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)

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1 and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50% with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].
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PMID:Pectinase production by Neurospora crassa: purification and biochemical characterization of extracellular polygalacturonase activity. 183 96

The finding of a powerful inhibitor of pectin methylesterase in ripe kiwi fruit is reported. The inhibitor was revealed to be a glycoprotein. It was purified to homogeneity and found to have a molecular mass of about 28 kDa, as estimated by gel filtration chromatography, SDS/PAGE and analytical ultracentrifugation. The sugar portion is composed of galactose, arabinose and rhamnose, the latter being much less represented. The amino acid composition showed a very high content of acidic residues compared to basic ones, which is the reason for the very low isoelectric point of the protein (less than 3.5). The kind of inhibition on kiwi pectin methylesterase was found to be competitive with an apparent Ki of 0.22 microM, using citrus pectin as a substrate. Moreover, the inhibitor is effective in inhibiting pectin methylesterase in the pH range 3.5-7.5. Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuch as it was found to be ineffective against other polysaccharide-degrading enzymes, such as polygalacturonase and amylase. Conversely, it appears to be completely aspecific as far as the pectin methylesterase source is concerned. In fact, it was found to inhibit this enzyme effectively from all the sources we assayed, i.e. orange, tomato, apple, banana, potato.
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PMID:A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis). 222 35

The precipitation of alginate by Ca2+ at pH 3.8 was found to occur concomitantly with the precipitation of endo-polygalacturonase from Aspergillus niger. Under optimum conditions, 92% of the enzyme activity was precipitated. The enzyme could be recovered from the precipitate by washing with 0.5 M NaCl/0.2 M Ca2+. All the precipitated endo-polygalacturonase activity could be recovered in this way. The enzyme thus obtained was purified 10-fold. A comparison of SDS/PAGE gel patterns of the crude preparation and enzyme purified by the affinity precipitation also showed a significant purification of the enzyme.
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PMID:Purification of endo-polygalacturonase by affinity precipitation using alginate. 829 9

In a culture filtrate of Trichosporan penicillatum B2, which is a gamma-ray irradiation mutant induced from T. penicillatum SNO3, we found three kinds of pectin-releasing enzymes, protopectinases SE1, SE2, and SE3, that have endo-polygalacturonase activity. These enzymes were purified to homogeneity with cation-exchange and size exclusion chromatographies. The major PPase in the culture filtrate was PPase SE1, which accounted for 75% of total activities in the culture filtrate, and the two others were 0.15% (PPase SE2) and 0.007% (PPase SE3). Their molecular masses were approximately 41, 41, and 42 kDa on SDS-PAGE, respectively. They had similar enzymatic properties but different PPase activity and pH- and thermo-stability. Antibody against PPase S, which is produced by strain SNO3, inhibited the activities of PPase SE1, SE2, and SE3. However PPase SE1 was completely inhibited by treatment with the anti-PPase S antibody, but the activities of PPases SE2 and SE3 remained at 20 and 50% of the original activity, respectively.
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PMID:Purification and characterization of three extracellular protopectinases with polygalacturonase activities from Trichosporon penicillatum. 882 24

An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M(r) of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 degrees C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identify to other known polygalacturonases.
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PMID:Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici. 896 70

Genetic control of polygalacturonase (PG) activity from Fusarium oxysporum f.sp. radicis lycopersici was analyzed on pectin and glucose cultures. One exopolygalacturonase from F. oxysporum f.sp. radicis lycopersici was strongly induced, in stationary culture, when the fungus was grown on apple pectin, while on glucose no extracellular PG activity could be detected. Although SDS-PAGE detected the presence of a putative PG band (66 kDa) in both conditions, specific antibodies obtained against the purified PG only detected it in PG-inducing conditions, that is to say, when apple pectin was used as the carbon source. Northern blot analysis of RNA of two isolates of F. oxysporum f.sp. radicis lycopersici (r6 and r2) confirmed that this regulation of PG synthesis was exerted at the transcriptional level. Only one single mRNA species of around 1400 nucleotides was detected on the cultures containing pectin and was absent in glucose-grown cultures. Southern blot analysis of genomic DNA indicated that pg gene seems to be present in a single copy in the genomes of F. oxysporum f.sp. radicis lycopersici r6 and r2 and Fusarium oxysporum f.sp. lycopersici, showing similar hybridization patterns in all species. The partial sequence of this pg gene from F. oxysporum f.sp. radicis lycopersici r6, which is also reported, showed high similarity to diverse PGs already reported. Exopolygalacturonase of F. oxysporum f.sp. radicis lycopersici r6 is heavily glycosylated; its deglycosylated form had a molecular mass of 50 kDa.
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PMID:Control of polygalacturonase synthesis in Fusarium oxysporum f.sp. radicis lycopersici. 943 11

