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Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T19:09:12Z : No. of bitstreams: 1 covizzi_lg_me_sjrp.pdf: 1482206 bytes, checksum: 2f1c1f77dc261f160aba2bc3a1d1ffea (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O uso de células microbianas imobilizadas para aumentar a produção de metabólitos fúngicos em processos fermentativos tem mostrado altos rendimentos. Nesse trabalho foi avaliado pela primeira vez, a imobilização de células do Botryosphaeria rhodina, um fungo ligninolitico produtor constitutivo de lacases. Três suportes foram avaliados: Fibra Acrílica Fina (FAF); Espuma de Poliuretano Expandido (EPE); Espuma de Poliuretano Fibroso (EPF). O EPF foi o melhor suporte por ter mostrado uma imobilização mais homogenia das células. Um planejamento fatorial foi desenvolvido para otimizar a produção de lacases por células livres, na presença de álcool veratrílico (AV). A análise da superfície de resposta mostrou que 18mM como a melhor concentração de AV para a produção de lacases, usando-se 3 mL de homogeinato de células como inóculo (DOλ400nm 0.4-0.6) para 25 mL de meio de cultura em frascos de 125mL, a 180 rmp, durante 126 horas a 28ºC. O perfil de crescimento do fungo, associado a produção de lacase foram comparados na presença e na ausência de AV, usando-se células livres e células imobilizadas do B. rhodina. A imobilização aumentou aproximadamente 3 vezes a produção de lacases e manteve estável o nível de produção durante 6 reciclos. A imobilização de células do B. rhodina mostrou-se útil uma vez que economizou 72horas para atingir a maior produção de lacase, quando comparada com células livres e também aumentou a tolerância do fungo a concentrações mais altas de AV (500mM) / The use of microbial immobilized cells to increase the production of fungal metabolites in fermentation processes has showed higher yields. This work evaluated by the first time, the immobilization of Botryosphaeria rhodina cells, a ligninolytic fungus that produces laccase. Three carriers were evaluated: acrylic fine fiber (FAF), expanded polyurethane foam (EPE), and fiber polyurethane foam (EPF). The EPF was the best carrier because showed a homogeneous immobilization cells. A factorial design was developed in order to optimize the laccase production by free cells in the presence of the laccase inducer veratryl alcohol (VA). The analysis by response surface answer showed 18 mM as the best VA concentration to produce laccase using 3 mL of a cell homogenate (ODλ400nm 0.4-0.6) as inoculum, to 25 mL of culture medium in shaked flasks (125 mL) at 180 rpm, during 126 hours at 28 °C. A growth profile for laccase and fungal biomass production were compared with and without VA using free and immobilized cells of B. rhodina. The cell immobilization increased approximately 3 folds the laccase production and maintained it stable during 6 consecutive recycles. The cell immobilization of B. rhodina showed to be useful once saved 72 hours to achieve the higher laccase production when compared to the one with free cells, and also increased the fungal cell tolerance at higher VA concentrations (500 mM)

Les biopiles enzymatiques, capables de convertir le glucose présent dans le fluide physiologique en électricité, sont une source d’alimentation pour les dispositifs implantables. Cependant, les faibles puissances délivrées ne permettent pas d’alimenter actuellement des organes artificiels implantables. Une nouvelle architecture de biocathode tirant profit des technologies d’impression a été testée en vue d’améliorer les performances des Biopiles implantables. Ce travail démontre la pertinence des procédés d’impression tels que le spray ultrasonique et l’héliogravure dans l’élaboration de biocathodes homogènes, fines et flexibles. Ainsi, des encres fonctionnelles, dont la formulation à base de nanotubes de carbone et de surfactant a été optimisée, ont pu être déposées sur un substrat flexible hydrophobe (feuilles de carbone). Les problèmes d’imprimabilité du substrat ont été surmontés et des couches actives flexibles ont été obtenues (épaisseur entre 5 et 10 µm). Enfin, une technique d’immobilisation non-covalente des laccases (via le pyrène adamantane) a été testée et un courant catalytique de l’ordre de 130 mA.cm-2 a été obtenu. / Enzymatic Biofuel Cells, capable of converting efficiently the glucose from extracellular fluid into electrical energy, are a power source for implantable devices. However, the power output generated by these cells is not sufficient to fulfill the energy required by implantable artificial organs. Therefore, a new packaging architecture design based on flexible materials derived from printing technologies has been explored in order to enhance the power output of this cell. This work demonstrates the relevance of printing processes such as ultrasonic spray and gravure to develop homogeneous, thin and flexible biocathodes. During this work, a carbon nanotubes / surfactant suspensions were deposited on a hydrophobic flexible substrate (carbon paper). Despite the poor printability of the substrate, flexible active layers were obtained (thickness between 5 and 10 µm). Finally, a non-covalent immobilization of laccases (via adamantane pyrene) was tested and a catalytic current of approximately 130 µA.cm-2 was obtained. mA.cm-2 was obtained.

