Figure 4. Comparison of XynA with XynA E182A/E280A in terms of binding, catalysis activity and structures. (A) Effect of XynA concentration on its xylanase activity. (B) XynA and XynA E182A/E280A activities for degrading substrates at different concentrations. Each experiment was repeated three times. XynA and XynA E182A/E280A were analyzed for significance, ns means no significant difference, * means P <0.05, ** means P <0.01, *** means P <0.0002, and **** means P <0.0001. (C) XynA binding experiment with BWX; XynA E182A/E280A binding experiment with BWX. Different colored lines in the figure represent different concentrations of BWX. The binding constants are shown in the table. (D) Protein thermal shift assay of XynA and XynA E182A/E280A. RFU of the Y-axis represents relative fluorescence units. The Tm value represents the denaturation or exposure temperature of the hydrophobic residues of the protein. ΔTm indicates the temperature change after adding BWX. (E) Interaction between two residues Glu182 and Glu280 and surrounding residues of XynA. (F) The interaction between the Ala182 and Ala280 residues and the surrounding residues of XynA E182A/E280A. Color cyan and pink indicate the crystal structure of WT and double mutant respectively. The red dotted line represents hydrogen bonds. The criterion for hydrogen bond judgment is that the distance between the acceptor and donor atoms should be less than 4 Å.