Computational design of a highly active and stable alkaline pectate lyase: Unraveling the molecular mechanisms for superior performance

Pectate lyases are essential for valorizing pectin-rich biomass. However, their industrial application is often limited by low stability and efficiency under harsh operational conditions. To overcome these challenges, we implemented a multifaceted computational design strategy, which integrated molecular dynamics simulations at multi-temperature (310.15 K, 343.15 K, and 363.15 K) to identify distal flexible regions. This was coupled with free energy-guided optimization of the proximal active sites, utilizing tools such as FireProt, PROSS, and HotSpot, to achieve a synergistic enhancement of enzyme properties. From an initial screening of 46 single-point mutants, the top-performing variant, D262E, was combined with S184A, resulting in the double mutant S184A/D262E. This variant demonstrated exceptional improvements in catalytic efficiency, with a 8.19-fold increase in catalytic efficiency (k_cat/K_m), a melting temperature of 80.90 °C, and a half-life of 61.74 min at 70 °C and pH 10.0. The observed stabilization was attributed to enhanced hydrophobic packing, optimized H-bond networks, and reduced solvent accessibility, all of which contribute to its robust performance under extreme conditions. When applied in fabric bioscouring, the S184A/D262E variant significantly enhanced hydrophilicity, increasing the wetting area by 1.28-fold compared to the wild-type enzyme. This performance demonstrates the variant's superior efficacy in industrial applications. This study highlights a generalizable approach for simultaneously improving both enzyme activity and stability, making it highly suitable for efficient biomass refining under demanding industrial conditions.