Study on the optimal production and partial purification of alkaline protease from alkalotolerant Bacillus sp. B12: Feasibility as a commercial detergent additive

Abstract

              Microorganisms, especially the genus of Bacillus show important roles on the production of commercial alkaline protease. Preliminary study revealed that alkalotolerant Bacillus sp. B12 can produce alkaline protease with the highest activity at 0.44 Unit/ml. The objects of this this study were (1) to investigate the optimal condition to produce alkaline protease by alkalotolerant Bacillus sp. B12 and (2) to study the effect of saturated ammonium sulfate concentration on partial purification of crude alkaline protease (CP), as well as study the compatibilities of CP and partially purified protease (PPP) with commercial detergents. Alkaline protease was produced by alkalotolerant Bacillus sp. B12 and the optimal parameters, including carbon and nitrogen sources, the amount of potassium nitrate, pH, temperature and shaking speed were determined. CP was partial purified by precipitation with various concentrations of saturated ammonium sulfate. The optimal pH and temperature of PPP were investigated. Moreover, the compatibilities of the alkaline protease (CP and PPP) and commercial detergents at 50⁰C for 90 min were determined.

              The results showed that the most suitable medium for the alkaline protease production was a mixture of 0.5% soybean meal as carbon source and 0.5% KNO₃ as nitrogen source with initial pH 9.0. The most suitable condition to produce the highest yield of the alkaline protease was an incubation at 37⁰C for 24 hr with the agitation at 200 rpm. With that condition, alkalotolerant Bacillus sp. B12 produced the highest alkaline protease at 0.63 Unit/ml which was a 1.4-fold increase in protease activity. The most suitable concentration of saturated ammonium sulfate for partial purification of the CP was 40-60% with a 6.9-fold increase in protease purity. The optimal pH and temperature of PPP were pH 8.0 - 11.0 and 40 - 70⁰C, respectively. The highest stability of PPP was at pH 8.0 and 10.0, 30⁰C and 50⁰C for 1 hr. For the compatibility of the alkaline proteases and commercial detergents, PPP was active in Baby best, Baby mild and Essence detergents but it was not stable in St’Luke’s and Fineline detergents. While CP was active in Baby best, Baby mild, Essence, St’Luke’s and Fineline and it was highly stable in 1.0 - 5.0% Baby best, 3.0% Baby mild and 1.0% Essence detergents. Therefore, the crude alkaline protease from alkalotolerant Bacillus sp. B12 could be a potential candidate as an additive in detergent formulations.