L’induction de la synthèse d’une acide férulique estérase (AFE) a été étudiée chez Streptomyces ambofaciens ATCC 23877. L’activité la plus élevée a été détectée en présence de son de blé désamidonné ou de xylane d’avoine (0,22, 0,21 mU/mg protéine, respectivement). Des productions d’AFE en bioréacteur ont également été réalisées en utilisant 1% (p/v) de son de blé comme inducteur. Le niveau de production de l’AFE a été trois fois plus important en bioréacteur qu’en fiole d’Erlenmeyer. L’AFE de Streptomyces ambofaciens ATCC 23877 et celle de Humicola sp., présente dans un mélange enzymatique commercial (DepolTM 740L), ont été partiellement purifiées et caractérisées. A l’issue de la purification, l’activité AFE de Streptomyces ambofaciens ATCC 23877 a été trop faible pour pouvoir être utilisée ultérieurement en synthèse. Par contre, le potentiel de l’AFE de Humicola sp., concentrée par précipitation à l’acétone, pour la synthèse de différents esters phénoliques a été testé. Les meilleurs rendements de conversion ont été observés lors de l’absence de substitutions sur le cycle aromatique de l’acide phénolique ou en présence de groupements hydroxyles. Les synthèses en milieu non aqueux (M2B2) se sont montrées infructueuses en raison, peut-être, d’un effet néfaste du solvant sur l’enzyme / The induction of the ferulic acid esterase (FAE) synthesis was studied with Streptomyces ambofaciens ATCC 23877. The highest activity was detected in the presence of either destarched wheat bran or oat spelt xylan (0,22, 0,21 mU/mg protein, respectively). FAE productions in bioreactor were also carried out using 1% (w/v) of wheat bran as inducer. The FAE production level was three times higher in bioreactor than in Erlenmeyer flask. FAE of Streptomyces ambofaciens ATCC 23877 and that of Humicola sp., present in an enzymatic commercial mixture (DepolTM 740L), were partially purified and characterised. At the end of the purification, FAE activity of Streptomyces ambofaciens ATCC 23877 was too weak to be used later in synthesis. However, the FAE potential of Humicola sp., concentrated by acetone precipitation, for the synthesis of various phenolic esters was tested. The best conversion yields were observed in the absence of substitution on the phenolic acid aromatic cycle or in the presence of hydroxyl groups. The synthesis in non-aqueous medium (M2B2) were unsuccessful maybe because of an harmful effect of the solvent on the enzyme

Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.

With increasing environmental awareness and natural resource limitations, researchers must begin to incorporate sustainability into their process and product designs. One target for green engineering is in reaction and separation design. This is typically done in a wasteful and often toxic manner with organic solvents and lack of recycle. The following thesis discusses alternatives to these costly separations by means of ionic liquids, benign extraction, separation with carbon dioxide, and near critical water. Ionic liquids are combined with carbon dioxide to induce melting point depressions of up to 124 degrees Celsius. Using this system as a reaction medium will offer control over the reaction phases while utilizing green solvents. Benign extractions are performed on both ferulic acid and on proteins from biomass by replacing alkaline solvents and costly protein separation techniques with simple liquid-liquid extraction. This means simpler systems and less waste than from previous methods. This thesis also discusses an opportunity for more efficient separation and recycle of a pharmaceutical catalyst, Mn-Salen. Using carbon dioxide with the organic aqueous tunable solvent system, the reaction can be run homogeneously and the product and catalyst separated heterogeneously, thus creating an extremely efficient process. Lastly, near critical water is used as an extraction and reaction medium by extracting ferulic acid from Brewers Spent Grain and then catalyzing its transformation to 4-vinylguaiacol. In this manner a simple, benign process is used to turn waste into valuable chemicals. Although somewhat different, each of the studied processes strives to eliminate waste and toxicity of many commonly used reaction and separation techniques, thus creating safe and sustainable processes.

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.

Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.

