Purification and characterization of endo-xyloglucan transferas from apoplast of plants

本研究的目的是纯化和鉴定内切木聚糖转移酶，这是一种能够催化一段木聚糖分子转移到另一段木聚糖分子的新型酶。建立了一种新的定量方法：将具有一定分子量分布的纯化的木聚糖及其荧光衍生物分别作为酶反应的供体和受体底物。这一新工艺成功地用于首次从苦豆质外体中分离纯化内切木糖葡聚糖转移酶。该酶是一种分子质量约为33000的糖蛋白，既催化木葡聚糖分子的内切型裂解，又催化新生成的木聚糖分子的还原末端连接到另一分子的非还原末端，从而介导木聚糖之间的分子接枝。它不显示糖苷酶或葡聚糖酶活性。转移酶需要基本的木葡聚糖结构，即带有木糖基侧链的β(1-4)-葡糖主链，才能获得受体和供体的活性。以相对分子质量较高的木聚糖为供体底物时，酶的反应速率较高。这种酶是第一种被发现的酶，它介导高相对分子质量片段在多糖分子之间的转移，从而产生嵌合聚合物。我们认为，内切木糖葡聚糖酒石酸酶是木葡聚糖的一种重建酶，参与了细胞壁基质的交织或重建，这是导致细胞壁形态变化的化学蠕变的原因。

The goal of this research is purificatin and characterization of endo-xyloglucan transferase, a novel enzyme capable of catalyzing transfer of a segment of one xyloglucan molecule to another.A new approach was developed for quantification of this new enzyme : Purified xyloglucans with defined molecular weight distributions and their fluorescent derivatives were used as donor and acceptor substrates, respectively, for the enzyme reaction. This new procedure was successfully exploited for the first purification of endo-xyloglucan transferase from apoplast of the Vigna angularis. The enzyme is a glycoprotein with a molecular weight of about 33000 and catalyzes both endo-type splitting of a xyloglucan molecule and linking of a newly generated reducing end of the xyloglucan molecule to the non-reducing terminus of another molecule, thereby mediating molecular grafting between xyloglucans. It exhibits no glycosidase or glycanase activity. The transferase required basic xyloglucan structure, i. e. a beta(1-4)-glucosyl backbone with xylosyl side chains, for both acceptor and donor activity. The enzyme exhibited higher reaction rates when xyloglucans with higher molecular weight were used as donor substrates. This enzyme is the first enzyme identified that mediates the transfer of a high molecular weight segment between polysaccharide molecules to generate chimeric polymers. We conclude that endo-xyloglucan tarnsferase functions as a reconstructing enzyme for xyloglucans and is involved in the interweaving or reconstruction of cell wall matrix, which is responsible for chemical creepage that leads to morphological changes in the cell wall.