Multifaceted Engineering of Xylanase Guided by Structural Insights to Enhance Thermal Stability and Catalytic Functionality

β-1,4-Xylanase is crucial for lignocellulosic biomass bioprocessing by degrading xylan, the major hemicellulose component. However, industrial applications are hindered by its inherent thermostability limitations. To overcome this challenge, we developed a comprehensive C–S–E strategy combining computational design, structural analysis, and experimental verification to first identify a novel thermotolerant xylanase (XynT) from Streptomyces calidiresistans. Subsequently, through a sequential design workflow encompassing flexible region analysis, virtual saturation mutagenesis, threshold-based mutant screening, iterative combinatorial mutagenesis, and strategic disulfide bond introduction, we successfully obtained high-performance variant M12 (A7C/P210H/W277P/G304C). The engineered M12 exhibited significant improvements, showing 2.1-fold enhanced specific activity (22,341.7 U/mg) and 7.6-fold increased thermal stability (t1/2 = 215 min) at 55 °C and pH 8.0 compared to wild-type XynT. Beechwood xylan hydrolysis assessment confirmed M12’s highly catalytic efficiency and thermostability, highlighting its potential for industrial applications, particularly in pulp prebleaching processes.