Mechanism and Application of Thermostable Superoxide Dismutase from <i>Alcaligenes faecalis</i> HNGD-HTN06 to Degrade AFB ₁ /ZEN

Mycotoxin contamination poses a serious risk to human and animal health. Here, we characterized AfSOD, a novel enzyme from <i>Alcaligenes faecalis</i> HNGD-HTN06, exhibiting dual activity against aflatoxin B₁ (AFB₁) and zearalenone (ZEN). AfSOD showed optimal degradation at pH 7.0 and 50 °C for AFB₁ (2.5 μg/mL) and pH 8.0 and 60 °C for ZEN (5 μg/mL), achieving 92.92% and 83.26% degradation within 24 h. The enzyme retained over 70% activity at 80 °C. LC-MS/MS identified AFQ₁/epi-AFQ₁, AFB₁-diol, 13-OH-ZEN, and 15-OH-ZEN as the main products, which were confirmed by zebrafish assays to be less toxic than parent compounds. Site-directed mutagenesis revealed H27, H81, H168, and D164 as key catalytic residues, and Y170 as crucial for thermostability. AfSOD efficiently lowered AFB₁ and ZEN in contaminated peanut and corn flours, demonstrating strong application potential. This is the first report on AfSOD's biochemical features and dual mycotoxin-degrading capacity, highlighting its promise for food and feed safety.