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1.
Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0. 总被引:14,自引:9,他引:5
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J D Marugg C F Gonzalez B S Kunka A M Ledeboer M J Pucci M Y Toonen S A Walker L C Zoetmulder P A Vandenbergh 《Applied microbiology》1992,58(8):2360-2367
The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
J D Marugg C F Gonzalez B S Kunka A M Ledeboer M J Pucci M Y Toonen S A Walker L C Zoetmulder P A Vandenbergh 《Applied and environmental microbiology》1992,58(8):2360-2367
The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
4.
Beaulieu L Tolkatchev D Jetté JF Groleau D Subirade M 《Canadian journal of microbiology》2007,53(11):1246-1258
Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trx-pedA) expression approach. Trx-PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications. 相似文献
5.
A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial alpha-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1. 相似文献
6.
Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase
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Gi-Seong Moon Yu-Ryang Pyun Myeong Soo Park Geun Eog Ji Wang June Kim 《Applied microbiology》2005,71(9):5630-5632
A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1. 相似文献
7.
Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products. 相似文献
8.
The Bactericidal Activity of Pediocin PA-1 Is Specifically Inhibited by a 15-mer Fragment That Spans the Bacteriocin from the Center toward the C Terminus 总被引:3,自引:2,他引:1
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Gunnar Fimland Ralph Jack Günther Jung Ingolf F. Nes Jon Nissen-Meyer 《Applied microbiology》1998,64(12):5057-5060
A 15-mer peptide fragment derived from pediocin PA-1 (from residue 20 to residue 34) specifically inhibited the bactericidal activity of pediocin PA-1. The fragment did not inhibit the pediocin-like bacteriocins sakacin P, leucocin A, and curvacin A to nearly the same extent as it inhibited pediocin PA-1. Enterocin A, however, was also significantly inhibited by this fragment, although not as greatly as pediocin PA-1. This is consistent with the fact that enterocin A contains the longest continuous sequence identical to that of pediocin PA-1 in the region spanned by the fragment. The fragment inhibited pediocin PA-1 to a much greater extent than did the other 29 possible 15-mer fragments that span pediocin PA-1. The results suggest that the fragment—by interacting with the target cells and/or pediocin PA-1—interferes specifically with pediocin-target cell interaction. 相似文献
9.
Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family. 相似文献
10.
Johnsen L Fimland G Eijsink V Nissen-Meyer J 《Applied and environmental microbiology》2000,66(11):4798-4802
Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4 degrees C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity. 相似文献
11.
M. Chikindas E. Emond A. J. Haandrikman J. Kok K. Leenhouts S. Pandian G. Venema K. Venema 《Probiotics and antimicrobial proteins》2010,2(2):66-76
The bacteriocins pediocin PA-1 and lactococcin A are synthesized as precursors carrying N-terminal extensions with a conserved cleavage site preceded by two glycine residues in positions -2 and -1. Each bacteriocin is translocated through the cytoplasmic membrane by an integral membrane protein of the ABC cassette superfamily which, in the case of pediocin PA-1, has been shown to possess peptidase activity responsible for proteolytic cleavage of the pre-bacteriocin. In each case, another integral membrane protein is essential for bacteriocin production. In this study, a two-step PCR approach was used to permutate the leaders of pediocin PA-1 and lactococcin A. Wild-type and chimeric pre-bacteriocins were assayed for maturation by the processing/export machinery of pediocin PA-1 and lactococcin A. The results show that pediocin PA-1 can be efficiently exported by the lactococcin machinery whether it carries the lactococcin or the pediocin leader. It can also compete with wild-type lactococcin A for the lactococcin machinery. Pediocin PA-1 carrying the lactococcin A leader or lactococcin A carrying that of pediocin PA-1 was poorly secreted when complemented with the pediocin PA-1 machinery, showing that the pediocin machinery is more specific for its bacteriocin substrate. Wild-type pre-pediocin and chimeric pre-pediocin were shown to be processed by the lactococcin machinery at or near the double-glycine cleavage site. These results show the potential of the lactococcin LcnC/LcnD machinery as a maturation system for peptides carrying double-glycine-type amino-terminal leaders. 相似文献
12.
