Characterization of Endolysin LysG77YL from <i>Bacillus licheniformis</i> -Infecting Bacteriophage G77YL and Application as an Antimicrobial Agent

<i>Bacillus licheniformis</i> is a spore-forming, Gram-positive bacterium widely found in nature and frequently associated with spoilage in dairy products due to its extracellular enzyme production. To develop a targeted biocontrol strategy, a novel lytic phage, G77YL, was isolated from the Gyeongchun Line Forest Trail in South Korea. Host range analysis revealed that G77YL specifically infects several strains of <i>B. licheniformis</i>. Genome analysis indicated that it is a virulent phage devoid of lysogeny-related genes and encodes a glycosyl hydrolase family 25 endolysin, designated as LysG77YL. The endolysin (LysG77YL) and its enzymatically active domain (LysG77YL_EAD) were cloned, expressed, and purified. Lytic assays demonstrated that LysG77YL_EAD exhibited broader and more potent antibacterial activity than the full-length enzyme, targeting all <i>Bacillus</i> strains tested, including eight <i>B. licheniformis</i> strains, while showing no activity against non-target Gram-positive and Gram-negative bacteria. Stability testing showed that LysG77YL_EAD retained high lytic activity under various pH (7-9), salt (100-200 mM NaCl), and temperature (4-40°C) conditions. In a food application using sterilized milk contaminated with <i>B. licheniformis</i>, treatment with 4 μM LysG77YL_EAD resulted in a 4 log CFU/ml reduction in bacterial count within 30 min at 4°C. These findings highlight the potential of LysG77YL_EAD as a stable and efficient endolysin-based biocontrol agent for controlling <i>B. licheniformis</i> contamination in dairy products, providing an attractive alternative to conventional antimicrobial agents.