Proteoma extracelular de Kluyveromyces lactis em cultura contínua sob limitação de nitrogênio

• Autor(a) principal: Santos, Agenor Valadares
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1532
• topic: Kluyveromyces lactis; Proteína extracelular; Proteoma; Kluyveromyces lactis; Extracellular protein; Proteome; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::BIOLOGIA E FISIOLOGIA DOS MICROORGANISMOS

The identification of proteins secreted by yeast, in continuous culture, offers information about the influence of nutritional status in the nutrient capture alternative via. This analysis assists the optimization of conditions needed for the production of these proteins with the highest yield. In addition, it offers a strategy for the construction of a system of proteins expression and secretion in yeast. The present work shows the profile of extracellular proteins of Kluyveromyces lactis cultures submitted to stress by nitrogen. The continuous culture technique associated to the proteomic analysis was utilized in order to investigate the response of K. lactis culture in two growth velocities established as a function of nitrogen concentration, respectively, 0,03 h-1 and 0,09 h-1. The 0,09 h-1 growth velocity showed the major yield in the production of extracellular proteins (1,54 mg.L-1), a better conversion of glucose to product (3,3 x 10-4 g.g-1) and a greater biomass yield (0,13 g.g-1). Differences in proteomic cultures of K. lactis could be initially distinguished through the analysis of protein profiles in SDS-PAGE sampling gradient of extracellular extracts in the two growth velocities. The 0,09 h-1 velocity presented a larger number of bands when compared to the profile obtained for 0,03 h-1. In the proteomic identification of the bands excised from the gel by mass spectrometry in MALDI-TOF-TOF-MS, the spectrums acquired were analyzed using the software MASCOT®. Proteins suggested by this analysis are related to process such as cell cycle, replication, transcription, post translational changes, carbohydrates metabolism, ubiquitination and cellular degradation. They are commonly found inside the cell. Extracellular extracts of K. lactis were also analyzed by bi- dimensional liquid chromatography (LC-2D), in which the 0,09 h-1 specific growth velocity presented larger numbers of chromatographic peaks. This result indicates the presence of a larger number of proteins in this velocity when compared to the 0,03 h-1. In the several analysis performed through mass spectrometry in MALDI-TOF-TOF-MS, made from samples obtained by 2D liquid chromatography, it was acquired a total of 95 molecular masses in the two specific growth velocities. From these masses, about 30% belongs to the 0,03 h-1 specific growth velocity and 70% belongs to the 0,09 h-1 specific growth velocity. The analysis by mass spectrometry also evidenced the presence of glycosylations (molecules of N-acetylglucosamine and from 8 to 15 molecules of hexoses per protein) and of other possible post translational changes in samples resulting from LC-2D of both specific growth velocities. In the extracellular protein identification, the samples obtained from LC-2D of both specific growth velocities were treated with trypsin and analyzed once more by mass spectrometry. Proteins suggested by the analysis were K. lactis proteins connected to the traffic of peptides and to the metabolism of carbohydrates, transcription enzymes and other enzymes, such as tranferases, kinases, hydrolases, decarboxylases, oxidases and mutarotases, as well as in the previous evaluation; nevertheless, these proteins did not obtain the minimal scores to the homology identification be considered statistically significant.