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|Stability:||> 2 years under recommended storage conditions|
|Monosaccharides (%):||Arabinose: Xylose = 38: 62|
|Main Chain Glycosidic Linkage:||β-1,4|
|Substrate For (Enzyme):||endo-1,4-β-Xylanase|
High purity Arabinoxylan (Wheat Flour; Medium Viscosity) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Recommended substrate for viscometric and reducing-sugar assays of endo-β-D-xylanase activity.
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Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
endo-1,4-β-Xylanase (EC 18.104.22.168) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.Hide Abstract
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.Hide Abstract
The secretome of Agaricus bisporus: temporal dynamics of plant polysaccharides and lignin degradation.
Duran, K., Magnin, J., America, A. H., Peng, M., Hilgers, R., de Vries, R. P., Baars, J. J. P., Berkel, W. J. H., Kuyper, T. W. & Kabel, M. A. (2023). iScience, 26(7): 107087.
Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom Agaricus bisporus develop and whether they modify lignin models in vitro. To clarify these aspects, A. bisporus secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised A. bisporus endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased. Laccases appeared from day 6 onwards. From day 10 onwards, many oxidoreductases were found, with numerous multicopper oxidases (MCO), aryl alcohol oxidases (AAO), glyoxal oxidases (GLOX), a manganese peroxidase (MnP), and unspecific peroxygenases (UPO). Secretomes modified dimeric lignin models, thereby catalyzing syringylglycerol-β-guaiacyl ether (SBG) cleavage, guaiacylglycerol-β-guaiacyl ether (GBG) polymerization, and non-phenolic veratrylglycerol-β-guaiacyl ether (VBG) oxidation. We explored A. bisporus secretomes and insights obtained can help to better understand biomass valorization.Hide Abstract
Development of a Multi-Enzymatic Approach for the Modification of Biopolymers with Ferulic Acid.
Giannakopoulou, A., Tsapara, G., Troganis, A. N., Koralli, P., Chochos, C. L., Polydera, A. C., Katapodis, P., Barkoula, N. & Stamatis, H. (2022). Biomolecules, 12(7), 992.
A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet–visible (UV–Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries.Hide Abstract
Novel bi-modular GH19 chitinase with broad pH stability from a fibrolytic intestinal symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.
Bai, L., Kim, J., Son, K. H., Chung, C. W., Shin, D. H., Ku, B. H., Kim, D. Y. & Park, H. Y. (2021). Biomolecules, 11(11), 1735.
Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.Hide Abstract
Partial acid-hydrolysis of TEMPO-oxidized arabinoxylans generates arabinoxylan-structure resembling oligosaccharides.
Pandeirada, C. O., Speranza, S., Bakx, E., Westphal, Y., Janssen, H. G. & Schols, H. A. (2021). Carbohydrate Polymers, 275, 118795.
Arabinoxylans (AXs) display biological activities that depend on their chemical structures. To structurally characterize and distinguish AXs using a non-enzymatic approach, various TEMPO-oxidized AXs were partially acid-hydrolysed to obtain diagnostic oligosaccharides (OS). Arabinurono-xylo-oligomer alditols (AUXOS-A) with degree of polymerization 2-5, comprising one and two arabinuronic acid (AraA) substituents were identified in the UHPLC-PGC-MS profiles of three TEMPO-oxidized AXs, namely wheat (ox-WAX), partially-debranched WAX (ox-pD-WAX), and rye (ox-RAX). Characterization of these AUXOS-A highlighted that single-substitution of the Xyl unit preferably occurs at position O-3 for these samples, and that ox-WAX has both more single substituted and more double-substituted xylose residues in its backbone than the other AXs. Characteristic UHPLC-PGC-MS OS profiles, differing in OS abundance and composition, were obtained for each AX. Thus, partial acid-hydrolysis of TEMPO-oxidized AXs with analysis of the released OS by UHPLC-PGC-MS is a promising novel non-enzymatic approach to distinguish AXs and obtain insights into their structures.Hide Abstract
Development of a prebiotic blend to influence in vitro fermentation effects, with a focus on propionate, in the gut.
Collins, S. M., Gibson, G. R., Kennedy, O. B., Walton, G., Rowland, I., & Commane, D. M. (2021). FEMS Microbiology Ecology, 97(8), fiab101.
