Preparation,characterization And Application Of Endolysins LysWL59 And LysWL60

Salmonella is one of the main foodborne pathogens.Bacteriophage endolysins,as a new method to control pathogenic bacteria,have the characteristics of high efficiency,strong specificity and low resistance,showing a good application prospect in the field of food safety.In this study,based on the functional gene annotation of bacteriophage LPST10 infecting S.Typhimurium ATCC14028,the basic characteristics of endolysins were analyzed through bioinformatics,and the cleavage mechanism was predicted;endolysins LysWL59 and LysWL60 were expressed in the Escherichia coli expression system through optimization;the activity of the recombinant protein was identified and the effects of partial environmental conditions on the relative lytic activity was studied;the synergies between endolysins and outer membrane permeabilizers and their application in different foods were studied.The results were as follows:1.ORF59 and ORF60 of bacteriophage LPST10 encoded two different endolysins LysWL59 and LysWL60,respectively,and their theoretical p I were 9.30 and 8.58.There were no signal peptides,transmembrane regions,and no hydrophobicity.The secondary structure of these two proteins had a similar proportion of?-helix and?-sheet,but the tertiary structure was quite different.Their conservative structure belonged to lysozyme-like domains,which might be glycosidases containing only globular domain.LysWL59 and LysWL60 require the holin or spanin protein Rz for"lysis from within".2.The optimal expression condition for LysWL59 was inducing E.coli C41 for 4 h at37°C,while for LysWL60 was inducing E.coli BL21 for 4 h at 30°C.The molecular weight of LysWL59 and LysWL60 were approximately 18 and 11.6 k Da,respectively.3.LysWL59 and LysWL60 had a wide lytic spectrum,and showed the best lytic activity at the concentration of 0.25 and 30?mol/L,respectively.Zn²⁺and high concentration of Mn²⁺(1?10 mmol/L)significantly inhibited the relative lytic activity of LysWL59 and LysWL60.LysWL59 could maintain high activity under the conditions of p H 8.0?10.0,4?90°C,10?500 mmol/L Na Cl,and its thermal stability,Na Cl tolerance,preservation stability and lytic activity were all better than LysWL60.Therefore,LysWL59was more suitable for application in food.4.The duplicate effect of LysWL59 with EDTA was better than malic acid and citric acid,and the optimal combination was 0.25?mol/L LysWL59 with 0.50 mmol/L EDTA,in which Salmonella was reduced by 97.50%within 1 h in the culture medium.When 2.5?mol/L LysWL59 and 0.50 mmol/L EDTA was used in different foods for 1 h,the best bactericidal effect was observed in lettuce,and S.Typhimurium ATCC14028 was reduced by 93.03%,followed by duck intestines.The bactericidal effects on cantaloupe,pork and skimmed milk powder were poor,which may be caused by the complexity of the food matrix.The present study provides insights into the prevention of the infection of S.Typhimurium ATCC14028,establishing a theoretical basis for the development of G^-endolysin products and a reference for solving actual production and application issues.