O presente trabalho teve como objetivo verificar a potencialidade do óleo de macaúba como matéria-prima para síntese de biodiesel pela rota enzimática. Utilizou-se como proposta a transesterificação do óleo de macaúba com etanol mediada pela enzima lipase em fluxo contínuo empregando reator de leito fixo, visando obter amostras de biodiesel com propriedades adequadas à sua utilização como biocombustível. A enzima selecionada para desenvolvimento experimental foi a lipase microbiana de Burkholderia cepacia imobilizada em suporte híbrido não comercial (SiO2-PVA). Foram testados reatores de leito fixo de um estágio e dois estágios. A primeira etapa do trabalho foi direcionada para testes visando avaliar a influência da razão entre a altura (l) e o diâmetro (d) do reator de leito fixo na etanólise do óleo de macaúba. As reações foram operadas continuamente por 20 dias, utilizando óleo: etanol numa razão molar de 1:12 na ausência de solvente e tempo espacial de 14h. Dois reatores foram testados: Reator A (l = 55 mm e d = 15 mm) e Reator B (l = 210 mm e d = 14 mm), apresentando relação geométrica (l/d) de 3,7 e 15, respectivamente. Os dados obtidos indicaram influência das dimensões da coluna empacotada na produção de biodiesel e nas condições testadas, a maior razão altura/ diâmetro não interferiu na transferência de massa do fluido através da coluna. O melhor desempenho foi obtido no sistema experimental que empregou o reator B, atingindo 89,7 ± 4,8% de rendimento e 40,4 ± 2,2 mgéster.gmeio -1.h--1 de produtividade. Na sequência o trabalho foi direcionado para a execução de testes empregando reatores de leito fixo (Reator B) duplo estágio incorporando uma coluna empacotada com resina catiônica (Lewatit® GF 202) para remover o glicerol formado e proporcionar um incremento na formação de ésteres de etila em relação ao primeiro estágio. O desempenho do reator foi avaliado para diferentes tempos espaciais (10 a 16h), mantendo fixas as demais condições operacionais (substrato constituído de óleo de macaúba e etanol na razão molar óleo: etanol de 1:12 e temperatura de 50 ºC). O funcionamento do sistema foi comprovado quantitativamente para tempos espaciais no sistema igual a 16h, resultando em valores médios de produtividade de 36,7 ??2,4 mgéster.gmeio -1.h-1com perdas mínimas de matéria-prima (rendimento de transesterificação = 96,3 ??2,1%), sem redução de eficiência durante 25 dias de operação. As amostras de biodiesel purificadas apresentaram baixos teores de monoacilgliceróis (3,8%) ausência de diacilgliceróis e viscosidade cinemática média de 5,8 ± 0,3 mm2.s-1, atendendo as normas vigentes pela resolução ANP n°14/2012, que estabelece viscosidade cinemática do B100 na faixa entre 3,0 - 6,0 mm2.s-1. O biocatalisador foi estável quanto suas características morfológicas e catalíticas, revelando tempo de meia-vida de 423 h. Desta forma, a configuração do sistema reacional constituído por reator de leito fixo duplo estágio com a remoção simultânea de glicerol tem grande potencial para atingir elevado rendimento de transesterificação, aumentando a produtividade de biodiesel e consequentemente diminuindo o custo do processo industrial. Em geral, os resultados foram promissores e mostraram o potencial do óleo de macaúba para ser usado como matéria-prima para a produção de biodiesel em fluxo contínuo. / The present study aimed at assessing the potential of macaw palm oil as a raw material for the synthesis of biodiesel by enzymatic route. The proposed experimental was to develop a process that was able to transesterify the macaw palm oil with ethanol by immobilized lipase in packed bed reactor under continuous flow, in order to obtain biodiesel having suitable properties to be used as a fuel. The enzyme chosen for the development of this work was the microbial lipase from Burkholderia cepacia immobilized on non-commercial hybrid matrix SiO2-PVA. Single and two stages packed bed reactors were tested. Initially the influence of the reactor dimensions and ratio between height (l) and diameter (d) in the performance of the ethanolysis of macaw palm oil was assessed. Tests were carried out using two reactors (A and B) having different geometric relations: Reactor A (l = 55 mm and d = 15 mm) and Reactor B (l = 210 mm and d = 14) which corresponded to height/diameter (l/d = 3.7 and l/d = 15), respectively. Runs were performed continuously for 20 days using substrate containing oil to ethanol molar ratio of 1:12 in a solvent-free system and fixed space time of 14h. Data suggested that the dimensions of the packed column had a slight influence on the biodiesel production and under the conditions tested, the highest relation (l/d = 15) did not affect the fluid mass transfer throughout the reactor column. Under these conditions runs carried out in the reactor B provided average yields of 89.7 ± 4.8% and productivities of 40.4 ± 2.2 mgester?g-1?h-1. Following this, a two-stage packed bed reactor incorporating a column with cationic resin (Lewatit® GF 202) to remove the glycerol formed as by-product was used. The reactor performance was quantified for four different flow rates corresponded to spatial times from 10 to 16 h. For each condition, the influence of spatial times in the ethyl esters formation, transesterification yields and productivities were determined. The reactor operation was demonstrated for spatial time igual to 16 h, attaining ethyl ester formation of 58.1?2.1 wt%, transesterification yields of 96.3 ??2.1% and productivities of 36.7 ??2.4 mgester?g-1?h-1 with no significant reduction in the efficiency during 25 days. The purified samples showed residual levels of monoglycerides (3.8 wt %), absence of diglycerides and average viscosity values of 5.8 mm2/s which can be considered appropriated according to Brazilian resolution ANP n° 14/2012. The immobilized lipase on SiO2-PVA was found to be stable regarding its morphological and catalytic characteristics, showing half-life time (t1/2) higher than 423 h. Therefore, the continuous packed-bed reactor connected in series with simultaneous glycerol removal has a great potential to attain high level of transesterification yields, raising biodiesel productivity, consequently decreasing industrial process cost. Overall, the results were promising and showed the potential of macaw palm oil to be used as feedstock for biodiesel production under continuous flow.

