FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Dentro das possibilidades terapÃuticas atuais para o tratamento de pacientes com problemas congÃnitos de metabolismo, a dieta constitui o pilar mais importante e a D-tagatose tem atraÃdo uma grande atenÃÃo nos Ãltimos anos devido a seus benefÃcios à saÃde humana, bem como à semelhanÃa de suas propriedades com a sacarose. Dentre as suas muitas aplicaÃÃes, ressalta-se o potencial em auxiliar no controle de peso, uma preocupaÃÃo crescente no Brasil, uma vez que a obesidade cresce a ritmos alarmantes. No entanto, a L-arabinose isomerase, enzima que catalisa isomerizaÃÃo de D-galactose em D-tagatose, ainda nÃo està disponÃvel comercialmente e, portanto, estudos visando à obtenÃÃo deste biocatalisador se fazem necessÃrios de maneira a viabilizar a implantaÃÃo do processo industrial. Portanto neste trabalho, estudou-se a obtenÃÃo da enzima L-arabinose isomerase utilizando uma cepa de Enterococcus faecium. A enzima produzida por fermentaÃÃo foi caracterizada e imobilizada em suportes a base de quitosana. Os resultados de estabilidade tÃrmica, operacional e de estocagem obtidos para a enzima imobilizada covalentemente sobre quitosana em meio alcalino (pH 10), confirmou a importÃncia do controle do pH durante a imobilizaÃÃo, uma vez que a formaÃÃo de uniÃes multipontuais à favorecida quando comparado ao pH 7,0. No entanto, baixas concentraÃÃes de proteÃna eram obtidas na etapa de fermentaÃÃo, portanto, estudou-se a produÃÃo da enzima L-AI de forma heterÃloga em Escherichia coli. As proteÃnas recombinantes expressas foram purificadas por cromatografia de afinidade em uma Ãnica etapa, e visualizadas em SDS-PAGE. O sucesso na construÃÃo do gene e na clonagem em vetores de expressÃo em E. coli resultou em quantidade satisfatÃria de expressÃo das proteÃnas recombinantes, pois apresentaram-se na forma solÃvel, facilmente purificadas e ativas, permitindo suas caracterizaÃÃes. Por ultracentrifugaÃÃo analÃtica foi possÃvel descobrir que a enzima L-AI recombinante tem uma tendÃncia para formaÃÃo de estruturas com maior tamanho (oligÃmeros). A seguir, suportes multifuncionais foram preparados para a imobilizaÃÃo da L-AI e observou-se uma rÃpida imobilizaÃÃo, apresentando um elevado rendimento de imobilizaÃÃo, superior a 75%. Devido à baixa estabilidade tÃrmica dos derivados, estudos futuros serÃo necessÃrios para a estabilizaÃÃo da estrutura quaternÃria desta enzima. / Within the current therapeutic options for treatment of patients with congenital metabolic problems, diet is the most important pillar and D-tagatose has attracted great attention in recent years because of its benefits to human health and due to its similarities with sucrose. Among its many applications, it emphasizes the potential to assist in weight management, a growing concern in Brazil, since obesity is growing at alarming rates. However, L-arabinose isomerase, the enzyme that catalyses the isomerization of D-galactose into D-tagatose, is not yet commercially available and therefore studies in order to obtain this biocatalyst are necessary in order to enable the implementation of the industrial process. Therefore, in this work, the production of L-arabinose isomerase by Enterococcus faecium was investigated. The produced enzyme was characterized and immobilized onto chitosan. Results of thermal, operational stability and self-life obtained by using L-AI, covalently immobilized onto chitosan in an alkaline medium (pH 10), confirmed the importance of the pH during immobilization, since multipunctuality is favored compared to pH 7.0. Nevertheless, enzyme concentration after fermentation was low and, therefore, we have studied the production of heterologous enzyme in Escherichia coli. The expressed recombinant proteins were purified by affinity chromatography by a single step, and displayed as a single band on SDS-PAGE. The successful construction of the gene and cloning into expression vectors in E. coli resulted in higher amount of the recombinant proteins, which are soluble, easily purified and active, allowing their characterization. Through analytical ultracentrifugation, it was possible to find that the recombinant L-AI has a tendency to form larger structures (oligomers). Multifunctional supports were prepared to L-AI immobilization, allowing achieving high yields (more than 75%) at short contact times. Due to the low thermal stability of the immobilized enzyme, future studies will be needed to stabilize its quaternary structure.

