Biochemical and structural features of the catalytic domain of cellulase CelE2 and its relation with the Calx-beta accessory domain

Author(s)

Agnes Cristina Pimentel

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-09-14

Examining board members

Fabio Marcio Squina; Fernando Segato; Thamy Lívia Ribeiro Corrêa

• Advisor: Fabio Marcio Squina; Thabata Maria Alvarez

Abstract

Glycoside hydrolases (GHs) comprise a class of enzymes widely used in the enzymatic processes, acting in the hydrolysis of the polysaccharides glycosidic bonds. In addition to the catalytic domain, several GHs present accessory domains, which may influence the biochemical and structural aspects of the enzymes. The CelE2, a metagenomic-derived cellulase, recently characterized, harbors in its structure an N-terminal catalytic domain characteristic of the GH5 family, and a C-terminal domain with identity for the Calx-betadomain, previously identified in other glycoside hydrolases but without clearly defined function in these cases. In order to investigate the biochemical and structural characteristics of the CelE2 domains separately, truncated derivations were constructed regarding the catalytic domain and the accessory domain Calx-beta. The results showed that the deletion of Calx-beta domain (CelE2382-477) did not cause alterations in the biochemical characteristics of CelE2, since the catalytic domain (CelE21-381) presented similar values of enzymatic activity in beta-glucan, carboxymethylcellulose and lichenan, optimal pH and temperature values near 5,3 and 45 ºC, respectively, thermal stability at 40 ºC and 50 ºC up to 360 minutes, and increase of enzymatic activity in the presence of CaCl2. The cleavage pattern and evidence of transglycosylation were also maintained after Calx-beta deletion from CelE2. However, the catalytic domain showed a 4-fold increase in the enzymatic activity of Avicel hydrolysis, compared to CelE2. Circular dichroism (CD) analysis of CelE21-381 indicated a predominant alpha-helix profile, decrease in the denaturation temperature around 4.6 ºC and an increase of these values in the presence of CaCl2, both for the complete construct and for CelE21-381. As well as CelE2, small angle X-ray scattering (SAXS) analyzes showed that the addition of CaCl2 resulted in a decrease in the protein aggregation tendency for CelE21-381. The binding assays for soluble and insoluble polysaccharides indicated that CelE2382-477 did not show the binding ability for the substrates tested. The three-dimensional structure of the catalytic domain CelE21-381 was determined at a resolution of 2.1 Å which allowed to identify an (alpha/beta)8 barrel fold. The results showed that the presence of the accessory domain Calx-beta is not fundamental for the enzymatic activity of CelE2, so that the most significant change concerns the increase of the enzymatic activity of the catalytic domain in Avicel, comparing to the whole construct. These results may contribute to future studies about this understudied domain among cellulases and information for the family 5 of glycoside hydrolases (AU)

• FAPESP's process: 16/01926-2 - Influence of an accessory domain in biochemical and structural characteristics of the cellulase CelE2
• Grantee: Agnes Cristina Pimentel
• Support Opportunities: Scholarships in Brazil - Master