Düsseldorf, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Düsseldorf, Universiẗat, Diss., 2003.

Marburg, Universiẗat, Diss., 2001.

Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Bacillus licheniformis é uma bactéria versátil, e tem sido utilizada atualmente na produção de diversos produtos bitecnológicos. As lipases (E.C.3.1.1.3 ) são enzimas encontradas na natureza, podendo ser obtidas de fontes animais, vegetais e microbianas, sendo essas últimas bastante utilizadas devido a série de vantagens apresentadas frente as demais. A utilização de rejeitos industriais para produção e formulação de meios de produção de diversos bioprodutos, tem surgido como uma alternativa de minimizar o descarte de rejeitos das diversas indústrias de produção, principalmente as de origem alimentícia. Sorvetes são produtos alimentícios obtidos a partir de uma emulsão de gorduras e proteínas, com ou sem a adição de outros ingredientes e substâncias que tenham sido submetidas ao congelamento, a sua produção em larga escala gera resíduos sólido-líquidos com uma grande quantidade de gorduras e outros resíduos orgânicos. Neste trabalho, inicialmente foram realizados estudos para selecionar meio alternativo para produção de lipase por B. licheniformis (UCP 1014). E a seguir a formulação de um meio alternativo de produção, utilizando um resíduo da indústria de sorvete através de um planejamento fatorial de 24. Os ensaios ocorreram durante 96h, a 37oC Os resultados obtidos indicaram que o meio denominado de C (glicose 1,0%, peptona, 2,0%, extrato de levedura 0,5%, óleo de oliva, 1,0%, NaNO3 0,1%, KH2PO4 0,1%, MgSO4. 7H2O 0,05%), apresentando uma atividade lipolítica de 256 (U/ml)/min. Os ensaios referentes ao planejamento fatorial, indicaram que o ensaio 11 apresentou a maior atividade de 480 U/mL, para a lipase produzida. Os resultados obtidos sugerem o reaproveitamento de resíduos oleosos provenientes da indústria de sorvete para formulação e produção de lipase microbiana. / The bacterium Bacillus licheniformis is a versatile, and has been used currently in production of various bitecnológicos. Lipases ( E.C.3.1.1.3) are enzymes found in nature and may be obtained from animal sources, plants and microbes, the latter being widely used due to several advantages presented against the other. The use of industrial wastes for production and formulation of means of production of various bioproducts, has emerged as an alternative to minimize the disposal of tailings from various manufacturing industries, especially those from food. Ice creams are food products obtained from an emulsion of fats and proteins, with or without the addition of other ingredients and substances which have been subjected to freezing their production generates large-scale solid-liquid waste with a large amount of fat and other organic wastes. In this work I study was conducted to select an alternative means for lipase production by B. licheniformis (UCP 1014). And then the formulation of an alternative means of production, using waste from ice cream industry through a factorial design, 24. The tests occurred during 96h at 37oC The results indicated that the middle called C (glucose 1.0%, peptone 2.0%, 0.5% yeast extract, olive oil, 1.0%, NaNO3 0.1%, 0.1% KH2PO4, MgSO4. 7H2O 0.05%), indicating a lipase activity of 256 (U / ml) / min. Tests of the factorial design, the test indicated that 11 had the highest activity of 480 U / mL for lipase production. The results suggest the reuse of waste oil from the ice cream industry for the development and production of microbial lipase.

