Ancestral sequence reconstruction of a robust β-1,4-xylanase and efficient expression in Bacillus subtilis

Xylanases are a class of glycoside hydrolases commonly used in the food, papermaking, and textile industries. However, most xylanases are rapidly inactivated under harsh industrial conditions. Here, a unique and robust GH11 xylanase, AncXyn18, was designed using an ancestral sequence reconstruction strategy, sequence analysis, structure prediction, and experimental verification. It displayed desirable robustness with high alkali resistance and thermostability, retaining >50 % of the initial activity after incubation at pH 10.0 or 70 °C for 10 h. Furthermore, the engineered strain Bs-AncXyn18-Du12 based on the dual promoter P_sigW-P₄₃ increased the enzyme activity of AncXyn18 7.5-fold, reaching 58.2 U/mL. This work offers a theoretical basis for the improvement of xylanases, which will benefit the enzymatic bioconversion of xylan-containing agricultural waste into high-value oligosaccharide products and promote green industrial development.