Dissertations / Theses on the topic 'Laccase (Trametes versicolor)'

Dans ce travail de recherche, des monolithes en silice présentant une très grande porosité (83 %), une double distribution de taille de pores (des macropores de diamètre 20 μm et des méseopores de diamètre 20 nm) et une très grande surface spécifique (370 m2 g-1) ont été utilisés comme supports pour immobiliser une laccase de Trametes versicolor par greffage covalent avec du glutaraldéhyde. Les monolithes enzymatiques ont été utilisés pour dégrader la tétracycline (TC) en solution aqueuse dans un réacteur de configuration tubulaire type "Flow Through Reactor" avec recyclage. Au cours des 5 premières heures de réaction à pH 7 ; 40 à 50 % de la TC a été dégradée, puis un seuil a été atteint. Une des hypothèses pouvant expliquer ce comportement est un éventuel manque de co-substrat (oxygène) à proximité des sites catalytiques. Des monolithes enzymatiques ont été utilisés pendant 75 h de fonctionnement séquentiel sans perte d'activité. L'efficacité de la dégradation de la TC a pu être simulée à travers un modèle mathématique construit en couplant la cinétique de la réaction (Michaelis-Menten) avec un bilan matière en régime dynamique. Les résultats de la simulation ont révélé que le procédé global est contrôlé par la cinétique enzymatique mais que la taille des monolithes pouvait être adaptée pour dégrader 100 % de la CT en un seul passage à travers un monolithe<br>In this research work, silica monoliths with high porosity (83 %), double pore size distribution (20 μm and 20 nm macro- and mesopores diameters, respectively) and high surface area (370 m2 g-1) have been used as solid supports to immobilize a laccase from Trametes versicolor by covalent grafting with glutaraldehyde. Enzymatic monoliths were applied to degrade tetracycline (TC) in aqueous solutions in a tubular “Flow Through Reactor” configuration with recycling. During the first 5h of reaction at pH 7, 40–50% of TC was degraded, and then a threshold was reached. One of the hypotheses explaining this behaviour is a possible co-substrate lack (oxygen) near catalytic sites. Enzymatic monoliths were used during 75 h of sequential operation without losing activity. A mathematical model built coupling the Michaelis-Menten reaction kinetics with a dynamic mass balance allowed computing TC degradation efficiency. Simulation results revealed that the global process is controlled by the enzymatic kinetics but the monolith size could be adapted to degrade 100 % TC in a single pass

Ce travail a pour objectif de mettre au point un procédé d’oligomérisation de rutine et d’esculine par la laccase de Trametes versicolor. Un procédé de synthèse en parallèle et d’analyse en ligne par SEC-UV et par MALDI-TOF a été mis au point. L’analyse par MALDI-TOF a révélé la formation d’un simple pontage, allant jusqu’au degré d’oligomérisation 6 pour la rutine et 9 pour l’esculine. Un pontage par liaison éther a été observé par FTIR dans le cas des oligorutines. L’analyse par RMN a démontré la mise en place de liaisons tant C-C que C-O localisées sur la partie phénolique et la partie sucre des monomères. De faibles pH et températures favorisent l’allongement de la chaîne, alors que l’augmentation de la constante diélectrique du solvant ou de la température augmente la production des oligomères de rutine. La limitation de la masse de ces oligomères serait due à une inhibition de l’enzyme, provoquée par les capacités chélatantes des oligomères. Une diminution du pouvoir antioxydant et une augmentation du pouvoir inhibiteur de la xanthine oxydase ont pu être observées lors de l’accroissement de la masse des oligomères de rutine. Ces deux activités sont améliorées lors de l’accroissement de la masse des oligomères d’esculine. Pour ces deux types d’oligomères, la solubilité dans l’eau est fortement accrue. Dans le cas des oligorutines, cette forte augmentation a été corrélée à la mise en place d’un réseau dense de liaisons hydrogène observé par modélisation moléculaire. Globalement, l’approche par modélisation moléculaire dans le vide et dans le solvant a permis de dégager des relations structure-activité, reliant notamment