Purification and characterization of thermostable β-N- acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost

K. Sakai, M. Narihara, Y. Kasama, M. Wakayama, M. Moriguchi

研究成果: ジャーナルへの寄稿学術誌査読

41 被引用数 (Scopus)

抄録

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 β-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-β- N-acetyl-D-galactosaminide (26% of the activity of pNP-β-B-acetyl-D- glucosaminide). These results indicated that the enzyme is a β-N- acetylhexosaminidase (EC 3.2.1.52). K(m)s for acetylchitooligosaccharides were 1 x 10-4 to 6 x 10-4 M, while those for the pNP derivatives were 4 x 10-3 to 8 x 10-3 M. The optimum temperature of the enzyme was 75°C, and it retained 100 and 28% reactivity after heating at 60 and 80°C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5, Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N- Acetyl-D-glucosamine inhibited the enzyme competitively (K(i) = 4.3 x 10-4 M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.

本文言語英語
ページ(範囲)2911-2915
ページ数5
ジャーナルApplied and environmental microbiology
60
8
DOI
出版ステータス出版済み - 1994
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • 食品科学
  • 応用微生物学とバイオテクノロジー
  • 生態学

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