cDNA cloning of enzymes involved in the synthesis and degradation of cell wall-matrix polysaccharides

细胞壁的力学性质是由细胞壁构成分子，特别是基质多糖决定的。本研究的目的是克隆与木葡聚糖和阿拉伯木聚糖合成和降解有关的酶的cDNA，这些酶决定细胞壁的可扩展性。结果表明：(1)基质多糖降解酶：用NaCl对木葡聚糖降解酶进行了增溶。采用Con A-Sepharose、Mono S、Superdex 75和Phenyl Superose色谱柱对酶进行分离纯化。酶在单S柱上被分成3个峰。消除木葡聚糖聚焦侧链的α -聚焦酶被分离成单s结合和不结合部分。(2)基质多糖修饰酶：利用高尔基囊泡组分，筛选阿魏酸与阿拉伯糖聚糖中阿拉伯糖基团酯连接的酶。以燕麦阿拉伯木聚糖和合成阿魏酰辅酶a为底物。不幸的是，由于酶制剂中阿魏酰辅酶a酯酶的高活性，酶活性未被检测到。目前，人们已经做了大量的努力来消除酶制剂中的酯酶。目前的研究将很快导致负责合成和降解基质多糖决定细胞壁力学性质的酶的cDNA克隆，尽管cDNA克隆在3年的研究期间没有完成。

The mechanical properties of cell walls are determined by cell wallconstituting molecules, particularly matrix polysaccharides. The goal of this study is to clone cDNA of enzymes involved in the synthesis and degradation of xyloglucans and arabinoxylans, which are responsible for determining cell wall extensibility. Results are as follows :(1) Matrix polysaccharide-degrading enzymes : Xyloglucan degrading enzymes were solubilized with NaCl from Vigna epicotyl cell walls. The enzymes were fractionated and purified using Con A-Sepharose, Mono S, Superdex 75, and Phenyl Superose column. The enzymes are separated into 3 peaks on Mono S colum. alpha-Fucosidases which eliminate fucos-sede chain of xyloglucans were separated into Mono S-binding and -not binding fractions.(2) Matrix polysaccharide-modifying enzymes : Using Golgi-vesicle fraction, the screening of the enzyme that ester-links ferulic acid to arabinose moieties of arabinoxylans. Oat arabinoxylans and synthesitic feruloyl-CoA were used as the substrates. Unfortunately, the enzyme activity was not detected, because of the high activity of feruloyl-CoA esterase in the enzyme preparation. At present, extensive effort has been done to eliminate the esterase from the enzyme preparation.The present srudy will soon lead to the cloning of cDNA of the enzymes responsible for the synthesis and degradation of matrix polysaccharides determing the mechanical properteis of cell walls, although the cDNA cloning was not done within the research period of 3 years.