scholarly journals Isolation Detection and Characterization of Syringolin A produced from the Probiotic Strain Bacillus Cereus Isolated from Donkey Milk

2018 ◽  
Vol 1 (2) ◽  
Author(s):  
Rashmi D ◽  
Sharmila T ◽  
Sushama Patil ◽  
Onkar Apine ◽  
Srinivas Sistla ◽  
...  
Keyword(s):  
Probiotic Strain ◽  
Uv Spectra ◽  
Organic Layer ◽  
Characteristic Peak ◽  
Antitumor Compound ◽  
Solvent Systems ◽  
Culture Supernatants ◽  
Layer Chromatography ◽  
Economical Process

Syringolin A is a non-ribosomal virulence factor secreted by few Pseudomonas strains. Syringolin A is an well known irreversible proteasome inhibitor and antitumor compound. The present study is focused on the extraction of Syringolin A through a non-tedious and economical process. Syringolin A is extracted from culture supernatants by the immiscible organic layer by mixing of dichloromethane or chloroform (trichloromethane). Syringolin A was identified by the characteristic peak at 350 nm by UV spectra. The compound was further characterized by Thin Layer Chromatography (TLC) with the retention value, Rf was found to be in the range of 0.78-0.83 run using a combination of solvent systems water and methanol.  The molecular weight of the compound was found to be 492.2614 g mol-1 identified and analyzed by UHPLC–QTOF-MS analysis. Due to its significant pharmacological importance in proliferative diseases, further studies on production and optimization of these compounds are necessary.   

scholarly journals Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

2020 ◽  
Author(s):  
Pashupati Bhandari ◽  
Jeffrey P. Tingley ◽  
D. Wade Abbott ◽  
Janet E. Hill
Keyword(s):  
Microbial Community ◽  
Phylogenetic Analyses ◽  
Anaerobic Bacteria ◽  
Glycoside Hydrolase Family ◽  
Vaginal Microbiome ◽  
Multistep Process ◽  
Ph Range ◽  
Culture Supernatants ◽  
Layer Chromatography

AbstractGardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up by bacteria requires the combined activities of several enzymes collectively known as “amylases”. In the present study, glycogen degrading abilities of Gardnerella spp. were assessed. We found that Gardnerella spp. isolates and filtered culture supernatants had amylase activity. Phylogenetic analyses predicted conserved Glycoside Hydrolase family 13 (GH13) members among Gardnerella spp. including a putative α-glucosidase. The gene for this enzyme was cloned and expressed, and recombinant protein was purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4 - 8) with an optimum activity at pH 6-7. Glucose was released from maltose, maltotriose and maltopentose, however, no products were detected on thin layer chromatography (TLC) when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce a secreted α-glucosidase enzyme that can contribute to the complex and multistep process of glycogen breakdown by degrading smaller oligosaccharides into glucose, contributing to the pool of nutrients available to the vaginal microbiota.

scholarly journals Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

2005 ◽  
Vol 71 (7) ◽  
pp. 3942-3950 ◽  
Author(s):  
S. Kralj ◽  
E. Stripling ◽  
P. Sanders ◽  
G. H. van Geel-Schutten ◽  
L. Dijkhuizen
Keyword(s):  
Lactobacillus Reuteri ◽  
Activity Ratio ◽  
Probiotic Strain ◽  
Transferase Activity ◽  
Strain Atcc ◽  
Isolation And Characterization ◽  
Branching Points ◽  
Probiotic Lactobacillus ◽  
Culture Supernatants

ABSTRACT Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (∼70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.

OPTIMIZATION OF THIN LAYER CHROMATOGRAPHY METHODS FOR SEPARATION AND IDENTIFICATION OF ANTIDEPRESSANTS IN THEIR MIXTURE

2014 ◽  
Vol 21 (1) ◽  
pp. 11-15
Author(s):  
Daiva Kazlauskienė ◽  
Guoda Kiliuvienė ◽  
Palma Nenortienė ◽  
Giedrė Kasparavičienė ◽  
Ieva Matukaitytė
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
Solvent System ◽  
Ammonia Solution ◽  
Relative Standard ◽  
Rf Values ◽  
Solvent Systems ◽  
Paroxetine Hydrochloride ◽  
Fluvoxamine Maleate ◽  
Layer Chromatography

