Enhancing the Substrate Adaptability of Laccase through Ancestral Sequence Reconstruction for Applications in Mycotoxin Detoxification

Toxic mycotoxins such as aflatoxin B1 (AFB1) and zearalenone (ZEN) in moldy grains pose significant health risks. Fungal laccases, such as LCC5 from <i>Coprinopsis cinerea</i>, have shown potential for converting these mycotoxins. In the present study, ancestral sequence reconstruction was employed to generate LCC5 variants with improved substrate adaptability and stability. Through molecular docking, ancestral laccase LacANC278 was identified with high activity toward both AFB1 and ZEN. Compared to LCC5, LacANC278 exhibited 23% higher in conversion rate for AFB1 and 61% higher for ZEN. In addition, the half-life of LacANC278 extended from 4 h for LCC5 to 16 h. Under the optimal conditions, compared to LCC5, LacANC278 demonstrated significantly higher conversion efficiencies for AFB1 and ZEN in corn powder, achieving 88% and 89% reduction of these mycotoxins, respectively. The major conversion products of AFB1 and ZEN, AFQ1 and 2-OH-ZEN, were tentatively identified. The safety of the conversion products was also evaluated.