In silico analysis, expression, and characterization of LysStp1 endolysin with l-alanyl-d-glutamate carboxypeptidase activity
摘要
An increased manifestation of antibiotic resistance in bacterial pathogens necessitates reduced usage of the current generation of antibiotics and warrants the search for novel antimicrobial agents. Peptidoglycan hydrolytic enzymes like endolysins are a promising alternative to antibiotics, resulting in a quest for novel endolysins. The analysis of Salmonella bacteriophage Stp1 complete genome revealed an ORF encoding an endolysin with l-alanyl-d-glutamate carboxypeptidase activity and was designated as LysStp1. The promising results obtained with molecular docking prompted us to carry out cloning, expression, and purification of LysStp1. Purified LysStp1 showed strong muralytic activity against multiple Gram-negative bacteria, following chloroform permeabilization of the outer membrane. LysStp1 activity ranged from pH 4 to 10, with maximal activity at pH 9 and stability [> 70%] at pH 6–9. Similarly, LysStp1 exhibited significant activity [> 80%] from 20 to 70 °C with optimum activity at 60 °C and retained 50% stability for up to 30 min of incubation at 100 °C. Interestingly, Ca2+ ions increased the activity of LysStp1 at lower concentrations. LysStp1 retained up to 50% activity at 500 mM NaCl concentration, with a broad antimicrobial spectrum against Gram-negative bacteria. These findings suggest that LysStp1 endolysin is a promising antibiotic alternative with rapid action, broad bactericidal activity and, high thermal stability. However, the antibacterial activity of LysStp1 against Gram-negative bacteria was observed only under outer-membrane permeabilized conditions, underscoring a limitation in direct applicability and the need for compatible outer membrane-disrupting strategies, such as antimicrobial peptides or related agents, to enable its translational potential.