le nombre de liaisons hydrogène à la solubilité<br>The aim of this work is the elaboration of rutin and esculin oligomerization process by the laccase from Trametes versicolor. A parallel synthesis process and on-line analysis of reaction media by SEC-UV and MALDI-TOF have been elaborated. The MALDI-TOF analysis has revealed the formation of simple bridges between rutin and esculin units, up to degree of oligomerization of 6 and 9 respectively. An ether bond has been observed by FTIR spectrometry for the rutin oligomers. Finally, the NMR analysis has revealed the formation of C-C and C-O bridges both on phenolic and the sugar parts of the flavonoids. At low pH and temperature, the elongation of the chain is favored, whereas increasing the dielectric constant of the solvent or the temperature favors the production of rutin oligomers. The limitation of oligomers mass is explained by the inhibition of the enzyme, probably due to the highest chelation properties of oligomers. In the case of oligorutin, a decrease of antiradical activity and an increase of xanthine oxidase inhibitory activity have been observed when the oligomers molecular mass increases. In the case of esculin oligomers, these two activities increase with the increase of the oligomers mass. For these two types of oligomers, the water solubility is considerably increased. For the oligorutins, this augmentation has been correlated to a dense network of H-bonds, which has been demonstrated by molecular modeling. Globally, the molecular modeling approach in vacuum and in solvent has allowed to establish structure-activity relationship

Les flavonols et les flavan-3ols représentent deux sous classes importantes tant pour les végétaux que pour les humains. Ces propriétés sont toutefois affectées par les phénomènes d’oxydation enzymatique. Les laccases constituent un des principaux groupes d’enzymes impliqués dans ces réactions. Peu d’études se sont intéressées aux mécanismes précis de ces oxydations ainsi qu’aux structures des produits obtenus. Le but de cette étude est de déterminer les structures des produits d'oxydation des flavonols et des flavan-3ols par la lacasse de Trametes versicolor et de comparer leurs mécanismes de formation. Après une première partie bibliographique qui montre clairement les fortes relations entre la structure et l’activité antioxydante des flanoïdes, nous exposons dans la deuxième partie les différents produits d’oxydation obtenus dans un premier temps avec la laccase d’Arabidopsis thaliana et avec la laccase de Trametes versicolor. L’étude de la structure des produits d’oxydation en fonction de la structure du substrat a permis de déterminer le mécanisme de ces réactions d’oxydation et de mettre en avant les spécificités de structure qui déterminent l’issue de la réaction<br>Flavonols and flavan-3-ols are two important sub-classes of flavonoïds, they are characterized by their biological properties which are important for both humans and plants. However, these properties are affected by the oxidation phenomenon. Laccases are among the enzymes involved in these enzymatic reactions. A few studies have been dedicated to the oxidation mechanisms and to the structures of the reaction products. The present study aims to identify and elucidate the structure of the oxidation products of flavanols and flavan-3-ols by the laccase of Trametes versicolor and compare their formation mechanisms. The first part is a review which clearly shows the strong structure-antioxidant activity relationship of flavonoïds. In the second part, we present the oxidation products generated by Arabidopsis thaliana laccase and Trametes versicolor laccase. The study of the structure of the oxidation products according to the substrate structure allowed the identification of the oxidation mechanisms. It also allowed to put forward that the structure specificities of the substrate affect the reaction’s outcome

