Made available in DSpace on 2017-06-01T18:20:31Z (GMT). No. of bitstreams: 1 dissertacao_rosileide_andrade.pdf: 37140920 bytes, checksum: 0822d27802efcfa2ece791f37a06d878 (MD5) Previous issue date: 2010-02-08 / The biosurfactants are amphipathic compounds produced by microorganisms with several important environmental and industrial applications, whose metabolites has been investigated, mainly by the advantages of biodegradability, production from renewable resources and functionality under extreme conditions. However, its use depends on the cost of production and purification, and compared to synthetic surfactants. Thus, in this work the use of resources consisting of industrial waste (Manipueira, corn steep liquor and whey) as low-cost alternative in the production of biosurfactants by the strains of the genus Candida. The studies were carried out initially with the strain of Candida lipolytica, maintained in the cultures of the Center for Research in Environmental Science UNICAP, to establish the formulation of a new medium for biosurfactant production using cassava wastewater as substrate according to factorial design 23. The results showed a reduction in surface tension of water 72mN / m to 26.35 mN / m by the biosurfactant produced by Candida lipolytica in medium formulated with 10% of Manipueira, 0.2% of ammonium sulfate and 0.1% of urea after 96 hours of culture. The biosurfactant produced was characterized as polymer composition due to high in carbohydrates, proteins and lipids, and shows high stability in a wide range of temperatures, showing efficiency in bioremediation processes by removing 81.7% of burnt motor oil is present in soil. The investigations were continued by a wild strain of Candida glabrata, isolated from soil in semi-arid, PE, Brazil. The isolated yeast was identified by morphology, by biochemical and molecular as C. glabrata, demonstrating the potential of producing biosurfactant using an experimental design type DCCR (central composite rotational design), using industrial waste corn steep liquor and whey, with significant reduction in surface tension of water from 72 to 28.53mN / m The isolated biosurfactant produced by C. glabrata, was described as due to high polymeric composition of lipids, proteins and carbohydrates demonstrating stable in high concentrations of sodium chloride, and remove 95% of burned motor oil in sandy soil. Because of the potential reduction of surface tension by C. lipolytica and C. glabrata, the process of production of these biosurfactants become economically viable due to the use of industrial waste to obtain these biopolymers, making the economic process with great potential for application in environmental processes / Os biossurfactantes são compostos anfipáticos produzidos por microrganismos com várias e importantes aplicações industriais e ambientais, cujos metabólitos vem sendo investigado, principalmente pelas vantagens de biodegradabilidade, produção a partir de fontes renováveis e funcionalidade sob condições extremas. Entretanto, seu uso depende do custo de produção e purificação, sendo comparados aos surfactantes sintéticos. Deste modo, neste trabalho foi estudada a utilização de meios constituídos por rejeitos industriais (manipueira, milhocina e soro de leite) como alternativa de baixo custo na produção de biossurfactantes pelas linhagens do Gênero Candida. Os estudos foram realizados, inicialmente com a linhagem de Candida lipolytica, mantida no banco de culturas do Núcleo de Pesquisa em Ciências Ambientas da UNICAP, visando estabelecer a formulação de um novo meio de produção de biossurfactante utilizando manipueira como substrato de acordo com planejamento fatorial de 23. Os resultados demonstraram a redução da tensão superficial da água de 72mN/m para 26,35mN/m pelo biossurfactante produzido por Candida lipolytica, em meio formulado com 10% da manipueira, 0,2% do sulfato de amônio e 0,1% da uréia após 96 horas de cultivo. O biossurfactante produzido foi caracterizado como polimérico devido a alta composição em carboidratos, proteínas e lipídeos, além de apresentar alta estabilidade em ampla faixa de temperaturas, demonstrando eficiência em processos de biorremediação removendo 81,7% do óleo queimado de motor presente em solo. As investigações foram continuadas por uma linhagem selvagem de Candida glabrata, isolada de solo do semi-árido, PE, Brasil. A levedura isolada foi identificada pela morfologia, por testes bioquímicos e moleculares como C. glabrata, demonstrando potencial de produção do biossurfactante, utilizando um planejamento experimental do tipo DCCR (Delineamento Composto Central Rotacional), empregando os resíduos industriais milhocina e soro de leite, havendo significativa redução da tensão superficial da água de 72 para 28, 53mN/m. O biossurfactante isolado produzido por C. glabrata, foi caracterizado como polimérico devido à alta composição em lipídeos, proteínas e carboidratos demostrando ser estável em altas concentrações de cloreto de sódio, além de remover 95% do óleo queimado de motor em solo arenoso. Devido o potencial de redução da tensão superficial por C. lipolytica e C. glabrata, o processo de produção destes biossurfactantes tornam-se economicamente viáveis devido a utilização de rejeitos industriais para obtenção destes biopolimeros, tornando o processo econômico com grande potencialidade de aplicação em processos ambientais

