Endoxylanase (Tx-Xyl) 3D-structure, quenching rate constants (
K
Q) and binding constants (
K
A) to phenolic compounds.
Display omitted
► Phenolic compounds inactivate endoxylanase by non competitive ...multi-site inhibition mechanism. ► The microenvironment of tryptophan residues of the enzyme changes through phenolic compound binding. ► Enzyme–phenolics interaction affinity was higher with increasing phenolic hydroxyl content.
Phenolic compounds generated from lignin degradation during the pre-treatment step in the process of producing bioethanol from lignocellulosic biomass are known to be inhibitory to enzymatic hydrolysis and fermentation. The inactivation mechanism of a GH11 endoxylanase (Tx-Xyl) by several phenolic compounds varying in their hydroxyl and methoxyl radical content was investigated. Apparent kinetic inactivation parameters were measured as an approximate index of the inhibitory effects. All the tested aromatic compounds had strong negative impact on enzyme activity and kinetic analysis revealed non competitive multi-site inhibition mechanism. The interactions between Tx-Xyl and the phenolic compounds were further studied by steady-state (tryptophan) fluorescence spectroscopy. Changes in
λ
max of emission and quenching of fluorescence intensity indicated changes in the microenvironment of tryptophan residues. In agreement with the kinetic parameters, the fluorescence derived binding constants evidenced higher enzyme–phenolics interaction affinity with increasing phenolic hydroxyl radical content, suggesting clear correlations of such radicals with the inhibitory effects. Results indicated that the inhibitory effects of phenolic compounds on Tx-Xyl activity are most likely brought about by conformational alterations of the enzyme protein inducing steric inactivation.
► Nanocomposites of xylan-model lignin were reconstituted in vitro. ► Nanocomposites with increased lignin content had different particle sizes. ► GH11 xylanase action on xylan was negatively ...correlated with lignin content of nanocomposites.
The effects of lignin content on the activity and action pattern of GH11 endoxylanase from Thermobacillus xylanilyticus were investigated using in vitro reconstituted non-covalent glucuronoarabinoxylan-model lignin (GAX-DHP) nanocomposites. Four types of nanocomposites were prepared, each displaying different lignin contents. Variations in the DHP (model lignin) polymerization process were induced by increasing the coniferyl alcohol concentration. Examination of the morphology of the nanocomposites revealed globular particles enrobed in a matrix. The size of these particles increased in line with the lignin concentration. Physicochemical characterization of the in vitro reconstituted GAX-DHPs strongly suggested that increased particle size is directly related to the solubility and reactivity of coniferyl alcohol, as reflected by changes in the amount of β-O-4 linkages. Evaluation of the impact of the GH11 endoxylanase on the GAX-DHP nanocomposites revealed a negative correlation between the proportion and organization patterns of DHP in the nanocomposites and enzyme activity.
Endo-β-1,4-xylanases (EC 3.2.1.8) are the main enzymes involved in the hydrolysis of xylans, the most abundant hemicelluloses in plant biomass. However, the development of efficient endoxylanases for ...use in biorefinery processes is currently hampered by insufficient knowledge regarding the impact of the cell wall network organization on the action of the enzyme at the supramolecular level. The action pattern of a GH11 endoxylanase from Thermobacillus xylanilyticus (Tx-xyl) was investigated by means of in vitro reconstituted model systems which can mimic certain cell wall structures. The action of Tx-xyl was evaluated on polymer assemblies displaying increasing complexity using delignified glucuronoarabinoxylan (GAX), then GAX-DHP model complexes obtained by oxidative polymerization of coniferyl alcohol into dehydrogenation polymers (DHP: lignin model compounds) in the presence of GAX. At a high concentration of GAX, interchain associations are formed leading to high molecular weight aggregates. These structures did not appear to affect the action of endoxylanase, which induces disaggregation of the self-aggregates along with polymer depolymerization. To mimic lignin−carbohydrate interactions, two different GAX-DHP nanocomposites were prepared and incubated with endoxylanase. In both cases, free GAX was hydrolyzed, while the GAX-DHP complexes appeared to be resistant. In the case of the noncovalently linked GAX-DHPZL complexes, enzyme action favored a decrease in particle size, owing to the removal of their relatively exposed carbohydrate chains, whereas the complex supramolecular organization of the covalently linked GAX-DHPZT complexes severely hampers the enzyme’s access to carbohydrate. Overall, these results establish the negative impact of DHP on the endoxylanase action and provide new knowledge regarding the limitations of the enzyme action in the lignocellulose bioconversion processes.
GH-11 xylanases are highly specific and possess a thumb-shaped loop, a unique structure among enzymes with a jelly-roll scaffold. To investigate this structure, in vitro mutagenesis was performed on ...a GH-11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus. Targets were the conserved amino acids Pro114-Ser115-Ile116 that are located at the thumb's tip and Thr121 and Tyr111, linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro114-Gly115-Cys116), not found in naturally occurring GH-11 xylanases. The kcat value for xylan hydrolysis catalysed by this mutant was increased by 20%. Re-positioning of the thumb through the deletion of the linker residues produced different effects. As predicted by in silico analyses, deletion of Thr121 had drastic consequences on activity, whereas deletion of Tyr111 only affected (4-fold decrease) kcat. Finally, deletion mutagenesis was used to create a thumbless variant that was almost catalytically inactive. Fluorescence titration with xylotetraose and xylopentaose revealed that this thumb-deleted xylanase retained the ability to bind substrates. This binding was comparable to that of the wild-type enzyme. Additionally, unlike wild-type Tx-Xyl, the thumb-deleted xylanase efficiently bound cellotetraose, although no cellulose hydrolysing activity was detected. Overall, these data show that the thumb is a key determinant for substrate selection and support previous data that suggest that it plays a role in the catalytic process.
The substrate specificity of an arabinofuranosidase (AbfD3) from family 51 of glycoside hydrolase classification was investigated in order to precisely evaluate its catalytic abilities. AbfD3 ...activity on destarched wheat bran was poor and less than 1% of total arabinose was released. AbfD3 was also tested on arabinoxylans derived from destarched wheat bran that present different degrees of polymerization, A/X ratios, ferulic acid content and solubility. Results indicated that AbfD3 can hydrolyze polymeric arabinoxylans, even if this action was moderate when compared to the efficient hydrolysis of oligosaccharides. The limited action of AbfD3 on polymeric arabinoxylans is discussed with regard to the heterogeneous distribution of the arabinose residues along the xylan main chain, the insolubility of arabinoxylans and to the presence of disubstituted xylose or feruloylated arabinose.
A polyphasic approach was used to characterize a bacterium, HAN-85T, isolated from thermal water in natural thermal spring at Tozeur, an oasis in southwest Tunisia. The novel isolate was ...thermophilic, strictly aerobic and amylolytic bacterium, which stained Gram negative. Cells were short rods motile by means of a single polar flagellum. Their optimum temperature and pH required for growth were 55°C and pH 7, respectively. Comparative 16S rRNA gene sequence analyses showed that strain HAN-85T belonged to the genus Caldimonas, with highest sequence similarity to the type strains Caldimonas manganoxidans and Caldimonas taiwanensis. DNA-DNA hybridization measurements revealed low DNA relatedness (35.2-44.5%) between the novel isolate and its closest relative, C. manganoxidans. The major cellular fatty acid components were 16:0, 17:0 cyclo and summed feature 3. The DNA G+C content was 68.3 mol%. Taken together, the results of DNA-DNA hybridization, fatty acids profile, physiological tests and biochemical analyses have allowed the genotypic and phenotypic differentiation of the isolate from currently recognized Caldimonas species. Therefore, we suggest that this isolate is a novel species within the genus Caldimonas and propose that it should be named Caldimonas hydrothermale sp. nov. The type strain is HAN-85T (=DSM 18497T =LMG 23755T). The Gen Bank/Embl/DDBJ accession number for the 16S rRNA gene sequence of strain DSM 18497T is AM283038.
The development of enzymatic technologies offers an alternative, environmentally-friendly interesting strategy for controlled fractionation and upgrading of lignocellulosic biomass (biofuels, ...biopolymers, industrially-relevant chemicals...). The effectiveness of these biocatalysts is, nevertheless, limited by multiple factors related to their structural and functional characteristics, but also to the complex nature of the lignocellulosic biomass (rich in lignified secondary cell walls). In order to identify the key parameters for an effective bioconversion of hemicelluloses, the major components of lignocelluloses, we have focused our study on the endoxylanase (Tx-Xyl) of Thermobacillus xylanilyticus, a family 11 glycoside- hydrolase (GH11). The effectiveness of hemicellulases within the lignified cell walls is strongly dependent on the structural diversity and organisational complexity of the cell wall networks. In order to specify lignocellulose structural limiting factors and to get more knowledge regarding the impact of the cell wall network organization on the enzyme action of at supramolecular level, we have studied, in a biomimetic approach, the action pattern of Tx-Xyl on different substrates displaying increasing complexity (isolated heteroxylans, in vitro reconstituted copolymer assemblies...). The use of nano-composites of heteroxylans - lignins (DHPs) synthesized in vitro according to two polymerization methods ("Zulaufverfahren", ZL and "Zutropfverfahren", ZT) has enabled us to reveal different organizations, morphologies and physicochemical characteristics of the polysaccharides - lignins associations and to underline the negative effect of lignins (DHPs) on heteroxylan hydrolysis by the xylanase (Tx-Xyl). This effect would be irrespective to the nature of heteroxylans - lignins associations (covalent or not). Nevertheless, the complex supramolecular organization of the covalent complexes (LCC) would severely hamper the enzyme's access to carbohydrates. Otherwise, lignins would interfere directly with the action of the enzyme through nonspecific or not productive interactions. A direct correlation has been, indeed, established between the increase in the lignin content of the nanocomposites and the decrease of the enzyme activity. In addition, the study of the interactions of Tx-Xyl with various hydroxycinamic acids (p-coumaric, ferulic, caffeic acids...) has revealed a non-competitive inhibition of the enzyme by these phenolic compounds. In addition to substrate parameters, understanding the structural properties of the enzymes determining their action in situ on lignocelluloses is essential. As a member of GH11endoxylanase family, Tx-Xyl is a non modular enzyme comprising only a catalytic module (20 kDa) and no Carbohydrate Binding Module (CBM). Using protein engineering, we have developed a strategy which aims at modifying the Tx-Xyl architecture and/or specificity by grafting, through "linker" sequences, different protein modules: the CBM1 of the cellulase Cel7A from Trichoderma reesei binding specifically crystalline cellulose and the GFP (Green Fluoerescent Protein). The chimeric fusion proteins Tx-Xyl-CBM1 and Tx-Xyl-GFP obtained have been less effective on soluble xylans (low Kcat) than Tx- Xyl. However, their efficiency on lignocellulosic substrates (such as wheat by products; straw and bran) was different. Indeed, modestly enhanced hydrolysis rates were obtained in the case of Tx-Xyl-CBM1, suggesting that the CBM1 may potentiate in situ action of the enzyme, contrary to Tx-Xyl-GFP whose size would be a factor limiting its diffusion/action within the cell wall network
