| In recent years,there have been frequent food safety incidents,among which the food spoilage caused by microorganisms and foodborne diseases constitute a large proportion.The addition of chemical preservatives can effectively inhibit the spoilage microorganisms in foods.However,artificial preservatives tend to accumulate in the human body.Long-term intake is potentially harmful to the human body and it is urgent to find new natural food preservatives as substitutes.The bacteriocin produced by Lactobacillus has no harm to human body,can be digested by protease in human gastrointestinal tract,and has application value as natural preservative.Piece of coccus,PA-1 is a bacteria produced by Pediococcus pentosaceus,compared with Nisin in pH6.0 has the characteristics of more than 90% of the loss of activity,whose nature is more stable and has a wide antimicrobial spectrum.The inhibitory effect on Listeria monocytogenes is especially obvious,and it has a wider application prospect in the field of food preservation and preservation.In this study,based on the Pediococcus pentosaceus C-2-1 strain that produced pediocin PA-1 from Inner Mongolian cheese in the early stage of the laboratory,the whole genome sequencing study was conducted on this strain.The immobilized cell fermentation conditions of pediocin PA-1 were optimized,and the prokaryotic expression and protein purification of pediocin pedA gene was performed.Further studied the mechanism of signal peptide in the soluble expression of pediocin PA-1.The specific research methods are as follows:(1)Whole genome sequencing and analysis of P.pentosaceus C-2-1.The whole genome sequencing technology was used to study the Pediococcus pentosaceus C-2-1 genome,and its genome components were predicted and corresponding functional genes were annotated.Then the metabolic pathway of Pediococcus pentosaceus C-2-1 was further explored.The total length of the P.pentosaceus C-2-1 genome is 1,643,106 bp and contains 1,946 genes.GC content was 37.87%,a total of 84 Scaffolds and 93 Contigs;A total of 1371 genes were successfully obtained for COG function annotation.The most functional category is carbohydrate transport and metabolism,with 144 genes accounting for 10.5%.There were 140 genes involved in translation,ribosome structure and biosynthesis,accounting for 10.07%;1007 genes were successfully annotated in the GO database and included three major subclasses of biological processes(20 branches),cellular components(12 branches),and molecular functions(11 branches).The cellular processes and metabolic processes are most active in biological processes.The most involved genes in the cell component category are cells and cell parts,and the binding and catalytic activity in the molecular functional class play an important role.KEGG enrichment analysis showed that there were 1102 genes corresponding to KEGG pathway in the gene of Pediococcus pentosaceus C-2-1,which existed in 126 metabolic pathways,and 113 genes involved in biosynthesis of secondary metabolites.AntiSMASH software predicted that 13 gene clusters were obtained from Pediococcus pentosaceus C-2-1 genome,and a single bacteriocin synthesis gene cluster was predicted,located in the Cluster 4 gene cluster,and located on the genome of Pediococcus pentosaceus.C-2-1 Scaffold1,the starting gene locus is 405542-411580.Five carbohydrate synthesis gene clusters,two fatty acid synthesis gene clusters,and five unknown gene clusters were also predicted.Finally,using ARDB and VFDB databases to evaluate the safety of the strains,after comparing the ARDB antibiotic resistance gene database and the VFDB database,it was not found that Pediococcus pentosaceus C-2-1 does contain antibiotic resistance gene and invasive virulence factors.It could basically conclude that the strain of P.pentosaceus C-2-1 was safe.(2)Optimization of immobilized cell fermentation conditions of pediocin PA-1.Using calcium alginate as a raw material,the immobilized fermentation of Pediococcus pentosaceus C-2-1 cells was performed by means of an embedding method.First,single-factor experiments were used to determine the fermentation time and temperature of the immobilized cells,the results were 42 h and 30? respectively.Under these culture conditions,the response surface methodology was used to determine the optimal factors for the immobilized cell fermentation to produce pediolite: The amount of calcium chloride was 2.41%,the amount of sodium alginate was 1.68%,the concentration of immobilized particle inoculation was 11.68%,and the amount of bacterial embedding was 0.066.Under the predicted conditions,the inhibitory activity was close to the predicted value,which showed that the model obtained in this experiment could be used to predict the ability of Pediococcus pentosaceus C-2-1 immobilized cells to produce pediocin.After optimization of immobilized cell preparation and various factors in the fermentation process,the antibacterial activity of Pediococcus pentosaceus C-2-1 immobilized cell pediocin increased by 36.9%.(3)Prokaryotic expression and protein purification of pediocin pedA gene.Based on the total DNA of Pediococcus pentosaceus C-2-1,we designed primers to amplify the structural gene pedA of pediocin PA-1.The recombinant plasmid pET28a-pedA was constructed and transformed into E.coli.After the expression,the purified recombinant protein was obtained by Ni-NTA affinity chromatography,and the molecular size of recombinant protein was determined to be about 7.8 kD via Tricine-SDS-PAGE,which was consistent with the predicted value by ProtParam.The antibacterial test of Listeria monocytogenes showed that the recombinant pediocin has obvious antibacterial activity.The soluble recombinant pediocin PA-1 successfully expressed in this experiment was not added with molecular chaperones and fusion proteins,making the extraction and purification of recombinant PA-1 more simple,which laid a good foundation for the future application of pediocin PA-1 in the field of food preservation and preservation.The bioinformatics method was used to analyze the structural information of recombinant pediocin PA-1.The ?-helix structure contained in the structure may be related to the soluble expression of the recombinant protein in E.coli.(4)Study on the mechanism of signal peptide in the soluble expression of pediocin PA-1.Firstly,bioinformatics methods were used to compare and analyze the physicochemical properties and secondary structures of the two kinds of PA-1.It was found that the pediocin PA-1 without signal peptide contains no ?-helical structure,while the pediocin PA-1 with signal peptide accounts for 23.53% of ?-helix structure,and the hydrophobicity of the recombinant protein is increased.After using the total DNA of the P.pentosaceus C-2-1 strain as a template,the two primers were designed to amplify the full length of the structural pedA gene of pediocin PA-1 and the structural pedA gene of pediocin PA-1 without signal peptide gene,constructed two kinds of recombinant plasmids pET28a-pedA,and expressed two kinds of recombinant pediocin PA-1 in E.coli.After the inclusion bodies were renatured and purified,two recombinant pediocin PA-1 with antibacterial activity were obtained.It can be basically concluded that the hydrophobic core signal peptide contained in the PA-1 protein may promote the secretion of the recombinant protein,thus a stable recombinant protein was obtained,which made the extraction and purification easier. |
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