Purification and Characterization of Alkaline Protease from a Mutant Bacillus licheniformis Bl8

Alkaline protease from a mutant of Bacillus licheniformis Bl8 was purified from the culture supernatant by employing the methods such as ammonium sulphate precipitation, DEAE cellulose chromatography followed by Gel filtration using Sephadex G-100. The yield of the enzyme after purification was found to be 10%. Protease was found to be homogenous when examined by SDS-PAGE and the enzyme showed that it has a molecular weight of 28 KDa. Characterization studies were carried out using the purified enzyme. While the optimum pH and temperature for the activity of alkaline protease was found to be 10 and 50°C and stable in the pH range 5.0 12.0. The thermo stability exhibited by protease ranged from 30-70°C. Among various protease inhibitors PMSF strongly inhibited the enzyme activity revealing that the enzyme in the present study is serine alkaline protease. Ca and Mn had a slight enhancing effect on the activity of the 2+ +2 enzyme. High level of hydrolytic activity was shown by casein and also found that purified alkaline protease digested the human blood clot, coagulated white egg to soluble form and also digested chicken skin upon the prolonged incubation of enzyme with chicken skin. The protease showed good compatibility and stability in the presence of CaCl and glycine with Nirma, Surf excel and Wheel. The enzyme retained 20-40% activity with 2 most of the detergents tested even after 3hrs. The supplementation of the enzyme preparation in detergent completely removed the blood stain of the cloth. The enzyme followed a typical Michaelis-Menten kinetics. Apparent Km value was found to be 3.2mgml . 1