Dissertations / Theses on the topic 'Lipase'
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Sias, Barbara. "Etude de deux lipases apparentées aux lipases pancréatiques : lipase pancréatique humaine apparentée de type 2 et la lipase du plasma seminal caprin." Aix-Marseille 2, 2005. http://www.theses.fr/2005AIX22003.
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El, Kouhen Karim. "Identification et caractérisation d'une lipase chez Arabidopsis thaliana." Aix-Marseille 2, 2005. http://theses.univ-amu.fr.lama.univ-amu.fr/2005AIX22046.pdf.
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Dridi, Kaouther. "Evolution moléculaire et structurale des membres de la famille génique des lipases pancréatique." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4019.
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Helicoverpa armigera et Epiphyas postvittana, deux insectes nuisibles pour l'agriculture, ont développé des résistances contre la plupart des insecticides connus. Les lipides étant les constituants majeurs de leur régime alimentaire, les fonctions digestives des lipases deviennent alors une cible privilégiée pour l'élaboration de nouveaux insecticides. L'identification récente de gènes codant pour l'expression de protéines potentiellement actives et inactives apparentées aux lipases pancréatiques (PLRP) dans le tractus digestif de ces deux insectes et dont le niveau de transcription varie en fonction de leur régime alimentaire a ouvert un nouveau champ de recherche. Dans le but de contribuer à cette thématique, nous avons construit cinq lipases recombinantes d'E. postvittana (EpLIPs) et testé leur expression dans trois systèmes différents (E.coli, P.pastoris et bacculovirus). Le tractus digestif de Helicoverpa armigera a été étudié par chromatographie échangeuse d'ions et les différentes protéines séparées ont été testées en spectrométrie de masse et sur pHstat pour l'activité. Les résultats obtenus ont permis de mettre en évidence, pour la première fois, la présence à la fois d'une lipase active et d'une lipase inactive dans le tractus digestif d'un lépidoptère. Par ailleurs, la caractérisation biochimique d'un mutant GPLRP2-Δ β9 a été faite dans le but de comprendre l'effet de cette boucle, partiellement délétée dans les lipases d'insectes, dans la spécificité du substrat. Nous avons pu montrer que cette boucle β9 est essentielle pour la stabilisation de la chaîne acyle durant la réaction de lipolyseHelicoverpa armigera and Epiphyas postvittana, two major pest crops, have developed resistances against most of the known insecticides. Lipids being a major component of insect diet, digestive function of lipase are a target of choice for new insecticide design. The recent identification of active and inactive pancreatic lipase related protein (PLRP) genes in those two insects midgut, with a level of transcription depending on the diet, opened the field of insect digestive lipase study. In order to contribute to this thematic, we built five recombinants lipase from E. postvittana (EpLIPs) and tested their expression in three different systems (E.coli, P.pastoris and bacculovirus). Protein structure prediction of EpLIPs allowed us to develop some functional hypothesis enlightening the role of inactive lipase in lipid transport. H. armigera midgut contents were separated through a one step purification chromatography and the different fractions were tested for activity and mass spectrometry. The results obtained gave the first evidence of the presence of both an active and an inactive lipase in lepidopteran midgut. In addition to this work, a biochemical characterisation of a β9 GPLRP2 mutant was carried out to understand the effect of this loop, partially deleted in insect lipase, in substrate specificity. The result shows that β9 loop is essential for stabilizing the leaving acyl chain during the lipolysis reaction
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Fernandez, Sylvie. "Lipolyse d'excipients lipidiques destinés à l'administration par voie orale de substances actives hydrophobes." Aix-Marseille 2, 2008. http://theses.univ-amu.fr.lama.univ-amu.fr/2008AIX22024.pdf.
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Le Labrasol® et le Gelucire® 44/14 sont des macrogolglycérides utilisés pour l’administration par voie orale des substances actives hydrophobes. Ils sont composés d’acylglycérols et d’esters de PEG, substrats potentiels des lipases digestives. Nous avons étudié la lipolyse in vitro de ces excipients par les lipases digestives. Nous avons mis en évidence que la lipase pancréatique humaine (HPL), principale lipase impliquée dans la lipolyse des triacylglycérols alimentaires, n’était pas capable d’hydrolyser ces excipients contrairement à la lipase gastrique de chien (DGL), la lipase pancréatique apparentée de type 2 (HPLRP2) et la carboxyl ester hydrolase (CEH). L’étude de la spécificité des lipases digestives vis-à-vis des différents substrats contenus dans ces excipients montre que les esters de PEG sont de mauvais substrats pour la HPL et la DGL présentant une spécificité marquée pour les di- et triacylglycérols. En revanche, la HPLRP2 et la CEH hydrolysent les esters de PEG et ne sont pas spécifiques vis-à-vis des différents composés contenus dans ces excipients. Nous avons développé une méthode de simulation in vitro de la lipolyse gastro-intestinale de ces excipients prenant en compte la lipolyse gastrique puis la lipolyse duodénale. La composition des excipients lipidiques est significativement modifiée à la fin de la phase gastrique montrant l’importance de la lipolyse gastrique in vivo. Nous avons aussi étudié l’influence de la lipolyse gastro-intestinale du Labrasol® et du Gelucire® 44/14 sur la solubilité apparente de deux substances actives hydrophobes, le piroxicam et la cinnarizine. Il apparaît que la lipolyse gastro-intestinale de l’excipient n’entraîne pas de précipitation du piroxicam et permet de maintenir la cinnarizine en solution aqueuse lorsque celle-ci formulée avec le Labrasol®Labrasol® and Gelucire® 44/14 are macrogolglycerides which are used for the oral drug delivery of poorly water-soluble drugs. They are composed of acylglycerols and PEG esters potential substrates of digestive lipases. We studied the in vitro lipolysis of these excipients by digestive lipases. We showed that the human pancreatic lipase (HPL), the main lipase involved in the lipolysis of dietary triacylglycerols, was not able to hydrolyze either of these excipients contrary to dog gastric lipase (DGL), human pancreatic lipase-related protein 2 (HPLRP2), and carboxyl ester hydrolase (CEH). The study of digestive lipases specificity showed that HPL and DGL possessed specificity toward di- and triacylglycerols, whereas HPLRP2 and CEH hydrolyzed PEG esters but did not present a marked specificity. We developed an in vitro method to simulate the gastrointestinal lipolysis of these excipients. At the end of the gastric phase, the composition of both of these excipients was significantly modified underlining the importance of gastric lipolysis in vivo. We also studied the influence of excipients’ lipolysis on the concentration of two poorly water-soluble drugs, piroxicam and cinnarizine, in the aqueous phase. It seems that the gastrointestinal lipolysis of these excipients did not undergo piroxicam precipitation whereas it was a prerequisite to maintain cinnarizine in aqueous solution when formulated with Labrasol®
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Qiu, Guosong. "Function of lipoprotein lipase and endothelial lipase in human macrophages." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31471.
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Lipoprotein lipase (LPL) and endothelial lipase (EL) are expressed in atherosclerotic lesions, mainly in macrophages. However, the functional roles of LPL and EL in macrophages are not well characterized. In the present thesis, the effects of these lipases on cholesterol efflux, low density lipoprotein (LDL) catabolism, and proinflammatory cytokine secretion in human macrophages were investigated. Lentivirus transduction successfully induced EL suppression or over-expression in macrophages. LPL suppression was mediated by lentivirus transduction whereas dexamethasone was used to stimulate LPL expression. Apolipoprotein AI- (apoAI-) mediated cholesterol efflux was modestly reduced after LPL and EL suppression, but significantly increased in lipase-overexpressing macrophages as well as transfected 293 cells. This effect was partially inhibited after the elimination of either catalytic or non-catalytic lipase function, but completely abolished when both functions were blocked. The observed effect on cholesterol efflux was mediated partially by an increased apoAI binding, an effect dependent on cell surface lipase. Lipase expression was inversely associated with phosphatidylcholine and sphingomyelin levels, but positively with lysophosphatidylcholine production, the later was shown to promote apoAI-mediated cholesterol efflux dose-dependently. EL expression was positively correlated with both native and oxidized LDL binding and association via non-catalytic function as observed in both EL-suppressed and over-expressed macrophages. By contrast, the catalytic activity of EL did not have a significant role in oxidized LDL metabolism with the exception of a positive correlation with native LDL association, which also partially depended on the LDL receptor. The concentration of interleukin-1β and 6, macrophage chemoattractant protein-1, and tumor necrosis factor-α was reduced after LPL and EL suppression, The lipase suppression also amplified the inhibitory effect of oxidized LDL in macrophages. Microarray analysis indicated that >50 genes, mainly proinflammatory ones, had marked expression changes after lipase suppression. Atorvastatin treatment reduced LPL and EL expression as well as Rho, the liver X receptor (LXR), and nuclear factor-κB (NF-κB) levels. Mechanistic studies identified LXR and NF-κB to be involved in atorvastatin-induced suppression of LPL and EL, respectively. In summary, by promoting apoAI-mediated cholesterol efflux, lipoprotein binding and uptake, and proinflammatory cytokine expression in macrophages, EL and LPL may influence the atherogenic potential of macrophages.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Infanzón, Ramos Belén. "Novel Lipases: Expression and Improvement for Applied Biocatalysis = Nuevas lipasas: expresión y mejoras para biocatálisis aplicada." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/456674.
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This thesis is focused in the identification and improvement of lipases for biotechnological application. The importance of lipases is increasing in several industries. However, the commercial use of lipases is still a drawback in the economics of the lipase-based industrial applications. There are many tools for improving and adapting the enzyme properties to the desired requirements of a process that could lead lipase catalysis through a cost-effective process. In this context, the main objective of this work was: “To characterize, express and to improve novel bacterial lipases for sustainable industrial processes”.
The first activity done was to explore and to characterize a new esterase from P. barcinonensis. It was isolate from P. barcinonensis the gene corresponding to Est23, and its cloning in a proper vector to perform expression and purification for biochemical characterization. Est23 showed preference for mid-chain substrates and having maximum activity at 37 °C and pH 7. It also includes in silico analysis of the 3D model structure and phylogeny. Moreover, a phylogenetic tree was constructed to assign Est23 to one of the bacterial hydrolase families described by Arpingy and Jaeger. Est23 could not be assigned to any bacterial hydrolases described till that moment, suggesting that Est23 could be part of a new group of bacterial lipases. Because Est23 displays a GGG(A)X-type putative oxyanion hole, widely described as a motif involved in tertiary alcohol resolution, the ability of Est23 for conversion and resolution of tertiary alcohols was evaluated. However, no conversion was detected using the esters linalyl and terpinyl acetate alcohols as substrates.
