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PubMed2000

Sequential synthesis and secretion of pectinases by Penicillium frequentans.

Penicillium frequentans synthesized eleven polygalacturonases and three pectinesterases when grown in the presence of pectin, sodium polypectate or monogalacturonic acid. When glucose was the sole carbohydrate source in the medium two of these polygalacturonases and one pectinesterase were produced. The enzymes produced under any of these conditions degraded pectic substrates to monogalacturonic acid, suggesting that this monosaccharide or its metabolites should induce the pectinolytic complex. All pectinesterases and most of the extracellular polygalacturonases were synthesized after the 2nd hour of incubation. The pectinases produced by Penicillium frequentans were not secreted at the same time but after 5 hours of incubation all of them could be detected outside the cell those detected only inside the cell were probably membrane-associated or unglycosylated forms of the extracellular pectinases.

Carbon↗

PubMed1984

Isolation, purification and assay of the macerating enzyme produced by Penicillium oxalicum Curie and Thom.

Penicillium oxalicum produced two isozymes of polygalacturonase (PG) and a pectate lyase (PL). The enzymes were separated and purified following ammonium sulphate precipitation, ion exchange chromatography, ultrogel column chromatography and isoelectric focusing. The first isozyme of polygalacturonase (PGI) was rather unstable hence its properties could not be much assayed. PGII macerated and killed yam tissue in 4 hours but PL was unable to do so. Enzyme assay for the end-products of degradation of sodium polypectate and yam tissue showed that PGI was an exo-enzyme while PGII and PL were endo-enzymes. Endo-polygalacturonase (PGII) appears to play the major role (as the macerating enzyme) in the pathogenesis of yam tissue infected by P. oxalicum.

Cell Wall↗

PubMed2005

Characterization and in vitro expression patterns of extracellular degradative enzymes from non-pathogenic binucleate Rhizoctonia AG-G.

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source beta-1,3 and beta-1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The beta-(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.

Carboxylic Ester Hydrolases↗

PubMedDate not supplied

Modeling the inhibitor activity and relative binding affinities in enzyme-inhibitor-protein systems: application to developmental regulation in a PG-PGIP system.

Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions.

Computer Simulation↗

PubMed2000

Effect of high-pressure treatment on the texture of cherry tomato.

The effect of high-pressure treatment (200-600 MPa for 20 min) on the texture of cherry tomatoes and on the key softening enzymes (pectinmethylesterase and polygalacturonase) was investigated. When subjected to high-pressure treatment whole cherry tomatoes showed increasing textural damage with increasing pressures up to 400 MPa. However, treatment at pressures above 400 MPa (500-600 MPa) led to less apparent damage than treatment at 300 and 400 MPa; the tomatoes appearing more like the untreated samples. These visual changes were reflected in the texture (firmness) and amount of cell rupture in the tomatoes, with the least firmness and the most cell rupture being seen after treatment at 400 MPa. Light and scanning electron microscopy supported these observations. Although a sample of purified commercial pectinmethylesterase was partially inactivated at pressures above 200 MPa, irrespective of pH (4-9), in the whole cherry tomatoes no significant inactivation was seen even after treatment at 600 MPa, presumably because other components in the tomato offered protection or the isoenzymes were different. Polygalacturonase was more susceptible to pressure, being almost totally inactivated after treatment at 500 MPa. It is concluded that the textural changes in tomato induced by pressure involve at least two related phenomena. Initially, damage is caused by the greater compressibilty of the gaseous phase (air) compared to liquid-solid components, giving rise to a compact structure which, on pressure release, is damaged as the air rapidly expands, leading to increases in membrane permeability. This permits egress of water, and the damage also enables enzymatic action to increase, causing further cell damage and softening. The major enzyme involved in the further softening is polygalacturonase, which is inactivated at 500 MPa and above, and not pectinmethylesterase, which in the whole fruit, is barotolerant.

Solanum lycopersicum↗

PubMed2001

Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution.

