Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase.
McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.
A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.
Novel and selective substrates for the assay of endo-arabinanase.
McCleary, B. V. (1989). "Gums and Stabilisers for the Food Industry, Vol 5”, (G. O. Phillips, D. J. Wedlock and P. A.Williams, Eds.), IRL Press, pp. 291-298.
Substrates and assay procedures for the measurement of endo-1,5-α-L-arabinanase in crude, technical pectinase preparations have been developed. The method of choice employs carboxymethy1-debranched beet araban as substrateT and rate of hydrolysis is measured using the Nelson-Somogyi reducing-sugar procedure with arabinose as the standard. The substrate is physically and chemically stable in solution, and the assay procedure is simple, reliable and specific. Other assay procedures for the measurement of endo-arabinanase which employ dyed debranched araban substrates, are also briefly described.
Comparison of endolytic hydrolases that depolymerize 1,4-β-D-mannan, 1,5-α-L-arabinan and 1,4-β-D-galactan.
McCleary, B. V. (1991). “Enzymes in Biomass Conversion”, (M. E. Himmel and G. F. Leatham, Eds.), ACS Symposium Series 460, Chapter 34, pp. 437-449. American Chemical Society, Washington.
Hydrolysis of mannan-type polysaccharides by β-mannanase is dependent on substitution on and within the main-chain as well as the source of the β-mannanase employed. Characterisation of reaction products can be used to define the sub-site binding requirements of the enzymes as well as the fine-structures of the polysaccharides. Action of endo-arabinanase and endo-galactanase on arabinans and arabinogalactans is described. Specific assays for endo-arabinanase and arabinan (in fruit-juice concentrates) are reported.
Adsorption of arabinoxylan on cellulosic surfaces: influence of degree of substitution and substitution pattern on adsorption characteristics.
Köhnke, T., Östlund, Å. & Brelid, H. (2011). Biomacromolecules, 12(7), 2633-2641.
This study presents results that show that the fine structure of arabinoxylan affects its interaction with cellulosic surfaces, an important understanding when designing and evaluating properties of xylan–cellulose-based materials. Arabinoxylan samples, with well-defined structures, were prepared from a wheat flour arabinoxylan with targeted enzymatic hydrolysis. Turbidity measurements and analyses using NMR diffusometry showed that the solubility and the hydrodynamic properties of arabinoxylan are determined not only by the degree of substitution but also by the substitution pattern. On the basis of results obtained from adsorption experiments on microcrystalline cellulose particles and on cellulosic model surfaces investigated with quartz crystal microbalance with dissipation monitoring, it was also found that arabinoxylan adsorbs irreversibly on cellulosic surfaces and that the adsorption characteristics, as well as the properties of the adsorbed layer, are controlled by the fine structure of the xylan molecule.
Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-β-D-xylanase.
Rantanen, H., Virkki, L., Tuomainen, P., Kabel, M., Schols, H. & Tenkanen, M. (2007). Carbohydrate Polymers, 68(2), 350-359.
Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-β-D-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase‘s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as α-L-Araf-(1→3)- β-D-Xylp-(1→4)-D-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-β-D-xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before.
Production and characterisation of recombinant α-L-arabinofuranosidase for production of xylan hydrogels.
Chimphango, A. F. A., Rose, S. H., Van Zyl, W. H. & Görgens, J. F. (2012). Applied Microbiology and Biotechnology, 95(1), 101-112.
A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-β-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd P ) and glucoamylase terminator (glaA T ) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g-1 (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40–55°C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60°C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.
Effect of nanocoating with rhamnogalacturonan‐I on surface properties and osteoblasts response.
Gurzawska, K., Svava, R., Syberg, S., Yihua, Y., Haugshøj, K. B., Damager, I., Ulvskov, P., Christensen, L. H., Gotfredsen, K. & Jørgensen, N. R. (2012). Journal of Biomedical Materials Research Part A, 100(3), 654-664.
Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.
Pectin Metabolism and Assembly in the Cell Wall of the Charophyte Green Alga Penium margaritaceum.
Domozych, D. S., Sørensen, I., Popper, Z. A., Ochs, J., Andreas, A., Fangel, J. U., Pielach, A., Sacks, C., Brechka, H., Ruisi-Besares, P., Willats, W. G. & Rose, J. K. C. (2014). Plant Physiology, 165(1), 105-18.
The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its co-localization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens and Arabidopsis (Arabidopsis thaliana) further suggested conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of Penium, a member of the Charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. The calcium-HG gel at the cell surface may therefore represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.
Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species.
Koutaniemi, S., Guillon, F., Tranquet, O., Bouchet, B., Tuomainen, P., Virkki, L., Petersen, H. L., Willats, W. G. T., Saulnier, L. & Tenkanen, M. (2012). Planta, 236(2), 739-751.
Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.
Enrichment of ginsenoside Rd in Panax ginseng extract with combination of enzyme treatment and high hydrostatic pressure.
