Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2016 (2016), Article ID 2906484, 8 pages
http://dx.doi.org/10.1155/2016/2906484
Research Article

Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

1School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China
2Guangdong Province Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China

Received 25 August 2016; Revised 13 October 2016; Accepted 19 October 2016

Academic Editor: Sujit S. Jagtap

Copyright © 2016 Yi Ma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (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 However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits.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 in E. 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 Gly310 by alanine or leucine and residue Asp540 by 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.