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Arabinoxylo-oligosaccharides (AXOS) are non-digestible dietary fibers that potentially confer a health benefit by stimulating beneficial bacteria in the gut. Still, a detailed overview of the diversity of gut bacteria and their specificity to utilize structurally different AXOS has not been provided to date and was aimed for in this study. Moreover, we assessed the genetic information of summarized bacteria, and we extracted genes expected to encode for enzymes that are involved in AXOS hydrolysis (based on the CAZy database). The taxa involved in AXOS fermentation in the gut display a large variety of AXOS-active enzymes in their genome and consequently utilize AXOS to a highly different extent. Clostridia and Bacteroidales are generalists that consume many structurally diverse AXOS, whereas Bifidobacterium are specialists that specifically consume AXOS with a low degree of polymerization. Further complexity is evident from the fact that the exact bacterial species, and in some cases even the bacterial strains (e.g. in Bifidobacterium longum ) that are stimulated, highly depend on the specific AXOS molecular structure. Furthermore, certain species in Bifidobacterium and Lactobacillaceae are active as cross-feeders and consume monosaccharides and unbranched short xylo-oligosaccharides released from AXOS. Our review highlights the possibility that (enzymatic) fine-tuning of specific AXOS structures leads to improved precision in targeting growth of specific beneficial bacterial species and strains in the gut.

Identification of arabinoxylo-oligosaccharides (AXOS) within complex mixtures is an ongoing analytical challenge. Here, we established a strategy based on hydrophilic interaction chromatography coupled to collision induced dissociation-mass spectrometry (HILIC-MS n ) to identify a variety of enzyme-derived AXOS structures. Oligosaccharide reduction with sodium borohydride remarkably improved chromatographic separation of isomers, and improved the recognition of oligosaccharide ends in MS-fragmentation patterns. Localization of arabinosyl substituents was facilitated by decreased intensity of Z ions relative to corresponding Y ions, when fragmentation occurred in the vicinity of substituents. Interestingly, the same B fragment ions (MS 2 ) from HILIC-separated AXOS isomers showed distinct MS 3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MS n identification of AXOS was strengthened by using specific and well-characterized arabinofuranosidases. The detailed characterization of AXOS isomers currently achieved can be applied for studying AXOS functionality in complex (biological) matrices. Overall, the present strategy contributes to the comprehensive carbohydrate sequencing., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry. Hence, we studied the fragmentation behaviours of non-, C1- and C4-oxidised cello-oligosaccharides, including their sodium borodeuteride-reduced forms, by using hydrophilic interaction chromatography and negative ion mode collision induced dissociation - mass spectrometry. Non-oxidised cello-oligosaccharides showed predominantly C- and A-type cleavages. In comparison, C4-oxidised ones underwent B-/Y- and X-cleavage close to the oxidised non-reducing end, while closer to the reducing end C-/Z- and A-fragmentation predominated. C1-oxidised cello-oligosaccharides showed extensively A-cleavage. Reduced oligosaccharides showed predominant glycosidic bond cleavage, both B-/Y- and C-/Z-, close to the non-reducing end. Our findings provide signature mass spectrometric fragmentation patterns to unambiguously elucidate the catalytic behaviour and classification of LPMOs., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest. All authors contributed to this study. Peicheng Sun, Matthias Frommhagen, Willem J.H. van Berkel and Mirjam A. Kabel contributed to the conception and design. Peicheng Sun, Matthias Frommhagen, Maloe Kleine Haar and Gijs van Erven developed the methodology and carried out the experiments. Peicheng Sun and Edwin J. Bakx performed the data analysis. Peicheng Sun and Mirjam A. Kabel prepared the original draft. All authors were involved in critically reviewing all data and in writing the final manuscript. All authors read and approved the final manuscript., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enzyme hydrolysed (hemi-)celluloses from oil palm empty fruit bunches (EFBs) are a source for production of bio-fuels or chemicals. In this study, after either peracetic acid delignification or alkaline extraction, EFB hemicellulose structures were described, aided by xylanase hydrolysis. Delignification of EFB facilitated the hydrolysis of EFB-xylan by a pure endo-β-1,4-xylanase. Up to 91% (w/w) of the non-extracted xylan in the delignified EFB was hydrolysed compared to less than 4% (w/w) of that in untreated EFB. Alkaline extraction of EFB, without prior delignification, yielded only 50% of the xylan. The xylan obtained was hydrolysed only for 40% by the endo-xylanase used. Hence, delignification alone outperformed alkaline extraction as pretreatment for enzymatic fingerprinting of EFB xylans. From the analysis of the oligosaccharide-fingerprint of the delignified endo-xylanase hydrolysed EFB xylan, the structure was proposed as acetylated 4-O-methylglucuronoarabinoxylan., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Corn fiber, a byproduct from the corn industry, would be a good source for bioethanol production if the hemicellulose, consisting of polymeric glucoronoarabinoxylans, can be degraded into fermentable sugars. Structural knowledge of the hemicellulose is needed to improve the enzymatic hydrolyses of corn fiber. Oligosaccharides that resisted a mild acid pretreatment and subsequent enzymatic hydrolysis, representing 50% of the starting material, were fractionated on reversed phase and size exclusion material and characterized. The oligosaccharides within each fraction were highly substituted by various compounds. Oligosaccharides containing uronic acid were accumulated in two polar fractions unless also a feruloyl group was present. Feruloylated oligosaccharides, containing mono- and/or diferulic acid, were accumulated within four more apolar fractions. All fractions contained high amounts of acetyl substituents. The data show that complex xylan oligomers are present in which ferulic acid, diferulates, acetic acid, galactose, arabinose, and uronic acids were combined within an oligomer. Hypothetical structures are discussed, demonstrating which enzyme activities are lacking to fully degrade corn glucuronoarabinoxylans.

