High purity Debranched Arabinan (Sugar Beet) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Purity ~ 95%. 1,5-α-L-Arabinan. Ara: Gal: Rha: GalUA = 88: 4: 2: 6

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. Link to Article Read Abstract 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 A^2,3XX, 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.

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. Link to Article Read Abstract 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.

Exo-arabinanase of Penicillium chrysogenum able to release arabinobiose from α-1, 5-L-arabinan.

Sakamoto, T. & Thibault, J. F. (2001). Applied and Environmental Microbiology, 67(7), 3319-3321. Link to Article Read Abstract An exo-arabinanase, designated Abnx, was purified from a culture filtrate of Penicillium chrysogenum 31B by ammonium sulfate precipitation, anion-exchange chromatography, and hydrophobic chromatography. Abnx had an apparent molecular mass of 47 kDa. The enzyme released only arabinobiose from the nonreducing terminus of α-1,5-L-arabinan and showed no activity towards p-nitrophenyl-α-L-arabinofuranoside and α-1,5-L-arabinofuranobiose. Abnx is the first enzyme with this mode of action.

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged.

Zykwinska, A., Thibault, J. F. & Ralet, M. C. (2007). Journal of Experimental Botany, 58(7), 1795-1802. Link to Article Read Abstract The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called ‘natural’ composites), rich in pectic neutral sugar side chains and cellulose, as well as ‘artificial’ composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.

Evidence for in vitro binding of pectin side chains to cellulose.

Zykwinska, A. W., Ralet, M. C. J., Garnier, C. D. & Thibault, J. F. J. (2005). Plant Physiology, 139(1), 397-407. Link to Article Read Abstract Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.

Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles.

Skjøt, M., Pauly, M., Bush, M. S., Borkhardt, B., McCann, M. C. & Ulvskov, P. (2002). Plant Physiology, 129(1), 95-102. Link to Article Read Abstract Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-α-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing α-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment.

Characterization of a modular enzyme of exo-1,5-α-L-arabinofuranosidase and arabinan binding module from Streptomyces avermitilis NBRC14893.

Ichinose, H., Yoshida, M., Fujimoto, Z. & Kaneko, S. (2008). Applied Microbiology and Biotechnology, 80(3), 399-408. Link to Article Read Abstract A gene encoding an α-L-arabinofuranosidase, designated SaAraf43A, was cloned from Streptomyces avermitilis. The deduced amino acid sequence implies a modular structure consisting of an N-terminal glycoside hydrolase family 43 module and a C-terminal family 42 carbohydrate-binding module (CBM42). The recombinant enzyme showed optimal activity at pH 6.0 and 45°C and was stable over the pH range of 5.0–6.5 at 30°C. The enzyme hydrolysed p-nitrophenol (PNP)-α-L-arabinofuranoside but did not hydrolyze PNP-α-L-arabinopyranoside, PNP-β-D-xylopyranoside, or PNP-β-D-galactopyranoside. Debranched 1,5-arabinan was hydrolyzed by the enzyme but arabinoxylan, arabinogalactan, gum arabic, and arabinan were not. Among the synthetic regioisomers of arabinofuranobiosides, only methyl 5-O-α-L-arabinofuranosyl-α-L-arabinofuranoside was hydrolyzed by the enzyme, while methyl 2-O-α-L-arabinofuranosyl-α-L-arabinofuranoside and methyl 3-O-α-L-arabinofuranosyl-α-L-arabinofuranoside were not. These data suggested that the enzyme only cleaves α-1,5-linked arabinofuranosyl linkages. The analysis of the hydrolysis product of arabinofuranopentaose suggested that the enzyme releases arabinose in exo-acting manner. These results indicate that the enzyme is definitely an exo-1,5-α-L-arabinofuranosidase. The C-terminal CBM42 did not show any affinity for arabinogalactan and debranched arabinan, although it bound arabinan and arabinoxylan, suggesting that the CBM42 bound to branched arabinofuranosyl residues. Removal of the module decreased the activity of the enzyme with regard to debranched arabinan. The CBM42 plays a role in enhancing the debranched arabinan hydrolytic action of the catalytic module in spite of its preference for binding arabinofuranosyl side chains.

A family 51 α-L-arabinofuranosidase from Penicillium purpurogenum: purification, properties and amino acid sequence.

