| Lysozyme is a kind of alkaline enzyme that can hydrolyze bacterial cell walls. It is a kindof natural protein that harmless to the human body function and can take the place of manychemical preservatives, is widely used in food, medicine, animal husbandry and otherindustries.The ion exchange method were used to extract lysozyme from egg white, the resultsshowed that the most optimum experiment conditions of ion exchange method were the resin35%of the egg white liquid, adsorption time6.5h, eluent17.5%(NH4)2SO4solution, elutiontime1h, elution times for3times，lysozyme final yield was0.365%, the enzyme activity of16500U/mg, enzyme activity yield of94%, the enzyme specific activity and enzyme activityyield increased.Cinnamic acid, caffeic acid and coumaric acid were used as modifier, the results showedthat the best condition of cinnamic acid modified enzyme preparation were organic acidcontent60mg, pH7.5, reaction time22h, EDAC usage130mg, the bacteriostatic circlediameter of modified enzyme to Escherichia coli was14.1mm. the best condition of caffeicacid modified enzyme preparation were organic acid content45mg, pH7.5, the reaction time20h, EDAC usage120mg, the bacteriostatic circle diameter of modified enzyme toEscherichia coli was16.3mm. The best condition of coumaric acid modified enzymepreparation were the organic acid content30mg, pH7.5, reaction time18h, EDAC usage130mg, the bacteriostatic circle diameter of modified enzyme to Escherichia coli was16.8mm.The minimal inhibitory concentration of cinnamic acid, caffeic acid, coumaric acid onEscherichia coli and Pseudomonas aeruginosa were1.25mg/mL, natural enzyme was1.50mg/mL, while caffeic acid and coumaric acid modified enzyme were0.5mg/mL and cinnamicacid modified enzyme were0.75mg/mL. The minimal inhibitory concentration of cinnamicacid, caffeic acid on Staphylococcus aureus and Micrococcus lysodeikticus was1.00mg/mL,coumaric acid was0.75mg/mL and1.00mg/mL, natural enzyme was1.00mg/mL, whilecaffeic acid and coumaric acid modified enzyme were1.25mg/mL, cinnamic acid modifiedenzyme were1.25mg/mL and1.50mg/mL. The inhibition effect of modified enzyme ongram-negative bacteria reach to the inhibition effect of natural enzyme on gram-positivebacteria. Temperature and pH all had a certain influence on bacteriostatic effect of natural andmodified enzyme.Enzymology properties were studied, the optimal reaction pH of natural enzyme was7.0while the three kinds of modified lysozyme was6.0, good acid stability and easy deactivationin alkaline condition. The optimal reaction temperature of natural enzyme was60℃while thethree kinds of modified lysozyme was50℃, good thermal stability. Na+、K+、Ca2+onenzyme activation function while Cu2+、Fe2+、Zn2+and Ba2+on enzyme activity inhibition.Surfactants glycerol, Span20, Span40, Span80, Tween20, Tween40, Tween80all had a slightinhibition on enzyme activity.Hydrophobic property and structure of the enzyme change study showed that the surfacehydrophobic index increased significantly, natural enzyme was78.27, and cinnamic acid, caffeic acid and coumaric acid modified enzyme were160.7,195.9and197.5. the contents ofeach secondary structure certainly changed, compared with natural enzyme, the α-helixcontent decreased while random curly content increased, the β-sheet content of caffeic acidmodified enzymedeclined while cinnamic acid and coumaric acid modified enzymes were onthe rise, the β-turn content of caffeic acid modified enzyme increased while cinnamic acid andcoumaric acid modified enzymes dropped. Compared with the natural enzyme, the orderlystructure of modified enzyme decreased, disordered structure increased, the surfacehydrophobic index increased, the hydrophobic group exposed, the enzyme molecule peptidestructure tend to be loose which was not conducive to the stability of the enzyme, this changeof the structure can be led to its bacteriostatic ability enhancement of gram-negative bacteriaand the cause of the positive ability become weak. |
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