Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.     

Acidic Arabinoxylan as an Exfracellular Polysaccharide of Suspension-Cultured Soybean Cells

An extracellular acidic arabinoxylan was obtained from the mediumof suspension-cultured soybean cells. The polysaccharide hada linear ß-1,4-linked D-xylopyranosyl backbone withboth neutral and acidic side chains. The acidic side chainswere glucuronosyl and 4-O-methyl glucuronosyl residues, andthe neutral side chains were arabinosyl and xylosyl residues. Although the amount of acidic arabinoxylan increased with theage of cultures, the percentage of acidic arabinoxylan in thewhole extracellular polysaccharides was almost constant duringthe growth period (2 to 8 days). (Received June 15, 1984; Accepted November 27, 1984)     

Molecular characterization and solution properties of enzymatically tailored arabinoxylans

Two α-l-arabinofuranosidases with different substrate specificities were used to modify the arabinose-to-xylose ratio of cereal arabinoxylans: one enzyme (AXH-m) removed the l-arabinofuranosyl substituents from the monosubstituted xylopyranosyl residues and the other (AXH-d3) the (1 → 3)-linked l-arabinofuranosyl units from the disubstituted xylopyranosyl residue. In this study, we noticed that not only the arabinose-to-xylose ratio but also the position of the arabinofuranosyl substituents affects the water-solubility of arabinoxylans. The AXH-d3 treatment had no significant effect on the solution conformation of arabinoxylans, but the density of the arabinoxylan molecules decreased in DMSO solution after AXH-m modification. The possible heterogeneity of arabinoxylans complicated the interpretation of data describing the macromolecular properties of the enzymatically modified samples.     

Material properties of films from enzymatically tailored arabinoxylans

Rye arabinoxylan, with an initial arabinose to xylose (Ara/Xyl) ratio of 0.50, was enzymatically modified with alpha-L-arabinofuranosidase. Different enzyme dosages were used to prepare arabinoxylan samples with a gradient of arabinose content varying from Ara/Xyl ratio 0.50 to 0.20. The degree of polymerization of the arabinoxylans was not affected by the enzymatic treatment, as detected with SEC-MALLS. Arabinoxylan samples with an Ara/Xyl ratio of 0.30 and below agglomerated in a water solution as seen by changes in light scattering. All samples, however, formed cohesive films upon drying, without addition of external plasticizers. The film from untreated arabinoxylan was completely amorphous; whereas films of the enzyme-treated arabinoxylans were semicrystalline with an increasing degree of crystallinity with decreasing arabinose content as determined by WAXS. Oxygen permeability measurements of the films showed that decreased arabinose content also resulted in lower oxygen permeability of the films. All films were strong and relatively stiff, but showed variations in strain at break. The moderately debranched film with an Ara/Xyl ratio of 0.37 had highest strain at break among all the films tested, yet was stiff and strong. This material also exhibited yielding and had stress/strain behavior similar to synthetic semicrystalline polymers, with a tendency to strain-induced crystallization. Such a combination of mechanical properties combined with oxygen barrier properties is very attractive for packaging applications.     

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.     

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-d-glucurono-d-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-l-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.     