This paper describes the development of two chemically modified electrodes (CME). The two systems were developed using multi-walled carbon nanotubes (MWCNT). The first system was developed on glassy carbon electrodes (GCE) with MWCNT and film of cobalt oxides (GCE/MWCNT/Co) for determination of hydrogen peroxide (H2O2) and the second system with MWCNT and ferulic acid (FA) (GCE/MWCNT/FA) for determination of epinephrine (EP). The techniques herein employed were cyclic voltammetry, cronoamperometry, which were used to study the electrochemical behavior of modified electrodes, for obtaining kinetic parameters and for the analytical characterization of systems. In addition, hydrodynamic and cronoamperometric studies were carried out with the aim of obtaining information about the H2O2 and EP oxidation process. This device (GCE/MWCNT/Co) was built through the electrodeposition of cobalt oxides on the surface of GCE/MWCNT, and presented a redox pair with E0 = + 0.175 V vs. Ag/AgCl, rate constant of heterogeneous electron transfer (ks) equal to 8.2 s-1 and the analytical figures of merit for H2O2 determination were: linear response range of 3 - 13344 µmol L-1, limit of detection (LOD) of 0.9 µmol L-1, limit of quantification (LOQ) of 3 mol L-1, sensitivity of 4.37 nA L mol-1 and catalytic constant (µobs) of 1,38 x 104 L mol-1 s-1. The system (GCE/MWCNT/FA) was built through the activation of FA on the (GCE/MWCNT), with detection potential for epinephrine of + 0.200 V vs. Ag/AgCl. It presented kobs of 6.99 x 104 L mol-1 s-1, linear response range of 73 - 1406, LOD, LOQ and sensitivity of 22.28 µmol L-1, 73 µmol L-1 and 3.69 nA L µmol-1, respectively. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho, descreveu-se o desenvolvimento de dois eletrodos quimicamente modificados (EQM). Os dois sistemas foram desenvolvidos utilizando eletrodo de carbono vítreo (ECV) e nanotubos de carbono de paredes múltiplas (MWNTC). O primeiro sensor foi desenvolvido com MWCTN e filmes de óxidos de cobalto (ECV/MWCNT/Co) para determinação de peróxido de hidrogênio (H2O2) e o segundo sensor com MWCNT e ácido ferúlico (AF) (ECV/MWCNT/AF) para determinação de epinefrina (EP). As técnicas empregadas para a realização deste trabalho foram voltametria cíclica e cronoamperometria, as quais foram utilizadas para avaliar o comportamento eletroquímico dos eletrodos modificados, obtenção dos parâmetros cinéticos e caracterização analítica dos sistemas. Além disso, os estudos hidrodinâmicos e cronoamperométricos foram realizados com o objetivo de se obter informações sobre o processo de oxidação de H2O2 e EP. O dispositivo (ECV/MWCNT/Co) foi construído através da eletrodeposição de óxidos de cobalto na superfície de ECV/MWCNT, apresentou um par redox com Eo´ = + 0,175 V vs. Ag/AgCl, constante de velocidade de transferência heterogênea de elétrons (ks) igual a 8,2 s-1. Na quantificação de H2O2, as figuras de mérito foram: faixa linear de resposta igual a 3 - 13344 µmol L-1, limite de detecção (LD) de 0,9 µmol L-1, limite de quantificação (LQ) de 3 µmol L-1, sensibilidade de 4,37 nA L µmol-1 e constante catalítica (kobs) de 1,38 x 104 L mol-1 s-1. O sistema (ECV/MWCNT/AF) foi construído através da ativação de AF sobre o (ECV/MWCNT), com potencial de detecção para epinefrina de + 0,200 V vs. Ag/AgCl. Apresentou kobs de 6,99 x 104 L mol-1 s-1, faixa linear igual a 73 - 1406, LD, LQ e sensibilidade de 22,28 µmol L-1 e 73 µmol L-1, e 3,69 nA L µmol-1, respectivamente.

