Purification and Characterization ef a Themiostable Alkaline Protease from Alkalophilic 1 : hermoactinomyces sp . HS 682

Protease secreted into the culture medium by alkalephili ¢ 11thermoactinonryees sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relatiye molecular mass of 25,OOO according to gel filtration on a Sephadex G-75 co)umn and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2' and Hg2". The enzyme was stable toward some detergents, sodium perborate, sodium niphosphate, sodium-n-dodecylbenzer)esu}fonate, and sodium dodecyl sulfate, at a concentration of O.1% and pH 11.5 and 370C for 60 min. The optimum pH was pH 11.5-13.0 at 37eC and the optimum temperature was 700C at pH 11.5. Calcium diyalent catioR raised the pH and heat stabilities of the enzyme. In the presence of 5mM CaCl2, it showed maximum proteolytic activity at 800C and stability from pH 412.5 at 600C and below 75eC at pH 11.5. The stabilization by Ca2' was obseryed in secendary conformation deduced from the circular dichroic spectrum ef the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ster well. The amino acid terminal sequence of the enzyme showed high hornology with those of microbial serine protease, although alanine of the NH,-terminal amino acid was deleted.