Functional Characterization and Mechanism of Two Zearalenone-Degrading Enzymes from <i>Bacillus velezensis</i> B.26

Mycotoxins threaten human and animal health, necessitating effective detoxification strategies. This study demonstrated that <i>Bacillus velezensis</i> B.26 efficiently degraded zearalenone (ZEN), achieving 91.64% reduction at 70 °C within 24 h. After verification, the degradation active components were found to mainly exist in the fermentation supernatant, which was sensitive to metal ions. Under treatment with proteinase K and sodium dodecyl sulfate, the degradation activity was significantly reduced, but it was not affected by heating treatment. Genome mining revealed two candidate genes, a laccase with 66.12% amino acid sequence agreement with CotA from <i>Bacillus licheniformis</i> and another with 42.65% amino acid sequence agreement with peroxiredoxin (Prx) from <i>Acinetobacter nosocomialis</i>. Heterologously expressed CotA (62 kDa) degraded 91.94% of 5 μg/mL ZEN at 70 °C, pH 8.0, within 6 h, while Prx (22 kDa) eliminated 59.74% at pH 11.0 under identical thermal conditions. Na⁺, Cu²⁺, and K⁺ enhanced CotA activity, whereas Mn²⁺ inhibited both enzymes. LC-MS confirmed nontoxic degradation pathways yielding low-toxicity metabolites C₁₇H₂₄O₄ and C₁₂H₁₆O₄, without hazardous byproducts (α-ZEL/α-ZAL). These findings indicated that both enzymes have the potential to serve as detoxification agents in food and feed applications.