Purification and characterization of an extracellular alkaline serine protease from bacillus subtilis nr18

An alkaline serine protease producing strain was isolated from local soil samples and identified based on morphological and biochemical characteristics as Bacillus subtilis NR18. The enzyme was purified in three step procedure involving ammonium sulfate precipitation, followed by gel filtration and ion-exchange chromatography. Through the process 13.7-fold increase in purity with a specific activity of 283.1 U/mg proteins was obtained. The molecular weight of the purified enzyme was found to be 21 kDa by SDS-PAGE. The enzyme was most active at 500C and pH 9.0. It was relatively stable between pH 7.0-10.0 and temperature between 40 and 500C. Influence of metal ions on enzyme activity revealed that, Ca2+, Mg2+ and Mn2+ slightly enhanced the enzyme activity; whereas Co2+, Fe2+, Hg2+ and Zn2+ strongly inhibited the enzyme activity. Among the protease inhibitors that were tested, the PMSF and DFP completely inhibited the enzyme activity, indicating that the protease is a serine protease. The enzyme retained more than 50% activity after 60 min incubation at 500C in the presence of commercial detergents indicating its suitability for application in detergent industry.