Purification and characterization of neutral protease enzyme from Bacillus Subtilis

Isolation and partial purification of neutral protease from Bacillus subtilis was the main aim of this study. The production medium for Bacillus subtilis was optimized by using different pH, Temperature, Carbon, Nitrogen sources and Agitation Speed for 48 hours fermentation culture. The protease enzyme was highly active and stable from pH 6.0 to 9.0 with an optimum at pH 7. Its optimum temperature was 37°C and, it proved stable up to 30- 60°C.The best carbon and nitrogen sources were starch and whey protein. The best buffer for production of protease enzyme was phosphate buffer. Protease was partially purified by ammonium sulphate precipitation, dialysis. Protease activity was very high in 180 rpm agitation speed. The proteolytic activity was also detected by casein zymography. The molecular weight of purified enzyme was characterized by SDS- PAGE as 50 kDa. This result showed that Bacillus subtilis under study is a good producer of extra cellular protease, which can be beneficial for industries. Keywords: Agitation, Bacillus subtilis, Carbon, Nitrogen, Protease, Zymography.