Improving Thermostability of GH11 Xylanase XynASP by the Design of Loop Region

Highly flexible loop regions can affect the structure-function relationship of enzymes. In this study, to reveal the effect of the loop on the thermal stability of GH11 xylanase XynASP from Aspergillus saccharolyticus JOP 1030-1, three mutants (T41V, A79Y, T81Q) located in the loop region were predicted by the FireProt web server and constructed by site-directed mutagenesis. Heat tolerance experiments were performed in the mutants and wild-type XynASP, as well as for previously reported mutant T41W, indicating that the thermostability of enzymes from weak to strong was wild-type XynASP, T41V, T81Q, A79Y and T41W. Novel hydrophobic contacts and hydrogen bonds in the single mutants were found and explained for enhanced thermostability. T41W, A79Y and T81Q were combined by site-directed mutagenesis. The combined double mutants T41W/A79Y and A79Y/T81Q displayed significantly higher thermostability than that of the wild-type, yet lower than that of the robust mutant T41W after 30 min of incubation at 35-60 degrees C. But the triple mutant T41W/A79Y/T81Q displayed a slight improvement in thermal stability compared to T41W after 30 min of incubation at 35-60 degrees C. Meanwhile, T41W/A79Y/T81Q exhibited substantially improved thermostability with a half-life of inactivation enhanced from 20.7 min to 127.0 min at 45 degrees C. Furthermore, the optimum temperature increased by 10 degrees C compared to the wild-type XynASP. The pH stability of T41W/A79Y/T81Q at pH 3.0-8.0 all obtained more than 88% residual activities, displaying a significant improvement compared to the wild-type XynASP (40-70% residual activities) at the same conditions. This study confirms that designing GH11xylanases in the loop region is an effective strategy for obtaining thermostabilizing xylanases.

Automated summary

This study investigates the effect of mutations in the loop region on the thermal stability of GH11 xylanase. The results show that certain mutations can significantly enhance the thermostability of the enzyme, and the mechanisms for improved stability are elucidated through structural analysis.