Computation-Aided Phylogeny-Oriented Engineering of β‑Xylosidase: Modification of “Blades” to Enhance Stability and Activity for the Bioconversion of Hemicellulose to Produce Xylose

Hemicellulose is a highly abundant, ubiquitous, and renewable natural polysaccharide, widely present in agricultural and forestry residues. The enzymatic hydrolysis of hemicellulose has generally been accomplished using β-xylosidases, but concomitantly increasing the stability and activity of these enzymes remains challenging. Here, we rationally engineered a β-xylosidase from Bacillus clausii to enhance its stability by computation-aided design combining ancestral sequence reconstruction and structural analysis. The resulting combinatorial mutant rXYLO^{M25I/S51L/S79E} exhibited highly improved robustness, with a 6.9-fold increase of the half-life at 60 °C, while also exhibiting improved pH stability, catalytic efficiency, and hydrolytic activity. Structural analysis demonstrated that additional interactions among the propeller blades in the catalytic module resulted in a much more compact protein structure and induced the rearrangement of the opposing catalytic pocket to mediate the observed improvement of activity. Our work provides a robust biocatalyst for the hydrolysis of agricultural waste to produce various high-value-added chemicals and biofuels.