| Full text | |
| Author(s): |
Pereira, Mariana Rangel
;
Maester, Thas Carvalho
;
Mercaldi, Gustavo Fernando
;
de Macedo Lemos, Eliana Gertrudes
;
Hyvonen, Marko
;
Balan, Andrea
Total Authors: 6
|
| Document type: | Journal article |
| Source: | Applied Microbiology and Biotechnology; v. 101, n. 12, p. 4935-4949, JUN 2017. |
| Web of Science Citations: | 10 |
| Abstract | |
Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of alpha/beta hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-C resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met(213) and Phe(217)) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues. (AU) | |
| FAPESP's process: | 11/09136-7 - Functional and Structural Characterization of Lipolitic Enzymes of Consortium Specialized on Diesel Oil Degradation |
| Grantee: | Mariana Rangel Pereira |
| Support Opportunities: | Scholarships in Brazil - Doctorate |
| FAPESP's process: | 12/20490-0 - Characterization of substrate binding to novel thermostable lipases/esterases and rational design to change their substrate specificity for biotechnological applications |
| Grantee: | Mariana Rangel Pereira |
| Support Opportunities: | Scholarships abroad - Research Internship - Doctorate |