title: Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.
abstract: Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.
authors.affiliation: Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, Av. Arlindo Béttio, 1000 São Paulo, Brazil.
authors.forename: Gabriel Stephani
authors.initials: GS
authors.lastname: de Oliveira
authors.affiliation: Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, Av. Arlindo Béttio, 1000 São Paulo, Brazil.
authors.forename: Patricia Pereira
authors.initials: PP
authors.lastname: Adriani
authors.affiliation: Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, Av. Arlindo Béttio, 1000 São Paulo, Brazil.
authors.forename: Flavia Garcia
authors.initials: FG
authors.lastname: Borges
authors.affiliation: Instituto Butantan, Av. Vital Brasil, 1500 São Paulo, Brazil.
authors.forename: Adriana Rios
authors.initials: AR
authors.lastname: Lopes
authors.affiliation: Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, Av. Arlindo Béttio, 1000 São Paulo, Brazil.
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Campana
authors.affiliation: Escola de Artes, Ciências e Humanidades, Universidade de São Paulo, Av. Arlindo Béttio, 1000 São Paulo, Brazil. Electronic address: fscha@usp.br.
authors.forename: Felipe S
authors.initials: FS
authors.lastname: Chambergo
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D003001:Cloning, Molecular; D004851:Epoxide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D004852:Epoxy Compounds; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases
keywords: Dichroism spectroscopy; Enantioselectivity; Epoxide hydrolase; Trichoderma reesei