This diploma thesis is focused on the study of the preparation for fat separators and wastewater pipes that contains the microorganisms with lipolytic activity. Theoretical part of this thesis describes lipases, microorganisms producing this enzymes and usage of lipases. In this part possibilities of identification of microorganisms are presented too. The practical part is concerned with the study of commercial preparation Sany Duo Spezial with proven presence of microorganisms with lipolytic activity. These microorganisms were identified by means of the PCR method. This method identified mictoorganisms like genus Bacillus sp. Next characterization of the preparation was focused on the determination of COD and the investigation of the influence of various conditions of culture medium on the lipases production and their activity. The effect of temperature, ions and pH was studied. Lipolytic activity was determine spectrophotometricaly with usage of p-nitrophenyllaurate whitch dissociates to yellow product p-nitrophenol.

Submitted master’s thesis is focused on the study of possibilities of lipids degradation in particular wastewaters originating in food industry or restaurants. The effort is given to the employment of lipolytic activity presenting microorganisms. In the literature review, wastewater treatment with aim on the sludge management and fat separators are described, as a way how to pre-treat these wastewaters. In this part the enzymes lipases of microbial origin are researched from point of view of their production conditions and possible applications. The experimental part is dedicated to the research of optimization of cultivation conditions for lipases production employing selected microorganisms (Bacillus subtilis, Geobacillus thermodenitrificans, G. thermocatenulatus and mixed bacterial culture Thermus and Bacillus) and a commercial formulation (Sany Duo Spezial). Lipases production and growth of microorganisms are determined spectrofotometrically on various concentrations of lipids. Moreover, employing the solid nutrition medium, the effect of detergents onto the Bacillus subtilis culture was assessed, since detergents are generally abundant in this particular wastewaters. As a conclusion, vide supra mentioned microorganisms were characterized according to their abilities to degrade triacylglycerols.

Les polyesters aliphatiques, comme le poly(acide malique) et ses dérivés, sont une famille de polymères aux propriétés de bio(comptabilité) et de bio(dégradabilité) remarquables, qui en font des candidats de choix pour l'élaboration de systèmes de vectorisation de principes actifs. Généralement, ces polymères sont synthétisés via des réactions de polymérisation utilisant des amorceurs, voir des catalyseurs, organiques, organométalliques ou métalliques. La présence de ces molécules, même à l'état de traces, peut être à l'origine d'une toxicité non souhaitée. Par conséquent, l'utilisation de biocatalyseurs, comme les lipases, se développe pour apporter une solution à cet inconvénient. Cependant, cette voie de synthèse enzymatique fait face à d'autres problèmes, tels qu'une polymérisation moins bien maîtrisée et des polymères de masses molaires faibles. Cette thèse a donc pour objectif de mettre au point une voie de polymérisation du malolactonate de benzyle utilisant la lipase de pancréas de porc (PPL) comme amorceur. Dans un premier temps, nous avons optimisé certains paramètres réactionnels permettant d'obtenir des poly(malate de benzyle) , PMLABe, de masses molaires suffisamment élevées pour que ces polymères puissent être utilisés dans la formulation de vecteurs de principes actifs, grâce à l'utilisation et l'extrapolation d'un plan d'expérience. Nous nous sommes ensuite intéressés à la compréhension du mécanisme réactionnel de la polymérisation enzymatique du malolactonate de benzyle, une β-lactone β-substituée. Les différentes études menées ont permis d'approfondir notre connaissance dans ce domaine. Deux mécanismes ont été proposés et des expériences sont en cours pour confirmer l'un d'entre eux. Finalement, comme l'objectif initial est de proposer une méthode de synthèse de dérivés du PMLA plus biocompatibles conduisant à des polymères sans résidus d'amorceurs chimiques toxiques, nous avons comparé les activités biologiques de nanoparticules préparées à partir de PMLABe synthétisés par voie chimique et par voie enzymatique. Pour cela, nous avons mesuré la captation de ces nanoparticules, encapsulant une sonde de fluorescence, par des cellules hépatiques HepaRG. Puis, nous avons évalué la toxicité aiguë et la toxicité chronique de ces nanoparticules vis-à-vis des cellules HepaRG. Ces études ont permis de mettre en