Characterization of β-N-acetylglucosaminidase from turnip roots

• Main Authors: Ju-Fong Yuan, 袁如鳳
• Other Authors: Chen-Tien Chang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/34565649689378635775

碩士 === 靜宜大學 === 食品營養學系 === 88 === Abstract Chitosan coating treated turnip seeds were cultivated in field for about 2 months. After harvest, the specific activity of N-acetyl-β-D-glucosaminidase in roots showed no significant change as compared with control, which was not treated with chitosan. N-acetyl-β-D-glucosaminidase was purified from turnip roots by sequential steps of buffer extraction, ammonium sulfate fractionation, Sephacryl S-100 HR gel filtration, PBE-94 chromatofocusing and Mono Q ion-exchange chromatography. Using these steps, the purity of the enzyme was increased 260 fold, with a yield of 10%. The purified enzyme was almost homogeneous, as examined by native-PAGE and activity staining. The N-acetyl-β-D-glucosaminidase had an optimal pH of 4, an optimal temperature of 60℃ and a Km of 1.05 mM for hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The molecular mass for the enzyme was 127 kDa, as estimated by Superose 6 HR gel filtration, the isoelectric point of the enzyme was 5.5, as estimated by isoelectrofocusing electrophoresis. SDS-PAGE and Hill plot kinetic analysis showed that enzyme belong to oligomeric enzyme and had one substrate binding site. Heavy metal ions Hg2+( 0.25 mM ) and Ag+( 0.25 mM ), significantly inhibited the activity of the enzyme. N-Bromosuccinimide ( 0.25 mM ), p-hydroxymercuribenzoic acid ( 0.25 mM), diethyl pyrocarbonate ( 7.5 mM ) and Woodward¢s reagent K ( 7.5 mM ) also significantly inhibited the activity of the enzyme. The enzyme showed activity for hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide and p-nitrophenyl-N-acetyl-β-D-galactosaminide but had no action on p-nitrophenyl-N-acetyl-α-D-glucosaminide and p-nitrophenyl-N-acetyl-α-D-galactosaminide. From the chemical modification and Dixon-Webb kinetic studies, the amino acid residues tryptophan, cysteine, histidine, aspartic acid and glutamic acid are probably located at or near the active sites of the enzyme.