Doctor Scientiae / An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.

La caractérisation de l'activité enzymatique des (phospho)lipases requiert des tests enzymatiques spécifiques, continus, utilisant des substrats lipidiques et adaptés au criblage à haut débit des activités et/ou des inhibiteurs de (phospho)lipases. Afin de développer de tels tests, la synthèse de glycérophosphatidylcholine (PC) estérifiée en position sn-1 et/ou sn-2 par l'acide alpha-éléostéarique (acide 9Z, 11E, 13E, octadécatriénoïque) a été effectuée. La triple insaturation conjuguée présente au sein de cet acide gras constitue un chromophore intrinsèque qui confère une forte absorption dans le domaine de l'ultra-violet à cet acide gras et aux lipides le contenant. Les PC contenant l'acide alpha-éléostéarique ont été adsorbées par « coating » au fond des puits d'une microplaque de titration. L'hydrolyse du substrat lipidique par une phospholipase A1 (PLA1) ou phospholipase A2 (PLA2), injectée dans le milieu réactionnel, est suivie en continu par l'augmentation de l'absorbance à 272 nm, due à la transition de l'acide alpha-éléostéarique de la phase adsorbée à la phase aqueuse. Des PC hétérogènes ont été synthétisées à partir de rac-glycidol pour effectuer un marquage sélectif de la PC par l'acide alpha-éléostéarique sur la position sn-1 (EOPC) ou sn-2 (OEPC). Pour empêcher la migration de la chaîne acyle, un lien éther non hydrolysable par les PLA1 ou PLA2 a été introduit sur l'autre position sn de la PC avec une chaîne alkyl (C18). Ces PC chimiquement définies ont permis d'élaborer une méthode de dosage en continu de l'activité enzymatique et discriminant les activités PLA1 ou PLA2, ce qui représente un caractère innovant par rapport à toutes les méthodes existantes / The characterization of the catalytic activity of (phospho)lipases requires specific assays, that are continuous, sensitive, use lipidic substrates and could be applied to high throughput screening. In order to perform these tests, several tailor-made alpha-eleostearic (9Z, 11E, 13E-octadecatrienoic acid) containing glycerophosphatidylcholines (PC) have been synthetized with the alpha-eleostearic acid at the position sn-1 and/or sn-2. The conjugated triene present in this fatty acid constitutes an intrinsic chromophore and, consequently, confers strong UV absorption properties of the fatty acid and the lipids harboring it. PC substrates were coated onto a microplate well and the phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activity was measured continuously by the increase in absorbance, at 272 nm, due to the transition of alpha-eleostearic acid from the adsorbed to the soluble state. Moreover, two structured analogues of PC labeled at the sn-1 (EOPC) or sn-2 (OEPC) position with the alpha-eleostearic acid have been synthetized from rac-glycidol. A non-absorbing and non-hydrolysable by PLA1 and PLA2 O-ether alkyl(C18) was introduced at the other sn position to prevent intramolecular acyl chain migration during the synthesis and the lipolysis. These structured PC were coated onto a microplate and used in a continuous assay, to discriminate, with excellent accuracy, between PLA1 or PLA2 activities. The development of a sensitive enzymatic method using coated substrates analogues to natural lipid is a relevant improvement from current assays for measuring continuously (phosphor)lipases activities and/or their inhibitors due to the alpha-eleostearic acid UV spectroscopic properties

Cette étude a porté sur le développement de procédés continus performants de production d’esters à partir de l’huile de tournesol hautement oléique vierge ou raffinée en réacteurs enzymatiques à lit fixe, très productifs et stables dans le temps. Un procédé de transestérification continue en réacteur à lit fixe utilisant Novozyme 435 (lipase B de candida antarctica immobilisée sur Lewatit VP OC 1600), biocatalyseur non régio-spécifique, a été optimisé pour transformer de l’huile vierge de tournesol hautement oléique en esters butyliques. Les phénomènes de partition des composés polaires (phospholipides présents initialement dans l’huile, du glycérol co-produits etc.) entre milieu réactionnel et support enzymatique ont été gérés grâce à l’utilisation de tert-butanol, un solvant polaire. Les conditions assurant le meilleur compromis entre stabilité, productivité et rendements de production d’esters ont été obtenues pour une concentration initiale en huile de 500mM et un rapport molaire entre substrats de 5. De telles conditions permettent une productivité de 13,8 tonnes.an-1.kg de Novozyme 435-1. Le réacteur ainsi dimensionné s’est avéré stable pendant 50 jours consécutifs sans aucune perte d’activité, permettant de minimiser le coût élevé de l’enzyme. L’originalité du procédé est l’utilisation d’huiles vierges contenant des antioxydants naturels (phospholipides, tocophérols etc.). Nous avons démontré que ces composés mineurs sont préservés au cours du procédé de transestérification. Cela confère aux esters formés de remarquables propriétés de résistances à l’oxydation.La pertinence économique du procédé a été améliorée grâce au développement d’un nouveau biocatalyseur sur support hydrophobe (l’Accurel MP) permettant d’éviter toute adsorption de composés polaires. Une analyse économique du procédé (maximisation