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Résolution de mélanges complexes d'oligosaccharides sulfatés par chromatographie 2D et spectrométrie de masse : application aux héparines thérapeutiques / Resolution of complex mixtures of sulfated oligosaccharides by 2D-chromatography and mass spectrometry : application to heparin-like drugsJaffuel, Aurore 20 September 2016 (has links)
Résumé confidentiel / Résumé confidentiel
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Synthesis Of 2-Deoxy-1-Thioglycosides And Establishing Their Efficient Glycosyl Donor Properties To Prepare Aryl 2-Deoxy Glycosides And 2-Deoxy OligosaccharidesPaul, Somak 01 May 2008 (has links)
Carbohydrates are a family of polyfunctional natural products and can be chemically modified in numerous ways. The primary significance of carbohydrates rests in their importance in biological functions. A particular class of sugars, namely, 2-deoxy or C-2 modified sugars has received a special attention, due to their importance in biological functions. These sugars are defined as carbohydrates carrying a hetero-atom, other than the hydroxyl group, and their derivatives. There is an ever-leading requirement to synthesize various carbohydrates-containing natural and un-natural products, such as, oligonucleotides, glycopeptides, antitumor drugs and cardiac glycosides, having C-2 modified sugars. Chapter 1 describes various synthetic modifications, particularly at the C-2 of a monosaccharide, as relevant to the work presented in this Thesis.
1, 2-Unsaturated glycopyranosides, namely, glycals, are versatile synthetic intermediates for the elaboration to a number of functionalized glycosyl derivatives. A major utility of the glycals is their conversion to the 2-deoxy glycosyl derivatives. In a programme, it was desired to identify a synthetic method to prepare 2-deoxy sugar derivatives that are endowed with an anomeric activation. In particular, a thioglycoside activation was desired. In the event, a methodology was identified, which allowed the synthesis of activated 2-deoxy-1-thioglycosides.The method involved reaction of a glycal with EtSH, in the presence of ceric ammonium nitrate (CAN) as the catalyst. The reaction was applicable to different epimeric glycals. Apart from the 2-deoxy-1-thioglycosides, formation of the 2, 3-unsaturated enoses, corresponding to the Ferrier product, also observed. Optimal conditions for the formation of the 2-deoxy-1-thioglycosides were identified (Scheme 1) and the reaction was proposed to proceed through a radical oxocarbenium ion and a thiolate intermediate.
(Fig)
Scheme1
Upon synthesis of 2-deoxy-1-thioglycosides, few glycosylation reactions with both aglycosyl and glycosyl acceptors were performed and the α-anomeric 2-deoxy glycosides were obtained exclusively.
Chapter 2 summarizes synthesis, characterization of 2-deoxy-1-thioglycosides and their glycosyl donor properties towards several glycosyl acceptors.
Many naturally-occurring antibiotic and antitumor drugs contain 2-deoxy glycosides as important structural components. For example, 2,6-dideoxy-hexopyranoses are common structural units of chromomycin A3, olivomycin A and mithramycin. The most common structural features of these molecules are: (i) the presence of 2-deoxy sugar residues and (ii) the sugar residues are connected to the aromatic moiety, through a β-glycosidic linkage. The synthesis of these biologically important 2-deoxy glycosides encounters difficulties, due to the absence of stereoelectronic influences at C-2 of the 2-deoxy glycosyl derivatives.
Direct glycosylation of phenols and naphthols with activated 2-deoxy-1-thio-glycosides, in the presence of the thiophilic activator N-iodosuccinimide/triflic acid (NIS/TfOH), lead to the formation of the α-anomer, as the major glycosylated product (Scheme 2).
(Fig)
An effort was under taken to identify methods to prepare the 2-deoxy aryl glycosides, in the β-anomeric configuration. A nucleophilic substitution reaction was anticipated to lead to the formation of β-anomeric glycosides. A halide substitution at C-1 for an effective nucleophilic substitution was adopted. Thus, conversion of the activated 2-deoxy-1-thioglycosides with Br2 in the first step, followed by reaction of the resulting bromide with aryloxy anions, led to the facile conversion to 2-deoxy glycosides in a nearly quantitative f-anomeric configuration at C-1(Scheme 3).
Scheme 3 (Fig)
Chapter 3 presents details of the methodologies that allow a facile preparation of each of the anomers of aryl 2-deoxy-D-glycosides from a common precursor, namely, 2-deoxy-1-thio-glycosides.
