Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

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The N-terminal domain of rhamnosyltransferase EpsF influences exopolysaccharide chain length determination in Streptococcus thermophilus 05-34

PeerJ ◽

10.7717/peerj.8524 ◽

2020 ◽

Vol 8 ◽

pp. e8524

Author(s):

Guohong Wang ◽

Jiaxi Li ◽

Shuxin Xie ◽

Zhengyuan Zhai ◽

Yanling Hao

Keyword(s):

Molecular Mass ◽

Chain Length ◽

Streptococcus Thermophilus ◽

Monosaccharide Composition ◽

Truncated Form ◽

Terminal Domain ◽

Homologous Overexpression ◽

Expression Of Genes ◽

Length Determination

Glycosyltransferases are key enzymes involved in the assembly of repeating units of exopolysaccharides (EPS). A glycosyltransferase generally consists of the N-terminal and the C-terminal domain, however, the functional role of these domains in EPS biosynthesis remains largely unknown. In this study, homologous overexpression was employed to investigate the effects of EpsFN, a truncated form of rhamnosyltransferase EpsF with only the N-terminal domain, on EPS biosynthesis in Streptococcus thermophilus 05-34. Reverse transcription qPCR and Western blotting analysis confirmed the successful expression of epsFN in 05-34 at the transcription and translation level, respectively. Further analysis showed that the monosaccharide composition and yield of EPS were not affected by the overexpression of epsFN, whereas the molecular mass decreased by 5-fold. Accordingly, the transcription levels of genes involved in EPS biosynthesis, including chain-length determination gene epsC, were down-regulated by 5- to 6-fold. These results indicated that the N-terminal domain of EpsF alone could influence the molecular mass of EPS, probably via lowering the concentration of sugar precursors, which may lead to decreased expression of genes responsible for chain-length determination.

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Purification and Structure Study on Exopolysaccharides Produced by Lactobacillus paracasei KL1-Liu from Tibetan Kefir

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.781-784.1513 ◽

2013 ◽

Vol 781-784 ◽

pp. 1513-1518 ◽

Cited By ~ 2

Author(s):

Hui Liu ◽

Yuan Hong Xie ◽

Tao Han ◽

Hong Xing Zhang

Keyword(s):

Molecular Weight ◽

High Performance ◽

Monosaccharide Composition ◽

Gradient Elution ◽

Structure Study ◽

Lactobacillus Paracasei ◽

Molar Ratio ◽

Test Results ◽

Sample Concentration ◽

Multi Level

To investigate the pure conditions of exopolysaccharides produced by Lactobacillus paracasei KL1-Liu from Tibetan Kefir, and to analyse the structure, we used Multi-level single-factor test to purify EPS by Sepharose CL-6B. And the purity of EPS was detected by UV scan and high performance liquid chromatogram (HPLC). EPS molecular weight and monosaccharide composition were determined by HPLC. Results: Adoption phosphate buffer gradient elution 0.02-0.10 mol/L, the velocity 0.25 mL/min, the sample concentration 1.0 mg/mL, the sample capacity 1.0 mL. Under this purification conditions, components EPSa and EPSb were obtained. The purities of EPSa and EPSb were 82.82% and 91.74% respectively, which were 1.4 and 1.5 times of the pre-purification. Purity Test results showed that EPSa and EPSb polysaccharide were single components, basically no nucleic acid and protein in them. Structural analysis revealed that the molecular weight of EPSa and EPSb were 4.60×104 Da and 2.12×104 Da detected by HPLC. EPSa monosaccharide components were glucose and rhamnose, and the molar ratio was 1:0.68. EPSb were composed of glucose, xylose and rhamnose, and the molar ratio was 1:0.77:0.69.

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Studies on the Preliminary Characterization of a Novel Exopolysaccharide Produced by Streptococcus thermophilus Strain from Tibetan Kefir Grain

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.690-693.1374 ◽

2013 ◽

Vol 690-693 ◽

pp. 1374-1377 ◽

Cited By ~ 3

Author(s):

Shu Juan Jiang ◽

Fang Qian ◽

Xiao Hui Ren ◽

Guang Qing Mu

Keyword(s):

High Performance ◽

Deae Cellulose ◽

Gel Permeation Chromatography ◽

Streptococcus Thermophilus ◽

Structural Features ◽

Molar Ratio ◽

Partial Hydrolysis ◽

The Novel ◽

Average Molecular Mass ◽

Kefir Grain

The novel exopolysaccharide (EPS) was produced by a streptococcus thermophilus strain isolated from Tibetan kefir grain, and it was purified using DEAE cellulose 52 and DEAE Sepharose CL-6B column chromatography. Then it was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular mass was estimated to be 30,092Da. Infrared spectrum analysis indicated it had characteristic glycosidic structures. Gas chromatography showed that the EPS is composed of mannose and glucose in a molar ratio of 2.6:1 with trace galactose. The structural features of EPS were investigated by partial hydrolysis with acid, methylation analysis and NMR spectroscopy analysis. The results revealed that the main backbone chain of EPS was (23)-β-D-Manp, (34)-β-D-Manp, (23,6)-α-D-Glcp and trace amounts of galactose.