O hidrogênio (H2) tem sido considerado uma fonte de energia limpa bastante promissora, pois sua combustão origina apenas moléculas de água, sendo uma alternativa ao uso de combustíveis fósseis. Entretanto, os métodos atuais de produção de H2 demandam matérias-primas finitas e uma grande quantidade de energia, tornando a sua obtenção não sustentável. Mais recentemente, a via fermentativa tem sido considerada para a produção de H2, utilizando como matérias-primas efluentes industriais, materiais lignocelulósicos e biomassa de algas, denominado de bio-hidrogênio de primeira, segunda e terceira geração, respectivamente. Neste trabalho foi isolada uma bactéria anaeróbia a partir de uma cultura mista (lodo) de um sistema de tratamento de vinhaça, após pré-tratamento do lodo a pH 3 por 12 horas. Este microrganismo foi identificado com 99% de similaridade como Clostridium beijerinckii com base na sequência do gene RNAr 16S denominado de C. beijerinckii Br21. A temperatura e o pH mais adequados para o crescimento e produção de H2 por esta cultura foi 35 °C e pH inicial 7,0. A bactéria possui a capacidade de utilizar ampla variedade de fontes de carbono para a produção de H2 por fermentação, especialmente, monossacarídeos resultantes da hidrólise de biomassa de algas, tais como glicose, galactose e manose. Foram realizados ensaios em batelada para a produção de H2 com a bactéria isolada empregando diferentes concentrações de glicose e galactose, visando a sua futura utilização em hidrolisados de alga. Os parâmetros cinéticos dos ensaios de fermentação estimados pelo modelo de Gompertz modificado, como a velocidade máxima de produção (Rm), a quantidade máxima de hidrogênio produzido (Hmáx) e o tempo necessário para o início da produção de hidrogênio (fase lag) para a glicose (15 g/L) foram de: 58,27 mL de H2/h, 57,68 mmol de H2 e 8,29 h, respectivamente. Para a galactose (15 g/L), a Rm, Hmáx e foram de 67,64 mL de H2/h, 47,61 mmol de H2 e 17,22 horas, respectivamente. O principal metabólito detectado ao final dos ensaios de fermentação, foi o ácido butírico, seguido pelo ácido acético e o etanol, tanto para os ensaios com glicose, como com galactose. C. beijerinckii é um candidato bastante promissor para a produção de H2 por fermentação a partir de glicose e galactose e, consequentemente, a partir de biomassa de algas como substratos. / Hydrogen (H2), considered an alternative to fossil fuels, is a promising source of clean energy because its combustion originates water molecules only. However, the current H2 production methods require finite raw materials and a large amount of energy, which makes them unsustainable. The fermentative pathway has been considered for H2 production from renewable raw materials such as industrial wastewater, lignocellulosic materials, and algal biomass, the so-called first, second, and third bio-hydrogen generation, respectively. In this work, after pre-treatment at pH 3 for 12 h, a H2-producing bacterium was isolated from a mixed culture (sludge) collected from an anaerobic bioreactor used to treat sugarcane vinasse. The microorganism was identified as Clostridium beijerinckii based on the sequence of the 16S rRNA gene; it was named C. beijerinckii Br21. The most appropriate temperature and initial pH to achieve H2 production by this strain was 35 °C and 7, respectively. The bacterium was able to use a wide variety of carbon sources, especially the monosaccharides glucose, galactose, and mannose resulting from hydrolysis of algal biomass. Batch assays using different concentrations of glucose and galactose were performed to produce H2. The kinetic parameters of the tests were estimated by the Gompertz modified model. The maximum production rate (Rm), the maximum amount of produced H2 (Hmáx), and the phase lag () for glucose and galactose, both at 15 g/L, were 58.27 and 67.64 mL of H2/h, 57.68 and 47.61 mmol of H2, and 8.29 and 17.22 h, respectively. The main metabolite detected at the end of fermentation tests was butyric acid, followed by acetic acid and ethanol. The results indicated that the new C. beijerinckii isolate is a promising candidate for fermentative H2 production from algal biomass.