Les biofilms représentent une contrainte technologique dans le secteur industriel et sont à l'origine de nombreux cas d'infections chroniques dans le domaine médical. De ce fait, la compréhension des processus biologiques qui s'opèrent au sein de ces communautés microbiennes est un défi permanent. Des méthodes spécifiques, sensibles et diversifiées s'avèrent essentielles pour apporter une connaissance approfondie de l'organisation des biofilms en réponse à des facteurs environnementaux. La matrice extracellulaire qui englobe les microorganismes constitue un réseau complexe, et la description de sa composition et de sa structure constitue un enjeu majeur. Des outils de caractérisation in situ et non-destructifs des Substances ExoPolymériques (SEP) ont pu être proposés lors de ce travail de thèse : des méthodes de quantification ciblant spécifiquement chaque composant majeur de la matrice extracellulaire ont ainsi été développées et validées sur des biofilms modèles. Ces biofilms, choisis pour leur diversité en terme de matrice extracellulaire sont formés par les souches Pseudomonas aeruginosa ATCC 15442, Bacillus licheniformis CIP 110824 et Weissella confusa LBAE-UPS C39-2. Des critères communs ont été retenus pour guider le choix des marqueurs retenus pour développer les dosages : spécificité pour la détection de toutes les formes moléculaires d'une même famille biochimique, sensibilité pour la détection de faibles quantités de polymères dans des biofilms en microplaque, et possibilité d'observation en microscopie basée sur la détection de fluorescence. Une stratégie commune a été adoptée pour la quantification des exoprotéines (ePN), exopolysaccharides (ePS) et fibres amyloïdes (FA) de la matrice et a consisté à : (i) définir une molécule standard pour calibrer le test ; (ii) analyser d'éventuelles interférences en solution ; (iii) valider la faisabilité et la fiabilité du test in situ par la méthode des ajouts dosées réalisée sur chacun des biofilms modèles. Un composé naturel pro-fluorescent, l'épicocconone, a été retenu pour la quantification des exoprotéines. Le test a montré une limite de détection de 0,2 µg par puits, sans aucune interférence significative des autres composants majeurs du biofilms (ePS, ADNe, cellules). D'autre part, les protéines sous forme amyloïdes ont pu être détectées avec la même sensibilité que les non amyloïdes par ce marqueur. Par la suite, les exopolysaccharides ont été dosés en exploitant la réaction de Schiff, et la méthode a permis d'obtenir une limite de détection de 0,3 µg par puits. Les protéines amyloïdes bactériennes (FA) ont également été quantifiées au moyen du marqueur Thioflavine T. La k-caséine a été utilisée comme étalon, après fibrillation de la protéine native in vitro. / Biofilms are detrimental in many industrial and medical areas and understanding of biofilms processes has been a challenge for decades. Specific, sensitive and rapid methods for monitoring biofilm formation are essential for a deep knowledge of biofilm organization and response to environmental factors. In such complex network that constitutes the biofilm matrix, it has become a major issue to succeed in describing its composition and local structure to determine the interactions that govern the biofilm formation. Non-destructive and in situ methods for Exopolymeric Substances (EPS) characterization were proposed along this PhD work. The first aim of the study was to propose some quantitative tools that would specifically target each major compound of the biofilm matrix. Some performance criteria were commonly established to guide the choice of each dye and to validate each step of the analytical development. These requirements can be displayed in terms of biochemical specificity, sensitivity and applicability on fluorescence-based microscopy. A common experimental strategy was carried out to quantify the exoproteins (ePN), exopolysaccharides (ePS) and amyloid fibrils (AF) and aimed at (i) choosing a calibration standard; (ii) analyzing in vitro interferences; (iii) validating the practicability and the reliability of each proposed method for an in situ implementation on biofilms. This last criteria was verified by implementing the Standard Addition Method (SAM). For that purpose, a first method was developed to take advantage of a natural pro-fluorescent dye, called epicocconone, to quantify the ePN of three model bacterial biofilms formed by the strains Bacillus licheniformis CIP 110824, Pseudomonas aeruginosa ATCC 15442 and Weissella confusa LBAE-UPS C39.2. The three bacterial models were chosen because of their contrasted EPS matrix composition. This method showed a detection limit of about 0.2µg per well and no significant interference were revealed. Moreover the epicocconone assay was able to quantify the AF with the same sensitivity as the other proteins. Subsequently, exopolysaccharides from the same strains were assayed by applying the Periodic acid-Schiff reaction in a microplate format. This chemical reaction targets the majority of the hydroxyl groups of carbohydrate and allows to give an exhaustive estimation of the sugar content of the matrix with a detection limit of 0.3µg per well. Bacterial amyloids were also specifically quantified by the using of the benzothiazole dye Thioflavine T (ThT). An amyloidogenic protein, the ?-casein, was used as standard after an in vitro fibrillation of the native form.

