Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design.

To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A, was selected from the mutagenesis library, E. coli/Aoxyn11A (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A or AoXyn11A, was designed by replacing Tyr in AoXyn11A or AoXyn11A with Phe. Finally, three variant-encoding genes, Aoxyn11A , Aoxyn11A and Aoxyn11A , were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T ) of recombinant (re) AoXyn11A was 60 °C, being 5 °C higher than that of reAoXyn11A or reAoXyn11A, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t ) of reAoXyn11A at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11A and reAoXyn11A. The melting temperature (T ) values of reAoXyn11A, reAoXyn11A, reAoXyn11A and reAoXyn11A were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k /K ) of reAoXyn11A was 473.1 mL mg s, which was 1.65-fold higher than that of reAoXyn11A.