[Enzyme electrophoresis in inclusion polymerisates of acrylamide. B. Polygalacturonases (pectinases)].
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Acrylic Resins↗
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[Research on the degradation mechanism of a purified polygalacturonase from Aspergillus niger].
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Aspergillus↗
Polygalacturonase of Erwinia carotovora.
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Chromatography, Paper↗
Inhibition of Verticillium polygalacturonase by oxidation products of polyphenols.
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Enzymes↗
Polygalacturonase activity of Penicillum sp 7/4B/EI 1 mutant grown on some by-products of food industry.
Pectolytic activity of the Penicillum sp 7/4B/E11 mutant, which synthesized effectively PG and was not capable of producing PE, was studied on some by-products of food industries (apple pulp, beet pulp, and mixtures of apple pulp or beet pulp with wheat bran) supplemented in some experiments with mineral salts of the Czapek's medium. The strain showed good growth but a low PG activity when grown on by-products without mineral salts and an increase in PG activity after cultivation on these by-products (mainly on beet pulp) enriched with mineral salts. The highest PG activity was obtained after 6-8 days cultivation on beet pulp (3%) with mineral salts. Among the tested salts, NaNO3 influenced PG activity most effectively, K2HPO4 and MgSO4 to a smaller degree whereas others were ineffective. The mutant did not synthesize PE under these culture conditions.
Culture Media↗
Isolation and properties of pectinases from the fungus Aspergillus japonicus.
Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI 5.6 and 3.3), pectin lyase (50 kD, pI 3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI (3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50 degrees C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30 degrees C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.
Aspergillus↗
The Tomato Fruit Cell Wall : II. Polyuronide Metabolism in a Nonsoftening Tomato Mutant.
A nonsoftening tomato (Lycopersicon esculentum L.) variety, dg, was examined to assess the physiological basis for its inability to soften during ripening. Total uronic acid levels, 18 milligrams uronic acid/100 milligrams wall, and the extent of pectin esterification, 60 mole%, remained constant throughout fruit development in this mutant. The proportion of uronic acid susceptible to polygalacturonase in vitro also remained constant. Pretreatment of heat-inactivated dg fruit cell walls with tomato pectinmethylesterase enhances polygalacturonase susceptibility at all ripening stages. Pectinesterase activity of cell wall protein extracts from red ripe dg fruit was half that in extracts from analogous tissue of VF145B. Polygalacturonase activities of cell wall extracts, however, were similar in both varieties. Diffusion of uronic acid from tissue discs of both varieties increased beginning at the turning stage to a maximum of 2.0 milligrams uronic acid released/gram fresh weight at the ripe stage. The increased quantity of hydrolytic products released during ripening suggests the presence of in situ polygalacturonase activity. Low speed centrifugation was employed to induce efflux of uronide components from the cell wall tree space. In normal fruit, at the turning stage, 2.1 micrograms uronic acid/gram fresh weight was present in the eluant after 1 hour, and this value increased to a maximum of 8.2 micrograms uronic acid/gram fresh weight at the red ripe stage. However, centrifuge-aided extraction of hydrolytic products failed to provide evidence for in situ polygalacturonase activity in dg fruit. We conclude that pectinesterase and polygalacturonase enzymes are not active in situ during the ripening of dg fruit. This could account for the maintenance of firmness in ripe fruit tissue.
Journal Article↗
Pectinolytic enzymes secreted by yeasts from tropical fruits.
Three hundred yeasts isolated from tropical fruits were screened in relation to secretion of pectinases. Twenty-one isolates were able to produce polygalacturonase and among them seven isolates could secrete pectin lyase. None of the isolates was able to secrete pectin methylesterase. The pectinolytic yeasts identified belonged to six different genera. Kluyveromyces wickerhamii isolated from the fruit mangaba (Hancornia speciosa) secreted the highest amount of polygalacturonase, followed by K. marxianus and Stephanoascus smithiae. The yeast Debaryomyces hansenii produced the greatest decrease in viscosity while only 3% of the glycosidic linkages were hydrolysed, indicating that the enzyme secreted was an endo-polygalacturonase. The hydrolysis of pectin by polygalacturonase secreted by S. smithiae suggested an exo-splitting mechanism. The other yeast species studied showed low polygalacturonase activity.
Carboxylic Ester Hydrolases↗
Release of protein from normal and mutant tomato cell walls.
The nitrogen content of cell wall preparations from normal tomato (cv Ailsa Craig) fruit remained constant during ripening, whereas salt-soluble protein increased throughout this process. Tomato polygalacturonase released about twice as much protein from the preparations as salts did, with a maximum at the orange stage of development. Polygalacturonase-solubilized protein from the tomato mutant ;ripening inhibitor' (rin) was less, and that from the mutant ;Never ripe' (Nr) cell walls was more than that from normal wall preparations. Release of protein by fungal cellulase was limited, but was increased by the addition of polygalacturonase from the same source. Salt-solubilized protein contained a range of enzymic activities but these were distributed between fewer multimolecular forms than is the case for whole cell preparations. The results suggest that metabolically active protein, removable by strong salt solutions, cellulase, or polygalacturonase, remains attached to the cell walls of tomato fruit until late in ripening. The unusual amounts of protein attached to the cell walls of mutant fruit appear to be a reflection of the absence of some or all of the isoenzymes of polygalacturonase that are associated with normal ripening.
