Developmental complexity of arabinan polysaccharides and their processing in plant cell walls.
Verhertbruggen, Y., Marcus, S. E., Haeger, A., Verhoef, R., Schols, H. A., McCleary, B. V., McKee, L., Gilbert, H. J. & Paul Knox, J. (2009). The Plant Journal, 59(3), 413-425.
Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.
Cellulose microfibril angles and cell-wall polymers in different wood types of Pinus radiata.
Brennan, M., McLean, J. P., Altaner, C. M., Ralph, J. & Harris, P. J. (2012). Cellulose, 19(4), 1385-1404.
Four corewood types were examined from sapling trees of two clones of Pinus radiata grown in a glasshouse. Trees were grown either straight to produce normal corewood, tilted at 45° from the vertical to produce opposite corewood and compression corewood, or rocked to produce flexure corewood. Mean cellulose microfibril angle of tracheid walls was estimated by X-ray diffraction and longitudinal swelling measured between an oven dry and moisture saturated state. Lignin and acetyl contents of the woods were measured and the monosaccharide compositions of the cell-wall polysaccharides determined. Finely milled wood was analysed using solution-state 2D NMR spectroscopy of gels from finely milled wood in DMSO-d6/pyridine-d5. Although there was no significant difference in cellulose microfibril angle among the corewood types, compression corewood had the highest longitudinal swelling. A lignin content >32% and a galactosyl residue content >6% clearly divided severe compression corewood from the other corewood types. Relationships could be drawn between lignin content and longitudinal swelling, and between galactosyl residue content and longitudinal swelling. The 2D NMR spectra showed that the presence of H-units in lignin was exclusive to compression corewood, which also had a higher (1→4)-β-D-galactan content, defining a unique composition for that corewood type.
Purification and characterization of a xylanase and an arabinofuranosidase from Bacillus polymyxa.
Morales, P., Madarro, A., Flors, A., Sendra, J. & Pérez-González, J. (1995). Enzyme and Microbial Technology, 17(5), 424-429.
Two hemicellulases from Bacillus polymyxa were purified and characterized: a xylanase with a molecular mass of 61 kD and pl of 4.7 and an arabinofuranosidase with a molecular mass of 166 kD and pl of 4.7. The xylanase, which showed increased thermostability in the presence of MgCl2, showed a typical endo-action mode on xylans from several sources. The arabinofuranosidase was only active on (1→5)-α-L-arabinooligosaccharides but not on linear (1→5)-α-L-arabinan, arabinogalactan, and arabinoxylan. However, it was able to release arabinose from arabinoxylan when an active endoxylanase was also present in hydrolysis assays.
α-L-Arabinofuranosidases from Aspergillus terreus with potential application in enology: induction, purification, and characterization.
Le Clinche, F., Piñaga, F., Ramón, D. & Vallés, S. (1997). Journal of Agricultural and Food Chemistry, 45(7), 2379-2383.
In the presence of L-arabitol as sole carbon source, Aspergillus terreus CECT 2663 produces three α-L-arabinofuranosidases (ABFs) named ABF1, ABF2, and ABF3, with molecular masses of 90 000, 82 000, and 78 500 Da, respectively. The synthesis of these enzymes is under carbon catabolite repression. Western blotting revealed that ABF2 is immunologically related to the α-L-arabinofuranosidase B previously isolated from Aspergillus niger. The three A. terreus proteins have been purified to homogeneity. They are acidic proteins with optimal pHs of 5.0 for ABF1 and ABF2 and 5.5 for ABF3 and optimal temperatures of 50, 60, and 65°C, respectively. Kinetic constants for the purified enzymes on p-nitrophenyl α-L-arabinofuranoside (pNPA) as substrate have been determined. The three enzymes maintain elevated activities in the presence of ethanol or glucose at those concentrations normally present in must or wine.
Near-infrared Fourier-transform Raman spectroscopy of flax (Linum usitatissimum L.) stems.
Himmelsbach, D. S. & Akin, D. E. (1998). Journal of Agricultural and Food Chemistry, 46(3), 991-998.
Samples of flax (Linum usitatissimum L.) stem and its anatomical parts were studied by near-infrared Fourier transform Raman (NIR-FT-Raman) spectroscopy to determine if the major chemical components of each could be detected by this method. The Raman spectra of reference compounds from relatively pure materials served as models for the chemical components. Bands for cellulose were greatest in the fibers. Hemicellulosic polysaccharides were observed to be prevalent in bast tissue and fibers. Weak signals for pectins were observed in the bast, cuticle/epidermis, fibers, and stem. Bands for aromatic rings were detectable in all materials. Bands from waxes/fatty acid esters were detectable in the cuticle/epidermal tissue. The results indicated that NIR-FT-Raman could be used detect the major chemical components in flax in situ and provide a simple, rapid, and noninvasive assessment of their relative amounts and location within the tissues of the flax plant.
Purification, functional characterization, cloning, and identification of mutants of a seed-specific arabinan hydrolase in Arabidopsis.
Minic, Z., Do, C. T., Rihouey, C., Morin, H., Lerouge, P. & Jouanin, L. (2006). Journal of Experimental Botany, 57(10), 2339-2351.
