Novel Robust-Thermostable Zearalenone Hydrolase Engineered via Ancestral Sequence Reconstruction: In Silico Design,...

Despite the high efficiency of zearalenone (ZEN) hydrolases in detoxifying ZEN, the limited thermostability restricts their industrial application. This study employed ancestral sequence reconstruction to enhance the thermostability of a novel ZEN hydrolase, ZHDX6, while simultaneously adopting rational design as a parallel comparative strategy. The ancestral enzyme An48 achieved an 18.83 °C increase in melting temperature and a 1328.46-fold increase in thermal half-life (t_1/2) at 40 °C compared to ZHDX6. Notably, An48 retained a t_1/2 of 2.30 min even at 80 °C, whereas ZHDX6 and rational design mutant S158Y/G157C were already inactivated. Molecular dynamics simulations revealed that An48 achieved superior stability through an increased number of hydrogen bonds, coordinated domain motions, and a more concentrated free energy minimum. Furthermore, in vivo tests confirmed that An48 effectively degrades ZEN in the gastrointestinal tract of mouse. These results indicate that ancestral enzyme An48 shows promising industrial applications.