Degrees Of Methyl Esterification Research Articles

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3–9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1→4)-β- d-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase

A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers — up to 10 galacturonic acid residues — could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer.

PgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger

The nucleotide sequence data for pgaA and pgaB have been deposited with the EMBL, GenBank and DDBJ Databases under accession numbers Y18804 and Y18805 respectively. pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillus niger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB were characterized with respect to pH optimum, activity on polygalacturonic acid, and mode of action and kinetics on oligogalacturonates of different chain length (n=3-7). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 ?kat?mg-1 and 8.3 ?kat?mg-1 respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.

PgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger

The nucleotide sequence data for pgaA and pgaB have been deposited with the EMBL, GenBank and DDBJ Databases under accession numbers Y18804 and Y18805 respectively. pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillus niger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB were characterized with respect to pH optimum, activity on polygalacturonic acid, and mode of action and kinetics on oligogalacturonates of different chain length (n=3-7). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 mu+kat.mg(-1) and 8.3 mu+kat.mg(-1) respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.

Phase transition of pectin with sorbed water

The phase-transition behaviour of pectin having various degrees of methyl esterification (DE), in the presence of sorbed water, was investigated by differential scanning calorimetry (DSC). In the first run DSC curves of pectin in the dry state, a melting endothermic peak ( T m) was observed at about 152°C. In the second run DSC curves, glass transition ( T g) was observed at about 35°C. The above facts indicate that the original pectin formed a regular molecular arrangement. However, amorphous glass is formed when the crystalline pectin is melted once. In pectin–water systems, the T m of pectin decreases with increasing water content ( W c=mass of sorbed water/mass of dry pectin (g/g)). When W c is greater than 0.4 g/g, melting of free water and the glass transition of the pectin–water system were observed. The T g of the pectin–water system decreased with increasing W c in the bound water region. After reaching a minimum value, T g slightly increased and approached a constant value. Water content at the minimum T g ( W c min ) decreased with increasing DE. This suggests that the number of water molecules required to break the hydrogen bonds between –OH and –COOH groups decreases with increasing DE.

Changes in the pectic substances of apples during development and postharvest ripening. Part 1: Analysis of the alcohol-insoluble residue

Cell wall material in the form of an alcohol-insoluble residue (AIR) was prepared from apples during growth and maturation on the tree and at different stages of ripening during postharvest storage. The galacturonic acid, neutral sugar, starch content as well as the degree of acetylation of the AIR were determined for each stage as was the degree of methyl esterification of the pectic substances. During the first weeks of development, the AIR content of the fruit decreased; it then stayed constant until the end of storage. The galactose and arabinose content of the AIR decreased with time while the galacturonic acid content stayed almost constant. Hardly any changes were observed in the degrees of methyl esterification and acetylation. The unripe fruit contained appreciable amounts of starch whereas none could be determined shortly after harvest. The significance of the losses of arabinose and galactose residues will be examined in a further paper.

Changes in the pectic substances of apples during development and postharvest ripening. Part 2: Analysis of the pectic fractions

Pectic material was extracted from the alcohol-insoluble residues of apples during growth on the tree and at different stages of ripening during postharvest storage. Three pectic fractions, soluble in the chelating agent trans-1,2-diaminocyclohexane- N, N, N′, N′-tetraacetic acid (CDTA) in cold dilute sodium carbonate and in sodium carbonate at room temperature, were isolated. The galacturonic acid, neutral sugar and starch content of the fractions were determined as well as the degrees of methyl esterification and acetylation of the CDTA-soluble fraction. In addition, the molecular weight distribution was examined by gel filtration chromatography. The high galacturonic acid content of the CDTA-soluble fraction was consistent with the polysaccharide being essentially linear and originating in the middle lamella. The sodium carbonate-soluble pectin had a higher rhamnose to galacturonic acid ratio, indicating that it was more highly branched. Galactose and arabinose residues were lost in the polymers from all three fractions during ripening, with the greatest loss occurring from the sodium carbonate-soluble fractions. At the same time, the pectins became less polydisperse and shifted towards higher molecular weights. It seems that during development there is a dynamic turnover with new pectic polymers, which contain fewer neutral sugars, but are of high molecular weight, appearing in the middle lamella and primary cell wall.

Extraction and characterisation of water-soluble pectic substances from guava ( Psidium guajava L.)

