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 Escher...
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Veröffentlicht in: | Journal of biotechnology 2009-06, Vol.142 (2), p.97-106 |
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Sprache: | eng |
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Zusammenfassung: | 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. |
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ISSN: | 0168-1656 1873-4863 |
DOI: | 10.1016/j.jbiotec.2009.03.019 |