Enhancing the Thermostability and Catalytic Activity of a Multicopper Oxidase from Bacillus megaterium for Aflatoxin B 1 and Zearalenone Degradation

Mycotoxins are toxic secondary metabolites commonly found in cereal crops, posing significant health risks to humans and animals. In this study, a multicopper oxidase from Bacillus megaterium (BmCueO) has been identified as an effective catalyst for degrading Aflatoxin B1 (AFB1) and Zearalenone (ZEN). Through semi-rational design, a triple-point mutant (D373P/Q144Y/Q350F) was constructed to enhance its thermostability and catalytic performance. The engineered variant exhibited a 3.96-fold increase in half-life at 60 °C, along with improvements of 22.6% and 48.2% in AFB1 and ZEN degradation activities, respectively. LC-MS/MS analysis confirmed the structural transformation of AFB1 and ZEN into less toxic derivatives, while zebrafish and Ames assays verified reduced the toxicity and mutagenicity of the degradation products. The variant efficiently degraded AFB1 and ZEN in feed matrices, demonstrating its potential for application. These results highlight the promise of the engineered BmCueO as a safe and efficient biocatalyst for mycotoxin detoxification in feed systems.