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

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 (Asp³⁸⁹, Glu⁵⁸⁹, Gln¹⁸⁵, Cys³⁹⁰, Tyr³⁵⁴, and Tyr⁵²⁶) 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.