Merlo, Rosa (2021) Thermostable DNA repair enzymes for novel biotechnological applications. [Tesi di dottorato]
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| Tipologia del documento: | Tesi di dottorato |
|---|---|
| Lingua: | English |
| Titolo: | Thermostable DNA repair enzymes for novel biotechnological applications |
| Autori: | Autore Email Merlo, Rosa rosamerlo91@gmail.com |
| Data: | 11 Aprile 2021 |
| Numero di pagine: | 181 |
| Istituzione: | Università degli Studi di Napoli Federico II |
| Dipartimento: | Biologia |
| Dottorato: | Biologia |
| Ciclo di dottorato: | 33 |
| Coordinatore del Corso di dottorato: | nome email Cozzolino, Salvatore cozzolin@unina.it |
| Tutor: | nome email Ricca, Ezio [non definito] Perugino, Giuseppe [non definito] |
| Data: | 11 Aprile 2021 |
| Numero di pagine: | 181 |
| Parole chiave: | AGT; thermostable enzymes; protein-tags; biotechnology |
| Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/10 - Biochimica |
| Depositato il: | 21 Apr 2021 08:43 |
| Ultima modifica: | 07 Giu 2023 10:33 |
| URI: | http://www.fedoa.unina.it/id/eprint/13902 |
Abstract
SNAP-tag® is a powerful tool with wide applications; however, this technology has some limitations: this technology is suitable only for mesophilic model organisms and mild reaction conditions, and the synthesis of ad hoc substrates needs tedious and complex purification procedures. This thesis is directed to get over these issues and to expand the biotechnological application potential of the SNAP-tag®. This aim was pursued by two strategies: acting on the enzyme, by the exploitation of homologous activities from extremophilic sources; and acting on the substrate, by the set-up of a novel approach. Acting on the enzyme: In part of my thesis (Chapter I and Chapter III), I addressed my work to the identification and characterization of a novel AGT from the hyperthermophilic organism P. furiosus, in order to expand the biotechnological potential of the SNAP-technology from "mesophilic" applications to in vivo and in vitro studies in extreme model organisms or in those processes that require non-moderate reaction conditions, and to the characterization of a novel immobilization and labelling tool. Acting on the substrate: In another part of my thesis (Chapter II), I focused my activity to the set-up of a novel approach to the SNAP-tag® reaction, by a chemo-enzymatic approach, which splits the reaction in two fast and extremely specific steps. The enzymatic reaction is performed by use of a selected azide-based BG-substrate, which allows the chemical step with a DBCO-based molecule via cycloaddition (or click-chemistry) reaction. I demonstrate that all the problems related to purification procedures can be bypassed by the utilization of a universal azide-derivative substrate, which is sufficiently exposed to the solvent to link any DBCO-molecules without affecting the reaction rate of the SNAP-tag®.
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