Abstract
A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (__Tk__NOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of __Tk__NOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP^+^ and NAD^+^ in alcohol dehydrogenase (ADH)‐catalyzed enantioselective oxidation of racemic 1‐phenylethanol. NADP^+^ regeneration at 30°C was performed by __Tk__NOX coupled with (R)‐specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)‐1‐phenylethanol. The use of __Tk__NOX with moderately thermostable (S)‐specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD^+^ regeneration at high temperatures. Optically pure (R)‐1‐phenylethanol was successfully obtained by this system after a shorter reaction time at 45–60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of __Tk__NOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air. Biotechnol. Bioeng. 2012;109: 53–62. © 2011 Wiley Periodicals, Inc.