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

Abstract

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, Gly21 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, AoXyn11AG21I, was selected from the mutagenesis library, E. coli/Aoxyn11AG21X (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11AY13F or AoXyn11AG21I–Y13F, was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe. Finally, three variant-encoding genes, Aoxyn11AG21I, Aoxyn11AY13F and Aoxyn11AG21I–Y13F, were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (Topt) of recombinant (re) AoXyn11AG21I–Y13F was 60 °C, being 5 °C higher than that of reAoXyn11AG21I or reAoXyn11AY13F, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) of reAoXyn11AG21I–Y13F at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11AG21I and reAoXyn11AY13F. The melting temperature (Tm) values of reAoXyn11A, reAoXyn11AG21I, reAoXyn11AY13F and reAoXyn11AG21I–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 (kcat/Km) of reAoXyn11AG21I–Y13F was 473.1 mL mg−1 s−1, which was 1.65-fold higher than that of reAoXyn11A.