Substrate recognition and stage-dependent transformation of aflatoxin B1 by mediator-free Bacillus laccase frL103

Enzyme-based mitigation of aflatoxin B1 (AFB1) is commonly evaluated by parent-toxin depletion. This study characterized the stage-dependent, mediator-free transformation of AFB1 by recombinant Bacillus laccase frL103 and assessed chemical, biological, and structural outcomes. UHPLC-HRMS profiling at 0, 30, and 60 h yielded 2846 filtered features and showed that the AFB1 response decreased to 23.8% and 14.8% of the initial value at 30 and 60 h, respectively. A feature at m/z 329.0660 increased at 30 h and declined to 17.7% of that response at 60 h; accurate mass and MS/MS fragmentation supported its putative identification as AFQ1. A lower-abundance feature at m/z 327.0504 was putatively assigned as Dehydro-AFQ1, a proposed dehydrogenated AFQ1-related product. The 60 h reaction mixture produced higher viability than intact AFB1 in HepG2 and 293T cells, structure-based predictions retained endpoint-dependent hazard signals for the proposed products. In molecular dynamics simulations, AFB1 and its proposed products differed in hydration, distance distributions relative to the T1 copper center and binding-pocket retention. These findings establish a stage-resolved, product-centered framework for evaluating enzymatic AFB1 transformation. This approach links bioprocess performance with product evolution, cytotoxicity attenuation, and substrate recognition, while identifying the need for product confirmation, quantitative mass balance.