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.

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 Structural analyses of the deduced amino acid sequences of a novel type heme–copper terminal oxidase, cytochrome aco3, from alkalophilic Bacillus YN-2000

2001 ◽  
Vol 47 (12) ◽  
pp. 1075-1081 ◽  
Author(s):  
Kimitoshi Denda ◽  
Akira Oshima ◽  
Yoshihiro Fukumori
Keyword(s):  
Amino Acid ◽  
Cytochrome C ◽  
Basic Amino Acid ◽  
Structural Features ◽  
Thermophilic Bacterium ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Bacterium Bacillus ◽  
Alkalophilic Bacillus

Cytochrome aco3 from a facultatively alkalophilic bacterium, Bacillus YN-2000, was found to be alkaline- and heat-tolerant. To better understand the structural features of Bacillus YN-2000 cytochrome aco3, the gene encoding this enzyme was cloned and sequenced. Nucleotide sequence analyses of the region neighboring the acoI (subunit I) gene revealed that the acoII (subunit II) and acoIII (subunit III) genes were concomitantly clustered upstream and downstream of the acoI gene, respectively, forming an operon with transcriptional polarity. The deduced amino acid sequence of subunit I was highly similar to that of cytochrome caa3 from thermophilic bacterium Bacillus PS3 in which the heme a3 could be replaced with heme o. Furthermore, a marked paucity of basic amino acid residues was found in the cytochrome c-binding subunit II, which might be a result of the adaptation to a highly alkaline external milieu.Key words: cytochrome c oxidase, alkalophile, thermostability, heme o, Bacilli.

scholarly journals Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

2002 ◽  
Vol 184 (3) ◽  
pp. 777-784 ◽  
Author(s):  
Naeem Rashid ◽  
Joel Cornista ◽  
Satoshi Ezaki ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Anion Exchange Chromatography ◽  
Soluble Starch ◽  
Binding Activity ◽  
Exchange Chromatography ◽  
Cyclodextrin Glucanotransferase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Terminal Domain ◽  
Catalytic Function

ABSTRACT A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT Tk ) was identified and characterized. The gene (cgt Tk ) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the α-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively. The presence of Ca2+ enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C. With the addition of Ca2+, the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C. CGT Tk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT Tk harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with β-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT Tk ΔC) with 23 C-terminal amino acid residues deleted. CGT Tk ΔC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT Tk . In contrast, the cyclization activity of CGT Tk ΔC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT Tk to properly catalyze the cyclization reaction.

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader,Rhodococcus sp. Strain RHA1

1998 ◽  
Vol 64 (6) ◽  
pp. 2006-2012 ◽  
Author(s):  
Akihiro Yamada ◽  
Hidekazu Kishi ◽  
Katsumi Sugiyama ◽  
Takashi Hatta ◽  
Kanji Nakamura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polychlorinated Biphenyl ◽  
Blot Hybridization ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Gene Encoding ◽  
Amino Terminal ◽  
Gene Assay ◽  
Amino Terminal Sequence

ABSTRACT The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes,etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. TheetbD2 gene was located in the vicinity of bphAgene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD,cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphDgenes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product ofbphD was very specific to HPDA, and the products ofetbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against themeta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphDgene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

scholarly journals Two globin strains in the giant annelid extracellular haemoglobins

1987 ◽  
Vol 241 (2) ◽  
pp. 441-445 ◽  
Author(s):  
T Gotoh ◽  
F Shishikura ◽  
J W Snow ◽  
K I Ereifej ◽  
S N Vinogradov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lumbricus Terrestris ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Reverse Phase ◽  
Terminal Amino ◽  
Polypeptide Chains

The constituent polypeptide chains I, II, III and IV of the giant extracellular haemoglobin of the oligochaete Lumbricus terrestris were isolated by mono Q ion-exchange chromatography and C8 reverse-phase chromatography. The N-terminal amino acid sequences of Lumbricus chains I, III and IV were determined and aligned with those of Lumbricus chain II and the four chains of the extracellular haemoglobin of the polychaete Tylorrhynchus heterochaetus. Three invariant amino acid residues, Cys-7, Val-15 and Trp-19, were found to occur in the N-terminal segments (17-22 residues) of the eight chains of Lumbricus and Tylorrhynchus haemoglobins. In addition, it was found that the eight sequences could be separated into two groups: ‘A’, consisting of Lumbricus chains I and II and Tylorrhynchus chains I and IIA, having invariant Lys-14 and Lys-16, and ‘B’, consisting of Lumbricus chains III and IV and Tylorrhynchus IIB and IIC, having invariant Cys-6, Ser-8 and Asp-11. This result suggests that there are two strains of globin chain in the annelid extracellular haemoglobins.

