Pisanelli Ines, Kujawa Magdalena, Spadiut Oliver, Kittl Roman, Halada Petr, Volc Jindrich, Mozuch Michael D, Kersten Philip, Haltrich Dietmar, Peterbauer Clemens
Department of Food Sciences and Technology, BOKU, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
J Biotechnol. 2009 Jun 15;142(2):97-106. doi: 10.1016/j.jbiotec.2009.03.019. Epub 2009 Apr 5.
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.
本研究报道了从担子菌黄孢原毛平革菌中分离出编码黄素蛋白吡喃糖2-氧化酶（P2Ox）的p2ox基因并进行异源表达。将p2ox cDNA插入细菌表达载体pET21a(+)，并在大肠杆菌中成功表达。我们获得了活性、完全黄素化的重组P2Ox，产量约为270 mg/l培养基。重组酶带有N端T7标签和C端His(6)标签，便于一步简单纯化。我们获得了一种表观均一的酶制剂，比活性为16.5 U/mg。对来自黄孢原毛平革菌的重组P2Ox的物理和催化特性进行了较为详细的表征，包括电子供体（糖类底物）以及首次对替代电子受体（1,4-苯醌、取代醌、2,6-二氯靛酚和铁离子）的表征。从催化效率k(cat)/K(m)判断，其中一些替代电子受体是比氧气更好的底物，这可能对所提出的吡喃糖2-氧化酶的体内功能有影响。
