• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 75
  • 73
  • 24
  • 9
  • 6
  • 3
  • 1
  • 1
  • Tagged with
  • 238
  • 69
  • 41
  • 38
  • 32
  • 31
  • 29
  • 28
  • 19
  • 18
  • 18
  • 18
  • 16
  • 16
  • 16
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
121

Enzyme-activated growth: development of a nuclear receptor based genetic selection system for engineering biocatalysts

Rood, Michael K. 12 January 2015 (has links)
Beyond their physiological roles, nuclear receptors have been exploited for their ability to act as intracellular sensors of small molecules. Accordingly, yeast two- and three-hybrid systems have been developed, exploiting them to control reporter gene expression. These systems may be used to identify nuclear receptor ligand interaction, or for protein engineering applications, particularly of the nuclear receptor ligand binding domain. In this work, the use of estrogen receptors as sensors for enzyme catalysis is explored, where expression of a reporter gene is induced in the presence of the product from an enzymatic reaction. This system, which we have called enzyme-activated growth, has applications for the engineering of biocatalysts. Biocatalytic routes are currently being explored in industrial applications since they often have financial and environmental benefits over traditional heterogeneous catalysis. Enzyme-activated growth is designed to serve as a system to select for engineered enzymes capable of catalyzing the desired reaction. For this work, a new yeast two-hybrid strain has been developed and characterized to allow for detection of both agonist and antagonist compounds. To increase the sensitivity of this assay, a variant of the estrogen receptor was created through random mutation, which responded to ligand concentrations an order of magnitude lower than the wild type receptor. The five mutations identified in the best variant were previously unknown in the literature and the roles of each of these are investigated, as is the mechanism by which they alter ligand sensitivity. As a proof-of-principle, the enzymatic production of genistein, an estrogenic metabolite from plants, using the enzyme isoflavone synthase, as well as the production of estrogen from testosterone, is explored. Synthesis of genistein from the starting material naringenin in vivo was detected in the yeast two-hybrid strain; however, attempts at pairing this with estrogen receptor activation and cell growth were met with limited success. Lastly, targeting the estrogen receptor with a series of novel anti-cancer therapeutics is explored. These compounds were designed to both bind and (in)activate the estrogen receptor while inhibiting histone deacetylase activity. The (anti-)estrogenic properties were analyzed as well as their potency as histone deacetylase inhibitors. These properties were compared to their anti-proliferative effects against various cancerous and healthy cell lines to determine their potential as selective anti-cancer therapeutics.
122

Αξιοποίηση αγροτοβιομηχανικών απορριμάτων για παραγωγή βιοκαταλυτών για συνεχή ερυθρά οινοποίηση σε χαμηλές θερμοκρασίες και μηλογαλακτική ζύμωση των οίνων

Αγουρίδης, Νικόλαος Α. 03 September 2010 (has links)
- / -
123

Dihydrogen driven cofactor recycling for use in bio-catalysed asymmetric organic synthesis

Lonsdale, Thomas January 2017 (has links)
Asymmetric reductions are used to produce chiral molecules, which are important precursors for the pharmaceutical industry. Bio catalytic reductions often display high enantioselectivity without the cost and toxicity associated with metal catalysis. However, unlike metal catalysts which use H<sub>2</sub> directly, many useful redox-enzymes require the hydride donor NADH. NADH is expensive; therefore for a bio-catalytic process to be viable it must be recycled, usually by using a sacrificial carbon based substrate, generating super-stoichiometric amounts of waste. Two different methods for H<sub>2</sub>-driven NADH recycling are explored in this project: using soluble hydrogenases (SH) and, carbon particles modified with a hydrogenase and an NAD<sup>+</sup>-reductase moiety. The conductive carbon particles allow electrons from H<sub>2</sub>-oxidation to be channelled from the hydrogenase to the NAD<sup>+</sup> reductase for reduction of NAD<sup>+</sup>. This project focuses on four main areas. The first looks at using the enzyme-modified particles for the production of high value chiral amines. A yield of &GT;98% was achieved using the enzyme-modified particles with an L alanine dehydrogenase for H<sub>2</sub> driven conversion of pyruvate to L-alanine. Moreover, a faster rate of reaction was demonstrated with the L-alanine dehydrogenase immobilised on particles versus with the L-alanine dehydrogenase in solution. The second section focuses on elevated temperature NADH recycling: an SH and an NAD<sup>+</sup>-reductase from a thermophilic organism were characterised. The NAD+-reductase was subsequently used as part of a system for recycling NADH at &GT;35 &deg;C. When demonstrated in combination with an enoate-reductase a 62 % yield was obtained for the reduction of 2 methyl 2 cyclopentenone. In the third strand SHs and enzyme-modified particles were investigated as recycling systems for NADH analogues. In summary, this thesis expands the scope for application of H<sub>2</sub>-driven biocatalytic reduction reactions.
124

