Endolysin LysPEF52H from Enterococcus faecalis phage PEF-SWUN52H: a potential antibacterial agent

Abstract Objectives This study depicted and characterized the phage-derived endolysin LysPEF52H, providing a new alternative antimicrobial strategy for controlling bacterial contamination and ensuring food safety. Materials and Methods The molecular weight of endolysin LysPEF52H was predicted using an online platform, and its three-dimensional (3D) structure was modeled and validated through a Ramachandran plot. Subsequently, LysPEF52H was expressed via a prokaryotic expression system, followed by purification and concentration determination. Its secondary structure and protein conformation were analyzed using circular dichroism spectroscopy and fluorescence spectroscopy. Moreover, molecular docking was used to preliminarily investigate the interactions between this endolysin and small molecules/metal ions. Finally, the physicochemical properties and antibacterial activity of LysPEF52H were examined, and its application for bacterial inactivation on chicken samples was evaluated. Results The predicted molecular weight of endolysin LysPEF52H was 46808.1 Da, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed an apparent molecular weight of approximately 45 kDa. The target protein was successfully eluted using elution buffer containing 200 mmol/L imidazole, and the original concentration was quantified as 0.107 mg/mL. The secondary structure of LysPEF52H predominantly consisted of β-sheets (46%) and random coils (48%), with a minor contribution from α-helixes (6%). Conformational changes in LysPEF52H occurred between 25 °C and 45 °C, accompanied by an increase in fluorescence intensity. Above 45 ℃, the protein gradually denatured, leading to a decrease in fluorescence intensity. LysPEF52H exhibited metal ion affinity. Additionally, ʟ-alanine exhibited the strongest docking affinity to LysPEF52H. Antimicrobial spectrum assays and scanning electron microscopy demonstrated that LysPEF52H exhibited antimicrobial activity against Escherichia coli 105H and Staphylococcus aureus 8M. LysPEF52H also exhibited wide pH tolerance and good antibacterial activity after heat treatment. Its antimicrobial activity was influenced by salt concentration, metal ions, and surfactants. In addition, measurable antibacterial effects were observed on chicken artificially contaminated with E. coli 105H and S. aureus 8M treated with LysPEF52H at 4, 25, and 37 ℃. Conclusions The phage-derived endolysin LysPEF52H shows potential as an antimicrobial agent for future applications.