de la valeur nette actualisée) a permis de rationaliser les conditions optimales d’immobilisation. Une économie de l’ordre de 50% sur les coûts générés tout au long du temps de vie du procédé a pu ainsi être obtenue. En conditions de transestérification continue, aucune différence dans le profil de produits par rapport à Novozyme 435 n’a été observée. Finalement, une alternative à la transestérification directe de l‘huile a été envisagée. Une première phase d’hydrolyse de l’huile est suivie d’un procédé de récupération des acides gras qui sont dans un second temps estérifiés enzymatiquement. Pour réaliser cette dernière étape, le meilleur système réactionnel s’est avéré être le milieu sans solvant. Un réacteur continu d’estérification de l’acide oléique avec l’isobutanol a été optimisé. Cela a permis un réacteur stable pendant 54 jours consécutifs et respectant les critères des biotechnologies blanches. Une productivité annuelle de 126 tonnes.kg de Novozyme 435-1 a été atteinte. Cela représente une amélioration de la productivité d’un facteur 9,2 par rapport au procédé de transestérification développée précédemment / This work focused on the development of efficient continuous processes for the production of esters from crude or refined high oleic sunflower oil with enzymatic packed bed reactor presenting high levels of productivity and stability. A process of continuous transesterification in packed bed reactor using Novozyme 435 (lipase B from Candida antarctica immobilized onto Lewatit VP OC 1600), a non-specific biocatalyst, was optimized to transformation of high-oleic sunflower oil into butylic esters. The phenomena of partition of polar compounds (phospholipids found in crude oils, produced glycerol etc.) between the reaction medium and the enzymatic support were managed using tert-butanol, a polar solvent. The conditions that enabled the best compromise between stability, productivity and production yields were obtained with an initial oil concentration of 500 mM and a molar ratio between co-substrates of 5. Such conditions enabled a productivity of 13.8 tons.kg-1.kg of Novozyme 435-1 to be reached. The reactor exhibited great stability for 50 consecutive days without any loss of activity. That enabled to minimize the high costs of the enzyme. The novelty of the process was the use of crude oils, containing high levels of natural antioxidants (phospholipids, tocopherols etc.). We demonstrated that these minor components of oils were preserved during the transesterification process. It conferred the synthesized esters some remarkable properties of oxidative resistance.The economic relevance of the process was improved thanks to the development of a new biocatalyst onto a very hydrophobic support (Accurel MP) in order to avoid any adsorptions of polar compounds. An economic analysis (maximisation of the net present value) enabled to rationalize the optimal immobilisation conditions. Over the whole process, it enabled a 50% saving on the global expenses.__ In continuous transesterification conditions, no difference in the product profile was noticed between the new biocatalyst and Novozyme 435.Finally, an alternative to direct transesterification of oil was considered. A first stage of oil hydrolysis is followed by a process of fatty acid recovery and a stage of enzymatic esterification into esters. In order to realize/complete this last stage, the best reaction system was a solvent-free medium. A continuous reactor for the esterification of oleic acid with isobutanol was optimized. It enabled a reactor stable/a stable reactor for 54 consecutive days, respecting the conditions of white biotechnologies. An annual productivity of 126 tons.year-1.kg of Novozyme 435-1 was reached. That represented a productivity improvement by a factor of 9.2 in comparison with the transesterification process.

Très récemment, les solvants de type « mélanges eutectiques profonds (MEP)» ont été décrits comme une alternative sérieuse et économiquement plus réaliste aux liquides ioniques. En effet, ces solvants qui consistent en un mélange d'un sel organique (ammonium ou phosphonium) et d'un donneur de liaison hydrogène peuvent également être liquides à température ambiante, non volatils et présentant une excellente stabilité thermique. De plus, contrairement aux liquides ioniques, ces nouveaux solvants sont très facilement préparés et leur innocuité ainsi que leur bonne biodégradabilité sont sensiblement améliorées. Dans le domaine des procédés enzymatiques, si la biocatalyse en milieu liquide ionique est très documentée, il n'existe que très peu de publications décrivant des réactions de biotransformation en MEP. Concernant les lipases en particulier, outre leurs applications dans le biofaçonnement des corps gras, ces enzymes sont également utilisées dans des réactions dites de lipophilisation pour la synthèse de nouvelles molécules à haute valeur ajoutée (tensioactifs, antioxydant lipophilisés). Au travers cette étude nous nous sommes investis à tester le potentiel des MEP en tant que nouveaux milieux réactionnels « verts » pour la synthèse lipasique. Ce travail n'a pas eu comme objectif de faire l'éloge de ces solvants pour leur utilisation dans le domaine de la biocatalyse, mais surtout d'évaluer leur capacité à favoriser ou non des synthèses lipasiques. Par