Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-21Bitstream added on 2014-06-13T19:40:51Z : No. of bitstreams: 1 giese_ec_dr_sjrp.pdf: 879284 bytes, checksum: e28308bffc3e725de127885e5249a777 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As β-glucanas fúngicas apresentam uma série de respostas biológicas. Estas glucanas apresentam baixa solubilidade em meio aquoso, sendo necessário fragmentá-las em moléculas de menor peso molecular. O Botryosphaeria rhodina MAMB-05 é um ascomiceto ligninolítico produtor de uma β-(1→3)(1→6)-glucana denominada botriosferana (EPS). Estudos preliminares demonstraram que o fungo Trichoderma harzianum Rifai é capaz de crescer em EPS como única fonte de carbono e de produzir β-glucanases específicas. As condições para a produção de -1,3-glucanases pelo T. harzianum Rifai em fermentador de bancada utilizando EPS como única fonte de carbono foram otimizadas através do uso de um planejamento fatorial e análise por superfície de resposta, o qual mostrou que a produção máxima de -1,3-glucanases ocorreu em 5 dias de cultivo, com pH inicial igual a 5,5 e com aeração de 1,5 vvm. As enzimas do complexo -glucanolítico extracelular foi parcialmente purificado e utilizado para a hidrólise de botriosferana, laminarina, paramilo e pustulana. Duas frações com atividade para -glucanase (F-I e F-II) foram obtidas a partir da cromatografia de filtração em gel, as quais apresentaram diferentes modos de ação sobre o botriosferana e a laminarina. O botriosferana foi hidrolisado em cerca de 66 % pela fração F-I e em 98 % pela fração F-II, em 30 min. Os produtos de hidrólise foram principalmente constituídos por gluco-oligossacarídeos (GP ≥ 4), e menor quantidade de glucose, di- e trissacarídeos. A ação enzimática das frações F-I e F-II sobre a laminarina resultaram em 15 % de conversão do polímero em glucose, enquanto a porcentagem de sacarificação foi totalmente diferente (70 % para F-I e 25 % para F-II). Em paramilo, ambas as frações promoveram a degradação de aproximadamente 20 % do polissacarídeo após 30 min, onde somente... / Fungal β-D-glucans presents a variety of biological response. These glucans presents low solubility in water and being necessary to obtain fragments with small molecular weight. A ligninolytic ascomyceteous Botryosphaeria rhodina MAMB-05 produces a (1→3)(1→6)-β-D-glucan named botryosphaeran (EPS). Preliminary studies verified that Trichoderma harzianum Rifai is able to grown on EPS as sole carbon source and secrete specific β-glucanases to act on this subtract. Conditions for -1,3-glucanase production by T. harzianum Rifai in bench-fermenter using EPS as sole carbon source were developed using a statistical factorial design, and analysis by response surface method, which showed maximal enzyme production at 5 days growth in a minimum Vogel salts medium, with initial pH 5.5 under 1.5 vvm aeration. A -glucanolytic extracellular complex was partially-purified and used to hydrolyze fungal botryosphaeran, algal laminarin, algal paramylon and the liquen pustulan. Two -glucanase fractions (F-I and F-II) were obtained by gel permeation chromatography, which presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was 66 % hydrolyzed by the F-I fraction, and 98 % by fraction F-II, within 30 min. The main products of hydrolysis were gluco-oligosaccharides (DP ≥ 4), and lower amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted 15 % of conversion to glucose, while the percentage of saccharification was radically different (70 % for F-I and 25 % for F-II). On paramylon, both fractions promoted approximately 20 % degradation after 30 min, and only 0.5 % corresponded to gluco-oligosaccharides (DP ≥ 4). Only F-I fraction could acted on pustulan resulting 25 % of glucose, gentiobiose and oligosaccharides (not identified), within 30 min. The difference in the hydrolysis... (Complete abstract click electronic access below)

<p>An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase ¹⁹F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. </p><p>The influence of resin structures for seven commercial resins upon resolution of gel-phase ¹⁹F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.</p>

An efficient and versatile non-destructive method to analyze the progress of solid-phase glycoconjugate synthesis with gel-phase 19F NMR spectroscopy is described. The method relies on use of fluorinated linkers and building blocks carrying fluorinated protective groups. Commercially available fluorinated reagents have been utilized to attach the protective groups. The influence of resin structures for seven commercial resins upon resolution of gel-phase 19F NMR spectra was investigated. Two different linkers for oligosaccharide synthesis were also developed and successfully employed in preparation of α-Gal trisaccharides and a n-pentenyl glycoside. Finally, reaction conditions for solid-phase peptide glycosylations were established.

Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

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.

Les structures sialylées sont présentes à la surface des cellules sous forme de glycoconjugués,couplés à des protéines ou des lipides. Ces structures jouent un rôle important dans divers processusbiologiques que ce soit à travers l’interaction avec des lectines, ou de par leurs propriétés physicochimiques.Ces structures sont également impliquées dans diverses pathologies et on constatenotamment une forte augmentation du taux d’acides sialiques chez les individus atteints de cancer,due à une surexpession de structures naturelles mais aussi à l’apparition de nouveaux motifs,naturellement absent chez l’individu sain. L’ensemble de ces structures sialylées présente un intérêtsoit par leur rôle biologique soit à cause de leur expression spécifique dans les cancers. Leurobtention est très difficile par voie chimique et la synthèse enzymatique in vitro est efficace mais trèscoûteuse en nucléotide-sucre et ne sont pas adaptées à une production à l'échelle préparative.Dans un premier temps, ces travaux de thèse s’intéressent à la synthèse bactérienne par ingénieriemétabolique d'acides polysialiques fonctionalisés. Ces polysaccharides présentent divers intérêts.Tout d’abord il est possible de les coupler à des protéines actives pour en augmenter le temps dedemi-vie in vivo. Mais ces polysaccharides peuvent également être utilisés dans le cadre de thérapievaccinale, soit contre des bactéries pathogènes de types Neisseria meningitidis qui le présententcomme polysaccharide capsulaire, soit contre les cellules cancéreuses surexprimant cette structure.Ensuite nous avons cherché à obtenir des oligosaccharides spécifiques des cancers, les motifssialylTn, et siallTF, toujours par ingénierie métabolique d’E. coli. Le sialylTn a été couplé à uneplateforme peptidique immunogène afin de construire un candidat vaccin qui a été testé in vitro et invivo sur la souris. / Sialylation is an important feature of glycolipids and glycoproteins of animal cell surfaces. Sialylatedmotifs are involved in many biological processes through lectin interactions or because of theirphysico-chemical properties. There is a great variety of sialylated structural motifs, and in manycases, there is a structure-relationship between the sialylated profile of and some pathologicprocesses. In cancer, there is an increase of sialylation including the apparition of newly andspecifically related sialylated structures belonging to the so-called tumor-associated carbohydrateantigens (TACA). Those structures present a particular interest, but their chemical or chemoenzymaticsynthesis is costly and quite unappropriated for preparative scale.This work addresses to the bacterial synthesis of sialylated motifs through the metabolic engineeringof Escherichia coli. The first part of the thesis deals with the biosynthesis of polysialylatedconjugatable motifs. Those motifs present various biological properties, such as an increase of thelife-time of therapeutic proteins; they also belong to the TACA family since over-expressed incancers. In addition, some of them are bacterial-specific motifs such as in pathogenic Neisseriameningitidis. Altogether, polysialylated conjugates can be useful for the synthesis of therapeuticdrugs and vaccines. The second part of the thesis describes a new way of producing sialylated Tn andTF carbohydrate antigens by metabolic engineering. The sialylTN motif was coupled to a peptidic andimmunogenic scaffold being a potential vaccine candidate, and its ability of raising specific antibodieswas assayed in mouse.

