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1
Salgueiro, Alexandra Amorim. "Lipase production in Candida lipolytica 1055." Thesis, University of St Andrews, 1994. http://hdl.handle.net/10023/14328.
Full textAbstract:
Microbial lipases are now increasingly employed in the redesign of animal fats and vegetable oils. Commercial scale manufacture of microbial lipases, which is limited, tends to be surrounded by secrecy and much remains to be clarified in terms of optimization of fermentation conditions. C. lipolytica metabolizes cheap carbon sources (from wastes and by-products), in particular non- carbohydrate substrates, accumulating lipid during the growth. As all potent producers of lipase are also amongst the lipid-accumulating microorganisms, this yeast shows potential as a major lipase producer. The future for lipase fermentation industries look promising due to these raw material being abundant and inexpensive. Lipase activity (triolein as a substrate) and esterase activity (p-nitrophenylpalmitate as a substrate) were measured throughout this work. Different operational strategies (batch, fed-batch, chemostat and continuous transient technique) have xi been investigated aiming lipase and esterase production by C. lipolytica 1055. Nutritional parameters (carbon and nitrogen sources, limiting substrates: glucose, Tween-80 and olive oil) under steady-state conditions at different dilution rates, as well as oleic acid and olive oil square wave oscillations have been studied. Pulsing experiments with triolein, olive oil, oleic acid and Tween-80 suggest that the oleyl residue may be responsible for the induction effect in C. lipolytica 1055 lipase and esterase production. However, the low values of lipase productivities (< 1 U/mg dry weight.h) under different cultivation methods appear to limit the advantages in using C. lipolytica 1055 as a lipase producer. Extracellular esterase productivity by C. lipolytica 1055 reached 5.3 - 6.5 U/mg dry weight.h in the presence of Tween-80 under batch and fed-batch culture subjected to oleic acid input. The esterase production phase under fed-batch process was prolonged suggesting that this operation mode could be a route towards obtaining esterase by C. lipolytica 1055. For example, a two-phase bioreactor operation may be devised with high yeast cell concentration promoted by a carbohydrate waste under batch conditions in the first stage, and a high enzyme production under fed-batch operation in the second one.
2
Smith, Catherine J. "Production of a lipase from a Pseudomonad species." Thesis, Cranfield University, 1991. http://dspace.lib.cranfield.ac.uk/handle/1826/4183.
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A lipase-producing micro-organism was found to be a strain of Pseudomonas aeruginosa. When it was grown batchwise in a stirred 2 dM3 fermenter in a simple defined minimal salts medium containing yeast extract and glucose, it produced lipase at a level of 0.173 LU/cm3 without the need for a lipid substrate. Batch cultures of cells were fed glucose at constant rates. An optimum rate was found to be 0.50 g/dm3/h and this produced a maximum lipase activity of 0.80 LU/cm3. Medium composition was changed until it was possible to produce 25 g/dm3 dry weight of cells. Evidence of linear growth due to nutient, limitations was obtained. A microcomputer controlled protocol was used successfully to feed glucose solutions at exponentially increasing rates. Problems were encountered with the production of lipase from the interaction with pyocyanin production and from possible trace nutrient limitations.
3
Ventura, Sónia Patrícia Marques. "Production of lipase by extractive fermentation with ionic liquids." Doctoral thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7023.
Full textAbstract:
Doutoramento em Engenharia Química<br>Novos processos fermentativos, designados por processos de Fermentação
Extractiva, são caracterizados por apresentarem etapas de produção e
extracção em simultâneo. A extracção líquido-líquido como técnica de
separação é amplamente usado na indústria química pela sua simplicidade,
baixo custo e facilidade de extrapolação de escala. No entanto o uso de
solventes orgânicos nestes processos potencia os riscos ocupacionais e
ambientais. Neste contexto, o uso de sistemas de duas fases aquosas
baseados em líquidos iónicos, apresenta-se como uma técnica eficaz para a
separação e purificação de produtos biológicos.
Este trabalho apresenta um estudo integrado sobre o uso de líquidos iónicos
não aromáticos foram determinados. A capacidade para a formação de
sistemas de duas fases foi estudada para uma vasta gama de líquidos iónicos
hidrofílicos com diferentes aniões, catiões e cadeias alqúilicas. A capacidade
de separação e purificação de um largo conjunto de líquidos iónicos foi
posteriormente investigada, recorrendo-se ao uso de várias biomoléculas
modelo de diferentes graus de complexidade, um amino-acido (L-triptofano) e
duas enzimas lipolíticas (enzima produzida pela bactéria Bacillus sp. e
Candida antarctica lipase B – CaLB). Esta última foi ainda usada para um
estudo de biocompatibilidade, tendo sido determinado o efeito de diferentes LIs
hidrofílicos na sua actividade enzimática. Este trabalho mostra um estudo
ecotoxicológico duma vasta gama de líquidos iónicos e espécies aquáticas,
inseridas em diversos níveis tróficos. A bioacumulação foi investigada através
do estudo dos coeficientes de distribuição 1-octanol-água (Dow).<br>Novel fermentation processes characterized by the simultaneously production
and separation are known as Extractive Fermentations. Liquid-liquid extraction
is a separation technique that can be used for this purpose. These processes
are widely used in the chemical industry owing to its simplicity, low costs and
ease of scale-up. However, most extraction processes include the use of
organic solvents, which have both environmental and occupational risks
associated to their use. This makes these extraction systems inappropriate for
the development of environmental-friendly technologies. In this context, the use
of aqueous two-phase systems (ATPS) based in Ionic Liquids (ILs) appears as
an effective and viable method for the separation and purification of biological
products.
The present work reports an integrated study on the use of ILs as alternative
solvents in Fermentation processes. Original data on the solubility of nonaromatic
ILs in water are reported. The ability of several ILs for the formation of
aqueous two-phase systems was studied for a wide range of hydrophilic ILs
with different anions, cations and alkyl chains. The separation and purification
capacity of a large set of ILs was then investigated, using different
biomolecules as models with distinct structural complexities, one amino-acid (Ltryptophan)
and two lipolytic enzymes (an enzyme produced by the bacterium
Bacillus sp. and Candida antarctica lipase B – CaLB). CaLB was also used on
a study of biocompatibility, where the effect of various hydrophilic ILs on its
enzymatic activity was addressed.
Finally, this work reports an ecotoxicological study for a large number of ILs
and aquatic species, included in different trophic levels. The bioaccumulation
data was investigated by the study of the 1-octanol-water distribution
coefficients (Dow).
4
Lee, Seoung Yong. "Production and characterization of esterase-lipase from Lactobacillus casei subspecies." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61842.
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5
Smerdon, Gary Randall. "Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe." Thesis, University of Exeter, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261145.
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6
Rodrigues, Roberta da Silva Bussamara. "Produção e caracterização de um biocatalisador heterogêneo para ser utilizado em aplicações industriais." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2009. http://hdl.handle.net/10183/17834.
Full textAbstract:
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.<br>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.
7
McCarthy, Conor Neil, and n/a. "Regulatory Elements Controlling Lipase and Metalloprotease Production in Pseudomonas fluorescens B52." Griffith University. School of Health Science, 2003. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20031015.124744.
Full textAbstract:
Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d<SUB>2</SUB>, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.
8
KERBER, TATIANA DE MATTOS. "EXPERIMENTAL DESIGN AND METROLOGICAL EVALUATION OF LIPASE PRODUCTION BY YARROWIA LIPOLYTICA." PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO, 2007. http://www.maxwell.vrac.puc-rio.br/Busca_etds.php?strSecao=resultado&nrSeq=10700@1.
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PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO<br>Objetivo: Otimização da produção de lipases por células de
Yarrowia
lipolytica, através do planejamento experimental e
avaliação dos aspectos
metrológicos envolvidos no processo. Motivação: Embora
existam muitos
trabalhos na literatura que relatem a produção de lipases
por microrganismos,
poucos abordam a otimização do processo através do
planejamento experimental e
nenhum deles considera o rigor metrológico e a estimativa
da incerteza de
medição. Contextualização: As enzimas lipases serão objeto
de estudo por
apresentarem grande versatilidade de aplicações e por
serem produzidas por
muitos microrganismos. As leveduras, no entanto,
apresentam inúmeras vantagens
operacionais sobre os demais microrganismos e entre elas
foi selecionada uma
cultura de Yarrowia lipolytica, isolada no Brasil. A
produção de lipases depende
de muitas variáveis, como temperatura, pH, concentração da
fonte de carbono e
nitrogênio, inóculo, entre outras. Nos processos de
fermentação, onde há interação
entre estas variáveis e a influência de cada uma é
importante, é essencial
estabelecer um método que considere estas interações, e
otimize as condições
experimentais para obtenção de melhores resultados. A
confiabilidade
metrológica do processo é de fundamental importância uma
vez que diferenças
significativas nos resultados são evidenciadas,
principalmente em escala
industrial. Metodologia: Dez experimentos foram planejados
através do método
D-optimal nos quais foram variadas as concentrações da
fonte de carbono, da
fonte de nitrogênio e de inóculo. A estimativa da
incerteza de medição da
atividade lipásica foi feita com base nas recomendações do
Guia para Expressão
da Incerteza de Medição (GUM) e do Guia CG 4 publicado
pela EURACHEM.
Resultados: A maior atividade lipásica obtida foi de 4815
U/L (80,3 (mi)kat/L) com
incerteza expandida de 137 U/L (2,3 (mi)kat/L), nas
seguintes condições: 0,5% de
óleo de oliva (fonte de carbono), 0,7% de peptona (fonte
de nitrogênio) e
10 mg/mL de inóculo. Em termos de produtividade
volumétrica, tais condições
forneceram o valor de 69,28 U/L h. Conclusões: Com estes
resultados, foi possível concluir que a utilização do
planejamento D-optimal favoreceu a
produção em agitador de frascos, já que foi atingida uma
atividade lipásica
superior às alcançadas anteriormente por outros autores
sem otimização, em
frascos agitados (2700 U/L) ou em fermentador (4240 U/L).
Sugere-se a produção
de lipases por células de Yarrowia lipolytica, nas
condições citadas acima, com
vistas à obtenção de materiais de referência certificados,
desde que estudos
posteriores de purificação e estabilidade da lipase obtida
sejam feitos.<br>Objective: Optimization of lipase production by Yarrowia
lipolytica cells
through experimental design and evaluation of metrological
aspects related to
process. Motivation: Although several published papers are
found describing
lipase production by microorganisms, few of them refer to
process optimization
through experimental design and none of them refer to
metrological evaluation
and the estimate of the uncertainty in measurement.
Context: Microbial lipases
are an important group of biotechnologically valuable
enzymes that present
widely diversified catalytic properties. The interest in
production of these versatile
enzymes increased significantly, due to the vast amplitude
of their industrial
applications. There are many microorganisms able to
produce lipases, but yeasts
present some operational advantages compared to other
microorganisms, and for
this reason a strain of Yarrowia lipolytica was used in
this work. A serie of
factors, individually or in association, can affect
production of such substances
leading to different levels of product concentration and
productivity. Empirical
studies have been traditionally used to determine the
effect of these factors on
process parameters. A possible approach is to vary one
factor while keeping the
other at a constant level, but this approach is time
consuming, does not include the
interaction effects among variables, and does not
necessarily lead to optimized
results. Nowadays, different strategies are employed to
optimize production
parameters. These strategies not only allow process
optimization but can also
establish the dependent and independent variables.
Methodology: Ten
experiments were planned with the D-optimal design in
which concentrations of
inoculum, carbon source and nitrogen source were changed.
The estimate of the
uncertainty in measurement of the lipase activity was
developed based on the
Guide to the Expression of Uncertainty in Measurement
(GUM) and on the Guide
CG 4 published by EURACHEM. Results: The highest lipolytic
activity obtained
was 4815 U/L (80,3 (mi)kat/L) with an expanded uncertainty
of 137 U/L
(2,3 (mi)kat/L), under the following conditions: 0.5% of
olive oil (carbon source),0.7% of peptone (nitrogen
source) and 10 mg/mL of inoculum. In terms of
volumetric productivity, these conditions provided 69.28
U/L h. Conclusions: It
is possible to conclude that the D-optimal design usage
enhanced lipase
production in shaken-flasks. Moreover the activity
obtained in this work was
higher than others previously reported with the same
microorganism, without
experimental design, in shaken-flasks (2700 U/L) or even
in bench-fermenter
(4240 U/L). The production of lipases by Yarrowia
lipolytica cells aiming the
classification as certified reference material can be
recommended after further
purification and stability studies.
9
May, Michael 1972. "Production of Lipase by Candida bombicola in a self-cycling fermenter (SCF)." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27242.
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The Self-Cycling Fermentation (SCF) technique and an agar diffusion assay were used to follow the production of lipase by Candida bombicola. Repeatable lipase production profiles were determined for two different media. Each medium gave a considerably different profile.<br>When the broth contained oil, 60 to 80% of the lipase activity remained after freezing. All activity was lost when the broth did not contain oil.<br>The lipase was found to be associated with the cell. Up to a certain concentration of lipase, the presence of agar caused the lipase to disassociate from the cell and diffuse into the agar. Beyond this concentration of lipase the agar was found to saturate.<br>A new technique of level control in the cyclone reactor was used. A pressure transducer was found to be an accurate and inexpensive device for level control.
10
Aryee, Alberta. "Immobilization of lipase and biodiesel production from fishery and animal processing waste." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110391.
