Purification and Characterization of β-N-Acetylhexosaminidase from the Liver of a Prawn, Penaeus japonicus

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概要

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• β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeusjaponicus,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 pl was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH_2-Thr-Leu-Pro-PCo-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℃, respectively. The enzyme was stable from pH 4 to 11, and below 55℃. It was 39% inhibited by 10 mM HgCl_2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAc_n, n=2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAc_n; n=1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the nonreducing end of the substrate. The parameters of K_m and k_<cat> at 25℃ and pH 5.5 were 0.137mM and 598s^<-1> for pNp-β-GlcNAc,0.117mM and 298s^<-1> for GlcNAc_2,0.055mM and 96.4s^<-1> for GlcNAc_3,0.044 mM and 30.1s^<-1> for GlcNAc_4, 0.045 mM and 14.7 S^<-1> for GlcNAc_5, and 0.047 mM and 8.3 S^<-1> for GlcNAc_6, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.

• 社団法人日本農芸化学会の論文
• 1996-02-23

著者

• KONO Michiko

  Fisheries Research Laboratory, Graduate School of Agricultural and Life Sciences, The University of

• Kono M

  Fisheries Rsearch Laboratory Faculty Of Agriculture The University Of Tokyo

• KOGA Daizo

  Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi Univ

• Kono M

  Univ. Tokyo Shizuoka Jpn

• Kono M

  Fisheries Research Laboratory Faculty Of Agriculture The University Of Tokyo

• Kono Michiko

  Fisheries Research Labolatory Graduate School Of Agricultural And Life Sciences University Of Tokyo

• TANAKA Atsushi

  Laboratory of Dairy Science, Research Group of Animal Product Science, Division of Bioresources and

• HOSHIKA Hironori

  Laboratory of Biochemistry, Faculty of Agriculture, Yamaguchi University

• MATSUSHITA Maki

  Laboratory of Biochemistry, Faculty of Agriculture, Yamaguchi University

• IDE Akio

  Laboratory of Biochemistry, Faculty of Agriculture, Yamaguchi University

• Ide A

  Kihara Institute For Biological Research Yokohama City University

• Ide Akio

  Laboratory Of Biochemistry Faculty Of Agriculture Yamaguchi University

• Koga D

  Department Of Biological Science Faculty Of Agriculture Yamaguchi University

• Koga Daizo

  Laboratory Of Biochemistry Department Of Biological Science Faculty Of Agriculture Yamaguchi Univers

• Matsushita M

  Okayama Univ. Okayama Jpn

• Hoshika H

  Yamaguchi Univ. Yamaguchi Jpn

• Tanaka Atsushi

  Laboratory Of Biochemistry Faculty Of Agriculture Yamaguchi University

• Hoshika Hironori

  Laboratory Of Biochemistry Department Of Biological Science Faculty Of Agriculture Yamaguchi Univers

• Kono Michiko

  Fisheries Rsearch Laboratory Faculty Of Agriculture The University Of Tokyo

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