PROSS-assisted engineering enhances the thermal and storage stability of an AFB1-degrading superoxide dismutase

Aflatoxin B 1 (AFB 1 ) contamination threatens feed safety, highlighting the need for enzymatic detoxification strategies. In this study, an AFB 1 -degrading strain, HNGD-122, was isolated and identified as a Priestia megaterium -related strain. Genome-guided mining identified a superoxide dismutase (SOD) with AFB 1 -degrading activity. To improve enzyme stability, PROSS-assisted design was applied, yielding the M163K mutant. M163K retained AFB 1 -degrading activity and exhibited enhanced thermostability, with half-lives at 70 and 80 °C extended to 32.2 and 20.7 h, respectively. It exhibited higher AFB 1 degradation rates than wild-type (WT)-SOD during storage at 4 °C and following freeze-drying. Molecular dynamics simulations suggested that M163K maintained SOD–AFB 1 interactions while improving thermostability through enhanced local stabilizing interactions. In zebrafish assays, it reduced developmental toxicity and oxidative stress after M163K treatment. In naturally contaminated corn flour and corn bran, M163K degraded approximately 66.5% and 61.9% of AFB 1 , respectively, supporting its potential for AFB 1 control in corn-based feed ingredients.