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Biodiesel (BD) is conventionally produced by the transesterification of vegetable oils or animal fats with a monohydric alcohol and base catalyst, and known for its many technical and environmental advantages over petrodiesel. However, these oils are not economically viable feedstock due to their value as edible oils. Alternative, inexpensive feedstocks with minimal to no food value such as the large quantities of fishery and animal processing by-products which are often discarded as waste were explored in this project. Lipase was investigated as an alternative to chemical catalyst due to the tolerance of the former to a wide variety of feedstocks and simpler post-production processes among other advantages. Approximately 23.32-61.53% (on dry weight basis) of salmon skin oil (SSO) was recovered by the various solvent extraction systems evaluated. The quality of SSO stored at 25, 4, -18, and -80oC were assessed over time (1- 45 days) with respect to changes in the fatty acid profile, free fatty acid (FFA) content, peroxide value, and thiobarbituric acid reactive substances. A Fourier transform infrared (FTIR) spectroscopic method was assessed as an alternative to the conventional AOCS titrimetric method for the determination of FFA content. With modifications, the new method was found to be capable of responding linearly to oleic acid (0-6.5%) addition, producing a FFA calibration equation with a S.D. of ±0.014% FFA. Based on the results from the initial assessment of the effects of temperature (25-65oC), oil:alcohol molar ratio (1:1-1:6), alcohol type (ethanol/methanol), and time (8-120 h) on Lipozyme®-IM-catalyzed transesterification of the recovered SSO, a commercial blend of yellow grease and rendered animal fat, and olive oil to fatty acid ethyl ester (FAEE) for use as BD, the process was considered for optimization. In three experiments, the linear, quadratic, and bilinear effects of the reaction variables on FAEE yield were assessed with response surface methodology (RSM) based on central composite rotatable design (CCRD). In each experiment, second-order polynomial models fitted to FAEE yield provided response surfaces at the various reaction times (8-48 h). These models were generally significant and produced reliable and stable predictions. The optimum conditions were found to be close to the centre point of the reaction variables. A high performance liquid chromatography unit equipped with a size exclusion column, and a refractive index detector was used for the simultaneous separation, identification, and quantitation of the reaction components; FAEE, unreacted triacylglycerol, residual diacyl- and monoacylglycerol, and alcohol as well as FFA. The transesterified oils were also tested for various fuel properties. To expand the uses of lipase recovered from fish processing discards to include catalyst for BD production, lipase from crude preparations of delipidated grey mullet (Mugil cephalus) viscera were isolated on para-aminobenzamidine agarose and immobilized on octyl Sepharose CL-4B (o-Sep). A signal in the amide I absorption region of the FTIR spectrum was attributed to the protein layer on o-Sep. Immobilized grey mullet lipase (GMLi) had a 10°C higher optimum temperature compared to the free enzyme (GML) for the hydrolysis of para-nitrophenyl palmitate. Immobilization lowered the enthalpy of activation (ΔH*), and free energy of activation (ΔG*) by more than 313 and 1315 cal/mol, respectively, while it enhanced the reusability, thermal, storage, and organic solvent stabilities of GML.<br>Le biodiesel (BD), ou des esters d'alkyle, est classiquement produit par la transestérification d'huiles végétales ou de graisses animales avec un monoalcool et un catalyseur de base, et est également connu pour ses nombreux avantages techniques et environnementaux par rapport au pétrodiesel. Toutefois, ces huiles BD ne sont pas des matières premières économiquement viables en raison de leur valeur principale en tant qu'huiles comestibles. Ce projet a exploré des matières premières de substitution, peu coûteuses avec peu ou pas de valeur alimentaire telle que les grandes quantités de sous-produits detransformation du poisson et de sous-produits animaux qui sont souvent jetés avec les déchets. La lipase a été étudiée comme une alternative aux catalyseurs chimiques en raison de la tolérance de la lipase à une grande variété de matières premières ainsi que son processus de post-production beaucoup plus simple entre autres avantages. Environ 23,32 et 61,53% (sur la base du poids sec) de l'huile de peau de saumon (SSO) a été récupéré selon les différents systèmes de solvants évalués. Pour la détermination de la teneur en FFA, une méthode de spectroscopie infrarouge à transformée de Fourier (FTIR) a été évaluée comme une alternative au procédé AOCS conventionnel. Avec des modifications, la nouvelle méthode a été jugée capable de répondre de façon linéaire à l'addition d'acide oléique (0 à 6,5%), avec la production d'une équation d'étalonnage FFA avec une SD de ±0,014% FFA. Sur la base des résultats de l'évaluation initiale des effets de la température de réaction (25-65°C), un rapport l'huile:alcool molaire (1:1-1:6), le type d'alcool (éthanol ou méthanol), et le temps de réaction (8-120 h) sur du la transestérification catalysée Lipozyme®-IM, un mélange commercial de graisse animale jaune et de graisses fondues (RC), et d'huile d'olive (OO) à ester éthylique d'acide gras (EEAG) pour une utilisation comme BD, le procédé a été considéré pour optimisation. Dans trois expériences, les effets linéaires, quadratiques et bilinéaires des variables de la réaction sur le rendement EEAG ont été évalués avec la méthode de réponse de surface (RSM) basée sur la conception centrale composite rotative (CCRD). Dans chaque expérience, des modèles polynomiaux du second ordre équipés d'EEAG ont modelé le rendement des surfaces de réponse fournis aux divers temps de réaction (8-48 h). Ces modèles sont généralement importants et produisent des prévisions fiables et stables. Les conditions optimales ont été trouvées être proche du point de centre des variables de réaction (50°C, charge de l'enzyme 39.06 U, et l'huile:rapport molaire de l'alcool 1:2), et simultanément identifiés, et quantifiés. Les différents composants de la réaction (par exemple: EEAG, triacylglycérol n'ayant pas réagi (TAG), diacyle et résiduelle monoacyle-glycérol (DAG et MAG), et l'alcool ainsi que la FFA), ont été séparés, identifiés et quantifiés en utilisant la chromatographie liquide à haute performance équipé d'unité de colonne d'exclusion de taille, et un détecteur par indice de réfraction. Pour élargir les usages de la lipase récupérée à partir de rejets de transformation du poisson pour inclure un catalyseur pour la production de BD, de la lipase à partir de préparations brutes de délipidé mulet (Mugil cephalus) les viscères ont été isolées sur le para-aminobenzamidine agarose (p-ABA) et immobilisées sur Sepharose CL-octyle 4B (o-Sep). Un signal dans la région d'absorption amide I du spectre FTIR a été attribué à la couche de protéine sur o-Sep. La lipase de mulet immobilisée (GMLi) a eu une température optimale de 10°C plus élevée par rapport à l'enzyme libre (GML) pour l'hydrolyse de para-nitrophényl palmitate (p-NPP). L'immobilisation a abaissé l'enthalpie d'activation (AH*), et l'énergie libre d'activation (AG*) de plus de 313 et 1315 cal/mol, respectivement, alors qu'alla améliorer la capacité thermique, la, réutilisabilité, et la stabilité des solvants des GML.
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May, Michael John. "Production of lipase by Candida bombicola in a self-cycling fermenter, SCF." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ29615.pdf.
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Ngom, Marie Odile. "Induction and production of specific extracellular lipases from selected microorganisms." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0031/MQ64416.pdf.
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Monforte, Mercado Sergi. "Systems metabolic engineering for recombinant protein production in Pichia pastoris." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669385.
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El llevat metilotròfic Pichia pastoris (Komagataella sp.) és un dels sistemes d’expressió més atractius per a la producció de proteïna recombinant, mercat contínuament en expansió. El fort promotor del gen de l’alcohol oxidasa 1 (PAOX1), induït per metanol però reprimit per glucosa, glicerol o etanol, és un dels més emprats per aquest propòsit. No obstant, existeixen encara diversos colls d’ampolla fisiològics que limitant el procés.
En aquest context, diferents estratègies han estat proposades i provades per tal de millorar la producció heteròloga de molts tipus diferents de proteïna. Les aproximacions més habituals inclouen increment en el nombre de còpies de gen heteròleg, enginyeria de promotors i modificació dels mecanismes de plegament i secreció. L’objectiu d’aquesta tesi ha estat el desenvolupament de noves estratègies per incrementar la producció de proteïna recombinant, emprant la lipasa de Rhizopus oryzae (Rol) com a proteïna model en el sistema d’expressió basat en el PAOX1.
Primerament, els gens del factors de transcripció de PAOX1, MXR1 i MIT1, es van sobreexpressar constitutivament per tal de millorar la transcripció de ROL. Això es va confirmar degut a una millora en la capacitat assimilatòria de metanol i un increment en els nivells relatius de mRNA de ROL i varis gens relacionats amb el metabolisme del metanol, i.e. revertint l’efecte de titulació causat per la transcripció de múltiples cassettes d’expressió de ROL. Tot i aquestes millores, els nivells extracel·lulars d’activitat lipàsica no van augmentar de forma significativa en cultius en quimiòstat, apuntant a colls d’ampolla addicionals limitant la producció de Rol.
En segon lloc, es van explorar possibles dianes d’enginyeria metabòlica en el metabolisme cel·lular de P. pastoris emprant el model metabòlic a escola genoma (GEM) consens iMT1026 v3.0. Aquest pas in silico va proporcionar diversos knock-outs prometedors que serien experimentalment testats fent servir el sistema d’edició genòmica CRISPR/Cas9. Les simulacions apuntaven a la disponibilitat de NADPH i una limitada aportació de determinats aminoàcids (serina i cisteïna) com a potencials factors limitants de la producció de Rol. Una reducció en el fitnes cel·lular que afecta a la viabilitat de les soques que es buscaven obtenir va impedir la verificació de la majoria dels knock-outs proposats.
Finalment, donat que les nostres anàlisis i estudis prèviament publicats identificaven el NADPH com un cofactor important limitant la producció de proteïna recombinant, els nostres esforços es van dirigir a incrementar la seva disponibilitat a través d’estratègies de knock-in de gens. Específicament, vam sobreexpressar dos gens que codificaven per enzims redox, una NADH quinasa i una NADH oxidasa, amb l’objectiu de pertorbar directament l’equilibri redox de la cèl·lula. A més, es va comprovar l’efecte fisiològic d’aquests enzims fent servir diferents mescles co-substrat/metanol com a font de carboni. En resum, vam observar un increment en la producció de proteïna recombinant amb diferents graus de millora depenent de la font de carboni provada. També vam realitzar anàlisis transcriptòmiques i una avaluació in silico dels nostres resultats per tal de presentar una interpretació millor de l’estat fisiològic de la cèl·lula. Dins del nostre coneixement, aquest és el primer estudi dirigit a incrementar la generació de NADPH en un sistema d’expressió basat en PAOX1, en condicions de creixement en metanol.
A grans trets, noves estratègies d’enginyeria de soques han estat proposades i provades durant l’execució d’aquest estudi. A més a més, s’han aplicat GEMs i aproximacions relacionades amb biologia de sistemes, demostrant que són eines potents i prometedores per al disseny racional d’organismes industrials.<br>The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process.
In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system.
Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production.
Second, possible metabolic engineering targets in P. pastoris’ cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs.
Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell’s redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions.
Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms.