Genetic analysis of water-extractable arabinoxylans in bread wheat endosperm

 Two mapping populations were used for the analysis of the water-extractable arabinoxylans. One originated from a cross between the hexaploid cultivars ‘Courtot’ and ‘Chinese Spring’ and the other from a cross between an amphiploid (Synthetic) and cv ‘Opata’. Arabinose (Ara), and xylose (Xyl) contents were quantified for the 91 and 76 lines obtained from the two crosses, respectively. Relative viscosity (η_rel) of the wheat flour aqueous extract was evaluated by capillary viscometry. Both crosses gave similar correlation coefficients between sugar contents and relative viscosity. There were strong positive relationships between arabinose, xylose and arabinoxylan contents. The relative viscosity was strongly and positively related to the arabinoxylan content and strongly and negatively related to the Ara/Xyl ratio (arabinose content to xylose content). For one of the two crosses two measurements of relative viscosity were generated from 2 years of consecutive harvesting. As a strong correlation was observed between these two measurements, an important genotypic effect can be deduced for the relative viscosity of water-extractable arabinoxylans. QTL (quantitative trait locus) research did not reveal any chromosomal segments that were strongly implicated in variations in sugar content. However, a QTL was found for relative viscosity values and the Ara/Xyl ratio on the long arm of the 1B chromosome for the two crosses considered. This QTL explained 32–37% of the variations in relative viscosity and 35–42% of the variations in the Ara/Xyl ratio. Genes located at this QTL controlled relative viscosity through modifying the Ara/Xyl ratio. Variations in the Ara/Xyl ratio were supposedly related to differences in the molecular structure of water-extractable arabinoxylans. Minor QTLs were also obtained for relative viscosity and Ara/Xyl ratio, but the chromosomes concerned were different for the two populations evaluated. Received: 5 January 1998 / Accepted: 15 May 1998     

Monoclonal antibodies to plant cell wall xylans and arabinoxylans.

Two rat monoclonal antibodies have been generated to plant cell wall (1-->4)-beta-D-xylans using a penta-1,4-xylanoside-containing neoglycoprotein as an immunogen. The monoclonal antibodies, designated LM10 and LM11, have different specificities to xylans in relation to the substitution of the xylan backbone as indicated by immunodot assays and competitive-inhibition ELISAs. LM10 is specific to unsubstituted or low-substituted xylans, whereas LM11 binds to wheat arabinoxylan in addition to unsubstituted xylans. Immunocytochemical analyses indicated the presence of both epitopes in secondary cell walls of xylem but differences in occurrence in other cell types.     

Structural characteristics of the cold-water-soluble arabinoxylans from the white flour of the soft wheat variety Kadet

Cold-water-soluble arabinoxylan polymers from the soft wheat variety Kadet were isolated by a stepwise (NH₄)₂SO₄ precipitation and freed from co-precipitating proteins by pronase digestion. The purified arabinoxylans, representing 0·5% of the dry weight of the white flour, were fractionated by graded precipitation with ethanol. Monosaccharide analysis revealed the major carbohydrate fractions to contain arabinoxylans only. The arabinoxylans are heterogeneous in molecular size. Determination of the carbohydrate composition in conjunction with methylation analysis and ¹³C-NMR spectroscopy, indicated that they can be divided into specific groups, based on characteristic differences with respect to -xylopyranose to -arabinofuranose ratio and in 2,3,4-tri-:3,4-di-:4-mono-substituted -xylopyranose ratio.     

Hydration of the left spiral of the poly-L-proline type. Study by the Monte Carlo method]

The paper exhibits results of hydration shell Monte Carlo calculations in poly-L-proline II and extended helix conformation and in alpha-helical and beta-structural conformations for comparison. It was found that left-handed helix of poly-L-proline II type as well as epsilon-helix are characterized by very favorable hydration. Therefore this conformation has preference as compared to other standard conformations of the main polypeptide chain. This determined inevitability of cold denaturation of protein.     

Specificity of monoclonal antibodies generated against arabinoxylans of cereal grains

Xylooligosaccharides substituted by arabinose have been produced by degradation of wheat flour arabinoxylans with an endoxylanase. These oligosaccharides were coupled to carrier proteins (KLH and BSA) and three monoclonal antibodies were isolated. The specificity of antibody recognition was studied using arabino-xylo-oligosaccharides exhibiting different pattern of substitution by arabinose.

