Methods for obtaining neutral and acid oligosaccharides from flax pectins

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, ¹H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.Revisions requested 24 September 2004; Revisions received 4 November 2004     

Uronic acid-containing oligosaccharins: Their biosynthesis,degradation and signalling roles in non-diseased plant tissues

Pectic oligosaccharides, produced by microbial enzymes, are well-known oligosaccharins, eliciting defence responses in diseased plants. Regulatory roles in non-diseased plants have also been proposed: e.g. oligosaccharides, generated in vitro by hydrolysis of pectins, promote ripening in tomato (Lycopersicon esculentum L.) and other fruits. Endo-polygalacturonase (endo-PG; EC 3.2.1.15) occurs in many fruits and theoretically could generate such oligosaccharides. However, the mere occurrence of an enzyme does not prove that it acts; the proposed products (oligosaccharides) must be sought in vivo. Recent evidence indicates that fruit tissues do naturally produce pectic oligosaccharides, some of which promote ripening when added to unripe fruits. Such studies, complementing earlier work on in-vitro generated oligosaccharides, provide a means of discovering novel oligosaccharins. Unlike fruits, cell cultures of rose (Rosa sp.) produce no pectic oligosaccharides. Their `absence' is not due to excessively rapid turnover: when [<sup>14</sup>C]-oligogalacturonides are added, they undergo only slow hydrolysis. The hydrolysis is by exo-polygalacturonases (exo-PG; EC 3.2.1.67), yielding free galacturonic acid (GalA). If no oligogalacturonides are added, no GalA accumulates in the medium; therefore, exo-PG does not normally operate in healthy rose cells. Exo-PG is presumably `in reserve', available to trim oligogalacturonides made by phytopathogens. We conclude that the in-vivo action of a wall enzyme is best studied at the level of carbohydrate metabolism in vivo rather than by assay of extracted enzymes in vitro. The major uronic acid-containing oligosaccharide produced by rose cell cultures is α-D-mannopyranosyl-(1→4)-α-D-glucuronopyranosyl-(1→2)-myo-inositol, which may be a novel phytoglycolipid-derived oligosaccharin related to the inositolphosphoglycans that mediate insulin action.     

Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation

Wall-associated kinase 1 (WAK1) is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell walls. In order to characterize further the interaction of WAK1 with pectin, a 564 bp DNA sequence corresponding to amino acids 67-254 of the extracellular domain of WAK1 from Arabidopsis thaliana was cloned and expressed as a soluble recombinant peptide in yeast. Using enzyme-linked immunosorbent assays (ELISA), we show that peptide WAK(67-254) binds to polygalacturonic acid (PGA), oligogalacturonides, pectins extracted from A. thaliana cell walls and to structurally related alginates. Our results suggest that both ionic and steric interactions are required to match the relatively linear pectin backbone. Binding of WAK(67-254) to PGA, oligogalacturonides and alginates occurred only in the presence of calcium and in ionic conditions promoting the formation of calcium bridges between oligo-and polymers (also known as 'egg-boxes'). The conditions inhibiting the formation of calcium bridges (EDTA treatment, calcium substitution, high NaCl concentrations, depolymerization and methylesterification of pectins) also inhibited the binding of WAK(67-254) to calcium-induced egg-boxes. The relevance of this non-covalent link between WAK(67-254) and cell wall pectins is discussed in terms of cell elongation, cell differentiation and host-pathogen interactions.     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

Analysis of methylated and unmethylated polygalacturonic acid structure by polysaccharide analysis using carbohydrate gel electrophoresis

