title: A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.
abstract: A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.
authors: Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.
authors: Lilian L
authors: LL
authors: Beloti
authors: Bruna Z
authors: BZ
authors: Costa
authors: Marcelo A S
authors: MA
authors: Toledo
authors: Clelton A
authors: CA
authors: Santos
authors: Aline
authors: A
authors: Crucello
authors: Marianna T P
authors: MT
authors: Fávaro
authors: André S
authors: AS
authors: Santiago
authors: Juliano S
authors: JS
authors: Mendes
authors: Anita J
authors: AJ
authors: Marsaioli
authors: Anete P
authors: AP
authors: Souza
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism
chemical_list: D004852:Epoxy Compounds; D005656:Fungal Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; C013690:styrene oxide; D004851:Epoxide Hydrolases