Palaniyandi, S. A., Damodharan, K., Lee, K. W., Yang, S. H. & Suh, J. W. (2015). Biotechnology and Bioprocess Engineering, 20(3), 608-613.
Ginsenoside Rd, a minor ginseng saponin, has several pharmacological activities. Traditionally, saponins are extracted using organic solvents or hot water extraction. However, both of these methods have disadvantages such as formation of artefacts and compound decomposition. Additionally, the use of organic solvents for extraction is hazardous to the environment. Therefore, we aimed to produce ginsenoside Rd without using organic solvents or hot extraction. We developed a simultaneous extraction and transformation process for higher yields of ginsenoside Rd using a combination of high hydrostatic pressure (HHP) and enzymes. Several commercial glycosidases in various combinations were studied for the enrichment of ginsenoside Rd from major ginsenosides by enzymatic transformation and HHP. We found that treatment with a combination of cellulase (2 U/mL), cellobiase (4 U/mL) and HHP of 100 Mpa at pH 4.8 and 45°C for 24 h resulted in a ginsenoside Rd content of 3.47 ± 0.35 mg/g of fresh ginseng. This yield is 2.1-fold higher than that of the corresponding enzyme treatment at atmospheric pressure (AP, 0.1 Mpa) at pH 4.8, 45°C and for 24 h. This simultaneous extraction and transformation process can be used for the preparation of Rd enriched ginseng beverage without using hazardous organic solvents.
Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin–carbohydrates complexes.
Huang, C., He, J., Li, X., Min, D. & Yong, Q. (2015). Bioresource Technology, 192, 471-477.
Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin–carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility.
Relationship between structure and immunological activity of an arabinogalactan from Lycium ruthenicum.
Peng, Q., Liu, H., Lei, H. & Wang, X. (2016). Food Chemistry, 194, 595-600.
An immunologically active arabinogalactan (LRGP3) was selectively degraded by acetolysis, mild acid hydrolysis and enzymatic digestion. After exo-α-L-arabinofuranosidase digestion, 56% of the arabinosyl chains were released. The resistant product (LRGP3-AF) had markedly increased complement fixating activities. The acid hydrolysis product (LRGP3-T) contained (1 → 3)-linked (17.6%), (1 → 6)-linked (23.1%), (1 → 3,6)-linked (30.1%) and terminal (29.2%) galactosyl residues, and its complement fixating activity was lower than that of LRGP3-AF. The side chains (Oligo-S) consisted of arabinose, galactose, and rhamnose in the molar ratios 16.8:1.4:1.0. The complement fixating activity of Oligo-S was weak, but Oligo-S had potent macrophage stimulation activity. Degradation of arabinosyl residues in LRGP3 decreased the macrophage stimulation activity, but the galactan backbone still expressed partial activity. The results demonstrated that the galactan backbone of the polymer might be essential for the expression of complement fixating activity and the arabinosyl side chains could be more responsible for the macrophage activation activity. There may be several structurally different active sites involved in the immunological activity of LRGP3.
Multi-layer mucilage of Plantago ovata seeds: Rheological differences arise from variations in arabinoxylan side chains.
Yu, L., Yakubov, G. E., Zeng, W., Xing, X, Stenson, J., Bulone, V. &Stokes, J. R. (2017). Journal of the Science of Food and Agriculture, 165(1), 132-141.
Mucilages are hydrocolloid solutions produced by plants for a variety of functions, including the creation of a water-holding barrier around seeds. Here we report our discovery of the formation of three distinct mucilage layers around Plantago ovata seeds upon their hydration. Each layer is dominated by different arabinoxylans (AXs). These AXs are unusual because they are highly branched and contain β-1,3-linked xylose in their side chains. We show that these AXs have similar monosaccharide and linkage composition, but vary in their polymer conformation. They also exhibit distinct rheological properties in aqueous solution, despite analytical techniques including NMR showing little difference between them. Using enzymatic hydrolysis and chaotropic solvents, we reveal that hydrogen bonding and side chain distribution are key factors underpinning the distinct rheological properties of these complex AXs.
Reduced-molecular-weight derivatives of frost grape polysaccharide.
Leathers, T. D., Price, N. P., Vaughn, S. F. & Nunnally, M. S. (2017). International Journal of Biological Macromolecules, In Press.
A new Type II arabinogalactan was recently described as an abundant gum exudate from stems of wild frost grape (Vitus riparia Michx.). The purpose of the current study is to more thoroughly characterize the physical properties of this frost grape polysaccharide (FGP), and develop methods to modify the molecular weight of FGP for potential new applications. Specifically, native FGP was modified by heat treatment, digestion with the enzyme L-arabinosidase, and ultrasonication. Results showed that native FGP was progressively and irreversibly denatured by heat treatment, while the polymer remained largely resistant to enzymatic digestion. However, ultrasonication reduced the molecular weight of FGP from 1.6 × 107 Da to about 3.0 × 105 Da. Reduced-molecular-weight FGP exhibited modified solution viscosity properties, which could be useful in food and prebiotic applications.