Le développement de technologies enzymatiques constitue un enjeu majeur pour le fractionnement maîtrisé et la valorisation des ressources lignocellulosiques (biocarburants, biopolymères, synthons…). L’efficacité de ces biocatalyseurs est cependant limitée par de multiples facteurs liés à la fois à leurs caractéristiques structurales et fonctionnelles, mais également à la nature complexe de la biomasse lignocellulosique (riche en parois secondaires lignifiées). Dans le but d’identifier les paramètres clés pour une conversion efficace des hémicelluloses, constituants majeurs des lignocelluloses, nous avons centré notre étude sur l’endoxylanase (Tx-Xyl) de Thermobacillus xylanilyticus appartenant à la famille 11 des glycosidehydrolases (GH11). L’efficacité d’une hémicellulase au sein des parois lignifiées est fortement dépendante de la diversité structurale et la complexité organisationnelle des réseaux pariétaux. Afin de préciser les facteurs limitants propres aux lignocelluloses et de mettre en évidence les niveaux d’organisation des polymères lignocellulosiques susceptibles d’entraver ou de limiter l’action de l’enzyme, nous avons étudié, dans une approche biomimétique, l’action de la xylanase GH11 (Tx-Xyl) sur des substrats différents et de complexité croissante (hétéroxylanes isolés, assemblages de copolymères reconstitués in vitro...). L’emploi de nano-composites hétéroxylanes extraits - lignines (DHPs) synthétisés in vitro selon deux modes de polymérisation ("Zulaufverfahren", ZL et "Zutropfverfahren", ZT) nous a permis de révéler différents niveaux d’organisation, morphologies et caractéristiques physico-chimiques des associations polysaccharides - lignines et de souligner l’effet négatif des lignines (DHPs) sur l’hydrolyse des hétéroxylanes par la xylanase (Tx- Xyl). Cet effet se manifeste indépendamment de la nature des associations hétéroxylanes -lignines (covalentes ou non), mais serait accentué par l’agencement tridimensionnel des complexes covalents (LCC) qui limiterait l’accessibilité des hétéroxylanes à l’enzyme. Au-delà des limitations de l’accessibilité, les lignines interféreraient directement avec l’action de l’enzyme via des interactions non spécifiques ou non productives. Une corrélation directe a pu, en effet, être établie entre l’augmentation du contenu en lignines des nano-composites et la baisse de l’activité de l’enzyme. Par ailleurs, l’étude des interactions de Tx-Xyl avec divers acides hydroxycinamiques (p-coumarique, férulique, cafféique…) a permis de mettre en évidence un phénomène d’inhibition non compétitif de l’enzyme par ces composés phénoliques. En plus des paramètres inhérents au substrat, la compréhension des propriétés structurales des enzymes régissant leur action in situ sur les lignocelluloses est indispensable. A l’instar des endoxylanases de la famille GH11, Tx-Xyl est une enzyme constituée d’un domaine catalytique d’environs 20 kDa et elle ne comporte pas de domaine dédié à la fixation au substrat (Carbohydrate Binding Module ou CBM). Moyennant des outils de biologie moléculaire, nous avons développé une stratégie qui vise à modifier l’architecture protéique et/ou la spécificité de fixation de Tx-Xyl en la fusionnant via des séquences "linker" à des modules protéiques différents: le CBM1 de la cellulase Cel7A de Trichoderma reesei fixant spécifiquement la cellulose cristalline et la GFP (Green Fluoerescent Protein). Les protéines chimériques Tx-Xyl-CBM1 et Tx-Xyl-GFP obtenues sont moins efficaces sur les xylanes solubles (faible Kcat) comparées à Tx-Xyl. Cependant, leurs modes d’action sur des substrats lignocellulosiques (tels que les coproduits du blé : paille et son de blé) semblent différents. En effet, des rendements d’hydrolyse légèrement augmentés sont obtenus dans le cas de Tx-Xyl-CBM1, suggérant un impact positif du CBM1 sur la migration et/ou l’action de l’enzyme in situ, contrairement à Tx-Xyl-GFP dont la taille serait un facteur limitant sa diffusion/pénétration au sein des parois végétales.
