Theoretical insights into the mechanism underlying aflatoxin B₁ transformation by the BsCotA-methyl syringate system

Global concern exists regarding the contamination of food and animal feed with aflatoxin B₁ (AFB₁), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB₁ in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB₁ transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q₁ (AFQ₁) resulting from the direct oxidation of AFB₁ by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ₁, AFB₁-, and AFD₁-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB₁ through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB₁. Finally, we proposed a set of different AFB₁-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB₁-transformation mechanism of the laccase-mediator system.