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Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

Hydrogels were prepared at high solid contents (70-100 g/L) with cellulose nanocrystals (CNC) and very short xyloglucans (XGs). At 70 g/L, CNCs form cholesteric liquid crystals regularly spaced by a distance of 30 nm. This structure was preserved after adsorption of XG with a molar mass (

Les enzymes capables de dégrader les mannanes appartiennent à plusieurs familles d’hydrolases qui se distinguent par les sites de coupure sur ces polysaccharides. La plus connue est de loin la « β-mannanase », seule autorisée en alimentation animale en Europe et qui est spécifique des liaisons β-(1,4) entre deux mannoses. Si on ne se limite pas aux seules enzymes aujourd’hui homologuées pour l’alimentation animale, les mannosidases, les β-glucosidases, les α-galactosidases, et les mannane acétyl-estérases peuvent participer, seules ou en association, à l’hydrolyse des polymères de mannose. L’ajout d’une β-mannanase dans les aliments des volailles et des porcs permet de réduire en grande partie les effets antinutritionnels, en particulier inflammatoires, des β-mannanes. L’épargne de nutriments qui en résulte peut alors être directement valorisée en formulation en déconcentrant l’énergie du régime. Au-delà de l’économie réalisée sur le coût de l’aliment, des améliorations de la performance (indice de consommation en particulier) et de la santé digestive sont très souvent observées.

The fine-tuning of pectins by polygalacturonases (PGs) plays a key role in modulating plant cell wall chemistry and mechanics, impacting plant development. In plants, the high number of PGs encoded in the genome questions the regulation of pectin depolymerization and the roles of distinct isozymes in the control of development. Here we report the first crystal structures of two PGs from Arabidopsis, PGLR and ADPG2 whose expression overlap in roots. Albeit having overall conserved folds and active sites, PGLR and ADPG2 differed in the structure of their binding grooves and in the amino-acids of the subsites. We determined the structural features that explain the absence of inhibition of the plant PGs by endogenous PG-Inhibiting Proteins (PGIPs). By combining molecular dynamic simulations, analysis of enzymes’ kinetics and hydrolysis products, we showed that subtle differences in PGLR and ADPG2 structures translated into distinct enzyme-substrate dynamics and enzymes’ processivities. Using the plant root as a developmental model, exogenous application of purified enzymes showed that these distinct PGLR/ADPG2 processivities ultimately translated into different impacts on development. The highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. Our study suggests that, in plants, gene redundancy is unlikely to reflect redundant biochemical specificities. Isozymes of distinct specificities and processivities are likely to be of major importance for the fine spatial and temporal regulation of pectin structure.Significance StatementPlant polygalacturonases (PG) are enzymes that play a key role in the regulation of cell wall pectin chemistry by controlling the degree of polymerization of the HG chains. The high number of genes encoding PG in Arabidopsis questions the rationale for such abundance. We solved the crystal structure of two PG (PGLR and ADPG2) whose expression overlap in roots and showed, using combined computational and experimental approaches, that they differ in their enzyme-substrate dynamics, leading to distinct processivities. The highly processive ADPG2 can generate digestion products of shorter degree of polymerization, and upon exogenous application on developing roots, induced drastic developmental defects. Our study suggests that gene redundancy is unlikely to reflect redundant biochemical specificities of isozymes.

International audience; The high fiber content of cereal coproducts used in animal feed reduces the digestibility and nutrient availability. Therefore, the aim of this study was to elucidate the ability of two carbohydrase complexes to degrade the cell wall of wheat, maize, and rice during in vitro digestion. One complex was rich in cell-wall-degrading enzymes (NSPase 1), and the other was similar but additionally enriched with xylanases and arabinofuranosidases (NSPase 2). Degradation of arabinoxylan, the main cereal cell wall polysaccharide, was followed directly by gas-liquid chromatography (GLC) and indirectly through phenolic acid liberation as quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effect was additionally visualized using a unique multispectral autofluorescence approach. Wheat fractions, in particular aleurone, were susceptible to degradation as judged from the redistribution of arabinoxylan (25% reduction in insoluble arabinoxylan), whereas the highest relative liberation of ferulic acid was observed in rice bran (6%). All cereal fractions, except for maize, had a higher release of ferulic acid with NSPase 2 than NSPase 1 (38% in rice and wheat bran, 30% in wheat whole grain, and 28% in wheat aleurone). Thus, the carbohydrase complexes were able to degrade important cell wall components during in vitro digestion but apparently through different mechanisms in wheat, maize, and rice.