Cette étude a pour objectif d'évaluer la relation entre la structure (au niveau physique et / ou moléculaire) de son de blé et de la couche à aleurone et leurs effets santé. En gardant la composition du son et de l'aleurone constante, leur structures ont été modifiés par des traitements mécaniques et / ou enzymatiques de façon à casser leur structure matricielle complexe et d'augmenter la bioaccessibilité des leurs fibres alimentaires et composés phénoliques. La capacité antioxydante des ces fractions a été évaluée par une méthode standard in vitro et par un modèle de digestion gastrique in vitro. La déstructuration physique du son et aleurone par le broyage a augmenté leur surface spécifique conduisant à une plus grande exposition du groupement acide férulique (AF) qui a augmenté leur capacité antioxydante. Le traitement enzymatique réalisée sur aleurone a modifié son organisation moléculaire, qui a largement libéré l'AF des arabinoxylanes (AX). Cette destructuration a apporté une amélioration plus importante sur la capacité antioxydante de l'aleurone que celle mené par le broyage. Comme l'aleurone est riche en AX fermentescibles, les effets de sa structure plus accessible et avec une plus grande dépolymérisation ont été évaluées en utilisant un modèle in vitro en mimant le côlon humain. La déstructuration physique de l'aleurone par le broyage n'a pas amélioré leur pouvoir fermentescible, c'est à dire que le métabolisme de composés phénoliques et de la formation des chaînes courtes d'acides gras ont été comparable à ceux obtenus pour l'aleurone natif. Néanmoins, la dégradation enzymatique de l'aleurone en apportant une plus grande quantité des AX soluble et de l'AF biodisponible a augmenté le métabolisme de l'AF dans le colon dans des métabolites avec propriétés anti-inflammatoires. Une étude in vivo avec des souris sur un régime riche en graisses a été fait pour évaluer le potentiel de l'aleurone physiquement et moléculaire déstructuré pour neutraliser les désordres métaboliques tels que l'obésité, le stress oxydatif et l'inflammation. La déstructuration physique de l'aleurone n'a pas eu d'effet positif sur les désordres métaboliques. La fraction d'aleurone qui a présenté la plus grande quantité d'AX soluble et de l'AF biodisponible a bien réduit l'obésité (gain de poids, l'adiposité et la sécrétion de leptine) et la résistance à l'insuline chez la souris. Par contre, aucune différence significative n'a été observée dans le stress oxydatif et l'inflammation des souris nourries avec des régimes enrichis en aleurone. En conclusion, nous avons démontré que les effets santé de l'aleurone sont clairement liées à l'intégrité de sa structure. Ses effets santé ont été accrues par une modification de sa structure principalement au niveau moléculaire en dégradant ses parois cellulaire pour augmenter la bioaccessibilité et la biodisponibilité des composés nutritionnellement intéressants, tels que l'AX et l'FA. / The objective of this study was to evaluate the relationship between the structure (at physical and/or molecular level) of wheat bran and aleurone and their health effects. Keeping the composition of bran and aleurone constant, their structure was modified by mechanical and/or enzymatic treatments in order to disrupt their complex matrix structure and increase the bioaccessibility of their dietary fibre and phenolic compounds. The antioxidant capacity of the bran and aleurone fractions was evaluated by a standard in vitro test and by an in vitro gastric digestion model. The grinding disrupted the physical structure of bran and aleurone increasing their specific surface leading to a greater exposition of ferulic acid moiety (FA) which increased their antioxidant capacity. Enzymatic treatment performed on aleurone acted on its molecular organisation releasing its FA from arabinoxylans (AX). This destructuration improved the antioxidant capacity of aleurone even more than the one allowed by grinding. As aleurone is rich in highly fermentable AX, the effects of its better accessibility and depolymerisation were evaluated using an in vitro model mimicking the human colon. The physical destructuration of aleurone by grinding did not improve its fermentability, i.e. the colonic metabolism of phenolic compounds and the formation of short-chain fatty acids were similar compared to the native aleurone. Nevertheless, the enzymatic degradation of aleurone produced higher amount of soluble AX and bioavailable FA improving the metabolism of colonic FA in metabolites with anti-inflammatory properties. An in vivo mouse study with a high-fat diet was used to evaluate the potential of physically and molecularly destructured aleurone to counteract the metabolic disorders, such as obesity, oxidative stress and inflammation. The physical destructuration of aleurone did not have any positive effect on the metabolic disorders. The aleurone fraction, which presented the highest level of soluble AX and bioavailable FA, reduced the obesity (body weight gain, adiposity, and leptin secretion) and insulin resistance in mice. But no significant differences were observed in the oxidative stress and inflammation status of mice fed with any of the aleurone-based diets. In conclusion, we demonstrated that the aleurone health effects were clearly linked to the integrity of its structure. Its health effects have been increased by modification of its structure mainly at molecular level by degrading its cell wall to increase the bioaccessibility and bioavailability of nutritionally interesting compounds, such as AX and FA.