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Filho, Edilson Holanda Costa. "Study of enzymatic production of biodiesel using residual oil and ethanol." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2779.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico<br>Biodiesel is a mixture of fatty acid alkyl esters produced by the reaction between vegetable oils and short chain alcohols, like methanol and ethanol, using a catalyst that can be acid, basic or enzymatic. However, the high cost of the raw material when refined vegetable oil is used, have made biodiesel production economically unattractive. Therefore, research with waste oils has increased, showing the technical viability of the production of biodiesel using the residential and industrial residues as raw material. Another variable that has influenced this type of reaction is the type of alcohol. In Brazil, the use of ethanol is interesting because the country has become one of the top worldwide producers of ethanol from vegetables sources, a cheaper and less toxic product than methanol, decreasing our petroleum dependence. The type of catalyst also influences biodiesel production. Alkali is the catalysts that is more often used in industry, but when the vegetable oil has a high acid value, it can not be used because soap is produced, diminishing the esters yield. In this case an acid or an enzyme is used as a catalyst. Based on the previous explanation, the results of this work correspond to the study of enzymatic production of biodiesel using waste oil and ethanol. The immobilized Candida antarctica lipase (Novozym 435) behavior was studied in the oleic acid esterification, studying the effect of the variables that has influence in the process. The variables chosen were: temperature (30 â 50oC), molar ratio acid:alcohol (1:1 â 1:6) and water content (0 â 20%). The reaction were performed in closed reactors with a capacity of 250 mL containing 10 g of oil, a known amount content of alcohol, pre-determined by experimental design and enzyme content of 5% p/p, based on the oil mass. The reaction medium was kept under constant stirring, 200 rpm. Maximum conversion of 88,36% was achieved when high molar ration, the lower temperature and water content values were used. However, by the kinetic study, it can be concluded that it is not necessary to use an alcohol excess to achieve good conversions. After that, the behavior of Candida Antarctica lipase B immobilized in chitosan was studied in acid oleic esterification. A slower initial rate of reaction was observed in comparison to Novozym 435. The behavior of both lipases was also studied in the esterification of waste coconut oil, showing good stability and giving a conversion of about 80% in 60 minutes. Both biocatalyst could be reused 10 times, keeping the same activity. In order to compare the behavior of Novozym 435 in two different mediums, an experimental design was performed with waste cotton oil, which had a low acid value. The same negative influence of the temperature and molar ratio was observed, but with a high reaction time, getting a maximum conversion of 82,66% in 72 hours of reaction. To calculate the conversions, the decreasing of the acid value was used when the raw material had a high acid value, and when the raw material had a low acid value the glycerol production was used.<br>O biodiesel à uma mistura de Ãsteres alquÃlicos de Ãcidos graxos resultante da reaÃÃo entre Ãleos vegetais e Ãlcoois de cadeia curta, como metanol ou etanol, auxiliada por um catalisador, que pode ser Ãcido, bÃsico ou enzimÃtico. Entretanto, o alto custo da matÃria-prima quando se utiliza Ãleo vegetal de grau alimentÃcio tem inviabilizado economicamente a produÃÃo desse biocombustÃvel. Por isso, as pesquisas com Ãleo residual tem aumentado, mostrando a viabilidade tÃcnica da produÃÃo de biodiesel a partir de resÃduos residenciais e industriais. Outro fator que influencia a reaÃÃo de produÃÃo de biodiesel à o tipo de Ãlcool. No Brasil, o uso do etanol à interessante desde que o nosso paÃs se tornou um dos maiores produtores mundiais de etanol vegetal, um produto mais barato e menos tÃxico que o metanol, diminuindo assim a nossa dependÃncia do petrÃleo. O catalisador tambÃm exerce influÃncia nesse tipo de reaÃÃo. Os catalisadores mais usados industrialmente sÃo as bases, mas quando o Ãleo vegetal tem um alto teor de Ãcidos graxos livres, o que acontece, geralmente, com os Ãleos residuais, nÃo à possÃvel usar catalisador bÃsico por favorecer a formaÃÃo de sabÃo e diminuir o rendimento em Ãsteres. Nesse caso, usa-se um catalisador Ãcido ou enzimÃtico. Partindo dessa premissa, os resultados constantes nessa dissertaÃÃo correspondem ao estudo da produÃÃo enzimÃtica de biodiesel utilizando Ãleo residual e etanol. Avaliou-se o comportamento da lipase comercial imobilizada de CÃndida antarctica tipo B (Novozym 435) na esterificaÃÃo do Ãcido olÃico comercial, estudando as variÃveis que influenciam no processo. As variÃveis escolhidas foram: temperatura (30-50o C), razÃo molar Ãcido:Ãlcool (1:1-1:6) e a concentraÃÃo de Ãgua presente no meio (0-20%). As reaÃÃes foram conduzidas em erlenmeyers de 250 mL fechados contendo 10 g de Ãleo e a quantidade de Ãlcool prÃ-determinada pelo planejamento de experimentos, mantendo-se a agitaÃÃo fixa em 200rpm e a concentraÃÃo de enzima em 5% m/m baseada na massa de Ãleo medida, obtendo-se uma conversÃo mÃxima de 88,36% na condiÃÃo de maior razÃo molar, menor temperatura e menor concentraÃÃo de Ãgua. Entretanto, pelo estudo cinÃtico concluÃ-se que nÃo à necessÃrio um excesso de Ãlcool para conseguir boas conversÃes. Em seguida, avaliou-se o comportamento de uma lÃpase do tipo B de CÃndida antarctica imobilizada em quitosana na esterificaÃÃo do Ãcido olÃico, observando um comportamento semelhante ao da Novozym 435 mas com uma taxa inicial de reaÃÃo mais lenta. Avaliou-se tambÃm o comportamento das duas lÃpases na esterificaÃÃo do Ãleo de coco residual Ãcido, observando uma boa estabilidade para ambos os biocatalisadores que forneceram uma conversÃo acima de 80% com 60 minutos de reaÃÃo e puderam ser reutilizados por no mÃnimo 10 vezes consecutivas sem perda considerÃvel de atividade. Para comparar o comportamento da Novozym 435 em dois meios distintos, realizou-se um planejamento experimental fatorial com um Ãleo de algodÃo residual de baixa acidez livre, observando a mesma influÃncia negativa da temperatura e da razÃo molar entre reagentes, mas com um tempo de reaÃÃo maior, pois uma conversÃo mÃxima de 82,66% sà foi atingida com 72 horas de reaÃÃo. Para o cÃlculo da conversÃo, utilizou-se a reduÃÃo do Ãndice de acidez quando a matÃria-prima tinha um alto teor de Ãcidos graxos livres e o mÃtodo do periodato de sÃdio na determinaÃÃo da glicerina quando a matÃria-prima tinha uma baixa acidez livre.
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Volpato, Giandra. "Produção, purificação e imobilização de lipases de Staphylococcus warneri EX17 produzidas em glicerol." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2009. http://hdl.handle.net/10183/15543.
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Lipases (EC 3.1.1.3) são um grupo de enzimas que catalisam a hidrólise e síntese de triacilgliceróis. Estas enzimas apresentam estabilidade em diversos solventes orgânicos, podendo ser aplicadas como biocatalisadores em vários processos anteriormente realizados apenas por catalisadores químicos. Este trabalho teve como objetivo produzir, purificar e imobilizar lipases de Staphylococcus warneri EX17, cepa capaz de utilizar glicerol como fonte de carbono. Inicialmente, as condições de cultivo para produção de lipases foram otimizadas através de duas ferramentas de planejamento experimental: delineamento Placket Burman (P-B) e delineamento composto central rotacional (DCCR). Determinou-se que as melhores condições para produção desta enzima são: temperatura de 36 °C; pH 8,1; 30 g/L de glicerol; 3,0 g/L de óleo de oliva e 2,5 g/L de óleo de soja. Também se verificou ser possível a utilização de glicerol residual, oriundo da síntese enzimática de biodiesel como fonte de carbono. O extrato enzimático mostrou-se estável em três solventes orgânicos testados (metanol, etanol e η-hexano). Ainda visando a otimização das condições de cultivo, foram realizados cultivos submersos em biorreatores a fim de estudar a influência da taxa volumétrica de transferência de oxigênio (kLa) e do controle do pH, na produção da enzima. A maior produção de lipases ocorreu quando aplicado um kLa de 38 h-1 e com o pH controlado em 7,0 ao longo do cultivo, o que permitiu aumentar a produção da enzima em 5 vezes, em relação ao obtido nas condições anteriormente empregadas. A purificação da lipase foi realizada baseando-se no mecanismo de ativação interfacial destas enzimas sobre superfícies hidrofóbicas. Duas resinas foram testadas, octil-Sepharose e butil- Toyopearl. A lipase produzida foi purificada em apenas um passo utilizando esta última resina. Foi estudada a hiperativação da lipase purificada na presença de detergentes, a atividade lipolítica foi aumentada em 2,5 vezes na presença de 0,1% de Triton X-100. A lipase purificada foi imobilizada através de três estratégias: adsorção em suporte hidrofóbico; união covalente unipontual e união covalente multipontual. A influência da imobilização na modulação das propriedades da enzima foi estudada. A lipase apresentou maior estabilidade quando imobilizada multipontualmente. A hidrólise de distintos ésteres quirais pelos diferentes biocatalisadores obtidos também foi estudada. Os ésteres utilizados foram: (±) mandelato de metila, (±)-2-O-butiril-2-fenilacético e (±)-2-hidroxi-4-fenilbutirato de etilo. A especificidade da enzima foi muito dependente do método de imobilização, sendo que a lipase imobilizada unipontualmente foi mais específica para o substrato (±)-2-hidroxi-4-fenilbutirato de etilo, enquanto que para os outros dois substratos foi a lipase adsorvida hidrofobicamente. Este estudo demonstrou que a lipase de S. warneri EX17 pode ser produzida utilizando glicerol residual como fonte de carbono, levando a diminuição do custo na produção da enzima, que apresenta propriedades bastante interessantes para sua aplicação em biocatálise.<br>Lipases (EC 3.1.1.3) constitute a group of enzymes that catalyze the hydrolysis and synthesis of triacylglycerols. These enzymes show stability in many organic solvents, being able to be used as biocatalysts in some processes that were once carried out only by chemical catalysys. The aim of this research was the production, purification, and immobilization of lipase by Staphylococcus warneri strain EX17 using glycerol as carbon source. Initially, the cultivation conditions for the production of lipases have been optimized through two statistical procedures, Plackett-Burman statistical design (PB) and central composite design (CCD). It was determined that the best conditions for this enzyme production are: temperature, 36 °C; pH, 8.1; glycerol, 30 g/L; olive oil, 3.0 g/L; and soybean oil, 2.5 g/L. It was also studied the use of raw glycerol from enzymatic synthesis of biodiesel as carbon source, and stability studies showed that this lipase from S. warneri EX17 was stable in methanol, ethanol and nhexane. Moreover, experiments were conducted in submerged bioreactors in order to study the influence of oxygen volumetric mass transfer rate (kLa) and the control of pH in the production of the enzyme. The higher lipase production occurred when the microorganism was submitted to a kLa of 38 h-1 and the pH controlled at 7.0 during the cultivation, which improved 5-fold the enzyme production, compared to the results obtained in shaker flasks. The lipase purification was carried out based on mechanisms of interfacial activation of these enzymes on hydrophobic surface. Two supports were tested, octyl-Sepharose and butyl-Toyopearl. The lipase produced was purified 20-fold in only one step of purification. The purified lipase was immobilized on cyanogens bromide activated agorese and its hyperactivation in the presence of detergents was studied. The lipolytic activity increased 2.5-fold in presence of 0.1% of Triton X-100. After this, lipase was immobilized by three strategies: adsorption on hydrophobic support, mild covalent attachment, and multipoint covalent attachment. The stability over thermal, organic solvent and detergent inactivation was verified, as well as the influence of the immobilization protocol in the modulation of the properties of the enzyme. The lipase showed higher stability when multipointly immobilized on glyoxyl agarose. The hydrolysis of different chiral esters by the three biocatalysts obtained was also studied. The esters used were: (±) methyl mandelate, ((±) methyl mandelate, (±)-2-O-butyryl-2-phenylacetic acid, (±)-2-hydroxy-4-phenyl-butyric acid ethyl ester. The specificity of the enzyme was highly dependent of the protocol of immobilization, and the lipase mildly immobilized on cyanogen bromide agarose was more specific to the hydrolysis of (±)-2-hydroxy-4-phenyl-butyric acid ethyl ester, while for the other two substrates was the lipase adsorbed on octyl agarose. This study demonstrated that the lipase from S. warneri EX17 can be produced using raw glycerol as carbon source, contributing to the reduction in production costs of the enzyme, and the enzyme, when immobilized on different supports, presented quite interesting properties that may be usefull as biocatalysts.
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Rodrigues, Joana Mendes Madeira. "Biodiesel production from Jathropha curcas l. oil using chemical or enzymatic catalysts." Doctoral thesis, ISA, 2016. http://hdl.handle.net/10400.5/12012.
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Doutoramento em Engenharia dos Biossistemas - Instituto Superior de Agronomia - UL<br>Jatropha curcas L. is a tropical shrub, belonging to the Euphorbiaceae family, well adapted to marginal lands, not competing for arable land and food chain. The seeds are rich in non-edible oil, suitable for biodiesel production.
In this work, variation in oil content and composition of Jatropha seeds from 12 origins, grown in Mozambique under the same edapho-climatic and agronomic conditions, was assessed. Oil contents varied from 37%-45% (d.w.). The major fatty acids identified in Jatropha oil were oleic (41.1%) and linoleic acids (38.8%). The oil presented low acidity and low levels of oxidation products, proving adequate for biodiesel production. Also high levels of gamma-tocopherol were found (69-182 mg/kg). The presence of this antioxidant may explain the high oxidative stability presented by the oil during the storage tests performed under controlled humidity and temperature, simulating tropical climate conditions. Conversely, seeds storage under the same conditions promoted the growth of fungi responsible for oil degradation. Therefore, it is better to extract the oil and store it in closed containers, than to store the seeds.
Dry conditioning of seeds to be extracted by screw pressing was optimized via Response Surface Methodology (RSM). The highest extraction oil yields (88%, w/w) were obtained with seeds dried at higher temperatures for shorter periods of time (e.g., 90◦C/10-20 min; 80◦C/30 min) or at lower temperatures for longer periods of time (e.g., 60◦C/50-60 min).
RSM was also applied for the optimization of fatty acid methyl esters (FAME, biodiesel) production from Jatropha oil, using sodium methoxide as catalyst.
Finally, the feasibility of enzymatic production of Jatropha FAME, using recombinant sn-1(3)-regioselective Rhizopus oryzae lipase and Carica papaya lipase and Candida parapsilosis lipase/acyltransferase, immobilized on different synthetic resins, was evaluated. All biocatalysts proved to be efficient and environmentally friendly alternatives to the conventional chemical process.
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Maldonado, Rafael Resende 1981. "Aplicação da lipase de geotrichum candidum para biocatálise de óleos vegetais com vistas à produção de biodiesel = Application of lipase from geotrichum candidum to biocatalysis of vegetable oils to biodiesel production." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/255037.
