A Novel Copper-Containing Nitrite Reductase for Simultaneous Detoxification of Aflatoxin B1 and Zearalenone: Catalytic Mechanism and Application in Edible Oils

Abstract

Co-contamination of aflatoxin B1 (AFB1) and zearalenone (ZEN) poses a major food safety risk. In this study, the copper-containing nitrite reductase AfNiR was identified as a novel biocatalyst for AFB1 and ZEN degradation, achieving maximum efficiencies of 99.22% and 93.53%, respectively, under optimal conditions of 40 °C, pH 7.0–8.0, 20 μg/mL (0.48 μM) enzyme dosage, and 24 h incubation for 5 μg/mL (16.01 μM) AFB1, and 50 °C, pH 8.0, 20 μg/mL (0.48 μM) enzyme dosage, and 24 h incubation for 10 μg/mL (31.41 μM) ZEN. UPLC–MS/MS identified AFQ1 and 15-OH-ZEN as the main transformation products, both exhibiting substantially reduced toxicity indicators in zebrafish assays. Molecular docking, site-directed mutagenesis, and molecular dynamics simulations indicated that both toxins bind near the T2 copper center and that copper-coordinating residues are essential for catalysis. To improve stability, AfNiR was immobilized in Cu3(PO4)2 nanoflowers and SA/SDS microspheres, achieving efficient detoxification in vegetable oil without affecting oil quality.