To improve LipR activity by protein engineering LipR was then desired. LipR was isolated from Rhodococcus sp. strain CR-53 in a previous and showed an unusual fungal-like oxyanion-hole never found among bacterial lipases, close to the Y-type oxyanion hole described for Candida antartica lipase A (CalA), a lipase widely used in industry. In order to improve LipR activity on long-chain substrates, several enzyme-engineering approaches were done to change the amino acids constituting the rare oxyanion hole of LipR for further industrial application. These mutations also allowed studying the role of the amino acids forming the oxyanion hole of LipR. Hydrolytic activity over short, mid and long- chain substrates was assayed with the variants obtained. The LipR variant Asp111Gly produced a change on the chain- length- substrate preference of LipR, displaying a 5.6 fold increase of activity on muf-oleate. This improvement of activity on longer chain length substrates makes of this LipR variant a very attractive candidate for testing activity on biodiesel synthesis, a process requiring activity on long-chain substrates. Nevertheless, LipR and LipR_YGS variant need a clear expression enhancement in order to apply them to transesterification reactions using oily feedstocks.
The stabilization Pseudomonas lipases LipA, LipC and LipCmut was improved by immobilization in order to applied these enzymes in transesterification reactions. Therefore, a fast and economic immobilization procedure by adsorption was set up.
Finally, the three immobilized lipase preparations and a commercial lipase were used to test alternative feedstocks for triglyceride transesterification. A total of four oils were tested: commercial triolein, degummed soybean oil, waste cooking oil, and Mucor circinelloides oil. Moreover, the characterization of the tested raw materials in terms of FFAs, tri, di and monoglyceride contents measure was also of interest. In a global analysis, a good increase of FAMEs percent was obtained with LipA, LipC and LipCmut immobilized on Accurel MP1000. But better results were achieved when the reactions were catalyzed by Novozym® 435 commercial enzyme.Esta tesis se centra en la identificación y mejora de lipasas para aplicaciones biotecnológicas. El objetivo principal de este trabajo fue: "Caracterizar, expresar y mejorar las nuevas lipasas bacterianas para procesos industriales sostenibles". La primera actividad realizada fue explorar y caracterizar una nueva esterasa, Est23, de P. barcinonensis. Se aisló de P. barcinonensis el gen correspondiente a Est23 y su clonación en un vector adecuado para realizar la expresión y purificación para caracterización bioquímica. Además, se construyó un árbol filogenético para asignar Est23 a una de las familias de hidrolasas bacterianas descritas por Arpingy y Jaeger, y debido a que Est23 tiene un oxyanion-hole de tipo GGG (A) X, ampliamente descrito como motivo implicado en la resolución del alcohol terciario, se evaluó la capacidad de Est23 en dichas reacciones. Luego se buscó mejorar la actividad sobre sustratos de cadena larga de la lipasa LipR de Rhodococcus sp. por ingeniería de proteínas. Diferentes enfoques de ingeniería enzimática se realizaron para cambiar los aminoácidos que forman parte del atípico oxyanion-hole de LipR. Estas mutaciones también permitieron estudiar el papel de los aminoácidos que forman este motivo. La actividad hidrolítica de las variantes obtenidas fue ensayada sobre sustratos de cadena corta, media y larga. La variante LipR Asp111Gly produjo un cambio en la preferencia de LipR de longitud de cadena. Sin embargo, LipR y LipR_YGS necesitan un aumento de expresión para aplicarlos a reacciones de transesterificación. La estabilización de tres lipasas de Pseudomonas, LipA, LipC y LipCmut, se mejoró por inmovilización con el fin de aplicar estas enzimas en las reacciones de transesterificación. Por lo tanto, se estableció un procedimiento de inmovilización por adsorción rápido y económico. Finalmente, se usaron las tres lipasa inmovilizadas y una lipasa comercial para probar materias primas alternativas para la transesterificación de triglicéridos. Se probaron un total de cuatro aceites: trioleína comercial, aceite de soja desgomado, aceite de cocina de desecho y aceite de Mucor circinelloides. Además se realizó la caracterización de las materias primas ensayadas en términos de la medida de los ácidos grasos, tri, di y monoglicéridos.
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Frenken, Leo G. "Pseudomonas glumae lipase : characterization, biogenese and protein engineering = Pseudomona glumae lipase /." [S.l. : s.n.], 1993. http://www.gbv.de/dms/bs/toc/131132261.pdf.
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Allouche, Maya. "Etude des interactions protéine-lipide : exemple du système lipase/colipase pancréatique." Aix-Marseille 2, 2008. http://www.theses.fr/2008AIX20674.
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Büchner, Susanne. "Bestimmung mikrobieller und gewebseigener Lipasen mit dem Reflectoquant® Lipase – Test (Merck KGaA)." Doctoral thesis, Universitätsbibliothek Leipzig, 2007. http://nbn-resolving.de/urn:nbn:de:bsz:15-20071024-093300-7.
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Fetthaltige Lebensmittel tierischer Herkunft sind aufgrund ihres hohen Lipidgehaltes besonders anfällig für Lipolyse. Ausschlaggebend hierfür ist die Wirkung mikrobieller und gewebseigener Lipasen. Für die Beurteilung der Haltbarkeit und Qualität derartiger Lebens-mittel werden vorwiegend die sensorische Prüfung und mikrobiologische Untersuchungen eingesetzt. Eine Einschätzung lipolytischer Aktivitäten erfolgt derzeit nicht routinemäßig. Von Seiten der Lebensmittelindustrie und der Lebensmittelüberwachung besteht, besonders im Zusammenhang mit Qualitätssicherungssystemen, großes Interesse an einer schnellen Verfügbarkeit von Ergebnissen, um das Verderbnispotential der Lebensmittel effizienter abschätzen zu können. Schwerpunkte dieser Arbeit bildeten daher: die Entwicklung von Applikationsvorschriften zur Bestimmung bakterieller und gewebs-eigener Lipasen mit dem Reflectoquant ® Lipase – Test, einem für Milchlipasen konzi-pierten Schnelltest der Fa. Merck, die Messung synthetisierter Lipasekonzentrationen ausgewählter Verderbniserreger (n= 168) bzw. gewebseigener Lipasen (n= 127) in Fleisch (Schwein, Rind, Hähnchen, Kaninchen), Fisch (Kabeljau, Forelle, Hering), Wurst- und Fischerzeugnissen sowie Leber (Schwein, Pute), die Erfassung von Konzentrationen nach 5-minütiger Erhitzung bei 50, 60, 70 und 80 °C, um eventuelle Unterschiede in der Thermoresistenz von bakteriellen und gewebseigenen Lipasen aufzuzeigen sowie der Vergleich der Substratverwertbarkeit verschiedener bakterieller Lipasen gegenüber Caprylat, Tributyrin und Tween 60. Folgende Ergebnisse wurden erzielt: Der Anwendungsbereich des Reflectoquant ® Lipase – Tests konnte um 5 Applikations-vorschriften zur Messung bakterieller Lipasekonzentrationen in Nährbouillon und zur Messung gewebseigener Lipasekonzentrationen in den oben aufgeführten Produkten erweitert werden. Dabei wurden Lösungsvorschläge zur Beseitigung von Störeinflüssen wie z.B. Ascorbinsäure in Wursterzeugnissen, erarbeitet. Unter Verwendung des Reflectoquant ® Lipase – Tests konnte das lipolytische Synthesepotential von 56 Bakterienstämmen der Spezies Ps. aeruginosa, Ps. fluorescens, A. hydrophila, A. caviae, S. aureus, S. epidermidis, B. subtilis, P. mirabilis und Ser. marcescens nach Anzüchtung unter Optimalbedingungen bestimmt werden. Als besonders starke Lipasebildner mit Konzentrationen von > 50 µg/l, fielen Stämme von Ps. aeruginosa, Ps. fluorescens, A. hydrophila und S. aureus auf. Für die Lebensmittel Fleisch, Fisch und Leber wurden erstmals Orientierungswerte zu natürlich enthaltenen Lipasekonzentrationen (gewebseigene Lipasen) ermittelt. Die Lipasekonzentrationen in Fleisch bewegten sich zwischen 44 µg/kg (Schwein) und 370 µg/kg (Kaninchen). In Fisch lagen sie zwischen 228 µg/kg (Kabeljau) und 1200 µg/kg (Forelle). Die höchsten Konzentrationen wurden in Schweineleber mit 122100 µg/kg gemessen. Die nach Erhitzung gemessenen Konzentrationen bestätigen, dass bakterielle Lipasen wesentlich hitzestabiler sind als gewebseigene. Besonders hitzestabile Lipasen mit Restaktivitäten zwischen 16 % und 31 % nach 5-minütiger Erhitzung bei 70 °C bildeten die Stämme Ps. aeruginosa 5x und 1, Ps. fluorescens 9 und S. aureus 4. Die geringe Hitzestabilität der gewebseigenen Lipasen (keine Restaktivitäten in Fisch, Rind-, Schweine- und Geflügelfleisch nach 5 min bei 70 °C) ist die Ursache für den fehlenden Lipasenachweis in den geprüften Brüh- und Kochwürsten (z.B. Bierschinken). Aufgrund dieser Unterschiede ist eine Abgrenzung bakterieller von gewebseigenen Lipasen möglich. Nachgewiesene Lipasekonzentrationen in erhitzten Produkten könnten daher ein Hinweis auf einen hohen Ausgangskeimgehalt mit Lipasesynthese und/oder mangelnde Erhitzung sein. Zu berücksichtigen ist allerdings, dass in lipasereichen Geweben, z.B. Leber, noch sehr geringe Lipasekonzentrationen nach Erhitzung auf 70 °C nachweisbar waren. Von den drei Substraten wurden besonders gut Tributyrin und Caprylat von den getesteten bakteriellen Lipasen hydrolysiert. Die Prüfung der Substratverwertbarkeit hat insbesondere Bedeutung für die Einschätzung der Wirkung von Lipasen und die Voraussage der Haltbarkeit fetthaltiger Lebensmittel. Mit der nunmehr breiteren Anwendung des Reflectoquant ® Lipase – Tests und den neugewonnenen Kenntnissen über Lipasekonzentrationen in Lebensmitteln könnte möglicherweise, unter Einbeziehung der Gesamtkeimzahlbestimmung und sensorischen Eindrücken, die Einschätzung des Verderbs bzw. der Haltbarkeit präzisiert werden. Welche konkreten Lipasekonzentrationen lebensmittelhygienisch in Bezug auf Haltbarkeit und Qualität relevant sind, muss für die einzelnen Lebensmittel in Lagerungsversuchen und begleitenden mikrobiologischen Untersuchungen noch geprüft werden.
