Ingénierie des glucane-saccharases de la famille 70 des glycoside-hydrolases pour la synthèse de nouveaux biopolymères

Les glucane-saccharases sont des transglucosidases de la famille 70 des glycoside-hydrolases. A partir de saccharose, ces enzymes catalysent la synthèse d alpha -glucanes, polymères de haute masse molaire formés d unités glucosyle. Elles sont aussi capables de synthétiser des oligosaccharides ou glucoconjugués par réaction de transglucosylation sur des accepteurs exogènes de natures variées. De par la diversité de leurs spécificités, tant au niveau des liaisons osidiques synthétisées [alpha (1->2) ; alpha (1->3) ; alpha (1->4) ou alpha (1->6)] que de l organisation de ces liaisons au sein des produits formés, ces biocatalyseurs peuvent être mis à profit pour produire des hydrates de carbones d'intérêt pour les secteurs de l'alimentation, de la santé et de l'environnement. L'objectif de ces travaux de thèse était de générer par ingénierie enzymatique de nouvelles glucane-saccharases capables de synthétiser des alpha -glucanes et des gluco-oligosaccharides de structures et propriétés innovantes, afin d élargir le panel d'applications de ces molécules. Sur le plan fondamental, l'enjeu était aussi d'améliorer la compréhension des relations entre structure et spécificité des glucane-saccharases. Pour atteindre nos objectifs, nous avons utilisé une stratégie d'ingénierie combinatoire de la dextrane-saccharase DSR-S de Leuconostoc mesenteroides NRRL B-512F, qui catalyse la synthèse d un dextrane formé à 95 % de liaisons alpha (1->6) et 5 % alpha (1->3). Le travail a tout d'abord consisté à développer une méthode de criblage multi-étapes et à haut-débit, pour isoler et trier les variants de spécificités de liaisons variées. La stratégie comprend une première étape de sélection in vivo sur milieu solide suivie d un criblage par RMN 1D 1H automatisé au format microplaque. Il s'agit de la première méthode de criblage à haut-débit de la spécificité de liaison des transglucosidases et glycosyltransferases. Cette stratégie a ensuite été appliquée au criblage de deux banques de variants de la DSR-S, totalisant plus de 36 000 clones obtenus par mutagénèse de saturation et recombinaison simultanée de 8 résidus du domaine catalytique. Au total, 82 mutants capables de synthétiser entre 2 et 8 fois plus de liaisons alpha (1->3) par rapport à l enzyme parentale ont été isolés. La caractérisation des propriétés catalytiques de sept mutants représentatifs de la diversité de spécificité générée a permis d identifier un nouveau motif peptidique 460DYVHT464 impliqué dans la spécificité de DSR-S. Enfin, la caractérisation de la structure et des propriétés rhéologiques et mécaniques des dextranes synthétisés par ces 7 mutants a mis en évidence les différences de taille et de conformation de ces macromolécules en solution et révélé l aptitude du polymère synthétisé par le variant H463R/T464V/S512T à former des films bio-sourcés innovants, dont les propriétés mécaniques sont remarquables en comparaison de celles d'autres biopolymères extraits de plantes ou produits par fermentationGlucansucrases are transglucosidases from glycoside hydrolase family 70. From sucrose, these enzymes catalyse the synthesis of alpha -glucans, high molecular weight polymers formed of glucosyl units. Glucansucrases are also able to synthesize oligosaccharides or glucoconjugates by transglucosylation reaction onto various exogenous acceptors. Due to the large specificity, in terms of osidic linkages synthesized [alpha (1->2); alpha (1->3); alpha (1->4) or alpha (1->6)] and their organization within the formed products, these biocatalysts can be used to produce carbohydrates of interest in food, health and environment fields. The aim of this research work was to generate, by enzyme engineering, new glucansucrases able to synthesize alpha -glucans and gluco-oligosaccharides with novel structures and properties, to enlarge applications of these molecules. At fundamental level, the work was to improve the understanding of the relationships between structure and specificity of glucansucrases. To reach our objectives, we used a combinatorial engineering strategy on the dextransucrase DSR-S of Leuconostoc mesenteroides NRRL B-512F, which catalyses the synthesis of a dextran formed by 95 % of alpha (1->6) and 5 % alpha (1->3) linkages. The work consisted first in the development of a multi-step high throughput screening methodology to sort out and isolate variants with altered linkage specificities. The strategy includes a first stage of in vivo selection on solid medium followed by an automated 1D 1H NMR-based screening using microplates. To date, this is the first high-throughput screening method for linkage specificity determination of transglucosidases and glycosyltransferases. This strategy was applied to the screening of two DSR-S variants libraries, totalizing more than 36,000 clones obtained by saturation mutagenesis and simultaneous recombination of 8 residues from the catalytic domain. Eighty two mutants able of synthesize 2 to 8 times more alpha (1->3) linkages compared to the parental enzyme were isolated. The characterization of the catalytic properties of 7 representative mutants enabled the identification of a new peptide motif 460DYVHT464, involved in DSR-S specificity. Finally, the characterization of structural, rheological and mechanical properties of dextrans synthetized by these 7 mutants highlighted the differences in size and conformation of these macromolecules in solution and revealed the capacity of the polymer synthesized by H463R/T464V/S512T variant to form biofilms, whose mechanical properties are remarkable in comparison to those of other biopolymers extracted from plants or produced by fermentation.TOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

