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Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase

文献类型： 外文期刊

作者： Xiong, Ai-Sheng 1 ; Peng, Ri-He 2 ; Zhuang, Jing 2 ; Liu, Jin-Ge 2 ; Xu, Fang 3 ; Cai, Bin; Guo, Zhao-Kui; Qiao, Yu 1 ;

作者机构： 1.Nanjing Agr Univ, Coll Hort, Nanjing 210095, Peoples R China

2.Nanjing Agr Univ, Coll Hort, Nanjing 210095, Peoples R China; Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China; Heilongjiang Tobacco Res Inst, Mudanjiang 157011, Peoples R China; Yangzhou Univ, Coll Biosci & Biotechnol, Yangzhou 225009, Peoples R China

3.Nanjing Agr Univ, Coll Hort, Nanjing 210095, Peoples R China; Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 2

关键词： beta-galactosidase;beta-glucuronidase;directed evolution;DNA shuffling;enzyme properties;structure-function analysis

期刊名称：JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY （ 影响因子：2.021； 五年影响因子：2.1 ）

ISSN： 1225-8687

年卷期： 2007 年 40 卷 3 期

页码：

收录情况： SCI

摘要： In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S-193N, T266A, Q267R, V411A, D448G G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high P-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

  • 相关文献

[1]Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Li, Xian,Xue, Yong,Liu, Jin-Ge,Gao, Feng,Cai, Bin,Chen, Jian-Min,Yao, Quan-Hong.

[2]Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability. Xiong, Ai-Sheng,Peng, Ri-He,Cheng, Zong-Ming,Li, Yi,Liu, Jin-Ge,Zhuang, Jing,Gao, Feng,Xu, Fang,Qiao, Yu-Shan,Zhang, Zhen,Chen, Jian-Min,Yao, Quan-Hong. 2007

[3]A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences. Xiong, Ai-Sheng,Peng, Ri-He,Zhuang, Jing,Liu, Jin-Ge,Gao, Feng,Xu, Fang,Cai, Bin,Yao, Quan-Hong.

[4]High efficiency and throughput system in directed evolution in vitro of reporter gene. Xiong, Ai-Sheng,Peng, Ri-He,Liu, Jin-Ge,Zhuang, Jing,Qiao, Yu-Shan,Xu, Fang,Cai, Bing,Zhang, Zhen,Chen, Jian-Min,Yao, Quan-Hong.

[5]Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance. Tian, Yong-Sheng,Xu, Jing,Peng, Ri-He,Xiong, Ai-Sheng,Xu, Hu,Zhao, Wei,Fu, Xiao-Yan,Han, Hong-Juan,Yao, Quan-Hong.

[6]Improved glyphosate resistance of 5-enolpyruvylshikimate-3-phosphate synthase from Vitis vinifera in transgenic Arabidopsis and rice by DNA shuffling. Tian, Yong-Sheng,Xu, Jing,Zhao, Wei,Fu, Xiao-Yan,Peng, Ri-He,Yao, Quan-Hong,Tian, Yong-Sheng,Tian, Yong-Sheng,Xing, Xiao-Juan.

[7]Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity. Tian, Yong-Sheng,Peng, Ri-He,Xu, Jing,Zhao, Wei,Gao, Feng,Fu, Xiao-Yan,Xiong, Ai-Sheng,Yao, Quan-Hong.

[8]Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis. Xiong, Ai-Sheng,Wang, Feng,Jiang, Hai-Hua,Peng, Ri-He,Jin, Xiao-Fen,Zhu, Bo,Yao, Quan-Hong,Zhuang, Jing,Zhang, Jian.

[9]Characterization of a thermostable beta-glucuronidase from Thermotoga maritima expressed in Arabidopsis thaliana. Xu, Jing,Tian, Yong-Sheng,Peng, Ri-He,Zhu, Bo,Gao, Jian-Jie,Yao, Quan-Hong. 2012

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