Directed evolution of a glycosynthase via chemical complementation
Directed evolution of a glycosynthase via chemical complementation
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DOI：
10.1021/ja046238v
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发表时间：
2004-11-24
影响因子：
15
通讯作者：
Cornish, VW
中科院分区：
文献类型：
--
作者：
Lin, HN;Tao, HY;Cornish, VW
Recently, we reported a general assay for enzyme catalysis based on the yeast three-hybrid assay, Chemical Complementation, which is intended to expand the range of chemical reactions to which directed evolution can be applied. Here, Chemical Complementation was applied to a glycosynthase derived from a retaining glycosidase, an important class of enzymes for carbohydrate synthesis. Using the yeast three-hybrid assay, the glycosynthase activity of the E197A mutant of the Cel7B from Humicola insolens was linked to transcription of a LEU2 reporter gene, making cell growth dependent on glycosynthase activity in the absence of leucine. Then the LEU2 selection was used to isolate the most active glycosynthase from a Glul 97 saturation library, yielding an E197S Ce17B variant with a 5-fold increase in glycosynthase activity. These results not only establish Chemical Complementation as a platform for the directed evolution of glycosynthases, but also show the generality of this approach and the ease with which it can be applied to new chemical reactions.