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Wannerberger, Kristin. "Lipases at solid surfaces an adsorption and activity study /." [Lund : Dept. of Food Technology, Lund University], 1996. http://catalog.hathitrust.org/api/volumes/oclc/38950353.html.
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Zallot, Rémi. "Identification et caractérisation d'une lipase exprimée pendant l'hydrolyse des réserves chez Arabidopsis thaliana." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21840/document.
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Les réserves d’huile de la graine d’Arabidopsis thaliana sont hydrolysées par des lipases au cours de la croissance post-germinative de la plantule. Une protéine capable de fixer un inhibiteur de lipase a été identifiée à partir d’un extrait de plantules de colza. La séquence de cette protéine ressemble à celles de lipases connues. L’expression transitoire du gène orthologue d’Arabidopsis chez Nicotiana benthamiana induit l’apparition d’une activité lipase. Ces données suggèrent que cette protéine est une lipase. Une étude de la localisation in vivo de cette enzyme chez Nicotiana benthamiana indique qu’elle est localisée au niveau des peroxysomes. Chez Arabidopsis, le gène codant pour cette lipase est exprimé essentiellement lors de la croissance des plantules, quand l’hydrolyse de l’huile est maximale. L’analyse d’un mutant montre que ce gène est responsable de l’essentiel de l’activité lipase mesurée pendant la mobilisation de l’huile de réserve. Ces données suggèrent que cette lipase pourrait être impliquée dans la mobilisation des réserves lipidiques pendant la croissance post-germinative. Néanmoins, l’hydrolyse des réserves n’est pas diminuée chez le mutant. Cela pourrait être lié à une compensation par d’autres lipasesIn germinating seedlings of Arabidopsis thaliana, fat storage breakdown is initiated by lipases. A protein capable to bind to a lipase inhibitor was identified from an extract of rape seedlings and its amino acid sequence found to resemble that of known lipases. Transient expression of the Arabidopsis orthologous gene led to a 100-fold increase in lipase activity in Nicotiana bethamiana leaves. Taken together, these data strongly suggest that this protein is indeed a lipase. In vivo localization studies using a GFP fusion protein in Nicotiana benthamiana as a transcient expression host showed a peroxisomal localization. In Arabidopsis, the gene coding for this lipase was found to be mainly expressed in seedlings during fat storage breakdown. Most lipase activity was abolished in germinating seedlings of an Arabidopsis mutant for this gene. These data suggest that this lipase is likely involved in the breakdown of fat storage in germinating seedlings of Arabidopsis. However, oil mobilization was not affected in Arabidopsis mutant plants. This might suggest that the effect of the mutation could be compensated for by other lipases
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Lambert, Jean-François. "Effet fondateur et origine de la mutation D9N du gène de la lipase lipoprotéique au sein de la population du Saguenay-Lac-St-Jean /." Thèse, Chicoutimi : Université du Québec à Chicoutimi, 2002. http://theses.uqac.ca.
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Souza, Maria Cristiane Martins de. "ImobilizaÃÃo de lipase de Candida antarctica do tipo B em nanopartÃculas magnÃticas visando a aplicaÃÃo na sÃntese de Ãsteres." Universidade Federal do CearÃ, 2013. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=9381.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorNeste trabalho, nanopartÃculas magnÃticas de ferro (Fe3O4) (NPM) foram avalia- das como suporte para a imobilizaÃÃo de lipase de Candida antarctica do tipo B (CALB). O biocatalisador (CALB-NPM) foi analisado na catÃlise dos Ãsteres: oleato de etila (biodiesel), butirato de metila e etila. NanopartÃculas magnÃticas sÃo particularmente interessantes para imobilizaÃÃo enzimÃtica devido as suas propriedades magnÃticas favorecerem a fÃcil separaÃÃo da mistura reacional atravÃs do uso de magnetismo. A enzima CALB à uma enzima capaz de atuar em diversas reaÃÃes, como, hidrÃlises e transesterificaÃÃes. Contudo, um dos problemas do uso de enzimas como catalisadores homogÃneos à a sua recuperaÃÃo. Assim, à necessÃrio o uso de suportes que retenham a enzima, mantendo suas caracterÃsticas catalÃticas. As na- nopartÃculas foram produzidas pelo mÃtodo de co-precipitaÃÃo. Determinou-se o tamanho das nanopartÃculas (11 nm) atravÃs da tÃcnica de difraÃÃo de raios-X (DRX) com posterior refi- namento das fases obtidas pelo mÃtodo Rietveld. Espectros de infravermelho foram obtidos para anÃlise de presenÃa de hidroxilas usando pastilhas de KBr das ferritas magnÃticas. O espectro foi medido na regiÃo entre 400 e 4000 cm−1. ModificaÃÃes foram realizadas na su- perfÃcie das mesmas com γ-aminopropiltrietoxissilano (APTS) e glutaraldeÃdo. No processo de imobilizaÃÃo, a influÃncia da velocidade de agitaÃÃo (20-250 rpm), carga enzimÃtica (45-200 UpNPB.g−1), tempo de contato enzima-suporte (0,5-5 h), concentraÃÃo de glutaraldeÃdo (2,5 e 25 % (m/v)), aditivo dodecil sulfato de sÃdio (SDS 0,23 %) e reutilizaÃÃo do biocatalisador fo- ram avaliadas. A imobilizaÃÃo foi realizada na presenÃa de 100 mM de tampÃo bicarbonato de sÃdio, pH 10, a 25 ÂC. ApÃs a imobilizaÃÃo, a enzima imobilizada exibiu melhor estabilidade tÃrmica e operacional do que na forma solÃvel. As condiÃÃes Ãtimas de imobilizaÃÃo foram: velocidade de agitaÃÃo de 45 rpm, carga enzimÃtica (80 UpNPB.g−1), tempo de imobilizaÃÃo de 1 h, soluÃÃo de glutaraldeÃdo (25 % (m/v)), possibilitando um rendimento de imobilizaÃÃo de 41,8 % e atividade enzimÃtica do derivado de 29,1 UpNPB/g. AlÃm disso, o biocatalisador manteve aproximadamente, 53% de atividade catalÃtica inicial apÃs cinco ciclos consecutivos de reaÃÃo hidrolÃtica. ApÃs a imobilizaÃÃo, a estabilidade tÃrmica dos derivados foi realizada a partir da reaÃÃo de hidrÃlise com 0,01 g de CALB-NPM. atividade catalÃtica da enzima livre e imobilizada foi analisada a 60 ÂC. A produÃÃo de esteres foi realizada com o biocatalisador na melhor condiÃÃo catalÃtica. AlÃm das nanopartÃculas foram analisadas a bioconversÃo de esteres por resinas acrÃlicas comerciais (CALB imobilizada). A conversÃo de oleato de etila foi de aproximadamente 90% para os biocatalisadores testados. Os ciclos de reaÃÃo consecutivos (14) mostram a manutenÃÃo da produÃÃo de biodiesel. A mÃxima conversÃo de buitrato de etila (96,8%) e metila (93,9%) foram obtidos apÃs 8 h de reaÃÃo a 25 ÂC com CALB imobilizada em nanopartÃculas magnÃticas. Os ciclos de reaÃÃo consecutivos (12) mostram a manutenÃÃo da produÃÃo dos Ãsteres (aproximadamente 76% para as nanopartÃculas e 79% para a resina acrÃlica).
In this work, magnetic nanoparticles of iron (Fe3O4) (NPM) were evaluated as a support for the immobilization of lipase Candida antarctica B (CALB). The biocatalyst (CALB- NPM) was analyzed in the catalysis of esters: ethyl oleate (biodiesel), methyl and ethyl buty- rate. Magnetic nanoparticles are particularly interesting for enzyme immobilization due to their magnetic properties favoring the easy separation from the reaction mixture by use of magne- tism. The CALB enzyme is an enzyme capable of acting in various reactions, such as hydrolysis and transesterifications. However, one problem of using enzymes as homogeneous catalysts is their recovery. Thus, it is necessary to use brackets that retain the enzyme while maintaining its catalytic characteristics. Nanoparticles were produced by co-precipitation method. We de- termined the size of the nanoparticles (11 nm) using the technique of X-ray diffraction (XRD) with subsequent refining of the phases obtained by the Rietveld method. Infrared spectra were obtained for analysis of the presence of hydroxyls using KBr pellets of magnetic ferrites. The spectrum was measured in the region between 400 and 4000 cm −1. Modifications were car- ried out on the nanoparticlesâ surfaces with γ-aminopropyltriethoxysilane (APTS) and glutaral- dehyde. The influence of stirring speed (20-250 rpm), enzyme load (45-200 UpNPB/gsupport), immobilization time (0.5-5 h), glutaraldehyde solution (2.5 and 25%), additive (SDS 0.23%) and reuse of the biocatalyst (six hydrolytic cycles reactions) were evaluated. The immobiliza- tion was performed in the presence of 100mMsodium bicarbonate buffer, pH 10, at 25 ÂC. After immobilization, CALB exhibited improved thermal and operational stabilities. The best result (Immobilization yield: 53% and immobilized enzyme activity: 29.1 UpNPB/gsupport) was obtained at 45 rpm, using 200 UpNPB/gsupport and 1h of immobilization. Furthermore, immo- bilized Calb maintained approximately 41.8 % of initial activity after five cycles of hydrolysis. The ethyl oleate production was analyzed with the best condition and compared to commercial acrylic resins (CALB immobilized). The ethyl oleate conversion was approximately 90 % for the two biocatalyst at 48 h. The consecutive reaction cycles (14) show the maintenance in the production of biodiesel. Maximum conversion of methyl butyrate (93.9 %) and ethyl butyrate (96.8 %) were achieved after 8 h of reaction at 25 ÂC for CALB immobilized onto magnetic nanoparticles. The consecutive reaction cycles (12) show the maintenance in the production of esters (approximately 76 % for nanoparticles and 79 % for acrylic resin).
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Zschenker, Oliver. "Zielgerichtete Mutagenese der lysosomalen Lipase." [S.l.] : [s.n.], 2001. http://www.sub.uni-hamburg.de/disse/429/Disse.pdf.
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Hedfors, Cecilia. "Lipase chemoselectivity - kinetics and applications." Licentiate thesis, KTH, School of Biotechnology (BIO), 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10232.
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A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.
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Rip, Jacob. "Lipoprotein lipase, hypertriglyceridemia and atherosclerosis." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2006. http://dare.uva.nl/document/28665.
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Zhang, Liyan. "Lipoprotein lipase - unstable on purpose? /." Doctoral thesis, Umeå : Department of Medical Biosciences, Physiological Chemistry, University of Umeå, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1058.
