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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Directed Evolution of Beta-galactosidase from Escherichia coli into Beta-glucuronidase

  • Xiong, Ai-Sheng (College of Horticulture, Nanjing Agricultural University) ;
  • Peng, Ri-He (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences) ;
  • Zhuang, Jing (College of Horticulture, Nanjing Agricultural University) ;
  • Liu, Jin-Ge (College of Horticulture, Nanjing Agricultural University) ;
  • Xu, Fang (College of Horticulture, Nanjing Agricultural University) ;
  • Cai, Bin (College of Horticulture, Nanjing Agricultural University) ;
  • Guo, Zhao-Kui (Heilongiiang Tobacco Research Institute) ;
  • Qiao, Yu-Shan (College of Horticulture, Nanjing Agricultural University) ;
  • Chen, Jian-Min (College of Bioscience and Biotechnology, Yangzhou University) ;
  • Zhang, Zhen (College of Horticulture, Nanjing Agricultural University) ;
  • Yao, Quan-Hong (Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences)
  • Published : 2007.05.31
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Abstract

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: S193N, 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 $\beta$-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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References

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