Purification and Characterization of an Extracellular Protease from Alkaliphilic and Thermophilic Bacillus sp.PS719

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An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp.PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and α-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75℃. The enzyme activity was stimulated by Ca^<2+> but was inhibited in the presence of Fe^<2+> or Cu^<2+>. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80℃ in the absence of Ca^<2+>. Since phenylmethylsulfonyl fluoride(PMSF)and 3, 4-dichloroisocoumarin(DCI)in addition to N-α-p-tosyl_L-lysine chloromethyl ketone(TLCK)completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy(CBZ)_L-arginine-p-nitroanilide with the K_m and V_<max> values of 0.6 mM and 1.0 μmol・min^<-1>・mg protein^<-1> respectively.
社団法人日本生物工学会の論文
1999-05-25