Yang_2025_Catalysts_15_92

Feruloyl esterases (FAEs) play critical roles in industrial applications such as food processing, pharmaceuticals, and paper production by breaking down plant cell walls and releasing ferulic acid. However, most bacterial FAEs function optimally in acidic environments, limiting their use in alkaline industrial processes. Additionally, FAEs with alkaline activity often lack the thermal stability required for demanding industrial conditions. In this study, an alkaline feruloyl esterase, PhFAE, from Pandoraea horticolens was identified that exhibits high catalytic activity but suffers from thermal instability, restricting its broader industrial applications. To address this limitation, molecular dynamics simulations were used to analyze enzyme stability, and FireProt, an automated computational tool, was employed to design stabilizing mutations. The engineered S155F mutant demonstrated a 7.8-fold increase in half-life at 60 C and a 1.72-fold improvement in catalytic efficiency (Kcat/Km), corresponding to 680% and 72% enhancements, respectively, compared to the wild-type enzyme. Molecular docking and dynamics simulations revealed that these enhancements were likely due to increased hydrophobic interactions and altered surface charge, which stabilized the enzymes structure. This study provides an effective strategy for improving the functional properties of FAEs and other industrial enzymes, broadening their applicability in diverse industrial processes