Academic literature on the topic 'Enzymatic acylation'

Covalent attachment of a variety of lipid groups to proteins is now recognized as a major group of post-translational modifications. S-acylation of proteins at cysteine residues is the only modification considered dynamic and thus has the potential for regulating protein function and/or localization. The activities that catalyse reversible S-acylation have not been well characterized and it is not clear whether both the acylation and the deacylation steps are regulated, since in principle it would be sufficient to control only one of them. Both apparently enzymatic and non-enzymatic S-acylation of proteins have previously been reported. Here we show that a synthetic myristoylated c-Yes protein tyrosine kinase undecapeptide undergoes spontaneous S-acylation in vitro when using a long chain acyl-CoA as acyl donor in the absence of any protein. The S-acylation was dependent on myristoylation of the substrate, the length of the incubation period, temperature and substrate concentration. When COS cell fractions were added to the S-acylation reaction no additional peptide:S-acyltransferase activity was detected. These results are consistent with the possibility that membrane-associated proteins may undergo S-acylation in vivo by non-enzymatic transfer of acyl groups from acyl-CoA. In this case, the S-acylation-deacylation process could be controlled by a regulated depalmitoylation mechanism.

We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.

Ce travail a consisté, dans un premier temps, à extraire et à identifier les flavonoïdes majeurs contenus dans les feuilles de Nitraria retusa et à évaluer leurs activités biologiques. Quatre flavonoïdes ont été identifiés dans les extraits et les fractions obtenus : l’isorhamnétine, l’isorhamnétine-3-O-glucoside et les deux isomères isorhamnétine-3-O-rutinoside et isorhamnétine-3-O-robinobioside. L’étude des activités biologiques des extraits et des fractions de N. retusa a permis d’établir une relation linéaire entre leur teneur en flavonoïdes et leurs activités antioxydantes et antiprolifératives, les milieux les plus riches présentant les activités les plus importantes. Ces activités dépendent également de la nature des flavonoïdes présents ; ainsi, la très forte activité d’inhibition de la xanthine oxydase relevée pour la fraction au chloroforme et sa grande capacité à piéger le radical DPPH ont été attribuées à sa teneur élevée en isorhamnétine, flavonoïde aglycone présentant une grande analogie structurale avec la quercétine, molécule bien connue pour ses activités antioxydantes. Dans un deuxième temps, l’acylation enzymatique de l’isoquercitrine, flavonoïde modèle, et de l’isorhamnétine-3-O-glucoside a été étudiée pour tenter d’améliorer leurs propriétés. L’acylation enzymatique de l’isoquercitrine par des esters éthyliques d’acides gras de différentes longueurs de chaîne, catalysée par la lipase B de Candida antarctica, a montré que les performances de la réaction sont inversement proportionnelles à la longueur de la chaîne du donneur d’acyle. Des résultats similaires ont été obtenus lors de l’acylation de l’isorhamnétine-3-O-glucoside. Les activités des esters d’isoquercitrine et d’isorhamnétine-3-O-glucoside ont été évaluées et comparées à celles des flavonoïdes non acylés. Les esters ont montré des activités antiprolifératives vis-à-vis de cellules Caco2 et d’inhibition de la xanthine oxydase plus importantes que celles des molécules d’origine. Finalement, ce travail a permis d’apporter des éléments de compréhension de la relation structure-activité de flavonoïdes et de leurs dérivés acylés
The present work firstly consisted in studying the extraction and the identification of major flavonoids contained in Nitraria retusa leaves and evaluating their biological activities. Four flavonoids were identified in extracts and fractions: isorhamnetin, isorhamnetin-3-O-glucoside and the two isomers isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-robinobioside. The evaluation of the biological activities of extracts and fractions of N. retusa allowed to establish a linear relationship between their antioxidant and antiproliferative activities and their total flavonoids content, the most enriched exhibiting the highest activities. The nature of the flavonoids present in the extracts and fractions was shown to be important too. Thus, the strong xanthine oxidase inhibition activity and the high DPPH radical scavenging capacity observed for the chloroform fraction can be attributed to its high content in the aglycone flavonoid isorhamnetin, a structural analogue of quercetin which is well known for its antioxidant activities. In a second part, the enzymatic acylation of isoquercitrin as a model compound and isorhamnetin-3-O-glucoside was studied in order to improve their properties. The enzymatic acylation of isoquercitrin by fatty acid ethyl esters of different chain lengths, catalyzed by the lipase B of Candida antarctica, showed that the performance of the reaction is inversely proportional to the acyl donor chain length. Similar results were obtained when acylating the isorhamnetin-3-O-glucoside. The activities of isoquercitrin and isorhamnetin-3-O-glucoside esters were determined and compared to that of initial flavonoids. Esters exhibited higher antiproliferative towards Caco2 cells and xanthine oxidase inhibition activities than original compounds. Finally, this work led to a better understanding of the structure-activity relationship of flavonoids and their acylated derivatives

