Presentation Information
[1ENZ-05]Ternary Natural Deep Eutectic Solvents Enhance Laccase Activity and Thermal Stability through Microenvironment Engineering
○Evanildo Francisco de Souza Jr.1, Xuhai Zhu1, Fang Lu1 (1. State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 110623, China. (China))
Keywords:
biocatalyst,Ternary NADES,Biocatalysis,Enzyme stabilization,Molecular dynamics
Purpose
Laccases (EC 1.10.3.2) are versatile multicopper oxidases with strong potential for lignin valorization, polymer functionalization, biorefinery integration, and environmental remediation. However, their industrial application remains limited by low operational robustness under non-conventional processing conditions. Natural deep eutectic solvents (NADES) have emerged as promising media for enzyme catalysis, but the molecular basis by which ternary formulations modulate laccase behavior remains poorly understood. This study investigated whether rationally designed ternary NADES (T-NADES) could enhance laccase catalytic performance, thermal stability, and structural robustness.
Method
Binary (Gly–Bet, 2:1) and ternary NADES, denoted as Gly–POL–Bet (2:1:1 or 1:1:1), were formulated using glycerol as the primary hydrogen bond donor, betaine as the hydrogen bond acceptor, and structurally distinct secondary polyhydroxylated donors (glucose, xylitol, sorbitol, erythritol, arabinose, and mannose). These systems were evaluated in aqueous solution at 10–85% w/w and characterized by FTIR and thermogravimetric analysis (TG). Enzyme activity (ABTS oxidation), pH response, catalytic efficiency, and thermal inactivation at 70 °C were assessed. Circular dichroism, AutoDock Vina docking, and 200 ns GROMACS molecular dynamics simulations were performed using the selected 50% w/w formulation.
Results
Intermediate T-NADES concentrations (25–50% w/w) yielded the best catalytic performance, indicating that balanced hydration and solvent structuring are critical for enzyme activation. The optimal condition, Gly–Sor–Bet (2:1:1, 50% w/w), increased relative laccase activity to ~190% at pH 7.0 and enhanced catalytic efficiency by more than twofold compared to aqueous buffer. Thermal stability was markedly improved, with the D-value increasing from ~30 min in buffer to ~150 min, while total catalytic output increased approximately fourfold. Circular dichroism suggested enhanced conformational stabilization. Computational analyses indicated that T-NADES reorganized the laccase solvation shell by partially replacing bulk water with a structured hydrogen-bonding network, reducing conformational fluctuations and stabilizing a more compact native-like state.
Consideration
These results show that ternary NADES are not merely passive green solvents, but active solvent-engineering systems capable of modulating the catalytic microenvironment of laccase. Formulations containing sorbitol and glucose demonstrated superior performance, underscoring the importance of hydrogen-bond donor selection in solvent structuring and enzyme stabilization.
Conclusion
Ternary NADES significantly improved laccase activity, thermal stability, and conformational robustness while providing mechanistic insight into solvent-mediated enzyme stabilization. These findings support T-NADES as a promising reaction medium for sustainable biocatalysis, biomass valorization, and advanced biobased processes.
Laccases (EC 1.10.3.2) are versatile multicopper oxidases with strong potential for lignin valorization, polymer functionalization, biorefinery integration, and environmental remediation. However, their industrial application remains limited by low operational robustness under non-conventional processing conditions. Natural deep eutectic solvents (NADES) have emerged as promising media for enzyme catalysis, but the molecular basis by which ternary formulations modulate laccase behavior remains poorly understood. This study investigated whether rationally designed ternary NADES (T-NADES) could enhance laccase catalytic performance, thermal stability, and structural robustness.
Method
Binary (Gly–Bet, 2:1) and ternary NADES, denoted as Gly–POL–Bet (2:1:1 or 1:1:1), were formulated using glycerol as the primary hydrogen bond donor, betaine as the hydrogen bond acceptor, and structurally distinct secondary polyhydroxylated donors (glucose, xylitol, sorbitol, erythritol, arabinose, and mannose). These systems were evaluated in aqueous solution at 10–85% w/w and characterized by FTIR and thermogravimetric analysis (TG). Enzyme activity (ABTS oxidation), pH response, catalytic efficiency, and thermal inactivation at 70 °C were assessed. Circular dichroism, AutoDock Vina docking, and 200 ns GROMACS molecular dynamics simulations were performed using the selected 50% w/w formulation.
Results
Intermediate T-NADES concentrations (25–50% w/w) yielded the best catalytic performance, indicating that balanced hydration and solvent structuring are critical for enzyme activation. The optimal condition, Gly–Sor–Bet (2:1:1, 50% w/w), increased relative laccase activity to ~190% at pH 7.0 and enhanced catalytic efficiency by more than twofold compared to aqueous buffer. Thermal stability was markedly improved, with the D-value increasing from ~30 min in buffer to ~150 min, while total catalytic output increased approximately fourfold. Circular dichroism suggested enhanced conformational stabilization. Computational analyses indicated that T-NADES reorganized the laccase solvation shell by partially replacing bulk water with a structured hydrogen-bonding network, reducing conformational fluctuations and stabilizing a more compact native-like state.
Consideration
These results show that ternary NADES are not merely passive green solvents, but active solvent-engineering systems capable of modulating the catalytic microenvironment of laccase. Formulations containing sorbitol and glucose demonstrated superior performance, underscoring the importance of hydrogen-bond donor selection in solvent structuring and enzyme stabilization.
Conclusion
Ternary NADES significantly improved laccase activity, thermal stability, and conformational robustness while providing mechanistic insight into solvent-mediated enzyme stabilization. These findings support T-NADES as a promising reaction medium for sustainable biocatalysis, biomass valorization, and advanced biobased processes.
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