Cloning,Expresssion And Mechanism Of Antimaicrobial From Lactobacillus Bacteriocin

The food safety problems caused by pathogenic microorganism are becoming more and more serious,it seriously affects people's health and quality of life.One of the way that solve this problem is to find the high efficiency,safety and stability bacteriocins produced by lactic acid bacteria as natural preservatives instead of chemical preservatives.Lactic acid bacteria strains are the Security strains recognized by the international(GRAS).In the metabolic process of lactic acid bacteria,a class of peptides protein or protein complexes with antibacterial activity can be synthesized by the ribosome,and the growth of gram-positive bacteria and some pathogenic bacteria breeding can be inhibited by these products synthesized by lactic acid bacteria effectively.One of lactic acid bacteria strain that has stronger antibacterial activity was screened from more than 60 of lactic acid bacteria were isolated from fermented cream of Inner Mongolia,Heilongjiang,Hebei etal.This strain has obvious advantages in the aspect of bacteriocin synthesis and antibacterial compared with other lactic acid bacteria strains.But the research of the bacteriocin was limited by the purification of natural bacteria element.The recombinant bacteriocin was heterologous expression in E.coli.The study was divided into several parts:1)Identification of the strain and the construction of the prokaryotic expression vector of recombinant bacteriocin;2)Heterologous expression in E.coli and purification of the recombinant bacteriocin;3)The research about the antibacterial properties of recombinant bacteriocin;4)The research about the antibacterial mechanism of recombinant bacteriocin.An experimental support about the industrial application of lactobacillus casei bacteriocin was provided by this study.The lactobacillus casei was identified by observating the colonial morphology,testing the physiological and biochemical,sequencing the 16S rDNA and pheS housekeeping gene.The bacteriocin synthesized by this strain was identified by eliminating the organic acid and hydrogen peroxide and protease sensitive experiment.The coding genes of bacteriocin LacA and LacB were screened by PCR.The recombinant bacteriocin gene was obtained by Overlap extension PCR.The recombinant bacteriocin gene was cloned into the pMD18-T plasmid and sequenced,and then the right gene fragment was cloned into the prokaryotic expression vector pGEX-4T-1,and the positive clones were screened with ampicillin.The prokaryotic expression vector that contains the recombinant bacteriocin gene was obtained and named as pGEX-4T-1-LacAB.The pGEX-4T-1-LacAB was converted into E.coli TOP 10,and the recombinant bacteriocin was expressed after induced with IPTG.The recombinant protein GST-LacAB was purified with GST fusion protein purification kits,and then the GST label was removed by digesting with clotting enzyme.The digestive juices were purified with GST fusion protein purification kits and the pure LacAB protein was obtained.The protein concentration was tested with BCA protein concentration detection kit and the concentration was 1.85 mg/mL.The antimicrobial activity of the LacAB protein was tested with the oxford cup agar diffusion method and the result showed that the LacAB protein had obvious inhibitory effect to Staphylococcus aureus and Escherichia coli,and the acteriostatic circle diameter were 12.15mm and 10.57mm,respectively.The express conditions of LacAB protein in E.coli were optimized.The result showed that the optimal conditions for the expression of LacAB protein were as followsed:the induction temperature was 30?,the IPTG concentration was 0.8 mmol/L,the induced time was 3 h and the host strain was E.coli BL21.The antibacterial activity of the LacAB protein was analyzed.The result showed that LacAB protein had a broad antibacterial spectrum,it not only significantly inhibited the growth of gram-positive bacteria,such as Staphylococcus aureus,Gambogic micrococcus and Listeria,but also significantly inhibited the growth of a part of gram-negative bacteria,such as Escherichia coli and Salmonella.The stability of LacAB protein was analyzed,the result showed that the LacAB protein had the stronger heat,acid and alkali,and freezing and thawing resistance,and the surfactant such as SDS,tween-20,tween-80,urea and TritonX-100,could not affect its antibacterial activity,but the EDTA could enhance its antibacterial activity.The minimum inhibition concentration(MIC)of LacAB protein was analyzed,and it was 62.5?g/ml and125?g/ml for Staphylococcus aureus and Escherichia coli,respectively.The amino acid sequence of LacAB was analyzed with bioinformatics technology.The result showed that the properties of physical and chemical properties,protein secondary structure and tertiary structure prediction,transmembrane region,signal peptide of LacAB were conform to the structure characteristics of bacteriocin Class ?b.According to the GxxxG structure of antibacterial active site of class ? b,the 40 valine(V)and 57 Isoleucine(I)in the amino acid sequence of were mutated into alanine(A)with amino acid point mutation technique,and the antimicrobial activity of mutant LacAB was tested.The result showed that the antimicrobial activity was significant reduction after mutating any of these two amino acid,it suggested that these two sites could be the key amino acid sites for the antimicrobial activity of Lactobacillus casei bacteriocin.