Purification and characterization of alkaline, thermostable and organic solvent stable protease from a mutant of Bacillus sp.

An alkaline protease produced from wild Bacillus sp. with potential in many industrial applications and its hyper-protease producer mutant obtained through random mutagenesis was purified from the culture supernatant by using ethanol precipitation (0-70% saturation) and DEAE ion-exchange chromatography. The yield of the purified protease after purification in wild strain was found 7.5% with specific activity of 4729.0 U/mg and purification fold of 5.6. Under similar conditions the yield of purified protease in mutant strain was 8.8% with a specific activity of 6584.6 U/mg and purification fold of 7.8. The purified proteases fractions were found to be homogenous in SDS-PAGE in both the cases with a molecular weight of 17 kDa. Both mutant and wild strain showed optimum ativity at 65 °C and pH-9.0. Mutant strain retained 97% of activity even at pH-10.0 while wild strain showed decrease in activity (62%) at this pH. Both strain showed substrate specificity toward casein with Km and Vmax value (0.294 mg/ml, 3703.7 mg/ml/min and 0.167 mg/ml, 4761.9 mg/ml/min) in wild and mutant strain respectively. The protease activity in both wild and mutant showed inhibition in enzyme activity in the presence of EDTA (5 mM), thus confirming the presence of metal residues at the active site. In both wild and mutant strain Ca2+ (5 mM) showed stabilizing effect but presence of Cu2+ (5 mM) exhibited inhibitory effect on the activity of the purified protease. Purified protease of both strains showed stability in the presence of various detergents but decreased activity was noticed in the presence of NaCl. MALDI-TOF-MS analysis also confirmed that the purified protein was protease in wild and peptidase in mutant strain respectively.