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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2020.tde-01082022-100116
Document
Author
Full name
Camila Cristina Coelho
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Winter, Carlos Eduardo (President)
Guimaraes, Margareth de Lara Capurro
Portaro, Fernanda Calheta Vieira
Prado, Lucila Bizari Fernandes do
Title in Portuguese
Proteases de Panagrolaimus sp. CEW2 (Nematoda, Rhabditida).
Keywords in Portuguese
Panagrolaimus
extremófilo
nematoide
protease
Abstract in Portuguese
Nematoides de vida livre do gênero Panagrolaimus se caracterizam por apresentar hábitos extremófilos, sobrevivendo a condições de baixa temperatura e baixa umidade relativa. A linhagem dióica que utilizamos em nossos estudos (Panagrolaimus sp. CEW2) foi isolada em Rondônia e tem se mostrado resistente a dessecação, congelamento e alta força iônica/pressão osmótica. Proteases têm sido estudadas do ponto de vista bioquímico principalmente em nematoides parasitas. Apesar de 2% dos genes de C. elegans serem anotados como protease pouco se sabe sobre suas proteases digestivas. Os ensaios de atividade encontrados no extrato total de CEW2 foram realizados utilizando substratos sintéticos para tripsina, quimotripsina, aminopeptidase, elastases e catepsina. Desenvolvemos um método de crescimento em massa de Panagrolaimus sp. CEW2 utilizando meio S, E. coli linhagem NA22 e um substrato sólido feito com esponja de poliuretano. Este método aumentou em 3x a quantidade de nematoides obtidos quando comparados ao crescimento em placa de Petri com meio NGM. As atividades enzimáticas detectadas foram caracterizadas em relação ao seu pH ótimo, temperatura ótima e sensibilidade a íons metálicos e inibidores. Uma atividade elastásica detectada no homogeneizado de vermes era dependente de cálcio e inesperadamente ativada por sulfato de cobre. Atividades tipo tripsina e quimotripsina não foram detectadas nos homogeneizados.
Title in English
Proteases from Panagrolaimus sp. CEW2 (Nematoda, Rhabditida). 2020
Keywords in English
Panagrolaimus
extremophile
nematode
protease
Abstract in English
Free-living nematodes of the genus Panagrolaimus are characterized by having extremophilic habits, surviving conditions of low temperature and low relative humidi-ty. The dioecious lineage that we used in our studies (Panagrolaimus sp. CEW2) was isolated in Rondônia-BR and is resistant to desiccation, freezing and high ionic strength/osmotic pressure. Proteases have been studied from a biochemical point of view mainly in parasitic nematodes. Although 2% of C. elegans genes are noted as pro-teases, little is known about their digestive proteases. The proteolytic activity tests made in the total extract of CEW2 were carried out using synthetic substrates for trypsin, chymotrypsin, aminopeptidase, elastase, cathepsin. We developed a mass growth meth-od for Panagrolaimus sp. CEW2 using a liquid medium, E. coli strain NA22 and a solid substrate composed of polyurethane sponge. This method increased three times the wet mass of nematodes obtained when compared to growth in Petri dishes. The enzymatic activities detected were characterized as to their optimum pH, optimum temperature and sensitivity to metal ions and inhibitors. An elastase activity present in the worm homog-enates was shown to be calcium dependent and unexpectedly activated by copper sul-fate. Chymotrypsin- or trypsin-like activities were not detected in CEW2 homogenates.
 
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Release Date
2025-03-12
Publishing Date
2023-04-06
 
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