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Previous issue date: 2014 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / A produ??o biotecnol?gica de enzimas por fungos filamentosos requer o conhecimento das suas caracter?sticas de crescimento, fisiologia e metabolismo porque a compreens?o das respostas biol?gicas e nutricionais contribui para o ajuste dos bioprocessos. Neste trabalho foram analisados o crescimento apical e alguns par?metros bioqu?micos e fisiol?gicos da resposta das linhagens Aspergillus tubingensis AN1257 e Talaromyces trachyspermus T10.5 a fontes de carbono complexas indutoras de carboidrases. Foi utilizada uma abordagem polif?sica de identifica??o baseada em caracter?sticas fenot?picas e moleculares. As linhagens foram cultivadas em meio suplementado com glicose (controle), amido e carboximetilcelulose (CMC). O crescimento apical foi avaliado quanto aos eventos iniciais, hidrata??o, polariza??o e extens?o do tubo germinativo, bem como foi investigado o papel da via de PKC nestes processos. As linhagens diferiram quanto ao potencial de produ??o de carboidrases sendo o T. trachyspermus T10.5 o mais eficiente para a express?o de amilase. A produ??o de celulase foi verificada apenas em meio s?lido. Os eventos iniciais do crescimento apical de T. trachyspermus T10.5 foram atrasados pelos carboidratos polim?ricos em meio s?lido: ap?s 14h de cultivo a propor??o de con?dios apolares, polares e com tubo germinativo foi de 31,0 ? 5,7%; 34,0 ? 0,0% e 35,0 ? 5,7% (glicose) contra 40,0 ? 0,0%; 45,0 ? 0,8% e 14,0 ? 1,6% (amido) ou 90,0 ? 4,9%; 6,0 ? 1,6% e 4,0 ? 3,3% (CMC). A biomassa de T. trachyspermus T10.5 foi formada exponencialmente no per?odo de 24 a 48h em cultivo submerso e n?o foi diminu?da por nenhuma fonte de carbono. Nas culturas submersas, a fase exponencial de crescimento foi simult?nea ao consumo exponencial do substrato; o esgotamento das fontes de carbono precedeu o in?cio da fase estacion?ria; a atividade de ?-amilase (239,2 ? 11 U/min.mL) induzida por amido coincidiu com o crescimento exponencial, permaneceu est?vel durante a fase estacion?ria e foi reprimida por glicose. O crescimento apical de A. tubingensis AN1257 respondeu mais rapidamente ? fonte de carbono: 36,0 ? 4,3% dos con?dios cultivados em meio suplementado com glicose estavam polarizados ap?s 6h, contra 1,3 ? 0,9% (amido) e 2,7 ? 1,9% (CMC). A extens?o do tubo germinativo da linhagem AN1257 tamb?m foi atrasada e reduzida por amido e CMC. A forma??o de biomassa de A. tubingensis AN1257 nas culturas submersas foi fortemente reduzida por CMC, que apresentou um efeito tamponante no meio. Nas demais culturas submersas, o crescimento exponencial de A. tubingensis AN1257 ocorreu entre 0 a 24h (glicose) ou 12 a 24h (amido) coincidindo com forte acidifica??o do meio e consumo acentuado de substrato. O efeito da ativa??o de PKC foi distinto nas duas linhagens: a ativa??o dessa enzima n?o parece modular a germina??o em A tubingensis AN1257, mas inibe fortemente a germina??o de T. trachyspermus T10.5. Assim, as duas esp?cies apresentaram respostas diferentes ? fonte de carbono e ? ativa??o de PKC, com fisiologia, germina??o e crescimento apical peculiares, cuja compreens?o permitir? direcionar a aplica??o biotecnol?gica das linhagens AN1257 e T10.5 de forma mais eficiente. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2014. / ABSTRACT
The biotechnological production of enzymes by filamentous fungi requires the knowledge of its characteristics of growth, physiology and metabolism because the comprehension of the biological and nutritional responses contributes to the adjustment of the bioprocesses. In this work, it were analyzed the apical growth, and some biochemical and physiological parameters of the response of the strains Aspergillus tubingensis AN1257 and Talaromyces trachyspermus T10.5 to complex carbon sources that induce carbohydrases. It was utilized a polyphasic approach of identification based on phenotypic and molecular characteristics. The strains were cultivated in media supplemented with glucose (control), starch and carboxy-methylcellulose (CMC). The apical growth was evaluated regarding the initial events, hydration, polarization and germtube extension, and the role of the PKC pathway in these processes was investigated as well. The strains differed according to their potential for carbohydrase production and T. trachyspermus T10.5 was the more efficient for amylase expression. Cellulase production was verified only in solid media. The initial events of T. trachyspermus T10.5 apical growth were delayed by the polymeric carbohydrates in solid media: after 14h of cultivation, the proportion of non-polar, polar and germtube-containing conidia was 31.0 ? 5.7%; 34.0 ? 0.0% and 35.0 ? 5.7% (glucose) versus 40.0 ? 0.0%; 45.0 ? 0.8% and 14.0 ? 1.6% (starch) or 90.0 ? 4.9%; 6.0 ? 1.6% and 4.0 ? 3.3% (CMC). The biomass of T. trachyspermus T10.5 was formed exponentially from 24 to 48h during submerged cultivation, and it was not decreased by any carbon source. In the submerged cultures, the exponential growth phase was simultaneous to the exponential consumption of the substrate; depletion of the carbon sources preceded the stationary phase; the ?-amylase activity (239.2 ? 11 U/min. mL) induced by starch coincided with the exponential growth, remained stable during the stationary phase and was repressed by glucose. A. tubingensis AN1257 apical growth responded more promptly to the carbon source: 36.0 ? 4.3% of the conidia cultivated in medium supplemented with glucose were polarized after 6h, versus 1.3 ? 0.9% (starch) and 2.7 ? 1.9% (CMC). Germtube extension by strain AN1257 was also delayed and reduced by starch and CMC. Biomass formation by A. tubingensis AN1257 in submerged cultures was strongly reduced by CMC, which presented a buffering effect in the medium. In other submerged cultures, the exponential growth of A. tubingensis AN1257 occurred from 0 to 24h (glucose) or 12 to 24h (starch), concomitant to a strong acidification of the medium and to intense substrate consumption. The effect of PKC activation was different in the two strains: activation of this enzyme does not seem to modulate germination in A. tubingensis AN1257, but it strongly inhibits T. trachyspermus T10.5 germination. Thus, the two species presented different responses to the carbon source and to PKC activation, with peculiar physiology, germination and apical growth, whose comprehension will allow direct the biotechnological application of strains AN1257 and T10.5 more efficiently.
| Identifer | oai:union.ndltd.org:IBICT/oai:acervo.ufvjm.edu.br/jspui:1/358 |
| Date | 25 February 2014 |
| Creators | Silva, Tiago Jos? da |
| Contributors | Said, Suraia, Pantoja, Lilian de Ara?jo, Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), Vanzela, Ana Paula de Figueiredo Conte |
| Publisher | UFVJM |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da UFVJM, instname:Universidade Federal dos Vales do Jequitinhonha e Mucuri, instacron:UFVJM |
| Rights | A concess?o da licen?a deste item refere-se ao ? termo de autoriza??o impresso assinado pelo autor, assim como na licen?a Creative Commons, com as seguintes condi??es: Na qualidade de titular dos direitos de autor da publica??o, autorizo a Universidade Federal dos Vales do Jequitinhonha e Mucuri e o IBICT a disponibilizar por meio de seus reposit?rios, sem ressarcimento dos direitos autorais, de acordo com a Lei n? 9610/98, o texto integral da obra disponibilizada, conforme permiss?es assinaladas, para fins de leitura, impress?o e/ou download, a t?tulo de divulga??o da produ??o cient?fica brasileira, e preserva??o, a partir desta data., info:eu-repo/semantics/openAccess |
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