Tese de doutoramento em Engenharia Quimica e Biológica<br>O principal objetivo deste trabalho consistiu em selecionar e avaliar diferentes estirpes de fungos da podridão branca da madeira (Fpb) quanto à capacidade de descoloração, em condições similares às apresentadas na indústria têxtil, utilizando o corante azo Reativo Preto 5 (RP5). O corante Poly R-478 (PR478) foi utilizado como parâmetro comparativo por ser considerado um indicador da atividade de enzimas existentes no sistema ligninolítico dos Fpb. O trabalho inicial foi realizado com doze estirpes de Fbp em meio de cultura sólido e permitiu a seleção das estirpes aptas em descolorir estes corantes em condições restritivas. A extensão da descoloração dos dois corantes e a atividade das enzimas ligninolíticas envolvidas neste processo foram avaliadas, utilizando-se as estirpes Trametes versicolor MUM 94.04, 04.100 e 04.101 para além da estirpe modelo de estudo Phanerochaete chrysoporium MUM 94.15 (ATCC 24725), em meio de cultura líquido (MCL) alcalino sob agitação. As estirpes T. versicolor MUM 94.04 e MUM 04.100 apresentaram uma taxa de descoloração de 100 % em MCL na presença do corante RP5. Definido o valor de pH, foram realizados novos ensaios com diferentes concentrações de NaCl adicionado à composição do MCL com o corante RP5. Os melhores resultados de descoloração e atividade enzimática foram obtidos em MCL contendo 15 g·l-1 de NaCl na sua composição, onde a estirpe T. versicolor MUM 04.100 foi a única que apresentou uma taxa de descoloração de 100 %. Esta estirpe apresentou atividade de lacase (Lcc) e lignina-peroxidase (LiP) em valores superiores aos obtidos nas melhores condições de alcalinidade. Em MCL agitado e com os parâmetros pH e concentração de NaCl otimizados, comparou-se a capacidade da estirpe T. versicolor imobilizada em dois suportes sintéticos: espuma de poliuretano (EPO) e esponja de “nylon” (EN), com os resultados obtidos com células livres. Esta estirpe imobilizada em EPO e EN manteve uma taxa de descoloração do corante RP5 igual ou superior a 85 % por 18 (6 ciclos) e 21 (7 ciclos) dias. A ampliação da escala neste estudo foi feita em dois tipos de reatores: de leito fixo e de bancada. O conjunto de experimentos realizados no reator de leito fixo com células livres e imobilizadas proporcionou uma taxa de descoloração de 100 %. A adição de glicerol ao meio de cultivo promoveu uma maior atividade da enzima lacase. Em contrapartida, o controle rígido do pH nos ensaios com reator de bancada denotou a inibição do microrganismo, necessitando-se assim uma maior investigação dos parâmetros de operação. Além disso, verificou-se que indução da expressão genética da lacase foi superior quando o MCL possuía glicerol como co-substrato. Em conclusão, os excelentes resultados obtidos com a estirpe T. versicolor MUM 04.100, proporcionará boas perspectivas futuras quanto a estudos de avaliação dos mecanismos metabólicos e de compostos intermediários que são formados a partir deste corante. Do mesmo modo, o aprofundamento do estudo do processo degradativo do corante com esta estirpe poderá ser avaliado com fontes alternativas de carbono, que poderão potencializar a síntese e consequente aumento da atividade enzimática Lcc.<br>The main goal of this work was to select and evaluate different strains of white rot fungi (Wrf) to the capability to decolourise azo dyes in similar conditions to those presented in the textile industry, using the Reactive Black 5 (RB5). The dye Poly R-478 (PR478) was used as a comparative parameter for being considered as an indicator of the activity of the ligninolytic system of the Wrf. The initial assays were performed with twelve strains of Wrf in solid medium which allowed the selection of strains able to decolourise these dyes in restringent conditions. The extension of the decolourisation for both dyes and the activity of the ligninolytic enzymes involved in this process were evaluated, using the strains Trametes versicolor MUM 94.04, 04.100 and 04.101, and the Phanerochaete chrysoporium MUM 94.15 (ATCC 24725) as reference strain, in alkaline liquid culture medium (LCM) under stirring conditions. The strains T. versicolor MUM 94.04 and MUM 04.100 showed a decolourising rate of 100 % in LCM with the presence of RB5. A optimal value of pH was defined and new assays were made with different concentrations of NaCl added to the composition of the MCL with the dye RB5. The