Pyranose 2-oxidase from Phanerochaete chrysosporium--expression in E. coli and biochemical characterization.
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). As judged from the catalytic efficiencies k(cat)/K(m), 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.
Pisanelli
Ines
I
Department of Food Sciences and Technology, BOKU, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Kujawa
Magdalena
M
Spadiut
Oliver
O
Kittl
Roman
R
Halada
Petr
P
Volc
Jindrich
J
Mozuch
Michael D
MD
Kersten
Philip
P
Haltrich
Dietmar
D
Peterbauer
Clemens
C
D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008031:Lignin / Q000378:metabolism; D008969:Molecular Sequence Data; D020075:Phanerochaete / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
D016227:Benzoquinones; D011994:Recombinant Proteins; C036909:lignocellulose; C004532:quinone; D008031:Lignin; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase