ObjectiveThe objective of this study was to develop a novel endolysin (PanLys.1) for the specific killing of the ruminal hyper-ammonia-producing bacteriumPeptostreptococcus anaerobius(P. anaerobius).MethodsWhole genome sequences ofP. anaerobiusstrains and related bacteriophages were collected from the National Center for Biotechnology Information database, and the candidate gene for PanLys.1 was isolated based on amino acid sequences and conserved domain database (CDD) analysis. The gene was overexpressed using a pET system inEscherichia coliBL21 (DE3). The lytic activity of PanLys.1 was evaluated under various conditions (dosage, pH, temperature, NaCl, and metal ions) to determine the optimal lytic activity conditions. Finally, the killing activity of PanLys.1 againstP. anaerobiuswas confirmed using anin vitrorumen fermentation system.ResultsCDD analysis showed that PanLys.1 has a modular design with a catalytic domain, amidase-2, at the N-terminal, and a cell wall binding domain, from the CW-7 superfamily, at the C-terminal. The lytic activity of PanLys.1 againstP. anaerobiuswas the highest at pH 8.0 (p<0.05) and was maintained at 37°C to 45°C, and 0 to 250 mM NaCl. The activity of PanLys.1 significantly decreased (p<0.05) after Mn2+or Zn2+treatment. The relative abundance ofP. anaerobiusdid not decrease after administration PanLys.1 underin vitrorumen conditions.ConclusionThe application of PanLys.1 to modulateP. anaerobiusin the rumen might not be feasible because its lytic activity was not observed inin vitrorumen system.