布氏锥虫来源内切-β-N-乙酰氨基葡糖苷酶的异源表达和活性鉴定

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  • (江南大学 生物工程学院,江苏 无锡,214122)

网络出版日期: 2018-09-13

基金资助

国家自然科学基金(21778023);国家自然科学基金(21576118)

Expression, purification and characterization of a novel Endo-β-N-acetylglucosaminidase from Trypanosoma brucei

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  • (School of Biotechnology,Jiangnan University, Wuxi 214122, China)

Online published: 2018-09-13

摘要

内切-β-N-乙酰氨基葡萄糖苷内切酶(Endo-β-N-acetylglucosaminidase,ENGase)是一类可以水解N-糖链中核心壳二糖之间β-1,4-糖苷键的糖苷内切酶,其中糖苷水解酶85家族(glycoside hydrolasefamilies 85, GH85)的ENGase除了水解活性之外还具有转糖基活性,能够以切除后再将均一的寡糖链转移到N-糖蛋白的β-N-乙酰氨基葡萄糖(N-acetylglucosamine, GlcNAc)上的方式,改造医药糖蛋白的N-糖基化修饰。通过数据库检索,在布氏锥虫(Trypanosoma brucei)的基因组中找到了GH85家族ENGase的同源序列,并命名为Endo-Tb基因,对其克隆后在带有Nus融合蛋白的原核(E.coli)表达载体上成功表达并纯化。检测到融合蛋白Nus-Endo Tb能够水解高甘露糖型和双天线的复合型糖链,但不能作用于三天线的复合型糖链。同时Nus-Endo Tb可以水解核糖核酸酶B和人类转铁蛋白上的N糖链。当用唾液酸糖肽(SGP)作为糖基供体,MU-GlcNAc作为糖基受体时,通过荧光基质底物四甲基伞形酮(MU)检测到Nus-Endo Tb具有转糖基活性。

本文引用格式

崔娟, 喜多岛敏彦, 王宁, 等 . 布氏锥虫来源内切-β-N-乙酰氨基葡糖苷酶的异源表达和活性鉴定[J]. 食品与发酵工业, 2018 , 44(8) : 8 -13 . DOI: 10.13995/j.cnki.11-1802/ts.016879

Abstract

Endo-β-N-acetylglucosaminidase (ENGase) belonging to the glycoside hydrolase family 85 (GH85) is a series of dual-functional enzymes that not only hydrolyze the glycosidic bonds of the N,N′-diacetylchitobiose moieties of N-glycan cores but also transfer chemically synthesized-homogeneous glycans onto glycoproteins. This unique transglycosylation activity enables them to modify proteins with a defined N-glycan structure. In this study, we identified ENGase homolog in Trypanosoma brucei genome by database search. The gene encoding ENGase was cloned into an expression vector with an N-terminal Nus tag, and expressed in Escherichia coli cells. The recombinant ENGase (Nus-Endo Tb) exhibited hydrolytic activity for high-mannose, bi-antennary N-linked oligosaccharides. Such hydrolytic activity can be applied to hydrolyze N-glycans attached to RNase B and human transferrin. Moreover, Nus-Endo Tb also can transfer a sialobiantennary type complex oligosaccharide onto MU-GlcNA, which indicated its transglycosylation activity.
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