V. and Société française de biochimie et biologie Moléculaire (SFBBM). The use of bacteriophage endolysins as specific antibacterial agents is a prospective strategy to treat bacterial infections caused by antibiotic-resistant pathogens. In case of Gram-negative species this strategy has limited applications since outer membrane shields the enzyme target and prevents bacteria lysis. We aimed to obtain and characterize the endolysin of the newly discovered anti-Salmonella bacteriophage S-394 (Lys394) and to choose an appropriate permeabilizing agent to disrupt Escherichia coli cells suspended in buffer solution and grown on agar surface. Lys394 synthesized in E. coli C41(DE3) was obtained as an electrophoretically homogenous protein. The protein of 18 kDa molecular weight shows high muralytic activity against various genera of chloroform treated Gram-negatives. Maximum of enzyme activity was observed at pH 8.5 and low ionic strength. In silico analysis of amino acid sequence identified Lys394 as an endopeptidase. Various outer membrane permeabilizers were analyzed in combination with Lys394 to degrade laboratory strain of E. coli CR63. Permeabilizing activity was evaluated using a periplasmic β-lactamase leakage test with untreated E. coli cells as a substrate. The highest rate of planktonic E. coli lysis was reached for Lys394 applied together with 25 μg/ml of poly-l-arginine with molecular weight distribution from 5 to 15 kDa or 20 μg/ml PGLa peptide. Lawn E. coli colony forming ability was decreased by 4 orders of magnitude after 30 min treatment with 25 μg of Lys394, 1 mM EDTA and 50 μg/ml of PGLa peptide at a room temperature.