Effect of introducing hydrophobic amino acids residues on the thermostability of family GH11 xylanases

The active sites of xylanase Xyn ZF- 2 from Aspergillus niger XZ- 3S was predicted by the software of bioinformatics.Simultaneously,to improve thermostability of the Xyn ZF-2,hydrophobic amino acids residues were introduced by site- directed mutagenesis. The active sites were substituted by site- directed mutagenesis( E103 D and E194D),and the amino acids residues( Val125、Lys178 and Gly180) in the α- helix domains were substituted into the hydrophobic amino acids residues( Ala、Met and Ala),respectively. The mutated active sites xyn ED- and the mutated hydrophobic xyn MA-encoding genes were constructed and expressed in Escherichia coli BL21( DE3).Compared to the native xylanase,it was found that the mutated xylanase Xyn ED was 0.17%. The optimum temperature of the variant Xyn MA was increased from 40 ℃ to 48 ℃,and the variant Xyn MA retained about 74.71%activity( the Xyn ZF-2 retained 44.36% activity) after treatment at 40 ℃ for 60 min.t45 ℃1 /2of the variant Xyn MA was also increased from 7 to 19 min. The result of the mutated xylanase indicated that the active center of Xyn ZF- 2 was mainly consisted of two catalytic residues( Glu103 and Glu194),and the introduction of hydrophobic amino acids residues in the α- helix domains could improve the thermostability of Xyn ZF-2.