Mono e diacilgliceróis são produtos empregados na indústria alimentícia, farmacêutica, cosmética e química como emulsificantes e melhoradores de viscosidade de produtos alimentícios, cosméticos e farmacêuticos. No entanto, a forma mais usual de obtê-los é por síntese química, o que acaba rendendo produtos finais caros e com atributos de qualidade, rendimento e de aplicabilidade tecnológica inferiores aos esperados. A busca por formas de obtenção mais racionais, eficientes e com melhor padrão de qualidade destes produtos foi o objetivo principal do trabalho, por meio de hidrólise parcial enzimática, que necessita de condições de reação mais brandas. Foram avaliadas a hidrólise enzimática descontína, empregando como substrato a trioleína técnica, e a hidrólise enzimática descontínua-alimentada, usando como substrato o óleo de girassol médio oléico. Foi utilizada, em ambos processsos, a lipase imobilizada sn-1,3 específica Lipozyme RM IM (de Rhizomucor miehei). A caracterização dos padrões e dos substrados, bem como o acompanhamento da formação dos produtos da hidrólise enzimática foram feitos por determinação da porcentagem de hidrólise, cromatografia em camada delgada (TLC), dos perfis das curvas de fusão e cristalização por calorimetria diferencial de varredura (DSC), cromatografia gasosa (CG) e cromatografia de exclusão de tamanho de alto desempenho (HPSEC). Os parâmetros de hidrólise descontínua foram o tempo de reação, a temperatura e a concentração inicial de substrato. Os parâmetros de hidrólise descontínua-alimentada foram tempo de enchimento e intervalo de alimentação de substrato. Para as respostas analíticas de porcentagem de hidrólise e de composição de frações lipídicas foi aplicado um modelo de regressão múltipla com base em metodologia de superfície de resposta. Os resultados experimentais observados nas reações de hidrólise enzimática descontínua de trioleína técnica mostraram de 24,7 a 34,2% de mono e diacilgliceróis (para 5% de óleo na emulsão) e de 21,4 a 33,6% de mono e diacilgliceróis (para 20% de óleo na emulsão). Os resultados experimentais observados nas reações de hidrólise enzimática descontínua-alimentada de óleo de girassol médio oléico (para 15% de óleo na emulsão), mostraram de 7,9 a 31,8% de mono e diacilgliceróis. Os modelos de superfície de resposta foram considerados significativos e preditivos. As hidrólises obtidas no formato descontínuo e descontínuo-alimentado permitiram efetivamente a obtenção de frações de mono/ diacilgliceróis com vários graus de eficiência de conversão e com corretas identificação e quantificação das frações de lipídios procuradas. As correlações feitas entre porcentagem de hidrólise e entalpias de cristalização e fusão, corroboradas com os resultados qualitativos e/ou quantitativos diretos obtidos na cromatografia de camada delgada (TLC) e de HPSEC, demonstraram que estes atributos podem positivamente indicar a ocorrência efetiva de reação de hidrólise, além de auferir uma escala de desempenho de reação alinhada com o previsto na literatura, à medida que são aumentadas a temperatura, o tempo de hidrólise e a porcentagem inicial de substrato oleoso, sob regime descontínuo, e que puderam ser melhoradas, de forma inovadora, sob parâmetros de tempo total de alimentação e de intervalo de alimentação, sob regime descontínuo-alimentado. A hidrólise parcial enzimática de triacilgliceróis utilizando lipase imobilizada sn-1,3 específica pode ser considerada uma alternativa às vias químicas para a produção de misturas de mono e diacilgliceróis para utilização como aditivos químicos. / Mono and diacylglycerols are products used in the food, pharmaceutical, cosmetic and chemical industries as emulsifiers and viscosity improvers for food products, cosmetics and pharmaceuticals. However, the most usual forms of obtaining them are by chemical synthesis, which ends up yielding expensive final products with attributes of quality, yield and technological applicability lower than expected. The search for more rational, efficient and better quality standards of these products was the aim of the work, through partial enzymatic hydrolysis, which requires milder reaction conditions. Discontinuous enzymatic hydrolysis was evaluated using technical triolein as substrate and discontinuous-fed enzymatic hydrolysis using as the substrate the mid oleic sunflower oil. In both processes, immobilized lipase sn-1,3 specific Lipozyme RM IM (from Rhizomucor miehei) was used. The characterization of the patterns and substrates, as well as the monitoring of the formation of the products from the enzymatic hydrolysis were made by determining the percentage of hydrolysis, thin layer chromatography (TLC), profiles of the melting and crystallization curves by differential scanning calorimetry ( DSC), gas chromatography (GC) and high performance size exclusion chromatography (HPSEC). The parameters of discontinuous hydrolysis were the reaction time, the temperature and the initial substrate concentration. The parameters of discontinuous-fed hydrolysis were filling time and substrate feed interval. For the analytical responses of hydrolysis percentage and composition of lipid fractions a multiple regression model was applied based on response surface methodology. The experimental results observed in the reactions of discontinuous enzymatic hydrolysis of technical triolein indicated amounts of mono- and diacylglycerols from 24.7 to 34.2% (for 5% of oil in the emulsion) and from 21.4 to 33.6% for mono and diacylglycerols with 20% oil in the emulsion. The experimental results observed in the reactions of discontinuous-fed enzymatic hydrolysis of mid oleic sunflower oil (for 15% oil in the emulsion), showed from 7.9 to 31.8% of mono and diacylglycerols. Response surface models were considered significant and viii predictive. The hydrolysis obtained in the discontinuous and discontinuous-fed form allowed to obtain fractions of mono / diacylglycerols with various degrees of conversion efficiency and with correct identification and quantification of the lipid fractions sought. The correlations between the percentage of hydrolysis and enthalpies of crystallization and fusion, corroborated with the qualitative and / or quantitative direct results obtained in thin layer chromatography (TLC) and HPSEC, showed that these attributes can positively indicate the effective occurrence of reaction of Hydrolysis, in addition to achieving a reaction performance scale in line with the literature, as the temperature rate, the hydrolysis time and the initial percentage of oily substrate are increased under a discontinuous regime and can be improved, in a innovative form, under parameters of total filling time and feeding interval, under a fed-batch regime. The partial enzymatic hydrolysis of triacylglycerols using specific sn-1,3-specific immobilized lipase may be considered an alternative to the chemical pathways for the production of mono- and diacylglycerol blends for use as chemical additives.

A DOW é uma empresa que busca continuamente por alternativas para agregar maior valor aos seus produtos através da avaliação das tendências apresentadas pela indústria química. Dentro desta dinâmica, identificou-se uma grande necessidade do mercado de fluidos de corte por inibidores de corrosão mais eficientes e adequados às novas questões regulatórias. Desta avaliação surgiu o tema deste Mestrado Profissional, no qual se estudou a síntese e aplicação de alcanolamidas em fluidos de corte como inibidores de corrosão. Optou-se pela síntese enzimática na ausência de solventes orgânicos por se tratar de uma nova tecnologia à DOW e estar alinhada aos preceitos de sustentabilidades promovidos pela empresa. A escolha pela avaliação das alcanolamidas surgiu de um estudo realizado pela companhia sobre novas tecnologias utilizadas neste segmento e a possibilidade de aplicação destes compostos em outros mercados de atuação da empresa. Foram sintetizadas quatro alcanolamidas, RMEA, RMIPA, RDIPA e RAEPD, obtidas respectivamente da reação entre o ácido ricinoléico e os aminoálcoois: 2-hidroxietilamina, 1-amino-2-propanol, bis-(2-hidroxipropil)amina e 2-amino-2-etil-1,3-propanodiol. O catalisador Novozym 435 (lipase) foi utilizado em todas as sínteses, e estas realizadas de acordo com um planejamento fatorial completo 23. Os produtos sintetizados foram caracterizados por RMN 13C, 1H e dept 135, Espectroscopia no Infravermelho e Espectroscopia de Massas. O rendimento das reações foi mensurado através da técnica de HPLC. Com base nos resultados obtidos foi possível, através do planejamento fatorial, determinar as condições reacionais nas quais o rendimento é maximizado (T = 80 °C; Catalisador = 15 mol/g de ácido ricinoléico; rotação = 600 rpm). A única desvantagem deste processo de síntese foi o custo inerente ao catalisador utilizado. Após o término do planejamento fatorial foram formulados oito fluidos de corte semissintéticos com as alcanolamidas sintetizadas e dois fluidos com o inibidor convencionalmente utilizado. Após verificar a estabilidade térmica destes fluidos, a eficiência à inibição da corrosão foi mensurada através da técnica de manchamento em ferro fundido. Os fluidos contendo as alcanolamidas apresentaram melhor desempenho à inibição da corrosão, porém, não foi possível mensurar quantitativamente as diferenças observadas através desta técnica. Com