Probióticos são suplementos alimentares de microrganismos vivos com efeitos benéficos no hospedeiro animal pela melhora do balanço intestinal. Dentre os microrganismos considerados probióticos, somente aqueles microrganismos classificados como bactérias ácido lácticas (LAB) são considerados importantes em relação à alimentação. Cultivos de altas densidades de células são cada vez mais importantes do ponto de vista industrial para a obtenção de LABs, pois produtos adicionados com esses suplementos apresentam alto valor agregado. Este trabalho tem por objetivo a produção de Lactobacillus plantarum em cultivo submerso em biorreator, avaliação da sua resistência na forma livre e imobilizada frente às condições de armazenamento sob refrigeração e trânsito gastrointestinal e elaboração de produto fermentado com adição de probióticos. Inicialmente foram realizados experimentos para selecionar Lactobacillus que apresentassem alta produtividade de biomassa. Em seguida foram realizados experimentos para avaliar a utilização de soro de queijo como ingrediente base da formulação do meio de cultivo e sua suplementação com diferentes fontes de nitrogênio. Com o microrganismo selecionado, L. plantarum, realizou-se a seleção de variáveis através do delineamento experimental Plackett Burman (P-B). A otimização das condições de cultivo foi realizada utilizando um delineamento composto central rotacional (DCCR). Paralelamente, foram testadas a sobrevivência de L. plantarum em armazenamento sob refrigeração e à exposição a meios que simulem a passagem pelo aparelho digestivo. Estas avaliações foram realizadas comparativamente entre os microrganismos na forma livre e na forma microencapsulada utilizando como polímeros alginato de sódio e pectina e recobrimentos com alginato de sódio e quitosana. Os resultados mostraram que a temperatura, pH, taxa de aeração, concentração de lactose e peptona foram os parâmetros que mais influenciaram a produção de biomassa. O DCCR para temperatura e taxa de aeração mostraram que o máximo de produção de biomassa predita foi de 14,30 g L-1 de L. plantarum, nas condições otimizadas. No ponto central do DCCR, atingiu-se a produção de biomassa de L. plantarum de 10,2 g L-1, como taxa de conversão de 0,10 g de células g-1 de lactose e 1,08 g de ácido láctico g-1 lactose (m/m) com as seguintes condições de cultivos: 140 g L-1 de lactose; 15 g L-1 peptona; 5 g L-1 extrato de levedura; pH 5,2; velocidade de agitação de 200 rpm; 34 ºC e 3,5 vvm. O meio intestinal simulado não interferiu na viabilidade dos microrganismos em relação ao meio controle. Já o meio gástrico simulado diminui drasticamente a viabilidade dos microrganismos nas condições testadas não havendo diferença significativa entre os diferentes materiais imobilizantes utilizados e o controle sem imobilização. No armazenamento sob refrigeração houve aumento da viabilidade em relação às células não imobilizadas, sendo que os tratamentos em que houve menor perda de viabilidade foram imobilização em 4 % de pectina, 3 % de alginato de sódio recoberto com quitosana e mistura de 2 % de alginato de sódio e 2 % de pectina. Quando testada a viabilidade em iogurte de L. plantarum imobilizados em 3 % de alginato recoberto com quitosana houve perda de viabilidade de 0,55 ciclo logarítmico durante 38 dias de armazenamento. A cepa de L. plantarum estudada se mostra como um microrganismos potencial para utilização como probiótico em alimentos, uma vez que demonstrou alta produtividade de células e boa viabilidade frente às condições de estresse utilizadas. / Probiotics are live microorganisms feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance. Among the microorganisms considered probiotics, only those strains classified as latic acid bacteria - LAB are considered of importance regarding to the nutritional effects. High cell density cultivations of LABs are important from the industrial viewpoint, because products added with this supplement are of high value. The aims of this work were to investigate the biomass production of Lactobacillus plantarum in submerged bioreactor cultures, evaluate the resistance of free and immobilized L. plantarum when submitted to refrigerated storage, the viability in simulated gastrointestinal juices and in yoghurt. Initially, experiments were performed to select Lactobacillus that showed high productivity of biomass. Further experiments were performed to evaluate the use of cheese whey as a basic ingredient in the formulation of the medium and its supplementation with different nitrogen sources. The selected microorganism, L. plantarum, was used for the selection of variables of the Plackett Burman (PB) design. The optimization of culture conditions was performed using a central composite rotational (CCD) design. In parallel, it was tested the survival of L. plantarum in refrigerated storage and exposure to media that simulated the passage through the digestive tract. These evaluations were performed comparatively between microorganisms in the free and microencapsulated form using as polymers sodium alginate and pectin, coated with sodium alginate or chitosan. Results have shown that temperature, pH, aeration rate, lactose, and peptone were the most influential over biomass formation. The CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g L-1 (dw) of L. plantarum under the optimized conditions. At central point of CCD, it was obtained a biomass production of 10.2 g L-1 (dw), with conversion rates of 0.10 g of cell g-1 lactose and 1.08 g lactic acid g-1 lactose (w/w), with the following conditions: 140 g L-1 of lactose; 15 g L-1 peptone; 5 g L-1 of yeast extract; pH 5.2; stirred agitation of 200 rpm; 34 ºC and 3.5 vvm. The simulated intestinal medium did not affect the viability of microorganisms in relation to the control medium. However, the simulated gastric medium drastically reduces the viability of microorganisms in the conditions tested with no significant difference between the different materials used and the control without immobilization. In refrigerated storage there was an increase in the viability compared to free microorganisms, and the treatments with lower loss of viability were those of 4% pectin, 3% sodium alginate coated with chitosan and a mixture of 2% alginate sodium and 2% pectin. When tested the viability in yogurt of L. plantarum immobilized in 3% alginate coated with chitosan, the viability loss was 0.55 log cycle during 38 days of storage. The strain of L. plantarum studied was shown as a potential organism for use as probiotics in food, since it has shown high yield and good cell viability in the face of stress conditions used.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Helena Badalcová Title of Thesis: Immobilization of protein macromolecules onto polymer carriers: An overview Since the 70s, the immobilised enzymes have been getting the attention of not only scientific and laboratory workers, but also industrial companies. Enzymes are unique biocatalysts, which are distinguished by their specificity, environment-friendliness and the ability to react under mild conditions can be easily subject of denaturation or inhibition. With regard to the usually high cost of purchase, the use of these enzymes could often be disadvantageous. Immobilization techniques offer an efficient solution to this problem and greatly simplify the use of enzymes in industry and research. Compared to the free forms, immobilized enzymes show greater activity, stability and allow repeated use as well as easier separation from products. This thesis contains an overview of the basic methods of immobilization - physical absorption and covalent bonds to the carrier, entrapment, encapsulation and carrier- free techniques using cross-linking. Finally, we outline possible biomedical applications as well as the use of immobilised enzymes in biosensors.

