Abstract

Bacteriophage endolysin is one of the most promising anti-biotic substitutes, but in Gram-negative bacteria, the outer membrane prevents the lysin from hydrolyzing peptidogly-cans and blocks the development of lysin applications. The prime strategy for new antibiotic substitutes is allowing lysin to access the peptidoglycan from outside of the bacteria by reformation of the lysin. In this study, the novel Escherichia coli (E. coli) phage lyase lysep3, which lacks outside-in cata-lytic ability, was fused with the N-terminal region of the Bacillus amyloliquefaciens lysin including its cell wall bind-ing domain D8 through the best manner of protein fusion based on the predicted tertiary structure of lysep3-D8 to ob-tain an engineered lysin that can lyse bacteria from the out-side. Our results showed that lysep3-D8 could lyse both Gram- negative and Gram-positive bacteria, whereas lysep3 and D8 have no impact on bacterial growth. The MIC of lysep3-D8 on E. coli CVCC1418 is 60 μg/ml; lysep3-D8 can inhibit the growth of bacteria up to 12 h at this concentration. The bac-tericidal spectrum of lysep3-D8 is broad, as it can lyse of all of 14 E. coli strains, 3 P. aeruginosa strains, 1 Acinetobacter baumannii strain, and 1 Streptococcus strain. Lysep3-D8 has sufficient bactericidal effects on the 14 E. coli strains tested at the concentration of 100 μg/ml. The cell wall binding do-main of the engineered lysin can destroy the integrity of the outer membrane of bacteria, thus allowing the catalytic do-main to reach its target, peptidoglycan, to lyse the bacteria. Lysep3-D8 can be used as a preservative in fodder to benefit the health of animals. The method we used here proved to be a successful exploration of the reformation of phage lysin.

• Keywords: endolysin lysep3; endolysin binding domain D8; antibacterial activity; fusing expression