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PubMed2004

Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen.

BACKGROUND: Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens. OBJECTIVE: We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifolia. METHODS: P acerifolia pollen extract was fractionated by using ion-exchange and gel-permeation chromatography. Analyses were carried out by using ELISA, SDS-PAGE, isoelectrofocusing, and immunoblotting. Partial amino acid sequence was obtained by means of Edman sequencing of cyanogen bromide-digested peptides. Specific cDNA was cloned by using reverse transcription, followed by PCR, with amino acid sequences from peptides of the allergen. RESULTS: The allergen isolated from P acerifolia pollen, Pla a 2, is a glycoprotein with an observed molecular mass of 43 kd and an isoelectric point value of 9.3. It is involved in the allergic responses of 84% of patients with planetree-induced pollinosis and represented 52% of the total IgE-binding capacity of the P acerifolia extract. Pla a 2 displays polygalacturonase (PG) activity, being the first PG with functional enzyme activity from an angiosperm plant pollen described as an allergen. The cDNA allergen sequence codified for a 372-residue protein with 56% and 42% sequence identity to PGs from pollen and fruits, respectively. Western blot analysis showed that Pla a 2 is present in pollen and stems and has IgG cross-reactivity with a PG from tomato and pectate lyases from Cupressaceae pollen. CONCLUSION: Pla a 2, a major allergen of P acerifolia pollen with PG activity has been purified, characterized, and cloned.

Adult↗
PubMed2004

Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii.

An endo-polygalacturonase, named PGI, was purified to homogeneity from the culture filtrate of Aspergillus kawachii IFO 4033 grown in a glucose-tryptone medium. The molecular mass of PGI was estimated to be 60 kDa by SDS-PAGE and 40 kDa by gel filtration on Sephacryl S-100. The isoelectric point was 3.55 as determined by isoelectic focusing. PGI exhibited binding properties to ConA-Sepharose suggesting that the protein is glycosylated. The N-terminal amino acid sequence was also determined as S-T-C-T-F-T-D-A-A-T-A-S-E-S-K. The remarkable property of PGI was its high activity in the pH range 2.0-3.0 towards soluble and insoluble substrates, while being inactive at pH 5.0. Enzyme stability at low pHs was markedly enhanced by different compounds, such as proteins, polysaccharides, simple sugars and the substrate pectin. PGI was very efficient to extract pectin from lemmon protopectin and to macerate carrot tissues at pH 2.0. These properties make PGI an interesting biocatalyst for industrial applications under highly acidic conditions.

Amino Acid Sequence↗
PubMed2006

Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum.

An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.

Enzyme Stability↗
PubMed2006

Separation of pectin methylesterases and polygalacturonases on monolithic columns.

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M(r) 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M(r) 82 kDa) and a basic protein with M(r) 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M(r) 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH(3)CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.

Carboxylic Ester Hydrolases↗
PubMed2005

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper.

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.

Capsicum↗
PubMed2006

Inactivation of polygalacturonase and pectate lyase produced by pH tolerant fungus Fusarium moniliforme NCIM 1276 in a liquid medium and in the host tissue.

Fusarium moniliforme NCIM 1276 produced pH dependent an extracellular polygalacturonase (PG) and pectate lyase (PL) at pH 5 and pH 8, respectively. In the extracellular medium about 20.3% PG and 54% of PL protein concentrations were present in the active state at pH 5 and pH 8, respectively, whereas in intracellularly, more than 86% of both protein contents remained in the active state at all pH tested. We found two possible reasons, end-product inhibition and effect of environmental pH on conformation of the proteins after their release into the medium. Additionally, in infected tomato and cauliflower plants, the fungus secreted similar proteins which were located near to the epidermal and vascular regions of the hypocotyls. In infected tissues, between 26.9% and to 41.5% of PG and only 0.84%-13.4% of PL protein concentrations were present in active state. Thus, the medium/cell sap pH and concentrations of substrate/end products seem to play an important role in fungal invasion during plant pathogenesis are discussed with current literature.

Brassica↗
PubMed2005

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.

Amino Acid Sequence↗
PubMed2006

A pollen-specific polygalacturonase from lily is related to major grass pollen allergens.