Thesis (MSc)--Stellenbosch University, 2001 / ENGLISH ABSTRACT: The effect of various organic reducing agents, in the. form of monosaccharide reducing sugars, on the rate of atom transfer radical polymerization (ATRP) of n-butyl methacrylate and methyl methacrylate is reported in this study. The addition of the reducing sugars has a positive effect on the rate of ATRP. Up to 100% increase in the rate of polymerization was recorded, in some cases. These organic reducing agents have little effect on the molecular weight and molecular weight distribution of the polyin-butyl methacrylate) and polydispersity indexes remain well below 1.2. The molecular weight of the poly(methyl methacrylate), when glucose and galactose are added to the reaction mixture, compares well with the theoretical expected values. An explanation for these observations is the ability of the reducing sugars to reduce part of the Cu(II) species, that serves to deactivate the growing radicals, to Cu(I), thereby ensuring a shift in the equilibrium between active and dormant chains in the direction of the former and a resulting increase in the rate of polymerization. uvNIS spectroscopy and cyclic voltammetry were used to investigate the mechanism behind the polymerization rate enhancement. / AFRIKAANSE OPSOMMING: In hierdie studie word die effek van verskeie organiese reduseermiddels, in die vorm van monosakkaried reduserende suikers, op die tempo van polimerisasie van ATRP gerapporteer. Hierdie reduserende suikers het 'n positiewe effek op die polimerisasie tempo. In sommige gevalle word 'n toename van 100% in die polimerisasie tempo waargeneem. Die organiese reduseermiddels het 'n minimale effek op die molekulere massa en molekulere massa verspreiding (in meeste gevalle minder as 1.2) van die poly(n-butiel metakrielaat). In die geval van die poly(metiel metakrielaat), wanneer glukose en galaktose by die reaksie mengsel gevoeg word, stem die molekulere massas goed ooreen met die teoreties voorspelde molekulere massas. Die waargenome toename in die polimerisasie tempo kan toegeskryf word aan die vermoe van die reduserende suikers om die Cu(II), wat dien om die groeiende radikale te deaktiveer, gedeeltelik te reduseer na Cu(l). Hierdeur word verseker dat die ewewig tussen die aktiewe en dormante kettings in die rigting van die eersgenoemde verskuif word, wat dus aanleiding gee tot 'n toename in die polimerisasie tempo. Ultraviolet spektroskopie en sikliese voltammetrie is ook gebruik om lig te werp op die meganisme agter die toename in die tempo van polimerisasie.

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.

Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.

Cost-effective processing of dairy whey permeates is important to the environment and economics of the agriculture industry in Canada. Bioconversion of whey permeates is an attractive means of obtaining value-added adjuncts with improved nutritional and functional properties. In the past, cost-effective technologies to recover additional value from whey permeates at a low cost were lacking. Currently, such a technological platform is now feasible with the introduction of new modern bioconversion technologies that incorporate batch or continuous bioreactors, and use ultra- and nano-filtration membranes for the separation of whey permeate components. In this dissertation, a novel processing methodology is described. This methodology, which is a desirable configuration for food manufacturers includes a stirred batch nanomembrane bioreactor equipped with a crossflow nanomembrane and offers lactose bioconversion with an immobilized biocatalyst, product separation, and biocatalyst recovery in a batch operation. The major focus of this research was on: a) the development of a new analytical methodology for carbohydrate measurement during the lactose bioconversion process, b) the selection, testing and integration of highly selective nanomembranes to separate the desired substrates, whey permeate carbohydrates, from the reaction mixture, and c) the production of a stable and highly active and specific immobilized biocatalyst. Noticeably, this methodology was designed, developed and tested for the bioconversion of lactose, but could also be used for the bioconversion of other carbohydrate feedstocks. The food industry in Canada needs an integrated approach to achieve complete lactose reclamation and use. This research project offers such a solution. The research described in this dissertation presents an integrated model of a stirred batch bioreactor that may support not only current, but also future research, and may economically impact the development and bioconversion of whey permeates containing lactose. This may lead to the development of a continuous processing methodology for low cost recovery of lactose from whey permeates and simultaneous conversion to value-added products. / Bioresource and Food Engineering

No description available.

Reproduction de : Thèse de doctorat : Médecine. Biologie cellulaire et moléculaire : Nantes : 2009. / Bibliogr.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1968. / Includes bibliographical references (p. 48-50).

published_or_final_version / abstract / toc / Anatomy / Doctoral / Doctor of Philosophy