International audience; Heteroxylans (HX) from vitreous and floury parts of maize endosperm were isolated. Structural analysis showed a xylan backbone with few unsubstituted xylose residues (

This study focuses on the use of tomato (Solanum lycopersicum L.) by-product biomass from industrial plants as reinforcement for designing a range of new degradable and biobased thermoplastic materials. As a novel technique, this fully circular approach enables a promising up-cycling of tomato wastes. After an in-depth morphological study of the degree of reinforcement through SEM and dynamic analysis, mechanical characterization was carried out. Our mechanical results demonstrate that this circular approach is of interest for composite applications. Despite their moderate aspect ratio values (between 1.5 and 2), the tomato by-product-reinforced materials can mechanically compete with existing formulations; PBS-Tomato fiber, for example, exhibits mechanical performance very close to that of PP-flax, especially regarding strength (+11%) and elongation at break (+6%). According to the matrix and particle morphology, a large range of products—biobased and/or degradable, depending on the targeted application—can be designed from tomato cultivation by-products.

International audience; Structural elucidation of plant cell wall xyloglucan through the analysis of enzymatically produced fragmentsrequires detailed knowledge of enzymes hydrolytic mechanism. In this note, the mode of action and cleavage siteof commercial recombinant xyloglucanases (GH74, Paenibacillus sp.) was studied on native and fluorescent-tagged tamarind xyloglucan. In complement to information provided by the manufacturer, GH74 hydrolysiswas shown dual endo/exo- and exo-processive with low affinity towards labelled reducing-ends. GH74 accom-modated X/G in its subsite --1 and X/L in its subsite +1. Moreover, hydrolysis kinetic indicated a GH74 activityinhibition by excess products. These results will help for application of this enzyme in xyloglucans structuralanalysis or for processing cell walls.

The high fiber content of cereal coproducts used in animal feed reduces the digestibility and nutrient availability. Therefore, the aim of this study was to elucidate the ability of two carbohydrase complexes to degrade the cell wall of wheat, maize, and rice during

BACKGROUNDCereal co‐products rich in dietary fibres are increasingly used in animal feed. The high fibre content decreases the digestibility and reduces the nutrient and energy availability, resulting in lower nutritive value. Therefore, this study investigated the ability of two carbohydrase complexes to solubilize cell‐wall polysaccharides, in particular arabinoxylan (AX), from different cereal fractions of wheat, maize, and rice using an in vitro digestion model of the pig gastric and small intestinal digestive system. The first complex (NSPase 1) was rich in cell‐wall‐degrading enzymes, whereas the second complex (NSPase 2) was additionally enriched with xylanases and arabinofuranosidases. The extent of solubilization of insoluble cell‐wall polysaccharides after in vitro digestion was evaluated with gas–liquid chromatography and an enzymatic fingerprint of the AX oligosaccharides was obtained with high‐performance anion‐exchange chromatography with pulsed amperometric detection.RESULTSThe addition of carbohydrase increased the digestibility of dry matter and solubilized AX in particular, with the greatest effect in wheat fractions and less effect in maize and rice fractions. The solubilization of AX (expressed as xylose release) ranged from 6% to 41%, and there was an increased effect when enriching with xylanases and arabinofuranosidases in wheat aleurone and bran of 19% and 14% respectively. The enzymatic fingerprint of AX oligosaccharides revealed several non‐final hydrolysis products of the enzymes applied, indicating that the hydrolysis of AX was not completed during in vitro digestion.CONCLUSIONThese results indicate that the addition of a carbohydrase complex can introduce structural alterations under in vitro digestion conditions, and that enrichment with additional xylanases and arabinofuranosidases can boost this effect in wheat, maize, and rice. © 2020 Society of Chemical Industry

The properties of pectinolytic enzymes have drawn a great attention from many research teams as potential biological catalysts in diverse industrial processes for plant transformation. Pectin-related enzymes are ubiquitous, produced by plants, fungi and bacteria. Due to the highly complex structure described in Chap. 2, pectin is the substrate for many modifying and degrading enzymes belonging to three families of Carbohydrate Active enZymes (CAZy): glycoside hydrolases, polysaccharide lyases and carbohydrate esterases. They are distinguished by their different mechanisms (hydrolysis or β-elimination), action patterns (endo/exo) and specificities (homogalacturonan, rhamnogalacturonan, neutral side-chains, oligogalacturonates). In the 90s, literature dealt with their biochemical characterisation while more recently, progress in protein biotechnologies allowed investigating the production of recombinant and mutated enzymes to study the role of key amino acids. This chapter reviews the state of knowledge of pectin-processing enzymes to illustrate their structural and functional diversities. Their physico-chemical and biological properties are presented, as well as the occurrence of inhibiting proteins.

