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

Abstract

AbstractA 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 O2 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.