Protease production using Bacillus licheniformis by submerged fermentation

Abstract     Enzymes, such as protease, occupy a pivotal position in the world of enzymes with respect to their applications in both physiological and commercial applications. Bacillus, like Bacillus licheniformis, produce protease and was used throughout the present study. The Bacillus licheniformis is a potential microorganism for producing proteins with various functions, including protease with substantial activity, effective pH, and temperature tolerance, at laboratory and industrial scales. This study investigated the effect of several temperatures ranging from ۲۵-۵۰ οC, pH ranging from ۷-۱۰.۵, shaker speeds of ۱۰۰-۲۰۰, and inoculum concentrations of ۳-۱۲ %V/V on enzyme production. Results showed that cultivating the medium at about pH (۸.۵), a temperature of ۳۵ οC, and a shaker gyratory speed of ۱۸۰ rpm influenced protease production. Firstly, it was found that using yeast extract, soybean meal corn, and steep liquor (powder) as nitrogen sources also enhanced protease production. Furthermore, cornstarch and glucose of ۳ %W/V were found to be effective as a carbon source for protease production. The protease stability was retained for almost ۳۵ minutes, heating at ۶۰ οC and pH ۸.۵, and as heating continued at the same temperature and pH, the enzyme had ۴۰% of its activity. Secondly, the effect of different metal ions types on the stability of the partially purified protease is reported. It was found that the protease activity at ۶۰ ± ۰.۵ οC, pH ۹.۵, and the presence of ۱۵ mM CaCl۲ and ۱۰ mM NaCl during an hour of treatment retained approximately ۴۰ percent of its activity. Since a batch period of ۲۴ protease production showed similar proteases activity as a  ۴۸ h operation,  the following fermentation was conducted within ۲۴ h. The partially purified protease had sound stability in the alkaline pH of ۹-۹.۵ and temperature range of ۶۰ ±۰.۵ οC in the presence of certain ions like calcium and sodium salt.

Keywords:

Bacillus licheniformis , protease , proteolytic activity , submerged fermentation , shaker’ s speed