ailleurs, nous nous sommes engagés à essayer de comprendre, d'un point de vue fondamental, l'organisation supramoléculaire de ce type de milieux pour déterminer les paramètres qui influencent le plus la réactivité et la stabilité enzymatique dans ce type d'environnement. Les variations des conditions réactionnelles (solvants et biocatalyseurs) ont permis de mettre en évidence la très nette supériorité de deux MEP (Chlorure de cholinium:Urée et Chlorure de cholinium:glycérol) pour la réalisation de réactions d'alcoolyses biocatalysées par la lipase B de Candida antarctica. Toutefois, les résultats ont montré que les réactions de biotransformations de composés phénoliques dans ces MEP sont extrêmement difficiles à réaliser sans l'addition d'eau. De profondes études (pH, activité thermodynamique de l'eau, activité et stabilité de la lipase, composition du solvant, etc.) réalisées sur des mélanges du type MEP-eau ont permis de finement adapter les conditions de réaction pour optimiser la catalyse enzymatique dans ce type de solvant. Compte tenu des difficultés rencontrées pour la lipophilisation de composés phénoliques, nous sommes toutefois parvenus à synthétiser toute une gamme de dérivés lipophiles d'acides férulique et coumarique de C4 à C16 (chaîne aliphatique) avec des rendements élevés. / With the emergence of the green chemistry concept in the 90s, many studies have been dedicated to the discovery of new reactions media both suitable and efficient for chemical/enzyme catalysis. Up to now, the main efforts have focused on the development of ionic liquids. However, recently a novel class of solvent called "deep eutectic mixtures (DES)", have been described as a serious alternative and economically stronger than ionic liquids. Such solvents are formed by mixing an organic salt (ammonium or phosphonium) with a hydrogen-bond donor. Just like ionic liquid, DES may also be liquid at room temperature, non-volatile and have excellent thermal stability. However, unlike most ionic liquids, these new solvents are biodegradable, inexpensive, and very easy to prepare. In the field of biocatalysis, whereas the studies in ionic liquid are deeply documented, the published papers describing biotransformation reactions in DES are very low, especially in lipase-catalyzed processing, where these enzymes may be used in so-called "lipophilisation reactions", for the synthesis of new molecules with high added value (surfactants or lipophilized antioxidants).The main objective of this work was to assess and test the potential of DES as new "green" reaction media for lipase-catalyzed synthesis. On a fundamental point of view, this study provides valuable information to understand how the different components involved in these mixtures could contribute to their functional properties in order to enhance their use in various applications. Changes in reaction conditions (solvents and biocatalysts) allowed us to highlight the clear superiority of two DES (chloride cholinium:Urea and chloride cholinium:glycerol) to carry out lipase-catalyzed reactions using the lipase B from Candida antarctica as biocatalyst. However, our results showed that the biotransformations of dissolved substrates (such as phenolic compounds) in DES are extremely difficult to achieve without the addition of water. Studying DES-water mixtures (pH, thermodynamic activity of water, activity and stability of lipase, mixtures composition, etc ...) we were able to fine-tune the reaction conditions to optimize the performance of the lipasic catalysis. Thus, given the difficulties encountered when performing lipase-catalyzed reactions with substrates of two different polarities, it was still possible to synthesize high yields of a full range of lipophilic derivatives of ferulic and coumaric acids from C4 to C16 (aliphatic chain).

Les lipases présentent un grand intérêt pour la synthèse du Biodiesel, carburant alternatif au gasoil, généralement obtenu d’une transestérification des triacylglycérols avec un alcool, la plupart du temps le méthanol. Pour avoir un ester issu totalement de la biomasse végétale, l’éthanol peut être utilisé comme accepteur d’acyle. L’objectif de cette étude est de développer des procédés enzymatiques de synthèses d’esters éthyliques catalysés par les lipases végétales sous leur forme brute avec des intrants (huile et alcool) d’origine végétale. D’abord, elle a consisté à la mise en évidence d’une activité lipasique pour des réactions d’éthanolyse et d’hydrolyse par les graines d’A. suarezensis, d’A. grandidieri, de J. curcas, de J. mahafalensis, de M. oleifera et de M. drouhardii. Ensuite, les influences de certains facteurs sur la capacité des extraits le(s) plus actif(s) à réaliser des réactions d’éthanolyse en milieux non aqueux, aqueux et en utilisant comme substrat leurs lipides natifs ont été étudiées. Enfin, des essais de combustion ont été menés sur un moteur monocylindre à injection directe pour l’étude des performances, des émissions et de la combustion du biodiesel produit et de ses mélanges avec le gasoil. Toutes les graines germées sont dotées d’une activité en hydrolyse et éthanolyse. La poudre d’A. grandidieri est la plus active en éthanolyse. Avec cette dernière, deux procédés ont pu être développés : un en milieu non aqueux et un en milieu aqueux (respectivement un rendement de 96,2 % et 96,3 %). Elle est aussi capable de transformer ses lipides natifs sans extraction au préalable en esters éthyliques (rendement de 91,6%). Les performances et la combustion du biodiesel et de ses mélanges sont similaires à celle du gasoil. Une réduction significative des émissions de CO, NOX, CO2 et SO2 au cours de la combustion du biodiesel et de ses mélanges est observée. Ces résultats montrent que les lipases végétales exploitées sous leurs formes brutes peuvent être une alternative aux lipases microbiennes et aux catalyseurs chimiques. / There is a great interest in the use of lipase in the production of Biodiesel, alternative diesel fuel, usually obtained from a transesterification of triacylglycerol with an alcohol which is mostly methanol. To have a biodiesel derived totally from vegetable biomass, ethanol must be explored as acyl acceptor. The objective of this work is to develop enzymatic processes for the synthesis of ethyl esters catalyzed by plant lipases in their crude form with all inputs (oil and alcohol) of origin plant. Firstly, the hydrolysis and ethanolysis activities of A. suarezensis, A. grandidieri, J. curcas, J. mahafalensis, M. oleifera and M. drouhardii seeds were assessed. Subsequently, the most active(s) plant lipase(s) was selected to study the effects of some factors on their ability to carry out ethanolysis reactions in nonaqueous, aqueous media and using as substrate their lipids. Finally, combustion tests were carried out on a single cylinder direct injection engine to study the performance, emissions and combustion of biodiesel and its mixture with diesel. All germinated seeds have hydrolysis and ethanolysis activity. The most active in ethanolysis is the powder from A. grandidieri seed. With this powder, two processes were developed: one in nonaquous medium and the other in aqueous medium (yield of 96.2 % and 96.3 %, respectively). Lipase from A. grandidieri seed is able to transesterify its oils without an extraction thereof into ethyl ester. Performance and combustion characteristics of biodiesel and its mixtures are similar to that of diesel fuel. A significant reduction in CO, NOX, CO2 and SO2 emissions during the combustion of biodiesel and its mixtures is observed. These results show that plant lipases exploited in their crude form can be an alternative to microbial lipases and chemical catalysts.

O Cerrado é o segundo maior bioma da América do Sul, abrigando 11.627 espécies de plantas nativas já catalogadas. A grande demanda industrial por novas fontes de lipases com diferentes características tem estimulado a procura de novas cepas de micro-organismos lipolíticos. O presente estudo objetiva otimizar a produção de lipases em cultivo submerso por leveduras pouco estudadas. Foi utilizado um delineamento Plackett-Burman para avaliar o efeito de dez variáveis (pH, agitação, sulfato de amônio, peptona, triton X-100, Tween 20, azeite de oliva, extrato de levedura, sulfato de magnésio e fosfato monopotássico) na produção de lipases por duas linhagens de leveduras, considerando o nível de significância p < 0,1. Para a produção de lípase pela linhagem TAQ 616 a variável significativa foi a concentração de azeite de oliva e para biomassa foram as concentrações de azeite de oliva, triton X-100 e KH2PO4. A atividade enzimática da linhagem ABRT 461 sofreu a influência das variáveis pH, agitação e concentração de (NH4)2SO4, e para a produção de biomassa nenhuma das variáveis foi estatísticamente significativa. A linhagem ABRT 461 foi escolhida para realização de um planejamento de Delineamento Composto Central Rotacional (DCCR) 2³. As condições ótimas de atividade enzimática geradas pelo modelo proposto foram de 114 rpm, pH 5,0 e 25 g.L-1 de concentração de (NH4)2SO4. A atividade enzimática de 84,4 U.mL-1 foi obtida após o cultivo da linhagem ABRT 461 durante 72 horas nas condições ótimas estimadas pelo modelo. / The Cerrado is the second largest biome in South America, housing 11.627 native plant species already cataloged. The great industrial demand for new sources of lipases with different characteristics has stimulated the search for new strains of lipolytic microorganisms. The purpose of this study is to optimize the lipase production in submerged culture for little studied yeast. A Plackett-Burman design was used to evaluate the effect of ten variables (pH, agitation, ammonium sulfate, peptone, Triton X-100, Tween 20, olive oil, yeast extract, magnesium sulfate and potassium dihydrogenphosphate) in the lipase production by two yeast strains, considering a significance level of p < 0,1. The significant variable for strain TAQ 616 lipases production was the olive oil concentration and for biomass were olive oil, triton X-100 and KH2PO4. The enzymatic activity of lineage ABRT 461suffered the influence of pH, agitation and KH2PO4, and for biomass production any variable was statistically significant. The ABRT 461 lineage was selected to do a central composite rotational design (CCRD)2³. The optimum conditions for enzyme activity generated by the proposed model was 114 rpm, pH 5.0 and 25 g.L-1 concentration of (NH4)2SO4. The enzymatic activity of 84,4 U.mL-1 was obtained after the cultivation of ABRT 461 lineage for 72 hours in optimal conditions estimated by the model.