International audience; According to the high interest in agro-industrial waste reutilisation, underutilised lignocellulosic materials, such as walnut shell (WS) and pea pod (PP), come in focus. The aim of this paper was to evaluate WS and PP as sources for the production of xylooligosaccharides (XOS). Hemicelluloses from WS and PP were recovered by combining varying parameters of delignification and alkaline extraction. At optimal recovery conditions, the fractions were further hydrolysed to XOS using GH11 endo-xylanase, by varying time and enzyme concentration. Xylose was predominant in the monomeric composition of the obtained hemicelluloses, building low-branched (arabino) glucuronoxylan, in WS exclusively, while in PP some xyloglucan as well. Delignification was essential for high recovery of total xylose from the materials, up to at least 70 %. High xylan conversions were obtained for 24 h hydrolysis, resulting in xylobiose and xylotriose when using low enzyme concentration, while in xylose and xylobiose with high enzyme concentration.

International audience; To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a beta-xylosidase, a alpha-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

International audience; Wood recycling is common practice in most countries. Currently, recovered wood is used for relatively low value uses including energy generation, particleboard manufacture, animal beddings and landscape uses. This paper gives an overview of two research projects that aim to generate high-value products from recovered wood and thereby encourage even higher rates of wood recycling. One project is concerned with the extraction of nano-crystalline cellulose from waste MDF and the other produces laminated beams from recovered wood.

Sugar beet pectin is a byproduct of the sugar industry with a particularly low gelling power which hinders its application as gelling agent and thickener. Here we consider the use of freeze casting to shape sugar beet pectin into lightweight foams. Freeze casting processing conditions such as the applied thermal gradient and the polysaccharide concentration were explored to obtain macroporous lightweight foams with different textures. The precise control over the foams texture and pore anisotropy was decisive for their performance as liquid transport devices by capillary ascension and for their mechanical performance. Overall, the obtained results show that the formation of highly anisotropic structures using freeze casting can be instrumental in the upcycling of polysaccharide industrial byproducts., Comment: 24 pages, 7 figures

International audience; Carbohydrate-degrading multi-enzyme preparations (MEP) are used to improve broiler performances. Their mode of action is complex and not fully understood. In this study, we compared the effect of water-soluble fractions isolated at the pilot scale from wheat grain incubated with (WE) and without (WC) MEP. The fractions were incorporated in a wheat-based diet (0.1% w/w) to feed Ross PM3 broilers and compared with a non-supplemented control group (NC). The body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) until d 14 were determined. At d 14, ileal and cecal contents and tissue samples were collected from euthanized animals. The intestinal contents were used to measure the short-chain fatty acids (SCFA) concentration using gas chromatography and to determine the abundance and composition of microbiota using 16S sequencing. Villi length of ileal samples was measured, while L-cell and T-cell densities were determined using immuno-histochemistry. The MEP treatment increased the amount of water-soluble arabinoxylans (AX) and reduced their molecular weight while retaining their polymer behavior. The WE fraction significantly (P < 0.05) increased FI by 13.8% and BWG by 14.7% during the first wk post hatch when compared to NC. No significant effect on FCR was recorded during the trial. The WE increased the abundance of Enterococcus durans and Candidatus arthromitus in the ileum and of bacteria within the Lachnospiraceae and Ruminococcaceae families, containing abundant butyrate-producing bacteria, in the ceca. It also increased the concentration of SCFA in the ceca, decreased the T-lymphocyte infiltration in the intestinal mucosa, and increased the glucagonl-ike-peptide-2 (GLP-2)-producing L-cell density in the ileal epithelium compared with WC and NC. No significant effects were observed on villi length. These results showed that AX present in the WE fraction altered the microbiota composition towards butyrate producers in the ceca. Butyrate may be responsible for the reduction of inflammation, as suggested by the decrease in T-lymphocyte infiltration, which may explain the higher feed intake leading to improved animal growth.