A pollen-specific gene from lily (Lilium longiflorum Thunb. cv. Snow Queen), designated LLP-PG, was characterized. Southern blots of lily genomic DNA indicated that LLP-PG is a member of a small gene family. A thorough sequence analysis revealed that the LLP-PG gene is interrupted by two introns and encodes a protein of 413 amino acids, with a calculated molecular mass of 44 kDa, and a pI of 8.1. Evaluation of the hydropathy profile showed that the protein has a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. A sequence similarity search showed a significant homology of the encoded protein to pollen polygalacturonases (PGs) from various plant species and to an important group (group 13) of grass pollen allergens. The LLP-PG transcript is pollen-specific and it accumulates only at the latest stage during pollen development, in the mature pollen. In contrast to other "late genes" LLP-PG transcript can neither be induced by abscisic acid (ABA) nor by dehydration. Immunoblot analyses of pollen protein extracts from lily, timothy grass and tobacco with IgG antibodies directed against LLP-PG and against the timothy grass pollen allergen, Phl p 13, indicated that lily LLP-PG shares surface-exposed epitopes with pollen PGs from monocotyledonous and dicotyledonous plants. Enzyme-linked immunosorbent assay (ELISA) analyses and inhibition ELISA assays with patients' IgE demonstrated a very low IgE reactivity of lily rLLP-PG and a lack of cross-reactivity between rLLP-PG and the timothy grass pollen allergen, rPhl p 13. These data demonstrated that despite the significant sequence homology and the conserved surface-exposed epitopes LLP-PG represents a low-allergenic member of pollen PGs.

Allergens↗
PubMed2002

Effect of infiltrated polyamines on polygalacturonase activity and chilling injury responses in zucchini squash (Cucurbita pepo L.).

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.

Cold Temperature↗
PubMed2003

New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins.

Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases.

Acetylation↗
PubMed2002

A polygalacturonase of animal origin isolated from the root-knot nematode Meloidogyne incognita.

The first animal polygalacturonase (PG, EC 2.1.15) encoding cDNA, Mi-pg-1, was cloned from the plant parasitic nematode Meloidogyne incognita. The enzymatic activity of MI-PG-1 was confirmed after heterologous expression in Escherichia coli. The presence of a predicted signal peptide on the MI-PG-1 sequence together with the specific localization of the transcripts of the Mi-pg-1 gene in the oesophageal glands of infective juveniles imply that MI-PG-1 could be secreted into plant tissues. The potential role of MI-PG-1 in parasitism is discussed.

Animals↗
PubMed1997

Isolation and characterization of new C-terminal substitution mutations affecting secretion of polygalacturonase in Erwinia carotovora ssp. carotovora.

An intact C-terminus was previously shown to be required for stability and secretion of the polygalacturonase (PehA) in Erwinia carotovora ssp. carotovora. Here we have analyzed the effects of amino acid (aa) substitutions generated to five C-terminal positions of PehA. Conservation of two hydrophobic and one non-hydrophobic residue (V372, V374 and N371, respectively) was found to be essential for maintenance of the protein stability. As an exception, one of the mutants (V372G) did not show major effects on protein stability, as determined by immunoblots and enzyme activity assay, yet it prevented the secretion completely. We conclude that the C-terminus of PehA is directly involved in the formation or stabilization of a conformation-sensitive structure needed for recognition of the protein as secreted.

Binding Sites↗
PubMed2001

Separation of endo-polygalacturonase using aqueous two-phase partitioning.

The partitioning of endo-polygalacturonase (endo-PG) in polyethylene glycol (PEG)-polyvinyl alcohol (PVA10000) and PEG-hydroxypropyl starch (Reppal PES100) aqueous two-phase systems was studied, and revealed the possibility of using aqueous two-phase extraction to purify and concentrate endo-PG from its clarified fermentation broth. For the PEG8000-PVA10000 system, endo-PG presented in the fermentation broth (at concentration that is more than 40% of total protein) mainly dominates in the top phase with a partitioning coefficient of 6, while total protein concentrates in the bottom phase. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: a first extraction in polyethylene glycol (PEG8000)-polyvinyl alcohol system, followed by a second extraction in PEG8000-(NH4)2SO4 system. This allowed the separation of endo-PG from polymer and the recycling of PEG polymer, since endo-PG was very strongly partitioned into the bottom phase of the PEG8000-(NH4)2SO4 system. Laboratory-scale experiments were performed to test the efficiency of this scheme. It was found that enzyme recovery was up to 91% with a total purification factor of about 1.9 and a concentration factor of more than 5. About 90% of the total PEG added into the systems can be recovered, and no reduction was obtained in the purification factor using recycled PEG.

Culture Media↗
PubMed2000

Purification and characterization of polygalacturonase from banana fruit.

Polygalacturonase isoenzyme 3 (PG-3) was purified to homogeneity with a specific activity of 0.7 mu katal mg-1 protein from banana fruit pulp. The purified enzyme was a glycoprotein with ca. 8% carbohydrate. The molecular weight of the native enzyme was found to be 90 +/- 10 kDa with a subunit molecular weight of 29 +/- 2 kDa. The enzyme exhibited optimum activity at pH 4.3 and temperature 40 degrees C with activation energy 35.4 kJ mol-1. A unique property of the enzyme was the requirement of -SH groups for the enzyme activity. The enzyme was inhibited by p-CMB and activated by 2-ME and DTT. The inhibition of p-CMB could be reversed by DTT. The enzyme contained eight free -SH groups. The Km of the enzyme was 0.15% for polygalacturonic acid.

