van Loo_2004_Chem.Biol_11_981

Reference

Title : Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling - van Loo_2004_Chem.Biol_11_981
Author(s) : van Loo B , Spelberg JH , Kingma J , Sonke T , Wubbolts MG , Janssen DB
Ref : Chemical Biology , 11 :981 , 2004
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.

PubMedSearch : van Loo_2004_Chem.Biol_11_981
PubMedID: 15271356
Gene_locus related to this paper: agrra-echA

Related information

Substrate pNPGE    Epichlorohydrin    Styrene-oxide
Gene_locus agrra-echA

Citations formats

van Loo B, Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB (2004)
Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling
Chemical Biology 11 :981

van Loo B, Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB (2004)
Chemical Biology 11 :981