International audience; Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast (R) preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

International audience; Xyloglucan (XG) is believed to act as a cementing material that contributes to the cross-linking and mechanical properties of the cellulose framework in plant cell walls. XG can adsorb to the cellulose nanocrystal (CNC) surface in vitro in order to simulate this in vivo relationship. The target of our work was to investigate the sorption behavior of tamarind seed XG on CNC extracted from cotton linters at different XG/CNC concentration ratios, that is, different adsorption regimes regarding the XG-CNC complex organization and the enzymatic susceptibility of XG. First, we determined the adsorption isotherm. Second, XG-CNC complexes were enzymatically hydrolyzed using a xyloglucan-specific endoglucanase in order to quantify the different XG fractions involved in binding to CNC and to determine adsorption regimes, that is, presence of loops, tails, and trains. Finally, the architecture of the XG-CNC complex was investigated by transmission electron microscopy imaging of negatively stained XG-CNC suspensions and XG immunolabeled suspensions at different XG/CNC concentration ratios, both before and after xyloglucanase hydrolysis process. This study revealed that an increasing XG/CNC concentration ratio led to a change in the XG binding organization to CNC. At low XG/CNC concentration ratios, almost all XG chains were bound as trains to the CNC surface. In contrast, at increasing XG/CNC concentration ratios, the proportion of loops and tails increases. The organization change induces CNC aggregation to form a cellulose/XG network at low XG/CNC regimes, whereas CNC remains in the form of individual particles at higher XG/CNC regimes. Results are discussed both regarding the biological role of XG in plant cell walls and in the perspective of designing new biobased materials.

The filamentous fungus Talaromyces versatilis produces a wide range of cellulolytic and hemicellulolytic enzymes such as xylanases. The recent accessibility to the T. versatilis genome allows identifying two new genes, xynE and xynF, encoding glycoside-hydrolases from family GH11. Both genes were cloned and expressed in the methylotrophic yeast Pichia pastoris in order to compare these new xylanases with two other GH11 xylanases from T. versatilis (XynB and XynC) that were previously reported. High-level expression of recombinant enzymes was obtained for the four enzymes that were purified to homogeneity. The XynB, XynC, XynE and XynF enzymes have molecular masses of 34, 22, 45 and 23 kDa, an optimal pH between 3.5 and 4.5 and an optimal temperature between 50 °C and 60 °C. Interestingly, XynF has shown the best thermal stability at 50 °C for at least 180 min with a weak loss of activity. The four xylanases catalysed hydrolysis of low viscosity arabinoxylan (LVAX) with K m(app) between 11.5 and 23.0 mg.mL−1 and k cat/K m(app) 170 and 3,963 s−1 mg−1.mL. Further investigations on the rate and pattern of hydrolysis of the four enzymes on LVAX showed the predominant production of xylose, xylobiose and some (arabino)xylo-oligosaccharides as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing degree of polymerisation of oligomer up to 6, suggesting that the specificity region of XynE and XynF spans at least six xylose residues. Because of their attractive properties, T. versatilis xylanases might be considered for biotechnological applications.

International audience; Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

International audience; Retting is an important step in traditional cassava processing that involves tissue softening of the roots to transform the cassava into flour and various food products. The tissue softening that occurs during retting was attributed to the degradation of cell wall pectins through the action of pectin-methylesterase and pectate-lyase that possibly originated from a microbial source or the cassava plant itself. Changes in cell wall composition were investigated during retting using chemical analysis, specific glycanase degradation and immuno-labelling of cell wall polysaccharides. Pectic 1,4-beta-D-galactan was the main cell wall polysaccharide affected during the retting of cassava roots. This result suggested that better control of pectic galactan degradation and a better understanding of the degradation mechanism by endogenous endo-galactanase and/or exogenous microbial enzymes might contribute to improve the texture properties of cassava products.

International audience; The use of exogenous cell wall degrading enzymes to improve the nutritional values of cereal grains has increased during the last decades. In this study, products from enzymatic hydrolysis of wheat grain were isolated and their ability to induce short-chain fatty acids was investigated using in vitro fermentation with cecal microbiota. Water-soluble fractions were obtained following incubation without and with a multicomponent enzyme preparation (MEP) (2 mL/kg) containing essentially endoxylanase and endoglucanase activities. These were further fractionated by size exclusion chromatography into high molecular weight (HMw) and low molecular weight (LMw) sub-fractions. The MEP treatment increased the content in water-soluble arabinoxylan (AX) and decreased the degree of polymerization (DP) of the xylan-backbone. In vitro fermentation assays using broiler cecal content as inoculum demonstrated that low substituted AX with reduced molecular weight isolated after MEP treatment yielded higher concentrations of acetate and butyrate. This result indicates that cecal fermentation of enzymatic degradation products into short-chain fatty acids, particularly butyrate, is one of the important mechanisms of MEP action on wheat-based diets that can contribute to the improvement of intestinal health and animal performance.

