Parallel Multidimensional Engineering of Acetylxylan Esterase Enables Concurrent Enhancement of Thermostability and Catalytic Efficiency

Simultaneously enhancing the thermostability and catalytic activity of acetylxylan esterases (AxEs) remains a significant challenge due to the inherent stability-activity trade-off. Here, a previously uncharacterized CE7 family acetylxylan esterase (TsAxE) from Thermoclostridium stercorarium was identified and engineered through a parallel multidimensional engineering strategy integrating consensus motif engineering, molecular docking, and interface engineering. The best-performing mutant BCF (D218L/D245P/G183Y) exhibited a 44.10 ± 0.44% increase in activity toward p -nitrophenyl acetate ( p NPA). Notably, the thermostability of BCF was substantially improved, with the half-life ( t 1/2 ) at 60 ° C extended from 0.63 ± 0.04 h to 43.82 ± 3.90 h. In addition, BCF showed improved catalytic efficiency toward p -nitrophenyl butyrate ( p NPB). Molecular docking and molecular dynamics analyses suggested that these mutations may reshape substrate-binding pocket and improve structural stability. This study demonstrates the effectiveness of integrating complementary engineering strategies for the rational improvement of AxEs activity and thermostability.