A novel fluorescence-based assay for the transglycosylation activity of endo-β-N-acetylglucosaminidases

被引：12

作者：

Hauser, S ^{[1
]}

Song, HJ ^{[1
]}

Li, HG ^{[1
]}

Wang, LX ^{[1
]}

机构：

[1] Univ Maryland, Inst Biotechnol, Inst Human Virol, Baltimore, MD 21201 USA

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2005年 / 328卷 / 02期

关键词：

endo-beta-N-acetylglucosaminidase; endoglycosidase; fluorescence-based assay; transglycosylation activity; chitinase; glycoconjugate; oligosaccharide;

D O I：

10.1016/j.bbrc.2005.01.012

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases (ENGases), was developed. The assay was based on the findings that a coupled chitinase can specifically capture and hydrolyze the fluorogenic intermediate that is formed by the ENGase-catalyzed transglycosylation to release a fluorophore, but does not hydrolyze the donor asparagine-linked N-glycan and the acceptor 4-methylumbelliferyl N-acetylglucosaminide. The assay method was verified by detecting the transglycosylation activities of the known ENGases. Its application for assessing the effects of organic solvents on transglycosylation activity was demonstrated. The novel coupled assay provides a highly sensitive, easy, and quantitative method for screening endo-beta-N-acetylglucosaminidases with transglycosylation activities useful for glycoconjugate synthesis. (C) 2005 Elsevier Inc. All rights reserved.

引用

页码：580 / 585

页数：6

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