International audience; Pectins account for one of the major polysaccharides of plant cell walls and are commonly used in the food industry as gelling and stabilising agents. Pectin methylesterase (PME) can alter pectin structure and affect their gelling properties by de-esterifying galacturonic acid. Previously, we reported different behaviours of plant and fungal PMEs on high methylated pectins in solution or during the gelling process (Slavov et al., 2009). In the present study, the behaviour of the two PMEs is studied on a medium methylated pectin in solution, i.e. in the absence of calcium, or during the gelling process, i.e. in the presence of calcium. Only slight modifications were observed for Aspergillus aculeatus PME behaviour between the two conditions. On the opposite we show a role of calcium on the processivity of orange PME: the processivity decreases in the presence of calcium. (C) 2011 Elsevier Ltd. All rights reserved.

International audience; We purified a novel, thermoactive pectate lyase (Pel), designated Pel 1 from the hyperpectinolytic mutant (CT1) of Penicillium occitanis using single-step, anion-exchange chromatography. The purified Pel 1 had a pl of 6.5 and a molecular mass of approximately 39 kDa as estimated by SDS-PAGE. The activity of Pel 1 was assayed in the presence of several divalent cations at different concentrations. Although we noted that the tested divalent cations are required for maximum activity, we found that none of them could substitute for the effects of 2 mM Ca(2+). Using three different carbon sources, our results showed that the highest level of Pel 1 activity (434 U/ml) was obtained in a medium supplemented with apple pectin. With polygalacturonic acid, Pel1 exhibited a Km of 0.0199 mg/ml and a V(max) of 17.19 mu mol/min/mg at pH 7.0. However, at pH 9.0. the K(m) and V(max) values were 0.0136 mg/ml and 19.23 mu mol/min/mg, respectively. The optimum temperature and pH value required for maximal enzymatic activity were found to be 60 degrees degrees C and pH 9.0. respectively. At this pH, the half-life time stability at 60 degrees C was measured to be 16.4 min. The novel Pel1 is very thermally stable and presents alkalophilic parameters; such features are rarely exhibited by fungal Pels. (c) 2011 Elsevier Ltd. All rights reserved.

International audience; Functional properties of “green labelled” pectins extracted with enzymes from chicory root, citrus peel and cauliflower by-products were assessed. Chicory and citrus pectins were selected to study their gelling properties, while cauliflower pectin was chosen to investigate its emulsifying ability. High methoxy chicory and citrus pectins were shown to gel in the presence of sucrose at acidic pH, whereas their corresponding low methoxy pectins were able to gel in the presence of calcium. Additionally, HM cauliflower pectin exhibited emulsifying ability. Atomic Force Microscopy was used to better understand the gelling mechanism of pectins and particularly the first steps of gel setting. The present work demonstrates that “green labelled” enzyme-extracted pectins can be successfully used as gelling or emulsifying agents. The present study allows enhancing the value of “green” extraction of pectins, since such extraction leads to products with good functional properties that can directly be used for food and non-food purposes.

International audience; High methoxy pectin was submitted to various amounts of a fungal pectin methylesterase (PME) from Aspergillus aculeatus and of a plant PME from orange in the presence of calcium. The systems were characterized by rheological means during the gelation process. By the way of in situ demethoxylation with low amount of orange PME, it was possible to gel pectin from the beginning of the reaction although its high degree of methylation around 70. To understand this unusual properties, the behaviour of the two enzymes was investigated in pectic gels and in solution through the analysis of content and distribution of the remaining methyl esters. In the gel, the degree of methylation decreased slowly with orange PME and rapidly with Aspergillus PME. The degree of methylation and degree of blockiness after treatment with each PME in solution or in gels were slightly different. Possible explanations for this are evolving visco-elastic properties, including gel formation or influence of calcium on the enzyme–substrate complex.