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Orientadores: Maria Isabel Rodrigues, Gabriela Alves Macedo<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-20T23:36:07Z (GMT). No. of bitstreams: 1
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Previous issue date: 2012<br>Resumo: A crescente necessidade por energia e o avanço das preocupações com problemas ambientais devido ao uso de combustíveis fósseis tem levado a busca de alternativas para a área energética. Dentro deste contexto, a produção de biocombustíveis como etanol e biodiesel mostram um grande potencial de avanço e despertam amplo interesse na área de pesquisas.O biodiesel é comumente obtido por reações de transesterificação com catálise alcalina, no entanto a via enzimática constitui-se em uma forma promissora para a produção deste tipo de combustível. Este trabalho analisou as etapas de produção, purificação e imobilização da lipase de Geotrichum candidum NRRLY-552 e aplicação dessa enzima na biocatálise das reações de hidrólise, esterificação e transesterificação de óleos vegetais em diferentes sistemas reacionais. Os resultados obtidos demonstraram que a lipase de Geotrichum candidum NRRLY-552 pode ser obtida a baixo custo, com a utilização de um resíduo agro-industrial (água de maceração de milho) com atividade lipolítica de cerca de 20 U/mL. A purificação parcial desta enzima foi realizada por dois métodos ¿ precipitação com sulfato de amônio e precipitação com etanol, seguido de liofilização em ambos os casos. Tais métodos proporcionaram a obtenção de um preparado enzimático com atividade lipolítica de cerca de 500 U/g em duas ou três etapas. O pequeno número de etapas e a elevada atividade enzimática propiciam a aplicação desta enzima para biocatálise de reações com óleos vegetais para uso na produção de biocombustíveis. A enzima selecionada apresentou um bom desempenho para a reação de hidrólise de óleos vegetais, atingindo taxas de hidrólise de cerca de 80% após 24 horas de reação, em frascos agitados a 210 rpm, 45oC e 15% m/m de água no meio reacional com uma concentração de enzima de 5% m/m da lipase. A enzima não apresentou ação de esterificação suficiente para produção de biodiesel na presença de etanol, no entanto o sistema reacional desenvolvido permite que a associação com outras lipases de boa ação de esterificação possibilite a obtenção do biodiesel a partir do hidrolisado obtido neste trabalho<br>Abstract: The growing energy necessity and the advancement of environmental concerns due to the use of fossil fuels have led the search for alternatives to the energy area. In this context, the production of biofuels like ethanol and biodiesel shows a great potential for advancement and arouse widespread interest in the area of research. Biodiesel is commonly obtained by transesterification with alkaline catalysis; however, the enzymatic pathway is a promising way to produce this type of fuel. This work studied the stages of production, purification and immobilization of lipase from Geotrichum candidum NRRLY-552 and application of this enzyme in the biocatalysis of the hydrolysis, esterification and transesterification of vegetable oils in different reaction systems. The results showed that the lipase from Geotrichum candidum NRRLY-552 can be obtained at a low cost, using a residue of the agro-industrial (corn steep liquor), with lipolytic activity of about 20 U/mL. Partial purification of this enzyme was performed by two methods - precipitation with ammonium sulphate and precipitation with ethanol, followed by lyophilization in both cases. Such methods provided to obtain an enzyme preparation with lipolytic activity of 500 U/g in two or three steps. The small number of steps and the high enzyme activity provide the application of this enzyme to catalysis of reactions with vegetable oils for the use in biofuel production. The selected enzyme showed a good performance for the hydrolysis of vegetable oils, reaching rates of hydrolysis of about 80% after 24 hours of reaction, in shaken flasks at 210 rpm, 45°C and 15% w/w of water in the reaction with enzyme concentration of 5% w/w of the lipase. The enzyme did not show enough action esterification for biodiesel production in presence of ethanol; however, the reaction system developed permits the association with other lipases with good deed esterification with allows obtainable biodiesel from the hydrolysed produced in this work<br>Doutorado<br>Engenharia de Alimentos<br>Doutor em Engenharia de Alimentos
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Casas, Godoy Leticia. "Lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids and production of structured lipids." Thesis, Toulouse, INSA, 2012. http://www.theses.fr/2012ISAT0057/document.
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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)<br>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)
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Godoy, Leticia Casas. "Lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids and production of structures lipids." Doctoral thesis, ISA/UTL, 2012. http://hdl.handle.net/10400.5/5200.
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Marmontel, Oriane. "Dysrégulations de la production et de la clairance des lipoprotéines riches en triglycérides." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1220/document.
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L’hypertriglycéridémie (HTG) correspond à une accumulation des lipoprotéines riches en triglycérides (LRTG) dans la circulation plasmatique, conséquence d’une augmentation de leur synthèse ou plus classiquement décrit, d’une diminution de leur catabolisme. Dans près de 50% des cas, aucune cause génétique n’est identifiée chez les patients présentant une présentant une HTG sévère, aussi bien dans le cadre du syndrome de chylomicronémie familiale (FCS) que dans celui du syndrome de chylomicronémie multifactorielle (MCS). Pour améliorer nos connaissances et la caractérisation de ces patients, la conduction de corrélations phénotypes-génotypes précises grâce à une collaboration clinico-biologique étroite, ainsi que le développement d’outils de diagnostic moléculaire performants, demeurent un enjeu majeur. Premièrement, l’évaluation de la concentration pré-héparinique en LPL et l’activité post-héparinique 60 minutes après l’injection d’héparine chez 62 patients MCS caractérises génétiquement a permis la mise en évidence deux sous-groupes chez ces patients. Deuxièmement, le développement d’une stratégie séquençage de nouvelle génération permettant d’explorer simultanément les 9 gènes les plus prévalents dans les hypercholestérolémies, les hypocholestérolémies et les hypertriglycéridémies, a permis de détecter les variants nucléotidiques avec une sensibilité équivalente au séquençage Sanger mais aussi de détecter des grands réarrangements. L’ensemble des résultats souligne la complexité des mécanismes de régulation du métabolisme des LRTG et l’intérêt de l’étude des interactions gène-gène. Ainsi, ces travaux ont permis de mettre en évidence de nouvelles hypothèses à explorer pour la compréhension des mécanismes physiopathologiques des HTG sévères et d’améliorer les outils disponibles pour les études de corrélation génotype-phénotype<br>Hypertriglyceridemia (HTG) correspond to an increase of triglyceride-rich lipoproteins (TGRL) circulating concentration, as a consequence of an increase in the synthesis of or a decrease in their catabolism, most classically described. In nearly 50% of patients with severe hypertriglyceridemia (HTG), no genetic cause is identified, either in familial chylomicronemia syndrome (FCS) or in multifactorial chylomicronemia syndrome (MCS). To gain new insights and to improve patient’s characterization, it remains important to conduct accurate phenotype-genotype association studies through close collaboration with referent lipidologists, and to develop high-performance tools for molecular diagnosis. Firstly, the assessment of pre-heparin LPL concentration as well as LPL activity 60 minutes after heparin injection, enabled the identification of two subgroups within 62 genotyped MCS patients Secondly, the development of a new sequencing generation workflow exploring simultaneously the 9 most prevalent genes in dyslipidemia, allowed the detection of single nucleotide variations with sensitivity equivalent to Sanger sequencing, but also allowed the detection of copy number variations. Collective consideration of the results underlines the complexity of the regulation mechanisms of TGRL metabolism and the interest of gene-gene interactions study. Thus, the studies presented herein bring new hypothesis to explore for understanding the pathophysiological mechanisms of severe HTG and to improve molecular diagnosis tools available for phenotype-genotype association studies
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Berendsen, Wouter Robert. "Model based development of continuous processes for production of chiral glycol ethers by biocatalysis Modellbasierte Entwicklung kontinuierlicher Prozesse zur Herstellung chiraler Glykolether durch Biokatalyse /." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-34445.
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Sams, Laura. "Production et mise en oeuvre d'une lipase gastrique pour le développement de modèles de digestion in vitro." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0082.
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L’objectif de ce travail était le développement d’une lipase gastrique pour des modèles de digestion in vitro reproduisant les conditions physiologiques du tractus digestif. Nous avons produit, caractérisé et mis en oeuvre un extrait d’estomacs de lapin contenant la lipase gastrique et la pepsine. Cet extrait, a permis de simuler le potentiel enzymatique du suc gastrique lors de digestions in vitro. La digestion d’émulsions d’huile de colza-β-caséine a permis d’appréhender l’impact des lipides et de la lipolyse sur la protéolyse de la β-caséine.Une lipase gastrique humaine recombinante (rHGL) a également été produite chez la levure Pichia pastoris grâce à un vecteur d’expression constitutive contenant le peptide de sécrétion α. Cette production a nécessité l’optimisation des conditions de pH pour éviter la dégradation de la protéine sécrétée, active et glycosylée. Une fraction importante de la rHGL sécrétée étant accrochée à la paroi des levures, un protocole de purification incluant une étape de décrochage a été développé en jouant de nouveau sur le pH. La rHGL purifiée a été caractérisée en fonction du pH, de divers substrats et de la présence de sels biliaires, et ses propriétés sont proches de celles de la HGL native. Nous avons ensuite étudié par mutagénèse dirigée le rôle de résidus (K4, E225, R229) impliqués dans des ponts salins stabilisant le volet de la rHGL dans sa conformation ouverte. Ce volet contrôle l’accès au site actif de la lipase et participe au site de reconnaissance interfaciale. La mutation de ces résidus modifie l’activité en fonction du pH de la rHGL et suggère qu’ils jouent un rôle important dans l’activité optimale de la HGL à pH acide<br>The aim of this work was the development of a gastric lipase for in vitro digestion models reproducing the physiological conditions of the digestive tract.We produced, characterized and implemented an extract of rabbit stomachs containing gastric lipase and pepsin. This extract was further used to simulate the enzymatic potential of the gastric juice during in vitro digestion assays. The digestion of rapeseed oil-β-casein emulsions allowed investigating the impact of lipids and lipolysis on the proteolysis of β-casein.A recombinant human gastric lipase (rHGL) was also produced in the yeast Pichia pastoris using a constitutive expression vector containing the α secretion peptide. This production required the optimization of pH conditions to avoid degradation of the secreted, active and glycosylated protein. A large fraction of the secreted rHGL remaining bound to the yeast cell wall, a purification protocol including a release step was developed by shifting the pH. The purified rHGL was characterized as a function of pH, various substrates and the presence of bile salts, and its properties were found to be similar to those of native HGL. We then studied by site-directed mutagenesis the role of residues (K4, E225, R229) involved in salt bridges stabilizing the rHGL lid in its open conformation. This lid controls the access to the lipase active site and is involved in the interfacial recognition site. The mutation of these residues modifies the activity of rHGL as a function of pH and suggests that these amino acid residues play an important role in the optimum activity of HGL at acid pH
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Rodriguez, De Rodriguez Maria Del Pilar. "Production de biodiesel à partir d'une huile modèle de microalgues par voie de catalyse enzymatique hétérogène." Mémoire, Université de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/111.
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Le biodiesel, considéré comme une solution pour remplacer le pétrodiesel est un sujet de recherche mondial. Un des principaux problèmes associés au développement industriel du biodiesel est la source de matière première ainsi que le procédé de transformation. Ainsi, la présente étude a pour but de trouver une source de matière première durable pour la production industrielle de biodiesel et de déterminer le procédé de transformation de la matière première, le plus approprié, ainsi que les meilleures conditions opératoires. Durant la première étape de ce projet, une source de matière première durable a été sélectionnée : les microalgues. Le procédé de transformation étudié est la transestérification enzymatique. L’huile d’olive, huile ayant une composition en acides gras similaires à celle de l’huile de la microalgue Chlorella protothecoides, a été choisie pour effectuer les réactions. Pendant la deuxième étape, le procédé de standardisation de la réaction (bioréacteur de 5 mL) consistait à faire varier : le type de catalyseur (lipases de Candida antarctica (Novozym® 435) et de Thermomyces lanuginosus (TL I150)), la concentration du catalyseur (7 à 14 % m/mhuile), la température de réaction (25 à 50 °C) et le ratio molaire alcool:huile (3:1 à 4:1) ; la vitesse d’agitation étant de 150 rpm pour toutes les réactions. Des techniques d’optimisation telle que la preincubation de l’enzyme ont été également essayées. Le rendement en esters alkyliques de la réaction de transestérification enzymatique de l’huile en fonction du temps est la variable de contrôle pour toutes les réactions. La standardisation des variables du procédé a été faite en fonction de la réduction du temps de réaction et du rendement en esters alkyliques. Un rendement élevé en esters alkyliques de 92 % (m/m) a été obtenu sous les conditions opératoires suivantes : une concentration de catalyseur (TL I150) de 7 % (m/mhuile), une température de réaction de 25 °C, un ratio molaire alcool:huile de 3:1 et un temps de réaction de 4 h ; la lipase a été preincubée pendant 6 h avant la réaction de transestérification. Le temps de réaction, un des paramètres importants lors du procédé de standardisation des variables, a été réduit de 24 à 4 h. Un autre paramètre significatif de la réaction est la température : une température de 25 °C a été utilisée; cette température de réaction est faible et rend le procédé au niveau industriel plus attrayant.
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Kouteu, Nanssou Paul. "Mise en œuvre des lipases végétales issues des graines dans la catalyse enzymatique d’esters éthyliques d’huiles végétales pour la production de biodiesel." Thesis, Montpellier, SupAgro, 2017. http://www.theses.fr/2017NSAM0011.
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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<br>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
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Rodriguez, De Rodriguez Maria Del Pilar. "Production de biodiesel ?? partir d'une huile mod??le de microalgues par voie de catalyse enzymatique h??t??rog??ne." Mémoire, Universit?? de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/111.
