Engineering xylanase from Trichoderma harzianum enhances xylan hydrolysis to produce xylooligosaccharides

Saccharification of xylan from lignocellulosic wastes into xylooligosaccharide prebiotics, specifically xylobiose and xylotriose, is of great economic importance and relies on specific endo-β-1,4-xylanases. Our previous studies identified endo-β-1,4-xylanase TXyn11A from Trichoderma harzianum that mainly produces xylobiose and/or xylotriose from hydrolysis of beechwood and sugarcane bagasse xylan. However, its catalytic mechanisms remain unclear, and yields from native T. harzianum are very low. In the present study, recombinant TXyn11A (rTXyn11A) purified from Pichia pastoris catalysed the conversion of xylan into xylobiose and xylotriose via its combined endo-β-1,4-xylanase and β-1,4-xylosidase activities, and transglycosidation. Random mutagenesis and point mutation based on auto-docking identified eight key residues essential for specific activity and hydrolysis ability. Notably, glutamine 156 and aspartic acid 201 respectively control hydrolysis of xylotriose and xylotetraose to generate xylobiose. Mutant enzymes Q156A and D201I, and double mutant Q156A-D201I, displayed enhanced specific activity and ability to hydrolyze xylan into xylooligosaccharide. Auto-docking analysis revealed that the active centres of the three mutant enzymes bound xylotriose and xylotetraose more strongly than wild-type enzyme via more hydrogen bonds. These results provide novel insights into the mechanism of xylanase action to produce mainly xylobiose from hydrolysis of xylan, and engineered xylanases with improved catalytic activity.