Nesse trabalho foram produzidas lipases da levedura Pseudozyma hubeiensis (HB85A) em reator de 14 L. Após produção da enzima, a lipase foi imobilizada por adsorção em suporte hidrofóbico por processo contínuo em reator de leito fixo. As melhores condições de imobilização foram: tempo de imobilização de 2 h e 29 min., pH de 4,76 e quantidade de enzima livre adicionada por grama de suporte de 1282 U/ g de suporte, sendo que, a máxima atividade da lipase imobilizada obtida foi de 143 U/g de suporte. O sobrenadante contendo lipase e o biocatalisador heterogênio foram caracterizados por planejamento fatorial. A máxima atividade da enzima imobilizada (71 U/g de suporte) foi obtida em pH 6,0 à temperatura de 52 °C. A imobilização da lipase resultou em um aumento na estabilidade dessa enzima em temperaturas altas, pH ácidos e neutros, presença de detergentes não-iônicos e altas concentrações de solventes orgânicos como iso-propanol, metanol e acetona. Foi possível a reutilização da lipase imobilizada por apenas uma vez na reação de hidrólise, havendo uma perda de 72 % da atividade após o primeiro reuso. Analisou-se ainda a estabilidade da lipase livre e imobilizada durante 40 dias de armazenamento a 4 °C. Durante o período de armazenamento, a lipase imobilizada manteve 50 % de sua atividade original e a lipase livre apresentou 80 %. O catalisador heterogêneo foi testado quanto a sua eficácia na produção de biodiesel. A reação de transesterificação foi realizada na ausência de co-solvente utilizando-se como matérias-primas metanol, etanol e iso-propanol e quatro fontes diferentes de triglicerídeo (óleo de soja, óleo de mamona, óleo residual de restaurante e a gordura bovina). A partir dos testes realizados, obteve-se um rendimento máximo quanto à produção de biodiesel de 3,15 % utilizando-se óleo de mamona e iso-propanol como matéria-prima pelo período de 24 h. A produção de biodiesel utilizando diferentes quantidades de lipase imobilizada e também a lipase livre como catalisador foi testada na presença de hexano, iso-propanol e óleo de mamona pelo período de 24 h nas temperaturas de 40, 50 e 60 °C. No entanto, não houve produção de biodiesel nas condições analisadas. / In this work, lipases from yeast Pseudozyma hubeiensis (strain HB85A) were produced in a 14 L reactor. After lipase from yeast P. hubeiensis (strain HB85A) production, the enzyme was immobilized by adsorption in polyestyrene divinylbenzene hydrophobic support in a packet bed column. The best conditions for lipase immobilization were: 2 h and 29 min. immobilizing time, pH 4.76 and rate of free enzyme added per gram of support equal to 1282 U/g. The maximum activity of immobilized lipase was reached of 143 U/g. The lipases of P. hubeiensis (HB85A) supernatant culture and the heterogeneous catalyst were characterized through response surface methodology by factorial design. The maximum activity of immobilized lipase was reached for a support rate of 71 U/g, with pH 6.0 and temperature of 52 °C. It was detected that lipase immobilization increased enzyme stability under high temperatures, neutral and acid pH levels, non-ionic detergent and high concentration of organic solvent like iso-propanol, methanol and acetone. The reuse of immobilized lipase was possible only once for hydrolysis reaction, with activity losses of 72 % after first re-use. Also, it was tested lipase stability in a period of 40 days, under 4 °C storage conditions. During storage period, immobilized lipase kept 50 % of its original activity. Free lipase kept 80 %. After the development of heterogeneous catalyst, its efficiency as catalyst for biodiesel production was analyzed in this study. The transesterification reaction was tested in co-solvent absence using as raw material three differents sources of alcohols (methanol, ethanol and iso-propanol) and four differents triglicerides source (soybean oil, castor oil, waste cooking oil and bovine fat) and as catalystis the immobilized lipase. Based in test results, the maximum biodiesel production yield was 3.15 % using castor oil and methanol as raw material for 24 h. The biodiesel production was also tested with different amount of immobilized lipase and with free lipase as catalystis at the presence of methanol, castor oil and the co-solvent hexane for 24 h at 40, 50 e 60 °C. However there was no biodiesel production at the tested conditions.

