Asymmetric reduction of ($\it R$)‐carvone through a thermostable and organic‐solvent‐tolerant ene‐reductase

Abstract

Ene‐reductases allow regio‐ and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene‐reductase $\it F$OYE‐1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel $\it F$OYE‐1 to produce valuable compounds. A maximum activity of (319.7$\pm$3.2) U mg$^{−1}$ with NADPH or of (206.7$\pm$3.4) U mg$^{−1}$ with 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) for the reduction of $\it N$‐methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: $\it F$OYE‐1 showed excellent performance in most systems with up to 20 vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200 mL biotransformation of 750 mg ($\it R$)‐carvone afforded 495 mg of (2$\it R$,5$\it R$)‐dihydrocarvone (>95 % $\it ee$), demonstrating the simplicity of handling and application of $\it F$OYE‐1