évidence certaines propriétés des nanoparticules ayant une influence sur la survie cellulaire et le métabolisme des cellules HepaRG. De la compréhension théorique aux applications potentielles, cette thèse apporte des connaissances sur la polymérisation enzymatique des lactones substituées, un domaine peu décrit dans la littérature. / Aliphatic polyesters, like poly(malic acid)and its derivatives, are a family of polymers with outstanding properties, such as bio(degradability) and bio(compatibility). Therefore, these polyesters can be considered as excellent candidates for the design of drug carriers. These kinds of polymers are usually synthesized thanks to polymerization reactions using organic, organometallic or metallic initiators or catalysts. The presence of such molecules, even in trace amounts, can cause undesired toxicities. Therefore, the use of biocatalysts, like lipases, is attracting more and more interest and research work to circumvent this problem. However, this enzymatic polymerization method has to face to other issues, such as a lower controlled of the polymerization process and polymers with lower molar masses. Therefore, this PhD research work aimed at setting up the enzymatic polymerization of benzyl malolactonate, using porcine pancreatic lipase (PPL). Firstly, we have optimized some reactional parameters allowing to obtain poly(benzyl malate), PMLABe, with molar masses adapted to their uses for the design of drug carriers, thanks to a Design of Experiments (DoE) and its extrapolation. We were then interested by the comprehension of the enzymatic polymerization mechanism of the benzyl malolactonate. The different studies we carried out allowed us to deepen our knowledges of such enzymatic polymerization. Two non-canonical mechanisms were proposed and further experiments are in progress to confirm the one which is the more probable. Finally, because our initial goal was to propose a more biocompatible polymerization method to obtain PMLABe free of traces of chemical initiator, we compared biologic activities of different nanoparticles prepared from PMLABe synthesized using chemical or enzymatic pathway. For that, we have first measured the uptake of these nanoparticles encapsulating a fluorescent dye, by the hepatic cells HepaRG. Then, we have studied the acute and chronic toxicity of the nanoparticles on the HepaRG cells. Results of these studies have highlighted that certain properties of the nanoparticles and/or of the polymers which constituted them have an influence on the cells viability and on the cells metabolism. From the theoretical mechanism to the probable applications, this thesis brings knowledge about the enzymatic polymerization of substituted lactone, a field poorly described in the literature.

Les matériaux utilisés pour les applications biomédicales doivent être biocompatibles, et idéalement biodégradables. Les acides biliaires proviennent de sources naturelles et sont présents dans le corps humain. De plus, les polyetsers composés en partie de ces molécules possèdent des liens hydrolysables, une mémoire de forme thermique, et leur flexibilité peut être variée. Jusqu’à présent, la synthèse de ces matériaux exigeait l’utilisation de catalyseurs contenant des métaux de transition lourds pour l’étape de macrocyclisation. Puisque la polymérisation par ouverture de cycle nécessite des précurseurs cycliques, l’étape de lactonisation fut réalisée par voie enzymatique, au lieu d’utiliser des catalyseurs à plus grande toxicité. De plus, une seule étape enzymatique a pu remplacer deux étapes de synthèse organique, avec un rendement de 58 % et l’obtention d’un matériel transparent. Ces macrocycles nouvellement obtenus ont par la suite été polymérisés par ouverture de cycle, de façon similaire à la technique élaborée par notre groupe en 2013, tout en optimisant la durée de réaction. En 15 heures, une masse molaire relativement grande de 40 000 g/mol fut obtenue, tout en maintenant la dispersité sous 1.4 et la température de transition vitreuse à 12 °C. Pour valider le principe de cyclisation et de polymérisation enzymatique, les conditions optimales pour combiner l’acide thapsique et le 1,10-decanediol furent préalablement déterminées. Entre autres, la durée de réaction et la quantité d’enzyme nécessaire furent analysées. Les polymères semi-crystallins obtenus possèdent aussi de grandes masses molaires et de basses dispersités. Or, il est possible d’utiliser un enzyme à la fois pour la fermeture et pour l’ouverture de cycle de molécules rigides à cœur stéroïdal, telles que les acides biliaires. Cette synthèse permet la production de matériaux plus biocompatibles, tout en favorisant plusieurs principes