Baker´s yeast (Saccharomyces cerevisiae) were magnetically modified by three different methods, namely, surface modification by magnetic fluid, entrapment of cells into alginate and covalent immobilization on particles of magnetic chitosan. The ability of H2O2 decomposition was tested for all types of modification. It is apparent that the most amount of hydrogen peroxid was degraded by magnetic fluid - modified cells (84-95%), while the efficiency of cell which were modified by other methods was much lower (40-60%). Thanks to immobilization on particles of magnetic chitosan, we made completely new type of magnetic material, which was tested for adsorption of Crystal violet and Safranin O. It was founded that magnetic chitosan adsorbs no dyes, so all adsorption belongs to immobilized yeast. The maximum adsorption capacities were determined using Langmuire isotherm at 69,4 mg/g for Crystal violet and 99,0 mg/g for Safranin O.

Approche multi-échelle pour l’étude de la réaction de N-acylation enzymatique d’acides aminés La réaction de N-acylation d’acides aminés ou de peptides permet l’obtention de dérivés de ces molécules présentant des propriétés bioactives et/ou techno-fonctionnelles, avec une biodisponibilité, une hydrophobie et une stabilité accrue. Les acides aminés acylés ont été largement décrits comme constituant une classe d'agents tensioactifs avec d'excellentes propriétés de surface, des activités biologiques intéressantes, un faible potentiel de toxicité et un faible impact environnemental. Actuellement réalisée de manière chimique à l’échelle industrielle, l’acylation de ces acides aminés ou peptides présente des contraintes en termes de sélectivité réactionnelle et d’innocuité vis-à-vis de l’environnement ainsi qu’en termes de coût de retraitement des effluents polluants. Une alternative à cette voie chimique est l’utilisation d’enzymes capables de catalyser ces réactions d’acylation. Dans la littérature, différents couples d’enzymes et de solvants ont déjà été décrits. Néanmoins, les performances réactionnelles de ces systèmes demeurent parfois limitées. L’objectif de cette thèse a donc été l’amélioration du procédé d’acylation par une approche à différentes échelles. À l’échelle moléculaire, une étude a été réalisée avec la lipase B de Candida antarctica (CALB). Une approche de modélisation moléculaire a été utilisée afin de mettre au point une méthodologie associant des simulations de docking et des calculs d’interaction permettant d’améliorer la compréhension et permettre la prédiction de la régiosélectivité de CALB lors de l’acylation de la lysine par différents acides gras. Des études ont également été conduites à l’échelle réactionnelle, notamment avec la recherche de nouveaux biocatalyseurs de type aminoacylases dans l’extrait brut de Streptomyces ambofaciens. La régiosélectivité et les performances de la réaction catalysée par ces enzymes ont été comparés à celles de CALB. Les résultats ont mis en évidence un potentiel très prometteur des aminoacylases de S. ambofaciens concernant la synthèse d’acide aminés/peptides acylés. En effet, en plus de leur aptitude à réaliser la réaction d’acylation en milieu aqueux, ces enzymes possèdent une régio-sélectivité qui diffère de celle de CALB. Cette régio-sélectivité orientée vers les groupements N-terminaux est un atout très peu décrit à ce jour, car elle permet d’acyler ces molécules sans modifier les chaînes latérales des acides aminés ou des peptides et donc leurs fonctionnalités. Dans la dernière partie de ces travaux, des études à l’échelle procédé ont été menées. Tout d’abord, l’immobilisation des aminoacylases sur des matériaux mésoporeux silicatés a été réalisée et différentes méthodes d’immobilisation ont pu être comparées. Cette étude a permis de proposer une méthode d’immobilisation des aminoacylases de S. ambofaciens par physisorption, permettant de conserver l’activité spécifique pendant au moins 3 cycles. Puis, dans une dernière partie, l’intensification de la réaction d’acylation en réacteur micro-ondes ou microstructurés a été abordée. Les expérimentations réalisées dans un réacteur chauffé par irradiation micro-onde ont montré que ce type de réacteur était adapté à la réaction d’acylation catalysé par CALB sous sa forme immobilisée commerciale (Novozym435®) en solvant organique, ce qui n’est pas le cas avec des aminoacylases de S. ambofaciens libres, en milieux aqueux. Pour cette réaction, d’autres méthodes d’intensification ont été envisagées, notamment en réacteur microstructuré de type microfluidique. L’efficacité du mélange étant primordiale notamment en milieu biphasique, celle-ci a pu être améliorée avec un taux de conversion supérieur dans ce réacteur comparativement à un réacteur classique agité mécaniquement / N-acylation of amino acids or peptides results in bioactive and/or functional molecules showing increased