Dong_2025_Int.J.Biol.Macromol__143101

This study presents a simple and efficient approach for enzyme immobilization using magnetic aldehyde-functionalized ionic liquids, with acetylcholinesterase (AChE) as a model enzyme. We synthesized a new magnetic particle, Fe(3)O(4)@SiO(2)@[ImBa][Cl], by modifying silica-coated Fe(3)O(4) with the ionic liquid 4-(imidazol-1-yl)benzaldehyde hydrochloride ([ImBa][Cl]), whose structure was characterized by SEM, TEM, FT-IR, EDS, TGA and VSM. Immobilization of AChE occurred through both physical adsorption and covalent bonding, driven by electrostatic interaction between cations of the ionic liquid and AChE, as well as Schiff base reaction between the aldehyde groups and protein amines. We identified the optimal immobilization conditions including solution pH (7), AChE concentration (0.8 mg/mL) and incubation time (90 min), under which the immobilization yield reached 16.38 microg/mg. Compared to free AChE, the immobilized AChE exhibited superior substrate affinity and catalytic activity based on kinetic study, also outperforming traditional covalent immobilization methods that utilized glutaraldehyde as cross-linker. Furthermore, the immobilized AChE demonstrated excellent reusability, as well as enhanced storage and thermal stability compared to free AChE. Additionally, it was employed for evaluation of drug inhibitory activity, which could be used for relevant drug discovery. This method represents a pioneering application of magnetic aldehyde-functionalized ionic liquids for simple and efficient immobilization of enzymes.