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Os lipídios microbianos apresentam composição similar aos óleos vegetais, e tem despertado o interesse de muitos pesquisadores, uma vez que podem suprir a demanda por óleos e gorduras. Além disso, estudos demonstram que ao submeter um microrganismo a baixas doses de radiação gama, estímulos são promovidos, podendo aumentar a função, a resistência celular, e o crescimento. Objetivou-se com esse estudo: avaliar os efeitos de baixas doses de radiação gama no crescimento e na síntese lipídica de Yarrowia lipolytica. A levedura foi submetida às doses de: (controle) 0,3; 0,5; 0,75; 1,0; 1,25 e 2,5 kGy de radiação gama, sob taxa de dose de 0,259 kGy/h. Foi utilizada câmara de Neubauer para determinação da viabilidade celular da levedura. Uma curva de crescimento e outra de sobrevivência foi determinada por densidade óptica em um espectrofotômetro. Posteriormente, a levedura foi inoculada em meio composto por xarope de cana-de-açúcar e vinhaça, e incubada por 7, 14 e 21 dias para a obtenção da biomassa seca, por liofilização, e da síntese lipídica, por metodologia de extração. A irradiação não interferiu na produção de biomassa seca, no crescimento e na sobrevivência da levedura Y. lipolytica. O período de incubação afetou a produção final de biomassa, sendo a maior produção obtida no 14° dia (13,46 g/L). Para os resultados de síntese lipídica, observou-se interferência na produção final, durante 21 dias de incubação e dose de 0,5 kGy, com produção de 4,17 g/L (34,94%). Conclui-se que não foi observado o efeito hormético durante as análises de crescimento, sobrevivência e produção de biomassa, quando submetidas às doses de radiação. Este efeito foi observado apenas durante a síntese lipídica, quando avaliadas as junções entre dose de radiação e período de incubação / Microbial lipids present similar composition to vegetable oils and has aroused great interest among researchers. In addition, studies show that by submitting a microorganism to low doses of gamma radiation, stimuli are promoted, which could increase the function, cell resistance, and the growth. Therefore, this study aimed to evaluate the effects of low doses of gamma radiation on the growth and lipid synthesis of Yarrowia lipolytica. The yeast received irradiation doses of: (control); 0.3; 0.5; 0.75; 1.0; 1.25 and 2.5 kGy gamma radiation dose rate in 0.259 kGy/h. Neubauer Chamber was used to analyze cell viability of the yeast. A growth curve and survival curve was determined by spectrophotometry. Then, the yeast was inoculated in the medium composed of syrup sugarcane and vinasse and incubated for 7, 14 and 21 days for the obtaining of dry biomass, by lyophilization, and lipid synthesis, by extraction methodology. The low doses of irradiation had no effect on the growth, survival, and production of dry biomass of Y. lipolytica. For the incubation periods, there was interference in the final production; the highest production was obtained on day 14 (13.46 g/L). The results for lipid synthesis revealed interference with the final production of lipids, being better at a dose of 0.5 kGy to 21 days incubation with yield of 4.17 g/L (34.94%). In conclusion, the low doses of irradiation had no effect on the growth, survival and biomass production of Y. lipolytica. Hormetic effect was observed only in the lipid synthesis, when the radiation dose was associated with the incubation time

In der Hefe Yarrowia lipolytica führen Mutationen im GPR1-Gen zu Essigsäuresensitivität. Die Deletion dieses Genes hat demgegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein näher charakterisiert. Es zeigte sich, dass GPR1-Mutantenstämme wesentlich schneller Acetat akkumulierten als der Wildtyp. Außerdem konnte bestetigt werden, dass Gpr1p ein integrales Membranprotein ist. Mittels Ortspezifischer Analyse wurden verschiedene funktionelle Bereiche untersucht. Das Protein unterliegt zudem einer Phosphorylierung/Dephosphorylierung. Auf der Grundlage der dargelegten Ergebnisse wurde ein Funktionsmodell für Gpr1p erstellt.

Trans-dominante Mutationen im GPR1-Gen der Hefe Yarrowia lipolytica führen zur Sensitivität der Hefezellen gegenüber Essigsäure. Die Deletion dieses Genes hat dem gegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein aus Y. lipolytica und dessen Orthologe Ycr010cp, Ydr384cp und Ynr002cp von S. cerevisiae weiter charakterisiert. S. cerevisiae-Transformanden, welche die Mutantenallele GPR1-1 bzw. GPR1-2 exprimierten, zeigten bei gleichzeitiger Anwesenheit von Glucose eine erhöhte Sensitivität gegenüber Essigsäure. Mittels Ort-spezifischer und zufälliger Mutagenese konnten funktionell wichtige Bereiche in den Proteinen Ycr010cp und Ynr002cp identifiziert werden. Die GPR1-Orthologen in S. cerevisiae werden durch verschiedene C-Quellen und voneinander unabhängig reguliert. Die Expression von YCR010c und YDR384c wird weiterhin durch allgemeinen Stress induziert. Die Deletion von zwei oder allen drei Homologen hatte eine Verringerung der Ammoniumproduktion zur Folge. Aufgrund der geringen Ähnlichkeit der Gpr1p-Orthologen zu Ammoniumtransportern wird davon ausgegangen, daß sie selber keine Ammoniumtransporter darstellen. Es wird angenommen, dass die Gpr1p-orthologen Proteine eine regulatorische Funktion haben bzw. Bestandteil einer bisher nicht bekannten Signaltransduktionskette sind.

Trans-dominante Mutationen im GPR1-Gen der Hefe Yarrowia lipolytica führen zur Sensitivität der Hefezellen gegenüber Essigsäure. Die Deletion dieses Genes hat dem gegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein aus Y. lipolytica und dessen Orthologe Ycr010cp, Ydr384cp und Ynr002cp von S. cerevisiae weiter charakterisiert. S. cerevisiae-Transformanden, welche die Mutantenallele GPR1-1 bzw. GPR1-2 exprimierten, zeigten bei gleichzeitiger Anwesenheit von Glucose eine erhöhte Sensitivität gegenüber Essigsäure. Mittels Ort-spezifischer und zufälliger Mutagenese konnten funktionell wichtige Bereiche in den Proteinen Ycr010cp und Ynr002cp identifiziert werden. Die GPR1-Orthologen in S. cerevisiae werden durch verschiedene C-Quellen und voneinander unabhängig reguliert. Die Expression von YCR010c und YDR384c wird weiterhin durch allgemeinen Stress induziert. Die Deletion von zwei oder allen drei Homologen hatte eine Verringerung der Ammoniumproduktion zur Folge. Aufgrund der geringen Ähnlichkeit der Gpr1p-Orthologen zu Ammoniumtransportern wird davon ausgegangen, daß sie selber keine Ammoniumtransporter darstellen. Es wird angenommen, dass die Gpr1p-orthologen Proteine eine regulatorische Funktion haben bzw. Bestandteil einer bisher nicht bekannten Signaltransduktionskette sind.

The thesis is aimed at understanding of molecular mechanisms of autophagic degradation of peroxisomes (pexophagy) in the yeast Yarrowia lipolytica. This microorganism has been extensively used to explore peroxisome biogenesis (Titorenko and Rachubinski, 2000). Gunkel et al. (1999) intoduced Y. lypolitica into pexophagy studies. However, the field of pexophagic research on this yeast remains quite unexplored. This work involved following tasks: (1) the development and optimization of Y. lipolytica as a model system to study peroxisome degradation; (2) Y. lipolytica genes and proteins implicated in pexophagy should be found and characterized; (3) a proper easy-to-handle selection procedure to isolate novel peroxisome degradation-deficient(pdd) mutants of Y. lipolytica should be devised.

In der Hefe Yarrowia lipolytica führen Mutationen im GPR1-Gen zu Essigsäuresensitivität. Die Deletion dieses Genes hat demgegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein näher charakterisiert. Es zeigte sich, dass GPR1-Mutantenstämme wesentlich schneller Acetat akkumulierten als der Wildtyp. Außerdem konnte bestetigt werden, dass Gpr1p ein integrales Membranprotein ist. Mittels Ortspezifischer Analyse wurden verschiedene funktionelle Bereiche untersucht. Das Protein unterliegt zudem einer Phosphorylierung/Dephosphorylierung. Auf der Grundlage der dargelegten Ergebnisse wurde ein Funktionsmodell für Gpr1p erstellt.

La sécrétion de lipase, la lipolyse extracellulaire et l’absorption des acides gras (AGL) ont été étudiés chez Yarrowia lipolytica (YL) en présence d’huile d’olive et/ou de sucrose. Des mesures d’activité lipase et d’immuno-révélation ont montré que l’activité lipase présente dans le milieu de culture provenait principalement de la lipase YLLIP2. L’huile d’olive induit la production de lipase qui est principalement associée aux cellules pendant les premières heures de cultures. YLLIP2 est ensuite libérée dans le milieu de culture avant d’être totalement dégradée par les protéases. Les triglycérides (TG) sont dégradés alors que la lipase est encore attachée aux cellules. Les produits de lipolyse présents dans le milieu de culture et à l’intérieur des cellules ont été quantifiés par chromatographie TLC-FID et GC. Les niveaux intracellulaires d’AGL et de TG augmentent transitoirement et dépendent de la source de carbone utilisée. Une accumulation maximum de 37,8 % w/w de lipides est observée avec l’huile d’olive seule. Cette étude montre que la levure YL est un modèle intéressant pour étudier la lipolyse extracellulaire et l’absorption des acides gras par les cellules / Lipase secretion, extracellular lipolysis and fatty acid (FFA) uptake were quantified in Yarrowia lipolytica (YL) grown in the presence of olive oil and/or sucrose. Lipase assays and western blot analysis indicated that the lipase activity measured in YL cultures mainly resulted from YLLIP2 lipase. Lipase production was triggered by olive oil and YLLIP2 remained associated with the yeast cells during the first hours of culture. It was then released in the culture medium before it was totally degraded by the alkaline protease. Olive oil triglycerides (TG) were degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell were investigated by quantitative TLC-FID and GC analysis. Intracellular levels of FFA and TG increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w was observed with olive oil alone. The present study shows that yeasts are interesting models for studying extracellular lipolysis and fat uptake by the cell

L’objectif de notre travail était d’étudier les bascules métaboliques chez Yarrowia li-polytica d’un métabolisme purement oxydatif vers l’accumulation de lipides puis à l’excretion d’acide citrique.Le développement d’un procédé D-stat et d’un mode de conduite fed-batch nous a permis, dans un premier temps, de quantifier les ratios N/C caractéristiques pour chacune des bascules étudiées. Nos résultats montrent que les ratios rN/rC critiques aux bascules métaboliques sont de 0,085 molN.Cmol-1 et de 0,018-0,022 molN.Cmol-1 pour l’accumulation de lipides et production de citrate, respectivement.L’analyse systémique des cultures réalisées nous a permis de mettre en évidence des mécanismes de co-régulation de certaines enzymes du métabolisme lipidique ainsi qu’une prépondérance de mécanismes post-transcriptionnels dans l’établissement des bascules étudiées.Enfin, l’utilisation de souches génétiquement modifiées au niveau de l’ATP citrate lyase, la malate déshydrogénase et de la glycérol-3-phosphate déshydogénase a permis d’évaluer l’impact de ces enzymes sur le métabolisme lipidique / This thesis aimed at studying the metabolic shifts in Yarrowia lipolytica from the pure oxidative metabolism to lipid accumulation and citric acid excretion.The development of a D-stat culture and of a monitoring fed-batch strategy allowed us to determine the N/C ratio characteristic for each of metabolic shifts. rN/rC ratio were determined equal to 0,085 molN.Cmol-1 and 0,018-0,022 molN.Cmol-1 for the lipid accumu-lation and the citric acid production, respectively.Systemic analysis of the cultivations showed coregulation phenomena among some enzymes of the lipidic metabolism and post-transcriptional modifications in the onset of the metabolic shifts.Finally, the impact of enzymes (ATP citrate lyase, malate dehydrogenase and gly-cérol-3-phosphate dehydrogenase) on the lipidic metabolism was evaluated through systemic analysis of 3 genetically modified strains