Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Appl Microbiol Biotechnol. 1995 Jul;43(3):473-81. doi: 10.1007/BF00218452.

Abstract

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, 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 Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg- Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55 degrees C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl fluoride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40 degrees C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like crystals.

MeSH terms

  • Amino Acid Sequence
  • Bacillus / classification
  • Bacillus / enzymology*
  • Endopeptidases / chemistry
  • Endopeptidases / isolation & purification*
  • Enzyme Stability
  • Molecular Sequence Data
  • Substrate Specificity
  • Surface-Active Agents / pharmacology

Substances

  • Surface-Active Agents
  • Endopeptidases