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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.
T Kobayashi, and
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S Ito
March 1995,
Acta crystallographica. Section D, Biological crystallography,
T Kobayashi, and
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September 1996,
World journal of microbiology & biotechnology,
T Kobayashi, and
Y Hakamada, and
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K Koike, and
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S Ito
October 1993,
Bioscience, biotechnology, and biochemistry,
T Kobayashi, and
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K Koike, and
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S Ito
September 1995,
Bioscience, biotechnology, and biochemistry,
T Kobayashi, and
Y Hakamada, and
S Adachi, and
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T Yoshimatsu, and
K Koike, and
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S Ito
March 1994,
Bioscience, biotechnology, and biochemistry,
T Kobayashi, and
Y Hakamada, and
S Adachi, and
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T Yoshimatsu, and
K Koike, and
S Kawai, and
S Ito
April 1995,
Biochimica et biophysica acta,
T Kobayashi, and
Y Hakamada, and
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J Hitomi, and
T Yoshimatsu, and
K Koike, and
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S Ito
January 2002,
Letters in applied microbiology,
T Kobayashi, and
Y Hakamada, and
S Adachi, and
J Hitomi, and
T Yoshimatsu, and
K Koike, and
S Kawai, and
S Ito
August 1998,
Wei sheng wu xue bao = Acta microbiologica Sinica,
T Kobayashi, and
Y Hakamada, and
S Adachi, and
J Hitomi, and
T Yoshimatsu, and
K Koike, and
S Kawai, and
S Ito
June 1995,
Current microbiology,
T Kobayashi, and
Y Hakamada, and
S Adachi, and
J Hitomi, and
T Yoshimatsu, and
K Koike, and
S Kawai, and
S Ito
February 1992,
Bioscience, biotechnology, and biochemistry,
