Rational molecular design for improved ZHD101 thermal stability based on the introduction of disulfide bonds at the dimer interface and B-factor analysis

AbstractZearalenone hydrolase 101 (ZHD101), derived from Clonostachys rosea, is known to effectively degrade the main contaminant (zearalenone) in animal feed, but the thermal instability of ZHD101 limits its industrial application. In this study, we successfully enhanced the thermal stability of ZHD101 through two iterative rounds of rational molecular design. First, after the prediction of disulfide bond sites, ZHD101T229C was obtained, and a new disulfide bond was formed between two single ZHD101 subunits to construct a dimer ZHD101. Then, based on ZHD101T229C, two high-vibration hotspot amino acids N137 and D170 were selected by analyzing atomic position fluctuations and dynamic information A small and precise mutant library containing three mutants (ZHD101T229C/N137L, ZHD101T229C/D170L, and ZHD101T229C/D170C) was obtained by saturation mutagenesis and calculation of binding energy in silico. Compared with the wild type, the thermal half-inactivation temperature (T50) of ZHD101T229C/D170C increased by 7{degrees}C, its half-life (t1/2) increased by 200% at 50{degrees}C, and its melting temperature (Tm) increased by 18.1{degrees}C. Molecular docking suggested that new covalent bond formation and shorter bond distance may contribute to the improved thermal stability of ZHD101. The rational design strategy proposed in this work can provide a reference for the thermal stability modification and optimization of other proteins. ImportanceZearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin that poses a significant threat to animal feed safety. ZEN hydrolase 101 (ZHD101) catalyzes the conversion of ZEN into a nontoxic product, offering a promising detoxification strategy. However, the thermal instability of ZHD101 severely limits its industrial application. In this study, the thermal stability of ZHD101 was significantly improved through two rounds of rational design, resulting in the ZHD101T229C/D170C mutant. ZHD101T229C/D170C exhibited the improved half-inactivation temperature (T50), half-life (t1/2) and the highest melting temperature (Tm) reported thus far. Overall, the iterative combinatiorial mutation strategy involved the introduction of a disulfide bond between two single subunits and the application of B-factor analysis to identify hotspot amino acids residues. This approach effectively enhanced the enzymes structural stability, paving the way for its industrial application. ZHD101T229C/D170C and the rational design strategy presented in this work provide a robust framework for the thermal stability optimization of similar biocatalysts, advancing the practical use of ZHD101 in the feed industry.