| Due to people's awareness of food safety and demand increasing in recent years, research and development of natural preservatives wasing a hot. Bacteriocin is a protein produced by certain bacteria antibacterial substances. Bacteriocins as a "green preservative" has been paid an attention. Bacteriocin-producing strains in the application is limited mainly because of low output and instability of gene expression. Bacteriocins isolated from lactic acid bacteria (LAB) have been studied by many researchers due to their bactericidal activities against food-borne pathogens and food-spoilage bacteria. Pediocin was paid more attention because of its strong anti-Listeria activity. We have isolated a lactic acid bacteria producing pediocin from bovine colostrum, Pediococcus acidilacticii LH31. Object of this research is about the purification of pediocin PA-1 from P. acidilacticii LH31, identifying their physical and chemical properties, and Cloning Pediocin genes for heterologous expression, studying the expression characteristics, investigating the optimal high cell-density culture procedure and feeding strategy, to obtain high-level expression.The bacteriocin from P. acidilacticii LH31, dsignated as pediocin PA-1, was rapidly purified to homogeneity by the pH mediated cell adsorption-desorption method and semi-preparative reversed-phase HPLC. It gave a single peak on tricine-SDS-PAGE, the protein molecular weight was determined as 5000 Da. Characteristics of pediocin PA-1 was identified by use of the ideas, experience and methods for characterization of cellulases and xylanases from Cellulosimicrobium cellulans. The activity of pediocin PA-1 against Enterococcus faecalis was determined, optimum temperature at 25?and optimum pH at 6.0. Pediocin treated in boiling water bath for some time remained active, but its activity gradually reduced as time growing. After treated in wide range of pH, pediocin remained stable activity; stability is higher in lower pH. Its stability is higher as concentration of pediocin increasing, High concentration of pediocin (>50?g/ml) showed good resistance to extremes of pH (13) and temperature (121?). By a series of gradient pediocin against E. faecalis experiments its minimum inhibitory concentration (MIC) was 50?g/mL, formulating time bactericidal curve with different doses of pediocin.To effectively express pediocin PA-1 (PED) in Escherichia coli, the primers specific for PED coding sequence was designed and synthesized according to the NCBI. E. coli BL21(DE3)/pET expression system was used to produce recombinant pediocin, Pediocin PA-1 fused with thioredoxin (PED-N). The gene fragment of PED was amplified from genomic DNA of Pediococcus acidilacticii LH31 by PCR. The plasmid pET32c-PED, encoding PED fused with His-tagged thioredoxin protein, was constructed and introduced into Escherichia coli BL21 (DE3) strain. The plasmid stabilities is higher in BL21(ED3)plysS than in BL21(DE3). The fusion protein was expressed in the strain after induction of isopropyl-?-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active PED, the purified fusion protein was cleaved by enterokinase and the liberated PED was finally purified by ultrafiltration with a 78% yield. The fusion protein, which did not have bactericidal activity against Enterococcus faecalis, was cleaved by enterokinase and the cleaved PED recovered its own bactericidal activity. The paper could be of great value for effective expression of a fusion-typed PED in E. coli, obtaining biologically active PED, and providing some basis to study application of PED, and also provides some approach to study the expression of bacteriocin.The research was conducted to investigate the characteristics of PED-N expression in BL21 (DE3) and BL21 (DE3) pLysS. For the BL21 (DE3), PED-N expression levels was maximum 3 hours after the induction; for the BL21 (DE3) pLysS, PED-N expression levels was maximum 5 hours after the induction. For the BL21 (DE3), PED-N expression levels was maximum when IPTG concentration was 0.2 mmol/L; for the BL21 (DE3) pLysS, PED-N expression levels was maximum when IPTG concentration was 2.0 mmol/L. Biomass and PED-N yield of BL21 (DE3) pLysS, were higher than those of BL21 (DE3) in the same culture conditions. At 37?or 30?, PED-N are effectively expressed in both strains, mainly in form of inclusion bodies; At 37?, expression level was higher than at 30?; At 30?, the proportion of soluble protein was higher than at 37?. At 37?, expression level in BL21 (DE3) pLys was higher than BL21 (DE3), but the proportion of soluble protein of BL21 (DE3) was slightly higher than that of BL21 (DE3) pLys. At 30?, expression level in BL21 (DE3) pLys was lower than the BL21 (DE3), while the proportion of soluble protein of BL21 (DE3) pLysS was slightly higher than BL21 (DE3). The fusion proteins were very sensitive to 1.0% Triton X-100, most of fusion protein became soluble when inclusion bodies were washed with 1.0% Triton X-100. Approximately half of the inclusion body was redissolved in 3 moI/L urea; when increasing the concentration of the urea to 5 mol/L, most of proteins were redissolved.Optimization of the high cell-density culture procedure of fusion-typed pediocin PA-1 (PED-N) in recombinant E. coli was carried out in 250 mL shaking flasks to investigate the effects of the composition of the culture medium, the range of pH, and induction condition on the growth of bacteria and the expression yield of PED-N and then transferred to a 5 L DO feed-back fed-batch culture system to analyze the optimal dissolved oxygen and specific growth rates. The results indicate that by keeping dissolved oxygen at 30%-40%and controlling nutrient feeding rate with DO feed back strategy, keeping pH at 6.5-7.0, culturing in optimized semi-synthetical medium and inducing for 5 h in the presence of 1 g/L lactose at 37?during fed-batch cultivation, a high cell density (A600=16) and 1.09 g PED-N per liter of broth were obtained under constant (0.2 h-1) specific growth rates in shorter fermentation periods.Excessive carbon source in medium may easily lead to acetic acid production, inhibit cell growth and exogenous protein expression. Cell concentration reached the maximum when C/N ratio was 1.0 in shake-flask, while soluble PED-N has the largest proportion. C/N ratio was too high or too low, not conducive to cell growth, the low percentage of soluble PED-N, a high proportion of inclusion bodies. New feeding strategies based on feeding balance between carbon source and nitrogen source were designed. Using a multistage carbon-nitrogen ratios feeding strategy to achive real-time feeding balance of carbon and nitrogen during fermentation process, a high density of biomass (maximum biomass density of wet weight of up to 81.8 g L-1) and high expression of fusion protein (fusion-typed pediocin PA-1) (content of up to 0.463 g L-1) and soluble protein (content of up to 0.157 g L-1) were obtained. The accumulation of acetate, which usually occurs during the process of high-density fermentation of recombinant Escherichia coli, was effectively avoided. |
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