de chimie verte. / Materials used in biomedical applications need to be biocompatible and ideally biodegradable. Bile acids are natural occurring compounds found in humans, and their polyesters possess hydrolyzable bonds, thermal shape memory and tunable flexibility. Until now, the synthetic pathway to obtain such materials required transition metal catalysts for the macrocyclization step, which is necessary to perform ring-opening polymerization (ROP). To circumvent the need for such catalysts, enzymatic ring closing was performed using lipases. Conveniently, two synthetic steps were replaced with a single step, using a renewable and reusable catalyst, with 58 % yield and a colorless product. The bile acid-containing macrocycles were then enzymatically polymerized as described in our previous work, while optimizing the reaction time. In 15 hours, relatively high Mw of 40 000 g/mol were obtained, while maintaining the dispersity ≤ 1.4 and a glass transition temperature of about 12 °C. As a proof-of-concept, conditions for the enzymatic ring closure of thapsic acid with 1,10-decanediol were determined beforehand. While optimizing for enzyme amount and reaction time, enzymatic ROP conditions to obtain di- and tetralactones from these monomers were established. The resulting semi-crystalline polymers also possess relatively high molecular weight and low dispersity. Hence, the use of lipases for both ring-closing and ring-opening reactions now shows potential for large, rigid moieties in addition to more mobile structures, using the same enzyme. This is a step towards the production of more biocompatible polymers, with a synthetic pathway that follows many green chemistry principles.

O presente trabalho teve como objetivo o aprimoramento da transesterificação do óleo de palmiste por rota etílica, por meio da atuação simultânea de lipases de diferentes fontes microbianas, visando a produção de biodiesel com teores residuais de monoacilglicerol (MAG) e diacilglicerol (DAG) dentro dos limites exigidos pela legislação ANP no 45/2014 para biocombustíveis. Para tal propósito, as lipases foram imobilizadas em suporte híbrido sílica-hidroxietilcelulose (SiO2- HEC) e os ensaios foram iniciados em regime descontínuo em reator de tanque agitado, temperatura fixa de 45 °C e razão molar óleo:álcool de 1:8. As lipases de Burkholderia cepacia, Pseudomonas fluorescens e Thermomyces lanuginosus, foram utilizadas de maneira isolada e em associação, com base no conhecimento prévio do bom desempenho destas enzimas para a produção de biodiesel. Conforme a capacidade de atuar em diferentes posições da molécula de triacilglicerol, as associações formaram combi-lipases de mesma especificidade (B. cepacia e P. fluorescens) e de diferente especificidade (B. cepacia e T. lanuginosus), com proporções de cada lipase variando de 75, 50 e 25% em relação ao seu par enzimático. Os melhores resultados foram observados quando utilizadas ambas as combinações em proporções equivalentes. Nestas condições, foram obtidas conversões similares (?98%) e ausência de DAG, com redução da viscosidade cinemática do óleo de palmiste de 30,35 para aproximadamente 4,00 mm2/s. Embora as conversões tenham sido próximas, a combi-lipase de diferente especificidade apresentou menor teor de MAG (1,68%, m/m), sendo escolhida como biocatalisador para o estudo em reatores de leito fixo em regime contínuo. Nesta etapa, foram realizados ensaios para testar os efeitos da razão molar óleo:álcool (1:12, 1:10 e 1:8) e do tempo espacial (16 e 14 h) na conversão, qualidade e produtividade do biodiesel. O melhor desempenho foi apresentado pela razão molar de 1:8 com tempo espacial de 16 h, proporcionando teor em MAG residual de 0,62% (m/m), conversão de 98% e produtividade de 30,9 mmolésteres.gcatalisador -1.h-1. Com relação à qualidade do biodiesel formado em termos de viscosidade cinemática (4,22 mm2/s), massa específica (870 kg/m3) e teor residual de MAG (0,62%, m/m), foi verificada adequação aos parâmetros exigidos pela ANP (3-6 mm2/s, 850-900 kg/m3 e <= 0,7%, m/m, respectivamente). / The objective of the present work was to improve the transesterification of palm kernel oil by ethanolic route through simultaneous action of lipases from different microbial sources, aiming to produce biodiesel with residual levels of monoacylglycerol (MAG) and diacylglycerol (DAG) within the limits required by ANP regulation 45/2014 for biofuels. For this purpose, lipases were immobilized on silica-hydroxyethylcellulose hybrid support (SiO2-HEC) and the assays were first carried out in a stirred tank batch reactor, at fixed temperature of 45 oC and oil -to-alcohol molar ratio of 1:8. The lipases from Burkholderia cepacia, Pseudomonas fluorescens and Thermomyces lanuginosus isolated and in association were used, based on the previous knowledge of the good performance of these enzymes for the biodiesel production. According to the ability to act at different positions of the triacylglycerol molecule, the associations formed combilipases of the same specificity (B. cepacia and P. fluorescens) and different specificities (B. cepacia and T. lanuginosus) at proportion of each lipase ranging from 75, 50 and 25% relative to their enzymatic pair. The best results were attained when both combinations were used at equivalent proportions. Under these conditions, similar conversions (?98%) and absence of DAG were obtained, which was consistent with the kinematic viscosity reduction of the palm kernel oil from 30.35 to 4.00 mm2/ s, approximately. Although similar conversions were attained, the combi-lipase of different specificities provided lower MAG contentes (1.68 wt%), being chosen as a biocatalyst for performing continuous runs in packed-bed reactor. In this step, the effects of the molar ratios oil-to-alcohol (1:12, 1:10 and 1:8) and space-times (16 and 14 h) on biodiesel production, quality and productivity were evaluated. The best performance was achieved by using molar ratio of 1:8 and space-time of 16 h, yielding residual MAG contents of 0.62 wt%, conversion of 98% and productivity of 30.9 mmolester.gcatalyst-1.h-1. The quality of biodiesel produced in terms of kinematic viscosity (4.22 mm2/s), specific mass (870 kg/m3) and residual MAG content (0.62 wt%) was found in accordance of ANP resolution (3-6 mm/ s, 850-900 kg/m3 and <= 0.7 wt%, respectively).

Ce travail étudie l’obtention de phospholipides structurés enrichis en DHA et en acide caprylique (PC DHA-C8) par voie enzymatique. Deux voies de synthèse sont étudiées, l’acidolyse et l’estérification. Suite à un criblage enzymatique, la lipase retenue pour les deux voies de synthèse est TL-IM. Une optimisation des paramètres de la réaction d’acidolyse a été réalisée entre l’acide caprylique (C8:0) et la phosphatidylcholine de tournesol (PC) par le biais d’un plan d’expériences. Les conditions optimales déterminées sont une température de 38°C, une activité de l’eau de 0,7, une quantité d’enzyme de 15% de la masse en substrat ainsi qu’un rapport molaire C8:0/PC de 18. Ces conditions ont ensuite été utilisées pour l’acidolyse de phospholipides microalgaux riches en DHA issus de la microalgue Tisochrysis lutea afin d’obtenir de la PC DHA-C8. Les résultats n’ont pas été concluants. L’autre voie de synthèse étudiée est l’estérification par des lipases de la GPC, de l’acide caprylique et du DHA en milieu fondu. Cette réaction a été optimisée par la technique du pas par pas. Les paramètres étudiés sont la température, la quantité d’enzyme, le rapport molaire GPC/C8:0/DHA et l’application d’un vide. Pour l’obtention de PC DHA-C8, il faut fixer chacun de ces paramètres respectivement de la sorte : 45°C, 20% d’enzyme, un rapport molaire de 1/3/15 et un vide de 100 mbar. La production de PC DHA-C8, bien qu’optimisée ne dépasse pas 2% de rendement. Cependant, durant cette expérience, il a été constaté une forte production de LPC DHA, atteignant 16% sans optimisation des paramètres de synthèse. / The enzymatic synthesis of structured phsopholipids enriched in DHA and caprylic acid (PC DHA-C8) is studied. Two different ways are studied, acidolysis and esterification. An enzymatic screening led to the choice of the immobilized lipase from Thermomyces lanuginosa (TL-IM) for the 2 reactions. Parameters of the acidolysis reaction between carpylic acid (C8:0) and sunflower phosphatidylcholine (PC) were optimized by means of an experimental design. The optimum conditions determined are a temperature of 38°C, an aw of 0.7, an amount of enzyme of 15% of the mass of substrate and a molar ratio of C8:0/PC of 18. These conditions were applied to the acidolysis of microalgal phospholipids from T. lutea, rich in DHA, in order to produce PC DHA-C8. The other studied reaction is the lipase catalyzed esterification of GPC with C8:0 and DHA in a solvent-free medium This reaction has been optimized by studying each factor independently. The parameters studied are the temperature, the amount of lipase, the molar ratio GPC/C8:0/DHA and the use of reduced pressure. In order to obtain PC DHA-C8, each of theses parameters are respectively set at: 45°C, 20% of enzyme, a molar ratio of 1/3/15 and a pressure of 100 mbar. The production of PC DHA-C8, although optimized, does not exceed a yield of 2%. However, during this experiment, a high production of LPC DHA is observed, up to 16% without optimization of the synthesis parameter.