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Alkaline Extraction, Structural Characterization, and Bioactivities of (1→6)-β-d-Glucan from Lentinus edodes

Molecules ◽

10.3390/molecules24081610 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1610 ◽

Cited By ~ 3

Author(s):

Jia Li ◽

Chao Cai ◽

Mengmeng Zheng ◽

Jiejie Hao ◽

Ya Wang ◽

...

Keyword(s):

High Performance ◽

Antioxidant Activities ◽

Gel Permeation Chromatography ◽

Monosaccharide Composition ◽

Biological Properties ◽

Orthogonal Experimental Design ◽

Lentinus Edodes ◽

Anion Exchange Column ◽

Strong Anion Exchange ◽

Fast Flow

The purpose of this study is to develop a robust approach to obtain β glucans from Lentinus edodes and to characterize their structural and biological properties for sustainable utilization. The alkali extraction was optimized with an orthogonal experimental design, and a concise process for obtaining specific targeting polysaccharides from Lentinus edodes was developed in this study. After purification with a Q-Sepharose Fast Flow strong anion-exchange column, the monosaccharide composition, a methylation analysis, and NMR spectroscopy were employed for their structural characterizations. LeP-N2 was found to be composed of (1→6)-β-d-glucans with minor β-(1→3) glucosidic side chains. Atomic force microscopy (AFM) and high-performance gel permeation chromatography–refractive index–multi-angle laser light scattering (HPGPC-RI-MALLS) also revealed LeP-N2 exhibiting a compact unit in aqueous solution. This (1→6)-β-d-glucan was tested for antioxidant activities with IC50 at 157 μg/mL. Moreover, RAW 264.7 macrophage activation indicated that the release of nitric oxide (NO) and reactive oxygen species (ROS) was markedly increased with no cytotoxicity at a dose of 100 μg/mL. These findings suggest that the (1→6)-β-d-glucans obtained from Lentinus edodes could serve as potential agents in the fields of functional foods or medicine.

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A COMPARISON OF THE BINDING OF ANTITHROMBIN III AND HEPARIN COFACTOR II TO HEPARINS, NATURALLY OCCURRING GLYCOSAMINOGLYCANS AND OTHER SULPHATED POLYMERS

10.1055/s-0038-1642827 ◽

1987 ◽

Author(s):

J Dawes ◽

D S Pepper

Keyword(s):

Molecular Weight ◽

Chain Length ◽

Antithrombin Iii ◽

Molar Ratio ◽

Heparin Binding ◽

Dextran Sulphate ◽

Heparin Cofactor Ii ◽

Wide Range ◽

Pentosan Polysulphate ◽

Binding Sequence

Antithrombin III (ATIII) and heparin cofactor II (HCII) are currently thought to be the most important protein mediators of the anticoagulant and antithrombotic activities of glycosamino-glycans. A simple, quantitative method for assessing the affinity of a protein for a sulphated polymer in the liquid phase, based on competition with immobilised heparin, has been developed. Using this technique, the binding of ATIII and HCII to a wide range of glycosaminoglycans and other sulphated polymers have been compared, and the contributions to binding of size, degree of sulphation and backbone structure of the polymers analysed.In the presence of the high protein concentrations found in plasma, unfractionated heparin inhibited the binding of ATIII to immobilised heparin with a Ki of 1 x 10-6. Binding was destroyed by N-desulphation. 1 Results with a range of low molecular weight (LMW) heparins and heparan sulphates are consistent with the view that they all contain the ATIII-binding sequence, but at a lower molar ratio than heparin. Highly sulphated synthetic polymers such as dextran sulphate bound ATIII by a different mechanism, which was molecular weight-dependent.The affinity of HCII for heparins increased markedly with heparin chain length. Binding was largely, but not entirely, mediated by sulphate residues. HCII bound to heparan and dermatan sulphates with lower affinities than to heparin, and to synthetic sulphated polymers with similar or higher affinities. Pentosan polysulphate (SP54) bound HCII as effectively as did heparin. Binding of HCII to dextran sulphate was highly dependent on molecular weight. The affinity of HCII for a sulphated polymer appears to depend both on its chain length and density of sulphation.Thus the profiles of binding of ATIII and HCII to glycosaminoglycans and other sulphated polymers are quite different. This technique is useful both for investigating the interactions of existing therapeutic anticoagulants and assessing new products.

