[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].
The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.
Xiong
Ai-Sheng
AS
Shanghai Key Laboratory of Agricultural Genetics And Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China. aishengxiong@yahoo.com
Yao
Quan-Hong
QH
Peng
Ri-He
RH
Chen
Jian-Min
JM
Li
Xian
X
Fan
Hui-Qin
HQ
D000595:Amino Acid Sequence; D001483:Base Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D005966:Glucuronidase / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D005966:Glucuronidase