Enhanced Biosynthesis and Purification of Proteases from Bacillus sp. AI-5 by SmF: A Green Approach for Degradation of Peptide Bonds in Complex Proteins

Proteases are theprotein degrading commercial enzymes with considerable importance for many industrial applications such as paper, leather, food industries as well as used in detergent formulation, toxic waste removal, pharmaceutical and drilling for oil. The focus of the current study was to isolate the novel protease producing bacterial sp. from environment, to optimize the submerged fermentation parameters in order to design the best supportive media for maximum enzyme yield and to purify the enzyme to increase its utilization in various industries, most importantly the pharmaceutical industry.Five pure cultures were isolated from soil of coastal area, Karachi and the bacterial strain that was proved to be the most potent producer of protease was selected for research purposeand named as Bacillus sp AI-5 after series of morphological and biochemical tests. The fermentation conditions and media composition were optimized using the starter medium that was selected among the three previously reported media for protease production. It was found that protease production from Bacillus sp AI-5 reached to maximum when media was supplemented with 0.4 gm % casein as carbon source, the combination of 0.5 gm % yeast extract and 0.5 gm % peptone as nitrogen source and 0.05 gm CaCl₂ as inducer and stabilizer of proteases. The optimum pH and temperature for maximum production of protease were found to be pH 5 and 45 and#176;C respectively after 24 hours of incubation. The protease of the Bacillus sp. AI-5 was purified to homogeneity by salt fractionation method using 60 gm % ammonium sulfate, dialysis and Sephadex G-100 Gel filtration chromatography. The specific activity of purified protease was found to be 322.25 U/mg with 13.80 fold purification as compared to crude enzyme. The purified enzyme gave a single band on Sodiumdodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)corresponding to a molecular weight of 66 kDa.