Transformation of aflatoxins by a thermostable and mediator-independent laccase from Thermincola ferriacetica.

Abstract

Aflatoxin, a potent carcinogenic mycotoxin, commonly contaminates crops and poses a serious threat to global food and feed security. In recent years, enzymatic degradation of aflatoxins (AFs) has attracted considerable attention, with microbial laccases being the most extensively investigated. Here, we identified a thermostable laccase from Thermincola ferriacetica (Thfe-Lac) that is capable of transforming AFB1 in the absence of mediators. Under mediator-free condition, Thfe-Lac removed over 95% of 20 μg/mL AFB1 within 12 h. The optimal pH of Thfe-Lac against ABTS and AFB1 were pH 4.0 and pH 8.0, indicating distinct oxidation mechanisms for these two substrates. Thfe-Lac exhibited its optimal temperature at 70 °C and retained over 60% of residual activity after 2 h of incubation at 70-80 °C. The addition of the natural mediator acetosyringone (AS) further enhanced the AFB1-transforming ability of Thfe-Lac. In the presence of AS, Thfe-Lac was also able to transform other mycotoxins, including zearalenone (ZEN) and deoxynivalenol (DON). Moreover, several key residues within the substrate-binding pocket were identified through molecular docking. Further quantum mechanics calculations on residue-substrate interaction models revealed that dispersion interactions played an important role in AFs binding. Notably, elimination of unfavored π-π interaction and steric hindrance significantly improved the activity of the F232A mutant. Meanwhile, the enlarged substrate binding pocket facilitate AFB1 entry and promotes closer positioning toward the His500-Cys501-His502 motif. Finally, Thfe-Lac and its mutant F232A represent efficient mediator-free laccase for decontamination of AFB1 in food and feed systems.