Bioengineering GH10 xylanase through rational design and MD simulation: Enhanced thermal stability and bioenergy synergies with cellulase

Xylanase plays a crucial role in lignocellulose degradation. Industrial processing demands enzymes with ideal thermostability as well as sufficient catalytic efficiency, especially at the reaction temperature of 50℃. However, existing xylanases often lack these characteristics. This study employed the rational design (FoldX) method to analyze the wild-type GtXyn10 from Gloeophyllum trabeum for mutation prediction, with a focus on ΔΔG. Two mutants, G29L and H218A, were screened and then combined to generate a combined mutant (G29L/H218A), designated as M3. The mechanism of the heat-resistant mutant was investigated through molecular dynamics simulation. The results showed that the rigidity of M3 was enhanced due to the formation of new salt bridges and hydrogen bonds, along with an increase in ΔΔG. Besides its remarkable pH resistance within the range of 2.0 10.0, the dominant mutant M3 demonstrated a higher catalytic efficiency (4.8 times; 240 vs. 50 mLꞏs −1 ꞏmg −1 ) and improved thermostability (the half-life was extended by 8.5 h at 50℃). Compared with cellulase alone (330.8 mg/g), the synergistic hydrolysis of M3 and cellulase (834.4 mg/g) increased the yield of reducing sugar by 152 % after pretreatment with “Seawater + Feton”. This research successfully obtained an industry-oriented xylanase with optimized thermostability, further facilitating more efficient biomass processing. • This study provides an industry-oriented xylanase for bagasse degradation. • Computer-aided design increased loop rigidity and remote interaction of GtXyn10. • Thermostability and catalytic efficient of the mutant were boosted simultaneously. • The bagasse biorefining achieved higher efficiency with the dominate mutant.