The development of enzymatic technologies offers an alternative, environmentally-friendly interesting strategy for controlled fractionation and upgrading of lignocellulosic biomass (biofuels, ...biopolymers, industrially-relevant chemicals...). The effectiveness of these biocatalysts is, nevertheless, limited by multiple factors related to their structural and functional characteristics, but also to the complex nature of the lignocellulosic biomass (rich in lignified secondary cell walls). In order to identify the key parameters for an effective bioconversion of hemicelluloses, the major components of lignocelluloses, we have focused our study on the endoxylanase (Tx-Xyl) of Thermobacillus xylanilyticus, a family 11 glycoside- hydrolase (GH11). The effectiveness of hemicellulases within the lignified cell walls is strongly dependent on the structural diversity and organisational complexity of the cell wall networks. In order to specify lignocellulose structural limiting factors and to get more knowledge regarding the impact of the cell wall network organization on the enzyme action of at supramolecular level, we have studied, in a biomimetic approach, the action pattern of Tx-Xyl on different substrates displaying increasing complexity (isolated heteroxylans, in vitro reconstituted copolymer assemblies...). The use of nano-composites of heteroxylans - lignins (DHPs) synthesized in vitro according to two polymerization methods ("Zulaufverfahren", ZL and "Zutropfverfahren", ZT) has enabled us to reveal different organizations, morphologies and physicochemical characteristics of the polysaccharides - lignins associations and to underline the negative effect of lignins (DHPs) on heteroxylan hydrolysis by the xylanase (Tx-Xyl). This effect would be irrespective to the nature of heteroxylans - lignins associations (covalent or not). Nevertheless, the complex supramolecular organization of the covalent complexes (LCC) would severely hamper the enzyme's access to carbohydrates. Otherwise, lignins would interfere directly with the action of the enzyme through nonspecific or not productive interactions. A direct correlation has been, indeed, established between the increase in the lignin content of the nanocomposites and the decrease of the enzyme activity. In addition, the study of the interactions of Tx-Xyl with various hydroxycinamic acids (p-coumaric, ferulic, caffeic acids...) has revealed a non-competitive inhibition of the enzyme by these phenolic compounds. In addition to substrate parameters, understanding the structural properties of the enzymes determining their action in situ on lignocelluloses is essential. As a member of GH11endoxylanase family, Tx-Xyl is a non modular enzyme comprising only a catalytic module (20 kDa) and no Carbohydrate Binding Module (CBM). Using protein engineering, we have developed a strategy which aims at modifying the Tx-Xyl architecture and/or specificity by grafting, through "linker" sequences, different protein modules: the CBM1 of the cellulase Cel7A from Trichoderma reesei binding specifically crystalline cellulose and the GFP (Green Fluoerescent Protein). The chimeric fusion proteins Tx-Xyl-CBM1 and Tx-Xyl-GFP obtained have been less effective on soluble xylans (low Kcat) than Tx- Xyl. However, their efficiency on lignocellulosic substrates (such as wheat by products; straw and bran) was different. Indeed, modestly enhanced hydrolysis rates were obtained in the case of Tx-Xyl-CBM1, suggesting that the CBM1 may potentiate in situ action of the enzyme, contrary to Tx-Xyl-GFP whose size would be a factor limiting its diffusion/action within the cell wall network
Le développement de technologies enzymatiques constitue un enjeu majeur pour le fractionnement maîtrisé et la valorisation des ressources lignocellulosiques (biocarburants, biopolymères, synthons…). L’efficacité de ces biocatalyseurs est cependant limitée par de multiples facteurs liés à la fois à leurs caractéristiques structurales et fonctionnelles, mais également à la nature complexe de la biomasse lignocellulosique (riche en parois secondaires lignifiées). Dans le but d’identifier les paramètres clés pour une conversion efficace des hémicelluloses, constituants majeurs des lignocelluloses, nous avons centré notre étude sur l’endoxylanase (Tx-Xyl) de Thermobacillus