Production and Characterization of an Alkaline Protease from Bacillus licheniformis MH31

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.