The price of informality : how informal finance schemes defaulted in China, 1989–2015.

The default of a large number of informal finance schemes in China has caused enormous financial losses, and therefore has potential social and political significance. Analysing 354 defaulted schemes from 1989 to 2015, this study defines how they differ from other types of informal finance. It also produces an ideal-type representation of the default process and concludes that the default results from greed, increasing financial pressure at the individual level and private enterprises’ restricted access to state bank loans at the institutional level. China’s financial system should be more flexible in order to prevent further financial losses through informal financial relations

Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1→4) also (α1→6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)–DP7 (-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.

Structures, physico-chemical properties, production and (potential) applications of sucrose-derived α-d-glucans synthesized by glucansucrases

Glycoside hydrolase family 70 (GH70) glucansucrases produce α-d-glucan polysaccharides (e.g. dextran), which have different linkage composition, branching degree and size distribution, and hold potential applications in food, cosmetic and medicine industry. In addition, GH70 branching sucrases add single α-(1→2) or α-(1→3) branches onto dextran, resulting in highly branched polysaccharides with "comb-like" structure. The physico-chemical properties of these α-d-glucans are highly influenced by their linkage compositions, branching degrees and sizes. Among these α-d-glucans, dextran is commercially applied as plasma expander and separation matrix based on extensive studies of its structure and physico-chemical properties. However, such detailed information is lacking for the other type of α-d-glucans. Aiming to stimulate the application of α-d-glucans produced by glucansucrases, we present an overview of the structures, production, physico-chemical properties and (potential) applications of these sucrose-derived α-d-glucan polysaccharides. We also discuss bottlenecks and future perspectives for the application of these α-d-glucan polysaccharides

Engineering glucansucrases from family 70 of glycoside-hydrolases for the synthesis of novel polysaccharides