xylanilyticus appartenant à la famille 11 des glycosidehydrolases (GH11). L’efficacité d’une hémicellulase au sein des parois lignifiées est fortement dépendante de la diversité structurale et la complexité organisationnelle des réseaux pariétaux. Afin de préciser les facteurs limitants propres aux lignocelluloses et de mettre en évidence les niveaux d’organisation des polymères lignocellulosiques susceptibles d’entraver ou de limiter l’action de l’enzyme, nous avons étudié, dans une approche biomimétique, l’action de la xylanase GH11 (Tx-Xyl) sur des substrats différents et de complexité croissante (hétéroxylanes isolés, assemblages de copolymères reconstitués in vitro...). L’emploi de nano-composites hétéroxylanes extraits - lignines (DHPs) synthétisés in vitro selon deux modes de polymérisation ("Zulaufverfahren", ZL et "Zutropfverfahren", ZT) nous a permis de révéler différents niveaux d’organisation, morphologies et caractéristiques physico-chimiques des associations polysaccharides - lignines et de souligner l’effet négatif des lignines (DHPs) sur l’hydrolyse des hétéroxylanes par la xylanase (Tx- Xyl). Cet effet se manifeste indépendamment de la nature des associations hétéroxylanes -lignines (covalentes ou non), mais serait accentué par l’agencement tridimensionnel des complexes covalents (LCC) qui limiterait l’accessibilité des hétéroxylanes à l’enzyme. Au-delà des limitations de l’accessibilité, les lignines interféreraient directement avec l’action de l’enzyme via des interactions non spécifiques ou non productives. Une corrélation directe a pu, en effet, être établie entre l’augmentation du contenu en lignines des nano-composites et la baisse de l’activité de l’enzyme. Par ailleurs, l’étude des interactions de Tx-Xyl avec divers acides hydroxycinamiques (p-coumarique, férulique, cafféique…) a permis de mettre en évidence un phénomène d’inhibition non compétitif de l’enzyme par ces composés phénoliques. En plus des paramètres inhérents au substrat, la compréhension des propriétés structurales des enzymes régissant leur action in situ sur les lignocelluloses est indispensable. A l’instar des endoxylanases de la famille GH11, Tx-Xyl est une enzyme constituée d’un domaine catalytique d’environs 20 kDa et elle ne comporte pas de domaine dédié à la fixation au substrat (Carbohydrate Binding Module ou CBM). Moyennant des outils de biologie moléculaire, nous avons développé une stratégie qui vise à modifier l’architecture protéique et/ou la spécificité de fixation de Tx-Xyl en la fusionnant via des séquences "linker" à des modules protéiques différents: le CBM1 de la cellulase Cel7A de Trichoderma reesei fixant spécifiquement la cellulose cristalline et la GFP (Green Fluoerescent Protein). Les protéines chimériques Tx-Xyl-CBM1 et Tx-Xyl-GFP obtenues sont moins efficaces sur les xylanes solubles (faible Kcat) comparées à Tx-Xyl. Cependant, leurs modes d’action sur des substrats lignocellulosiques (tels que les coproduits du blé : paille et son de blé) semblent différents. En effet, des rendements d’hydrolyse légèrement augmentés sont obtenus dans le cas de Tx-Xyl-CBM1, suggérant un impact positif du CBM1 sur la migration et/ou l’action de l’enzyme in situ, contrairement à Tx-Xyl-GFP dont la taille serait un facteur limitant sa diffusion/pénétration au sein des parois végétales.
The development of enzymatic technologies offers an alternative, environmentally-friendly interesting strategy for controlled fractionation and upgrading of lignocellulosic biomass (biofuels, ...biopolymers, industrially-relevant chemicals...). The effectiveness of these biocatalysts is, nevertheless, limited by multiple factors related to their structural and functional characteristics, but also to the complex nature of the lignocellulosic biomass (rich in lignified secondary cell walls). In order to identify the key parameters for an effective bioconversion of hemicelluloses, the major components of lignocelluloses, we have focused our study on the endoxylanase (Tx-Xyl) of Thermobacillus xylanilyticus, a family 11 glycoside- hydrolase (GH11). The effectiveness of hemicellulases within the lignified cell walls is strongly dependent on the structural diversity and organisational complexity of the cell wall networks. In order to specify lignocellulose structural limiting factors and