Rational design of an engineered zearalenone lactone hydrolase with enhanced thermostability and acid tolerance for zearalenone degradation in food

Zearalenone (ZEN), a prevalent and stable Fusarium mycotoxin in cereals, threatens food safety and resists conventional processing. Natural ZEN hydrolases are often inadequate under industrial thermal and acidic conditions. Here, we engineered a robust variant, R76Q/T217C-G242C, via a hybrid computational strategy integrating energy calculations, electrostatic optimization, consensus analysis, and disulfide bond design. The resulting biocatalyst completely degraded 16.6 mg/L ZEN within 30 min, retained 71 % activity after 5 min at 55 °C (10-fold improvement), and exhibited >75 % activity at pH 5.0 with a 192.5-min half-life at 40 °C. It also eliminated 95.13 % of naturally occurring ZEN in moldy distillers' grains. Structural and dynamic analyses revealed stabilization mechanisms mediated by a designed disulfide bond and optimized interaction networks. This study provides a high-performance enzymatic solution for ZEN degradation in challenging food-processing environments.