ABSTRACTIn bacteriophage (phage) therapy against Gram-positive bacteria, such asStaphylococcus aureus,Listeria monocytogenes, andEnterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infectingE. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) ofE. faecalisphage φEF24C was analyzedin silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein isN-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 againstE. faecaliswere confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn2+ions for its activity, contrary to the bioinformatics prediction of a Zn2+ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) ofE. faecalisstrains to 60% (6 out of 10 strains) ofEnterococcus faeciumstrains. Fifth,N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.