Cette thèse porte sur la fonctionnalisation de la pectine de citron par des composés phénoliques. Une première stratégie a consisté à greffer les produits issus de l’oxydation de l’acide férulique (AF), en milieu aqueux (pH 7,5, 30 ° C), en présence de la laccase de Myceliophthora thermophila (obtention de pectine F). L’enjeu était de démontrer le greffage covalent de composés phénoliques exogènes sur le polysaccharide, de recueillir des informations sur la structure et les propriétés du polymère fonctionnalisé et de les comparer aux caractéristiques du polymère natif. Une deuxième stratégie de fonctionnalisation de la pectine a été envisagée, basée sur l'adsorption des produits d'oxydation de l'AF sur la pectine (obtention de pectine POX). Quel que soit le mode de fonctionnalisation, des analyses biochimiques ont montré l'incorporation de composés phénoliques dans la pectine. La structure et les propriétés des pectines modifiées dépendent du type de fonctionnalisation subie par le polysaccharide (greffage covalent ou adsorption). Des analyses structurales suggèrent que le greffage covalent de phénols fait intervenir les fonctions carboxyles de la pectine (formation de liaisons ester) sur lesquelles des oligomères d'acide férulique viendraient se lier. Les propriétés des pectines fonctionnalisées (POX et F) et de la pectine native ont été étudiées et comparées afin de mettre en évidence les changements apportés. L'étude des propriétés thermiques des différentes poudres de pectine suggèrent une structure moins organisée et moins compacte pour les polymères fonctionnalisés par rapport à la pectine native. L’activité antioxydante des pectines fonctionnalisées est améliorée tandis que leur caractère hygroscopique est diminué en raison de l'incorporation de composés phénoliques hydrophobes. De même que la pectine native, les pectines POX et F présentent un profil rhéofluidifiant. Toutefois, la viscosité et la vitesse de gélification mesurées dans le cas de la pectine F sont significativement diminuées par rapport à celles obtenues pour la pectine native. La pectine POX présente un comportement intermédiaire. Des résultats préliminaires d'assemblages ont montré qu'il est possible d'associer la pectine native ou modifiée à un autre polysaccharide, le chitosane. Les microparticules obtenues ont montré leur capacité à encapsuler un principe actif tel que la curcumine / This dissertation concerns the functionalization of the citrus pectin with phenolic compounds. A first strategy consisted in grafting the products issued from the oxidation of ferulic acid (FA), in aqueous medium (pH 7,5, 30 ° C), in the presence of the laccase of Myceliophthora thermophila (pectin F). The main objectives were to demonstrate the covalent grafting of exogenous phenols onto the polysaccharide, to collect information about the structure and the properties of the modified polymer and to compare them with the characteristics of the native one. A second strategy of functionalization was applied, based on the adsorption of FA oxidation products onto the pectin (pectin POX). Whatever the functionalization pathway, biochemical analyses showed the incorporation of phenolic compounds into the pectin. The structure and the properties of the modified pectins depended on the type of modification undergone by the polysaccharide (covalent grafting or adsorption). Structural analyses suggested that the covalent grafting of phenols involved the carboxyl groups of the pectin (ester bound) on which FA oligomers were bound. The properties of native and modified pectins (POX and F) were studied and compared aiming to highlight the changes brought by functionalization. The study of the thermal properties of pectin POX and F suggested less organized and less compact structures compared to the native one. The antioxidant activity of the modified pectins was improved whereas their hygroscopic character was decreased because of the incorporation of hydrophobic phenolic compounds. As the native pectin, the pectins POX and F presented a shear-thinning profile. However, the viscosity and the gelation rate measured for the pectin F were significantly decreased, compared with those obtained for the native pectin. The pectin POX presented an intermediate behavior. Preliminary results of assemblies demonstrated the possibility to associate the native or modified pectin to another polysaccharide, the chitosan, leading to microparticles capable to encapsulate an active ingredient such as the curcumin

