title: Laboratory evolution of an epoxide hydrolase - towards an enantioconvergent biocatalyst.
abstract: We performed a laboratory evolution study with the epoxide hydrolase from Aspergillus niger M200. This enzyme exhibits no enantioconvergence with the substrates styrene oxide or para-chlorostyrene oxide, i.e. racemic vicinal diols are produced from the racemic substrates. After saturation mutagenesis, screening by chiral gas chromatography revealed enzyme variants with improved enantioconvergence as manifested by an increased enantiomeric excess of the diol product. Nine amino acid exchanges accumulated in the active site and the substrate access tunnel over the course of 5 productive rounds of iterative saturation mutagenesis, resulting in an enantioconvergent epoxide hydrolase variant. The final mutant enzyme transformed racemic styrene oxide and para-chlorostyrene oxide to (R)-diol enantiomers with enantiomeric excesses of 70%. Sequential bi-enzymatic reactions using the wild-type EH and/or its evolved variants enabled preparation of the chiral building blocks (R)-phenyl-1,2-ethanediol and (R)-para-chlorophenyl-1,2-ethanediol from inexpensive racemic epoxides with enantiomeric excesses of 91% and 88%, respectively.
authors.affiliation: Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic. kotik@biomed.cas.cz
authors.forename: Michael
authors.initials: M
authors.lastname: Kotik
authors.forename: Alain
authors.initials: A
authors.lastname: Archelas
authors.forename: Veronika
authors.initials: V
authors.lastname: Faměrová
authors.forename: Pavla
authors.initials: P
authors.lastname: Oubrechtová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D004852:Epoxy Compounds; C013690:styrene oxide; D004851:Epoxide Hydrolases