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International audience; Background: The filamentous fungus Talaromyces versatilis is known to improve the metabolizable energy of wheat-based poultry diets thanks to its ability to produce a pool of CAZymes and particularly endo-beta(1,4)-xylanases. In order to appreciate their in vivo mode of action, the supplementation effect of two of its xylanases, XynD and XynB from families GH10 and GH11 respectively, have been evaluated on two different wheat cultivars Caphorn and Isengrain, which were chosen amongst 6 varieties for their difference in non starch polysaccharides content and arabinoxylan composition. Results: Polysaccharides digestion was followed during 6 h along the digestive tract using the TNO gastrointestinal model-1, to mimic monogastric metabolism. Polysaccharide degradation appeared to occur mainly at the jejunal level and was higher with Isengrain than with Caphorn. For both cultivars, XynD and XynB supplementation increased notably the amount of reducing end sugars into the jejuno-ileal dialysates, which has been confirmed by a valuable increase of the soluble glucose into the jejunal dialysates. Conclusions: The amounts of arabinose and xylose into the dialysates and ileal deliveries increased consequently mainly for Caphorn, suggesting that XynD and XynB supplementation in wheat-based diet could alleviate the anti-nutritional effects of arabinoxylans by limiting the physical entrapment of starch and could increase the available metabolizable energy.

� Background and Aims The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremulaalba). � Methods Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested. � Key Results In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance. � Conclusions Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical proper- ties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these in- fluence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettabil- ity, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.

pdf; International audience; Pectin gels and pectin–cellulose binary gels were used as cell wall-mimicking systems to investigate the diffusion ability of a fungal pectin methylesterase. Increasing content of cellulose in the gel appears to result: (i) in longer demethylated blocks thus favouring AaPME processivity, and (ii) in accelerated enzyme kinetics. To better understand this unexpected behaviour, a method was set up to investigate the gel porosity as a function of the cellulose content by following the passive diffusion of three pullu- lans having different hydrodynamic volumes. Like the enzyme, the pullulans diffused more efficiently in the gels containing the highest proportions of cellulose. Altogether, these results suggest that the gel settled differently during formation according to the respective proportions of the two polysaccharides. With cellulose present, a fraction of pectin would form close interactions with the microfibrils result- ing in a larger volume accessible to diffusing molecules. This volume would be related to the cellulose concentration.

This work was supported by a grant from the Conseil Regional de Picardie, France and the European Regional Development Fund (PECTINHIB project) through a studentship awarded to ML' and from the Agence National de la Recherche (GALAPAGOS project, ANR-12-BSV5-0001; International audience; Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of similar to 3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity. (C) 2015 Elsevier B.V. All rights reserved.

International audience; The objective of the present work was to relate the physical evolution quantified by image analysis to the chemical transformation of beet pulp particles during enzymatic degradation. Beet pulps were degraded into a torus reactor equipped for visualisation. Pectinolytic and cellulolytic enzymes were used separately or in combination. Two global image analysis techniques were tested to characterise the size distribution of overlapping particles. Granulometric curves were extracted by mathematical morphology and a regularisation dimension was assessed by fractal analysis. Both techniques proved efficient to follow particle size evolution during degradation. When using cellulolytic enzymes alone, no chemical or physical evolution was observed. When using pectinolytic enzymes, a chemical modification occurred without any physical evolution. Particles physically disappeared when both enzymes were used. The chemical and physical evolutions of particles during degradation were interpreted taking into account the current model of molecular arrangement of primary cell walls.

Forty microbial strains isolated from raw milk samples and black and green olives were grown in MP5 (mineral pectin 5) medium containing 0.5% lemon pectin. All strains synthesized an extracellular polygalacturonase. Rhodotorula sp. ONRh9 (0.44 U·mL–1) and Leuconostoc sp. LLn1 (0.16 U·mL–1), which had a more active poly galacturonase in MP5 medium, were studied in MAPG5 medium containing polygalacturonic acid. Highest biomass and polygalacturonase production by these two strains were observed for polygalacturonic acid concentrations of 10 g·L–1(Rhodotorula sp. ONRh9) and 5 g·L–1(Leuconostoc sp. LLn1) and for initial pH values of 6 (Rhodotorula sp. ONRh9) and 5.5 (Leuconostoc sp. LLn1). The two strains grown in fermenters in MAPG5 medium generated the following results: with controlled initial pH, Rhodotorula sp. produced maximum biomass (DO) and polygalacturonase (PG) after 20 h (DO, 3.86; PG, 0.24 U·mL–1) of growth, and this level was sustained until the end of the culture; Leuconostoc sp. LLn1 synthesized more cells and polygalacturonase between 4 h (DO, 1.80; PG, 0.17 U·mL–1) and 24 h (DO, 3.90; PG, 0.27 U·mL–1) of culture. With uncontrolled initial pH, the cultures produced maximum biomass and poly galacturonase after 20 h (DO, 3.30; PG, 0.26 U·mL–1) for Rhodotorula sp. ONRh9 and 10 h (DO, 2.84; PG, 0.17 U·mL–1) for Leuconostoc sp. LLn1.Key words: Leuconostoc, Rhodotorula, polygalacturonic acid, polygalacturonase, cell growth.[Journal translation]

