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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. [ABSTRACT FROM AUTHOR]

Highlights: [•] In all phases studied, xylan structures show a similar degree of substitution. [•] Water solubility of xylan and glucan structures increased 20% during mycelium growth. [•] Xylan alkaline extractability remained the same over the whole process studied. [•] During mycelium growth part of the xylan decreases in size. [ABSTRACT FROM AUTHOR]

Abstract: Pressed potato fibre (PPF) has a high water holding capacity (WHC) affecting its processing as an animal feed. The aim of this study was to characterize cell wall polysaccharides (CWPs) in PPF and investigate their WHC. This was done via sequential extractions. Half of all CWPs were recovered in the hot buffer soluble solids extract as pectins (uronic acid and rhamnose) and galactans wherein most pectins (76%) from PPF were water soluble. Most likely, the network of CWPs is loosened during processing of potatoes. PPF showed a WHC of 7.4 expressed as the amount of water held per g of dry matter (mL/g). Reconstituting hot buffer soluble solids with buffer insoluble solids in water gave a WHC comparable to that of PPF. Removal of alkali soluble solids, which mainly comprised xyloglucans, lowered the WHC of the final residue. The results indicated that interactions between CWPs could affect the WHC of PPF. [Copyright &y& Elsevier]

Abstract: The effects of hydrothermal conditions for pretreating wheat bran on the quality of residual protein for animal feeding, and on monosaccharide release for ethanol production were studied according to a 4×2×2 design with the factors, temperature (120, 140, 160, and 180°C), acidity (pH 2.3 and 3.9), and retention time (5 and 10min). Temperature affected the quality of residual protein for animal feeding. Pretreatment at 120 and 140°C did not affect O-methylisourea-reactive lysine in protein-rich wheat bran residue, although total lysine decreased with increasing temperature at pH 2.3. At temperatures higher than 140°C, reactive lysine decreased and melanoidins, furfural and 5-HMF increased. Lower acidity during pretreatment at 120 and 140°C increased the digestibility of the residual wheat protein in vitro by 36%. Pretreatment conditions did not substantially affect the release of monomeric xylose and arabinose by hemicellulases, which suggests that arabinoxylans in wheat bran are well accessible for enzymes. [Copyright &y& Elsevier]

Abstract: The effect of process conditions used for wheat straw pretreatments on the liquor- and residue-composition was studied. Hereto, the pretreatment conditions were expressed in a ‘combined severity -factor’. The higher the combined severity factor () the more xylan was released from the wheat straw, but the more xylan decomposed and furfural formation occurred. The percentage of residual xylan present after pretreatment appeared to be a good indicator concerning cellulose degradability or bio-ethanol production. Namely, cellulose degradation by using commercial enzymes was higher at higher severities corresponding to a lower amount of residual xylan. The xylan release and degradation was studied in more detail by using HPSEC and MALDI-TOF mass spectrometry. The more severe the treatment the more (acetylated) xylose oligomers with a DP lower than nine were analysed. The presence of (acetylated) xylans with a DP of 9–25 increased slightly from low to medium severity. The quantification of the DP-distribution of the (acetylated) xylans released proved to be a good tool to predict cellulose degradability. [Copyright &y& Elsevier]

The affinity of xylan to cellulose is an important aspect of many industrial processes, e.g. production of cellulose, paper making and bio-ethanol production. However, little is known about the adsorption of structurally different xylans to cellulose. Therefore, the adsorption of various xylans to bacterial cellulose (BC) was studied. Also, the relationship between xylan size and adsorption was analysed. BC was used as cellulosic material, because of its high specific surface area and homogeneous structure. In general, unsubstituted linear xylan parts favoured adsorption to BC. Xylan affinity for BC was also related to xylan size. The presence of arabinosyl and O-acetyl substituents to xylan decreased the adsorption of xylan to BC considerably. Removing substituents resulted in higher amounts of adsorbed material. Most likely, increasing the number of unsubstituted xylosyl residues induced the formation of xylan–xylan interactions, which contributed to adsorption to BC. Schematic models are proposed showing the adsorption of structurally different xylans to BC. [Copyright &y& Elsevier]

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)-1H 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. [Copyright &y& Elsevier]

