Substituting Both the N-Terminal and “Cord” Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics

To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and “cord” regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A41, possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A41 (or AoXyn11A), another mutant, named ATX11A41/cord, was designed by substituting the cord region (93GTYNPGSGG101) of ATX11A41 with the corresponding one (93GTYRPTG99) of pXYL11. Both mutant-encoding genes, ATx11A41 and ATx11A41/cord, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A41, and ATX11A41/cord were 2916.7, 2667.6, and 2457.0 U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (Topt) of reATX11A41 or reATX11A41/cord was 65 °C, which was 15 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t1/2) values of reATX11A41 and reATX11A41/cord at 60 °C were 55 and 83 min, respectively, whereas that of reAoXyn11A was only 18 min at 50 °C. The melting temperature (Tm) values of reAoXyn11A, reATX11A41, and reATX11A41/cord were 54.2, 66.7, and 71.9 °C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.