Carbohydrates↗
PubMed2000

Pectin esterase, polygalacturonase and gel formation in peach pectin fractions.

Peaches (Prunus persica cv. Hermoza) were stored at 0C in regular air (RA) or in controlled atmosphere (CA 10% CO2, 3% O2) for 4 weeks and then ripened for 4 days at 20 degrees C. Woolliness developed in the regular air stored fruit while the controlled atmosphere stored fruit ripened normally. In the woolly fruit symptoms of the disorder were greater in the inner mesocarp than in the outer. Polygalacturonase (PG) and pectin esterase (PE) activities differed in the outer and inner mesocarp of the affected fruit. PG activity was low and PE activity was high in the inner mesocarp of the woolly fruit during ripening relative to the outer mesocarp, while in the healthy fruit, activities were similar in both areas. Cell wall fractions of water-soluble, CDTA-soluble and carbonate-soluble pectins were prepared from freshly harvested peaches and incubated with PE and PG from ripe peaches at different ratios. Only the CDTA-soluble fraction formed a gel with peach enzymes, and the rate of gelation increased with increasing amounts of PE relative to PG. Both water-soluble and CDTA-soluble pectin fractions formed gels with commercial PE (extracted from orange peel). The PE extracted from peaches was stable when stored at 0 degrees C for 9 days, while PG activity was stable only for 1 day. We suggest that PE, acting on pectins in the cell wall in vivo may cause gel formation and that the CDTA-soluble polymers have the capacity to bind apoplastic water and create the dry appearance observed in woolly fruit.

Carboxylic Ester Hydrolases↗
PubMed1997

Inhibitors of polygalacturonase in calli of Orobanche aegyptiaca.

The presence of at least three distinct polygalacturonases (PGase) in callus of Orobanche was demonstrated. The PGase activity is labile and at pH 4.5 does not require activation by cations. It can be partially purified on Biogel P 100 columns and can be resolved by PAGE into several bands. Broomrape callus tissue also contains inhibitors of PGase activity. One of these is a low M(r) compound, stable to boiling and removable by dialysis. An additional inhibitor precipitable by ammonium sulphate is also present.

Cells, Cultured↗
PubMed1998

Multiple forms of polygalacturonase from banana fruits.

Three multiple forms of polygalacturonase (PG) in ripe and two in unripe banana (Musa acuminata) fruits were separated by DEAE-cellulose and further purified using Sephadex G-150 chromatography. The multiple forms can be differentiated from each other on the basis of their properties. PG1 and PG3 were identified as endo-PG and PG2 as exo-PG on the basis of decrease in viscosity, increase in reducing sugar and the reaction product. PG2 and PG3 increased with the ripening of fruits. PG1, PG2 and PG3 exhibited optimum activity at pH 3.3, 3.7 and 4.3, respectively. Complete loss of PG2 and PG1 activities occurred at 60 and 70 degrees, but PG3 retained 60 and 50% activity respectively. The three forms showed a different response towards divalent metal ions. Ca2+ activated PG1 activity only. Teepol 0.1%, inhibited PG1 activity by 25%, but PG2 and PG3 activities were completely inhibited. CTAB, 0.1%, had no effect on PG1 and PG2 activities, but inhibited PG3 activity by 40%. 2-ME stimulated PG2 and PG3 activities but had no effect on PG1 activity. Gel filtration through Sephacryl indicated M(r) of 23,200, 58,000 and 130,000, respectively, for PG1, PG2 and PG3. The substrate saturation curve for PG1 and PG2 were Michaelian, while PG3 showed biphasic curve. The Km values of PG1 and PG2 were 0.22% and 0.14%, respectively.

Cations↗
PubMed2002

Flax-retting by polygalacturonase-containing enzyme mixtures and effects on fiber properties.

Enzyme-retting of flax was accomplished via individual treatment with four polygalacturonase (PGase) containing solutions of various fungal sources and the resulting fibers were characterized. The retting solutions were equilibrated to contain 2.19 U of PGase activity as determined via a dinitrosalicylic acid (DNS) reducing sugar assay. As compared with the buffer control, treatment with the various enzyme solutions increased the yield of fine fibers. Treatment with Aspergillus niger PGase resulted in a 62% increase in fine fiber yield as compared with the buffer control and fiber strength did not statistically differ (P</=0.05) between these treatments. Retting via PGases of Rhizopus origin produced the weakest fibers. These results illustrate that the crude PGases differ in their ability to ret flax and that under the defined experimental conditions the A. niger PGase is a better retting agent. Light microscopy demonstrated the ability of all enzymes to separate fiber from shive and epidermal tissues. Enzyme profiles of the solutions were determined via viscometric assays. Pectinolytic activity was the predominant activity of all enzymes tested. Activity against carboxymethyl cellulose (CMC) was a minor component of all solutions except A. niger PGase for which no activity was detected.

Aspergillus niger↗