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.
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.
Complete genome of a new Firmicutes species belonging to the dominant human colonic microbiota (‘Ruminococcus bicirculans’) reveals two chromosomes and a selective capacity to utilize plant glucans.
Wegmann, U., Louis, P., Goesmann, A., Henrissat, B., Duncan, S. H. & Flint, H. J. (2014). Environmental Microbiology, 16(9), 2879–2890.
The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially β-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD+, NADP+ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.
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.
L-Arabinose production from sugar beet arabinan by immobilized endo-and exo-arabinanases from Caldicellulosiruptor saccharolyticus in a packed-bed reactor.
Kim, Y. S., Lim, Y. R. & Oh, D. K. (2012). Journal of Bioscience and Bioengineering, 113(2), 239-241.
The immobilized endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus produced continuously an average of 16.5 g l-1 L-arabinose from 20 g l-1 sugar beet arabinan at pH 5.0 and 75°C for 216 h, with a productivity of 9.9 g l-1 h-1 and a conversion yield of 83%.
Mapping the polysaccharide degradation potential of Aspergillus niger.
Andersen, M. R., Giese, M., de Vries, R. P. & Nielsen, J. (2012). BMC Genomics, 13(1), 313.
Background: The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. Results: Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. Conclusions: The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.
A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases.
Park, Y. B. & Cosgrove, D. J. (2012). Plant Physiology, 158(4), 1933-1943.
Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this “tethered network” model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.
Structural basis for entropy-driven cellulose binding by a type-A cellulose-binding module (CBM) and bacterial expansin.
Georgelis, N., Yennawar, N. H. & Cosgrove, D. J. (2012). Proceedings of the National Academy of Sciences, 109(37), 14830-14835.
Components of modular cellulases, type-A cellulose-binding modules (CBMs) bind to crystalline cellulose and enhance enzyme effectiveness, but structural details of the interaction are uncertain. We analyzed cellulose binding by EXLX1, a bacterial expansin with ability to loosen plant cell walls and whose domain D2 has type-A CBM characteristics. EXLX1 strongly binds to crystalline cellulose via D2, whereas its affinity for soluble cellooligosaccharides is weak. Calorimetry indicated cellulose binding was largely entropically driven. We solved the crystal structures of EXLX1 complexed with cellulose-like oligosaccharides to find that EXLX1 binds the ligands through hydrophobic interactions of three linearly arranged aromatic residues in D2. The crystal structures revealed a unique form of ligand-mediated dimerization, with the oligosaccharide sandwiched between two D2 domains in opposite polarity. This report clarifies the molecular target of expansin and the specific molecular interactions of a type-A CBM with cellulose.
Prioritization of a plant polysaccharide over a mucus carbohydrate is enforced by a Bacteroides hybrid two‐component system.
Lynch, J. B. & Sonnenburg, J. L. (2012). Molecular Microbiology, 85(3), 478-491.
Bacteroides is a dominant genus within the intestinal microbiota of healthy humans. Key adaptations of the Bacteroides to the dynamic intestinal ecosystem include a diverse repertoire of genes involved in sensing and processing numerous diet- and host-derived polysaccharides. One such adaptation is the carbohydrate-sensing hybrid two-component system (HTCS) family of signalling sensors, which has been widely expanded within the Bacteroides. Using Bacteroides thetaiotaomicron as a model, we have created a chimeric HTCS consisting of the well-characterized sensing domain of one HTCS, BT1754, and the regulatory domain of another HTCS, BT0366, to explore the regulatory capabilities of these molecules. We found that the BT0366 regulatory region directly binds to and mediates induction of the adjacent polysaccharide utilization locus (PUL) using whole-genome transcriptional profiling after inducing signalling through our chimeric protein. We also found that BT0366 activation simultaneously leads to repression of distal PULs involved in mucus carbohydrate consumption. These results suggest a novel mechanism by which an HTCS enforces a nutrient hierarchy within the Bacteroides via induction and repression of multiple PULs. Thus, hybrid two-component systems provide a mechanism for prioritizing consumption of carbohydrates through simultaneous binding and regulation of multiple polysaccharide utilization loci.
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.
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.
Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls.
Levigne, S., Ralet, M. C., Quéméner, B. & Thibault, J. F. (2004). Carbohydrate Research, 339(13), 2315-2319.
After degradation of sugar beet cell walls with Driselase® and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1→5)-linked arabinose units esterified by a central 8-O-4′ ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.
Role of (1,3)(1,4) β-glucan in cell walls: Interaction with cellulose.
Kiemle, S. N., Zhang, X., Esker, A. R., Toriz, G., Gatenholm, P. & Cosgrove, D. J. (2014). Biomacromolecules, 15 (5), 1727-1736.
(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.
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.
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 KM 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.
Optimization of Arundo donax Saccharification by (Hemi) cellulolytic Enzymes from Pleurotus ostreatus.
Liguori, R., Ionata, E., Marcolongo, L., Vandenberghe, L. P. D. S., La Cara, F. & Faraco, V. (2015). BioMed research International, 2015, Article ID 951871.
An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 33 factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes.
Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis.
Larsbrink, J., Tuveng, T. R., Pope, P. B., Bulone, V., Eijsink, V. G., Brumer, H. & McKee, L. S. (2017). Journal of Proteomics, 156, 63-74.
Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this study: Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.
The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.
Thieme, N., Wu, V. W., Dietschmann, A., Salamov, A. A., Wang, M., Johnson, J., Singan, V. R., Grigoriev, I. V., Glass, N. L., Somerville, C. R., & Benz, J. P. (2017). Biotechnology for Biofuels, 10(1), 149.
Background: Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. Results: The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of L-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. Conclusions: PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.