Purification et fonctionnalisation d acides gras polyinsaturés Oméga-3 par des lipases et production de lipides structurés

Les lipases sont des enzymes présentant un grand intérêt industriel. L intérêt de ces enzymes a conduit à caractériser ces enzymes, à mieux comprendre leur mécanisme réactionnel et leur cinétique, et à établir des méthodes efficaces de production en système d expression homologue et hétérologue. Plus récemment, l ingénierie enzymatique permet d améliorer les caractéristiques des enzymes. Ce thèse s est fixé deux objectifs principaux: premièrement, la purification et la fonctionnalisation d acides gras poly-insaturés de type Omega-3 (PUFAs), et spécialement l acide cis-4, 7, 10, 13, 16, 19-docosahexaénoique (DHA) et deuxièmement la production de lipides structurés (SL). Un premier objectif fut de produire une molécule pharmaceutique, le nicotinyl DHA ester. Le co-substrat du DHA est le nicotinol, un alcool qui après absorption, il est rapidement converti en acide nicotinique (Vitamine B3). La trans-esterification enzymatique entre l ester éthylique du DHA et le nicotinol a été optimisée dans le but de synthétiser un ester présentant les propriétés cumulatives des deux réactants. Après la sélection de l enzyme optimale (lipase immobilisée de Candida antarctica; Novozyme 435) et le choix du milieu réactionnel (milieu sans solvant), le procédé a été optimisé. Une conversion supérieure à 97 % a été obtenu en 4 heures avec 45 g.L-1 d enzyme. Dans ces conditions, une productivité de 4.2 g de produit .h-1.g d enzyme-1 a été obtenue. Ce projet nécessite une haute pureté en DHA. Un procédé de purification enzymatique a été choisi. Les lipases sont capables de discriminer entre les acides gras en fonction de la longueur de chaine et du degré d insaturation. Les lipases agissent par résolution cinétique, en réagissant plus efficacement avec les acides gras saturés et mono-insaturés qu avec les PUFAs résistants. La lipase YLL2 de Yarrowia lipolytica apparait comme un bon candidat car elle est homologue à une des lipases les plus efficaces, la lipase de Thermomyces lanuginosus. YLL2 a permis d obtenir une discrimination très efficace. Les raisons de la sélectivité de l enzyme ont été identifiées : il s agit du positionnement de la double liaison la plus proche de la fonction carboxylique. La concentration en DHA la plus élevée a été obtenue avec YLL2 (73%) avec un pourcentage de récupération du DHA-EE de 89%. YLL2 est par conséquent l enzyme décrite la plus efficace pour la purification du DHA.La mutagénèse ciblée dans le site actif de YLL2 a été utilisée pour améliorer la sélectivité de cette enzyme. L analyse de la structure 3D et les alignements avec des lipases homologues a permis de choisir les cibles de mutagénèse dirigée. Les acides aminés cibles ont été changés de manière à restreindre ou élargir le site actif. De ce premier screening de variantes deux positions ont permis d améliorer la spécificité de l enzyme, les positions I100 et V235. Finalement la saturation de ces 2 positions a été réalisée. Le dernier objectif de la thèse était la production de SL par acidolysis enzymatique entre l'huile d'olive vierge et les acides caprylic ou capric utilisant la lipase YLL2 immobilisé. Le SL obtenu devrait être riche en acide oléique à la position sn-2 tandis que les C8:0 et C10:0 devraient être principalement estérifiés aux positions sn-1,3. YLL2 immobilisé sur Accurel 1000 a été testé dans un système sans solvant. La réaction d acidolysis d'huile d'olive avec C8:0 ou C10:0 a été optimisée avec la méthodologie de surface de réponse (RSM).Lipases are enzymes with applications extended to a wide variety of industries. The variety of lipases applications led to increased research to characterize them and better understand their kinetics and reaction mechanisms and to establish methods for lipase production in homologous and heterologous expression systems. Lately enzymatic engineering allowed the improvement of lipase characteristics. This thesis project studies the use of lipases for two main objectives: lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids (PUFAs), especially cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA) and production of structured lipids (SL). DHA was used for the synthesis of a pharmaceutical molecule, the nicotinyl DHA ester. The co-substrate of the reaction was nicotinol, an alcohol from the group B pro-vitamin, which after absorption is rapidly converted into nicotinic acid (Vitamin B3). The enzymatic trans-esterification of DHA ethyl esters with nicotinol was optimised to synthesise an ester presenting the cumulative properties of the two reactants. After enzyme (immobilized lipase from Candida antarctica; Novozym 435) and reaction medium (solvent-free system) selection, the process was optimised. A conversion to nicotinyl-DHA superior to 97 % was obtained in 4 hours using 45 g.L-1 of enzyme. With a productivity of 4.2 g of product .h-1.g of enzyme-1.This project requires DHA of high purity. Enzymatic purification was chosen for the production of DHA concentrates. Lipases can discriminate between fatty acids in function of their chain length and saturation degree. Lipases react more efficiently with the bulk of saturated and mono-unsaturated fatty acids than with the PUFAs. The objective was the discovery of more specific enzymes for DHA purification. The lipase Lip2 from Yarrowia lipolytica (YLL2) appears as a good candidate since it is homologous to one of the most efficient lipase, the lipase from Thermomyces lanuginosus. YLL2 enables a high discrimination to be obtained, enzyme selectivity being principally due to the positioning of the double-bond the closest from the carboxylic group. The highest concentration of DHA was obtained with YLL2 (73%) with a recovery percentage of DHA-EE of 89%. YLL2 is the most efficient described lipase for DHA purification.Site directed mutagenesis was used to improve YLL2 from Y. lipolytica. Using its three dimensional structure and alignment with homologous lipases, targets for site directed mutagenesis were chosen. Chosen amino acids were substituted by two amino acids of different sizes. From the screening of variants two positions with promising specificities where chosen, positions I100 and V235. Finally saturation of both positions and the analysis of their performances in the selected reactions were carried out. The last objective was the production of SL by enzymatic acidolysis between virgin olive oil and caprylic or capric acids using immobilized Lip2 from Y. lipolytica. The SL obtained should be rich in oleic acid at the sn-2 position while C8:0 and C10:0 should be mainly esterified at the sn-1,3 positions. Lip2 from Y. lipolytica immobilized on Accurel MP 1000 was tested in a solvent-free system. The acidolysis reaction of olive oil with C8:0 or C10:0 was optimized by response surface methodology (RSM)TOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Enzyme-catalyzed production of biodiesel as alternative to chemical-catalyzed processes : advantages and constraints

