scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis ofN-acetylmannosamine kinase from methicillin-resistantStaphylococcus aureus

2014 ◽  
Vol 70 (5) ◽  
pp. 643-649 ◽  
Author(s):  
Rachel A. North ◽  
Simona Seizova ◽  
Anja Stampfli ◽  
Sarah A. Kessans ◽  
Hironori Suzuki ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Bacterial Pathogens ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Sialic Acids ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
E Coli ◽  
Exclusion Chromatography

N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such asEscherichia coli,Staphylococcus aureus,Vibrio choleraeandV. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the `Nan-Nag cluster', which have proven to be essential forS. aureusgrowth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzymeN-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5′-triphosphate to the C6 position ofN-acetylmannosamine to generateN-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed inE. coliBL21 (DE3) cells and purifiedviaanion-exchange chromatography, hydrophobic interaction chromatography and size-exclusion chromatography. PurifiedN-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space groupP2. Understanding the structural nature of this enzyme from methicillin-resistantS. aureuswill provide insights necessary for the development of future antimicrobials.

scholarly journals Chromatographic resolution of chicken phosvitin. Multiple macromolecular species in a classic vitellogenin-derived phosphoprotein

1986 ◽  
Vol 240 (3) ◽  
pp. 871-878 ◽  
Author(s):  
R A Wallace ◽  
J P Morgan
Keyword(s):  
Hydrophobic Interaction Chromatography ◽  
Egg Yolk ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Yolk Granules ◽  
Minor Satellite ◽  
Exclusion Chromatography ◽  
Original Procedure ◽  
Chromatographic Resolution

Chicken phosvitin was prepared from egg yolk by a variety of published methods, including a modification of our own original procedure. Yolk granules and all phosvitin preparations have been previously found to contain major phosphoproteins at Mr 40,000 and 33,000 and minor satellite components when electrophoresed on polyacrylamide gradient gels and stained with Stains-all. However, only our current preparation contained three additional phosphoproteins (Mr 18,000, 15,000 and 13,000) that are also present in yolk granules. Our current phosvitin preparation also appeared to have additional components when compared with other preparations by size-exclusion and anion-exchange chromatography. Particularly complex but entirely reproducible patterns were obtained by hydrophobic-interaction chromatography. However, a cross-referencing of fractions eluted by size-exclusion chromatography to the other procedures employed, including gel electrophoresis, reinforced the notion that unfractionated chicken phosvitin contains at least five major components, designated B, C, E1, E2 and F for the Mr 40,000, 33,000, 15,000, 18,000, and 13,000 phosphoproteins, respectively. Stoichiometric considerations lead us to suggest that vitellogenin I gives rise to phosvitins C and F, vitellogenin II gives rise to phosvitin B, and vitellogenin III gives rise to either phosvitin E1 or E2, but not both. Thus, a fourth, as yet undetected, vitellogenin may exist for the chicken.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11 691

2021 ◽  
Author(s):  
Ken-ji Yokoi ◽  
Sosyu Tsutsui ◽  
Gen-ya Arakawa ◽  
Masakazu Takaba ◽  
Koichi Fujii ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Medium ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Reading Frame ◽  
Exclusion Chromatography ◽  
Chain Lengths

Abstract Information about the inulosucrase of non-lactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0–9.0 and 50 °C–55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multi-angle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3,806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3–27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

scholarly journals Prebiotic Activity of Poly- and Oligosaccharides Obtained from Plantago major L. Leaves

2020 ◽  
Vol 10 (8) ◽  
pp. 2648 ◽  
Author(s):  
Paolina Lukova ◽  
Mariana Nikolova ◽  
Emmanuel Petit ◽  
Redouan Elboutachfaiti ◽  
Tonka Vasileva ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Galacturonic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Plantago Major ◽  
Prebiotic Activity ◽  
Molecular Fraction ◽  
Exclusion Chromatography

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO− group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

Analysis of the substituent distribution along the chain of water-soluble methyl cellulose by combination of enzymatic and chemical methods

Holzforschung ◽  
2004 ◽  
Vol 58 (1) ◽  
pp. 97-104 ◽  
Author(s):  
B. Saake ◽  
S. Lebioda ◽  
J. Puls
Keyword(s):  
Anion Exchange ◽  
Size Exclusion Chromatography ◽  
Methyl Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Degree Of Substitution ◽  
Water Soluble ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
C Nmr

Abstract Four methyl cellulose samples in the degree of substitution range from 0.5 to 2.0 were characterised by combination of different analytical methods. Samples were analysed regarding their partial degree of substitution by hydrolysis and anion exchange chromatography with pulsed amperometric detection. For calibration of the chromatographic system, standard substances were isolated by preparative HPLC and their structure was confirmed by 13C-NMR spectroscopy. For two methyl cellulose samples per-acetylation and 13C-NMR with inverse gated decoupling was carried out for comparison with the chromatographic analysis. Endoglucanase fragmentation of methyl celluloses was performed and water-soluble and insoluble fractions were analysed separately. A preparative size exclusion chromatography system for enzymatic-degraded water-soluble methyl cellulose was developed and the molar masses of the individual fractions were examined by analytical size exclusion chromatography. By combination of endoglucanase fragmentation, preparative chromatography, hydrolysis and anion exchange chromatography an approach for the analysis of the substitutent distribution along the polymeric chain of water-soluble methyl cellulose could be established.