Nikki Horn María I. Martínez José M. Martínez Pablo E. Hernández Michael J. Gasson Juan M. Rodríguez Helen M. Dodd 《Applied and environmental microbiology》1998,64(3):818-823
The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.Bacteriocins of lactic acid bacteria have received considerable attention in recent years due to their potential application in the food industry as natural preservatives. Most interest has focused on lantibiotics (class I bacteriocins), e.g., nisin, and small heat-stable non-lanthionine-containing bacteriocins (class II) (22, 23). A major subgroup of class II bacteriocins (IIa) has been given the generic name of pediocin family (28) after its most extensively studied member, pediocin PA-1. Members of this class have a number of features in common, including a very strong antimicrobial activity against Listeria species (28). The food-borne pathogen Listeria monocytogenes is a major concern in the dairy industry since it can grow in a variety of dairy products at low temperature and pH (13). Although a pediocin PA-1-producing Lactobacillus plantarum strain has recently been isolated (12), this bacteriocin is generally produced by Pediococcus acidilactici strains of meat origin (3, 16, 18, 29, 31). Because of its antilisterial activity, the expression of pediocin PA-1 in strains of dairy origin would be highly desirable.Pediocin PA-1 production, immunity, and secretion are determined by an operon containing four genes (26). The structural gene, pedA, encodes the pediocin PA-1 precursor, pedB specifies immunity, and the pedC and pedD gene products are membrane-bound proteins required for secretion of the active peptide (39). Homologs of these genes have been described for related peptides. Biosynthesis of the well-characterized class II bacteriocin, lactococcin A, produced by strains of Lactococcus lactis also involves four genes (20, 36, 40). In addition to the structural gene (lcnA) and immunity gene (lciA), there are two genes (lcnC and lcnD) whose products together form a transport system dedicated to the translocation of lactococcin through the host membrane. The LcnC protein belongs to the family of ATP-binding cassette transporter proteins (40), and LcnD acts as an accessory protein (14). These two proteins have considerable homology to PedD and PedC, respectively (39), suggesting that the latter proteins play a similar role in the transport of active pediocin. The two bacteriocins also share the double glycine-processing site found in many lactic acid bacteria class II bacteriocins, some lantibiotics, and the Escherichia coli bacteriocin, colicin V (17).Van Belkum et al. (38) have recently investigated the role of leader sequences of the class II bacteriocins in the recognition of the precursor peptide by the dedicated translocation machinery of the host organism. By constructing hybrid genes, they demonstrated that the leader peptides of leucocin A, lactococcin A, and colicin V, which are cleaved at the Gly-Gly (positions −2 and −1) site, can direct the secretion of the nonrelated bacteriocin divergicin A. Our studies have focused on the class II bacteriocins pediocin PA-1 and lactococcin A. Since these peptides have a number of features in common, it might be expected that a pediocin PA-1 precursor could be secreted and processed by using the lactococcin A translocation machinery. L. lactis IL1403 is a plasmid-free strain that does not produce bacteriocin but contains chromosomal copies of genes analogous to lcnC and lcnD (33, 40). In addition, the natural resistance of this strain to pediocin PA-1 (8) makes it an ideal candidate for a production host to investigate the expression of pediocin PA-1 in lactococci.This paper describes the development of an expression system geared to the production of heterologous peptides in L. lactis. Testing the system with pediocin PA-1 involved the construction of a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and the structural part of mature pediocin PA-1. The hybrid genes were introduced into L. lactis IL1403, and the ability of these strains to produce and secrete pediocin PA-1 was investigated. 相似文献
13.
Line Johnsen Gunnar Fimland Vincent Eijsink Jon Nissen-Meyer 《Applied microbiology》2000,66(11):4798-4802
Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4°C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity. 相似文献
14.
Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application 总被引:2,自引:0,他引:2
The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20g/l of glucose or lactose at 20 and 24h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×10(5)AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15μg from 20μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1. 相似文献
15.
Santosh Kumar Tiwari Katia Sutyak Noll Veronica L. Cavera Michael L. Chikindas 《Applied and environmental microbiology》2015,81(5):1661-1667
Two hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such as Micrococcus luteus, Salmonella enterica serovar Enteritidis 20E1090, and Escherichia coli O157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics. 相似文献
16.
Prakash M. Halami A. Ramesh A. Chandrashekar 《World journal of microbiology & biotechnology》2005,21(8-9):1351-1358
Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was
active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to
proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence
of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented
cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin.
These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It
was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization
could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic
strains. 相似文献
17.
Replacement of Trifluoroacetic Acid with HCl in the Hydrophobic Purification Steps of Pediocin PA-1: a Structural Effect 总被引:2,自引:0,他引:2
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Hlne Gaussier Hlne Morency Marc C. Lavoie Muriel Subirade 《Applied microbiology》2002,68(10):4803-4808
Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1. 相似文献
18.
Gaussier H Morency H Lavoie MC Subirade M 《Applied and environmental microbiology》2002,68(10):4803-4808
Trifluoroacetic acid (TFA) is a purification contaminant associated with pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of pediocin. Consequently, we propose a new pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of pediocin PA-1. 相似文献
19.
Generation of Polyclonal Antibodies of Predetermined Specificity against Pediocin PA-1 总被引:4,自引:4,他引:0
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Jos M. Martínez María I. Martínez Ana M. Surez Carmen Herranz Pilar Casaus Luis M. Cintas Juan M. Rodríguez Pablo E. Hernndez 《Applied microbiology》1998,64(11):4536-4545
Polyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)—a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)—and by immunodotting. All immunoassays indicated the existence of pedA1-specific antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 μg/ml by immunodotting and 1 μg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 μg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 μg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 μg/ml, and the 50% binding inhibition value was reduced to 0.5 μg of pedA1/ml. All immunoassays and the slot dot assay detected the presence of pedA1 in the supernatant of the producing strain Pediococcus acidilactici 347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches taken for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the food industry. 相似文献
20.
Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles. 总被引:6,自引:3,他引:3
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The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe. Based on a three-dimensional model (Y. Chen, R. Shapira, M. Eisenstein, and T. J. Montville, Appl. Environ. Microbiol. 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes. Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum. The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer. The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out. Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide. The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch. Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues. These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane. 相似文献