Improving the Thermostability and pH Stability of Aspergillus niger Xylanase by Site-directed Mutagenesis

Abstract To increase the thermostability and pH stability of the recombinant xylanase MxynB from the Aspergillus niger nl-1, site-directed mutagenesis method was selected by rational approaches based on the three-dimensional structures using a homology modeling of mxynB constructed by SWISS-MODEL and BLAST. The effect of disulphide in the structure of xylanase was studied to increase the optimum temperature and thermostability of enzyme, the variant MxynB-116-135 which was created with amino acid residues substitutions G116C+Y135C by site-directed mutagenesis had its reaction temperature for the maximum activity at 50oC, which corresponded to a basic 10oC unit increase compared with the wild-type enzyme. Moreover, the thermostability of A. niger nl-1 xylanase after mutation MxynB-G116C-Y135C-D76R-N28H-N29D-Y45M-N47L was clearly increased by 570% as compared with the wild-type enzyme under 70oC. In addition, the optimal activity pH of the enzyme after the mutations MxynB-G116C-Y135C-D76R-N28H-N29D-Y45M-N47L and MxynB-G116C-Y135C-S58H-D76R-N28H-N29D-Y45M-N47L raised from 4.5 to 6.0, meanwhile, the pH stabilities were increased from 120 to 194% compared with the wild-type enzyme under pH 5.0 to 7.0. All the results suggest that these amino acid residues are important in determining the thermostability and pH stability of MxynB from Aspergillus niger nl-1, which will raise its potential interest for the industrial applications.