isso, os compostos foram submetidos a ensaios de perda de massa e polarização potenciodinâmica em ácido clorídrico, além de microscopia atômica para avaliar o efeito dos inibidores na superfície metálica. Através destes estudos foi possível comprovar que os produtos RDIPA e RAEPD possuem maior eficiência à inibição da corrosão. O mecanismo de inibição destes compostos, determinado através de isotermas de Langmuir, ocorre por fisissorção. Após a comprovação das propriedades anticorrosivas dos compostos sintetizados, foram analisadas as seguintes propriedades dos fluidos produzidos: viscosidade, formação de espuma, ângulo de contato, desgaste Reichert, alcalinidade e contaminação microbiológica. Observou-se um aumento da viscosidade e formação de espuma do fluido concentrado. Entretanto, comprovou-se que não há impacto significativo destas propriedades durante a aplicação destes fluidos. As alcanolamidas impactaram positivamente no aumento da lubricidade e reserva alcalina dos fluidos, além de diminuir a taxa de corrosão e a susceptibilidade dos fluidos à contaminação microbiológica, e facilitar o tratamento do resíduo gerado no processo de usinagem devido a maior biodegradabilidade das alcanolamidas. / DOW is a company that continuously searches for alternatives to add greater value to their products through the assessment of trends presented by the chemical industry. Within this dynamic it was identified a great need for more efficient and suitable (to new regulatory issues) corrosion inhibitors applied in metalworking fluids. This Master Thesis came up from this evaluation, where it was studied the synthesis and application of alkanolamides in metalworking fluids as corrosion inhibitors. The enzymatic synthesis in the absence of organic solvents was the chosen production process of alkanolamides because it\'s a new technology to DOW and it\'s aligned with sustainable precepts promoted by the company. The choice for the evaluation of alkanolamides emerged from a study conducted by the company on new technologies applied in metalworking fluids and the possibility of application of these compounds in other markets. It was synthesized four alkanolamides, RMEA, RMIPA, RDIPA and RAEPD, respectively obtained from the reaction between ricinoleic acid and following amino alcohols: 2-hydroxyethylamine, 1-amino-2-propanol, bis(2-hydroxypropyl)amine and 2-amino-2-ethyl-1,3-propanediol. The Novozym 435 catalyst (lipase) was used for all syntheses, and these were carried out according to a full factorial design for three factors. The synthesized products were characterized by NMR 13C, 1H and dept 135, Infrared and Mass Spectroscopy. The yield of the reactions was measured by HPLC technique. Based on the results it was possible, through the factorial design, determine the reaction conditions in which the yield is maximized (T = 80 ° C; Catalyst = 15 mol / g of ricinoleic acid; Speed = 600 rpm). The only disadvantage found of this synthesis process was the cost of the catalyst used. After the factorial design eight semi-synthetic metalworking fluids were formulated with the synthesized alkanolamides and two with the corrosion inhibitor conventionally used. After checking the thermal stability of these fluids, the corrosion inhibition efficiency was measured by staining technique of cast iron. Fluids containing alkanolamides performed better corrosion inhibition, however, was not possible to measure quantitatively the differences observed using this technique. Thus, the compounds were subjected to weight loss and potentiodynamic polarization tests in hydrochloric acid, besides the atomic microscopy to evaluate the effect of the inhibitors on the metal surface. Through these studies it was possible to demonstrate that RDIPA and RAEPD products were more efficient at inhibiting corrosion. The mechanism of inhibition of these compounds, as determined by Langmuir isotherms, is by physisorption. After checking the anticorrosive properties of the synthesized compounds, the following properties were analyzed from the formulated fluids: viscosity, foaming, contact angle, Reichert friction, alkalinity and microbiological contamination. It was observed an increase in viscosity and foaming on the concentrated fluids. However, it was found that there is no significant impact of these properties during the application of these fluids. Alkanolamides enabled an increase in lubricity and alkalinity of the formulated fluids. Additionally they reduced the corrosion rate and the susceptibility of fluids to microbiological contamination, and would make easier the treatment of the waste generated in cutting process due to their higher biodegradability.

