title: Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I fromGeobacillus stearothermophilusby Site-Directed Mutagenesis at the Active Site.
abstract: The large fragment of DNA polymerase I fromGeobacillus stearothermophilusGIM1.543 (Bst DNA polymerase) with 5'-3' DNA polymerase activity while in absence of 5'-3' exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified inE. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310by alanine or leucine and residue Asp540by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Ma
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Beilei
authors.initials: B
authors.lastname: Zhang
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Yanghui
authors.initials: Y
authors.lastname: Ou
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jufang
authors.identifier: 0000-0002-9908-1001
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China; Guangdong Province Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Shan
authors.identifier: 0000-0002-5211-0872
authors.initials: S
authors.lastname: Li
mesh_terms: D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D056506:Geobacillus / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
chemical_list: D004256:DNA Polymerase I