bioavailability, hydrophobicity and stability. Acylated amino acids have been broadly described as being a kind of surfactant with great surface chemistry properties, interesting biological activities, weak toxicity and low environmental impact. Acylation of amino acids or peptides is being performed chemically at industrial scale. It creates constraints in term of reaction selectivity, environmental safety and cost of polluted wastewater treatment. Enzymatic catalysis is an alternative to chemical acylation reaction. Several enzyme/solvent pairs have already been described in the literature. Their performance are however somewhat limited. The objective of this thesis work was thus to improve the capacity of acylation processes at different scales. At the molecular scale, a study was performed using Candida antarctica’s (CALB) lipase B. Molecular modeling was used to create a methodology coupling docking simulation and interaction calculus that would allow for a better understanding of CALB regioselectivity during lysine acylation by different fatty acids. Studies were also conducted at the reaction level, especially by searching for new aminoacylase-type of biocatalysts in Streptomyces ambofaciens raw extract. Regioselectivity and performance of these enzyme’s catalytic reactions were compared to those of CALB. Results brought into light a promising potential from S. ambofaciens’ aminoacylases in synthesizing acylated amino acids/peptides. Indeed, on top of their ability to catalyse acylation reaction in aqueous solution, these enzymes have a different regioselectivity compared to CALB’s. Regioselectivity targeting N-terminal groups is a rarely researched phenomenon allowing acylation to be performed without modifying amino acids or peptides lateral chains and hence their functionality. In the last part part of this work, studies at process scale were performed. Aminoacylase were first immobilized on mesoporous silicates and several immobilisation methods were compared. Using physisorption, a method for the immobilisation of S. ambofaciens’ aminoacylases was developed to reach a conserved specific activity during 3 cycles. Finally, intensification of acylation reaction was examined in microwave or microstructured reactors. First, an experimental set up was performed in an heated reactor using microwaves irradiation. This kind of reactor was demonstrated as being adapted to acylation reaction using a commercial immobilized form of CALB (Novozym435®) as catalyst in organic solvent. The microwave reactor was however not suited for free S. ambofaciens aminoacylase in aqueous solution. For that latter reaction, intensification had to be approached through other aspects of the process. Hydrodynamic appeared indeed as an important aspect for this reaction occurring in a biphasic medium composed of fatty acids and aqueous solution. A microstructured microfluidic reactor was hence tested. Conversion yield were increased with this system. This study demonstrated how mixing quality was an important factor for acylation reaction and could be a way to intensify the enzymatic process at larger scale

Probióticos são suplementos alimentares de microrganismos vivos com efeitos benéficos no hospedeiro animal pela melhora do balanço intestinal. Dentre os microrganismos considerados probióticos, somente aqueles microrganismos classificados como bactérias ácido lácticas (LAB) são considerados importantes em relação à alimentação. Cultivos de altas densidades de células são cada vez mais importantes do ponto de vista industrial para a obtenção de LABs, pois produtos adicionados com esses suplementos apresentam alto valor agregado. Este trabalho tem por objetivo a produção de Lactobacillus plantarum em cultivo submerso em biorreator, avaliação da sua resistência na forma livre e imobilizada frente às condições de armazenamento sob refrigeração e trânsito gastrointestinal e elaboração de produto fermentado com adição de probióticos. Inicialmente foram realizados experimentos para selecionar Lactobacillus que apresentassem alta produtividade de biomassa. Em seguida foram realizados experimentos para avaliar a utilização de soro de queijo como ingrediente base da formulação do meio de cultivo e sua suplementação com diferentes fontes de nitrogênio. Com o microrganismo selecionado, L. plantarum, realizou-se a seleção de variáveis através do delineamento experimental Plackett Burman (P-B). A otimização das condições de cultivo foi realizada utilizando um delineamento composto central rotacional (DCCR). Paralelamente, foram testadas a sobrevivência de L. plantarum em armazenamento sob refrigeração e à exposição a meios que simulem a passagem pelo aparelho digestivo. Estas avaliações foram realizadas comparativamente entre os microrganismos na forma livre e na forma microencapsulada utilizando como polímeros alginato de sódio e pectina e recobrimentos