Mono e diacilgliceróis são produtos empregados na indústria alimentícia, farmacêutica, cosmética e química como emulsificantes e melhoradores de viscosidade de produtos alimentícios, cosméticos e farmacêuticos. No entanto, a forma mais usual de obtê-los é por síntese química, o que acaba rendendo produtos finais caros e com atributos de qualidade, rendimento e de aplicabilidade tecnológica inferiores aos esperados. A busca por formas de obtenção mais racionais, eficientes e com melhor padrão de qualidade destes produtos foi o objetivo principal do trabalho, por meio de hidrólise parcial enzimática, que necessita de condições de reação mais brandas. Foram avaliadas a hidrólise enzimática descontína, empregando como substrato a trioleína técnica, e a hidrólise enzimática descontínua-alimentada, usando como substrato o óleo de girassol médio oléico. Foi utilizada, em ambos processsos, a lipase imobilizada sn-1,3 específica Lipozyme RM IM (de Rhizomucor miehei). A caracterização dos padrões e dos substrados, bem como o acompanhamento da formação dos produtos da hidrólise enzimática foram feitos por determinação da porcentagem de hidrólise, cromatografia em camada delgada (TLC), dos perfis das curvas de fusão e cristalização por calorimetria diferencial de varredura (DSC), cromatografia gasosa (CG) e cromatografia de exclusão de tamanho de alto desempenho (HPSEC). Os parâmetros de hidrólise descontínua foram o tempo de reação, a temperatura e a concentração inicial de substrato. Os parâmetros de hidrólise descontínua-alimentada foram tempo de enchimento e intervalo de alimentação de substrato. Para as respostas analíticas de porcentagem de hidrólise e de composição de frações lipídicas foi aplicado um modelo de regressão múltipla com base em metodologia de superfície de resposta. Os resultados experimentais observados nas reações de hidrólise enzimática descontínua de trioleína técnica mostraram de 24,7 a 34,2% de mono e diacilgliceróis (para 5% de óleo na emulsão) e de 21,4 a 33,6% de mono e diacilgliceróis (para 20% de óleo na emulsão). Os resultados experimentais observados nas reações de hidrólise enzimática descontínua-alimentada de óleo de girassol médio oléico (para 15% de óleo na emulsão), mostraram de 7,9 a 31,8% de mono e diacilgliceróis. Os modelos de superfície de resposta foram considerados significativos e preditivos. As hidrólises obtidas no formato descontínuo e descontínuo-alimentado permitiram efetivamente a obtenção de frações de mono/ diacilgliceróis com vários graus de eficiência de conversão e com corretas identificação e quantificação das frações de lipídios procuradas. As correlações feitas entre porcentagem de hidrólise e entalpias de cristalização e fusão, corroboradas com os resultados qualitativos e/ou quantitativos diretos obtidos na cromatografia de camada delgada (TLC) e de HPSEC, demonstraram que estes atributos podem positivamente indicar a ocorrência efetiva de reação de hidrólise, além de auferir uma escala de desempenho de reação alinhada com o previsto na literatura, à medida que são aumentadas a temperatura, o tempo de hidrólise e a porcentagem inicial de substrato oleoso, sob regime descontínuo, e que puderam ser melhoradas, de forma inovadora, sob parâmetros de tempo total de alimentação e de intervalo de alimentação, sob regime descontínuo-alimentado. A hidrólise parcial enzimática de triacilgliceróis utilizando lipase imobilizada sn-1,3 específica pode ser considerada uma alternativa às vias químicas para a produção de misturas de mono e diacilgliceróis para utilização como aditivos químicos. / Mono and diacylglycerols are products used in the food, pharmaceutical, cosmetic and chemical industries as emulsifiers and viscosity improvers for food products, cosmetics and pharmaceuticals. However, the most usual forms of obtaining them are by chemical synthesis, which ends up yielding expensive final products with attributes of quality, yield and technological applicability lower than expected. The search for more rational, efficient and better quality standards of these products was the aim of the work, through partial enzymatic hydrolysis, which requires milder reaction conditions. Discontinuous enzymatic hydrolysis was evaluated using technical triolein as substrate and discontinuous-fed enzymatic hydrolysis using as the substrate the mid oleic sunflower oil. In both processes, immobilized lipase sn-1,3 specific Lipozyme RM IM (from Rhizomucor miehei) was used. The characterization of the patterns and substrates, as well as the monitoring of the formation of the products from the enzymatic hydrolysis were made by determining the percentage of hydrolysis, thin layer chromatography (TLC), profiles of the melting and crystallization curves by differential scanning calorimetry ( DSC), gas chromatography (GC) and high performance size exclusion chromatography (HPSEC). The parameters of discontinuous hydrolysis were the reaction time, the temperature and the initial substrate concentration. The parameters of discontinuous-fed hydrolysis were filling time and substrate feed interval. For the analytical responses of hydrolysis percentage and composition of lipid fractions a multiple regression model was applied based on response surface methodology. The experimental results observed in the reactions of discontinuous enzymatic hydrolysis of technical triolein indicated amounts of mono- and diacylglycerols from 24.7 to 34.2% (for 5% of oil in the emulsion) and from 21.4 to 33.6% for mono and diacylglycerols with 20% oil in the emulsion. The experimental results observed in the reactions of discontinuous-fed enzymatic hydrolysis of mid oleic sunflower oil (for 15% oil in the emulsion), showed from 7.9 to 31.8% of mono and diacylglycerols. Response surface models were considered significant and viii predictive. The hydrolysis obtained in the discontinuous and discontinuous-fed form allowed to obtain fractions of mono / diacylglycerols with various degrees of conversion efficiency and with correct identification and quantification of the lipid fractions sought. The correlations between the percentage of hydrolysis and enthalpies of crystallization and fusion, corroborated with the qualitative and / or quantitative direct results obtained in thin layer chromatography (TLC) and HPSEC, showed that these attributes can positively indicate the effective occurrence of reaction of Hydrolysis, in addition to achieving a reaction performance scale in line with the literature, as the temperature rate, the hydrolysis time and the initial percentage of oily substrate are increased under a discontinuous regime and can be improved, in a innovative form, under parameters of total filling time and feeding interval, under a fed-batch regime. The partial enzymatic hydrolysis of triacylglycerols using specific sn-1,3-specific immobilized lipase may be considered an alternative to the chemical pathways for the production of mono- and diacylglycerol blends for use as chemical additives.

