Development of Novel Catalytic Deep Eutectic Solvent for Esterification and Elucidation of Dynamic Solvent-Enzyme-Substrate Adaptability.

Abstract

Biocatalytic ester synthesis often encounters bottlenecks like vast substrate polarity differences and reliance on toxic solvents or activated acyl donors, severely limiting industrial expansion. To address this, a novel catalytic deep eutectic solvent (CDES) system composed of cyclohexanone, stearic acid, and N-methylimidazole (CS-NMI) was developed. This study utilized an extreme esterification, sucrose stearate synthesis, as a model reaction due to the immense substrate sizes and polarity differences. After optimization, the conversion for sucrose stearate synthesis reached 98.2± 0.7%, remaining at 90.5± 0.8% even after 10 reuses of the lipase. More crucially, enzymatic esterification of sucrose with inexpensive stearic acid was achieved, yielding 42.8± 0.5% conversion. To verify the system's broad applicability, the synthesis of vitamin E succinate, menthyl acetate, and propyl laurate were performed, all achieving yields over 85.0% without optimization. To further expand the enzymatic universality and industrial application potential of CS-NMI, an in-house Candida antarctica lipase B (CALB) mutant, X1, was employed to synthesize sucrose stearate in CS-NMI. Following scale-up in a 5 L fermenter (achieving a fermentation enzyme activity of 6.7± 1.9 U/mL without concentration) and kilogram-scale immobilization, this non-commercial enzyme X1 attained a remarkable 98.1± 1.3% conversion for sucrose stearate synthesis. Molecular dynamics (MD) simulations indicated that CS-NMI maintained the enzyme's dynamic conformation, reconstructed the hydrophobic microenvironment of the binding pocket, and enhanced mass transfer within the tunnel. Overall, the highly adaptable solvent-enzyme-substrate synergistic system constructed in this study provides a sustainable and universal strategy for complex biocatalytic esterification.