L’encrassement microbiologique des membranes de filtration constitue une problématique majeure au sein de l’industrie laitière. La présence de bactéries sporulantes dans les biofilms se formant à la surface des membranes d’ultrafiltration (UF) peut engendrer une contamination des co-produits de la transformation laitière ayant des répercussions négatives sur leur conservation. L’environnement, la température de filtration ainsi que les propriétés de surface des membranes influencent l’adhésion des microorganismes sur celles-ci. Des travaux précédents ont suggéré qu’une plus grande rugosité du matériau membranaire favoriserait la présence de bactéries sporulantes du genre Bacillus. L'objectif de cette étude était de caractériser l'influence de l’usure membranaire sur la performance du procédé d’UF du lactosérum et sur la persistance des bactéries sporulantes à la surface des membranes. Des filtrations de lactosérum inoculé par Bacillus licheniformis ont été réalisées à l’échelle pilote, afin d’étudier l’adhésion de la bactérie à la surface de deux membranes d’UF en polyéthersulfone (PES) présentant un stade d’usure différent. Par la suite, une méthode qPCR ciblant le gène spo0A de la bactérie a été développée pour quantifier B. licheniformis à la surface des membranes. Les résultats de ces travaux ont montré que l’état d’usure des membranes à l’étude n’a pas d’impact sur la baisse de performance en cours de filtration du lactosérum. Bien qu’il eût été suggéré que B. licheniformis persiste à la surface des membranes suivant un cycle de nettoyage standard après une filtration de lactosérum sur une période de 20h à 50°C, il n’a pas été possible de démontrer une relation entre la rugosité membranaire et la rétention de bactéries sporulantes.Cependant, nos travaux ont permis la mise au point et l’optimisation de méthodologies de caractérisation des surfaces membranaires et de l’adhésion de bactéries sporulantes à la surface des membranes représentant des avancées importantes visant le développement de stratégies efficaces pour prévenir la contamination des spores dans les produits laitiers. / Biofouling of filtration membranes is a major issue in the dairy industry. Bacterial spores in biofilms adhering on membrane surfaces can induce a contamination of dairy fluids and ingredients. The presence of sporulating bacteria in biofilms is influenced by the dairy plant environment, the nature of the dairy fluid, the filtration parameters, and characteristics of membrane surfaces. Recent studies suggested a potential adhesion of sporulating Bacillusgenus to rougher membrane materials after whey ultrafiltration.The objective of the present study was to evidence the impact of membrane ageing on the persistence of sporulating bacteria on the surface of aged membranes. Ultrafiltration experiments with whey inoculated with Bacillus licheniformis were then performed to evaluate the impact of two polyethersulfone membranes at different state of ageing on bacterial adhesion. A real-time PCR method was developed targeting the sporulating gene spo0A of B. licheniformis to quantify the bacteria on membrane surface. Results suggest that membrane aging did not impact the filtration performance during whey processing. Although it was suggested that the persistence of the bacteria on membrane surfaces was observed following a cleaning-in-place procedure performed after 20h of filtration at 50°C,it was not possible to evidence that membrane roughness influenced the persistence of bacterial spores at the membrane surface.Moreover, the present study enabled the development and optimization of methods for characterization of surface properties and bacterial spore adhesion which will help to develop and improve strategies to prevent spore contamination in dairy fluids and products.

As mudanças climáticas estão causando prejuízos em vários setores da economia mundial. Na reunião da COP21, que teve como foco estas mudanças climáticas, participantes do mundo todo decidiram tomar atitudes urgentes para tentar conter aumento da temperatura média global. Dentro deste cenário, a produção e o consumo de energia têm uma importância central, onde fontes de energia renováveis vêm sendo preferidas às fontes de energias fósseis. O Brasil tem uma participação importante na geração de energia renovável mundial aportando um 40% do total de sua matriz energética. A degradação dos componentes da parede celular vegetal tem um vasto potencial na geração de biocombustíveis e outros compostos verdes a partir da celulose, hemicelulose e lignina. Para isto estudos das enzimas capazes de degradas estes componentes vem sendo realizados, com ênfase nas enzimas hidrolases de glicosídeos. Dentre as hidrolases, encontram-se as arabinanases, enzimas capazes de hidrolisar o arabinano, componente polissacídeo da hemicelulose, em L-arabinose. Neste trabalho, estudos envolvendo duas arabinanases de Bacillus licheniformis foram realizados, iniciando na etapa de clonagem dos genes. Os produtos foram transformados em Escherichia coli e expressos e purificados. A avaliação da estabilidade térmica indicou uma afinidade das enzimas por metais divalentes. Tentativas de cristalização resultaram na formação de um cristal, que possibilitou a determinação da estrutura uma das arabinanases. Através de ensaios bioquímicos, foi determinada a especificidade por substrato, temperatura e pH ótimos e a atividade frente a metais. Foi observado que as enzimas são seletivas para arabinano não ramificado, tem temperatura ótima em 45 e 40 graus, para BlAbn-1 e BlAbn-2, respectivamente, e pH ótimo em 8 e 7. Por último, foram realizados ensaios complementares de sinergismo e atividade oxidativa. Embora os ensaios de atividade oxidativa tenham sido inconclusivos, os ensaios de sinergismo mostraram que a enzima BlAbn-1 é capaz de aumentar em 30% a atividade do coquetel enzimático Accellerase 1500 sobre biomassa pré-tratada e sobre celulose pura. Este efeito é ainda maior na presença de sulfato de níquel. / Climate change is causing losses in different sectors of the world economy. At the meeting of COP21, focused on climate changes, participants from around the world decided to take urgent actions to try