Characterization of a multifunctional enzyme from Trichoderma harzianum and its application in enhanced enzymatic hydrolysis.

Efficient saccharification of lignocellulose to fermentable sugars is crucial for bioconversion, yet the process is often hindered by insufficient β-glucosidase, β-xylosidase, and α-L-arabinofuranosidase activities in enzyme cocktails from Trichoderma reesei. This study addresses this gap by identifying BX1, a multifunctional enzyme from the underexplored fungus Trichoderma harzianum EM0925, which demonstrates a triad of activities targeting hemicellulose-derived oligosaccharides preferentially. We used structural analysis, molecular docking, and mutation studies to elucidate the roles of specific residues (Asp, Glu, Gln, Cys, Tyr, and Tyr) in BX1's multifunctionality. The enzyme showed synergistic effects with cellulase and xylanase, leading to a 90.23% increase in fermentable sugar yields at 2% (w/v) solid substrate loads and a 22.14% improvement at 15% (w/v) loads when added to Celluclast 1.5L. These findings highlight BX1's potential to enhance lignocellulosic bioconversion efficiency and reduce associated costs, paving the way for more cost-effective saccharification processes and future enzyme engineering advancements.