An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal amplification applications

作者：Li, Jiaxuan; Li, Yang; Li, Yanmei; Ma, Yi; Xu, Wei^*; Wang, Jufang^*

来源：Applied Microbiology and Biotechnology, 2023, 107(21): 6527-6540.

DOI：10.1007/s00253-023-12751-6

• Site-directed mutagenesis
• Chimeric enzyme
• Bst DNA polymerase
• Loop-mediated isothermal amplification (LAMP)
• Salmonella typhimurium detection
• Thermostability

Loop-mediated isothermal amplification (LAMP) is a widely used method for clinical diagnosis, customs quarantine, and disease prevention. However, the low catalytic activity of Bst DNA polymerase has made it challenging to develop rapid and reliable point-of-care testing. Herein, we developed a series of Bst DNA polymerase mutants with enhanced activity by predicting and analyzing the activity sites. Among these mutants, single mutants K431D and K431E showed a 1.93- and 2.03-fold increase in catalytic efficiency, respectively. We also created a chimeric protein by fusing the DNA-binding domain of DNA ligase from Pyrococcus abyssi (DBD), namely DBD-K431E, which enabled real-time LAMP at high temperatures up to 73 degrees C and remained active after heating at 70 degrees C for 8 h. The chimeric DBD-K431E remained active in the presence of 50 U/mL heparin, 10% ethanol, and up to 100 mM NaCl, and showed higher activity in 110 mM (NH4)(2)SO4, 110 mM KCl, and 12 mM MgSO4. Notably, it generated a fluorescence signal during the detection of Salmonella typhimurium at 2x10(2) ag/mu L of genomic DNA and 1.24 CFU/mL of bacterial colony, outperforming the wild type and the commercial counterpart Bst 2.0. Our results suggest that the DBD-K431E variant could be a promising tool for general molecular biology research and clinical diagnostics.

• 单位
  南方医科大学