Immobilization and application of protein kinase B (Akt) by magnetic dual-aldehyde-functionalized ionic liquids

Immobilized kinases are capable of catalyzing the in vitro phosphorylation of substrates, offering the advantages of recoverability and reusability. Nevertheless, existing immobilization strategies are often limited by intricate procedures and suboptimal biocompatibility. In this work, we selected Akt (protein kinase B) as a model enzyme and developed an efficient immobilization strategy based on a magnetic aldehyde-functionalized ionic liquid support. Key advantages of this approach include a short immobilization time, avoidance of toxic crosslinkers, and applicability to protein-level phosphorylation. A dual-aldehyde-functionalized ionic liquid was synthesized using 4-(imidazol-1-yl)benzaldehyde as the cation and 3-bromo-4-aldehyde-benzoic acid as the anion. This ionic liquid was anchored onto Fe 3 O 4 to yield the novel magnetic nanocomposite Fe 3 O 4 @SiO 2 @[ImBa][BABa], which was characterized by SEM, TEM, IR, EDS, TGA and VSM analyses. Akt was immobilized onto this carrier via Schiff base covalent linkage between aldehyde and amino groups. The optimal immobilization parameters were identified, and the immobilized Akt exhibited enhanced catalytic activity relative to conventional immobilization approaches. Furthermore, the immobilized enzyme demonstrated superior substrate affinity and storage stability compared with its free counterpart, as well as proof-of-concept reusability over four cycles (>80% residual activity). Importantly, the immobilized Akt retained its ability to catalyze the phosphorylation of both peptide and protein (GSK3β) substrates, underscoring its practical potential. To the best of our knowledge, this study represents the first application of magnetic ionic liquids for kinase immobilization and the first establishment of an immobilization protocol for Akt.