Orientador: Cláudio Cabello / Banca: Fernando Broetto / Banca: Magali Leonel / Banca: Ana Paula Cerino Coutinho / Banca: Mariana Schimidt Rechsteiner / Resumo: Os produtos das hidrólises de amido são glicose, maltose e uma série de oligossacarídeos e polissacarídeos que encontram utilização principalmente na indústria de alimentos. Neste grupo enquadram-se os adoçantes que aditam sabores a produtos que são demandados por consumidores específicos. Atualmente o açúcar mais utilizado no Brasil é a sacarose, produto extraído da cana-de-açúcar, e o mais utilizado mundialmente é a frutose obtida a partir da hidrólise do milho e posterior isomerização da glicose para frutose. A frutose apresenta capacidade edulcorante 30% maior que a sacarose, 2,5 vezes maior que a glicose e 2 vezes mais solúvel que a glicose, com isso, pode ser utilizada em menor quantidade, diminuindo o poder calórico do alimento e viabilizando sua utilização no tratamento da obesidade. Levando em consideração a importância dos adoçantes para o mercado de alimentos, o presente trabalho teve como objetivo realizar estudos sobre o processo de obtenção da frutose a partir de amido de mandioca. Para a execução dos ensaios utilizou-se fontes comerciais de α- amilase e amiloglucosidase, Liquozyme Supra 2.2X e Saczyme 750 AGUg-1, respectivamente, aplicadas em substrato de amido de mandioca em reator agitado com temperatura controlada. Após o processo de hidrólise enzimática, o hidrolisado passou por um processo de purificação utilizando terra diatomácea e carvão ativado em quatro temperaturas (30, 40, 50 e 60°C), com a finalidade de remoção de contaminantes originários da matéria prima, que levam a odor, cor e sabores indesejáveis. Após o tratamento com carvão ativo e terra diatomácea foram realizados ensaios para estabelecer os melhores parâmetros para a realização do processo de isomerização, buscando a conversão de parte da glicose à frutose, utilizando a enzima... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The products of starch hydrolyses are glucose, maltose and a series of oligosaccharides and polysaccharides which have their main utilization in food industry. This group comprises sweeteners that add flavor to products demanded by specific consumers. Currently the most used sugar in Brazil is sucrose, a product extracted from sugarcane, while the most used sugar worldwide is fructose obtained from maize hydrolysis and subsequent glucose isomerization to fructose. The sweetening capacity of fructose is 30% higher than that of sucrose and 2.5-fold higher than that of glucose; in addition, fructose is 2-fold more soluble than glucose and thus can be used in smaller quantities, decreasing the food's caloric potential and making its use viable in obesity treatment. Considering the importance of sweeteners for the food market, the present study aimed to investigate the process of fructose production from cassava starch. The assays were performed by using commercial sources of α-amylase and amyloglucosidase, Liquozyme Supra 2.2X and Saczyme 750 AGUg-1, respectively, applied to cassava starch substrate in an agitated reactor at controlled temperature. Following the process of enzymatic hydrolysis, the hydrolysate underwent a purification process using diatomaceous earth and activated charcoal at four temperatures (30, 40, 50 and 60°C), in order to remove contaminants originated from the raw material, which lead to undesirable smell, color and flavor. After the treatment with activated charcoal and diatomaceous earth, assays were carried out to establish the best parameters for the isomerization process, aiming at the conversion of part of glucose into fructose, using the enzyme isomerase. The process selected for the studies was in a continuous system where the glucose syrup, previously purified, was continuously pumped... (Complete abstract click electronic access below) / Doutor