Nesse trabalho foram produzidas lipases da levedura Pseudozyma hubeiensis (HB85A) em reator de 14 L. Após produção da enzima, a lipase foi imobilizada por adsorção em suporte hidrofóbico por processo contínuo em reator de leito fixo. As melhores condições de imobilização foram: tempo de imobilização de 2 h e 29 min., pH de 4,76 e quantidade de enzima livre adicionada por grama de suporte de 1282 U/ g de suporte, sendo que, a máxima atividade da lipase imobilizada obtida foi de 143 U/g de suporte. O sobrenadante contendo lipase e o biocatalisador heterogênio foram caracterizados por planejamento fatorial. A máxima atividade da enzima imobilizada (71 U/g de suporte) foi obtida em pH 6,0 à temperatura de 52 °C. A imobilização da lipase resultou em um aumento na estabilidade dessa enzima em temperaturas altas, pH ácidos e neutros, presença de detergentes não-iônicos e altas concentrações de solventes orgânicos como iso-propanol, metanol e acetona. Foi possível a reutilização da lipase imobilizada por apenas uma vez na reação de hidrólise, havendo uma perda de 72 % da atividade após o primeiro reuso. Analisou-se ainda a estabilidade da lipase livre e imobilizada durante 40 dias de armazenamento a 4 °C. Durante o período de armazenamento, a lipase imobilizada manteve 50 % de sua atividade original e a lipase livre apresentou 80 %. O catalisador heterogêneo foi testado quanto a sua eficácia na produção de biodiesel. A reação de transesterificação foi realizada na ausência de co-solvente utilizando-se como matérias-primas metanol, etanol e iso-propanol e quatro fontes diferentes de triglicerídeo (óleo de soja, óleo de mamona, óleo residual de restaurante e a gordura bovina). A partir dos testes realizados, obteve-se um rendimento máximo quanto à produção de biodiesel de 3,15 % utilizando-se óleo de mamona e iso-propanol como matéria-prima pelo período de 24 h. A produção de biodiesel utilizando diferentes quantidades de lipase imobilizada e também a lipase livre como catalisador foi testada na presença de hexano, iso-propanol e óleo de mamona pelo período de 24 h nas temperaturas de 40, 50 e 60 °C. No entanto, não houve produção de biodiesel nas condições analisadas. / In this work, lipases from yeast Pseudozyma hubeiensis (strain HB85A) were produced in a 14 L reactor. After lipase from yeast P. hubeiensis (strain HB85A) production, the enzyme was immobilized by adsorption in polyestyrene divinylbenzene hydrophobic support in a packet bed column. The best conditions for lipase immobilization were: 2 h and 29 min. immobilizing time, pH 4.76 and rate of free enzyme added per gram of support equal to 1282 U/g. The maximum activity of immobilized lipase was reached of 143 U/g. The lipases of P. hubeiensis (HB85A) supernatant culture and the heterogeneous catalyst were characterized through response surface methodology by factorial design. The maximum activity of immobilized lipase was reached for a support rate of 71 U/g, with pH 6.0 and temperature of 52 °C. It was detected that lipase immobilization increased enzyme stability under high temperatures, neutral and acid pH levels, non-ionic detergent and high concentration of organic solvent like iso-propanol, methanol and acetone. The reuse of immobilized lipase was possible only once for hydrolysis reaction, with activity losses of 72 % after first re-use. Also, it was tested lipase stability in a period of 40 days, under 4 °C storage conditions. During storage period, immobilized lipase kept 50 % of its original activity. Free lipase kept 80 %. After the development of heterogeneous catalyst, its efficiency as catalyst for biodiesel production was analyzed in this study. The transesterification reaction was tested in co-solvent absence using as raw material three differents sources of alcohols (methanol, ethanol and iso-propanol) and four differents triglicerides source (soybean oil, castor oil, waste cooking oil and bovine fat) and as catalystis the immobilized lipase. Based in test results, the maximum biodiesel production yield was 3.15 % using castor oil and methanol as raw material for 24 h. The biodiesel production was also tested with different amount of immobilized lipase and with free lipase as catalystis at the presence of methanol, castor oil and the co-solvent hexane for 24 h at 40, 50 e 60 °C. However there was no biodiesel production at the tested conditions.

Made available in DSpace on 2016-06-02T19:55:25Z (GMT). No. of bitstreams: 1 2620.pdf: 1689550 bytes, checksum: 4fa47e9cd5f6de2ff9af48027193823e (MD5) Previous issue date: 2009-05-25 / Universidade Federal de Minas Gerais / The objective of this thesis was to prepare and select immobilized lipase derivatives with high catalytic activity and thermal stability to mediate the biodiesel synthesis from palm and babassu oils by ethanolic route. The experimental work was carried out in two steps. In the first, different lipases sources, including lipases from Thermomyces lanuginosus (TLL), Candida antarctica type B (CALB), porcine pancreas (PPL), Bacillus thermocatenulatus (BTL2), Pseudomonas fluorescens (LPF) and Lipex® 100L were immobilized on different supports activated by several protocols using two immobilization methods, such as physical adsorption and multipoint covalent attachment. The following matrixes were used: agarose, Toyopearl, chitosan, alginate-chitosan, octyl-agarose, hexyltoyopearl and PHB and activating agents were: glutaraldehyde, epichlorohydrin and glycidol. As expected the immobilization procedure, support and lipase source affected the catalytic properties of the immobilized derivatives and their suitability for the proposed reaction. With an exception of PPL, all lipase preparations (TLL, PFL, Lipex® 100L and CALB) showed high alkaline stability under the immobilization conditions (72 h at pH 10.05) resulting in immobilized derivatives having high hydrolytic activities. The highest hydrolytic activities were