Purification and characterization of a protease from Thermophilic bacillus strain HS08

The purification and characterization of a thermophilic neutral protease from Thermophilic bacillus strain HS08, originally isolated from a soil sample collected from the Tulufan Crater of China, is presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion exchange chromatography and Sephacryl S-100HR on AKTA purifier 100 protein liquid chromatography. The method gave a 4.25 fold increase of the specific activity and had a yield of 5.1%. The molecular weight of the protease was found to be around 30.9 kDa by SDS-PAGE technique. The optimal pH and optimal temperature of the protease were at pH 7.5 and 65 degrees C, respectively. The protease was found stable during the 1 h incubation at 50 degrees C. The protease activity showed wide range of variation in the presence of different reagents: it was inhibited remarkably by EDTA or PMSF and was almost activated by 2 mM Zn2+, even though it was only marginally inhibited by other inhibitors. We concluded that the protease was a Zn2+-acitived serine protease. Substrates specificity tests indicated that azocasein was the best substrate among the three substrates tested (azocasein, casein, and BSA).