Phd thesis_XXVI cycle Giuliana Donadio.pdf

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Tipologia del documento: Tesi di dottorato
Lingua: English
Data: 25 Marzo 2014
Numero di pagine: 114
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Biologia
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 26
Coordinatore del Corso di dottorato:
Di Donato, Alberto[non definito]
Data: 25 Marzo 2014
Numero di pagine: 114
Parole chiave: Oxydative stress,BIOSYNTHESIS,ANTIOXIDANTS, monoxygenase, ROS and RNS, Phenols and catechols, DPPH, Bacterial Multicomponent Monooxygenases, Toluene o-xylene monooxygenase, computational model,2-phenylethanol, TYROSOL, hydroxytyrosol, Baeyer Villiger monooxygenases, H9C2,Bacillus subtilis in industrial biotransformations
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Area 05 - Scienze biologiche > BIO/19 - Microbiologia generale
Area 03 - Scienze chimiche > CHIM/01 - Chimica analitica
Aree tematiche (7° programma Quadro): BIOTECNOLOGIE, PRODOTTI ALIMENTARI E AGRICOLTURA > "Fork to farm" - Prodotti alimentari (inclusi prodotti ittici), salute e benessere
Depositato il: 08 Apr 2014 11:03
Ultima modifica: 26 Gen 2015 11:40


Reactions in which organic compounds are oxygenated or hydroxylated are of great value for organic synthesis. The “oxyfunctionalization” of aromatic compounds is a modification of primary interest for the pharmaceutical and food industries allowing to obtain high-value-added compounds, characterized by a wide array of biological activities, starting from cheap and commercially available molecules. Selective oxyfunctionalization of organic substrates, however, can be a significant problem in organic synthesis, as these reactions are often carried out with strong oxidizing agents and occur with little chemo-, regio-, and enantio- selectivity. Thus, growing attention has been dedicated in the last years to the development of biotransformations, also known as bioconversions, which make use of the metabolic versatility of either purified enzymes or whole microorganisms to perform the oxyfunctionalization of organic substrates of industrial interest. These methodologies, compared with already established chemical processes, are appealing alternatives to obtain active aromatic compounds under mild experimental conditions and without employing toxic reagents. In this thesis project several aspects of this kind of biotransformation were analyzed; more in detail the attention was focused on: - The use of the bacterial multicomponent monooxygenase ToMO from Pseudomonas sp. OX1 for the production of novel hydroxylated antioxidants starting from commercially available aromatic precursors such as 2-phenoxyethanol, 2,3-dihydrobenzofuran, 2-indanol and phtalan. The antioxidant potential of the hydroxylated compounds obtained in ToMO-catalyzed bioconversion was assessed both in vitro, by using the DPPH assay, and ex vivo on the embryonic rat cardiomyoblast cell line H9c2 subjected to oxidative stress induced by sodium arsenite. Not all compounds showed antioxidant potential in the DPPH assay; however, when cells where incubated with any of them, a differential protective effect towards the oxidative stress induced by sodium arsenite was observed. - The recombinant expression of ToMO in the GRAS host microorganism Bacillus subtilis to analyze the potential use of this bacterium for the industrial scale-up of ToMO-catalyzed hydroxylation of aromatic substrates of interest. To this purpose, ToMO gene cluster was cloned in two different shuttle vectors, a non-integrative plasmid indicated as pHT01, and Pr19, a vector that allows instead the direct integration of the recombinant gene in B.subtilis chromosomal DNA through single crossing-over. In this latter case, no integration was observed. When using a non-integrative shuttle vector, the ToMO system was efficiently expressed in E.coli, but RT-PCR experiments showed that almost no mRNA corresponding to the tou gene cluster appeared to be transcribed when the plasmid was inserted in B.subtilis. - The expression and purification of the Baeyer Villiger 3,6-diketocamphane 1,6 monooxygenase (3,6 DKCMO) and its flavin reductase component from Pseudomonas putida NCIMB 10007. The optimization of the expression and the purification of these proteins will pave the way to the future biochemical characterization of this flavoenzyme and to its biotechnological use for the oxyfunctionalization of aromatic compounds of industrial interest.

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