Baeyer-Villiger monooxygenase immobilized on magnetic nanoparticles: reusable biocatalytic system for drug metabolite synthesis

: Drug metabolites are critical for assessing drug efficacy, pharmacokinetics, and safety. Biocatalysis offers a selective and sustainable route to their synthesis. In this study, a Baeyer-Villiger monooxygenase from Acinetobacter radioresistens (Ar-BVMO) was immobilized onto bare iron oxide nanoparticles (BIONs), producing a reusable biocatalytic system (BVMO@BION) for drug metabolite production. The magnetic properties of BIONs facilitated easy recovery and reuse of the biocatalyst, making the system practical for repeated use. High loading efficiency (0.14 mg enzyme per mg of BIONs) was achieved through histidine-tag-mediated binding. The immobilized enzyme exhibited enhanced thermostability, increasing its melting temperature from 46.3 °C to 54.9 °C, and reduced nanoparticle aggregation. The system demonstrated robust activity for Baeyer-Villiger and S/N-oxidation reactions. Notably, BVMO@BION achieved over 95 % conversion efficiency for the N-oxidation of tozasertib (an anti-cancer drug) across nine reaction cycles (2 h each) over 3 days, while activity recovery values ranged from 81 % to 127 %. For S-oxidation of ethionamide (an antibiotic used in multidrug-resistant tuberculosis) approximately 26 % conversion was consistently achieved across eight 1-hour cycles. This work demonstrates that BVMO@BION is a robust, magnetically recoverable platform for repeated and selective drug metabolite synthesis, supporting greener and more efficient pharmaceutical development.