Thermo-assisted NaOH pretreatment to deconstruct xylan and lignin in sugar cane bagasse (SCB) is poorly understood. Hence, in this research it is was aimed to study the effect of NaOH pretreatment on the insoluble remaining lignin structures. Hereto, SCB milled fibres were pretreated using different dosages of NaOH at different temperatures and residence times. Of untreated SCB about 63% of the lignin compounds were assigned as p -coumarates and ferulates, analysed by pyrolysis-GC/MS as 4-vinyl phenol and 4-vinyl guaiacol, and designated as non-core lignin (NCL) compounds. More severe NaOH pretreatments resulted in lower xylan and lower lignin recoveries in the insoluble residues. Especially, the relative abundance of NCL decreased and this decrease followed a linear trend with the decrease in xylan. Core lignin compounds, analysed as phenol, guaiacol and syringol, accumulated in the residues. The decrease in residual xylan and NCL correlated positively with the enzymatic hydrolysis of the residual glucan. [ABSTRACT FROM AUTHOR]

Pressed potato fibres (PPF), a by-product of starch production, has a high water-holding capacity (WHC). In this study, it is shown that the WHC is caused by a network of mainly insoluble, non-cellulosic cell wall polysaccharides (CWPs). Despite the solubilization of one-fourth of the CWPs from PPF, representing 40–60 w/w% of pectic CWPs (rhamnosyl, uronyl, galactosyl and arabinosyl residues) present in PPF, the insoluble residues still had similar WHCs as PPF. Only after enzymatic hydrolysis of mainly non-cellulosic CWPs, the WHC decreased substantially (by 61%). Combining the cellulose-rich residue obtained after enzyme hydrolysis with a polymeric homogalacturonan (HG)-rhamnogalacturonan-I (RG-I)-arabinogalactan (AG) extract increased the WHC. This increased hydration is suggested to result from the observed adsorption of the soluble HG-RG-I-AG to the insoluble cellulose-rich residue. No adsorption was observed of the HG-RG-I-AG to an insoluble residue enriched in non-cellulosic CWPs. [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]

Highlights: [•] Polysaccharides identified in Brassica napus were partly similar to Brassica campestris. [•] Different from B. campestris, linear arabinan and XXGG-type xyloglucan were found. [•] Next to cellulose, the final residue still contained xyloglucan and pectins. [ABSTRACT FROM AUTHOR]

Abstract: Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached. [Copyright &y& Elsevier]

• Fungal genome sequencing aids functionality prediction of carbohydrate-active enzymes. • Genome comparison revealed significant differences in the enzyme sets of fungi. • An expansion of CAZyme-encoding genes was observed in specific fungi. • Xylanolytic enzymes have high potential in sustainable bioprocesses. As important polysaccharide degraders in nature, fungi can diversify their extensive set of carbohydrate-active enzymes to survive in ecological habitats of various composition. Among these enzymes, xylanolytic ones can efficiently and sustainably degrade xylans into (fermentable) monosaccharides to produce valuable chemicals or fuels from, for example relevant for upgrading agro-food industrial side streams. Moreover, xylanolytic enzymes are being used in various industrial applications beyond biomass saccharification, e.g. food, animal feed, biofuel, pulp and paper. As a reference for researchers working in related areas, this review summarized the current knowledge on substrate specificity of xylanolytic enzymes from different families of the Carbohydrate-Active enZyme database. Additionally, the diversity of enzyme sets in fungi were discussed by comparing the number of genes encoding xylanolytic enzymes in selected fungal genomes. Finally, to support bio-economy, the current applications of fungal xylanolytic enzymes in industry were reviewed. [ABSTRACT FROM AUTHOR]

Abstract: 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, β-xylose-α-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. [Copyright &y& Elsevier]

Abstract: Fibre-specific (1→4)-β-galactan extracted from bast fibre peels of developing flax (Linum usitatissimum L.) stem has been studied to elucidate its structural details. The polysaccharide was characterized by NMR and subjected to partial degradation protocols, including chemical and enzymatic approaches. The oligosaccharide fragments obtained were fractionated by gel permeation chromatography and analyzed for their molecular mass with MALDI-TOF MS. The obtained data show that this flax galactan is a complex RG-I polysaccharide with variable side chain structures. The backbone is composed of the common GalA-Rha repeats with a high degree of branching. These side chains are mainly composed of β-1,4-linked Gal oligomers: (1) short branches of only one or two Gal residue(s); (2) long (linear) branches of up to 26 Gal residues; (3) mixed branches of between 3 and 12 Gal residues (possibly derived from longer linear side chains), that are resistant to galactanase cleavage; (4) side chains of at least 17 Gal residues, decorated with single Ara moieties. The linkage between RG backbone and galactan side chains was confirmed by the presence of fragments with (Rha-GalA) n Hex m structure type. Neither chemical, nor enzymatic hydrolysis yielded oligomeric GalA residues, indicating that RG-I blocks are not interrupted by HGA regions. The polymer can be cleaved only partially by the rhamnogalacturonan hydrolase used, while the remaining part is resistant, probably due to peculiarities of side chain structure. Novel Rha-GalA oligomers were liberated by RG-hydrolase containing two or three Gal attached to Rha near the cleavage site. The native polymer is decorated by acetyl groups, with yet unknown distribution patterns. Treatment with purified and well-characterized galactanase does not change the hydrodynamic volume of flax galactan (despite considerable cleavage of Gal moieties), suggesting a complex “secondary” structure of the polymer. [Copyright &y& Elsevier]

