Cofactor Regeneration of NAD⁺from NADH: Novel Water‐Forming NADH Oxidases

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co-substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD+ can be considered solved, the inverse problem of regenerating NAD+ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD+ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.-.-) which can reduce O2 either to undesirable H2O2 or to innocuous H2O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine-annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O2 to H2O.