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Improving the Stability and Catalyst Lifetime of the Halogenase RebH By Directed Evolution
Author(s) -
Poor Catherine B.,
Andorfer Mary C.,
Lewis Jared C.
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300780
Subject(s) - halogenation , directed evolution , catalysis , chemistry , enzyme , combinatorial chemistry , stereochemistry , organic chemistry , biochemistry , mutant , gene
Abstract We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3‐LR, with a T opt over 5 °C higher than that of wild‐type, and 3‐LSR, with a T m 18 °C higher than that of wild‐type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non‐natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.