Approche multi-échelle pour l’étude de la réaction de N-acylation enzymatique d’acides aminés La réaction de N-acylation d’acides aminés ou de peptides permet l’obtention de dérivés de ces molécules présentant des propriétés bioactives et/ou techno-fonctionnelles, avec une biodisponibilité, une hydrophobie et une stabilité accrue. Les acides aminés acylés ont été largement décrits comme constituant une classe d'agents tensioactifs avec d'excellentes propriétés de surface, des activités biologiques intéressantes, un faible potentiel de toxicité et un faible impact environnemental. Actuellement réalisée de manière chimique à l’échelle industrielle, l’acylation de ces acides aminés ou peptides présente des contraintes en termes de sélectivité réactionnelle et d’innocuité vis-à-vis de l’environnement ainsi qu’en termes de coût de retraitement des effluents polluants. Une alternative à cette voie chimique est l’utilisation d’enzymes capables de catalyser ces réactions d’acylation. Dans la littérature, différents couples d’enzymes et de solvants ont déjà été décrits. Néanmoins, les performances réactionnelles de ces systèmes demeurent parfois limitées. L’objectif de cette thèse a donc été l’amélioration du procédé d’acylation par une approche à différentes échelles. À l’échelle moléculaire, une étude a été réalisée avec la lipase B de Candida antarctica (CALB). Une approche de modélisation moléculaire a été utilisée afin de mettre au point une méthodologie associant des simulations de docking et des calculs d’interaction permettant d’améliorer la compréhension et permettre la prédiction de la régiosélectivité de CALB lors de l’acylation de la lysine par différents acides gras. Des études ont également été conduites à l’échelle réactionnelle, notamment avec la recherche de nouveaux biocatalyseurs de type aminoacylases dans l’extrait brut de Streptomyces ambofaciens. La régiosélectivité et les performances de la réaction catalysée par ces enzymes ont été comparés à celles de CALB. Les résultats ont mis en évidence un potentiel très prometteur des aminoacylases de S. ambofaciens concernant la synthèse d’acide aminés/peptides acylés. En effet, en plus de leur aptitude à réaliser la réaction d’acylation en milieu aqueux, ces enzymes possèdent une régio-sélectivité qui diffère de celle de CALB. Cette régio-sélectivité orientée vers les groupements N-terminaux est un atout très peu décrit à ce jour, car elle permet d’acyler ces molécules sans modifier les chaînes latérales des acides aminés ou des peptides et donc leurs fonctionnalités. Dans la dernière partie de ces travaux, des études à l’échelle procédé ont été menées. Tout d’abord, l’immobilisation des aminoacylases sur des matériaux mésoporeux silicatés a été réalisée et différentes méthodes d’immobilisation ont pu être comparées. Cette étude a permis de proposer une méthode d’immobilisation des aminoacylases de S. ambofaciens par physisorption, permettant de conserver l’activité spécifique pendant au moins 3 cycles. Puis, dans une dernière partie, l’intensification de la réaction d’acylation en réacteur micro-ondes ou microstructurés a été abordée. Les expérimentations réalisées dans un réacteur chauffé par irradiation micro-onde ont montré que ce type de réacteur était adapté à la réaction d’acylation catalysé par CALB sous sa forme immobilisée commerciale (Novozym435®) en solvant organique, ce qui n’est pas le cas avec des aminoacylases de S. ambofaciens libres, en milieux aqueux. Pour cette réaction, d’autres méthodes d’intensification ont été envisagées, notamment en réacteur microstructuré de type microfluidique. L’efficacité du mélange étant primordiale notamment en milieu biphasique, celle-ci a pu être améliorée avec un taux de conversion supérieur dans ce réacteur comparativement à un réacteur classique agité mécaniquement
N-acylation of amino acids or peptides results in bioactive and/or functional molecules showing increased bioavailability, hydrophobicity and stability. Acylated amino acids have been broadly described as being a kind of surfactant with great surface chemistry properties, interesting biological activities, weak toxicity and low environmental impact. Acylation of amino acids or peptides is being performed chemically at industrial scale. It creates constraints in term of reaction selectivity, environmental safety and cost of polluted wastewater treatment. Enzymatic catalysis is an alternative to chemical acylation reaction. Several enzyme/solvent pairs have already been described in the literature. Their performance are however somewhat limited. The objective of this thesis work was thus to improve the capacity of acylation processes at different scales. At the molecular scale, a study was performed using Candida antarctica’s (CALB) lipase B. Molecular modeling was used to create a methodology coupling docking simulation and interaction calculus that would allow for a better understanding of CALB regioselectivity during lysine acylation by different fatty acids. Studies were also conducted at the reaction level, especially by searching for new aminoacylase-type of biocatalysts in Streptomyces ambofaciens raw extract. Regioselectivity and performance of these enzyme’s catalytic reactions were compared to those of CALB. Results brought into light a promising potential from S. ambofaciens’ aminoacylases in synthesizing acylated amino acids/peptides. Indeed, on top of their ability to catalyse acylation reaction in aqueous solution, these enzymes have a different regioselectivity compared to CALB’s. Regioselectivity targeting N-terminal groups is a rarely researched phenomenon allowing acylation to be performed without modifying amino acids or peptides lateral chains and hence their functionality. In the last part part of this work, studies at process scale were performed. Aminoacylase were first immobilized on mesoporous silicates and several immobilisation methods were compared. Using physisorption, a method for the immobilisation of S. ambofaciens’ aminoacylases was developed to reach a conserved specific activity during 3 cycles. Finally, intensification of acylation reaction was examined in microwave or microstructured reactors. First, an experimental set up was performed in an heated reactor using microwaves irradiation. This kind of reactor was demonstrated as being adapted to acylation reaction using a commercial immobilized form of CALB (Novozym435®) as catalyst in organic solvent. The microwave reactor was however not suited for free S. ambofaciens aminoacylase in aqueous solution. For that latter reaction, intensification had to be approached through other aspects of the process. Hydrodynamic appeared indeed as an important aspect for this reaction occurring in a biphasic medium composed of fatty acids and aqueous solution. A microstructured microfluidic reactor was hence tested. Conversion yield were increased with this system. This study demonstrated how mixing quality was an important factor for acylation reaction and could be a way to intensify the enzymatic process at larger scale

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

Croton é o segundo maior gênero de Euphorbiaceae, com aproximadamente 1.300 espécies, sendo 300 delas existentes no Brasil em diversos biomas. Várias espécies apresentam um característico látex vermelho-sangue, chamado \"sangue-de-dragão\", sendo usadas mundialmente na medicina tradicional. Estudos químicos indicam a presença de múltiplas classes de compostos, sendo as principais: diterpenos (clerodanos, labdanos, kauranos e traquilobanos), óleos voláteis, esteroides e triterpenoides, alcaloides, proantocianidinas e flavonoides. Estes últimos são metabólitos secundários com grande variedade estrutural, possuindo atividades biológicas reconhecidas e de potencial interesse medicinal. Croton floribundus Spreng. e Croton urucurana Baill., por apresentarem várias atividades biológicas de interesse medicinal, são utilizadas na medicina tradicional. Entretanto, poucos estudos têm sido desenvolvidos no sentido de se conhecer melhor aquela classe de substâncias fenólicas. Um dos objetivos deste trabalho corresponde ao isolamento e identificação de flavonoides de folhas de Croton floribundus e Croton urucurana. O material pulverizado foi extraído por refluxo com metanol 80% e concentrado. O extrato seco foi tratado com tolueno e diclorometano. A fase metanólica resultante foi fracionada e subfracionada por meio de cromatografia em coluna de PVPP e Sephadex LH-20, e cromatografia em papel. Depois de fracionadas, as amostras foram analisadas por cromatografia líquida de alta eficiência (CLAE) e os compostos identificados por ressonância magnética nuclear de hidrogênio (1H) e carbono (13C) ou por CLAE com co-injeção de amostras autênticas. Em ambas as espécies foi identificado o flavonoide acilado tilirosídeo (5). Este parece ser um flavonoide característico do gênero. É um derivado acilado de kaempferol, isolado de um grande número de espécies de Croton. Em C.floribundus foram identificados três triglicosídeos: alcesefolisídeo (1), mauritianina (2), e isoramnetina-3-O-(2,6-di-ramnosil)-galactosídeo (3). Foram identificados também: quercetina-3-O-glucosídeo (4), helicrisosídeo-3\'-metil-éter (6), kaempferol (7), isoramnetina (8) 3-O-metil-kaempferol (9) e 3-O-metil isoramnetina (10). Em C.urucurana, foram identificados: orientina (11), rutina (12), vitexina (13), quercetina-7-O-ramnosídeo (14), ramnetina-3-O-ramnosídeo (15) e quercetina (16). Os flavonoides 1, 3, 14 mostraram-se inéditos, tanto para o gênero, quanto para a família. O flavonoide 15 foi inédito para o gênero, tendo sido encontrado na família, porém, na subfamília Euphorbioideae. Com a detecção do tilirosídeo, foi verificada a possibilidade de se utilizar os extratos proteicos de folhas