Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

Abstract

Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic <i>Bacillus</i> sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca²⁺ and Mn²⁺ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn²⁺, K⁺, Ba² ⁺, Co² ⁺, Hg²⁺ and Cu²⁺ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride) completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80) and other commercial detergents (SDS, Triton X-100).