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Doctoral Thesis
DOI
https://doi.org/10.11606/T.60.2020.tde-19122019-084515
Document
Author
Full name
Rafael Pedezzi
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Cabral, Hamilton (President)
Russo, Elisa Maria de Sousa
Silva, Roberto da
Gomes, Eleni
Oliveira, Arthur Henrique Cavalcante de
Polizeli, Maria de Lourdes Teixeira de Moraes
Title in Portuguese
Prospecção de enzimas no fungo Purpureocillium lilacinum utilizando as técnicas de proteoma: produção recombinante de enzimas com potencial biotecnológico e avaliação de antifúngicos
Keywords in Portuguese
Expressão recombinante
Peptidases fúngicas
Purpureocillium lilacinum
Resistência a antifúngicos
Transcriptoma
Abstract in Portuguese
O fungo ascomiceto Purpureocillium lilacinum pode ser encontrado em solo, vegetação em decomposição, insetos, nematoides e outros locais. Apresenta grande importância agrícola, visto que pode ser utilizado como controle biológico por ser capaz de infectar e matar nematoides pragas de culturas, graças às suas enzimas, como peptidases e quitinases. Além disso, o fungo apresenta importância médica, pois é capaz de gerar infecção em humanos. A terapêutica das doenças infecciosas causadas por P. lilacinum é dificultada pelo fato de ele apresentar resistência a antifúngicos "azólicos", principais moléculas aplicadas na clínica médica atualmente. O presente trabalho teve como objetivo explorar molecularmente o fungo P. lilacinum com relação à sua capacidade de resistir a antifúngicos e à sua capacidade de produzir peptidases robustas, com potencial de aplicação biotecnológica. Ferramentas moleculares como transcriptômica e proteômica foram aplicadas para acessar os dados moleculares do fungo e viabilizar análises de expressão diferencial para verificar as respostas aos antifúngicos fluconazol e itraconazol e anotação gênica para prospecção de peptidases. Foi observado que P. lilacinum promove um aumento de expressão dos genes alvo dos antifúngicos e dos genes que codificam bombas de efluxo. Com esse mecanismo, não há acúmulo de antifúngico intracelular e há compensação numérica da enzima que está sendo inibida pelo antifúngico com mais expressão da mesma. Dessa forma, o fungo consegue manter a síntese de ergosterol normalizada. As enzimas prospectadas e expressas de maneira recombinante em Pichia pastoris foram uma serino peptidase (rPl_SerPep) e uma metalo peptidase (rPl_MetPep). A rPl_SerPep se mostrou alcalina, com temperatura ótima aparente de 60 °C e termoestável. Já a rPl_MetPep se mostrou ativa em pH neutro e com temperatura ótima aparente de 40 °C. Essas enzimas se mostraram promissoras para futuros testes de aplicação biotecnológica.
Title in English
Prospecting of enzymes in Purpureocillium lilacinum using proteomic technique: recombinant production of enzymes with biotechnological potential and evaluation of antifungals
Keywords in English
Antifungal resistance
Fungal peptidases
Purpureocillium lilacinum
Recombinant expression
Transcriptome
Abstract in English
The ascomicota fungus Purpureocillium lilacinum can be found in soil, decaying vegetation, insects, nematodes and other places. It has great agricultural importance, since it can be used as biological control by being able to infect and kill nematode crop pests, thanks to its enzymes such as peptidases and chitinases. In addition, the fungus is of medical importance because it can generate infection in humans. The treatment of infectious diseases caused by P. lilacinum is hampered by the fact that it is resistant to "azolic" antifungals, the main molecules applied in medical practice today. The present work aimed to molecularly explore the fungus P. lilacinum in relation to its ability to resist antifungal and its ability to produce robust peptidases with potential for biotechnological application. Molecular tools such as transcriptomics and proteomics were applied to access the molecular data of the fungus and enable differential expression analyzes to verify the responses to the antifungal fluconazole and itraconazole and gene annotation to prospect for peptidases. It has been observed that P. lilacinum promotes an increase in expression of antifungal target genes and genes encoding efflux pumps. With this mechanism, there is no accumulation of intracellular antifungal and there is numerical compensation of the enzyme that is being inhibited by the antifungus with more expression of the same. In this way, the fungus can maintain standard ergosterol synthesis. The serine peptidases prospected were recombinantly expressed in Pichia pastoris. There were a serine peptidase (rPl_SerPep) and a metal peptidase (rPl_MetPep). The rPl_SerPep was alkaline, with an apparent optimum temperature of 60 ° C and showed thermostable characteristics. The rPl_MetPep was neutral and with an apparent optimum temperature of 40 ° C. These enzymes have proven to be promising for future biotech application tests.
 
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Release Date
2021-12-18
Publishing Date
2020-05-12
 
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