Orientador: Rubens Monti / Banca: José Roberto Ernandes / Banca: Eleonora Cano Carmona / Banca: Hamilton Cabral / Banca: Sandra Helena da Cruz / Resumo: As endoglicanases EC 3.2.1.4 são enzimas capazes de hidrolisar as ligações glicosídicas β-1,4 da celulose, resultando na liberação de glicose, celobiose e celo-oligossacarídeos. Empregadas mundialmente em diversos processos industriais, por exemplo, como amaciante de tecidos de algodão e bioestonagem de jeans, na extração de sucos de frutas, na adição em detergentes comerciais, na fabricação de cerveja, e na produção de papel e de etanol. Atualmente, há um grande interesse em enzimas que hidrolisem biomassa lignocelulósica para obtenção de etanol de segunda geração. A imobilização de enzimas em matrizes sólidas oferece muitas vantagens, entre as quais, reuso da enzima, a fácil separação do produto e o aumento da estabilidade operacional. Os objetivos deste trabalho foram: produzir CMCases secretadas por Aspergillus niger e Humicola grisea var. thermoidea em cultivo sólido e submerso; imobilizar CMCases em suporte agarose com diferentes ativações químicas; determinar a estabilidade térmica e as propriedades cinéticas dos derivados obtidos, comparando-os com as enzimas livres; avaliar a capacidade de reuso dos derivados ativos e estáveis e analisar qualitativamente o produto de hidrólise dos substratos celulósicos. O fungo A. niger secretou grandes quantidades de CMCases, 34,1 U.mg-1, em meio Vogel, utilizando pó de bagaço de cana-de-açúcar como fonte de carbono. O pH 5 foi determinado como o ótimo para a enzima, apresentando instabilidade ao pH 10. Com a utilização de aditivos estabilizante, a trealose a 10% (m/v), manteve, em pH 10, 80% de atividade residual, por 25 horas. Em relação à temperatura, apresentou uma faixa ampla de atividade entre 45°C a 75°C, tendo como temperatura ótima 65°C. O derivado enzimático que ofereceu melhor estabilidade foi aquele em que a imobilização foi conduzida juntamente com o substrato carboximeticelulose (CMC 1%), em... / Abstract: The endoglucanases EC 3.2.1.4 are enzymes that hydrolyze the β-1, 4 glycosidic linkages of the cellulose producing glucose, cellobiose and cello-oligosaccharides. Used worldwide in many industrial processes such as fabric softener and bioestonagem cotton jeans, extraction of fruit juices, in the addition of commercial detergents, in brewing, and the production of paper and ethanol. Currently, there is great interest in enzymes that hydrolyze lignocellulosic biomass to obtain second-generation ethanol. The immobilization of enzymes on solid arrays provides many advantages as the enzyme reuse, easy separation of the product and increased operational stability. The aims of this study were to produce secreted: CMCases produce secreted by Aspergillus niger and Humicola grisea var. thermoidea in solid and submerged cultivation; CMCases immobilized on agarose support with different chemical activations; determine the thermal stability and kinetic properties of the derivatives obtained comparing them with the free enzyme; evaluate the reuse capacity assets and stable derivatives and qualitatively analyze the product of hydrolysis of cellulosic substrates. The fungus A. niger secreted large amounts of CMCases, 34.1 U.mg-1 amid Vogel, using powdered sugarcane bagasse as a carbon source. The pH of 5 was determined as the optimum for the enzyme, presenting instability to pH 10. With the use of stabilizing additives, the 10% trehalose (w / v), kept at pH10, 80% residual activity for 25 hours. Regarding temperature there was a broad activity range from 45 °C to 75 °C and the optimum temperature as 65 °C. The enzyme derivative which offered better stability was one in which immobilization was conducted with the carboxymethycellulose substrate (1% CMC) in agarose support Glutaraldehyde (70% of reactive groups), preserving catalytic activity of 35% at 60 °C for 250 hours. Through qualitative TLC analysis, it... / Doutor

Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.

