title: Heterologous expression and physicochemical characterization of a fungal dye-decolorizing peroxidase from Auricularia auricula-judae.
abstract: An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5mg per liter of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60°C, but the former was more stable in the 62-70°C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family.
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Linde
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Coscolín
authors.affiliation: TU Dresden - International Institute Zittau, Markt 23, 02763 Zittau, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Liers
authors.affiliation: TU Dresden - International Institute Zittau, Markt 23, 02763 Zittau, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hofrichter
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain. Electronic address: fjruiz@cib.csic.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ruiz-Dueñas
mesh_terms: D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology*; D004396:Coloring Agents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D004396:Coloring Agents; D009195:Peroxidase
keywords: Biphasic kinetics; Dye-decolorizing peroxidase; Escherichia coli expression; In vitro activation; Kinetic constants; Stability properties