Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis

2018 ◽  
Vol 28 (1) ◽  
pp. 14-27 ◽  
Author(s):  
Carlos Eduardo Serrano-Maldonado ◽  
Israel García-Cano ◽  
Augusto González-Canto ◽  
Eliel Ruiz-May ◽  
Jose Miguel Elizalde-Contreras ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Heterologous Protein ◽  
Biochemical Characterization ◽  
Ph Values ◽  
Food Borne ◽  
Sds Page ◽  
Exclusion Chromatography

The <i>atlD</i> gene from<i></i> an <i>Enterococcus faecalis</i> strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in <i>Escherichia coli</i> in order to perform a biochemical characterization<i>.</i> A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62–75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6–7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn<sup>2+</sup>. It showed antibacterial activity against <i>Listeria monocytogenes</i>,<i> Staphylococcus aureus</i>, and enterococcal<i></i> strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals Purification and biochemical characterization of an extracellular lipase produced by a new strain of Rhizopus sp.

2006 ◽  
Vol 30 (3) ◽  
pp. 494-502 ◽  
Author(s):  
Maria Gabriela Bello Koblitz ◽  
Gláucia Maria Pastore
Keyword(s):  
Molecular Mass ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Extracellular Lipase ◽  
Optimum Activity ◽  
Ph Values ◽  
Sds Page ◽  
Rhizopus Sp

The present study had as a goal to purify and characterize the lipolytic fraction secreted by a strain of Rhizopus sp. Only 3 steps of purification were necessary to achieve SDS-PAGE homogeneity. One band with 37.5 KDa molecular mass and with 1446 U/mg specific activity was obtained. The purified fraction presented 2 lipase isoforms; both showed optimum activity at 50ºC, and were stable between 6.5 and 7.5 pH values and at temperatures below 50ºC and also kept their activity in hexane. The lipase was inactivated by Hg+2 and by n-bromosuccinimide and activated by Na+.

scholarly journals Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 582
Author(s):  
Cristian Franco-Servín ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
Ramsés Alejandro Rosales-García ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Biochemical Characterization ◽  
Two Dimensions ◽  
Size Exclusion ◽  
Muscle Twitch ◽  
Amino Terminal ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Main Components

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

scholarly journals Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product

2021 ◽  
Vol 12 (1) ◽  
pp. 85-93
Author(s):  
Wallapat Phongtang ◽  
Ekachai Chukeatirote
Keyword(s):  
Bacillus Cereus ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Morphological Analysis ◽  
Food Poisoning ◽  
Fermented Soybean ◽  
Foodborne Pathogenic Bacteria ◽  
Ph Range ◽  
Potential Use

Abstract Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

scholarly journals Isolation and Characterization of Bacteriophage ZCSE6 against Salmonella spp.: Phage Application in Milk

Biologics ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 164-176
Author(s):  
Abdallah S. Abdelsattar ◽  
Anan Safwat ◽  
Rana Nofal ◽  
Amera Elsayed ◽  
Salsabil Makky ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Raw Milk ◽  
Salmonella Spp ◽  
Isolation And Characterization ◽  
Food Borne ◽  
Wide Range ◽  
Almost All ◽  
And Control ◽  
Milk And Dairy Products

Food safety is very important in the food industry as most pathogenic bacteria can cause food-borne diseases and negatively affect public health. In the milk industry, contamination with Salmonella has always been a challenge, but the risks have dramatically increased as almost all bacteria now show resistance to a wide range of commercial antibiotics. This study aimed to isolate a bacteriophage to be used as a bactericidal agent against Salmonella in milk and dairy products. Here, phage ZCSE6 has been isolated from raw milk sample sand molecularly and chemically characterized. At different multiplicities of infection (MOIs) of 0.1, 0.01, and 0.001, the phage–Salmonella interaction was studied for 6 h at 37 °C and 24 h at 8 °C. In addition, ZCSE6 was tested against Salmonella contamination in milk to examine its lytic activity for 3 h at 37 °C. The results showed that ZCSE6 has a small genome size (<48.5 kbp) and belongs to the Siphovirus family. Phage ZCSE6 revealed a high thermal and pH stability at various conditions that mimic milk manufacturing and supply chain conditions. It also demonstrated a significant reduction in Salmonella concentration in media at various MOIs, with higher bacterial eradication at higher MOI. Moreover, it significantly reduced Salmonella growth (MOI 1) in milk, manifesting a 1000-fold decrease in bacteria concentration following 3 h incubation at 37 °C. The results highlighted the strong ability of ZCSE6 to kill Salmonella and control its growth in milk. Thus, ZCSE6 is recommended as a biocontrol agent in milk to limit bacterial growth and increase the milk shelf-life.

