Fe₃O₄ Nanoparticle-Polyacrylonitrile Composite Magnetic Beads Decorated with Candida antarctica Lipase B for Long-Term Reusability in the Hydrolysis of Esters

The reusability of immobilized lipases, including their hydrolysis activity, is greatly affected by lipase leakage, mass transfer channel blockage, and compound deposition on the outside surface. In this study, Candida antarctica lipase B (CALB) was immobilized on Fe3O4 nanoparticle-polyacrylonitrile (PAN) composite magnetic beads through phase inversion (L/FP). The hydrolysis activity of lipase was investigated using p-nitrophenyl palmitate (p-npp) as a model ester. To overcome the limitations of immobilized lipase in ester hydrolysis, three modification strategies were employed. First, lipase was covalently immobilized on Fe3O4 nanoparticles modified with methallyl (M9) and chloro (M13) silane modifiers, which exhibited faster immobilization with reduced lipase leaching and higher activity expression and stability. Second, poly(ethylene glycol) (PEG6000) and polylactic acid (PLA) were added to the precursors to physically modify the composite structures of L/FP, resulting in modified beads with larger specific surface area (15.26 m(2)g(-1)) and smaller average pore size (19 nm). The modified beads, L/FP-9-M and L/FP-13-M, demonstrated superior reusability in long-term hydrolysis activity over 250 cycles. L/FP-9-M retained 63 and 34% of the initial activity at the 100th cycle and the 250th cycle, respectively. L/FP-13-M retained 55 and 36% of the initial activity at the 100th cycle and the 250th cycle, respectively. Furthermore, even after 250 repeated uses, the unit activities (Ug(support)(-1)) of both L/FP-9-M and L/FP-13-M remained higher than the initial activity of Novo 435.