com alginato de sódio e quitosana. Os resultados mostraram que a temperatura, pH, taxa de aeração, concentração de lactose e peptona foram os parâmetros que mais influenciaram a produção de biomassa. O DCCR para temperatura e taxa de aeração mostraram que o máximo de produção de biomassa predita foi de 14,30 g L-1 de L. plantarum, nas condições otimizadas. No ponto central do DCCR, atingiu-se a produção de biomassa de L. plantarum de 10,2 g L-1, como taxa de conversão de 0,10 g de células g-1 de lactose e 1,08 g de ácido láctico g-1 lactose (m/m) com as seguintes condições de cultivos: 140 g L-1 de lactose; 15 g L-1 peptona; 5 g L-1 extrato de levedura; pH 5,2; velocidade de agitação de 200 rpm; 34 ºC e 3,5 vvm. O meio intestinal simulado não interferiu na viabilidade dos microrganismos em relação ao meio controle. Já o meio gástrico simulado diminui drasticamente a viabilidade dos microrganismos nas condições testadas não havendo diferença significativa entre os diferentes materiais imobilizantes utilizados e o controle sem imobilização. No armazenamento sob refrigeração houve aumento da viabilidade em relação às células não imobilizadas, sendo que os tratamentos em que houve menor perda de viabilidade foram imobilização em 4 % de pectina, 3 % de alginato de sódio recoberto com quitosana e mistura de 2 % de alginato de sódio e 2 % de pectina. Quando testada a viabilidade em iogurte de L. plantarum imobilizados em 3 % de alginato recoberto com quitosana houve perda de viabilidade de 0,55 ciclo logarítmico durante 38 dias de armazenamento. A cepa de L. plantarum estudada se mostra como um microrganismos potencial para utilização como probiótico em alimentos, uma vez que demonstrou alta produtividade de células e boa viabilidade frente às condições de estresse utilizadas. / Probiotics are live microorganisms feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance. Among the microorganisms considered probiotics, only those strains classified as latic acid bacteria - LAB are considered of importance regarding to the nutritional effects. High cell density cultivations of LABs are important from the industrial viewpoint, because products added with this supplement are of high value. The aims of this work were to investigate the biomass production of Lactobacillus plantarum in submerged bioreactor cultures, evaluate the resistance of free and immobilized L. plantarum when submitted to refrigerated storage, the viability in simulated gastrointestinal juices and in yoghurt. Initially, experiments were performed to select Lactobacillus that showed high productivity of biomass. Further experiments were performed to evaluate the use of cheese whey as a basic ingredient in the formulation of the medium and its supplementation with different nitrogen sources. The selected microorganism, L. plantarum, was used for the selection of variables of the Plackett Burman (PB) design. The optimization of culture conditions was performed using a central composite rotational (CCD) design. In parallel, it was tested the survival of L. plantarum in refrigerated storage and exposure to media that simulated the passage through the digestive tract. These evaluations were performed comparatively between microorganisms in the free and microencapsulated form using as polymers sodium alginate and pectin, coated with sodium alginate or chitosan. Results have shown that temperature, pH, aeration rate, lactose, and peptone were the most influential over biomass formation. The CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g L-1 (dw) of L. plantarum under the optimized conditions. At central point of CCD, it was obtained a biomass production of 10.2 g L-1 (dw), with conversion rates of 0.10 g of cell g-1 lactose and 1.08 g lactic acid g-1 lactose (w/w), with the following conditions: 140 g L-1 of lactose; 15 g L-1 peptone; 5 g L-1 of yeast extract; pH 5.2; stirred agitation of 200 rpm; 34 ºC and 3.5 vvm. The simulated intestinal medium did not affect the viability of microorganisms in relation to the control medium. However, the simulated gastric medium drastically reduces the viability of microorganisms in the conditions tested with no significant difference between the different materials used and the control without immobilization. In refrigerated storage there was an increase in the viability compared to free microorganisms, and the treatments with lower loss of viability were those of 4% pectin, 3% sodium alginate coated with chitosan and a mixture of 2% alginate sodium and 2% pectin. When tested the viability in yogurt of L. plantarum immobilized in 3% alginate coated with chitosan, the viability loss was 0.55 log cycle during 38 days of storage. The strain of L. plantarum studied was shown as a potential organism for use as probiotics in food, since it has shown high yield and good cell viability in the face of stress conditions used.

