Characterization of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

2020 ◽  
Author(s):  
S Reid ◽  
Ian Sims ◽  
LD Melton ◽  
AM Gane
Keyword(s):  
Monosaccharide Composition ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Neutral Fraction ◽  
Extracellular Polysaccharides ◽  
Neutral Sugar ◽  
Diffusion Test ◽  
Selective Precipitation ◽  
Acidic Fraction

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG;>90 mol%) which was purified by selective precipitation with Fehling's solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.

Characterization of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

2020 ◽  
Author(s):  
S Reid ◽  
Ian Sims ◽  
LD Melton ◽  
AM Gane
Keyword(s):  
Monosaccharide Composition ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Neutral Fraction ◽  
Extracellular Polysaccharides ◽  
Neutral Sugar ◽  
Diffusion Test ◽  
Selective Precipitation ◽  
Acidic Fraction

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG;>90 mol%) which was purified by selective precipitation with Fehling's solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

scholarly journals Enzymatic Synthesis and Characterization of Different Families of Chitooligosaccharides and Their Bioactive Properties

2021 ◽  
Vol 11 (7) ◽  
pp. 3212
Author(s):  
Noa Miguez ◽  
Peter Kidibule ◽  
Paloma Santos-Moriano ◽  
Antonio O. Ballesteros ◽  
Maria Fernandez-Lobato ◽  
...  
Keyword(s):  
High Performance ◽  
Chemical Characterization ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enzymatic Production ◽  
Bioactive Properties ◽  
Substrate Properties ◽  
Chitin Hydrolysis

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry, is critical for structure–function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharides.

Aniline hydroxylation and 7-ethoxycoumarin O-deethylation activities in solubilized and partially resolved liver cytochromes P-450 from ethanol-fed rats

1984 ◽  
Vol 62 (3) ◽  
pp. 266-271 ◽  
Author(s):  
V. Plourde ◽  
C. Hétu ◽  
J.-G. Joly
Keyword(s):  
Liver Microsomes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Female Rats ◽  
Ethanol Administration ◽  
Molar Activity ◽  
Acidic Fraction ◽  
Chronic Ethanol Administration ◽  
And Control ◽  
Cytochrome P 450

Chronic ethanol administration in female rats enhances the apparent molar activity of liver microsomes for aniline hydroxylation and 7-ethoxycoumarin O-deethylation. Microsomal cytochromes P-450 from ethanol-fed and control rats have been solubilized and partially resolved in six fractions by anion-exchange chromatography. Induction of aniline hydroxylase activity by ethanol was associated with marked increases in the turnover numbers of the more basic cytochrome P-450 containing fractions in a reconstituted aniline hydroxylation system. Cytochrome P-450, exhibiting by far the highest 7-ethoxycoumarin O-deethylase activity, was eluted in a relatively acidic fraction; its turnover number with 7-ethoxycoumarin after ethanol consumption, however, did not differ significantly from that of the corresponding fraction from control microsomes. These observations suggest that induction of liver microsomal mixed function oxidases by ethanol may reflect the contribution of more than one cytochrome P-450 isozyme.

Purification and characterization of a Ca2+-dependent novel lectin from Nymphaea nouchali tuber with antiproliferative activities

2011 ◽  
Vol 31 (6) ◽  
pp. 465-475 ◽  
Author(s):  
Syed Rashel Kabir ◽  
Md. Abu Zubair ◽  
Md. Nurujjaman ◽  
Md. Azizul Haque ◽  
Imtiaj Hasan ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Deae Cellulose ◽  
Ehrlich Ascites Carcinoma ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Neutral Sugar ◽  
The Stability

A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5–9 and temperatures of 30–60°C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC50 value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl2 indicated the requirement of Ca2+ for the stability of NNTL.

scholarly journals Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301
Author(s):  
Huiqin Wang ◽  
Guanzhen Gao ◽  
Lijing Ke ◽  
Jianwu Zhou ◽  
Pingfan Rao
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scutellaria Baicalensis ◽  
Dependent Manner ◽  
Scutellaria Baicalensis Georgi ◽  
Isolation And Characterization ◽  
Pas Staining ◽  
Ph Range ◽  
Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

scholarly journals Methodology for quantitative determination of the carbohydrate composition of brown seaweeds (Laminariaceae)

RSC Advances ◽  
2014 ◽  
Vol 4 (49) ◽  
pp. 25736-25746 ◽  
Author(s):  
D. Manns ◽  
A. L. Deutschle ◽  
B. Saake ◽  
A. S. Meyer
Keyword(s):  
High Performance ◽  
Monosaccharide Composition ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Saccharina Latissima ◽  
Carbohydrate Composition ◽  
Laminaria Digitata ◽  
Brown Seaweeds ◽  
Cellulase Treatment

The monosaccharide composition of four different samples of brown seaweeds Laminaria digitata and Saccharina latissima were compared by different high performance anion exchange chromatography (HPAEC) methods after different acid hydrolysis treatments or a cellulase treatment.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

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