Large-scale preparation of α-D-(14)-oligogalacturonic acids from pectic acid

2002 ◽  
Vol 80 (8) ◽  
pp. 900-903 ◽  
Author(s):  
Hong-Ni Fan ◽  
Mei-Zheng Liu ◽  
Yuan C Lee
Keyword(s):  
Galacturonic Acid ◽  
Large Scale ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Inexpensive Method ◽  
Pectic Acid ◽  
Large Scale Preparation ◽  
Exclusion Chromatography ◽  
Scale Preparation

An efficient and inexpensive method for large-scale preparation of α-D-(1[Formula: see text]4)-oligogalacturonic acids (oligo-GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure – size-exclusion chromatography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2-5. Key words: pectic acid, galacturonic acid, galabiose, galatriose, pectinase.

scholarly journals Structure of a Rhamnogalacturonan Fragment from Apple Pectin: Implications for Pectin Architecture

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Xiangmei Wu ◽  
Andrew Mort
Keyword(s):  
Galacturonic Acid ◽  
2D Nmr ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Direct Connection ◽  
Apple Pectin ◽  
Nmr Spectrometry ◽  
Exclusion Chromatography ◽  
Rhamnogalacturonan Hydrolase

A commercial apple pectin was sequentially digested with the cloned enzymes endopolygalacturonase, galactanase, arabinofuranosidase, xylogalacturonase, and rhamnogalacturonan hydrolase. The rhamnogalacturonan hydrolase-generated oligosaccharides were separated by ultrafiltration, anion exchange, and size-exclusion chromatography. Fractions from the ion exchange chromatography were pooled, lyophilized, and screened by MALDI-TOF MS. An oligosaccharide (RGP14P3) was identified and its structure, α-D-GalpA-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA, determined by 1D and 2D NMR spectrometry. This oligosaccharide probably represents a direct connection between homogalacturonan and rhamnogalacturonan in pectin. Alternatively, it could indicate that the nonreducing end of rhamnogalacturonan starts with a galacturonic acid residue.

scholarly journals Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis

2016 ◽  
Vol 63 (3) ◽  
Author(s):  
Marcin Grąz ◽  
Kamila Rachwał ◽  
Radosław Zan ◽  
Anna Jarosz-Wilkołazka
Keyword(s):  
Oxalic Acid ◽  
Ph Value ◽  
Enzyme Reaction ◽  
Oxalate Oxidase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Optimum Temperature ◽  
Intracellular Enzyme ◽  
Exclusion Chromatography

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min-1, respectively.

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.

scholarly journals Structure of the omalizumab Fab

2015 ◽  
Vol 71 (4) ◽  
pp. 419-426 ◽  
Author(s):  
Rasmus K. Jensen ◽  
Melanie Plum ◽  
Luna Tjerrild ◽  
Thilo Jakob ◽  
Edzard Spillner ◽  
...  
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Structural Basis ◽  
Hek 293 ◽  
Functional Studies ◽  
293 Cells ◽  
Exclusion Chromatography ◽  
Ige Mediated ◽  
High Affinity Ige Receptor

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

scholarly journals Purification and Characterization of Transglutaminase from Streptomyces sp. TTA 02 SDS 14

2016 ◽  
Vol 11 (3) ◽  
Author(s):  
Lia Siti Nur'amaliyah ◽  
Dewi Seswita Zilda ◽  
Nisa Rachmania Mubarik
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Phenacyl Bromide ◽  
Optimum Temperature ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Exclusion Chromatography ◽  
Sulfonyl Fluoride

Streptomyces sp. TTA 02 SDS 14 is a transglutaminase producing bacteria which previously had been  screened along with more than one hundred isolates. This research aimed to purify and characterize transglutaminase from this strain. Transglutaminase was purified from crude enzyme by ultrafiltration, Q-Sepharose ion exchange chromatography and Sepacryl S200 size exclusion chromatography sequentially, obtaining yield and purification fold of  1.36%  and 27 folds, respectively. The molecular weight of the purified transglutaminase was 72 kDa detected by zymogram gel electrophoresis. The optimum temperature and pH were 50°C and 6. The transglutaminase was stable at 45°C and could be activated in the presence of 5 mM and 10 mM of Na+, K+, Li+,Ca2+, Mg2+, BPB (4-bromo-phenacyl bromide), and IAA (iodo acetamide acid), but the activity was inhibited by  the presence of Cu+, Zn2+, and PMSF (phenyl methyl sulfonyl fluoride).

scholarly journals Peroxidase from infected fruit of Solanum sp. grown in Nsukka

2019 ◽  
Vol 54 (2) ◽  
pp. 131-138
Author(s):  
Omeje KO ◽  
Eze SOO ◽  
FC Chilaka
Keyword(s):  
Molecular Weight ◽  
Quality Control ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Optimum Temperature ◽  
Ammonium Sulfate Precipitation ◽  
Optimum Ph ◽  
Exclusion Chromatography ◽  
The Ideal

In this study, we characterized the activity of peroxidase a quality control enzyme from the infected fruit of Solanum sp. Peroxidase was purified to homogeneity by ammonium sulfate precipitation, dialysis, ion exchange chromatography and size exclusion chromatography. The molecular weight of the native enzyme was 63000 da. The enzyme was shown to have two iso-enzymes with distinct optimum pH of 4.5 and 7.0 and optimum temperature of 40 and 70⁰C. The purified enzyme had broad substrate specificity with o-dianisidine being the ideal substrate. Na+, Ca2+, Mg2+, Mn2+, Cu2+, Al3+ were shown to be activators of the enzyme, while the peroxidase activity was severely inhibited by Co2+. Bangladesh J. Sci. Ind. Res.54(2), 131-138, 2019

scholarly journals Expression of Monomeric Insulin Precursor in Pichia pastoris and Its Conversion into Monomeric B27 Lys Destripeptide Insulin by Tryptic Hydrolysis

2005 ◽  
Vol 37 (4) ◽  
pp. 234-240 ◽  
Author(s):  
Jin-Guo Ding ◽  
Jian Fei ◽  
Da-Fu Cui ◽  
You-Shang Zhang
Keyword(s):  
Pichia Pastoris ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Exclusion Chromatography ◽  
Native Gel ◽  
Tryptic Hydrolysis

Abstract Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition, and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.

Large-scale preparation of galacturonic acid oligomers by matrix-bound polygalacturonase

1974 ◽  
Vol 34 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Frans E.A. van Houdenhoven ◽  
Pierre J.G.M. de Wit ◽  
Jaap Visser
Keyword(s):  
Galacturonic Acid ◽  
Large Scale ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Matrix Bound

scholarly journals Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva

1991 ◽  
Vol 280 (2) ◽  
pp. 341-352 ◽  
Author(s):  
N Ramasubbu ◽  
M S Reddy ◽  
E J Bergey ◽  
G G Haraszthy ◽  
S D Soni ◽  
...  
Keyword(s):  
Large Scale ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Proline Rich Peptide ◽  
Purification Protocol

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

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