Structure-Guided Design and Rapid Cell-Free Screening of a Superior Restriction Endonuclease DpnI Mutant to Enhance the Specific Activity and Efficiency of Site-Directed Mutagenesis

Restriction endonuclease DpnI is a critical tool for eliminating parental templates in site-directed mutagenesis (SDM); however, the wild-type enzyme exhibits low efficiency at low substrate concentrations. In this study, we introduced a lysine mutation into the C-terminal winged helix (wH) domain of DpnI via structure-guided design to enhance electrostatic interactions and established a rapid screening platform using rolling circle amplification (RCA) coupled with cell-free protein synthesis (CFPS). Results showed that the Q235K variant achieved a specific activity of 640,000 U/mg, four-fold higher than the wild-type, and increased the positive mutation rate in SDM from 82.7% to 92.3% while significantly reducing background plasmid residue. This study not only yielded a high-performance tool enzyme but also demonstrated the high efficiency of CFPS for rapid iteration and optimization in enzyme engineering.