PURIFICATION AND PROPERTIES OF AN ALKALINE PROTEASE FROM ALKALOPHILICBACILLUS SP KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in deterge nts, was detected in the alkaline culture medium of Bacillus sp. KSM-K 16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was l-Gln-Ala-Pr o-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the prote ase was 28 kDa, and its isoelectric point was close to pH 10.6. Maximu m activity toward casein was observed at 55 degrees C and at pH 12.3 i n 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmet hylsulfonyl fluoride and chymostatin. The enzyme was very stable in lo ng-term incubation with liquid detergents at 40 degrees C. The enzyme cleaved the oxidized insulin B chain initially at Leu(15)-Tyr(16) and efficiently at ten more sites. Among various oligopeptidyl p-nitro-ani lides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hyd rolyzed by M protease. M protease was precipitated in (NH4)(2)SO4-satu rated acetate buffer (pH 5.0) as plank-like crystals.