Otimização do processo fermentativo e análise do secretoma de Kluyveromyces marxianus UFV-3 em meios contendo lactose em diferentes condições de cultivo

• Ano de defesa: 2013
• Autor(a) principal: Diniz, Raphael Hermano Santos
• Orientador(a): Passos, Flávia Maria Lopes
• Banca de defesa: Santos, Agenor Valadares , Ribeiro, Cleberson , Nascimento, Antonio Galvão do
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Microbiologia Agrícola
• Departamento: Associações micorrízicas; Bactérias láticas e probióticos; Biologia molecular de fungos de interesse
• País: BR
• Palavras-chave em Português: Kluyveromyces marxianus; Secretoma; Metologia de superfície resposta; Etanol; Soro de queijo
• Palavras-chave em Inglês: Kluyveromyces marxianus; Secretome; Response surface method; Ethanol; Cheese whey
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::MICROBIOLOGIA AGRICOLA
• Link de acesso: http://locus.ufv.br/handle/123456789/1561

Kluyveromyces marxianus UFV-3 is yeast that has respirofermentative metabolism, i.e., respiration and fermentation coexists, however fermentative or respiratory pathway can be favored depending on the oxygen and carbohydrate concentration in the culture medium. Considering this characteristic of yeasts of the genus Kluyveromyces the objective of this study was to optimize the production of ethanol from cheese whey permeate (CWP) and characterize the secretome of K. marxianus UFV-3 in view of the use of this yeast as a host for producing heterologous proteins. To optimize the production of ethanol we used the response surface method (RSM) with a central composite rotational design (CCRD) to evaluate the effects of pH (4.5-6.5), temperature (30-45 ºC), lactose concentration (50-250 g L-1), and dry cell mass concentration (1-2 g L-1). We performed 29 fermentations under hypoxia in CWP and 7 fermentations for the validation of the equation obtained via RSM. Temperature was the most significant factor in optimizing ethanol production, followed by pH, cell biomass concentration, and lactose concentration. The conditions for producing ethanol at yields above 90% were as follows: temperature between 33.3-38.5 ºC, pH between 4.7-5.7, dry cell mass concentration between 1.26-1.68 g L-1, and lactose concentration between 50-108 g L-1. The equation generated from the optimization process was validated and exhibited excellent bias and accuracy values for the future use of this model in scaling up the fermentation process. To analyze how culture conditions influencing protein secretion in K. marxianus UFV-3, this yeast was cultivated in five different conditions: batch, fed-batch (initial feed rate of 0.05 and 0.1 h-1) and continuous culture (0.3 and 0.1 h-1) in medium containing lactose. The supernatant of the cultures were analyzed on SDSPAGE and all the samples had low diversity of proteins. However, in continuous cultures with dilution of 0.1 h-1 and in fed batch 0.05 h-1 detected a higher concentration and diversity of proteins in other cultures, and in both cultures, we obtained a protein secreted in greater proportion than the others. To identify proteins secretome of K. marxianus was cultivated in continuous culture because it has more reproducible results and control of fermentation parameters when compared with fed batch. Samples of the continuous culture supernatant were subjected to protein size fractioning and in-gel digestion, followed by reversed-phase peptide separation in the nano-LC system and subsequent peptide identification by MALDI-TOF/TOF. Eight proteins were identified as structural proteins or membrane proteins, and the protein was secreted differentially was identified as an endopolygalacturonase - endoPG - (EC 3.2.1.15). EndoPG was characterized with optimum activity enzymatic at a temperature of 59.5 ºC and pH 5.1. Furthermore, it was found that apparently is repressed by glucose and shows intense secretion in batch cultures in medium containing glycerol as the carbon source, and cultures on lactose and galactose.