L'objectif est de produire un antioxydant puissant, l'acide 3,4-dihydroxyphénylacétique (3,4-DHPA) à partir de la L-tyrosine chez l'archée hyperthermophile et acidophile, Sulfolobus solfataricus 98/2. Les microorganismes extrêmophiles possèdent des enzymes particulièrement résistantes et intéressantes pour l'industrie.Des cultures ont donc été réalisées en fermenteur contrôlé dans 4 conditions : (i) en présence de glucose avec ou sans L-tyrosine, (ii) en présence de phénol avec ou sans L-tyrosine. Il a été montré que le 3,4-DHPA est synthétisé seulement en présence de phénol et de L-tyrosine. Les gènes codant pour les enzymes impliquées dans cette voie métabolique et potentiellement responsables de la synthèse du 3,4-DHPA ont été identifiés par homologie de séquence chez cette archée.Des études transcriptomiques et protéomiques ont donc été initiées pour confirmer ces hypothèses et tenter de caractériser les enzymes impliquées dans ces voies métaboliques. Plusieurs toluène-4-monooxygénases (T4MO) et une catéchol 2,3-dioxygénase, impliquées dans le métabolisme du phénol et potentiellement dans la voie de dégradation de la L-tyrosine ont été identifiées. Leur production est soumise à une régulation transcriptionnelle dépendant de la présence de phénol. L'analyse des régions génomiques correspondantes a permis de mettre en évidence une région consensus qui pourrait être impliquée dans la fixation d'un facteur de transcription lors de la régulation par le phénol. Ces différentes études ont permis, de déterminer d'une part dans quelles conditions le 3,4-DHPA est synthétisé, d'autre part d'identifier les enzymes qui interviendraient dans le métabolisme de la L–tyrosine. / The aim is to produce a powerful antioxidant, 3,4-dihydroxyphenylacetic acid (3,4-DHPA) from L-tyrosine in the hyperthermophilic and acidophilus archaea, Sulfolobus solfataricus 98/2. Extremophiles microorganisms have resistant enzymes and interesting for industry. Cultures have been carried out in controlled bioreactor four conditions: (i) in the presence of glucose with or without L-tyrosine, (ii) in the presence of phenol with or without L-tyrosine. It has been shown that 3,4-DHPA is synthesized only in the presence of phenol and L-tyrosine. The genes encoding enzymes involved in the metabolic and potentially responsible for the synthesis of 3,4-DHPA pathway have been identified by sequence homology in S. solfataricus.Des transcriptomic and proteomic studies have therefore been initiated to confirm these hypothesis and attempt to characterize the enzymes involved in these pathways. Several toluene-4-monooxygenase (T4MO) and catechol 2,3-dioxygenase involved in the metabolism of phenol and potentially in the degradation pathway of L-tyrosine were identified. Their production is subjected to a dependent transcriptional regulation of the presence of phenol. The analysis of the corresponding genomic regions has highlighted a consensus region that could be involved in the binding of a transcription factor in the regulation of phenol. These studies helped to determine the one hand the conditions under which 3,4-DHPA is synthesized, secondly to identify enzymes that intervene in the metabolism of L-tyrosine.