obtained by TLL immobilized on glyoxyl-agarose, glyoxyl-chitosan-alginate-TNBS, epoxy-chitosan-alginate and Lipex® 100L immobilized on epoxy-chitosan-alginate and glyoxyl-agarose. Under non-aqueous media using butyl butyrate synthesis as a model system, TLL and PFL immobilized on glyoxyl-agarose and glyoxyl-amine-toyopearl showed similar conversions. The highest thermal stability were obtained for non-aminated BTL2 immobilized on glyoxyl-agarose 10BCL (Stability Factor- SF =2648) and chemically aminated (SF=4360), followed by aminated CALB immobilized on glyoxyl-agarose BCL (SF=290) and TLL immobilized on glyoxyl-agarose BCL (SF~300). Using chitosan-alginate, the highest thermal stability was obtained for TLL immobilized on chitosan-alginate-TNBS activated with glyoxyl groups and glutaraldehyde (SF=45). The immobilization of lipases BTL2, CALB and TLL on hydrophobic supports such as octyl-agarose and hexyl-toyopearl by physical adsorption allowed obtaining thermal stable derivatives. In addition, immobilized derivatives on poly-(hydroxybutyrate) (PHB) showed high catalytic activity in both hydrolysis and esterification reactions. In the second step and based on their catalytic properties under both aqueous and non-aqueous media as well as thermal stabilities the following immobilized derivatives: TLL and PFL immobilized on glyoxyl-agarose and glyoxyl-amine-Toyopearl, TLL immobilized on chitosan-alginate-TNBS activated with glyoxyl and glutaraldehyde; TLL, PFL, Lipex® 100L and CALB immobilized by physical adsorption on PHB were selected to mediate the synthesis of biodiesel from palm and babassu oils. For derivatives prepared by multipoint covalent attachment and under the conditions used, total conversion in ethyl esters was achieved within 24 to 48 h, depending on the vegetable oil. For immobilized derivatives prepared by physical adsorption on PHB, a slight higher reaction time (72 h) was needed to attain total conversion in ethyl esters. Despite its high esterification activity, BTL2 immobilized on PHB failed to mediate the ethanolysis of both vegetable oils. The viscosity values for the biodiesel samples (3.4-4.5 cSt) are in accordance with specifications recommended by the Brazilian Petroleum Agency (ANP) to be used as biofue. / Este trabalho teve como objetivo preparar e selecionar derivados imobilizados de lipases com elevada atividade catalítica e estável termicamente para mediar a síntese de biodiesel a partir dos óleos de palma e babaçu pela rota etílica. O trabalho experimental foi desenvolvido em duas etapas. Na primeira etapa, diferentes fontes de lipases incluindo as lipases de Thermomyces lanuginosus (LTL), Candida antarctica tipo B (CALB), pâncreas de porco (LPP), Bacillus thermocatenulatus (BTL2), Pseudomonas fluorescens (LPF) e Lipex® 100L foram imobilizadas em diferentes suportes ativados por diferentes protocolos usando dois procedimentos de imobilização, adsorção física e ligação covalente multipontual. As seguintes matrizes foram testadas: agarose, Toyopearl, complexos polieletrolíticos de quitosana, octil-agarose, hexil-toyopearl e PHB e os agentes de ativação testados foram: glutaraldeído, epicloridrina e glicidol. Como esperado o procedimento de imobilização, suporte e fonte de lipase afetaram as propriedades catalíticas dos derivados imobilizados e adequação para mediar a síntese proposta. Com exceção da LPP, todas as preparações de lipase (LTL, PFL, Lipex® 100L e CALB) mostraram elevada estabilidade em meio alcalino (72 h em pH 10,05) e resultaram em derivados com elevada atividade hidrolítica. As atividades hidrolíticas mais elevadas foram obtidas pela LTL imobilizada em glioxil-agarose, glioxil-quitosana-alginato-TNBS, epóxi-quitosana-alginato e Lipex® 100L imobilizada em epóxi-quitosana-alginato e em glioxil-agarose. Na síntese de butirato de butila, as reações catalisadas por LTL e LPF imobilizadas em glioxil-agarose e glioxil-amino-toyopearl apresentaram conversões superiores a 65%. As estabilidades térmicas mais elevadas foram obtidas para BTL2 não-aminada imobilizada em glioxil-agarose 10BCL (FE=2648) e aminada quimicamente (FE=4360), seguido de CALB aminada imobilizada em glioxil-agarose 6BCL (FE=290) e LTL imobilizada em glioxil-agarose 6BCL (FE~300). Usando quitosana-alginato, as estabilidades térmicas mais elevadas foram obtidas para LTL imobilizada em quitosanaalginato- TNBS ativados com grupos glioxil e glutaraldeído (FE=45). A imobilização de lipases BTL2, CALB e LTL em suportes hidrofóbicos octil-agarose e hexil-toyopearl por adsorção física resultaram em derivados estáveis termicamente com elevada atividade catalítica em reações de hidrólise. Lipases também foram imobilizadas em poli- (hidróxibutirato) (PHB) por adsorção física e os derivados preparados apresentaram alta atividade catalítica em meio aquoso e orgânico. Tomando por base as propriedades catalíticas em meio aquoso e orgânico, bem como a estabilidade térmica foram selecionados para mediar a síntese de biodiesel os derivados de LTL e LPF imobilizados em géis glioxil-agarose e glioxil-amino-toyopearl e LTL imobilizada em quitosana-alginato-TNBS por ativação com glicidol, derivados preparados das lipases LTL, LPF, Lipex® 100L e CALB em PHB. Para as lipases imobilizadas por ligação covalente multipontual e nas condições testadas, a conversão total em ésteres de etila foi alcançada entre 24 a 48 h, dependendo do óleo vegetal. Para os derivados preparados por adsorção física em PHB, um maior tempo de reação (72 h) foi necessário para atingir a conversão total em ésteres de etila. Apesar da elevada atividade de esterificação, BTL2 imobilizada em PHB não catalisou a reação de etanólise de ambos os óleos vegetais, mas mostrou alta atividade de esterificação. Os valores de viscosidade das amostras de biodiesel purificadas (3.4-4.5 cSt) atendem as espeficacões recomendadas pela Agência Brasileira de Petróleo (ANP) para ser usado como biocombustível.

