Thesis (Ph. D.)--Columbia University. / Vita. Bibliography: p. 37.

Thesis (Ph. D.)--Columbia University, 1934. / Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: p. 38.

Orientador: Yong Kun Park / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-02T01:58:47Z (GMT). No. of bitstreams: 1 Passos_LucianaMariaLiboni_D.pdf: 22421623 bytes, checksum: e98e183502d09b07df170798fe1542d8 (MD5) Previous issue date: 2002 / Resumo: o yacon é uma raiz andina, rica em frutooligossacarídeos, e de fácil cultivo. Além da presença de grande quantidade de açúcares, há evidências da presença de enzimas do tipo beta-frutofuranosidases, como invertases, em seu metabolismo. Raízes de yacon (Smallanthus sonchifolia) foram colhidas e armazenadas em diferentes condições. Foram acompanhados o conteúdo de carboidratos e a atividade enzimática de invertases destas raízes. Em termos de carboidratos as maiores mudanças observadas, nas raízes de yacon, foram no conteúdo de frutose e 1-kestose. A atividade enzimática variou bastante durante os diferentes armazenamentos, sendo a atividade máxima de invertase obtida após 15 dias de armazenamento a 4°C. As concentrações dos açúcares variaram bastante, sendo que os açúcares que se encontraram em maiores concentrações foram frutose e 1-kestose. As concentrações dos açúcares como um todo aumentaram durante os diferentes armazenamentos. Enzimas do tipo beta-frutofuranosidase foram extraídas de yacon. A otimização da extração foi realizada utilizando diferentes tampões, com diferentes valores de pH, e com diferentes tipos de fracionamento de proteínas. O extrato enzimático de yacon apresentou invertase com pH ótimo igual a 5,5 e temperatura ótima igual a 40° C, e valores Km e Vmáx, para substrato sacarose, iguais a 0,0258M e O,1265 umol/mL/mim, respectivamente. O extrato apresentou atividade de invertase detectada por açúcares redutores resultantes da reação enzimática. As enzimas apresentaram estabilidade a valores de pH entre 5,0 e 5,5, e estabilidade à temperatura entre 25° C e 40° C. As invertases de yacon extraídas foram semipurificadas e caracterizadas. O perfil cromatográfico e SDS-PAGE indicaram a presença de isoformas na fração final da purificação enzimática, bem como o seu comportamento com relação a temperatura e pH. O pH ótimo de atuação da invertase semipurificada foi de 5,5, e a temperatura ótima de atuação foi a 60° C. O peso molecular da invertase foi estimado em 68 KDa por eletroforese SDS-PAGE e filtração em gel Sephacril S200. A enzima apresentou baixa estabilidade ao pH, e estabilidade a temperatura somente entre 35° C e 40° C. / Abstract: Yacon is an Andean root, rich in fructooligosaccharides, and easy to grow. It contains great amount of sugars, and evidence of the presence of enzymes of the beta-fructofuranosidase type, such as invertases, in its metabolism. Yacon (Smallanthus sonchifolia) roots were harvested and stored under different conditions. The sugar contents and invertase activity of these roots were followed. The greatest changes in the sugar contents occurred with fructose and 1-kestose. The enzymatic activity varied with the different storage conditions, and the maximum activity was after 15 days storage at 4°C. The sugar concentrations varied a lot, and the highest sugar concentrations were found for fructose and 1-kestose. All the sugar concentrations increased during the different storage conditions and times. Enzymes of the type p-fructofuranosidase were extracted from yacon. The extraction optimization was accomplished using different buffers, different pH values and different types of protein precipitation. The enzymatic extract was biochemically characterized, showing an optimum pH of 5.5, optimum temperature of 40° C and the following kinetic parameters: Km= 0.0258 and Vmax = 0.1265. The extract presented invertase activity, detected by the production of reducing sugars from the enzyme reaction. The enzymes showed pH stability between 5,0 and 5,5, and temperature stability between 25° C and 40° C. The yacon invertases were semipurified and characterized. The chromatographic profile and SDS-PAGE indicated the presence of isoforms in the final fraction of the purification, as well as its behavior with respect to temperature and pH. The optimum pH of the semipurified invertase was 5.5 and the optimum temperature was 60° C. The molecular weight of the invertase was 68 Kda, determined by eletroforese SDS/PAGE and gel filtration in Sephacril S-200. The enzyme presented low stability to pH, and temperature stability only between 30° C and 40° C. / Doutorado / Doutor em Ciência de Alimentos

Plant invertases (EC 3.2.1.26) represent a multi-gene family of β-fructofuranosidases that perform integral roles in several biochemical processes. The central importance of this family of enzymes to plant growth and development has made them a primary target of investigation in plant biology. Research has principally focused on sink-source interactions, and the potential to increase sink capacity in several economically important crop species, including potato and tomato. However, studies exploring the impacts of invertase mis-regulation on cellulose and lignin, the two most abundant biopolymers on earth, had not been conducted. Consequently, we investigated the effects of overexpressing yeast-derived invertases in tobacco and hybrid poplar. Transgenic tobacco expressing the yeast-derived invertases showed reduced height and interference in sink-source metabolism. In addition, some transgenic lines showed significant changes in cellulose and lignin content, providing evidence that sink capacity can be altered via the overexpression of this class of enzyme. In contrast, hybrid poplar expressing foreign invertase genes showed no visible phenotype, with only minor changes to the structural polymers cellulose and lignin, suggesting the mechanism of carbohydrate transport differs between tobacco and hybrid poplar. However, there was evidence for post-translational modification of the foreign invertases in hybrid poplar, which may also explain the difference in phenotypes observed. We suggest that the yeast-derived invertases may not be the most effective target to alter sink biopolymers, and that mis-regulating endogenous invertases may be a more suitable alternative. Consequently, we identified three cell-wall invertase genes in hybrid poplar and investigated their spatial and temporal expression profiles during the complete first year of growth. In addition, we heterologously expressed and characterized two hybrid poplar cell-wall invertase genes involved in vegetative growth. Collectively, the expression and functional characterization data suggest that one floral-specific and two vegetative cell-wall invertases exist in hybrid poplar. Of the two vegetative cell-wall invertases, one (PaxgINV1) appears to be involved in processes relating to dormancy, while the other (PaxgINV2) appears to be involved in phloem unloading and the seasonal reallocation of carbohydrate. We therefore hypothesize that PaxgINV2 may be a suitable target for future mis-regulation studies aimed at altering sink capacity. / Forestry, Faculty of / Graduate

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Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-28Bitstream added on 2014-06-13T20:21:36Z : No. of bitstreams: 1 goncalves_hb_dr_araiq_parcial.pdf: 153628 bytes, checksum: 3d4ba64426023c8a7714053c2f4e83d3 (MD5) / As β-D-frutofuranosidases, também conhecidas como invertases, são hidrolases que catalisam a hidrólise da sacarose em uma mistura equimolar de β-D-glicose e β-D-frutose que recebe o nome de açúcar invertido. Estas enzimas podem ser encontradas em diversos organismos, como vegetais, animais, bactérias, leveduras e em fungos filamentosos, sendo que os últimos merecem destaque pela alta produção enzimática e estabilidade. O objetivo deste trabalho foi estudar a β-D-frutofuranosidase produzida por Fusarium graminearum em Fermentação Submersa (FSbm) e Fermentação em Substrato Sólido (FSS) purificando-a e caracterizando-a bioquimicamente, além de submeter esta enzima a secagem em Spray dryer e imobilização em suportes de baixo custo. O fungo F. graminearum foi selecionado como bom produtor da enzima, com maiores níveis de produção encontrados quando cultivado em substrato sólido com farelo de trigo umidificado com água de torneira (1:1; m/v) por 9 dias (150 U/g substrato). A β-D-frutofuranosidase extracelular obtida foi purificada 8,41 vezes com recuperação de 14%, obtendo-se em PAGE 7% uma única banda protéica, e em SDS-PAGE 12%, 2 bandas proteicas (94 kDa e 70 kDa) supondo-se um heterodímero, uma vez que a enzima nativa, estimada pela coluna de filtração Sephacryl S200, apresentou 159 kDa. A temperatura ótima de atividade ficou na faixa de 55-60ºC e o pH ótimo 4,5. Foi totalmente estável em temperaturas entre 30oC e 50oC por 1 hora, e com atividade residual acima de 80% entre pH 3,0 e 8,0 por 30 minutos. A β-D-frutofuranosidase extracelular foi ativada pelos íons Mn2+ e K+. A enzima foi capaz de hidrolisar sacarose e rafinose, mas não a inulina, com maior afinidade para a rafinose, com Km de 21,16 mM e Vmax maior quando utilizada a sacarose como substrato... / The β-D-fructofuranosidases, also known as invertase, are hydrolases which catalyze the hydrolysis of sucrose in an equimolar mixture of β-D-glucose and β-D-fructose that is called invert sugar. These enzymes can be found in many organisms, such as plants, animals, bacteria, yeasts and filamentous fungi, and the latter must be highlighted for high enzyme production and stability. The objective of this work was to study the β-D-fructofuranosidase produced by Fusarium graminearum in Submerged Fermentation (SbmF) and Solid Substrate Fermentation (SSF) purifying and characterizing them biochemically, and submit this enzyme to drying using Spray dryer and immobilization on low cost supports. The F. graminearum was selected as a good enzyme producing strain, with higher production levels found when the fungus was grown on wheat bran as solid substrate moistened with tap water (1:1, w/v) for 9 days (150 U/g substrate). The extracellular β-D-fructofuranosidase obtained was purified 8.41-fold with recovery of 14%. A single protein band was obtained in 7% PAGE, and two protein bands (94 kDa and 70 kDa) in 12% SDS-PAGE indicating a heterodimeric structure, since the native enzyme, estimated by gel filtration using Sephacryl S200 column presented 159 kDa. The optimum temperature for activity was 55-60°C and optimum pH 4.5. It was completely stable at temperatures between 30oC and 50oC for 1 hour, and residual activity above 80% between pH 3.0 and 8.0 for 30 minutes was observed. This β-D-fructofuranosidase was activated by Mn2+ and K+. The enzyme was able to hydrolize sucrose and raffinose, but not inulin, with a higher affinity for raffinose, with a Km of 21.16 mM and Vmax was higher when sucrose was used as substrate (1639.34 U/mg protein). The crude extract containing the extracellular enzyme was... (Complete abstract click electronic access below)

Orientador: José Roberto Ernandes / Banca: Rubens Monti / Banca: João Atílio Jorge / Resumo: Para o aprimoramento do processo industrial de produção de bioetanol, é fundamental o conhecimento da bioquímica e fisiologia dos microrganismos envolvidos. Assim, este estudo tem o objetivo de obter informações bioquímicas de leveduras industriais, principalmente ao que se refere à hidrólise da sacarose e maltose pela enzima invertase (β-frutofuranosidase, EC 3.2.1.26) e maltase (α- glicosidase, EC 3.2.1.20) respectivamente. As linhagens industriais Ethanol RedTM (Fermentis/Lasaffre - linhagem francesa) PE-2, SA-1, CAT-1 e BG (linhagens brasileiras) foram cultivadas em meios contendo diferentes fontes de carbono (sacarose, glicose, frutose, maltose e galactose), suplementados com peptona e extrato de levedo, e avaliadas com relação a produção de biomassa, consumo da fonte de carbono, viabilidade celular e níveis de atividade invertásica e maltásica. Resultados mostraram que todas as linhagens apresentam crescimento rápido e intenso em sacarose, glicose e frutose, porém apresentam comportamento diferente em meios contendo maltose e galactose. Todas as linhagens crescem lentamente em galactose. Ethanol RedTM é mais adaptada para o crescimento em maltose do que as linhagens brasileiras PE-2 e SA-1. As outras linhagens brasileiras (CAT-1 e BG) não crescem em maltose devido à ausência de atividade maltásica. Com relação aos níveis de atividade invertásica, foi identificada a presença de três categorias de linhagens: uma com altos níveis de atividade (Ethanol RedTM), outra com níveis mais baixos (PE-2, CAT-1), e uma terceira categoria com atividade intermediária entre estas duas primeiras categorias (SA-1 e BG). Além do valor acadêmico, os resultados obtidos têm importância aplicada na medida em que indicam que as linhagens industriais produtoras de etanol combustível apresentam diferentes características fisiológicas, que podem ser exploradas para aperfeiçoar o processo de fermentação alcoólica / Abstract: For the improvement of the industrial ethanol fuel-producing process, it is crucial to understand the biochemistry and physiology of the microorganisms involved. This study aims to obtain biochemical information of industrial yeasts, especially when it refers to the hydrolysis of sucrose and maltose by the enzyme invertase (β-frutofuranosidase, EC 3.2.1.26) and maltase (α-glicosidase, EC 3.2.1.20) respectively. The industrial strains Ethanol RedTM (Fermentis/Lasaffre - French strain) PE-2, SA-1, CAT-1 and BG (Brazilian strains) were cultured in media containing different carbon sources (sucrose, glucose, fructose, maltose and galactose) supplemented with peptone and yeast extract, and evaluated relative to biomass production, carbon source consumption, cell viability and levels of invertase and maltase activities. Results showed that all strains exhibit rapid and intense growth in presence of sucrose, glucose and fructose, but they have differing behavior in media containing maltose and galactose. All strains grow slowly on galactose. Ethanol RedTM is more adapted for growing in maltose than Brazilian PE-2 and CAT- 1. The remaining Brazilian strains (BG and CAT-1) do not grow in maltose, due to the absence of maltase activity. As far as the levels of invertase activity is concerned, three categories of strains have been identified: with high activity levels (Ethanol RedTM), with lower levels (PE-2, CAT-1), and a third category with intermediate activity levels between these first two categories (SA-1 and BG). Besides academic value, the results obtained have applied significance in indicating that industrial ethanol fuel-producing strains exhibit different physiological characteristics that can be exploited to improve the fermentation process / Mestre

Thesis (Ph. D.)--Columbia University, 1923. / Vita.

Thesis (Ph. D.)--Columbia University, 1931. / Vita. eContent provider-neutral record in process. Description based on print version record. "References": p. [49-52].

Thesis (Ph. D.)--Columbia University, 1925. / Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: p. [46].