Cajnko_2025_Int.J.Biol.Macromol__149523

Deep eutectic solvents (DES), a new generation of (green) solvents, are increasingly used in enzymatic reactions as stabilization media. However, the roles of individual components and water content remain poorly understood. We investigated the activating and thermostabilizing effects of various DES compositions and their water dilutions on immobilized lipase B from Candida antarctica. Our results show that enzyme thermostabilization strongly depends on both factors. Certain hydrogen-bond donor (HBD) and acceptor (HBA) combinations act synergistically even at high dilutions, while others exhibit inhibitory effects dominated by a single component. The highest relative activity of 13.1 was observed in choline chloride (ChCl):Glycerol (1:1 ratio; 10 wt% water), whereas a 75 wt% aqueous solution of ChCl exhibited relative activity of 10.7. Molecular docking simulations with individual DES components showed binding of choline near the enzyme's catalytic triad, suggesting stabilization without competing with substrate binding. Our findings highlight the importance of HBD/HBA selection, water content, and component-specific enzyme interactions, and offer experimental and atomistic insights for efficient and targeted design of DES formulations for biotechnological applications.