Nesse trabalho foram produzidas lipases da levedura Pseudozyma hubeiensis (HB85A) em reator de 14 L. Após produção da enzima, a lipase foi imobilizada por adsorção em suporte hidrofóbico por processo contínuo em reator de leito fixo. As melhores condições de imobilização foram: tempo de imobilização de 2 h e 29 min., pH de 4,76 e quantidade de enzima livre adicionada por grama de suporte de 1282 U/ g de suporte, sendo que, a máxima atividade da lipase imobilizada obtida foi de 143 U/g de suporte. O sobrenadante contendo lipase e o biocatalisador heterogênio foram caracterizados por planejamento fatorial. A máxima atividade da enzima imobilizada (71 U/g de suporte) foi obtida em pH 6,0 à temperatura de 52 °C. A imobilização da lipase resultou em um aumento na estabilidade dessa enzima em temperaturas altas, pH ácidos e neutros, presença de detergentes não-iônicos e altas concentrações de solventes orgânicos como iso-propanol, metanol e acetona. Foi possível a reutilização da lipase imobilizada por apenas uma vez na reação de hidrólise, havendo uma perda de 72 % da atividade após o primeiro reuso. Analisou-se ainda a estabilidade da lipase livre e imobilizada durante 40 dias de armazenamento a 4 °C. Durante o período de armazenamento, a lipase imobilizada manteve 50 % de sua atividade original e a lipase livre apresentou 80 %. O catalisador heterogêneo foi testado quanto a sua eficácia na produção de biodiesel. A reação de transesterificação foi realizada na ausência de co-solvente utilizando-se como matérias-primas metanol, etanol e iso-propanol e quatro fontes diferentes de triglicerídeo (óleo de soja, óleo de mamona, óleo residual de restaurante e a gordura bovina). A partir dos testes realizados, obteve-se um rendimento máximo quanto à produção de biodiesel de 3,15 % utilizando-se óleo de mamona e iso-propanol como matéria-prima pelo período de 24 h. A produção de biodiesel utilizando diferentes quantidades de lipase imobilizada e também a lipase livre como catalisador foi testada na presença de hexano, iso-propanol e óleo de mamona pelo período de 24 h nas temperaturas de 40, 50 e 60 °C. No entanto, não houve produção de biodiesel nas condições analisadas. / In this work, lipases from yeast Pseudozyma hubeiensis (strain HB85A) were produced in a 14 L reactor. After lipase from yeast P. hubeiensis (strain HB85A) production, the enzyme was immobilized by adsorption in polyestyrene divinylbenzene hydrophobic support in a packet bed column. The best conditions for lipase immobilization were: 2 h and 29 min. immobilizing time, pH 4.76 and rate of free enzyme added per gram of support equal to 1282 U/g. The maximum activity of immobilized lipase was reached of 143 U/g. The lipases of P. hubeiensis (HB85A) supernatant culture and the heterogeneous catalyst were characterized through response surface methodology by factorial design. The maximum activity of immobilized lipase was reached for a support rate of 71 U/g, with pH 6.0 and temperature of 52 °C. It was detected that lipase immobilization increased enzyme stability under high temperatures, neutral and acid pH levels, non-ionic detergent and high concentration of organic solvent like iso-propanol, methanol and acetone. The reuse of immobilized lipase was possible only once for hydrolysis reaction, with activity losses of 72 % after first re-use. Also, it was tested lipase stability in a period of 40 days, under 4 °C storage conditions. During storage period, immobilized lipase kept 50 % of its original activity. Free lipase kept 80 %. After the development of heterogeneous catalyst, its efficiency as catalyst for biodiesel production was analyzed in this study. The transesterification reaction was tested in co-solvent absence using as raw material three differents sources of alcohols (methanol, ethanol and iso-propanol) and four differents triglicerides source (soybean oil, castor oil, waste cooking oil and bovine fat) and as catalystis the immobilized lipase. Based in test results, the maximum biodiesel production yield was 3.15 % using castor oil and methanol as raw material for 24 h. The biodiesel production was also tested with different amount of immobilized lipase and with free lipase as catalystis at the presence of methanol, castor oil and the co-solvent hexane for 24 h at 40, 50 e 60 °C. However there was no biodiesel production at the tested conditions.