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Additional file 1: Figure S1. Sequence alignment of XylE and XYL10C. The fragments of M1 to M10 in XYL10C are marked in different colors. Figure S2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the purified recombinant wild-type XylE and its mutants. Lanes: M, the standard protein molecular weight markers; A, C, E, G, I, K, N, and P: XylE, XylE-M3, XylE-M6, XylE-M9, XylE-M3/M6, XylE-M3/M9, XylE-M6/M9, and XylE-M3/M6/M9; B, D, F, H, J, L, O, and Q: the deglycosylated enzymes. Figure S3. Graph showing the Lineweaver and Burk regression and the equation that was used to calculate Km and Vmax for each enzymatic construction. A to J represent XylE, XylE-M3, XylE-M6, XylE-M9, XylE-M3/M6, XylE-M3/M9, XylE-M6/M9, and XylE-M3/M6/M9, respectively. Figure S4. High-performance liquid chromatography (HPLC) analysis of the hydrolysis products of beechwood xylan produced by the XylE (A) and its mutants. X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; and X6, xylohexaose. Figure S5. Root mean square deviation (RMSD) values of the wild-type XylE and its hybrid mutants XylE-M6, XylE-M9, and XylE-M6/M9 during the molecular dynamics (MD) simulation.

Additional file 1: Table S1. X-Ray data collection and structure refinement statistics. Table S2. Temperature and pH optima of XYL10C-ΔN and its mutants with beechwood xylan as the substrate. Table S3. Specific activity of XYL10C-ΔN and its mutants with beechwood xylan as the substrate under standard conditions (80 °C and pH 4.0). Table S4. Temperature and pH optima of XylE and its mutants with beechwood xylan as the substrate. Table S5. Specific activity of XylE and its mutants with beechwood xylan as the substrate under standard conditions (70 °C and pH 5.0). Table S6. Primers used in this study. Figure S1. Multiple sequence alignments of XYL10C-ΔN and other GH10 xylanases using the ClustalW and ESPript. Strictly conserved residues are marked in red. Similar residues are shown in black bold characters and marked in yellow. The catalytic residues are indicated by red diamonds. Some important amino acids are indicated by red triangles. α-Helices and β-strands are represented by black coils and arrows, respectively. Figure S2. SDS-PAGE analysis of the purified recombinant XYL10C, XYL10C-ΔN, XylE, XynE2, and their mutants. Lanes: M, the standard protein molecular weight markers; 1, 3, 10, and 45, the crude enzyme of wild-type XYL10C, XYL10C-ΔN, XylE and XynE2; 2, 4, and 11, the deglycosylated XYL10C, XYL10C-ΔN, and XylE; 5-9 and 13-26, the saturated mutants of XYL10C-ΔN; 12 and 27-44, the saturated mutants of XylE; and 46, the mutant XynE2-Q85E. Figure S3. Comparison of thermostability of XYL10C and XYL10C-ΔN. A, the kinetic stability assayed at 80 °C and 90 °C; B, the thermodynamic stability (Tm values). Figure S4. pH stability of XYL10C and XYL10C-ΔN. Figure S5. HPLC analysis of the hydrolysis products by XYL10C (A) and XYL10C-ΔN (B). X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; beechwood, beechwood xylan. Figure S6. The X-ray diffraction pattern of the XYL10C-ΔN crystal. The resolution edges are shown by different concentric circles. Figure S7. The dimeric structure (Chains A and B) of the XYL10C-ΔN. A, The hydrogen bonds between monomers. The residues involved in forming hydrogen bonds are indicated, and the red dotted line represents hydrogen bond. B, The van der Waals force network between monomers. The residues in blue are from chain A, and those red are from chain B. The residues involved are represented by yellow dashed lines. Figure S8. The XYL10C-ΔN docked with xyloheptaose. The enzyme and substrate interactions are indicated by yellow dotted lines. Figure S9. The active-site architecture of mutant XylE-Q116E.