Gene

Hfr cell

Xylanase

Protein engineering

Thermophile

Biochemistry

Food science

Saturated mutagenesis

Mutant

Enzyme

Escherichia coli

Aspergillus niger

Thermostability

Chemistry

Journal of Agricultural and Food Chemistry

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 EvXyn11TS 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.

Significantly Enhanced Thermostability of <i>Aspergillus niger</i> Xylanase by Modifying Its Highly Flexible Regions

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