Development of Novel Catalytic Deep Eutectic Solvent for Esterification and Elucidation of Dynamic...

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.