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(21) 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(G21I), was selected from the mutagenesis library, E. coli/Aoxyn11A (G21X) (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A(Y13F) or AoXyn11A(G21I–Y13F), was designed by replacing Tyr(13) in AoXyn11A or AoXyn11A(G21I) with Phe. Finally, three variant-encoding genes, Aoxyn11A (G21I), Aoxyn11A (Y13F) and Aoxyn11A (G21I–Y13F), were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T (opt)) of recombinant (re) AoXyn11A(G21I–Y13F) was 60 °C, being 5 °C higher than that of reAoXyn11A(G21I) or reAoXyn11A(Y13F), and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t (1/2)) of reAoXyn11A(G21I–Y13F) at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11A(G21I) and reAoXyn11A(Y13F). The melting temperature (T (m)) values of reAoXyn11A, reAoXyn11A(G21I), reAoXyn11A(Y13F) and reAoXyn11A(G21I–Y13F) 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 (cat)/K (m)) of reAoXyn11A(G21I–Y13F) was 473.1 mL mg(−1) s(−1), which was 1.65-fold higher than that of reAoXyn11A. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-017-0399-9) contains supplementary material, which is available to authorized users.