Exploring peptide space for enzyme modulators

January 2010 (has links)
abstract: Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry. / Dissertation/Thesis / Ph.D. Chemistry 2010
125

Directed evolution of amino acid dehydrogenases for biocatalysis of chiral amines

Hours, Raphaelle January 2018 (has links)
By applying the principles of Darwinian natural selection in the laboratory, directed evolution has become a powerful practical approach to study enzymes and optimize them to catalyze industrially relevant transformations. In this thesis, I applied this strategy to the engineering of amino acid dehydrogenases for biocatalysis of chiral amines, focusing on two crucial features for successful directed evolution experiments. A first key aspect is the development of technologies allowing the screening of large libraries of enzyme variants to explore sequence space efficiently. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsions has recently led to the development of ultrahigh-throughput screening platforms that allow sorting of more than 106 variants per hour. So far, these microfluidic droplet sorters have relied exclusively on fluorescent readouts. To further extend the range of applications toward enzymes for which no fluorescent assays are available, I successfully developed a sorting module based on absorbance detection. Using this new module, microdroplets could be sorted based on an absorbance readout at rates of up to 1 million droplets per hour. To demonstrate the utility of this module for protein engineering, three rounds of directed evolution were performed to improve a poorly stable NAD+ dependent phenylalanine dehydrogenase (PheDH) toward its native substrate. Five hits showed increased activity (improved up to 10-fold in lysate; kcat increased >3.5-fold), soluble protein expression levels (>2.5-fold) and thermostability (Tm, 8 °C higher). To increase the sensitivity of the device (3–4 orders of magnitude lower than fluorescence assays) for detection of enzymes with limited stability and low turnovers, an extra step of growth in droplets from single cell encapsulation, followed by piconinection of substrates and lysis agents was implemented. As a result, a fivefold signal enhancement over background was achieved, for an amine dehydrogenase (AmDH) reaction shown to be undetectable in a droplet single cell assay. Second, I investigated how mutational robustness may correlate with protein stability and lead to successful hits after mutagenesis and screening. To examine this issue, I initially investigated various approaches (including ancestral resurrection and computational design) to identify stabilized PheDH variants. One such variant (dubbed Pross 4) showed increased expression levels (>3.3-fold) and thermostability (Tm, 13 °C higher) compared to the wild-type PheDH. I further compared the mutational tolerance and the hit rate between PheDH and Pross 4 by generating variant libraries focused on key active site residues and screening them for improved AmDH activity. The Pross 4 background generated 6.4 times more active variants than the PheDH background, the best hits displaying increased activity (up to 2.5-fold in lysate; kcat/KM increased up to 8-fold) compared to previously engineered AmDHs with the PheDH scaffold. In conclusion, this work highlights how directed evolution experiments could be designed for increased success rates, by combining reliable high-throughput screens with careful choice of evolutionary robust starting points.
126

The exploitation of thermophiles and their enzymes for the construction of multistep enzyme reactions from characterised enzyme parts

Finnigan, William John Andrew January 2016 (has links)
Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.
127

Développement d'outils enzymatiques pour la synthèse de protéines S-glycosylées / Novel enzymatic tools for S-glycosylated proteins synthesis

