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 aminoterminal 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 (GlcNAc(n), n=2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAc(n), 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 K-m and k(cat) at 25 degrees C and pH 5.5 were 0.137 mM and 598s(-1) for pNp-beta-GlcNAc, 0.117 mM and 298s(-1) for GlcNAc(2), 0.055 mM and 96.4s(-1) for GlcNAc(3), 0.044 mM and 30.1s(-1) for GlcNAc(4), 0.045 mM and 14.7s(-1) for GlcNAc(5), and 0.047 mM and 8.3s(-1) for GlcNAc(6), respectively, These results suggest that this beta-N-acetylhexosaminidase is an eso-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.