As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)-(1)H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC-NMR was performed in order to study the structural features of the acetylated oligomers 'on-line' avoiding O-acetyl migration. Finally, the precise location of the 2-O- or 3-O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.

Xylo-oligosaccharides (XOS) with various substituents were fermented in vitro by fecal inocula (FI) from four human volunteers to study the influence of substitution on the ability and rate of fermentation and on the production of short-chain fatty acids (SCFA) and lactate. By all FI used nonsubstituted XOS (nXOS) and arabino-XOS (AXOS) were fermented more quickly than the more complex structures of acetylated XOS (AcXOS) and XOS containing a 4-O-methylglucuronic acid group (GlcA(me)XOS). In the first stage (0-40 h) of the fermentations of nXOS and AXOS mainly acetate and lactate were formed. The fermentations of AcXOS and GlcA(me)XOS resulted in a lower lactate production, whereas the concentration of propionate and butyrate increased. These results put emphasis on the detailed elucidation of the structural features of nondigestible oligosaccharides in general to understand their fermentation mechanisms more precisely.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard‐to‐convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello‐oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H218O, it was confirmed that the C6 gem‐diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem‐diol, could not be excluded. These findings can be extended to how the reactive LPMO‐cosubstrate complex is positioned towards the substrate. [ABSTRACT FROM AUTHOR]

Background: The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-)glucuronic acids (UAme). Hence, α-glucuronidases play an important role in the degradation of UAmexylan structures facilitating the complete utilisation of plant biomass. The characterisation of α-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UAmexylan structures. Results: The mode-of-action of two α-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 °C. ReGH67 was able to release UAme from UAme-substituted xylo-oligosaccharides (UAmeXOS), but only the UAme linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UAmeXOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UAmeXOS (DP 3-4). On the contrary, ReGH115 was able to release UAme from both polymeric UAmexylan and UAmeXOS. ReGH115 cleaved UAme from both internal and non-reducing end xylosyl residues, with the exception of UAme attached to the non-reducing end of a xylotriose oligosaccharide. Conclusion: In this research, and for the first time, we define the mode-of-action of two α-glucuronidases from two different GH families both from the ascomycete R. emersonii. To date, only four α-glucuronidases classified in GH115 are characterised. ReGH67 showed limited substrate specificity towards only UAmeXOS, cleaving UAme only when attached to the non-reducing end xylosyl residue. ReGH115 was much less substrate specific compared to ReGH67, because UAme was released from both polymeric UAmexylan and UAmeXOS, from both internal and non-reducing end xylosyl residues. The characterisation of the mode-of-action of these two α-glucuronidases helps understand how R. emersonii attacks UAmexylan in plant biomass and the knowledge presented is valuable to improve enzyme cocktails for biorefinery applications. [ABSTRACT FROM AUTHOR]

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOSFA,Ac) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOSFA,Ac, a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose. [ABSTRACT FROM AUTHOR]

Abstract: Various plant polysaccharide derived mono- and oligosaccharides were derivatized with the fluorescent 9-aminopyrene-1,4,6-trisulfonate (APTS) and subjected to capillary electrophoresis (CE) in combination with laser induced fluorescence (LIF) detection. CE-LIF was suitable for mol-based quantification of various APTS-monosaccharides. CE-LIF of APTS-oligosaccharides showed high resolutions, while analysis times were at maximum 15min. The coupling of CE to electrospray-iontrap mass spectrometery (MS) with online UV detection showed to be a powerful technique in the identification of APTS-oligosaccharides. For the first time, various APTS-xylo-oligosaccharides, having either no, O-acetyl, arabinosyl or xylosyl substitutions at varying positions, were identified by using CE-LIF and CE-MS n . [Copyright &y& Elsevier]

