Search

Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus, inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC-MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for β-|O-4' ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Insoluble fiber degradation by supplemented enzymes was previously shown to improve fermentation in poultry, and has been further postulated to disrupt the cereal cell wall matrix, thus improving nutrient digestion. Here, we characterized insoluble feed-derived polysaccharides and lignin in digesta from broilers fed wheat-soybean and maize-soybean diets without or with xylanase/glucanase supplementation. Enzyme supplementation in wheat-soybean diet increased the yield of water-extractable arabinoxylan (AX) in the ileum. Still, most AX (> 73 %) remained insoluble across wheat-soybean and maize-soybean diets. Analysis of so-far largely ignored lignin demonstrated that a lignin-rich fiber fraction accumulated in the gizzard, while both insoluble AX and lignin reaching the ileum appeared to be excreted unfermented. More than 20 % of water-insoluble AX was extracted by 1 M NaOH and 11-20 % was sequentially extracted by 4 M NaOH, alongside other hemicelluloses, from ileal digesta and excreta across all diets. These findings showed that enzyme-supplementation did not impact AX extractability by alkali, under the current experimental conditions. It is, therefore, suggested that the degradation of insoluble AX by dietary xylanase in vivo mainly results in arabinoxylo-oligosaccharide release, which is not accompanied by a more loose cell wall architecture., Competing Interests: Declaration of competing interest Natalia Soares is employed by the funder. All other authors declare that they have no competing interest., (Copyright © 2022. Published by Elsevier B.V.)

Penicillium subrubescens has an expanded set of genes encoding putative endoxylanases (PsXLNs) compared to most other Penicillia and other fungi. In this study, all GH10 and GH11 PsXLNs 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, PsXLNs released distinct oligosaccharides from WAX, and showed significant cooperative action with P. subrubescens α-L-arabinofuranosidases (PsABFs) 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 PsXLNs, 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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. 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.)

Unprocessed and acid-extruded rapeseed meal (RSM) was fed to broiler chickens, with and without addition of commercial pectolytic enzymes. Nonstarch polysaccharide (NSP) fermentability and unfermented NSP structures from RSM were studied in the excreta in detail. From unprocessed RSM, 24% of the nonglucose polysaccharides could be fermented. Acid treatment did not have a significant effect, but enzyme addition did improve fermentability to 38%. Most likely, the significant increase in NSP fermentability can be ascribed to the addition of pectolytic enzymes, which decreased branchiness of the water-soluble arabinan. Mainly xyloglucan, (glucurono-)xylan, (branched) arabinan, and cellulose remained in the excreta. The proportion of unextractable carbohydrates increased in excreta from broilers fed acid-extruded RSM. Probably, acid extrusion resulted in a less accessible NSP matrix, also decreasing the accessibility for pectolytic enzymes added in the diet. During alkaline extraction of the excreta, 39 to 52% (wt/wt) of the insoluble carbohydrates was released as glucosyl- and uronyl-rich carbohydrates, probably originally present via ester linkages or hydrogen bonding within the cellulose-lignin network. These linkages are expected to hinder complete NSP fermentation and indicate that digestibility of RSM may benefit substantially from an alkaline treatment or addition of esterases.

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MS(n). The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an α-l-galactopyranosyl-(1→2)-β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose side chain attached to the O-3 position of the β-1-4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxylan., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.

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 15 min. 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).

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.

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.