Purification and Characterization of β-N-Acetylhexosaminidase from Rice Seeds
| Autor: | Kil Yong Kim, Yu Young Jo, Yu Lan Jin, Ro-Dong Park, Yong Woong Kim, Jae-Han Shim |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
β n acetylhexosaminidase
Acetylgalactosamine Biochemistry Thiol group Acetylglucosamine Substrate Specificity Tosyl Compounds chemistry.chemical_compound N-Acetyl-beta-D-hexosaminidase Acetylglucosaminidase Enzyme Stability Isoelectric Point Enzyme Inhibitors Binding site Molecular Biology chemistry.chemical_classification biology Chloramines Oryza General Medicine Hydrogen-Ion Concentration beta-N-Acetylhexosaminidases Enzyme assay Molecular Weight Kinetics Hexosaminidases Enzyme chemistry Mercuric Chloride Seeds Chloramine-T N-acetyl-beta-D-glucosaminidase biology.protein Silver Nitrate Disinfectants |
| Zdroj: | BMB Reports. 35:313-319 |
| ISSN: | 1976-6696 |
| DOI: | 10.5483/bmbrep.2002.35.3.313 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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