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Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus.
Koga, D; Hoshika, H; Matsushita, M; Tanaka, A; Ide, A; Kono, M.
Afiliação
  • Koga D; Laboratory of Biochemistry, Yamaguchi University, Yoshida, Japan.
Biosci Biotechnol Biochem ; 60(2): 194-9, 1996 Feb.
Article em En | MEDLINE | ID: mdl-9063965
Beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS-PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS-PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The amino-terminal amino acid sequence was NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly- Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50 degrees C, respectively. The enzyme was stable from pH 4 to 11, and below 55 degrees C. It was 39% inhibited by 10 mM HgCl2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAcn, n = 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat at 25 degrees C and pH 5.5 were 0.137 mM and 598 s-1 for pNp-beta-GlcNAc, 0.117 mM and 298 s-1 for GlcNAc2, 0.055 mM and 96.4 s-1 for GlcNAc3, 0.044 mM and 30.1 s-1 for GlcNAc4, 0.045 mM and 14.7 s-1 for GlcNAc5, and 0.047 mM and 8.3 s-1 for GlcNAc6, respectively. These results suggest that this beta-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.
Assuntos
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Base de dados: MEDLINE Assunto principal: Beta-N-Acetil-Hexosaminidases / Penaeidae / Fígado Limite: Animals Idioma: En Revista: Biosci Biotechnol Biochem Assunto da revista: BIOQUIMICA / BIOTECNOLOGIA Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Japão
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Base de dados: MEDLINE Assunto principal: Beta-N-Acetil-Hexosaminidases / Penaeidae / Fígado Limite: Animals Idioma: En Revista: Biosci Biotechnol Biochem Assunto da revista: BIOQUIMICA / BIOTECNOLOGIA Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Japão