Endo -xylanase and endo -glucanase are supplemented to poultry diets in order to improve nutrient digestion and non-starch polysaccharide (NSP) fermentation. Here, the action of these enzymes on alcohol insoluble solids (AIS) from wheat and maize grains as well as its implications for starch digestion in milled grains were evaluated in vitro , under conditions mimicking the poultry digestive tract. For wheat AIS, GH11 endo -xylanase depolymerized soluble arabinoxylan (AX) during the gizzard phase, and proceeded to release insoluble AX under small intestine conditions. At the end of the in vitro digestion (480 min), the endo -xylanase, combined with a GH7 endo -β-1,4-glucanase, released 30.5 % of total AX and 18.1 % of total glucan in the form of arabinoxylo- and gluco-oligosaccharides, as detected by HPAEC-PAD and MALDI-TOF-MS. For maize AIS, the combined enzyme action released 2.2 % and 7.0 % of total AX and glucan, respectively. Analogous in vitro digestion experiments of whole grains demonstrated that the enzymatic release of oligomers coincided with altered grain microstructure, as examined by SEM. In the present study, cell wall hydrolysis did not affect in vitro starch digestion kinetics for cereal grains. This study contributes to understanding the action of feed enzymes on cereal NSP under conditions mimicking the poultry digestive tract. [Display omitted] [ABSTRACT FROM AUTHOR]

Highlights • LLA treatment of wheat straw results in high carbohydrate recovery. • LLA treatment of wheat straw decreases relative abundance of hydroxycinnamic acids. • Treatment time, together with NH 3 concentration, affects enzymatic hydrolysis. • Low NH 3 loadings loosen the lignocellulose architecture. Abstract Ammonia treatment of lignocellulose improves carbohydrate degradability, however, low ammonia dose treatment effects and mechanisms are hardly considered. This study describes low dose ammonia treatment of wheat straw in a statistical design of experiments (Taguchi design) to evaluate the effects of ammonia concentration, treatment time and the Solid:Liquid ratio on structure, composition and enzymatic degradability of the residual fractions. The results showed that low ammonia concentration (≤2 w/w % NH 3) resulted in a high carbohydrate recovery (>80%) coupled enzymatic hydrolysis of 50% of xylan and 40% of glucan of the treated material using a (hemi-) cellulase enzyme cocktail. This effect coincidences with the relative decrease in ferulic acid by 10% and coumaric acid by more than 50% analysed via pyrolysis-GC–MS, measured as 4-vinyl-phenol and 4-vinyl-guaiacol, respectively. Our findings show that lowering ammonia concentration increased the effect of treatment time on the enzymatic degradability of the residual fraction. [ABSTRACT FROM AUTHOR]

