Production of a thermostable and alkaline Protease by Bacillus Subtilis MTCC 9226

A thermostable alkaline protease producing strain identified as Bacillus subtilis MTCC 9226 was isolated from cow dung compost sample. Culture conditions were optimized for protease production. Maximum enzyme production was detected in stationary phase with Bacillus subtilis MTCC 9226 at 45 degrees C, pH 9.0 with 5% inoculum after 32 h incubation. The optimum temperature and pH for the activity of this protease were 45 degrees C and 9.0 respectively. Out of various carbon and nitrogen source tested, glucose (1%) and yeast extract (0.5%)+peptone (5%) proved to be very good source/s of carbon and nitrogen respectively for the production of protease by this organism. The enzyme was stable from pH 7.0 to 11.0 at temperature 30 degrees C to 60 degrees C. Activity of protease increased in presence of 4mM Mg(2+) (107%) and was slightly effected by EDTA 50mM (79%) and completely inhibited by SDS. The enzyme activity was stable up to in 15% of H(2)O(2) and sodium hypochlorite and inhibited at higher concentrations. The enzyme was very stable in benzene (97%) followed by methanol (86%). Complete removal of gelatin from X-ray films was achieved in 25 min at 45 degrees C.