jovens dessas duas espécies para acilar enzimaticamente flavonoides de interesse, pois estudos na literatura indicam que a acilação pode aumentar a estabilidade e biodisponibilidade de flavonoides, e também melhorar suas atividades biológicas. Para isso, folhas jovens foram coletadas e mantidas em N2 líquido, trituradas e extraídas com tampão de extração. O extrato obtido foi concentrado, sua concentração proteica foi determinada e, posteriormente, foi utilizado para acilar os substratos quercetina-3-O-glucosídeo, kaempferol-3-O-glucosídeo, quercetina-3-O-galactosídeo e quercetagetina-7-O-glucosídeo. p-Cumaroil-CoA e uma mistura de ácido p-cumárico e Coenzima A, foram utilizados como agentes acilantes. Foi verificado que o rendimento do extrato proteico de C.floribundus foi menor que o de C.urucurana. Enquanto o extrato de C.urucurana acilou os substratos glicosilados na posição 3, (kaemferol-3-O-glucosídeo, quercetina-3-O-glucosídeo e quercetina-3-Ogalactosídeo), não acilou o mono-glicosilado na posição 7 (quercetagetina-7-Oglucosídeo), indicando que as aciltransferases dessa espécie são regiosseletivas quanto à posição do resíduo de açúcar. Também demonstrou capacidade de acilação usando como agentes acilantes p-cumaroil-CoA e a combinação ácido p-cumárico + CoA + ATP. O extrato proteico de C.floribundus, talvez por ter apresentado um menor rendimento de extração, acilou apenas o kaempferol-3-O-glucosídeo, resultando no tilirosídeo, o flavonoide acilado característico do gênero
Croton is the second largest genus of Euphorbiaceae, with approximately 1,300 species, 300 among them native from Brazilian biomes. Several species of the genus, used worldly in traditional medicine, possess a characteristic blood colored latex, called \"dragon\'s blood\". Chemical studies about Croton species have uncovered multiple classes of secondary metabolites, such as diterpenes (clerodanes, labdanes, kauranes and trachylobanes), volatile oils, steroids and triterpenoids, alkaloids, proanthocyanidins and flavonoids. The latter are secondary metabolites with high structural diversity and recognized as having biological activities with medicinal potential. Croton floribundus Spreng. and C. urucurana Baill. have shown several medicinally promising biological activities and are used in traditional medicine. However, few investigations have been performed aiming the flavonoid chemistry of any of the two species. One of the objectives of the present study is the isolation and identification of flavonoids from leaves of C. floribundus and C. urucurana. Powdered material from both species was extracted by reflux with 80% methanol. The dry extracts were treated with toluene and dichloromethane, lyophilized and solubilized in methanol. The methanol solution was analyzed by polyvinylpolypyrrolidone column chromatography (PVPP-CC). The fractions obtained were further analyzed by PVPP-CC, Sephadex LH-20 column chromatography and paper chromatography. The fractions and isolated compounds obtained were analyzed by HPLC. Isolated compounds were identified by 1H and 13C NMR and HPLC co-chromatography with authentic samples. The acylated flavonol tiliroside (5) was obtained from extracts of both species. It seems to be a characteristic marker of the genus, having been reported for a high number of Croton species. From the leaf extract of C. floribundus three triglycosides were obtained in the present work: alcesefoliside (1), mauritianin (2) and isorhamnetin-3-O-(2,6-dirhamnosyl)-galactoside (3). Other glycosides identified were quercetin-3-O-glucoside (4), 3\'-helichrysoside-3-O-methyl ether (6), kaempferol (7), isorhamnetin (8), 3-O-methyl-kaempferol (9) and 3-O-methyl-isorhamnetin (10). The following compounds were obtained from C. urucurana: orientin (11), rutin (12), vitexin (13), quercetin-7-O-rhamnoside (14), rhamnetin-3-O-rhamnoside (15) and quercetin (16). Flavonoids 1, 3 and 14 are new regarding genus Croton and family Euphorbiaceae. Flavonoid 15 was previously found in subfamily Euphorbioideae and is now reported for the first time in Croton. Taking into account the detection of tiliroside in the material analyzed, and that acylation increases both stability and bioavailability of flavonoids, while enhancing their biological activity, an approach was planned to use protein extracts of young leaves of both species aiming the enzymatic acylation of several flavonoids. Young leaves were maintained in liquid N2, ground and treated with extraction buffer. The extract obtained was concentrated and mixed with p-coumaroyl-CoA and a mix of p-coumaric acid and Coenzyme A. The extract was used in assays aiming the acylation of quercetin-3-O-glucoside, kaempferol-3-O-glucoside, quercetin-3-O-galactoside and quercetagetin-7- O-glucoside. The concentration of the protein extract from C. floribundus was lower than that of C. urucurana. The extract from C. urucurana acylated the 3-O-glycosilated substrata kaempferol-glucoside, quercetin-glucoside and quercetin-galactoside, but was ineffective toward quercetagetin-7-O-glucoside. These results suggest that acyltransferases in the extract are regioselective about the position of attachment of the sugar moiety. They were shown to be effective using either p-coumaroyl or the combination p-coumaroyl-CoA + ATP. C. floribundus protein extract acylated only kaempferol-3-O-glucoside, yielding tiliroside, the characteristic acylated flavonoid of Croton

