Thermostable Oxidoreductases CotA and Prx Enable Synergistic and Peroxide-Enhanced Degradation of Aflatoxin B₁

Aflatoxin B₁ (AFB₁) is a highly stable mycotoxin that can persist during conventional food processing and therefore poses a serious risk to food and feed safety. In this study, two enzymes (CotA and Prx) were heterologously expressed in Bacillus subtilis, purified by Ni-NTA affinity chromatography, and evaluated for their ability to degrade AFB₁. Both enzymes exhibited remarkable thermostability and distinct catalytic optima. CotA exhibited its highest activity at 80 °C with an AFB₁ removal of 38.4%, whereas Prx showed its highest activity at 90 °C with a removal of 82.6%. The optimal pH values were near neutral, with CotA performing best at pH 7.0 and Prx at pH 7.5, and both reactions approached maximal conversion within approximately 10 h. When the two enzymes were combined, a clear cooperative effect was observed. The mixed system achieved 91.0% AFB₁ removal at 80 °C after 10 h, with the best degradation activity occurring at a CotA to Prx ratio of 1:3. At 50 °C, neither enzyme alone caused appreciable AFB₁ degradation, but the addition of hydrogen peroxide markedly enhanced catalytic activity. Both enzymes also retained substantial activity after boiling and autoclaving. In a maize flour model, the mixed-enzyme system showed strong AFB₁ degradation capacity, and peroxide-assisted treatment further improved activity. These results establish a thermostable and peroxide-responsive enzymatic platform for AFB₁ degradation and support future development of enzyme-based detoxification strategies for food and feed applications. Product identification and toxicological validation will be needed to confirm the safety of the treated products.