An easy access to activated 2-deoxy-1-thioglycosides from the 1, 2-unsaturated sugar and their synthetic utility towards various glycosyl and aglycosyl acceptors led towards synthesis of 2-deoxy disaccharides. Synthesis of six new 2-deoxy-arabino-hexopyranosyl and 2-deoxy-lyxo-hexopyranosyl sugar containing disaccharides were accomplished. These are: (i) 2-deoxy-α-D-arabino-hexopyranosyl-(1→4)-D-glucopyranose (2'-deoxy maltose); (ii) 2-deoxy-α-D-lyxo-hexopyranosyl-(1→4)-D-glucopyranose; (iii) 2-deoxy-α-D-arabino-hexopyranosyl-(1→4)-2-deoxy-D-arabino-hexopyranose (2,2'-dideoxy maltose); (iv) 2-deoxy-α-D-lyxo- hexopyranosyl-(1→4)-2-deoxy-D-arabino-hexopyranose; (v) α-D-glucopyranosyl-(1→4)-2 deoxy-D-arabino-hexopyranose (2-deoxy maltose) and (vi) β-D-galactopyranosyl-(1→4)- deoxy-D-arabino-hexopyranoside (2-deoxy lactose).
The 2'-deoxy and 2, 2'-dideoxydisaccharides were synthesized using a 2-deoxy glycosyl donor and a normal glycosyl acceptor (in case of 2'-deoxy disaccharides) and a 2-deoxy glycosyl acceptor (in case of 2, 2'-dideoxy disaccharides) with a free OH group at C-4, while the remaining hydroxyl groups protected suitably (Scheme 4).
Scheme 4 (Fig)
On the other hand, the syntheses of 2-deoxy disaccharides were initiated from a D-maltose and D-lactose, respectively. The conversion of these disaccharides to a disaccharide glycals was targeted first and conversion of these glycals to a 2-deoxy-1-thioglycosides or a 2-deoxy-1-acetates, followed by a hydrolysis of the thiol moiety or the acetate group, afforded the 2-deoxy disaccharides (Scheme 5). (Fig)
Chapter 4 describes synthesis, characterization of 2-deoxy, 2,2'-dideoxy and 2'-deoxy disaccharides.
Continuing the efforts to establish the utility of 2-deoxy-1-thioglycosides as potential glycosyl donor, synthesis of 2-deoxy cyclic and linear oligosaccharides was undertaken. Prominent among cyclic oligosaccharides are the cyclodextrins. Due to their unique structural and physical properties, cyclodextrins find manifold applications. Known methods to synthesize cyclic oligosaccharides are (i) the cyclization of linear oligosaccharides to produce the cyclic oligosaccharides and (ii) the synthesis of designed monomers and subjecting them to cyclooligomerization protocols. The cyclooligomerization was adopted to synthesize new types of 2-deoxy cyclic-and linear oligosaccharides. After a series of trials, a disaccharide monomer, namely, ethyl 4-O-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-2-deoxy-3,6-di-O-methyl-arabino-hexopyranoside (1), was identified as a suitable monomer for thecyclooligomerization protocol. For an effective oligomerization, the concentration of the monomer and the choice of the reagents are important.
The reaction was conducted at three different monomer concentrations, 2 mM, 10 mM and 25 mM, using two thiophilic activators, namely, (i) NIS/TfOH and (ii) NIS/AgOTf. Better yields of the cyclic oligosaccharides, namely, the cyclic tetrasaccharide (2) (40 %) and cyclic hexasaccharide (3) (25 %), were isolated when the monomer (1) concentration was 25 mM and NIS/TfOH acid was used as the promoter (Scheme 6). The formation of linear disaccharide (4) (10 %) and tetrasaccharide (5) (18 %) was also observed at this concentration.
On the other hand, when the reaction of the monomer was performed in the presence of NIS/AgOTf, the oligomerization reaction led to the formation of linear oligosaccharides, consisting of di-to eicosa-saccharides. Synthesis of different monomers, their characterization and oligomerization reaction using these monomers through a polycondensation protocol are
discussed in Chapter 5.
Scheme 6(fig)
In summary, the Thesis establishes the chemistry of 2-deoxy sugars, formation of activated 2-deoxy sugars, formation of alkyl and aryl glycosides, 2-deoxy disaccharides, 2-deoxy cyclic and linear oligosaccharides. Routine physical methods were used to characterize the newly formed 2-deoxy sugars and the oligosaccharides. Single crystal X-ray structural determination was performed for an aryl 2-deoxyglycosides, which provided the solid state configurational features of the 2-deoxy pyranose.
