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

Abstract

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 βxylosidase 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.

Simultaneously Enhanced Catalytic Activity and Thermostability of a Baeyer–Villiger Monooxygenase from Oceanicola granulosus by Reshaping the Binding Pocket

“…Subsequently, 500 ps isothermal–isochoric (NVT) and isothermal–isobaric (NPT) equilibrations were performed at 303 K. Finally, MD simulations were run on the equilibrated system, with conformations saved every 10 ps. The trajectory analyses were performed on the MD simulation data using the cpptraj to obtain root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and dynamics cross-correlation map (DCCM) …”