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At least 793 records · Page 44Linked to original sources
PubMed2005

Post-translational modifications of recombinant B. cinerea EPG 6.

The fungus Botrytis cinerea is a ubiquitous plant pathogen that infects more than 200 different plant species and causes substantial economic losses in a wide range of agricultural crops and harvested products. Endopolygalacturonases (EPGs) are among the first array of cell-wall-degrading enzymes secreted by fungi during infection. Up to 13 EPG glycoforms have been described for B. cinerea. The presence of multiple N-linked glycosylation modifications in BcPG1-6 is predicted by their deduced amino acid sequences. In this work, the glycosylation sites and the attached oligosaccharide structures on BcPG6 were analyzed. The molecular mass of the intact glycoprotein was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis. BcPG6 contains seven potential N-linked glycosylation sites. Occupancy of these glycosylation sites and the attached carbohydrate structures were analyzed by tryptic digestion followed by liquid chromatography/mass spectrometry (LC/MS) using a stepped orifice voltage approach. Five out of seven potential N-linked sites present in BcPG6 were determined to be occupied by high-mannose-type oligosaccharides. Four of them were readily determined to be at Asn58 (T3 peptide), Asn198 (T7 peptide), Asn237 (T9 peptide) and Asn256 (T11 peptide), respectively. Another was located on the T8 peptide, which contained two potential N-linked sites, Asn224 and Asn227 (SNNN224VTN227ITFK). LC/MS/MS of a sample treated with N-glycanase placed the glycan in this peptide at Asn224 rather than at Asn227. The potential glycosylation site on Asn146 (T6 peptide) was not glycosylated. In addition, two disulfide bonds were observed, linking the Cys residues within the T13 and T16 peptides.

Amino Acid Sequence↗
PubMed2001

On-target endoglycosidase digestion matrix-assisted laser desorption/ionization mass spectrometry of glycopeptides.

The digestion of glycopeptides with endoglycosidases can be used in the process of their structural characterization, and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is often used to analyze the products of these digestions. In the currently accepted protocol for the endoglycosidase digestion of glycopeptides on the MALDI target, the target must be incubated at 37 degrees C, and an hour or more is needed for digestion. We have modified the procedure so that the process can be performed at room temperature in 5 to 15 min, and digestions are performed in the presence of a MALDI matrix. The endoglycosidases used for digestion were endoglycosidase H and peptide-N-glycosidase F. Glycopeptides from asialofetuin and endopolygalacturonase (EPG) II were used as standards because their glycan structures have been previously characterized. Glycopeptides with unknown glycan structures were also digested, including glycopeptides from pectate lyase, EPG I, and pectin methylesterase from Aspergillus niger.

Amidohydrolases↗
PubMed2002

Studying protein-carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry.

Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.

Amides↗
PubMed1994

Continuous determination of endopolygalacturonase activity by means of rotational viscosimeters.

A continuous viscosimetric method for the determination of endopolygalacturonase activity has been developed from theoretical considerations. The method is based upon the decrease in the specific viscosity of a sodium polygalacturonate solution caused by the action of the enzyme and measured by means of a rotational viscosimeter. The assay has advantages over other viscosimetric methods previously described and over those which rely on the reducing groups assay. The suitability of the newly developed method for measuring the heat resistance of endopolygalacturonase samples has been tested and proved to be satisfactory.

Chemistry↗
PubMed1998

Analysis of partially methyl-esterified galacturonic acid oligomers by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Two methods were developed to detect partially methyl-esterified galacturonic acid oligomers, generated by endopolygalacturonase treatment of a 30% methyl-esterified pectin. The enzyme digest was shown, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to contain sodiated galacturonic acid oligomers with a degree of polymerization of 2-12, containing 0-6 methyl esters. Galacturonic acid (monomer) could not be detected because of matrix ions interference in the low mass region. Using high-performance anion-exchange chromatography, with a sodium acetate gradient at pH 5.0 and postcolumn sodium hydroxide addition to allow pulsed amplified detection, a complex elution profile was obtained with the endopolygalacturonase-treated 30% methyl-esterified pectin. All the components eluted before nonesterified tetragalacturonic acid. The partially methyl-esterified oligogalacturonic acids eluted in a discernible series of oligomers with an identical number of nonesterified carboxylic acid groups; the large, more esterified oligomers eluted before small, less esterified oligomers. The methyl esters may hinder the interaction of the neighboring carboxylic acid groups with the anion-exchange resin, thereby giving the components an apparent lower overall negative charge.

