Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T18:41:33Z (GMT). No. of bitstreams: 1 Dias_ElisangelaFrancisconGuimaro_D.pdf: 1126460 bytes, checksum: 218340ed688851cd46f637d8f8b3cd16 (MD5) Previous issue date: 2010 / Resumo: Azocorantes são compostos aromáticos com um ou mais grupos azo (-N=N-). São os maiores e a mais importante classe de corantes sintéticos usados em aplicações industriais. Eles são considerados compostos xenobióticos recalcitrantes aos processos de biodegradação, e a presença destes azocorantes nos ecossistemas aquáticos é a causa de sérios problemas ambientais e relacionados com a saúde. Neste trabalho, a habilidade em degradar azocorantes de 62 linhagens bacterianas previamente isoladas de efluente industrial foi investigada. A seleção das linhagens foi realizada através de testes de descoloração visual em meio líquido com azocorantes suplementados com diferentes fontes de carbono. O processo de descoloração foi realizado sob condições microaerofílicas ou estáticas até que nenhuma cor fosse observada, seguido de agitação para promover a biodegradação dos metabólitos produzidos. A descoloração e a biodegradação dos azocorantes bem como dos metabólitos produzidos foram monitoradas por análises de UV-vis, Carbono Orgânico Total (COT), Espectroscopia no Infravermelho com Transformadas de Fourier (FTIV) e Cromatografía Líquida de Alta Eficiência e Espectrometria de Massa (CLAE-EM). A atividade de enzimas oxidativas (peroxidase, lacase and tirosinase) foram analisadas para verificar se estas estavam envolvidas no metabolismo de biodegradação dos azocorantes. Análises de toxicidade foram realizadas antes e após a degradação dos azocorantes utilizando o organismo teste Daphnia magna.As aminas aromáticas geradas da biodegradação dos azocorantes foram testadas com o propósito de obter a polimerização. A enzima lacase de Myceliophthora Thermophila foi usada para catalizar reações de acoplamento entre as aminas aromáticas produzidas. As análises de UV-vis mostraram que os corantes foram descoloridos (80%) em condições microaerofílicas ou estáticas. Não houve nenhuma mudança significativa na cor, no estágio aeróbio seguinte. O tempo de descoloração mostrou relação com o meio de cultura utilizado e com a estrutura química dos corantes. Os corantes monoazo foram descoloridos entre 8 a 120 hs. Os diazo e os triazo foram descoloridos após 120 e 168 hs. As linhagens bacterianas descoloriram os corantes somente quando o meio foi suplementado com glicose e piruvato ou extrato de levedura. Na ausência destes compostos, as culturas foram incapazes de descolorir, indicando um requerimento obrigatório de uma fonte suplementar de carbono para alcançar a descoloração. Resultados mostraram que, quando o meio foi incubado em condições microaerofílicas ou estáticas a redução no COT (Carbono Orgânico Total) foi menor do que em condições aeróbias, onde 70% de redução foi observado. A presença de altas concentrações de aminas aromáticas em condições microaerofílicas ou estáticas confirma que houve redução das ligações azo. Porém, houve a confirmação da oxidação destas aminas no estágio aeróbio, indicando que um proceso oxidativo foi responsável pela biodegradação dos metabólitos. Foi observada atividade de tirosinase para na linhagem de Brevibacterium sp, sugerindo o papel desta enzima no processo de descoloração dos azocorantes, não sendo observada atividade de lacase e peroxidase. Nas análises de Espectroscopia no Infravermelho com Transformadas de Fourier (FTIV) após condições microaerofílicas ou estáticas foram observadas bandas em regiões atribuidas a grupamentos amina. Estas bandas desapareceram no estágio aeróbio e foram observadas novas bandas nas regiões associadas com ácido carboxílicos e íons NH3+, confirmando mineralização parcial dos produtos de degradação dos azocorantes, bem como dos metabólitos do meio de cultura. Os metabólitos produzidos pela biodegradação do azocorante RR 198 foram analisados por CLAEEM, para tentar identificar alguns metabólitos desconhecidos. Entre os possíveis compostos produzidos da biodegradação do RR198, 4-cloro-N-o-toluil-1,3,5-triazina-2-amino; sódio 4-aminonaftaleno-2-sulfonado e 3,6 -dimetil-7-(o-toluildiazenil) naftaleno-1-amino, tiveram razoável semelhança com os metabólitos aromáticos encontrados na amostra. Após condições aeróbias, a intensidade dos íons presentes nestes metabólitos foram reduzidos. Estes resultados também foram confirmados pelas análises de FTIV e poderia ser explicado pela diminuição dos compostos aromáticos gerados nas condições microaerofílicas ou estáticas. Foi observado que após um longo período de tempo, a lacase catalizou a polimerização das aminas aromáticas presentes nas soluções descoloridas. Os produtos gerados precipitaram e adquiriram cor, como confirmado pelas análises de UV-Vis. Os tamanhos das partículas foram significativamente maiores após o tratamento com lacase, como mostra as análises de Espectroscopia de Correlação de Fótons. Todas as linhagens bacterianas usadas neste estudo foram capazes de descolorir e degradar os azocorantes em condições