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Sonesson, Andreas. "Lipase diffusion on solid surfaces /." Stockholm, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-381.
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Sindelar, Pavel J. "Hepatic lipase and dolichol esterification /." Stockholm, 1997. http://diss.kib.ki.se/1997/91-628-2772-3.
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Carulla, i. Sanmartí Pere. "Isoformes de pI de la lipoproteïna lipasa: Origen, distribució i funció." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/462027.
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Un dels principals aspectes que hem descrit ha estat la presència i la distribució de les isoformes de pI de l’LPL. Inicialment hem treballat amb teixits de rata adulta: cor, TAB, TAM i múscul. Els resultats han demostrat que tots els teixits de rata dels quals s’ha pogut purificar parcialment l’LPL presenten isoformes de pI de l’LPL. També hem treballat en una de les poques situacions en què el fetge expressa LPL: en cries de rata. Lamentablement, l’LPL ha resultat ser una proteïna difícilment purificable. A les cries de rata, però, hem pogut descriure clarament la presència d’isoformes de l’LPL al cor i al TAM.
A partir de tota la informació obtinguda de la descripció d’isoformes en teixits de rata, hem dissenyat un mètode per modelitzar el patró d’isoformes de cada teixit, determinat pel nombre d’isoformes, i el pI i l’abundància relativa de cada isoforma. Aquesta modelització ens ha permès comparar els patrons obtinguts en cada purificació i determinar la similitud dels patrons d’isoformes entre diferents teixits i diferents espècies.
La presència de les isoformes en sí ja és un fet molt important pel coneixement de l’LPL. Però el fet d’haver descrit diferències entre els patrons d’isoformes entre els teixits de rata, va posar de manifest la rellevància de la funció diferencial de l’LPL en cada teixit. A partir d’aquesta idea hem estudiat la funció de les isoformes de pI de l’LPL. Com està àmpliament descrit, l’expressió i l’activitat de l’LPL és depenent de cada teixit i de la situació fisiològica en què es trobi l’animal.
Hem descrit els patrons de l’LPL de diferents teixits de rata (cor, TAB i TAM) en situacions fisiològiques en les quals és sabut que l’activitat LPL varia notablement (fred, dejuni i realimentació) respecte a la situació control.
Mitjançant el pI i la abundància relativa de les isoformes de tots els teixits de rata, hem dissenyat un sistema de classificació de les isoformes en poblacions segons clústers. Aquesta caracterització de les isoformes en clústers permet descriure les variacions que hi ha entre diferents situacions o teixits des d’un enfocament totalment diferent al tractat fins aleshores. Hem pogut detectar importants variacions en les poblacions de les isoformes de pI segons la situació fisiològica.
Paral·lelament, hem estudiat l’afinitat de les isoformes de pI de l’LPL tant per l’ancoratge (heparina) com pel substrat. Entre les diferents isoformes no hem descrit diferències d’afinitat per heparina i totes presenten activitat LPL. Tot i això, no podem descartar petites diferències d’afinitat pel substrat.
Del TAB visceral de macaco, n’hem descrit la presència d’isoformes de pI de l’LPL i el seu patró de distribució. Totes les isoformes de pI del TAB d’aquesta espècie també presenten afinitat pel substrat i, per tant, activitat LPL. A més, també són degudes, parcialment, a la glicosilació de la proteïna. Ens hem plantejat determinar quines són les característiques que determinen l’origen les isoformes de pI de l’LPL.
A partir de la purificació del TAB de macaco, hem determinat de novo d’un 74% de la seqüència de l’LPL. En aquesta cobertura, hem pogut confirmar la presència de l’asparagina 44 i les tirosines 95 i 165. Aquests aminoàcids estan descrits en altres espècies com a objectius de modificacions posttraduccionals.
A continuació, hem treballat per separar les isoformes de pI, les unes de les altres, mitjançant l’isoelectroenfocament en líquid (off-gel electrophoresis). L’objectiu d’aquest treball ha estat poder treballar amb les isoformes per separat i determinar quina combinació de modificacions posttraduccionals és la responsable de cada isoforma. Novament, probablement a causa de les característiques de l’LPL, ens ha resultat impossible obtenir la purificació de cada isoformaWe have described has been the presence and distribution of the pI isoforms of LPL. We have worked with adult rat tissues: heart, WAT, BAT and muscle. The results have shown that all tissues have LPL isoforms. In 15-days old rats, the LPL expressed in the liver LPL has turned out to be a protein that is hard to purify. However, we have been able to clearly describe the presence of isoforms of LPL in heart and BAT. We have designed a method for modelling the isoform pattern of each tissue, determined by the number of isoforms, and the pI and the relative abundance of each isoform. This modelling has allowed us to compare the patterns and to determine the similarity of isoform patterns. We have studied the function of the pI isoforms of LPL. The expression and activity of the LPL is dependent on each tissue and the physiological situation in which the animal is found. We have described the LPL patterns of different rat tissues (heart, TAB and TAM) in physiological situations in which LPL activity is known to vary markedly (cold, fasting and refeeding) compared to the control animals. By means of the pI and the relative abundance of the isoforms of all the rat tissues, we have designed a system of classification of the isoforms in populations according to clusters. This characterization of isoforms in clusters allows describing the variations that exist between different situations or tissues from a totally different approach to the one treated until now. At the same time, we have studied the affinity of the pI isoforms of LPL for the anchor (heparin) and the substrate. Among the different isoforms we have not described differences in affinity and all isoforms are active. We have described the presence of pI isoforms of LPL and its distribution pattern of the WAT of Macaca fascicularis. In addition, they are also partially due to glycosylation of the protein. We have described 74% of the sequence of the LPL. In this coverage, we have been able to confirm the presence of asparagine 44 and tyrosine 95 and 165. These amino acids are described in other species as targets for posttranslational modifications.
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Paques, Fernanda Wiermann. "Extração e caracterização da fração lipolitica de residuos de processamento de mamão formosa." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256634.
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Orientador: Gabriela Alves MacedoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Mestrado
Ciência de Alimentos
Mestre em Ciência de Alimentos
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Martins, Mariana Provedel. "Biotransformação de epóxidos com fungos de origem marinha e síntese de cloroidrinas." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/75/75131/tde-14102008-095435/.
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Neste trabalho realizou-se uma triagem com os fungos de origem marinha Trichoderma sp Gc1, Penicillium miczynskii Gc5, Penicillium raistrickii Ce16 e Aspergilus sydowii Gc12 para catalisar a abertura do (RS)-2-(benziloximetil)oxirano (2). O melhor resultado foi obtido com o fungo Trichoderma sp Gc1, pois forneceu o (R)-(-)-2-(benziloximetil)oxirano (2) com excesso enantiomérico de 60 % e rendimento isolado de 39 %; o diol (S)-(+)-1,2-propanodiol-3-fenilmetóxi (2a) com excesso enantiomérico de 32 % e rendimento de 19 %. Posteriormente otimizou-se as condições experimentais com o epóxido 2 e o fungo Trichoderma sp Gc1, variando-se a massa de biocatalisador, o meio de cultura e o tempo de reação. Os melhores resultados sob essas condições foram aplicadas para os epóxidos 3-5 fornecendo o (S)-(+)-2-[4-metoxifenoxi)metil]oxirano (3a), (S)-(+)-2-(propeniloxi)oriano (4), (R)-(+)-1-alilóxi-2,3-propanodiol (4a) e o (-)-9-deceno-1,2-diol (5a). Nesses estudos embora ocorreu a abertura seletiva dos epóxidos com as células totais do fungo Trichoderma sp Gc1, não obteve-se altas purezas enantioméricas dos produtos. Ainda nesse trabalho realizou-se a síntese das cloroidrinas racêmicas, a (RS)- 1-cloro-2-propanol- 3-fenilmetóxi (2b), (RS)- 1-cloro-2-propanol- 3-(4-metoxifenóxi) (3b) e (RS)- 1-alilóxi-3-cloro-2-propanol (4b) em bons rendimentos e uma metodologia sintética ambientalmente apropriada, pois os compostos foram preparados em meio aquoso na presença de íons cloreto. Em seguida realizou-se uma resolução enzimática da (RS)-1-alilóxi-3-cloro-2-propanol (4b) com a lipase de Candida antarctica onde obteve-se a clorodrina 4a (e.e. 72 %) e o seu correspondente produto acetilado 4c (e.e. 82 %) em bons excessos enantioméricos. Conclui-se que os fungos de origem marinha utilizados neste trabalho são potenciais fontes de epóxido-hidrolases para promover a abertura seletiva de epóxidos.In this work carried out itself the first study biocatalytic involving reactions of reduction of cetonas with fungi of marine origin. They were utilized 7 cetonas commercial as substratos and 8 fungi derived little seas like biocatalisadores. The fungi were isolated of the sponges little seas Geodia corticostylifera (Trichoderma sp Gc1, Penicillium miczynskii Gc5, Aspergillus sydowii Gc12) and Chelonaplysylla erect (Bionectria sp Ce5, Aspergillus sydowii Ce15, Penicillium raistrickii Ce16 and Aspergillus sydowii Ce19). The reduction 2-chloro-1-phenylethanone (1) was studied under several conditions of reaction (changes of pH, addition or absence of glucose) and the best result was with fungus P. miczynskii Gc5, therefore itself obteve an isolated performance of 60% and excess enantiomeric of 50% for the (S)- 2-chloro-1- phenylethanol (1a). The interesting one in these studies was that all of the fungi utilized in the selection with the 2-chloro-1-phenylethanone (1) presented selectivity anti- Prelog. In the literature is common obtain reduction enzymatic with selectivity Prelog. To 2-bromo-1-phenylethanone (2) was biotransformaded by the fungus A. sydowii Ce19 you correspond composed: (S)-2-bromo-1-phenylethanol (2a), (S)-2-cloro-1- phenylethanol (1a), whereas to (2c), 2-chloro-1-phenylethanone (1) and the 2- phenyloxirane (2b) were obtained by reactions not enzymatic. To 2-bromo-1-(4- bromophenyl)ethanone (3) and to 2-bromo-1-(4-nitrophenyl)ethanone (4) were entirely biodegradadas by the fungus A. sydowii Ce19. The reduction biocatalytic of the 1-(2- iodophenyl)ethanol (5) and 1-(3-iodophenyl)ethanol (6) with the fungus Trichoderma sp Gc1 supplied the 1-(2-iodophenyl)ethanol (5a) and the 1-(3-iodophenyl)ethanol (6a) with excellent excesses enantiomeric (e.e. > 99%). It stayed verified also that the fungi derived little seas for promote the reactions of reduction by biocatalysis are going to be cultivated in water of the artificial sea.