Short chain fatty acids (SCFAs) derived from the human gut microbiota, and in particular propionate, may beneficially influence metabolic processes such as appetite regulation. Development of prebiotics that induce high propionate levels during fermentation is desirable. A total of 11 candidate prebiotics were screened to investigate their fermentation characteristics, with a focus on propionate production in mixed anaerobic batch culture of faecal bacteria. Further to this, a continuous 3-stage colonic fermentation model (simulating the human colon) was used to evaluate changes in microbial ecology, lactate and SCFA production of three 50:50 blends, comprising both slow and rapidly fermented prebiotics. In mixed batch culture: xylo-oligosaccharide, polydextrose and α-gluco-oligosaccharide were associated with the greatest increase in propionate. Polydextrose, α-gluco-oligosaccharide, β-1,4 glucan and oat fibre induced the greatest reductions in the acetate to propionate ratio. The most bifidogenic prebiotics were the oligosaccharides. Fermentation of a 50:50 blend of inulin and arabinoxylan, through the continuous 3-stage colonic fermentation model, induced a substantial and sustained release of propionate. The sustained release of propionate through the colon, if replicable in vivo, could potentially influence blood glucose, blood lipids and appetite regulation, however, dietary intervention studies are needed. Bifidogenic effects were also observed for the inulin and arabinoxylan blend and an increase synthesis of butyrate and lactate, thus indicating wider prebiotic potential.Hide Abstract
Extraction of sugarcane bagasse arabinoxylan, integrated with enzymatic production of xylo-oligosaccharides and separation of cellulose.
Khaleghipour, L., Linares-Pastén, J. A., Rashedi, H., Siadat, S. O. R., Jasilionis, A., Al-Hamimi, S., Sardari, R. R. R. & Karlsson, E. N. (2021). Biotechnology for Biofuels, 14(1), 1-19.
Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121°C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121°C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0-0.1 M NaOH) in the time and temperature range 10-30 min and 50-150°C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121°C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.Hide Abstract
Wheat cell walls and constituent polysaccharides induce similar microbiota profiles upon in vitro fermentation despite different short chain fatty acid end-product levels.
Lu, S., Mikkelsen, D., Yao, H., Williams, B. A., Flanagan, B. M. & Gidley, M. J. (2021). Food & Function, In Press.
Plant cell walls as well as their component polysaccharides in foods can be utilized to alter and maintain a beneficial human gut microbiota, but it is not known whether the architecture of the cell wall influences the gut microbiota population. In this study, wheat flour cell walls (WCW) were isolated and compared with their major constituents - arabinoxylan (AX), mixed linkage (1,3)(1,4)-β-glucan (MLG) and cellulose - both separately and as a physical mixture of polysaccharides (Mix) equivalent in composition to WCW. These samples underwent in vitro fermentation with a faecal inoculum from pigs fed a diet free of cereals and soluble-fibre to avoid prior adaptation to substrates. During fermentation, samples were collected for DNA extraction and 16S rRNA gene amplicon sequencing. Bioinformatics analyses revealed that the microbial communities promoted during fermentation by AX, MLG, Mix and WCW were similar at the genus level, but differed from the microbiota observed for the cellulose substrate. Differences in proportions of propionate and butyrate end-products were associated with differences in the relative levels of genera. These findings show that, in this experiment, the microbes that flourished were able to utilize diverse WCW polysaccharides alone, in mixtures or in intact cell walls in a similar way, but that different fermentation end-products were associated with AX (propionate) or MLG (butyrate) polysaccharides.Hide Abstract
TEMPO/NaClO2/NaOCl oxidation of arabinoxylans.
Pandeirada, C. O., Merkx, D. W., Janssen, H. G., Westphal, Y. & Schols, H. A. (2021). Carbohydrate Polymers, 259, 117781.
TEMPO-oxidation of neutral polysaccharides has been used to obtain polyuronides displaying improved functional properties. Although arabinoxylans (AX) from different sources may yield polyuronides with diverse properties due to their variable arabinose (Araf) substitution patterns, information of the TEMPO-oxidation of AX on its structure remains scarce. We oxidized AX using various TEMPO:NaClO2:NaOCl ratios. A TEMPO:NaClO2:NaOCl ratio of 1.0:2.6:0.4 per mol of Ara gave an oxidized-AX with high molecular weight, minimal effect on xylose appearance, and comprising charged side chains. Although NMR analyses unveiled arabinuronic acid (AraAf) as the only oxidation product in the oxidized-AX, accurate AraA quantification is still challenging. Linkage analysis showed that > 75 % of the β-(1→4)-xylan backbone remained single-substituted at position O-3 of Xyl similarly to native AX. TEMPO-oxidation of AX can be considered a promising approach to obtain arabinuronoxylans with a substitution pattern resembling its parental AX.Hide Abstract
Formation of Cellulose-Based Composites with Hemicelluloses and Pectins Using Komagataeibacter Fermentation.
Mikkelsen, D., Lopez-Sanchez, P., Wang, D. & Gidley, M. J. (2020). The Plant Cell Wall, 73-87.
