Feruloylated xyloglucan and p-coumaroyl arabinoxylan oligosaccharides from bamboo shoot cell-walls.

A novel feruloylated xyloglucan disaccharide and p-coumaroylated arabinoxylan trisaccharide were isolated from cell walls of growing bamboo (Phyllostachys edulis) shoots. On the basis of chemical and spectral data, their structures were determined to be O-(4-O-trans-feruloyl-alpha-D- xylopyranosyl)-(1----6)-D-glucopyranose and O-[5-O-(trans-p-coumaroyl)- alpha-L-arabinofuranosyl]-(1----3)-O-beta-D-xylopyranosyl-(1----4)-D- xylopyranose, respectively. This is the first reported evidence of a phenolic acid covalently associated with the cell wall hemicellulose, xyloglucan.     

Intracellular feruloylation of arabinoxylan in wheat: evidence for feruloyl-glucose as precursor

Incorporation of [(3)H]arabinose and [(14)C]ferulic acid into soluble and polymeric fractions from suspension-cultured wheat (Triticum aestivum L.) cells and the corresponding extracellular medium was studied. The major part of these products was identified as arabinoxylan and two proteins of 40 and 100 kDa. The time course suggests an intracellular synthesis of feruloylated arabinoxylan with feruloyl-glucose as substrate. In contrast, synthesis of feruloylated proteins appears to occur with feruloyl-CoA as precursor. Intracellular formation of ferulic acid dimers is limited to 8,5'-diferulic acid, while other dimers appear to be formed extracellularly. [(3)H]Arabinose was incorporated into polymeric material in both the cellular and in the medium fraction while [(14)C]ferulic was only found in polymers from the cellular fraction, indicating synthesis of both feruloylated and non-feruloylated arabinoxylan by the cells.     

Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure making material more accessible to glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : III. Structural Changes in the Feraxan during Coleoptile Elongation

Changes in structural features of feraxan (feruloylated arabinoxylans) in cell walls during development of maize (Zea mays L.) coleoptiles were investigated by analysis of fragments released by feraxanase, a specific enzyme purified from Bacillus subtilis. The following patterns were identified: (a) The total quantity of carbohydrate dissociated from a given dry weight of cell wall by feraxanase remained rather constant throughout the initial 10 days of coleoptile development. However, during the same period the proportion of ferulic acid in the fraction increased 12-fold. The absolute amount of ferulic acid per coleoptile also increased rapidly during this developmental phase. (b) Fragments dissociated by the enzyme were resolved into feruloylated and nonferuloylated components by reversed phase chromatography. While the quantity of feruloylated fractions represented a small portion of the total arabinoxylan during the phase of maximum coleoptile elongation (days 2-4) this component increased in abundance to reach a plateau (after 8-10 days). In contrast, nonferuloylated fractions were most abundant during the stage of maximum elongation but declined to a constant level by day 6. (c) Glycosidic linkage analysis of carbohydrate reveals that substitution of the xylan backbone of feraxan by arabinosyl residues decreased during coleoptile growth. We conclude that significant incorporation of ferulic acid occurs and/or more feruloyated domains are added to the arabinoxylan during development. This augmentation in phenolic acids is accompanied by a concerted displacement of arabinosyl residues and/or a reduction in the incorporation of regions enriched in arabinosyl sidechains.     

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

Arabinoxylan and mono- and dimeric ferulic acid release from brewer’s grain and wheat bran by feruloyl esterases and glycosyl hydrolases from<Emphasis Type="Italic"> Humicola insolens</Emphasis>

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewers grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5 benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewers grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : II. Fractionation and Partial Characterization of Feraxan Fragments Dissociated by a Bacillus subtilis Enzyme (Feraxanase)

