Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling

Bert Van Loo, Jeffrey H.Lutje Spelberg, Jaap Kingma, Theo Sonke, Marcel G. Wubbolts, Dick B. Janssen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

105 Citations (Scopus)

Abstract

The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.

Original languageEnglish
Pages (from-to)981-990
Number of pages10
JournalChemistry and Biology
Volume11
Issue number7
Early online date23 Jul 2004
DOIs
Publication statusPublished - 23 Jul 2004
Externally publishedYes

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