(For structural formula pl see the pdf file)
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Préparation et modification d'oligosaccharides de cellulose par chimie douce bio-inspirée / Soft bio-inspired chemistry for the preparation and modification of cellulose oligosaccharidesClaisse, Nathalie 13 December 2012 (has links)
La valorisation de la biomasse saccharidique pour la production de dérivés biosourcés d'intérêt est un enjeu important. La cellulose est le polysaccharide le plus abondant sur Terre et représente une source de matière première considérable. Dans ce travail, de nouveaux procédés de dépolymérisation de la cellulose pour l'obtention contrôlée de cellodextrines sont décrits. Ils proposent une approche alternative plus douce aux procédés de production actuels en privilégiant l'utilisation d'enzymes, et de liquides ioniques comme solvants alternatifs. Ce travail rapporte l'élaboration de deux méthodes d'obtention contrôlée d'oligosaccharides à partir de cellulose et de cellulose acétate par combinaisons successives d'hydrolyses acide et enzymatique. Ces procédés ont permis l'obtention de cellodextrines de tailles ciblées avec de bons rendements, et constituent une voie prometteuse pour la valorisation de la cellulose en dérivés biosourcés. La deuxième partie de ce travail consiste en la modification chimio-enzymatique des oligosaccharides de cellulose produits pour leur valorisation en biomolécules d'intérêt, plus particulièrement dans le domaine de l'agrochimie. Les cellodextrines sont utilisées en tant que base saccharidique pour la synthèse d'analogues de lipo-chitooligosaccharides comme potentiels fertilisants verts. Deux méthodes de préparation ont été élaborées à l'aide des glycoside-hydrolases comme outils de synthèse. Les stratégies développées permettent un accès efficace à la synthèse d'analogues et peuvent être adaptées pour la production d'autres molécules. / Valorisation of biomass is a timely challenge and its bio-conversion raises a growing interest from academics and industrials. Cellulose is the most abundant biopolymer on Earth and offers a wide range of applications including value added bio-derived compounds. In this work we report the design of new methods to produce cellodextrins from cellulose and from cellulose acetate, by a combination of successive hydrolyses. These strategies imply the use of ionic liquids and enzymes to perform the depolymerisation. They represent a soft alternative to the current processes of production. The controlled hydrolysis of cellulose was realised in two steps involving first the partial fragmentation of cellulose in ionic liquids by acid catalysis. Then, the selective hydrolysis of those cellulose fragments was performed by an endoglucanase to produce the targeted sizes of cellodextrins. The second method deals with the depolymerisation of water soluble cellulose acetate by enzymatic hydrolysis. Those two methods have led to the controlled production of cellodextrins of interest, with significantly improved yields. In the second part of this work, cellodextrins were used as building block for the synthesis of analogs of lipo-chitooligosaccharides as potential natural fertilizers. Several chemo-enzymatic routes were investigated to produce these biomolecules, and two methods of synthesis were elaborated using glycoside-hydrolases as coupling tools. Those methods offer an easy access to analogs and can be adapted for the production of further molecules.
