Immobilization of cytochrome P450 enzymes onto magnetic beads: An approach to drug metabolism and biocatalysis

• Published in: Talanta open Vol. 7; p. 100181
• Main Authors: Furlani, Izadora Liranço, Oliveira, Regina Vincenzi, Cass, Quezia Bezerra
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.08.2023; Elsevier
• Subjects: Biocatalysts; CYP450 immobilization; Drug metabolism; In-vitro assay; Phenotyping; Drug metabolism; Biocatalysts; In-vitro assay; CYP450 immobilization; Phenotyping
• ISSN: 2666-8319; 2666-8319
• DOI: 10.1016/j.talo.2023.100181

•Immobilization of human microsomal fractions onto magnetic beads (Hum-Mbs).•Application of Hum-Mbs in enzymatic activity, stationary kinetic study, and inhibition assays.•Boost in metabolite production, enzymatic stability, and reuse of Hum-Mbs.•Production of a dual biocatalyst by the junction of G6PDH-Mbs and Hum-Mbs and application in in vitro drug metabolism. Cytochrome P450 (CYP, P450) presents a wide range of applicability in drug metabolism studies and in biocatalysis as a great alternative to synthesizing compounds. Nonetheless, their lack of stability is one of their major drawbacks. Aiming the possibility of enhancing the catalytic activity and promoting higher stability, liver microsomal fractions have been immobilized on magnetic beads (Mb). The immobilized procedure was modulated by using rat liver microsomal fractions (RLM-Mb). The optimal condition achieved was further employed for the immobilization of the human microsomal fractions on magnetic beads (HLM-Mb). In vitro metabolism assays were conducted using albendazole (ABZ) as a model drug, and the formation of albendazole sulfoxide (ABZSO) was monitored. Biotransformation reactions applying the produced HLM-Mb were examined for the best temperature to increase the production of metabolites and their reuse cycles. A kinetic study was carried out for HLM-Mbs monitoring the production of ABZO by the oxidation reaction of ABZ by CYP3A4. The Km value was 25.6 µmol L−1 and Vmax was 121.0 µmol L−1. Inhibition assays were conducted in the presence of ketoconazole and the production of ABZSO decreased by 46.8 ± 2.5%. Enzymatic activities for CYP2C9 and CYP2D6 on HLM-Mbs were evaluated by monitoring the hydroxylation reactions of diclofenac and bufuralol as substrates. The immobilization of CYP P450 on magnetic beads increased not only the production of ABZSO metabolites but also the stability of CYP. The use of HLM-Mbs jointly with immobilized glucose-6-phosphate dehydrogenase (G6PDH-Mbs) as a unique dual generator system to produce NADPH has established the one-pot conditions for biocatalysis in a greener approach with reuse of the biocatalyst (G6PDH-HLM-Mbs). To this end, the herein reported HLM-Mbs and G6PDH-HLM-Mbs are excellent analytical tools to be explored either in biocatalysis reactions or in in vitro metabolism studies. Dual biocatalyst by the junction of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads and human microsomal fractions immobilized onto magnetic beads to promote a unique bioreactor for the production of NADPH and simultaneous drug metabolism studies. [Display omitted]