Abstract:A β-xylosidase Xln-DT has been cloned and heterologously expressed from the thermophilic bacterium Dictyoglomus thermophilum in the previous studies. A mutant of β-xylosidase Xln-DT with high enzyme activity and excellent enzymatic properties was obtained, in order to improve its vital application value in bio-fuel, food and medicine industries. The key amino acid mutation sites of β-xylosidase Xln-DT were obtained through bioinformatics methods to improve the specific enzyme activity. Amino acids HIS/LEU, PHE/LEU and TRP were introduced at sites 161, 202 and 231, respectively, and the constructed mutant enzymes Xln-DT-202PHE and Xln-DT-202LEU obtained significantly increased β-xylosidase activity. In addition, the enzymatic properties before and after the mutation were analyzed and compared. The expressed enzyme activities of Xln-DT-202PHE and Xln-DT-202LEU were 3.28 and 2.97 times higher than that of Xln-DT, respectively, while their specific activities were 2.86 and 2.54 times higher than that of Xln-DT, respectively. The optimal pH of mutant enzyme Xln-DT-202PHE and Xln-DT-202LEU decreased significantly from pH 6.0 to pH 4.5 and pH 5.0, respectively. The enzyme activity of Xln-DT-202LEU remained unchanged when incubated at 75 ℃ for 2 h, which indicated significantly improved thermal stability compared with that of Xln-DT under 75 ℃. Xln-DT-202PHE and Xln-DT-202LEU could maintain more than 80% of the residual enzyme activity in the range of pH 4.0~7.0 after 24 h incubation. The mutant enzyme not only significantly improved the enzyme activity, but also greatly improved its thermal stability, laying a foundation for its wide application in food thermal processing and other fields.
