Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (Imaxapp/KMapp) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.

中文翻译：

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用于生物电催化应用的吡喃糖氧化酶变体的表征。

吡喃糖氧化酶 (POx) 催化 d-葡萄糖氧化为 2-酮葡萄糖，同时将氧气还原为 H2O2。来自 Trametes ochracea (ToPOx) 的 POx 已知会与替代电子受体反应，包括 1,4-苯醌 (1,4-BQ)、2,6-二氯苯酚靛酚 (DCPIP) 和二茂铁离子。在这项研究中，具有改善的电子受体转换和减少的氧转换的酶变体被表征为潜在的阳极生物催化剂。使用停流分光光度法评估 ToPOx 变体 T166R、Q448H、L545C 和 L547R 与这些替代电子受体的氧化半反应的稳态前动力学。对于一些变体，与野生型 ToPOx 相比，观察到更高的动力学常数。随后，将变体固定在玻碳电极上。在 1,4-BQ、DCPIP 或二茂铁甲醇作为氧化还原介质的情况下，进行循环伏安法测量以测量这些变体以葡萄糖作为底物的电化学响应。与野生型 POx 相比，高催化效率 (Imaxapp/KMapp) 证明了这些变体在未来生物电催化应用、生物传感器或生物燃料电池中的潜力。在变体中，L545C 显示出最理想的特性，如动力学和电化学测定。在生物传感器或生物燃料电池中。在变体中，L545C 显示出最理想的特性，如动力学和电化学测定。在生物传感器或生物燃料电池中。在变体中，L545C 显示出最理想的特性，如动力学和电化学测定。