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 ATX11A, 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 ATX11A (or AoXyn11A), another mutant, named ATX11A, was designed by substituting the cord region (GTYNPGSGG) of ATX11A with the corresponding one (GTYRPTG) of pXYL11. Both mutant-encoding genes, ATx11A and ATx11A, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A, and ATX11A were 2916.7, 2667.6, and 2457.0 U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (T) of reATX11A or reATX11A was 65 °C, which was 15 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t) values of reATX11A and reATX11A at 60 °C were 55 and 83 min, respectively, whereas that of reAoXyn11A was only 18 min at 50 °C. The melting temperature (T) values of reAoXyn11A, reATX11A, and reATX11A 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.