Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.15 (pectinase)
2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation. 757 47

Extensin, a major hydroxyproline (Hyp)-rich glycoprotein in walls of cultured cells of dicotyledonous plants, is very difficult to solubilize. To learn about the nature of the insolubilization, we have tested the ability of a variety of selective hydrolytic methods, and combinations of them, to liberate extensin or fragments of extensin from suspension-culture cell walls. After the complete deglycosylation of cotton (Gossypium hirsutum L.) walls, trypsinization solubilized 80% of the Hyp. The sequences of three abundant peptides were: (a) serine-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-serine-Hyp-Hyp-lysine, (b) serine-Hyp-Hyp-Hyp-Hyp-valine-lysine, and (c) serine-Hyp-Hyp-serine-alanine-Hyp-lysine. After a sequential treatment of walls with endopolygalacturonase, cellulase, -73 degrees C anhydrous hydrogen fluoride solvolysis, and ammonium bicarbonate extraction, only sugars indicative of rhamnogalacturonan I and protein remained insoluble. Trypsin treatment of this residue liberated 50% of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fell into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0 degrees C anhydrous hydrogen fluoride treatment, the first class was little affected in its electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides in both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectin-protein cross-links in addition to the protein-protein (or protein-phenolic-protein) cross-links that have been repeatedly suggested.
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PMID:Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures. Evidence for a covalent cross-link between extensin and pectin. 765 56

E.atroseptica 36A cells were transformed by the recombinant plasmids p27-1 and pEA364 (derivatives of the vector plasmid pUC19) containing pectate lyase genes of E.carotovora 17A and E.atroseptica 36A, respectively. The synthesis of pectate lyases determined by the cloned genes of bacteria of both subspecies, as well as the synthesis of the native enzymes, were induced by sodium poly pectate. Increase of the dose of pectate lyase genes did not result in alteration of pectate lyase secretion by E.atroseptica 36ApEA364 cells. At the same time, the efficiency of secretion of heterologous pectate lyases by E.atroseptica 36Ap27-1 cells was lower. The synthesis and secretion of the resident isoenzymes are as efficient as those of the parental cells. The results indicate a high specificity of the pectinase secretory system in Erwinia of different species and, moreover, subspecies.
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PMID:[Expression of pectate lyase genes of Erwinia carotovora subsp. carotovora 17A and Erwinia carotovora subsp. atroseptica 36A in Erwinia carotovor substp. atroseptica 36A cells]. 773 92

A simple method for the immobilization of Aspergillus niger mycelium producing polygalacturonase (PG) and pectinesterase (PE) is described. Fungal conidia were immobilized on wheat, rye, barley, peas, buckwheat and mustards seeds. Spongy mycelia overgrowing the seed surfaces on mineral medium with pectin produced extracellular PG and PE; the highest production was reached on the wheat carrier. Some of the variables influencing the enzymatic activity have been optimized. After every 24 h, a culture liquid with 6.8-7.8 U of PG ml-1 and 7.0-10.1 U of PE ml-1 was obtained. This procedure also made possible repeated batch enzyme production and, as many as eight subsequent 24-h batches could be fermented by using the same carrier without any loss of PG activity. The addition of sodium orthovanadate (1 mmol) into the medium with pectin caused a significant increase in PG and PE activity produced by free cells of A. niger (by 1.59-fold and 1.67-fold respectively), and only 0.47-fold of PG activity in case of the immobilized mycelium.
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PMID:Seeds as natural matrices for immobilization of Aspergillus niger mycelium producing pectinases. 774 27

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.
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PMID:Continuous determination of endopolygalacturonase activity by means of rotational viscosimeters. 797 77

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.
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PMID: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. 951 88