The electrophoretic migration in polyacrylamide gels of oligogalacturonic acids (OGAs) derivatized by a fluorophore (2-aminoacridone) was studied. We found conditions such that OGAs can be separated up to a degree of polymerization (DP) of 40. The migration was dependent on degree of methylation and DP, because the OGA mobility relies on the charge of the galacturonic acid residues. Since both methylated and unmethylated oligosaccharides can be resolved, polysaccharide analysis using carbohydrate gel electrophoresis (PACE) is a powerful method for studying the fingerprint of pectin hydrolysis. It can be used to characterize endopolygalacturonase (Endo-PG) tolerance of methylation. Furthermore, using an Endo-PG that can distinguish low and highly methylated pectin, PACE can be used to investigate the blockwise or nonblockwise distribution of methylation of polygalacturonic acid. We show that the method can be applied to crude cell wall preparations of Arabidopsis inflorescence stems. Using chemical deesterification before or after Endo-PG digestion, we show that in the Arabidopsis cell wall, the pectins have both nonesterified and highly esterified regions.     

Oligogalacturonans production by free radical depolymerization of polygalacturonan

The large production of acidic oligosaccharides was investigated by non-enzymatic depolymerization of polygalacturonic acid (PGA) using free hydroxyl radical hydrolysis process from H(2)O(2)/copper (II) system. A large amount of oligogalacturonides (OGAs) with degrees of polymerization up to 6 were fractionated, and characterized by ESI-Q/TOF-MASS spectrometry and (1)H NMR spectroscopy. This efficient production of uronic oligosaccharides from PGA constitutes an original process to produce bioactive compounds in large scale up.     

Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight

Gentiooligosaccharides and alternansucrase gentiobiose acceptor products were fractionated by their degree of polymerization (DP) on a Bio-Gel P2 column. Fractions were characterized by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, and incubated with human faecal bacteria under anaerobic conditions at 37 degrees C. The growth of predominant gut bacteria on the oligosaccharides was evaluated by fluorescence in situ hybridization and a prebiotic index (PI) was calculated. Lower DP gentiooligosaccharides (DP2-3) showed the highest selectivity (PI of 4.89 and 3.40, respectively), whereas DP4-5 alternansucrase gentiobiose acceptor products generated the greatest values (PI of 5.87). The production of short-chain fatty acids was also determined during the time course of the reactions. The mixture of DP6-10 alternansucrase gentiobiose acceptor products generated the highest levels of butyric acid but the lowest levels of lactic acid. Generally, for similar molecular weights, alternansucrase gentiobiose acceptor products gave higher PI values than gentiooligosaccharides.     

Mode of action of pectin lyase A of Aspergillus niger on differently C(6)-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.     

L-Altruronic acid formed by epimerization of D-galacturonic acid methyl esters during saponification of citrus pectin

While searching for oligosaccharides containing rhamnose residues in the endopolygalacturonase (EPG) digest of saponified citrus pectin, we found several oligomers containing, in addition to galacturonic acid, a sugar previously unreported in pectin. The 1- and 2-D 1H NMR spectra of the oligosaccharides were consistent with the sugar being a uronic acid with its 2- and 3-hydroxyls being axial and 4-hydroxyl being equatorial. MALDI-TOF mass spectrometry indicated that the oligomers consisted solely of uronic acids. Reduction of the uronic acids in the oligosaccharides converted them to galactose and altrose. The altrose was found to be the L enantiomer by comparison of its trimethylsilyl (-)-2-butyl glycosides to those of authentic D-altrose and a racemic mixture. The sugar was not found in oligosaccharides prepared from EPG digestion of citrus pectin deesterified with pectin methylesterase rather than saponification. Thus, it appears that during saponification, a small proportion of the methylesterified galacturonic acid residues in pectins is epimerized at C-5 leading to formation of L-altruronic acid residues.     

Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosaccharides

AIMS: To compare the in vitro fermentation properties of pectins and oligosaccharides derived from them in pure and mixed faecal cultures. METHODS AND RESULTS: Specific growth rates of selected bacterial genera were calculated in pure culture. Bifidobacterium angulatum, B. infantis and B. adolescentis had higher growth rates on pectic oligosaccharides (POS I) derived from high methylated pectin (HMP) than on HMP and B. pseudolongum and B. adolescentis on pectic oligosaccharides (POS II) derived from low methylated pectin than on HMP. Controlled pH batch mixed faecal cultures were then carried out and a prebiotic index was calculated as a mean to compare the fermentation properties of the different substrates. In general, greater fermentation selectivity was obtained with lower degrees of methylation (PI24(-HMP) = -0.11, PI24(-LMP) = 0.033; PI24(-POS I) = 0.071 and PI24(-POS II) = 0.092). An effect of size on prebiotic potential was observed, with the oligosaccharides having more selective fermentation properties than the pectins they derived from. CONCLUSIONS: The degree of methylation plays an important role in the fermentation properties of pectins. Pectic-oligosaccharides are a better prebiotic candidate than the pectins, although their bifidogenic effect is low compared to oligofructose. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of size on prebiotic potential was demonstrated. Non-selectively fermented polysaccharides like pectin can have their bifidogenic properties improved by partial hydrolysis.     

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

High-performance anion-exchange chromatography DAD as a tool for the identification and quantification of oligogalacturonic acids in pectin depolymerisation

High-performance anion-exchange chromatography (HPAEC) coupled with a diode array detector (DAD) was used to identify and quantify oligogalacturonic acid components in pectins. Purified pectin lyase and polygalacturonase were used to generate unsaturated and saturated oligomers from pectins and sodium polygalacturonate, respectively. This method resulted in a good separation of saturated and unsaturated oligomers up to DP 13. It allowed us to follow polygalacturonase and pectate lyase depolymerisation pathways simultaneously.     

两步法硫酸水解热凝胶生产β-1,3-葡寡糖

对硫酸水解热凝胶制备β-1,3-葡寡糖进行了研究。首先建立了葡寡糖的分析方法,采用半制备HPLC分离热凝胶水解液得到β-1,3-葡寡糖,利用ESI-MS和TLC技术对分离组分进行了成分鉴定。考察了反应温度和反应时间对热凝胶寡糖收率的影响,结果表明,两步法硫酸水解获取低聚合度β-1,3-热凝胶寡糖(DP 2~6)的较优水解条件为:1.0 mol/L硫酸于70℃水解1%热凝胶6h,回收水解残留物后于80℃继续水解3 h;对于较高聚合度寡糖(DP 7~10)两步水解时间分别为4 h和1 h较为合适。     

Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides

This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.     

Effect of successive acid and enzymatic hydrolysis on the structure and antioxidant activity of pectins

It has been shown that the treatment of pectins under conditions close to those in an artificial gastroenteral medium results in the destruction of their carbohydrate chain. The degree of pectin destruction depends on the structural features of their macromolecules. During successive acid and enzymatic hydrolysis of pectins, an increase in the number of molecules with molecular masses of 300–400 and 100–300 kDa and cleavage of mono- and oligosaccharides occurred. It was found that comaruman, bergenan, potamogetonan, pectins from marsh cinquefoil, Siberian tea, and broad-leaved pondweed possess a high antioxidant activity and contain large amounts of common phenols. Treatment with hydrochloric acid and pectinase led to a significant decrease in their antioxidant activity and simultaneously to a decrease in the amount of common phenols.     

Linear beta-1,3 glucans are elicitors of defense responses in tobacco

Laminarin, a linear beta-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 microg mL(-1) laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H(2)O(2). After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor beta-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight beta-1, 3-1,6 glucans from fungal cell walls, and the beta-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with beta-glucans are specific to linear beta-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 microg mL(-1) laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.     

Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.     

New monolithic enzymatic micro-reactor for the fast production and purification of oligogalacturonides

Fast production and purification of alpha-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 degrees C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.     

Elicitor and resistance-inducing activities of beta-1,4 cellodextrins in grapevine, comparison with beta-1,3 glucans and alpha-1,4 oligogalacturonides

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.