Abstract
Protease secreted into the culture medium by alkalophilic Thermoactinomyces 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 relative molecular mass of 25, 000 according to gel filtration a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DEP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodyecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5-13.0 at 37°C the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5mM CaCl2, it showed maximum proteolytic activity at 80°C and stability from pH 4-12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.