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Le biodiesel, consid??r?? comme une solution pour remplacer le p??trodiesel est un sujet de recherche mondial. Un des principaux probl??mes associ??s au d??veloppement industriel du biodiesel est la source de mati??re premi??re ainsi que le proc??d?? de transformation. Ainsi, la pr??sente ??tude a pour but de trouver une source de mati??re premi??re durable pour la production industrielle de biodiesel et de d??terminer le proc??d?? de transformation de la mati??re premi??re, le plus appropri??, ainsi que les meilleures conditions op??ratoires. Durant la premi??re ??tape de ce projet, une source de mati??re premi??re durable a ??t?? s??lectionn??e : les microalgues. Le proc??d?? de transformation ??tudi?? est la transest??rification enzymatique. L???huile d???olive, huile ayant une composition en acides gras similaires ?? celle de l???huile de la microalgue Chlorella protothecoides, a ??t?? choisie pour effectuer les r??actions. Pendant la deuxi??me ??tape, le proc??d?? de standardisation de la r??action (bior??acteur de 5 mL) consistait ?? faire varier : le type de catalyseur (lipases de Candida antarctica (Novozym?? 435) et de Thermomyces lanuginosus (TL I150)), la concentration du catalyseur (7 ?? 14 % m/mhuile), la temp??rature de r??action (25 ?? 50 ??C) et le ratio molaire alcool:huile (3:1 ?? 4:1) ; la vitesse d???agitation ??tant de 150 rpm pour toutes les r??actions. Des techniques d???optimisation telle que la preincubation de l???enzyme ont ??t?? ??galement essay??es. Le rendement en esters alkyliques de la r??action de transest??rification enzymatique de l???huile en fonction du temps est la variable de contr??le pour toutes les r??actions. La standardisation des variables du proc??d?? a ??t?? faite en fonction de la r??duction du temps de r??action et du rendement en esters alkyliques. Un rendement ??lev?? en esters alkyliques de 92 % (m/m) a ??t?? obtenu sous les conditions op??ratoires suivantes : une concentration de catalyseur (TL I150) de 7 % (m/mhuile), une temp??rature de r??action de 25 ??C, un ratio molaire alcool:huile de 3:1 et un temps de r??action de 4 h ; la lipase a ??t?? preincub??e pendant 6 h avant la r??action de transest??rification. Le temps de r??action, un des param??tres importants lors du proc??d?? de standardisation des variables, a ??t?? r??duit de 24 ?? 4 h. Un autre param??tre significatif de la r??action est la temp??rature : une temp??rature de 25 ??C a ??t?? utilis??e; cette temp??rature de r??action est faible et rend le proc??d?? au niveau industriel plus attrayant.
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Speranza, Paula 1976. "Production of special lipids by enzymatic interesterification of Amazonian oils and influence on the biological activity = Produção de lipídios especiais por interesterificação enzimática de óleos da Amazônia e influência na atividade biológica." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256645.
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Orientadores: Gabriela Alves Macedo, Ana Paula Badan Ribeiro<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-25T18:56:33Z (GMT). No. of bitstreams: 1
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Previous issue date: 2014<br>Resumo: Este trabalho teve como objetivos produzir, caracterizar e avaliar as propriedades antimicrobianas de bases lipídicas interesterificadas produzidas com óleos e gorduras da Amazônia utilizando diferentes lipases. No trabalho foram utilizadas duas misturas de óleo e gordura da Amazônia para a produção das bases lipídicas, sendo a primeira delas composta pelo óleo de buriti e a gordura de murumuru e a segunda composta pelo óleo de patauá e a estearina de palma. As reações de interesterificação foram catalizadas por duas lipases em três sistemas enzimáticos diferentes: lipase comercial Lipozyme TL-IM (Novozymes), lipase do micro-organismo Rhizopus sp. e a mistura de ambas as enzimas (comercial + Rhizopus sp.). Em ambas as misturas, os lipídios produzidos apresentaram diferentes características dependendo da enzima utilizada. Na mistura buriti: murumuru, a lipase de Rhizopus sp., além de ser específica pelas posições sn-1,3 do triacilglicerol, foi específica para o tipo de ácido graxo (insaturado). A lipase comercial foi específica apenas pelo tipo de ácido graxo (insaturado), enquanto que a utilização de ambas as enzimas não apresentou efeito sinérgico nesta mistura, os resultados obtidos foram intermediários aos obtidos com as enzimas individualmente. Na mistura patauá: estearina de palma, a lipase de Rhizopus sp. foi específica para o tipo de ácido graxo (insaturados), enquanto que a lipase comercial não demonstrou específicidade para esta mistura. No sistema catalisado por ambas as enzimas também não foi observado efeito sinérgico; os resultdos obtidos foram similiares aos obtidos com a enzima de Rhizopus sp. Para ambas as misturas, com os três sistemas enzimáticos, houve redução nos triacilglceróis trisaturados e tri-insaturados após as reações, com a formação de bases lipídicas predominantemente mono e di-insaturados. Estes lipídios formados mantiveram a concentração elevada de tocoferóis, carotenos e fenóis, indicando que a reação não influenciou na concentração dos compostos minoritários. Na avaliação antimicrobiana, as misturas antes e após a interesterificação foram emulsificadas, produzindo diferentes respostas. Emulsões produzidas com os lipídios interesterificados apresentaram menor tamanho de partícula e maior potencial antimicrobiano, exibindo efeito bactericida; emulsões produzidas com as misturas não-interesterificadas, apresentaram maior tamanho de partícula e menor potencial antimicrobiano, exibindo efeito bacteriostático. Portanto, as lipases foram capazes de catalisar as reações de interesterificação entre os óleos da Amazônia, indicando o potencial destes catalisadores nestas reações. As frações lipídicas obtidas apresentaram atividade antimicrobiana, o que abre precedentes para que estudos biológicos mais aprofundados sejam realizados<br>Abstract: This study aimed to produce, characterize and evaluate the antimicrobial properties of interesterified Amazonian oils produced by different lipases. Two blends of Amazonian oils were subjected to enzymatic interesterification: the first one was composed by buriti oil and murumuru fat and the second one was composed by patauá oil and palm stearin. The interesterification reactions were catalyzed by two microbial lipases in three different enzymatic systems: one with a commercial lipase Lipozyme-TL-IM (Novozymes); a second with a lipase from the microorganism Rhizopus sp.; and the third with a mixture of both lipases (commercial and Rhizopus sp.). In both blends, depending on the enzyme used, the lipids produced presented different characteristics. In the buriti: murumuru blend, the lipase from Rhizopus sp. besides being specific for the sn-1,3 positions of triacylglycerol was specific for the type of fatty acids (unsaturated). The commercial lipase was specific only for the type of fatty acids (unsaturated), while the use of both enzymes showed no synergistic effect in this blend; the results were intermediate to those obtained with the individual enzymes. In the patauá: palm stearin blend, the lipase from Rhizopus sp. is specific for the type of fatty acid (unsaturated), while commercial lipase showed no specificity to this blend. In the system with both enzymes no synergistic effect was also observed; the results obtained were similiar to those obtained using only the enzyme from Rhizopus sp. In both blends, with the three enzymatic systems, there was a reduction in the proportions of triacylglycerols of the types trisaturated and tri-unsaturated after the reactions, with the formation of predominantly mono -and di- unsaturated lipids. These lipids produced maintained the high concentration of tocopherols, carotenoids and phenolics, indicating that the reaction did not influence the concentration of minor compounds. In the antimicrobial evaluation, the blends before and after interesterification were emulsified, producing different responses. Emulsions produced with interesterified lipids showed lower droplet size and higher antimicrobial activity (bactericidal effect); emulsions produced with non-interesterified blends showed larger droplet size and lower antimicrobial activity (bacteriostatic effect). Therefore, lipases were able to catalyze the interesterification reactions between Amazonian oils, indicating the potential of these catalysts in these reactions. Lipids obtained showed antimicrobial activity, which encourages more detailed biological studies<br>Doutorado<br>Ciência de Alimentos<br>Doutora em Ciência de Alimentos
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Barrigón, de San Marcos José Manuel. "Model-based design and development of operational strategies for Rhizopus oryzae lipase production in Pichia pastoris under the AOX1 promoter." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/287984.
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Pichia pastoris es identificada como una de las factorías celulares más eficientes para
la producción de proteínas recombinantes. Más de 500 proteínas han sido expresadas
usando este sistema de expresión. P. pastoris combina la habilidad de crecer en
medios mínimos a altas densidades celulares con la de secreción de proteína
heteróloga, ayudando a su recuperación.
En este trabajo, la proteína modelo seleccionada es la lipasa recombinante de Rhizopus
oryzae (ROL). La producción heteróloga de ROL en cultivos fed-bach de P. pastoris
PAOX1 (Mut+) ha sido estudiada desde el punto de vista de la monitorización y control
del bioproceso, la modelización cinética y el diseño y desarrollo de estrategias
operacionales.
El primer paso en la investigación ha sido la estimación de la concentración de
biomasa, sustrato y velocidad específica de crecimiento (μ) mediante dos observadores
no lineales y uno lineal. El objetivo de este estudio ha sido comparar las prestaciones
de los diferentes algoritmos en bioproceso de P. pastoris.
La producción de proteína recombinante está estrechamente relacionada con la μ. Por
lo tanto, debido a su elevada relevancia en el bioproceso, μ fue estimada utilizando el
análisis de los gases de salida en línea o las medidas de la concentración de sustrato.
La biomasa y el sustrato fueron obtenidos directamente de sus correspondientes
balances de materia.
La estrategia de cultivo más frecuentemente utilizada para conseguir altas densidades
celulares y elevada producción de proteína heteróloga con el sistema PAOX1 (Mut+) es
la operación fed-batch. Las estrategias operacionales estándar están basadas en el
control de la concentración de sustrato próximas a cero (estrategias limitantes) o manteniendo la concentración a un valor contante (estrategias no limitantes). En
consecuencia se estudió el efecto de las estrategias operacionales fed-batch de metanol
no limitante (MNLFB) y metanol limitante (MLFB) en la producción de ROL. Éstas
son las estrategias de control más comunes que tienen como objetivo mantener
contantes las velocidades específicas claves: crecimiento celular (μ), consumo de
sustrato (qs) y producción de proteína (qp) a partir de la hipótesis de estado quasiestacionario
para el sustrato. Los resultados se analizaron con el objetivo de
determinar la condición más apropiada en función de rendimientos y productividades.
Las velocidades específicas medias y las variables de estado para varios cultivos fedbatch,
bajo condiciones de metanol limitante y no limitante, se usaron para el
desarrollo de un modelo macrocinético no estructurado para la producción heteróloga
de la ROL por el sistema P. pastoris PAOX1. Posteriormente, se ha realizado un metaanálisis
comparativo sobre la producción de varias proteínas heterólogas modelo para
P. pastoris bajo el promotor AOX1 y se ha desarrollado una estrategia general para
mejorar la producción de proteínas a partir de la cinética como clave para la
optimización del proceso.
Adicionalmente se ha estudiado y caracterizado la capacidad de transferencia de
oxigeno en diferentes biorreactores a escala laboratorio y piloto. El modelo de
transferencia de oxigeno ha sido también desarrollado y validado en la producción de
ROL en P. pastoris bajo el promotor AOX1. Finalmente, el modelo cinético
previamente desarrollado para la producción heteróloga de ROL y el modelo de
transferencia de oxigeno han sido aplicados para definir estrategias alternativas de
operación basadas en operaciones con oxígeno limitante (OLFB) comparándose con
las estrategias estándar.<br>Pichia pastoris is recognized as one of the most efficient cell factories for the
production of recombinant proteins. More than 500 proteins have been expressed
using this system. P. pastoris combines the ability of growing on minimal medium at
very high cell densities with secreting the heterologous protein, aiding to their
recovery.
In this work, the selected target protein has been is the recombinant Rhizopus oryzae
lipase (ROL). The heterologous ROL production in P. pastoris PAOX1 (Mut+) fed-batch
cultures has been studied for bioprocess monitoring and control, kinetic modelling,
and the design and development operational strategies.
The first step in the research has been the estimation of biomass, substrate and specific
growth rate (μ) by means of two non-linear observers and a linear estimator. The aim
of this study has been to compare the performance of the different algorithms in P.
pastoris bioprocesses.
Heterologous protein production is closely related to μ. So, due to its high relevance in
the bioprocess, μ has been estimated by on-line gas analyses or substrate concentration
measurements. Biomass and substrate have been straightforwardly obtained solving
their corresponding mass balances.
The most frequently used cultivation strategy to achieve high cell densities and high
heterologous protein production levels with PAOX1-(Mut+)-based system is the fedbatch
operation. The standard operational strategies are based on the control of the
substrate concentration close to zero (limiting strategies) or keeping the concentration
at a constant value (non-limiting strategies). Consequently, the effect of methanol nonlimiting
fed-batch (MNLFB) and methanol limited fed-batch (MLFB) operational strategies on ROL production has been studied. These most commonly applied control
strategies allow maintaining rather constant key specific rates: cell growth (μ),
substrate uptake (qs), and protein production (qp) through the quasi-steady state
hypothesis for substrate. Results have been analyzed in order to determine the most
suitable operating conditions in terms of yields and productivities.