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.

scholarly journals Identification and Characterization of pantocin wh-1, a Novel Cyclic Polypeptide Produced by Pantoea dispersa W18

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 485 ◽  
Author(s):  
Tieshan Teng ◽  
Xianghui Li ◽  
Lei Zhang ◽  
Yanzhang Li
Keyword(s):  
Amino Acid ◽  
Hydrophobic Interaction Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Heat Stable ◽  
Pantoea Dispersa ◽  
Natural Peptide ◽  
Cyclic Polypeptide ◽  
Identification And Characterization

Pantoea dispersa W18, isolated from contaminated soil, was found to exert antimicrobial activity against Mycobacterium species, including Mycobacterium tuberculosis, an important human pathogen. Here, the anti-mycobacterial compound produced by Pantoea dispersa W18 was purified by a combination of hydrophobic interaction chromatography, cation exchange chromatography, and reverse phase HPLC. Active compounds from Pantoea dispersa W18 were identified as a natural peptide named pantocin wh-1 with a 1927 Da molecular weight. The primary structure of this compound was detected by N-terminal amino acid sequencing. The amino acid sequence of pantocin wh-1 consisted of 16 amino acid residues with a cyclic structure. The pantocin wh-1 could be inactivated by protease K, but was heat stable and unaffected by pH (2–12). However, the activity was not completely inactivated by trypsin and pepsin. This is the first report of a cyclic polypeptide purified from a strain of Pantoea dispersa.

The Amino Acid Sequence of Streptomyces griseus Protease A: The Peptic Peptides

1971 ◽  
Vol 49 (10) ◽  
pp. 1083-1097 ◽  
Author(s):  
P. Johnson ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Streptomyces Griseus ◽  
Disulfide Bridge ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Homologous Proteins ◽  
Acidic Peptide ◽  
Dowex 1 ◽  
Peptide Fractions

The peptic peptides of Streptomyces griseus Protease A (excluding the previously characterized disulfide bridge peptic peptides) were fractionated into basic, neutral, and neutral plus acidic peptide fractions by chromatography on Dowex 1 × 2. These three peptide fractions were then fractionated by cation-exchange chromatography on Chromobead P resin using the Technicon autoanalyzer system. Following further purifications on paper, the amino acid compositions and sequences of the peptic peptides were determined. The N-terminal sequence of Protease A has been identified as Ile–Ala–Gly–Gly–Glu–Ala. The numbers of amino acid residues obtained from the amino acid sequences reported are in agreement with those numbers obtained from amino acid analysis of the total protein in the cases of tryptophan, methionine, histidine, proline, phenylalanine, and glutamic acid. Some of the results suggest either the presence of nonidentical but highly homologous proteins in the Protease A preparation or the possibility of repeating sequences in a single molecular species.

scholarly journals Cloning, Overexpression, and Mutagenesis of theSporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

1999 ◽  
Vol 65 (12) ◽  
pp. 5207-5211 ◽  
Author(s):  
Keiko Kita ◽  
Takanobu Fukura ◽  
Koh-Ichi Nakase ◽  
Kenji Okamoto ◽  
Hideshi Yanase ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Inhibition ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Binding Motif ◽  
Aldehyde Reductase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
E Coli

ABSTRACT We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolorAKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced inEscherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed inE. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G19-X-X-G22-X-X-A25, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G19→A and G22→A mutant enzymes by 4-COBE did not occur. The A25→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

scholarly journals Cloning and Targeted Disruption of MLG1, a Gene Encoding Two of Three Extracellular Mixed-Linked Glucanases ofCochliobolus carbonum

1998 ◽  
Vol 64 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Jenifer M. Görlach ◽  
Esther Van Der Knaap ◽  
Jonathan D. Walton
Keyword(s):  
Amino Acid ◽  
Cation Exchange ◽  
High Performance ◽  
Extracellular Enzymes ◽  
Pathogenic Fungus ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Culture Filtrates

ABSTRACT Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze β1,3-1,4-glucans (also known as mixed-linked glucans or cereal β-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Mlg1a and Mlg1b also hydrolyze β1,3-glucan (laminarin), whereas Mlg2 does not degrade β1,3-glucan but does degrade β1,4-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively. The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b, MLG1, was isolated by using PCR primers based on amino acid sequences of Mlg1b. The product ofMLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes. An internal fragment of MLG1 was used to create mlg1 mutants by transformation-mediated gene disruption. The total MLGase and β1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1. The growth of mlg1 mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.

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