Abstract

Bst DNA polymerase has been widely used in isothermal nucleic-acid amplification platforms for pathogen detection and molecular diagnostics. Improving the catalytic efficiency and operational robustness of Bst DNA polymerase through protein engineering is therefore of substantial interest. In this study, we combined domain fusion with the introduction of a homologous site transplantation in the large fragment of Bst DNA polymerase (BstLF). Specifically, the double-stranded DNA-binding domain Sso7d was fused to BstLF via a flexible linker, while the F496H mutation was introduced. The resulting mutant, Sso7d-BstLF(F496H), demonstrated improved catalytic efficiency in isothermal amplification techniques of denaturation bubble-mediated strand exchange amplification (SEA). Compared with the wild-type, Sso7d-BstLF(F496H) reduced the SEA amplification time by approximately 50 %. In addition, the engineered polymerase retained robust amplification activity at 71°C and showed improved tolerance to pH fluctuations and to common inhibitory components (NaCl, EDTA, urea, ethanol, and SDS). Molecular docking and mutation energy calculations suggested that improved performance might be associated with additional hydrogen bonds between the R group and DNA, consistent with increased protein-DNA affinity. Molecular dynamics simulations further indicated that F496H preserves global structural stability while reducing local flexibility, providing a plausible structural basis for the observed activity enhancement. Collectively, these findings identify Sso7d–BstLF(F496H) as a promising polymerase for improved isothermal amplification–based molecular diagnostics.