A highly conserved residue unlocks thermostability in β-glucuronidases

β-Glucuronidase, a glycosyl hydrolase that cleaves β-glucuronic acid residues from glycosides, is widely used in the production of pharmaceutically active compounds. However, the intrinsically low thermal stability of most native enzymes restricts their industrial application. In this study, we developed an efficient screening workflow coupled with directed evolution to identify thermostability-enhancing mutant. To further characterize the variant, we identified the key motif and residue that regulate thermal stability. Site-directed mutagenesis of β-glucuronidases from three distinct sources revealed that the conserved residue "N" within the "WNFADF" motif is critical for thermostability. Substitution of this residue with threonine (N→T) increased the melting temperature by 6.6-12.61 °C and extended the half-life by 4.26-33.6fold relative to the wild-type enzymes. The characterization of the kinetic parameters revealed that all these variants exhibited the typical "activity-stability" trade-off phenomenon. Molecular dynamics simulations and structural analyses indicate that the improved thermostability of the N→T variant results from reduced local flexibility and enhanced structural compactness, as evidenced by lower RMSF, Rg, and SASA values and an increased number of stabilizing hydrogen bonds. Overall, this work offers mechanistic insight into thermostability engineering and provides a promising strategy for enhancing the robustness of β-glucuronidases for industrial biocatalysis.