Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-25Bitstream added on 2014-06-13T20:56:25Z : No. of bitstreams: 1 oliveira_dm_me_rcla.pdf: 453316 bytes, checksum: bb92218b6015fc38766c9207299c3f70 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Isolou-se uma cepa de levedura do tratamento biológico do efluente da Refinaria de Paulínia – SP. Esta levedura foi submetida a alguns ensaios preliminares e mostrou-se capaz de biodegradar lipídios vegetais, gorduras animais e hidrocarbonetos. Diante destas características metabólicas providenciou-se a sua taxonomia resultando após análise molecular do genoma na espécie Candida viswanathii. Pelas suas propriedades hidrolíticas, procurou-se verificar nesta pesquisa a sua capacidade da em degradar biodiesel de origem animal, mediante o emprego do método respirométrico de Bartha e Pramer. Como suporte para esta avaliação utilizou-se solo arenoso, pH 4,0, 8,0g/dm3 de matéria orgânica, coletado na Estação Ecológica de Itirapina, coordenadas geográficas (S 21°13’V e 0 47º 49’W). Procurou-se avaliar a capacidade de biodegradar biodiesel desta levedura com a quantidade de água sugerida no próprio método de respirometria isto é, de 70% da capacidade de campo (CC) e com quantidade adicional para 90% da CC. Constatou-se nos experimentos realizados que capacidade de campo diminui a eficiência da respirometria no tratamento em que havia biodiesel e o solo apresentava 90% da CC. Nos tratamentos com levedura e biodiesel tanto em 70 como 90% da CC houve maior evolução de CO2. Comparando-se os ensaios com biodiesel e adição da levedura, verificou-se que na ausência da C. viswanathii a produção de CO2 foi 4% menor. Nos tratamentos em que apenas o biodiesel foi acrescentado verificou-se que a microbiota autóctone do solo também promove a biodegradação e conseqüente biorremediação indicando que, mesmo os microrganismos existentes no solo possuem a capacidade de degradar biodiesel e promover a biorremediação. / A stream of yeast was isolated from biological treatment of effluent from the refinery in Paulinia - SP. This yeast has been subjected to some preliminary tests and was capable of biodegradability vegetable oil, animal fat and hydrocarbon. Facing these metabolic characteristics, has provided its taxonomy and after molecular analysis of the genome resulted in Candida viswanathii species. For its hydrolytic properties, this research tried to verify its ability to degrade biodiesel from animal fat, by the use of the of Bartha and Pramer’s respirometry method. As support for this research, was utilized a sandy soil, pH 4,0; 8,0 g/dm3 of organic material, collected from Itirapina’s Ecologic Station, geographic co-ordinates (S 21° 13’V and 0 47º 49’W). C. viswanathii was isolated from oil refinery effluent after biological treatment. The research evaluated the yeast’s capacity of biodiesel degradation with the same quantity of water suggested by respirometric method, which means, at 70% of field capacity (FC) and with additional quantity to 90% of FC. It was found on the control experiments realized that the respirometric’s efficiency was lower in the treatment with biodiesel and the soil was 90% of FC (with more quantity of water). On treatments those containing the yeast and biodiesel, either at 70 or 90% FC there were more production of CO2. Comparing the treatments with biodiesel and yeast’s addition, it was found that the production of CO2 decreases 4% in the yeast absence. On treatments where only biodiesel was added it was observed that the soil’s autochthon microbiota also promoted the biodegradation and consequent bioremediation. This result indicates that even microorganisms from soil have the capacity to degrade biodiesel and promote the bioremediation of contaminated areas.

Resumo: Isolou-se uma cepa de levedura do tratamento biológico do efluente da Refinaria de Paulínia - SP. Esta levedura foi submetida a alguns ensaios preliminares e mostrou-se capaz de biodegradar lipídios vegetais, gorduras animais e hidrocarbonetos. Diante destas características metabólicas providenciou-se a sua taxonomia resultando após análise molecular do genoma na espécie Candida viswanathii. Pelas suas propriedades hidrolíticas, procurou-se verificar nesta pesquisa a sua capacidade da em degradar biodiesel de origem animal, mediante o emprego do método respirométrico de Bartha e Pramer. Como suporte para esta avaliação utilizou-se solo arenoso, pH 4,0, 8,0g/dm3 de matéria orgânica, coletado na Estação Ecológica de Itirapina, coordenadas geográficas (S 21°13'V e 0 47º 49'W). Procurou-se avaliar a capacidade de biodegradar biodiesel desta levedura com a quantidade de água sugerida no próprio método de respirometria isto é, de 70% da capacidade de campo (CC) e com quantidade adicional para 90% da CC. Constatou-se nos experimentos realizados que capacidade de campo diminui a eficiência da respirometria no tratamento em que havia biodiesel e o solo apresentava 90% da CC. Nos tratamentos com levedura e biodiesel tanto em 70 como 90% da CC houve maior evolução de CO2. Comparando-se os ensaios com biodiesel e adição da levedura, verificou-se que na ausência da C. viswanathii a produção de CO2 foi 4% menor. Nos tratamentos em que apenas o biodiesel foi acrescentado verificou-se que a microbiota autóctone do solo também promove a biodegradação e conseqüente biorremediação indicando que, mesmo os microrganismos existentes no solo possuem a capacidade de degradar biodiesel e promover a biorremediação. / Abstract: A stream of yeast was isolated from biological treatment of effluent from the refinery in Paulinia - SP. This yeast has been subjected to some preliminary tests and was capable of biodegradability vegetable oil, animal fat and hydrocarbon. Facing these metabolic characteristics, has provided its taxonomy and after molecular analysis of the genome resulted in Candida viswanathii species. For its hydrolytic properties, this research tried to verify its ability to degrade biodiesel from animal fat, by the use of the of Bartha and Pramer's respirometry method. As support for this research, was utilized a sandy soil, pH 4,0; 8,0 g/dm3 of organic material, collected from Itirapina's Ecologic Station, geographic co-ordinates (S 21° 13'V and 0 47º 49'W). C. viswanathii was isolated from oil refinery effluent after biological treatment. The research evaluated the yeast's capacity of biodiesel degradation with the same quantity of water suggested by respirometric method, which means, at 70% of field capacity (FC) and with additional quantity to 90% of FC. It was found on the control experiments realized that the respirometric's efficiency was lower in the treatment with biodiesel and the soil was 90% of FC (with more quantity of water). On treatments those containing the yeast and biodiesel, either at 70 or 90% FC there were more production of CO2. Comparing the treatments with biodiesel and yeast's addition, it was found that the production of CO2 decreases 4% in the yeast absence. On treatments where only biodiesel was added it was observed that the soil's autochthon microbiota also promoted the biodegradation and consequent bioremediation. This result indicates that even microorganisms from soil have the capacity to degrade biodiesel and promote the bioremediation of contaminated areas. / Orientador: Roberto Naves Domingos / Coorientador: Dejanira de Francheschi de Angelis / Banca: Cassiana Maria Reganhan Coneglian / Banca: Jorge José Correa Lopes / Mestre

Les lipases/acyltransférases présentent des propriétés intermédiaires entre les lipases et les acyltransférases. Capables de se comporter comme des hydrolases, elles catalysent cependant la réaction de transfert d'acyle préférentiellement à l'hydrolyse même en milieu aqueux à forte activité thermodynamique de l'eau en présence de divers nucléophiles. La recherche de nouvelles lipases/acyltransférases, soit sécrétées par des levures sauvages, soit identifiées parmi les séquences protéiques disponibles dans des bases de données, nous a permis d'identifier deux nouvelles enzymes de ce type : CvisL2 de Candida viswanathii et CtroL4a de C. tropicalis. Cette dernière, produite par expression hétérologue, a été plus particulièrement étudiée en comparaison avec les deux lipases/acyltransférases déjà connues, CpLIP2 de C. parapsilosis et CaLIP4 de C. albicans, ainsi qu'avec des enzymes plus éloignées (AflaL0a d'Aspergilus flavus, isolée dans ce travail, et CaLA de C. antarctica, qui présentent respectivement 35 % et 31 % d'identité avec CpLIP2). Le caractère spécifique des acyltransférases semble relié à leur degré d'homologie et à leurs relations phylogénétiques. En effet, les trois lipases/acyltransférases étudiées appartiennent à un sous-groupe phylogénétique distinct composé de diverses autres protéines actuellement non-caractérisées présentant plus de 57 % d'identité avec CpLIP2. En plus de leur activité acyltransférase plus ou moins prononcée, ces nouveaux biocatalyseurs diffèrent par leur spécificité de substrat, leur stabilité en présence de fortes concentrations en alcool ou leur activité à basse température, élargissant ainsi le spectre des applications potentielles des lipases et lipases/acyltransférases. / Lipases/acyltransferases have intermediate properties between lipases and acyltransferases. Although being active hydrolases, they catalyze acyltransfer reactions preferentially to hydrolysis even in an aqueous medium with a high thermodynamic activity of water in the presence of various nucleophiles. Searching for new lipases/acyltransferases, either secreted by wild yeast strains or identified in protein sequences databases, allowed us to identify two new enzymes of this type: CvisL2 from Candida viswanathii and CtroL4a from C. tropicalis. The latter, produced by heterologous expression, has been more particularly studied and compared with the two already known, closely related, lipases/acyltransferases, CpLIP2 from C. parapsilosis and CaLIP4 from C. albicans, and with two more distantly related lipases (a new lipase AflaL0a from Aspergillus flavus and CaLA from C. antarctica, with 35 % and 31 % identity with CpLIP2, respectively). The specific catalytic behavior of the acyltransferases seems to be associated with sequence homology and phylogenetic relationships. Indeed, the three lipases/acyltransferases studied are part of a phylogenetic subgroup composed of various proteins (identity with CpLIP2 higher than 57 %), currently not characterized. Besides their acyltransfer activity, these new biocatalysts differ in properties such as their substrate selectivity, their stability in the presence of high alcohol concentration or their activity at low temperature, opening the way to new applications.

As lipases (EC 3.1.1.3) hidrolisam ligações éster de triacilgriceróis numa interface água-óleo. Por possuírem diversas aplicações industriais, são as enzimas mais utilizadas em sínteses orgânicas e mais de 20% de biotransformações são realizadas com lipases. Os objetivos deste trabalho foram avaliar as condições nutricionais de meios de cultivo (fontes de óleos vegetais e animais, nitrogênio e carbono) para melhorar a produção de lipase por Candida viswanathii além de purificar a enzima em sistema aquoso bifásico e realizar a caracterização parcial da lipase. Entre os meios de cultivos analisados o meio de Vogel proporcionou a maior produção de lipase após 36 horas de cultivo (11,73 U/mL). Após analisar as fontes de carbono lipídicas e não-lipídicas e fontes de nitrogênio, um planejamento experimental de misturas foi aplicado para analisar o efeito de glicerol, lactose e sorbitol sobre a produção de lipase. Os resultados obtidos mostraram que a maior produção de lipase (20,41 U/mL) foi observada com 0,5% de lactose (p/v) e 0,5% (p/v) de sorbitol, sendo observado um aumento de 74% da produção de enzima. Nesse estudo observou-se que a lactose e sorbitol foram utilizados como adjuntos para a produção de lipase. A lipase produzida nas condições anteriores foi purificada em sistema aquoso bifásico (SAB) formado por polietilenoglicol (PEG) e fosfato de potássio. O maior coeficiente de partição (1,34) foi encontrado no ensaio com PEG 4000/fosfato a Temperatura de 40 °C e pH 7,0, bem como o maior balanço de atividade (50,73%). Na caracterização parcial, a lipase não sofreu influência da temperaturana faixa de 20 – 60 °C e obteve atividade máxima em pH 8,1. A enzima apresentou também elevada estabilidade em solventes orgânicos como metanol e etanol, sendo estas propriedades consideradas importantes para aplicações em processos biotecnológicos. / Lipases (EC 3.1.1.3) hydrolyze ester bonds of triacylglycerols at a water-oil interface. By they have several industrial applications, they are the enzymes most used in organic synthesis and more than 20% of biotransformations are performed with lipases. The objectives of this work were to evaluate the nutritional conditions of culture media (plant and animal oils, nitrogen and carbon) to improve the production of lipase by Candida viswanathii in addition to purifying the enzyme in a biphasic aqueous system and to realize the partial characterization of lipase. Among the culture media analyzed, the Vogel medium provided the highest lipase production after 36 hours of culture (11,73 U/mL). After analyzing the lipid and non-lipid carbon sources and nitrogen sources, an experimental design of blends was applied to analyze the effect of glycerol, lactose and sorbitol on lipase production. The results showed that the highest production of lipase (20,41 U/mL) was observed with 0.5% lactose (w/v) and 0.5% (w/v) sorbitol, with an increase of 74% of enzyme production. In this study it was observed that lactose and sorbitol were used as adjuncts for the production of lipase. The lipase produced under the above conditions was purified in a aqueous two-phase systems (ATPS) consisting of polyethylene glycol (PEG) and potassium phosphate. The highest partition coefficient (1,34) was found in the PEG 4000/phosphate assay at temperature of 40 °C and pH 7,0, as well as the highest activity balance (50,73%). In the partial characterization, the lipase was not influenced by the temperature range of 20 - 60 °C and obtained maximum activity at pH 8,1. The enzyme also showed high stability in organic solvents such as methanol and ethanol, and these properties are considered important for applications in biotechnological processes.