Optimization of Alkaline Protease Production by Alkaliphilic Bacillus sp. KW2 in Low Cost Medium using Statistical Approaches

Abstract

Objective: Isolation of alkaline protease producing alkaliphilic bacteria and optimization of protease production in low cost medium. Methods: Optimization of protease production was performed by one-variable-at-a-time (OVAAT) approach and statistical approaches using Design Expert 6 software. Results: Nine (9) morphologically distinct alkaliphilic bacterial strains were isolated. Of these isolates 6 strains were found to be positive for protease production. Among these proteolytic strains, Bacillus sp. KW2 was selected for protease production optimization studies. The optimal protease production was observed at 30°C and pH 10.7. Among the various carbon and nitrogen sources studied rice bran and soybean meal were found to be the best Carbon and Nitrogen sources respectively. Plackett-Burman design (PBD) was used to screen signal factors that influenced the protease production. Rice bran, soybean meal, inoculum age, CaCl2 and inoculum size gave positive effects whereas KH2PO4 and MgSO4 gave negative effects on protease production. The four most critical factors viz. rice bran, soybean meal, calcium chloride and potassium dihydrogen phosphate were selected for optimization studies by Response Surface Methodology (RSM). The response surface graphs showed significant interactions among the factors and a final optimized medium (FOM) was obtained after PBD and RSM experiments. Overall, a 4.8 fold increase in protease production was observed after optimization. Studies of the time course of protease production by Bacillus sp. KW2 revealed that maximal protease production occurred at the stationary phase (84 h) of growth. Conclusions: The present study shows that protease production is greatly influenced by cultural conditions and media constituents. This study also established that the strain KW2 could produce alkaline protease in a low cost medium containing rice bran and soybean meal as major carbon and nitrogen sources. KW2 and its protease may be promising agents for biotechnological applications.