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Ingénierie des protéines pour la synthèse d'oligosaccharides d'intérêts biologique et industriel / Protein engineering for the synthesis of oligosaccharides with biological and industrial interestsChambon, Remi 20 November 2014 (has links)
Le but du projet est de mettre au point de nouveaux outils enzymatiques permettant la production de chitinoligosaccharides de taille et de degré d'acétylation parfaitement contrôlés pour l'étude d'enzymes impliquées dans la biosynthèse, la biodégradation et la modification de la chitine, et pour l'étude de récepteurs protéiques d'origine animale ou végétale. Des études récentes ont montré que les oligomères de la chitine et leurs dérivés sont des molécules qui interviennent dans les phénomènes symbiotiques et de reconnaissance hôte-pathogène dans le règne végétal. Ces molécules sont utilisées en agrochimie comme biofertilisants, et potentiellement en phytosanitaire. Ils sont connus également pour posséder de nombreuses activités biologiques dans le domaine de la santé (effets antimicrobiens, anticancéreux, anti-inflammatoires, immunostimulants...). Si les activités de cette classe d'oligosaccharides sont parfaitement reconnues, leurs modes d'actions restent encore à éclaircir, ce qui nécessite de disposer de molécules pures aux structures chimiques parfaitement contrôlées. La production d'oligomères de la chitine nécessite traditionnellement la mise en œuvre d'une chimie fastidieuse, qui peut être facilitée par une approche chimio-enzymatique.Dans le cadre de cette thèse, nous souhaitons donc développer des outils enzymatiques permettant la synthèse d'une bibliothèque de molécules de taille et de degré d'acétylation contrôlés en vue d'études structure-activité biologique. Pour cela, nous chercherons à produire des N-désacétylases et des chitinases dans différents systèmes d'expression et à caractériser leur activité afin de générer une panoplie de molécules de structure moléculaire parfaitement définie à partir de fragments saccharidiques issus de la biomasse. Les molécules ainsi préparées pourront ensuite être modifiées de façon chimio-sélective afin d'obtenir des sondes photoactivables et/ou biotinylées pour la caractérisation de récepteurs, des substrats fluoro- ou chromogéniques pour le dosage spécifique d'activités enzymatiques ou encore des lipochitinoligosaccharides capables de favoriser la croissance des plantes. Les approches utilisées pour mener à bien ce projet pluridisciplinaire seront : l'ingénierie et la production de protéines recombinantes, la caractérisation biochimique d'activités enzymatiques ainsi que la synthèse chimio-enzymatique et la modification chimique d'oligosaccharides qui devront être caractérisés d'un point de vue physicochimique. Il s'agit d'un projet intégré dans une collaboration nationale financée par l'ANR réunissant des équipes de l'Université de Grenoble, de Lyon, d'Orsay, et l'entreprise Bayer CropScience. / The aim of the project is to develop new tools for enzymatic production of chitooligosaccharides size and degree of acetylation perfectly controlled for the study of enzymes involved in biosynthesis, biodegradation and modification of chitin and for the study of protein receptors of animal or vegetable origin. Recent studies have shown that oligomers of chitin and its derivatives are molecules involved in the phenomena of symbiotic and host-pathogen recognition in plants. These molecules are used as agrochemicals biofertilizers. They are also known to possess numerous biological activities in the field of health (anti-microbial, anti-cancer, anti-inflammatory, immunostimulant ...). If the activities of this class of oligosaccharides are well recognized, their modes of action remain to be clarified, which requires having pure molecules with chemical structures perfectly controlled. The production of chitin oligomers traditionally requires the implementation of a tedious chemistry, which can be facilitated by a chemoenzymatic approach.As part of this thesis, we want to develop enzymatic tools for the synthesis of a library of molecules of size and degree of acetylation controlled studies for structure-biological activity. For this, we will seek to produce N-deacetylase and chitinases in different expression systems and characterize their activity to generate a variety of molecules well-defined molecular structure from saccharide fragments derived from biomass. The molecules thus prepared can then be modified so as to achieve chemoselective photoactivatable probes and / or biotinylated for the characterization of receptors, substrates or fluoro-chromogenic assay for specific enzyme activities or lipo-chitooligosaccharides can promote plant growth. The approaches used to complete this multidisciplinary project are: engineering and production of recombinant proteins, the biochemical characterization of enzymatic activities and chemoenzymatic synthesis and chemical modification of oligosaccharides to be characterized from the point of physicochemical view. This is an integrated project in a national collaboration funded by the ANR with teams from the University of Grenoble, Lyon, Orsay, and Bayer CropScience.
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Temperature-dependent Regulation of Sugar Metabolism During Cold Stress ResponsesZhao, Lu 07 July 2017 (has links)
No description available.
