Made available in DSpace on 2014-06-11T19:23:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-28Bitstream added on 2014-06-13T19:29:24Z : No. of bitstreams: 1 aragon_cc_me_arafcf.pdf: 397051 bytes, checksum: e29ac6a4f5baf629041e6e9442044f9e (MD5) / Universidade Estadual Paulista (UNESP) / No presente trabalho, a atividade da enzima glicerol quinase (GK; EC 2.7.1.30; ATP: glicerol 3-fosfotransferase), proveniente de extratos de levedura seca de panificação, foi otimizada. A melhor preparação enzimática da GK foi obtida por rompimento celular com esferas de vidro, durante sete minutos, com lise de 54,2% das células. O extrato celular foi parcialmente purificado com 1% de sulfato de estreptomicina, antes da precipitação com igual volume de solução a 30% (m/v) de polietilenoglicol 3350, e posteriormente dialisado. A atividade máxima da GK foi obtida em pH 10,0, a 60ºC e 50mM de substrato, por metodologia clássica. A enzima apresentou alta estabilidade térmica ― a atividade foi completamente mantida até 50ºC, durante uma hora ― e em pH entre 6,0 e 8,0. Além disso, manteve-se estável, por quatro meses, a 4°C, na presença de azida de sódio 0,05% e cloreto de cobalto 10mM, e, por até oito meses, com o extrato liofilizado. Calculados pelos métodos de Lineweaver-Burk, Hanes-Woolf e Eadie-Hofstee, o valor da constante de Michaelis (Km) da enzima variou entre 1,99mM e 3,11mM, e a Vmax, entre 1,14U/mL e 1,19U/mL. Utilizou-se a metodologia de superfície de resposta (MSR) para melhor definição dos parâmetros da reação enzimática, observando-se valores ótimos de atividades a temperaturas entre 52ºC e 56ºC, pH entre 10,2 e 10,5 e concentração de substrato de 150mM a 170mM. A MSR mostrou-se adequada para modelar a reação e maximizar a atividade da glicerol quinase. Este método, de baixo custo, dosa a glicerol quinase em uma seqüência de reações, sendo de grande importância para diversas indústrias, como a de alimentos, açúcar e álcool. / In the present study, the activity of the enzyme glycerol kinase (GK; EC 2.7.1.30; ATP: glycerol 3-phosphotransferase) from dry baker´s yeast, was optimized. The best enzymatic preparation of GK was obtained by cell disruption with glass beads, for seven minutes, with 54.2% of lysed cells. Cell extract was partially purified with 1% of streptomycin sulphate, before the precipitation with equal volume of a 30% solution (m/v) of polyethylene glycol 3350, and then it was dialyzed. The maximum activity of GK was obtained with pH 10.0, at 60ºC and 50mM of substrate, by the classic methodology. The enzyme presented high thermal stability ― the activity was completely maintained up to 50ºC, during one hour ― and at pH between 6.0 and 8.0. Besides, it was stable, for four months, at 4°C, in the presence of sodium azide 0.05% and cobalt chloride 10mM, and, for up to eight months, with the lyophilized extract. The value of the Michaelis constant (Km) of the enzyme was calculated by the methods of Lineweaver-Burk, Hanes-Woolf and Eadie-Hofstee,and it varied between 1.99mM and 3.11mM, and Vmax, between 1.14U/mL and 1.19U/mL. Response surface methodology (RSM) was used for better definition of the parameters of the enzymatic reaction, being observed higher activity values at temperatures between 52ºC and 56ºC, pH between 10.2 and 10.5 and substrate concentration from 150mM to 170mM. RSM showed to be an adequate approach for modeling the reaction and maximizing the glycerol kinase activity. This low cost method doses glycerol kinase in a sequence of reactions, being of great importance for many industries, like food, sugar and alcohol.

<p>Bioethanol is a fuel of tomorrow, and progress in the use of enzymes and reduction of non-fermentable materials by debranning will probably be a part to make it more economical with low environmental impact.</p><p> </p><p>Ethanol production based on debranned wheat was optimized in this study by batch experiments as well as continuous experiments in laboratory scale. Enzymes from Novozymes and Genencor were compared and no significant differences were discovered between the different set of enzymes. The yeast strains Ethanol Red and AmyloFerm were compared with traditional baker’s yeast and baker’s yeast were surprisingly the fastest to ferment, but Ethanol Red had higher viability during fermentation. Protease addition during saccharification does not seem to improve fermentation with baker’s yeast. Prolonged liquefaction and saccharification time does probably not have any large impact on glucose yield. The continuous lab-scale process has a potential to be a realistic model but the stirring has to be improved and the pipe diameter increased.</p>

Bioethanol is a fuel of tomorrow, and progress in the use of enzymes and reduction of non-fermentable materials by debranning will probably be a part to make it more economical with low environmental impact.   Ethanol production based on debranned wheat was optimized in this study by batch experiments as well as continuous experiments in laboratory scale. Enzymes from Novozymes and Genencor were compared and no significant differences were discovered between the different set of enzymes. The yeast strains Ethanol Red and AmyloFerm were compared with traditional baker’s yeast and baker’s yeast were surprisingly the fastest to ferment, but Ethanol Red had higher viability during fermentation. Protease addition during saccharification does not seem to improve fermentation with baker’s yeast. Prolonged liquefaction and saccharification time does probably not have any large impact on glucose yield. The continuous lab-scale process has a potential to be a realistic model but the stirring has to be improved and the pipe diameter increased.

Proteins in Escherichia coli were compared in terms of essentiality, centrality, and conservation. The hypotheses of this study are: for proteins in Escherichia coli, (1) there is a positive, measureable correlation between protein conservation and essentiality, (2) there is a positive relationship between conservation and degree centrality, and (3) essentiality and centrality also have a positive correlation. The third hypothesis was supported by a moderate correlation, the first with a weak correlation, and the second hypotheis was not supported. When proteins that did not map to orthologous groups and proteins that had no interactions were removed, the relationship between essentality and conservation increased to a strong relationship. This was due to the effect of proteins that did not map to orthologus groups and suggests that protein orthology represented by clusters of orthologus groups does not accurately dipict protein conservation among the species studied.

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