Komagataeibacter xylinussynthesizes cellulose in an analogous fashion to plants. Through fermentation of K. xylinus in media containing cell wall polysaccharides from the hemicellulose and/or pectin families, composites with cellulose can be produced. These serve as general models for the assembly, structure, and properties of plant cell walls. By studying structure/property relationships of cellulose composites, the effects of defined hemicellulose and/or pectin polysaccharide structures can be investigated. The macroscopic nature of the composites also allows composite mechanical properties to be characterized. The method for producing cellulose-based composites involves reviving and then culturing K. xylinu in the presence of desired hemicelluloses and/or pectins. Different conditions are required for construction of hemicellulose- and pectin-containing composites. Fermentation results in a floating mat or pellicle of cellulose-based composite that can be recovered, washed, and then studied under hydrated conditions without any need for intermediate drying.Hide Abstract
Structural modifications to water-soluble wheat bran arabinoxylan through milling and extrusion.
Demuth, T., Betschart, J. & Nyström, L. (2020). Carbohydrate Polymers, 240, 116328.
Feruloylated arabinoxylan (AX) is one of the most predominant dietary fiber in cereal grains. In recent decades, soluble AX has gained interest, as a result of its well-established health benefits. Apart from enzymatic degradation during cereal storage, food processing causes AX degradation. These reactions lead to structural modifications and influence both the AX functionalities and its health promoting effects. The aim of this study was to investigate the structural modifications and related property changes of health promoting water-extractable (WE) wheat bran AX through grain milling and extrusion. Multi-detector HPSEC revealed a correlation between Mw, conformational changes and the related viscosity behaviour depending on the processing type. Processing caused molecular degradation of insoluble high Mw AX, which increased the solubility significantly. Moreover, extrusion leaded to a more heterogenic AX fine structure. The detailed characterization of processed dietary fiber may help to facilitate the optimized incorporation of AX in health-promoting foods.Hide Abstract
Barley α‐amylase/subtilisin inhibitor shows inhibitory activity against endogenous xylanase isozyme I of malted barley: A novel protein function.
Sun, J., Xu, F. & Lu, J. (2020). Journal of food Biochemistry, 44(6), e13218.
Mashing process had little influence on the arabinoxylan content in the finished wort. In this paper, a protein with inhibitory activity against the endogenous xylanase isozyme I (X‐I) of malted barley was extracted and purified using a combination of ion‐exchange and size‐exclusion chromatography. The protein was identified as barley α‐amylase/subtilisin inhibitor (BASI). According to the amino acid sequence analysis, BASI was completely different from the previous reported xylanase inhibitors. BASI showed dosage‐dependent inhibitory activity. BASI exhibited a maximum inhibitory activity at 50°C and pH 6.0. BASI inhibited X‐I as a competitive manner.Hide Abstract
A Glycoside Hydrolase Family 62 AL-Arabinofuranosidase from Trichoderma Reesei and Its Applicable Potential during Mashing.
Sun, J., Xu, F. & Lu, J. (2020). Foods, 9(3), 356.
Arabinoxylan is the second most abundant component in the endosperm cell wall of barley and it has been shown to have negative effects on the viscosity and filtration rate of wort and beer. In this study, a glycoside hydrolase (GH) family 62 α-L-arabinofuranosidase (AFase), termed as TrAbf62A, was purified from the culture filtrate of Trichoderma reesei CICC 41495 by a combined chromatographic method. The preferred substrates of the purified TrAbf62A were soluble, highly substituted arabinoxylan oligosaccharides and polymers, similar to the type found in barley grain. TrAbf62A exhibited activity towards oligomeric and polymeric arabinoxylans, as well as colorimetric arabinose-based substrates, thus meeting the criteria to be classified as a type B AFase. TrAbf62A released mainly arabinose and xylose from soluble wheat arabinoxylan, thus indicating a dual lytic enzyme activity. Supplementation of TrAbf62A during mashing, with a loading of 12 mU/g malt, resulted in a 36.3% decrease in arabinoxylan polymer content, a 5.6% reduction in viscosity, and finally, a 22.1% increase in filtration rate. These results revealed that TrAbf62A has a high potential value in improving lautering performance during mashing.Hide Abstract
Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.
Kapsokalyvas, D., Loos, J., Boogers, I. A. L. A., Appeldoorn, M. M., Kabel, M. A. & Zandvoort, M. V. (2019). Biopolymers, 111(3), e23347.
Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two‐photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti‐inhibitory agents.Hide Abstract
Comparison of Japanese and Indian intestinal microbiota shows diet-dependent interaction between bacteria and fungi.
Pareek, S., Kurakawa, T., Das, B., Motooka, D., Nakaya, S., Rongsen-Chandola, T. et al. (2019). NPJ Biofilms and Microbiomes, 5(1), 1-13.
The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and Candida. Candida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.Hide Abstract
Comparative prebiotic activity of mixtures of cereal grain polysaccharides.