Non starch polysaccharide hydrolyzing enzyme preparations analyzed in this study were composed of up to nine (1-3, 1-4)-beta-glucanase activities and up to six xylanase activities with different molecular weights in the range from 100 kD down to 18 kD as determined with SDS/PAGE zymograms. Partially purified enzyme fractions differed in terms of pH-optima, isoelectric point and thermal stability in aquaeous solutions. Different beta-glucanase activities were found in different production strains, although some enzymes were conserved over genus boundaries. Enzyme preparations from the same or related strains exhibited different patterns of enzyme activity, indicating modification of strain and/or fermentation conditions. Some enzyme preparations contained significant amounts of polygalacturonase and/or galactomannase activity. The pH profiles of whole enzyme preparations resulted from pH optima of isoenzyme fractions. Temperature optima for all preparations were between 50 and 60 degrees C. Thermal stability of high molecular weight components tended to be lower than for low molecular weight fractions. Fractions with cellulase activity were most stable, followed by (1-3, 1-4)-beta-glucanase activities, while fractions with xylanase activities exhibited low thermal stabilities. Incubation of enzyme preparations and their respective active fractions in digesta supernatants revealed only small differences in residual xylanase activity. Digesta from gizzard samples led to the highest inactivation. It is concluded that commercial enzyme preparations display different modes of action and that the development of improved enzyme preparations depends not only on thermal stability, but also on pH profile, substrate specificity and proteolytic stability within the digestive tract.
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PMID:Biochemical characteristics of non starch polysaccharide hydrolyzing enzyme preparations designed as feed additives for poultry and piglet nutrition. 1054 72

The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encode polygalacturonase. Cloning of the PGL1 open reading frame behind the ADH1 promoter allowed overexpression of polygalacturonase activity in S. cerevisiae. This enzyme was purified to apparent homogeneity from cultures of recombinant S. cerevisiae on synthetic medium using one-step purification by anionic exchange chromatography. The enzyme, named Pgl1P, had an apparent M(r) of 42 kDa as shown by SDS-PAGE. Pgl1P was active from pH 3 to 5.5, with an optimum temperature at 25 degrees C. This enzyme hydrolyzed polygalacturonic acid as an endo-polygalacturonase as demonstrated by independent methods. The purified protein was N-glycosylated. However, the activity remained in the N-deglycosylated form. The N-terminal amino acid sequence was also determined as D-S-C-T-L-T-G-S-S-L.
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PMID:Purification and characterization of acidic endo-polygalacturonase encoded by the PGL1-1 gene from Saccharomyces cerevisiae. 1065 Feb 15

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver-leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI-MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N-binding sugar chains matches that of a high-mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C-terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3-Cys17, Cys175-Cys191, and Cys300-Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.
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PMID:Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry. 1075 64

Salivary proteins (SPs) of Schizaphis graminum, Acyrthosiphon pisum and Myzus persicae were studied after probing and feeding on different artificial diets. Salivary sheaths as well as apical lumps of saliva were found, presumably representing subsequently excreted saliva of different types. Phenoloxidase, pectinase and peroxidase activities were detected by staining the enzyme-converted products, thus confirming these enzyme activities found earlier by others. Proteinase and cellulase were not found. SPs in three major SDS-PAGE bands, at 154 and 66/69 kDa, were collected in fluid diets (soluble fraction) and as sheath material (solid fraction) attached to the membranes covering these diets. Proteins of both fractions presumably represented the enzymatic activities found, although this could not be proven. The lack of electrophoretic mobility of the undenaturated (isoelectrofocusing and PAGE) active proteins meant that they could not be separated, whereas the mobile denaturated (SDS-PAGE) proteins had lost their enzyme activity. Polyclonal antibodies, anti-SP154 and anti-SP66/69, both cross-reacted to most salivary proteins in Western blots. They also reacted to sheath material and to the principal salivary glands. For further studies of saliva some monoclonal antibodies were developed. The complexity of salivation and the relation of the results obtained to the behaviourally known secretion periods is discussed.
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PMID:Salivary proteins of aphids, a pilot study on identification, separation and immunolocalisation. 1081 45


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