La lipase de Yarrowia lipolytica (EC 3.1.1.3) est une enzyme appartenant à la classe des hydrolases. La non pathogénicité et le caractère hyperproducteur en lipase de cette levure lui confèrent une place de choix au sein de lunité de Bio-industries du Centre Wallon de Biologie Industrielle. Ce présent travail sinscrit dans le cadre général du développement industriel de la lipase de Yarrowia lipolytica et concerne plus particulièrement le traitement post-culture de lenzyme afin de réaliser des formes liquides, poudres atomisées, immobilisées et enrobées à laide des polymères acryliques. Latomisation de la lipase en présence ou en absence de poudre de lait a permis lacquisition de poudres fluentes, stables à 4 et 20°C et présentant des températures de transition vitreuse comprises entre 51 et 79°C. Lactivité deau de conservation des poudres était ≤ 0.4. La stabilisation de lenzyme sous forme de liquide concentré réalisée avec le monopropylène glycol (MPG), les inhibiteurs de protéases et lirradiation aux rayons gamma ont révélé que le MPG à 50% et la technique dirradiation au rayon gamma permettaient la stérilisation et la préservation de lactivité enzymatique. Par ailleurs, limmobilisation de cette enzyme par trois techniques (adsorption, inclusion et liaison covalente) a révélé une amélioration de ses propriétés caractéristiques telles que la thermostabilté et la résistance aux solvants. La technique dimmobilisation par adsorption et par liaison covalente a permis une utilisation multiple de lenzyme. Létude préliminaire de faisabilité des formes galéniques à base de la lipase de Y. lipolytica a montré la capacité de cette enzyme à être mise sous forme de comprimés et de poudres encapsulées. La comparaison réalisée in vitro entre le Créon 150mg (produit pharmaceutique) et les formes galéniques à base de la lipase a montré des temps de gastro-résistance et de délitage similaires. Ces différentes formules de la lipase posent des jalons nécessaires pour leurs applications dans des secteurs agroalimentaires, environnementaux et pharmaceutiques. Yarrowia lipolytica lipase (EC.3.1.1.3) is an enzyme which belongs to the class of hydrolases. Nonpathogenicity and the high-lipase producing character of this yeast have emphasised its use within the laboratory of Bio-industry of the Walloon Center of Industrial Biology. The present work lies within the general scope of the industrial development of the lipase from Yarrowia lipolytica. More particularly it relates to the post-culture treatment of the enzyme in order to obtain liquid forms, atomized powders, immobilized and coated enzymes using acrylic polymers. The atomization of lipase in presence or absence of milk powder allowed the achievement of flowing, stable powders at 4 and 20°C, with glass transition temperatures ranging between 51 and 79°C. The water activity of preservation of the powders was ≤ 0.4. Stabilization of the enzyme under the form of concentrated liquid carried out with monopropylen glycol (MPG), proteases inhibitors and gamma irradiation revealed that MPG (50%) and gamma irradiation allowed sterilization and conservation of the enzymatic activity. In addition, the immobilization of the enzyme through three techniques (adsorption, inclusion and covalent bond) revealed an improvement of some properties such as thermostability and resistance to solvents. Immobilization by adsorption and covalent bond allowed multiple uses of the enzyme. The preliminary study of feasibility of galenic forms containing the lipase from Y. lipolytica showed the capacity of this enzyme to be put under the form of tablets and encapsulated powders. The in vitro comparison of Creon 150mg (pharmaceutical product) and galenic forms containing the lipase, showed similar times of acid-resistance and of disintegration. These various formulas of the lipase constitute milestones necessary for their applications in food, environmental and pharmaceutical industries.

No description available.

