Tove M.I.E. Christensen

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)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger.

A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.

Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillusniger

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.

Chemically methylated and reduced pectins: preparation, characterisation by 1H NMR spectroscopy, enzymatic degradation, and gelling properties

The gelling properties of pectins are known to be closely related to the degree of methylation (DM) and the distribution of the ester groups. In order to investigate this dependency, a natural citrus pectin (DM 64%) has been methylated to pectins with higher DM or saponified to achieve pectins with lower DM. A simple method for determination of DM by 1H NMR spectroscopy is presented. New modified pectins have been prepared by treatment of pectins having different DM with NaBH(4) to reduce selectively the methyl esters to primary alcohols in the presence of free acids. The degree of reduction (DR) and the DM of the remaining carboxylic acids could likewise be determined by 1H NMR spectroscopy. The new reduced pectins are recognized by the pectin degrading enzymes polygalacturonase PGI and PGII as well as by pectin lyase, all from Aspergillus niger, but the enzymes exhibit lower specific activities as compared with unmodified pectin. The new reduced pectins exhibit high gelling properties.

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Abstract. Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with Km values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.

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.

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme.

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.

Distribution of pectin methyl esterase and acetylesterase in the genus Citrus visualized by tissue prints and chromatography

The distribution of pectin methyl esterase (PME) and acetylesterase (AE) in orange, lime, lemon, grapefruit and clementine fruits has been investigated. Isoforms from PME and AE have been separated by cation exchange chromatography. PME isoforms separated in two main groups for all citrus extracts. For AE isoforms, only one major AE isoform was seen in orange, whereas several isoforms were separated in extracts from grapefruit, lime and lemon. The overall localization of AE and PME in the different citrus fruits was investigated by tissue print immunolocalization. In orange, grapefruit and clementine, PME was immunolocalized in the juice vesicles, whereas the PME localization in the juice vesicles was significantly weaker in lemon and lime. The distribution of AE in the juice vesicles differed from the distribution of PME in the sense that PME was more restricted to the periphery of the juice vesicles, whereas AE was also found in the more central parts of the juice vesicles. The immunological detection indicated that PME is involved in the ripening process, but this is probably not the only function for AE. The distribution of AE in paraffin sections of developing lime fruits showed that AE was present in almost all parts of the fruit deposited in the cell wall and intracellularly.

Application of mass spectrometry to determine the activity and specificity of pectin lyase A.

Electrospray ionization (ESI) with quadrupole ion-trap mass spectrometry was used to assess the activity and specificity of the enzyme pectin lyase A (PLA) (EC 4.2.2.10) on model pectins with varying degrees and patterns of methyl esterification. PLA is a pectinase which cleaves alpha-(1-->4)-glycosidic linkages in pectin by a trans-elimination process. Using pectins with different degrees and patterns of methyl esterification, there was a significant variation in the activity rate of PLA. The enzymatic products generated at various time intervals were structurally analyzed by mass spectrometry to determine the specificity of PLA. Although the preferred substrate for PLA is fully methyl esterified polygalacturonate, cleavage was also observed with a non-methyl esterified galacturonic acid residue on either the non-reducing end or the reducing end. The current study shows that although PLA prefers fully methyl esterified substrates it can also accept partially esterified ones. It also demonstrates the suitability of ESI ion-trap mass spectrometry in determining enzyme specificities.

Isolation, characterization and immuno localization of orange fruit acetyl esterase

Abstract Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with K M of 39 mM and K M of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. Immuno localization using polyclonal antibodies raised against AE showed that AE was widely distributed in orange fruit but with more intensive immunological detection in the outer part of the peels e.g. albedo and flavedo and in the segments (juice vesicles). The results indicate that AE is located at the site where the major fraction of pectin is deposited.