Background: Biodiesel represents an interesting alternative to fossil fuels. Traditionally the standard method for biodiesel production from oils is alkaline-catalyzed transesterification. Chemical catalysis can be replaced by enzymatic catalysis using lipases (EC 3.1.1.3, triacylglycerol acyl hydrolases), obtained from plants, animals or microorganisms. Enzymatic catalysis has important advantages compared to chemical catalysis, as lower energy consumption and undesirable side-reactions do not occur originating pure compounds. However, some problems should be overcome to achieve a real alternative to chemical catalysis, such as the prize, the stability, and the reutilization of the biocatalyst. -Results: The review consists of an update of the state of the art of enzymatic biodiesel production, including legislation, feedstocks, lipases used for biodiesel synthesis, the role of acyl acceptors and strategies to avoid lipase inactivation, the mechanisms proposed for biocatalysis and the enzymatic bioreactors used. In addition, the economics of the bioprocess is also presented. - Conclusion: Nowadays, there are already some examples of enzymatic processes for biodiesel production implemented at industrial scale and the number of pilot and industrial scale plants has greatly increased in the recent years. In spite of this trend, the chemical catalysis process still remains the most popular on an industrial scale mainly due to the high cost of commercial lipases. Thus, it is necessary to improve the enzymatic technology, increasing the productivity of the bioprocess and reducing the cost of the bioprocess. To attain this goal, it is necessary to act in a multidisciplinary approach of Genetic engineering, Bioprocess engineering, including the production of recombinant lipase in the most adequate cell factory, Enzyme engineering and applied Biocatalysis. It is a fact that the approach to "create" by genetic engineering, a lipase with a high tolerance to methanol, high biocatalyic performance and high resistance to work at higher temperatures and under harsh conditions is not corresponding with the important advances as compared to the other aspects. Also, the use of low cost non-commercial biocatalysts, presenting both high transesterification activity and operational stability, as an alternative to commercial biocatalysts, is a solution to reduce enzymatic biodiesel production costs, making it competitive with chemical processes. The price of biodiesel is highly affected by the market price fluctuation of oil feedstock. Thus the commercial efficiency and competitiveness of biodiesel market needs the development of high-valued product from the FAMEs as rawmaterial, under the concept of a biodiesel refinery. Other approach to minimize the cost of the global process is the production of heterologous lipases, using the crude glycerol obtained in the same biodiesel industry, without high purification, as carbon source. The presence of low methanol concentration and other possible contaminants jointly in the matrix of crude glycerol is not a problem for P. pastoris, one of the most popular cell factories to produce recombinant lipases. In conclusion, enzymatic biodiesel, as a green alternative to chemical biodiesel, has a potential economic growth in the near future

Lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids and production of structures lipids

Doutoramento em Engenharia Alimentar - Instituto Superior de Agronomi

Purification et fonctionnalisation d’acides gras polyinsaturés Oméga-3 par des lipases et production de lipides structurés

Lipases are enzymes with applications extended to a wide variety of industries. The variety of lipases applications led to increased research to characterize them and better understand their kinetics and reaction mechanisms and to establish methods for lipase production in homologous and heterologous expression systems. Lately enzymatic engineering allowed the improvement of lipase characteristics. This thesis project studies the use of lipases for two main objectives: lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids (PUFAs), especially cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA) and production of structured lipids (SL). DHA was used for the synthesis of a pharmaceutical molecule, the nicotinyl DHA ester. The co-substrate of the reaction was nicotinol, an alcohol from the group B pro-vitamin, which after absorption is rapidly converted into nicotinic acid (Vitamin B3). The enzymatic trans-esterification of DHA ethyl esters with nicotinol was optimised to synthesise an ester presenting the cumulative properties of the two reactants. After enzyme (immobilized lipase from Candida antarctica; Novozym 435) and reaction medium (solvent-free system) selection, the process was optimised. A conversion to nicotinyl-DHA superior to 97 % was obtained in 4 hours using 45 g.L-1 of enzyme. With a productivity of 4.2 g of product .h-1.g of enzyme-1.This project requires DHA of high purity. Enzymatic purification was chosen for the production of DHA concentrates. Lipases can discriminate between fatty acids in function of their chain length and saturation degree. Lipases react more efficiently with the bulk of saturated and mono-unsaturated fatty acids than with the PUFAs. The objective was the discovery of more specific enzymes for DHA purification. The lipase Lip2 from Yarrowia lipolytica (YLL2) appears as a good candidate since it is homologous to one of the most efficient lipase, the lipase from Thermomyces lanuginosus. YLL2 enables a high discrimination to be obtained, enzyme selectivity being principally due to the positioning of the double-bond the closest from the carboxylic group. The highest concentration of DHA was obtained with YLL2 (73%) with a recovery percentage of DHA-EE of 89%. YLL2 is the most efficient described lipase for DHA purification.Site directed mutagenesis was used to improve YLL2 from Y. lipolytica. Using its three dimensional structure and alignment with homologous lipases, targets for site directed mutagenesis were chosen. Chosen amino acids were substituted by two amino acids of different sizes. From the screening of variants two positions with promising specificities where chosen, positions I100 and V235. Finally saturation of both positions and the analysis of their performances in the selected reactions were carried out. The last objective was the production of SL by enzymatic acidolysis between virgin olive oil and caprylic or capric acids using immobilized Lip2 from Y. lipolytica. The SL obtained should be rich in oleic acid at the sn-2 position while C8:0 and C10:0 should be mainly esterified at the sn-1,3 positions. Lip2 from Y. lipolytica immobilized on Accurel MP 1000 was tested in a solvent-free system. The acidolysis reaction of olive oil with C8:0 or