Chromatography, Ion Exchange↗
PubMed2002

Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases.

A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative.

Arabidopsis↗
PubMed2002

Analysis of partially methyl-esterified galacturonic acid oligomers by capillary electrophoresis.

Recent progress in the determination of the intramolecular distribution of methyl-esterified residues in pectic substrates has been made using a fragmentation approach in which endopolygalacturonase is used to digest the polysaccharide and its subsequent (methyl-ester sequence-dependent) digest pattern is determined. This has been facilitated by the separation of partially methyl-esterified enzyme digest fragments using high-performance anion-exchange chromatography at pH 5. Here we demonstrate that capillary electrophoresis can be used as an additional method for separation and quantification of such digest patterns. The technique offers improvements in speed and economy of materials and a straightforward quantification procedure.

Electrophoresis, Capillary↗
PubMed2001

Botrytis cinerea endopolygalacturonase genes are differentially expressed in various plant tissues.

Botrytis cinerea, the causal agent of blight, rot, and gray mold on many plant species, secretes various endopolygalacturonases during all stages of infection. The expression pattern of the encoding genes (Bcpg 1-6) was studied on four hosts: tomato, broad bean, apple, and courgette (also known as zucchini). All gene family members are differentially expressed, depending on the stage of infection and the host. Bcpg1 is expressed in all tissues tested although differences in transcript levels occur. Bcpg2 expression is detected early in the infection of three of four plant tissues tested. Bcpg3 and Bcpg5 are expressed in apple fruit tissue, although probably as a result of different regulatory mechanisms. The expression patterns of Bcpg4 and 6 are in agreement with their inducibility by monogalacturonic acid. The pattern of Bcpg gene expression indicates that B. cinerea is equipped with a flexible enzymatic pectate degradation machinery. The studies pinpoint new targets for gene disruption studies.

Blotting, Northern↗
PubMed1994

Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis.

The Erwinia chrysanthemi kdgR gene encodes a repressor that negatively regulates the expression of genes involved in pectinolysis and in pectinase secretion. The cloned kdgR gene was overexpressed in Escherichia coli by using a phage T7 system. Overproduced repressor was purified to homogeneity by two chromatographic steps. Gel retardation and DNase I protection experiments demonstrated the specific binding of the KdgR protein to the operators of pectinase genes (pelA, pelB, pelC, pelE), to the operator of genes involved in pectin catabolism (kdgT, ogl, kduI-kdgF) and to that of the outT gene involved in pectinase secretion. These interactions involved one (pelA, pelB, kduI-kdgF, outT) or several operator sites (pelC, pelE, ogl, kdgT) that generally overlap the promoter. Despite the presence of potential KdgR binding sites (KdgR-box) in the regulatory regions of four genes involved in pectin catabolism (kdgC, kduD, pem, kdgA) and in a pectinase secretion gene outC, no DNA-repressor complex could be observed by in vitro experiments. By using a missing contact experiment on the coding strand of ogl and pelE regulatory regions, a new KdgR-binding consensus was proposed. This new consensus, constituted by two half motifs (AATGAAAACT)N(NTCGATTTCTA), is well conserved in the operators which interact in vitro with the KdgR repressor. In contrast, this repressor-recognized motif is degenerated in the other operators that cannot interact in vitro with the repressor. These results suggest the existence of different regulation mechanisms mediated by the KdgR protein for the two classes of operators.

Base Sequence↗
PubMed2002

Combined use of regulatory elements within the cDNA to increase the production of a soluble mouse single-chain antibody, scFv, from tobacco cell suspension cultures.

