title: Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution.
abstract: We describe here a strategy to improve the expression efficiency and enantioselectivity of Aspergillus niger epoxide hydrolase (ANEH) by directed evolution. Based on a blue-colony screening system using the LacZα (β-galactosidase α peptide) complementation solubility reporter, several ANEH variants out of 15 000 transformants from a random-mutagenesis library were identified that show improved recombinant expression in E. coli. Among them, Pro221Ser was subsequently used as a template for iterative saturation mutagenesis (ISM) at sites around the ANEH binding pocket. Following four rounds of ISM, a highly enantioselective mutant was identified that catalyzes the hydrolytic kinetic resolution of racemic glycidyl phenyl ether with a selectivity factor of E=160 in favor of the (S)-diol compared to WT ANEH characterized by E=4.6. Expression of this mutant is 50 times higher than that of WT ANEH. It also serves as an excellent stereoselective catalyst in the hydrolytic kinetic resolution and desymmetrization of several other structurally diverse epoxides.
authors.affiliation: Department of Synthetic Organic Chemistry, Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr, Germany. reetz@mpi-muelheim.mpg.de
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.forename: Huabao
authors.initials: H
authors.lastname: Zheng
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008958:Models, Molecular; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001616:beta-Galactosidase; D004851:Epoxide Hydrolases