β-Xylosidases are often inhibited by its reaction product xylose or inactivated by high temperature environment, which limited its application in hemicellulosic biomass conversion to fuel and food processing. Remarkably, some β-xylosidases from GH39 family are tolerant to xylose. Therefore, it is of great significance to elucidate the effect mechanism of xylose on GH39 β-xylosidases to improve their application. In this paper, based on the homologous model and prediction of protein active pocket constructed by I-TASSA and PyMOL, two putative xylose tolerance relevant sites (283 and 284) were mutated at the bottom of the protein active pocket, where xylose sensitivity and thermostability of Dictyoglomus thermophilum β-xylosidase Xln-DT were improved by site-directed mutagenesis. The Xln-DT mutant Xln-DT-284ASP and Xln-DT-284ALA showed high xylose tolerance, with the K_i values of 4602 mM and 3708 mM, respectively, which increased by 9-35% compared with the wildtype Xln-DT. The thermostability of mutant Xln-DT-284ASP was significantly improved at 75 and 85 °C, while the activity of the wild enzyme Xln-DT decreased to 40-20%, the activity of the mutant enzyme still remained 100%. The mutant Xln-DT-284ALA showed excellent stability at pH 4.0-7.0, but Xln-DT-284ASP showed slightly decreased activity. Furthermore, in order to explore the key sites and mechanism of xylose’s effect on β-xylosidase activity, the interaction between xylose and enzyme was simulated by molecular docking. Besides binding to the active sites at the bottom of the substrate channel, xylose can also bind to sites in the middle or entrance of the channel with different affinities, which may determine the xylose inhibition of β-xylosidase. In conclusion, the improved xylose tolerance of mutant enzyme could be more advantageous in the degradation of hemicellulose and the biotransformation of other natural active substances containing xylose. This study supplies new insights into general mechanism of xylose effect on the activity of GH 39 β-xylosidases as well as related enzymes that modulate their activity via feedback control mechanism.

β-木糖苷酶常常被其反应产物木糖抑制或在高温环境下失活，这限制了其在半纤维素生物质转化为燃料和食品加工中的应用。值得注意的是，来自 GH39 家族的一些 β-木糖苷酶对木糖具有耐受性。因此，阐明木糖对GH39 β-木糖苷酶的作用机制，对提高其应用具有重要意义。本文基于 I-TASSA 和 PyMOL 构建的蛋白质活性口袋的同源模型和预测，在蛋白质活性口袋的底部发生了两个假定的木糖耐受相关位点（283 和 284）突变，其中木糖敏感性和热稳定性嗜热粘菌属通过定点诱变改进了β-木糖苷酶Xln-DT。Xln-DT 突变体 Xln-DT-284ASP 和 Xln-DT-284ALA 表现出较高的木糖耐受性，K _i值分别为 4602 mM 和 3708 mM，与野生型 Xln-DT 相比增加了 9-35%。突变体Xln-DT-284ASP的热稳定性在75和85°C时显着提高，而野生酶Xln-DT的活性下降到40-20%，突变酶的活性仍保持在100%。突变体 Xln-DT-284ALA 在 pH 4.0-7.0 下表现出优异的稳定性，但 Xln-DT-284ASP 的活性略有下降。此外，为了探索木糖影响β-木糖苷酶活性的关键位点和机制，通过分子对接模拟了木糖与酶的相互作用。木糖除了与底物通道底部的活性位点结合外，还可以与通道中部或入口处的位点以不同的亲和力结合，这可能决定了木糖对β-木糖苷酶的抑制作用。总之，突变酶提高的木糖耐受性可能更有利于半纤维素的降解和其他含有木糖的天然活性物质的生物转化。本研究为木糖对 GH 39 β-木糖苷酶以及通过反馈控制机制调节其活性的相关酶的活性的一般机制提供了新的见解。