scholarly journals Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5553
Author(s):  
Loreto Robles-Hernández ◽  
Nora A. Salas-Salazar ◽  
Ana C. Gonzalez-Franco
Keyword(s):  
Antibacterial Activity ◽  
Ganoderma Lucidum ◽  
Pathogenic Bacteria ◽  
Plant Pathogens ◽  
Heat Stability ◽  
Limited Information ◽  
Dialysis Tubing ◽  
Molecular Weights ◽  
Exclusion Chromatography

Previous studies of Ganoderma lucidum have focused on its medicinal applications. Limited information is available about its antibacterial activity against plant pathogens. Thus, the goal of this study was to purify and characterize the antibacterial activity against plant pathogenic bacteria from culture fluids of G. lucidum. The nature of the bioactive components was determined using heat boiling, organic solvents, dialysis tubing, gel exclusion chromatography (GEC), proteinase sensitivity, HPLC, HPLC-APCI-MS, and GC-MS. The bioactive compounds were neither lipid, based on their solubility, nor proteic in nature, based on proteinase digestion and heat stability. The putative-bioactive polysaccharides have molecular weights that range from 3500 to 4500 Daltons as determined by dialysis tubing, GEC and APCI-MS analysis. The composition of the antibacterial compounds was determined by GC-MS. This is the first report of small polysaccharides produced by G. lucidum with activity against bacterial plant pathogens.

scholarly journals Physical, physicochemical and taxonomic characterization of Psidium araça Raddi

2016 ◽  
Vol 1 (1) ◽  
pp. 106-110 ◽  
Author(s):  
Maria do Rosário Fátima Padilha ◽  
Neide Kazue Sakugawa Shinohara ◽  
Emanuella de Paula Rodrigues Pereira ◽  
Rejane Magalhães Mendonça Pimentel ◽  
Samara Alvachian Cardoso Andrade ◽  
...  
Keyword(s):  
Food Industry ◽  
Fruit Trees ◽  
Average Weight ◽  
Ph Values ◽  
In The Wild ◽  
Promising Application ◽  
New Research ◽  
Agricultural Regions ◽  
Taxonomic Characterization

New research has stimulated a large reevaluation of species with regards to the use of fruits from native plants. Araçá (a small Brazilian guava-like fruit) is found scattered in the wild in Pernambuco/Brazil. There is a scarcity of detailed studies on such regional fruit trees, especially the Psidium araçá Raddi species, whereas there is an enormous possibility to explore the potential offered by these fruits, such as in the field of gastronomy, where the search for new ingredients and exotic flavors associated with functional properties has been increasing. This article aimed to evaluate the physical, physicochemical, and taxonomic characterization of araçá obtained from different agricultural regions of Pernambuco in order to investigate the possibility of developing new products from this native fruit. The taxonomic identification confirmed that all collected material belonged to the species P. araçá Raddi. The average weight of 7.45 g/fruit was observed. With respect to pH, values between 3.17 and 3.48 were found, and the acidity as a percentage of citric acid was on the order of 0.96% to 0.99%. It was shown that the P. araçá Raddi fruit has a desirable quality for the food industry, presenting excellent conditions for the development of formulations of high commercial value and promising application in the national gastronomy.

