Applied Microbiology and Biotechnology

, Volume 43, Issue 3, pp 473–481

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

  • T. Kobayashi
  • Y. Hakamada
  • S. Adachi
  • J. Hitomi
  • T. Yoshimatsu
  • K. Koike
  • S. Kawai
  • S. Ito
Biochemical Engineering Original Paper

DOI: 10.1007/BF00218452

Cite this article as:
Kobayashi, T., Hakamada, Y., Adachi, S. et al. Appl Microbiol Biotechnol (1995) 43: 473. 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°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°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 cyrstals.

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • T. Kobayashi
    • 1
  • Y. Hakamada
    • 1
  • S. Adachi
    • 1
  • J. Hitomi
    • 1
  • T. Yoshimatsu
    • 1
  • K. Koike
    • 1
  • S. Kawai
    • 1
  • S. Ito
    • 1
  1. 1.Tochigi Research Laboratories of Kao CorporationTochigiJapan