In order to facilitate production and secretion of a soluble form of a small, single-chain antibody ScFv (32 kDa) in tobacco cell suspension culture, several modifications were made simultaneously to the antibody cDNA that included elements that have been shown to regulate the expression of proteins in plants. The scFv cDNA was initially ligated into a binary vector under the control of the CaMV 35S promoter and the T7 terminator for expression in tobacco suspension culture. Subsequently, modifications were engineered into the cDNA for enhancement of scFv production. These included the following: (i) the signal peptide (SP) of the tobacco pathogenesis-related protein PR1a which was added in-frame to the N-terminal end of scFv cDNA; (ii) a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence), which was fused to the N-terminal end of the SP; and (iii) the endoplasmic reticulum retention signal peptide KDEL, which was added to the C-terminal end of the scFv protein. Using a modified disruption method involving pectinase, the highest expression of total scFv (344 ng scFv/g cell) occurred when the plant leader sequence, the TEV sequence, and the KDEL peptide were all present in the expression construct. Although the addition of the KDEL sequence significantly increased the total yield of protein 5.4-fold, it did not increase the overall amount of protein secreted. These studies indicate that while the SP is very important in promoting secretion of the scFv, it had little influence on increasing scFv secretion levels even when both the TEV and the KDEL sequences significantly increased overall protein levels.

Animals↗
PubMed1996

Characterization of pectinases and pectin methylesterase cDNAs in pods of green beans (Phaseolus vulgaris L.).

Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity. To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development. Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development. These results do not favour a possible synergistic action of PE and PG. For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification. At a molecular level, one partial chromosomal clone of 210 bp (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv. verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv. Masai were isolated. The identity of the CDNA clones was confirmed by expression in Escherichia coli and immunodetection with antibodies directed towards a tomato fruit PE. Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length. In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.

Amino Acid Sequence↗
PubMed1980

Biochemical changes in mango after infection with Rhizoctonia bataticola.

Rhizoctonia bataticola is responsible for the spoilage of mango fruits (Mangifera india) during post-harvest preservation and storage. Culture of R. bataticola exhibited significant pectinase and cellulase activity. In Rhizoctonia-infected fruits an increase of protease and cellulase activity, and a decrease in certain enzymes of carbohydrate metabolism, were observed in comparison to healthy fruits.

Cellulase↗
PubMed1992

Characteristics of juice from palmyrah palm (Borassus) fruit.

The ancestral palmyrah palm (Borassus aethiopum Mart.) produces in the V-Baoulé region of Côte d'Ivoire, sweet and fragrant fruits in large numbers. Most of these spoil in the bush when ripe. The present work was to develop an efficient process for the extraction of juice from these fruits using a pectinase (Pectinex Ultra SP-L) at different concentrations (0.2, 0.4 and 0.6 mg/kg), temperatures (25 degrees C, 35 degrees C and 45 degrees C), and for different times (30, 60, 90 and 120 min). Some physico-chemical characteristics of the juice were determined in order to evaluate the quality of the product. Treatment at 45 degrees C for 120 min at an enzyme concentration of 0.6 mg/kg was selected for highest juice yield (73.14%). The colour parameters L; a; b) fluctuated in intensity between batches of rehydrated material. Juice consistency was found to be stable at 45 degrees C at the enzyme concentrations used. There was a slight variation in pH among the different treatments. In general, the yield and quality of the juice obtained were affected by treatment temperature, enzyme concentration and maceration time.

Beverages↗
PubMed1995

Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation.

Three extracellular pectinases were produced by Aspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (Km values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but the Km values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number of protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced by A. niger.

Aspergillus niger↗
PubMed1997

Enzymatic treatment to enhance carotenoid content in dehydrated marigold flower meal.

The effect of enzymatic treatments using a commercial enzyme (Econase-cep at pH 5.0 and 0.1% w/w concentration) at different levels of dehydrated marigold meal (5, 10, 15 and 20% dry weight), to enhance carotenoid extraction, was evaluated. The AOAC method consisting of a simultaneous extraction and saponification by using hexane-ethanol-acetone-toluene (10:6:7:7 v/v) and metanolic KOH (40% w/v) was used to evaluate the carotenoid content. The measurement of carotenoids was also carried out in samples in which the water soluble compounds were previously eliminated (AOAC-H2O). Total carotenoids ranged from 11.4 to 17.4 g/kg and 18 to 24.7 g/kg of control and treated marigold meal, respectively. Highest amount of carotenoids were noted when 5% level of treated meal was used.

Carotenoids↗