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Knapp, Andreas Verfasser], Karl-Erich [Akademischer Betreuer] Jaeger, and Dieter [Akademischer Betreuer] [Willbold. "Regulation der Bildung einer extrazellulären Lipase LipA und des Lipase-spezifischen Chaperons LipB in Burkholderia glumae / Andreas Knapp. Gutachter: Karl-Erich Jaeger ; Dieter Willbold." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1052993753/34.
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Knapp, Andreas [Verfasser], Karl-Erich Akademischer Betreuer] Jaeger, and Dieter [Akademischer Betreuer] [Willbold. "Regulation der Bildung einer extrazellulären Lipase LipA und des Lipase-spezifischen Chaperons LipB in Burkholderia glumae / Andreas Knapp. Gutachter: Karl-Erich Jaeger ; Dieter Willbold." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://nbn-resolving.de/urn:nbn:de:hbz:061-20140630-085540-5.
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Ben, Ameur Villain Sawsen. "Conception et étude d'un réacteur enzymatique à membrane fonctionnant en milieu supercritique : application à la synthèse enzymatique d’esters." Thesis, Montpellier, Ecole nationale supérieure de chimie, 2012. http://www.theses.fr/2012ENCM0001/document.
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Ce travail de thèse vise à développer un procédé de synthèse d'esters dans un réacteur enzymatique membranaire fonctionnant en milieu CO2 supercritique. Un tel procédé représente une alternative intéressante à la synthèse chimique classiquement utilisée en industrie car il permet, d'une part, d'utiliser le CO2 supercritique comme solvant et de substituer ainsi les solvants organiques généralement utilisés et, d'autre part, d'avoir un produit final doté d'un label naturel grâce à l'utilisation d'un catalyseur biologique. Dans cette étude, une membrane enzymatique de taille industrielle a été développée. Un pilote spécifique permettant la conduite de réactions enzymatiques en milieu supercritique a été conçu. Le procédé a été testé à l'aide d'une réaction modèle : la synthèse d'acétate d'anisyle à partir d'alcool et d'acétate de vinyle. L'influence de divers paramètres opératoires tels que la température, la pression, ou le débit sur les performances du procédé a été évaluée. Les performances du procédé ont été également comparées à celles d'un réacteur à lit fixeThis study deals with the development of an enzymatic membrane reactor working in supercritical carbon dioxide for ester synthesis. This process is an alternative to the chemical synthesis classically used in industry because it allows, on the one hand, the use of supercritical carbon dioxide as a solvent instead of organic solvents and on the other hand it leads to natural label ester thanks to the use of a biological catalyst. In this work, an industrial size enzymatic membrane was developed. A special pilot plant was designed to achieve enzymatic reactions in supercritical carbon dioxide medium. The process was studied with a model reaction : the anisyl acetate synthesis from anisic alcohol and vinyl acetate. The impact of several operating conditions like temperature, pressure and flow rate on the process performances was studied. The enzymatic membrane developed in this study was active and showed an interesting conversion rate. The performances were compared to those obtained with a packed bed reactor
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Østerlund, Torben. "Structure-function relationships of hormone-sensitive lipase." Lund : Section for Molecular Signalling, Dept. of Cell and Molecular Biology, Lund University, 1998. http://catalog.hathitrust.org/api/volumes/oclc/39103640.html.
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Svedendahl, Maria. "Lipase and ω-Transaminase : Biocatalytic Investigations." Doctoral thesis, KTH, Biokemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-13279.
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In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition.QC20100609
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Eriksson, Magnus. "Lipase-Catalyzed Syntheses of Telechelic Polyesters." Doctoral thesis, KTH, Biokemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-12101.
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Telechelic polyesters have successfully been synthesized with lipase-catalyzed polymerization. The produced telechelics had a high degree of difunctionalization, high purity (requiring little or no workup) and controlled degree of polymerization. The syntheses were performed in one-pot one-step reaction systems. The use of protection/deprotection chemistry was not necessary, since the lipase selectivity was utilized in the syntheses. Two different types of lipase-catalyzed polymerizations were applied – ring-opening polymerization and polycondensation. In ring-opening polymerization telechelics were produced by a combination of initiation, α-functionalization, and linking through termination, w-functionalization. In polycondensation different types of end-cappers were used to synthesize telechelics. Several exampels of functional groups were used for end-functionalization - epoxide, methacrylate and tetraallyls. Enzyme kinetic schemes describing the different functionalization methods of polyesters are presented and discussed. Stoichiometry and different reaction conditions have been studied to understand the effects these functions have on the final structure of the synthesized telechelics. Polyesters are classified as biodegradable, and can also be synthesized from materials that can be extracted or fermented from renewable sources like plants. Lipase-catalysts have several beneficial attributes, like high selectivity, they are renewable and biodegradable, are non-toxic and metal-free and can operate under mild reaction conditions. The focus of this thesis has been on lipase-catalyzed syntheses and characterization of the produced telechelics, in addition some materials have been produced. Some uses of telechelics are surface modification, materials for block co-polymers, functional films and biomedical applications.QC20100726
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Hedfors, Cecilia. "Lipase selectivity in functional polyester synthesis." Doctoral thesis, KTH, Biokemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-34023.
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Enzyme selectivity means that the enzyme´s preferences towards competing substrates will be different. In this thesis, the enzyme selectivity has been studied for utilization in synthesis of functionalized macromonomers. The aim was to study how the inherent –or introduced – selectivity of lipases can be used to introduce thiol‐ or enefunctionalities into short polyesters. Thiol‐ and ene‐functionalized renewable organic precursor molecules in combination with thiol‐ene click chemistry opens up for a sustainable material production. Lipases do not normally affect ene‐moieties and the preference towards thiols is low, enabling introduction of these functional groups for further modifications. In addition, lipases have been shown to be good catalysts in the formation of polyesters, both via ring‐opening and polycondensation polymerization. In paper I Candida antarctica lipase B was used to end‐functionalize poly(ε‐caprolactone) with free thiols in a one‐pot reaction. The advantage of using achemoselective lipase as catalyst was that no protection of the thiol was needed. The chemoselectivity displayed by Candida antarctica lipase B turned out to be 88 000 in favour of the alcohol (paper II). Rhizomucor miehei lipase showed less pronounced chemoselectivity. The largest contribution to the selectivities was derived from the more than two orders of magnitude higer KM towards the thiol compared to the alcohol. Thiols can be cross‐linked with enes in radical reactions to form networks, enabling formation of materials. One promising renewable molecule containing an acrylate moiety is itaconic acid. In paper III the selectivity towards the two esters in dimethyl itaconate was investigated and the active site of Candida antarctica lipase B was redesigned to generate variants with increased and decreased selectivity. One variant showed 14‐fold higher selectivity and could regioselectively add dimethyl itaconate onto a diol. This variant could be used in end‐functionalizations of polymers, introducing acrylate‐ester end‐groups. The enzyme selectivity towards lactones and their corresponding polyesters is of importance when designing a ring‐opening polymerization reaction. In paper IV Candida antarctica lipase B was found to prefer ω‐pentadecalactone and polyesters over ε‐caprolactone ten‐fold, while Humicola insolens cutinase preferred ε‐caprolactone and its corresponding polyester four‐fold over ω‐pentadecalactone and its polyester. From a selectivity point of view, Candida antarctica lipase B and Humicola insolens cutinase would be equally good in ring‐opening polymerization of ω‐pentadecalactone, while in the case of ε‐caprolactone Humicola insolens cutinase would be the preferred choice.QC 20110608
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Kikuchi, Hirofumi. "Ring-Opening Polymerizations with Lipase Catalysis." 京都大学 (Kyoto University), 2001. http://hdl.handle.net/2433/150688.
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Ramsamy, Tanya A. "The regulation of hepatic lipase activity." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/29156.
Full textAbstract:
Human hepatic lipase (HL) is a 66-kDa glycoprotein that plays an important role in the metabolism of apoB-containing lipoproteins and high density lipoproteins (HDL). Both the lipid and apolipoprotein composition of lipoproteins have been shown to modulate HL activity. In this study, very low density lipoproteins (VLDL) were found to be the best lipoprotein substrates for HL followed by low density lipoproteins (LDL) and HDL. Only 1% of the fatty acids released from HDL3 by HL are derived from triglycerides (TG) whereas the remainder of fatty acids produced are from the lipolysis of diglycerides (DG (49%)) and phospholipids (PL (50%)). In order to further study the role of lipoprotein composition on HL activity, HDL and LDL fractions were isolated from subjects with familial combined hyperlipidemia (FCHL) and matched controls and used as substrates for the enzyme. HL-mediated hydrolysis of patient and control LDL and HDL fractions is similar despite elevated serum TG levels in subjects with FCHL. In both patient and control samples, the most buoyant LDL and HDL fractions are better substrates for HL than the corresponding denser fractions when normalized for total protein content. Although differences are observed in the acylglycerol and PL hydrolysis of the two patient groups, these differences are not related to the DG content of the lipoproteins.
The association of HL with pure heparan sulfate proteoglycans (HSPG) has little effect on hydrolysis of HDL particles, but significantly inhibits (>80%)the hydrolysis of LDL and VLDL. Lipolytic inhibition is associated with a differential ability of the lipoproteins to remove HL from the HSPG. LDL and VLDL are unable to displace HL, while HDL readily displaces the enzyme from the HSPG. This is consistent with the view that HSPG-bound HL is inactive. HDL also displaces HL from the surface of the hepatoma cell line, HepG2, and Chinese Hamster Ovary (CHO) cells stably overexpressing human HL. Purified apolipoprotein (apo) A-I is more efficient than HDL at liberating HL from both pure and cell surface HSPG. Furthermore, different HDL fractions vary in their abilities to displace the enzyme from the cell surface. The buoyant HDL2 has a greater capacity to remove HL from the cell surface and intracellular compartments when compared to the smaller denser HDL particles.
Displacement of HL by apoA-I does not enhance the hydrolysis of VLDL. This somewhat paradoxical finding appears to result from the direct inhibition of HL by apoA-I, as both apoA-I and HDL are able to inhibit VLDL-lipid hydrolysis. The different HDL subspecies also uniquely affect the activity of the enzyme. A detailed evaluation of different HDL fractions shows that HDL2 stimulates HL-mediated hydrolysis of VLDL-TG, while the smaller HDL3 is inhibitory. Inhibition of VLDL hydrolysis appears to result from a decreased interlipoprotein shuttling of HL between VLDL and the smaller more dense HDL particles. These findings suggest that high HDL2 levels are positively related to efficient TG hydrolysis by their ability to enhance the liberation of HL into the plasma compartment and by a direct stimulation of VLDL-TG hydrolysis.
