Purification and properties of beta-N-acetylglucosaminidase from mung bean seedlings

• Main Authors: Chen, yi-ching, 陳怡靜
• Other Authors: Ching-San Chen
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/94279080333169634710

碩士 === 國立海洋大學 === 生物技術研究所 === 85 === ABSTRACTb-N-acetylglucosaminidase (b-GlcNAcase) is involved in the metabolism of b-N-acetylglucosamine-containing polysaccharides in seeds and the biosynthesis and processing of N-linked glycoproteins in plants. We found that the specific ativity of b-GlcNAcase (EC 3.2.1.30) in mung bean seedlings reached the highest at 6th day after germination. These 6-day- old mung bean seedlings, therefore, were used this study.The 6-day-old mung bean seedlings were homogenized in 25 mM sodium acetate buffer, pH 5.0. The homogenate was centrifuged, and the supernatant, designated as crude extract was sequentially subjected to purification procedure including ConA Sepharose affinity chromatography, Chromatofocusing (pH 5.0~7.0), Sephadex G-150, Superdex 75 HR gel filtration and MonoQ anion exchange chromatography. Three isozymes of b-N-acetylglucosaminidases, named III, Ib and Ia were separated through these purification steps. The b-GlcNAcases III, Ib and Ia were isolated 315, 333, 661 folds, respectively. The purified b-GlcNAcases Ib and III were appeared to be homogeneous as examined by coomassie brilliant blue staining and activity staining of the Native- PAGE. The molecular weights of b-GlcNAcase III, Ib and Ia determined by Superdex 200 HR gel filtration were 135、127、110 kDa, respectively. However, smaller molecular sizes of 67, 60 and 48 and 48 kDa for the isoforms III, Ib and Ia, respectively, were observed on the sodium dodecyl sulfate PAGE. These results suggested that these three b-GlcNAcases were oligomeric enzymes containing two subunits.The pI values of the isozymes III, Ib and Ia were 6.3, 6.1, 5.9, respectively. The Km values of forms III, Ib, Ia were 4.71、4.52、3.76 mM and the Vmax values were 1.109，0.769，0.3 unit using p-Nitrophenyl-b-D-N- acetylglucosaminide as substrate. Assays of b-GlcNAcase activity in various tissue of mung bean seedlings revealed that this enzyme is most abundant in cotyledons, followed by roots, stems and leaves.