Biosynthesis optimization and purification of a solvent stable alkaline serine protease from Halobacterium sp.

Three halotolerant bacterial isolates that were isolated from the salt pan of coastal Tamilnadu, India, were screened on skimmed milk agar plates containing 1–25% (w/v) NaCl for protease secretion. Among the three positive isolates, Halobacterium sp. showed the maximum protease secretion (4.5 mm diameter zone) and was hence selected for this study. The enzyme was a serine protease as evidenced by complete inactivation of enzyme after treatment with a serine protease inhibitor (phenylmethylsulfonyl fluoride). Culture conditions which optimize protease production that included pH, fermentation period, NaCl in addition to different factors such as carbon and nitrogen sources, casein concentration were investigated. Optimum bacterial growth and proteolytic activity was achieved after 48 h of incubation at 37°C, pH 10.0 with 5% (w/v) NaCl concentration and 2.5% (w/v) casein. Initial experiments revealed that the culture medium containing 1.0% (w/v) lactose as the carbon source and 1.0% (w/v) ammonium chloride as the nitrogen source supported maximized enzyme production. Analysis carried out using SDS-PAGE revealed that the extracellular enzyme consisted of a single polypeptide chain of 43 kDa which was purified by a combination of ammonium sulphate fractionation, DEAE cellulose and Sephadex G-75 gel filtration chromatography. This protease was stable for a wide range of temperatures (40–60°C), NaCl concentrations (1–5%, w/v) and pH (8.0–10.0) with maximal activity observed at 60°C, 5% NaCl and pH 10.0. Proteolytic activity was enhanced by 1.0 mM of Ca2+ (137%) and strongly inhibited by Mn2+ (67%). Most importantly, the proteolytic activity remained stable in alkaline pH and was found to be active at higher temperatures. The broad range of temperature optima with respect to enzyme activity was indicative of this enzymes suitability in fish sauce fermentation, which was carried out at 35–45°C. This enzyme was stable in organic solvents such as toluene and hexane and the relative enzyme activity was 103 and 114%, respectively, which mainly finds use in the paint industry.