L’hypertriglycéridémie (hyperTG) est une dyslipidémie fréquente, caractérisée par une augmentation de la concentration plasmatique en triglycérides (TG). L’hyperTG est considérée comme un facteur de risque indépendant de la maladie cardiovasculaire, particulièrement de la maladie coronarienne athérosclérotique. Plusieurs facteurs environnementaux et génétiques ont été associés avec l’hyperTG. Cependant, près de 90% des cas d’hyperTG primaire sont encore incomplètement caractérisés au niveau moléculaire. Dernièrement, la protéine GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1), qui a un rôle clef dans le métabolisme des TG, a été associée à l’expression d’hyperTG sévère et rare chez l’humain. Ce mémoire présente les résultats de nos travaux qui ont été effectués afin d’identifier de nouvelles bases moléculaires associées à l’expression de l’hyperTG dans le locus du gène GPIHBP1. Nous avons observé que le polymorphisme GPIHBP1 g.-469G>A (rs72691625), dont la fréquence de l’allèle mineure a été évaluée à 19,6% dans notre échantillon, serait associé à l’expression d’hyperTG (TG ≥ 2mmol/L) dans une population canadienne-française. Ce polymorphisme est associé à un risque 1,67 fois plus grand d’exprimer une triglycéridémie ≥ 2mmol/L chez les porteurs hétérozygotes et 5,7 fois plus grand chez les porteurs homozygotes, comparativement aux non-porteurs. Ce risque d’hyperTG serait exacerbé par la présence concomitante d’une mutation hypertriglycéridémiante dans le gène codant pour la lipoprotéine lipase. La présence de ce polymorphisme serait particulièrement associée à l’expression de la dysbêtalipoprotéinémie familiale et de l’hypertriglycéridémie familiale endogène. GPIHBP1 g.-469G>A est le premier polymorphisme fréquent identifié dans le promoteur du gène à être associé avec l’expression d’hyperTG. GPIHBP1 émerge de plus en plus comme un gène candidat intéressant pour la recherche de nouvelles bases moléculaires pouvant expliquer certaines formes d’hyperTG primaire fréquente. / Hypertriglyceridemia (hyperTG) is a frequent dyslipidemia referring to an increased fasting plasma triglyceride (TG) level ≥ 2 mmol/L. HyperTG is an independent risk factor for cardiovascular disease, such as coronary artery diseases. Several environmental and genetic factors have been associated with hyperTG. Although several gene factors were associated with hyperTG, nearly 90% of cases of primary hyperTG are still incompletely characterized at the molecular level. Recently, few cases of rare and severe hyperTG have been associated with some rare polymorphisms in the gene coding for GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1). This manuscript resumes our research regarding the identification of new molecular bases associated with the expression of frequent hyperTG subtypes in the gene locus GPIHBP1. Our results show that the GPIHBP1 g.-469G>A polymorphism (rs72691625), whose the minor allele frequency was estimated to 19.6% in our sample, was associated with the expression of hyperTG (TG ≥ 2 mmol/L) in a French-Canadian population. Subjects heterozygous and homozygous for this polymorphism respectively had a 1.67-fold and 5.70-fold increased risk to exhibit plasma TG levels ≥ 2mmol/L as compared to non-carriers. This increased risk of hyperTG observed in g.-469A carriers seems to be exacerbated by the concomitant presence of a frequent loss-of-function lipoprotein lipase gene variant. This polymorphism seems also particularly associated with dysbetalipoproteinemia and familial hypertriglyceridemia. The g.-469G>A polymorphism is the first common polymorphism in the GPIHBP1 gene promoter to be associated with the expression of hyperTG. GPIHBP1 emerges as a significant candidate for the molecular based of primary hyperTG.

Les peptides sont des molécules pouvant posséder une activité biologique intéressante (antibiotique, anti-oxydante, antivirale, anti-hypertensive…). Ce sont cependant des molécules difficiles à utiliser car elles possèdent un faible temps de demi-vie in vivo et sont peu bio-disponibles. Le greffage d’un acide gras permet de les protéger et d’accroître leur potentiel d’action. Cette réaction appelée acylation peut être catalysée par des enzymes. A l’heure actuelle, peu de recherches sont faites sur l’acylation de peptides par voie enzymatique et sur la recherche de nouveaux biocatalyseurs adaptés pour cette réaction. Les objectifs de cette thèse ont été, dans un premier temps, de comprendre les mécanismes de la sélectivité d’acylation de peptides de la lipase B de Candida antarctica par une approche de modélisation moléculaire combinant docking et dynamique moléculaire, couplée à une approche expérimentale. Cette étude a permis d’identifier des interactions enzyme-substrats impliquées dans la sélectivité enzymatique et a permis de construire un modèle expliquant la régio- et chimio-sélectivité de l’acylation peptidique catalysée par cette enzyme. Dans un deuxième temps, une étude préliminaire a été menée afin d’identifier de nouvelles enzymes de type acylases présentes dans des surnageants de culture de différentes espèces de Streptomyces. Ces enzymes sont capables de catalyser des réactions d’acylation de peptides en milieux aqueux. Une méthode de semi-purification a été établie et une étude comparative a été menée sur la sélectivité d’acylation de la lipase B de C. antarctica et celle de nouvelles enzymes de type aminoacylases présentes dans un extrait protéique de surnageant de culture de Streptomyces ambofaciens. Ces nouvelles enzymes présentent une spécificité différente de celle de la lipase B de C. antarctica, permettant notamment, une acylation des acides aminés sur leur fonction amine en position α. Une caractérisation partielle des activités amino-acylase du surnageant de culture de S. ambofaciens a été réalisée. Dans une troisième et dernière