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KMID : 0380220070400030419
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 3 p.419 ~ p.425
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Directed Evolution of Beta-galactosidase from Escherichia coli into Beta-glucuronidase
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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
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Abstract
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In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli &bgr;-galactosidase and &bgr;-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 &bgr;-glucuronidase activity than wild-type &bgr;-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 &bgr;-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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KEYWORD
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β-galactosidase, β-glucuronidase, Directed evolution, DNA shuffling, Enzyme properties, Structure-function analysis
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