A tale of two pectins: Diverse fine structures can result from identical processive PME treatments on similar high DM substrates.

The effects of a processive pectin-methylesterase (PME) treatment on two different pectins, both possessing a high degree of methylesterification (DM), were investigated. While the starting samples were purportedly very similar in fine structure, the intermolecular DM distributions arising from their PME treatments were strikingly different. Herein, a simulation that illuminates the origin of this phenomenon is described. It is concluded that: (1) very different low-DM samples (with the same average DM) can be generated using the same processive PME, simply by a judicious choice of the high DM starting material; (2) observing the intermolecular DM distribution of the products of processive-PME-processing is an extremely sensitive discriminator of the fine structure of high DM starting materials; and (3) for PMEs with unknown action patterns the processive nature of the enzyme is most simply revealed by studying the changes it induces in the intermolecular DM distribution of very-highly-methylesterified homogalacturonans.

Recovery and fine structure variability of RGII sub-domains in wine (Vitis vinifera Merlot).

BACKGROUND AND AIMS: Rhamnogalacturonan II (RGII) is a structurally complex pectic sub-domain composed of more than 12 different sugars and 20 different linkages distributed in five side chains along a homogalacturonan backbone. Although RGII has long been described as highly conserved over plant evolution, recent studies have revealed variations in the structure of the polysaccharide. This study examines the fine structure variability of RGII in wine, focusing on the side chains A and B obtained after sequential mild acid hydrolysis. Specifically, this study aims to differentiate intrinsic structural variations in these RGII side chains from structural variations due to acid hydrolysis. METHODS: RGII from wine (Vitis vinifera Merlot) was sequentially hydrolysed with trifluoroacetic acid (TFA) and the hydrolysis products were separated by anion-exchange chromatography (AEC). AEC fractions or total hydrolysates were analysed by MALDI-TOF mass spectrometry. KEY RESULTS: The optimal conditions to recover non-degraded side chain B, side chain A and RGII backbone were 0·1 m TFA at 40 °C for 16 h, 0·48 m TFA at 40 °C for 16 h (or 0·1 m TFA at 60 °C for 8 h) and 0·1 m TFA at 60 °C for 16 h, respectively. Side chain B was particularly prone to acid degradation. Side chain A and the RGII GalA backbone were partly degraded by 0·1 m TFA at 80 °C for 1-4 h. AEC allowed separation of side chain B, methyl-esterified side chain A and non-methyl-esterified side chain A. The structure of side chain A and the GalA backbone were highly variable. CONCLUSIONS: Several modifications to the RGII structure of wine were identified. The observed dearabinosylation and deacetylation were primarily the consequence of acidic treatment, while variation in methyl-esterification, methyl-ether linkages and oxidation reflect natural diversity. The physiological significance of this variability, however, remains to be determined.

Microwave heating for the rapid generation of glycosylhydrazides.

Conditions for simple derivatization of reducing carbohydrates via adipic acid dihydrazide microwave-assisted condensation are described. We demonstrate with a diverse set of oligo- and polysaccharides how to improve a restrictive and labor intensive conventional conjugation protocol by using microwave-assisted chemistry. We show that 5 min of microwave heating in basic or acidic conditions are adequate to generate, in increased yields, intact and functional glycosylhydrazides, whereas hours to days and acidic conditions are generally required under conventional methods.

Monoclonal antibodies to rhamnogalacturonan I backbone.

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides--BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [-->2)-alpha-L-rhamnosep-(1-->4)-alpha-D-galacturonic acid p-(1-->](7) structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose-galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.

Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.

The phenolic fraction of maize bran: evidence for lignin-heteroxylan association.

Maize bran heteroxylan samples were extracted in various conditions of severity. Their ferulate and diferulate content was investigated by GC-MS of methyl ester-TMSi derivatives. When extracted by 0.5 M NaOH in mild conditions, the heteroxylan sample contained a low level of ferulic acid (0.032% by wt.) and the main diferulate surviving alkaline extraction was found to be the 8-8' diferulate. On peroxidase treatment, this sample nevertheless produced a firm and brittle gel without any change in the diferulate profile. Typical lignin structures, mainly comprising syringyl units interconnected through beta-O-4, beta-1 and beta-beta interunit bonds, were evidenced in the maize bran sample. More importantly, these lignin structures were found to be tightly associated with the alkali-extracted heteroxylans. Thioacidolysis revealed the occurrence of 0.1-0.5% (by wt.) lignin structures in heteroxylan fractions extracted in mild or severe conditions, before and after purification of the polysaccharides. The gelling potential of the heteroxylan fractions was not only dependent on their ferulate level, but also influenced by associated lignin structures. These results argue for the occurrence of covalent linkages between heteroxylan chains and lignin structures which could participate in the peroxidase-driven gelation of feruloylated polysaccharides. They demonstrate the role of low lignin levels in the organization of native or reconstructed polysaccharide networks.

Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

Soybean beta-conglycinin alpha subunit is phosphorylated on two distinct serines by protein kinase CK2 in vitro.

Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean beta-conglycinin alpha subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. Beta-conglycinin alpha subunit (68 kDa) presents seven consensus sequences, but only 0.5-1 mol P/mol alpha subunit was incorporated by CK2. [32P]Phosphorylated beta-conglycinin alpha subunit was cleaved either by cyanogen bromide or by trypsin. 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70-89, 116-126, and 116-127 sequences. Over the seven consensus sequences of beta-conglycinin alpha subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites.

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry.

Beta-conglycinin alpha subunit has been phosphorylated using a cAMP-independent protein kinase (CK2) purified from the yeast Yarrowia lipolytica. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Asp/Glu. Only 0.5 to 1 mol P/mol alpha subunit was incorporated although seven consensus sequences are present. Phosphorylated beta-conglycinin alpha subunit (P-alpha) was digested by trypsin. The resulting peptides were analysed by RP-HPLC coupled to electrospray ionisation mass spectrometry (LC-ESMS). Two phosphopeptides were identified corresponding to 70-89 and 116-127 sequences with Ser 75 and Ser 117 phosphorylated respectively. Ser 75 is one of the predicted phosphorylation sites according to the consensus sequence criteria. Ser 117 is inside a very acidic peptide but does not belong to a previously described consensus sequence.

Specificity of an esterase (XYLD) from Pseudomonas fluorescens subsp. cellulosa.

Activity of an esterase from Pseudomonas fluorescens subsp. cellulosa (XYLD) on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran) was dependent on the source of added endo-xylanase. The esterase exhibited high selectivity for the nature, position of linkage and size of the feruloylated oligosaccharides generated by hydrolysis of the hemicellulose. Increased affinity of XYLD with increasing size of the oligosaccharide substrate suggests that optimal activity is observed on substrates with at least 4 sugars.

Isolation and purification of feruloylated oligosaccharides from cell walls of sugar-beet pulp.

Cell walls from sugar-beet pulp contain some feruloyl groups linked to the pectic neutral side-chains. Enzymic as well as chemical hydrolysis of the pulp yielded a series of feruloylated oligosaccharides, which have been purified by Sephadex LH-20 and Biogel P-2 chromatography in aqueous solvents. Feruloylated arabinose di-, tri-, hexa-, hepta-, and octa-saccharides as well as feruloylated galactose disaccharides were obtained after hydrolysis of the pulp with a mixture of fungal carbohydrases (Driselase). Feruloylated arabinose and galactose monosaccharides were obtained through mild acid hydrolyses. Both arabinose and galactose residues in the side-chains are feruloylated, 50-55% of the feruloyl groups being linked to arabinose residues and 45-50% to galactose residues. It is concluded that 1 out of 56 arabinose residues and 1 out of 16 galactose residues present as pectic side-chains in sugar-beet pulp carry a feruloyl group.

Structure identification of feruloylated oligosaccharides from sugar-beet pulp by NMR spectroscopy.

1D NMR (1H and 13C) and 2D NMR spectroscopy have been used to determine the structure of feruloylated oligosaccharides obtained by enzymic degradation or mild acid hydrolysis of sugar-beet pulp. Feruloylated oligosaccharides derived from pectic neutral side-chains containing arabinose or galactose residues were identified. In the feruloylated arabinose oligosaccharides, feruloyl groups were linked to O-2 of L-Ara f residues. The structure of the feruloylated arabinose disaccharide was identified as O-[2-O-(transferuloyl)-alpha-L-Ara f]-(1-->5)-L-Ara f and that of the feruloylated arabinose trisaccharide as O-alpha-L-Ara f-(1-->3)-O-[2-O-(trans-feruloyl)-alpha-L-Ara f]-(1-->5)-L- Ara f. The structure of the feruloylated galactose disaccharide was identified as O-[6-O-(trans-feruloyl) -beta-D-Gal p]-(1-->4)-D-Gal p. From our results, we suggest that the feruloyl groups present in sugar-beet pulp are linked to the arabinofuranosyl residues of the main core of alpha-(1-->5)-linked arabinan chains and to the galactopyranosyl residues of the main core of beta-(1-->4)-linked type I galactan chains.

Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger.

The activity of two forms of ferulic acid esterase (FAE) from Aspergillus niger on a synthetic feruloylated substrate (methyl ferulate) and on 11 different feruloylated oligosaccharides from sugar-beet pulp and wheat bran was determined. The enzymes exhibited different specificities for the various feruloylated substrates and were more active on certain substrates of cell-wall origin than on methyl ferulate. Both enzymes preferred the arabinose residue to which ferulic acid is attached in the furanose form. FAE-I had no clear preference for the type of linkage involved between the ferulic acid units and the oligosaccharide chain. In contrast, FAE-III had a clear requirement for ferulic acid to be attached to O-5 of the Ara f ring while no catalysis was observed when ferulic acid was attached to O-2. Both enzymes showed maximum activity on feruloylated trisaccharides. An increase in the length of the oligosaccharide chain did not preclude catalysis, but feruloylated oligosaccharides of a dp > 3 were hydrolysed at a reduced rate. Our results support the hypothesis that different kinds of ferulic acid esterases exist with different specificities for the oligosaccharide chain of the feruloylated substrates.