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Type: Artigo de periódico
Title: A Novel And Enantioselective Epoxide Hydrolase From Aspergillus Brasiliensis Cct 1435: Purification And Characterization.
Author: Beloti, Lilian L
Costa, Bruna Z
Toledo, Marcelo A S
Santos, Clelton A
Crucello, Aline
Fávaro, Marianna T P
Santiago, André S
Mendes, Juliano S
Marsaioli, Anita J
Souza, Anete P
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.
Subject: Amino Acid Sequence
Chromatography, Affinity
Circular Dichroism
Epoxide Hydrolases
Epoxy Compounds
Escherichia Coli
Fungal Proteins
Molecular Sequence Data
Recombinant Fusion Proteins
Sequence Alignment
Citation: Protein Expression And Purification. v. 91, n. 2, p. 175-83, 2013-Oct.
Rights: fechado
Identifier DOI: 10.1016/j.pep.2013.08.001
Date Issue: 2013
Appears in Collections:Unicamp - Artigos e Outros Documentos

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