Les protéines extraites des végétaux sont des matériaux relativement peu coûteux, non toxiques, biocompatibles et biodégradables. Elles représentent une bonne alternative aux protéines d’origine animale et aux polymères dérivés du pétrole. Dans le cadre de cette étude, les protéines extraites de graines de soja et de tournesol ont été utilisées en tant que matériaux enrobants pour la microencapsulation de la matière active hydrophobe (α-tocophérol) ou hydrophile (acide ascorbique) par le procédé d’atomisation. Les protéines de soja sont largement utilisées dans les applications alimentaires et non-alimentaires, notamment en microencapsulation. Elles sont donc étudiées dans ce travail comme matériau enrobant de référence. Les protéines de tournesol n’ont quant à elles pas d’application industrielle concrète, si ce n’est sous la forme de tourteaux dans l’alimentation animale. C’est pourquoi il nous semble pertinent de trouver des nouvelles voies de valorisation pour ce coproduit d’origine agricole. Plusieurs modifications des protéines, telles que l’hydrolyse enzymatique, l’acylation, la réticulation enzymatique et la cationisation ont été étudiées dans le but d’améliorer les propriétés encapsulantes du matériau enrobant. Dans le contexte de la chimie verte, toutes les modifications ont été effectuées sans utilisation de solvants organiques ni de catalyseurs chimiques. L’influence des modifications chimiques et enzymatiques des protéines, et des paramètres du procédé (pression d’homogénéisation, ratio matériau enrobant/matière active et concentration en protéines) sur les différentes caractéristiques des préparations liquides et des microparticules (viscosité, taille des gouttelettes dans le cas des émulsions, morphologie et taille des microparticules), ainsi que sur les paramètres liés au procédé d’atomisation (rendement et efficacité de microencapsulation) a été particulièrement étudiée au cours de ce travail. Les résultats obtenus confirment que l’extrait protéique de tournesol est tout à fait pertinent comme matériau enrobant et permet d’obtenir des efficacités de microencapsulation significativement plus élevées par rapport à celles obtenues avec l’extrait protéique de soja
Proteins extracted from vegetables are relatively low-cost, non-toxic, biocompatible and biodegradable raw materials. They represent a good alternative to animal-based proteins and petroleum-extracted polymers. In this study, proteins derived from soybean and sunflower seeds were used as wall materials for microencapsulation of hydrophobic (-tocopherol) or hydrophilic (ascorbic acid) active material by spray-drying technique. Soybean proteins are widely used in food and non-food applications, especially in microencapsulation. They were studied in this work as wall material of reference. Sunflower proteins are not actually used in industrial application, but only in the form of oil-cake for animal feeding. That’s why new ways of valorization of this agricultural by-product should be investigated. Several proteins’ modifications such as enzymatic hydrolysis, acylation, cross-linking and cationization were studied in order to improve encapsulating properties of wall material. In the context of green chemistry, all the modifications and preparations were performed without use of organic solvents and chemical catalysts. The effect of protein chemical and enzymatic modifications, and process parameters (homogenization pressure, wall/core ratio and protein concentration) on different characteristics of liquid preparations and microparticles (viscosity, emulsion droplet size, microparticle size and morphology) and on parameters related to the spray-drying process (yield and efficiency of microencapsulation) was particularly investigated in this study. The obtained results confirmed that sunflower proteins are quite suitable as encapsulating agent and provide the microencapsulation efficiencies significantly higher compared to those obtained with soy proteins

Ce travail a eu pour objectif de déterminer les conditions optimales de production d’un caroténoïde original, la torularhodine, par Sporobolomyces ruberrimus, cultivée en réacteur discontinu. Cette souche est capable d’utiliser le glycérol technique comme source de carbone et d’énergie pour sa croissance et pour la production de caroténoïdes. D’abord, il s’est agi d’identifier les facteurs opératoires majeurs qui sont susceptibles d’avoir une influence sur la production de la torularhodine, au travers d’une étude préliminaire. L’identification expérimentale de ces facteurs d’action - la température, le taux d’oxygène dissous et la supplémentation en acide oléique - a été validée statistiquement, à des degrés divers, avant d’engager une étape d’optimisation par la construction d’un plan d’expériences multicritère. Celui-ci