Ectoine enhances the catalytic performance of lipase in the solvent-free synthesis of feruloylated acylglycerols.

In a solvent-free system, Candida rugosa lipase (CRL) catalyzes the transesterification reaction between ethyl ferulate and triolein to yield feruloyl oleoyl glycerol, a lipophilic food antioxidant. However, this reaction is limited by temperature, time, and water activity, posing significant challenges to improving enzymatic efficiency and reaction yield. Inspired by this, this study added the enzyme protector ectoine to the reaction system. The results showed that low concentrations (2.0 mmol/L) of ectoine can significantly enhance the catalytic performance of CRL, achieving a maximum conversion rate of 63.6% under optimized conditions (50 °C, 96 h, Aw = 0.54), substantially higher than the control (42.7%). After 9 batch cycles, the conversion rate remained at 12.5%, whereas the control lost activity after 6 cycles (<5%). Mechanistically, ectoine improves CRL's thermal stability, ethanol tolerance, reusability, and selectivity through two complementary pathways: the priority exclusion model and the water substitution hypothesis. Kinetic and thermodynamic analyses revealed that ectoine increased the activation energy for thermal denaturation from 20.60 to 24.35 kJ/mol and extended the half-life at 50 °C from 17.20 to 20.44 h. Spectroscopic characterization indicated that the conformational changes induced by ectoine are consistent with the retention of α-helix content under both thermal stress (from 25.4% to 29.1% at 70 °C) and low-Aw conditions (from 35.6% to 39.6% at Aw = 0.11). These findings demonstrate the potential of ectoine as an effective additive for improving lipase performance in solvent-free transesterification systems and provide mechanistic insights into the stabilization of enzyme conformation under non-aqueous conditions.