scholarly journals Characterization of Garvicin ML, a Novel Circular Bacteriocin Produced byLactococcus garvieaeDCC43, Isolated from Mallard Ducks (Anas platyrhynchos)

2010 ◽  
Vol 77 (1) ◽  
pp. 369-373 ◽  
Author(s):  
Juan Borrero ◽  
Dag A. Brede ◽  
Morten Skaugen ◽  
Dzung B. Diep ◽  
Carmen Herranz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Genetics ◽  
De Novo ◽  
Leader Peptide ◽  
Nucleotide Sequencing ◽  
Amino Acid Sequencing ◽  
Circular Bacteriocin ◽  
Amino Acid Precursor ◽  
Acid Precursor

ABSTRACTLactococcus garvieaeDCC43 produces a bacteriocin, garvicin ML (GarML), with a molecular mass of 6,004.2 Da. Data fromde novoamino acid sequencing by tandem mass spectrometry and nucleotide sequencing by reverse genetics suggested that the bacteriocin is synthesized as a 63-amino-acid precursor with a 3-amino-acid leader peptide that is removed by cleavage. Subsequently, a covalent linkage between the N and C termini forms the mature version of this novel 60-amino-acid circular bacteriocin.

scholarly journals Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21

2020 ◽  
Author(s):  
Aida Golneshin ◽  
Mian Chee Gor ◽  
Ben Vezina ◽  
Nicholas Williamson ◽  
Thi Thu Hao Van ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Lactobacillus Plantarum ◽  
De Novo ◽  
Peptide Sequencing ◽  
Exchange Chromatography ◽  
Leader Peptide ◽  
Proteolytic Resistance ◽  
Wide Range ◽  
Circular Bacteriocin

AbstractLactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.

The Pediocin AcH Precursor Is Biologically Active

1999 ◽  
Vol 65 (6) ◽  
pp. 2281-2286 ◽  
Author(s):  
Bibek Ray ◽  
Robin Schamber ◽  
Kurt W. Miller
Keyword(s):  
Amino Acid ◽  
Membrane Damage ◽  
Biologically Active ◽  
Leader Peptide ◽  
Chimeric Proteins ◽  
Cell Protection ◽  
E Coli ◽  
Amino Acid Precursor ◽  
Insight Into ◽  
Acid Precursor

ABSTRACT The properties of the pediocin AcH precursor, prepediocin AcH, have been studied to gain insight into how producer cells may protect themselves from the activity of intracellular prebacteriocins. The native 62-amino-acid precursor and the 44-amino-acid mature species were expressed in Escherichia coli host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the growth of the test bacterium Listeria innocua Lin11, indicating that the native precursor is biologically active. The two species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was ∼80% as active as the chimeric mature species. Of relevance to cell protection and pediocin AcH production, it was determined that the precursor is strongly susceptible to inactivation by reducing agents and to degradation by chymotrypsin and endogenous E. coliproteases. Taken together, the results indicate that the activity of prepediocin AcH may have to be controlled prior to secretion to prevent toxicity to the host. Perhaps producer cells avoid membrane damage by maintaining the precursor in a reduced inactive state or by degrading molecules whose secretion is delayed.

scholarly journals Synthesis and Characterization of a Novel Biheterocyclic -amino Acid Precursor of the Triazole-Tetrazole Type, via the Copper (I) Catalyzed Alkyne-Azide Cycloaddition Reaction (CuAAC)

2021 ◽  
Vol 2 (2) ◽  
pp. 7-15
Author(s):  
Khadim Dioukhane ◽  
Younas Aouine ◽  
Salaheddine Boukhssas ◽  
Asmae Nakkabi ◽  
Hassane Faraj ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cycloaddition Reaction ◽  
Sodium Ascorbate ◽  
Dipolar Cycloaddition ◽  
Resonance Spectroscopy ◽  
Compound A ◽  
Amino Acid Precursor ◽  
Acid Precursor

