Structural insights into xylanase mutant 254RL1 for improved activity and lower pH optimum

Xylanases degrade xylan to valuable end products. In our previous study, the alkaline xylanase S7-xyl from Bacillus halodurans S7 was engineered by rational design and the best mutant xylanase 254RL1 exhibited 3.4-fold improvements in specific activity at pH 9.0. Further research found that the enzyme activity at pH 6.0 was almost 2-fold than that at pH 9.0. To elucidate the reason of enhanced performance of 254RL1 at decreased pH optimum, we determined the X-ray crystal structure of 254RL1 at 2.21 angstrom resolution. The structural analysis revealed that the mutations enlarged the opening of the access tunnel and shortened the tunnel. Moreover, the mutations changed the hydrogen bond network around the catalytic residue and decreased the pK(a) value of acid-base catalyst E159 which reduced the pH optimum of the xylanase. The result provided the basis for the acid-alkaline engineering of the glycoside hydrolases.

Automated summary

The engineered xylanase showed improved activity at a lower pH, with structural analysis revealing mutations that enlarged and shortened the access tunnel, changed the hydrogen bond network and decreased the pK(a) value, leading to a decreased pH optimum. This provides a basis for acid-alkaline engineering of glycoside hydrolases.