scholarly journals Structural Analysis and Characterization of Lacticin Q, a Novel Bacteriocin Belonging to a New Family of Unmodified Bacteriocins of Gram-Positive Bacteria

2007 ◽  
Vol 73 (9) ◽  
pp. 2871-2877 ◽  
Author(s):  
Koji Fujita ◽  
Shiro Ichimasa ◽  
Takeshi Zendo ◽  
Shoko Koga ◽  
Fuminori Yoneyama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
High Performance ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Methionine Residue ◽  
New Family

ABSTRACT Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A.

A NEW PEPTIDE THROMBIN INHIBITOR FROM STREPTOMYCES GRISEUS

1987 ◽  
Author(s):  
Hua-zhen Liu ◽  
Wei Chen ◽  
Qi-ying Liu ◽  
Xia Zhang ◽  
Li-xiu Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Antithrombin Iii ◽  
Streptomyces Griseus ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Thrombin Inhibitor ◽  
Amino Acid Residues ◽  
Macroporous Resin ◽  
Terminal Residue

A new peptide thrombin inhibitor was found in the Streptomyces griseus strain 254 isolated from a soil sample from Tongan, Fujian province, China, the inhibitor being a secondary metabolic product. The production of the inhibitor reached a maximum after 3 days culture of bacteria at 28°C in a rotary shaker. The inhibitor excreted in the culture filtrate was purified by absorption on macroporous resin, followed by ion exchange chromatography on DEAE-52, CM-32 cellulose, affinity chromatography on the immobilized thrombin and high performance liquid chromatography. The amino acid composition of the inhibitor was determined to be Val(2), Met(l), Ile(l), Leu(2) and Arg (1), similar to that of the amino acid residues around the reactive site of human antithrombin III, the critical plasma inhibitor of thrombin. The NH2-terminal residue of the inhibitor seems to be blocked by the alkyl group due to the negative reaction to ninhydrin, whereas the COO-terminal residue is most likely to be arginal because of that Arg was not found in the amino acid analysis, unless the peptide was oxidized by performic acid before acid hydrolysis. The chromogen substrates Bz-Phe-Val-Arg-PNA and Bz-Gly-Pro-Lys-PNA were used to determine the thrombin and plasmin activities, respectively. Besides thrombin, the purified inhibitor also exhibits a weak inhibitory activities on trypsin and much weak on plasmin, but not on chymotrypsin and other protein-ases.

scholarly journals Identification and Characterization of Lactocyclicin Q, a Novel Cyclic Bacteriocin Produced by Lactococcus sp. Strain QU 12

2009 ◽  
Vol 75 (6) ◽  
pp. 1552-1558 ◽  
Author(s):  
Naruhiko Sawa ◽  
Takeshi Zendo ◽  
Junko Kiyofuji ◽  
Koji Fujita ◽  
Kohei Himeno ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Hydrophobic Interaction Chromatography ◽  
Heat Stability ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
High Level

ABSTRACT Lactococcus sp. strain QU 12, which was isolated from cheese, produced a novel cyclic bacteriocin termed lactocyclicin Q. By using cation-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography, lactocyclicin Q was purified from culture supernatant, and its molecular mass was determined to be 6,062.8 Da by mass spectrometry. Lactocyclicin Q has been characterized by its unique antimicrobial spectrum, high level of protease resistance, and heat stability compared to other reported bacteriocins of lactic acid bacteria. The amino acid sequence of lactocyclicin Q was determined chemically, and this compound is composed of 61 amino acid residues that have a cyclic structure with linkage between the N and C termini by a peptide bond. It showed no homology to any other antimicrobial peptide, including cyclic bacteriocins. On the basis of the amino acid sequences obtained, the sequence of the gene encoding the prepeptide lactocyclicin Q was obtained. This is the first report of a cyclic bacteriocin purified from a strain belonging to the genus Lactococcus.

Thermitase from Thermoactinomyces vulgaris; amino acid sequence of the large N-terminal cyanogen bromide peptide

1985 ◽  
Vol 50 (4) ◽  
pp. 885-896 ◽  
Author(s):  
Bedřich Meloun ◽  
Miroslav Baudyš ◽  
Manfred Pavlík ◽  
Vladimír Kostka ◽  
Gert Hausdorf ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
High Performance ◽  
Cyanogen Bromide ◽  
Sequential Data ◽  
Amino Acid Residues ◽  
Methionine Residue ◽  
Large N ◽  
Thermoactinomyces Vulgaris

The large cyanogen bromide fragment (CB1) represents the N-terminal part of the molecule of thermitase and contains 226 amino acid residues. Its molecular weight calculated from sequential data is 22 932 (the C-terminal residue is regarded as a methionine residue in the calculations). The amino acid sequence of fragment CB1 was determined by analysis of peptides obtained by tryptic hydrolysis of the fragment; these data were complemented by sequence analysis of the chymotryptic digest of fragment Mf (residues 75 through 226) and of chymotryptic fragment ET3 (residues 103 through 226) isolated from the limited tryptic digest of fragment CB1. The peptides were purified by high performance liquid chromatography and by thin layer techniques. The sequence analysis of the large peptides was effected in the sequenator, small peptides were sequenced manually by the DABITC/PITC double coupling technique. The results obtained in this study together with those of previous work5 permitted the complete amino acid sequence of fragment CB1 to be determined.

scholarly journals Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Mengxin Geng ◽  
Frank Austin ◽  
Ronald Shin ◽  
Leif Smith
Keyword(s):  
Amino Acids ◽  
Antibacterial Activity ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Linear Peptide ◽  
Gram Positive Bacteria ◽  
Lipid Ii ◽  
Covalent Structure ◽  
Terminal Amino

ABSTRACTLantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain,Streptococcus salivariusK12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCEThe amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

1989 ◽  
Vol 54 (3) ◽  
pp. 803-810 ◽  
Author(s):  
Ivan Kluh ◽  
Ladislav Morávek ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Cyanogen Bromide ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Mild Acid Hydrolysis ◽  
Methionine Residue ◽  
Cyanogen Bromide Fragment ◽  
Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

scholarly journals In Vitro Characterization of the Bacillus subtilis Protein Tyrosine Phosphatase YwqE

2005 ◽  
Vol 187 (10) ◽  
pp. 3384-3390 ◽  
Author(s):  
Ivan Mijakovic ◽  
Lucia Musumeci ◽  
Lutz Tautz ◽  
Dina Petranovic ◽  
Robert A. Edwards ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Class Ii ◽  
Gram Positive ◽  
Protein Tyrosine ◽  
Gram Positive Bacteria ◽  
New Family ◽  
In Vitro Characterization

ABSTRACT Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains one such CpsB-like PTP, YwqE, in addition to two class II Cys-based PTPs, YwlE and YfkJ. The substrates for both YwlE and YfkJ are presently unknown, while YwqE was shown to dephosphorylate two phosphotyrosine-containing proteins implicated in UDP-glucuronate biosynthesis, YwqD and YwqF. In this study, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE.

scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

Purification of large peptides using Chemoselective tags

1999 ◽  
Author(s):  
Paolo Mascagni
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Exchange Chromatography ◽  
Target Sequence ◽  
Coupling Reactions ◽  
Amino Acid Residues ◽  
Quaternary Structures ◽  
Specific Stationary Phase

In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.

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