Purification and characterization of protease enzyme from native isolate Bacillus subtilis and its compatability with commercial detergents

Author(s): Raj Kumar, G.Adarsh, Ramesh Malothu

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 ammoniumsulfate precipitation, followed by gel filtration and ion-exchange chromatography. Through the process 13.7-fold increase in puritywith 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 enzymewasmost active at 60Cand pH 9.0. It was relatively stable between pH 7.0-10.0 and temperature between 40 and 60°C 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 incubation at different time intervals at 60°C in the presence of commercial detergents indicating its suitability for application in detergent industry.

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