Arabinan (Sugar Beet)

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.

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.

Sugar beet pulp is a byproduct of white sugar production, and it is quite significant in terms of volume. Every year, tens of millions of tons of beet pulp are produced around the world. However, only a fraction of it is currently used, mainly as animal feed. The composition of beet pulp includes plant polysaccharides, such as cellulose, arabinan, and pectin. Through the process of enzymatic hydrolysis, these polysaccharides are converted into technical C6/C5 sugars, which can be further used as a substrate for the microbial synthesis of various substances, including biofuels, organic acids, and other green chemistry molecules. The current study was designed with a primary objective that focused on the development of a strain that had the potential for enhanced productivity and the capacity to produce enzymes suitable for beet pulp hydrolysis. The pelA and abfA genes, which encode pectin lyase and arabinofuranosidase, respectively, in the fungus Penicillium canescens (VKPM F-178), were cloned and successfully expressed in the recipient strain Penicillium verruculosum B1-537 (VKPM F-3972D). New recombinant strains were created using the expression system of the mycelial fungus P. verruculosum B1-537, which is capable of simultaneously producing pectin lyase and arabinofuranosidase, as well as homologous cellulases. The screening of strains for increased enzymatic activity towards citrus pectin, sugar beet branched arabinan, and microcrystalline cellulose revealed that a B4 clone of P. verruculosum exhibited the greatest potential in sugar beet pulp cake hydrolysis. This clone was selected as the basis for the creation of a new enzyme preparation with enhanced pectin lyase, arabinase, and cellulase activities. The component composition of the enzyme preparation was determined, and the results indicated that the enzyme content comprised approximately 11% pectin lyase, 40% arabinofuranosidase, and 40% cellulases. The primary products of the enzymatic hydrolysis of the unpretreated beet pulp cake were arabinose and glucose. The degree of arabinan and cellulose conversion was observed to be up to 50% and 80%, respectively, after a period of 48 to 72 h of hydrolysis. The new B4 preparation was observed to be highly efficacious in the hydrolysis of beet cake at elevated concentrations of solids (up to 300 g/L) within the reaction mixture. The newly developed strain, as a producer of pectin lyase, arabinofuranosidase, and cellulase complexes, has the potential to be utilized for the bioconversion of sugar beet processing wastes and for the efficient generation of highly concentrated solutions of technical sugars for further implementation in processes of microbial synthesis.

Prebiotic oligosaccharides are dietary supplements that modulate the intestinal gut microbiome by selectively nourishing subsets of the microbial community with a goal to enhance host health. To date, the diversity of polysaccharide compositions in the fiber consumed by humans is not well represented by the limited scope of oligosaccharide compositions present in current commercial prebiotics. Recently, our UC Davis group developed a novel method to generate oligosaccharides from any polysaccharide fiber, termed Fenton's Initiation Toward Defined Oligosaccharide Groups (FITDOG). Using this method, sugar beet pulp (SBP) was transformed into sugar beet oligosaccharides (SBOs) composed of arabinose- and galactose-containing oligosaccharides. Fecal fermentations of SBO and SBP produced similar shifts in donor-specific bacterial communities and acid metabolite profiles with a general enrichment of Bacteroides and Bifidobacterium. However, in vitro tests revealed more Bifidobacterium strains could consume SBO than sugar beet arabinan, and specific strains showed differential consumption of arabinofuranooligosaccharides or galactooligosaccharide (GOS) portions of the SBO pool. Genomic and glycomic comparisons suggest that previously characterized, arabinan-specific, extracellular arabinofuranosidases from Bifidobacterium are not necessary to metabolize the arabino-oligosaccharides within SBO. Synbiotic application of SBO with an SBO-consuming strain Bifidobacterium longum subsp. longum SC596 in serial fecal enrichments resulted in enhanced persistence among 9 of 10 donor feces. This work demonstrates a novel workflow whereby FITDOG creates novel oligosaccharide pools that can provide insight into how compositional differences in fiber drive differential gut fermentation behaviors as well as their downstream health impacts. Moreover, these oligosaccharides may be useful in new prebiotic and synbiotic applications.

Arabinoxylan is a prevalent hemicellulose type, notably heterogeneous and resistant to biodegradation. Arabinofuranosidases (Abfs) remove arabinofuranosyl decorations of arabinoxylan, thus enabling hydrolysis by xylanases. However, a variety of Abf and xylanase specificities have emerged in recent years, necessitating a deeper understanding of their role in biomass degradation. This work investigates the biochemical features of TtAbf43 from Thermothelomyces thermophila, which specifically removes the O-3-linked arabinofuranose moieties from di-substituted xylopyranoses. The enzyme also exhibited secondary hydrolytic activity on o-nitrophenyl-β-d-xylopyranoside and arabinan. The hydrolysis of pretreated wheat and corn bran substrates was assessed using TtAbf43 and AnAbf51, two enzymes with distinct catalytic specificities. The Abfs enhanced the performance of endo-xylanases TmXyn10 and AnXyn11, promoting the release of xylo-oligomers, while the xylanases, in turn, stimulated arabinose release by the Abfs. Additionally, the Abfs facilitated the endo- and exo-activities of the bifunctional xylobiohydrolase/glucuronoxylanase TtXyn30A for the release of xylobiose and short aldouronic acids from biomass. AnAbf51 also acted in synergy with the acetyl xylan esterase OCE6 and the exo-deacetylase TtCE16B in debranching enzymatically derived oligomers from lignocellulose, whereas TtAbf43 remained unaffected by the esterases. These diverse synergistic relationships among different hemicellulases could assist the development of new enzymatic approaches for efficient biomass valorization.

There is growing interest in members of the genus Segatella (family Prevotellaceae) as members of a well-balanced human gut microbiota (HGM). Segatella are particularly associated with the consumption of a diet rich in plant polysaccharides comprising dietary fiber. However, understanding of the molecular basis of complex carbohydrate utilization in Segatella species is currently incomplete. Here, we used RNA sequencing (RNA-seq) of the type strain Segatella copri DSM 18205 (previously Prevotella copri CB7) to define precisely individual polysaccharide utilization loci (PULs) and associated carbohydrate-active enzymes (CAZymes) that are implicated in the catabolism of common fruit, vegetable, and grain polysaccharides (viz. mixed-linkage β-glucans, xyloglucans, xylans, pectins, and inulin). Although many commonalities were observed, several of these systems exhibited significant compositional and organizational differences vis-à-vis homologs in the better-studied Bacteroides (sister family Bacteroidaceae), which predominate in post-industrial HGM. Growth on β-mannans, β(1, 3)-galactans, and microbial β(1, 3)-glucans was not observed, due to an apparent lack of cognate PULs. Most notably, S. copri is unable to grow on starch, due to an incomplete starch utilization system (Sus). Subsequent transcriptional profiling of bellwether Ton-B-dependent transporter-encoding genes revealed that PUL upregulation is rapid and general upon transfer from glucose to plant polysaccharides, reflective of de-repression enabling substrate sensing. Distinct from previous observations of Bacteroides species, we were unable to observe clearly delineated substrate prioritization on a polysaccharide mixture designed to mimic in vitro diverse plant cell wall digesta. Finally, co-culture experiments generally indicated stable co-existence and lack of exclusive competition between S. copri and representative HGM Bacteroides species (Bacteroides thetaiotaomicron and Bacteroides ovatus) on individual polysaccharides, except in cases where corresponding PULs were obviously lacking.

Gum arabic is widely used in the food and beverage industries for its emulsifying, stabilizing, and prebiotic effects, which promote Bifidobacterium growth. The two commercially approved varieties of gum arabic, namely, Acacia senegal gum and A. seyal gum, predominantly consist of arabinogalactan protein (AGP), albeit with different side chain modifications. We previously characterized two enzymes belonging to glycoside hydrolase (GH) family 39 in bifidobacteria involved in the release of α-d-Gal-(1→3)-α-l-Ara and β-l-Arap-(1→3)-α-l-Ara from the side chains of A. senegal gum. Although the carbohydrate structure of A. senegal gum is being increasingly explored, limited information is available on A. seyal gum. In this study, we discovered a novel GH39 β-arabino-oligosaccharide 3-O-β-l-arabinopyranosyl-α-l-arabinofuranosidase from Bifidobacterium catenulatum and revealed the accurate structure of β-l-arabino-oligosaccharides released from A. seyal gum as [β-l-Araf-(1→2)-]_n-β-l-Arap-(1→3)-α-l-Araf-(1→) (n = 0–3). Growth assays and intracellular enzyme activity assessments using B. catenulatum revealed that β-l-arabino-oligosaccharides were degraded to l-arabinose by GH127 β-l-arabinofuranosidase and GH36 β-l-arabinopyranosidase. This study provides new insights into the diversity of AGP structures and the utilization mechanisms of A. seyal gum in bifidobacteria.

The presence of diverse carbohydrate-active enzymes (CAZymes) is crucial for the direct bioconversion of lignocellulose. In this study, various anaerobic microbial consortia were employed for the degradation of 10 g/L of minimally pretreated corncob. The involvement of lactic acid bacteria (LAB) and a CAZyme-rich bacterium (Bacteroides cellulosilyticus or Paenibacillus lautus) significantly enhanced the lactic acid production by Ruminiclostridium cellulolyticum from 0.74 to 2.67 g/L (p < 0.01), with a polysaccharide conversion of 67.6%. The supplement of a commercial cellulase cocktail, CTec 2, into the microbial consortia continuously promoted the lactic acid production to up to 3.35 g/L, with a polysaccharide conversion of 80.6%. Enzymatic assays, scanning electron microscopy, and Fourier transform infrared spectroscopy revealed the substantial functions of these CAZyme-rich consortia in partially increasing enzyme activities, altering the surface structure of biomass, and facilitating substrate decomposition. These results suggested that CAZyme-intensified consortia could significantly improve the levels of bioconversion of lignocellulose. Our work might shed new light on the construction of intensified microbial consortia for direct conversion of lignocellulose.

Dietary oligo- and polysaccharides modulate gut microbiota and thus exert prebiotic activity, which is determined by their heterogeneous structure. To explore the correlations between monosaccharide profile and microbial community, simulated gut fermentation of different glycans, including arabinan (ArB), galactooligosaccharide (GOS), arabinogalactan (ArG), rhamnogalacturonan (RhG), and xyloglucan (XyG) that are characterized by typical sugar residues were performed. Results showed that RhG displayed high contents of galacturonic acid (344.79 mg/g), rhamnose (171.70 mg/g), and galactose (151.77 mg/g), and the degradation ratio of them after fermentation was 73.87 %, 84.96 %, and 87.11 %, respectively. Meanwhile, the relative abundance of glycan-degrading bacteria Bacteroides in the RhG was boosted from 4 h (4.97 %) to 48 h (36.45%). Butyrate-generating bacteria Megasphaera (56.69 %) and Bifidobacterium (28.02 %) are dominant genera in the ArB, which generated the highest concentration of carbohydrate-metabolite (94.58 mmol/L) in terms of acetate, propionate, butyrate and valerate, followed by the ArG (87.36 mmol/L). However, ammonia generation of the ArG increased rapidly, representing the highest content of protein-metabolite (66.36 mmol/L) including ammonia, isobutyrate, and isovalerate. As compared, metabolites generated from protein and carbohydrates grow steadily at a low level during the XyG fermentation. Correlation analysis further indicated that Bacteroides was positively correlated with propionate (p < 0.001), galacturonic acid (p < 0.001), and rhamnose (p < 0.05), while Bifidobacterium has positive correlation with butyrate and arabinose (p < 0.01). Overall, monosaccharides composition in the different oligo- and polysaccharides induces distinct responses of the dominant microbiota and thus modulates the subsequent fermentation metabolites of carbohydrate and protein, promoting a deep understanding of the structure-fermentation relationship of dietary glycans.

Recent investigations have highlighted, both experimentally and clinically, that probiotic strains equipped with arabinofuranosidase, in particular abfA and abfB, favor regular intestinal motility, thus counteracting constipation. By analyzing the gene expression and the proliferative response in the presence of arabinan of the probiotic B. longum W11, a strain previously validated as an anti-constipation probiotic, we have speculated that its response mechanism to arabinan can effectively explain its clinical action. Our approach could be used in the future to select probiotics endowed with arabinofuranosidase-related anti-constipation effects.