PURIFICATION AND PROPERTIES OF THE HIGHLY THERMOSTABLE ALKALINE PROTEASE FROM AN ALKALIPHILIC AND THERMOPHILIC BACILLUS SP

Thermostable alkaline protease from an alkaliphilic thermophile Bacillus sp. B18' was purified by using DEAE- and CM-Toyopearl 650M column chromatographies. Molecular weights of the enzyme determined by SDS-PAGE and gel filtration were 30000 and 28000, respectively. The optimum pH and temperature toward the hydrolysis of casein were pH 12-13 and 85-degrees-C, both of which are higher than those of a mesophilic alkaline protease from an alkaliphile, Bacillus sp. B21-2. The enzyme was stable at pH 5.0-12.0 and about 60% of the initial enzymatic activity was retained after a 60 min incubation period at pH 10.0 and 70-degrees-C. Thermostability of the enzyme was enhanced by Ca2+. The enzyme activity was inhibited by DFP, suggesting that the enzyme is a serine protease. The NH2-terminal amino acid is Gln, which is that of many subtilisin-type proteases. The 20 residues of the NH2-terminal amino acid sequence have a comparative high homology with those of other alkaline proteases from alkaliphiles (40-50%), especially thermostable alkaline protease from Bacillus sp. No. AH-101 (95%) and Thermoactinomyces sp. HS682 (95%).