Production, Purification and Characterization of an Alkaline Protease from an Alkaliphilic Bacillus Sp

An extracellular serine like alkaline protease was purified and characterized from alkaliphilic Bacillus sp. The enzyme was purified to the homogeneity by a combination of aqueous two-phase system and affinity chromatographic techniques. The apparent molecular weight was estimated as 26-29 kDa based on SDS-PAGE and gel filtration chromatography and the enzyme appeared to be a monomer. The Km and Vmax for the casein were 4.7mgml-1 and 24.5μgml-1 respectively. The enzyme was active over a wide range of temperature (10-65°C) and pH (8-12.5); the optimum activity being at 35°C and pH 11.0. The purified enzyme was quite stable at 35°C up to 1 h of incubation, while 80 and 50% residual activities were detected at 55 and 65°C respectively after 20 min of incubation. Similarly, enzyme exhibited 100% stability in alkaline pH range, which sharply decreased in acidic range. The enzyme lost its activity in the presence of PMSF, Hg+2 and Cu+2 and remained unaffected with EDTA, Mg+2 and Ba+2.