Enhancing the catalytic performance of xylanase XynASP through semi-rational design in the cord region to promote its application in juice clarification.

In the GH11 family of xylanases, the cord region, a dynamic peptide linker connecting the "thumb" and "palm" regions, exhibits remarkable flexibility. To reveal the structure-function relationship in this region, saturation mutagenesis was performed on the cord segment of XynASP, a xylanase derived from Aspergillus saccharolyticus JOP 1030-1 GH11. Among the generated mutants, two variants, D116S and E119V, showed superior enzymatic properties and were subsequently combined to generate XynASP-SV. XynASP-SV exhibited a 3.05-fold increase in specific enzyme activity compared to the wild type, a 6 °C rise in T value, and a 4.62-fold extension in half-life at 50 °C. When beechwood xylan was used as the substrate, the k/K of XynASP-SV increased 27.69-fold compared to the wild type. Molecular dynamics simulations revealed that the synergistic effects of the D116S and E119V mutations, along with amino acids in the "thumb" region, significantly enhanced the structural rigidity of XynASP-SV, thereby improving its thermostability. In the clarification experiments with mango and pitaya juices, XynASP-SV demonstrated substantial potential for industrial applications. This study highlights the enhanced catalytic performance of xylanase achieved by controlling its flexibility in the cord region.