Furthermore, mean specific rates and state variables for various fed-batch cultures,
under methanol limited and non-limited conditions were used for modeling. Hence, an
unstructured macrokinetic model for heterologous ROL production by a P. pastoris
PAOX1 based system has been developed. Then, a comparative meta-analysis of
heterologous protein production of various target proteins by P. pastoris under AOX1
promoter has been conducted and a general strategy for improving protein production
from process kinetics was developed as a key to bioprocess optimization.
Additionally, the characterization of oxygen transfer capacity in different
laboratory/pilot scale bioreactors has been studied. The oxygen transfer model was
also developed and validated in heterologous ROL production by P. pastoris under
AOX1 promoter. Finally, the previous kinetic model for heterologous ROL production
and oxygen transfer model have been applied to define alternative operational
strategies based on oxygen limited fed-batch operations (OLFB) and have been
compared to the standard strategies.
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Skoczinski, Pia [Verfasser], Karl-Erich [Akademischer Betreuer] Jaeger, and Holger [Gutachter] Gohlke. "Single amino acid substitutions in lipase A affect its production and secretion by Bacillus subtilis / Pia Skoczinski ; Gutachter: Holger Gohlke ; Betreuer: Karl-Erich Jaeger." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1127200097/34.
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Pascot, Agnès. "La déficience familiale en lipoprotéine lipase, LPL, caractérisation d'une cohorte de patients lipémiques et étude de la production in vitro de LPL des macrophages." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ31772.pdf.
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Teixeira, Mayra Ferreira Netto. "Produção de lipase por Candida viswanathii: otimização das condições de cultivo, purificação em sistema aquoso bifásico e propriedades bioquímicas." Universidade Federal do Tocantins, 2017. http://hdl.handle.net/11612/436.
Full textAbstract:
As lipases (EC 3.1.1.3) hidrolisam ligações éster de triacilgriceróis numa interface água-óleo. Por possuírem diversas aplicações industriais, são as enzimas mais utilizadas em sínteses orgânicas e mais de 20% de biotransformações são realizadas com lipases. Os objetivos deste trabalho foram avaliar as condições nutricionais de meios de cultivo (fontes de óleos vegetais e animais, nitrogênio e carbono) para melhorar a produção de lipase por Candida viswanathii além de purificar a enzima em sistema aquoso bifásico e realizar a caracterização parcial da lipase. Entre os meios de cultivos analisados o meio de Vogel proporcionou a maior produção de lipase após 36 horas de cultivo (11,73 U/mL). Após analisar as fontes de carbono lipídicas e não-lipídicas e fontes de nitrogênio, um planejamento experimental de misturas foi aplicado para analisar o efeito de glicerol, lactose e sorbitol sobre a produção de lipase. Os resultados obtidos mostraram que a maior produção de lipase (20,41 U/mL) foi observada com 0,5% de lactose (p/v) e 0,5% (p/v) de sorbitol, sendo observado um aumento de 74% da produção de enzima. Nesse estudo observou-se que a lactose e sorbitol foram utilizados como adjuntos para a produção de lipase. A lipase produzida nas condições anteriores foi purificada em sistema aquoso bifásico (SAB) formado por polietilenoglicol (PEG) e fosfato de potássio. O maior coeficiente de partição (1,34) foi encontrado no ensaio com PEG 4000/fosfato a Temperatura de 40 °C e pH 7,0, bem como o maior balanço de atividade (50,73%). Na caracterização parcial, a lipase não sofreu influência da temperaturana faixa de 20 – 60 °C e obteve atividade máxima em pH 8,1. A enzima apresentou também elevada estabilidade em solventes orgânicos como metanol e etanol, sendo estas propriedades consideradas importantes para aplicações em processos biotecnológicos.<br>Lipases (EC 3.1.1.3) hydrolyze ester bonds of triacylglycerols at a water-oil interface. By they have several industrial applications, they are the enzymes most used in organic synthesis and more than 20% of biotransformations are performed with lipases. The objectives of this work were to evaluate the nutritional conditions of culture media (plant and animal oils, nitrogen and carbon) to improve the production of lipase by Candida viswanathii in addition to purifying the enzyme in a biphasic aqueous system and to realize the partial characterization of lipase. Among the culture media analyzed, the Vogel medium provided the highest lipase production after 36 hours of culture (11,73 U/mL). After analyzing the lipid and non-lipid carbon sources and nitrogen sources, an experimental design of blends was applied to analyze the effect of glycerol, lactose and sorbitol on lipase production. The results showed that the highest production of lipase (20,41 U/mL) was observed with 0.5% lactose (w/v) and 0.5% (w/v) sorbitol, with an increase of 74% of enzyme production. In this study it was observed that lactose and sorbitol were used as adjuncts for the production of lipase. The lipase produced under the above conditions was purified in a aqueous two-phase systems (ATPS) consisting of polyethylene glycol (PEG) and potassium phosphate. The highest partition coefficient (1,34) was found in the PEG 4000/phosphate assay at temperature of 40 °C and pH 7,0, as well as the highest activity balance (50,73%). In the partial characterization, the lipase was not influenced by the temperature range of 20 - 60 °C and obtained maximum activity at pH 8,1. The enzyme also showed high stability in organic solvents such as methanol and ethanol, and these properties are considered important for applications in biotechnological processes.
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Guenot, Sophie. "Développement d’un modèle prédictif de l’énantiosélectivité des lipases." Toulouse, INSA, 2007. http://eprint.insa-toulouse.fr/archive/00000347/.
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L’étude a porté sur le développement d’un modèle prédictif de l’énantiopréférence de la lipase de Burkholderia cepacia ATCC 21808 et plus particulièrement sur la relation pouvant exister entre l’accessibilité au site actif des énantiomères dérivés du (R,S)-alpha-bromo-phényl acétate d’éthyle et l’énantiosélectivité. Une approche mixte, couplant des techniques de modélisation moléculaire classiques à de nouvelles techniques algorithmiques de planification de mouvements issues de la robotique, a été utilisée pour modéliser les trajectoires de différents esters dérivés de l’alpha-bromo-phényl acétate d’éthyle. Les résultats obtenus in silico ont montré que les recherches de trajectoires sont plus rapides avec l’énantiomère (R) qu’avec l’énantiomère (S) pour 7 substrats synthétisés par voie chimique. De manière qualitative, ces résultats corrèlent avec l’énantiosélectivité de l’enzyme, obtenue expérimentalement, indiquant que celle-ci pourrait être associée à une plus ou moins grande difficulté d’accès. De plus, l’analyse des trajectoires a permis d’identifier des cibles privilégiées de mutagénèse (points de collision fréquemment rencontrés pour tous les substrats) parmi lesquels trois positions ont été retenues : Leu-17, Val-266 et Leu-287. Afin de générer et caractériser ces mutants ponctuels, un système d’expression recombinante de la lipase de B. Cepacia chez E. Coli et un protocole de purification par chromatographie d’affinité ont été développés. Les gènes lip et hp codant respectivement pour la lipase et sa protéine chaperonne ont été clonés dans différents vecteurs d’expression sous le contrôle de différents promoteurs. Le plus haut niveau d’activité lipase sous forme soluble jamais décrit dans la littérature concernant cette enzyme a été obtenu en utilisant le vecteur pFLAG-ATS sous le contrôle du promoteur tac (6679 U/Lculture). Les mutants ponctuels ont été construits par mutagénèse dirigée en remplaçant chaque position par les 19 autres acides aminés possibles. Les mutants V266, présentant les plus hautes activités lipase lors d’un crible au para-nitrophényl butyrate, ont été sélectionnés dans un premier temps afin de mesurer leurs énantiosélectivités lors de l’hydrolyse de l’α-bromo phényl acétate de 2-chloro éthyle. Parmi ces mutants, le V266G, présente une inversion d’énantiosélectivité en faveur de l’énantiomère (S) (E=18). L’absence de chaîne latérale de la glycine diminue l’encombrement stérique au niveau de la position 266 permettant ainsi à l’énantiomère (S) soit d’accéder plus facilement au site catalytique, soit de mieux se positionner pour former l’intermédiaire tétraédrique. En parallèle, un protocole de production de la lipase de B. Cepacia à l’échelle microplaque a été développé et validé sur la banque de mutants V266, afin de disposer, à terme, d’un outil de criblage pour de plus larges banques de mutants créées par évolution dirigée<br>The study focused on the development of a predictive model of Burkholderia cepacia ATCC 21808 lipase enantioselectivity and more specifically on the relationship that may exist between enantioselectivity and the accessibility to the active site of (R/S)-alpha-bromo phenyl acetic acid ethyl ester derivatives. A mixed approach, combining molecular modelling techniques and path planning algorithms issued from robotics research, was used to compute the geometrically allowed trajectories of various alpha-bromo phenyl acetic acid ethyl ester derivatives. In silico results indicated that the pathways are computed faster for the (R) enantiomer than the (S) enantiomer for 7 substrates synthesized by chemical synthesis. These results are in qualitative agreement with the enzyme enantioselectivity experimentally determined, indicating that enantioselectivity could be associated with a greater or lesser difficulty of access. In addition, the analysis of trajectories allowed to identify targets for mutagenesis (collision points frequently encountered for all substrates), amongst which three positions were selected: Leu-17, Val-266 and Leu-287. In order to generate and characterize these rational mutants, a recombinant expression system of B. Cepacia lipase in E. Coli and a purification protocol by affinity chromatography have been developed. Genes lip and hp encoding respectively for the lipase and its chaperone protein were cloned in different expression vectors under the control of different promoters. The highest level of lipase activity in soluble form ever described in the literature for this enzyme was obtained by using the vector pFLAG-ATS under the control of the tac promoter (6679 U / Lculture). Rational mutants were constructed by site-directed mutagenesis by replacing each position by the 19 other possible amino acids. Mutants V266, displaying the highest lipase activities in screening with para-nitrophenyl butyrate, were selected to measure their enantioselectivity during hydrolysis of the (R,S)- a-bromo phenyl acetic acid 2-chloro ethyl ester. Among these mutants, V266G displayed an inverted enantioselectivity in favour of the (S)-enantiomer (E=18). The absence of side chain in glycine decreases the steric hindrance at position 266, allowing thus the (S)-enantiomer to (a) access more easily to the catalytic site or (b) adopt a more easily productive conformation in order to form the tetrahedral intermediate. In parallel, a protocol for the production of B. Cepacia lipase at microplate scale has been developed and validated on the library of V266 mutants, in order to dispose of a screening tool for larger mutants libraries created by directed evolution
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Harchouni, Seddik. "Manipulation des voies de signalisation de l'énergie pour améliorer la production des biocarburants chez les organismes photosynthétiques." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0498.
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Les triacylglycérol (TAG) est un métabolite hautement énergétique qui peut être facilement converti en biodiesel. Les TAG peuvent être produits à partir de plantes et de microalgues. L'étude des voies de signalisation de l'énergie peut offrir de nouvelles stratégies pour améliorer l'accumulation de biomasse et de TAG sans compromettre la croissance. Dans cette thèse, j'ai étudié le rôle de deux voies principales de signalisation énergétique: la voie du de guanosine ppGpp (guanosine penta(tétra) phosphate) dans le chloroplaste et la voie de TOR (Target of rapamycin) dans le cytosol. J'ai choisi de travailler sur la mousse Physcomitrella patens, un modèle d'eucaryote photosynthétique en raison de sa position évolutive entre les algues et les plantes vasculaires. Pour étudier le rôle du ppGpp, nous avons créé des lignées transgéniques exprimant de manière inductible une ppGpp synthase et sur-accumulant le ppGpp. J'ai trouvé que l'induction de SYN provoque une forte inhibition de la capacité photosynthétique en raison de l'inhibition de l'expression des protéines clés codées par les chloroplastes et aussi une réorganisation des membranes thylakoïdes. Pour l’étude de TOR nous avons traité la mousse avec des inhibiteurs de TOR et montré que cela provoque l’inhibition de la croissance de manière dose dépendante et l’accumulation de TAG. L’utilisation des marqueurs lipidiques a révélé la perte de petites vésicules associées à la croissance et l'accumulation de plus grandes structures de corps lipidiques. Des études supplémentaires permettront de développer des stratégies pour améliorer la production de biocarburants chez les organismes photosynthétiques<br>Triacylglycerol (TAG) is a highly energetic metabolite that can be easily converted into biodiesel. TAG can be produced from both plants and microalgae. However, plants have low TAG yields in their dominant vegetative tissues. Microalgae can accumulate high amounts of TAG, but only under stress, leading to growth inhibition and limiting yield. The study and manipulation of stress and energy signaling pathways can offer new strategies to improve biomass and TAG accumulation without compromising growth. In this thesis, I studied the role of two major energy signaling pathways: the guanosine penta(tetra) phosphate (ppGpp) pathway in the chloroplast and the target of rapamycin (TOR) pathway in the cytosol. I chose to work on the moss Physcomitrella patens which is an interesting model of photosynthetic eukaryote because of its evolutionary position between algae and vascular plants. To study the role of ppGpp we created transgenic lines that inducibly express a ppGpp synthase and over-accumulate ppGpp. I found that ppGpp accumulation causes a strong inhibition of photosynthetic capacity due to the inhibition of the expression of key chloroplast encoded proteins, and also reorganization of the thylakoid membrane system into super grana. For the TOR pathway, we treated P. patens protonema with active site TOR inhibitors and showed that this cause growth inhibition in a dose dependent manner and is accompanied by TAG accumulation. The use of lipid dyes reveals a shift from small growth associated vesicles to a larger oil body structures after treatment. Further studies will allow the development of new strategies for improving biofuel production in photosynthetic organisms
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Laboret, Françoise. "Biosynthèse d'esters naturels à propriétés aromatisantes." Université Joseph Fourier (Grenoble), 1996. http://www.theses.fr/1996GRE10116.