Taken together, these results demonstrate that the lipid and apolipoprotein composition of lipoproteins, hence lipoprotein structure, in addition to interlipoprotein interactions are central to the regulation of HL activity.
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Neuger, Lucyna. "Aspects on lipoprotein lipase and atherosclerosis." Doctoral thesis, Umeå : Univ, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-564.
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Svensson, B. Martin. "Lipase catalysis in lecithin-stabilised microemulsions." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406836.
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Rees, Gareth David. "Lipase catalysis in microemulsion-based systems." Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236927.
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Sani, Halimah Abdullah. "Mechanisms of control of lipoprotein lipase." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386912.
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Admans, Gary David. "Asymmetric transformations catalysed by lipase enzymes." Thesis, University of Exeter, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240288.
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Salgueiro, Alexandra Amorim. "Lipase production in Candida lipolytica 1055." Thesis, University of St Andrews, 1994. http://hdl.handle.net/10023/14328.
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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.
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Pastore, Glaucia Maria 1953. "Produção e caracterização bioquimica de monoacilglicerol lipase microbiana e aplicação de lipases na hidrolise e esterificação enzimatica." [s.n.], 1992. http://repositorio.unicamp.br/jspui/handle/REPOSIP/255846.
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Orientador: Yong Kun ParkTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Um mil duzentas e oitenta e cinco linhagens de microrganismos foram isoladas de amostras de solo, frutas, resíduos industriais e testados quanto a capacidade de hidrolisar glicerídeos. Oito linhagens de Fungos foram relacionadas preliminarmente como produtoras de enzimas lipolíticas. Entre estas, uma linhagem identificada como Trichoderma sp apresentou alta produção de lipase. A enzima lipolítica de Trichoderma sp hidrolizou as ligações éster de monoleína, dioleína e do substrato paranitrofenillaurato. A enzima pode ser classificada como uma lipase (glicerol-mono éster hidrolase E.C. 3.1.1.23). Estudou-se a produção de lipase extracelular pela linhagem selecionada de Trichoderma sp e a enzima foi purificada através de fracionamento com sulfato de amônio, cromatografia em coluna de DEAE-celulose e CM-celulose. A enzima purificada foi caracterizada e apresentou atividade ótima em pH 5.6 a 40 - 45°C. A atividade enzimática foi severamente afetada pela presença de MgSO4, MnSO4, FeSO4, na concentração de 1 mM de sal em relação ao volume final da mistura de reação, enquanto que KCl, CaCl2, NiSO4, inibiram moderamente a atividade de lipase.O reagente cisteína na concentração de 1 mM em relação ao volume final da mistura de reação inibiu significativamente a atividade enzimática. Verificou-se que a lipase de Trichoderma sp hidrolisa eficientemente monoleína e paranitrofenillaurato. A enzima não tem afinidade por trioleína. A hidrólise de óleo de oliva por via enzimática foi estudada utilizando-se lipases combinadas. Foi verificado que o sistema composto de lipase de Cândida rugosa e lipase de Penicillium sp hidrolisou 95,9% do óleo com o tempo de reação de 20 horas. A esterificação enzimática de ácido graxo e glicerol por lipase de Penicillium sp foi examinada e verificou-se que a enzima esterificou cerca de 71% de monoglicerídeo após 40 horas de reação
Abstract: The screening of lipase producing microorganisms was performed using 1,283 strains of microorganism which were isolated from samples of soil, fruits, and industrial residues. It was found that eight strains of fungi produced high activity of extracellular lipase and one of them produced exceptionally high lipase activity. This strains was identified as Trichederma sp, and he lipase from the strain hydrolised only monoolein, diolein and p-nitrephenyl laurate. For this reason, the lipase was classified as monoglyecride lipase (monoacylglycerol lipase E.C. 3.1.1.23). The lipase from Tric:hederma sp, was produced by solid state fermentation and submerse fermentation. The enzyme extracts were purified by fractionation with ammonium sulfate, DEAE-cellulose and CM-cellulose column chromatography. The purified enzyme was e:haracteri2ed and found that optimum pH and temperature were 5.6 and 40-45°C. MgSO4, MnSO4 and FeSO4, 1 mM, markably inhibited enzyme activity and KCl, CaCl2 and NiS04, 1 mM, slightly inhibited, Cysteine 1 mM significantly inhibited the enzyme activity. The enzyme also efficiently hydrolised monoolein and p-nitrophenyl laurate but not triolein. It was studied a combined enzyme system, which was a mixture of triacylglycerol lipase (from Candida ruqosa) and monoacylglycerol lipase (from Penicillium sp.), for hydrolyses of olive oil. It was found that the combined enzyme system was able to hydrolyse 95.9% the oil, when the reaction time was 20 hours. Enzymatic esterification using glycerol and fatty acid by lipase from Penicillium sp was also examined. It was found that the reaction system esterified 71% monoglyceride after 40 hours of reaction time
Doutorado
Doutor em Ciência de Alimentos
39
Burkert, Janaina Fernandes de Medeiros. "Otimização das condições de produção da lipase por Geotrichum candidum NRRL-Y552." [s.n.], 2003. http://repositorio.unicamp.br/jspui/handle/REPOSIP/255036.
Full textAbstract:
Orientador: Maria Isabel RodriguesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O interesse na produção de lipases microbianas tem aumentado significativamente nas últimas décadas, devido ao seu amplo potencial de aplicações industriais, seja na indústria de alimentos como aditivos (modificação de aromas), química fina (síntese de ésteres), detergentes (hidrólise de gorduras), tratamento de efluentes (decomposição e remoção de substâncias oleosas), couro (remoção de lipídios das peles dos animais), farmacêutica e na área médica (remédios, digestivos e enzimas para diagnósticos). A produção de lipase pode ser influenciada por diferentes variáveis, como o microrganismo produtor da enzima, as fontes de carbono, nitrogênio e lipídio, as condições de aeração e agitação, o tipo do impulsor, e até mesmo a geometria do reator. Na primeira etapa deste trabalho foram realizados testes com diferentes indutores (óleo de soja, óleo de milho, óleo de girassol, óleo de canola e óleo de oliva) para produção de lipase utilizando o microrganismo Geotrichum candidum NRRL- Y552, obtendo como melhor indutor o óleo de soja. Em seguida, um estudo para padronização do inóculo foi realizado possibilitando o início da otimização do meio de cultura, em frascos agitados, obtendo como meio otimizado 3,58% de peptona e 0,64% de óleo de soja, com pH inicial de 7,0 a 30°C e 250 rpm, alcançando 16 U/mL em 48 horas de fermentação. Utilizando as condições de produção otimizadas, a enzima foi caracterizada quanto a pH ótimo, temperatura ótima e de estabilidade, a influência de sais minerais na atividade enzimática e a determinação dos parâmetros cinéticos KM e Vmáx. Seqüencialmente, com o meio de cultura otimizado, foi verificada a influência dos impulsores turbina de Rushton, hélice naval e pás inclinadas na produção da lipase em fermentadores de bancada, atingindo em tomo de 25 g/L de biomassa para todos os impulsores e 19 U/mL em 30 horas, 12 U/roL em 30 horas e 22 U/mL em 54 horas, respectivamente, de atividade lipolítica. Utilizando o agitador tipo pás inclinadas foi investigado o efeito clã agitação e aeração no processo fermentativo, sendo obtida as condições de 300 rpm e 1 vvm a 30°C como ótimas para produção da lipase, alcançando aproximadamente 22 U/mL em 54 horas de processo. Ainda foi investigado o efeito de diferentes taxas de aeração em um reator não convencional "air lift" que permitiu obter cerca de 20 U/mL de atividade lipolítica em 30 horas de fermentação, possibilitando nesta geometria de reator uma produtividade 40% maior em relação aos reatores convencionais. Estes resultados para o processo de produção da lipase foram superiores em relação aos relatados na literatura para o mesmo microrganismo
Abstract: The interest in microbial lipase production increased significantly in the last decades, because of the large potential in industrials applications such as: additives in foods (flavor modification), fine chemicals (synthesis of esters), detergents (hydrolysis of fats), waste water treatment (decomposition and removal of oily substances), leather (removal of lipids of animal skins), pharmaceutical and medical areas (remedies, digestives and enzymes for diagnostics). The lipase production can be influenced by different variables such as the microorganism, the carbon sources, nitrogen and lipid, the aeration and agitation conditions, the impeller type, and also including the geometry of the reactor. In the first stage of this work tests was carried out with different inductors (soy oil, com oil, sunflower oil, canola oil and olive oil) for lipase production by Geotrichum candidum NRRL- Y552, getting as the best inductor the soy oil. After that, a study for standardization of inoculum was carried out, making possible the beginning of the optimization of the culture medium, in shaker-flasks, getting as half optimized 3.58% of peptona and 0.64% of soy oil, with initial pH of 7,0, 30°C and 250 rpm that conditions allow reaching 16 U/mL in 48 hours of fermentation. Using the optimized conditions of production, the enzyme was characterized concerning to optimum pH, optimum temperature and stability temperature, the influence of salts in the enzymatic activity and the determination the kinetic parameters KM and Vmáx, Sequentially, with the optimized medium culture, the influence of the impellers it was verified for Rushton turbine, helix naval and pitched blade up in the production of lipase in fermenter, reaching around 25 g/L of biomass for all impellers and 19 U/mL in 30 hours, 12 U/mlL in 30 hours and 22 U/mlL in 54 hours, respectively. Using the impeller type pitched blade up the effect of the agitation and aeration in the of lipase production was investigated, being the optimum conditions 300 rpm and 1 vvm at 30°C for enzyme production, reaching approximately 21 U/mL in 54 hours of fermentation. The effect of different aeration rates in a not conventional reactor air lift was also investigated, resulting in about 20 U/mL of lipolytic activity ín 30 hours of fermentation, making possible to obtain with this geometry of reactor a larger productivity (40% greater ín comparison to the conventional reactors). These results for the process of lipase production are larger than the reported ones in the literature for the same microorganism
Doutorado
Doutor em Engenharia de Alimentos
40
Silva, Érica Benjamim da 1990. "Isolamento e seleção de fungos silvestres com potencial para produção das enzimas lipase e tanase extracelulares." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256641.