Harris, S., Monteagudo-Mera, A., Kosik, O., Charalampopoulos, D., Shewry, P. & Lovegrove, A. (2019). AMB Express, 9(1), 203.
The main components of the non-starch polysaccharide (NSP) fraction of wheat flour are arabinoxylan (AX) and β-glucan. These are also present in other cereal grains, but their proportions vary with AX being the major component in wheat and rye and β-glucan in barley and oats. Therefore, it was hypothesised that these NSPs could act synergistically when fermented in vitro at the ratios present in the major foods consumed, resulting in increased prebiotic activity. AX and β-glucan were therefore tested in in vitro fermentation studies to assess their prebiotic activity when used individually and/or in different ratios. Short-chain fatty-acids (SCFAs) produced from in vitro fermentation were measured using HPLC and bacterial populations were measured using flow cytometry with fluorescence in situ hybridisation (Flow-FISH). Fermentation of AX alone resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate, after 8-24 h of fermentation, however β-glucan alone did not show prebiotic activity. The greatest prebiotic activity, based on concentration of total SCFAs and increases in total bacteria as well as beneficial Bifidobacterium and Clostridium coccoides /Eubacterium groups, was observed when AX and β-glucan were combined at a 3:1 ratio, which corresponds to their ratios in wheat flour which is major source of cereal fibre in the diet. This indicates that the population of bacteria in the human GI tract may be modulated by the composition of the fibre in the diet, to maximise the prebiotic potential.Hide Abstract
High molecular weight mixed-linkage glucan as a mechanical and hydration modulator of bacterial cellulose: Characterization by advanced NMR spectroscopy.
Muñoz-García, J. C., Corbin, K. R., Hussain, H., Gabrielli, V., Koev, T., Iuga, D., Round, A. N., Mikkelsen, D., Gunning, P., Warren, F. J. & Khimyak, Y. Z. (2019). Biomacromolecules, 20(11), 4180-4190.
Bacterial cellulose (BC) consists of a complex three-dimensional organization of ultrafine fibers which provide unique material properties such as softness, biocompatibility, and water-retention ability, of key importance for biomedical applications. However, there is a poor understanding of the molecular features modulating the macroscopic properties of BC gels. We have examined chemically pure BC hydrogels and composites with arabinoxylan (BC–AX), xyloglucan (BC–XG), and high molecular weight mixed-linkage glucan (BC–MLG). Atomic force microscopy showed that MLG greatly reduced the mechanical stiffness of BC gels, while XG and AX did not exert a significant effect. A combination of advanced solid-state NMR methods allowed us to characterize the structure of BC ribbons at ultra-high resolution and to monitor local mobility and water interactions. This has enabled us to unravel the effect of AX, XG, and MLG on the short-range order, mobility, and hydration of BC fibers. Results show that BC–XG hydrogels present BC fibrils of increased surface area, which allows BC–XG gels to hold higher amounts of bound water. We report for the first time that the presence of high molecular weight MLG reduces the density of clusters of BC fibrils and dramatically increases water interactions with BC. Our data supports two key molecular features determining the reduced stiffness of BC–MLG hydrogels, that is, (i) the adsorption of MLG on the surface of BC fibrils precluding the formation of a dense network and (ii) the preorganization of bound water by MLG. Hence, we have produced and fully characterized BC–MLG hydrogels with novel properties which could be potentially employed as renewable materials for applications requiring high water retention capacity (e.g. personal hygiene products).Hide Abstract
Microbial enzymatic degradation of tamarind galactoxyloglucan and wheat arabinoxylan by a porcine faecal inoculum.
Feng, G., Flanagan, B. M., Mikkelsen, D., Williams, B. A. & Gidley, M. J. (2019). Bioactive Carbohydrates and Dietary Fibre, 18, 100183.
This study investigated the enzymatic degradation of solubilised tamarind galactoxyloglucan (GXG) and wheat arabinoxylan (AX), as models for abundant polysaccharides in the primary cell walls of fruits/vegetables and cereal grains, respectively. Using a porcine faecal inoculum, it was demonstrated that the microbial consortia produced surface-located endo-β-xyloglucanase and endo-β-xylanase, to enable the degradation of these polymers into oligosaccharides, close to the microbial surface. Subsequently, these oligosaccharides were completely degraded into monosaccharides by exo-degrading enzymes located both within and on the microbial cell wall, while no enzymatic activities were detected in the cytoplasm. This study reveals a competitive utilisation of GXG and AX by a porcine faecal microbiota, and contributes to our understanding of the mechanisms by which fibre polysaccharides are degraded by the gut microbial community.Hide Abstract
A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.
Wang, K., Cao, R., Wang, M., Lin, Q., Zhan, R., Xu, H. & Wang, S. (2019). Biotechnology for Biofuels, 12(1), 48.
Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.Hide Abstract