOD shaker revolving at 150 rpm, protease was partially purified using the isopropanol. Factors affecting the enzyme production viz incubation temperature, initial pH of the shake flask medium. and inoculum size were optimized. Protease having the maximum enzyme activity of 521.739 IU was obtained at an incubation temperature of 30oC, an initial pH of the basal medium as 5.5 and an inoculum size of to 1x105 spores ml-1 of Tween-80. Protease deactivated at 80 oC. Introduction Enzymes are proteins composed of 20 amino acids. They are produced by cellular anabolism, the naturally occurring biological process of making more complex molecules from simpler ones. Enzymes increase the rate of biochemical reaction and decrease the time for those reactions to reach equilibrium. They are not consumed in the chemical reaction, and thus their action is catalytic (Nielson, 1991). Enzymes used in food processing are produced by bacteria, fungi, higher plants and animals. Most of the organisms that produce enzymes are fungi. Mycolytic enzymes are primarily originated as a desire of microbiologists to understand the chemical nature and ultra structure of fungal cell wall. A number of enzymes can be classified under mycolytic enzymes viz; protease, cellulase, xylanase etc. Protease enzyme conducts proteolysis by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain. Proteases are the most valuable commercial enzymes and account for 60% of the total enzyme market (Rao et al, 1998). They find increased application in food, pharmaceutical, detergent, leather, tanning industry and to some extent in silver recovery and peptide synthesis (Godfrey and West, 1996; Kumar and Takagi, 1999; Oberoi et al, 2001). Though plants and animals also produce extra cellular proteases, microorganisms are the preferred source because of their rapid growth, limited space required for their cultivation, longer shelf life and the ease with which they can be genetically manipulated to generate improved enzymes (Rao et al, 1998). Extra cellular proteases help in hydrolysis of protein in the cell free environment and their cellular uptake (Kalitz, 1988). Materials and methods Microorganism and inoculun preparation Rhizopus microsporus var oligospous was procured from the institute of Microbial Technology, Chandigarh and was maintained at 4°C on potato dextrose agar (PDA). Spore suspension was made from five days old cultures that had been grown on PDA slants at 30°C. It was prepared by suspending the spores from one tube in 10 ml of sterilized distilled water containing 0.1ml Tween–80. The spore count was adjusted to105 spores ml-1 (Smith et al, 1996). One ml of inoculum was used per flask to carry out submerged fermentation. Shake flask cultivation media The enzymes were produced using basal medium (Manomani et al, 1983). The basal medium contained the following ingredients (g l -1): (NH4)SO4 - 1.4, CO(NH2)2 - 0.3, KH2PO4 - 4.0, K2HPO4 – 0.84, CaCl2.2H2O – 0.3, MgSO4.7H2O – 0.3, FeSO4.7H2O – 0.005, MnSO4.H2O – 0.00156, ZnCl2 – 0.00167, Peptone 0.25, Yeast extract – 0.10, Rice Bran – 4%. All the components of the basal medium except rice bran were dissolved in distilled water. Flasks having 250 ml capacity were taken and 50 ml of this solution was poured in each flask. To each flask 2.0 g of rice bran was added (Thenawadjaja et al, 1990) and autoclaved at 1.1kg/cm2 for 20 minutes. Flasks were cooled and inoculated with 1ml of the spore suspension. The inoculated flasks were incubated at 30°C on a rotary shaker cum BOD incubator revolving at 150 rpm (Seyis and Aksoz, 2005; Meenaksui et al, 2005). After 96 hours of growth, the contents of each flask were centrifuged at 4°C and this supernatant was used as the source of crude enzyme (Nehra et al, 2002). Partial purification of fungal pro