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Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterizationAuthor(s): Ines Pisanelli; Magdalena Kujawa; Oliver Spadiut; Roman Kittl; Petr Halada; Jindrich Volc; Michael D. Mozuch; Philip Kersten; Dietmar Haltrich; Clemens Peterbauer
Source: Journal of biotechnology. Vol. 142, no. 2 (June 15, 2009): pages 97-106.
Publication Series: Miscellaneous Publication
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DescriptionThe presented work reports the isolation and heterologous expression of the p2ox gene encoding the flavoprotein pyranose 2-oxidase (P2Ox) from the basidiomycete Phanerochaete chrysosporium. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in Escherichia coli. We obtained active, fully flavinylated recombinant P2Ox in yields of approximately 270 mg/lmedium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His6-tag to facilitate simple one-step purification. We obtained an apparently homogenous enzyme preparation with a specific activity of 16.5 U/mg. Recombinant P2Ox from P. chrysosporium was characterized in some detail with respect to its physical and catalytic properties, both for electron donor (sugar substrates) and – for the first time – alternative electron acceptors (1,4-benzoquinone, substituted quinones, 2,6-dichloroindophenol and ferricenium ion). As judged from the catalytic efficiencies kcat/Km, some of these alternative electron acceptors are better substrates than oxygen, which might have implications for the proposed in vivo function of pyranose 2-oxidase.
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CitationPisanelli, Ines; Kujawa, Magdalena; Spadiut, Oliver; Kittl, Roman; Halada, Petr; Volc, Jindrich; Mozuch, Michael D.; Kersten, Philip; Haltrich, Dietmar; Peterbauer, Clemens. 2009. Pyranose 2-oxidase from Phanerochaete chrysosporium : expression in E. coli and biochemical characterization. Journal of biotechnology. Vol. 142, no. 2 (June 15, 2009): pages 97-106.
KeywordsPyranose 2-oxidase, Phanerochaete chrysosporium, lignocellulose degradation, heterologous expression, lignocellulose, biodegradation, enzymes, Basidiomycetes, molecular genetics, fungi, genetics, wood-decaying fung, industrial applications, gene expression, biotechnology, pyranose, Escherichia coli, Bacteria, flavoproteins, recombinant DNA, sugars, microbial metabolism, chemical kinetics, oxygen
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