Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling.
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.
van Loo
Bert
B
Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Spelberg
Jeffrey H Lutje
JH
Kingma
Jaap
J
Sonke
Theo
T
Wubbolts
Marcel G
MG
Janssen
Dick B
DB
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D019020:Directed Molecular Evolution; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics*; D013237:Stereoisomerism
D017931:DNA Primers; D004851:Epoxide Hydrolases