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

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Abstract

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.

Original languageEnglish
Pages (from-to)2911-2915
Number of pages5
JournalApplied and environmental microbiology
Volume60
Issue number8
DOIs
Publication statusPublished - 1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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