Cofactor regeneration of both NAD+ from NADH and NADP+ from NADPH:NADH oxidase from Lactobacillus sanfranciscensis

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 H2O, 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.