Highlights • Ensiling does not change the cell wall degradation of maize stems. • Fungal treatment does not decrease the lignin content of lower maize stems. • Fungal treatment does not improve the degradability of lower maize stems. Abstract The effect of ensiling and fungal treatment on the chemical composition and on in vitro gas production, an indication of rumen degradability, of maize stems was studied. Stems from two maize cultivars (LG30211 and MZP8057) were separated into three parts: upper (above internode 12 up to and including internode 15), middle (above internode 8 up to and including internode 11), and lower stem (above internode 5 up to and including internode 7). The three parts of the stems were ensiled for 1, 2, 4, and 8 weeks and the lower stem was treated with the white-rot fungi Lentinula edodes and Pleurotus eryngii for 3, 6, and 9 weeks. The dry matter content decreased and the acid detergent fibre (ADF) and acid detergent lignin (ADL) content increased from the top to the bottom in the stem. Upon ensiling, the neutral detergent fibre, ADF and ADL content increased. The gas production between 3 and 20 h, a measure of cell wall degradability in rumen fluid, did not change after ensiling, but gas production within 3 h and within 72 h decreased after ensiling. The acetic acid content and the proportion of ammonia-N in total N increased, and pH decreased, after 1 week of ensiling and did not show a clear trend from 1 to 8 weeks. The ADL and cellulose content of the lower part of the stem of both maize cultivars increased after fungal treatment and the hemicellulose content decreased except for the lower stem of MZP8057 treated with P. eryngii. The ratio of syringyl to guaiacyl compounds increased after fungal treatment. The gas production between 3 and 20 h and within 72 h decreased after fungal treatment. Both ensiling and fungal treatment cannot improve the degradability of the investigated maize stems. [ABSTRACT FROM AUTHOR]

In this study, two acid catalysts, acetic acid (HAc) and sulfuric acid (H 2 SO 4 ), were compared in thermal pretreatments of corn stover, in particular to assess the less understood fate of lignin. HAc-insoluble lignin, analyzed by pyrolysis GC–MS, showed decreasing levels (%) of Cα-oxidized (from 3.7 ± 0.2 to 1.8 ± 0.1), propenyl (from 2.5 ± 0.1 to 1.0 ± 0.1), vinyl-G (from 34.5 ± 1.8 to 28.4 ± 0.9), vinyl-S (from 4.2 ± 0.2 to 3.7 ± 0.1) and methylated (from 4.9 ± 0.04 to 2.8 ± 0.1) lignin units at increasing HAc amounts. Concurrently, unsubstituted and vinyl-H units increased (from 7.5 ± 0.5 to 11.3 ± 0.2 and from 40.5 ± 1.9 to 49.9 ± 0.9, respectively). Similar trends were seen for residual lignin in H 2 SO 4 catalyzed pretreatments, although the composition differed from that of residual HAc-lignin. In particular, H 2 SO 4 -lignin showed slightly lower values (%) for unsubstituted (9.9 ± 0.2) and vinyl-H (45.7 ± 4.1) units, while Cα-oxidized (3.4 ± 0.4), propenyl (1.9 ± 0.1), vinyl-G (28.5 ± 0.9), vinyl-S (4.4 ± 0.6) and methylated (4.6 ± 0.2) lignin units remained higher compared to HAc-catalysis at similar pH values. Xylan yields and corresponding enzymatic conversions of the solids were similar regardless the type of acid. Our findings show that HAc in pretreatments decreased lignin complexity, possibly due to cleavage reactions, although subsequent recondensation reactions increased solid lignin yields, more than H 2 SO 4 , while removal of xylan and enzymatic conversion of solids were equal. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Techno-economic parameters of this pretreatment are presented. • Plant cell wall and composition of biomass are shown. • Environmental aspects of sustainability for hydrothermal are discuses. • Process design integration for hydrothermal pretreatment is presented. The development and sustainability of second-generation biorefineries are essential for the production of high added value compounds and biofuels and their application at the industrial level. Pretreatment is one of the most critical stages in biomass processing. In this specific case, hydrothermal pretreatments (liquid hot water [LHW] and steam explosion [SE]) are considered the most promising process for the fractionation, hydrolysis and structural modifications of biomass. This review focuses on architecture of the plant cell wall and composition, fundamentals of hydrothermal pretreatment, process design integration, the techno-economic parameters of the solubilization of lignocellulosic biomass (LCB) focused on the operational costs for large-scale process implementation and the global manufacturing cost. In addition, profitability indicators are evaluated between the value-added products generated during hydrothermal pretreatment, advocating a biorefinery implementation in a circular economy framework. In addition, this review includes an analysis of environmental aspects of sustainability involved in hydrothermal pretreatments. [ABSTRACT FROM AUTHOR]

The ability of Agaricus bisporus to degrade xylan in wheat straw based compost during mushroom formation is unclear. In this paper, xylan was extracted from the compost with water, 1 M and 4 M alkali. Over the phases analyzed, the remaining xylan was increasingly substituted with (4- O -methyl-)glucuronic acid and arabinosyl residues, both one and two arabinosyl residues per xylosyl residue remained. In the 1 M and 4 M KOH soluble solids of spent compost, 33 and 49 out of 100 xylosyl residues, respectively, were substituted. The accumulation of glucuronic acid substituents matched with the analysis that the two A. bisporus genes encoding for α-glucuronidase activity (both GH115) were not expressed in the A. bisporus mycelium in the compost during fruiting. Also, in a maximum likelihood tree it was shown that it is not likely that A. bisporus possesses genes encoding for the activity to remove arabinose from xylosyl residues having two arabinosyl residues. [ABSTRACT FROM AUTHOR]

