<br>工程设计与电催化 FAD 辅因子再生集成的不依赖氧的 NADH 氧化酶,JACS Au

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工程设计与电催化 FAD 辅因子再生集成的不依赖氧的 NADH 氧化酶
JACS Au ( IF 8.5 ) Pub Date : 2024-08-21 , DOI: 10.1021/jacsau.4c00528
Mengjie Hou _{1,

2} , Jing Yuan ₂ , Xinyu Dong ₂ , Yingjie Wang ₂ , Shihe Yang _{3,

4} , Jiali Gao _{1,

2,

5}

Affiliation

开发了一种用于烟酰胺腺嘌呤二核苷酸 (NADH) 氧化和生物传感的电化学介导酶过程，其中野生型 NADH 氧化酶的氧依赖性活性被活性位点黄素腺嘌呤二核苷酸 (FAD) 辅因子的电化学再生所取代。因此，目前的生物电催化在生物合成应用中不依赖于通过鼓泡空气或纯氧的连续供氧，这降低了酶的稳定性。耦合电化学和酶催化是通过电极上的酶固定和电子转移介体二茂铁羧酸 (FcCA) 介导的活性位点 FADH ₂的电化学氧化相结合来实现的。此外，为了最大限度地减少电催化过程暴露在空气中时溶解氧的影响，我们成功地在肠系膜明串珠菌( Lm NOx) 的 Leu40 和 Cys42 位点设计了突变，以阻止氧气进入活性位点并消除天然 FAD辅因子再生半反应。这些工程酶的活性在溶液中显着降低或失活，但对 NADH 转化为 NAD ⁺具有电催化活性，证明通过电化学在活性位点成功再生 FAD 辅因子。最后，我们开发了两种用于 NADH 检测的高响应电化学生物传感器，其具有针对使用金属电极的标准检测器的卓越底物，以及相当的检测范围和检测限（1-3 μM）。

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Engineering Oxygen-Independent NADH Oxidase Integrated with Electrocatalytic FAD Cofactor Regeneration

An electrochemically mediated enzyme process for nicotinamide adenine dinucleotide (NADH) oxidation and biosensing has been developed in which the oxygen-dependent activities of wild-type NADH oxidase are replaced by electrochemical regeneration of the flavin adenine dinucleotide (FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis does not rely on a continuous oxygen supply through bubbling air or pure oxygen in biosynthetic applications, which reduces enzyme stability. The coupled electrochemical and enzymatic catalysis is achieved through a combination of enzyme immobilization on the electrode and electrochemical oxidation of FADH₂ in the active site mediated by the electron transfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to minimize the effect of dissolved oxygen when the electrocatalytic process is exposed to air, we successfully designed mutations at the Leu40 and Cys42 sites of Leuconostoc mesenteroides (LmNOx) to block the oxygen passage into the active site and to eliminate the native FAD cofactor regeneration half-reaction. The engineered enzymes, whose activities are significantly reduced or inactive in solution, are electrocatalytically active toward conversion of NADH to NAD⁺, demonstrating successful FAD cofactor regeneration in the active site via electrochemistry. Finally, we developed two highly responsive electrochemical biosensors for NADH detection which has a superior substrate specific to standard detectors using metal electrodes, and comparable detection range and detection limit (1–3 μM).

更新日期：2024-08-21

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