to get more knowledge regarding the impact of the cell wall network organization on the enzyme action of at supramolecular level, we have studied, in a biomimetic approach, the action pattern of Tx-Xyl on different substrates displaying increasing complexity (isolated heteroxylans, in vitro reconstituted copolymer assemblies...). The use of nano-composites of heteroxylans - lignins (DHPs) synthesized in vitro according to two polymerization methods ("Zulaufverfahren", ZL and "Zutropfverfahren", ZT) has enabled us to reveal different organizations, morphologies and physicochemical characteristics of the polysaccharides - lignins associations and to underline the negative effect of lignins (DHPs) on heteroxylan hydrolysis by the xylanase (Tx-Xyl). This effect would be irrespective to the nature of heteroxylans - lignins associations (covalent or not). Nevertheless, the complex supramolecular organization of the covalent complexes (LCC) would severely hamper the enzyme's access to carbohydrates. Otherwise, lignins would interfere directly with the action of the enzyme through nonspecific or not productive interactions. A direct correlation has been, indeed, established between the increase in the lignin content of the nanocomposites and the decrease of the enzyme activity. In addition, the study of the interactions of Tx-Xyl with various hydroxycinamic acids (p-coumaric, ferulic, caffeic acids...) has revealed a non-competitive inhibition of the enzyme by these phenolic compounds. In addition to substrate parameters, understanding the structural properties of the enzymes determining their action in situ on lignocelluloses is essential. As a member of GH11endoxylanase family, Tx-Xyl is a non modular enzyme comprising only a catalytic module (20 kDa) and no Carbohydrate Binding Module (CBM). Using protein engineering, we have developed a strategy which aims at modifying the Tx-Xyl architecture and/or specificity by grafting, through "linker" sequences, different protein modules: the CBM1 of the cellulase Cel7A from Trichoderma reesei binding specifically crystalline cellulose and the GFP (Green Fluoerescent Protein). The chimeric fusion proteins Tx-Xyl-CBM1 and Tx-Xyl-GFP obtained have been less effective on soluble xylans (low Kcat) than Tx- Xyl. However, their efficiency on lignocellulosic substrates (such as wheat by products; straw and bran) was different. Indeed, modestly enhanced hydrolysis rates were obtained in the case of Tx-Xyl-CBM1, suggesting that the CBM1 may potentiate in situ action of the enzyme, contrary to Tx-Xyl-GFP whose size would be a factor limiting its diffusion/action within the cell wall network
Le développement de technologies enzymatiques constitue un enjeu majeur pour le fractionnement maîtrisé et la valorisation des ressources lignocellulosiques (biocarburants, biopolymères, synthons…). L’efficacité de ces biocatalyseurs est cependant limitée par de multiples facteurs liés à la fois à leurs caractéristiques structurales et fonctionnelles, mais également à la nature complexe de la biomasse lignocellulosique (riche en parois secondaires lignifiées). Dans le but d’identifier les paramètres clés pour une conversion efficace des hémicelluloses, constituants majeurs des lignocelluloses, nous avons centré notre étude sur l’endoxylanase (Tx-Xyl) de Thermobacillus xylanilyticus appartenant à la famille 11 des glycosidehydrolases (GH11). L’efficacité d’une hémicellulase au sein des parois lignifiées est fortement dépendante de la diversité structurale et la complexité organisationnelle des réseaux pariétaux. Afin de préciser les facteurs limitants propres aux lignocelluloses et de mettre en évidence les niveaux d’organisation des polymères lignocellulosiques susceptibles d’entraver ou de limiter l’action de l’enzyme, nous avons étudié, dans une approche biomimétique, l’action de la xylanase GH11 (Tx-Xyl) sur des substrats différents et de complexité croissante (hétéroxylanes isolés, assemblages de copolymères reconstitués in vitro...). L’emploi de nano-composites hétéroxylanes extraits - lignines (DHPs) synthétisés in vitro selon deux modes de polymérisation ("Zulaufverfahren", ZL et "Zutropfverfahren", ZT) nous a permis de révéler différents niveaux d’organisation, morphologies et caractéristiques physico-chimiques des associations polysaccharides - lignines et de souligner l’effet négatif des lignines (DHPs) sur l’hydrolyse des hétéroxylanes par la xylanase (Tx- Xyl). Cet effet se manifeste indépendamment de la nature des associations hétéroxylanes -lignines (covalentes ou non), mais serait accentué par l’agencement tridimensionnel des complexes covalents (LCC) qui limiterait l’accessibilité des hétéroxylanes à l’enzyme. Au-delà des limitations de l’accessibilité, les lignines interféreraient directement avec l’action de l’enzyme via des interactions non spécifiques ou non productives. Une corrélation directe a pu, en effet, être établie entre l’augmentation du contenu en lignines des nano-composites et la baisse de l’activité de l’enzyme. Par ailleurs, l’étude des interactions de Tx-Xyl avec divers acides hydroxycinamiques (p-coumarique, férulique, cafféique…) a permis de mettre en évidence un phénomène d’inhibition non compétitif de l’enzyme par ces composés phénoliques. En plus des paramètres inhérents au substrat, la compréhension des propriétés structurales des enzymes régissant leur action in situ sur les lignocelluloses est indispensable. A l’instar des endoxylanases de la famille GH11, Tx-Xyl est une enzyme constituée d’un domaine catalytique d’environs 20 kDa et elle ne comporte pas de domaine dédié à la fixation au substrat (Carbohydrate Binding Module ou CBM). Moyennant des outils de biologie moléculaire, nous avons développé une stratégie qui vise à modifier l’architecture protéique et/ou la spécificité de fixation de Tx-Xyl en la fusionnant via des séquences "linker" à des modules protéiques différents: le CBM1 de la cellulase Cel7A de Trichoderma reesei fixant spécifiquement la cellulose cristalline et la GFP (Green Fluoerescent Protein). Les protéines chimériques Tx-Xyl-CBM1 et Tx-Xyl-GFP obtenues sont moins efficaces sur les xylanes solubles (faible Kcat) comparées à Tx-Xyl. Cependant, leurs modes d’action sur des substrats lignocellulosiques (tels que les coproduits du blé : paille et son de blé) semblent différents. En effet, des rendements d’hydrolyse légèrement augmentés sont obtenus dans le cas de Tx-Xyl-CBM1, suggérant un impact positif du CBM1 sur la migration et/ou l’action de l’enzyme in situ, contrairement à Tx-Xyl-GFP dont la taille serait un facteur limitant sa diffusion/pénétration au sein des parois végétales.
Abstract
Background
The aim of this study was to evaluate the structural retinal vascular integrity using optical coherence tomography angiography (OCTA) in treatment-naïve eyes with diabetic macular ...edema (DME) and to compare it with findings in diabetic eyes without DME.
Methods
In this prospective study, 70 eyes with diabetic retinopathy were included (37 eyes with DME and 33 eyes without DME). The medical records, including swept-source optical coherence tomography and 9 × 9 mm swept-source OCTA images were reviewed and compared between DME and non-DME groups. Microaneurysms, intraretinal microvascular abnormalities (IRMA), areas of capillary non perfusion, foveal avascular zone (FAZ), and capillary vascular density (CVD) were analyzed in the superficial capillary plexus (SCP) and the deep capillary plexus (DCP).
Results
Compared to the non-DME eyes, DME eyes had more microaneurysms in the SCP and the DCP (
p
= 0,039 and
p
= 0,024 respectively), more IRMA in the SCP (
p
= 0,005), larger areas of capillary non perfusion in the SCP and the DCP (
p
= 0,026 and
p
= 0,02 respectively) and larger FAZ in both plexuses (
p
= 0,048 in the SCP and
p
= 0,012 in the DCP). The CVD in the DCP was lower in DME eyes compared to non-DME eyes (
p
= 0,007). The severity of DME was significantly correlated to the number of microaneurysms and to the FAZ surface. Central macular thickness was significantly correlated with the number of microaneurysms in the DCP, the surface of capillary non perfusion areas and the FAZ area in both plexuses.
Conclusions
OCTA with a 9 × 9 mm field of view showed that the retinal vascular integrity regarding the number of microaneurysms, the number of IRMA, the surface of capillary non perfusion areas, the FAZ area and the CVD, was significantly more impaired in DME eyes compared to diabetic eyes without DME. The DCP seemed to be more affected in diabetic eyes with and without DME than the SCP.
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