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Avaliação do desempenho de Suínos Alimentados com Mananoligossacarídeos (MOS) / Effects of mannan oligosaccharides on gilts and litters performanceHorta, Felipe de Conti 21 August 2009 (has links)
O presente estudo buscou avaliar os efeitos dos Mananoligossacarídeos (MOS) como aditivo alimentar no desempenho de primíparas suínas em final de gestação e em lactação, bem como no desempenho e na saúde dos leitões até os 65 dias de idade. O experimento foi realizado no Laboratório de Pesquisa em Suínos (VNP-FMVZ-USP) Pirassununga SP. O delineamento experimental foi inteiramente casualizado em arranjo fatorial de tratamentos, sendo um fator o fornecimento dos MOS para fêmeas e o segundo para os leitões. Foram utilizadas 17 primíparas prenhes, tratadas a partir de 81 ±1,36 dias de gestação com 0,2% de MOS na dieta. Aos 82 ± 1,36 dias de gestação as fêmeas foram submetidas à coleta de sangue e à vacinação contra rinite atrófica progressiva, sendo pesadas quinzenalmente até a transferência para a maternidade, aos 109 ± 1,36 dias de gestação. Na segunda quinzena as fêmeas tratadas apresentaram uma vantagem numérica no ganho de peso médio diário (P=0,063). Durante o parto foram colhidas amostras de sangue e colostro para titulação de anticorpos contra o antígeno vacinal, sendo consideradas positivas 66,67% das fêmeas MOS e 42,86% das fêmeas controle. Os leitões aleitados por fêmeas tratadas tiveram um maior ganho numérico de peso na primeira semana (p=0,0614), significativo na segunda semana (p=0,047). Na terceira semana foi introduzido o segundo fator através do oferecimento de alimento sólido (0,4% de MOS). No período total, do nascimento aos 21 dias, foi observada uma vantagem numérica (p=0,0989) no ganho de peso a favor dos leitões de fêmeas MOS. As fêmeas, contudo, não diferiram quanto à variação de peso ou consumo durante a fase de lactação. Aos 23 ± 1,91 dias de idade dos leitões foi realizado o desmame abrupto com a transferência dos leitões para unidade de creche, onde foram alojados em gaiolas para 4 animais. O peso ao desmame foi maior nos leitões aleitados por fêmeas tratadas (p<0,0001), bem como em todas as pesagens semanais até a quinta semana pós-desmame. O consumo na primeira semana sofreu influência da suplementação de MOS nas fêmeas e do maior peso à desmama se mostrando superior nesses animais (P=0,049) influenciando diretamente o ganho de peso, superior na segunda semana (p=0,002). O MOS para os leitões aumentou o consumo de alimento (p=0,0784), e a conversão alimentar na segunda semana (p=0,0103). Aos 14 dias pós-desmame foram colhidas amostras de sangue para hemograma e realizada a aplicação oral de 108 UFC de Salmonella Typhimurium. Foram observadas diariamente a consistência das fezes por 28 dias e aferidas as temperaturas retais por 9 dias. Leitões tratados com MOS e os leitões oriundos de fêmeas MOS tenderam a apresentar o pico de hipertermia mais cedo que os demais (p=0,0629 e p=0,0976, respectivamente) e os leitões alimentados com MOS tenderam a ter uma temperatura de pico mais baixa (p=0,0989). Na última semana os leitões de fêmeas MOS apresentaram um maior consumo (p=0,0007) e uma incidência de Salmonella numéricamente inferior em linfonodos mesentéricos e nas fezes colhidas aos 36 dias pós-desafio. Pode-se concluir que a suplementação de MOS para primíparas prenhes e lactentes pode melhorar o desempenho e a saúde entérica de seus leitões nas fases de aleitamento e creche. / The present study evaluated the effects of mananoligossacarides (MOS) as a feed additive on the performance of primiparous sows in late gestation and lactation, as well as on performance and health status of their progeny up to 65 days of age. The experiment was conducted in the Laboratory of Swine Research (FMVZ/USP) Pirassununga SP. For this purpose a completely random factorial design was used, factors which corresponded to (1) feeding sows with MOS and (2) feeding piglets with MOS, characterizing 4 treatments: MM Feeding MOS to both sows and piglets, MC Feeding MOS only to the sows, CM Feeding MOS only to piglets, CC Control diets for both sows and piglets. A total of seventeen pregnant gilts were used, and divided into 2 groups: MOS (n=9) and Control (n=8). MOS gilts had 0,1% MOS added to their diets from 81 ± 1,36 days of gestation onward. On day 82 ± 1,36 blood was collected and vaccination against Progressive Atrophic Rhinitis was conducted. Animals were weighted biweekly until 109 ± 1,36 days of gestation, when transference to the farrowing unit was conducted. Between the first and second weighing, MOS gilts show a numerical advantage in daily weight gain (p=0,063). At farrowing, blood and colostrum samples were collected for determination antibodies titles against vaccine antigen, being considered positive 66,67% and 42,86% of MOS and Control sows, respectively. Piglets nursed by MOS sows had a numerical advantage in weight gain in the first week (p=0,0614), with statistical significance in the second week (p=0,047). On the third week, the second factor was introduced by the offering of solid feed (0,4% MOS). During the suckling