Preparation，Isolation And Identification Of Lysozyme Antibacterial Peptide

Lysozyme is antibacterial protein, with the advantages of strong activity, natural sourceand non-toxicity. But its immunogenicity and poor thermal stability in non-acidic conditionsseriously effect its applications. After hydrolysed by pepsin, it not only can reduceimmunogenicity, but also can strengthen the thermalstability. This paper studies thepreparation, antibacterial activity and stability, isolation and purification of lysozymeantimicrobial peptide. At the end, the antibacterial mechanism was simplely studied．First of all, preparing lysozyme antimicrobial peptide was studied. By single factorexperiment and orthogonal test, the optimum parameters were obtained as follows:enzymeofpepsin, lysozyme concentrations of10mg/mL, the ratio of pepsin and lysozyme1:100, temperature38℃, pH2.0, hydrolysis time2h. Under the optimal conditions, thedegree of hydrolysis was5.46％. The inhibitory activity of autoclaved lysozyme hydrolysiswas53.2％.Secondly, molecular weight distribution, antimicrobial activity and stability of lysozymehydrolysates digested pepsin digestion (Lys-Pe) was studied. The molecular mass of Lys-Pedistributed mainly in1000~4000. The enzyme activity of Lys-Pe was1228U/mg, accountingfor7.08%of lysozyme. Lys-Pe showed great inhibition effect on Gram-positive bacteria,especially staphylococcus aureus. Lys-Pe was more thermostable than lysozyme. Without heattreatment, the antimicrobial activity of Lys-Pe on S.aureus was weaker than lysozyme. Butafter heat treatment, Lys-Pe was more antibacterial than lysozyme. The thermal stability ofLys-Pe decreased with the increase of pH. At acidic conditions, Lys-Pe had good storagestability. Repeated freezing and thawing would reduce the antibacterial activity of Lys-Pe.Finally, the purification procedure and the antibacterial mechanism were studied. Bytwo-step purifiying method of semi-preparative RP-HPLC followed by analytical RP-HPLC,a antimicrobial peptide (F4-h) was found. F4-h has better thermal stability than the unpurifiedLys-Pe. Through LC/MC-MC identification and software analys, the amino acid sequence ofF4-h was MKRHGLDNY, located at positions12to20of lysozyme. F4-h was strongly activeagainst Gram-positive bacteria, but inactive against Gram-negative bacteria. F4-h kill bacteriaby destroying the bacterial cell membrane, leading the leak of material inside cell. However,the unique structure of outer membrane of Gram-negative bacteria hindered F4-h through thecell wall to reach the cell membrane.