ELISA competition tests and molecular modelling suggest that the conformation adopted by beta-(1→4) linked xylose residues is an antigenic determinant recognized by the different antibodies. Arabinose was not specifically involved in the interaction of antibody and epitope.     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Antitumor and immunomodulatory activity of arabinoxylans: a major constituent of wheat bran

We aimed to investigate the antitumor activity of wheat bran arabinoxylans, including the role of its immunostimulatory effect. In S180 tumor-bearing mice arabinoxylan administration significantly inhibited the growth of mouse transplantable tumors and remarkably promoted thymus and spleen indexes, splenocyte proliferation, natural killer cell and macrophage phagocytosis activity, interleukin 2 production, and delayed-type hypersensitivity reaction. In addition, it increased peripheral leukocyte count, and bone-marrow cellularity in tumor-bearing mice. As the antitumor activity of arabinoxylans may be mediated via the improvement in the immune response, they can be considered an antitumor agent with immunomodulatory activity.     

Purification and mode of action of two different arabinoxylan arabinofuranohydrolases from Bifidobacterium adolescentis DSM 20083

Two novel arabinofuranohydrolases (AXH-d3 and AXH-m23) were purified from Bifidobacterium adolescentis DSM 20083. Both enzymes were induced upon growth of Bi. adolescentis on xylose and arabinoxylan-derived oligosaccharides. They were only active with arabinoxylans and therefore denoted as arabinoxylan arabinofuranohydrolases. Their optimal activity was at pH 6 and 30–40 °C. They were very specific in their mode of action and were clearly different from AXH-m from Aspergillus awamori. AXH-m23 released only arabinosyl groups, which were linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan oligomers. AXH-d3 hydrolysed C-3-linked arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arab- inoxylan-derived oligosaccharides. No activity was observed with C-2-linked arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against C-2- and C-3-linked arabinofuranosyl residues of singly substituted xylopyranosyl residues. The combination of AXH-d3 and AXH-m showed low debranching activity with highly substituted glucurono-arabinoxylans. However, arabinoxylan from wheat flour was debranched almost completely. Received: 12 January 1999 / Accepted: 17 January 1999     

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase-d3 and endo-xylanase activities and its application in the hydrolysis of cereal arabinoxylans

Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the −1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure–function relationship of GH43 multifunctional enzymes.     

Effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral balance (Ca, Mg) in rats

The effects of soluble corn bran arabinoxylans on cecal digestion, lipid metabolism, and mineral utilization [calcium (Ca) and magnesium (Mg)] were investigated in rats adapted to semipurified diets. The diets provided either 710 g/kg wheat starch alone (control) or 610 g/kg wheat starch plus 100 g/kg corn soluble fiber (arabinoxylans) and either 0 or 2 g/kg cholesterol (control + cholesterol and arabinoxylans + cholesterol, respectively). Compared with rats fed the control diets, rats fed the arabinoxylan diets had significant cecal hypertrophy (+50% after 3 days of the fiber adaptation) and an accumulation of short-chain fatty acids, especially propionic acid (up to 45% in molar percentage). Arabinoxylans enhanced the cecal absorption of Ca and Mg (from 0.07 to 0.19 micromol/min for Ca and from 0.05 to 0.23 micromol/min for Mg). Mg balance was enhanced by arabinoxylans (+25%). The arabinoxylan diet markedly reduced the cholesterol absorption from 50% of ingested cholesterol in controls up to approximately 15% in rats adapted to the arabinoxylans diet. Arabinoxylans were effective in lowering plasma cholesterol (approximately -20%). There was practically no effect of the diets on cholesterol in d > 1.040 lipoproteins (high density lipoproteins) whereas arabinoxylans were very effective in depressing cholesterol in d < 1.040 lipoproteins (especially in triglyceride-rich lipoproteins). Corn fermentable fiber decreased the accumulation of cholesterol in the liver. In parallel, the arabinoxylan diet counteracted the downregulation of 3-hydroxy-3-methylglutaryl-CoA by cholesterol. These data suggest that arabinoxylans may have a great impact on intestinal fermentation, mineral utilization, and cholesterol metabolism.     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.     

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.