a conduit à l’établissement de modèles polynômiaux du second degré pour représenter l’effet conjugué des facteurs retenus et permettre la prédiction des valeurs de µmax et de concentration de torularhodine rapportée à la biomasse. Cette étude a alors été consacrée à un essai de fonctionnalisation de la torularhodine, à partir de sa fonction carboxylique, en vue de la stabilisation de la molécule dont l’activité antioxydante est élevée. L’acylation enzymatique de la lysine par la torularhodine a été envisagée. Les conditions d’acylation par la lipase B de C. antarctica ont été déterminées avec un caroténoïde modèle, la bixine. Le produit dérivé obtenu après transacylation a été purifié et a montré une activité antiradicalaire supérieure à celle de la bixine. Ces résultats permettent d’envisager la synthèse de peptides acylés avec ce type de caroténoïdes
The aim of this work was to determine the optimum of an original carotenoid, the torularhodin, produced by Sporobolomyces ruberrimus, in batch culture. A very interesting characteristic of this strain is its ability to consume raw glycerol as a carbon and energy source for microbial growth and carotenoid production. In the fist part of this study, the identification of operating parameters that have an influence on the optimum torularhodin production, was achieved. Experimental assays reinforced by a statistical study allowed to identify temperature, dissolved oxygen pressure and oleic acid supplementation, as the major parameters of influence, and then the integration of these data was performed for the construction of a multiobjective optimization based on a multicriteria experimental design. The establishment of a mathematical model of a second degree polynomial type was developed for the prediction of the values of µmax and of the torularhodin concentration reported to biomass. In the last part, considering that torularhodin has an important antioxidant property and it exhibits a free carboxyl acid function which can be used as acyl agent, a study of its structure modifying by an enzymatic way as a stabilization pattern was started. The experimental conditions of lysine acylation by the lipase B of Candida antarctica were determined using a model carotenoid, the bixin. The resulting product of the synthesis of bixin derivative was purified and showed an antiradical activity of 2.5 times higher than that of bixin. This result showed the ability of the acylation reaction of peptides with this kind of carotenoids

Gli esteri svolgono un ruolo rilevante nell'industria alimentare, sono tra i composti più importanti e versatili di aromi e fragranze naturali in alimenti, bevande e cosmetici. La loro preparazione da substrati naturali e l'utilizzo di bioprocessi (eg. fermentazione o reazioni enzimatiche) è attrattivo, perché il prodotto finale può essere etichettato e commercializzato in UE e USA come naturale. Pertanto, nuovi approcci biotecnologici per ottenere aromi sono desiderati, una volta che siano efficienti e sostenibili. Molti esteri con proprietà aromatiche possono essere ottenuti enzimaticamente usando lipasi che catalizzano reazioni di esterificazione, transesterificazione o interesterificazione. In questa tesi di dottorato abbiamo sviluppato due sistemi per la produzione di esteri con proprietà aromatiche: 1) Un metodo biocatalitico per la preparazione enzimatica di diversi esteri con proprietà aromatiche da alcoli primari (isoamilico, n-esilico, geranilico, cinnamilico, 2-feniletilico e benzilico) ed esteri etilici naturalmente disponibili (formiato, acetato, propionato e butirrato). Le biotrasformazioni sono catalizzate da un'aciltransferasi di Mycobacterium smegmatis (MsAcT) e precedute da eccellenti rese (80- 97%) e brevi tempi di reazione (30-120 minuti), anche quando sono state utilizzate concentrazioni di substrato elevate (fino a 0,5 M). Questa strategia enzimatica rappresenta un'alternativa efficace all'applicazione delle lipasi nei solventi organici e un miglioramento significativo rispetto ai metodi già noti in termini di uso ridotto di solventi organici, aprendo la strada a una preparazione sostenibile ed efficiente degli agenti aromatizzanti naturali. 2) Un metodo biocatalitico con la lipasi legata al micelio liofilizzato di Aspergillus oryzae che catalizza l'esterificazione diretta di alcoli e acido acetico in solvente organico. Ha mostrato un'elevata stabilità verso substrati e prodotti. L'acqua prodotta durante l'esterificazione non ha influenzato in modo significativo l'equilibrio della reazione, consentendo conversioni elevate. Queste caratteristiche sono state sfruttate per preparare esteri con proprietà aromatiche dell'acetato (isoamil e cinnamil acetato) in sistemi in batch e continui. È stato sviluppato un continuous stirred tank membrane reactor (CST-MR) ovvero un reattore continuo a membrana sotto agitazione per garantire una buona produttività e un'elevata stabilità del biocatalizzatore. Entrambi i sistemi di produzione sono promettenti, rappresentano due diverse alternative e possono essere ulteriormente ottimizzati e scalati per gli interessi del settore.