C10:0 was optimized by response surface methodology (RSM)Les lipases sont des enzymes présentant un grand intérêt industriel. L’intérêt de ces enzymes a conduit à caractériser ces enzymes, à mieux comprendre leur mécanisme réactionnel et leur cinétique, et à établir des méthodes efficaces de production en système d’expression homologue et hétérologue. Plus récemment, l’ingénierie enzymatique permet d’améliorer les caractéristiques des enzymes. Ce thèse s’est fixé deux objectifs principaux: premièrement, la purification et la fonctionnalisation d’acides gras poly-insaturés de type Omega-3 (PUFAs), et spécialement l’acide cis-4, 7, 10, 13, 16, 19-docosahexaénoique (DHA) et deuxièmement la production de lipides structurés (SL). Un premier objectif fut de produire une molécule pharmaceutique, le nicotinyl DHA ester. Le co-substrat du DHA est le nicotinol, un alcool qui après absorption, il est rapidement converti en acide nicotinique (Vitamine B3). La trans-esterification enzymatique entre l’ester éthylique du DHA et le nicotinol a été optimisée dans le but de synthétiser un ester présentant les propriétés cumulatives des deux réactants. Après la sélection de l’enzyme optimale (lipase immobilisée de Candida antarctica; Novozyme 435) et le choix du milieu réactionnel (milieu sans solvant), le procédé a été optimisé. Une conversion supérieure à 97 % a été obtenu en 4 heures avec 45 g.L-1 d’enzyme. Dans ces conditions, une productivité de 4.2 g de produit .h-1.g d’enzyme-1 a été obtenue. Ce projet nécessite une haute pureté en DHA. Un procédé de purification enzymatique a été choisi. Les lipases sont capables de discriminer entre les acides gras en fonction de la longueur de chaine et du degré d’insaturation. Les lipases agissent par résolution cinétique, en réagissant plus efficacement avec les acides gras saturés et mono-insaturés qu’avec les PUFAs résistants. La lipase YLL2 de Yarrowia lipolytica apparait comme un bon candidat car elle est homologue à une des lipases les plus efficaces, la lipase de Thermomyces lanuginosus. YLL2 a permis d’obtenir une discrimination très efficace. Les raisons de la sélectivité de l’enzyme ont été identifiées : il s’agit du positionnement de la double liaison la plus proche de la fonction carboxylique. La concentration en DHA la plus élevée a été obtenue avec YLL2 (73%) avec un pourcentage de récupération du DHA-EE de 89%. YLL2 est par conséquent l’enzyme décrite la plus efficace pour la purification du DHA.La mutagénèse ciblée dans le site actif de YLL2 a été utilisée pour améliorer la sélectivité de cette enzyme. L’analyse de la structure 3D et les alignements avec des lipases homologues a permis de choisir les cibles de mutagénèse dirigée. Les acides aminés cibles ont été changés de manière à restreindre ou élargir le site actif. De ce premier screening de variantes deux positions ont permis d’améliorer la spécificité de l’enzyme, les positions I100 et V235. Finalement la saturation de ces 2 positions a été réalisée. Le dernier objectif de la thèse était la production de SL par acidolysis enzymatique entre l'huile d'olive vierge et les acides caprylic ou capric utilisant la lipase YLL2 immobilisé. Le SL obtenu devrait être riche en acide oléique à la position sn-2 tandis que les C8:0 et C10:0 devraient être principalement estérifiés aux positions sn-1,3. YLL2 immobilisé sur Accurel 1000 a été testé dans un système sans solvant. La réaction d’acidolysis d'huile d'olive avec C8:0 ou C10:0 a été optimisée avec la méthodologie de surface de réponse (RSM)

