• DOI:10.1007/BF00218452
• Corpus ID: 6077293

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

@article{Kobayashi2004PurificationAP,
  title={Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16},
  author={T. Kobayashi and Yoshihiro Hakamada and S Adachi and Jun Hitomi and Tadashi Yoshimatsu and Kenzo Koike and Shuji Kawai and Susumu Ito},
  journal={Applied Microbiology and Biotechnology},
  year={2004},
  volume={43},
  pages={473-481}
}

• T. Kobayashi, Y. Hakamada, +5 authors S. Ito
• Published 1 July 1995
• Biology, Chemistry
• Applied Microbiology and Biotechnology

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.