Engineering a Substrate-Binding Chain Assisting the Balance of Thermostability and Activity Trade-Off for Esterase

Esterases provide an ecofriendly route for malathion degradation, but their limited thermostability restricts industrial application. Here, a previously identified esterase was engineered using a combinatorial strategy integrating folding energy optimization, net charge modification, and consensus design, achieving a 40.4% success rate in thermostability enhancement. The double mutant S275Y-S326 M retained a 92.3% activity after 20 min at 50 °C, corresponding to a 3.7-fold improvement over the wild type but exhibited reduced catalytic efficiency toward 4-nitrophenyl butyrate. Molecular dynamics simulations indicated that excessive rigidification impaired substrate positioning. Subsequent activity recovery was achieved by introducing aromatic or charged residues to enhance the substrate binding. The resulting quadruple mutant S275Y-S326M-L236 K-F372 K showed a 1.9-fold higher catalytic efficiency than S275Y-S326 M and fully degraded malathion within 25 min at 50 °C. This work demonstrates a practical strategy for balancing thermostability and activity through substrate-binding-oriented design with potential applicability to other enzyme systems.