Purification and characterization of β-N-acetylglucosaminidase from Mung Bean Seedlings

• Main Authors: Liou Wei-Liang, 劉韋良
• Other Authors: Chen Ching-San ,Li Yung-An
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/76563809355969407108

碩士 === 輔仁大學 === 生物學研究所 === 83 === Beta-N-acetylglucosaminidase (β-glcNAcase) is involved in the biosynthesis and processing of N-linked glycoproteins in plants, and the metabolism of β-N-acetylglucosamine- containing polysaccharides in seeds. We found that the specific activity ofβ-glcNAcase in mung bean seedlings approached the highest at 6th day after germination. These 6-day- germinating mung beans, therefore, were used for study of the structure and function of plant β-glcNAcases. The 6-day- germinating mung beans were homogenized in 25 mM sodium acetate buffer, pH 5.0 and centrifuged . The supernatant (crude extract) was fractionationated with ammoniun sulfate and chromatographed sequentially on CM-cellulose, TSK-DEAE, Con A Sepharose and chromatofocusing medium ( pH 6.2~4.8 , polybuffer 74 ).Three isoforms of β-glcNAcases, named Ia , Ib, III , were isolated through these purification steps. The β-glcNAcases Ib and III were purified to apparent homogeneity as examined by silver staining and activity staining of the native PAGE gels.β-glcNAcases Ia was partially purified .Antisera were raised against the purified β-glcNAcases Ib and III .Activity staining for β- glcNAcases in the crude extract separated by two- dimensional electrophoresis revealed four isoforms of this enzyme. These three isoforms exhibited acidic pI, in the range of 5.7~5.2 .Their optimal pH for enzymatic reaction was between 4.4 and 5.2 .The three isofoms were inhibited by Hg2+ ,DTNB and high concentration of diacetylchitobiose .The molecular weights ofβ -glcNAcases Ia, Ib and III determine by S-200 gel filtration were 106,132, and 160 kD , respectively. And there km values were 1.76 , 1.04 , 0.67 mM for PNP-N- acetyl-β-D-glucosaminide at pH 4.6.