Humicola grisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichia pastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (k_cat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

Humicola grisea var. thermoidea是一种需氧和嗜热的真菌，在甘蔗渣存在的情况下会分泌 GH11 木聚糖酶 HXYN2。在这项研究中，HXYN2 在毕赤 酵母中表达，并在存在山毛榉木聚糖底物和阿魏酸 (FA) 的情况下进行生化和结构表征。HXYN2 是一种热稳定的蛋白质，如圆二色性所示，在 40-50°C 和 pH 5.0-9.0 范围内具有更大的活性，最佳温度和 pH 分别为 50°C 和 6.0。FA 导致酶活性增加 75%，催化速度、催化效率和催化速率常数（k _cat)，酶对底物的亲和力没有改变。荧光猝灭表明 FA 与 HXYN2 与溶剂暴露的色氨酸残基相互作用形成复合物。结合常数范围从中等（pH 7.0 和 9.0）到强（pH 4.0）亲和力。等温滴定量热法、结构模型和分子对接表明，氢键和疏水相互作用发生在糖苷配基区域，分别由初始和最终焓和熵过程驱动的活性位点发生构象变化。这些结果表明 HXYN2 具有生物技术应用的潜力，例如在富含阿魏酸的植物残留物的生物转化中。