Nov 19, 2004

Directed evolution of a glycosynthase via chemical complementation

Journal of the American Chemical Society
Hening LinVirginia W Cornish

Abstract

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 Glu197 saturation library, yielding an E197S Cel7B 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.

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Citations

Mentioned in this Paper

Directed Evolution
Endoglucanase 1
DLEU2 wt Allele
Enzymes, antithrombotic
CD8A wt Allele
Glycosidase Activity
Transcription, Genetic
T-CELL ANTIGEN LEU2 GENE
Cell Growth
Isolate compound

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