引入中性氨基酸对木聚糖酶XynZF-2热稳定性的影响

Effect of introducing neutral amino acids residues on the thermostability of xylanases XynZF-2

通过对黑曲霉（Aspergillus niger） XZ-3S木聚糖酶XynZF-2进行生物信息学分析,在N-端区域引入半胱氨酸,定点突变E27C,构建突变基因xyn-E27C,并在大肠杆菌BL21 （DE3）中表达.酶学性质分析发现,突变酶Xyn-E27C的最适温度为45℃,与原酶XynZF-2相比提高了5℃.在40℃条件下,突变酶Xyn-E27C的半衰期t1/2^40℃为100 min,相比原酶XynZF-2 （t1/2^45℃=55 min）提高了45 min.在45℃条件下,突变酶Xyn-E27C的半衰期t1/2^45℃为24 min,相比原酶XynZF-2（t1/2^45℃=7 min）提高了17 min;突变酶Xyn-E27C的最适pH值由5.0提高至5.5,而pH稳定范围均为5.0-9.0.因此,定点突变E27C对木聚糖酶XynZF-2的热稳定性以及最适pH值均有重要影响.

The xylanase XynZF-2 from Aspergillus nigerXZ-3S was analyzed by bioinformatics. Neutral amino acids residues （Cys） were introduced at the N-terminus. The mutated gene xyn-E27C was amplified by site-directed mutagenesis of E27C and expressed in Escherichia coil BL21 （DE3）. According to the enzyme properties analysis and compared to the recombinant XynZF-2, results showed that the optimum temperature of mutant Xyn-E27C was 45℃, which was increased by 5 ℃. At 40 ℃, compared to XynZF-2 （t1/2^45℃=55 min）, the t1/2^℃ of the mutated Xyn-E27C was 100 min, which was increased by 45 min. At 45 ℃, compared to XynZF-2（t1/2^45℃=7 min）, the half-life of the mutated Xyn-E27C was 24 min, which was increased by 17 rain. The optimum pH of mutated Xyn-E27C was increased from 5.0 to 5.5, while the pH stability range was from pH 5.0 to 9.0. There- fore, E27C site-directed mutagenesis had important effect on the thermal stability and pH of xylanase XynZF-2.