Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase.
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.
Xiong
Ai-Sheng
AS
College of Horticulture, Nanjing Agricultural University, Nanjing, China.
Peng
Ri-He
RH
Zhuang
Jing
J
Liu
Jin-Ge
JG
Xu
Fang
F
Cai
Bin
B
Guo
Zhao-Kui
ZK
Qiao
Yu-Shan
YS
Chen
Jian-Min
JM
Zhang
Zhen
Z
Yao
Quan-Hong
QH
D032621:DNA Shuffling / Q000379:methods; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001616:beta-Galactosidase; D005966:Glucuronidase