Research On The End Modification And Bacteriostatic Mechanism Of LKantimicrobial Peptides And Their Application In Latex Membranes

With the emergence of drug-resistant bacteria and the post antibiotic era,there is an increasingly urgent need for a class of antibiotics.Antimicrobial peptide is one of the most potential substitutes.Antimicrobial peptide is a kind of polypeptide compound with antibacterial activity and short sequence,which makes pathogenic bacteria produce drug resistance much faster than antibiotics.However,antimicrobial peptides also have some urgent problems to be solved,such as poor stability,hemolytic activity and cytotoxicity.Rational design and modification of antimicrobial peptides is one of the effective ways to solve the above problems.In this paper,（LLKK）₃ antibacterial peptide was synthesized by solid-phase synthesis method,and its end was modified as a template.The antibacterial activity,hemolytic activity,cytotoxicity,stability,antibacterial mechanism and its application in latex membrane of the modified antibacterial peptide were studied.The main research contents and results are as follows:（1）Using（LLKK）₃ as template,Gly and Leu were added to its N-terminal and C-terminal.The four antimicrobial peptide sequences were P1:（LLKK）₃,P2:G（LLKK）₃,P3:（LLKK）₃L and P4:G（LLKK）₃L.The actual molecular weights of the four peptides were 1466.35,1523.40,1580.40 and 1631.20,which were consistent with the theoretical molecular weights.The hydrophobic values and hydrophobic moments of the four antimicrobial peptides were（LLKK）₃L>G（LLKK）₃L>（LLKK）₃>G（LLKK）₃L.The results of bacteriostatic circle experiment,MIC/MBC and CFU showed that the four antimicrobial peptides were effective to E.coli、S.aureus and C.albicans have obvious antibacterial activity;Among them,G（LLKK）₃L has the best bacteriostatic effect,while（LLKK）₃ and G（LLKK）₃ have relatively poor bacteriostatic effect.（2）The temperature,pH,pepsin and time stability,hemolytic activity and cytotoxicity of the four antimicrobial peptides were analyzed.The results showed that the four antimicrobial peptides had good thermal stability,pH stability and time stability.However,their antibacterial activity was affected by gastric juice,and the antibacterial activity of G（LLKK）₃L was less affected by gastric juice.In addition,the hemolytic activity and cytotoxicity of G（LLKK）₃L and G（LLKK）₃ were lower than those of（LLKK）₃ and（LLKK）₃L,indicating that the addition of Gly at the N-terminal could reduce the hemolytic activity and cytotoxicity.（3）The effects of G（LLKK）₃L on cell wall components of E.coli、S.aureus and C.albicans cell membrane damage DNA block and protein synthesis were analyzed,and the antibacterial mechanism of G（LLKK）₃L on pathogenic bacteria was investigated.SEM results showed that E.coli from smooth and complete rod-shaped surface to shrinkage and collapse;S.Aureus shrinks inward from round to flat and collapses inward;C.albicans changed from smooth oval shape to shuttle shape.The FT-IR absorption transmittance of E.coli、S.aureus and C.albicans cell wall was significantly lower than that of the control,indicating that G（LLKK）₃L antimicrobial peptide inhibited the synthesis of cell wall components.The results of fluorescence microscopic staining and flow cytometry showed that with the increase of G（LLKK）3L concentration,the cell membrane damage rate of E.coli、S.aureus and C.albicans also increased.The damage of G（LLKK）₃L on the membrane of E.coli and S.aureus was greater than that of C.albicans.The results of DNA and protein electrophoresis showed that G（LLKK）₃L could block DNA migration and inhibit E.coli and S.aureus had no effect on the genomic DNA of C.albicans.G（LLKK）₃L antimicrobial peptide can inhibit the synthesis or degradation of some proteins in E.coli、S.aureus and C.albicans,resulting in the loss of some bands and the decrease of protein content in the cells.（4）Natural latex film was modified by glycerin,and G（LLKK）3L antibacterial peptide was added into the modified latex film to prepare latex antibacterial film,and the characterization and characteristics of natural latex glycerin antibacterial peptide film were analyzed.The results show that the addition of 10%glycerol can effectively change the softness of the latex film,and excessive addition will lead to the precipitation of glycerol in the latex film.The addition amount of G（LLKK）₃L antibacterial peptide has no effect on the tensile properties of the membrane,but too high addition amount of antibacterial peptide will produce cytotoxicity.The optimal addition level of antimicrobial peptides is 600μg/ml of membrane forming solution.The results of Scanning Electron Microscope showed that glycerol could change the density of latex film and make dense holes appear on the surface of latex glycerol film and latex glycerol antibacterial peptide film.FT-IR spectrum analysis showed that latex,glycerol and antimicrobial peptides were not covalently bound,which was conducive to the release of antimicrobial peptides from the membrane.The modified latex glycerin antibacterial peptide film has good air permeability,water absorption,water retention and sustained release properties,and has certain inhibitory effect on E.coli、S.aureus、C.albicans and Pseudomonas aeruginosa.