The effect of hemicellulose and lignin solubilisation by H 2 SO 4 and NaOH catalysed pretreatments was correlated to the extent of subsequent enzymatic cellulose hydrolysis. Three different grass-type feedstocks, palm empty fruit bunch, sugarcane bagasse and barley straw, were investigated. Soluble fractions after catalysis were characterised for mono- and oligosaccharides contents, while the residues were analysed for constituent monosaccharides composition. Alkali pretreatment resulted into extensive lignin removal. This removal resulted in up to 90% (w/w) conversion of glucan into glucose by enzymes. But, the alkaline conditions also provoked up to 50% unwanted xylan losses. Acid pretreatment resulted into solubilisation (70–80% (w/w)) of xylan with almost no losses, while lignin remained. Although moderate xylan solubilisation increased enzymatic cellulose hydrolysis of residual glucan, extensive removal of xylan decreased it. Therefore, under the treatment conditions, the alkali treatments were the most efficient in terms of enzymatic release of xylose and glucose from the insoluble residues. [ABSTRACT FROM AUTHOR]

Previously, arabinoxylan (AX) depolymerization by dietary endo-xylanase was observed in the broiler ileum, but released arabinoxylo-oligosaccharides (AXOS) were not characterized in detail. This study aimed at extracting and identifying AXOS released in vivo in broilers, in order to delineate the influence of endo-xylanase on AX utilization. Hereto, digesta from the gizzard, ileum, ceca and excreta of broilers fed a wheat-soybean diet without (Con) or with endo-xylanase supplementation (Enz) were assessed. Soluble AX content in the ileum was higher for Enz diet (26.9%) than for Con diet (18.8%), indicating a different type and amount of AX entering the ceca. Removal of maltodextrins and fructans enabled monitoring of AX depolymerization to AXOS (Enz diet) using HPSEC-RI and HPAEC-PAD. A recently developed HILIC-MSn methodology allowed AXOS (DP 4–10) identification in ileal digesta and excreta. Xylanase-induced AXOS formation coincided with decreased total tract AX recovery, which indicated improved AX hindgut utilization. [Display omitted] [ABSTRACT FROM AUTHOR]

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-MSn) 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 (MS2) from HILIC-separated AXOS isomers showed distinct MS3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MSn 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new Mt LPMO9F was characterized for its substrate (xyloglucan) specificity, and Mt LPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both Mt LPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that Mt LPMO9F, having a short active site segment 1 and a long active site segment 2 (−Seg1+Seg2), followed a "substitution-intolerant" xyloglucan cleavage profile, while for Mt LPMO9H (+Seg1−Seg2) a "substitution-tolerant" profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of Mt LPMO9F and Mt LPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations. [Display omitted] [ABSTRACT FROM AUTHOR]

Highlights: [•] Water-soluble carbohydrates were almost completely fermented. [•] Residual pectins were mainly rhamnogalacturonan and branched arabinan. [•] Residual hemicelluloses were mainly xyloglucan, xylan and galactomannan. [•] Carbohydrates could be linked via alkali-labile interaction in cellulose-lignin network. [ABSTRACT FROM AUTHOR]

Unprocessed and acid-extruded rapeseed meal (RSM) was fed to broiler chickens, with and without addition of commercial pectolytic enzymes. Non-starch 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. [ABSTRACT FROM AUTHOR]

Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-β-d-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase‘s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as α-l-Araf-(1→3)-β-d-Xylp-(1→4)-d-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-β-d-xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before. [Copyright &y& Elsevier]

• LPMO-generated C1- and C4-oxidised cello-oligomers have distinct MS/MS patterns. • C4-oxidised cello-oligomers showed B-/Y- and X-type fragments at the oxidised end. • Diagnostic 2,4X n fragments were obtained for C4-oxidised cello-oligomers. • Extensive A-type cleavage was typical for C1-oxidised cello-oligomers. • Reduced and non-reduced cello-oligomers yield distinct fragmentation behaviours. 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. [ABSTRACT FROM AUTHOR]