Harnessing Xylanase Potential in Thermothelomyces fergusii: Insights from Computational and Functional Analysis

Xylanases are crucial for the breakdown of hemicellulose, enabling the conversion of lignocellulosic biomass into fermentable sugars for biofuels and other industrial applications. For the first time, we investigated the biochemical and genetic characteristics of 22 xylanase genes from <i>Thermothelomyces fergusii</i> within glycoside hydrolase (GH) families GH10, GH11, and GH43. Xylanase genes structural diversity clustered the phylogenetic tree into GH10, GH11, GH43-I, and GH43-II groups. Structural analysis revealed that all <i>TfGH10</i> and <i>TfGH11</i> genes contained conserved GH domains, with CBM1 present in <i>TfGH10-5</i> and <i>TfGH11-4</i>. Secondary domains, including CBM35, CBM42, and CBM91, were found in the GH43 gene family. The presence of key glutamic (Glu) and aspartic (Asp) residues in active sites is essential for substrate binding and catalysis. RT-qPCR analysis revealed substrate-dependent gene expression, with peak upregulation on day three in beechwood xylan (BWX) cultures and day two in corncob xylan (CCX) and rice straw (RS) cultures. Consistent with these findings, enzymatic assays demonstrated the highest xylanase activity in BWX-induced cultures, followed by RS and CCX, underscoring the differential regulation of these enzymes in response to distinct hemicellulosic substrates. These findings provide valuable insights into the structural, functional, and regulatory mechanisms of <i>T. fergusii</i> xylanases, facilitating their industrial application.