Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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Purification and characterization of enterocin MC13 produced by a potential aquaculture probiontEnterococcus faeciumMC13 isolated from the gut ofMugil cephalus

Canadian Journal of Microbiology ◽

10.1139/w11-092 ◽

2011 ◽

Vol 57 (12) ◽

pp. 993-1001 ◽

Cited By ~ 26

Author(s):

R. Satish Kumar ◽

P. Kanmani ◽

N. Yuvaraj ◽

K.A. Paari ◽

V. Pattukumar ◽

...

Keyword(s):

Molecular Mass ◽

Enterococcus Faecium ◽

Vibrio Vulnificus ◽

Proteolytic Enzymes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Probiotic Bacterium ◽

Mass Spectrometry Analysis ◽

Native Page ◽

Spectrometry Analysis

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium . The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus , and Vibrio vulnificus . This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi , and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

Applied and Environmental Microbiology ◽

10.1128/aem.00409-09 ◽

2009 ◽

Vol 75 (14) ◽

pp. 4661-4667 ◽

Cited By ~ 26

Author(s):

Alejandro Hernández-Soto ◽

M. Cristina Del Rincón-Castro ◽

Ana M. Espinoza ◽

Jorge E. Ibarra

Keyword(s):

Bacillus Thuringiensis ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Microbial Control ◽

Control Agent ◽

Mass Spectrometry Analysis ◽

Parasporal Crystal ◽

Spectrometry Analysis ◽

Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

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Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

Applied and Environmental Microbiology ◽

10.1128/aem.02484-06 ◽

2007 ◽

Vol 73 (7) ◽

pp. 2247-2250 ◽

Cited By ~ 75

Author(s):

Sirinat Srionnual ◽

Fujitoshi Yanagida ◽

Li-Hsiu Lin ◽

Kuang-Nan Hsiao ◽

Yi-sheng Chen

Keyword(s):

Proteolytic Enzymes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Band ◽

Proteinase K ◽

Mass Spectrometry Analysis ◽

Fermented Fish ◽

Spectrometry Analysis ◽

Weissella Cibaria ◽

Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

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Simple Method for Repurification of Endotoxins for Biological Use

Applied and Environmental Microbiology ◽

10.1128/aem.02452-06 ◽

2007 ◽

Vol 73 (6) ◽

pp. 1803-1808 ◽

Cited By ~ 35

Author(s):

Alina Tirsoaga ◽

Alexey Novikov ◽

Minou Adib-Conquy ◽

Catherine Werts ◽

Catherine Fitting ◽

...

Keyword(s):

Mass Spectra ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

Ionization Mass ◽

Toll Like Receptor ◽

Simple Method ◽

Spectrometry Analysis ◽

Toll Like Receptor 2 ◽

Oligomerization Domain

ABSTRACT A method for obtaining highly purified endotoxin (lipopolysaccharide [LPS]) in a few hours by repurification of commercial or laboratory preparations was devised. It avoids the use of phenol, which is not suitable for phenol-soluble lipopolysaccharides nor for some industrial purposes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry analysis confirmed the integrity of the purified LPSs. The purified products did not activate Toll-like receptor 2 (TLR2), nuclear oligomerization domain 1 (NOD1), or NOD2 but did activate TLR4. Applied to different lipopolysaccharides, the method also improved their mass spectra, thus facilitating their structural analysis.

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The content of immunomodulatory glycoepitopes in seminal plasma glycoproteins of fertile and infertile men

Reproduction Fertility and Development ◽

10.1071/rd18124 ◽

2019 ◽

Vol 31 (3) ◽

pp. 579

Author(s):

Anna Kałuża ◽

Mirosława Ferens-Sieczkowska ◽

Beata Olejnik ◽

Justyna Kołodziejczyk ◽

Mariusz Zimmer ◽

...

