Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus.
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.
Koga
D
D
Laboratory of Biochemistry, Yamaguchi University, Yoshida, Japan.
Hoshika
H
H
Matsushita
M
M
Tanaka
A
A
Ide
A
A
Kono
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D002686:Chitin / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D033561:Penaeidae / Q000201:enzymology*; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification* / Q000378:metabolism*
D002413:Cations, Divalent; D009844:Oligosaccharides; D002686:Chitin; D001619:beta-N-Acetylhexosaminidases