Present project focuses on the study of protease from the indigenously isolated strain Bacillus. Effects of different substrate levels, ionic concentrations (MgCl2, CaCl2, K2HPO4, (NH4)2SO4, Urea), cane molasses and yeast sludge on the production of proteases by Bacillus subtilis. Various conditions like incubation period, mode of fermentation, pH, temperature etc. were optimized. After optimization of conditions, the proteases were produced on large scale. The proteases thus produced were subjected to the purification through various steps like ammonium sulphate precipitation, ion exchange chromatography and gel filtration. Kinetic study of proteases was also carried out in order to calculate Km and Vmax of the enzymes. Thermal inactivation was studied in order to calculate the Kd, energy of activation (Ea), Gibbs free energy (∆G), Enthalpy (∆H) and entropy (∆S). Activation or inhibition effect of different metal ions and chelating agents like EDTA, EGTA and PMSF on the protease was examined. Maximum activity of the crude proteases ( 502 U) was obtained at pH 7.5, temperature 45 °C after 48 hours of continuous shaking at 220 rpm using Bacillus subtilis cultured on skim milk (2%) in the optimized medium containing MgCl2 ( 0.01%), (NH4)2SO4 (0.3%) , CaCl2 ( 0.03%), K2HPO4 ( 0.5%), yeast sludge (300 μL) and cane molasses (0.03%). Specific activity of the purified protease was (80 U/mg), yield is 3.18 % and 3 folds increase in activity. Kinetic investigation revealed that that Km and Vmax were 0.2mg/mL and 60 U/mg. Energy of activation Ea , Free energy of activation ∆G , Activaton Enthalpy ∆H, Activation entropy (∆S) were -403 KJ/mol, 91.4 KJ/mol, -403.37 KJ/mol and -407.80 J/mol. Thermo stabilization of proteases is mostly accompanied by a decrease in ∆H and ∆S. A metal ions effect on the activity of the proteases was also studied. PMSF inhibited the enzyme activity, but EDTA and EGTA would not had much pronounced effect on the activity of enzyme. That proved the class of proteases to be the serine due to the inhibition of PMSF. It was concluded that Bacillus subtilis had significant potential for the production of proteases. With respect to the kinetic study of proteases, small vales of Km than the substrate concentration showed large affinity of the enzyme with the substrate and found good source of for the production on proteases on large scale. The nature of the reaction mechanism was found to be first order because [S] was much less than Km. Activation enthalpy had negative values indicates that the enzyme was of endothermic nature. Economically cheaper metals enhanced the enzyme activity. So the Bacillus subtilis was found good catalytic agent for the production of proteases.
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