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Les esters de faible poids moleculaire sont utilises dans l'industrie des aromes et parfums pour leurs odeurs fruitees caracteristiques. Leur production par procedes biotechnologiques, a partir de substrats naturels et d'enzyme presente un potentiel economique important, les produits pouvant obtenir le label naturel. Un procede complet de bioconversion d'esters par catalyse enzymatique en milieu organique a ete developpe a partir du propionate de methyle, utilise comme modele d'optimisation des conditions reactionnelles. Les effets de l'hydrophobicite du solvant, de la polarite des substrats et de l'hydratation sur l'activite enzymatique ont ete mis en evidence. La lipase de mucor miehei a revele une large specificite de substrats lors de l'etude de la selectivite enzymatique. Le passage a la production a grande echelle a conduit a la mise au point d'un reacteur a lit fluidise triphasique multifonctionnel, fonctionnant en mode non continu. Differentes etudes ont ete realisees afin d'optimiser successivement la bioconversion, la separation et la purification de l'ester ainsi que le recyclage du solvant et du catalyseur. La maitrise des differentes etapes de production a facilite l'extension a la biosynthese d'autres esters naturels d'interet alimentaire et permis une nette amelioration des capacites de production, soulignant l'efficacite potentielle du procede a l'echelle industrielle
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Laborde, de Monpezat Thierry de. "Production de lipases fongiques spécifiques : étude par fluorimétrie." Toulouse 3, 1990. http://www.theses.fr/1990TOU30174.
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Les lipases catalysent l'hydrolyse des triglycerides en glycerol et acides gras. Elles presentent plusieurs types de specificite. Afin d'etudier la specificite de la nature des acides gras, nous avons mis au point une methode de dosage de l'activite lipasique utilisant la lipase de rhizopus delemar. Pour cela, sept esters de l'umbelliferone ont ete synthetises. Les conditions optimales d'hydrolyse ont ete etablies. Cette methode presente une sensibilite importante. Pour controler sa fiabilite, des dosages ont ete effectues apartir de deux champignons connus pour produire des lipases. L'hydrolyse des divers esters a permis de determiner des specificites comparables a celles citees dans la litterature. Puis, un test sur boites de petri a ete mis au point pour rechercher des souches productrices de lipases specifiques. Les esters sont incorpores dans le milieu de culture et les resultats ont permis de classer les souches en fonction du type de specificite des lipases produites. Botrytis cinerea a ete selectionne pour ses lipases specifiques des acides gras insatures. Cette enzyme produite en culture liquide est inductible par l'oleate de methyle. La production a ete augmentee d'un facteur 20 en optimisant les conditions de culture. Botrytis cinerea a ete teste pour l'hydrolyse de corps gras en milieu organique. La lipase presente une activite importante et une selectivite tres marquee vis-a-vis des acides gras insatures
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Hiol, Abel. "Contribution à l'étude de deux lipases extracellulaires issues de souches fongiques isolées à partir du fruit de palme." Aix-Marseille 3, 1999. http://www.theses.fr/1999AIX30034.
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L'acidification enzymatique de l'huile de palme est l'un des problemes cruciaux rencontres par cette huile tropicale qui occupe la premiere place dans les echanges mondiaux en oleagineux. Dans la premiere partie de ce travail, nous avons isole du fruit de palme plusieurs microorganismes lipolytiques. Notre attention s'est portee sur deux souches tres actives sur les triglycerides, qui se sont averees comme des contaminants majeurs de notre echantillonnage. Il s'agit de mucor hiemalis f. Hiemalis et rhizopus oryzae. Pour chacune des deux souches, nous avons optimise la production de la lipase extracellulaire en batch ou en fermenteur. Dans la seconde partie de ce travail, nous avons purifie la lipase extracellulaire de mucor hiemalis f. Hiemalis a l'homogeneite en trois etapes de chromatographie precedees d'une etape de fractionnement au sulfate d'ammonium. L'enzyme a ete purifiee 2200 fois avec une activite specifique sur tricaprilyne de 6153 moles/min/mg. L'enzyme est une glycoproteine de 49 kda, de phi 4,6 et active sous forme monomerique. Sur une colonne d'interactions hydrophobes, l'analyse de la lipase montre le profil d'une proteine hydrophile. La sequence n-terminale des 19 premiers residus a ete etablie et ne presente aucune identite de sequence significative avec celle des autres lipases connues. Notre enzyme est une triacylglycerol lipase avec une specificite de substrat large. Elle montre une preference pour les acides gras en position sn-1 et sn-3 et presente une activation interfaciale sur la tributyrine. Par ailleurs, nous avons purifie a l'homogeneite la lipase extracellulaire de rhizopus oryzae. Le facteur de purification obtenu est de 1243 avec une activite specifique de 8700 ui/mg. Notre enzyme a des caracteristiques proches des autres lipases de rhizopus sp. Purifiees. C'est une glycoproteine de 32 kda, avec un phi de l'ordre de 7,6. La sequence n-terminale est identique a celle des autres lipases du meme genre mais presente deux acides amines supplementaires qui pourraient provenir du propeptide. Nos resultats mettent en evidence les caracteristiques de deux lipases pures, tres actives sur les triacylglycerols et potentiellements impliquees dans l'acidification de l'huile de palme.
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Yan, Youchun. "Enzymatic production of sugar fatty acid esters." [S.l. : s.n.], 2001. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB9102241.
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El, Abbadi Najia. "Sur les lipases exogènes et endogènes d'une souche de Penecillium Cyclopium var. (syn : P. Aurantiogriseum Dierck) : production, caractérisation, localisation et étude relationnelle entre les différentes lipases." Aix-Marseille 3, 1991. http://www.theses.fr/1991AIX30021.
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Ce travail porte sur l'etude des lipases exogenes et endogenes, produites par une souche sauvage de penicillium cyclopium var. , isolee a partir de noix de grenoble; leurs caracteristiques physico-chimiques, les liens existant entre les deux types de lipases et leur localisation au niveau des cellules myceliennes. La lipase endogene presente les caracteristiques les plus interessantes grace a son immobilisation naturelle sur le mycelium qui lui confere une grande stabilite thermique et dans le temps. En outre elle est regioselective 1,3 et stereo-selective. Quelques essais d'application ont ete conduits avec cette lipase afin d'apprecier ses possibilites. La localisation des lipases au niveau des hyphes, a ete realisee en plusieurs temps. D'abord par centrifugation differentielle, puis par hydrolyses enzymatiques et enfin par des reactions cytochimiques et immunocytochimiques, debouchant sur une etude par microscopie optique et electronique. Ainsi il a ete etabli que la lipase endogene est une des deux formes exogenes, secretees dans le milieu de culture. Elle se trouve concentree au niveau de la paroi des cellules myceliennes, ainsi que dans l'espace periplasmique, et sa concentration est moins importante dans le cytoplasme
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Vanot, Guillaume Edwin. "Etude des lipases du genre fongique penicillium et maximisation de leur production pour l'espèce cyclopium." Aix-Marseille 3, 2002. http://www.theses.fr/2002AIX30079.
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En raison de leur mode d'action particulier, les lipases suscitent depuis longtemps un intérêt académique. En effet, bien que solubles en phase aqueuse, elles agissent préférentiellement sur des substrats insolubles. Ces biocatalyseurs suscitent également de plus en plus d'intérêt au niveau industriel, du fait de leurs spécificités variées. La connaissance structurale et fonctionnelle de ces enzymes a beaucoup progressée au cours de ces dix dernières années. C'est dans ce contexte que nous avons étudié les lipases d'une souche fongique : Penicillium cyclopium. Dans un premier temps, leur caractérisation biochimique, structurale et cinétique nous a permis de les comparer à celles produites par d'autres espèces de Penicillii. A la lumière de cette comparaison, P. Cyclopium apparaît comme un excellent modèle regroupant les différentes activités lipolytiques rencontrées au sein du genre Penicillium. Dans un second temps, à l'aide de la Méthodologie de la Recherche Expérimentale, nous avons maximisé la sécrétion de deux lipases distinctes produites par P. Cyclopium. Des essais ont également été réalisés pour produire celles-ci à une plus grande échelle en vue de potentielles applications industrielles<br>For many years, lipases have been an appealing subject for academic research since they are such unusual catalysts. Indeed, although they are water-soluble, they act on hydrophobic substrates. Because they display a wide spectrum of specificities, they have also become objects of industrial interest. The structural and functional knowledge of these enzymes has increased rapidly in the past ten years. Our study of the fungal lipases from Penicillium cyclopium is set in this background. Firstly, their biochemical, structural and kinetic characterisation enabled us to compare them with lipases produced by other Penicillium species. From this comparison, it appears P. Cyclopium is a good model of all Penicillii since it displays all the different lipolytic activities occurring in this genus. Secondly, using an optimal design methodology, we maximised the production of two different extracellular lipases from P. Cyclopium. Finally experiments were carried out to increase the production scale for potential industrial applications
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Mason, Susan Leigh. "Metabolism of triacylglycerol-rich lipoproteins in sheep." Lincoln University, 1991. http://hdl.handle.net/10182/1756.
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This thesis describes two approaches for studying of lipoprotein metabolism in sheep. The first approach involves the assay of lipoprotein lipase (LPL) activity to determine the role of lipoprotein-triacylglycerol fatty acids in fat deposition in sheep. This enzyme is the rate limiting enzyme in the hydrolysis of fatty acids from lipoprotein-triacylglycerol. The second approach was to characterize and quantify in vivo lipoprotein metabolism using iodinated very low density lipoprotein (¹²⁵I-VLDL) and low density lipoprotein (¹³¹I-LDL). Cross-bred lambs were divided into two treatment groups and either weaned early at 5 weeks of age or remained suckling. Lambs were slaughtered at 12 or 23 weeks at which time the body composition and adipose tissue LPL activity were determined. The differences in rearing led to differences in body composition. The suckled lambs were larger and fatter than weaned lambs. The increased fatness in the suckled lambs was associated with increased LPL activity (U/mg protein) in subcutaneous adipose tissue and was reflected in higher LPL activity in post-heparin plasma (PHP) taken 2 days prior to slaughter. The role of insulin in the regulation of LPL activity was investigated by either infusing a subset of the weaned and suckled lambs with insulin for 7 or 18 weeks or using the euglycemic clamp technique to study the effect of short insulin infusions. The long term infusion of insulin had no significant effect on PHP LPL or on adipose tissue LPL (U/g tissue). However, after infusing insulin for 6h at 6.3 mU.kg⁻·⁷⁵.h⁻¹ during the euglycemic clamps, a two fold increase in LPL activity in biopsied subcutaneous adipose tissue was observed. In the second approach, in vivo lipoprotein metabolism was investigated in 4 lambs using apolipoprotein B as a marker. Following the simultaneous injection of ¹²⁵I VLDL and ¹³¹I VLDL, the specific activities of apoB in VLDL, IDL and LDL fractions were determined. ApoB specific activity curves demonstrated that VLDL is metabolised to IDL and subsequently to LDL. The turnover of VLDL-B (3.45mg.d⁻¹.kg⁻¹) and LDL-B (4.8mg.d⁻¹.kg⁻¹) was calculated by fitting the VLDL-¹²⁵I-B and LDL-¹³¹I-B specific activity data to a mono-exponential equation. The metabolism of lipoproteins, inferred from the study of apoB, was shown to be similar in sheep to that reported in other animals although the amount of lipoprotein synthesised was low. A model to describe the kinetics of apoB metabolism in sheep was developed using SAAM. The proposed model features a three pool delipidation chain for VLDL, and subsystems containing two pools for IDL and LDL. IDL may be catabolised to LDL or cleared directly from the plasma. The developed model can now be used to compare the metabolism of lipoproteins in different physiological states and to design new experiments to study lipoprotein metabolism further.
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Pabai, Franknel Sandi Kouvea. "Production, purification, characterization of selected microbial lipases and their application for interesterification of butter fat." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0005/NQ30354.pdf.
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Drouet, Philippe. "Production d'arômes en milieu biphasique : application à la voie lipoxygénasique." Compiègne, 1994. http://www.theses.fr/1994COMPD729.