Submitted by Bruna Rodrigues (bruna92rodrigues@yahoo.com.br) on 2016-09-26T14:51:02Z No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-26T18:48:09Z (GMT) No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-26T18:48:18Z (GMT) No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) / Made available in DSpace on 2016-09-26T18:48:26Z (GMT). No. of bitstreams: 1 DissMAF.pdf: 3997675 bytes, checksum: 3238bd54da33e9d0c201deb533005fa2 (MD5) Previous issue date: 2015-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Xylooligosaccharides (XOS) are short-chain polymers of xylose (2 to 7 units) which can be produced by enzymatic hydrolysis of the xylan from the lignocellulosic feedstocks. XOS have a great potential as probiotic ingredients, and when they are incorporated in diets, they can provide many health benefits. The worldwide interest in the use of lignocellulosic residues is constantly growing, and in this scenario the soybean hull arises as a potential residue of the Brazilian agroindustry. The bioconversion of these residues to value-added products requires suitable pretreatments to deconstruct/disorganize the recalcitrant lignocellulosic complex, separating its main fractions: cellulose, hemicellulose, and lignin. In this context, this work did evaluate different biomass pretreatments aiming to produce XOS by the action of a Bacillus subtilis endoxylanase. Initially, the conditions for maximum catalytic activity of this enzyme were evaluated changing pH, buffer, and temperature. Among these parameters, 50 mM citrate buffer, pH 5.5, and 45 oC were the one that gave highest activity. The in nature soybean hull (previously chemically characterized) was hydrolyzed with soluble endoxylanase with different enzyme loads (40, 80, and 100 U/g biomass) under preestablished pH and temperature, producing around 55 mg RS/g dry biomass. This result, though little expressive, showed the viability of XOS production from soybean hull. However, this approach requires a suitable pretreatment of the lignocellulosic biomass to improve the endoxylanase accessibility to the C-5 fraction. Several pretreatments were performed in the soybean hulls, such as, enzymatic deproteinization, hydrogen peroxide/acetic acid pretreatment, and organosolv-ethanol pretreatment. For some pretreatments, reagent concentration and reaction time were evaluated, as well as, sequential pretreatment. Besides, enzymatic hydrolysis of the in nature soybean hull under microwave irradiation was also evaluated. The deproteinization of the soybean hull was not very efficient to the enzymatic hydrolysis of the remnant solid (production of 30 mg RS/dry biomass). However, this pretreatment allows the protein recovery as a high nutritional value hydrolysate. The pretreatment of the deproteinized soybean hulls with hydrogen peroxide solution (5 M, 1 h) removed 56% lignin without cellulose losses. However, this pretreatment did not contribute to an efficient action of the endoxylanase to the hemicellulose fraction (production of around 30 mg RS/g dry biomass). The organosolv-(50% v/v)ethanol pretreatment of the deproteinized soybean hulls promoted the removal of around 50% lignin, with low solubilization of hemicellulose (<17%), producing a poor substrate for the endoxylanase. The organosolv pretreatments with 50 and 70% (v/v) ethanol of the in nature soybean hull were able to solubilize around 30% hemicellulose, allowing the production of around 76 and 49 mg RS/g dry biomass, respectively, after hydrolysis with endoxylanase. Finally, the microwave action on the lignocellulosic biomass probably decreased the biomass recalcitrance, because the hydrolysis of the in nature soybean hulls catalyzed by the endoxylanase (100 IU / g of biomass) yielded approximately 100 mg of RS/g dry biomass. On the other hand, the hydrolysis performed in a reactor under conventional heating produced only 52 mg RS/g dry biomass. The results of this work did show that the combination of microwave irradiation and