The critical roles of exposed surface residues for the thermostability and halotolerance of a novel GH11 xylanase from the metagenomic library of a saline-alkaline soil.

Thermostable and halotolerant GH11 xylanases are highly desirable in various industrial applications but they were rarely characterized. In this study, we firstly revealed the genetic diversity of GH11 xylanases from the metagenomic DNA of a saline-alkaline soil by constructing a gene fragment library. Twelve distinct xylanase fragment sequences were identified, which shared 62-96% identity to known GH11 xylanases. Among them, Xyn22 with the highest relative abundance was cloned and found to exhibit excellent halotolerance. Xyn22 retained 80 and 58% relative activity in the presence of 3 and 5 M NaCl, respectively, and had no loss of activity after incubation in 3 M NaCl at 37 °C for 1 h. Additionally, the acidic amino acid residues E137 and E139 on the protein's surface were revealed to cooperatively play important roles in its excellent halotolerance and halostabilty. Furthermore, the thermostability of Xyn22 was significantly improved without compromising halotolerance by introducing an aromatic interaction between Y48 and F53. In conclusion, this study not only provides a thermostable and halotolerant GH11 xylanase, but also reveals the key roles of surface-exposed amino acids in its thermostability and halotolerance, which could be used for further molecular modification of other xylanases and similar enzymes.