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| Author(s): |
Aline Minali Nakamura
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Carlos. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT) |
| Defense date: | 2020-05-08 |
| Examining board members: |
Igor Polikarpov;
Ana Paula Ulian de Araujo;
Denise Maria Guimarães Freire;
Camila Alves de Rezende;
Fernando Segato
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| Advisor: | Igor Polikarpov |
| Abstract | |
Carboxylesterases comprise a major class of α/β-fold hydrolases and catalyze the cleavage and formation of ester bonds. They are widespread in nature, being expressed by animals, plants and microorganisms, and playing an essential role in the metabolism of the endogenous and exogenous carboxyl esters. Besides the important physiological functions, they compose one of the most important biocatalysts for biotechnology, being widely applied on a broad range of industrial applications and with many available commercial preparations. Moreover, B. licheniformis features a promising source of carboxylesterases. However, up to date, there is no structural information regarding carboxylesterases of this organism. This study aimed to analyze biochemically and structurally two B. licheniformis carboxylesterases, focusing on relevant features for biotechnological applications. BlEst1 presented the higher hydrolytic activity against p-nitrophenyl acetate at pH 7.0 and 40 ºC. Furthermore, BlEst1 showed to be stable in some organic solvent stability in high salt concentrations (0 – 3 M NaCl), while maintaining activity, with significantly increase of 17 °C on its melting temperature, revealing its halotolerant character. Structural analysis revealed an acidic electrostatic surface, indicating that BlEst1 may adopt the solvation-stabilization model, the most common theory for the halophilic adaptation. BlEst2 presented the core region with a typical α/β-hydrolase fold and an overall multidomain structure. The catalytic domain presented two insertions, which occupy conserved locations in α/β-hydrolase proteins and commonly made up the lid domain on lipases. The C-terminal domains compose the BlEst2 propeptide and their removal is required to enzyme activation. Besides this, they act like intramolecular chaperones, being required for properly folding. After activation, BlEst2 presented the higher hydrolytic activity against p-nitrophenyl butyrate (292 U/mg) at pH 8.0 and 45 ºC. For both enzymes, we found inconsistencies between classification and experimental data, indicating that the classification systems are not representative enough to explain the great diversity within this group of hydrolases. (AU) | |
| FAPESP's process: | 15/26041-0 - Functional and structural studies of Bacillus licheniformis carboxylesterases and their biotechnological potencial |
| Grantee: | Aline Minali Nakamura |
| Support Opportunities: | Scholarships in Brazil - Doctorate |