In this paper, we describe the regioselective synthesis of a novel tri-heterocyclic compound, a biheterocyclic amino acid precursor, derived from both triazole and tetrazole. The key step of our synthesis approach was the Huigsen 1,3-dipolar cycloaddition reaction, catalyzed by the copper (I) formed in situ by reduction of Cu(II) salts (CuSO4), 5H2O) by sodium ascorbate, and using as dipole the oxazoline azide derivative 4-(azidomethyl)-4-ethyl-2-phenyl-4,5-dihydrooxazole (4) and as dipolarophile 5-(4-methoxyphenyl)-2-(prop-2-yn-1-yl)-2H-tetrazole (3).  The Cu(I) catalysis allowed us to carry out the cycloaddition at room temperature during a reaction time of only 8 hours and also to selectively obtain the 1,4-regioisomer; one of the two possible isomers, with a yield of 90% after chromatography on a silica gel column (ether/hexane: 1/2), and recrystallization in an ether/acetone mixture. The desired compound, 4-ethyl-4-((4-((5-(4-methoxyphenyl)-2H-tetrazol-2-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-phenyl-4,5-dihydrooxazole (5) was analyzed by 1D magnetic resonance spectroscopy (1H, 13C), and characterized physico-chemically by mass spectrometry and elemental analysis.

scholarly journals Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Juan Borrero ◽  
Eoin Kelly ◽  
Paula M. O'Connor ◽  
Philip Kelleher ◽  
Colm Scully ◽  
...  
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Gene Cluster ◽  
Lactobacillus Plantarum ◽  
Leader Peptide ◽  
Alicyclobacillus Acidoterrestris ◽  
Content Type ◽  
Circular Bacteriocin ◽  
Spoilage Bacterium

ABSTRACTBacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain,Lactobacillus plantarumNI326, with activity against the beverage-spoilage bacteriumAlicyclobacillus acidoterrestris. Genome sequencing of NI326 enabled the identification of a gene cluster (designatedplc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant ofLb. plantarumNI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. Theplcgene cluster was cloned and expressed inLactococcus lactisNZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced byLb. plantarum. The inhibition displayed againstA. acidoterrestrishighlights its potential use as a preservative in food and beverages.IMPORTANCEIn this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by aLactobacillus plantarumstrain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, includingAlicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for aLactobacillus plantarumstrain.

scholarly journals The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (8) ◽  
pp. 2323-2329 ◽  
Author(s):  
Miguel Prudêncio ◽  
Robert R. Eady ◽  
Gary Sawers
Keyword(s):  
Amino Acid ◽  
Nitrite Reductase ◽  
Recombinant Enzyme ◽  
Leader Peptide ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

scholarly journals Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

2002 ◽  
Vol 68 (8) ◽  
pp. 3830-3840 ◽  
Author(s):  
Shinichi Kawamoto ◽  
Jun Shima ◽  
Rumi Sato ◽  
Tomoko Eguchi ◽  
Sadahiro Ohmomo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Characterization ◽  
Solid Phase ◽  
Exchange Chromatography ◽  
Nucleotide Sequencing ◽  
Lactobacillus Curvatus ◽  
Amino Acid Peptide ◽  
Enterococcus Mundtii

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

scholarly journals Roles of Prenyl Protein Proteases in Maturation of Saccharomyces cerevisiae a-Factor

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 95-101
Author(s):  
Victor L Boyartchuk ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Direct Role ◽  
Amino Acid Precursor ◽  
Carboxyl Terminal ◽  
Secreted Peptides ◽  
Pro Region ◽  
Acid Precursor

Abstract In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors. In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor. This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor. In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo. Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone. However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production.

A serum protease cleaves proANF into a 14-kilodalton peptide and ANF

1987 ◽  
Vol 252 (1) ◽  
pp. E147-E151
Author(s):  
K. D. Bloch ◽  
J. B. Zisfein ◽  
M. N. Margolies ◽  
C. J. Homcy ◽  
J. G. Seidman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Culture Medium ◽  
Rat Plasma ◽  
Biologically Active ◽  
Amino Terminal ◽  
Isolation And Characterization ◽  
Amino Acid Precursor ◽  
Acid Precursor

Proatrial natriuretic factor (proANF), the 126-amino acid precursor of ANF, is the major storage form in mammalian atria. In contrast, two ANF peptides containing the 28- and 24-carboxyterminal residues of proANF have been isolated from rat plasma. Whether the cleavage of proANF in vivo to these ANF peptides occurs during or after its release into the circulation has not been determined. The latter possibility was suggested by our previous study where, by using a cultured rat cardiocyte preparation, we demonstrated that proANF is secreted intact into the culture medium. We now report that serum, but not plasma, contains a protease that specifically cleaves the 17-kdalton proANF to a 14-kdalton amino-terminal peptide and the carboxyterminal 3-kdalton circulating forms of ANF. The role of this proANF-cleaving enzyme in the generation of the biologically active ANF peptides remains to be defined. Its isolation and characterization should provide insights into its site of production and whether in vivo it is involved in the processing of circulating proANF.

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