Abstract: In order to investigate the enzymatic activity of the CHAP domain from the Staphylococcus aureus phage endolysin LysAEJ79809.1, the protein was cloned and recombinantly expressed, and its potential as a food antibacterial agent was examined by evaluating its lytic activity under different conditions. The lytic activities of the CHAP and CHAP-Amidase against S. aureus were compared. Specifically, the CHAP gene was inserted into an expression vector using seamless cloning technology and expressed in Escherichia coli. Protein purification was performed via nickel affinity chromatography, with expression and purity verified by SDS-PAGE. The lytic activity of CHAP protein was measured using turbidity tests under various settings, including evaluations of the effects of metal ions, pH, temperature, and NaCl concentrations. Furthermore, the lysis efficiency of the CHAP protein against S. aureus in a milk matrix was investigated. At a concentration of 0.3 mg/mL, the highest lytic activity was demonstrated by the CHAP protein, with the S. aureus OD_{600 nm} value being lowered at a lysis rate of up to 70%. The highest lytic activity of CHAP-Amidase was observed at 0.5 mg/mL. Notably, CHAP has a smaller molecular weight than CHAP-Amidase, contributing to higher expression efficiency. A considerable effect on lytic activity was exerted by metal ions, with CHAP's activity being greatly increased by Mg²⁺ and Ca²⁺, whereas inhibitory effects were shown by Mn²⁺, Zn²⁺, and Cu²⁺, with its activity being almost totally suppressed by Zn²⁺ and Cu²⁺. Lytic activity increased at 100 mmol/L for the concentration of NaCl, but it decreased to half of its original level at 300 mmol/L. The highest activity of the CHAP protein was observed in alkaline settings, particularly at pH9. Additionally, over 40% activity was maintained by the protein in acidic conditions (pH3). According to temperature tests, CHAP was found to be inactivated above 50 ℃ while good activity was retained below 40 ℃. Significant lytic activity was exhibited by CHAP in a milk matrix. When compared with the CHAP-Amidase, stronger lytic activity against S. aureus was exhibited by the CHAP domain alone, along with a higher expression level, indicating easier large-scale production. The wide-ranging potential of the CHAP domain of endolysin LysAEJ79809.1 as an antimicrobial agent in food settings is demonstrated by these results.