STUDIES ON PRODUCTION AND APPLICATIONS OF ALKALINE PROTEASE FROM BACILLUS PSEUDOFIRMUS
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2010
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Abstract
Microbial alkaline proteases are one of the most important hydrolytic enzymes and hence are being studied extensively since last two decades. These enzymes constitute 60% to 65% of the total enzymes sales in the world. These enzymes have versatile applications in many industries viz., tannery, dairy, food, pharmaceutical, silk, detergent, waste management and effluent treatment. Microbial alkaline proteases, especially from Bacillus sp. are most widely exploited industrially. In general, the majority of the commercially available enzymes are not stable in the presence of oxidizing agents, organic solvents and wide range of pH as well as temperature. An efficient alkaline protease producer was isolated from a tannery waste soil, which grew in a wide range of pH. A new screening media was designed containing gelatin as a sole source of carbon and nitrogen. The culture broth (supernatant) was found to hydrolyze natural proteins viz., clotted blood, egg white and poultry feather. Very efficient dehairing was achieved using the culture broth (supernatant). The isolate was identified to be Bacillus pseudofirmus and designated as B. pseudofirmus SVB1 on the basis of 98% similarity in 16s rRNA sequence. Physical parameters viz., initial pH of the medium, temperature and rpm of the shaking incubator were optimized using central composite experimental design to enhance the production of alkaline protease and cell growth simultaneously. The individual optimum levels of parameters were found to be 9.2, 27DC and 195 for initial pH of the medium, temperature and rpm of the shaking incubator, respectively for the 210, respectively for cell growth. From multi-response analysis, the optimal levels were found to be 9.6, 28DC and 191 rpm for initial pH of the medium, temperature and rpm of the shaking incubator, respectively for both production and cell growth. The values of cell growth (A600), alkaline protease activity and specific activity obtained were 0.44, 40.03 U/ml, 6.37 U/mg, respectively, under these optimal conditions. Before optimization the values were 0.30, 30.56 U/ml, 4.7 U/mg, for cell growth (A600), alkaline protease activity and specific activity, respectively. The production of alkaline protease and cell growth were enhanced by 36% and 44%, respectively under optimal levels of the physical parameters. The results obtained from optimization of individual responses and multi-response were compared. Medium was developed to maximize the production of alkaline protease from B. pseudofirmus SVB1 in three steps. In the first step, various carbon/nitrogen source and salts were selected using one variable at a time (OVAT) method. Casein was found to be the most efficient source for both carbon and nitrogen for alkaline protease production along with MgSO4, CaCl2, NH4NO3, FeCl3, K2HPO4, KH2PO4 and NaCl. In the second step, Plakett-Burman experimental design was applied to screen the significantly influencing medium components, and casein, MgSO4, NH4NO3 and CaCl2 were selected for optimization of their levels. These medium components were optimized using central composite experimental design in the third step to improve the production. The optimal levels of casein, MgSO4, CaCl2 and NH4NO3 were found to be 7.636 g/l, 0.811 g/l, 0.086 g/l and 0.576 g/l, respectively. An overall 4.9 fold increase in alkaline protease production (specific activity) was achieved in th..........
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Supervisor: V. Venkata Dasu & B. Mandal
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ENVIRONMENT