Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose
Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose
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DOI：
10.1006/prep.1999.1043
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发表时间：
1999-07-01
影响因子：
1.6
通讯作者：
Farkas, V
中科院分区：
文献类型：
--
作者：
Sulová, Z;Farkas, V
木葡聚糖内糖基转移酶(XET)通过转糖基化机制催化木葡聚糖(XG)分子的裂解，包括两个步骤：(A)通过形成糖基酶中间体使木葡聚糖分子的β(1，4)连接的多葡糖基骨架内切；(B)将糖基残基从另一分子XG或XG衍生的寡糖的中间体转移到非还原末端葡糖单元的C-4位(Z，Sulova等人，1998，Biochem)。在目前的XET纯化方法中，利用了XET与XG形成相对稳定的活性络合物和木葡聚糖通过氢键与纤维素紧密结合的倾向，将玉米子叶粗提物和其他含有XG的植物来源混合，形成XET：XG络合物，并将其应用于纤维素上。通过洗涤去除未吸附的蛋白质，并通过将XET的糖基部分转糖基化到添加XG衍生的寡糖上，将XET从吸附的XET：XG复合物中释放出来。用Bio-Gel P30凝胶渗透层析法进一步纯化XET，可使XET的比活力一步提高100倍以上。类似的方法也可用于从其他来源的植物中提纯XET，如小扁豆种子、豌豆上胚轴和悬浮培养的长春花细胞培养上清液。(C)1999年学术出版社。
Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the C-4 position of the nonreducing end glucosyl unit of another molecule of XG or an XG-derived oligosaccharide with liberation of the enzyme (Z, Sulova et al., 1998, Biochem. J. 330, 1475-1480), The formation of a relatively stable active complex of XET with XG and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose were exploited in the present method of purification of XET, Crude extracts from nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the enzyme were mixed with XG in order to form the XET:XG complex, which was applied onto cellulose. Unadsorbed proteins were removed by washing and the XET was released from the adsorbed XET:XG complex by transglycosylation of its glycosyl moiety to added XG-derived oligosaccharides. The described procedure resulted in an over 100-fold increase in specific activity of XET in a single step, Further purification of the enzyme to homogeneity was achieved by gel-permeation chromatography on Bio-Gel P30. Similar procedure could be used for purification of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea (Pisum sativum) epicotyls, and supernatant of suspension-cultured Catharanthus roseus cells. (C) 1999 Academic Press.