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Orientador: Gabriela Alves MacedoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A bioprospecção de micro-organismos nativos capazes de produzir enzimas com diferentes propriedades catalíticas é essencial para o desenvolvimento do setor biotecnológico brasileiro. A lipase é uma enzima com vasta aplicação biotecnológica, utilizada na resolução de fármacos quirais, modificação de gorduras, produção de biocombustíveis, cosméticos, agroquímicos, oleoquímicos e realçadores de sabor. A tanase é outra enzima com grande potencial de aplicação industrial por ser capaz de catalisar a hidrólise de taninos hidrolisáveis e de ésteres de ácido gálico. Desse modo, o objetivo desse trabalho foi isolar novas linhagens de micro-organismos capazes de produzir as enzimas tanase e lipase extracelulares e caracterizar, bioquimicamente, as enzimas selecionadas. Foram isoladas 131 linhagens de fungos e 109 linhagens de leveduras da Região Amazônica e da região da Mata Atlântica. Tais linhagens fúngicas juntamente com outras linhagens da Coleção de Micro-organismos do Laboratório de Bioquímica FEA-UNICAMP (n=348) foram analisadas em meio sólido diferencial. Foram obtidas 26 linhagens positivas para lipase e 105 para tanase. As linhagens positivas foram testadas quanto à capacidade de produzir lipase e tanase por fermentação em estado sólido. A atividade enzimática da tanase foi quantificada por espectrofotometria e a da lipase por titulometria de neutralização. A lipase da linhagem de Colletotrichum sp. apresentou atividade enzimática específica igual a 25,97 U/mg; Km= 6,3 %; Vm= 19,5 U/mg; pH ótimo de atividade de 6,5 a 7,0 e temperatura ótima de atividade de 25 °C à 35 °C. A tanase da linhagem de Aspergillus niger apresentou atividade enzimática de 0,79 U/mL e a produzida pela linhagem de Paecilomyces sp. atingiu atividade específica de 2,14 U/mg, pH e temperatura ótima de atividade de 5,5 e 60°C, respectivamente
Abstract: The bioprospection of native microorganisms capable of producing enzymes with diverse catalytic properties is essential to the development of the Brazilian biotechnological sector. Lipase is an enzyme with wide biotechnological application, it is used on chiral drugs resolution, fat modification and on the production of biofuels, cosmetics and flavor enhancers. Tannase is another enzyme with large potential to be applied at industries, it is capable of catalyze the hydrolysis of hydrolysable tannins and galic acid esters. Therefore, this work goal is to isolate novel microorganisms strains capable of producing extracellular lipase and tannase, and to characterize biochemically the selected strains. 131 fungal strains and 109 yeast strains were isolated from Amazon and Atlantic Rainforest regions. These fungal strains summed with others strains from the Microorganisms Collection of the Biochemistry Laboratory/ FEA-UNICAMP (n=348) by differential solid media. It was found 26 lipase producing strains and 105 tannase producing strains. The positive strains were evaluated regarding their capacity to produce lipase and tannase by solid state fermentation. Tannase¿s enzymatic activity was measured by spectrophotometry and lipases¿s enzymatic activity by neutralization titration. Lipase from Colletotrichum sp. strain showed specific activity of 25.97 U/mg; Km= 6.3 %; Vmax= 19.5 U/mg; pH optimum between 6.5 to 7.0 and temperature optimum from 25 °C to 35 °C. Tannase from Aspergillus niger strain presented enzymatic activity of 0.79 U/mL and tannase from Paecilomyces sp. strain reached 2.14 U/mg, pH and temperature optimum of 5.5 and 60 °C, respectively
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos
41
Macedo, Gabriela Alves 1971. "Produção, purificação, caracterização bioquimica e aplicações de lipase de geotrichum sp." [s.n.], 1995. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256693.
Full textAbstract:
Orientador: Glaucia Maria PastoreDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Setecentas linhagens de leveduras foram isoladas de amostras de solo, frutas, água, resíduos industriais e testadas quanto à capacidade de hidrolisar glicerídeos através da produção de lipase extracelular. Destas linhagens, cinco foram pré selecionadas como produtoras de lipase. Foi encontrada uma linhagem identificada como Geotrichum sp que apresentou alta produção de lipase em meio de cultivo composto por 1.5 % de farinha de soja desengordurada, 1% de farinha de trigo, 3% de extrato de levedura, 0.2 % NH4NO3, quando incubada a 30 °C por 48 horas com agitação de 100 rpm. A lipase de Geotrichum sp hidrolisa preferencialmente ésteres de ácidos graxos de cadeia longa insaturada e não tem afinidade pelo substrato p-nitrofenil laurato. A lipase de Geotrichum sp foi purificada 16.5 vezes através de fracionamento com sulfato de amónio e Cromatografia em coluna de DEAE-Sephadex A-50. A enzima purificada foi caracterizada bioquimicamente verificando-se que possui duas subunidades de peso molecular estimado em 52000 e 57000, através de eletroforese vertical em gel SDS- poliacrilamida. A lipase de Geotrichum sp apresentou atividade ótima na faixa de pH 5 a 8 a 45°C. A atividade enzimática foi acrescida em 45% na presença de 1 mM MgS04 no sistema de reação. A enzima não sofreu ação inibitória na presença de p-cloromercuribenzoato quando pura. A esterificação enzimática de ácido graxo e glicerol pela lipase de Geotrichum sp foi examinada e verificou-se que a enzima catalisou a reação de esterificação de ácido oléico em glicerol, com incorporação de 63% do ácido oléico após 2 horas de reação a 40°C
Abstract: The screening of lipase producing yeasts was performed including 700 strains of microorganisms which were isolated from samples of soil, fruits, and industrial residues. It was found that five strains produced high activity of extra cellular lipase and one of them produced exceptionally higher lipase activity. This strain was identified as Geotrichum sp. The lipase from Geotrichum sp was produced by liquid fermentation in medium containing 1.5 % deflated soybean flour, 1% wheat flour, 3% yeast extract, 0.2 % NH4NO3, at 30°C for 2 days. The lipase from Geotrichum sp hydrolysed preferentialy esters of fatty acids with high insaturated chain and it did not hydrolysed the sinthetic substrate p-nitrophenil laurate. The lipase was purified by fractionation with ammonium sulfate and DEAE- Sephadex A-50 column cromatography.The purified lipase was characterized and was found that optimum pH and temperature were between 5-8, and 45°C, respectively. The enzyme activity increased by the presence of MgS04, 0.1mM and was not inhibited in the presence of enzyme nhibitors. Enzymatic esterification using glicerol and fatty acid by lipase from Geotrichum sp was also examined. It was found that reaction system esterified 63% triglyceride after 2 hours of reaction time at 40 °C. The enzyme was shown to be constituted by two subunits. The molecular weight of the subunits was estimate to be 52000 - 57000 daltons by SDS-poliacrylamide gel
Mestrado
Mestre em Ciência de Alimentos
42
Costa, Vinicius dos Santos Ribeiro da. "Produção, purificação e caracterização bioquimica de lipase de uma nova linhagem de Rhizopus Sp." [s.n.], 1997. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256700.
Full textAbstract:
Orientador: Glaucia Maria PastoreDissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos
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Resumo: Nas últimas décadas as Indústrias de Alimentos e Farmacêutica, têm demonstrado grande interesse na aplicação potencial de lipases de diferentes fontes como as de origem animal, as lipases de plantas e especialmente as lipases microbianas por sua capacidade de produção e especificidade. Deste modo, este trabalho teve como objetivo caracterizar bioquimicamente uma lipase fúngica de uma linhagem de Rhizopus ainda não relatada na literatura. A lipase de Rhizopus sp. linhagem n° 14, isolado no laboratório de Bioquímica de Alimentos do Departamento de Ciências de Alimentos (FEA/UNICAMP), foi produzida em meio semi-sólido constituído de farelo de trigo e água na proporção 60 / 40 (p/v). Esta linhagem foi escolhida por ter sido a que melhor produziu a enzima neste meio de fermentação. O extrato enzimático bruto ou lipase bruta foi extraído do meio após 120 horas de incubação a 30°C, por precipitação em sulfato de amônio. A atividade lipolítica foi testada contra os substratos gordura de côco, óleo de oliva, óleo de mamona, gordura do leite de cabra e o substrato sintético p-nitrofenil-laurato. A enzima mostrou maior atividade para hidrolisar gordura de côco, frente aos outros substratos. O extrato enzimático bruto foi parcialmente purificado em coluna de DEAE-Sephadex A-50, sendo obtidas duas frações, as quais foram submetidas a testes bioquímicos para a caracterização de suas propriedades em substrato sintético p-nitrofenil-laurato. As frações apresentaram um perfil de atividade em temperatura na faixa de 40 a 55°C e temperatura de estabilidade na faixa de 24 a 40°C, e, pH ótimo de 6,0 a 6,5 e mostraram-se estáveis na faixa de pH de 5,6 a 7,5, semelhante a enzima bruta.
Abstract: Interest on lipases from different sources (microorganisms, animals and plants), has markedly increased in the last decade due to the potential applications of lipases in Food and Pharmaceutical Industries. The objective of this work was to characterize the biochemical from the lipase of Rhizopus sp fungal. These lipase has not still reported. The lipase from Rhizopus sp,strain n.O 14, isolated in the Food Biochemistry Laboratory (DCA/ FEA/ UNICAMP),was produced in a solid state medium, wich consited of 60% wheat bran and 40% water. This strain was selected based on high activity of extracelular lipase in the broth. The cru de enzyme extract was obtained from the culture medium after 120 hours incubation at 30°C. The lipolytic activity was tested using as substrate cocconut fat, olive oil, castor oil, goat' s milk fat and p-nitrophenylaurate synthetic substrat. The enzyme demonstrated the highest activity on hydrolyzed coconut fat as compared to others substrates. The crude enzyme extract was purified on DEAE-Sephadex A-50 column. two fractions were obtained. These fractions were submitted to biochemical tests to characterize their properties on p-nitrophenylaurate synthetic substrate. The fractions presented activity in the range of 40°C to 55°C and thermostability in the range of 24 'DEGREE'C to 40 'DEGREE'C. Optimum pH of the enzymes was 6,5 - 7,5. It showed to be stable in pH's of 5,6- 7,5. These data were similar in the crude enzyme.
Mestrado
Mestre em Ciência da Nutrição
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Soberon, Lozano Maria Mercedes. "Estudo da propriedade de esterificação enantioseletiva da lipase de rhizopus sp." [s.n.], 2001. http://repositorio.unicamp.br/jspui/handle/REPOSIP/256681.