microaerofílicas ou estáticas. Em condições aeróbias, ocorreu parcial mineralização dos produtos de degradação dos azocorantes, bem como dos metabólitos do meio, como confirmado para o organismo teste Daphnia magna e Carbono Orgânico Total (COT). Após o estudo, estas bactérias foram identificadas através de análises de sequência de rDNA 16S como Staphylococcus arlettae, Klebsiella sp, Microbacterium sp, Leucobacter albus e Brevibacterium sp / Abstract: Azo dyes, which are aromatic compounds with one or more azo (-N=N-) groups, are the most important and largest class of synthetic dyes used in commercial applications. They are considered as xenobiotic compounds that are very recalcitrant to biodegradation processes. The presence of these dyes in the aqueous ecosystem are a cause of serious environmental and health concerns. In this work, the ability of 62 bacterial strains previously isolated from an industrial activated sludge process treating effluent containing azo dyes was investigated. The selection was undertaken, through visual decolorization, in liquid media with azo dyes supplemented with diferent carbon sources. Decolorization process was performed under microaerophilic or static conditions until no color was observed. The medium was then aerated to promote the biodegradation of the metabolites produced. The azo dyes decolorization and biodegradation and the aromatic amines produced were monitored by UV-Vis, Total Organic Carbon (TOC), Fourier Transformed Infra Red (FTIR) and High Performence Liquid Chromatography (HPLC- MS). Activity of the oxidoreductase enzymes (peroxidase, laccase and tyrosinase) was evaluated in cultures of the bacterial isolates. Acute toxicity tests with Daphnia magna (Crustacea, Cladocera) were carried out after and before microaerobic or static and aerobic conditions. The aromatic amines generated from the biodegradation of the azo dyes were tested for their ability to undergoing polymerization using a laccase from M. thermophila to catalyze the coupling reactions of the aromatic amines. The decolorization time showed a relationship with the culture medium and chemical structure of the dyes. The monoazo dyes were decolourized within 8 to 120 h. The diazo and triazo were decolourized required 120 to168 h, approximately. UV-Vis analysis showed complete decolorization (>80%) in the microaerophilic or static conditions. No significant color changes were detected in the following aerobic stage. However the bacterial strains could only decolourize the dyes effectively when the medium was supplemented with glucose and pyruvate or yeast extract. In the absence of these compounds, the cultures were unable to decolorize the dyes, thus indicating an obligate requirement for a supplementary carbon source for dye decolorization. When the medium was incubated under microaerophilic conditions, the reduction in TOC was low even after 7 days of incubation. Conversely, a significant increase in TOC reduction (>70%) was observed in the aerobic stage. The reduction of azo bonds is known to yield the production of high concentrations of amines in the microaerophilic or static stage therefore confirmed the azo bond was reduced. Therefore the oxidation of these aromatic amines was confirmed by the absence of amine in the aerobic stage indicating that an oxidative process was responsible by metabololite biodegradation. Tyrosinase activity was observed for Brevibacterium sp, suggesting the role of this enzyme in the decolorization process, but no-activity was observed for laccase and peroxydase. In the Fourier Transformed Infra Red (FTIR) analysis after microaerophilic or static decolorization, new bands were observed in region attributed to amine groups. These bands disappeared in the aerobic stage and a new broad region associated with carboxylic acid and NH3+ ions were observed. However, in the aerobic stage the partial mineralization of the dye degradation products and of the medium metabolites was confirmed. The decolorization products of the RR198 dye were analyzed by High Performence Liquid Chromatography (HPLC- MS) for tentative identification of the unknown metabolites tentating identifield compounds included, 4-chloro-N-o-tolyl-1,3,5-triazin-2-amine; sodium 4-aminonaphthalene-2-sulfonate and 3,6-dimethyl-7-(o-tolyldiazenyl) naphthalen-1-amine. After aerobic conditions the intensity of these metabolites was reduced. These results were also confirmed by FTIR and could be explained by degradation of these aromatics coumpounds previously generated in microaerophylic or static stage. After an extended period of time, laccase catalyzed polymerization of the aromatic amines in the destained solutions. The products generated precipitated spontaneously from the solution and acquired some color as confirmed by the UV-Vis analysis. The particle size was also significantly higher after laccase treatment as show by Photon Correlation Spectroscopy analysis (PCS). The bacterial strains used in this study were able to totally destain the azo dyes under microaerophilic or static condition. In the aerobic stage, partial mineralization of the dye decolorization products as well as of the medium metabolites was also confirmed by toxicity testing and TOC measurements. The strains were identified by 16S rDNA gene sequence analysis as Staphylococcus arlettae, Klebsiella sp, Microbacterium sp, Leucobacter albus e Brevibacterium sp. / Doutorado / Doutor em Ciência de Alimentos

