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PubMed1993

Purification of endo-polygalacturonase by affinity precipitation using alginate.

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.

Alginates↗
PubMed1997

The course of pectin degradation by polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici.

The production of polygalacturonases from Fusarium oxysporum f. sp. radicis lycopersici (FORL) occurred sequentially, and endo- and exo-polygalacturonases (PG) predominated at different times during growth. At first FORL produced a PG which did not release monomers from pectins and was unable to hydrolyse dimers and trimers. Later a complex was produced in which exo-PG seemed to be the predominant activity; the monomer was released rapidly and this activity was able to degrade dimers and trimers.

Fusarium↗
PubMed2003

The OsFOR1 gene encodes a polygalacturonase-inhibiting protein (PGIP) that regulates floral organ number in rice.

We have isolated a cDNA clone, OsFOR1, from the immature panicles of rice. The OsFOR1 (Oryza sativa floral organ regulator 1) gene encodes a protein that contains a leucine-rich repeat (LRR) domain. This domain comprises 10 tandem repeats of a canonical 24-amino acid LRR sequence. The structure and the number of LRRs for OsFOR1 are similar to those of polygalacturonase-inhibiting proteins (PGIPs) from various other plant species. Moreover, the OsFOR1 recombinant protein, when fused to maltose-binding protein (MBP), shows PGIP activity against the Aspergillus niger polygalacturonase. OsFOR1 is highly expressed in the calli and immature and mature panicles, while detectable at only low levels in seedling roots and mature stems. In situ hybridization experiments showed the transcripts of OsFOR1 are present in young spikelet primordia and in almost all of the young floral organs. Transgenic approaches were used to study in vivo functioning. Antisense expression of OsFOR1 resulted in an increase in the numbers of floral organs, including the stamen, carpel, palea/lemma, stigma, and lodicule. OsFOR1 transcript was not detected in the frizzy panicle mutant, which is defective in its spikelet formation but normal in inflorescence-meristem initiation and maintenance. Therefore, we suggest that OsFOR1 plays a role in the formation and/or maintenance of floral organ primordia.

Amino Acid Sequence↗
PubMed1997

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris.

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

Enzyme Inhibitors↗
PubMed1994

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit.

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.

Journal Article↗
PubMed1994

Tomato Fruit Polygalacturonase Isozyme 1 (Characterization of the [beta] Subunit and Its State of Assembly in Vivo).

Polygalacturonase isozyme 1 (PG1) is a heterodimer comprising a catalytic and noncatalytic or [beta] subunit, whereas polygalacturonase isozyme 2 (PG2) comprises only the catalytic subunit. To assess the state of assembly of PG1 in vivo, both subunits were purified to homogeneity and used to study assembly of the heterodimer. PG1 could be reconstituted in vitro from purified [beta] subunit and purified PG2 under a wide range of salt and pH conditions, and PG1 reconstituted in vitro was indistinguishable from PG1 isolated from tomato (Lycopersicon esculentum) fruit. Specific antibodies indicated that the [beta] subunit was present in fruit of all developmental stages, but absent in vegetative tissue. The state of assembly of PG1 in vivo was tested based on the differential thermal stability of PG1 and PG2 by heating segments of ripe fruit pericarp tissue. Temperatures well below those required to inactivate PG1 in vitro caused the loss of activity of both PG1 and PG2, suggesting that only heat-labile PG2 is present in vivo. In addition, when extracts of ripe fruit were rigorously maintained and analyzed at 4[deg]C, PG1 was absent or barely detectable. These results are consistent with the hypothesis that PG1 can assemble spontaneously and is essentially absent in intact tomato fruit but forms artifactually from PG2 and the [beta] subunit during the extraction of tomato fruit tissue when low temperatures are not rigorously maintained.

Journal Article↗
PubMed1982

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro.

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.Similar cell wall-bound protein extracts from ripe Nr fruit were not as effective and those from ripe rin fruit gave no cell wall degradation at all in vitro. This was correlated with the absence of polygalacturonase in rin and low activity of Nr extracts.Purified polygalacturonase was capable of in vitro cell wall degradation and it seems that this enzyme can account for the cell wall degradation observed with the total cell wall-bound protein extracts from ripe fruit.

Journal Article↗
PubMed1987

Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers.

Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not.

Journal Article↗
PubMed2004

Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit.

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

Journal Article↗
PubMed1996

Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici.

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.

The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.

Amino Acid Sequence↗
PubMed2001

Study of the methyl ester distribution in pectin with endo-polygalacturonase and high-performance size-exclusion chromatography.

A method was developed that enables the study of the methyl ester distribution in the polymers of pectin on a molecular level. Endo-polygalacturonase was used to extensively degrade three 70% methyl esterified pectins. The molecular weight distribution of the non- and enzymatically degraded pectins was determined with high-performance size-exclusion chromatography. Next, the molecular weight distribution was converted into a degree of polymerization distribution of galacturonan fragments. Monte Carlo methods were employed for the reconstruction of the parental polymers from their enzymatic degradation products. The results for the random methyl esterified pectin revealed that the enzyme-degradable sites were indeed randomly distributed, which confirmed the correctness of the procedure developed. The two other pectins studied differed greatly in the amount of non-, low-, and high-esterified regions present in the reconstructed pectic molecules of a given molecular mass. That the approach developed is able to reveal such detailed information makes it unique. The information on the fragmental composition of pectic polymers obtained is an important addition to the study of the methyl ester distribution and the functional properties of pectin.

