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

本研究的目的是纯化和表征内切木葡聚糖转移酶，一种能够催化木葡聚糖分子片段转移到另一分子的新型酶。我们开发了一种新的方法来定量这种新酶：纯化的具有确定分子量分布的木葡聚糖及其荧光衍生物分别作为酶反应的供体和受体底物。该方法首次成功地纯化了角藤蔓外质体的内切木葡聚糖转移酶。该酶是一种糖蛋白，分子量约为33000，既催化木葡聚糖分子的内型分裂，又催化新生成的木葡聚糖分子的还原端与另一分子的非还原端连接，从而介导木葡聚糖之间的分子接枝。它没有糖苷酶或糖聚糖酶活性。转移酶需要基本的木葡聚糖结构，即β(1-4)-葡萄糖基主链和木基侧链，以保证受体和供体活性。以分子量较大的木葡聚糖为底物时，酶的反应速率较高。该酶是第一个被鉴定的介导多糖分子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，参与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动的原因。

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.