This work describes the purification and characterization of an enzyme that exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) as a putative β-D-xylosidase that belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3 hydrolysed synthetic substrates such as p-nitrophenyl-α-L-arabinofuranoside and p-nitrophenyl-β-D-xyloside with similar catalytic efficiency. XYL3 released L-arabinose from (1→5)-α-L-arabinofuranobiose, arabinoxylan, sugar beet arabinan, and debranched arabinan. The enzyme hydrolysed both arabinosyl-substituted side group residues and terminal arabinofuranosyl residues (1→5)-α-linked to the arabinan backbone. This indicates that XYL3 is able to degrade all terminal arabinosyl residues and suggests that it participates in the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by semi-quantitative RT-PCR, in-situ hybridization and a promoter–GUS fusion demonstrated that AtBX3 was specifically expressed in the seed endosperm at the globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera found that arabinan, the XYL3 substrate, was also present in this seed tissue. T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the α-L-arabinofuranosidase and β-D-xylosidase activities corresponding to XYL3. Mutants showed reduced seed size and are delayed in seedling germination compared with the wild type.
Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC.
Wu, D. T., Cheong, K. L., Deng, Y., Lin, P. C., Wei, F., Lv, X. J., Long, Z, R., Zhao, J., Ma, S, C. & Li, S. P. (2015). Carbohydrate Polymers, 134, 12-19.
Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barbarum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.
Enzymatic pectic oligosaccharides (POS) production from sugar beet pulp using response surface methodology.
Babbar, N., Dejonghe, W., Sforza, S. & Elst, K. (2017). Journal of Food Science and Technology, 1-9.
Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM).
Cloning and expression of a novel α-1, 3-arabinofuranosidase from Penicillium oxalicum sp. 68.
Hu, Y., Yan, X., Zhang, H., Liu, J., Luo, F., Cui, Y., Wang, W. & Zhou, Y. (2018). AMB Express, 8(1), 51.
The discovery and creation of biocatalysts for plant biomass conversion are essential for industrial demand and scientific research of the plant cell wall. α-1,2 and α-1,3-L-arabinofuranosidases are debranching enzymes that catalyzing hydrolytic release of α-L-arabinofuranosyl residues in plant cell wall. Gene database analyses shows that GH62 family only contains specific α-L-arabinofuranosidases that play an important role in the degradation and structure of the plant cell wall. At present, there are only 22 enzymes in this group has been characterized. In this study, we cloned a novel α-1,3-arabinofuranosidase gene (poabf62a) belonging to glycoside hydrolase family 62 from Penicillium oxalicum sp. 68 and expressed it in Pichia pastoris. The molecular mass of recombinant PoAbf62A was estimated to be 32.9 kDa. Using p-nitrophenyl-α-L-arabinofuranoside (pNPαAbf) as substrate, purified PoAbf62A exhibited an optimal pH of 4.5 and temperature of 35°C. Results of methylation and 13C NMR analyses showed that PoAbf62A was exclusively α-1,3-arabinofuranosidase, specific for cleavage of α-1,3-arabinofuranosyl residues, and with the absence of activity towards α-1,2-arabinofuranose and α-1,5-arabinofuranose. Therefore, PoAbf62A exhibits high activity on sugar beet arabinan and wheat arabinoxylan, because their branched side chain are decorated with α-1,3-arabinofuranose. On the other hand, there is a lack of activity with linear-α-L-1,5-arabinan and xylan that only contained α-L-1,5-arabinofuranose or β-1,4-xylose. The α-1,3-arabinofuranosidase activity identified here provides a new biocatalytic tool to degrade hemicellulose and analyze the structure of plant cell walls.
Ruminiclostridium josui Abf62A-Axe6A: a tri-functional xylanolytic enzyme exhibiting α-L-arabinofuranosidase, endoxylanase, and acetylxylan esterase activities.
Wang, Y., Sakka, M., Yagi, H., Kaneko, S., Katsuzaki, H., Kunitake, E., Kimura, T. & Sakka, K. (2018). Enzyme and Microbial Technology, 117, 1-8.
Ruminiclostridium josui Abf62A-Axe6A is a modular enzyme comprising (in order from the N-terminus): an N-terminal signal peptide, a glycosidehydrolase family 62 (GH62) catalytic module, a family 6 carbohydratebinding module (CBM6), a dockerin module and an additional carbohydrate esterase family 6 catalytic module (CE6). In this study, three Abf62A-Axe6A derivatives were constructed, overexpressed in Escherichia coli, purified, and biochemically characterized: RjAbf62A-Axe6A, containing all four modules but lacking the signal peptide; RjAbf62A-CBM6, containing the GH62 and CBM6 modules; and RjAxe6A, containing only CE6. RjAbf62A-Axe6A was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, and released mainly arabinose. Analysis of the arabinoxylooligosaccharide hydrolysis products revealed that RjAbf62A-Axe6A released α-1,2- and α-1,3-linked arabinofuranose from both singly and doubly substituted xylosyl residues. Furthermore, RjAbf62A-Axe6A exhibited a weak activity toward linear 1,5-α-L arabinan and arabinooligosaccharides, indicating that it is capable of cleaving α-1,5-linkage. Surprisingly, RjAbf62A-Axe6A also demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. Although RjAbf62A-CBM6 exhibited a similar substrate specificity to RjAbf62A-Axe6A, RjAbf62A-CBM6 showed lower activities toward soluble arabinoxylans, birchwood and beechwood xylans and arabinoxylooligosaccharides but not toward insoluble arabinoxylan. RjAbf62A-Axe6A is the first reported GH62 enzyme with α-L-arabinofuranosidase and endoxylanase activities. Although both RjAbf62A-Axe6A and RjAxe6A had acetylxylan esterase activities, RjAbf62A-Axe6 exhibited a higher activity toward insoluble wheat arabinoxylan compared with RjAxe6.