Laccase stability and activity in diol-based deep eutectic solvents: An experimental and computational study

In pursuit of sustainable alternatives to conventional catalysts, laccase, a multicopper oxidoreductase enzyme, has gained significant attention due to its ability to oxidize a broad range of compounds using molecular oxygen. Its natural occurrence in fungi, plants, bacteria and insects, along with its applicability in bioremediation, wastewater treatment, organic synthesis and various industrial processes, makes it a valuable biocatalyst. However, laccase's stability and activity are often compromised under industrial conditions such as high temperature, variable pH and the presence of inhibitors. Recent advancements have explored enzyme engineering, immobilization, and solvent engineering to address these limitations, but these methods often involve complex, costly or unsustainable procedures. In this search, deep eutectic solvents (DESs), have emerged as promising green solvents to enhance laccase performance. Studies indicate that polyol-based DESs significantly improve both the activity and thermal stability of laccase. Despite this, the specific influence of polyol structure, particularly the number and position of hydroxyl groups remain unclear. This study investigates 15 betaine- and choline chloride-based DESs containing three different diols to elucidate their effect on laccase activity and stability. The findings indicate that only betaine-derived diol-based DESs enhance both the activity and stability of laccase. In particular, DESs with a 1:4 M ratio significantly improves laccase activity. Among them, Betaine:1,2-Propanediol DES exhibited higher laccase activity than Betaine:1,3-Propanediol DES. This suggests that the number and position of hydroxyl groups in the polyols play a crucial role in modulating laccase functionality. Molecular docking supported these findings by showing that DES components form stabilizing hydrogen bonds with amino acids near the catalytic cluster, enhancing activity. In addition, molecular dynamics (MD) simulations provided atomistic insights into these interactions, demonstrating that Betaine:1,2-Propanediol (1:4) maintained laccase compactness, optimal residue flexibility and stable hydrogen-bonding networks, in agreement with experimental trends. Overall, the combined experimental and computational results reveal that the structural features of polyols in DESs critically determine their ability to modulate laccase activity and stability. These findings provide a framework for rationally designing DESs as sustainable co-solvents to improve laccase biocatalysis for diverse industrial applications.