Enzymes for the valorization of agroindustrial Waste

Tema del mesLa industria agrícola y la alimentaria son de las más importantes a nivel mundial. El procesamiento de alimentos genera grandes cantidades de residuos agroindustriales que en la mayoría de los casos no son aprovechados. Estos residuos suelen utilizarse para la extracción y recuperación de compuestos bioactivos, la producción de enzimas, antibióticos, hongos comestibles, ácidos orgánicos y biocombustibles, como alimento para animales y para la producción de composta. Además, son ricos en compuestos orgánicos, muchos de ellos con alto valor agregado como fibras dietéticas, pigmentos, pectinas, oligosacáridos, flavonoides, carotenoides, compuestos fenólicos, tocoferoles y vitaminas. Las enzimas son una alternativa ecológicamente amigable para la extracción de estos compuestos. Los residuos más utilizados en la actualidad provienen de frutas y mariscos. A través de transformaciones enzimáticas, los compuestos bioactivos extraídos de los residuos son empleados para la síntesis de nutracéuticos. Estas moléculas son de interés ya que mejoran las propiedades de los compuestos bioactivos de los que se obtienen.Food and agricultural industries are two of the most important industries worldwide. Food processing generates large quantities of agroindustrial waste, which in most cases is not used as valuable resources. Most of this waste is used for the extraction and recovery of bioactive compounds, the production of enzymes, antibiotics, edible fungi, organic acids and biofuels, as animal feed and for the production of compost. It is also rich in organic substances, many of them with high added value such as dietary fibers, pigments, pectins, oligosaccharides, flavonoids, carotenoids, phenolic compounds, tocopherol and vitamins. Enzymes represent an ecologically friendly option for the extraction of these substances. Currently the most exploited waste is obtained from the fruit and seafood industries. Through enzymatic transformations, bioactive compounds extracted from the waste are converted into nutraceuticals. These molecules have improved properties compared to the original ones

Evaluation of Yarrowia lipolytica Oil for Biodiesel Production: Land Use Oil Yield, Carbon, and Energy Balance

Oils from yeasts have emerged as a suitable alternative raw material to produce biodiesel, due to their similar composition to common raw materials such as vegetable oils. Additionally, they have the advantage of not competing with human or animal feed, and, furthermore, they do not compete for arable land. In this work, a carbon and energy balance was evaluated for Yarrowia lipolytica as a model yeast, using crude glycerol from biodiesel as the only carbon source, which improves biodiesel overall yield by 6%. The process presented a positive energy balance. Feasibility of yeast oil as biodiesel substrate was also evaluated by determination of the lipid fatty acid profile and cetane number. Moreover, a comparison of oil yields, in terms of land use, between vegetable, microalgae, and yeast oils is also presented. The results showed that Y. lipolytica oil yield is considerably higher than vegetable oils (767 times) and microalgae (36 times)

Lipases: An Overview

International audienceLipases are ubiquitous enzymes, widespread in nature. They were first isolated from bacteria in the early nineteenth century and the associated research continuously increased due to the particular characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes

Production of carbohydrate fatty acid esters from Agave tequilana fructans by enzymatic acylation

Trabajo presentado en el VII Workshop on Biocatalysis and Biotransformations - 1º Simposio Latinoamericano de Biocatalisis y Biotransformaciones, celebrado en Búzios (Brasil) del 23 al 26 de septiembre de 2014.Peer Reviewe