Keyword(s):

Sialic Acid ◽

Seminal Plasma ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Specific Antigen ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Ulex Europaeus ◽

Maackia Amurensis ◽

Sambucus Nigra Agglutinin

According to a concept of fetoembryonic defence, protein–carbohydrate interaction may be involved in the regulation of maternal immunity that prevents rejection of allograft spermatozoa, embryo and fetus. In the present study we focussed on the evaluation of the expression of glycoepitopes that may be of crucial importance in this process: LewisY (LeY) and LewisX (LeX) as well as terminal sialylation. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate was used to separate seminal plasma samples of fertile (n=10) and infertile (n=103) men; these were then probed with lectins specific to fucose (Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin) and sialic acid (Sambucus nigra agglutinin and Maackia amurensis agglutinin). Differential expression of α2,3-bound sialic acid was found in six out of seven analysed bands, whereas differences in the other analysed glycoepitopes were found in fewer numbers of bands. Mass spectrometry analysis focussed on the identification of proteins carrying glycans with immunomodulatory epitopes, including fibronectin, lactoferrin, clusterin, zinc-α2-glycoprotein, prostate acid phosphatase and prostate-specific antigen; these should be submitted to further detailed analysis.

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Association of a Virus with Wheat Displaying Yellow Head Disease Symptoms in the Great Plains

Plant Disease ◽

10.1094/pd-89-0888 ◽

2005 ◽

Vol 89 (8) ◽

pp. 888-895 ◽

Cited By ~ 12

Author(s):

Dallas L. Seifers ◽

Tom L. Harvey ◽

T. J. Martin ◽

Steve Haber ◽

Y.-M. She ◽

...

Keyword(s):

Great Plains ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

High Plains ◽

Spectrometry Analysis ◽

Yellow Head Virus ◽

Flight Mass Spectrometry ◽

Sds Page ◽

Leaf Symptoms

Wheat with yellow head disease (YHD) (yellow heads and mosaic leaf symptoms) has been observed in Kansas since 1997. A pathogen was transmitted from the infected wheat to maize by vascular puncture inoculation and to Nicotiana benthamiana by rub inoculation. The original infected wheat and infected maize and N. benthamiana test plants all produced a unique 32- to 34-kDa protein when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Time-of-flight mass spectrometry analysis of the unique 32- to 34-kDa protein showed that the amino acid sequence was most closely related to the nucleoprotein of Rice hoja blanca virus, indicating that the virus causing YHD symptoms in wheat is a tenuivirus. Antiserum made to this protein failed to react with extracts made from healthy wheat or wheat infected with Wheat streak mosaic virus or the High Plains virus. The antiserum did react to extracts made from symptomatic wheat, maize, and N. benthamiana, shown by SDS-PAGE to contain the unique protein, and to extracts of wheat with YHD symptoms from Kansas, North Dakota, South Dakota, and Oklahoma. The name Wheat yellow head virus is proposed for this virus.

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Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Applied and Environmental Microbiology ◽

10.1128/aem.01362-10 ◽

2010 ◽

Vol 76 (22) ◽

pp. 7536-7540 ◽

Cited By ~ 33

Author(s):

Ariane Bisaillon ◽

Réjean Beaudet ◽

François Lépine ◽

Eric Déziel ◽

Richard Villemur

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

Activity Levels ◽

Chlorinated Phenols ◽

Quinone Oxidoreductase ◽

Spectrometry Analysis ◽

Reductive Dehalogenase ◽

Molecular Masses ◽

Desulfitobacterium Hafniense

ABSTRACT Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent Km value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.

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Structural Characterization of an Oligosaccharide Made by Neisseria sicca

Journal of Bacteriology ◽

10.1128/jb.01433-08 ◽

2009 ◽

Vol 191 (10) ◽

pp. 3311-3320 ◽

Cited By ~ 3

Author(s):

Ellen T. O'Connor ◽

Hui Zhou ◽

Kevin Bullock ◽

Karen V. Swanson ◽

J. McLeod Griffiss ◽

...

Keyword(s):

Mass Spectrometry ◽

Structural Characterization ◽

Lectin Binding ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

Ionization Mass ◽

Spectrometry Analysis ◽

Neisseria Sicca

ABSTRACT Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization—time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-d-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its β-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) β-l-rhamnose (1-3) β-N-acetyl-d-glucosamine (1-] with an N-acetyl-d-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

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Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.260.6.r1168 ◽

1991 ◽

Vol 260 (6) ◽

pp. R1168-R1175

Author(s):

L. Bosca ◽

K. B. Storey

Keyword(s):

Enzyme Activity ◽

Protein Kinase ◽

Molecular Mass ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Busycon Canaliculatum ◽

Native Molecular Mass ◽

Dependent Protein

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

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