enzymatic hydrolysis might be a promising alternative to produce XOS. Keywords: soybean hulls; xylo-oligosaccharides; pretreatments / Xilo-oligossacarídeos (XOS) são polímeros de xilose de cadeia curta (2 a 7 unidades) que podem ser obtidos por hidrólise enzimática da xilana presente na fração de hemicelulose dos materiais lignocelulósicos. XOS possuem um grande potencial como ingredientes prebióticos, e quando incorporados na dieta, podem fornecer muitos benefícios à saúde. O interesse mundial no aproveitamento de resíduos lignocelulósicos é cada vez maior, e no cenário nacional a casca de soja se destaca como um potencial resíduo da agroindústria brasileira. Para viabilizar a bioconversão desses resíduos em produtos de interesse comercial (etanol 2G e XOS, por exemplo) são necessários pré-tratamentos, que atuam desconstituindo/desorganizando a estrutura altamente recalcitrante do complexo lignocelulósico e separando as frações principais da biomassa: celulose, hemicelulose e lignina. Neste contexto, este trabalho teve por objetivo avaliar diferentes pré-tratamentos da biomassa para produzir sequencialmente XOS por ação de uma endoxilanase de Bacillus subtilis. Inicialmente as condições de máxima atividade catalítica dessa enzima foram avaliadas variando pH, tampão e temperatura. Dentre as variáveis estudadas, as que contribuíram para uma melhor atividade da endoxilanase foram tampão citrato de sódio (50mM) pH 5,5 e 45 °C. A casca de soja in natura (previamente caracterizada quimicamente) foi hidrolisada com endoxilanase solúvel com diferentes cargas enzimáticas (40, 80 e 100 U/g casca) nas condições de pH e temperatura pré-estabelecidas, produzindo em média 55 mg de AR/g biomassa seca. Esse resultado, embora pouco expressivo, demonstrou a viabilidade da produção de XOS a partir de casca de soja, requerendo, entretanto, um pré-tratamento adequado para melhorar a acessibilidade da endoxilanase à fração C-5 da biomassa. Os pré-tratamentos avaliados foram a desproteinização enzimática da casca, prétratamento com peróxido de hidrogênio e ácido acético e pré-tratamento organossolve-etanol, variando nestes, as concentrações de solventes, tempo de reação e pré-tratamentos sequenciais. Adicionalmente, realizou-se a hidrólise enzimática da casca de soja in natura em reator micro-ondas. A desproteinização da casca de soja mostrou-se ineficiente para a hidrólise da fração sólida remanescente com endoxilanase (produção de 30 mg de AR/g biomassa seca), embora esse pré-tratamento permita a recuperação de proteínas como um hidrolisado de alto valor nutricional. O pré-tratamento com peróxido de hidrogênio (5 M, 1 h) para casca de soja desproteinizada removeu 56% de lignina sem perdas de celulose, entretanto, este pré-tratamento não contribuiu para uma eficiente atuação da endoxilanase sobre a fração hemicelulósica (produção de aproximadamente 30 mg de AR/g biomassa seca). O pré-tratamento organossolve-etanol 50% (v/v) da casca de soja desproteinizada removeu em torno de 50% de lignina com baixa solubilização de hemicelulose (< 17%), gerando, portanto, um líquido com baixa concentração de substrato para a ação da endoxilanase. Os pré-tratamentos organossolve-etanol 50 e 70% (v/v) da casca de soja in natura foram capazes de solubilizar em torno de 30% da hemicelulose, sendo possível a produção de 76 e 49 mg de AR/g de biomassa seca, respectivamente, após hidrólise com endoxilanase. Por fim, a ação das microondas sobre a biomassa lignocelulósica provavelmente reduziu a recalcitrância da biomassa, pois a hidrólise da casca in natura com endoxilanase (100 U/g de casca) produziu aproximadamente 100 mg de AR/g de biomassa seca, ao contrário da hidrólise conduzida em reator com aquecimento convencional que produziu em torno de 52 mg de AR/g de biomassa seca. Os resultados deste trabalho indicam que a combinação de irradiação micro-ondas e hidrólise enzimática pode ser uma alternativa promissora para a produção de XOS.

