PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE THIOL PROTEASE FROM A NEWLY ISOLATED HYPERTHERMOPHILIC PYROCOCCUS SP

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfata ra at a wharf on Kodakara Island, Kagoshima, Japan. The growth tempera ture of the strain ranged from 65 to 100 degrees C, and the optimal te mperature was 95 degrees C. The anaerobic strain was an S-0-dependent heterotroph. Cells were irregular cocci and were highly motile with se veral polar flagella. The membrane lipid was of the ether type, and th e GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequ ence was 95% homologous to that of Pyrococcus abyssi. The optimum grow th pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respe ctively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Str ain KOD1 produced at least three extracellular proteases. One of the m ost thermostable proteases was purified 21-fold, and the molecular siz e was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees C, with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 deg rees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsu lfonyl fluoride. Proteolytic activity was enhanced threefold in the pr esence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.