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Studies on antimicrobial activity of arginine-based surfactants and chemo- enzymatic synthesis of novel amphiphiles based on L-arginine and D-fagomineCastillo Expósito, José Antonio 02 February 2007 (has links)
Los conjugados de lípidos y aminoácidos presentan excelentes propiedades tensioactivas, alta biodegradabilidad y baja toxicidad. De entre ellos, los derivados de arginina poseen un amplio espectro de actividad antimicrobiana.Con el objetivo de elucidar el modo de acción de los tensioactivos derivados de arginina, se ha investigado su interacción con modelos de membrana (liposomas y monocapas de fosfolípidos) y se ha evaluado los efectos que causan en Staphylococcus aureus y Escherichia coli. La eficacia antimicrobiana de los tensioactivos derivados de L-arginina previamente sintetizados en nuestro grupo de investigación se ha mejorado. Para ello, se ha preparado quimo-enzimáticamente una nueva familia de derivados de L-arginina, bis(fenilacetilargininas). Finalmente y con el propósito de preparar nuevos posibles agentes antimicrobianos, se ha sintetizado una familia de compuestos anfifílicos, derivados alquilados de D-fagomina, empleando fructosa-6-fosfato aldolasa (FSA) como biocatalizador. / Amino acid lipid conjugates possess excellent surface properties, high biodegradability and low toxicity. Among them, arginine-based surfactants show a broad antimicrobial activity.The mode of action of arginine-based surfactants was investigated. Thus, their interaction with membrane models (liposomes and phospholipid monolayers) and the effects caused on Staphylococcus aureus and Escherichia coli were studiedThe antimicrobial efficacy of the arginine-based surfactants synthesised previously in our research group was improved. To this end, novel bis(phenylacetylarginine) derivatives were prepared chemoenzymatically.Finally and with the aim of preparing novel antimicrobial agents, we prepared a new family of amphiphilic compounds, N-alkylated derivatives of D-fagomine, using fructose-6-phosphaste aldolase (FSA) as biocatalyst.
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Aldolases for Enzymatic Carboligation : Directed Evolution and Enzyme Structure-Function Relationship StudiesMa, Huan January 2015 (has links)
The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6-phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties. In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates.
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Purification and Characterization of glpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coliDonahue, Janet Lee 01 May 2000 (has links)
In Escherichia coli, the utilization of glycerol and sn-glycerol 3-phosphate is mediated by gene products of the glp regulon. The regulon encompasses five operons, including the glpFKX operon. Although glpF and glpK encode glycerol diffusion facilitator and glycerol kinase,respectively, the function of glpX was unknown. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP, ATP and phosphate and activated by PEP.
The attributes of the glpX-encoded FBPase were different from those of the previously characterized E. coli FBPase encoded by fbp. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. In contrast, a glpX mutant exhibited a growth-positive phenotype on glycerol, glucose or fructose media. Surprisingly, a double mutant strain glpX pfkA (6-phosphofructokinase I) was more inhibited in growth on glucose and glycerol media than the pfkA parent. Carbohydrate metabolism in the pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF. / Master of Science
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Nouveaux matériaux biohybrides multifonctionnels pour la biocatalyse / New multifunctional biohybrid materials for biocatalysisMahdi, Rima 11 December 2015 (has links)
Ces travaux de thèse pluridisciplinaires à l‘interface entre biocatalyse et nanomatériaux visent la conception de matériaux biohybrides innovants par assemblage dans des conditions douces de matériaux inorganiques de type hydroxydes doubles lamellaires (HDL) avec des enzymes. La première partie de ce mémoire est consacrée à la caractérisation des interactions physico-chimiques entre les HDL et la fructose-6-phosphate aldolase (FSA) catalysant la formation stéréosélective de liaisons C-C pour conduire à des polyols chiraux. Les structures lamellaires HDL permettent un confinement efficace de systèmes enzymatiques grâce à leur structure bidimensionnelle poreuse, leurs propriétés physico-chimiques favorables à l‘échange ionique et leur biocompatibilité. Différentes stratégies d‘immobilisation de la FSA dans des matrices d‘HDL ont été explorées, le taux d‘immobilisation et l‘activité biocatalytique étant fortement dépendant de la méthode d‘assemblage et de la nature des phases HDL. Le taux d‘immobilisation de l‘enzyme obtenu par coprécipitaton est supérieur à celui obtenu par adsorption. Dans une deuxième partie, un bioréacteur a été élaboré par un assemblage hiérarchisé constitué de la FSA, de nanoplaquettes d‘HDL et de billes de polysaccharide, ce dernier jouant le rôle de matrice macrostructurante. De façon notable, le taux d‘encapsulation de l‘enzyme dans la matrice macroscopique est amélioré lorsque le