Penicillium subrubescens has an expanded set of genes encoding putative endoxylanases (Ps XLNs) compared to most other Penicillia and other fungi. In this study, all GH10 and GH11 Ps XLNs were produced heterologously in Pichia pastoris and characterized. They were active towards beech wood xylan (BWX) and wheat flour arabinoxylan (WAX), and showed stability over a wide pH range. Additionally, Ps XLNs released distinct oligosaccharides from WAX, and showed significant cooperative action with P. subrubescens α- L -arabinofuranosidases (Ps ABFs) from GH51 or GH54 for WAX degradation, giving insight into a more diverse XLN and ABF system for the efficient degradation of complex hemicelluloses. Homology modeling analysis pointed out differences in the catalytic center of Ps XLNs, which are discussed in view of the different modes of action observed. These findings facilitate understanding of structural requirements for substrate recognition to contribute to recombinant XLN engineering for biotechnological applications. [Display omitted] • The P. subrubescens genome harbors an expanded set of genes encoding endoxylanases. • P. subrubescens endoxylanases (Ps XLNs) are xylan-specific and base-tolerant. • Ps XLNs display a diverse hydrolysis pattern and 3D-structure. • The diversity amongst Ps XLNs is attractive for various biotechnology applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fungal enzymes of GH30 are only present in four subfamilies based on phylogenetic analysis. • Products from fungal GH30 enzymes are mainly short non-digestible di- and oligosaccharides. • A novel xylobiohydrolase from GH30_SF7 shows potential for xylobiose production. Efficient bioconversion of agro-industrial side streams requires a wide range of enzyme activities. Glycoside Hydrolase family 30 (GH30) is a diverse family that contains various catalytic functions and has so far been divided into ten subfamilies (GH30_1-10). In this study, a GH30 phylogenetic tree using over 150 amino acid sequences was contructed. The members of GH30 cluster into four subfamilies and eleven candidates from these subfamilies were selected for biochemical characterization. Novel enzyme activities were identified in GH30. GH30_3 enzymes possess β-(1→6)-glucanase activity. GH30_5 targets β-(1→6)-galactan with mainly β-(1→6)-galactobiohydrolase catalytic behavior. β-(1→4)-Xylanolytic enzymes belong to GH30_7 targeting β-(1→4)-xylan with several activities (e.g. xylobiohydrolase, endoxylanase). Additionally, a new fungal subfamily in GH30 was proposed, i.e. GH30_11, which displays β-(1→6)-galactobiohydrolase. This study confirmed that GH30 fungal subfamilies harbor distinct polysaccharide specificity and have high potential for the production of short (non-digestible) di- and oligosaccharides. [ABSTRACT FROM AUTHOR]

Highlights: [•] 21 Enzyme recalcitrant oligosaccharides from corn fiber are structurally identified. [•] Common structural features: gal-xyl-ara side chains, acetyl-, and feruloyl groups. [•] Branched backbone xylose residues may be acetylated as well. [•] Ethyl glycosides of hemicellulosic structures identified may originate from SSF. [•] Recalcitrant structures identified clearly point to lacking enzyme activities. [Copyright &y& Elsevier]

Abstract: Different separation (HPAEC, RP–HPLC, CE) and identification (MALDI–TOF–MS, ESI–MS n ) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC–MALDI–TOF–MS) or online (CE–ESI–MS n , RP–HPLC–ESI–MS n ) connection provided complementary information on xyloglucan structure. Online CE–MS and RP–HPLC–MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains. [Copyright &y& Elsevier]

The enzymatic conversion of cellulosic-rich waste streams of white asparagus into cellobiose and cello-oligosaccharides (COS) is proposed for producing a natural carrier agent. The enzyme cocktail 'Celluclast' was used to steer towards maximum conversion and minimum formation of monosaccharides to obtain an enzymatic hydrolysate with a high glass transition temperature (T g). Different enzyme loadings and hydrolysis times were tested in combination with a sodium hydroxide pre-treatment of the asparagus fibre. A loading of 700 nkat/g substrate and 7 h of hydrolysis time resulted in the best yield/purity combination, namely a conversion of 36 g/100 g cellulose with 81% celllobiose/COS. The same hydrolysis conditions were tested in a larger bench-scale experiment (conversion of 45 g/100 g cellulose) and the soluble hydrolysates were concentrated and spray-dried. The high T g (108 °C) of the spray-dried hydrolysates of asparagus fibre proves its potential usage as a carrier agent for spray drying. [Display omitted] • The abundant asparagus waste stream is rich in cellulose. • Steering formation of cellobiose and cello-oligosaccharides (COS) was feasible. • Alkaline pre-treatment of asparagus fibre increased the hydrolysis yield. • Cellobiose and COS have potential as carrier agent for spray drying. [ABSTRACT FROM AUTHOR]