Orientador: José Geraldo Nery / Banca: Eleni Gomes / Banca: José Manoel Marconcini / Resumo: Os principais objetivos do presente trabalho de pesquisa são a síntese de novos catalisadores zeolíticos, a avaliação de seus usos como possíveis matrizes para imobilização enzimática, e o uso desse complexo zeólita/enzima como catalisador para produção de biodiesel. Inicialmente foram sintetizadas duas classes de zeólitas: faujasita (FAU) e gismondina (GIS), em suas formas sódicas, e estas zeólitas foram convertidas para as formas Cu2+, Ni2+ e Zn2+ por troca iônica. Na sequência foi avaliado o potencial desses materiais zeolíticos para imobilização e atividade lipolítica da enzima (Lipase de Rhizomucor miehei) na reação de hidrólise do p-nitrofenil palmitato (pNPP) a p-nitrofenol (pNP). Os estudos de imobilização foram feitos variando os parâmetros térmicos do pré-tratamento do suporte zeolítico e o meio catiônico dominante. Os resultados obtidos mostraram que dos 16 suportes avaliados para imobilização enzimática, todos se mostraram capazes de imobilizar a enzima em questão e produzir atividade catalítica, no entanto, o melhor compromisso entre imobilização e atividade enzimática foi obtido pelo complexo LIPASE/GIS/Ni2+ pré-tratada a 200ºC. Este complexo que imobilizou 11,2 ± 0,6% da quantidade de enzima da solução e obteve uma atividade de 13,278 U/mg-suporte. Para as reações de transesterificação foram usados como catalisadores as zeólitas puras (GIS/Ni2+ e GIS/Zn2+), enzimas livres (Lipases de Rhizomucor miehei) e enzimas imobilizadas sobre as zeólitas (LIPASE/GIS/Ni2+ e LIPASE/GIS/Zn2+) para a produção de biodiesel a partir do óleo vegetal (óleo de soja). Os resultados obtidos indicaram que os dois complexos produziram biodiesel, no entanto, o teor de ésteres metílicos de 56,2% produzidos pelo suporte LIPASE/GIS/Ni2+ mostrou que houve... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main goals of this research work are the syntheses of new zeolitic catalysts and their use as possible solid matrices for enzyme immobilization, and the study this complex zeolite/enzyme as a catalyst for biodiesel production. Initially, two different zeolites were synthesized: faujasite (FAU) and gismondine (GIS). The two zeolites were first prepared in their sodic forms and thereafter they were submitted to an ion exchange process in order to the replace the extra-framework Na+ by Cu2+, Ni2+ and Zn2+. The potential of these zeolitic materials for immobilization and lipolytic activity of the enzyme (Lipase from Rhizomucor miehei) was evaluated through the hydrolysis reaction from p-nitrophenyl palmitate (pNPP) to p-nitrophenol (pNP). Several immobilization studies were performed and two main parameters were singled out for this study: the thermal parameters or thermal stability of the zeolitic support and the main dominant cationic medium. The results showed that all of the 16 prepared zeolitic supports were not only able to immobilize the enzyme, but also they were able to show significant catalytic activity. However, the best compromise between immobilization and enzymatic activity was obtained with the complex LIPASE/GIS/Ni2+ pre-treated at 200°C, since it was able to immobilize 11.2 ± 0.6% of the amount of enzymes in solution and an activity of 13.278 U/mg-support. The transesterification reactions for the production of biodiesel from vegetable oils (soybean oil) were performed with the following catalysts: zeolite pure (GIS/Zn2+ and GIS/Ni2+), free enzymes (Lipases from Rhizomucor miehei) and immobilized enzymes on zeolites (LIPASE/GIS/Zn2+ and LIPASE/GIS/Ni2+). The results indicated that two zeolite/enzyme complexes were able to produce biodiesel, but the content... (Complete abstract click electronic access below) / Mestre