Family 28 belongs to the largest families of glycoside hydrolases. It covers several enzyme specificities of bacterial, fungal, plant and insect origins. This study deals with all available amino acid sequences of family 28 members. First, it focuses on the detailed analysis of 115 sequences of polygalacturonases yielding their evolutionary tree. The large data set allowed modification of some of the existing family 28 sequence characteristics and to draw the sequence features specific for bacterial and fungal exopolygalacturonases discriminating them from the endopolygalacturonases. The evolutionary tree reflects both the taxonomy and specificity so that bacterial, fungal and plant enzymes form their own clusters, the endo- and exo-mode of action being respected, too. The only insect (animal) representative is most related to fungal endopolygalacturonases. The present study brings further: (i) the analysis of available rhamnogalacturonase sequences; (ii) the elucidation of relatedness between the recently added member, the endo-xylogalacturonan hydrolase and the rest of the family; and (iii) revealing the sequence features characteristic of the individual enzyme specificities and the evolutionary relationships within the entire family 28. The disulfides common for the individual enzyme groups were also proposed. With regard to functionally important residues of polygalacturonases, xylogalacturonan hydrolase possesses all of them, while the rhamnogalacturonases, known to lack the histidine residue (His223; Aspergillus niger polygalacturonase II numbering), have a further tyrosine (Tyr291) replaced by a conserved tryptophan. Evolutionarily, the xylogalacturonan hydrolase is most related to fungal exopolygalacturonases and the rhamnogalacturonases form their own cluster on the adjacent branch.

Amino Acid Sequence↗

PubMed1995

Pectinolytic enzymes of anaerobic fungi.

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii, A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100-900 and 10-450 micrograms galacturonic acid h-1 mg protein-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).

Animals↗

PubMedDate not supplied

[Various characteristics of growth kinetics and exohydrolase synthesis in Aspergillus foetidus and Zygofabospora marxiana].

The kinetics of growth and synthesis of exohydrolases (polygalacturonases and proteinases) were studied in Aspergillus foetidus and Zygofabospora marxiana. The processes of growth and synthesis of polygalacturonases were found to be shifted in time. Endopolymethylgalacturonase of the micromycete and endopolygalacturonase of the yeast are not accumulated in the cells, but are mainly secreted into the growth medium. The maximal value of polygalacturonase bound to the cell coincides with the maximal rate of synthesis of the enzyme secreted into the medium and with the maximal level of cell-bound alkaline and acid proteinases. The activity of alkaline proteinase is not found in the growth medium of the cultures, and only traces of acid proteinase were detected. It is possible that proteinase are involved in processing of endopolygalacturonases.

Ascomycota↗

PubMed1996

Regulation by Galacturonic Acid of Pectinolytic Enzyme Production by Sclerotinia sclerotiorum

Production of polygalacturonases and pectinases from Sclerotinia sclerotiorum was induced in vitro by galacturonic acid. The inductive effect of galacturonic acid was abolished by the presence of glucose, leading to a basal enzyme production. Zymograms of extracellular enzymes showed that galacturonic acid induced the synthesis of six polygalacturonase and one pectin-methylesterase isoforms. Immunoblotting revealed that an exo-polygalacturonase and an exo-polymethylgalacturonase were secreted in all conditions. They are not glucose repressed and not regulated by galacturonic acid. These constitutive enzymes provide the pathogen with the inherent ability to release galacturonic acid from plant cell walls and to trigger inducible enzyme synthesis.

Journal Article↗

PubMed1996

Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

Amino Acid Sequence↗

PubMed1997

Cloning of a protopectinase gene of Trichosporon penicillatum and its expression in Saccharomyces cerevisiae.

A protopectinase (PPase)-encoding gene, PSE3, from Trichosporon penicillatum was cloned by colony hybridization using two oligonucleotide probes synthesized from the N-terminal amino acid sequences of native PPase SE1 and one peptide from a lysyl endopeptidase digest. Nucleotide sequencing revealed that PSE3 contains an ORF encoding a 367 amino acid protein. Mature PPase SE3 is composed of 340 amino acids and the N-terminus of the ORF appeared to correspond to a signal peptide and a propeptide processed by a KEX2-like proteinase. The deduced amino acid sequence of PSE3 was 65.4, 56.7, 58.1, 61.8 and 48.9% homologous to the polygalacturonases of Aspergillus oryzae, Aspergillus niger, Aspergillus tubigensis, Cochliobolus carbonum and Fusarium moniliforme, respectively. One domain, which might interact with polygalacturonic acid, is highly conserved not only in fungal polygalacturonases but also in bacterial and plant polygalacturonases. PSE3 was expressed in Saccharomyces cerevisiae, but three forms (the mature form, a glycosylated form and an uncharacterized processed form) of PPase SE3 were present among the PSE3 products.