A vinhaça e o bagaço de cana são os principais subprodutos oriundos do processamento da cana-de-açúcar nas indústrias sucroalcooleiras, sendo geradas grandes quantidades dos mesmos. O fungo basidiomiceto Pleurotus ostreatus tem a capacidade de degradar materiais lignocelulolíticos e produzir enzimas lignolíticas de interesse para as indústrias. Com o objetivo de avaliar a produção das enzimas lacases e peroxidase, o fungo Pleurotus ostreatus, foi cultivado em meio contendo bagaço pré-tratado e vinhaça, ou em meio contendo apenas vinhaça, em sistema de fermentação semissólido ou submerso; as enzimas extracelulares foram avaliadas após 7, 10 e 12 dias de cultivo. O bagaço peneirado foi considerado pré-tratado fisicamente (T1); para o pré-tratamento T2 o bagaço umedecido foi submetido a autoclave (121°C e 1 atm por 15 min); nos pré-tratamentos químicos, T3 e T4, o bagaço foi tratado com peróxido de hidrogênio e hidróxido de sódio nas seguintes concentrações: 0,75% H2O2 + 0,75% NaOH (T3) e 0,75% H2O2 + 1% NaOH (T4) na proporção 1:10 (p/v) e, em seguida foram submetidos à autoclave (121°C e 1 atm por 15min). A vinhaça utilizada foi proveniente de uma indústria sucroalcooleira (V1) e outra de destilaria (V2); a composição físico-química mostrou que a primeira possuía os índices de matéria orgânica e fósforo mais elevados que na vinhaça V2, enquanto que a relação C:N foi menor na vinhaça V1. Os extratos enzimáticos foram obtidos após filtração do meio submerso; para o meio semissólido foi necessário a adição de tampão citrato (1:5 p/v) antes da filtração. A atividade de lacasse e peroxidase em meio submerso, nos tratamentos com a vinhaça V1, foi superior ao observado em meio semissólido. A produção das enzimas em fermentação submersa, utilizando a vinhaça V1, apresentou valores de atividade de lacase, no tratamento TL1 e TL2, de 784,9 e 707,5 U.L-1, com atividade específica de 3,04 e 2,86 U.mg-1, respectivamente, e a amostra VL1, contendo apenas vinhaça, de 1,91 U.mg-1, no 12º dia de fermentação. Os valores mais altos de atividade de peroxidase foram obtidos nos tratamentos TL1, TL2, VL1, com 133,1; 131,2 e 126,1 U.L-1, respectivamente, após 12 dias de cultivo. A maior atividade específica obtida foi na VL1 (0,86 U.mg-1) no 7º dia de cultivo. O pré-tratamento físico do bagaço mostrou melhores condições para a produção das enzimas. Para a produção da lacase e da peroxidase é fundamental a composição da vinhaça. / The vinasse and bagasse are the principal by-products derived from the processing of sugarcane in the sugarcane industry, which generated large amounts of them. The basidiomycete fungus Pleurotus ostreatus, has the ability to degrade lignocellulolytic materials and produce lignolíticas enzymes of interest to industry. In order to evaluate the production of laccase and peroxidase enzymes, fungus P. ostreatus was grown in medium containing pre-treated bagasse and vinasse, or in medium containing only vinasse in semi-solid or submerged fermentation system; extracellular enzymes were evaluated after 7, 10 and 12 days of cultivation. The screened bagasse was considered pretreated physically (T1); for the pretreatment T2 moistened residue was subjected to autoclaving (121°C and 1 atm for 15 min). The chemical pretreatments, T3 and T4, the residue was treated with hydrogen peroxide and sodium hydroxide solution in the following concentrations 0,75% H2O2 + 0,75% NaOH (T3) and 0,75% H2O2 + 1% NaOH (T4) 1:10 (w/v) and then underwent autoclaving (121°C and 1 atm for 15 min). Vinasse used was coming from a sugar and alcohol industry (V1) and a distillery (V2); the physico-chemical composition showed that the former had the rates of organic matter and phosphorus higher than in V2 vinasse, whereas the C: N ratio was lower in V1 vinasse. The enzymatic extracts were obtained after filtration medium the submerged; to semisolid medium was necessary the addition of citrate buffer (1:5 w/v) prior to filtration. The activity of peroxidase and lacasse in submerged medium, in the treatments with the V1 vinasse, was higher than observed in semi-solid medium. The production of enzymes by submerged fermentation using the vinasse V1, presented laccase activity values, in the treatment TL1 and TL2, of 784,9 and 707,5 UI.L-1, with specific activity of 3,04 e 2,86 U.mg-1, on the 12th day of fermentation. Higher values peroxidase activity were obtained in the treatments TL1, TL2, VL1, with 133,1; 131,2 and 126,1 UI.L-1, respectively, after 12 days of culture. The highest specific activity was obtained at VL1 (0,86 U.mg-1) on the 7th day of culture. Physical bagasse pretreatment showed better conditions for the production of enzymes. For the production of the laccase and peroxidase is fundamental composition of vinasse.

