Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Preparative and Kinetic Analysis of ?-1,4- and ?-1,3-Glucan Phosphorylases Informs Access to Human Milk Oligosaccharide Fragments and Analogues Thereof.

ABSTRACT: The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild-type cellodextrin phosphorylase (CDP: ?-1,4-glucan linkage-dependent) and laminaridextrin phosphorylase (Pro_7066: ?-1,3-glucan linkage-dependent) to tolerate a number of sugar-1- phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1?% of the natural reactions, we demonstrate the utility of given phosphorylase-sugar phosphate pairs to access new-to-nature fragments of human milk oligosaccharides, or analogues thereof, in multi-milligram quantities.

SUBMITTER: Singh RP

PROVIDER: S-EPMC7187349 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Preparative and Kinetic Analysis of β-1,4- and β-1,3-Glucan Phosphorylases Informs Access to Human Milk Oligosaccharide Fragments and Analogues Thereof.

Singh Ravindra Pal RP Pergolizzi Giulia G Nepogodiev Sergey A SA de Andrade Peterson P Kuhaudomlarp Sakonwan S Field Robert A RA

Chembiochem : a European journal of chemical biology 20191230 7

The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild-type cellodextrin phosphorylase (CDP: β-1,4-glucan linkage-dependent) and laminaridextrin phosphorylase (Pro_7066: β-1,3-glucan linkage-dependent) to tolerate a number of sugar-1- phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficienc ...[more]

PMID: 31657512

Similar Datasets

β-Glucan phosphorylases in carbohydrate synthesis.

Project description:β-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of β-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of β-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 ( www.cazy.org ). Since the discovery of β-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of β-glucan and associated β-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of β-glucan phosphorylases. • β-Glucan phosphorylases in the production of functional carbohydrates.

| S-EPMC8140972 | biostudies-literature

Structure-Immunogenicity Relationship of α- and β-Tetrasaccharide Glycoforms from Bacillus anthracis Exosporium and Fragments Thereof.

Project description:The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-α-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rhamnose is unknown. Because the linkage of this residue to the GlcNAc bridging the glycan and the protein is lost during isolation of the tetrasaccharide, its α- and β-glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete α- and β-tetrasaccharide glycoforms, a 2-demethoxylated version of the α-tetrasaccharide, and the α- and β-trirhamnosides and CRM197. By immunization of mice, we showed that the anti α- and β-tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the α- and β-trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti α- and β-tetrasaccharide sera recognized Sterne spores in a comparable fashion. ΔBclA spores not expressing the major exosporium protein were also recognized by the same sera, while mutants that produced the carbohydrate antigen with deletion of either rhamnose or anthrose were not. The tetrasaccharide could, therefore, be expressed in proteins other than BlcA. This work proves that α- and β-tetrasaccharide are equally potent immunogens.

| S-EPMC6222408 | biostudies-literature

Thermus thermophilus nucleoside phosphorylases active in the synthesis of nucleoside analogues.

Project description:Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.

| S-EPMC3346467 | biostudies-literature

Action of an endo-β-1,3(4)-glucanase on cellobiosyl unit structure in barley β-1,3:1,4-glucan.

Project description:β-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single β-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-β-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley β-1,3:1,4-glucan. To find the minor structure on which the endo-β-1,3(4)-glucanase acts, we prepared oligosaccharides from barley β-1,3:1,4-glucan by endo-β-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-β-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as β-Glc-1,3-β-Glc-1,4-β-Glc-1,3-β-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-β-1,3(4)-glucanase acts on the cellobiosyl units of barley β-1,3:1,4-glucan in an endo-manner.

| S-EPMC4673573 | biostudies-literature

A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase.

Project description:Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the beta-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a beta-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

| S-EPMC1895987 | biostudies-literature

Synergic activity of Cel8Pa β-1,4 endoglucanase and Bg1Pa β-glucosidase from <i>Paenibacillus xylanivorans</i> A59 in beta-glucan conversion.