<p><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a>ABSTRACT</a></p> <p>Phenols are ubiquitous in our surroundings including biological molecules such as L-Dopa metabolites, food components, such as whiskey and liquid smoke, etc. This dissertation describes a new method for detecting phenols, by reaction with Gibbs reagent to form indophenols, followed by mass spectrometric detection. Unlike the standard Gibbs reaction which uses a colorimetric approach, the use of mass spectrometry allows for simultaneous detection of differently substituted phenols. The procedure is demonstrated to work for a large variety of phenols without <i>para</i>‐substitution. With <i>para</i>‐substituted phenols, Gibbs products are still often observed, but the specific product depends on the substituent. For <i>para</i> groups with high electronegativity, such as methoxy or halogens, the reaction proceeds by displacement of the substituent. For groups with lower electronegativity, such as amino or alkyl groups, Gibbs products are observed that retain the substituent, indicating that the reaction occurs at the <i>ortho</i> or <i>meta</i> position. In mixtures of phenols, the relative intensities of the Gibbs products are proportional to the relative concentrations, and concentrations as low as 1 μmol/L can be detected. The method is applied to the qualitative analysis of commercial liquid smoke, and it is found that hickory and mesquite flavors have significantly different phenolic composition.</p> <p>In the course of this study, we used this technique to quantify major phenol derivatives in commercial products such as liquid smoke (catechol, guaiacol and syringol) and whiskey (<i>o</i>-cresol, guaiacol and syringol) as the phenol derivatives are a significant part of the aroma of foodstuffs and alcoholic beverages. For instance, phenolic compounds are partly responsible for the taste, aroma and the smokiness in Liquid Smokes and Scotch whiskies. </p> <p>In the analysis of Liquid Smokes, we have carried out an analysis of phenols in commercial liquid smoke by using the reaction with Gibbs reagent followed by analysis using electrospray ionization mass spectrometry (ESI-MS). This analysis technique allows us to avoid any separation and/or solvent extraction steps before MS analysis. With this analysis, we are able to determine and compare the phenolic compositions of hickory, mesquite, pecan and apple wood flavors of liquid smoke. </p> <p>In the analysis of phenols in whiskey, we describe the detection of the Gibbs products from the phenols in four different commercial Scotch whiskies by using simple ESI-MS. In addition, by addition of an internal standard, 5,6,7,8-tetrahydro-1-napthol (THN), concentrations of the major phenols in the whiskies are readily obtained. With this analysis we are able to determine and compare the composition of phenols in them and their contribution in the taste, smokey, and aroma to the whiskies.</p> <p>Another important class of phenols are found in biological samples, such as L-Dopa and its metabolites, which are neurotransmitters and play important roles in living systems. In this work, we describe the detection of Gibbs products formed from these neurotransmitters after reaction with Gibbs reagent and analysis by using simple ESI‐MS. This technique would be an alternative method for the detection and simultaneous quantification of these neurotransmitters. </p> <p>Finally, in the course of this work, we found that the positive Gibbs tests are obtained for a wide range of <i>para</i>-substituted phenols, and that, in most cases, substitution occurs by displacement of the <i>para</i>-substituent. In addition, there is generally an additional unique second-phenol-addition product, which conveniently can be used from an analytical perspective to distinguish <i>para</i>-substituted phenols from the unsubstituted versions. In addition to using the methodology for phenol analysis, we are examining the mechanism of indophenol formation, particularly with the <i>para</i>-substituted phenols. </p> <p>The importance of peptides to the scientific world is enormous and, therefore, their structures, properties, and reactivity are exceptionally well-characterized by mass spectrometry and electrospray ionization. In the dipeptide work, we have used mass spectrometry to examine the dissociation of dipeptides of phenylalanine (Phe), containing sulfonated tag as a charge carrier (Phe*), proline (Pro) to investigate their gas phase dissociation. The presence of sulfonated tag (SO₃^-) on the Phe amino acid serves as the charge carrier such that the dipeptide backbone has a canonical structure and is not protonated. Phe-Pro dipeptide and their derivatives were synthesized and analyzed by LCQ-Deca mass spectroscopy to get the fragmentation mechanism. To confirm that fragmentation path, we also synthesized dikitopeparazines and oxazolines from all combinations of the dipeptides. All these analyses were confirmed by isotopic labeling experiments and determination and optimization of structures were carried out using theoretical calculation. We have found that the fragmentation of Phe*Pro and ProPhe* dipeptides form sequence specific b₂ ions. In addition, not only is the ‘mobile proton’ involved in the dissociation process, but also is the ‘backbone hydrogen’ is involved in forming b₂ ions. </p> <p> </p>