period, from birth to 21 days of age, a numerical advantage in weight gain was observed for MOS sows piglets (p=0,0989). The sows themselves did not differ in weight change or feed consumption. At 23 ± 1,91 days of age, weaning was conducted, as piglets were transferred to nursing facilities and allocated in pens of 4 animals. Weight of MOS sows piglets was higher at weaning (p<0,001) and until the 5th week port-weaning. Feed consumption was affected by MOS supplementation of sows and by the higher weaning weight (p=0,0049), directly influencing weight gain, which was superior in the 2nd week. Feeding MOS to piglets enhanced feed consumption (p=0,0784) and feed conversion (p=0,0103) on the 2nd week. At 14 days of age, blood samples were collected for hemogram analysis and an oral dose of 108 CFU of Salmonella Typhimurium administered. Fecal consistency and rectal temperature were evaluated for 28 and 9 days, respectively. Piglets treated with MOS and MOS sows\' piglets tended to show a peek of hyperthermia earlier (p=0,0629 e p=0,0976, respectively). Piglets fed with MOS tended to show a lower temperature peek (p=0.0989). In the last week, MOS sows\' piglets show a higher feed consumption (p=0.0007) and a numerically inferior Salmonella incidence in mesenteric lymph nodes and feces 36 days after the bacterial challenge. In conclusion, supplementing primiparous sows with MOS during gestation and lactation can enhance their body weight gain and the performance and intestinal health of the offspring during the suckling and nursing period.
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Inulinases produzidas por leveduras isoladas do semi?rido baiano: estudos de imobiliza??o e aplica??o na produ??o de frutose e fruto-oligossacar?deosRibeiro, Geise Camila de Araujo 18 December 2014 (has links)
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Previous issue date: 2014-12-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The exploration of the microorganisms species semiarid region has become a viable alternative for the industry, especially producing yeast inulinases which can be applied in the production of fructo-oligosaccharides and fructose from the hydrolysis of inulin. This work had as its theme the study of the immobilization inulinases enzymes produced by yeasts of the semiarid region of Bahia, with the overall objective of the work is to study the application of inulinases immobilized in the production of concentrated fructose and fructo-oligosaccharides. The strains of yeast Pseudozyma sp. (CCMB 306) and Rhodotorula mucilaginosa (CCMB 604) were obtained from the Culture Collection of Microorganisms of Bahia (CCMB), Universidade Estadual de Feira de Santana (UEFS). Immobilization was performed using as support eggshell, celite, Immobeads?, Sepabeads? and silica, and the results analyzed using the Origin version 8.5.1 program. Immobilization in solid supports inulinase shown to be a viable technique, since the non-inactivated enzyme, showing increased activity in the inulinase CCMB 306 samples immobilized on celite in organic medium. Optimization of fructo-oligosaccharides production was achieved the highest concentration of product in samples with inulin concentration of 25% and time of reaction 16 h. In conclusion, the application of inulinases enzymes produced by micro-organisms of the semiarid in the hydrolysis of inulin is of significant importance for biotechnology development in the region. / A explora??o de esp?cies de micro-organismos da regi?o semi?rida tem se tornado uma alternativa vi?vel para a ind?stria, destacando-se leveduras produtoras de inulinases, que podem ser aplicadas na produ??o de frutose e fruto-oligossacar?deos, a partir da hidr?lise da inulina. O presente trabalho teve como tema o estudo da imobiliza??o de enzimas inulinases produzidas por leveduras do semi?rido baiano, sendo o objetivo geral estudar a aplica??o de inulinases imobilizadas na produ??o de concentrados de frutose e fruto-oligossacar?deos. As linhagens de leveduras Pseudozyma sp. (CCMB 306) e Rhodotorula mucilaginosa (CCMB 604) foram obtidas da Cole??o de Cultura de Micro-organismos da Bahia (CCMB) da Universidade Estadual de Feira de Santana (UEFS). A imobiliza??o foi realizada utilizando-se como suporte casca de ovo, celite, Immobeads?, Sepabeads? e s?lica, sendo os resultados analisados com aux?lio do programa Origin vers?o 8.5.1. A imobiliza??o de inulinase nos suportes s?lidos demonstrou ser uma t?cnica vi?vel, uma vez que n?o inativou a enzima, apresentando a maior atividade nas amostras de inulinase de CCMB 306 imobilizada em celite em meio org?nico. Na otimiza??o da produ??o de fruto-oligossacar?deos obteve-se a maior concentra??o de produto nas amostras com concentra??o de inulina de 25% e tempo de rea??o de 16 h. Em conclus?o, a aplica??o de enzimas inulinases obtidas atrav?s de micro-organismos presentes no semi?rido na hidr?lise da inulina ? de significativa import?ncia para o desenvolvimento biotecnol?gico da regi?o, sendo interessante a otimiza??o dos m?todos de imobiliza??o e sele??o adequada dos suportes a serem utilizados para obten??o de melhores resultados.
