Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 126, Issue 46, Pages 15051-15059Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja046238v
Keywords
-
Categories
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available