Orientador: Ana Marisa Fusco Almeida / Resumo: As células eucarióticas desenvolveram diversas estratégias para combater os efeitos nocivos de uma variedade de condições estressantes. Linhagens de Saccharomyces cerevisiae tem uma capacidade inata de suportar altos níveis de etanol que se tornariam letais ou prejudiciais à fisiologia de outros organismos. A resposta de estresse sob alta concentração de etanol em S. cerevisiae é importante em reações de fermentação e opera através da superexpressão e subexpressão de genes, alterando o perfil proteico e podendo resultar em perturbações do bioprocesso. O objetivo deste estudo foi determinar as diferenças no metabolismo de cada cepa de levedura visando à obtenção de informações a respeito da resistência à alta concentração de etanol a 12%. Para isso, as linhagens industriais PE-2 e CAT-1, e a linhagem Y12632 de S. cerevisiae isolada de processo de produção de etanol, foram utilizadas para a realização de testes em meio sintético contendo etanol 12%, simulando uma situação de estresse ocorrente em processos industriais para produção de etanol. Sendo assim, para avaliar essa resposta durante o estresse, a caracterização do processo fermentativo e a análise de integridade da membrana celular foram relacionadas à análise proteômica por shotgun. Principalmente através da avaliação de integridade da membrana citoplasmática, a cepa PE-2 demonstrou elevada resistência, além de alto consumo de substrato durante a fermentação com estresse induzido em comparação com a cepa industrial CAT-... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Resumo: As leveduras são microrganismos importantes em diferentes processos biotecnológicos, sendo amplamente empregadas industrialmente. Apesar de se apresentarem em grande número em ambientes naturais, como na superfície de plantas (folhas, flores e frutos) e na rizosfera, pouco é conhecido sobre sua função nestes habitats. O controle de fitopatógenos tem sido estudado como um potencial papel das leveduras, principalmente inibindo fungos que causam podridões em frutas no período pós-colheita, e controlando doenças de diversas culturas de interesse econômico no campo, pois são ótimas competidoras por nutrientes e espaço. Neste contexto, o objetivo do presente trabalho foi isolar, avaliar e caracterizar leveduras de áreas agrícolas quanto ao controle de fungos fitopatogênicos in vitro e in vivo. Foram isoladas mais de uma centena de linhagens de leveduras, as quais foram avaliadas inicialmente quanto ao antagonismo à três fungos fitopatogênicos (Colletotrichum sublineolum, Colletotrichum graminicola e Thielaviopsis paradoxa) em testes in vitro. Os isolados com comportamento antagônico foram identificados através do sequenciamento da região ITS do rDNA e por fingerprinting utilizando-se a técnica de ISSR (Inter Simple Sequence Repeats). Foram realizados testes de antagonismo em meios de cultura sólidos (pela avaliação do crescimento micelial do fitopatógeno) e líquidos (pela avaliação da germinação dos esporos fúngicos), além de experimentos in vivo em plantas de sorgo e toletes de cana-de-açúcar, verificando-se o controle das doenças antracnose e podridão abacaxi, respectivamente. Foram realizadas análises para detecção de possíveis mecanismos de ação das leveduras, como competição por nutrientes; produção de toxina killer, compostos voláteis, enzimas hidrolíticas e sideróforos, além da avaliação de possíveis danos causados pela levedura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The yeasts are important microorganisms for different purposes in biotechnological processes and widely utilized industrially. Although in high numbers in natural environments, as plant surfaces (leaves, flowers and fruits) and rhizosphere, a little is known about their functions in the habitats. The control of phytopathogens by the yeasts has been studied, mainly inhibiting molds which cause fruit rots in the postharvesting period, and controlling diseases of economically important cultures in the field, once they are good competitors for nutrients and space. In this context, the aim here is the isolation, screening and characterization of yeasts from agricultural areas regarding their ability to control phytopathogenic molds in vitro and in vivo. More than a hundred of strains were isolated, which were initially screened for the in vitro antagonism against three phytopathogenic molds (Colletotrichum sublineolum, Colletotrichum graminicola and Thielaviopsis paradoxa). The isolates with antagonistic behavior were identified by the sequencing of ITS region in the rDNA and by fingerprinting with the ISSR (Inter Simple Sequence Repeats) technique. Antagonism tests in solid media (evaluating the mycelial growth of the phytopathogen) and liquid media (evaluating the fungal spore germination) were carried out, besides in vivo experiments with sorghum and sugar cane to verify the control of anthracnose and pineapple disease, respectively. Mechanisms of action by yeasts towards the molds were evaluated as competition for nutrients; production of killer toxin, volatile compounds, hydrolytic enzymes and siderophores. The damage caused by the yeast in the fungal hyphae was also approached. The results indicated three yeast species with good results as antagonists (Torulaspora globosa, Candida intermedia and Rhodotorula mucilaginosa). All the yeasts exhibited antagonism against the phytopathogenic... (Complete abstract click electronic access below) / Orientador: Sâmia Maria Tauk Tornisielo / Coorientador: Sandra Regina Ceccato-Antonini / Banca: Kátia Cristina Kupper / Banca: Fernando Alves de Azevedo / Banca: Carlos Renato Corso / Banca: Dejanira de Franceschi de Angelis / Doutor

Orientador: Francisco Maugeri Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T13:20:36Z (GMT). No. of bitstreams: 1 Goldbeck_Rosana_M.pdf: 985115 bytes, checksum: 293c51120ad7c4857c9bff41ab4e2724 (MD5) Previous issue date: 2008 / Resumo: Lipases (E.C.3.1.1.3) constituem um importante grupo de enzimas definidas como carboximetilesterases que catalisam a hidrólise de cadeias longas de acilgliceróis na interface água-óleo. Estão amplamente distribuídas na natureza estando presentes em animais e vegetais, podendo ser produzidas por microrganismos como fungos e bactérias. As lipases apresentam promissoras aplicações comerciais devido a sua estabilidade, seletividade, larga especificidade por substratos e capacidade de síntese orgânica. Embora os estudos científicos tenham se concentrado mais na aplicação das lipases, alguns grupos de pesquisa se dedicam também ao isolamento de microrganismos produtores de lipase, em busca de novas enzimas com diferentes propriedades e especificidades. Este trabalho teve como objetivo explorar uma classe de microrganismos pouco utilizada para a produção de lipases, que são as leveduras. Assim, a partir de leveduras silvestres isoladas de diversas regiões do país, selecionaram-se cepas produtoras de lipase, que foram em seguida estudas e caracterizadas. Primeiramente, realizou-se uma triagem inicial a partir de 372 leveduras isoladas e selecionaram-se aquelas que apresentam capacidade de produção de lipase. Esta seleção foi feita através da formação de um halo transparente ao redor das colônias quando cultivadas em placas contendo 0,5% de peptona, 0,3% de extrato de levedura, 2% de agar e 0,1% de tributirina, pH 6,0 a 30ºC por 48 horas. Após pré-selecionadas, as leveduras foram cultivadas em meio líquido contendo: 0,5% de peptona, 0,3% de extrato de levedura e 1% de óleo de oliva, pH 6,0, temperatura de 30ºC durante 48 horas sob agitação de 150 rpm para posterior determinação da atividade lipolítica. Em uma segunda etapa, após a seleção dessas leveduras, a lipase foi caracterizada quanto à especificidade de substrato, perfil de pH e temperatura, estabilidade térmica e de pH e capacidade biocatalítica de síntese orgânica. A especificidade de substrato da enzima foi realizada através da determinação da atividade lipolítica utilizando diferentes triglicerídeos (C4, C8, C10, C14, C18:1) como substratos. Para verificar a atividade catalítica, foi realizada síntese de ésteres em meio orgânico. Os ésteres formados durante a reação foram quantificados por cromatografia gasosa. Na etapa de seleção, das 372 leveduras silvestres estudadas, 3 cepas foram selecionadas com potencial para produção de lipase. Estas foram caracterizadas e apresentaram comportamentos distintos. Para a cepa AC02, temos como condições ótimas para a reação enzimática pH 7,0 e temperatura 44ºC, para o microrganismo AAV1, também temos como ótimo o pH 7,0, porém, uma temperatura um pouco mais elevada, 47ºC, já para a levedura AY3, as condições ótimas de temperatura e pH foram 37ºC e 6,6 respectivamente. Quanto à especificidade de substrato, a enzima proveniente do microrganismo AY3 apresentou atividade lipolítica superior em tributirina (C4) e tricaprilina (C8) quando comparada com óleo de oliva, demonstrando maior especificidade por triglicerídeos de cadeia curta e média. As três cepas estudadas apresentaram capacidade biocatalítica, no entanto foram registradas maiores porcentagens de esterificação quando utilizado etanol ao invés de heptanol como componente reacional. Portanto, a procura denovas lipases através de programas de seleção de microrganismos produtores é de fundamental importância para ampliar ainda mais o campo de aplicação dessas enzimas / Abstract: Lipases (E.C.3.1.1.3) consists of an important group of enzymes defined as carboxymethylesterases that catalyze the hydrolysis of long chains of acylglycerols in the water-oil interface. They are thoroughly distributed in the nature being present in animals and vegetables and can be produced by microorganisms such as fungal and bacteria. Lipase presents a promising commercial application because of their stability, selectivity, large specificity for substrates and capacity of organic synthesis. Although the scientific studies have been more concentrated in the application of lipases, some research groups are dedicated to the isolation of lipase producer microorganisms, with the goal of finding new enzymes with different substrate specificities. In face of that, this work had as main objective to select wild lipase producer yeasts isolated from different areas of the country, as well as study the specificity of the enzyme. At first, a selection of wild yeasts that presented lipase production capacity was accomplished. The strains that produce lipase were selected considering the formation of a transparent halo around of the colonies when cultivated in plates containing 0.5% of peptone, 0.3% of yeast extract, 2% of agar and 0.1% of tributyrin, at pH 6.0 and 30ºC for 48 hours. After being previously selected, the yeasts were then cultivated in shacked flasks in medium containing 0.5% of peptone, 0.3% of yeast extract and 1% of olive oil also at pH 6.0 and 30ºC for 48 hours, under agitation of 150 rpm for subsequent determination of the lipolytic activity. At a second stage, after the selection of the yeasts, the lipases were characterized concerning substrate specificity, pH and temperature profile, thermal and pH stability and the biocatalyst capacity for synthesis in organic media. The enzyme specificities for substrates were accomplished throughout the determination of the activity using different triglycerides (C4, C8, C10, C14, C18:1) as substrate. To verify the catalytic lipolytic activity, the synthesis of esters were accomplished in organic medium. The esters formed during the reaction were quantified by gas chromatography. In the selection stage, of the 372 studied wild yeasts, 3 strains were selected with potential for lipase production. These enzymes were characterized and they presented different behavior. For the strain AC02, the optimum conditions for the enzymatic reaction were pH 7.0 and 44ºC, for the microorganism AAV1, the pH 7.0 was also better however the optimum temperature was a little higher, 47ºC, and for the yeast AY3, the optimum conditions for temperature and pH were 37ºC and 6.6 respectively. For substrate specificity, the enzyme originated from the microorganism AY3 presented superior lipolytic activity in tributyrin (C4) and tricaprylin (C8) when compared with olive oil, demonstrating larger specificity for triglycerides of short and medium chain. The three strains studied presented biocatalyst capacity, however larger esterification percentages were registered when ethanol was used as component of the reaction instead of heptanol. Hence, the search of new lipases through programs of selection of microorganisms is of fundamental importance to enlarge the field of application of those enzymes / Mestrado / Mestre em Engenharia de Alimentos

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Yeasts form an important part of many ecosystems and significantly contribute to biodiversity. However, yeast biodiversity in the North West Province remains largely unexplored. The aim of this study was to determine the diversity and characteristics of yeasts from water sources in the North West Province, South Africa. Samples were collected over a two year period and included three rivers, a spruit and an inland lake. Temperature, pH, and electrical conductivity (EC) were measured on site using a multi-probe. Nitrate (NO3-N), nitrite (NO2-N) and phosphate (PO42-) levels were determined in the laboratory using Hatch kits and equipment. The pH ranged from 7.2 to 9.2. Elevated EC levels (36-70 mS) were detected especially at the Harts River and Barberspan (38-165 mS) sites. Physico-chemical parameter levels were higher during the cold dry sampling period compared to the warm rainy sampling period. Levels and diversity of yeasts were determined using the membrane filtration method. The highest level of yeasts was detected in the Mooi River and Schoonspruit during 2010 and 2011 sampling periods. Pigmented and non-pigmented yeasts were enumerated from all samples. Over the two year period the highest number of pigmented yeasts was detected in the Schoonspruit samples. In some cases there were significant (P<0.05) differences between pigmented and non-pigmented yeast levels among the sites. The diazonium blue B (DBB) test was carried out to distinguish between ascomycetous and basidiomycetous yeasts. These isolates were then identified using the API ID 32C system. Yeasts isolates were identified as belonging to the following genera: Candida, Cryptococcus, Pichia, Rhodotorula and Zygosaccharomyces. In addition using 26S rRNA gene sequencing Aureobasidium spp., Clavispora spp., Cystofilobasidium spp., Hanseniaspora spp., Meyerozyma spp., Sporidiobolus spp., and Wickerhamomyces spp.were also identified. The diversity and abundance of yeasts in the water sources demonstrated that opportunistic pathogens were present. This was supported by results that indicated some isolates could grow at 37°C and higher. In conclusion, our results provide preliminary information on the distribution and diversity of yeasts in water sources of the North West Province, South Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Yeasts form an important part of many ecosystems and significantly contribute to biodiversity. However, yeast biodiversity in the North West Province remains largely unexplored. The aim of this study was to determine the diversity and characteristics of yeasts from water sources in the North West Province, South Africa. Samples were collected over a two year period and included three rivers, a spruit and an inland lake. Temperature, pH, and electrical conductivity (EC) were measured on site using a multi-probe. Nitrate (NO3-N), nitrite (NO2-N) and phosphate (PO42-) levels were determined in the laboratory using Hatch kits and equipment. The pH ranged from 7.2 to 9.2. Elevated EC levels (36-70 mS) were detected especially at the Harts River and Barberspan (38-165 mS) sites. Physico-chemical parameter levels were higher during the cold dry sampling period compared to the warm rainy sampling period. Levels and diversity of yeasts were determined using the membrane filtration method. The highest level of yeasts was detected in the Mooi River and Schoonspruit during 2010 and 2011 sampling periods. Pigmented and non-pigmented yeasts were enumerated from all samples. Over the two year period the highest number of pigmented yeasts was detected in the Schoonspruit samples. In some cases there were significant (P<0.05) differences between pigmented and non-pigmented yeast levels among the sites. The diazonium blue B (DBB) test was carried out to distinguish between ascomycetous and basidiomycetous yeasts. These isolates were then identified using the API ID 32C system. Yeasts isolates were identified as belonging to the following genera: Candida, Cryptococcus, Pichia, Rhodotorula and Zygosaccharomyces. In addition using 26S rRNA gene sequencing Aureobasidium spp., Clavispora spp., Cystofilobasidium spp., Hanseniaspora spp., Meyerozyma spp., Sporidiobolus spp., and Wickerhamomyces spp.were also identified. The diversity and abundance of yeasts in the water sources demonstrated that opportunistic pathogens were present. This was supported by results that indicated some isolates could grow at 37°C and higher. In conclusion, our results provide preliminary information on the distribution and diversity of yeasts in water sources of the North West Province, South Africa. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: MAPK cascades in mammals & yeast. Includes bibliographical references (p. 122-141). Also available online through Digital Dissertations.

Thesis (Doctoral)--Ludwig-Maximilians-Universität zu München, 1935.