By conducting the toxicological analysis it is meaningful to determine the analytical system that could identify simultaneously several medicinal preparations quickly and precisely. The purpose of this work was to create and validate the method of thin-layer chromatography that would be suitable to separate the components of antidepressant mixture (amitriptyline hydrochloride, paroxetine hydrochloride, sertraline hydrochloride, fluvoxamine maleate and buspirone hydrochloride) and to identify them. The system was validated with regard to the sensitivity, repetition of data, resistance and particularity. The solvent systems with potential of high separation of components in their mixture were created: acetonitrile, methanol, ammonia solution 25 percent (85:10:5); acetonitrile, methanol, ammonia solution 25 percent (75:20:5); dichlormethane, 1,4-dioxane, ammonia solution 25 percent (50:45:5); dichlormethane, 1,4-dioxane, ammonia solution 25 percent (42:55:3); trichlormethane, 1,4-dioxane, ammonia solution 25 percent (25:70:5); trichlormethane, 1,4-dioxane, ammonia solution 25 percent (60:36:4). One of the most suitable solvent systems for separation of the analyzed mixture (sertraline, amitriptyline, paroxetine, buspirone, fluvoxamine) was determined – acetonitrile, methanol, ammonia solution 25 percent (85:10:5). When this solvent system was used, the average Rf values of the analyzed compounds differed the most. Validation was conducted – the relative standard deviation (RSD, percent) of the average Rf value of the analyzed compounds varied from 0,6 to 1,8 percent and did not exceed the permissible error of 5 percent. The sensitivity of methodology was determined by assessing the intensity of the mixture’s spots on the chromatographic plate. The detection limit of buspirone was 0,0012 µg; sertraline – 0,0008 µg; amitriptyline – 0,0004 µg; fluvoxamine – 0,0004 µg; paroxetine – 0,0008 µg. The resistance of results to the changed conditions – it was determined that when the amounts of the solvents acetonitrile and methanol were increased or decreased to two milliliters, the average Rf values of the analyzed compounds did not change statistically significantly

Comparison of UV- and Derivative-Spectrophotometric and HPTLC UVDensitometric Methods for the Determination of Amrinone and Milrinone in Bulk Drugs

2020 ◽  
Vol 16 (3) ◽  
pp. 246-253
Author(s):  
Marcin Gackowski ◽  
Marcin Koba ◽  
Stefan Kruszewski
Keyword(s):  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Low Cost ◽  
Uv Spectra ◽  
Derivative Spectrophotometry ◽  
Therapeutic Monitoring ◽  
Bulk Drug ◽  
Layer Chromatography ◽  
Standard Solutions

Background: Spectrophotometry and thin layer chromatography have been commonly applied in pharmaceutical analysis for many years due to low cost, simplicity and short time of execution. Moreover, the latest modifications including automation of those methods have made them very effective and easy to perform, therefore, the new UV- and derivative spectrophotometry as well as high performance thin layer chromatography UV-densitometric (HPTLC) methods for the routine estimation of amrinone and milrinone in pharmaceutical formulation have been developed and compared in this work since European Pharmacopoeia 9.0 has yet incorporated in an analytical monograph a method for quantification of those compounds. Methods: For the first method the best conditions for quantification were achieved by measuring the lengths between two extrema (peak-to-peak amplitudes) 252 and 277 nm in UV spectra of standard solutions of amrinone and a signal at 288 nm of the first derivative spectra of standard solutions of milrinone. The linearity between D252-277 signal and concentration of amironone and 1D288 signal of milrinone in the same range of 5.0-25.0 μg ml/ml in DMSO:methanol (1:3 v/v) solutions presents the square correlation coefficient (r2) of 0,9997 and 0.9991, respectively. The second method was founded on HPTLC on silica plates, 1,4-dioxane:hexane (100:1.5) as a mobile phase and densitometric scanning at 252 nm for amrinone and at 271 nm for milrinone. Results: The assays were linear over the concentration range of 0,25-5.0 μg per spot (r2=0,9959) and 0,25-10.0 μg per spot (r2=0,9970) for amrinone and milrinone, respectively. The mean recoveries percentage were 99.81 and 100,34 for amrinone as well as 99,58 and 99.46 for milrinone, obtained with spectrophotometry and HPTLC, respectively. Conclusion: The comparison between two elaborated methods leads to the conclusion that UV and derivative spectrophotometry is more precise and gives better recovery, and that is why it should be applied for routine estimation of amrinone and milrinone in bulk drug, pharmaceutical forms and for therapeutic monitoring of the drug.

scholarly journals Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
Author(s):  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Shuttle Vector ◽  
Open Reading Frame ◽  
Cosmid Library ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Degradation Activity ◽  
Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

Synthesis and chromatographic characterization of [Tc-99m]technetium-humic acid species

2000 ◽  
Vol 88 (2) ◽  
Author(s):  
D. Rößler ◽  
K. Franke ◽  
R. Süß ◽  
E. Becker ◽  
H. Kupsch
Keyword(s):  
Humic Acid ◽  
Stannous Chloride ◽  
Gel Permeation Chromatography ◽  
Paper Electrophoresis ◽  
Gel Permeation ◽  
Soil Humic Acid ◽  
Chromatographic Characterization ◽  
Ph Dependent ◽  
Layer Chromatography

A natural moor soil humic acid (HA) was labeled with Tc-99m via reduction of pertechnetate with stannous chloride. The humic acid species obtained were characterized by thin layer chromatography (TLC), gel permeation chromatography (GPC), sequential chromatographic analysis (SCA), paper electrophoresis and micropore filtration. Labeling was found to take place in all ranges of molecular weight. Due to the complex humic acid composition and the formation of hydroxo species the labeling yields strongly depend on the separation conditions, ranging from 42% to 80%. The pH-dependent distribution of mobile and immobile species was determined by SCA for HTcO

Characterization of Sorption of Endosulfan Isomers and Chlorpyrifos on Container Walls Using Mixed Solvent Systems

2010 ◽  
Vol 58 (13) ◽  
pp. 7902-7907 ◽  
Author(s):  
J. Wasswa ◽  
P. Nkedi-Kizza ◽  
B. T. Kiremire
Keyword(s):  
Mixed Solvent ◽  
Solvent Systems
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