Orientador: Glaucia Maria Postore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-03T17:17:03Z (GMT). No. of bitstreams: 1 Almeida_MareciMendesde_D.pdf: 1394335 bytes, checksum: aeb99b8a1fe68ffc5a8b6d83ce4b6809 (MD5) Previous issue date: 2003 / Resumo: Os galactooligossacarídeos são produzidos a partir da lactose por ação da B-galactosidase com atividade de transgalactosilação. São carboidratos não digeríveis, sendo resistentes às enzimas digestivas e fermentados por bifidobactérias. Os benefícios da ingestão de galactooligossacarídeos são de elevar a população de bifidobactérias no cólon e por efeito antagônico, suprimir a atividade de bactérias putrefativas e reduzir a formação de produtos tóxicos por fermentação. O microrganismo Scopulariopsis sp acumulou grande quantidade de B-galactosidase por fermentação semi-sólida e a enzima foi produzida constitutivamente. A enzima obtida foi fracionada com sulfato de amônio, acetona e etanol, sendo a precipitação com sulfato de amônio a que apresentou melhores resultados. O extrato enzimático foi caracterizado bioquimicamente, apresentando um pH ótimo de 4,5 e temperatura ótima de 65°C, e a estabilidade enzimática foi em pH 5,0 e 50°C. A enzima apresentou atividade de transgalactosilação quando avaliada por cromatografia de papel. A b-?galactosidase extracelular de Scopulariopsis sp foi semi-purificada e caracterizada. A enzima foi semi-purificada 13 vezes com um rendimento de aproximadamente 14%. O peso molecular da enzima foi estimado por filtração em gel em torno de 98 KDa. Algumas características da enzima foram determinadas usando o-nitrofenil-??galactopiranosideo (ONPG) como substrato. O pH e temperatura ótimos de atividade foram em torno de 4,5 e 60°C, respectivamente. A enzima foi estável em 40°C por 24 horas e pH 5,0. Os valores de Km e Vmax para ONPG foram 2,28mM e 123?moles/mL/min, respectivamente. Os cátions Fe+2, Fe+3 e Mn+2 ativaram a enzima e N-bromosuccinimida inibiu sua atividade. A atividade de transgalactosilação da ??galactosidase foi avaliada por cromatografia líquida de alta eficiência. A temperatura ótima para atividade de galactosiltransferase foi entre 35 e 45°C e o efeito do pH foi mínimo em alta concentração de lactose; a concentração de enzima para síntese de GOS foi 4-6 U/mL. Quando uma solução de lactose a 40% (p/v) em 0,1 M de tampão acetato de sódio pH 4,5 a 45°C foi incubada com 6 U/mL de ?-galactosidase, a enzima converteu 30% da lactose em galactooligossacarídeos. Foi utilizado planejamento fatorial completo de 2 níveis para estudar o efeito da enzima (4,0 e 8,0 U/mL), pH (5,0 e 7,0) e temperatura (35°C e 45°C) na formação enzimática de galactooligossacarídeos a partir da lactose. Uma diminuição da concentração de enzima e temperatura, e um aumento do valor de pH resultou uma maior produção de galactooligossacarídeos. O produto obtido em maior quantidade foi identificado com 4'galactosil-lactose por cromatografia líquida de alta eficiência / Abstract: Galactooligosaccharides are produced frem lactose by the action of B?-galactosidases which have transgalactosylation activity. They are non-digestible carbohydrates, which are resistant to gastrointestinal digestive enzymes and are fermented by bifidobacteria. The benefits of galactooligosaccharide ingestion arise from increased population of bifidobacteria in the colon which, by their antagonistic effect, suppress the activity of putrefactive bacteria and reduce the formation of toxic fermentation products. The strain of Scopulariopsis accumulated large amounts of the ??galactosidase by semi-solid fermentation and the enzyme was produced constitutively. The enzyme obtained was fractionated with ammonium sulphate, acetone and ethanol, the precipitation with ammonium sulphate showed better results. The enzymatic extract was biochemically characterized, showing an optimum pH of 4.5 and optimum temperature of 65°C, and the enzymatic stability was at pH 5.0 and 50°C. The enzyme showed transgalactosylation activity, when it was evaluated by paper chromatography. Extracellular ??galactosidase from Scopulariopsis sp was semi-purified. The enzyme was a semi-purified 13-fold with a yield of about 14%. The molecular weight of the enzyme was estimated to be about 98 KDa by gel filtration. Some enzyme characteristics were determined using o-nitrophenyl-??galactopyranoside (ONPG) as substrate. The pH and temperature optimum of the activity were about 4.5 and 60°C, respectively. The enzyme was stable at 40°C for 24 hours and pH 5.0. The Km and Vmax values for ONPG were 2,28mM and 123llmoles/mUmin, respectively. The cations Fe+2, Fe+3 and Mn+2 activated the enzyme and N- bromosuccinimide inhibited activity. Transgalactosylation activity of the ??galactosidase was evaluated by high pressure liquid chromatography. The optimum temperature for galactosyltransferase activity was in the range of 35 to 45°C and the pH effect was minimal at high lactose concentration; the enzyme concentration for GOS synthesis was 4-6 U/mL. When 40% (w/v) lactose solution (in 0.1 M sodium acetate buffer, pH 4.5, 45°C) was incubated with 6 U/mL of ??galactosidase, the enzyme converted 30% of the lactose in galactooligosaccharides. Two-Ievel full-factorial design experiments were designed to study effects of enzyme (4.0 and 8.0 U/mL), pH (5.0 and 7.0) and temperature (35°C e 45°C) on enzymatic formation of galactooligosaccharides from lactose. A decrease of enzyme concentration and temperature and an increase of pH value resulted in a higher galactooligosaccharides production. The major product was identified as 4'galactosyl-lactose by high performance liquid chromatography / Doutorado / Doutor em Ciência de Alimentos

Orientador: Gláucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T09:32:30Z (GMT). No. of bitstreams: 1 Fai_AnaElizabethCavalcante_D.pdf: 2054300 bytes, checksum: 82245332b8fef2de5e0f8a67509b102a (MD5) Previous issue date: 2011 / Resumo: Galactooligossacarídeos (GOS) são prebióticos sintetizados via transgalactosilação enzimática da lactose e existem vários modos de se obter os produtos sintetizados nesta reação. Com o objetivo de se produzir GOS e hidrolisar lactose, ß-galactosidase de Aspergillus oryzae (Sigma®) foi covalentemente imobilizada em celite e em quitosana em pó ativada com glutaraldeído, sendo as propriedades destas avaliadas e comparadas em relação à enzima livre. Uma coluna de leito empacotado foi utilizada para a produção de GOS e hidrólise da lactose nos sistemas imobilizados. Os oligossacarídeos foram obtidos com um máximo de produtividade de 14,42 e 3,50 g/L.h, partindo de uma solução de lactose 40% (p/v), na coluna de leito empacotado com quitosana e celite, respectivamente. A hidrólise da lactose foi de 50,00 e 84,74% após 24 horas utilizando quitosana e celite como suportes enzimáticos, respectivamente. A fim de verificar a habilidade de síntese de GOS por Pichia kluyveri e Pseudozyma tsukubaensis, isoladas de pêssego (Prunus persica) e nectarina (Prunus persica var. nucipersica), respectivamente, utilizou-se células íntegras e viáveis para fermentar um meio de lactose a 40% (p/v). Rendimento máximo de 14,01 e 15,71% (p/p) de GOS foi obtido desta fermentação em 24 horas por P. kluyveri e P. tsukubaensis, respectivamente. Uma estratégia sequecial de dois planejamentos experimentais foi utilizada para otimização de rendimento de GOS e, sob as condições validadas, a atividade de transgalactosilação de P. tsukubaensis resultou em 28,35% (p/p) de rendimento de GOS em 24 horas, com uma produção de 73,71 g/L e aproximadamente 50% de hidrólise de lactose, a partir de uma concentração inicial de lactose de 26% (p/v). Foi proposto, ainda, um processo associado de biossurfactante/biomassa para síntese de GOS a partir da lactose. O biossurfactante sintetizado por P. tsukubaensis em manipueira foi otimizado utilizando planejamento experimental e análise de superfície de resposta, considerando como resposta a tensão superficial e a concentração de biomassa. A mínima tensão superficial e a máxima concentração de biomassa observada foram de 26,87 mN/m e 10,50 g/L, respectivamente. A atividade de trasgalactosilação da biomassa de P. tsukubaensis, nas condições otimizadas do processo, resultou em uma produção de 73,12 g/L e em rendimento de 18,28% (p/p) em 24 horas, a partir de 40% (p/v) de lactose / Abstract: Galactooligosaccharides (GOS) are prebiotics synthesized by enzymatic lactose transgalactosylation and there are many modes in which GOS producing reactions can be performed. In order to produce GOS and to hidrolise lactose Aspergillus oryzae ß-galactosidase (Sigma®) was covalently immobilized on celite and on glutaraldehyde-treated chitosan powder and its properties were evaluated and compared with those of free enzyme. A packed bed column with lactose recycle was employed for production of GOS and lactose hydrolysis by immobilized systems. Oligosaccharides were obtained with a maximum productivity of 14.42 and 3.50 g/L.h, from 40% (w/v) lactose solution, in packed bed column with enzyme on chitosan and celite, respectively. Lactose was 50.00 and 84.74% hydrolyzed after 24 hours using chitosan and celite as enzyme support, respectively. In order to verify the ability to synthesize GOS from Pichia kluyveri, isolated from peach (Prunus persica), and Pseudozyma tsukubaensis, isolated from nectarine (Prunus persica var. Nucipersica), living whole cells were used to ferment 40% (w/v) lactose solution. A maximum yield of 14.01 and 15.71% (w/w) GOS was obtained from the fermentation in 24 hours by P. kluyveri and P. tsukubaensis, respectively. A sequential strategy of two experimental designs was used to optimize GOS yield and under the validated conditions the transgalactosylation activity of P. tsukubaensis resulted in 28.35% (w/w) of GOS yield at 24h, with a production of 73.71 g/L and approximately 50% of lactose hydrolysis, from 26% (w/v) initial lactose concentration. An associated process for biosurfactant/biomass for GOS synthesis from lactose was subsequently proposed. Biosurfactant synthesis by P. tsukubaensis on cassava wastewater was optimized using experimental design on the response of surface tension and biomass concentration. The minimum surface tension and maximum biomass concentration predicted and experimentally confirmed were 26.87 mN/m and 10.50 g/L, respectively. The transgalactosylation activity of P. tsukubaensis biomass at optimized conditions from 40% (w/v) lactose resulted in a GOS production of 73.12 g/L and a yield of 18.28% (w/w) in 24 hours / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Single enzymes of E. coli sourced B-galactosidase were analysed in effort to expand the wealth of knowledge in the area of heterogeneity. Static and dynamic heterogeneity was studied with respect to catalytic rate, electrophoretic mobility, and heat shock protein chaperone systems. Temperature was found to be a contributing factor to the observed range of dynamic heterogeneity, with the range increasing with temperature. The inhibitor dissociation constant was determined to be a heterogeneous property of B-galactosidase. A novel assay was developed in which a single enzyme molecule was subjected to three separate solutions while the enzyme itself remained free in solution. / October 2016

This research is directed towards developing a more sensitive and rapid electrochemical sensor for enzyme labeled immunoassays by coupling redox cycling at interdigitated electrode arrays (IDA) with the enzyme label b-galactosidase. Coplanar and comb IDA electrodes with a 2.4 mm gap were fabricated and their redox cycling currents were measured. ANSYS was used to model steady state currents for electrodes with different geometries. Comb IDA electrodes enhanced the signal about 3 times more than the coplanar IDAs, which agreed with the results of the simulation. Magnetic microbead-based enzyme assay, as a typical example of biochemical detection, was done using the comb and coplanar IDAs. The enzymes could be placed close to the sensing electrodes (~10 mm for the comb IDAs) and detection took less than 1 min with a limit of detection of 70 amole of b-galactosidase. We conclude that faster and more sensitive assays can be achieved with the comb IDA. A paramagnetic bead assay has also been demonstrated for detection of bacteriophage MS2, used as a simulant for biothreat viruses, such as small pox. The immunoassay was carried out in a microfluidic format with the IDA, reference and counter electrodes integrated on the same chip. Detection of 90 ng/mL MS2 or 1.5x1010 MS2 particles/mL was demonstrated.

Made available in DSpace on 2016-06-24T04:00:22Z (GMT). No. of bitstreams: 1 Sergio Araujo Figueiredo.pdf: 5051471 bytes, checksum: 845523d46a114f99e92e4c1f07b80e2a (MD5) Previous issue date: 2011-06-03 / ß-galactosidases are a class of glycosyl-hydrolases that act on the plant cell primary walls, hydrolyzing ß-D-galactose at the nonreducing ends of ß-D-galactosides present in several biological molecules. Initially a characterization of the monosaccharides present in the primary wall of the pericarp and endosperm of Coffea arabica fruits at different ripening stages was performed, identifying the polysaccharides present in these regions, along with the putative carbohydrate target for the ß-galactosidase. In parallel, a molecular and a biochemical characterization of ß-galactosidase was performed. A partial characterization of ß-galactosidase genomic DNA structure, along with a transcription analysis and an in vitro and in situ biochemical activity were performed, identifying peaks of expression in the early stages of growth and in fully ripe fruit. Finally, in order to evaluate the ß-galactosidase effects on coffee fruit ripening, C. arabica calli were transformed by biolistic using RNA interference approach, in order to obtain genetically modified coffee plants with a silenced ß-galactosidase expression. Three transgenic calli growing on selective medium containing ammonium glufosinate were obtained, two of which contained the ß-galactosidase gene fragment. These calli are under embryogenic regeneration and the resulting seedlings will be further analyzed in order to confirm the presence of the transgenes and to assess of the effects of ß-galactosidase gene silencing on coffee fruit ripening. / As ß-galactosidases sao uma classe de glicosil-hidrolases que atuam na parede primaria das celulas vegetais, hidrolisando residuos ß-D-galactosis de extremidades nao redutoras de ß-D galactosideos presentes em diversas moleculas biologicas. Inicialmente foi feita uma caracterizacao dos monossacarideos presentes na parede primaria do pericarpo e do endosperma de frutos de Coffea arabica em distintas fases de maturacao, identificando os polissacarideos presentes nesta regiao celular, juntamente com os provaveis carboidratos-alvo para as ß-galactosidases. Em paralelo, foi realizada uma caracterizacao molecular e bioquimica das ß-galactosidases. Foi realizada uma caracterização parcial da estrutura do seu DNA genomico, juntamente com uma analise do nivel de transcricao e da atividade bioquimica in vitro e in situ foram realizadas, identificando picos de expressao nas fases iniciais de crescimento e nos frutos completamente maduros. Por fim, visando avaliar os efeitos das ß-galactosidases na maturacao de frutos de cafe, calos embriogenicos de C. arabica foram transformados por biobalistica, utilizando a tecnica do RNA interferente, com a finalidade de obtencao de plantas geneticamente modificadas de cafeeiro para o silenciamento da expressao do gene das ß-galactosidases. Foram obtidos tres calos transgenicos crescendo em meio seletivo com glufosinato de amonio, dentre os quais dois continham o fragmento deste gene. Estes calos encontram-se em fase de formacao de embrioes somaticos e as plantulas resultantes desta regeneracao serao analisadas, a posteriori, para confirmacao da presenca dos transgenes e avaliacao dos efeitos do silenciamento do gene das ß-galactosidases sobre a maturacao de frutos de cafe.

Insulin regulated aminopeptidase (IRAP) was first identified in fat and muscle cells where it is believed to regulate GLUT4 translocation. It has since been found to be behind a variety of functions, many not yet fully understood. Preliminary research from Monash University suggested that IRAP may play a role in cellular senescence. Senescence is a term that describes arrested cell division and is a tumor repressive mechanism. Senescent cells have been shown to secrete, among other things, the growth hormone TGFβ1, which in turn plays an important role in the cell differentiation of fibroblasts to myofibroblasts. The potential link between IRAP and senescence was the basis of this work. Senescent fibroblasts from three different passages (n=3) in the BJ3 cell-line were cultured and treated with different IRAP inhibitors; ANG-4, AL06 and HFI-419 which were all compared to an untreated control group. They were marked with a β-galactosidase stain, a senescent cellmarker, and imaged. The study demonstrated that the IRAP inhibitors led to a certain decrease in % of senescent cells compared to the control groups. However, this reduction was not considered statistically significant. Similarly, inhibition of the enzyme did not indicate any influence over the differentiation of the cells. The lack of effect could be due to chance based on the low number of sample size, or the condition of the cells used in the trial as they were partially immortalized BJ fibroblasts well beyond the passage of their intended use. In order to further demonstrate an association between IRAP and senescence, further trials are required. / Insulin reglerad aminopeptidas (IRAP) introducerades till en början som ett markörprotein. Man har sedan dess funnit att den står bakom en rad olika funktioner, många ännu inte  fullt klarlagda. Preliminär forskning från laboratoriet i Monash University tydde på att IRAP kan ha en koppling till senescerande fibroblaster. Senescence är en term som beskriver upphörd celldelning och är en tumörrepressiv mekanism. Senescerande celler har påvisats utsekrera bland annat tillväxthormonet TGFβ1, som i sin tur spelar en viktig roll i celldifferentieringenav fibroblaster till myofibroblaster. Den potentiella kopplingen mellan IRAP och senescence låg som grund till detta arbete. Senescerande fibroblaster från tre olika kulturer (n=3) i BJ3-cellinjen odlades och behandlades med olika IRAP-inhibitorer; ANG-4, AL06 och HFI-419 som alla jämfördes med en kontrollgrupp. Därefter markerades de med en β-galaktosidas-markör, en markör för senescerande celler, och mikroskoperades. Studien påvisade att IRAP-inhibitorerna ledde till en viss procentuell minskning av senescerande celler jämfört med kontrollgrupperna. Dock bedömdes inte denna minskning som statistiskt signifikant i studien. Likväl fann man ingen procentuell minskning av differentierade fibroblaster. Hypotetiskt sett skulle man vilja se att reduktionen av senescerande celler motsvarade en nedreglering av TGFβ1-proteiner. Eftersom närvaron av TGFβ1 tros spela en ledande roll i celldifferentiering till myofibroblastfenotypen, bör den procentuella mängden differentierade cellerna minska med inhibitorbehandlingarna. Den bristande påverkan av enzyminhibitionen kan bero på en rad olika faktorer. Cellerna som användes under försökets gång var väl bortom deras brukliga användningscykel. För att vidare påvisa ett potentiellt samband mellan IRAP och senescence behöver vidare försök utföras.