Structural features of feruloylated arabinoxylan (feraxan) present in Zea mays L. (hybrid B 73 × Mo 17) coleoptile cell walls have been studied using a purified feraxan-dissociating enzyme (feraxanase) and an α-arabinofuranosidase. This experimental approach has demonstrated the following. (a) Feraxanase dissociated ca. 20% (dry weight basis) of the maize wall preparation. The predominant oligosaccharides enzymically liberated were allocated into seven major subfractions designated A-1 (0.8%), B-1 (1.6%), B-2 (2.4%), B-3 (4.6%), C-1 (1.0%), C-2 (4.2%), and C-3 (0.3%). Values in parentheses reflect the percentage of the wall associated with each subfraction. Subfractions represent samples enriched in different degrees of polymerization, sugar composition, linkage arrangements, and phenolic acid content. (b) B-1, B-2, and B-3 fractions are not feruloylated and have smaller molecular mass (less than 10⁴ kilodaltons) and consist chiefly of t-arabinosyl-5-arabinosyl, 4-xylosyl, 2,4/3,4-xylosyl, and glucuronosyl residues, suggesting that these fragments constitute nonferuloylated regions of arabinoxylan. (c) C-2 and C-3 fractions contain ferulic acid (6.2% and 12.1%, respectively) and are similar to the B series in their sugar linkage arrangements but were derived from feruloylated regions. (d) Alkali treatment of the C-2 fraction decreases the molecular size of the fragment and liberates phenolic acids. The results suggest the presence of alkaline-labile links, probably diferulate bridges. (e) A-1 and C-1 fractions are larger (more than 5 × 10⁵ kilodalton) and contain t-galactosyl-, 4-galactosyl, 2,4-rhamnosyl-residues, galacturonic acid, and the sugar linkage arrangements common to other fractions. The A-1 fraction is not feruloylated, whereas C-1 fraction contains 0.5% ferulic acid. The presence of galactose, rhamnose, and galacturonic acid suggests that pectic polymers, probably homopolygalacturonans and rhamnogalacturonans, are linked to nonferuloylated and feruloylated segments of arabinoxylans.     

Acetylation at O-2 of arabinofuranose residues in feruloylated arabinoxylan from bamboo shoot cell-walls.

Hydrolysis of bamboo shoot cell-walls with Driselase (a fungal enzyme preparation) gave an arabinoxylan trisaccharide with ferulic and acetic acids as ester groups. The structure of this oligosaccharide was determined to be O-[2-O-acetyl-5-O-[E)-feruloyl)-alpha-L-arabinofuranosyl]-(1----3) -O-beta- D-xylopyranosyl-(1----4)-D-xylopyranose, on the basis of spectroscopy and methylation analysis.     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Inhibition of Gibberellin-Induced Elongation Growth of Rice by Feruloyl Oligosaccharides

The biological activity of cell wall-derived feruloyl oligosaccharideswas investigated using a modified micro-drop bioassay. A feruloylarabinoxylan trisaccharide (FAXX) and a feruloyl arabinoxylantetrasaccharide (FAXXX) were found to inhibit the gibberellin-inducedelongation of dwarf rice (Oryza sativa L., cv, Tan-ginbozu)that had been treated with uniconazole (S-3307), an inhibitorof the biosynthesis of gibberellins. An arabinoxylan trisaccharide(AXX) was ineffective. The growth-inhibitory effecf of feruloyloligosaccharides depended on their feruloyl and glycosyl moieties.The amount of esterified diferulic acid residues in the cellwalls of the second leaf sheath of rice seedlings that had beentreated with FAXX was almost same as that of controls. Feruloyloligosaccharides did not inhibit the incorporation of [¹⁴C]leucineinto acid-precipitable proteins by suspension-cultured maizecells, whereas cinnamic acid and its derivatives strongly inhibitedsuch incorporation. (Received May 10, 1995; Accepted August 23, 1995)     

Isolation and structural identification of complex feruloylated heteroxylan side-chains from maize bran

Three complex heteroxylan side-chains acylated with ferulate and one arabinosyl ester of p-coumaric acid have been isolated from maize bran insoluble fibre after acidic hydrolysis and fractionation by gel permeation chromatography and semi-preparative RP-HPLC. The complete structural elucidation of all isolated compounds was achieved by 1D/2D NMR spectroscopy and HPLC-MS in combination with methylation analysis. The absolute configuration of the carbohydrate constituents was determined by chiral GC after acidic hydrolysis and trifluoroacetylation. The identified feruloylated tetrasaccharides alpha-d-xylopyranosyl-(1-->3)-alpha-l-galactopyranosyl-(1-->2)-beta-d-xylopyranosyl-(1-->2)-5-O-trans-feruloyl-l-arabinofuranose (FAXGX) and alpha-d-galactopyranosyl-(1-->3)-alpha-l-galactopyranosyl-(1-->2)-beta-d-xylopyranosyl-(1-->2)-5-O-trans-feruloyl-l-arabinofuranose (FAXGG) are the most complex heteroxylan side-chains from maize bran that have been isolated to date. The isolated trisaccharide alpha-l-galactopyranosyl-(1-->2)-beta-d-xylopyranosyl-(1-->2)-5-O-trans-feruloyl-l-arabinofuranose (FAXG) contributes to the complexity of heteroxylan side-chains from maize bran and 5-O-trans-p-coumaroyl-l-arabinofuranose represents the first p-coumaroylated heteroxylan side-chain isolated from cereal grains. Complex feruloylated heteroxylan side-chains are possibly, like ferulate cross-linking of the heteroxylans and binding of heteroxylans to lignin, a factor contributing to limited enzymatic degradation of fibre.     

