Solvent-Response-Guided Engineering Improves Urethanase Activity and Thermal Stability for Ethyl Carbamate Degradation

Microbial urethanases catalyze carbamate bond cleavage, offering biocatalytic tools for polyurethane recycling and ethyl carbamate (EC) removal from fermented foods. Realizing this potential requires enzymes combining high catalytic activity with thermal stability, yet identifying substitutions that improve both remains challenging. Here, we developed an engineering strategy integrating evolutionary conservation, local isothermal compressibility, and solvation analysis, using the metagenome-derived urethanase UMG-SP2 as a model. The UMG-SP2 variants H142F and Q415L showed specific activities toward the activated model substrate 4-nitrophenyl N-benzylcarbamate that were 3.48 and 2.72 times that of the wild type, respectively. Their apparent melting temperatures were 13.1 and 8.6 °C higher than that of the wild type, respectively. Molecular dynamics simulations associated these improvements with greater pocket hydration and increased sampling of substrate geometries compatible with nucleophilic attack. We then adapted the strategy to ethanol exposure and applied it to the EC-hydrolyzing urethanase UH to evaluate its application potential. All eight selected variants showed higher activity and melting temperatures than the wild type. N262P achieved 7.55 U mg−1, 3.68 times the wild-type specific activity, and increased the melting temperature by 14.64 °C. After 12 h in model wine, residual EC was 273.49 µg L−1 with N262P versus 406.73 µg L−1 with wild-type UH. These findings support solvent-informed urethanase engineering and suggest potential extension to other industrial biocatalysts.