Methyl-esterified Oligogalacturonides Research Articles

Environmentally Friendly Hydrothermal Processing of Melon by-Products for the Recovery of Bioactive Pectic-Oligosaccharides.

Melon by-products, that currently lack high value-added applications, could be a sustainable source of bioactive compounds such as polysaccharides and antioxidants. In this work, melon peels were extracted with water to remove free sugars, and the water-insoluble solids (WISs) were subjected to hydrothermal processing. The effect of temperature on the composition of the obtained liquors and their total phenolic content was evaluated. The selected liquors were also characterized by matrix assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF MS), fourier transform infrared spectroscopy (FTIR) and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and its phenolic compounds were identified and quantified by high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC–DAD–MS/MS). In addition, the spent solids from the hydrothermal treatment were characterized and their potential use was assessed. At the optimal conditions of 140 °C (severity 2.03), the total oligosaccharide yield accounted for 15.24 g/100 g WIS, of which 10.07 g/100 g WIS were oligogalacturonides. The structural characterization confirmed the presence of partially methyl esterified oligogalacturonides with a wide range of polymerization degrees. After precipitation, 16.59 g/100 g WIS of pectin were recovered, with a galacturonic acid content of 55.41% and high linearity.

Differential fragmentation patterns of pectin oligogalacturonides observed by nanoelectrospray quadrupole ion‐trap mass spectrometry using automated spectra interpretation

Oligogalacturonides of different degrees of polymerization (DP) and methyl esterification (DE) were structurally analyzed by nanoESI quadrupole ion-trap mass spectrometry. The fragmentation patterns of the oligogalacturonides were compared using the program 'Virtual Expert Mass Spectrometrist' (VEMS) for structural annotation. In the analyzed oligogalacturonides of lower DP, the generation of C/Y ions, i.e. ions retaining the glycosidic oxygen, was higher than that of B/Z ions. In general, with oligogalacturonides of higher DP, the B/Z ions were generated more abundantly. Oligogalacturonides with free carboxylic acid groups underwent higher water loss compared to fully methyl-esterified oligogalacturonides under the same fragmentation conditions. Cross-ring cleavage, in which fragmentation occurs across the ring system of the galacturonate residue and signified by unique mass losses, was observed to be higher in fully methyl-esterified oligogalacturonides than in non-methyl-esterified ones. This study demonstrates the different fragmentation patterns of oligogalacturonides as influenced by the presence or absence of methyl ester groups. For a detailed analysis of unknown oligogalacturonides, cross-ring fragmentation gives more structural information than glycosidic bond cleavage. One implication of this is that more structural information is obtained when analyzing methyl-esterified oligogalacturonides than non-methyl-esterified ones in an ion-trap instrument. This is of particular importance in pectin chemistry, where mass spectrometry has become the technique of choice for structural determination. Although this study was not designed to explain the mechanisms of oligogalacturonide fragmentation, possible explanations for why non-methyl-esterified oligogalacturonides undergo more water loss than methyl-esterified ones will be postulated. In addition, the VEMS program was extended to automatically interpret and assign the fragment ions peaks generated in this study.

Resolution of the structural isomers of partially methylesterified oligogalacturonides by polysaccharide analysis using carbohydrate gel electrophoresis

Pectins differing in their degree and pattern of methylesterification are important in diverse aspects of plant physiology and also in many industrial applications. Determination of methylesterification fine structure and knowledge of enzyme specificities in modification and fragmentation of pectin are key to understanding the relationship between structure and function. The development of methodologies for the detection, separation and sequencing of different partially methylesterified oligogalacturonides (Me-OGAs) is consequently very important. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) has been shown to be powerful for the quantitative resolution of species different in degree of polymerization (DP) and/or degree of methylesterification (DM). Mass spectrometry (MS) has, to date, been the only tool with which to obtain isomeric information. However, it is not quantitative, and the presence of isobaric species makes the interpretation of the fragmentation patterns complicated. Here, we present evidence that Me-OGAs with the same DP and DM but different patterns of methylesterification (structural isomers) can easily be separated and quantified using PACE.