scholarly journals Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

2019 ◽  
Author(s):  
MacLean G. Kohlmeier ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Ivan J. Oresnik
Keyword(s):  
Sinorhizobium Meliloti ◽  
Model Organism ◽  
Time Frame ◽  
Industrial Applications ◽  
Size Exclusion ◽  
Ligand Docking ◽  
X Ray Diffraction ◽  
E Coli ◽  
Exclusion Chromatography

AbstractSinorhizobium meliloti1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression inE. coliBL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 Å in the absence of substrate and 2.0 Å in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organismS. meliloti.

scholarly journals Isolation a Homogalacturonan from the Outer Seed Coat of Shaddock (Citrus grandis Osbeck)

2018 ◽  
Vol 13 (6) ◽  
pp. 1934578X1801300
Author(s):  
Heng Long Wang ◽  
Chin Wei Tu ◽  
Wei Zhi Wu ◽  
Chen Yi Lin ◽  
Su Yu Chen ◽  
...  
Keyword(s):  
Seed Coat ◽  
Room Temperature ◽  
Spectroscopic Analysis ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Infrared Spectroscopic Analysis ◽  
Low Degree ◽  
Exclusion Chromatography

An acidic mucilage was isolated from the outer-layer seed coat of shaddock (Citrus grandis Osbeck) by water extraction at room temperature, and purified by DE-52 anion-exchange chromatography using 0.26 – 0.37 M NaCl. This purified mucilage was almost entirely composed of galacturonic acid residues. Glycosyl-linkage analysis showed that the backbone was 1→4 linked. Fourier Transform Infrared Spectroscopic analysis and determination of the degree of methylation further revealed that the mucilage was a low-degree (11.94%) esterified homogalacturonan. Size exclusion chromatography showed that the major molecular weight distribution of 610.9 kDa.

Partial Purification and Some Properties of Brassica napus Lipase

1994 ◽  
Vol 49 (5-6) ◽  
pp. 293-301 ◽  
Author(s):  
Cornelia Fuchs ◽  
Gerd Hansen
Keyword(s):  
Brassica Napus ◽  
Size Exclusion Chromatography ◽  
Lipase Activity ◽  
Sodium Deoxycholate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Partial Purification ◽  
Exclusion Chromatography ◽  
Molecular Masses

Abstract Lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from rape (Brassica napus cv. Ceres) was isolated from cotyledons of dark-grown seedlings. The enzyme was partially purified by poly­ ethylene glycol precipitation. Delipidation of the lipase with n-hexane was required prior to further purification by anion exchange chromatography and size exclusion chromatography. A purification factor of 337 was ultimately achieved and the purification process was moni­tored by SDS-PAGE. Here, at least two protein bands with molecular masses of 62 and 64 kD a respectively were found in the active fraction obtained by size exclusion chromatography. Sodium deoxycholate was found to stimulate the lipase activity, but appeared to cause aggregation of the enzyme. It was not possible to estimate the isoelectric point of the dialyzed rape lipase due to the high molecular mass of the aggregates. Two simple methods to detect lipase activity directly on polyacrylamide gel were applied. No esterase activity was found by using p-nitrophenyl acetate as substrate.

scholarly journals Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6

2006 ◽  
Vol 395 (2) ◽  
pp. 319-329 ◽  
Author(s):  
Synnøve Holtan ◽  
Per Bruheim ◽  
Gudmund Skjåk-Bræk
Keyword(s):  
High Performance ◽  
Azotobacter Vinelandii ◽  
Block Size ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Mannuronic Acid ◽  
Enzyme Degradation ◽  
Exclusion Chromatography

AlgE1, AlgE5 and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by the bacterium Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyse the post-polymerization conversion of β-D-mannuronic acid (M) residues into α-L-guluronic acid residues (G). All enzymes show preference for introducing G-residues neighbouring a pre-existing G. They also have the capacity to convert single M residues flanked by G, thus ‘condensing’ G-blocks to form almost homopolymeric guluronan. Analysis of the length and distribution of G-blocks based on specific enzyme degradation combined with size-exclusion chromatography, electrospray ionization MS, HPAEC–PAD (high-performance anion-exchange chromatography and pulsed amperometric detection), MALDI (matrix-assisted laser-desorption ionization)-MS and NMR revealed large differences in block length and distribution generated by AlgE1 and AlgE6, probably reflecting their different degree of processivity. When acting on polyMG as substrates, AlgE1 initially forms only long homopolymeric G-blocks >50, while AlgE6 gives shorter blocks with a broader block size distribution. Analyses of the AlgE1 and AlgE6 subsite specificities by the same methodology showed that a mannuronan octamer and heptamer respectively were the minimum substrate chain lengths needed to accommodate enzyme activities. The fourth M residue from the non-reducing end is epimerized first by both enzymes. When acting on MG-oligomers, AlgE1 needed a decamer while AlgE6 an octamer to accommodate activity. By performing FIA (flow injection analysis)-MS on the lyase digests of epimerized and standard MG-oligomers, the M residue in position 5 from the non-reducing end was preferentially attacked by both enzymes, creating an MGMGGG-sequence (underlined and boldface indicate the epimerized residue).

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