Magnetically Controlled Wall-Loading of CALB-ImmobilizedFe3O4 Nanoparticles for Continuous-Flow Transesterificationin a Microreactor

Abstract The combination of immobilized enzymes and microreactors offers great advantages in green biomanufacturing. However, improving the catalytic loading and reusability of immobilized enzymes remains a challenge. This study developed a magnetically separable immobilized enzyme system for continuous-flow biocatalysis. Candida antarctica lipase B (CALB) was covalently immobilized onto polydopamine-modified Fe3O4 nanoparticles to prepare a magnetic nanoimmobilized enzyme (CALB@PNMNs). Compared to free enzymes, CALB@PNMNs exhibited enhanced stability in organic solvents and storage stability. CALB@PNMNs microreactor was constructed, with CALB@PNMNs immobilized on its inner walls by an external magnetic field. This layer of magnetic particles allows for the convenient recovery and reuse of the catalyst by removing the applied magnetic field. After optimizing the immobilization process, the actual CALB loading per unit area was 0.30 mg/cm2, and the adsorption yield reached 86.89%. The microreactor was applied to the transesterification of n-butanol and ethyl acetate, with the optimal conditions identified as an alcohol–ester ratio of 1:3, a total flow rate of 10 μL/min, and a reaction temperature of 60 °C, yielding an n-butanol conversion of 63.72%. After short-term continuous operation for 6 h, the microreactor retained 87.63% of the enzyme adsorption capacity and CALB@PNMNs maintained a n-butanol conversion above 50% after 8 reuse cycles. This work has established a stable magnetically controlled immobilized enzyme microreactor (IMER) platform, offering application potential for continuous-flow bioprocesses.