Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T18:00:04Z (GMT). No. of bitstreams: 1 Costa_BrunaZucolotoda_D.pdf: 6212345 bytes, checksum: ec8b84a214cbc377e0f9f13d33ddc561 (MD5) Previous issue date: 2011 / Resumo: Os processos biocatalíticos foram abordados nesta tese de doutorado deste a triagem de micro-organismos para a seleção de biocatalisadores adequados até o uso de enzimas isoladas em reações de interesse biotecnológico. O primeiro capítulo apresenta o isolamento, identificação e a triagem enzimática de bactérias heterotróficas isoladas de rejeitos de mineração de cobre. A partir destas amostras foi possível isolar 189 bactérias, as quais apresentaram uma diversificada atuação catalítica. As bactérias isoladas foram identificadas por MALDI-TOF e por sequenciamento do gene RNAr 16S sendo encontrados diversos gêneros como Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, entre outros. O segundo capítulo apresenta o estudo do potencial catalítico de uma nova epóxido hidrolase de Aspergillus brasiliensis CCT1435, recombinante e expressa em E. coli. Esta EH é ativa em uma ampla faixa de pH e temperatura, apresentando um desempenho ótimo em pH 6,0 e 30 °C. Este trabalho ainda permitiu uma avaliação detalhada da aplicação biocatalítica desta EH na hidrólise do óxido de estireno em meio aquoso e bifásico. Por fim, o terceiro capítulo apresenta resultados preliminares envolvendo um processo quimio-enzimático para a produção de alcalóides pirrolidínicos a partir das respectivas 1,4-dicetonas. Esta metodologia é promissora uma vez que diversas 1,4-dicetonas foram convertidas nas suas respectivas iminas cíclicas, sendo que a subsequente etapa de redução com NaBH3CN promoveu a formação das pirrolidinas de interesse. Portanto, esta tese de doutorado apresenta um estudo amplo e diversificado envolvendo processos biocatalíticos aplicados em uma série de reações de interesse biotecnológico / Abstract: Biocatalytic processes have been addressed in this thesis from the microorganism screening for the selection of suitable biocatalysts to the use of isolated enzymes in biotechnological reactions. The first chapter presents the isolation, identification and enzymatic screening of heterotrophic bacteria isolated from copper mine drainages. From these samples, 189 bacteria were isolated, which showed diverse catalytic activities. The isolated bacteria were identified by MALDI-TOF and 16S rRNA gene sequencing and several genera were found: Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, among others. The second chapter presents the catalytic potential of a new epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH), recombinant and expressed in E. coli. This EH is active in a wide pH and temperature range, with a great performance at pH 6.0 and 30 °C. This work also presents the AbEH biocatalytic application for the styrene oxide hydrolysis in aqueous and biphasic media. Finally, the third chapter presents preliminary results involving a chemo-enzymatic method for the production of pyrrolidine alkaloids from the corresponding 1,4-diketones. This approach is promising since several 1,4-diketones were converted to their respective cyclic imines, and the subsequent reduction with NaBH3CN promoted the pyrrolidines formation. Therefore, this thesis presents a broad and diverse study of biocatalytic processes applied in a number of interesting biotechnological reactions / Doutorado / Quimica Organica / Doutora em Ciências

La L-asparaginase est une enzyme d’intérêt thérapeutique pour le traitement des leucémies aigües lymphoblastiques participant à l’hydrolyse de son substrat naturel L-asparagine conduisant à l’apoptose des cellules cancéreuses. À ce jour, la L-asparaginase d’origine bactérienne fait partie intégrante des formulations car possédant des activités catalytiques élevées mais provoquant de nombreux effets secondaires liés à une immunogénicité. Trois enzymes avec une activité Lasparaginase produites chez l’homme ont été découvertes récemment mais possèdent des activités catalytiques qui sont 1000 à 2000 fois inférieures aux enzymes d’origine bactérienne. Augmenter l’activité catalytique de ces enzymes par évolution dirigée pourraient permettre leurs utilisations en thérapeutique en plus de potentiellement participer à la réduction de l’immunogénicité chez les patients. Ces travaux de doctorat décrivent le développement d’outils pour l’expression etla détection de l’activité L-asparaginase à l’échelle d’une cellule individuelle. La L-asparaginase d’E. coli, utilisée en thérapeutique, a servi de référence et a permis de démontrer que le test AUR est le plus adapté pour la mesure de l’activité en microfluidique. L’expression de l’enzyme à partir de différents vecteurs d’expression a montré que l’expression périplasmique semble la plus adaptée pour le ciblage permettant un bon rendement et une bonne accessibilité pour le substrat. La viabilité des cellules à l’issu des mesures a été aussi démontrée. Ces outils pourront être directement utilisés pour le criblage de banques de mutants de L-asparaginase d’origine humaine en microfluidique. Les propriétés de la L-asparaginase ont aussi été utilisées pour démontrer la potentielle utilisation de billes de silice en tant que biocatalyseurs où sont confinées des bactéries. Ces billes sont des excellents supports pour la croissance de microorganismes qui peuvent rester viables au-delà d’une semaine. L’expression d’enzymes peut être induite et l’activité catalytique être aisément contrôlée en faisant varier la concentration bactérienne au sein du matériau. La combinaison de différentes populations bactériennes offre la possibilité d’effectuer des réactions en cascade. Le recyclage de ces billes pour différents cycles de réactions a également été démontré. Ces matériaux bioactifs peuvent avoir de nombreuses applications dans le domaine des biotechnologies. / L-asparaginase is an enzyme of therapeutic value for the treatment of acute lymphoblastic leukemia. Ths enzyme catalyzes the hydrolysis of L-asparagine conducting to apoptosis of cancer cells. To date, L-asparaginase of bacterial origine is used in the treatments due to high catalytic activities but causing a number of side effects linked with an immunogenicity. The human produces three enzymes with L-asparaginase activity but their catalytic activities are 1000 to 2000 times lower than the bacterial enzymes. Increase the catalytic activity of these enzymes by directed evolution could allow their uses in therapeutic in addition to potentially reduce immunogenicity in patients. This PhD work describes the development of tools for expression and detection of L-asparaginase at the single cell level for their applications in the screening of human L-asparaginase libraries in microfluidic. E. coli L-asparaginase, used in therapy, served as a reference and allowed to demonstrate that AUR assay is most suitable for measuring activity in microfluidic. Expression of the enzyme from different expression vectors showed that the periplasmic expression seems to be the most successful for screening enabling a good yield and good accessibility for the substrate. The viability of the cells following the measures has been shown. These tools might be used for the screening of mutants libraries of human L-asparaginases in microfluidic. The properties of L-asparaginase were also used to demonstrate the potential use of silica beads as biocatalysts in which bacteria are confined. These beads are excellent supports for the growth of microorganisms which may remain viable beyond one week. The expression of the enzymes may be induced and the catalytic activity can be reliably controlled by varying the concentration of bacteria within the material. The combination of various bacterial populations provides the possibility to carry out cascades reactions. The recycling of these beads for several cycles of reactions was also demonstrated. These bioactive materials have many potential applications in the field of biotechnologies.