Structures of the Oligosaccharides from the Enzymic Hydrolyzate of Rice-straw Arabinoxylan by a Xylanase of Aspergillus niger

A trisaccharide consisting of two d-xylose units and one l-arabinose unit, and a tetrasaccharide consisting of three d-xylose units and one l-arabinose unit were isolated from the hydrolyzate of rice-straw arabinoxylan by the xylanase I produced by Asp. niger.

The structures of the trisaccharide and the tetrasaccharide were determined to be 3¹-α-l-arabinofuranosylxylobiose ([α]_d^? 80°) and 3¹-α-l-arabinofuranosylxylotriose ([α]_d^? 84°), respectively, by chemical and enzymic methods.

According to the structures of two arabinose-xylose mixed oligosaccharides, it was shown that the rice-straw arabinoxylan is composed of chain of 1,4-linked βd-xylopyranose residues and some of xylose residues have side-chain of 1,3-linked α-l-arabinofuranose.     

Enzymatic fingerprinting of arabinoxylan and β-glucan in triticale, barley and tritordeum grains

Enzymatic fingerprinting of arabinoxylan (AX) and β-glucan using endo-xylanase and lichenase, respectively, helps determine the structural heterogeneity between different cereals and within genotypes of the same cereal. This study characterised the structural features of AX and β-glucan in whole grains of eight triticale cultivars grown at two locations, 20 barley cultivars/lines with wide variation in composition and morphology and five tritordeum breeding lines. Principal component analysis (PCA) resulted in clear clustering of these cereals. In general, barley and tritordeum had a higher relative proportion of highly branched arabinoxylan oligosaccharides (AXOS) than triticale. Subsequent analysis of triticale revealed two clusters based on growing region along principal component (PC) 1, while PC2 explained the genetic variability and was based on mono-substitution and di-substitution in AX fragments. PCA of β-glucan features separated the three cereals based on β-glucan content. The molar ratio of trisaccharide to tetrasaccharide was 2.5-3.4 in triticale, 2.3-3.3 in barley and 2.8-3.4 in tritordeum. Barley showed a strong positive correlation (r=0.86) between β-glucan content and relative proportion of trisaccharide. The results show that structural features of AX and β-glucan vary between and within triticale, barley and tritordeum grains which might be important determinants of end-use quality of grains.     

Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn²⁺. Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.     

Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran

Feruloylated arabinoxylans isolated from wheat flour and wheat bran were compared in their cross-linking behaviour with respect to viscosity properties and cross-linking products formed when various oxidative agents were applied to dilute solutions. Optimal conditions for each oxidative agent were investigated. In case of hydrogen peroxide and peroxidase, similar conditions were found for both types of arabinoxylans but wheat bran arabinoxylans gave a larger viscosity increase upon cross-linking than those of wheat flour.

When glucose, glucoseoxidase and peroxidase or ammonium persulphate were used as oxidative agents, differences in the concentration of reagent needed to induce cross-linking and in viscosity increase were observed. The distribution of coupling products for both types of arabinoxylans and the different oxidative treatments was approximately 5 : 3 : 1 : 1 for 8-5, 8-O-4, 8-8 and 5-5, respectively. The low ferulate recovery after oxidative treatment was assumed to be caused by formation of unknown compounds, such as higher oligomers and lignin-linked products.

A 1 : 1 mixture of flour arabinoxylan and feruloylated pectin showed a maximum synergistic effect on viscosity upon oxidative treatment using hydrogen peroxide and peroxidase. Both polysaccharides were shown to participate in cross-linking.     

The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk)

The physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of Plantago ovata Forsk seed husk (psyllium seed) and some derived partial hydrolysis products were studied by compositional and methylation analysis and NMR spectroscopy. Resolving the conflicting claims of previous investigators, the material was found to be a neutral arabinoxylan (arabinose 22.6%, xylose 74.6%, molar basis; only traces of other sugars). With about 35% of nonreducing terminal residues, the polysaccharide is highly branched. The data are compatible with a structure consisting of a densely substituted main chain of beta-(1-->4)-linked D-xylopyranosyl residues, some carrying single xylopyranosyl side chains at position 2, others bearing, at position 3, trisaccharide branches having the sequence L-Araf-alpha-(1-->3)-D-Xylp-beta-(1-->3)-l-Araf. The presence of this sequence is supported by methylation and NMR data, and by the isolation of the disaccharide 3-O-beta-D-xylopyranosyl-L-arabinose as a product of partial acid hydrolysis of the polysaccharide.     

Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.     

Divergent selection for ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and degradability

Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (CLs). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8–8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged.     

The Structure of Plant Cell Walls: VII. Barley Aleurone Cells

The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls.