Purification and characterization of beta-N-acetylhexosaminidase from rice seeds.
N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) 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 S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'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-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), 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 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), 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.
Jin
Yu-Lan
YL
Department of Agricultural Chemistry and Institute of Agricultural Science and Technology, Chonnam National University, 500-757, Gwangju, Korea.
Jo
Yu-Young
YY
Kim
Kil-Yong
KY
Shim
Jae-Han
JH
Kim
Yong-Woong
YW
Park
Ro-Dong
RD
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000378:metabolism; D002700:Chloramines / Q000494:pharmacology; D004202:Disinfectants / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006596:Hexosaminidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology; D008970:Molecular Weight; D012275:Oryza / Q000201:enzymology*; D012639:Seeds / Q000737:chemistry; D012835:Silver Nitrate / Q000494:pharmacology; D013379:Substrate Specificity; D014105:Tosyl Compounds / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D002700:Chloramines; D004202:Disinfectants; D004791:Enzyme Inhibitors; D014105:Tosyl Compounds; C016300:chloramine-T; C014123:4-nitrophenyl-2-acetamido-2-deoxygalactopyranoside; C014124:4-nitrophenyl-N-acetyl-2-deoxyglucopyranoside; D008627:Mercuric Chloride; D012835:Silver Nitrate; C026646:4-nitrophenyl-alpha-N-acetylglucosaminidase; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D000116:Acetylgalactosamine; D000117:Acetylglucosamine