Orientador : Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T02:25:10Z (GMT). No. of bitstreams: 1 Kawaguti_HaroldoYukio_D.pdf: 24058276 bytes, checksum: a6cb1016d8d9d61e1418acf5a2867097 (MD5) Previous issue date: 2007 / Resumo: A isomaltulose é um dissacarídeo redutor, isômero da sacarose, que possui um sabor adocicado suave e propriedades físicas e sensoriais muito similares, que tem sido considerado um substituto promissor da sacarose na indústria de alimentos, devido a algumas características como baixo potencial cariogênico e baixo índice glicêmico, promoção do crescimento de bifidobactérias benéficas da microbiota intestinal, e por apresentar maior estabilidade em relação à sacarose em alimentos e bebidas acidificados, além de poder ser convertido para isomalte, um açúcar álcool dietético e não cariogênico aplicado na indústria de alimentos e farmacêutica. Os objetivos deste trabalho foram otimizar um meio de cultivo, de menor custo, para a produção da enzima glicosiltransferase pela linhagem Erwinia sp. D12 e estudar a produção de isomaltulose a partir de sacarose utilizando-se células livres e células imobilizadas em alginato de cálcio. Na otimização do meio de cultivo, em frascos sob agitação, a máxima atividade obtida foi de 12,4 UA de glicosiltransferase/mL de meio de cultivo após 8 horas de fermentação a 30ºC, em meio composto de 150 g/L de melaço de cana-de-açúcar, 20 g/L de água de maceração de milho- Milhocina®, 15 g/L de extrato de levedura Prodex Lac SDÒ, e pH ajustado a 7,5. No estudo da produção de glicosiltransferase, em fermentador de 6,6 litros, utilizando-se o meio de cultivo otimizado foi obtida máxima atividade de 22,5 UA de glicosiltransferase/mL de meio de cultivo, após 8 horas de fermentação a 27oC. No estudo da produção de isomaltulose por células íntegras imobilizadas de Erwinia sp. D12 em alginato de cálcio foi verificado que o tratamento dos grânulos de células imobilizadas com 0,06% de glutaraldeído, promoveu uma maior taxa de conversão, sendo obtido cerca de 72,3% de isomaltulose, após 12 horas de incubação em frascos sob agitação a 30ºC. As células íntegras imobilizadas e tratadas com 0,06% de glutaraldeído, em colunas de leito empacotado, apresentaram maior estabilidade do que àquelas imobilizadas sem tratamento com o aditivo, e mantiveram a conversão de sacarose em isomaltulose entre 50-60% por 10 dias, a partir de solução de sacarose 35% e fluxo de 0,56 mL/min a 30ºC. Foram estudados diferentes tratamentos para a preparação de células íntegras, células lisadas e extrato enzimático bruto imobilizados em alginato de cálcio. Os métodos que mostraram melhores resultados, em processo em batelada, foi o extrato enzimático bruto imobilizado em alginato de cálcio (EEI), em que foram obtidas taxas de conversão entre 59,7% e 63,3%; e células lisadas por sonicação e imobilizadas (CSI), com taxas de conversão entre 47,6% e 62,3%. A coluna de leito empacotado contendo grânulos de células lisadas imobilizadas (CSI) apresentou maior estabilidade do que a coluna contendo os grânulos de extrato enzimático bruto imobilizado (EEI). A coluna de leito empacotado de CSI converteu 53-59% de sacarose em isomaltulose durante sete dias, posteriormente houve queda lenta e gradual da conversão não havendo mais transformação em isomaltulose após 21 dias. No estudo da produção de isomaltulose utilizando-se células livres de Erwinia sp. D12, em processo em batelada, foi verificado o efeito do pH, da temperatura, da concentração do substrato sacarose e da concentração de massa celular em frascos agitados a 150 rpm e 30ºC. A conversão de sacarose em isomaltulose foi favorecida utilizando-se temperaturas superiores a 30ºC, pH entre 6,0-6,5, massa celular entre 7,5- 12,5% e solução de sacarose de 20-35%, obtendo-se rendimentos de isomaltulose acima de 50%. No estudo da vida útil das células livres em escala de bancada, utilizando-se frascos Erlenmeyers sob agitação, foi verificado que os parâmetros de conversão fixados a: temperatura de 35ºC, pH 6,5, concentração de substrato sacarose 35% e concentração de massa celular 10% foram os mais favoráveis, promovendo um alto rendimento em isomaltulose entre 70-75%, por 16 bateladas. Os ensaios realizados em escala piloto demonstraram a viabilidade da conversão de sacarose em isomaltulose por células livres, em que foram obtidos cerca de 114 litros de xarope com alto teor de isomaltulose (63,40%). Os cristais de isomaltulose, após clarificação e purificação do xarope convertido, apresentaram pureza de 96,5% / Abstract: Isomaltulose is a reducing disaccharide and a structural isomer of sucrose. It has a mild sweet flavour and very similar physical and sensorial properties and has been considered as a promising substitute for sucrose in the food industry, due to some of its characteristics such as a low cariogenic potential and low glycemic index and the promotion of beneficial bifid bacteria in the intestinal microbial flora. It also shows greater stability than sucrose in acidified foods and drinks, and can be converted into isomalt, a dietetic sugar alcohol with no cariogenic potential for use in the food and pharmaceutical industries. The objectives of this research were the optimisation of a culture medium with reduced costs for the production of the enzyme glucosyltransferase by the strain Erwinia sp. D12, and the study of isomaltulose production from sucrose by free and immobilized cells. In the optimisation of the culture medium in shaken flasks, the highest glucosyltransferase activity achieved was 12.4 UA/mL of culture medium after 8 hours of fermentation at 30ºC, in a medium composed of 150 g/L of sugar cane molasses, 20 g/L of corn steep liquor- Milhocina® and 15 g/L of yeast extract Prodex Lac SD®, with the pH adjusted to 7.5. In the study for glucosyltransferase production in a 6.6-liter reactor using the optimised culture medium, the highest glucosyltransferase production achieved was 22.5 UA/mL of culture medium, after 8 hours of fermentation at 27ºC. In the study for isomaltulose production using Erwinia sp. D12 cells immobilized in calcium alginate, it was shown that the addition of 0.06% glutaraldehyde during the immobilization process, promoted a higher conversion rate, reaching about 72.3% isomaltulose after 12 hours of incubation at 30°C in shaken flasks. The immobilized whole cells treated with 0.06% glutaraldehyde, used in packed-bed reactors, presented greater stability than those immobilized without the addition of the additive, and maintained the conversion of sucrose into isomaltulose between 50-60% for 10 days, using a 35% sucrose solution with a flow rate of 0.56 mL/min at 30ºC. Different treatments were studied for the preparation of whole cells, lysed cells and a crude enzyme extract immobilized in calcium alginate. The methods that showed the best results in batch processes were the crude enzyme extract immobilized in calcium alginate (EEI), where conversion rates between 59.7% and 63.3% were achieved; and immobilized lysed cells (CSI), with conversion rates between 47.6% and 62.3%. The packed bed column containing granules of immobilized lysed cells (CSI) presented greater stability than that containing granules of immobilized crude enzymatic extract (EEI). The packed bed column with CSI converted 53-59% of sucrose into isomaltulose during seven days, and then showed a gradual decline in conversion, ceasing completely after 21 days. In the study of isomaltulose production using free Erwinia sp. D12 cells in a batch process, the effects of pH, temperature, sucrose substrate concentration and cell mass concentration were determined in shaken flasks at 150 rpm and 30ºC. The following conditions favoured the conversion of sucrose into isomaltulose: temperatures above 30ºC, pH between 6.0-6.5, cell mass between 7.5-12.5% and a sucrose concentration between 20-35%; when isomaltulose yields above 50% were obtained. The half-life of the free cells was studied on a bench scale in shaken Erlenmeyers flasks and it was shown that the following fixed conversion parameters were the most favourable: temperature of 35ºC, pH 6.5, 35% sucrose substrate concentration and 10% cell mass concentration; promoting high isomaltulose yields between 70-75%, for 16 batches. The pilot scale assays demonstrated the viability of the conversion of sucrose into isomaltulose by free cells, obtaining about 114 liters of high isomaltulose syrup (63.40%). The isomaltulose crystals, after clarification and purification of the converted syrup, showed a purity of 96.5% / Doutorado / Mestre em Ciência de Alimentos

