Improvements in xylose stability and thermalstability of GH39 β-xylosidase from Dictyoglomus thermophilum by site-directed mutagenesis and insights into its xylose tolerance mechanism

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 Ki 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.