Novel Chemo‐Enzymatic Mimic of Hydrogen Peroxide‐Forming NAD(P)H Oxidase for Efficient Regeneration of NAD⁺ and NADP⁺

Abstract

Abstract The current methods for regeneration of NAD + and NADP + in dehydrogenase‐catalyzed organic synthesis are not technologically mature due to problems with operational stability, kinetic efficiency or thermodynamic equilibrium. We show here that Candida tenuis xylose reductase converts 9,10‐phenanthrenequinone with good turnover frequency when using NADH (12 s −1 ) or NADPH (1.6 s −1 ) as the coenzyme and exhibits high binding affinity for this quinone substrate ( K m ≤13 μM). Because chemical reaction of the hydroquinone product with dissolved molecular oxygen regenerates the quinone at a rate comparable to that of the enzymatic reduction, a chemo‐enzymatic process is established where the reductase, a catalytic concentration of 9,10‐phenanthrenequinone (25 μM) and molecular oxygen promote efficiently the production of NAD(P) + from NAD(P)H. Oxidation of the hydroquinone occurs via a radical chain reaction that involves superoxide as the propagating species and yields a molar equivalent of hydrogen peroxide for each 9,10‐phenanthrenequinone recycled. Using the NAD + ‐ or NADP + ‐dependent transformation of D ‐mannitol (25 mM) into D ‐fructose as a model transformation, the total turnover numbers for 9,10‐phenanthrenequinone and the coenzyme obtained in a single batchwise conversion were 1000 and ≤125, respectively. The yields of ketose product were quantitative, indicating that molecular oxygen reduction drives the thermodynamically unfavourable synthetic reaction. Oxygen transfer to the liquid phase was shown to be rate‐limiting for the overall process under conditions of surface aeration and bubble‐free molecular oxygen supply. Xylose reductase was fully stable during the reaction (25 °C, pH 8.0). The novel chemo‐enzymatic system should therefore be broadly applicable to biocatalytic synthesis with isolated dehydrogenases utilizing NAD + or NADP + . Abbreviations: Ct XR: xylose reductase from Candida tenuis (E.C. 1.1.1.21); M2DH: D ‐mannitol 2‐dehydrogenase (E.C. 1.1.1.67); OTR: O 2 transfer rate; PQ: 9,10‐phenanthrenequinone; PQH 2 : 9,10‐phenanthrene hydroquinone; SOD: superoxide dismutase; STY : space‐time yield; TTN: total turnover number.