Purification and Characterization of Alkaline Protease from Bacillus sp. HD292

Abstract

Proteolytic enzymes form a very significant group of enzymes which are used for a variety of purposes including as a detergent additive. In the present investigation, the protein hydrolyzing enzyme secreted by Bacillus sp. HD292 was purified and characterized. The purification of the proteolytic enzyme was undertaken by ammonium sulphate fractionation (20–50%) and gel filtration chromatography using Sephadex G-75 which resulted in 17.78 fold purification. On performing SDS-PAGE analysis, a single band of approx. 30 kD was observed. The purified protease displayed peak activity at pH 9.5 and 70 °C. The Km value of the enzyme was found to be 2.7 mg ml−1 with casein as the substrate. pBLAST analysis of the translated sequence resulting from the PCR product obtained from the conserved region of the gene, showed it to be similar to various intracellular and extracellular serine proteases. Also, the purified bacterial protease was found to retain its proteolytic activity at high pH as well as high temperature and was also observed to be quite compatible with the detergent Paras (R3 Organics).