title: [Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].
abstract: 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.
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics And Breeding, Agro-Biotechnology Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China. aishengxiong@yahoo.com
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Chen
authors.forename: Xian
authors.initials: X
authors.lastname: Li
authors.forename: Hui-Qin
authors.initials: HQ
authors.lastname: Fan
mesh_terms: 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
chemical_list: D005966:Glucuronidase