Chemical Modification Of Lysozyme With Ferulic Acid And Exhibiting An Enhanced Antimicrobial Action

Egg-white lysozyme (E.C.3.2.1.1), which can kill bacterial by decomposing the peptidoglycan of the cell wall, is very rich in nature. Lysozyme is a natural protein, so it has a negligible toxigenic potential. Many countries and organizations have authorized to use it as a food preservative, insteading of other traditional chemical preservative.However, the practical use of lysozyme as an effective preservative is limited by its narrow antimicrobial spectrum, almost lacking an antimicrobial capability against Gram-negative bacteria. Therefore it was the aim of this study to extend the bactericidal spectrum of lysozyme against Gram-positive bacteria and Gram-negative bacteria by slight chemical modification of the protein with ferulic acid. Linkage of the organic acid to lysozyme was achieved by the constitution of amide bindings between the carboxyl group of ligand and amino groups of the enzyme mediated by a carbodiimide (1-ethyl-3-(dimethyl amino propyl) carbodiimide EDAC).Firstly, synthesis the FA-Lys and analyze the degree of modification. The Lysozyme derivations, which are reacted according to the mass ratios of 1:4 and 1:1, were well dissolved. The modified lysozyme and the unmodified lysozyme wre isolated by cation-exchange chromatography, and the average degrees of modification were about 41.2% and 47.5% according to TNBS method.Then, compare to Natural lysozyme, the lytic activity of FA-Lys was little reduced. Under acidic condition, FA-Lys also had good thermal stability. But pH had a great effect on the lytic activity of FA-Lys. The optimal pH of activity was reduced due to the lowering of the isoelectric point of lysozyme through modification. During the study of the structure and character on the FA-Lys, it showed the hydrophobicity of the FA-Lys was increased, which indicated the theory of FA-Lys against Gram-negative bacteria. After modified by ferulic acid, the molecular structure of lysozyme changed according to the difference of UV-absorb spectra and the CD spectra of Natural lysozyme and FA-Lys. Theα-helix andβ-structure content decreased with modification. The random content increased, so the peptide chain extended. This might cause the decrease of lysozyme′s stability and the lytic activity.Finally, the bactericidal activity against Staphylococcus aueres,E.coli and Salmonella were measured for Natural lysozyme and FA-Lys. The results were showed: FA-Lys conjugate exhibited a significantly enhanced antimicrobial effect against E.coli and Salmonella. The MIC against E.coli was about 0.5mg/mL, and against Salmonella was 0.5mg/mL. However, making lysozyme effective against Gram-negative bacteria leaded to a partial decrease in its antimicrobial activity against Gram-positive strains. The MIC against Staphylococcus aueres was above 1mg/mL.