Guillotin, Laure 12 November 2015 (has links)
Dans la communauté scientifique, la glycosylation suscite un vif intérêt tant les relations existantes entre les sucres et les protéines sont étroites et sont impliquées dans de nombreux processus biologiques. De nombreuses méthodologies de synthèse ont été développées pour permettre la production de glycoprotéines sous forme homogènes nécessaires pour leur étude. Les formes S-glycosidiques sont d’un intérêt particulier grâce notamment à la réactivité de l’atome de soufre qui permet le couplage spécifique sucre – acide aminé et qui confère à la liaison une relative résistance à l’hydrolyse acido/basique et enzymatique. Dans le cadre de ce projet nous avons souhaité utiliser des outils biocatalytiques pour proposer une nouvelle voie d’accès aux protéines S-glycosylées en s’appuyant sur le concept des thioglycoligases. Pour répondre à ce challenge, nous nous sommes intéressés à la production d’une banque de glycosidases natives à partir du génome de la bactérie thermophile Dictyoglomus thermophilum. Trois protéines originales ont pu être produites, caractérisées et criblées en activité transglycosylase. Parmi ces dernières la β-glycosidase DtGly s’est révélée être un biocatalyseur efficace en favorisant la synthèse d’une variété de glycosides d’alkyle et de glycosyl glycérol. Par la suite, neuf mutants thioglycoligases ont été produits par mutagénèse dirigée à partir des glycosidases natives. A l’issue de leur criblage en activité thioligase, le mutant de DtGly E159Q a été retenu pour la synthèse de S-glycoconjugués. Une étude de relation structure/activité menée sur l’enzyme mutée nous a conduit à la synthèse d’un acide aminé non-naturel, la Thiotyrosine. L’essai de glycosylation de l’analogue estérifié de cette Thiotyrosine par DtGly E159Q a été suivi par LC-MS et a permis de mettre en évidence la formation du produit de thioglucosylation. Ce premier résultat préliminaire s’avère très prometteur pour la validation du concept de S-glycosylation d’un acide aminé catalysée par une thioglycoligase. / Glycosylation, one of the most complex co- and post-translationnal modifications of proteins, is of utmost importance for the scientific community as carbohydrates and proteins are closely related and involved in numerous diseases. The need to access homogeneous glycoproteins allows the development of a wide range of synthetic methods. Among them S-glycosidic forms have been attractive thanks to the reactivity of sulfur atom which allows the specific sugar – amino acid conjugation and confers a relative resistance through acido/basic or enzymatic hydrolysis. In this project we attempt to take advantage of natural tools to develop an enzymatic methodology to prepare S-glycosylated proteins through the thioglycoligase concept. In order to take up this challenge we first generate an enzymatic pool of wild type glycosidases from the thermophilic bacteria Dictyoglomus thermophilum. Activity screening of the three proteins produced and characterized allows the isolation of the β-glycosidase DtGly with good transglycosylation activity, thus affording a variety of alkyl glycosides and glyceroglycosides. Next we generate nine thioglycoligase mutants through site-directed mutagenesis and after screening of activity, the variant DtGly E159Q was selected to produce S-glycoconjugates. Finally, DtGly E159Q relation structure/activity studies led us to synthesize the unnatural amino acid Thiotyrosine. Enzymatic thioglucosylation assay catalyzed by DtGly E159Q was monitored by LC-MS and gave converging evidence of the successful formation of the S-glucosylated Thiotyrosine. This tremendous preliminary result is promising for further investigations of enzymatic S-glycosylation of amino acid using thioglycoligases as toolbox.
128

Aplicação da cenoura (Daucus carota) como biocatalisador : estudos visando a síntese assimétrica da Shahidina e da Marmelina