biocatalyseur est pré-encapsulé dans les nanoplaquettes d‘HDL. Ceci est attribué aux interactions électrostatiques favorables entre les chaînes de polysaccharide et les HDL, facilitant une charge de matière plus importante. L‘efficacité catalytique du bioréacteur obtenu et sa recyclabilité ont été démontrés. Dans la troisième partie de cette thèse, nous décrivons pour la première fois la conception de bionanoréacteurs enzymes@HDL par co-immobilisation de systèmes bi- ou tétra-enzymatiques dans les HDL permettant de réaliser des cascades multienzymatiques biomimétiques. L‘immobilisation des différentes enzymes prises séparément a d‘abord été optimisée afin de déterminer les conditions de co-immobilisation et de réaliser les cascades biocatalytiques en phase hétérogène. Ces bionanoréacteurs, dont nous avons démontré la recyclabilité, ont été appliqués pour la synthèse de sucres phosphorylés de série D. Enfin, une cascade multienzymatique a été conçue de novo en solution aqueuse et optimisée pour synthétiser différents sucres phosphorylés rares de série L. / This multidisciplinary thesis at the biocatalysis/nanomaterial interface perfectly aims at designing innovative biohybrid materials by the assembly of inorganic materials the Layered Double Hydroxides (LDH) with enzymes under mild conditions. The first part of this thesis is devoted to the characterization of physico-chemical interactions between the LDH and the fructose-6-phosphate aldolase (FSA) catalyzing the stereoselective C-C bond formation to provide chiral polyols. LDH structures allow the effective confinement of enzymatic systems thanks to their opened two-dimensional structure as well as their chemical surface properties at the nanoscale and their biocompatibility. The FSA immobilization in different LDH matrices by different methods was studied. Biocatalytic activity is highly dependent on the method of assembling, modulating the final amount of FSA. The retaining activity rate of co-precipitated material was higher than that obtained for the adsorbed enzyme. In a second part, a bionanoreactor was developed based on a hierarchized assembly of FSA, LDH nanoplatelets and polysaccharide beads acting as a macrostructuring matrices. Significantly, the encapsulated enzyme rate in the beads was improved when the biocatalyst was pre-encapsulated in LDH nanoplatelets. This is attributed to favorable electrostatic interactions between the polysaccharide chains and LDH, facilitating a higher catalyst loading. The catalytic efficiency of the prepared bioreactor and its recyclability were demonstrated. In the third part of this thesis, we describe for the first time the design of bionanoreactors ―enzymes@LDH‖ by co-immobilisation of two and four enzymes in LDH allowing biomimetic multienzymatic cascades. We first studied the immobilization of the different enzymes taken separately. Then we worked on the optimization of the biocatalytic cascades in heterogeneous phase. These bionanoreactors, for which we have shown the recyclability, have been applied to the synthesis of D-series phosphorylated sugars. Finally, a multienzymatic cascade was de novo designed in aqueous homogeneous solution. It was optimized for the synthesis of rare L-phosphorylated sugars.
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Novel pathogenic mechanisms of myasthenic disorders and potential therapeutic approachesZoltowska, Katarzyna Marta January 2014 (has links)
Congenital myasthenic syndrome (CMS) and myasthenia gravis (MG) are, respectively, inherited or autoimmunological disorders caused by aberrant neuromuscular transmission, which manifests as fatiguable muscle weakness. A novel subtype of CMS, resulting from mutations in GFPT1 and characterised by a limb girdle pattern of muscle weakness, has been described. The gene encodes L glutamine:D fructose-6-phosphate amidotransferase 1 (GFAT1) – a key rate limiting enzyme in the hexosamine biosynthetic pathway, providing building blocks for glycosylation of proteins and lipids. The research focused on the molecular bases of the CMS resulting from mutations in the ubiquitously expressed gene, but with symptoms largely restricted to the neuromuscular junction (NMJ). The work has established a link between the NMJ and GFPT1 CMS by demonstrating that the AChR cell surface is decreased in GFPT1 patient muscle cells and in GFPT1-silenced cell lines. The decrease is likely to be caused by reduced steady-state levels of individual AChR α, δ and ε, but not β, subunits. To optimise treatment for myasthenic disorders, a comparative in vivo trial of therapy with pyridostigmine bromide and salbutamol sulphate, and pyridostigmine bromide alone, was conducted. Supplementation of the AChE inhibitor-based therapy with the β2-adrenergic receptor agonist had a beneficial effect. This offers promise for more effective treatments for CMS and MG affected individuals. Molecular causes of MG were also investigated. The search for novel antibody targets was conducted with the use of a designed cell-based assay for the detection of anti COLQ autoimmunoglobulins in MG patient sera. The antibodies were detected in 24 out of 418 analysed samples, but their pathogenicity has not been determined.
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