Impact of xylanase and glucanase on oligosaccharide formation, carbohydrate fermentation patterns, and nutrient utilization in the gastrointestinal tract of broilers

This study aimed at determining how the degradation of cereal non-starch polysaccharides (NSP) by dietary enzymes during feed digestion can influence nutrient digestibility and NSP fermentability in broilers. Ninety-six one-day-old male broilers were assigned to 4 different treatments: control and enzyme-supplemented wheat-based (WC, WE) or maize-based (MC, ME) treatments. Enzyme supplementation with endo-xylanase and endo-glucanase occurred from day 20 onwards. On day 28, digesta samples were collected. Nutrient digestibility, NSP recovery, oligosaccharide profile, and short-chain fatty acids (SCFA) content were determined. Enzyme supplementation in WE resulted in a higher starch (3%; p = 0.004) and protein (5%; p = 0.002) digestion in the ileum compared to WC. Xylanase activity in WE led to in situ formations of arabinoxylan-oligosaccharides consisting of 5 to 26 pentose units in the ileum. This coincided with decreased arabinose (p = 0.059) and xylose (p = 0.036) amounts in the ceca and higher acetate (p = 0.014) and butyrate (p = 0.044) formation in WE compared to WC. Conversely, complete total tract recovery of arabinoxylan in MC and ME suggested poor maize NSP fermentability. Overall, enzyme action improved nutrient digestibility and arabinoxylan fermentability in the wheat-based diet. The lower response of the maize-based diet to enzyme treatment may be related to the recalcitrance of maize arabinoxylan as well as to the high nutritive value of maize

GH10 and GH11 endoxylanases in Penicillium subrubescens: Comparative characterization and synergy with GH51, GH54, GH62 α-L-arabinofuranosidases from the same fungus

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

GH10 and GH11 endoxylanases in Penicillium subrubescens : Comparative characterization and synergy with GH51, GH54, GH62 α-L-arabinofuranosidases from the same fungus

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

Strategy to identify reduced arabinoxylo-oligosaccharides by HILIC-MSn

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

In vivo formation of arabinoxylo-oligosaccharides by dietary endo-xylanase alters arabinoxylan utilization in broilers

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

Glycoside Hydrolase family 30 harbors fungal subfamilies with distinct polysaccharide specificities

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

Glycoside Hydrolase family 30 harbors fungal subfamilies with distinct polysaccharide specificities

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

Cereal type and combined xylanase/glucanase supplementation influence the cecal microbiota composition in broilers

Dietary fiber-degrading enzyme supplementation in broilers aims at off-setting the anti-nutritive effect of non-starch polysaccharides and at promoting broiler health. Recently, we demonstrated that xylanase/glucanase addition in wheat-based diet improved nutrient digestibility, arabinoxylan fermentability and broiler growth. Conversely, maize arabinoxylan was found to be recalcitrant to xylanase action. These findings suggested that enzyme-mediated improvement of nutrient digestion and carbohydrate fermentation depended on the cereal type present in the diet, and may have contributed to broiler growth. Hence, we aimed at further investigating the link between dietary enzymes and carbohydrate fermentation in broilers, by studying the impact of enzyme supplementation in cereal-based diets, to the microbial communities in the ileum and ceca of broilers. For that purpose, 96 one-day-old male broilers were randomly reared in two pens and received either wheat-based or maize-based starter and grower diets. At d 20, the broilers were randomly assigned to one out of four dietary treatments. The broilers received for 8 d the wheat-based or maize-based finisher diet as such (Control treatments; WC, MC) or supplemented with a xylanase/glucanase combination (Enzyme treatments; WE, ME). At d 28, samples from the digestive tract were collected, and the ileal and cecal microbiota composition was determined by 16S ribosomal RNA gene amplicon sequencing. A similar phylogenetic (alpha) diversity was observed among the four treatments, both in the ileal and the cecal samples. Furthermore, a similar microbial composition in the ileum (beta diversity) was observed, with lactobacilli being the predominant community for all treatments. In contrast, both cereal type and enzyme supplementation were found to influence cecal communities. The type of cereal (i.e., wheat or maize) explained 47% of the total variation in microbial composition in the ceca. Further stratifying the analysis per cereal type revealed differences in microbiota composition between WC and WE, but not between MC and ME. Furthermore, the prevalence of beneficial genera, such as Faecalibacterium and Blautia, in the ceca of broilers fed wheat-based diets coincided with arabinoxylan accumulation. These findings indicated that fermentable arabinoxylan and arabinoxylo-oligosaccharides released by dietary xylanase may play an important role in bacterial metabolism

GH10 and GH11 endoxylanases in Penicillium subrubescens: Comparative characterization and synergy with GH51, GH54, GH62 α-L-arabinofuranosidases from the same fungus

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