Esters play a significant role in the food industry, they are among the most important and versatile components of natural flavours and fragrances in food, drinks and cosmetics Their preparation starting from natural substrates and using bioprocesses (e.g., fermentation or enzymatic reactions) is appealing, since the final product can be labelled and commercialized in EU and USA as natural. Therefore, new biotechnological approaches for obtaining flavours are highly demanded as long as they are efficient and sustainable. Many flavour/fragrance esters can be enzymatically obtained using lipases that catalyse esterification, transesterification or interesterification reactions. In this PhD thesis we studied two systems for production of flavours esters: 1) A straightforward biocatalytic method for the enzymatic preparation of different flavour esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalysed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and preceded with excellent yields (80-97%) and short reaction times (30-120 minutes), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared to already known methods in terms of reduced use of organic solvents, paving the way to a sustainable and efficient preparation of natural flavouring agents. 2) Mycelium-bound lipase of dry mycelium of Aspergillus oryzae catalysed direct esterification of alcohols and acetic acid in organic solvent, showing high stability towards substrates and products. Water produced during the esterification did not significantly affect the equilibrium of the reaction, allowing for high conversions. These features were exploited for preparing flavour-active acetate esters (e.g., isoamyl and cinnamyl acetate) in batch and continuous systems. A continuous stirred tank membrane reactor (CST-MR) was developed securing good reactor productivity and high biocatalyst stability. Both production systems are promising, represent two different alternatives and can be further optimized and scaled up for the interests of the industry.

Triquentrinas A são produtos naturais marinhos com atividade biológica e alta complexidade estrutural. Estes fatores tornam estes alcaloides e compostos análogos, como os herbindóis, alvos para a síntese total e plataforma para o desenvolvimento de novas metodologias sintéticas. Nesta Dissertação de Mestrado, procurou-se completar a síntese estereosseletiva da (+)-cis-triquentrina A empregando-se intermediários sintéticos protegidos com o grupo benzila que poderão ser usados para a futura prospecção de novos compostos com atividade biológica. A rota proposta baseia-se na obtenção do ácido (S)-3-(1-benzil-4-etil-1H-indol-7-il)butanóico, um intermediário-chave já descrito por Silva e colaboradores no percurso da síntese total da (+)-trans-triquentrina A, e da finalização da síntese de acordo com a abordagem proposta pelo grupo de RajanBabu para conversão do análogo protegido com grupo tosila à (+)-cis-triquentrina A. A resolução enzimática do intermediário-chave com lipase de Pseudomonas cepacia imobilizada em terra diatomácea foi otimizada, resultando em rendimentos de 32% e 99% ee na metade do tempo descrito anteriormente. Na etapa-chave da síntese, o (S)-ácido foi submetido a uma acilação de Friedel-Crafts intramolecular na presença de anidrido trifluoroacético que produziu o produto de ciclização desejado com 40% de rendimento. Na etapa final da síntese, o intermediário sintético protegido com grupo benzila não pode ser convertido à (+)-cis-triquentrina A, empregando-se a metodologia desenvolvida para a redução do composto análogo tosilado, dada a diferença de reatividade imposta pela troca do grupo protetor. As etapas realizadas até o penúltimo intermediário sintético (S)-8, consta com 10 etapas e rendimento global de 1,3%. viii As diferenças eletrônicas e estruturais relacionadas a diferentes grupos protetores poderão ser refletidas em variações na atividade antiproliferativa de indóis relacionados a triquentrinas. Ainda no interesse de preparar moléculas para envio à análises de atividade antiproliferativa preparou-se um composto relacionado à síntese da trans-triquentrina A tendo como etapa-chave uma contração de anel mediada por I(III) em 21% de rendimento, cujo trabalho foi incluído nos anexos.
