Significantly Enhanced Thermostability of Aspergillus niger Xylanase by Modifying Its Highly Flexible Regions

In this study, the thermostability of an acid-resistant GH11 xylanase (xynA) from Aspergillus niger AG11 was enhanced through systematic modification of its four highly flexible regions (HFRs) predicted using MD simulations. Among them, HFR I (residues 92-100) and HFR II (residues 121-130) were modified by iterative saturation mutagenesis (ISM), yielding mutants G92F/G97S/G100K and T121V/A124P/I126V/T129L/A130N, respectively. For HFR III, the N-(residues 1-37) and C-termini (residues 179-188) were, respectively, substituted with the corresponding sequences from thermophilic EvXyn11^TS and Nesterenkonia xinjiangensis xylanase. N-Glycosylation was introduced into HFR IV (residues 50-70) through site-directed mutation (A55N/D57S/S61N) and the recombinant expression in A. niger AG11. Combining these positive mutations from each HFR yielded the variant xynAm1 with 137.6- and 1.3-fold increases in half-life at 50 °C and specific activity compared to the wild-type xynA, respectively. With the highest thermostability at 80 and 90 °C in reports, xynAm1 could be a robust candidate for industrial applications in functional foods, feed products, and bioethanol production.