La bactérie phytopathogène Dickeya dadantii est responsable de la pourriture molle de nombreux végétaux. Elle sécrète dans le milieu extérieur toute une batterie d’enzymes capables de dégrader les constituants des parois végétales. La première partie de mon travail concerne les féruloyl estérases FaeD et FaeT. Les féruloyl estérases sont responsables de l’hydrolyse de liaisons ester entre l’acide férulique et les chaînes de xylane ou de pectine. En clivant ces liaisons, elles favorisent une dégradation complète de la paroi végétale. L'importance de ces enzymes nous a conduit à rechercher si D. dadantii produit de telles estérases. Le criblage d’une banque de gènes par un test de détection de l’activité féruloyl estérase a permis d’identifier deux gènes qui ont été caractérisés. Alors que faeT est faiblement transcrit dans toutes les conditions, la transcription de faeD est fortement induite en présence d’acide férulique et contrôlée par le régulateur FaeR. Alors que FaeT est une protéine cytoplasmique, FaeD est sécrétée par le système Out qui permet la sécrétion de nombreuses pectinases. Les enzymes FaeD et FaeT ont été surproduites dans E. coli et leurs principales propriétés biochimiques ont été déterminées. La connaissance de la séquence complète du génome de D. dadantii permet d’aborder des études de génomique fonctionnelle. Cette séquence confirme la présence des gènes codant les pectinases déjà caractérisées et révèle que ce génome code de nouvelles pectinases potentielles. La deuxième partie de mon travail concerne le gène pelN identifié par analyse du génome. Les pectate lyases coupent les liaisons glycosidiques du polygalacturonate par une réaction de β-élimination, générant des produits insaturés. Leur mécanisme d’action nécessite des cations comme cofacteur, en général Ca2+. Après clonage du gène pelN, la protéine PelN a été surproduite dans E. coli. Son activité pectate lyase a été prouvée en montrant sa capacité à produire des dérivés insaturés à partir de polygalacturonate ou de pectines plus ou moins méthylées. Cette étude démontre que PelN est la première pectate lyase utilisant les ions Fe2+ comme cofacteur préférentiel. Chez D. dadantii, l’expression du gène pelN dépend de divers régulateurs affectant la synthèse des pectinases, comme PecS ou GacA. PelN est une protéine extracellulaire sécrétée par le système Out. Ces études contribuent à mieux comprendre le rôle respectif des différentes enzymes impliquées dans la dégradation de la paroi végétale et le fonctionnement coopératif de ce système pluri-enzymatique. / The phytopathogenic bacterium Dickeya dadantii is responsible for soft rot diseases of various plants. It secretes in the external medium a large array of enzymes which are able to degrade the constituents of the plant-cell wall. The first part of my work was related to the féruloyl esterases FaeD and FaeT. Feruloyl esterases are responsible for the hydrolysis of ester linkages between ferulic acid and the xylan or pectin chains. By cleaving these linkages, they improve the complete degradation of the plant-cell wall. The importance of these enzymes led us to search whether D. dadantii produces such esterases. A gene bank screening using a specific detection test for the feruloyl esterase activity allowed us to identify two genes which were characterized. While faeT is weakly transcribed in all the conditions, the faeD transcription is strongly induced in the presence of ferulic acid and it is controlled by the regulator FaeR. Whereas FaeT is a cytoplasmic protein, FaeD is secreted by the Out system responsible for the secretion of several pectinases. The enzymes FaeD and FaeT were overproduced in E. coli and their main biochemical properties were determined. The determination of the complete sequence of the D. dadantii genome makes it possible to develop functional genomic studies. This sequence confirms the presence of genes encoding the previously characterized pectinases and it reveals that this genome encodes new potential pectinases. The second part of my work was related to the gene pelN identified by genome analysis. Pectate lyases cleave the glycosidic bounds in the polygalacturonate chain by a β-elimination reaction, generating unsaturated products. This reaction mechanism requires cations as cofactor, generally Ca2+. After cloning of the gene pelN, the protein PelN was overproduced in E. coli. Its pectate lyase activity was demonstrated by its capacity to produce unsaturated derivatives from polygalacturonate or pectins. This study showed that PelN is the first pectate lyase that uses Fe2+ ions as the preferential cofactor. In D. dadantii, the pelN expression depends on various regulators controlling the pectinase synthesis, such as PecS or GacA. PelN is an extracellular protein secreted by the Out system. These studies contribute to increase the knowledge on the respective role of the different enzymes involved in the degradation of the plant-cell wall and the cooperative interactions in this pluri-enzymatic system.

