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

Abstract

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 Ctermini (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.

Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions

“…In addition to hydrogen bonds and hydrophobic interactions, electrostatic interactions may play a role in the structural rigidity and stability of FRAPD-TGm2A (Table ). Similarly, researchers also demonstrated the critical part of the newly formed hydrophobic interaction in stabilizing xylanase stability and that of electrostatic interactions in the stabilization of the glycoside hydrolases …”

“…However, saturated mutation or iterative mutation for all residues in the flexible region is a long process and a vast workload. 16 Although the prediction tools (e.g., FoldX 42 and Rosetta_Cartesian_ddg 20 ) based on folding free energy variation have been successfully applied in the thermostability enhancement, scanning whole protein sequences generally consumes many computing resources with a high false positive rate. 21 In addition, the ″trade-off″ between thermostability and catalytic activity is inevitable in many rational design strategies.…”

“…To date, flexible region modification is still an effective way to improve enzyme stability. However, saturated mutation or iterative mutation for all residues in the flexible region is a long process and a vast workload . Although the prediction tools (e.g., FoldX and Rosetta_Cartesian_ddg) based on folding free energy variation have been successfully applied in the thermostability enhancement, scanning whole protein sequences generally consumes many computing resources with a high false positive rate .…”

“…Flexible region modification strategies mainly include iterative saturation mutations, substitution with rigid sequences, flexible region deletion, and introduction of glycosylation . However, the flexible region usually consists of multiple amino acid residues, making the iterative saturation mutation within these regions a ton of work . On the other hand, substitution or deletion of the flexible regions does not always work smoothly .…”

“…It is generally believed that the flexible region is one of the key regions contributing to poor enzyme stability. Flexible region modification strategies mainly include iterative saturation mutations, substitution with rigid sequences, flexible region deletion, and introduction of glycosylation . However, the flexible region usually consists of multiple amino acid residues, making the iterative saturation mutation within these regions a ton of work .…”