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Physicochemical Characterization and Immunomodulatory Activity of a Novel Acid Polysaccharide from Solanum muricatum

Polymers ◽

10.3390/polym11121972 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1972 ◽

Cited By ~ 1

Author(s):

Heng Yue ◽

Qianqian Xu ◽

Xianheng Li ◽

Jeevithan Elango ◽

Wenhui Wu ◽

...

Keyword(s):

High Performance ◽

Average Molecular Weight ◽

Thermal Studies ◽

Physicochemical Characterization ◽

Monosaccharide Composition ◽

Molar Ratio ◽

Immunomodulatory Activity ◽

Composition Analysis ◽

Solanum Muricatum ◽

Acid Polysaccharide

To investigate the structure and immunomodulatory activity of polysaccharide from Solanum muricatum, a novel acid polysaccharide named SMP-3a was purified from Solanum muricatum pulp through DEAE-52 cellulose column and Sephadex G-200 chromatography. Monosaccharide composition analysis showed that SMP-3a was mainly composed of rhamnose, arabinose, galactose, and galacturonic acid with the molar ratio of 1.09:2.64:1.54:1. The average molecular weight was found to be 227 kDa by high performance gel permeation chromatography (HPGPC). Thermal studies revealed the SMP-3a was a thermally stable polymer. Based on the results of methylation and NMR analysis, the backbone chain of SMP-3a was composed of →2)-α-l-Rhap-(1→, →4)-α-d-GalpA-(1→ and →4)-α-d-Galp-(1→. The side chain was consisted of α-l-Araf-(1→ and →5)-α-l-Araf-(1→. Immunomodulatory assay indicated that SMP-3a could significantly promote the proliferation of macrophages and stimulate the secretion of cytokines, including TNF-α, IL-1β, and IL-6. Our results suggested that SMP-3a could be used as a novel potential immunomodulatory agent in functional food.

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Purification and Characterization of a New Lectin from Loach Skin Mucus

Journal of Chemistry ◽

10.1155/2019/3853646 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11

Author(s):

Peng-peng Sun ◽

Yuan-yuan Ren ◽

Jie Zheng ◽

Ai-jun Hu

Keyword(s):

Molecular Weight ◽

Carbohydrate Chain ◽

Acid Resistance ◽

Monosaccharide Composition ◽

Molar Ratio ◽

Pathogen Defense ◽

Hemagglutination Activity ◽

Purification And Characterization ◽

Skin Mucus

Lectin from loach skin mucus plays an important role in pathogen defense. However, hardly can any paper relevant to the character of lectin from loach skin mucus be found in recent years. In this study, a kind of new lectin (LML), with a high hemagglutination activity of 166.23 × 103 HU/mg, was successfully isolated and purified from loach skin mucus. LML was a kind of glycoprotein with a molecular weight of 245 kDa. Also, the monosaccharide composition suggested that its carbohydrate chain was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose with a molar ratio of 2.02 : 11.66 : 2.06 : 1.00 : 14.09 : 6.00. Besides, LML depended on Ca2+ to induce hemagglutination and was strongly inhibited by D-lactose. The lectin exhibited powerful resistance to alkali and kept about 30% hemagglutination activity at pH 14.0, whereas its capacity of acid resistance was weak. The maximum hemagglutination activity of LML maintained at a temperature range from 20°C to 50°C. Moreover, the structure of LML was preliminarily studied, indicating it contained abundant glutamic acid, histidine, and serine, and its secondary structure contained α-helix (4.97%), β-sheet (27.55%), turns structure (49.78%), and unordered structure (17.70%).

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Methylene-Bridged Tridentate Salicylaldiminato Binuclear Titanium Complexes as Copolymerization Catalysts for the Preparation of LLDPE through [Fe]/[Ti] Tandem Catalysis

Polymers ◽

10.3390/polym11071114 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1114 ◽

Cited By ~ 2

Author(s):

Yani Luo ◽

Jian Li ◽

Derong Luo ◽

Qingliang You ◽

Zifeng Yang ◽

...

