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No. of bitstreams: 1 inforsato_fj_me_rcla.pdf: 2106040 bytes, checksum: 5384cac1311ff16eee3bec56a6987c15 (MD5) Previous issue date: 2017-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os micro-organismos que habitam o continente Antártico podem apresentar produção de compostos de interesse industrial e ambiental com propriedades específicas por estarem adaptados às condições adversas da Antártica. Dentre os compostos produzidos por micro-organismos da Antártica podemos destacar as enzimas adaptadas ao frio. O presente trabalho teve como objetivo principal estudar a diversidade dos fungos filamentosos e leveduriformes isolados de cinco amostras de sedimentos marinhos da Antártica, bem como prospectar as enzimas lipase e protease. Para o isolamento dos fungos duas estratégias foram aplicadas (com e sem enriquecimento das amostras). Para ambas as estratégias, os sedimentos marinhos foram diluídos em solução salina e após homogeneização inoculados em placas de Petri contendo quatro diferentes meios de cultura (PDA, BSA, PDA diluído 10x, MA), as quais foram incubadas a 5 e 15ºC. Um total de 226 isolados foi recuperado, sendo 60 fungos filamentosos e 166 leveduras. A maioria dos fungos foi isolada a 15ºC (68%) De acordo com os dados de sequenciamento e análise filogenética, representantes de oito gêneros e quatro espécies de fungos filamentosos foram isolados das amostras de sedimentos marinhos. Os gêneros Penicillium e Pseudogymnoascus foram os mais abundantes (40,00%), seguidos pelo gênero Cadophora (6,67%), Cladosporium (5,00%), Toxicocladosporium, Pseudocercosporella, Pestalotiopsis e Paraconiothyrium (1,67%). Para as leveduras, representantes de nove gêneros e 13 espécies foram recuperadas das amostras, sendo a espécie Metschnikowia australis (45,18%) a mais abundante, seguida por representantes do gênero Mrakia (21,68%), Cryptococcus e Glaciozyma (7,83%), Meyerozyma e Holtermanniella (5,42%), Rhodotorula (4,21%), Cystobasidium (1,80%) e Phenoliferia (0,60%). Alguns dos fungos filamentosos identificados no presente trabalho não haviam sido ainda reportados em ambientes Antárticos (Toxicocladosporium, Pseudocercosporella e Paraconiothyrium). As análises de diversidade revelaram que a amostra A5 coletada no Refúgio 2 (20 m, 0,1ºC) apresentou a maior riqueza de espécies, enquanto a amostra A2 coletada no Botany Point (24,7 m, 0,6ºC) se mostrou mais diversa. Com relação à diversidade β, as amostras A5 e A6 coletadas na mesma região geográfica (Refúgio 2) apresentaram o maior compartilhamento de espécies (42,3%). Os isolados foram avaliados quando à capacidade de produção de lipase e protease. Os resultados revelaram que a levedura 6A-1C2II (M. australis) apresentou a maior atividade lipolítica (0,88 U.mL-1) e o fungo filamentoso 4A-1C615III (Pseudogymnoascus sp.) a maior atividade proteolítica (6,21 U.mL-1). Ambos os isolados foram submetidos aos estudos de avaliação de diferentes fatores na produção das enzimas visando maximização da produção enzimática. Após a condução de planejamentos experimentais do tipo Plackett-Burman (PB), a produção de lipase por M. australis 6A-1C2II aumentou 1,36 vezes e a produção de protease por Pseudogymnoascus sp. 4A-1C615III 4,72 vezes. Nos experimentos de validação das condições otimizadas, as médias das triplicatas apresentaram valores inferiores aos alcançados nos planejamentos. A condução do presente trabalho permitiu a ampliação do conhecimento sobre a diversidade de fungos e leveduras que habitam os sedimentos marinhos da Antártica. Em adição, resultou na obtenção de isolados de fungos filamentosos e leveduras de ambiente extremo, os quais estão sendo mantidos na coleção de pesquisa associada à Central de Recursos Microbianos da UNESP (CRMUNESP) e poderão ser utilizados em estudos futuros de prospecção e aplicação biotecnológica. / Microorganisms that inhabit the Antarctic continent can produce compounds of industrial and environmental interest with specific characteristics, since they are adapted to the adverse conditions in Antarctica. Among the compounds that are produce by Antarctic microorganisms we can enhance the cold-adapted enzymes. The main objective of this research was to study the diversity of filamentous fungi and yeasts from five Antarctic marine sediments samples, as well as to prospect lipase and protease enzymes. Two strategies were applied for fungal isolation (with and without enrichment of the samples). For both strategies, the sediments were diluted with saline solution and after homogenization the dilutions were inoculated in Petri dishes containing four different culture media (PDA, BSA, PDA diluted 10x, and MA), which were incubated at 5 and 15 ºC. The total amount of isolates recovered was 226, being 60 fungi and 166 yeasts. Most of the fungi were isolated at 15ºC (68%). According to sequencing data and phylogenetic analyses, eight genera and four species of filamentous fungi were isolated from the marine sediment samples. The genera Penicillium and Pseudogymnoascus were the most abundant (40.00%); followed by genus Cadophora (6.67%), Cladosporium (5.00%), Toxicocladosporium, Pseudocercosporella, Pestalotiopsis, and Paraconiothyrium (1.67%). Representatives of nine genera and thirteen yeast species were recovered from the sediment samples, the specie Metschnikowia australis was the most abundant (45.18%), followed by isolates from genus Mrakia (21.68%), Cryptococcus and Glaciozyma (7.83%), Meyerozyma and Holtermanniella (5.42%), Rhodotorula (4.21%), Cystobasidium (1.80%), and Phenoliferia (0.60%). Some of the fungi identified in the present work were not reported in Antarctic environments before (Toxicocladosporium, Pseudocercosporella e Paraconiothyrium). The diversity analyses showed that the sample A5 collected from Refúgio 2 (20 m, 0.1ºC) presented higher species richness, while sample A2 that was collect from Botany Point (24,7 m, 0.6ºC) was the most diverse. According to β diversity data, the samples A5 and A6 that were collect in the same geographic region (Refúgio 2) showed the higher species sharing (42.3%). The capacity to produce lipase and protease were evaluated. The results revealed that the yeast 6A-1C2II (M. australis) presented the best lipolytic activity (0.88 U.mL-1) and the fungus 4A- 1C615III (Pseudogymnoascus sp.) the best proteolytic activity (6.21 U.mL-1). Both isolates were submitted to the evaluation of different factors in the enzyme production process aiming the enzymatic production maximization. After the conduction of Plackett-Bruman (PB) experimental designs, the lipase production by M. australis 6A- 1C2II increased 1.36 times and the protease production by Pseudogymnoascus sp. 4A- 1C615III increased 4.72 times. In the optimized conditions validation experiments, results from the triplicates averages were lower than that one reached during the experimental designs. This work allowed the improvement in the knowledge of fungal diversity in the Antarctic marine sediments ecosystem. In addition, resulted in the obtaining of fungi and yeasts from extreme environment, which are being maintained in the research culture collection associated to the Central of Microbial Resources (CRMUNESP) and can be used in future studies related to prospection and biotechnology application. / FAPESP: 2013/19486-0

"2003". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2004. / Bibliography: leaves 136-160. / Introduction: The Antarctic environment; Antarctic inhabitants; Microfungi; Identification of microfungi; Physiological factors affecting Antactic microfungi; Flow cytometry and microfungi; Hydrolytic enzymes of industrial interest; Isolation of genes from microfungi; Aims of this study -- Materials and methods: Fungal strains and cultivation conditions; Molecular identification of fungal isolates; Fungal physiology; Hydrolase activity of secreted proteins; Gene cloning and expression -- Results and discussion: Microfungal identification; Physiological factors affecting Antarctic microfungi; Activity in microfungi when grown on solid media; Characterisation of hemicellulases from selected Antarctic microfungi; Cloning of an Antarctic Penicillium allii lipase gene and its expression in Trichoderma reesei -- Conclusions and future prospects. / The Antarctic occupies that region of the planet that falls below the 60th parallel of South latitude. Although it has been frequented by adventurers, journeyman scientists and tourists for the past 100 years, the Continent has remained virtually unoccupied. The intense cold, the absence of human occupation and the limited range of local higher animal species have combined to create the impression that the Continent is virtually devoid of life. -- Although the microbiota of the Antarctic has attracted some small level of attention in the past, the examination of filamentous microfungi has been largely overlooked and fallen to a small group of dedicated investigators. In this study it will be shown that far from being an insignificant component of the Antarctic network, microfungi represent a potentially large and so far untapped bioresource. -- From just 11 bryophyte samples collected at four sites in the Ross Sea/Dry Valleys region of Southern Antarctica, some 30 microfungal isolates were recovered. Using molecular techniques, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) was sequenced to reveal no less than nine unique microfungal species. For only two of these species did the ITS sequence data produce a 100% match with records held on the public databases. This investigation also highlighted the problems inherent in the traditional morphological identification system which are now being perpetuated in the molecular database records. -- A set of seven notionally identified isolates obtained from ornithogenic soil samples gathered in the Windmill Islands in Eastern Antarctica (offshore from the Australian Antarctic Division's Casey Station) were also subjected to molecular identification based on ITS sequence data. Each of the seven isolates was identified as a unique species; six were cosmopolitan in nature and the one remaining bore very little resemblance at the molecular level to any of the recorded species although it was provided with an epithet commonly used in the identification of Antarctic microfungal species. -- To evaluate their potential as a bioresource, samples of Antarctic microfungi were examined to determine if the same physiological factors common to mesophilic species also applied to their Antarctic analogues. It is known that when placed under stress, trehalose can act as a protectant against cold (cryoprotection) and dehydration in mesophilic yeasts and fungi. The level of trehalose produced by the Antarctic isolates and their mesophilic analogues when subjected to stress was compared. A similar comparison was made for the production of glycerol which is well established as a compatible solute providing protection to mesophilic species against osmotic stress. Only in the case of trehalose production by an Antarctic Embellisia was there any indication that either of these two compounds could play a significant role in providing protection to the Antarctic fungi against the rigours of their environment, which leaves open to question what in fact does. -- In the course of investigating the means by which Antarctic microfungi guard against the damage which can ensue when subjected to oxidative stress, flow cytometry was introduced as an investigatory tool. It was established that there is a window of opportunity during which flow cytometry can be used to undertake a detailed analysis of the early stages of fungal growth from germination through hyphal development. -- Of major significance in determining the potential of Antarctic microfungi as a resource is their ability to produce new and novel enzymes and proteins. The microfungal isolates were screened for hydrolytic activity on solid media containing indicative substrates and proved to be a fruitful source of enzymes active over a range of temperatures. A detailed characterisation of two hemicellulases, β-mannanase and xylanase, secreted into a liquid medium by a subset of the Antarctic fungi and a high producing mesophilic reference strain permitted direct comparisons to be made. It was shown that the maximum hemicellulase activity of the Antarctic strains occurred at least 10°C and as much as 30°C lower than that of the reference strain and that mannanase activity for two of the Antarctic isolates exceeded 40% of their maximum at 0°C. These assay results highlight the potential of Antarctic microfungi to yield novel cold-active enzymes. -- As a final measure of the capacity of the Antarctic to yield novel enzymes from its microfungal stock, a lipase gene was selected as a target for isolation and expression in a heterologous fungal host. Using PCR techniques, the gene of interest was isolated from an Antarctic isolate of Penicillium allii, transformed into the mesophilic production host Trichoderma reesei and the active protein successfully produced in the growth medium. The recombinant lipase was assayed and found to exhibit novel characteristics consistent with a cold-adapted enzyme. / Mode of access: World Wide Web. / 186 leaves ill