Amino Acid Sequence↗

PubMed1979

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening.

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato beta-galactosidase.

Journal Article↗

PubMed1980

Abscission of Citrus Leaf Explants: INTERRELATIONSHIPS OF ABSCISIC ACID, ETHYLENE, AND HYDROLYTIC ENZYMES.

The question whether abscisic acid (ABA) induces cellulase and polygalacturonase activity and, hence, abscission directly or whether its action is mediated by C(2)H(4) was studied in citrus (Osbeck var. Shamouti) leaf explants using aminoethoxyvinyl glycine (AVG), an inhibitor of C(2)H(4) biosynthesis. ABA in concentrations of 10 micromolar and higher induced C(2)H(4) production and accelerated abscission. AVG inhibited C(2)H(4) formation, activity of cellulase and polygalacturonase, and abscission in ABA-treated explants. AVG did not inhibit the increase in the activity of the cell-wall degrading enzymes or abscission in a saturating level of externally supplied C(2)H(4). This indicates that the effect of AVG resulted from inhibition of the formation of endogenous ethylene. The data indicate that in citrus leaf explants the induction of the activity of cellulase and polygalacturonase and abscission by ABA is mediated by C(2)H(4).

Journal Article↗

PubMed1984

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes.

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO(2), 6% O(2), and 88% N(2) for 14 weeks at 12 degrees C, exhibited a temporal separation of certain biochemical events associated with ripening.The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22 degrees C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

Journal Article↗

PubMed1989

Pectic enzymes in pectolyase: separation, characterization, and induction of ethylene in fruits.

The pectic enzymes in Pectolyase were separated by ion exchange chromatography on Q-Sepharose. Three pectin lyases, two polygalacturonases, and a pectinmethylesterase were resolved. The enzymes were further purified on Mono Q and/or Mono S columns to remove traces of cellulase. The enzymes had molecular weights ranging from 25,000 to 36,000 daltons. They were optimally active between pH 4.0 and 6.2 and were not greatly affected by ions. The pectin lyases and polygalacturonases were endo-enzymes. They solubilized uronic acids from washed cell wall fragments, but the lyases were much more effective than the polygalacturonases. The mixture of enzymes constituting Pectolyase increased ethylene production 15- to 25-fold when introduced into tomato and orange fruits. The enzymes purified from Pectolyase all increased ethylene production in the fruits but the lyases were generally more effective than the hydrolases.

Journal Article↗

PubMed2005

The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens.

The intercellular ascomycetous pathogen Cymadothea trifolii, causing sooty blotch of clover, proliferates within leaves of Trifolium spp. and produces a complex structure called interaction apparatus (IA) in its own hyphae. Opposite the IA the plant plasmalemma invaginates to form a bubble. Both structures are connected by a tube with an electron-dense sheath. Using immunocytochemistry on high-pressure frozen and freeze-substituted samples, we examined several plant and fungal cell wall components, including those in new host wall appositions at the interaction site, as well as a fungal polygalacturonase. Within the tube linking IA and host bubble, labelling was obtained for cellulose and xyloglucan but not for rhamnogalacturonan-I and homogalacturonans. The IA labelled for chitin and beta-1,3-glucans, and for a fungal polygalacturonase. Plant wall appositions reacted with antibodies against callose, xyloglucans and rhamnogalacturonan-I. Cymadothea trifolii partly degrades the host cell wall. Structural elements remain intact, but the pectin matrix is dissolved. A fungal polygalacturonase detected in the IA is probably a key factor in this process. Owing to the presence of chitin and beta-1,3-glucans, the IA itself is considered an apoplastic compartment.

Ascomycota↗

PubMed1986

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica.

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Erwinia↗

PubMed1985

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Bacterial Proteins↗