Made available in DSpace on 2017-07-10T18:08:17Z (GMT). No. of bitstreams: 1 Jamal Abd Awadallak.pdf: 7067148 bytes, checksum: 8e321c5aa3d2b9b3eeaafb2fd17b9d47 (MD5) Previous issue date: 2012-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diacylglycerol rich oils have its organoleptic characteristics very similar to those of conventional edible oils, but these oils do not tend to accumulate in the body, even when consumed in high quantities, making them a great resource in the fight against obesity. Palm oil ranks first the world production of edible oils mainly due to its low cost. This work aimed to propose a new technology for enzyme production using diacylglycerol lipase Lipozyme RM IM and ultrasound to promote water in oil emulsions, which increases the interfacial area of the system leading to higher reaction rates compared to conventional enzymatic processes. . The reactions were carried out at 55 °C with two different methods. First, the reaction system was exposed to ultrasonic waves for the whole reaction time, which led to enzymatic inactivation and water evaporation. Ultrasound was then used to promote emulsification of the water/oil system before the hydrolysis reaction, avoiding contact between the probe and the enzymes. Achieved conversions were superior to the conventional method further hydrolysis rate when the ultrasound is employed for emulsion formation was significantly greater. For 12 hours of reaction the conversion was 85% higher than the conventional method and 15% higher for a period of 24 hours of reaction. . An experimental design was used to optimize the ultrasound-related parameters and maximize the hydrolysis rate, and in these conditions, with a change in equilibrium, DAG production was evaluated.Better reaction conditions were achieved for the second method: 11.20 wt% (water+oil mass) water content, 1.36 wt% (water+oil mass) enzyme load, 12 h of reaction time, 1.2 min and 200 W of exposure to ultrasound. In these conditions diacylglycerol yield was 37.69 wt%. / Óleos enriquecidos com diacilglicerol possuem características organolépticas muito semelhantes às dos óleos comestíveis convencionais, porém, estes óleos não tendem a se acumular no organismo, mesmo quando consumidos em altas quantidades, tornando-os um grande recurso no combate à obesidade. O óleo de palma está no topo da produção mundial de óleos comestíveis principalmente devido ao seu baixo custo. Este trabalho teve como objetivo propor uma nova tecnologia para a produção enzimática de diacilglicerol empregando a lipase Lipozyme RM IM e utilizando ultrassom como gerador de emulsões de água em óleo, o que aumenta a área interfacial do sistema conduzindo a maiores taxas de reação em relação aos processos enzimáticos convencionais. A hidrólise parcial do óleo de palma foi realizada em meio livre de solventes a 55 °C em duas etapas distintas e comparadas com reações em condições semelhantes sem o uso do ultrassom. Primeiramente o sistema reacional foi exposto às ondas ultrassônicas, o que levou a taxas iniciais de reação elevadas, porém, as conversões obtidas foram baixas, em função da desativação enzimática e da evaporação de água, pelo longo período de exposição ao ultrassom. Posteriormente, utilizou-se o ultrassom para gerar emulsões antes a etapa reacional, não permitindo seu contato com o sistema contendo a enzima. As conversões obtidas foram superiores ao método convencional, além disso, taxa de hidrólise quando se empregou o ultrassom para a formação de emulsões foi significativamente maior. Para 12 horas de reação a conversão foi 85% superior ao método convencional e de 15% superior para um período de 24 horas de reação. Foi desenvolvido um planejamento fatorial, o delineamento central composto rotacional para avaliar os efeitos das variáveis tempo de exposição ao ultrassom, potência do ultrassom e razão água/óleo na conversão em ácidos graxos livres da reação, sendo que a razão água/óleo e o produto tempo x potência apresentaram os maiores efeitos. Nas melhores condições, foi produzido um óleo concentrado com 37,69% de DAG em de 12h de reação, exposto ao ultrassom por 1,2min à 200W.