Orientador: José Augusto Rosário Rodrigues / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T17:51:51Z (GMT). No. of bitstreams: 1 Zampieri_LuizArthur_D.pdf: 85903823 bytes, checksum: 1e986b4988d705babbd33a4f64b81704 (MD5) Previous issue date: 2013 / Resumo: Estudou-se e foram desenvolvidos biocatalisadores baseados em duas técnicas de imobilização: em gel de alginato (com/sem revestimento de quitosana) e em nanossílica funcionalizada. Foram preparados biocatalisadores com lacases (comercial e do caldo enzimático de crescimento do fungo Pycnoporus sanguineus, sob ação de indutores de atividade enzimática), utilizados em reações de oxidação de alcoóis, na decomposição de fármacos e em descoloramento de corantes azo. Também foram preparados biocatalisadores por imobilização de células íntegras de 4 microrganismos, utilizados em reações de redução de enonas. As lacases foram imobilizadas em esferas de alginato de cobre além de poderem ser revestidas com quitosana. A quitosana aumentou a resistência mecânica das esferas e possibilitou que fossem utilizadas por até 3 vezes sucessivas. As reações de oxidação de alcoóis feitas com alginato revestido com quitosana forneceram porcentagens de produto inferiores a relatado na literatura (~50% contra 80% da literatura) e, por isso, essa técnica foi substituída por outra, baseada em nanossílica funcionalizada. A técnica de imobilização de lacases em nanossílica funcionalizada forneceu maiores porcentagens de produtos de oxidação do que a de imobilização em gel, havendo 100% de conversão inicial do substrato (álcool para-metoxibenzílico) quando é utilizado o mediador TEMPO. É possível utilizar este biocatalisador por até 10 vezes, sendo esta a técnica de escolha para essa reação de oxidação de alcoóis com o sistema lacase mediador. A imobilização de lacases em nanossílica funcionalizada também mostrou-se capaz de decompor fármacos (diclofenaco, estradiol, ciprofloxacina, naproxeno e norfloxacina), se colocando como uma alternativa complementar para sistemas de tratamento de águas. O descoloramento de corantes azo pelas esferas de alginato contendo lacases foi estudado tanto com esferas contendo lacase comercial como com o caldo enzimático sob indução. Ambas demonstraram capacidade de descolorir todos os corantes testados, por até 4 ciclos, sendo que a utilização do mediador HBT ampliou as porcentagens de descoramento (~40/50% sem HBT contra ~70/85% com HBT). As esferas contendo caldo enzimático apresentaram resultados ligeiramente superiores, ambas com HBT (85% do caldo enzimático contra 70% da enzima comercial). O biocatalisador com células de microrganismos (S.cerevisiae, R.glutinis, C.albicans e G. candidum) foi preparado em esferas de alginato de cálcio e também em esferas de alginato de cálcio revestidas com quitosana, o que alterou as propriedades e forneceu resultados diversos daquelas sem quitosana, permitindo o controle quimiosseletivo do processo reacional para alguns dos substratos. Os biocatalisadores com células em gel apresentaram algumas vantagens em relação às células livres, já que não ocorrem as emulsões durante o processo de isolamento, não se observou desalogenação, contorna-se a morte das células possibilitando que as reações sejam mantidas por mais tempo ou com maior quantidade de substrato e, em alguns casos, houve aumento do excesso enantiomérico, mostrando que esta técnica tem grande versatilidade e ainda atribuiu características de controle do processo reacional, o que é difícil ou mesmo impossível de ser feito com células livres / Abstract: In this work biocatalysts based on two immobilization techniques were developed and studied: those based on alginate gel entrapping (with or without an outer chitosan layer) and those based on functionalized nanosilica linkage. Laccase-based biocatalysts were prepared and used in the oxidation of alcohols, degradation of pharmaceuticals and bleaching of azo dyes. Both commercial laccase and laccase obtained from the fungus Pycnoporus sanguineus under enzymatic activity inductors were used. Biocatalysts for the reduction of enones were also prepared by immobilization of whole cells from four different microorganisms in calcium alginate. Laccases were immobilized in copper alginate, in some experiments being covered by a layer of chitosan. The chitosan layer enhanced the spheres¿ mechanical resistance, making it possible for them to be reutilized up to three successive times. Alcohol oxidations carried out by laccases in alginate beads covered by chitosan yielded lower conversions to those reported in the literature (ca. 50% versus 80% from the literature), and, thus, this technique was replaced by another one based on functionalized nanosilica. This immobilization technique yielded a higher amount of the oxidation product than the gel immobilization, and up to 100% conversion was achieved for the model substrate (p-methoxybenzyl alcohol) when TEMPO was used as the mediator. It