In this study a strain of Bacillus sp. Capable of producing protease enzyme was isolated from leather processing tannery soil, upon characterization four strain which shows maximum protease activity on skimmed milk- agar plates were further analyzed by protease assay and only one strain which shows the the maximum protease activity was selected for further analysis. It was identified as Bacillus subtilis BS.HK  through 16SrRNA gene sequencing and submitted to GenBank under the accession #OP604265.1. Alkaline protease enzyme was produced from this strain bysubmerged fermentation technique. The highest yield of alkaline protease enzyme production was attained within a 24-hour fermentation period in a basic medium with glucose and Casein as the best Carbon and Nitrogen sources. The optimal conditions for the production of alkaline protease by BS.HK were determined as temperature 37°C, pH 9.5, and an agitation speed of 140 rpm. The enzyme was purified upto 7.40 folds by (NH4)₂SO₄ ammonium sulphate precipitation and then purified by Gel filtration chromatography upto 40.62 folds. The Kinetic parameter study revealed that the purified enzyme has molecular weight of 35KDa exhibits optimum activity at 50℃, pH 9.5, and shows high stability at 60℃   and pH 8-12 for 1 hour. The enzyme display remarkable activity with lower concentration of casein as substrate having Km 0.992 µM and Vmax 19.72 U/ml. The inhibitors PMSF and EDTA significantly reduce enzyme activity through different mechanisms. Different surfactant like Triton X-100 and Tween-80 have milder inhibitory effects. Metal ions can either enhance or inhibit enzyme activity, additionally,the anticipation of physicochemical characteristics indicated that the serine protease from BS.HK strain exhibits exception stability, and resilience to alkaline conditions. The purified enzyme from B. subtilis BS.HK completely dehaird cow skin without damiging the structural components of skin as compared to chemically dehaired skin. In conclusion, alkaline protease seems to be an important enzyme for leather processing industry because of its thermostability at high temperature and alkali stability at alkaline pH.

Keywords-Protease, Bacillus subtilis, Submerged Fermentation, Enzyme Purification, Kinetic Parameters, Alkaline Protease