Engineering Oxygen-Independent\nNADH Oxidase Integrated\nwith Electrocatalytic FAD Cofactor Regeneration

An electrochemically\nmediated enzyme process for nicotinamide adenine\ndinucleotide (NADH) oxidation and biosensing has been developed in\nwhich the oxygen-dependent activities of wild-type NADH oxidase are\nreplaced by electrochemical regeneration of the flavin adenine dinucleotide\n(FAD) cofactor in the active site. Consequently, the present bioelectrocatalysis\ndoes not rely on a continuous oxygen supply through bubbling air or\npure oxygen in biosynthetic applications, which reduces enzyme stability.\nThe coupled electrochemical and enzymatic catalysis is achieved through\na combination of enzyme immobilization on the electrode and electrochemical\noxidation of FADH<sub>2</sub> in the active site mediated by the electron\ntransfer mediator ferrocene carboxylic acid (FcCA). Furthermore, to\nminimize the effect of dissolved oxygen when the electrocatalytic\nprocess is exposed to air, we successfully designed mutations at the\nLeu40 and Cys42 sites of Leuconostoc mesenteroides (<i>Lm</i>NOx) to block the oxygen passage into the active\nsite and to eliminate the native FAD cofactor regeneration half-reaction.\nThe engineered enzymes, whose activities are significantly reduced\nor inactive in solution, are electrocatalytically active toward conversion\nof NADH to NAD<sup>+</sup>, demonstrating successful FAD cofactor\nregeneration in the active site via electrochemistry. Finally, we\ndeveloped two highly responsive electrochemical biosensors for NADH\ndetection which has a superior substrate specific to standard detectors\nusing metal electrodes, and comparable detection range and detection\nlimit (1–3 μM).