Purification and Characterization of Alkaline Protease Produced by a Mutant Strain of Bacillus Subtilis

The present study describes the production, purification and characterization of alkaline protease from mutant strain of Bacillus subtilis EMS-8. The enzyme was purified using ammonium sulphate precipitation which gave 2.64 fold purification with 81.5% yield at 70% saturation. The molecular weight of the enzyme was determined using SDS-PAGE and it was found to be 25 KDa. The optimum pH of enzyme activity was 8.5; however the enzyme remained stable up to pH 10 after 24 hrs of incubation. Similarly, the optimum temperature for enzyme activity was 40oC, whereas it remained stable up to 90oC with greatly reduced activity. Alkaline protease showed highest specificity towards casein. Among different inhibitors, Phenylmethylsulphonyl fluoride (PMSF) completely inhibited the enzyme activity indicating the serine nature of protease. Similarly, the protease activity was greatly reduced in the presence of MnCl2, whereas MgCl2 enhanced its activity. The shelf life of the protease was also determined and it was found that the activity of the enzyme came to an end after second week, when the enzyme was stored at room temperature.