Dual role of ionic liquid modified β-Cyclodextrin in dynamic conformational regulation and substrate enrichment for improved lipase immobilization

To address the limitations of free enzymes in industrial applications, immobilization technologies have garnered significant attention as a robust strategy for enhancing operational stability and reusability. In this work, a novel-designed immobilized lipase (Fe₃O₄@β-CD-IL-CRL) was successfully developed via the introduction of β-cyclodextrin (β-CD), Fe₃O₄ nanoparticles and ionic liquid (IL). The properties studies indicated that Fe₃O₄@β-CD-IL-CRL exhibited remarkable stability, maintaining 72.8 % residual activity after 7 successive catalytic cycles and retaining more than 80 % of its initial activity following 2 h incubation at 60 °C. Furthermore, to examine the practical applications of Fe₃O₄@β-CD-IL-CRL, it was used in the synthesis of phytosterol esters. The results suggested that a yield of 95.0 % can be obtained at 50 °C in 48 h, highlighting its exceptional catalytic efficiency. Molecular dynamics (MD) simulations showed that the immobilization of Fe₃O₄@β-CD-IL maintained an optimal pocket size for CRL, which was advantageous for substrate anchoring. Additionally, analyses of the lid structure distance and tunnel further supported this finding. Beyond the impact on the dynamic conformation of lipase, β-CD was also found to effectively enrich substrates, enhancing catalytic efficiency. This discovery offers new insights into the optimization of biocatalytic systems and highlights the potential of β-CD as a versatile carrier for enzyme immobilization.