Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16.
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.
Kobayashi
T
T
Tochigi Research Laboratories of Kao Corporation, Japan.
Hakamada
Y
Y
Adachi
S
S
Hitomi
J
J
Yoshimatsu
T
T
Koike
K
K
Kawai
S
S
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology
D013501:Surface-Active Agents; D010450:Endopeptidases