Antibacterial activity of Bacillus amyloliquefaciens phage endolysin without holin conjugation

To characterize the enzymatic activity and antibacterial activity of endoly sin encoded by a Bacillus amyloliquefaciens phage, the open reading frame e ncoding endolysin was amplified by PCR and cloned into the expression plasm id pET21d(+). The resultant plasmid was used to transform Escherichia coli JM109(DE3). Production of endolysin in the cytosol facilitated cell lysis w ithout coproduction of holin, which is considered to degrade or alter the c ytoplasmic membrane. The phage endolysin was overexpressed and purified. Al though the specific activity of the purified phage endolysin towards lyophi lized Micrococcus luteus cells was 1/11 of the activity of chicken egg whit e lysozymes, the endolysin showed stronger antibacterial activity towards E . coli W3110, E. coli JM109(DE3) and Pseudomonas aeruginosa PAO1 than chick en egg white lysozymes. The antibacterial activity of the endolysin towards these three bacterial strains was marked when EDTA was added to the endoly sin solution.