Mechanistic and Structural Analysis of Aflatoxin B1 Degradation by Bacillus safensis Multicopper Oxidase

Aflatoxin B1 (AFB1) is a potent mycotoxin threatening food and feed safety. Here, we report the identification and characterization of a Bacillus safensis-derived multicopper oxidase (BsaMCO) capable of efficient AFB1 detoxification. Recombinant BsaMCO exhibited robust in vitro activity, achieving >78% degradation of AFB1 under 24 h incubation at 37 °C. Optimization experiments revealed that enzyme concentration, pH, temperature, metal ions, and electron acceptors significantly influenced degradation efficiency, defining an operational window suitable for practical applications. LC-MS profiling suggested the presence of transformation products tentatively consistent with oxidative demethylation to aflatoxin P1 (AFP1) and with the formation of AFG2a-like products through subsequent hydration- and oxidation-related transformations. Molecular docking and 100 ns all-atom molecular dynamics (MD) simulations demonstrated stable binding of AFB1 in the T1 copper pocket. Van der Waals and electrostatic interactions, together with a persistent hydrogen bond at Gly323, facilitated single-electron transfer through the intramolecular T2/T3 copper cluster. Principal component and Gibbs free energy analyses confirmed a low-energy, stable conformational ensemble. HepG2 cell assays indicated that BsaMCO-degraded products substantially reduced cytotoxicity and apoptosis compared with native AFB1. Simulated feed experiments further validated enzymatic AFB1 degradation, with approximately 53% reduction after 24 h. Collectively, these findings establish BsaMCO as a safe and effective biocatalyst for AFB1 detoxification, providing mechanistic, structural, and cellular evidence supporting its application in food and feed safety.