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

文献类型： 外文期刊

第一作者： Xiong, Ai-Sheng

作者： Xiong, Ai-Sheng;Peng, Ri-He;Zhuang, Jing;Liu, Jin-Ge;Xu, Fang;Cai, Bin;Guo, Zhao-Kui;Qiao, Yu-Shan;Chen, Jian-Min;Zhang, Zhen;Yao, Quan-Hong

作者机构：

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.

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