| Texto completo | |
| Autor(es): |
Pimentel, Agnes C.
;
Liberato, Marcelo V.
;
Cairo, Joao Paulo L. Franco
;
Tomazetto, Geizecler
;
Gandin, Cesar A.
;
Neto, Mario de Oliveira
;
Alvarez, Thabata M.
;
Squina, Fabio M.
Número total de Autores: 8
|
| Tipo de documento: | Artigo Científico |
| Fonte: | Enzyme and Microbial Technology; v. 165, p. 10-pg., 2023-04-01. |
| Resumo | |
Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular archi-tectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-beta domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-beta domain. In this study, the GH5 and the Calx-beta domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 degrees C for the catalytic domain (CelE21-381) compared to CelE2 full-length. The CD analyses revealed that the Calx-beta domain (CelE2382-477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21-381 was determined at 2.1 angstrom resolution, showing a typical (alpha/beta)8-bar-rel fold and a narrow active site compared to other cellulases from the same family. The biochemical charac-terization showed that the deletion of the Calx-beta domain increased more than 3-fold the activity of the catalytic domain CelE21-381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21-381 protein solvency. This work contributed to funda-mental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production. (AU) | |
| Processo FAPESP: | 16/01926-2 - Influência de um domínio acessório nas características bioquímicas e estruturais da celulase CelE2 |
| Beneficiário: | Agnes Cristina Pimentel |
| Modalidade de apoio: | Bolsas no Brasil - Mestrado |
| Processo FAPESP: | 15/50590-4 - Valorização da lignina em plantas de etanol celulósico: a conversão biocatalítica via ácido ferúlico a produtos químicos de alto valor |
| Beneficiário: | Fábio Márcio Squina |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa BIOEN - Temático |
| Processo FAPESP: | 20/05784-3 - EMU concedido no processo 15/50590-4: sistema cromatográfico e detectores para análise de açúcares e monolignois de lignocelulose |
| Beneficiário: | Fábio Márcio Squina |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa Equipamentos Multiusuários |
| Processo FAPESP: | 14/04105-4 - Caracterização estrutural e funcional de novas celulases com foco na relação entre domínios catalíticos e CBMs |
| Beneficiário: | Marcelo Vizoná Liberato |
| Modalidade de apoio: | Bolsas no Brasil - Pós-Doutorado |
| Processo FAPESP: | 10/11469-1 - Desenvolvimento de uma biblioteca de enzimas a partir de metagenoma de solo |
| Beneficiário: | Thabata Maria Alvarez |
| Modalidade de apoio: | Bolsas no Brasil - Doutorado Direto |
| Processo FAPESP: | 16/09950-0 - Caracterização funcional, estrutural e aplicação biotecnológica de mono-oxigenases líticas de polissacarídeos do cupim inferior Coptotermes gestroi |
| Beneficiário: | João Paulo Lourenço Franco Cairo |
| Modalidade de apoio: | Bolsas no Brasil - Pós-Doutorado |
| Processo FAPESP: | 15/23279-6 - Análise metagenômica e metasecretômica de um consórcio microbiano enriquecido de fungos anaeróbicos degradador de bagaço de cana: prospecção de celulossomas e enzimas lignocelulolíticas |
| Beneficiário: | Geizecler Tomazetto |
| Modalidade de apoio: | Bolsas no Brasil - Pós-Doutorado |