Linkers in Action: Exploring Fusion Enzymes for Oxyfunctionlizations in Non‐Conventional Media Through Experiments and Simulations

Abstract Baeyer–Villiger monooxygenases (BVMOs) are key for the selective oxidation of ketones into diverse (cyclic) esters. However, challenges like oxygen and cofactor dependence and substrate/product inhibition hinder their broader application. To address some of these issues, nonconventional media have been applied; still, they lack certain water required for enzyme hydration and cofactor regeneration, reducing activity and/or stability. Fusion approaches enable efficient cofactor recycling by shortening the diffusion distance between enzyme active sites in cascades, especially under low‐water conditions. Trial‐and‐error linker design and time‐intensive construction of fusion enzymes substantially slow down the development of fusion enzymes. In this study, we present the work on the fusions of cyclohexanone monooxygenase (CHMO) and alcohol dehydrogenase (ADH) with linkers owing varying lengths and flexibility in both orientations in nonconventional media, focusing on understanding the effects of linkers on the structural and catalytic properties of fusion enzymes. As such, 12 new fusion enzymes were constructed and evaluated regarding the kinetics, specific activity, and stability, identifying the optimal ones for the linear oxyfunctionlization cascade in aqueous–organic biphasic systems. The conformation and flexibility of linkers and the spatial arrangement of fusion enzymes were studied with simulations, which provides a deep understanding of linkers’ influence and offers insights into the rational design of fusion enzymes.