Bonfim, Thiago Ramalho do January 2015 (has links)
Orientador: Prof. Dr. Álvaro Takeo Omori / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2015. / Este trabalho consistiu no estudo para a síntese dos compostos Shahidina 1 e Marmelina 2 ambos extraídos da planta Aegle marmelos amplamente cultivada no continente asiático. Ambos os compostos possuem centros quirais onde um dos objetivos é definir estes centros por biocatálise com o uso de cenoura (Daucus carota). Também foi explorada a formação de anéis oxazolínicos entre alcoóis e azidas pela reação de Boyer. Porém esta metodologia demonstrou alguns resultados imprevistos. Houve a formação de epóxido na redução química da cetona 17 para o álcool 19 e o método de biorredução teve que ser otimizado pois o método clássico apresentou baixas taxas de conversão. A reação de Boyer também se mostrou ineficaz quando realizada entre azidas e aldeídos conjugados. Houve a necessidade de se alterar a rota. / This work aimed the synthesis of Shahidine 1 and Marmeline 2 both extracted from Aegle marmelos plant (widely grown in Asia). Both compounds have chiral centers located next to the aromatic ring. One of the key steps was to resolve these stereogenic centers by biocatalysis with Daucus carota (carrot) root. Another key step was the formation of oxazoline ring between aldehydes and azido alcohols by the Boyer reaction. However some unexpected results were observed. For example, epoxide formation was observed in the chemical reduction of ketone 17. Also, bioreduction protocol showed low conversion rate. Besides, we observed that the the Boyer reaction between azido alcohol 19 and conjugated aldehyde were ineffective. It was necessary to change the route.
129

Etude de la cycloaddition 1,3-dipolaire des ylures de pyridinium pour le développement de nouvelles réactions de ligations fluorogéniques / Study of pyridinium ylide-alkyne cycloaddition for the development of new fluorogenic click reactions

Bonte, Simon 16 December 2013 (has links)
L'objectif de ce travail est la mise au point de réactions de chimie « click » fluorogéniques permettant le développement de nouveaux outils chimiques et/ou la conception d'édifices bioactifs par l'assemblage des sous-unités fonctionnalisées en présence de la cible biologique. La cycloaddition 1,3-dipolaire des ylures de pyridinium a ainsi été réalisé en conditions physiologiques (H2O, pH neutre, TA), pour la mise en œuvre de réactions de couplages pouvant être biocompatibles. En effet, les ylures de pyridinium ont l'avantage d'être facilement générés in situ à partir des sels correspondants et de mener régiosélectivement, sans catalyseur métallique, à des indolizines fluorescentes après cycloaddition. Pour cela, la synthèse et la réactivité des différents partenaires de la cycloaddition ont été étudiés, tout comme l'optimisation des conditions expérimentales. Tout d'abord, deux types de dipolarophiles se caractérisant par leur caractère déficient en électrons, ont été sélectionnés: les dérivés de l'acide propiolique (amides et esters) obtenus selon une synthèse biocatalysée utilisant les lipases (CAL B), et les alcynes conjugués à des structures hétérocycliques (quinoléine, pyridine). Concernant le dipôle, à partir d'une petite famille de sels de pyridinium (diversement fonctionnalisés) ou de cycles apparentés, l'influence de la nature et de la position des substituants a été évaluée au niveau du pKa, de la réactivité (selon une réaction modèle faisant intervenir le propiolate d'éthyle), et de la fluorescence du cycloadduit. Les résultats expérimentaux, complétés par une approche de chimie théorique, ne nous permettent pas à ce jour d'expliquer la très bonne réactivité à température ambiante des dipôles portant un groupement électro-attracteur en position 4 du sel de pyridinium (sélection des sels de cyano- et acétyl-pyridinium pour nos applications). Enfin, la fonctionnalisation des précurseurs a été effectuée par l'introduction de diverses fonctions réactives dont des amides, pour obtenir une réaction de chimie « click » facilement applicable et généralisable. La preuve de concept est en cours de réalisation au laboratoire, avec la synthèse de molécules dimériques dérivées de la tacrine, pour la conception d'inhibiteurs de l'acétylcholinestérase. La seconde application porte sur la post-fonctionnalisation des indolizines et la synthèse de structures trimériques (plateforme d'assemblage fluorescente) / In the course of investigations aimed at designing fluorescent metal-free click ligations for application in bioconjugation and drug design, we turned our attention to the use of azomethine-ylides as dipoles for [3+2] cycloadditions. In this regard, pyridinium-ylides, generated in situ from suitable pyridinium salts, are of great interest due to their good reactivity with activated alkynes, such as propiolic acid derivatives or conjugated alkynes. In addition, the corresponding fluorescent indolizines are formed in a regioselective manner and any catalyst is required. From the outset, we defined a specific set of requirements for these ligations, most notably that they be biocompatible (i.e. physiological conditions, pH 7, room temperature) and that the starting reagents could be readily functionalized with the reporter group(s) or biomolecule(s) of choice. To this end, we screened a series of pyridinium salts as suitable 1,3-dipoles. The reactivity patterns observed correlate well with the pKa of the pyridinium (ylide formation) and with the electron-withdrawing character of the pyridinium ring substituent. This enabled us to identify the 4-acetyl and 4-cyano pyridinium salts suitable for ligation with propiolic esters and amides in the desired biological conditions. The synthetic strategies used to prepare the “two partners” for this [3+2] cycloaddition are described, and our preliminary results on their applications as “click” reactions are reported in particular the formation of heterodimeric tacrine derivatives (acetylcholinesterase inhibitors) or the design of fluorescent tripodal scaffold useful in chemical biology.
130

Biocatalyse : aldolisation, acylation et oxydation - Applications synthétiques / Biocatalysis : aldolization, acylation and oxidation - Synthetic applications

Hiault, Florence 24 November 2017 (has links)
Les travaux présentés dans ce manuscrit s’inscrivent dans le contexte général de l’essor de la biocatalyse et de son utilisation en synthèse organique. Le thème principal porte sur l’étude et le développement de différentes voies d’accès stéréosélectives à des acides alpha-aminés bêta-hydroxylés substitués. L’utilisation d’un biocatalyseur permettant d’accéder à des acides alpha-aminés bêta-hydroxylés par une aldolisation entre la glycine et divers aldéhydes, en présence de phosphate de pyridoxal, a été étudiée. Des aldéhydes aliphatiques, aromatiques et hétéroaromatiques ont pu être impliqués avec succès comme partenaires électrophiles dans ces réactions qui permettent un excellent contrôle de la configuration du carbone asymétrique créé en alpha du groupe carbonyle mais s’effectuent généralement avec des diastéréosélectivités plus modestes. Par ailleurs, un dédoublement cinétique enzymatique d’esters alpha,bêta-dihydroxylés, précurseurs d’acides alpha-aminés bêta-hydroxylés substitués en alpha, a été étudié. La méthode développée repose sur la monoacylation d’esters alpha,bêta-dihydroxylés, acycliques ou cycliques, en présence d’une lipase et d’un donneur d’acyle. De façon indépendante, la mise au point de séquences réactionnelles monotopes faisant intervenir une étape d’oxydation biocatalytique a été étudiée pour accéder à des composés aminés hautement fonctionnalisés. / The research work presented in this manuscript pertains to the field of biocatalysis and some applications in organic synthesis. The main subject is the development of stereoselective synthetic methods allowing access to substituted alpha-amino beta-hydroxy acids. The use of a biocatalyst enabling the preparation of optically enriched alpha-amino beta-hydroxy acids in a single step from glycine by an aldol reaction, in the presence of pyridoxal phosphate, was investigated. Aliphatic, aromatic and heteroaromatic aldehydes could be successfully used as electrophilic partners in such reactions that allow an excellent control of the stereocenter created at the alpha position of the carbonyl group whereas moderate levels of diastereoselectivity were generally observed. The enzymatic kinetic resolution of acyclic or cyclic alpha,beta-dihydroxy esters, which are precursors of alpha-substituted alpha-amino beta-hydroxy acids, was also achieved by monoacylation in the presence of a lipase and an acyl donor. Independently, a one-pot sequence involving a biocatalytic oxidation was developed to access highly functionalized nitrogen containing compounds.

Page generated in 0.0789 seconds