Characterization and application of lysGPK against Escherichia coli O157:H7 on fresh lettuce, with molecular insights into its lytic activity

Escherichia coli O157:H7 is frequently associated with foodborne disease outbreaks, particularly on raw lettuce. In this study, an endolysin LysGPK derived from a Geobacillus phage was identified, recombinantly expressed and characterized for lysing Escherichia coli O157:H7. The antibacterial activity of LysGPK was evaluated using a turbidity reduction assay. Biochemical characterization revealed that LysGPK exhibited optimal lytic activity at 37 °C and pH 9.0, and remained stable at 70 °C. Notably, LysGPK exhibited broad-spectrum lytic activity against Gram-negative pathogens. Furthermore, the combination of LysGPK (15 μg/mL) and EDTA achieved a significant 99.4% reduction in Escherichia coli O157:H7 on lettuce within 2 h using the plate counting method. The subsequent molecular dynamics simulation displayed that the binding of LysGPK to peptidoglycan was stabilized by hydrogen bonds and hydrophobic interactions, and the MM-PBSA calculation showed residues Arg62 and Arg132 contributed critical binding energy. This study provides a theoretical basis for applying endolysins in controlling foodborne pathogens, and offers molecular insights into interactions between the LysGPK and bacterial peptidoglycan.