Project description:In the efficient bioconversion of polysaccharides from lignocellulosic biomass, endoglucanases and β-glucosidases are key enzymes for the deconstruction of β-glucans. In this work, we focused on a GH8 endoglucanase (Cel8Pa) and a GH1 β-glucosidase (Bg1Pa) from Paenibacillus xylanivorans A59. Cel8Pa was active on a broad range of substrates, such as β-glucan from barley (24.5 IU/mg), lichenan (17.9 IU/mg), phosphoric acid swollen cellulose (PASC) (9.7 IU/mg), carboxi-methylcellulose (CMC) (7.3 IU/mg), chitosan (1.4 IU/mg) and xylan (0.4 IU/mg). Bg1Pa was active on cellobiose (C2) and cello-oligosaccharides up to C6, releasing glucose as the main product. When both enzymes were used jointly, there was a synergic effect in the conversion rate of polysaccharides to glucose. Cel8Pa and Bg1Pa presented important properties for simultaneous saccharification and fermentation (SSF) processes in second generation bioethanol production, such as tolerance to high concentration of glucose and ethanol.

| S-EPMC7490527 | biostudies-literature

Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Project description:Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.

| S-EPMC1144599 | biostudies-other

beta-Glucan oligosaccharide enhances CD8(+) T cells immune response induced by a DNA vaccine encoding hepatitis B virus core antigen.

Project description:DNA vaccination can induce specific CD8(+) T cell immune response, but the response level is low in large mammals and human beings. Coadministration of an adjuvant can optimize protective immunity elicited by a DNA vaccine. In this study, we investigated the effect of a synthetic glucohexaose (beta-glu6), an analogue of Lentinan basic unit, on specific CD8(+) T cell response induced by a DNA vaccine encoding HBcAg (pB144) in mice. We found that beta-glu6 promoted the recruitment and maturation of dendritic cells, enhanced the activation of CD8(+) and CD4(+) T cells and increased the number of specific CD8(+)/IFN-gamma(+) T cells in lymphoid and nonlymphoid tissues in mice immunized by pB144. Immunization with pB144 and beta-glu6 increased the anti-HBc IgG and IgG2a antibody titer. These results demonstrate that beta-glu6 can enhance the virus-specific CTL and Th1 responses induced by DNA vaccine, suggesting beta-glu6 as a candidate adjuvant in DNA vaccination.

| S-EPMC2896713 | biostudies-literature

Purification and characterization of an endoglucanase (1,4-beta-D-glucan glucanohydrolase) from Clostridium thermocellum.

Project description:An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) was purified from Clostridium thermocellum by procedures that included centrifugation, ultrafiltration, selective precipitation, ion-exchange Sephadex chromatography and preparative gel electrophoresis. The 22-fold-purified enzyme behaved as a homogeneous protein under non-denaturing conditions. The enzyme represented a significant component (greater than 25%) of total extracellular endoglucanase activity, but was purified in low yield by the procedures employed. The native molecular weight of the endoglucanase was determined by ultracentrifugational analysis, amino acid composition and polyacrylamide-gel electrophoresis, and varied between 83000 and 94000. The enzyme contained 11.2% carbohydrate and was isoelectric at pH 6.72. The pH and temperature optima of the endoglucanase were 5.2 and 62 degrees C respectively. The enzyme lacked cysteine and was low in sulphur-containing amino acids. The purified endoglucanase displayed: high activity towards carboxymethylcellulose, celloheptaose, cellohexaose and cellopentaose; low activity towards Avicel microcrystalline cellulose and cellotetraose; no detectable activity towards cellotriose or cellobiose; increased activity towards cello-oligosaccharides with increasing degree of polymerization. The internal glycosidic bonds of cello-oligosaccharides were cleaved by the enzyme in preference to external linkages. The apparent Michaelis constant ([S]0.5V) and Vmax. for cellopentaose and cellohexaose hydrolysis were 2.30 mM and 39.3 mumol/min per mg of protein, and 0.56 mM and 58.7 mumol/min per mg of protein, respectively.

| S-EPMC1163377 | biostudies-other

A promiscuous glycosyltransferase generates poly-β-1,4-glucan derivatives that facilitate mass spectrometry-based detection of cellulolytic enzymes.

Project description:Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise cello-oligosaccharides with tailored functionalities on a scale suitable for employment in mass spectrometry-based assays. The resulting glycan structures enabled reporting of the activity and selectivity of celluloltic enzymes.

| S-EPMC8243248 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data