Abstract
Xylanase from glycoside hydrolases family 11 (GH 11) is widely applied because of their substrate selectivity and broad optimal pH. However, higher thermostability is always preferred in addition to other stress tolerance of the enzyme. Therefore, we first expressed and characterized a novel GH 11 xylanase from Hortaea werneckii (HWxyn11) tolerant to NaCl and extreme pH conditions although still unsatisfied thermostability. To achieve better thermostability, we then constructed mutants by applying N-terminal replacement or C-terminal carbohydrate binding module (CBM) fusion, considering that thermostability of GH 11 xylanase could be affected by structural differences between N-terminus and C-terminus significantly. The combined mutant HWxA31-CBM 6 had increased thermostability than that of HWxyn11. Molecular dynamics simulation analysis reveals that the N-terminal replacement or C-terminal CBM fusion resulted in declined flexibility, indicating the improvement of structural rigidity and thermostability. Structural analysis confirms that the removal of N-terminal propeptide and the fusion of CBM contributed to improved thermostability. Our findings prove that the combination of replacing N-terminal sequences and fusing with CBMs from thermophilic xylanases contributed to declined structural flexibility, providing a practicable method to improve the thermostability of GH 11 xylanases.