Purification and Characterization of β-N-Acetylhexosaminidase from Rice Seeds

N-Acetyl-β-D-hexosaminidase (β-HexNAc’ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (F1F7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-β-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of βHexNAc’ase. The ratio of the pNP-GlcNAc’ase activity to the pNP-GalNAc’ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-β-glucopyranoside, or pNPβ-galactopyranoside. The enzyme showed Km , Vmax and Kcat for pNP-GlcNAc of 1.65 mM, 79.49 mM min, and 4.79 × 10 6 min, respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl2 and AgNO3, suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.