Exploring DES‐Water Mixtures for Improved ADH‐CHMO Fusion Enzyme Performance in Linear Cascades

Abstract The application of multienzyme cascade reactions in nonaqueous media has garnered attention due to their potential in Green Chemistry. Deep eutectic solvents (DESs) have emerged as a novel alternative with high tunability. This study investigated the use of fusion enzymes, combining an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO), for a linear cascade stepwise oxidation of cyclohexanol to ɛ‐caprolactone with in situ cofactor regeneration in DES‐water mixtures. The stability of fusion enzymes was determined in the two DESs comprising glycerol and betaine or choline acetate at the same molar ratio (Bet–Gly, ChAc–Gly, 1:2) with various water contents (20–80 vol.%), as well as in pure buffer for comparison. Bet–Gly significantly improved enzyme thermostability at ≥40 vol.% water and doubled the half‐life time of CHMO, whereas ChAc–Gly showed a deleterious effect. The improved thermostability promoted the further optimization of the cascade reaction while addressing the high viscosity of DESs through increasing water ratios and temperatures. The reaction efficiency in DES‐buffer mixtures was comparable to the pure buffer system (97.0% yield in 24 h), while product hydrolysis was completely avoided. High substrate loadings (8 g/L cyclohexanol) and extended substrate ranges were accomplished. These findings highlight the potential of DES‐enriched media to improve catalytic efficiency and further advance sustainable biocatalytic processes.