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

Bacillus licheniformis 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 B. licheniformis. 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 Bacillus strains tested, including eight B. licheniformis 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 B. licheniformis, 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 B. licheniformis contamination in dairy products, providing an attractive alternative to conventional antimicrobial agents.