best decolourising results and the enzymatic activity were obtained in MCL containing 15g·l-1 of NaCl in its composition, where the strain T. versicolor MUM 04.100 presented a decolourising rate of 100 %. This strain presented higher values of laccase (Lcc) and lignin-peroxidase (LiP) activity when compared to those obtained in the best alkaline conditions. In stirred MCL with RB5 and under optimised parameters of pH and NaCl, the capacity of the immobilised strain in two synthetic carriers, polyurethane foam (EPO) and nylon sponge (EN), to decolourise the dye was studied and compared with the results obtained for the free cell cultures. This strain, immobilized in EPO and EN maintained a decolourising rate, for the dye RB5, equal or higher than 85 % for 18 (6 cycles) and 21 (7 cycles) days. The scale-up of this assay was performed using two types of reactors: fixed bed and stirred tank. The set of assays performed in the fixed bed reactor with free and immobilized cells provided a decolourising rate of 100 %. The addition of glycerol to the medium promoted a higher activity of laccase. However, the pH control on the assays with the stirred tank denoted the inhibition of the microorganism, thus requiring further research of the operation parameters. Moreover, it was found that the induction of the genic expression of the laccase was higher when using MCL with glycerol as a co-substrate. In conclusion, the excellent results obtained with strain T. versicolor MUM 04.100 will provide future prospects for studies of metabolic pathways and intermediates meatabolites that are formed from this dye under biodegradation conditions. Additionally, further study of degradation process of the dye with this strain can be assessed with alternative sources of carbon, which may stimulate the synthesis and consequent increase the Lcc activity.

In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...

>Magister Scientiae - MSc<br>Current water treatment technologies do not remove many endocrine disruptor compounds (EDCs) such as 17α-ethynylestradiol (EE2) in its entirety, and the amount of these pollutants that continues to enter the aquatic environment through wastewater effluents is still capable of causing harmful health effects. Therefore the development of simpler and more sensitive biosensor system for detection of EE2 must be developed which have high responsiveness, low cost and easy handling. Therefore the aim of this study was to work towards the development of rapid test system of polyaniline-laccase on gold enzyme nanobiosensor (PANI-PSSA/Lac/Glu) for water estrogens. Preliminary studies were first done on the materials used in this study: estrogens, laccase, gold nanoparticles (AuNPs), and electropolymerized PANI-PSSA. Laccase was shown to be active towards EE2 and the enzyme could be stored for over three months. EE2 solution also could be used for over three months. Buffer used in this study was found to be suitable. Phosphoric acid (H3PO4) was a suitable electrolyte than hydrochloric acid (HCl) to be used for the electropolymerization of aniline and was used because it has same ions as the McIlvaine buffer (McIlB) which the post-deposition CVs indicated the formation of electrochemically very stable film. AuNPs were successfully synthesized and its size was identified to be less than 22 nm. McIlB used for testing electrochemical properties of AuNP. CVs of GC/PANI-PSSA and GC/PANIPSSA/ Au showed no difference before and after exposure to aq. EE2 solution, an indication of being re-usable and could also serve as stable immobilising platform in laccase biosensor. When interrogating with electrochemical impedance spectroscopy (EIS), the charge transfer resistance (Rct) of both GC/PANI-PSSA and GC/PANI-PSSA/Lac/Glu showed an average increase by about 2.4% and 21% before and after exposure of EE2, respectively. This shows that the GC/PANI-PSSA/Lac/Glu was a functional EE2 biosensor and showing a positive step towards achieving a re-usable biosensor for EE2 as a model water estrogen. Future work Page | vi will focus on exploring different ways of improving the biosensor’s surface regeneration and its sensitivity to EE2.