Pyranose 2-oxidase from Phanerochaete chrysosporium--Expression in E.coli and Biochemical Characterization

0338755 - MBÚ 2010 RIV NL eng J - Journal Article
Pisanelli, I. - Kujawa, M. - Spadiut, O. - Kittl, R. - Halada, Petr - Volc, Jindřich - Mozuch, M. D. - Kersten, P. - Haltrich, D. - Peterbauer, C.
Pyranose 2-oxidase from Phanerochaete chrysosporium--Expression in E.coli and Biochemical Characterization.
Journal of Biotechnology. Roč. 142, č. 2 (2009), s. 97-106. ISSN 0168-1656. E-ISSN 1873-4863
Institutional research plan: CEZ:AV0Z50200510
Keywords : Pyranose 2-oxidase * Phanerochaete chrysosporium * Lignocellulose degradation
Subject RIV: CE - Biochemistry
Impact factor: 2.881, year: 2009

The 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/l medium. The recombinant enzyme was provided with an N-terminal T7-tag and a C-terminal His(6)-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)
Permanent Link: http://hdl.handle.net/11104/0182446