Rational Molecular Design for Improved ZHD101 Thermal Stability Based on the Introduction of Disulfide Bonds at the Dimer Interface and B-Factor Analysis

Zearalenone hydrolase, derived from <i>Clonostachys rosea</i>, effectively degrades zearalenone, a major contaminant in animal feed; however, its poor thermal stability significantly hinders industrial applications. We improved the thermal stability of ZHD101 through rational molecular design. First, disulfide bonds were introduced at the interface to construct dimers, with ZHD101^T229C demonstrating stable dimerization. Subsequently, two high-vibration hotspot amino acids were selected by analyzing atomic position fluctuations and dynamic information on the dimer crystal structure. A precise mutant library, containing ZHD101^T229C/N137L, ZHD101^T229C/D170L, and ZHD101^T229C/D170C, was obtained by virtual saturation mutagenesis and conformational free energy calculations. The ZHD101^T229C/D170C variant exhibited remarkable thermal stability enhancement, with thermal half-inactivation temperature (<i>T</i>_<i>50</i>) reaching approximately 54 °C (7 °C higher than wild-type) and half-life (<i>t</i>_1/2) at 50 °C extending to 10.5 min (110% increase). The melting temperature (<i>T</i>_m) of ZHD101^T229C/D170C increased from 49 ± 1.0 °C (wild-type) to 67.1 ± 0.95 °C, representing an increase of 18.1 °C, making it the highest Δ<i>T</i>_m value reported for ZEN hydrolases to date. Overall, a combination mutation strategy of introducing disulfide bonds between monomers and utilizing B-factor analysis was applied, resulting in improved thermal stability of ZHD101^T229C/D170C. The improved strategy and identification method reported herein provides a reference for the thermal stability modification and optimization of other proteins.