Directed evolution of new glycosynthases from Agrobacterium β-glucosidase:: a general screen to detect enzymes for oligosaccharide synthesis

被引:88
作者
Mayer, C
Jakeman, DL
Mah, M
Karjala, G
Gal, L
Warren, RAJ
Withers, SG
机构
[1] Univ British Columbia, Prot Engn Network, Ctr Excellence Canada, Dept Chem, Vancouver, BC V6T 1Z1, Canada
[2] Univ British Columbia, Prot Engn Network, Ctr Excellence Canada, Dept Microbiol & Immunol, Vancouver, BC V6T 1Z1, Canada
来源
CHEMISTRY & BIOLOGY | 2001年 / 8卷 / 05期
基金
加拿大自然科学与工程研究理事会;
关键词
directed evolution; enzymatic oligosaccharide synthesis; glycosynthase; glycosyl transfer;
D O I
10.1016/S1074-5521(01)00022-9
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
Background: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes. Results: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants. Conclusions: plate-based coupled-enzyme screens of this type are extremely valuable fur identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases. (C) 2001 Published by Elsevier science Ltd.
引用
收藏
页码:437 / 443
页数:7
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