partie, une comparaison des séquences génétiques a été réalisée entre treptomyces mobaraensis et S. ambofaciens afin d’identifier les gènes codant pour les acylases découvertes chez S. ambofaciens. Des mutants de S. ambofaciens délétés pour ces gènes ont été construits et la fonctionnalité des enzymes codées par ces gènes a été vérifiée ; enfin, une expression hétérologue de l’ε-lysine acylase a été initiée / Peptides exhibit various beneficial effects such as antioxidant, anti-hypertensive, neuroprotective, antiviral or antimicrobial activities. However, their use can be limited by their short half-life and their low biological availability. One solution to overcome these drawbacks is the acylation of peptides with fatty acids. This reaction called acylation can be catalyzed using enzymes. To date, very few studies focus on enzymatic acylation of peptides and on finding new enzymes catalyzing this reaction. The objectives of this work were, in a first time, to understand the selectivity mechanisms of the lipase B of Candida antarctica for peptides acylation combining experimental and molecular modeling approaches. This study highlighted enzyme/substrate interactions involved in the enzymatic selectivity and a modelexplaining the chemo- and regio-selectivity of this enzyme for peptide acylation reactions was built. In a second time, a preliminary study was carried out in order to identify new aminoacylase enzymes produced in the culture supernatant of various species of Streptomyces. These enzymes are able to catalyze acylation of peptides in aqueous media. A partial purification method was set and a comparative study was performed on the selectivity of C. antarctica lipase Band that of the new aminoacylases discovered in the culture supernatant of Streptomyces ambofaciens ATCC 23877. These enzymes presented a selectivity different from C. antarctica lipase B allowing the acylation of the N-terminal amino group of amino acids or peptides. A partial description of the aminoacylase activity of the supernatant crude extract of S. ambofaciens was performed. In a third and final part, a comparison of sequences of aminoacylases from Streptomyces mobaraensis with the genome of S.s ambofaciens ATCC 23877 was performed in order to identify genetic sequences encoding the new discovered aminoacylases from S. ambofaciens ATCC 23877. Each identified gene was deleted to correlate it with the aminoacylase activity observed in the crude extract of S. ambofaciens. Lastly, a heterologous expression of the ε-lysine acylase was initiated

Les lipases/acyltransférases présentent des propriétés intermédiaires entre les lipases et les acyltransférases. Capables de se comporter comme des hydrolases, elles catalysent cependant la réaction de transfert d'acyle préférentiellement à l'hydrolyse même en milieu aqueux à forte activité thermodynamique de l'eau en présence de divers nucléophiles. La recherche de nouvelles lipases/acyltransférases, soit sécrétées par des levures sauvages, soit identifiées parmi les séquences protéiques disponibles dans des bases de données, nous a permis d'identifier deux nouvelles enzymes de ce type : CvisL2 de Candida viswanathii et CtroL4a de C. tropicalis. Cette dernière, produite par expression hétérologue, a été plus particulièrement étudiée en comparaison avec les deux lipases/acyltransférases déjà connues, CpLIP2 de C. parapsilosis et CaLIP4 de C. albicans, ainsi qu'avec des enzymes plus éloignées (AflaL0a d'Aspergilus flavus, isolée dans ce travail, et CaLA de C. antarctica, qui présentent respectivement 35 % et 31 % d'identité avec CpLIP2). Le caractère spécifique des acyltransférases semble relié à leur degré d'homologie et à leurs relations phylogénétiques. En effet, les trois lipases/acyltransférases étudiées appartiennent à un sous-groupe phylogénétique distinct composé de diverses autres protéines actuellement non-caractérisées présentant plus de 57 % d'identité avec CpLIP2. En plus de leur activité acyltransférase plus ou moins prononcée, ces nouveaux biocatalyseurs diffèrent par leur spécificité de substrat, leur stabilité en présence de fortes concentrations en alcool ou leur activité à basse température, élargissant ainsi le spectre des applications potentielles des lipases et lipases/acyltransférases. / Lipases/acyltransferases have intermediate properties between lipases and acyltransferases. Although being active hydrolases, they catalyze acyltransfer reactions preferentially to hydrolysis even in an aqueous medium with a high thermodynamic activity of water in the presence of various nucleophiles. Searching for new lipases/acyltransferases, either secreted by wild yeast strains or identified in protein sequences databases, allowed us to identify two new enzymes of this type: CvisL2 from Candida viswanathii and CtroL4a from C. tropicalis. The latter, produced by heterologous expression, has been more particularly studied and compared with the two already known, closely related, lipases/acyltransferases, CpLIP2 from C. parapsilosis and CaLIP4 from C. albicans, and with two more distantly related lipases (a new lipase AflaL0a from Aspergillus flavus and CaLA from C. antarctica, with 35 % and 31 % identity with CpLIP2, respectively). The specific catalytic behavior of the acyltransferases seems to be associated with sequence homology and phylogenetic relationships. Indeed, the three lipases/acyltransferases studied are part of a phylogenetic subgroup composed of various proteins (identity with CpLIP2 higher than 57 %), currently not characterized. Besides their acyltransfer activity, these new biocatalysts differ in properties such as their substrate selectivity, their stability in the presence of high alcohol concentration or their activity at low temperature, opening the way to new applications.

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