International audience; Exogenous non-starch polysaccharide (NSP) enzymes do not always exhibit the same improvement in AMEn (apparent metabolisable energy corrected for nitrogen balance) in wheat-fed broiler chickens. In order to understand the factors that determine this variation, the in vitro mode of action of an NSP enzyme preparation (Rovabio (TM) Excel) was investigated in several wheats and wheat extracts. In vivo experiments were also performed in broilers fed seven different wheat cultivars. In vitro studies showed an important solubilisation of insoluble arabinoxylan (AX) in wheat flours, ranging from 65 to 85% depending on the wheat cultivar. The negative correlation observed between arabinose/xylose ratio and AX solubilisation (r(2) = 0.45, P = 0.001) suggests that the variation in solubilisation can be partly explained by the AX structure. NSP enzymes are also able to hydrolyse wheat bran AX by 30-50% depending on the enzyme dosage. Whereas rapid solubilisation of insoluble AX was confirmed in pig digestive physiological conditions (TIM-1), only a minor proportion of AX was hydrolysed as < SkDa oligomers and then absorbed. In vivo studies on wheat-based diets showed an average AMEn improvement of 108 kcal kg(-1) dry matter (DM) (P < 0.001), but a large variation in enzyme efficacy (up to 200 kcal kg(-1) DM) was observed. Among the wheat characteristics measured, only a significant negative correlation between AMEn improvement and wheat moisture content was found (r(2) = 0.71, P = 0.035). A tendency of positive correlation between AMEn improvement with Rovabio (TM) Excel and the arabinose/xylose ratio of water-soluble AXs of wheat (r(2) = 0.42, P = 0.068) and between the arabinose/xylose ratio of water-soluble AXs and the moisture content of wheat (r(2) = 0.52, P = 0.064) was also observed. It can be deduced from the results that the AX structure might influence the efficiency of enzyme supplementation. In feed, Rovabio (TM) Excel quickly solubilises the different AXs of various wheat cultivars as a result of its broad enzyme activity profile. However, relationships between wheat characteristics and the response of wheat batches to enzyme addition are not yet fully understood.

Two series of lime pectins with different degrees and patterns of methyl-esterification were successively fractionated by size exclusion followed by anion exchange chromatography. ‘Homogeneous’ populations with respect to molar mass and charge density were thereby obtained and their physico-chemical properties were investigated. Recovered fractions were characterised by a high galacturonic acid and a low neutral sugars content. The homogeneous population obtained for each pectin sample was treated with side-chain releasing enzymes (rhamnogalacturonan hydrolase, endo-β-(1→4)- d -galactanase, endo-α-(1→5)- l -arabinanase, α- l -arabinofuranosidase and β- d -galactosidase) and combinations of these enzymes. The reaction products were fractionated by anion exchange chromatography and their constitutive homogalacturonans were recovered. Each homogalacturonan was found homogeneous with respect to charge density and to molar mass (polydispersity index close to 1).

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.

The mode of action of the endopolygalacturonase from Fusarium moniliforme was studied towards a series of pectins with different amounts and distribution patterns of methyl-ester groups. The enzyme hydrolysed the linkages between two galacturonic acid residues according to a multi-chain attack mechanism, at least at the early stage of the reaction. The final percentage of hydrolysis decreased with increasing the degree of methylation. The distribution pattern of the methyl groups affected the rate of hydrolysis as well as the final percentage of hydrolysis, a blockwise distribution being more favourable than a random one. The final products, as analysed by mass spectrometry, included methyl-esterified oligogalacturonates. The detailed analysis of the structure of the oligomers showed that the enzyme was able to accommodate methylated galacturonic acid in its active site, but that methyl-esterification negatively affected the affinity of the enzyme.