Cofactor Regeneration of both NAD+ from NADH and NADP+ from NADPH:NADH Oxidase from Lactobacillus sanfranciscensis

Abstract A possible solution for the regeneration of NAD + from NADH is the oxidation of NADH with concomitant reduction of oxygen catalyzed by NADH oxidase (E. C. 1.6.‐.‐). We employ NADH oxidase from Lactobacillus sanfranciscensis , which reduces O 2 to innocuous H 2 O, and ( R )‐alcohol dehydrogenase [( R )‐ADH] from Lactobacillus brevis to perform enantioselective oxidation of racemic phenylethanol to acetophenone and ( S )‐phenylethanol with regeneration of either NADH or NADPH to their respective oxidized precursors. NADH oxidase from L. sanfranciscensis accepts both NADH and NADPH; in contrast, the wild‐type ( R )‐ADH only accepts NADP(+)(H) whereas its G37D mutant strongly prefers NAD(+)(H). Highly purified. NADH oxidase (221 U/mg, two‐step protocol) was coupled with wild‐type ADH from L. brevis on NADP(H) and mutant ADH from L. brevis on NAD(H) to achieve 50% conversion of racemic phenylethanol to ( S )‐phenylethanol and acetophenone. Depending on the relative concentration of alcohol to cofactor, up to more than 100 turnovers were observed. We believe that this is the first demonstration of a regeneration scheme for both NAD + from NADH and NADP + from NADPH with the same enzyme.