was possible to use this biocatalyst up to ten times, ultimately being the chosen technique for the alcohol oxidations using the laccase-mediator system. Functionalized nanosilica-immobilized laccases were also able to decompose pharmaceuticals (diclofenac, estradiol, ciprofloxacin, naproxen and norfloxacin), presenting itself as a complementary alternative to water treatment systems. Azo dye bleaching by alginate-entrapped laccases was studied using both commercial laccase and the enzymatic broth under induction. Both showed capability of decolorizing all inspected dyes, for up to four cycles, and the utilization of the mediator HBT enhanced the decolorizing percentage (ca. 40-50% without HBT versus ca. 70- 85% with HBT). Beads containing the enzymatic broth presented slightly superior results, both with HBT (85% for the enzymatic broth versus 70% with the commercial enzyme). Whole-cell biocatalysts were prepared with microorganisms (S. cerevisiae, R. glutinis, C. albicans and G. candidum) entrapped in calcium alginate beads with or without an outer chitosan layer. The chitosan layer altered the beads¿ properties, as well as the reduction outcomes, allowing the chemoselectivity control for some of the substrates. Gel-entrapped biocatalysts presented some advantages compared to those with free cells, since no emulsion was observed in the reaction workups, no dehalogenation was observed in the case of halogenated enones, cell death could be delayed, making it possible for reactions to be carried out for more time and with greater amounts of substrate, and in some cases, an enhancement of enantiomeric excess was observed. These results show that it is a very versatile technique that can be used as a strategy for the control of the biocatalytic reactions, which is harder to achieve when free cells are employed / Doutorado / Quimica Organica / Doutor em Ciências

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T10:24:06Z (GMT). No. of bitstreams: 1 Cesarino_Igor_D.pdf: 8868735 bytes, checksum: c2f0579fef15f4e7a7a37e4b77a434f3 (MD5) Previous issue date: 2012 / Resumo: A lignina é um heteropolímero complexo depositado principalmente na parede celular secundária de tipos celulares especializados, conferindo força mecânica e rigidez para as plantas se manterem eretas e também proporcionando hidrofobicidade para que células condutoras transportem água e nutrientes por longas distâncias. Embora a lignina seja essencial para o desenvolvimento da planta, este polímero é o principal componente da parede celular responsável pela recalcitrância da biomassa vegetal, sendo que sua presença afeta negativamente o uso do material lignocelulósico para a produção de biocombustíveis e biomateriais. Diversas evidências suportam um papel para peroxidases e laccases no processo de polimerização da lignina. No entanto, identificar genes/isoformas relacionados(as) com o processo de polimerização de lignina e caracterizar seu mecanismo de ação estão entre as tarefas mais desafiadoras acerca do metabolismo deste polímero fenólico. Neste trabalho, uma abordagem técnica abrangente foi aplicada com o objetivo de se identificar potenciais candidatos envolvidos na oxidação dos monômeros de lignina. No capítulo I, atividade enzimática e o perfil proteômico de peroxidases de classe III foram analisados durante o desenvolvimento do colmo de cana-de-açúcar. No capítulo II, células em suspensão foram usadas como valiosa ferramenta para isolar e caracterizar peroxidases de classe III potencialmente envolvidas na polimerização de lignina. Finalmente, no capítulo III, a combinação de análises de co-expressão, expressão tecido/tipo celular específica e complementação de um mutante de Arabidopsis thaliana permitiu a caracterização de uma laccase fortemente relacionada com a polimerização de lignina em cana-de-açúcar. Acreditamos que estes foram os primeiros trabalhos a caracterizar peroxidases e laccases em cana-de-açúcar, além de terem contribuído para aumentar o conhecimento acerca do metabolismo de lignina nesta importante cultura dedicada à bioenergia / Abstract: Lignin is a complex heteropolymer deposited in the secondarily thickened walls of specialized plant cells to provide strength and rigidity for plants to stand upright and hydrophobicity to conducting cells for long-distance water transport. Although lignin is essential for plant growth and development, this phenolic polymer is the major plant cell wall component responsible for biomass recalcitrance and its presence negatively affects the use of ligriocellulose as a source for biofuels and bio-based materiais. Several evidences support the role of peroxidases and laccases in lignin polymerization. However, the identification and characterization of peroxidases/laccases involved in lignin polymerization is still a major bottleneck. Here, we carried out a comprehensive approach to identify candidate genes related to the combinatorial coupling of lignin