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Doctoral Thesis
DOI
https://doi.org/10.11606/T.97.2020.tde-29012020-160134
Document
Author
Full name
Gislene Ferreira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Lorena, 2019
Supervisor
Committee
Segato, Fernando (President)
Cardoso, Patricia Gomes
Finardi Filho, Flavio
Milagres, Adriane Maria Ferreira
Title in Portuguese
Clonagem, expressão e caracterização de fitase do fungo termofílico Myceliophthora thermophila
Keywords in Portuguese
Myceliophtora thermophila
Expressao heterologa
Fitases
Fungos termofilicos
Abstract in Portuguese
Fitases são enzimas que hidrolisam o ácido fítico, substancias orgânicas complexas amplamente encontradas na natureza, principalmente em cereais e leguminosas. Nesses alimentos vegetais, o fitato e a principal fonte de fósforo, correspondendo entre cinquenta a oitenta por cento do conteúdo de fósforo orgânico. Entretanto, o fósforo do fitato e muito pouco utilizado por humanos e outros animais monogástricos, como suínos e aves, devido a ausência ou insuficiência das enzimas que o degradam. Assim, rações contém o fósforo orgânico do fitato e fósforo inorgânico adicionado, que é um mineral caro e não renovável. Em áreas de criação intensiva o excesso de fósforo e excretado no ambiente, causando problemas, tais como a eutrofização. Devido aos problemas ambientais e ao alto custo do fósforo inorgânico, a suplementação de enzimas, principalmente fitases, tem ganhado destaque, entre elas, as enzimas fúngicas, devido a sua estabilidade a diferentes temperaturas e pH. Além disso, técnicas de engenharia genética permitem a transferência de genes entre espécies, resultando em muitos benefícios, principalmente em relação ao aumento e melhoramento na produção de enzimas industriais. O objetivo deste trabalho foi realizar a clonagem, expressão heteróloga, purificação e caracterização bioquímica da enzima fitase (MtPhy) obtida a partir do fungo termofílico M. thermophila. O gene que codifica a enzima foi clonado no vetor pEXPYR e heterologamente expresso em A. nidulans. A proteína recombinante foi purificada e teve sua identidade confirmada por espectrometria de massas. Nas analises bioquímicas, apresentou pH ótimo de 5,0 e temperatura ótima de 60°C e manteve praticamente 100% de sua atividade a 50°C por 2 horas e pela análise de dicroísmo circular e ThermoFluor, apresentou desenovelamento de sua estrutura nas temperaturas de 63 e 67°C, respectivamente. Demonstrou atividade aumentada na presença de CaCl2 e MgCl2 e foi fortemente inibida por FeCl3, CuSO3 e SDS. Sua atividade especifica (954,8 U/mg) utilizando fitato de sódio foi bastante superior à de outras fitases descritas na literatura. Demonstrou-se efetiva na liberação do fósforo em rações e extremamente resistente as proteases, tripsina e pepsina, presentes no trato digestório de animais monogástricos, demonstrando dessa forma, seu elevado potencial para aplicação industrial.
Title in English
Cloning, expression and characterization of phytase produced by thermophilic fungus Myceliophthora thermophyla
Keywords in English
Myceliophthora thermophila
Heterologous expression
Phytases
Thermophilic fungal
Abstract in English
Phytases are enzymes that hidrolyse phytic acid, wich are complex organic substances, found widely in nature mainly in cereals and legumes. In plants, the phytate are the main source of phosphorus. It account for fifty to eighty percent of the total organic phosphorus. However, the phytate phosphorus is very poorly used by humans and other animals, such as swines and poultry due to the absence or insufficiency of enzymes that degrade phytate. Thus, animal feed contain phytate organic phosphorus and inorganic phosphorus added, wich is an expensive and not renovable mineral and in areas of intensive breeding, the excess phosphorus is excreted in the environment, causing phoblems, such as, eutrophication. Due to environmental problems and the high price of inorganic phosphorus, the enzyme supplementation, mainly phytases has been growing, among them, fungal enzymes, because of their stability at different temperature and pH. In addition, genetic engineering techniques allow the transfer of genes between species, resulting in many benefits, mainly in relation to the increase and improvement in the production of industrial enzymes. The aim of this work was to perform the cloning, heterologous expression, purification and biochemical characterization of the phytase enzyme (MtPhy) obtained from the thermofilic fungus Myceliophthora thermophila. The gene encoding this enzyme was cloned and heterologously expressed in A. nidulans A773, the MtPhy was purified and had its identity confirmed by mass spectrometry. In the biochemical analysis the MtPhy showed an optimum pH of 5.0 and optimum temperature of 60°, maintaining practically 100% of its activity at 50°C for 2 hours. The circular dichroism and ThermoFluor analysis showed a denaturation of its structure at temperatures of 63 and 67°C, respectively. The activity of MtPhy was increased in the presence of CaCl2 and MgCl2 and was highly affected by FeCl3, CuSO3 and SDS. The specific activity (954,8 U/mg) in sodium phytate was far superior to another phytases described in literature. It was effective in release of phosphorus in animal feed and extremely resistant to proteases, trypsin and pepsin, present in the digestive tract of monogastric animals, thus proving high potential for industrial application.
 
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Publishing Date
2020-01-29
 
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