Journal Article↗
Cry j 2, a major allergen of Japanese cedar pollen, shows polymethylgalacturonase activity.
We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity of PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by beta-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.
Allergens↗
Preparation and properties of pectic enzymes produced by Trichoderma lignorum.
Polygalacturonase and protein-methylesterase were isolated from shaken culture of Trichoderma lignorium. Isolation was carried out with various agents. Methanol was the most suitable precipitant for isolating polygalacturonase, yielding enzyme preparations 6.6 times more active than that of culture filtrate. Likewise, tannin afforded active fractions at pH 4 and 0.05% concentrations. Similarly, 50% ammonium sulphate saturation gave active fractions. The least polygalacturonase activity was obtained from ethanol. In any of the organic solvents used, highest enzymic activity was obtained when using only one volume. As regards pectin-methylesterase, no correlation existed between its activity and concentration of the precipitant used. A substrate concentration above 0.8% was a limiting factor for polygalacturonase activity, while optimum enzyme concentration was 40 microgram protein/ml at 40 degrees C and pH 4.45.
Esterases↗
The cleavable N-terminal domain of plant endopolygalacturonases from clade B may be involved in a regulated secretion mechanism.
Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant. By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain. We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain. We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod. We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod. Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.
Amino Acid Sequence↗
Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora.
A clone producing a polygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in PUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402-amino-acid (aa) polypeptide with an Mr of 42,849. In E. carotovora the polygalacturonase was synthesized with a 26-aa cleavable signal peptide. The mature 376-aa PehA had a calculated Mr of 40,064 and a pl of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35-45 degrees C. Analysis of the reaction products of polygalacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of erwinias. However, substantial similarity was detected within the C-terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic polygalacturonases may have a common origin.
Amino Acid Sequence↗
Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli.
Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.
Cell Wall↗
[Isoelectric characterization of extracellular polygalacturanases of various Aspergillus alliaceus strains].
The composition of pectin hydrolase complexes produced by various Aspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains of A. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.
Aspergillus↗
Production, characterization and properties of polysaccharide depolymerizing enzymes from a strain of Curvularia inaequalis.
Xylanase, beta-glucosidase, beta-xylosidase, endoglucanase and polygalacturonase production from Curvularia inaequalis was carried out by means of solid-state and submerged fermentation using different carbon sources. beta-Glucosidase, beta-xylosidase, polygalacturonase and xylanase produced by the microorganisms were characterized. beta-Glucosidase presented optimum activity at pH 5.5 whereas xylanase, polygalacturonase and beta-xylosidase activities were optimal at pH 5.0. Maximal activity of beta-glucosidase was determined at 60 degrees C, beta-xylosidase at 70 degrees C, and polygalacturonase and xylanase at 55 degrees C. These enzymes were stable at acidic to neutral pH and at 40-45 degrees C. The crude enzyme solution was studied for the hydrolysis of agricultural residues.
Ascomycota↗
Expression patterns of cell wall-modifying enzymes during grape berry development.
During ripening of grape (Vitis vinifera L.) berries, softening occurs concomitantly with the second growth phase of the fruit and involves significant changes in the properties of cell wall polysaccharides. Here, the activities of enzymes that might participate in cell wall modification have been monitored throughout berry development. Alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23) and pectin methylesterase (EC 3.1.1.11) activities were present, but no polygalacturonase (EC 3.2.1.15), cellulase (EC 3.2.1.4), xyloglucanase (xyloglucan-specific cellulase EC 3.2.1.4) or galactanase (EC 3.2.1.89) could be detected. The accumulation of mRNAs encoding wall-modifying enzymes was examined by northern hybridization analysis. Transcripts for beta-galactosidase, pectin methylesterase, polygalacturonase, pectate lyase (EC 4.2.2.2) and xyloglucan endotransglycosylase (EC 2.4.1.207) were present during ripening, although polygalacturonase activity had not been detected in berry extracts. Cellulases could not be detected in ripening berries, either at the enzyme or mRNA levels. The increase in beta-galactosidase activity and mRNA is consistent with the observed decrease in type-I arabinogalactan content of the walls during ripening, and the detection of polygalacturonase and pectate lyase mRNAs might explain the increased solubility of galacturonan in walls of ripening grapes. Thus, the modification of cell wall polysaccharides during softening of grape berries is a complex process involving subtle changes to different components of the wall, and in many cases only small amounts of enzyme activity are required to effect these changes.
Blotting, Northern↗
Glycosidases in Cell Wall-degrading Extracts of Ripening Tomato Fruits.
Enzyme preparations were obtained from cell wall debris of tomato (Lycopersicon esculentum L. cv. Tropic) fruits at various stages of ripeness and were assayed for glycosidase and polysaccharidase activities. In addition to polygalacturonase (mol wt 40,000), ripening fruits contain beta-galactosidase (mol wt 63,000) and beta-1, 3-glucanase (mol wt 12,000). The beta-glycosidases, unlike polygalacturonase, are active in extracts of green fruits. Placental tissue shows very low polygalacturonase but increasing beta-galactosidase and beta-1, 3-glucanase activities as ripening proceeds. A large change in the susceptibility of the walls to hydrolase action occurs before the stage in which the greatest polygalacturonase activity occurs. The possibility that the beta-glycosidases contribute to the wall modifications that lead to fruit softening is discussed.
Journal Article↗