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Investiga??o sobre um m?todo de detec??o de adulterantes em caf? por CLUE-EM-EM / Investigation about method of adulterants detection in coffee by UPLC-MS-MSMARTINS, V?ctor de Carvalho 14 February 2017 (has links)
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Previous issue date: 2017-02-14 / CAPES / Coffee is one of the most appreciated beverages in the world, due to its sensorial and functional characteristics. Currently, as the main producer and exporter in the world, it is estimated that in Brazil there will be growth in the domestic market in the coming years, in which roasted and ground coffee predominates. This type of product is impaired mainly by adulteration with other plant materials. The implementation of more sensitive and selective methods is necessary to ensure higher quality products. The objective of this dissertation was to develop a method to detect adulterants (rice, barley, corn and soybeans) in commercial samples of coffee, through the oligosaccharides profile and the techniques of Ultra Performance Liquid Chromatography coupled to Tandem Mass Spectrometry (UPLC-MS-MS). From standard solutions of five possible chemical markers (maltose, raffinose, stachyose, 1-kestose and nystose) in aqueous solution of 0.1% formic acid and acetonitrile (1:1) and in Milli-Q grade water, the operational parameters of the mass spectrometer and the chromatographic conditions were tested. The adopted chromatographic method consisted of chromatography by hydrophilic interaction and gradient elution of acetonitrile and aqueous solution of formic acid 0.1%, with injector temperature 20?C; injection volume 1.0 ?L; flow rate 0.5 mL/min; column temperature 35?C; and run time 10 min. For the spectrometric method, electrospray ionization was used in positive mode, with capillary, sampling and extraction cones voltages of, respectively, 3.0 kV, 50 V, 2.0 V; sample temperature 80?C; cone gas flow 40 L/h; temperature and flow of desolvation gas 300?C and 500 L/h; and collision energies of at least 15.0 V. The results indicated a good repeatability among analysis of standard solution, with selectivity by the real-time monitoring of the sequential mass spectra. Extracts from the previously roasted samples of adulterants were analyzed. It was confirmed the effect of temperature as an interfering factor for the detection of oligosaccharides. Only soybean presented as potential chemical markers raffinose and stachyose. For the grains of rice, barley and corn, another precursor ion of the same mass/charge ratio (m/z) of raffinose and 1-kestose was observed. It has been identified as maltotriose, in which isomeric differentiation can be ensured by different fragmentation profiles. The study of the mass spectra still ratified these results by the observation of a greater susceptibility of the rupture of ?, ? (1?2) bonds and the formation of ions fragments with saturated cyclic chain. However, through the use of the methodology, the chemical markers were not detected in the commercial samples of coffee, even with the samples previously disapproved for the presence of barley. The optimization of the extraction method or the inclusion of sodiated solvents may be necessary and, therefore, new experiments should still be performed, ensuring the applicability of the method by UPLC-MS-MS. / O caf? consiste em uma das bebidas mais apreciadas em todo o mundo, devido a suas caracter?sticas sensoriais e funcionais. Atualmente como principal produtor e exportador no mundo, estima-se que no Brasil haja crescimento no mercado interno para os pr?ximos anos, em que se predomina o caf? torrado e mo?do. Este tipo de produto ? prejudicado principalmente pela pr?tica de adultera??o com outras mat?rias-primas vegetais. A implementa??o de m?todos de maior sensibilidade e seletividade ? necess?ria para a garantia de produtos de maior qualidade. O objetivo desta disserta??o foi desenvolver um m?todo de detec??o de adulterantes (arroz, cevada, milho e soja) em amostras comerciais de caf?, atrav?s do perfil de oligossacar?deos e das t?cnicas de Cromatografia L?quida de Ultra Efici?ncia acoplada a Espectrometria de Massas Sequencial (CLUE-EM-EM). A partir de solu??es