to halt the increase in global average temperature. Within this scenario, the production and consumption of energy are of central importance, where renewable energy sources have been preferred to fossil fuels. Brazil has an important role in the global renewable energy generation by contributing 40% of its total energy mix. The degradation of the components of plant cell wall has a vast potential in the generation of biofuels and other green chemical from cellulose, hemicellulose and lignin. Thus, studies of enzymes that degrade these components have been carried out, with emphasis on glycoside hydrolases. Among the hydrolases are the arabinanases, enzymes capable of hydrolyzing arabinan, a polysaccharide component of hemicellulose, in L-arabinose. In this work, studies involving two arabinanases from Bacillus licheniformis were carried out, starting in gene cloning step. The products were transformed into Escherichia coli, expressed and purified. The evaluation of the thermal stability of the enzymes showed an affinity for divalent metals. Crystallization attempts resulted in the formation of a single crystal, which made it possible to determine the crystal structure of one arabinanase. Through biochemical assays, it was determined the substrate specificity, optimum temperature and pH and activity against metals. It was observed that the enzymes are selective for non-branched arabinan, have optimum temperature at 45 and 40 degrees, to BlAbn-1 and BlAbn-2, respectively, and optimum pH of 8 and 7. Finally, additional tests were performed to evaluate the possible synergism and oxidative activity. Although the oxidative activity assays were inconclusive, the synergism tests showed that BlAbn-1 is able to increase by 30% the activity of the enzymatic cocktail Accellerase 1500 on pre-treated biomass and on pure cellulose. This effect is even greater in the presence of nickel sulfate.

As mudanças climáticas estão causando prejuízos em vários setores da economia mundial. Na reunião da COP21, que teve como foco estas mudanças climáticas, participantes do mundo todo decidiram tomar atitudes urgentes para tentar conter aumento da temperatura média global. Dentro deste cenário, a produção e o consumo de energia têm uma importância central, onde fontes de energia renováveis vêm sendo preferidas às fontes de energias fósseis. O Brasil tem uma participação importante na geração de energia renovável mundial aportando um 40% do total de sua matriz energética. A degradação dos componentes da parede celular vegetal tem um vasto potencial na geração de biocombustíveis e outros compostos verdes a partir da celulose, hemicelulose e lignina. Para isto estudos das enzimas capazes de degradas estes componentes vem sendo realizados, com ênfase nas enzimas hidrolases de glicosídeos. Dentre as hidrolases, encontram-se as arabinanases, enzimas capazes de hidrolisar o arabinano, componente polissacídeo da hemicelulose, em L-arabinose. Neste trabalho, estudos envolvendo duas arabinanases de Bacillus licheniformis foram realizados, iniciando na etapa de clonagem dos genes. Os produtos foram transformados em Escherichia coli e expressos e purificados. A avaliação da estabilidade térmica indicou uma afinidade das enzimas por metais divalentes. Tentativas de cristalização resultaram na formação de um cristal, que possibilitou a determinação da estrutura uma das arabinanases. Através de ensaios bioquímicos, foi determinada a especificidade por substrato, temperatura e pH ótimos e a atividade frente a metais. Foi observado que as enzimas são seletivas para arabinano não ramificado, tem temperatura ótima em 45 e 40 graus, para BlAbn-1 e BlAbn-2, respectivamente, e pH ótimo em 8 e 7. Por último, foram realizados ensaios complementares de sinergismo e atividade oxidativa. Embora os ensaios de atividade oxidativa tenham sido inconclusivos, os ensaios de sinergismo mostraram que a enzima BlAbn-1 é capaz de aumentar em 30% a atividade do coquetel enzimático Accellerase 1500 sobre biomassa pré-tratada e sobre celulose pura. Este efeito é ainda maior na presença de sulfato de níquel. / Climate change is causing losses in different sectors of the world economy. At the meeting of COP21, focused on climate changes, participants from around the world decided to take urgent actions to try to halt the increase in global average temperature. Within this scenario, the production and consumption of energy are of central importance, where renewable energy sources have been preferred to fossil fuels. Brazil has an important role in the global renewable energy generation by contributing 40% of its total energy mix. The degradation of the components of plant cell wall has a vast potential in the generation of biofuels and other green chemical from cellulose, hemicellulose and lignin. Thus, studies of enzymes that degrade these components have been carried out, with emphasis on glycoside hydrolases. Among the hydrolases are the arabinanases, enzymes capable of hydrolyzing arabinan, a polysaccharide component of hemicellulose, in L-arabinose. In this work, studies involving two arabinanases from Bacillus licheniformis were carried out, starting in gene cloning step. The products were transformed into Escherichia coli, expressed and purified. The evaluation of the thermal stability of the enzymes showed an affinity for divalent metals. Crystallization attempts resulted in the formation of a single crystal, which made it possible to determine the crystal structure of one arabinanase. Through biochemical assays, it was determined the substrate specificity, optimum temperature and pH and activity against metals. It was observed that the enzymes are selective for non-branched arabinan, have optimum temperature at 45 and 40 degrees, to BlAbn-1 and BlAbn-2, respectively, and optimum pH of 8 and 7. Finally, additional tests were performed to evaluate the possible synergism and oxidative activity. Although the oxidative activity assays were inconclusive, the synergism tests showed that BlAbn-1 is able to increase by 30% the activity of the enzymatic cocktail Accellerase 1500 on pre-treated biomass and on pure cellulose. This effect is even greater in the presence of nickel sulfate.