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Orientador: Glaucia M. PartoreTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A propriedade enantioseletiva de uma lipase parcialmente purificada isoladade Rhizopussp foi estudadaatravésde umsistemade esterificaçãodo ácido láurico e o álcool quiral 2-octanol. Os experimentos foram realizados nas seguintes condições: proporção de substratos 1:1, 10 unidades hidrolíticas de lipase, presença de peneira molecular na reação, presença e ausência de hexano, e nas temperaturasde 45°C e 50°C respectivamente. Diferentes métodos foram utilizados para medir a enantioseletividade da lipase: reações usando o álcool 2-octanol na suas formas ( R ) e ( S ) puras, assim como a forma racêmica do álcool. Os experimentos foram analisados através de cromatografia gasosa convencional e cromatog rafia quiral. A enantioseletividadeda enzima foi estudada considerando os parâmetros de conversão de álcool ( 'dzeta' ), excesso enantiomérico do substrato residual ( ees ), excesso enantiomérico do produto ( eep ) e os valores de enantioseletividade (E ) obtidos com (ees) e (eep). Os resultadosmostraramque a enzima em estudo catalisou com maior eficiência a forma ( R )-enantiômero do 2-octanol do que a sua contraparte.Esta catálise enantioseletivada enzima aumentou com a presençade hexano, no sistemareacional. Os valores de ( 'dzeta' ), ( ees), ( eep), e ( E ) foram dependentesda temperaturana ausência de hexano no meio de reação. As constantescinéticas, constante de Michaelis-Menten ( Km), e velocidade máxima (Vmax), foram calculadaspara cada enantiômero observando-se diferenças significativas nos valores de Vmax.Os resultados obtidos com diferentes ácidos graxos mostraram uma melhor afinidade da enzima por o ácido octan6ico seguido do ácido láurico, indicando que o comprimento da cadeia alifática do ácido graxo é um fator importante na avaliação da esterificação enantioseletiva da lipase de Rhizopus sp.
Abstract: The enantioselective property of partially purified lipase from Rhizopus sp, was tested by esterification of lauric acid with 2-octanol alcohol media. The experiments were performed in equal molar proportion of substrates, 10 hydrolytic activity units of lipase both, in organic solvent and organic solvent free media at 45°C and 50°C of temperature.The presence of molecular sieves in the reaction was important for the synthesis of ester by lipase. Different methods of determining the enantios electivity was used: reaction using single enantiomers as well as racemic mixture. Experiments were analyzed by capillary GC with conventional and chiral columns.The enantioselectivity of the enzyme was studied measuring degree conversion ('dzeta'), substrate enantiomeric excess (ees), product enantiomeric excess (eep)and enantiomeric ratio (E). The results showed that (R)-2-octanol was better substrate than (S)-2-octanol for the enzyme. This enantio specificity preference of Rhizopus sp lipase toward (R)-2-octanol increasedwith the presenceof hexane in the reaction.'dzeta', ees, eep and E values were dependent on temperature, and presence of solvent in reaction media. Km and Vmax values were calculated for each enantiomer observing that highest enantioselectivity was reflected in the largest variance of Vmax values. Results obtained with different fatty acids showed that long chain acyl fatty acids were better substrates (e.g: octanoic, lauric, myristic acids). So, the size of fatty acid is an important factor to be considered in the study of enantio selective esterification of Rhizopus sp lipase.
Doutorado
Doutor em Ciência de Alimentos
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Tremblay, Eric. "Ontogénèse et mécanismes de régulation de la synthèse de la lipase dans l'estomac humain." Sherbrooke : Université de Sherbrooke, 1998.
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Perignon, Marlène. "Compréhension et maîtrise de l'interestérification enzymatique d'huiles végétales sur les plans nutritionnel et technologique." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20136.
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La nutrition constitue l'un des facteurs déterminants du développement ou de la prévention de diverses pathologies. Parmi les trois classes de nutriments majeurs, les lipides alimentaires occupent une place essentielle dans cette relation nutrition-santé puisque leur déséquilibre qualitatif est impliqué dans l'incidence des maladies cardiovasculaires. L'importance de l'équilibre alimentaire justifie le développement de produits fonctionnels au profil nutritionnel amélioré. En parallèle, suite à la prise de conscience de l'impact environnemental de la production alimentaire, il est aujourd'hui important d'inscrire le choix des matières premières et des procédés dans une démarche de développement durable.Dans ce contexte, les travaux menés durant cette thèse concernent tout d'abord l'amélioration des propriétés nutritionnelles d'une matière grasse végétale tartinable par réduction de sa teneur en acides gras (AG) saturés délétères, mais également la limitation de son impact environnemental par réduction des ingrédients issus de la filière palme. La mise au point d'un procédé d'interestérification enzymatique vise à ajuster les propriétés rhéologiques du produit sans modifier le profil global en AG, et sans utiliser de solvants ou produits chimiques grâce à l'action d'un biocatalyseur (lipase). Ces travaux ont conduit au développement jusqu'au stade pilote d'un substitut interestérifié par voie enzymatique permettant de réduire la teneur en AG saturés délétères de 65% et l'utilisation de dérivés d'huile de palme de 70% tout en maintenant une fonctionnalité similaire au produit actuel. En parallèle, un procédé enzymatique à également été exploité dans l'étude d'une nouvelle méthode d'analyse de la régiodistribution : l'évaluation des sélectivités de la lipase de Rhizopus oryzae a montré son potentiel pour l'analyse de la position sn-2 des triacylglycérols contenant des AG à chaines moyennesNutrition is one of the main factors contributing to various diseases occurrence or prevention. Among the three major types of nutrients, dietary lipids are essential in this nutrition-health relationship since lipid disorder is associated with an increased risk of cardiovascular diseases. Thus, the importance of a healthy diet explains the development of functional foods with improved nutritional properties. Furthermore, with environmental impact of food production being a growing concern for consumers, selection of raw materials and processes should meet the requirements for sustainable development. In this context, this work concerns the improvement of the nutritional properties of a vegetable oil spread by reducing its unhealthy fatty acids (FA) content, but also the limitation of its environmental impact by minimizing the use of palm oil products. An enzymatic interesterification process has been developed to adapt rheological properties without modifying FA profile nor using solvents and chemical products by action of a biocatalyst (lipase). This work led to the pilot-scale development of an enzymatically interesterified substitute allowing to reduce the unhealthy FA content by 65%, and the use of palm oil products by 70% while keeping a functionality similar to the current product.At the same time, an enzymatic approach has also been used in the investigation of a new method for regiodistribution analysis. Thus, Rhizopus oryzae lipase appeared to be a good candidate for the sn-2 position analysis of triacylglycerols containing medium chain fatty acids
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Mas, Eric. "Glycosylation de la lipase sels biliaires dépendante du pancréas : Glycosylation de la lipase sels biliaires dépendante du pancréas." Aix-Marseille 2, 1995. http://www.theses.fr/1995AIX22028.
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La lipase sels biliaires-dependante (bsdl) est une enzyme lypolitique de la secretion pancreatique. Dans un premier temps, nous avons montre que la n-glycosylation de la bsdl, issue d'un sujet sain, est de type biantennaire complexe et elle presente 12 a 14 structures o-glycosylees de type (gal betal-3 galnac)-ser/thr plus ou moins sialylees et/ou fucosylees. La n-glycosylation est aussi essentielle a l'expression d'une bsdl totalement active et a sa secretion. Cependant, les inhibiteurs de la maturation de la n-glycosylation n'influent ni sur l'activite ni sur la secretion de l'enzyme. La structure oligosaccharidique n-liee participerait donc a l'etablissement de la conformation correcte du site actif de la bsdl. Un glycovariant oncofoetal de la bsdl, la proteine foeto-acinaire pancreatique (fap) identifiee par l'epitope oncofoetal j28 a ete caracterise. Leur principale difference reside dans leurs structures glycosidiques: la fap montre une n-glycosylation immature de type oligomannosidique avec une diminution des motifs oligo-saccharidiques o-lies. La caracterisation de la structure de l'epitope j28 montre l'implication de residus de fucoses portes par la o-glycosylation. Nous avons aussi montre que, lors des pancreatites chroniques, seule la bsdl montre une alteration de la n-glycosylation qui reste immature. Enfin, nous avons mis au point un dosage elisa de la bsdl presente dans le serum. Ce dosage, couple a celui de l'antigene ca 19/9, permet de diagnostiquer les cancers pancreatiques et de les distinguer des pathologies pancreatiques
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Herd, Sara L. "Exercise, postprandial lipaemia and lipoprotein lipase activity." Thesis, Loughborough University, 1997. https://dspace.lboro.ac.uk/2134/28431.
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Impaired clearance of triacylglycerol-rich lipoproteins contributes to atherogenesis. It can be argued that exercise may decrease the risk of atherosclerotic diseases through its potential to improve the metabolic capacity for triacylgycerol and hence, clearance of triacylglycerol-rich lipoproteins. The investigations described in this thesis focused on the influence of exercise on postprandial lipid and lipoprotein metabolism.
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Morton, Jessica. "Characterization of a lipase in Arabidopsis defense." Thesis, Manhattan, Kan. : Kansas State University, 2007. http://hdl.handle.net/2097/392.
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Magnusson, Anders. "Rational redesign of Candida antarctica lipase B." Doctoral thesis, Stockholm, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-186.
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Moss, S. J. "The adsorption of lipase onto prepared surfaces." Thesis, Swansea University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638265.
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The separation of a commercially obtained lipase and a bacterially obtained lipase were investigated by examining their ability to adsorb to, and be desorbed from, lipid/non-lipid particles and silanised 'Ballotini' glass particles. Lipase P from Pseudomonas fluorescens SAM-2, which was later reclassified by the manufacturer as lipase PS from Pseudomonas cepacia, was used as a commercially purified lipase for initial adsorption and desorption experiments. Particles of coconut oil, paraffin wax and paraffin oil were produced by emulsification and lipase P was adsorbed and desorbed from them. The emulsified particles were considered unsatisfactory for further experimentation owing to an inability to calculate the specific surface area per unit weight of particles. 'Ballotini' particles with a mean diameter of 213 μm and a specific surface area of 9.53 ± 0.22 m2/kg were shown to be made to behave in the same manner as fat and oil emulsions. The adsorption onto the 'Ballotini' followed a Langmuir isotherm with p to 4,900 U/m2 of lipase PS at 65% adsorption, and 12,900 U/m2 at 25% adsorption. The adsorption was not highly pH dependent since there was good adsorption from pH 4 to pH 9. A wide range of potential desorption agents were tested and the non-ionic surfactants tween 20 and triton X-100 proved to be the best with up to 85% desorption of lipase. Lipase was produced from Pseudomonas aeruginosa L130 (NCIMB 12718) in nutrient broth and also in defined media. The lipase production was induced by glycerol, even in the absence of lipids giving a maximum of 5.9 ± 0.2 U/mL in nutrient broth, but only 0.3 ± 0.1 U/mL in the defined media. The adsorption and desorption of the bacterial lipase from silanised glass particles was very poor with only 536 U/m2 of lipase at 23% adsorption being achieved. The poor separation of the bacterial lipase was believed to result from microbially produced components that inhibited adsorption.