No description available.

El presente trabajo de Tesis tiene como finalidad analizar el potencial de los líquidos iónicos como medios de reacción y separación en reacciones de transesterificación enzimáticas. Los ILs inmiscibles con agua han resultado medios muy adecuados para llevar a cabo la síntesis de ésteres catalizada por la lipasa B de Candida antarctica (CALB), incrementando la actividad y selectividad enzimática con respecto a los valores obtenidos en disolventes orgánicos convencionales como n-hexano. El uso de líquidos iónicos miscibles con agua ha dado lugar a menores valores de actividad enzimática que n-hexano, aunque la selectividad en estos medios es mucho mayor. Los ILs se han revelado también como prometedores disolventes en su empleo en procesos de separación, más concretamente como fase líquida en membranas líquidas soportadas (SLMs). Se han desarrollado SLMs basadas en ILs altamente estables que permiten llevar a cabo la separación selectiva de sustratos y productos de reacciones de transesterificación. / The main objective of this thesis is to analyze the potential use of ionic liquids as reaction and separation media in lipase-catalysed transesterification reactions. The use of ionic liquids in biocatalytic processes has been shown to be an environmentally attractive alternative to classical organic solvents. Water-immiscible ionic liquids appeared as suitable media for the transesterification reactions catalysed by Candida antarctica lipase B (CALB), increasing the activity and selectivity with respect to the values obtained with a classical organic solvent, n-hexane. The use of water-miscible ionic liquids led to lower activities than obtained in n-hexane, although the selectivity values were much higher in these media. Ionic liquids have also been shown to be promising solvents for the use in separation processes, more specifically as liquid phase in supported liquid membranes (SLMs). Highly stable SLMs based on ionic liquids have been succesfully used for the selective separation of different organic compounds which are substrates and products of transesterification reactions.

La biodisponibilité des nutriments lipidiques dépend d'un processus physico-chimique et enzymatique complexe : digestion par les lipases dans l'estomac puis l'intestin, absorption par les entérocytes et transport vers les cellules utilisatrices. L'altération physiologique (nouveau-né, personne âgée) ou pathologique (mucoviscidose, pancréatite) de ce processus réduit la biodisponibilité des acides gras essentiels, indispensables au développement et au fonctionnement des cellules de l'organisme. Ce mémoire présente des stratégies permettant d'améliorer la biodisponibilité des nutriments lipidiques chez l'insuffisant pancréatique, en utilisant les propriétés physico-chimiques des lipides. En effet, elles déterminent des caractéristiques primordiales de l'interface lipidique (superficie, composition), au niveau de laquelle se déroule l'hydrolyse enzymatique. Nos travaux montrent, in vitro dans des conditions proche de la physiologie, que le type de phospholipide entrant dans la composition d'une émulsion de trioléine, ou l'ajout d'un type donné d'acide gras libre dans le mélange lipidique avant l'émulsification, modifie la taille et le potentiel zêta des globules lipidiques, influence l'action des lipases gastrique, pancréatique et stimulée par les sels biliaires, et module l'absorption des acides gras par des cellules Caco-2. Un effet majeur est obtenu avec le lysophosphatidylinositol, rendant ce lipide potentiellement utilisable en nutrition clinique. Il présente un comportement interfacial étonnant (aire moléculaire, compressibilité) et son mécanisme d'action associe des effets indirects (modifications de l'interface lipidique) et directs (interactions avec les lipases).