Prokaryotic Expression Of PlnE And PlnF Genes Derived From Lactobacillus Plantarum HD-23 And Study Of Antibacterial Mechanism Of Pln EF

Bacteriocins are ribosomally-synthesized peptides or proteins with antimicrobial activity,which produced by different groups of bacteria.Many lacticacid bacterias(LAB)produce bacteriocins with rather broad spectra of inhibition.Several LAB bacteriocins offer potential applications in food preservation,and the use of bacteriocins can help to reduce the addition of chemical preservatives as well as the intensity of heattreatments in the food industry,resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties.In this research,a bacteriocin-producing Lactobacillus plantarum HD-23 was isolated from some kinds of fermented foods.The bacteriocin genes plnE and plnF were cloned and expressed in Escherichia coli BL21(DE3),then the bacteriocin PlnEF was purified.In order to know the antibacterial mechainism of bacteriocin PlnEF and its receptor on cell membrane,the pure bacteriocin PlnEF was used to inhibit Salmonella typhimurium SL1344,the main results were as follow:1 Isolation and identification of bacteriocin-producing lactic acid bacteria.Eighty suspected lactic acid bacteria were isolated from the homemade pickles,bean curd,homemade yogurt,quinces and preserved fish.After biological features and antibacterial experiments,the bacteriocin-producing strain HD-23 was screened.The strain HD-23 was identified as Lactobacillus plantarum.2 Response surface optimization of corn flour medium composition.Core is the supporting crop of China's grain and oil industry.In order to use of grain resources efficiently,reduce the costs of bacteriocin production and fit the needs of industrial production,the core was used as a medium composition.The inhibition zone diameters were used as response value,The optimal culture medium was obtained by response surface experiments: glucose 3.13 g/L,peptone 1.22 g/L,sodium acetate 0.57 g/L,corn flour 8 g/L,beef extract 10 g/L,yeast extract1 g/L,diammonium hydrogen citrate 2 g/L,dipotassium bicarbonate 0.4 g/L,magnesium sulfate 0.58 g/L,manganese sulfate 0.25 g/L and tween-801 mL/L.3 Prokaryotic expression of bacteriocin genes plnE and plnF and purification of peptides.Homologous recombinase was used to connect pln E and plnF to plasmid pET-32 a.The recombinant plasmids were transferred to E.coli BL21(DE3)to obtained the recombinant strains pET-32a-plnE-E.coli BL21 and pET-32a-pln F-E.coli BL21.The peptides PlnE(3.6 kDa)and PlnF(3.7 kDa)were purified by Ni-NTA affinity chromatography,ultrafiltration and enterokinase digestion eventually.The bacteriocin PlnEF was further analysed for antimicrobial activity and found to be active against some gram-positive and gram-negative bacteria.4 The antibacterial mechanism of bacteriocin PlnEF.The mechanism on energized cells of Salmonella typhimurium SL1344 was studied with purified bacteriocin PlnEF.The cell lysis rate was increased with the increasing concentration of PlnEF.After adding bacteriocin PlnEF,leakage of intracellular ATP,LDH and UV-absorbing substances was observed,and the action resulted in cell death.5 Study on the binding sites of bacteriocin PlnEF to Salmonella typhimurium SL1344.Cell membrane protein was extracted from Salmonella typhimurium SL1344,where one of strain was treated with PlnEF and the other one was untreated.After two-dimensional electrophoresis and image analysis,the phosphoglycerate kinase(PGK)protein was determined by MALDI-TOF-MS.The open reading frame of PGK gene was knocked out by using the ?-red technique.The results can speculate that the PGK protein of Salmonella typhimurium SL1344 was affected by bacteriocin PlnEF,and then leading the cell death.In this research,Lactobacillus plantarum HD-23 was confirmed to be a good bacteriocin-producing strain.In order to reasonable use of grain resources,the corn flour was added to the MRS medium.The bacteriocin genes plnEF were cloned in E.coli,purified bacteriocin PlnEF could contribute to the effective study on the antibacterial mechanism.Salmonella typhimurium SL1344 was used as target bacteria,the results showed that the mechanism of PlnEF was leading to the leakage of intracellular substances and cell death.Futhermore,this study also research the function of PGK protein,speculate that bacteriocin PlnEF interaction with PGK protein,and leading the cell death of Salmonella typhimurium SL1344.The results of this study suggested a new way to study the bacteriocins' acting site and provided a theoretical basis for the further study on bacteriocins.