monomers. ln chapter I, we analyzed the enzymatic activity and proteomic profile of class III peroxidases during sugarcane stem development. ln chapter II, suspension cell culture was used as a tool for the characterization of class III peroxidases potentially involved in lignin polymerization. Finally, in chapter III, we provide evidence for the role of a laccase gene in lignin biosynthesis in sugarcane, by using a combination of co-expression analysis, tissue-specific expression analysis and genetic complementation of an Arabidopsis thaliana mutant. To our knowledge, these are the first reports on the characterization of peroxidases and laccases in sugarcane, which might ultimately improve our understanding of the lignin metabolism in this important bioenergy crop / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Abstract Laccases are members of the multicopper oxidases, having the ability to catalyze the oxidation of a wide spectrum of phenolic compounds. These enzymes are capable of oxidizing lignin-related compounds and highly resistant environmental pollutants, and hence can be used in wastewater treatment and detoxification. Laccases are mostly derived from fungi, bacteria, and plants. In general, fungal laccases are known for their high redox potential, while bacterial laccases have a better tolerance to temperature. A combination of these properties is ideal for industrial processes. Directed evolution and the consensus method are often used to engineer protein stability. However, they are time-consuming and expensive. Ancestral sequence reconstruction (ASR), an approach whereby probable ancestral sequences are obtained, is known to provide thermostable variants of modern proteins as the output. In this study, ASR was used to design a laccase with high thermostability and high redox potential. ASR was performed through the web tool Phylobot, where fungal laccases were used as the input to obtain a phylogenetic tree with ancestral variants of the fungal laccases. For rooting the phylogenetic tree, multiple outgroups were tested, and the ‘Three-outgroups’ scenario proved to be the most efficient. In all cases, the phylogenetic tree was branched into major clusters of thermostable clades and non-thermostable clades. Nodes representing ancestral sequences were selected based on their sequence length and proximity to the thermostable clades. Sequence alignment of the selected nodes to the fungal laccases showed that the selected nodes have a high percentage of identity to well-known, highly thermostable laccases like Trametes pubescens and Basidiomycete PM1. All selected nodes have some common conserved motifs in the vicinity of the copper ligands. It also shows that the number of prolines in nodes 229 and 345 is more than in other selected nodes. The gene for the most promising candidate, Node 345, was synthesized, cloned into the pPICZ A vector, and transformed into Pichia pastoris cells. In the future, the protein will be expressed and characterized. Furthermore, the engineered laccases from the directed evolution method and the ancestral sequence reconstruction method will be compared for their activity and thermostability. This work would pave the way for using ASR as a less resource-intensive and time-consuming method for protein engineering. 2.12.0.0 / <p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>2.12.0.0</p>

Yes / Phenol and its derivatives are hazardous, teratogenic and mutagenic, and have gained significant attention in recent years due to their high toxicity even at low concentrations. Phenolic compounds appear in petroleum refinery wastewater from several sources, such as the neutralized spent caustic waste streams, the tank water drain, the desalter effluent and the production unit. Therefore, effective treatments of such wastewaters are crucial. Conventional techniques used to treat these wastewaters pose several drawbacks, such as incomplete or low efficient removal of phenols. Recently, biocatalysts have attracted much attention for the sustainable and effective removal of toxic chemicals like phenols from wastewaters. The advantages of biocatalytic processes over the conventional treatment methods are their ability to operate over a wide range of operating conditions, low consumption of oxidants, simpler process control, and no delays or shock loading effects associated with the start-up/shutdown of the plant. Among different biocatalysts, oxidoreductases (i.e., tyrosinase, laccase and horseradish peroxidase) are known as green catalysts with massive potentialities to sustainably tackle phenolic contaminants of high concerns. Such enzymes mainly catalyze the o-hydroxylation of a broad spectrum of environmentally related contaminants into their corresponding o-diphenols. This review covers the latest advancement regarding the exploitation of these enzymes for sustainable oxidation of phenolic compounds in wastewater, and suggests a way forward.