Earths most abundant biomolecules, carbohydrates, offer tremendous potential forelectricity production. Carbohydrate fuel cells are electrical fuel cells that can harvest the stored electrons in carbohydrates and offer a cheap and efficient method that could help solve growing energy demands, while providing a renewable green energy source. Viologen-mediated carbohydrate fuel cells have demonstrated the ability to accelerate carbohydrate oxidation while decreasing partial or incomplete oxidation products reducing the electricity production. Subsequent studies suggested polymeric viologen compounds could improve the efficiency by increasing the local concentration of viologen. This thesis presents the utility of surface-immobilized viologen mediators for the oxidation of simple carbohydrates. Methyl viologen formed a self-assembled monolayer on a gold electrode surface to enhance its electrocatalytic oxidation of dihydroxyacetone, fructose, and glucose. The thiolated viologen formed surface adsorbed films on the gold electrodes that where consistent with monolayers and were characterized by quartz crystal microbalance and cyclic voltammetry. Cyclic voltammetry indicated that carbohydrates can generate electricity when combined with methyl viologen. Monolayer formation of methyl viologen indicates that immobilized mediators can be used to enhance oxidation of simple carbohydrates to generate electricity. This same tethered mediator strategy could be used for other mediators to increase their electrochemical efficiency in carbohydrate fuel cells.

Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a γ- aminopropyltriethoxysilane solution and ending with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeatability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5-10 μm and, depending on the length of the pipette inserted into the CNS, diameters of the pipette shaft will approach 40-50 μm. Once removed, the microprobe is then incubated with the radiolabeled peptide. Binding of the radiolabeled peptide will occur to the antibody sites not occupied by the endogenously released peptide. The images of the microprobes on sensitive autoradiographic film are analyzed for differences in the optical density along a specified length of probe. Areas of lighter density signify sites along the microprobe where endogenous peptide was biologically released during the physiological intervention. Knowing the exact location of the probe tip in vivo in the CNS permits identification of neurophysiological sites corresponding along the length of the microprobe where the peptide was released.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 370-377. / by James Chi-Yung Leung. / Ph.D.

Linear alkylbenzenes (LAB) are semi-products in the manufacture of linear alkylbenzene sulfonate surfactants that are active ingredients of various detergents. The use of traditional soluble acids (e.g. hydrofluoric acid or aluminum chloride) as catalysts for production of LAB results in the formation of large amounts of acidic toxic wastes. In this work, an efficient heterogeneous catalyst containing immobilized phosphotungstic acid (PTA) was synthesized and tested in the alkylation of benzene by higher alkenes. Sol–gel synthesis of silica gel from tetraethyl orthosilicate and PTA, as precursors, produced a mesoporous material containing covalently embedded PTA clusters. Obtained superacidic catalyst demonstrated high catalytic activity in liquid-phase alkylation of benzene by higher alkenes. Conversion of alkenes to corresponding phenylalkanes on this catalyst was significantly higher than on pure PTA. Covalent embedding of catalytically active HPA clusters prevented their leaching from the catalyst surface, which enabled its excellent catalytic properties.

No description available.

No description available.

A stable biofilm of Penicillium chrysogenum in a slab geometry was characterized with respect to penicillin production at various sugar and oxygen concentrations by studies using a novel bench scale bioreactor. The biofilm was submerged in aerated liquid media, mounted vertically in a tube and oriented so that the liquid media and sparged gas bubbles flow uniformly over the two slab faces. The conditions necessary for definite oxygen and sugar limitations were found by operating under various bulk nutrient concentrations. The periodic determination of total biofilm volume (rate of growth) and dry cell weight provided additional information. Definite oxygen mass transfer limitations were found to exist in the biofilm. These limitations could be overcome by increasing the oxygen supply rate to the biofilm. / Master of Science