Identification and Thermal Stability Modification of a Zearalenone Lactone Hydrolase ZHD30L

In this study, a previously uncharacterized zearalenone lactone hydrolase, ZHD30L, from Knufia peltigerae was identified and characterized, and semi-rational design was employed to enhance its thermostability. The results indicate that the optimal reaction conditions for this enzyme are 40 °C and pH 9.0, under which it catalyzes the ring-opening of the ZEN lactone to generate the non-toxic product HZEN. The mutant with five combined sites Mut5, (E11T-A127V-I180K-H235P-V156I), was obtained through semi-rational design combined with multiple rounds of iterative superposition and negative elimination strategies. Thermodynamic characterization revealed that, compared to the wild type, the t1/2 of Mut5 at 48 °C was significantly extended from 1.67 min to 796.8 min, and the Tm value increased from 39.56 °C to 50.04 °C. The markedly prolonged thermal half-life and increased apparent melting temperature may improve the operational robustness of Mut5 during moderate-temperature feed and food processing or post-processing applications, potentially reducing activity loss and the need for repeated enzyme additions. Concurrently, the kcat/KM improved from 19.08 μM−1·s−1 (95% Cl = 12.80 to 25.36 μM−1·s−1) to 33.29 μM−1·s−1 (95% Cl = 23.20 to 43.38 μM−1·s−1). Under optimal reaction conditions, WT and Mut5 degraded 76.51% ± 1.65% and 99.13% ± 0.71% of ZEN (10 μg/mL), respectively, within 3 min at equal enzyme concentrations. Molecular dynamics simulations indicate that the H235P and E11T mutations reduce local conformational entropy, the A127V and V156I mutations optimize internal hydrophobic stacking, and the I180K mutation reshapes the local charge microenvironment. Furthermore, Mut5 exhibited an overall RMSD at high temperatures that was 0.026 Å lower than that of the wild type, and a global free energy minimum that was 0.36 kcal/mol lower. These computational results offer possible structural interpretations, providing a theoretical reference for developing highly efficient enzyme preparations for the feed and food industries.