Abstract
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 NH2-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°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°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-β-GlcNAcn; 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 Km and kcat at 25°C and pH 5.5 were 0.137mM and 598s-1 for pNp-β-GlcNAc, 0.117mM and 298s-1 for GlcNAc2, 0.055mM and 96.4s-1 for GlcNAc3, 0.044 mM and 30.1s-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 β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.
