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

本研究以木葡聚糖内切转移酶为研究对象，建立了一种新的木葡聚糖内切转移酶的定量方法：以具有一定分子量分布的木葡聚糖及其荧光衍生物为供体和受体底物进行酶促反应。该方法首次成功地从赤豆质外体中分离纯化了内切木葡聚糖转移酶。所述酶是分子量约为33000的糖蛋白，并且催化木葡聚糖分子的内切型分裂和木葡聚糖分子的新产生的还原末端与另一分子的非还原末端的连接，从而介导木葡聚糖之间的分子接枝。它不表现出糖苷酶或聚糖酶活性。该酶需要基本的木葡聚糖结构，即：e.具有木糖基侧链的β（1-4）-葡糖基主链，用于受体和供体活性。该酶表现出较高的反应速率时，具有较高的分子量的木葡聚糖用作供体底物。这种酶是第一种鉴定的介导多糖分子之间高分子量片段转移以产生嵌合聚合物的酶。我们得出结论，内切木葡聚糖转移酶作为木葡聚糖的重建酶发挥作用，并参与细胞壁基质的交织或重建，这是负责化学蠕变，导致细胞壁的形态变化。

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.