Mechanistic insights into activity-enhancing mutations from comparative analysis of two Bacillus subtilis GH11...

GH11 Xylanases are valuable biocatalysts for xylooligosaccharides production. Enhancing catalytic activity is one of the primary goals in xylanase engineering. Leveraging evolutionary information through comparative analysis of homologous sequences offers a promising strategy to identify key residues for modification, thereby improving enzyme performance and providing insights for rational design. In this study, two GH11 xylanases from Bacillus subtilis (BsXyn1 and BsXyn2), which differ by eight amino acids, were screened. Three key substitution sites (Y79I, V81C, and S100G) in BsXyn1 significantly enhanced its enzymatic activity, but reduced thermal stability. Then, we systematically characterized the enzymatic properties and molecular mechanisms of BsXyn1 and its mutants BsXyn1^Y79I, BsXyn1^V81C, and BsXyn1^S100G. All enzymes exhibited optimal activity at 50 °C and pH 6.0, retaining strict xylan specificity. Kinetic analysis revealed that BsXyn1^V81C exhibited improved substrate affinity and catalytic turnover. Molecular dynamics simulations and MMPBSA calculations revealed that the V81C mutation modulated the activity-stability trade-off through long-range dynamic cooperativity. Specifically, it reshaped the interaction network and increased the conformational flexibility of the thumb region, thereby enhancing its functional role in substrate binding. Concurrently, this perturbation weakened overall structural stability by altering the folding process and reducing hydrophobic core efficiency. This study illustrated how naturally occurring sequence variations could guide the identification of functional hotspots, offering a strategy for developing tailored biocatalysts. However, given the observed trade-off between activity and stability, future engineering efforts should focus on improving thermal stability to better meet the requirements of industrial applications.