PROCESS OPTIMIZATION BY RESPONSE SURFACE METHODOLOGY FOR EXTRACELLULAR ALKALINE PROTEASE PRODUCTION FROM BACILLUS SUBTILIS

Abstract

Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37o C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran,7.5 g), MgSO4.7H2O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ionexchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, predominantly alkaline and optimally active at pH 8.0.