Purification and Properties of an Extra Cellular Alkaline Protease Produced By Bacillus Subtilis (MTTC N0-10110)

The purification and characterization of alkaline protease from a Bacillus subtilis (KHS-1) (MTTC No-10110), isolated from slaughter house soil sample have been reported in this paper. An alkaline protease from Bacillus Subtilis was purified by Ammonium Sulfate Precipitation and sephadex G-200. Proteolytic activity of the enzyme was detected by casein zymography, which gave a clear protease activity zone on gel that corresponding to the band on SDS-PAGE with a molecular weight nearly 20.5 kDa. Maximum activity of the protease with casein as substrate was observed at 60°C temperature and at pH 10.5. The purified protease was activated by Ca²⁺ (42%) and β-Mercapto ethanol (28%). However, Mg²⁺, and Mn²⁺ slightly activated the protease activity. PMSF completely inhibited the protease activity. Whereas, Hg²⁺, Zn²⁺and Al³⁺ slightly inhibited the proteolytic activity. The Purified protease was highly stable with liquid detergents on long-term incubation at 55°C. Purified protease has the potential application of dehairing of goat skin, destaining of blood and degradation of gelatinous coating of x-ray films.