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Applied Microbiology and Biotechnology

, Volume 43, Issue 3, pp 473–481 | Cite as

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

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.

Keywords

Enzyme Molecular Mass Column Chromatography Acetate Buffer Isoelectric Point 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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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

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