Keyword(s):

Molecular Weight ◽

High Temperature ◽

Gel Permeation Chromatography ◽

13C Nmr Spectroscopy ◽

Molar Ratio ◽

Tandem Catalysis ◽

Titanium Complexes ◽

Scanning Calorimetry ◽

Molecular Weights ◽

Branching Degree

A novel tandem catalysis system consisted of salicylaldiminato binuclear/mononuclear titanium and 2,6-bis(imino)pyridyl iron complexes was developed to catalyze ethylene in-situ copolymerization. Linear low-density polyethylene (LLDPE) with varying molecular weight and branching degree was successfully prepared with ethylene as the sole monomer feed. The polymerization conditions, including the reaction temperature, the Fi/Ti molar ratio, and the structures of bi- or mononuclear Ti complexes were found to greatly influence the catalytic performances and the properties of obtained polymers. The polymers were characterized by differential scanning calorimetry (DSC), high temperature gel permeation chromatography (GPC) and high temperature 13C NMR spectroscopy, and found to contain ethyl, butyl, as well as some longer branches. The binuclear titanium complexes demonstrated excellent catalytic activity (up to 8.95 × 106 g/molTi·h·atm) and showed a strong positive comonomer effect when combined with the bisiminopyridyl Fe complex. The branching degree can be tuned from 2.53 to 22.89/1000C by changing the reaction conditions or using different copolymerization pre-catalysts. The melting points, crystallinity and molecular weights of the products can also be modified accordingly. The binuclear complex Ti2L1 with methylthio sidearm showed higher capability for comonomer incorporation and produced polymers with higher branching degree and much higher molecular weight compared with the mononuclear analogue.

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Biosynthesis of glycoproteins by membranes of Acer pseudoplatanus. Incorporation of mannose and N-acetylglucosamine

Biochemical Journal ◽

10.1042/bj1920569 ◽

1980 ◽

Vol 192 (2) ◽

pp. 569-577 ◽

Cited By ~ 7

Author(s):

J Barr ◽

P Nordin

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

High Molecular Weight ◽

Gel Permeation Chromatography ◽

Monosaccharide Composition ◽

Paper Electrophoresis ◽

Acer Pseudoplatanus ◽

Gel Permeation ◽

Chloroform Methanol ◽

Insoluble Pellet

Membrane preparations from Acer pseudoplatanus suspension cultures were demonstrated to incorporate radioactivity from GDP-[U-14C]mannose and UDP-N-acetyl-[6-(3)H]glucosamine into high-molecular-weight polymers characterized as glycoprotein. From 20 to 25% of the 14C was incorporated as fucose with the remainder as mannose, whereas 90% of the 3H was incorporated as N-acetylglucosamine with the remainder as N-acetylgalactosamine. Pronase digestion yielded radioactive glycopeptides that were separated into four fractions by gel-permeation chromatography and paper electrophoresis. The isolated glycopeptides differed in molecular weight and isotopes incorporated, as well as in amino-acid and monosaccharide composition. The membrane preparation also incorporated radioactivity from the added nucleotides into chloroform/methanol (2:1, v/v)- and chloroform/methanol/water (10:10:3, by vol.)-soluble lipids, and into an insoluble pellet.

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Fractionation of lignosulphonates released during the early stages of delignification

Canadian Journal of Chemistry ◽

10.1139/v83-074 ◽

1983 ◽

Vol 61 (2) ◽

pp. 416-420 ◽

Cited By ~ 8

Author(s):

Norman G. Lewis ◽

David A. I. Goring ◽

Alfred Wong

Keyword(s):

Molecular Weight ◽

High Performance ◽

Black Spruce ◽

Gel Permeation Chromatography ◽

Bimodal Distribution ◽

High Yield ◽

Low Molecular Weight ◽

Lower Yield ◽

Spruce Wood ◽

Gel Permeation

High-yield spent bisulphite liquor (HY-SBL) from sulphonated black spruce wood (Piceamariana) was fractionated by gel permeation chromatography (GPC) and by high-performance liquid chromatography (HPLC). The GPC fractionation gave a wide bimodal distribution, whereas with HPLC, a more detailed resolution was seen with the bulk of the fraction giving several clearly defined peaks. The paucidisperse material was further concentrated by a bulk fractionation of the crude SBL which included complexing the lignosulphonates with dicyclohexylamine. The isolated paucidisperse material was found to be dialyzable and to constitute 90% of the lignosulphonate in the sample of SBL. If the bisulphite pulp obtained was recooked in fresh acid sulphite liquor to a lower yield, most of the lignosulphonate dissolved was widely polydisperse with no indication of the discrete components resolvable by HPLC. However, 25% of the lignin made soluble was in the form of the paucidisperse fractions. In all, we were able to obtain about 50% of the lignin in spruce wood as a relatively low molecular weight lignosulphonate resolvable into discrete fractions by HPLC.

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