Purification, immobilization, and characterization of protease from local Bacillus subtilis M-11

Protease from Bacillus subtilis M-11 was produced and purified using ammonium sulfate precipitation, gel filtration chromatography with 15.8-fold. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was determined that its molecular weight was 32kDa. The purified enzyme was immobilized on polysulfone membrane containing Silica gel-3 aminopropyltriethoxysilane (Si-APTS) by physical adsorption. The optimal immobilization conditions (pH, temperatures, and incubation time) were investigated. The properties of immobilized protease were determined and compared with free enzyme. The investigations on immobilized enzyme indicated that the optimum pH was shifted 0.5 units to alkaline pH (pH 8.0-8.5) after immobilization with physical adsorption. Immobilization improved pH stability of enzyme between pH 7.5 and 8.5. Immobilized enzyme sustained 77.3% of its activity after usage of 10 times. However, there is no change on optimum temperature (50 degrees C) after immobilization. All these results indicated that polysulfone membrane containing Si-APTS could be a proper support for protease immobilization, and immobilized protease by using physical adsorption could be used for industrial applications efficiently. (c) 2015 Curtin University of Technology and John Wiley & Sons, Ltd.