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La dégradation chez les plantes des acides gras polyinsaturés permet la production de molécules aromatiques. Cette voie métabolique est constituée de quatre étapes : l'hydrolyse des triglycérides insaturés par la lipase, l'oxydation des acides linoléique et linoléique par la lioxygénase, la scission des hydroperoxydes obtenus en aldéhydes et oxo-acides, par l'intermédiaire de l'hydroperoxyde-lyase et enfin la réduction de ces aldéhydes en alcools par l'alcool déshydrogénase. La synthèse à l'échelle préparative d'aldéhydes (hexanal, hexenal) et d'alcools (cis-3-hexenol, hexanol) est réalisée grâce à la synergie de deux démarches. D'une part, la mise en œuvre de systèmes compartimentés de type biphasique (octane/tampon pH 9,6) afin d'optimiser la biocatalyse d'un substrat hydrophobe (l'acide gras polyinsaturé) en un produit hydrophile (l'hydro peroxyde). Une augmentation du rendement de la réaction de lipoxyéenation est ainsi observée pour des concentrations élevées en substrat (23-40%). D'autre part, la découverte d'une hydroperoxyde-lyase possédant une bonne activité de scission et une stabilité conséquente permet d'obtenir à partir de 13(S)-HPOT, 457 mg/L de trans-2-hexénal et à partir de 13(S)-HPOD, 241 mg/L d'hexanal. En présence d'alcool-déshydrogénase de foie de cheval, 462 mg/L de cis-3-hexénol et 344 mg/L d'hexanol sont alors produits. La production de trans-2-hexénal à partir d'huile de lin riche en acide linoléique est également réalisée (368 mg/L). L'association d'une démarche fondamentale couplée à de nombreuses expériences de recherches appliquées permet ainsi d'apporter une certaine contribution à la valorisation des huiles riches en acides gras polyinsaturés.
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Borgard, Philippe. "L' alun de l'Occident Romain : production et distribution des amphores romaines de Lipari." Aix-Marseille 1, 2001. http://www.theses.fr/2001AIX10019.
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"Le terme alun désigne diverses substances de composition apparentée (et notamment le sulfate double d'aluminium et de potassium hydraté), utilisées jusqu'à une date avancée du XXe siècle, dans des domaines aussi variés que la médecine, la tannerie ou la teinturerie. L'importance de ce produit a été clairement démontrée pour les industries médiévales et modernes. Qu'en est-il pour des périodes plus anciennes ?. Une distinction fondamentale s'impose : seul l'alun natif, composé rare, généralement produit en faibles quantités et dans quelques points seulement du pourtour méditerranéen, semble avoir été exploité durant l'Antiquité. Les principaux districts miniers se situent en Orient. L'un d'eux, par exception, se trouve en Occident : celui de Lipari. Son rendement, comme la hauteur des profits que " les liparotes comme les romains " en tirent, est soulignée par Diodore (V, 10, 2). Différentes observations permettent de penser que cet alun - comme celui, sans doute, d'autres régions productrices - était conditionné en amphores : il s'agit, dans ce cas précis, de conteneurs longtemps restés apatrides que les anglo-saxons ont tout d'abord dénommés " Richborough 527 " et que l'on peut rebaptiser, compte tenu de leurs rapports exclusifs avec l'archipel éolien, Amphores Romaines de Lipari. La typo-chronologie de ces objets, dressée à partir de l'étude parallèle du matériel des sites consommateurs et des rebuts de l'atelier de Portinenti (Ile de Lipari), permet de suivre l'évolution de leur distribution - comme celle de leur contenu - depuis le deuxième quart du Ier s. Avant notre ère jusqu'au milieu du IIIe siècle. Elle révèle, avec l'appui des textes grecs et latins, un usage de l'alun aussi varié que pour l'époque moderne. Mais si l'alun de Lipari a vraisemblablement alimenté l'essentiel de la consommation occidentale, les quantités annuellement produites restent relativement modestes, inférieures sans doute, dans le meilleur des cas, à deux cents tonnes. "
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Cordova, Lopez Jesus Antonio. "Isolement, identification et physiologie des champignons thermophiles en vue de la production de lipases par fermentation en milieu solide." Montpellier 2, 1998. http://www.theses.fr/1998MON20037.
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La question initiale est : existe-t-il dans la nature des champignons filamenteux capables de se developper a des temperatures elevees et de produire des enzymes thermostables ayant des caracteristiques specifiques ? pour repondre a cette question, notre travail s'est deroule en trois etapes : 1. L'isolement et l'identification de nouvelles souches de champignons filamenteux thermophiles a partir de biotopes tropicaux ; 2. La description de leurs besoins nutritionnels et de leurs potentialites metaboliques en vue de selectionner une souche capable de produire des lipases thermostables par fermentation en milieu solide (fms). 3. La production de lipases dans differentes conditions de cultures en milieux liquides ou en milieux solides, en presence d'inducteurs de lipases, et en utilisant des sous-produits agricoles comme la bagasse de canne a sucre, les grignons d'olive ou le tourteau de coprah.
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Poisson, Laurent. "Valorisation enzymatique de lipides : synthèse de cires à partir de la matière grasse laitière : production et traitements d'acides gras polyinsaturés de microalgues." Le Mans, 1999. http://www.theses.fr/1999LEMAA002.
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Windbergs, Maike. "Towards a better understanding of lipid based matrices innovations in the production and analysis of physically stable solid lipid extrudates with tailor-made dissolution profiles." Göttingen Cuvillier, 2009. http://d-nb.info/996511962/04.
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Angius, Federica. "Molecular basis of membrane protein production and intracellular membranes proliferation in E. coli." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC217/document.
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Le système d’expression le plus utilisé pour la production des protéines membranaires, est le système basé sur l’ARN polymérase T7 (ARNpol T7) (Hattab et al., 2015). L'inconvénient de ce système est néanmoins que la vitesse de transcription de l’ARNpol T7 est dix fois plus rapide que celle de l’enzyme bactérienne. Depuis l’isolement de mutants spontanés, notamment C41 (DE3) et C43 (DE3) (Miroux et Walker, 1996) et l’identification de leurs mutations dans le génome, il apparaît clairement que la toxicité provoquée par la surproduction des protéines membranaires est liée à la quantité trop élevée d’ARNpol T7 dans la cellule (Wagner et al., 2008 ; Kwon et al., 2015). Les protéines membranaires ont besoin d’une vitesse de transcription/traduction plus basse pour se replier correctement dans la membrane de la bactérie. Le premier objectif de ma thèse était d’étendre l’amplitude du promoteur du système T7 sur laquelle est basée l’expression des protéines. Pour cela, nous avons isolé et caractérisé de nouvelles souches bactériennes dans lesquelles le niveau d’ARNpol T7 était efficacement régulé par un mécanisme non transcriptionnel très favorable à l’expression des protéines membranaires (Angius et al., 2016). Le deuxième objectif était de comprendre la prolifération des membranes intracellulaires chez E. coli suite à la surexpression de la protéine AtpF, une sous unité membranaire du complexe de l’ATP synthétase (Arechaga et al., 2000). Pour mieux comprendre les voies métaboliques impliquées dans la biogenèse, la prolifération et l’organisation des membranes, nous avons utilisé une approche de séquençage d’ARN à haut débit à différents temps après induction de la surexpression de la sous-unité AtpF dans la souche C43 (DE3). Ensuite, et en collaboration avec Gerardo Carranza and Ignacio Arechaga (Université de Cantabria, Espagne), nous avons construit et étudié des mutants de C43 (DE3) déficients pour les trois gènes codants pour des enzymes de la biosynthèse des cardiolipides afin d’évaluer leur participation dans la biogénèse des membranes intracellulaires<br>The most successful expression system used to produce membrane proteins for structural studies is the one based on the T7 RNA polymerase (T7 RNAP) (Hattab et al., 2015). However, the major drawback of this system is the overtranscription of the target gene due to the T7 RNAP transcription activity that is over ten times faster than the E. coli enzyme. Since the isolation of spontaneous mutants, namely C41(DE3) and C43(DE3) (Miroux and Walker, 1996) and the identification of their mutation in the genome, it becomes clear that reducing the amount of the T7 RNAP level removes the toxicity associated with the expression of some membrane proteins (Wagner et al., 2008; Kwon et al., 2015). Also, some membrane proteins require a very low rate of transcription to be correctly folded at the E. coli membrane. The first objective of my PhD was to extend the promoter strength coverage of the T7 based expression system. We used genetic and genomic approaches to isolate and characterize new bacterial strains (Angius et al., 2016) in which the level of T7 RNAP is differently regulated than in existing hosts. A second objective was to understand intracellular membrane proliferation in E. coli. Indeed it has been shown that over-expression of membrane proteins, like overexpression of AtpF of E. coli F1Fo ATP synthase is accompanied by the proliferation of intracellular membranes enriched in cardiolipids (Arechaga et al., 2000). To understand metabolic pathways involved in membrane biogenesis, proliferation and organization, we used a RNA sequencing approach at several time point upon over-expression of the F-ATPase b subunit in C43(DE3) host. On the other hand, in collaboration with Gerardo Carranza and Ignacio Arechaga (University of Cantabria, Spain) we studied C43(DE3) cls mutants, in which the cardiolipids genes A, B and C are deleted, to test how they participate to intracellular membranes structuration
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Menacho, Tito Paola Alessandra, and Mandujano Ariadne Guadalupe Prado. "Simi&Lips: Bálsamo natural hidrante para labios artesanal." Bachelor's thesis, Universidad Peruana de Ciencias Aplicadas (UPC), 2019. http://hdl.handle.net/10757/651747.
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En la actualidad, más son las personas que se preocupan por su cuidado personal y que los productos para este fin provengan de fuentes naturales. Tal es el caso de que muchas marcas de cosméticos, para satisfacer estas nuevas tendencias, crean nuevas líneas de productos de insumos naturales, con certificaciones orgánicas, libre de crueldad animal, entre otras.
Simi&Lips nace de la idea de cuatro alumnos de la Universidad Peruana de Ciencias Aplicadas de crear un producto que busca regresar a lo tradicional, es decir, utilizar las propiedades de insumos que se encuentran en la naturaleza para fabricar un bálsamo hidratante para labios artesanal, libre de incipientes, parabenos y metales pesados que a la larga causan repercusiones para la salud.
Simi&Lips utiliza insumos que se encuentran certificados como orgánicos entre ellos, el aceite de coco y la mantequilla de cacao, así como también esencias y aceites como la vainilla y la vitamina E, todos ellos reconocidos por sus propiedades humectantes y reparadoras.
Simi&Lips busca estar en equilibrio con el medio ambiente y que el cliente lo pueda comprender, es por ello que el concepto se basa en 3 pilares: producto 100% natural, excelente calidad y sobretodo un empaque que contribuye a la reducción de residuos plásticos porque está fabricado en su totalidad de cartón.<br>Nowadays, people are more concern about personal care and are looking for new products that come from natural sources. Such is the case, that cosmetics industries, to meet this new trend, are creating new product lines with organic inputs with international certifications, free of animal cruelty.
Simi&Lips is a four-student’s product idea from Universidad Peruana de Ciencias Aplicadas. This product seeks to go back to the traditional, which means, use the benefits of natural inputs to manufacture a moisturizing lip balm; incipient, heavy metals and parabens free.
Simi&Lips uses, on its majority, organic certified products such as cocoa butter and coconut oil, as well as beeswax, vainilla and vitamin E essential oils. All of our lip balm inputs are well-recognized by their moisturizing and repairing properties.
Finally, Simi&Lips seeks to be in balance with the environment and that our costumers be aware of it. Our concept is based in a 100% natural product, excellent quality and above all, we package the product in a paperboard tube which contributes to reduce plastic waste.<br>Trabajo de investigación
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Savonnière-Miccoli, Sandrine. "Implications de l'interleukine-6 et de lipides exogènes dans le contrôle de la croissance cellulaire et la sécrétion d'anticorps monoclonaux de deux lignées d'hybridomes : rôle des propriétés membranaires." Nancy 1, 1996. http://www.theses.fr/1996NAN10399.
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Frommholz, Gotz Harald. "Local embeddedness matters! : a study of the meaning of locality for the production process in the kitchen furniture industry in East Westphalia and Lippe." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8266.
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New institutionalism in sociology addresses how institutional pressure influences organisational behaviour. Its particular impact on “new economic sociology” is to establish a counter perspective to neo-classical economics by criticising the rational actor model of behaviour and emphasising cultural and cognitive references for business actions. Recent developments in new institutionalism increasingly focus on researching national and international contexts, which demonstrate a keen interest in non-local environments. Micro sociological research accordingly receives limited attention and the meaning of locality for production strategy in relation to markets is largely neglected. This thesis presents evidence from the kitchen-furniture industry of East Westphalia and Lippe (EWL) in Germany that, in an increasingly globalised economic system, local institutional contexts continue to influence business behaviour significantly. The thesis demonstrates the importance of locality for production organisation and business strategy in this case. The research aims to contribute to new institutionalist theory by establishing the relevance of “localness” and to encourage research to re-engage in meso-analysis on the sub-national level. The analysis presents results from a qualitative case study, which encompasses in-depth interviews, as well as results derived from contextual analysis of the industry’s structure and performance and statistical indicators provided by local institutions. The study tries to understand why about 70% of German produced kitchens, and about every fourth kitchen in Europe, originates from EWL. The findings demonstrate that managers’ evaluations of local production networks, regional cultural norms and values, shape managerial cognitive frameworks, which influence business behaviour significantly and can create meaning for locality of production sites.
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Mourey, André. "La lipolyse en milieux naturels et manipulés." Nancy 1, 1989. http://www.theses.fr/1989NAN10070.
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Étude de la dégradation des lipides dans les sols : numération des microorganismes en cause, mesure de la disparition de matière grasse in situ, mesure de l'activité lipolytique de différents sols. L'activité lipolytique est intense dans les sols où on rencontre une accumulation de matière organique et ne présente pas une étape limitante dans le processus de minéralisation. Étude des microorganismes responsables de la lipolyse dans les produits carnes : bactéries et levures. Identification des bactéries les plus actives. Production d'enzymes lipolytiques par bacillus pumilus, caractérisation et purification de ces enzymes