padr?o de cinco poss?veis marcadores qu?micos (maltose, rafinose, estaquiose, 1-kestose e nistose) em solvente solu??o aquosa de ?cido f?rmico a 0,1% e acetonitrila (1:1) e em ?gua grau Milli-Q, foram testados os par?metros operacionais do espectr?metro de massas e as condi??es cromatogr?ficas. O m?todo cromatogr?fico adotado consistiu no uso da cromatografia por intera??o hidrof?lica e elui??o em gradiente de acetonitrila e solu??o aquosa de ?cido f?rmico, com as condi??es de temperatura do injetor de 20?C; volume de inje??o de 1,0 ?L; fluxo de 0,5 mL/min; temperatura da coluna de 35?C; e tempo de corrida de 10 min. Para o m?todo espectrom?trico, empregou-se a ioniza??o por eletrospray em modo positivo, com voltagem no capilar, nos cones de amostragem e de extra??o de, respectivamente, 3,0 kV, 50 V, 2,0 V; temperatura da amostra de 80?C; fluxo do g?s do cone de 40 L/h; temperatura e fluxo do g?s de dessolvata??o de 300?C e 500 L/h; e energias de colis?o de, no m?nimo, 15,0 V. Os resultados indicaram uma boa repetitividade entre as an?lises das solu??es padr?o, com a seletividade sendo garantida pelo monitoramento em tempo real dos espectros de massas sequencial. Extratos das amostras previamente torradas dos adulterantes foram analisados, sendo confirmado o efeito da temperatura como um fator interferente para a detec??o dos oligossacar?deos. Apenas a soja apresentou como potenciais marcadores qu?micos rafinose e estaquiose. Para os gr?os de arroz, cevada e milho, foi observado outro ?on precursor de mesma rela??o massa/carga (m/z) da rafinose e da 1-kestose. Este foi identificado como a maltotriose, em que a diferencia??o isom?rica pode ser garantida pelos diferentes perfis de fragmenta??o. O estudo dos espectros de massas ainda ratificou estes resultados pela observa??o de uma maior facilidade da ruptura das liga??es ?,? (1?2) e de forma??o de ?ons fragmentos de cadeia c?clica saturada. Por?m, atrav?s do emprego da metodologia, n?o foram detectados os marcadores qu?micos nas amostras comerciais de caf? analisadas, mesmo com as amostras previamente reprovadas pela presen?a de cevada. A otimiza??o do m?todo de extra??o ou a inclus?o de solventes sodiados podem ser necess?rios e, por tal raz?o, novos experimentos ainda devem ser realizados, garantindo a aplicabilidade do m?todo por CLUE-EM-EM.
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Investigations into aspects of nod factor utilization for crop productionSupanjani January 2005 (has links)
No description available.
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Mesures parallélisées d'interactions oligosaccharides / protéines au moyen de biopucesMercey, Emilie 17 November 2005 (has links) (PDF)
Le but de cette thèse est de concevoir une puce à sucres, compatible avec un système optique d'imagerie en résonance des plasmons de surface (SPRi), afin d'analyser simultanément de multiples interactions protéine/oligosaccharide en temps réel. La réalisation d'un système modèle, ayant comme sucre sonde, l'héparine, est proposée. La modification du sucre par un pyrrole permet de le déposer sur une surface d'or par électrocopolymérisation. Cette chimie nécessite la construction de molécules « pyrrole - bras espaceurs » compatibles avec les propriétés des sucres utilisés. La puce utilisable en SPRi est ensuite fabriquée par électrocopolymérisations successives. Ces plots formés peuvent être caractérisés par MEB et par AFM. Des expériences préliminaires utilisant des traceurs fluorescents ont permis de valider le système ainsi que la chimie proposée ; la fluorescence observée est spécifique de l'interaction biologique grâce à la présence de négatifs. Les études d'interactions biologiques suivies par SPRi ont alors été réalisées ; la sensibilité de l'interaction est confirmée. Grâce à l'acquisition en temps réel des interactions, les cinétiques d'association et de dissociation du complexe oligosaccharide/protéine sont observées puis optimisées. De plus, les puces fabriquées sont régénérables mais aussi réutilisables sur plusieurs mois, ce qui permet l'étude des paramètres chimiques et biologiques. Cette approche est appliquée finalement à plusieurs problématiques biologiques.
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