scholarly journals Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 565
Author(s):  
Anne Tøndervik ◽  
Olav A. Aarstad ◽  
Randi Aune ◽  
Susan Maleki ◽  
Philip D. Rye ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Brown Algae ◽  
Amperometric Detection ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Proton Nuclear Magnetic Resonance ◽  
Drug Candidate ◽  
Low Molecular Weight ◽  
Laminaria Hyperborea ◽  
Mannuronic Acid

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

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).

Characterisation of Persistent Anti-Xa Activity Following Administration of the Low Molecular Weight Heparin Enoxaparin Sodium (Clexane)

1994 ◽  
Vol 72 (02) ◽  
pp. 275-280 ◽  
Author(s):  
David Brieger ◽  
Joan Dawes
Keyword(s):  
Molecular Weight ◽  
Enoxaparin Sodium ◽  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Active Material ◽  
Biologically Active ◽  
Size Exclusion ◽  
Low Molecular Weight ◽  
Exclusion Chromatography ◽  
Liver And Kidney

SummaryIt is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected 125I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

Comparison of exopolysaccharides from mucoid and nonmucoid strains of Clavibacter michiganensis subspecies sepedonicus

1993 ◽  
Vol 39 (3) ◽  
pp. 291-296 ◽  
Author(s):  
Paul J. Henningson ◽  
Neil C. Gudmestad
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Neutral Sugar ◽  
Clavibacter Michiganensis ◽  
Serological Reaction ◽  
Molecular Weights ◽  
Exclusion Chromatography

The exopolysaccharides produced by six strains of Clavibacter michiganensis ssp. sepedonicus were isolated and purified by liquid chromatography. Neutral sugar composition and molecular weights were determined for each polysaccharide fraction, using gas chromatography and high-performance size-exclusion chromatography. The serological reaction of each fraction was tested using enzyme-linked immunosorbent assay. Exopolysaccharide from nonmucoid strains contained only low molecular weight polysaccharides (1.5 × 103 to 1.1 × 104). Exopolysaccharide from mucoid and intermediate strains could be separated into low (4.0 × 103 to 1.1 × 104) molecular weight and high (5.0 × 105 to 1.6 × 106) molecular weight fractions. High molecular weight polysaccharides were composed almost exclusively of galactose, glucose, and fucose. The ratios of these sugars were highly variable among strains. Low molecular weight polysaccharides were primarily composed of galactose with significant and varying amounts of glucose, rhamnose, mannose, and ribose. All polysaccharide fractions except one, produced by a nonmucoid strain, reacted in the immunoassay test.Key words: exopolysaccharide, polysaccharide, Clavibacter, michiganensis, sepedonicus.

Structural features of low-molecular-weight heparins affecting their affinity to antithrombin

2009 ◽  
Vol 102 (11) ◽  
pp. 865-873 ◽  
Author(s):  
Antonella Bisio ◽  
Davide Vecchietti ◽  
Laura Citterio ◽  
Marco Guerrini ◽  
Rahul Raman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Nmr Spectra ◽  
Biological Activities ◽  
Anticoagulant Activity ◽  
2D Nmr ◽  
Structural Features ◽  
Size Exclusion ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
2D Nmr Spectra

SummaryAs part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin,Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT).The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions,molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*.Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.

scholarly journals HEMICELLULOSE APPARENT MOLECULAR SIZE CHANGES DURING SOFTENING OF PEACH FRUIT

HortScience ◽  
1994 ◽  
Vol 29 (7) ◽  
pp. 737d-737
Author(s):  
Supreetha Hegde ◽  
Niels Maness
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Apparent Molecular Weight ◽  
Weight Fraction ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Neutral Sugar ◽  
Fruit Softening ◽  
Peach Fruit ◽  
Cell Wall Polymers

Peach fruit softening appears to be associated with changes in cell wall polymers, particularly pectins and hemicelluloses. To determine changes of cell wall polymers associated with peach fruit softening, we conducted sequential extractions of pectin and hemicellulose from softening fruit. A more tightly bound hemicellulose fraction contained considerable amounts of pectin associated sugars. This fraction was separated into charged and neutral fractions, using anion exchange chromatography, and then fractionated into two apparent molecular weight classes by size exclusion chromatography. Virtually all of the charged fraction eluted in the higher apparent molecular weight fraction. The neutral sugar fraction segregated into both apparent molecular weight size classes, with a redistribution from the large to the small size class during softening. This redistribution was accompanied by changes in neutral sugar composition. A possible relationship between changes in this fraction and fruit softening will be discussed. Supported by USDA grant 92-34150-7190 and the Oklahoma Agricultural Experiment Station.

Sign in / Sign up

Export Citation Format

Share Document