On the separation, detection and quantification of pectin derived oligosaccharides by capillary electrophoresis

Having previously reported that capillary electrophoresis can be used as a tool for the analysis of partially methyl-esterified oligogalacturonides we now describe a method that improves the resolution of individual oligomers, and detail a more rigorous quantification scheme that uses an internal standard and takes into account the relative molecular absorbance of different partially methyl-esterified species. The internal consistency of the method is subsequently demonstrated by performing the quantification of an endo-polygalacturonase pectin digest before and after de-methylation of the resultant oligomers.

Structural characterisation by both positive- and negative-ion electrospray mass spectrometry of partially methyl-esterified oligogalacturonides purified by semi-preparative high-performance anion-exchange chromatography.

The off-line coupling of high-performance anion-exchange chromatography (HPAEC) to electrospray ionisation/ion trap mass spectrometry (ESI-ITMS) is described. The Dionex carbohydrate membrane desalter (CMD) has been assessed as an on-line chromatographic desalting system to remove the high sodium concentration necessary for the HPAEC separation of partially methyl-esterified oligogalacturonides. The developed HPAEC configuration proved to be suitable for indirect coupling with ESI-ITMS. This paper provides some interesting features of positive- and negative-ion multistage tandem mass spectrometry (MS(n)) analysis of these acidic oligosaccharides. The spectra acquired in both negative- and positive-ion modes show characteristic fragment ions resulting from glycosidic bond and cross-ring cleavages. Some new mass spectrometric fragmentation routes are also described. The positive-ion mode gave more complex spectra but was as informative as the negative-ion mode. ESI-ITMS was revealed to be, as previously reported from direct use on an unseparated enzymatic digest, a powerful sequencing technique for the determination of linkage type and the methyl ester distribution of partially methyl-esterified oligogalacturonides. Moreover, unlike matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS), it gives valuable information on the elution behaviour of these oligomers in relation to their structure, namely the HPAEC co-elution of isomeric structures.

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C(6)-substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several Delta 4,5 unsaturated products and their saturated counterparts. The Delta 4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 Delta 4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite +2, +4, or -1 to -4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite +1 and +3. So PLA is capable to cleave the bond between a methyl-esterified and a non-esterified galacturonic acid residue, where the newly formed Delta 4,5 unsaturated non-reducing end residue always contains a methyl-ester.

Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and/or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying concentrations of ammonia or methylamine and monitored in time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimized. Amidation was performed best under anhydrous conditions at a concentration of 4 M ammonia or methylamine at ambient temperature. Amidation using methylamine reached almost completeness (DAm 95) without hardly any hydrolysis of methyl-esters while amidation with ammonia reached a DAm of 70 on average. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueous solutions instead off anhydrous ammonia resulted in 6–10% lower DAm values due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagent for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties.

Characterisation of methyl-ester distributions in galacturonan regions of complement activating pectins from the roots of Angelica acutiloba Kitagawa

On digestion with endo-α-(1 → 4)-polygalacturonase the complement activating pectins (AR-2 IIa, IIb, IIc and IId) from the roots of Angelica acutiloba Kitagawa gave long (PG-2) and short (PG-3) oligogalacturonide fractions in addition to rhamnogalacturonan cores possessing neutral carbohydrate side chains. HPLC analysis of 2-aminopyridine-labelled derivatives indicated that PG-3 from the four pectins contained mono- to octa-galacturonides of which penta- to octa-galacturonides were suggested to contain some methyl-esterified GalA. Three major partially methyl-esterified oligogalacturonides (oligo-1, 2 and 3) were purified from PG-3 of AR-2 IIb, and oligo-1, 2 and 3 were suggested to be tetra-, hepta- and hexa-galacturonides which had one, three and two methyl-esters, respectively. Long oligogalacturonide fraction (PG-2) from AR-2 IId contained hepta- to tetradeca-galacturonides possessing some methyl-esters. Carboxyl-reduction of the methyl-esterified GalA followed by lithium degradation gave only galactitol as the final product from PG-2 of AR-2 IId, suggesting that methyl-esterified GalA in PG-2 of AR-2 IId was attached to another methyl-esterified GalA through unesterified GalA in a similar way to the short oligo-galacturonides in PG-3 of AR-2 IIa- IIc.