Resumo: As β-glucanas fúngicas apresentam uma série de respostas biológicas. Estas glucanas apresentam baixa solubilidade em meio aquoso, sendo necessário fragmentá-las em moléculas de menor peso molecular. O Botryosphaeria rhodina MAMB-05 é um ascomiceto ligninolítico produtor de uma β-(1→3)(1→6)-glucana denominada botriosferana (EPS). Estudos preliminares demonstraram que o fungo Trichoderma harzianum Rifai é capaz de crescer em EPS como única fonte de carbono e de produzir β-glucanases específicas. As condições para a produção de -1,3-glucanases pelo T. harzianum Rifai em fermentador de bancada utilizando EPS como única fonte de carbono foram otimizadas através do uso de um planejamento fatorial e análise por superfície de resposta, o qual mostrou que a produção máxima de -1,3-glucanases ocorreu em 5 dias de cultivo, com pH inicial igual a 5,5 e com aeração de 1,5 vvm. As enzimas do complexo -glucanolítico extracelular foi parcialmente purificado e utilizado para a hidrólise de botriosferana, laminarina, paramilo e pustulana. Duas frações com atividade para -glucanase (F-I e F-II) foram obtidas a partir da cromatografia de filtração em gel, as quais apresentaram diferentes modos de ação sobre o botriosferana e a laminarina. O botriosferana foi hidrolisado em cerca de 66 % pela fração F-I e em 98 % pela fração F-II, em 30 min. Os produtos de hidrólise foram principalmente constituídos por gluco-oligossacarídeos (GP ≥ 4), e menor quantidade de glucose, di- e trissacarídeos. A ação enzimática das frações F-I e F-II sobre a laminarina resultaram em 15 % de conversão do polímero em glucose, enquanto a porcentagem de sacarificação foi totalmente diferente (70 % para F-I e 25 % para F-II). Em paramilo, ambas as frações promoveram a degradação de aproximadamente 20 % do polissacarídeo após 30 min, onde somente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fungal β-D-glucans presents a variety of biological response. These glucans presents low solubility in water and being necessary to obtain fragments with small molecular weight. A ligninolytic ascomyceteous Botryosphaeria rhodina MAMB-05 produces a (1→3)(1→6)-β-D-glucan named botryosphaeran (EPS). Preliminary studies verified that Trichoderma harzianum Rifai is able to grown on EPS as sole carbon source and secrete specific β-glucanases to act on this subtract. Conditions for -1,3-glucanase production by T. harzianum Rifai in bench-fermenter using EPS as sole carbon source were developed using a statistical factorial design, and analysis by response surface method, which showed maximal enzyme production at 5 days growth in a minimum Vogel salts medium, with initial pH 5.5 under 1.5 vvm aeration. A -glucanolytic extracellular complex was partially-purified and used to hydrolyze fungal botryosphaeran, algal laminarin, algal paramylon and the liquen pustulan. Two -glucanase fractions (F-I and F-II) were obtained by gel permeation chromatography, which presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was 66 % hydrolyzed by the F-I fraction, and 98 % by fraction F-II, within 30 min. The main products of hydrolysis were gluco-oligosaccharides (DP ≥ 4), and lower amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted 15 % of conversion to glucose, while the percentage of saccharification was radically different (70 % for F-I and 25 % for F-II). On paramylon, both fractions promoted approximately 20 % degradation after 30 min, and only 0.5 % corresponded to gluco-oligosaccharides (DP ≥ 4). Only F-I fraction could acted on pustulan resulting 25 % of glucose, gentiobiose and oligosaccharides (not identified), within 30 min. The difference in the hydrolysis... (Complete abstract click electronic access below) / Orientador: Roberto da Silva / Coorientador: Aneli de Melo Barbosa / Banca: Maria de Lourdes Corradi Custódio da Silva / Banca: João Ruggiero Neto / Banca: Rubens Monti / Banca: Inês Conceição Roberto / Doutor

Made available in DSpace on 2014-12-17T15:01:46Z (GMT). No. of bitstreams: 1 MariaGBP.pdf: 1032728 bytes, checksum: 535260d4afcd2c2deeacab5b5006e73c (MD5) Previous issue date: 2008-10-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The obtaining of the oligosaccharides from chitosanase, has showed interest of the pharmaceutical area in the last years due their countless functional properties. Although, the great challenge founded out is how to keep a constant and efficient production. The alternative proposed by this present work was to study the viability to develop an integrated technology, with reduced costs. The strategy used was the obtaining of the oligomers through enzymatic hydrolysis using chitosanolitic enzymes obtained straight from the fermented broth, eliminating this way the phases involved in the enzymes purification. The two chitosanases producing strains chosen for the work, Paenibacillus chitinolyticus and Paenibacillus ehimensis, were evaluated according to the behavior in the culture medium with simple sugar and in relation to the pH medium variations. The culture medium for the chitosanases induction and production was developed through addition of soluble chitosan as carbon source. The soluble chitosan was obtained using hydrochloric acid solution 0.1 M and afterwards neutralization with NaOH 10 M. The enzymatic complexes were obtained from induction process in culture medium with 0.2% of soluble chitosan. The enzymes production was verified soon after the consumption of the simple sugars by the microorganisms and the maximum chitosanolitic activity obtained in the fermented broth by Paenibacillus chitinolyticus was 249 U.L-1 and by Paenibacillus ehimensis was 495U.L-1. These two enzymatic complexes showed stability when stored at 20?C for about 91 days. The enzymes in the fermented broth by Paenibacillus chitinolyticus, when exposed at temperature of 55?C and pH 6.0, where the activity is maximum, showed 50% lost of activity after 3 hours Meanwhile, for the complex produced by Paenibacillus ehimensis, after 6 days of exposure, it was detected 100% of the activity. The chito-oligosaccharides obtained by the hydrolysis of a 1% chitosan solution, using the enzymatic complex produced by Paenibacillus chitinolyticus showed larger quantity after 9 hours hydrolysis and using the complex produced by Paenibacillus ehimensis after 20 minutes was observed the chito-ligosacharides with polymerization degree between 3 and 6 units. Evaluating these results, it was verified that the production of chitosan-oligosaccharides is possible, using a simultaneous process / A obten??o de oligossacar?deos, a partir da quitosana, tem despertado interesse da ?rea farmac?utica nos ?ltimos anos devido as suas in?meras propriedades funcionais. Por?m, o grande desafio encontrado ? manter uma produ??o constante e eficiente. A alternativa proposta por este trabalho foi estudar a viabilidade de desenvolver uma tecnologia integrada e de baixo custo. A estrat?gia utilizada foi a obten??o dos olig?meros por meio de hidr?lise enzim?tica utilizando enzimas quitosanol?ticas obtidas diretamente do caldo fermentado, eliminando dessa forma as etapas envolvidas na purifica??o de enzimas. As duas cepas produtoras de quitosanases selecionadas para o trabalho, Paenibacillus chitinolyticus e Paenibacillus ehimensis, foram avaliadas quanto ao comportamento em meio de cultivo contendo a??cares simples e em rela??o as varia??es de pH do meio. O meio de cultivo para a indu??o e produ??o das quitosanases foi desenvolvido atrav?s da adi??o de quitosana sol?vel como fonte de carbono. A quitosana sol?vel foi obtida utilizando solu??o de ?cido clor?drico 0,1 M e posterior neutraliza??o com NaOH 10 M. Os complexos enzim?ticos foram obtidos a partir de processos de indu??o em meio de cultivo contendo 0,2% de quitosana sol?vel. A produ??o das enzimas foi observado logo ap?s o t?rmino do consumo dos a??cares simples pelos microrganismos e a m?xima atividade quitosanol?tica obtida no caldo fermentado pelo Paenibacillus chitinolyticus foi de 249 U.L-1 e pelo Paenibacillus ehimensis foi de 495 U.L-1. Esses dois complexos enzim?ticos apresentaram estabilidade quando armazenadas a -20?C por at? 91 dias. As enzimas presentes no caldo fermentado pelo Paenibacillus chitinolyticus, quando expostas ? temperatura de 55?C e pH 6,0, onde a atividade ? m?xima, apresentaram perda de 50% da atividade ap?s 3 horas. Enquanto que, para o complexo produzido pelo Paenibacillus ehimensis, ap?s 6 dias de exposi??o, 100% da atividade foi detectada. Os quito-oligossacar?deos obtidos a partir da hidr?lise de uma solu??o de 1% de quitosana, utilizando o complexo enzim?tico produzido pelo Paenibacillus chitinolyticus, apresentaram-se em maior quantidade ap?s 9 horas de hidr?lise e utilizando o complexo produzido pelo Paenibacillus ehimensis, ap?s 20 minutos pode-se observar os quito-oligossacar?deos com grau de polimeriza??o entre 3 e 6 unidades. Avaliando esses resultados, foi verificado que ? poss?vel a produ??o de quito-oligossacar?deos utilizando um processo simult?neo