Fritz, M., Ravanal, M. C., Braet, C. & Eyzaguirre, J. (2008). Mycological Research, 112(8), 933-942. Link to Article Read Abstract The soft rot fungus Penicillium purpurogenum secretes a wide variety of xylanolytic enzymes to the medium, among them three α-L-arabinofuranosidases. This work refers to arabinofuranosidase 2 (ABF 2). This enzyme was purified to homogeneity and characterized; it is a glycosylated monomer with a molecular weight of 70 000 and an isoelectric point of 5.3. When assayed with p-nitrophenyl α-L-arabinofuranoside (pNPAra) the enzyme followed Michaelis–Menten kinetics with a K_M of 0.098 mm. The optimum pH is 5 and the optimal temperature 60°C. ABF 2 showed weak activity on natural polymeric substrates, such as sugar beet arabinan, debranched arabinan, and arabinoxylan. These results, together with its low K_M (pNPAra) and its activity towards short arabinooligosaccharides, suggest that the enzyme belongs to the exo α-L-arabinosyl hydrolases not active on polymers. The abf2 gene and its cDNA were sequenced, and the gene was found to possess seven introns. The mature protein is 618 amino acids long with a calculated molecular weight of 67 212. Amino acid sequence alignments show that the enzyme belongs to family 51 of the glycosyl hydrolases, although it differs in some properties from other enzymes of this family.

A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis.

Ferreira, M. J. & de Sá-Nogueira, I. (2010). Journal of Bacteriology, 192(20), 5312-5318. Link to Article Read Abstract Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.

Purification, characterization and functional analysis of an endo‐arabinanase (AbnA) from Bacillus subtilis.

Leal, T. F. & Sá‐Nogueira, I. (2004). FEMS Microbiology Letters, 241(1), 41-48. Link to Article Read Abstract Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His₆-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-α-L-arabinan.

Purification and characterization of thermostable endo-1,5-α-L-arabinase from a strain of Bacillus thermodenitrificans.

Takao, M., Akiyama, K. & Sakai, T. (2002). Applied and Environmental Microbiology, 68(4), 1639-1646. Link to Article Read Abstract A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60°C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75°C. The enzyme had optimal activity at 70°C and pH 6.0. The enzyme had apparent K_m values of 8.5 and 45 mg/ml and apparent V_max values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (α-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.

Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

Pérez, R. & Eyzaguirre, J. (2016). Applied biochemistry and biotechnology, 179(1), 143-154. Link to Article Read Abstract The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with K_M of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37°C and for ABFII 42°C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

Screening of thermotolerant and thermophilic fungi aiming β-xylosidase and arabinanase production.

Benassi, V. M., Lucas, R. C. D., Jorge, J. A. & Polizeli, M. D. L. T. D. (2014). Brazilian Journal of Microbiology, 45(4), 1459-1467. Link to Article Read Abstract Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of β-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45°C, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50°C. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of β-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, β-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40°C), while the correspondent extracellular activities were favorably secreted from cultures at 30°C. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.

Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.

Mardones, W., Callegari, E. & Eyzaguirre, J. (2015). Fungal biology, 119(12), 1267-1278. Link to Article Read Abstract Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60-80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40°C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions.

Synergistic hydrolysis of xylan using novel xylanases, β-xylosidases, and an α-L-arabinofuranosidase from Geobacillus thermodenitrificans NG80-2.

Huang, D., Liu, J., Qi, Y., Yang, K., Xu, Y. & Feng, L. (2017). Applied Microbiology and Biotechnology, 1-15. Link to Article Read Abstract Lignocellulosic biomass from various types of wood has become a renewable resource for production of biofuels and biobased chemicals. Because xylan is the major component of wood hemicelluloses, highly efficient enzymes to enhance xylan hydrolysis can improve the use of lignocellulosic biomass. In this study, a xylanolytic gene cluster was identified from the crude oil-degrading thermophilic strain Geobacillus thermodenitrificans NG80-2. The enzymes involved in xylan hydrolysis, which include two xylanases (XynA1, XynA2), three β-xylosidases (XynB1, XynB2, XynB3), and one α-L-arabinofuranosidase (AbfA), have many unique features, such as high pH tolerance, high thermostability, and a broad substrate range. The three β-xylosidases were highly resistant to inhibition by product (xylose) accumulation. Moreover, the combination of xylanase, β-xylosidase, and α-L-arabinofuranosidase exhibited the largest synergistic action on xylan degradation (XynA2, XynB1, and AbfA on oat spelt or beechwood xylan; XynA2, XynB3, and AbfA on birchwood xylan). We have demonstrated that the proposed enzymatic cocktail almost completely converts complex xylan to xylose and arabinofuranose and has great potential for use in the conversion of plant biomass into biofuels and biochemicals.

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