Water-soluble pectic substances (CP-endo and CP-meso) were extracted from the alcohol-insoluble residues of fresh endocarp and mesocarp of white guavas (cv. Suprème) and fractionated by ion-exchange chromatography into minor neutral (NP-endo and NP-meso) and major acidic pectic substances (AP-endo and AP-meso). The neutral fractions contained mainly arabinose, galactose, and glucose, and methylation analysis revealed xyloglucans, highly branched arabinans, and type I arabinogalactans, together with arabino-3,6-galactan-proteins that interacted with the Yariv antigen. The APs contained 72–80% of galacturonic acid together with 16–19% of arabinose, galactose, and rhamnose, were heterogeneous in size-exclusion chromatography, differed greatly in their degrees of methyl esterification (21.9 and 88.0% for AP-endo and AP-meso, respectively) and acetylation (16.0 and 2.0%), and had typical rhamnogalacturonan structures with neutral chains of densely substituted arabinans and arabinogalactans of both types. Weak intrinsic viscosities [η] (13 and 26 mL/g for AP-endo and AP-meso, respectively) reflected low hydrodynamic volumes for guava pectins.

Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose

A procedure for the determination of the degree of methyl esterification of pectin in virtually any sample is described. Samples were dissolved or suspended in 1 m imidazole buffer, pH 7.0, cooled on ice, and reduced with sodium borohydride. Quantitative reduction of samples was accomplished after 1 h using at least 20 mg sodium borohydride/mg sample. The degree of methyl esterification was determined by either the increase in galactose content as determined by GLC of the sugar or by the change in galacturonic acid content by colorimetric uronic acid analyses. Sample requirements were at least as low as 100 μg per determination by GLC or 2 to 3 mg per determination by colorimetric uronic acid analysis compared to 5 mg or more per determination for other published procedures. The degrees of methyl esterification determined by the methods described have compared very favorably with those determined by established methods.

Isolation and characterization of the gel-forming polygalacturonide from seeds of Ficus awkeotsang.

To examine the spontaneous gel-forming property of the polysaccharide of seeds of Ficus awkeotsang Makino, seeds were extracted sequentially with cold and hot water, and then aqueous ammonium oxalate. The major acidic polysaccharide ([α]25D+239°; DE = 63.6%; M.W. 3.4 × 105), which was responsible for gel-formation, was obtained from the surface of heat-treated seeds and found to be composed mainly of galacturonic acid (96%). On fractionation of the major acidic polysaccharide by anion-exchange column chromatography, three kinds of methyl esterified components were obtained (P-1, DE=69.3%; P-2, 49.2% and P-3, 22.8%). Methylation analysis of the carboxylreduced polysaccharide indicated that the acidic polysaccharide is an essentially linear polysaccharide containing α-(1→4)-linked D-galacturonic acid residues and devoid of L-rhamnose residues, which confirmed it to be a polygalacturonide. Comparison of the IR spectra and the degrees of methyl esterification of the polygalacturonide extracted from heat-treated and heat-untreated seeds suggested that the methyl ester groups in the polygalacturonide must have been released during the seed extraction at room temperature. As a model test, furthermore, when the acid polysaccharide prepared from heat-treated seeds was incubated with pectinesterase from lemon peel in the presence of 1mM CaCl2-2H2O, the gel-forming behavior observed was closely similar to the spontaneous gel-formation of the awkeotsang extract. These results may support the possibility that the high amount of calcium ions (4.0-5.6 %) in an aqueous extract of the seeds contributes to the egg-box formation, by being placed between the de-esterified structurally regular polygalacturonide chains.

Extraction and characterization of pectic substances from pulp of grape berries

Pectic substances were successively extracted from the alcohol-insoluble residue (AIR) of the pulp of grape berries, by water (WSP), oxalate (OXP), hot dilute HCl (HP) and cold dilute NaOH (OHP). Pectins (WSP, OXP, HP) were purified by ion-exchange chromatography (DEAE-Sephacel) or precipitation with cupric ions (OHP). Total pectic substances represent 20·8% (w/w) of the AIR, WSP and HP being the main components (6% and 12% of the AIR, respectively). An alternative to oxalate extraction of the water-insoluble pectin was extraction with NaCl solutions of increasing concentrations, which had released small amounts of pectins. Each of the fractions contained mainly galacturonic acid, arabinose and galactose, lower amounts of rhamnose and xylose, and minor amounts of glucose and mannose. Ion-exchange chromatography was performed on DEAE-Sephacel, M w distribution was checked by gel permeation on Sepharose CL-2B, and M v was determined as well as degrees of methylesterification (DM), acetylation (DA), and protein content.