Computationally assisted combinatorial rational design of xylanase with improved thermostability and catalytic...

Lignocellulosic biomass has low utilization efficiency because of its complex structure. Therefore, xylanases with high thermostability and catalytic activity are critical for improving saccharification and biomass utilization. In this study, a rational engineering strategy was developed based on the Aspergillus niger-derived xylanase XynB expressed in Pichia pastoris GS115, integrating signal peptide optimization, disulfide bond engineering, ΔΔG-guided multisite mutagenesis, fermentation scale-up, and biomass application evaluation. Signal peptide screening identified HFBI as the optimal secretion signal; compared with the parental strain 9K-XynB, 9K-HFBI-XynB showed 1.42-fold and 1.60-fold increases in secretion level and catalytic activity, respectively. Combining disulfide bond engineering with ΔΔG-guided multisite mutagenesis generated the engineered variant 9K-XynB2 (HFBI-A52C/T205C-F31Y/A50F/A143P), which showed an approximately 11-fold longer half-life at 60℃ and a 7.7-fold increase in catalytic efficiency (kcat/Km) relative to 9K-XynB. In 5-L high-cell-density fermentation, 9K-XynB2 reached an activity of about 8.2 × 10³ U/mL, demonstrating scale-up potential. Using dilute alkali-pretreated lignocellulosic residues as substrates, 9K-XynB2 enhanced xylan hydrolysis, with corncob showing the highest saccharification efficiency. In combination with the commercial cellulase , 9K-XynB2 exhibited synergistic effects, with a maximum synergy index of 1.95 and a highest reducing sugar concentration of 6.21 ± 0.06 g/L. Residue composition analysis further confirmed improved substrate utilization. In summary, this study presents a combinatorial strategy for xylanase performance enhancement and targeted engineering, and yields an engineered xylanase with high thermostability and catalytic efficiency, supporting the development of efficient lignocellulose-degrading systems and cost-effective biomass conversion.