Les glucane-saccharases sont des transglucosidases de la famille 70 des glycoside-hydrolases. A partir de saccharose, ces enzymes catalysent la synthèse d’alpha -glucanes, polymères de haute masse molaire formés d’unités glucosyle. Elles sont aussi capables de synthétiser des oligosaccharides ou glucoconjugués par réaction de transglucosylation sur des accepteurs exogènes de natures variées. De par la diversité de leurs spécificités, tant au niveau des liaisons osidiques synthétisées [alpha (1→2) ; alpha (1→3) ; alpha (1→4) ou alpha (1→6)] que de l’organisation de ces liaisons au sein des produits formés, ces biocatalyseurs peuvent être mis à profit pour produire des hydrates de carbones d'intérêt pour les secteurs de l'alimentation, de la santé et de l'environnement. L'objectif de ces travaux de thèse était de générer par ingénierie enzymatique de nouvelles glucane-saccharases capables de synthétiser des alpha -glucanes et des gluco-oligosaccharides de structures et propriétés innovantes, afin d’élargir le panel d'applications de ces molécules. Sur le plan fondamental, l'enjeu était aussi d'améliorer la compréhension des relations entre structure et spécificité des glucane-saccharases. Pour atteindre nos objectifs, nous avons utilisé une stratégie d'ingénierie combinatoire de la dextrane-saccharase DSR-S de Leuconostoc mesenteroides NRRL B-512F, qui catalyse la synthèse d’un dextrane formé à 95 % de liaisons alpha (1→6) et 5 % alpha (1→3). Le travail a tout d'abord consisté à développer une méthode de criblage multi-étapes et à haut-débit, pour isoler et trier les variants de spécificités de liaisons variées. La stratégie comprend une première étape de sélection in vivo sur milieu solide suivie d’un criblage par RMN 1D 1H automatisé au format microplaque. Il s'agit de la première méthode de criblage à haut-débit de la spécificité de liaison des transglucosidases et glycosyltransferases. Cette stratégie a ensuite été appliquée au criblage de deux banques de variants de la DSR-S, totalisant plus de 36 000 clones obtenus par mutagénèse de saturation et recombinaison simultanée de 8 résidus du domaine catalytique. Au total, 82 mutants capables de synthétiser entre 2 et 8 fois plus de liaisons alpha (1→3) par rapport à l’enzyme parentale ont été isolés. La caractérisation des propriétés catalytiques de sept mutants représentatifs de la diversité de spécificité générée a permis d’identifier un nouveau motif peptidique 460DYVHT464 impliqué dans la spécificité de DSR-S. Enfin, la caractérisation de la structure et des propriétés rhéologiques et mécaniques des dextranes synthétisés par ces 7 mutants a mis en évidence les différences de taille et de conformation de ces macromolécules en solution et révélé l’aptitude du polymère synthétisé par le variant H463R/T464V/S512T à former des films bio-sourcés innovants, dont les propriétés mécaniques sont remarquables en comparaison de celles d'autres biopolymères extraits de plantes ou produits par fermentationGlucansucrases are transglucosidases from glycoside hydrolase family 70. From sucrose, these enzymes catalyse the synthesis of alpha -glucans, high molecular weight polymers formed of glucosyl units. Glucansucrases are also able to synthesize oligosaccharides or glucoconjugates by transglucosylation reaction onto various exogenous acceptors. Due to the large specificity, in terms of osidic linkages synthesized [alpha (1→2); alpha (1→3); alpha (1→4) or alpha (1→6)] and their organization within the formed products, these biocatalysts can be used to produce carbohydrates of interest in food, health and environment fields. The aim of this research work was to generate, by enzyme engineering, new glucansucrases able to synthesize alpha -glucans and gluco-oligosaccharides with novel structures and properties, to enlarge applications of these molecules. At fundamental level, the work was to improve the understanding of the relationships between structure and specificity of glucansucrases. To reach our objectives, we used a combinatorial engineering strategy on the dextransucrase DSR-S of Leuconostoc mesenteroides NRRL B-512F, which catalyses the synthesis of a dextran formed by 95 % of alpha (1→6) and 5 % alpha (1→3) linkages. The work consisted first in the development of a multi-step high throughput screening methodology to sort out and isolate variants with altered linkage specificities. The strategy includes a first stage of in vivo selection on solid medium followed by an automated 1D 1H NMR-based screening using microplates. To date, this is the first high-throughput screening method for linkage specificity determination of transglucosidases and glycosyltransferases. This strategy was applied to the screening of two DSR-S variants libraries, totalizing more than 36,000 clones obtained by saturation mutagenesis and simultaneous recombination of 8 residues from the catalytic domain. Eighty two mutants able of synthesize 2 to 8 times more alpha (1→3) linkages compared to the parental enzyme were isolated. The characterization of the catalytic properties of 7 representative mutants enabled the identification of a new peptide motif 460DYVHT464, involved in DSR-S specificity. Finally, the characterization of structural, rheological and mechanical properties of dextrans synthetized by these 7 mutants highlighted the differences in size and conformation of these macromolecules in solution and revealed the capacity of the polymer synthesized by H463R/T464V/S512T variant to form biofilms, whose mechanical properties are remarkable in comparison to those of other biopolymers extracted from plants or produced by fermentation

Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans

International audienc

A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-β 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies

Correction: A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

[This corrects the article DOI: 10.1371/journal.pone.0161209.]