Engineering and screening of novel β-1,3-xylanases with desired hydrolysate type by optimized ancestor sequence reconstruction and data mining

[Display omitted] •A novel integrative strategy for engineering β-1,3-xylanases with desired products.•AncXyl10 is the first successful example of ASR to shift the hydrolysate types.•The hydrolysates of AncXyl10 was only β-1,3-xylobiose and β-1,3-xylotriose.•The underlying mechanism laid a new groundwork towards hydrolase engineering. Engineering of hydrolases to shift their hydrolysate types has not been attempted so far, though computer-assisted enzyme design has been successful. A novel integrative strategy for engineering and screening the β-1,3-xylanase with desired hydrolysate types was proposed, with the purpose to solve problems that the separation and preparation of β-1,3-xylo-oligosaccharides was in high cost yet in low yield as monosaccharides existed in the hydrolysates. By classifying the hydrolysate types and coding them into numerical values, two robust mathematical models with five selected attributes from molecular docking were established based on LogitBoost and partial least squares regression with overall accuracy of 83.3% and 100%, respectively. Then, they were adopted for efficient screening the potential mutagenesis library of β-1,3-xylanases that only product oligosaccharides. The virtually designed AncXyl10 was selected and experimentally verified to produce only β-1,3-xylobiose (60.38%) and β-1,3-xylotriose (39.62%), which facilitated the preparation of oligosaccharides with high purity. The underlying mechanism of AncXyl10 may associated with the gap processing and ancestral amino acid substitution in the process of ancestral sequence reconstruction. Since many carbohydrate-active enzymes have highly conserved active sites, the strategy and their biomolecular basis will shield a new light for engineering carbohydrates hydrolase to produce specific oligosaccharides.