Made available in DSpace on 2017-06-01T18:20:38Z (GMT). No. of bitstreams: 1 dissertacao_ladiel_luiz_pedrozo_tavares.pdf: 1056271 bytes, checksum: 10d14c9baea2d68dab838c2177a14816 (MD5) Previous issue date: 2011-10-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterium Bacillus licheniformis is a versatile, and has been used currently in production of various bitecnológicos. Lipases ( E.C.3.1.1.3) are enzymes found in nature and may be obtained from animal sources, plants and microbes, the latter being widely used due to several advantages presented against the other. The use of industrial wastes for production and formulation of means of production of various bioproducts, has emerged as an alternative to minimize the disposal of tailings from various manufacturing industries, especially those from food. Ice creams are food products obtained from an emulsion of fats and proteins, with or without the addition of other ingredients and substances which have been subjected to freezing their production generates large-scale solid-liquid waste with a large amount of fat and other organic wastes. In this work I study was conducted to select an alternative means for lipase production by B. licheniformis (UCP 1014). And then the formulation of an alternative means of production, using waste from ice cream industry through a factorial design, 24. The tests occurred during 96h at 37oC The results indicated that the middle called C (glucose 1.0%, peptone 2.0%, 0.5% yeast extract, olive oil, 1.0%, NaNO3 0.1%, 0.1% KH2PO4, MgSO4. 7H2O 0.05%), indicating a lipase activity of 256 (U / ml) / min. Tests of the factorial design, the test indicated that 11 had the highest activity of 480 U / mL for lipase production. The results suggest the reuse of waste oil from the ice cream industry for the development and production of microbial lipase. / O Bacillus licheniformis é uma bactéria versátil, e tem sido utilizada atualmente na produção de diversos produtos bitecnológicos. As lipases (E.C.3.1.1.3 ) são enzimas encontradas na natureza, podendo ser obtidas de fontes animais, vegetais e microbianas, sendo essas últimas bastante utilizadas devido a série de vantagens apresentadas frente as demais. A utilização de rejeitos industriais para produção e formulação de meios de produção de diversos bioprodutos, tem surgido como uma alternativa de minimizar o descarte de rejeitos das diversas indústrias de produção, principalmente as de origem alimentícia. Sorvetes são produtos alimentícios obtidos a partir de uma emulsão de gorduras e proteínas, com ou sem a adição de outros ingredientes e substâncias que tenham sido submetidas ao congelamento, a sua produção em larga escala gera resíduos sólido-líquidos com uma grande quantidade de gorduras e outros resíduos orgânicos. Neste trabalho, inicialmente foram realizados estudos para selecionar meio alternativo para produção de lipase por B. licheniformis (UCP 1014). E a seguir a formulação de um meio alternativo de produção, utilizando um resíduo da indústria de sorvete através de um planejamento fatorial de 24. Os ensaios ocorreram durante 96h, a 37oC Os resultados obtidos indicaram que o meio denominado de C (glicose 1,0%, peptona, 2,0%, extrato de levedura 0,5%, óleo de oliva, 1,0%, NaNO3 0,1%, KH2PO4 0,1%, MgSO4. 7H2O 0,05%), apresentando uma atividade lipolítica de 256 (U/ml)/min. Os ensaios referentes ao planejamento fatorial, indicaram que o ensaio 11 apresentou a maior atividade de 480 U/mL, para a lipase produzida. Os resultados obtidos sugerem o reaproveitamento de resíduos oleosos provenientes da indústria de sorvete para formulação e produção de lipase microbiana.