Les biopiles enzymatiques constituent une alternative intéressante de production d'électricité renouvelable. On s'est intéressé dans ce travail au compartiment cathodique d'une biopile utilisant la laccase, une oxydase multi-cuivres, comme biocatalyseur pour la réduction de l'oxygène (ORR) par transfert direct des électrons. Plusieurs stratégies ont été mises en œuvre afin d'optimiser la cinétique de l'ORR sur électrode de graphite. Une des stratégies a consisté à déposer un film mince de nitrure de carbone amorphe (a-CNx) sur le graphite. La présence de groupements amines de surface a ensuite permis le greffage covalent de la laccase. Des groupements carboxyliques peuvent également être introduits par un traitement électrochimique. En alliant plusieurs techniques de caractérisation, notamment des mesures d'impédance, on a démontré que notre système se comporte comme un réseau de microélectrodes. Pour ce type d'électrode on a mesuré une densité de courant maximale de -44,6 µA/cm2. Dans une autre stratégie, la surface du graphite a été nanostructurée par formation de nanowalls de carbone (CNWs) par dépôt chimique en phase vapeur assisté par plasma. On a optimisé les conditions du traitement ultérieur de fonctionnalisation de la surface par APPJ en ayant recours à des plans d'expériences, ce qui a permis d'atteindre des densités de courants de l'ordre de -1 mA/cm2. On a également étudié l'orientation et la cinétique de greffage de l'enzyme sur une surface d'or en utilisant la technique PM-IRRAS. / Enzymatic biofuel cells are an attractive alternative for renewable electricity generation. In this work, we are focusing on the cathodic compartment of a biofuel cell using laccase, a multi-copper oxidase, as biocatalysts for the oxygen reduction reaction (ORR) by direct electron transfer of electrons. Several strategies have been used to optimize the kinetic of ORR on graphite electrode. One strategy was to deposit thin film of amorphous carbon nitride (a-CNx) on graphite. The presence of surface amine groups then allowed the covalent grafting of the laccase. Carboxylic groups can also be produced by an electrochemical treatment. By combining several characterisation techniques, especially impedance measurements, we have demonstrated that our system behaves like microelectrodes network. For this type of electrode, we have measured a maximal current density equal to -44,6 µA/cm2. In another strategy, the surface of graphite was nanostructured by forming carbon nanowalls (CNWs) using the plasma-enhanced chemical vapour deposition technique in a CO/H2 microwave discharge. We have optimized then the APPJ functionalization conditions using experiments design. We reached current densities of the order of -1 mA/cm2. We have also studied the orientation and the kinetic of enzyme immobilisation on gold surface using PM-IRRAS technique.

Ce travail a pour objectif la conception et l'étude d'un réacteur enzymatique à membrane (REM) en vue de la dégradation de composés phénoliques. Pour cela, des membranes actives ont été préparées par greffage covalent de la laccase de Trametesversicolor à la surface d'une membrane céramique Après avoir mis en évidence les potentialités du réacteur vis-à-vis de la dégradation du 2,6-diméthoxyphénol (DMP) choisi comme substrat modèle, l'impact de paramètres opératoires (débit d'alimentation, concentration en substrats) sur les performances du REM a été étudié. Pour résoudre des problèmes d'instabilité, différentes étapes du protocole de fabrication des membranes actives ont été revues. Puis les paramètres opératoires (pH et température) ont été étudiés afin d'optimiser les conditions de mise en œuvre du REM. Il a été établi que les performances épuratoires étaient maximales à pH acide (pH 4) mais restaient stables sur une large plage de températures (15 à 40°C). Enfin la dernière partie de la thèse a été consacrée à l'étude du colmatage et à la recherche de différentes stratégies visant à limiter l'impact de ce dernier sur les performances et la stabilité du REM. / This work was devoted to the design and the study of an enzymatic membrane reactor (EMR) for the degradation of phenolic compounds. For this, active membranes were synthesized by covalent grafting of laccase from Trametes versicolor on the surface of a porous ceramic membrane.The 2,6-dimethoxyphenol (DMP) was chosen as model substrate and the effect of operating parameters (feed flow rate, substrate concentration) on the performance of the EMR have been studied.Different stages of the active membrane preparation process were reviewed in order to improve the system stability. Moreover operating parameters (pH and temperature) were studied in order to optimize the performance of the EMR. It was shown that the depletion efficiency was maximal at relatively low pH (pH 4) but remained stable over a wide temperature range (15 to 40 ° C).Finally the last part of the work was devoted to the developmentof different strategies to limit the impact of fouling on the performance and stability of the EMR.

