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The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg after 3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01 IU mg) and total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms.

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Agradeço primeiramente a Deus por sustentar-me em minha caminhada. Agradeço a meus pais que sempre colocaram meus estudos em primeiro lugar e permitiram minha vinda para São Paulo. Obrigada pelo carinho e incentivo! Aos meus irmãos Radha Krishna e Endrigo Stêfanes pelo apoio e incentivo. Agradeço à FAPESP e à CAPES pelo apoio financeiro que auxiliou na minha pesquisa. Agradeço à orientação da Profa. Dra. Luiziana Ferreira da Silva e à co-orientação do Prof. Dr. José Gregório Cabrera Gomez a quem devo boa parte do aprendizado adquirido ao longo do meu trabalho. Agradeço também à Marilda Keiko Taciro pela orientação nas questões de engenharia bioquímica. Agradeço a meu companheiro André Luis Lopes Neves pelo apoio, paciência e carinho que me ajudaram na conclusão deste trabalho. Obrigada por compartilhar comigo noites a fio de experimentos infindáveis no laboratório. Agradeço aos que me ajudaram ao longo da caminhada: à equipe do laboratório, especialmente Daniela, pelas análises no HPLC e CG; Karen, Kelli, Karinna, Thatiane, Débora, Amanda e Rogério, que me auxiliaram nos experimentos e colaboraram com meu aprendizado. Obrigada pela disposição, paciência e amizade. Agradeço à equipe da Profa. Dra. Heloiza Ramos Barbosa, do Prof. Dr. Gabriel Padilla, do Prof. Dr. Beny Spira, do Prof. Dr. René Schneider e à equipe da Profa. Dra. Marilis do Valle Marques pela colaboração. Agradeço aos amigos que deixei em Natal, mas que até hoje guardo no coração: Larissa Klemig, Elizângela, Keith e Aleida. Agradeço também a Leonardo Nobre a quem devo em parte minha vinda a São Paulo e início do mestrado. Obrigada pelo apoio e incentivo! Palavras-chave: Xilose. Bactérias. Resíduos lignocelulósicos. Bagaço de cana-de-açúcar. Etanol. Polihidroxialcanoatos. Repressão catabólica. ABSTRACT FREIRE, R. K. B. Engineering recombinant bacteria to produce ethanol and biopolymers using sugars derived from sugarcane bagasse hydrolysate. 2012. 132f. [Masters thesis (Microbiology)] -Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2012.Lignocellulosic residues are remarkable substrates for biofuels production such as ethanol and for biopolymers such as polyhydroxyalkanoates (PHAs). The sugarcane bagasse has been indicated as the most promising raw material in Brazil due to increasing sugarcane production in this country. Xylose is one of the most important lignocellulose component, but its efficient utilization still represents a technical barrier. The aim of this work was to obtain bacterial strains more efficient in the xylose consumption. Multiple copies of the catabolism (xylAB) and transport (xylFGH) genes of xylose were introduced in the ethanol producer Escherichia coli KO11 strain and the poly-3-hydroxybutyrate (PHB) producer Burkholderia sacchari LFM 101. The recombinant strains were analysed for ability of xylose consumption and production in cultures containing xylose in the absence and presence of glucose. The inclusion of multiple copies of xylAB in E. coli KO11 reduced the rate of xylose consumption and increase...