title: Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme.
abstract: cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 degrees C in amount corresponding to 8% of the cell soluble protein (0.6 Umg(-1)). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 degrees C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.
authors.affiliation: Laboratory of Enzyme Technology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Maresová
authors.forename: Branislav
authors.initials: B
authors.lastname: Vecerek
authors.forename: Marcela
authors.initials: M
authors.lastname: Hradská
authors.forename: Nathalie
authors.initials: N
authors.lastname: Libessart
authors.forename: Stanislav
authors.initials: S
authors.lastname: Becka
authors.forename: Marie-Helene
authors.initials: MH
authors.lastname: Saniez
authors.forename: Pavel
authors.initials: P
authors.lastname: Kyslík
mesh_terms: D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D002479:Inclusion Bodies / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D017510:Protein Folding; D013045:Species Specificity
chemical_list: D005656:Fungal Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase