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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2020.tde-19012022-172820
Document
Author
Full name
Tania Geraldine Churasacari Vinces
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Carvalho, Cristiane Rodrigues Guzzo (President)
Favaro, Denize Cristina
Gomez, José Gregorio Cabrera
Ramos, Carlos Henrique Inacio
Title in Portuguese
Caracterização funcional de uma nova esterase de solo escuro da Amazônia com comportamento histerético.
Keywords in Portuguese
Caraterização bioquímica
Cinética enzimática
Quorum quenching
Abstract in Portuguese
No presente estudo, caracterizamos bioquimicamente uma nova enzima com atividade esterase membro da superfamília α/β-hidrolase obtida de uma biblioteca metagenômica de solo escuro da Amazônia. Essa enzima foi nomeada como Amazonian Dark Earth Esterase 1 (Ade1). Ade1 hidrolisa ligações éster de diferentes substratos como: tributirina (cadeia alifática com 3 carbonos), Tween 20 (cadeia alifática com 11 carbonos), p-nitrofenil butirato (cadeia alifática com 4 carbonos), e p-nitrofenil octanoato (cadeia alifática com 8 carbonos) e N-hexanoil-L-homoserina lactona (C6-HSL, cadeia alifática com 6 carbonos) onde Ade1 atua interrompendo o quórum sensing pela sua capacidade de hidrolisar o autoindutor N-hexanoyl homoserine lactone, processo conhecido como quórum quenching. Dessa forma, Ade1 mostra um grau de promiscuidade em sua atividade enzimática pela sua capacidade de hidrolisar ligações ésteres de diferentes moléculas cuja estereoquímica permita coloca-la/seu ingresso no bolsillo catalítico no não seja volumosa. Ade1 é um monómero que apresento um perfil sigmoidal evidente nos ensaios de cinética, conhecido como comportamento histerético com perfil transitório tipo burst, o que já foi reportado para outras enzimas lipolíticas. Analise de dinâmica molecular mostra que Ade1 tem dois estados conformacionais: E1 e E2 cujo equilíbrio foi dependente da concentração do substrato. Alem, a atividade enzimática de Ade1 foi influenciada pela presencia de cobalto. O estado E2 possui uma maior velocidade de hidrólise de p-nitrofenil octanoato do que o estado E1. Ade1 pode ser uma enzima com interesse biotecnológico por poder modular quorum quenching em algumas bactérias que tem C6-HSL como autoindutor.
Title in English
Functional characterization of a new dark soil esterase from Amazon with hysterical behavior.
Keywords in English
Biochemical characterization
Enzymatic kinetics
Quorum quenching
Abstract in English
In the present study, we biochemically characterized a new enzyme with esterase activity that is a member of the α/β-hydrolase superfamily from a metagenomic library of dark soil in the Amazon. This enzyme was named as Amazonian Dark Earth Esterase 1 (Ade1). Ade1 hydrolyses ester bonds from different substrate such as: tributyrin (aliphatic chain with 3 carbons), Tween 20 (aliphatic chain with 11 carbons), p-nitrophenyl butyrate (aliphatic chain with 4 carbons), and p-nitrophenyl octanoate (aliphatic chain with 8 carbons) and N-hexanoyl-L-homoserine lactone (C6-AHL, aliphatic chain with 6 carbons) where Ade1 interrupts the quorum sensing by its ability to hydrolyze C6-HSL autoinducer. Thus, Ade1 shows a degree of promiscuity in its enzymatic activity due to its ability to hydrolyze ester bonds of different molecules whose stereochemistry is not bulky. Ade1 is a monomer that has a sigmoidal profile evident in kinetic test, known as hysteretic behavior with a burst profile, which has already been reported for other lipolytic enzymes. Molecular dynamics analysis shows that Ade1 has two conformational states: E1 and E2 whose equilibrium depend on the substrate concentration. In addition, the enzymatic activity of Ade1 was influenced by the presence of cobalt. The E2 state has a higher hydrolysis rate of p-nitrophenyl octanoate than the E1 state. Ade1 can be an enzyme with biotechnological interest because it can modulate quorum quenching in some bacteria that have C6-HSL as an autoinducer.
 
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Release Date
2024-01-19
Publishing Date
2022-12-02
 
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