Conformational Dynamics- and Energy-Guided Hierarchical Combinatorial Engineering Achieves Cooptimization of Thermostability and Catalytic Efficiency in a GH11 Xylanase T-Xyn

GH11 xylanases are promising biocatalysts for xylan valorization, but improving thermostability without compromising catalytic efficiency remains challenging. Here, flexibility analysis, sequence-wide consensus free-energy screening, and iterative combinatorial design were integrated to engineer T-Xyn from Talaromyces thermophilus F1208. Beneficial substitutions were identified, whereas their combinations exhibited background-dependent and nonadditive effects. The optimal variant B3 (F1Q/D53P/N68P) exhibited an optimum temperature of 75 °C, a half-life of 203.87 min at 70 °C (5.9-fold that of T-Xyn), and a 2.4-fold increase in catalytic efficiency. B3 accelerated xylooligosaccharide production while retaining xylobiose and xylotriose as the predominant products. Structural inspection and molecular dynamics simulations supported a model in which increased global compactness and spatially redistributed flexibility were associated with improved thermostability, whereas subtle catalytic-cleft reshaping and enhanced enzyme–substrate contacts may facilitate substrate accommodation and catalysis. This work provides a thermostable variant for industrial use and a potentially transferable framework for engineering high-temperature active GH11 xylanases.