引入疏水性氨基酸对GH11家族木聚糖酶热稳定性的影响
Effect of introducing hydrophobic amino acids residues on the thermostability of family GH11 xylanases
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摘要: 生物信息学预测黑曲霉(Aspergillus niger)XZ-3S木聚糖酶Xyn ZF-2的催化活性中心位点,并在α-螺旋处引入疏水性氨基酸以提高木聚糖酶Xyn ZF-2的热稳定性。通过定点突变活性位点E103D、E194D,构建突变基因xyn ED。同时,将α-螺旋处氨基酸Val125、Lys178和Gly180分别突变为疏水性氨基酸Ala、Met和Ala,构建突变基因xyn MA。突变基因xyn ED和xyn MA在大肠杆菌BL21中表达发现,突变酶Xyn ED酶活性为原酶Xyn ZF-2的0.17%;突变酶Xyn MA最适温度由原来的40℃提高到了48℃;在40℃条件下保温1 h,突变酶Xyn MA酶活性仍高达74.71%(原酶Xyn ZF-2为44.36%);突变酶Xyn MA的半衰期t45℃1/2从原来的7 min提高到了19 min。因此,Glu103和Glu194为木聚糖酶Xyn ZF-2的催化活性中心位点,且在木聚糖酶Xyn ZF-2α-螺旋处引入疏水性氨基酸能大大提高其热稳定性。Abstract: 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.