摘要
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.
出处
《中国酿造》
CAS
北大核心
2015年第10期27-31,共5页
China Brewing
基金
国家级大学生创新创业训练计划项目(201410472028)
河南省教育厅科学技术研究重点项目(13A180861)
河南省高等学校青年骨干教师资助计划项目(2011GGJS-125)
新乡医学院科研项目培育基金(2013ZD113)
关键词
木聚糖酶
热稳定性
定点突变
中性氨基酸
xylanase
thermostability
site-directed mutagenesis
neutral amino acids residues