GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Penicillium funiculosum </it>produces a range of glycoside hydrolases (GH). The <it>XynD </it>gene, encoding the sole <it>P. funiculosum </it>GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast <it>Pichia pastoris</it>, in order to compare the results obtained with the <it>P. funiculosum </it>GH11 xylanases data.</p> <p>Results</p> <p>High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L^-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with <it>K</it>m_(app)and <it>k_cat</it>/<it>K</it>m_(app)of 3.7 ± 0.2 (mg.mL^-1) and 132 (s^-1mg^-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS.</p> <p>Conclusion</p> <p>Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.</p

Design participatif d'un tableau de bord enseignant

International audienceCe travail s'inscrit dans le cadre du projet E-FRAN METAL, dont l'objectif est de « concevoir, développer et évaluer un ensemble d'outils de suivi individualisé destinés aux élèves ou aux enseignants (Learning Analytics), et des technologies innovantes pour un apprentissage personnalisé des langues à l'écrit (grammaire française) et à l'oral (prononciation de langues vivantes) ». Une des actions de ce projet porte sur la réalisation d'un tableau de bord enseignant permettant de motiver le développement du travail personnel de l'élève. Ce tableau de bord doit comporter deux vues : une vue de l'activité globale des élèves, avec une attention particulière sur leur effort à effectuer les tâches éducatives qui leur sont proposées, et une vue individuelle des élèves qui inclut des recommandations engageantes dans le but d'aider l'enseignant

In vitro gastrointestinal digestion study of two wheat cultivars and evaluation of xylanase supplementation

International audienceBackground: 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

Contribution of image analysis to the description of enzymatic degradation kinetics for particulate food material

International audienceThe 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

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers

第1038回千葉医学会例会・平成13年度千葉大学大学院医学研究院胸部外科学・基礎病理学例会

Heatmap of data sets correlation. Heatmap of correlation between selected genes expression profiles and monosaccharide content of cell wall (%NSP) or oligosaccharides relative content in glucanase digest during apple development and ripening. Positive and negative correlations are respectively shown in red and blue colours. (XLSX 28 kb

Autophagy protein 5 controls flow-dependent endothelial functions

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing

Contribution of cell wall-modifying enzymes to the texture of fleshy fruits. The example of apple

The cell walls of fleshy fruits consist of polysaccharide assemblies (pectin, hemicelluloses and cellulose), the structure and interactions of which vary depending on the genetics of the fruit, and its stage and conditions of development. The establishment and the structural reorganization of the assemblies result from enzyme/protein consortia acting in muro. The texture of fleshy fruits is one of the major criteria for consumer choice. It impacts also post-harvest routes and transformation processes. Disassembly of fruit cell wall polysaccharides largely induces textural changes during ripening but the precise role of each polysaccharide and each enzyme remains unclear. The changes of cell wall polysaccharides during fruit ripening have mainly emphasized a modulation of the fine chemical structure of pectins by hydrolases, lyases, and esterases. This restructuring also involves a reorganization of hemicelluloses by hydrolases/transglycosidases and a modulation of their interactions with the cellulose by non-catalytic proteins, such as expansin. Apple is the third most produced fruit in the world and has been the subject of studies about fruit quality. This paper presents some of the results to date about the enzymes//proteins involved in fruit ripening with particular emphasis on apple

Les réseaux de soins gérontologiques en France

Le système de soins français est l'un des meilleurs, cependant il comporte des limites économiques et sociales. A la lumière de quelques pays étrangers (Royaume-Uni, Etats-Unis, Pays-Bas), on recherche des solution pour maîtriser les dépenses. Les réseaux de soins existent depuis très longtemps de manière informels. Plusieurs lois sont ensuite apparues permettant le développement des réseaux : Les ordonnances " Juppé " en 1996, le FAQSV (Fond d'aide à la qualité des soins de ville) en 1998?Ainsi plusieurs types de réseaux axés sur la gérontologie se sont développés. Les réseaux de soins gérontologiques apparaissent comme une solution pour résoudre le problème des seniors, mais ils ne peuvent se réaliser sans le pharmacien.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF