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Laccase-catalyzed oxidation of bisphenol A: kinetic study and fate of reaction productsHautphenne, Catherine 29 April 2016 (has links)
Since recent years, pollution of surface and drinking waters by chemical pollutants fromanthropogenic activities, in particular micropollutants, has become an important environmentalissue in industrialized countries. Indeed the majority of these molecules are toxic toliving organisms, even present at very low concentrations (pg.L−1 to μg.L−1). Some of themare referred as endocrine disrupting compounds. The existing conventional wastewater treatmentsare most of the time not adapted for the removal of these compounds. Among them,advanced oxidative processes give the better removal efficiencies, but with drawbacks such ashigh energetic costs, or the formation of reaction products, sometimes more toxic than theparent compounds. A promising solution for water decontamination could be bioremediation.This work is based on this technology, via the study of the use of lignolytic enzymes, such aslaccases, for the removal of micropollutants. Degradation studies of a lot of micropollutants(bisphenol A, nonylphenol, triclosan, 17-_-estradiol, etc.) have already been investigatedwith different reactor configurations, using free (solubilized) or immobilized enzymes (biocatalysts).However, a decrease in the performance of some continuous long-term degradationprocesses has been highlighted, most of the cases because of biocatalysts deactivation. Oneof the reasons to explain this deactivation was identified as being induced by the deposit ofreaction products onto the surface of the biocatalysts.The general objective of this work was to investigate the degradation of bisphenol A(BPA) by laccases oxidation in different reactor configurations. Especially, the focus wasmade on the study of BPA degradation and reaction products kinetics, and reaction productscharacterization and identification. In the continuity, a second objective was related tothe optimization of a packed-bed reactor developed in our laboratory, through the developmentof a new biocatalyst, and assessment of biocatalyst deactivation.First, a deep literature review has been done in order to summarize the knowledge regardingsoluble and insoluble reaction products formation during laccase oxidation of a listof the most studied recalcitrant phenolic compounds, and their risk assessment. This reviewreported that the majority of reaction products were insoluble, and corresponded to smalloligomers associated by C-C or C-O bonds. In most of the cases, the assessment of theirstructure identification, and evaluation of their toxicity and/or estrogenic activity was missing.Secondly, a research for the selection of a new suitable support for laccase immobilizationhas been investigated. Celite R648 supports were selected for the immobilization of laccasesfrom Trametes versicolor, based on physical, catalytic and economic properties. Characterizationof the packed-bed reactor developed at the TIPs was performed with this support,through evaluation of pressure drop and residence time distributions. This new biocatalystwas selected as a tool for further studies regarding BPA degradation and reaction productsformation kinetics after laccase oxidation.The third part of this work consisted in the kinetic study of BPA degradation, and solubleand insoluble reaction products formation. For BPA degradation kinetic study, experimentaldata were obtained by LC/MS analyses for different initial conditions and reactor configurations.A mathematical model was build, based on a modified Briggs-Haldane model, in orderto identify the limiting step(s) occurring during laccase-catalyzed oxidation of BPA in batchreactor with free enzymes. This model was validated on experimental data, and highlightedthat the limiting step during BPA degradation was the formation of the enzyme-substratecomplex. For the kinetic study of insoluble reaction products, a protocol was developed enablingthe monitoring of these products over time, through their mass, in batch reactor withfree enzymes, and for seven different experimental conditions according to a Doehlert design.A mathematical model was build, expressed by a first-order kinetics, and fitted experimentaldate from 30 minutes of reaction. The modeling of short reaction times was achieved with theuse of the model build to characterize BPA degradation kinetics, with the assumption thatall the BPA degraded was converted into insoluble reaction products. Combination of resultsfrom these two kinetic studies highlighted that insoluble reaction products formation waslimited by BPA conversion, limited itself by the formation of the enzyme-substrate complex.The study of soluble reaction products kinetics was only experimental, and showed majorkinetics differences in batch reactor depending on the use of free enzymes or biocatalysts.Mass transfer limitations could occur in the case of immobilized enzymes.In parallel, experiments have been done in order to characterize and/or identify reactionproducts. DSC, GPC and FTIR analyses were performed for the characterization of insolublereaction products, and LC/MS(-MS) analyses for the characterization of soluble reactionproducts. Results highlighted that our products were in majority polymers, and that theircomposition was varying depending on initial BPA concentration and reaction time.All the information gathered in this work were used and exploited in order to characterizebiocatalysts deactivation, and to propose solutions to optimize the packed-bed reactor andrecover biocatalysts activity. In parallel, we have developed a tool to monitor biocatalystsdeactivation, with the purpose of increasing the degradation performance of the reactor. / Depuis plusieurs années, la pollution des eaux potable et de surface par des polluants chimiquesprovenant d’activités anthropogéniques, en particulier les micropolluants, est devenueune problématique environnementale importante dans les pays industrialisés. En effet, la majoritéde ces substances sont toxiques pour les organismes vivants, même présentes en trèsfaibles concentrations (de l’ordre du pg.L−1 au μg.L−1). Un grand nombre de ces substancessont considérées comme des perturbateurs endocriniens. Les techniques conventionnelles actuellesde traitement des eaux usées ne sont la plupart du temps pas adaptées à l’abattementde ces substances. Parmi ces techniques, les procédés d’oxydation avancée sont ceux quidonnent les meilleurs rendements d’abattement. Cependant, ils présentent certains inconvénients,incluant des coûts énergétiques importants, et la formation de produits de réactionparfois plus toxiques que les molécules de départ. La bioremédiation pourrait constituer unesolution prometteuse. Ce travail est basé sur cette idée, au travers de l’étude de l’utilisationd’enzymes lignolytiques, comme les laccases, pour la dégradation des micropolluants. Desétudes sur la dégradation de nombreux micropolluants (bisphénol A, nonylphénol, triclosan,17-_-estradiol, etc.) ont déjà été menées pour différentes configurations de réacteurs, avecl’usage d’enzymes libres (solubilisées) ou immobilisées (biocatalyseurs). Cependant, à longterme, une diminution des performances de dégradation lors de certains procédés continusa été mise en évidence, la plupart du temps à cause d’une désactivation des biocatalyseurs.Une raison possible pouvant expliquer cette désactivation réside dans le dépôt de produits deréaction sur la surface des biocatalyseurs.L’objectif général de ce travail a été d’étudier la dégradation d’un micropolluant, le bisphénolA (BPA), via oxydation par les laccases, dans différentes configurations de réacteurs.Plus précisément, nous nous sommes intéressés à l’étude des cinétiques de dégradation dubisphénol A et de formation de ses produits de réaction, ainsi que leur caractérisation etindentification. Dans la continuité, un second objectif a consisté en l’étude de l’optimisationd’un réacteur à lit fixe développé dans notre laboratoire, au travers du développement d’unnouveau biocatalyseur, et l’évaluation de la désactivation enzymatique de ces biocatalyseurs.Premièrement, une revue détaillée de la littérature a été effectuée dans le but de résumerles connaissances acquises à propos de la formation des produits de réaction solubles etinsolubles après oxydation par les laccases des composés phénoliques récalcitrants les plusétudiés actuellement, et l’évaluation de leur toxicité. Cette revue a mis en évidence que lamajorité des produits de réaction sont insolubles, et correspondent à de petits oligomères dontles unités constitutives sont associées via des liaisons C-C ou C-O. Dans la plupart des cas,l’identification de leur structure, et l’évaluation de leur toxicité et/ou activité oestrogéniqueest manquante.Dans un second temps, une étude pour la sélection d’un nouveau support pour l’immobilisationde laccases a été menée. Le support Celite R648 a été sélectionné pour l’immobilisationde laccases de Trametes versicolor, sur bases de propriétés physico-chimiques, catalytiques etéconomiques. La caractérisation du réacteur à lit fixe a été réalisée avec ce support, au traversde l’évaluation des pertes de charges et des distributions de temps de séjour. Le nouveaubiocatalyseur développé a finalement été sélectionné comme outil pour l’étude des cinétiquesde dégradation du BPA, et de formation des produits de réaction, après oxydation par leslaccases.La troisième partie de ce travail a consisté en les études cinétiques de la dégradation du BPA, et de la formation des produits solubles et insolubles de réaction. Pour l’étude de la cinétiquede dégradation du BPA, des données expérimentales ont été obtenues via des analysesde LC/MS, pour différentes conditions expérimentales et pour différentes configurations deréacteurs. Un modèle mathématique a été construit, basé sur un modèle de Briggs-Haldanemodifié, dans le but d’identifier la(les) étape(s) limitante(s) se produisant durant l’oxydationdu BPA par les laccases, en réacteur batch et avec enzymes libres. Ce modèle a été validésur les données expérimentales, mettant en évidence que l’étape limitante dans la réactionde dégradation du BPA était la formation du complexe enzyme-substrat. Pour l’étude de lacinétique de formation des produits de réaction insolubles, un protocole a été développé pourle suivi de ces produits au cours du temps, au travers de leur masse, en réacteur batch etavec enzymes libres, dans sept conditions expérimentales différentes, selon le schéma d’undesign expérimental de Doehlert. Un modèle mathématique a été construit, exprimé commeune cinétique de premier ordre, et a été validé sur les données expérimentales à partir de 30minutes de réaction. La modélisation sur des temps de réactions inférieurs à 30 minutes a étéobtenue en utilisant le modèle construit pour la cinétique de dégradation du BPA, en supposantque tout le BPA dégradé était directement converti en produits de réaction insolubles.L’étude combinée des résultats obtenus a mis en évidence que la formation des produits deréaction insolubles était limitée par la dégradation du BPA, elle-même limitée par la formationdu complexe enzyme-substrat. L’étude cinétique de la formation de produits solubles deréaction a uniquement été expérimentale, et a permis de mettre en évidence des différencesmajeures dans les cinétiques de formation en réacteur batch, selon l’usage d’enzymes libresou de biocatalyseurs.En parallèle, des expériences ont été réalisées dans le but de caractériser et/ou d’identifierles produits de réaction. Des expériences de DSC, GPC et FTIR ont été faites pour la caractérisationdes produits insolubles de réaction, et des expériences de LC/MS(-MS) ont étéréalisées pour la caractérisation des produits solubles de réaction. Les résultats ont permisde mettre en évidence que les produits obtenus sont en majorité des petits oligomères, etque leurs compositions varient en fonction de la concentration initiale en BPA et le temps deréaction.Les informations récoltées dans ce travail ont été utilisées et exploitées pour tenter decaractériser la désactivation des biocatalyseurs, et de proposer des solutions pour optimiserle réacteur à lit fixe et récupérer l’activité initiale des biocatalyseurs. En parallèle, nousavons également développé une méthode pour détecter et suivre la désactivation, dans le butd’améliorer les performances de dégradation du réacteur. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished
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The degradation of the endocrine disrupting chemical, bisphenol-A : a comparative study between fungal and bacterial laccasesPrins, Alaric January 2015 (has links)
>Magister Scientiae - MSc / The degradation of endocrine disrupting chemicals (EDCs) is a topic of high importance and one that research efforts are continually being focused on. These harmful chemicals are known to cause adverse health effects in humans and animals. In particular, bisphenol-A (BPA), a high volume chemical which is mainly used in the manufacturing of polycarbonate plastics and epoxy resins have been shown to be implicated in the development of a variety of health problems. In this study, the ability of two fungal laccases [Trametes versicolor (TvL) and Trametes pubescens (TpL)], and two bacterial laccases [Streptomyces coelicolor (SLAC), and a mutant of SLAC (SLAC- VN)] to degrade or remove BPA from solution was investigated. The commercial preparation of TvL was used for the purposes of this study, while TpL was produced from the native strain. T. pubescens was cultured in shake-flasks, the supernatant harvested and subjected to ammonium sulphate precipitation. SLAC and SLAC-VN were produced from recombinant strains using a standard protocol and the enzymes purified by size-exclusion chromatography. The presence of the laccases were confirmed by the 2,6-dimethoxyphenol assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).The removal or degradation of BPA from solution was determined for the free enzymes, as well as the enzymes in immobilised form. For immobilisation, the enzymes were encapsulated in sodium alginate beads and cross-linked to form cross- linked enzyme aggregates (CLEAs).High levels of BPA removal was exhibited by the fungal laccase, TpL (100% removal)and the bacterial mutant laccase, SLAC-VN (96%) in their free form. When all four laccases were encapsulated in sodium alginate beads, a number of changes to the characteristics of the enzymes were observed. Overall, the level of BPA removal was reduced for all enzymes as when compared to the free laccases, while SLAC-VN removed more BPA than either of the fungal laccases (59% for SLAC-VN versus 57% TvL and 54% for TpL). The encapsulation of the laccases in alginate beads also led to changes in the optimal temperature for BPA removal, with all encapsulated laccase being able to remove BPA optimally at 40°C. The immobilisation of the laccases in CLEA form had the most significant effect on the BPA removal ability of the laccases. The pH range for both fungal laccases was extended beyond the acidic range [for TpL, optimal removal occurred at pH 8.5 compared to pH 4.5 (free) and pH 6.0 (encapsulated)]. Most remarkable, however, was that the formation of CLEAs greatly enhanced the BPA removal ability of SLAC (60% removal compared to 25% when encapsulated).
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Caractérisation du potentiel de dégradation de matières organiques naturelle (litière) et anthropique (HAP) par les communautés microbiennes issue du milieu littoral méditerranéenQasemian, Leila 02 February 2012 (has links)
Les écosystèmes méditerranéens littoraux sont soumis à divers stress environnementaux naturels (stress hydrique et halin) et anthropiques susceptibles de s'intensifier dans les prochaines décennies. Dans ce contexte, le fonctionnement des communautés microbiennes - encore très peu étudiés dans de tels milieux - était important à préciser. L'effet du stress halin sur la transformation de la matière organique dans la litière de pin d'Alep issues des calanques de Marseille a été estimé ainsi que le potentiel de biodégradation d'un polluant chronique, l'anthracène, un Hydrocarbures Aromatiques Polycycliques. Différents approches in situ, ex situ et in vitro ont été utilisées en associant différentes méthodes afin de mesurer l'état fonctionnel du milieu (activités enzymatiques, respirométrie basale), la diversité fonctionnelle microbienne (Catabolic Level Physiological Profile), la biomasse microbienne et l'évolution chimique de la matière organique (RMN du solide du 13C). En mésocosmes, les laccases, induites par la présence d'anthracène, ont contribué à son oxydation et ont été séquencées par LC/MS/MS afin de déterminer les espèces fongiques à l'origine de leur synthèse. Les résultats montrent que certaines activités enzymatiques du cycle du carbone sont peu affectées par la salinité et l'apport d'anthracène. Toutefois la diversité fonctionnelle des communautés autochtones de litière de pin d'Alep issues de ces environnements est modifiée à une échelle micro-locale par l'effet marin. Par ailleurs les réponses fonctionnelles face à l'apport d'anthracène des communautés microbiennes de litières de pin d'Alep en zone continentale sont différentes de celles des zones littorales / Mediterranean coastal ecosystems are subjected to various natural and anthropogenic environmental pressures which are supposed to be enhanced because of climatic changes. Little is known about microbial community functioning in such ecosystems. Our site of study is located in the Calanques of Marseille, a hot spot of biodiversity. The effect of salinity (via sea spray exposure) on microbial communities and their ability to transform organic matter in an Aleppo pine litter have been studied as well as the potential of autochthonous microorganisms to transform anthracene used as a polycyclic aromatic hydrocarbon model. To do so, different approaches (in situ, ex situ and in vitro experimental design) were used and we combined various methods such as enzyme activities (laccase, cellulase, phosphatase, lipase), CLPP (Biolog ECO and FF plates), respirometry (basal and induced) and litter chemical characterization (solid-state 13C NMR). Laccases were induced by anthracene in mesocosms and oxidized this compound (with anthraquinone as an intermediate). These enzymes were sequenced by LC/MS/MS to determine the fungal strains responsible for their production. We also found that enzyme activities were not strongly influenced by salinity or anthracene inputs. On the other hand, functional diversity was structured at a small-spatial scale. Moreover, functional responses of microbial communities from inland areas strongly differ from those of coastal areas regarding anthracene inputs since no laccase induction was observed in inland litter.
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Le système phénoloxydase : caractérisation biochimique et rôle dans la réponse immunitaire chez la palourde japonaise Venerupis philippinarum exposée à Vibrio tapetis / No titleLe Bris, Cédric 17 December 2013 (has links)
La palourde japonaise, Venerupis philippinarum, a été introduite en France au début des années 70 à des fins aquacoles. Depuis 1987, d’importants épisodes de mortalité, causés par la bactérie pathogène Vibrio tapetis, touchent cette espèce le long des côtes françaises et européennes. Cette vibriose, appelée Maladie de l’Anneau Brun (MAB), est considérée comme une maladie d’eau froide. L’interaction tripartite entre V. philippinarum, V. tapetis et l’environnement a été explorée à travers le rôle du système enzymatique des phénoloxydases (POs) dans le but de mieux comprendre la réponse immunitaire de la palourde japonaise, la virulence de l’agent pathogène mais aussi l’impact de l’environnement et plus particulièrement de la température. Les POs sont des oxydoréductases impliquées dans la synthèse de la mélanine et de ses dérivés mais aussi dans les processus de reconnaissance du non-soi et d’encapsulation chez les invertébrés. Dans un premier temps, l’activité PO du sérum d’hémolymphe a été caractérisée d’un point de vue biochimique comme étant majoritairement de type laccase ; une activité minoritaire de type tyrosinase a également été identifiée. Des infections de palourdes par trois souches de V. tapetis, à différentes températures, ont mis en évidence une modulation de la réponse du système PO en fonction du temps et du compartiment étudiés. De façon générale, l’infection bactérienne s’est traduite par une augmentation de l’activité PO. Toutefois, le niveau basal d’activité PO est variable d’une population à une autre et cette variabilité semble traduire une susceptibilité différente à la MAB. L’augmentation de la température de 15 à 22°C a entraîné une augmentation des capacités immunitaires de la palourde japonaise. La températurea également eu un impact sur la pathogénicité de V. tapetis et ce, de façon différentielle selon les souches. L’inhibition de l’activité PO observée in vitro en présence de produits extracellulaires bactériens souligne la complexité de l’interaction entre V. philippinarum et V. tapetis. Ainsi, le suivi de l’activité PO constitue un biomarqueur pertinent des capacités immunitaires des invertébrés marins dans l’interaction tripartite hôte-pathogène-environnement. / The manila clam, Venerupis philippinarum, was introduced in France in the early 70’s for aquaculture purposes. Since 1987, mass mortality events, caused by the pathogenic bacterium Vibrio tapetis, have occurred in this species along French and European coasts. This vibriose, called Brown Ring Disease (BRD), is considered as a cool water disease. The tripartite interaction between V. philippinarum, V. tapetis and the environment was explored through the role of the enzymatic system of phenoloxidases (POs) in order to better understand the manila clam immune response, the pathogen virulence and also the environment impact and particularly the temperature’s one. POs are oxidoreductase enzymes involved in the synthesis of the melanin and its derivatives and also in the processes of non-self recognition and encapsulation in invertebrates. Firstly, PO activity was biochemically characterized in the hemolymph serum as a laccase-like activity; a minority tyrosinaselike activity was also identified. Clam inoculation with three V. tapetis strains, at various temperatures, highlighted a modulation of the answer of the PO system according to time post-infection and studied compartments. Overall, bacterial infection resulted in an increase in PO activity. However, the PO activity basal level varies between populations and this variability seems to reflect different susceptibility to the BRD. An increase in temperature from 15 to 22°C led to an increase in manila clam immune abilities. The temperature also had an impact on the V. tapetis pathogenicity and this impact was variable according to the strains. PO activity inhibition observed, in vitro, in the presence of bacterial extracellular products underlines the complexity of the interaction between V. philippinarum and V. tapetis. Thus, the monitoring of the PO activity constitutes a relevant biomarker of marine invertebrate immune abilities in the tripartite interaction host-pathogen-environment.
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Elaboration de biocatalyseurs artificiels à deux composantesNpetgat Ngoutane, Eloïne Arlette 13 December 2012 (has links)
Depuis quelques années on assiste au développement de nouveaux bio-catalyseurs qui sont des hybrides combinant les avantages de la catalyse organométallique (système robustes, efficaces, mais couteux) à ceux de la catalyse enzymatique (écologique, spécifique mais peu flexible). Parmi les différentes stratégies mises en œuvre pour créer des enzymes artificielles, aucune d'elles n'a jusqu'à présent véritablement envisagé d'utiliser l'activité d'une enzyme et de la combiner à celle d'un composé organique. C'est la nouvelle approche proposée dans ce travail pour tenter d'orienter l'activité catalytique d'une oxydase à fort potentiel industriel, la laccase. Les produits d'oxydation de la laccase sont des radicaux phénoxyls qui se recombinent dans le milieu sans contrôle de l'enzyme. Chez certaines plantes, des petites protéines nommées « dirigent proteins » interagissent avec les radicaux phénoxyls pour conduire à la formation d'un seul produit optiquement pur. Dans ce travail, nous avons tenté de mimer ces « dirigent proteins » en greffant une molécule cage de type cyclodextrine à proximité du site actif de la laccase. Dans un premier temps, nous avons réalisé la synthèse de modules organiques dits « plateformes » possédants a) un point d'attachement à l'enzyme, b) un groupement pour la purification des protéines fonctionnalisées et c) une cyclodextrine qui permet d'encapsuler un grand nombre de molécules organiques. Par des mesures de fluorescences et d'immuno-détection, nous avons identifié les conditions optimales de fonctionnalisation pour la laccase et ainsi validé sa dérivatisation. / In recent years we are witnessing the development of new bio-catalysts which are hybrids combining the advantages of organometallic catalysis (robust and efficient system, but expensive) to those of enzymatic catalysis (ecological, specific but not very flexible). Among the various strategies used to create artificial enzymes, none of them has yet seriously considered to combine an enzyme activity with that of an organic compound. This is the new approach proposed in this work to try to orient the catalytic activity of the laccase, an oxidase with an industrial potential. The oxidation products of the laccase are phénoxy radicals which recombine in the medium regardless of the activity of the enzyme. In some plants, small proteins called "dirigent proteins" interact with phénoxy radicals leading to the formation of a single optically pure product. In this work, we tried to mimic the "dirigent proteins" by grafting a cyclodextrin-cage molecule near the active site of the laccase. As a first step, we performed the synthesis of organic modules called "platforms" with a) an anchor point to the enzyme, b) a group for functionalized protein purification c) a cyclodextrin that encapsulates a large number of organic molecules. By fluorescence and immunodetection measurements, we identified the optimal conditions for laccase functionalization and thus validated its derivatization. Monitoring the oxidation of coniferyl alcohol by the functionalized laccase with a cyclodextrined platform highlights a change in the kinetic profiles of the substrate and products. This difference appears due to the location of the cyclodextrin near the substrate oxidation site.
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Morfogênese do tegumento em Apis mellifera: construindo o exoesqueleto adulto / Morphogenesis of integument in Apis mellifera: building the adult exoskeletonElias Neto, Moysés 25 February 2008 (has links)
O exoesqueleto (ou cutícula) é um dos responsáveis pelo sucesso evolutivo dos insetos, não só pela proteção e suporte que lhes confere, mas também pela interface que representa entre o animal e o meio ambiente. A construção do exoesqueleto do inseto adulto envolve um processo de diferenciação denominado tanning, caracterizado pela melanização e pela esclerotização. Às enzimas lacases classicamente tem sido atribuído um papel fundamental no tanning cuticular, em particular na esclerotização. O presente trabalho consiste no estudo da função e regulação do gene Amlac 2 codificador da Lacase 2 de Apis mellifera, no âmbito da relação entre a expressão deste gene e a morfogênese do tegumento (cutícula e epiderme) do adulto. Através de RT-PCR semi-quantitativa, a abundância de transcritos foi contrastada entre diferentes estágios da ontogênese e entre distintas regiões do corpo (tórax, asas e abdome). A transcrição se acentua logo após a apólise pupal-imaginal, e se mantém alta durante todo o desenvolvimento do adulto farato. Embora haja um padrão de expressão comum entre as três regiões do corpo estudadas, nota-se um relativo atraso do início da transcrição desse gene no tegumento abdominal. Este resultado é consistente com o menor grau de esclerotização da cutícula do abdome em comparação com a do tórax e das asas. Uma análise comparativa entre abelhas operárias, zangões e rainhas revelou ainda padrões casta e sexo-específicos de diferenciação do tegumento adulto. Experimentos de silenciamento gênico pós-transcricional resultaram no comprometimento da melanização e da esclerotização cuticulares e não conclusão do ciclo de muda, efeitos que influenciaram drasticamente a viabilidade dos adultos. Análises histológicas do tegumento de abelhas submetidas ao silenciamento de Amlac 2 revelaram ainda má formação da cutícula, com alterações principalmente em sua espessura e arquitetura. Tais resultados evidenciam uma importante contribuição da enzima Lacase 2 na diferenciação do exoesqueleto adulto de Apis mellifera, fenômeno esse fundamental na ontogênese plena da abelha. Experimentos de ligadura abdominal em pupas iniciais, procedimento que impede o fluxo de hormônios ecdisteróides provenientes das glândulas protorácicas para o abdome, resultaram em inibição do aumento do título de ecdisteróides, repressão temporária da transcrição do gene Amlac 2 e bloqueio do processo de diferenciação cuticular. Tais efeitos sugerem fortemente que esses hormônios controlam a expressão do gene Amlac 2, por sua vez envolvido no processo de maturação da cutícula. Ainda, a detecção inesperada de quantidades crescentes de ecdisteróides no abdome de pupas, após o terceiro dia de ligadura, nos levou a propor um novo modelo endócrino para o desenvolvimento do adulto de Apis mellifera. / The evolutionary success of the insects is to a large extent due to the structural and mechanical properties of the integument, which is made up of an outer cuticle layer and the subjacent epidermis. As an effective interface between the insect soft body and the environment, the integument performs all the functions of a skin and of an exoskeleton. It not only supports the insect, but gives it its shape, means of locomotion, and provides protection against desiccation, besides being involved in defense strategies towards predators and pathogenic agents. Building and maturation of the adult exoskeleton include complex biochemical pathways where the enzymes Laccases (E.C. 1.10.3.2) may have a key role. Laccases have been characterized mainly in fungi and bacteria. In insects, the function of these enzymes has been linked to cuticle tanning (pigmentation and sclerotization) and stabilization of the protein-based exoskeleton. It was our aim to identify and investigate the function and regulation of the gene, Amlac 2, which encodes the enzyme Laccase 2 in the honeybee, Apis mellifera. Semi-quantitative RT-PCR analyses evidenced that Amlac 2 is highly expressed in the integument of pharate adults in correlation with cuticle pigmentation and sclerotization. Transcription increases in thoracic, abdominal and wing integuments immediately after pupal-imaginal apolysis, and remains abundant all through pharate adult development. Consistent with the different degree of sclerotization in cuticle areas recovering distinct body parts, the increase in the levels of Amlac2 transcripts occurs later in abdominal than in thoracic and wing integuments. A comparative approach using honeybee workers, queens and drones also revealed caste and sex-specific patterns of adult integument differentiation. Post-transcriptional Amlac2 gene silencing resulted in abnormalities in cuticle structure, melanization and sclerotization, as revealed by histological analyses, and drastically affected the adult molt. Such results clearly indicate a critical role of Laccase 2 in the differentiation of the adult exoskeleton in the honeybee. Experiments using a ligature to prevent the increase in ecdysteroid titer in abdomen resulted in inhibition of Amlac 2 transcription and severely impaired cuticular differentiation. These results strongly indicate that Amlac 2 expression is controlled by ecdysteroids, and has a crucial role in the differentiation and maturation of the adult cuticle. Moreover, a radioimmunoassay using hemolymph from ligated abdomens suggested the existence of an alternative source of ecdysteroids, in addition to prothoracic glands, thus leading us to propose a new endocrine model for differentiation of the adult honeybee.
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Dégradation enzymatique de micropolluants récalcitrants d'origine pharmaceutique / Enzymatic degradation of recalcitrant pharmaceutical micropollutantsParra Guardado, Ana Luisa 10 May 2019 (has links)
Ce travail concerne l'étude de la dégradation enzymatique de micropolluants pharmaceutiques récalcitrants présents dans l'eau. Tout d’abord, les efficacités de trois laccases différentes issues respectivement de : Pycnoporus sanguineus CS43, Trametes versicolor (Tv) et Myceliophtora thermophila ont été comparés lors d’essais de dépollution de solutions modèles renfermant trois antibiotiques (amoxicilline, ciprofloxacine et sulfaméthoxazole) et un antiépileptique (carbamazépine). Les essais ont été réalisés avec les laccases libres en présence ou non de médiateurs redox. L'impact de plusieurs paramètres opératoires sur les performances des enzymes a également été étudié. Puis, une nouvelle méthode d’immobilisation des laccases impliquant l’activation du support (microparticules à base de silice commerciales) par du glutaraldéhyde en phase vapeur a été mise au point et optimisée en utilisant la méthodologie de plans d’expériences. Après immobilisation, la laccase Tv s’est avérée être la plus active. Des essais de dégradation en présence de médiateurs redox ont confirmé l’efficacité de l’enzyme immobilisée et sa possible réutilisation lors de cycles successifs. La toxicité des solutions après traitement a été évaluée par des tests Microtox®. La laccase Tv a également été immobilisée sur des nanoparticules non commerciales à base de silice ou d’argile ainsi que sur des composites à base de silice et d’argile. La laccase Tv immobilisée sur les supports composites riches en silice a montré une plus grande réactivité et de meilleures performances pour l'élimination des composés cibles. / This work is focused on the study of the enzymatic depletion of recalcitrant pharmaceutical micropollutants in water. The potential degradation of three antibiotics (amoxicillin, ciprofloxacin and sulfamethoxazole) and one anti-epileptic (carbamazepine) was studied with three laccases: Pycnoporus sanguineus CS43, Trametes versicolor (Tv) and Myceliophtora thermophila. Free laccase systems were evaluated for pharmaceuticals depletion on model solutions in the presence or absence of redox mediators and the impact of several parameters on the performance of laccases for degradation were studied. The enzymes were then immobilized on different solid supports: commercial silica, laboratory synthetized nano-silica and clay based composite nanomaterials and used for degradation tests. A novel methodology for the covalent binding of laccases onto carriers was developed by using glutaraldehyde in vapour phase and the best immobilization conditions were determined through a 23 full factorial design. The immobilized Tv shown the highest activity and was tested in presence of redox mediators. Moreover, the reusability was evaluated in several degradation cycles and the toxicity of the solutions after treatment was assessed with the Microtox® test. In comparison to laccase immobilized on commercial silica, the Tv supported on laboratory synthetized materials showed higher activity and a better performance for the removal of target compounds.
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Identificação e caracterização molecular das lacases de eucalipto (Eucalyptus grandis) / The identification and characterization of laccases gene family in eucalyptus (Eucalyptus grandis)Arcuri, Mariana de Lara Campos [UNESP] 24 May 2017 (has links)
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Previous issue date: 2017-05-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As lacases, p-diphenol-O2-oxidoredutases, são enzimas que desempenham papel fundamental na oxidação de monolignóis durante a biossíntese de lignina, estando, portanto, associadas a processos de crescimento e tolerância a alguns tipos de estresses abióticos. As lacases podem ser encontradas em bactérias, fungos, plantas e insetos. Estudos apontam que as lacases vegetais apresentam comportamento similar às de origem fúngica, atuando de formar complementar à rota de lignificação, em resposta ao estresse oxidativo, promovendo a detoxificação celular. As lacases são geralmente codificadas por famílias multigênicas. Através de análises in silico no genoma de eucalipto, o presente estudo identificou 54 genes codificadores de lacases (denominados EgLAC) que, filogeneticamente, se distribuem em seis diferentes subgrupos. Com base em dados de RNA-Seq, padrões distintos de expressão das lacases identificadas foram observados, sendo algumas enriquecidas em um dado órgão/tecido e outras com expressão não detectável pelo método. Análises de RT–qPCR de alguns genes selecionados com base em um banco de sequências expressas confirmaram, por exemplo, a expressão raiz-específica do gene EgLAC52 bem como as expressões preferenciais em raiz e folha dos genes EgLAC4 e EgLAC32, respetivamente. Em paralelo, a expressão de alguns destes genes em reposta a estresses foi investigado, e alterações na expressão relativa em resposta aos estresses oxidativo e osmótico foram constatadas, sugerindo a participação destas lacases em respostas a estresses abióticos. / Laccases are p-diphenol-O2-oxidoreductases encoded by multigene families widely distributed throughout the plant kingdom. They exhibit important roles in the oxidation of monolignols during lignin biosynthesis and are reported to be functionally involved in plant development, tolerance and response to stress. Apart from plants, laccases can be also found in bacteria, fungi and insects. Here, a genome-wide survey of the eucalyptus genome revealed the presence of 88 putative laccases genes. However, after meticulous analyzes using different approaches, the redundant sequences were discarded and 54 laccases genes (referred as EgLAC) were retrieved. These genes were phylogenetically distributed in six different subgroups. Based on RNA-Seq data, distinct organ/tissue expression patterns of the identified EgLAC genes were ascertained. The vast majority showed organ/tissue-enriched expression, while certain genes exhibited no detectable expression. RT-qPCR analyzes confirmed the organ/tissue expression patterns of a representative set of genes such as, for example, the rootspecific expression of EgLAC52 and the root and leaf preferential expressions of genes EgLAC4 and EgLAC32. Further expression profiling of selected EgLAC genes in response to oxidative and osmotic stresses revealed differences in their relative expression, with some genes being stress-induced. These results suggest that certain laccases might be implicated in abiotic stress responses.
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Morfogênese do tegumento em Apis mellifera: construindo o exoesqueleto adulto / Morphogenesis of integument in Apis mellifera: building the adult exoskeletonMoysés Elias Neto 25 February 2008 (has links)
O exoesqueleto (ou cutícula) é um dos responsáveis pelo sucesso evolutivo dos insetos, não só pela proteção e suporte que lhes confere, mas também pela interface que representa entre o animal e o meio ambiente. A construção do exoesqueleto do inseto adulto envolve um processo de diferenciação denominado tanning, caracterizado pela melanização e pela esclerotização. Às enzimas lacases classicamente tem sido atribuído um papel fundamental no tanning cuticular, em particular na esclerotização. O presente trabalho consiste no estudo da função e regulação do gene Amlac 2 codificador da Lacase 2 de Apis mellifera, no âmbito da relação entre a expressão deste gene e a morfogênese do tegumento (cutícula e epiderme) do adulto. Através de RT-PCR semi-quantitativa, a abundância de transcritos foi contrastada entre diferentes estágios da ontogênese e entre distintas regiões do corpo (tórax, asas e abdome). A transcrição se acentua logo após a apólise pupal-imaginal, e se mantém alta durante todo o desenvolvimento do adulto farato. Embora haja um padrão de expressão comum entre as três regiões do corpo estudadas, nota-se um relativo atraso do início da transcrição desse gene no tegumento abdominal. Este resultado é consistente com o menor grau de esclerotização da cutícula do abdome em comparação com a do tórax e das asas. Uma análise comparativa entre abelhas operárias, zangões e rainhas revelou ainda padrões casta e sexo-específicos de diferenciação do tegumento adulto. Experimentos de silenciamento gênico pós-transcricional resultaram no comprometimento da melanização e da esclerotização cuticulares e não conclusão do ciclo de muda, efeitos que influenciaram drasticamente a viabilidade dos adultos. Análises histológicas do tegumento de abelhas submetidas ao silenciamento de Amlac 2 revelaram ainda má formação da cutícula, com alterações principalmente em sua espessura e arquitetura. Tais resultados evidenciam uma importante contribuição da enzima Lacase 2 na diferenciação do exoesqueleto adulto de Apis mellifera, fenômeno esse fundamental na ontogênese plena da abelha. Experimentos de ligadura abdominal em pupas iniciais, procedimento que impede o fluxo de hormônios ecdisteróides provenientes das glândulas protorácicas para o abdome, resultaram em inibição do aumento do título de ecdisteróides, repressão temporária da transcrição do gene Amlac 2 e bloqueio do processo de diferenciação cuticular. Tais efeitos sugerem fortemente que esses hormônios controlam a expressão do gene Amlac 2, por sua vez envolvido no processo de maturação da cutícula. Ainda, a detecção inesperada de quantidades crescentes de ecdisteróides no abdome de pupas, após o terceiro dia de ligadura, nos levou a propor um novo modelo endócrino para o desenvolvimento do adulto de Apis mellifera. / The evolutionary success of the insects is to a large extent due to the structural and mechanical properties of the integument, which is made up of an outer cuticle layer and the subjacent epidermis. As an effective interface between the insect soft body and the environment, the integument performs all the functions of a skin and of an exoskeleton. It not only supports the insect, but gives it its shape, means of locomotion, and provides protection against desiccation, besides being involved in defense strategies towards predators and pathogenic agents. Building and maturation of the adult exoskeleton include complex biochemical pathways where the enzymes Laccases (E.C. 1.10.3.2) may have a key role. Laccases have been characterized mainly in fungi and bacteria. In insects, the function of these enzymes has been linked to cuticle tanning (pigmentation and sclerotization) and stabilization of the protein-based exoskeleton. It was our aim to identify and investigate the function and regulation of the gene, Amlac 2, which encodes the enzyme Laccase 2 in the honeybee, Apis mellifera. Semi-quantitative RT-PCR analyses evidenced that Amlac 2 is highly expressed in the integument of pharate adults in correlation with cuticle pigmentation and sclerotization. Transcription increases in thoracic, abdominal and wing integuments immediately after pupal-imaginal apolysis, and remains abundant all through pharate adult development. Consistent with the different degree of sclerotization in cuticle areas recovering distinct body parts, the increase in the levels of Amlac2 transcripts occurs later in abdominal than in thoracic and wing integuments. A comparative approach using honeybee workers, queens and drones also revealed caste and sex-specific patterns of adult integument differentiation. Post-transcriptional Amlac2 gene silencing resulted in abnormalities in cuticle structure, melanization and sclerotization, as revealed by histological analyses, and drastically affected the adult molt. Such results clearly indicate a critical role of Laccase 2 in the differentiation of the adult exoskeleton in the honeybee. Experiments using a ligature to prevent the increase in ecdysteroid titer in abdomen resulted in inhibition of Amlac 2 transcription and severely impaired cuticular differentiation. These results strongly indicate that Amlac 2 expression is controlled by ecdysteroids, and has a crucial role in the differentiation and maturation of the adult cuticle. Moreover, a radioimmunoassay using hemolymph from ligated abdomens suggested the existence of an alternative source of ecdysteroids, in addition to prothoracic glands, thus leading us to propose a new endocrine model for differentiation of the adult honeybee.
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Enzimas lignocelulolíticas de basidiomicetos cultivados em biomassas vegetais oriundas da agroindústria do dendê e obtenção de açúcares fermentescíveisPeláez, Rubén Darío Romero 31 May 2017 (has links)
Três resíduos vegetais da agroindústria do dendê foram usados como substrato para
o cultivo de distintas linhagens de fungos da podridão-branca (basidiomicetos). Foram
avaliados o crescimento e produção de enzimas lignocelulolíticas por fermentação em
estado sólido e submerso. Os extratos enzimáticos obtidos das fermentações foram
utilizados no processo de hidrólise enzimática de bagaço de cana-de-açúcar e cacho
vazio do dendê previamente pré-tratados. Um total de 54 linhagens de macrofungos
foram cultivadas em três formulações de biomassa de dendê em placas Petri, onde
foram escolhidas 5 linhagens com crescimento mais rápido e denso. Estas cinco
linhagens de macrofungos foram cultivadas em fermentação em estado sólido e
avaliados pelas atividades enzimáticas dos extratos obtidos. Nestes extratos, houve
predominância das atividades de lacase, peroxidase, manganês peroxidase e
protease. Das cinco linhagens foram selecionadas três linhagens com predominância
nas atividades oxidativas nos cultivos em fermentação em estado sólido, para serem
avaliadas em fermentação submersa (monocultivos) usando meio sintético
suplementado com biomassa vegetal do dendê. As atividades enzimáticas das três
espécies de basidiomicetos foram comparadas com cinco linhagens fúngicas usadas
frequentemente na literatura. Houve diferenças significativas em função das
atividades oxidativas (lacase e peroxidases) e hidrolíticas (FPase e β-glicosidases)
entre as linhagens testadas. Estas diferenças foram usadas para estabelecer
subgrupos, os quais foram avaliados através da interação em placa e cocultivos em
fermentação submersa. As atividades enzimáticas dos extratos obtidos de
monocultivos e cocultivos apresentaram diferenças com interações
predominantemente positivas entre os três basidiomicetos e T. reesei ATCC® 60787.
Os monocultivos e cocultivos foram comparados em função da liberação de glicose
após da hidrólise enzimática de bagaço de cana-de-açúcar pré-tratado por autohidrólise.
As hidrólises enzimáticas aplicando os extratos dos cocultivos obtiveram
rendimento 44,7% superior quando comparadas com os monocultivos. Foi feita uma
análise de misturas simplex lattice, usando como componentes os extratos dos
monocultivos para a obtenção de um coquetel enzimático a fim de otimizar a liberação
de glicose do bagaço de cana pré-tratado. Os resultados demostraram que os extratos
com maiores atividades hidrolíticas estão correlacionadas com a maior liberação de
glicose, e que os extratos com enzimas oxidativas podem melhorar o rendimento, tendo assim uma mistura ou coquetel caracterizado com enzimas hidrolíticas e
oxidativas com potencial para obtenção de açúcares. Finalmente, este coquetel foi
utilizado na hidrólise de cacho vazio de dendê pré-tratado fisicamente, biologicamente
e biológica-físicamente (combinado) obtendo um rendimento máximo de 11,8 g.L-1 de
glicose a partir de biomassa vegetal de dendê quando pré-tratada biológicafisicamente,
o que correspondeu entre 40 - 60% do rendimento do rendimento obtido
quando empregadas as enzimas comerciais Cellic® Ctec3 e Cellic® Ctec2. / Three vegetable residues from the palm oil industry were used as substrate for
cultivation of different white rot fungi strains (basidiomycetes). The growth and
production of lignocellulolytic enzymes were evaluated by solid and submerged
fermentation. The enzymatic extracts obtained from fermentations were used in
enzymatic hydrolysis process of pretreated sugarcane bagasse and oil palm empty
bunch. Fifty-four macrofungal strains were cultivated in three formulations of oil palm
biomass on Petri dishes, where 5 strains with faster and dense growth were chosen.
These five macrofungal strains were cultivated in solid state fermentation and
evaluated by the enzymatic activities of the extracts obtained. In these extracts,
activities of laccase, peroxidase, manganese peroxidase and protease were found to
be predominant. Three of the five strains were selected with predominance in oxidative
activities on solid state fermentation cultures to be evaluated in submerged
fermentation (monocultures) using synthetic medium supplemented with oil palm
biomass. The enzymatic activities of the three basidiomycetes srtains were compared
with five fungal strains frequently used in the literature. There were significant
differences in function of the oxidative activities (laccase and peroxidases) and
hydrolytic activities (PFase and β-glucosidases) among the tested strains. These
differences were used to establish subgroups which were evaluated through the
interaction in plates and cocultures in submerged fermentation. The enzymatic
activities of the extracts obtained from monocultures and cocultures presented
differences, with predominantly positive interactions between the three basidiomycetes
and T. reesei ATCC® 60787. Monocultures and cocultures were compared as a
function of the glucose release after the enzymatic hydrolysis of sugarcane bagasse
pretreated by autohydrolysis. The enzymatic hydrolysis of the coculture extracts
obtained higher percentages (maximum value 44.7%) when compared to
monocultures. An analysis of simplex lattice mixtures was made using the monoculture
extracts as components to obtain an enzymatic cocktail in order to optimize the glucose
release of the pretreated sugarcane bagasse. The results showed that extracts with
higher hydrolytic activities are correlated with the higher glucose release and extracts
with oxidative enzymes can improve the sugar yield, thus having a mixture or cocktail
characterized by hydrolytic and oxidative enzymes with the potential to obtain sugars. Finally, this cocktail was used in the hydrolysis of untreated, physically, biologically and
biologically-physically (combined) pretreated oil palm empty bunch, obtaining a
maximum yield of 11.8 gL-1 of glucose in the biomass of palm oil when pretreated
biological-physically, which corresponded between 40-60% of the yield of the
commercial enzymes Cellic® Ctec3 and Cellic® Ctec2.
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