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this study, two tungsten-based oxides, iron tungstate (FeWO4) and zinc tungstate (ZnWO4), were synthesized by the routes microwave-assisted hydrothermal and solvo-hidrothermal, respectively. The iron tungstate oxide was used as a catalyst in heterogeneous photo-Fenton reaction for removal of Amaranth dye, whereas the zinc tungstate was used as a support for the immobilization of inulinase by adsorption process. Both materials produced were characterized by techniques such as X-ray diffraction (XRD), nitrogen adsorption-desorption analysis by Brunauer-Emmett-Teller method (BET), Fourier transform infrared spectroscopy (FTIR) and particle size distribution analysis by laser diffraction. For heterogeneous photo-Fenton reaction, an experimental design was used to study the effect of variables such as pH, hydrogen peroxide concentration and dye concentration on the degradation efficiency of Amaranth dye. The inulinase immobilization on the ZnWO4 oxide was investigated in two temperatures. According to the characterization results, both synthesized material has a porous structure and high crystallinity. The FeWO4 oxide showed a satisfactory ability to degrade amaranth dye, and under the optimum reaction conditions, 97% decolorization and 58% mineralization were obtained. Furthermore, the efficiency and stability of this catalyst were maintained high after five cycles of reuse. The ZnWO4 oxide showed a satisfactory inulinase adsorption, where the best result was found to be 605 U.g-1 at 30 oC. / Neste trabalho, dois óxidos a base de tungstênio, tungstato de ferro (FeWO4) e tungstato de zinco (ZnWO4), foram sintetizados pelos métodos hidrotérmico assistido por micro-ondas e solvo-hidrotérmico, respectivamente. O tungstato de ferro foi utilizado como catalisador na reação heterogênea de foto-Fenton para remoção do corante orgânico Amaranto, enquanto que o tungstato de zinco foi utilizado como suporte para imobilização da inulinase pelo processo de adsorção. Ambos os materiais produzidos foram caracterizados pelas técnicas tais como difração de raios-X (DRX), análise de adsorção-dessorção de nitrogênio pelo método de Brunauer-Emmett-Teller (BET), espectroscopia de infravermelho por transformada de Fourier (FTIR) e análise de distribuição do tamanho de partículas por difração laser. Para a reação foto-Fenton heterogênea, um planejamento experimental f oai empregado a fim de estudar os feitos das variáveis pH, concentração de peróxido e de corante sobre a eficiência de degradação do corante amaranto. A imobilização de inulinase sobre o óxido ZnWO4 foi investigada em duas temperaturas. De acordo com os resultados da caracterização, ambos os materiais sintetizados apresentaram uma estrutura porosa e com alta cristalinidade. O óxido FeWO4 demonstrou uma satisfatória habilidade para degradar o amaranto, sendo que nas condições ótimas de reação, 97 % de descoloração e 58% de mineralização foram obtidos. Além disso, a eficiência e estabilidade desse catalisador foram mantidas elevadas após cinco ciclos de reuso. O óxido ZnWO4 apresentou uma satisfatória adsorção de inulinase, onde o melhor resultado encontrado foi de 605 U.g-1 na temperatura de 30 oC.