PG1, the major endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum, was secreted during growth on pectin by 10 of 12 isolates belonging to seven formae speciales, as determined with isoelectric focusing zymograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A Southern analysis of genomic DNA and PCR performed with gene-specific primers revealed that the pg1 locus was highly conserved structurally in most isolates. Two PG1-deficient isolates were identified; one lacked the encoding gene, and the other carried a pg1 allele disrupted by a 3.2-kb insertion with sequence homology to hAT transposases. The virulence for muskmelon of different F. oxysporum f. sp. melonis isolates was not correlated with PG1 production in vitro. We concluded that PG1 is widely distributed in F. oxysporum and that it is not essential for pathogenicity.
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PMID:Endopolygalacturonase PG1 in Different Formae Speciales of Fusarium oxysporum 957 83

The hydrolysis of cell wall pectins by tomato (Lycopersicon esculentum) polygalacturonase (PG) in vitro is more extensive than the degradation affecting these polymers during ripening. We examined the hydrolysis of polygalacturonic acid and cell walls by PG isozyme 2 (PG2) under conditions widely adopted in the literature (pH 4.5 and containing Na+) and under conditions approximating the apoplastic environment of tomato fruit (pH 6.0 and K+ as the predominate cation). The pH optima for PG2 in the presence of K+ were 1.5 and 0.5 units higher for the hydrolysis of polygalacturonic acid and cell walls, respectively, compared with activity in the presence of Na+. Increasing K+ concentration stimulated pectin solubilization at pH 4.5 but had little influence at pH 6.0. Pectin depolymerization by PG2 was extensive at pH values from 4.0 to 5.0 and was further enhanced at high K+ levels. Oligomers were abundant products in in vitro reactions at pH 4.0 to 5.0, decreased sharply at pH 5.5, and were negligible at pH 6.0. EDTA stimulated PG-mediated pectin solubilization at pH 6.0 but did not promote oligomer production. Ca2+ suppressed PG-mediated pectin release at pH 4.5 yet had minimal influence on the proportional recovery of oligomers. Extensive pectin breakdown in processed tomato might be explained in part by cation- and low-pH-induced stimulation of PG and other wall-associated enzymes.
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PMID:Polygalacturonase-mediated solubilization and depolymerization of pectic polymers in tomato fruit cell walls . Regulation By ph and ionic conditions 970 84

High-performance anion-exchange chromatography (HPAEC) coupled with a diode array detector (DAD) was used to identify and quantify oligogalacturonic acid components in pectins. Purified pectin lyase and polygalacturonase were used to generate unsaturated and saturated oligomers from pectins and sodium polygalacturonate, respectively. This method resulted in a good separation of saturated and unsaturated oligomers up to DP 13. It allowed us to follow polygalacturonase and pectate lyase depolymerisation pathways simultaneously.
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PMID:High-performance anion-exchange chromatography DAD as a tool for the identification and quantification of oligogalacturonic acids in pectin depolymerisation. 1112 36

A collaborative trial was conducted to validate the effectiveness of a liquid chromatographic (LC) procedure for determination of patulin in both clear and cloudy apple juices and apple puree. The test portion of clear apple juice was directly extracted with ethyl acetate; cloudy apple juice and apple puree were treated with pectinase enzyme before extraction. After back-extraction into sodium carbonate to remove interfering acidic compounds, the extract was dried and concentrated, and patulin was determined by LC with UV detection. Clear and cloudy apple juices, apple puree test samples naturally contaminated with patulin, and blank test samples for spiking with patulin were sent to 14 collaborators in 12 different European countries. Test portions of each of the 3 test sample types were spiked with patulin at 75 ng/g. Recoveries of patulin ranged from 80 to 92%. Based on the results for spiked test samples (blind pairs) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) ranged from 8 to 35% and 11 to 36%, respectively. Although HORRAT values of <1.4 were obtained for all 3 matrixes at patulin levels ranging from 26 to 121 ng/g, better performance values (RSDr values 6-10% and RSDR values 11-25%) were obtained for clear and cloudy apple juice spiked above 50 ng/g, which is either the statutory limit or the advisory level for patulin contamination in apple juices in many countries.
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PMID:Liquid chromatographic method for determination of patulin in clear and cloudy apple juices and apple puree: collaborative study. 1112 42


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