Trikentrins A are marine natural products with biological activity and high structural complexity. These factors make these alkaloids and analogous compounds, such as herbidoles, targets for total synthesis and platform for the development of new synthetic methodologies. In this Master\'s Dissertation, we attempted to complete the stereoselective synthesis of (+)-cis-trikentrin A using synthetic intermediates protected with the benzyl group that could be used for the future prospection of new compounds with biological activity. The proposed route is based on the preparation of (S)-3-(1-benzyl-4-ethyl-1H-indol-7-yl) butanoic acid, a key intermediate already described by Silva et al., in the course of total synthesis (+)-trans-triquentrin A, and the final part of the synthesis according to the approach proposed by the RajanBabus group for conversion of the protected analogue with tosyl group to (+)-cis-trikentrin A. The enzymatic resolution of the key intermediate with Pseudomonas cepacia lipase immobilized on diatomaceous earth was optimized, resulting in 32% yield and 99% ee in half the time described above. In the key step of the synthesis, the (S)-acid was subjected to an intramolecular Friedel-Crafts acylation in the presence of trifluoroacetic anhydride which yielded the desired cyclization product in 40% yield. In the final step of the synthesis, the synthetic intermediate protected with benzyl group couldnt be converted to (+)-cis-trikentrin A, employing the methodology developed for the reduction of the tosylated analogous compound, given the difference of reactivity imposed by exchange of the protective group. The steps carried out up to the synthetic intermediate (S)-8, consists of 10 steps and overall yield of 1.3%. Electronic and structural differences related to different protective groups may be reflected in variations in the antiproliferative activity of indoles related to trikentrins. A compound related to the synthesis of trans-triquentrin A having as its key step an I(III) mediated ring contraction in 21% yield was also prepared in the interest of preparing molecules for antiproliferative activity analysis, whose work was included in the appendix.

Dissertação de Mestrado em Química Farmacêutica Industrial apresentada à Faculdade de Farmácia
Os compostos polifenólicos são um grupo de metabolitos secundários que ocorrem na Natureza. São considerados compostos bioativos pois apresentam múltiplas propriedades biológicas e farmacológicas, entre elas destaca-se a atividade antioxidante. A sua aplicação é reduzida devido à sua baixa solubilidade em água, fraca absorção intestinal e estabilidade limitada. A polidatina é um derivado glicosilado do resveratrol com uma grande variedade de actividades biológicas descritas em diversos estudos in vitro e in vivo.A modificação semi-sintética dos compostos polifenólicos glicosilados através da obtenção de derivados acilados visa melhorar as caraterísticas físico-químicas dos compostos e explorar a modulação das suas actividades biológicas. A acilação enzimática seletiva destes compostos é preferível em relação à catálise química, uma vez que se trata de moléculas complexas e lábeis, sendo necessárias reações seletivas e condições suaves, condições estas não oferecidas pela catálise química. Além disso, por via enzimática, as etapas de proteção /desproteção dos grupos funcionais são evitadas, reduzindo os passos de síntese. Tendo em consideração as vantagens que oferece a biocatálise, esta parece ser uma estratégia eficaz para a modificação estrutural dos compostos polifenólicos.De forma a melhorar as propriedades biológicas e farmacológias da polidatina, um derivado glicosilado do resveratrol, este trabalho teve como objetivo geral a obtenção de derivados acilados da polidatina através da biocatálise, recorrendo à lipase B de Candida antarctica (CALB) como biocatalisador. Neste trabalho estudou-se a síntese e caraterização estrutural de derivados acilados da polidatina, onde pela análise dos espetros de ressonância magnética nuclear foi possível verificar as posições de acilação da polidatina, bem como confirmar a estrutura dos derivados acilados obtidos.
Polyphenolic compounds are a group of secondary metabolites occurring in Nature. They are considered bioactive compounds because they have multiple biological and pharmacological properties, among them the antioxidant activity. Its application is reduced due to its low solubility in water, poor intestinal absorption and limited stability.Recently, the scientific interest has turned to the acylated derivatives, in order to improve the physicochemical characteristics of the polyphenolic compounds. The selective enzymatic acylation of these compounds is preferred over chemo-catalysis. There are several reasons for this assertion, because as they are complex molecules, soft conditions are required, conditions not offered by chemical catalysis. In addition, enzymatically, the protection / deprotection steps of the functional groups are avoided, reducing the steps of synthesis. In view of the advantages of biocatalysis, this appears to be an effective method for the structural modification of the polyphenolic compounds in order to improve their properties.In order to improve the biological and pharmacological properties of polydatin, this work had as general objective to obtain acylated derivatives of polydatin through biocatalysis, resorting to the lipase B of Candida antarctica (CALB) as biocatalyst.This work consisted in the synthesis and characterization of the acylated derivatives of polydatin, where by the analysis of nuclear magnetic resonance spectra it was possible to determine the acylation positions of polydatin, as well as to confirm the structure of the acylated derivatives obtained.

Vitamin B5 is the simple acid pantothenic acid (from the Greek pantos, meaning widespread). B5 needs to undergo five enzymatic steps to be converted to coenzyme A, in which the terminal thiol of a cysteamine moiety is the key reactive atom, once dissociated to the thiolate anion. The thiolate serves as nucleophile towards a variety of acids to yield acyl thioesters (acyl-CoAs). These are the cellular acylating agents in metabolic pathways. The two-carbon acyl group in acetyl CoA is doubly activated: as electrophile at C1 and as nucleophile at C2, and so ideally set up for C–C bond formations in fatty acid and steroid biosynthetic pathways, as well as the first step in the citrate metabolic cycle.