Chromatography, High Pressure Liquid↗
PubMed2004

Influence of beta-subunit on thermal and high-pressure process stability of tomato polygalacturonase.

Polygalacturonase (PG; E.C. 3.2.1.15) was extracted from tomato fruit and purified by cation-exchange chromatography. Two peaks containing PG activity were detected: the first denotes a thermolabile PG fraction (PG2) and the second a thermostable fraction (PG1). PG1 is a dimer of PG2 and a heat-stable protein called the beta-subunit. In contrast to its resistance to heat, PG is easily inactivated at elevated pressure. Although the thermal stability of purified tomato PG1 and PG2 is distinctly different, they show an identical pressure stability. To gain further insight into the thermal and pressure stability of both PG isoenzymes, the in vitro recombination of PG2 and beta-subunit was studied. After severe heat (up to 140 degrees C for 5 min) and pressure (up to 800 MPa for 15 min) treatments, the residual fractions containing the beta-subunit were able to convert PG2 into the heat-stable PG1, showing the extreme thermal and pressure stability of the beta-subunit. PG1 was detected in heat-treated tomato juice and, to a lesser extent, in tomato pieces. In contrast, as was the case for purified PG, no pressure-stable fraction was observed when tomato juice and pieces were treated under pressure. These data clearly show the differing behavior of the PG1-PG2-beta-subunit system under thermal and high-pressure treatments and offer the possibility of inactivating tomato PG using high pressure without the need for high temperatures.

Enzyme Activation↗
PubMed1980

[Effects of molecular parameters of galacturonan substrate on the activity of a polygalacturonase from tomatoes].

The activity of a major form of the tomato polygalacturonase (EC 3.2.1.15) depends of the origin of the galacturonan substrates (apple, citrus) as well as upon the molecular mass, the degree of esterification and the distribution of the ester methoxyl groups. Optimal substrates are citrus pectic acids with a degree of esterification < 1% and a molecular mass corresponding to a viscosity number [eta] = 90 ml/g galacturonan. In the [eta] range from 16 to 474 ml/g, the Km values decrease to constant amount of 15.6 mM galacturonic acid units, which corresponds to 0.27% galacturonan. In a statistical distribution of the ester methoxyl groups, the activity reaches zero in the range of the degree of esterification from 80 to 90%. Enzymatically de-esterified pectins with a degree of esterification < 32% and a block-like distribution of the ester methoxyl groups behave as comparable pectic acids. In summary, there is a good agreement between these enzymesubstrate interactions and those of endopolygalacturonases from Aspergillus spec. Differentiations manifested themselves only in the transition range between macromolecular galacturonan substrates and oligomeric substrates below the established critical molecular mass.

Glycoside Hydrolases↗
PubMed1987

Influence of culture conditions on mycelial structure and polygalacturonase synthesis of Aspergillus niger.

Biosynthesis of polygalacturonase (PG) by A. niger strain R 1/214 correlates with the morphology of mycelium in submerged culture. The mean specific PG-synthesis (PG-U.g-1.h-1) increases with the degree of compactness of mycelium. PG-production can be optimized by a precise adjustment of the culture conditions after direct spore inoculation (diffuse mycelium) but the high synthesis as by compact mycelium is never obtained. Different reasons for the higher enzyme production by the pellet mycelium are discussed. PG-synthesis is assumed to be strictly connected with a limitation of nutrient and oxygen supply.

Aspergillus niger↗
PubMed1994

Detection of polygalacturonase, pectin-lyase and pectin-esterase activities in a Saccharomyces cerevisiae strain.

The catalytic capacity of several excreted pectinolytic enzymes obtained from various yeast strains was examined using in vivo and biochemical techniques. Of the 33 yeast strains studied, 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectinolytic enzymes and was identified as Saccharomyces cerevisiae and designated SCPP. Pulsed-field gel electrophoresis and the polymerase chain reaction technique were used to characterize further this specific strain. Three types of pectinolytic enzymes were found to be excreted by SCPP: polygalacturonase, pectin-lyase and pectin-esterase. These enzymes allow pectin hydrolysis during cell growth.

Base Sequence↗
PubMed1995

Detection method for polygalacturonase-producing strains of Saccharomyces cerevisiae.

In the presence of glycerol or ethanol, SCPP (a strain of Saccharomyces cerevisiae that expresses pectinolytic activity) is capable of utilizing galacturonic acid or pectins for growth purposes. We now establish a relationship between the pectinolytic power of various strains of S. cerevisiae and their ability to grow on a pectin/glycerol-based medium. This property is further exploited for the detection of polygalacturonase-producing strains of S. cerevisiae.

Culture Media↗