Characterization Of Endolysin LysGH15 And Its Functional Domains From Staphylococcus Aureus Bacteriophage And Their Activities In Milk System

As a wildly found foodborne pathogen in natural environment,Staphylococcus aureus needs to be strictly controlled during the production of diary products.In this dissertation,the characteristics of S.aureus bacteripphage endolysin LysGH15 which is a highly specific peptidoglycan hydrolase towards S.aureus produced at the last phase of bacteriophage GH15multiplication cycle were studied.At the same time,the potential of LysGH15 to lyse S.aureus in milk was tested.The enzymatically active domains and cell wall binding domains of LysGH15 were expressed separately for a better understanding of the behavior of LysGH15 in milk system.Their properties were also studied and their potential applications in milk were explored.The main results are as follows.（1）LysGH15 had a strong ability to lyse S.aureus in the buffer system.Its optimum temperature and pH were 30³5^oC and 6.0,respectively.0.4 mg/mL of LysGH15 could bring the OD_600nm of S.aureus from 1.2 to 0.2 within 5 min,showing a great potential for application.However,its thermal stability is not optimistic.The half-life of LysGH15 at 60^oC was less than 30 s,which means it was not suitable to be used in food that needs high temperature processing.At the same time,the researches in the milk stimulation system found that most of the components in milk would inhibit the lysis activity of LysGH15,especially the environments that had high fat content and low concentration of salt.The addition of 250 mM of NaCl in whole milk made the activity of LysGH15 increased,which is more significant in skim milk.（2）In order to have a deeper understanding of the behavior of LysGH15 in milk against S.aureus,and to explain the obstacles between LysGH15 and S.aureus in milk,the fusion expression of enhance green fluorescent protein（EGFP）and LysGH15 cell wall binding domain SH3b was conducted and the fluorescence intensity was used as the indicator of the binding properties of SH3b towards S.aureus.The optimal temperature and pH of EGFP-SH3b were 30^oC and 4,respectively.In the buffer system,there was binding ability when the concentration of NaCl was 0,and the binding ability was the strongest when the concentration of NaCl was 100 mM to 400 mM.In the milk system,EGFP-SH3b had a weak binding ability to S.aureus without NaCl,but showed a strong binding ability when added with 500 mM NaCl.It indicated that one reason why LysGH15 was unable to lyse S.aureus in milk was that the environment with low concentration of salt hindered the binding between LysGH15 and S.aureus and NaCl was able to break that obstacle.（3）Since the properties of LysGH15 were not optimistic to be used in milk sterilization and most of the enzymatically active domains had certerin lytic activity,the antimicrobial activity of CHAP which is the enzymatically active domains of LysGH15 in milk was studied.The optimal expression condition for BL21（DE3）/pET28a-CHAP were as follows.IPTG was added to 0.6 mM when the OD_600nm of BL21（DE3）/pET28a-CHAP was 0.6.and the induction lasted for 6 h under 26^oC.Under these conditons,the soluble expression quanlity of CHAP was the highest to 25.3 mg/L.The optimum reaction temperature of CHAP was 30^oC,and the optimum pH was 6⁸.The activity of CHAP increased as the concentration of NaCl decreased,and reached to the highest when the content of NaCl was 0 in the buffer solution.The antibacterial effect of CHAP was limited by its low lytic activity and the rapid proliferation rate of S.aureus.However,under 0^oC,the Colony-Forming Unit（CFU）of S.aureus in milk reduced by 1.5 logs within 8 hours when added with 1.0 mg/mL of CHAP.Besides,CHAP showed significant lysis activity in different commencial diary products,with breakfast milk being the most obvious,reducing the total number of colonies by 3 logs within8 hours.