La laccase de Myceliophtora thermophila oxyde certains acides hydroxycinnamiques avec formation éphémère d’intermédiaires de différentes couleurs avant que ceux-ci n’évoluent ensuite spontanément en polymères brun noir. Quand cette oxydation est effectuée en milieu biphasique (tampon phosphate pH 7,5 / acétate d’éthyle), une partie de ces intermédiaires colorés peut être récupérée dans la phase organique et ainsi soustraite à la polycondensation ultérieure. Dans le cas de l’acide férulique, on peut ainsi isoler une fraction colorée en jaune orangé surtout composée de dimères résultant de la condensation des semiquinones initialement formées. La synthèse de ces colorants peut être favorisée et le rendement amélioré en contrôlant la réaction par un ajout minimum régulé d’oxygène. Ces colorants conservent le pouvoir antioxydant de l’acide férulique parent mais à concentration élevée (100 à 200 mg/ml), ils présentent une cytotoxicité plus élevée vis-à-vis de cellules humaines normales (HUVEC) et cancéreuses (Caco-2) ce qui limite éventuellement leur intérêt comme colorants naturels. Les semi quinones de l’acide férulique ne forment pas de dimères mixtes, quand on effectue l’oxydation laccasique de cet acide en présence d’autres acides phénoliques car la laccase de M. thermophila effectue une oxydation séquencée en fonction de sa plus ou moins grande affinité pour les différents substrats mis en jeu. En présence d’un polyoside aminé insoluble comme le chitosane, les intermédiaires d’oxydation laccasique de différents composés phénoliques (acides férulique, sinapique, syringique et catéchine) réagissent avec les groupements NH2 pour former des liaisons de covalence et conduire ainsi à des chitosanes colorés doués de nouvelles propriétés dues au greffage d’entités phénoliques. Ces chitosanes fonctionnalisés conservent les propriétés filmogènes du chitosane natif mais en plus, forment des solutions plus visqueuses et sont devenus solubles en milieux acide et basique. Ils permettent la croissance de cellules HUVEC. Ils ont surtout acquis des propriétés antioxydantes et forment des films imperméables à l’oxygène ce qui laisse entrevoir de multiples applications intéressantes / The laccase of Myceliophtora thermophila oxidizes some hydroxycinnamic acids with ephemeral formation of intermediates of different colors before these evolve spontaneously in dark brown polymers. When this oxidation is performed in biphasique medium (phosphate buffer pH 7.5 and ethyl acetate), a part of these colored intermediates can be recovered in the organic phase and it can be subtracted from an ulterior polycondensation. In the case of ferulic acid, we could isolate an orange yellow fraction, which is especially composed of dimers resulting from the condensation of the semiquinones initially formed. The synthesis of these colorants can be enhanced and their yield can be improved by controlling the reaction through a regulated minimum addition of oxygen. They keep the antioxidant power of related ferulic acid, but in high concentration (from 100 to 200 mg/ml), their cytotoxicity toward the human normal cells (HUVEC) and cancerous one (Caco-2) is important and consequently, their interest is limited as natural colorants. The semiquinone of ferulic acid doesn't form any mixed dimers, when the laccase-catalysed ferulic acid oxidation is performed in the presence of other phenolic acids. This can be explained by the fact that laccase from M. thermophila performes a multioxidation in the function of its high or little affinity for the involved substrates. In presence of an insoluble amino polyoside as chitosan, the intermediates of laccase-catalysed oxidation of different phenolic compounds (ferulic, sinapic, syringic acids and catechin) react with its NH2 groups forming covalent liaisons. Actually, this type of link leads to colored chitosans endowed of news properties due to the grafting of phenolic entities. These functionalized chitosans keep the filmogens properties of the native one. Moreover, also it can form more viscous solutions and become soluble in acidic and basic medium. It can permit the growth of HUVEC cells. They especially acquired some antioxidant properties and formed impermeable films to the oxygen which highlights multiple interesting applications

Ces travaux de thèse traitent de l’utilisation d’une molécule polycyclique, l’acide abiétique, issu de la colophane, et de dérivés phénoliques potentiellement dérivés de la lignine, pour la synthèse de polymères rigides bio-sourcés. Dans les deux cas, des monomères symétriques et difonctionnels sont élaborés par réaction de dimérisation des précurseurs bio-sourcés puis testés en polymérisation. D’une part, les dimères de l’acide abiétique obtenus par un mécanisme cationique possèdent des structures mal définies qui compliquent leur polymérisation. Ces dimères ont alors été estérifiés avec de l’undécénol afin d’obtenir un composé bis-insaturé qui est ensuite polymérisé par ADMET. D’autre part, un procédé de dimérisation de molécules phénoliques, potentiellement issues de la lignine, a été développé par voie enzymatique utilisant une laccase. L’avantage majeur de ce procédé ‘vert’ réside dans la séparation très simple entre le monomère, soluble, et son dimère, insoluble. Ces dimères ont ensuite été modifiés chimiquement afin de constituer une bio-plateforme de composés biphényles fonctionnels. Ces composés ont été utilisés pour la synthèse de polyesters, polyamides et résines époxy qui présentent des propriétés thermiques et thermomécaniques remarquables. / The aim of this thesis is to investigate new biobased rigid synthons for the synthesis of polymers with high thermomechanical properties. A polycyclic biobased molecule, i.e resinic acids, and phenolic compounds potentially derived from lignin, such as vanillin were selected. Both classes of substrates were dimerized in order to get difunctional symmetric synthons. On the one hand, abietic acid dimers synthesized via a cationic mechanism presented an ill-defined structure. To avoid reactivity issues, dimers with reactive terminal double bonds were successfully synthesized by esterification of abietic acid dimers with undecenol and polymerized by ADMET methodology. On the other hand, we developed a “green” process to dimerize phenolic compounds derived from lignin in large quantity and high yield via enzymatic catalysis using a laccase. The main advantage of this method is that the phenolic monomer is soluble in the reaction medium while the dimer precipitates. After chemical modifications of the dimers, we built a functional bio-platform of biphenyl derivatives. The latter were then used for the synthesis of polyesters, polyamides and epoxy resins which exhibited remarkable thermal and thermomechanical properties.