The Study On Antibacterial Mechanism Of Lysozyme Against Escherichia Coli And The Enhancement Of Its Activity

Lysozyme is a kind of murein hydrolase widely distributed in organisms. The properties of lysozymes are slightly different according to their sources. Egg white lysozyme can specifically hydrolyze theβ-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues in the polysaccharide chain of peptidoglycan, thus destroy the integrity of cell wall sacculus and cause cell lysis. Because of its abundant and security,hee egg white lysozyme (HEWL) becomes the most important lysozyme.This work focused on the antibacterial mechanism of HEWL on gram-negative bacteria of Escherichia coli and the strategies to improve antimicrobial activity of lysozyme. The main results are listed below.(1) Two strains of E. coli, ATCC25922 and DH5αwere selected as the models. By studying antibacterial activity of HEWL against E. coli, we found that, E. coli ATCC25922 was more sensitive to HEWL than DH5α. The muramidase activity of lysozyme reduced greatly after heated, but its antimicrobial activity only decreased slightly, showing that the antibacterial activity of lysozyme not depended absolutedly on its catalytic activity.(2) In order to realize the reasons for the difference of antibacterial activity of lysozyme on the two strains of E. coli, lysozyme inhibitor, outer-membrane material and surface properties of the two E. coli were analyzed. Lysozyme inhibitors were presented in both of the two strains and their expression level were also same, showing that the lysozyme inhibitor was not the important factor causing the different lysozyme sensitivity of two E. coli. The lipopolysaccharides (LPS) of two strains were different, with E. coli ATCC25922 having O-antigen and DH5αhaving not. But the lipid A structure had no difference in the two E. coli. The assay of fluorescence of 1-N-phenylnaphthylamine (NPN) showed that the outer membrane permeability of E. coli ATCC25922 is stronger than that of DH5α. The cell surface hydrophobicity of E. coli ATCC25922 was 24.5%, much higher than that of DH5α.(3)In order to enhance the antibacterial activity of HEWL against gram-negative bacteria E. coli ATCC25922 and DH5α, the combinations of two permeabilizers, glycine and ethylenediaminetetraacetate (EDTA), with lysozyme were analyzed. As combined with glycine or EDTA, the antimicrobial activity of lysoyme against both E.coli strains improved obviously. As in combination with glycine and EDTA, the antibacterial activity of lysozyme against E. coli ATCC25922 increased 281.8 times,while that against DH5αincreased 631 times, indicating the synergistic antibacterial action of lysozyme with these two permeabilizers. The assay of fluorescence of NPN showed that the outer membrane permeability of two E.coli strains increased as cells were treated with lysozyme and these two cell permeabilizers. TEM image of E. coli ATCC 25922 cells also revealed the synergistic destroy effect of lysozyme with these cell permeabilizers on cell surface structure. So improving the outer membrane permeability of E. coli would help to enhance the antibacterial effect of lysozyme against them.