scholarly journals Prevalence of Tonsillitis caused by Streptococcus spp. Among children and the effect of some Lactic acid bacterial (LAB) strain on it

2016 ◽  
Vol 13 (2) ◽  
pp. 218-227
Author(s):  
Baghdad Science Journal
Keyword(s):  
Inhibitory Activity ◽  
Pathogenic Bacteria ◽  
Biochemical Characterization ◽  
Bacterial Isolate ◽  
Inhibition Zone ◽  
Laboratory Culture ◽  
Diffusion Method ◽  
Inhibition Activity ◽  
Str System

In this study 100 samples were collected from infected children with acute and chronic tonsillitis who attended to Al-Yarmook Teaching Hospital (ENT consultation clinic) from 5/12/2013 to 1/3/2014. The result of laboratory culture was positive in 67 samples. Depending on their cultural, morphological and biochemical characterization of bacterial isolate of them were identified as (37.31%) belonged to Streptococcus pyogenes and the diagnosis is confirmed by the use of Remel Rapid STR System, (34.32%) belonged to S.parasanguinis, (11.94%) S.mitis, (11.94%) S.oralis and (4.47%) S.thoraltensis . Results confirmed that cup assay gave highest inhibition zone after 24 hrs compare with well diffusion methods for suspension of L.acidophilus gave highest inhibition zone after 48 hrs for incubation, while ahigh inhibition zone revealed for suspension of L.fermentum after 24 hrs incubation. the study included also the measurement of the inhibition activity for bacteriocins produced by L.acidophilus bacteria against pathogenic bacteria on nutrient agar by well diffusion method in which results revealed stability of the bacteriocins produced towards PH which kept its activity with PH 4-6 for 24 hrs, and the highest stability was with PH 4, however it lost a lot of its activity with acidic PH less than 2 and alkaline PH as 8. The treatment of bacteriocins with salts such as Nacl it revealed little effect in inhibition zone within 1 & 2% concetrations. The salt MgSo4 & Kcl showed reduction in the inhibitory activity in the low concentration, however the higher concentration of salt caused great reduction and 5% concentration led to loss of inhibitory activity for bacteriocins completely.

Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties

1989 ◽  
Vol 93 (1) ◽  
pp. 123-131
Author(s):  
NANCY J. LANE ◽  
STEPHEN M. DILWORTH
Keyword(s):  
Periplaneta Americana ◽  
Biochemical Characterization ◽  
Septate Junction ◽  
Septate Junctions ◽  
Molecular Weights ◽  
Thin Sectioning ◽  
Sds Page ◽  
High Concentrations ◽  
Membrane Components

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.

Characterization of a Bacteriocin Produced by Enterococcus faecalis N1-33 and Its Application as a Food Preservative

2009 ◽  
Vol 72 (3) ◽  
pp. 524-530 ◽  
Author(s):  
TOMOMI HATA ◽  
MELAKU ALEMU ◽  
MIHO KOBAYASHI ◽  
CHISE SUZUKI ◽  
SUNEE NITISINPRASERT ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Marked Reduction ◽  
Pathogenic Bacteria ◽  
Culture Supernatant ◽  
Amino Acid Residues ◽  
Bacteriocin Activity ◽  
Food Preservative ◽  
Fermented Bamboo Shoot ◽  
Potential Use

A bacteriocin-producing strain, N1-33, isolated from fermented bamboo shoot was identified as Enterococcus faecalis. The pH-adjusted culture supernatant of this strain consisted of several peptides with bacteriocin activity, and the supernatant inhibited the growth of pathogenic bacteria such as Listeria monocytogenes. The major peptide with bacteriocin activity was purified, and the first 39 amino acid residues of the bacteriocin were found to be identical to enterocin MR10A produced by E. faecalis MRR10-3. Addition of the pH-adjusted and concentrated culture supernatant of strain N1-33 caused a marked reduction in the growth of Bacillus cereus in custard cream and L. monocytogenes in pickled cucumber. These results suggest the potential use of the bacteriocin produced by strain N1-33 in food biopreservation.

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