Thesis (PhD (Chemical Engineering))--Cape Peninsula University of Technology, 2020 / The use of bioproducts in different commercial sectors such as medicine, agriculture, cosmetics, food, and chemical industries motivates the need for easy production and recovery techniques of bioproducts at laboratory and pilot scale. This study aims at the production and recovery of some selected bioproducts using supercritical fluid extraction techniques. Three products are chosen as case studies: these are ethanol, acetoin, and vanillin, since the ease of separation is influenced by the concentration of the product in the broth, these compounds were selected based on their concentration in the fermentation broth, according to literature sources. A standard method was developed in a spectrophotometer for quantifying the targeted product in the broth, while the product recovery studies was carried out using a supercritical fluid extraction pilot plant. Saccharomyces and Bacillus species were chosen for the bioproduction of the selected bioproducts. Experimental design and statistical analysis of results were carried out using response surface methodology (RSM) and artificial neural network (ANN). Studies on each of the selected bioproducts are as justified in the paragraphs below. Bioethanol production has recently become an increasing trend in research, with a focus on increasing its economic viability. Hence, the need to develop a low-cost fermentation medium with minimum redundant nutritional supplements, thereby minimizing the costs associated with nutritional supplements whereby inoculum preparation becomes necessary for ethanol production. Corn steep liquor (CSL) in glucose fermentation by Saccharomyces Type 1 (ST1) strain and Anchor Instant Yeast (AIY), which are low-cost media, are used as replacements for yeast extract (YE). The fermentation process parameters were optimized using artificial neural networks (ANN) and the response surface methodology (RSM). The study shows that for CSL, a maximum average ethanol concentration of 41.92 and 45.16 g/L representing 82% and 88% of the theoretical yield were obtained after 36 h of fermentation in a shake flask for ST1 and AIY respectively. For YE, ethanol concentration equivalent to 86% and 88% of theoretical yield were obtained with ST1 and AIY respectively after 48 h. Although, ANN predicted the responses of ethanol yield better than RSM, optimum conditions for ethanol production were better predicted by RSM. The consumers’ preference for ‘naturally’ produced aromas drives the development of bioproduction of acetoin from glucose with a view to optimize its production. The results revealed that by using a cheap nitrogen source, corn steep liquor, the yield of acetoin was similar to those of yeast and beef extracts. Furthermore, it was shown that by using Box-Behnken design, the optimum parameters such as glucose concentration, corn steep liquor, and inoculum size to maximize the concentration of acetoin produced were 78.40 g/L, 15.00% w/v and 2.70% v/v respectively. The validated concentration of acetoin produced in a triplicate analysis, 10.7 g/L, was 0.06% less than the predicted value. Increasing awareness of consumers of healthy, eco-friendly flavors and fragrances motivates the bioproduction of vanillin. The interactive effects of three variables on vanillin yield were evaluated by response surface methodology (RSM) with Box-Behnken design (BBD) model. The results showed the optimum conditions for the biotransformation of ferulic acid into vanillin can be achieved with maximum overall desirability (D) of 1.0 and a significant (p<0.05) quadratic model with regression coefficient (R2) of 0.995. Corn steep liquor, initial ferulic acid concentration and pH significantly influence the concentration of vanillin in the broth. The results in triplicate experiments confirmed vanillin yield of 386 mg/L after validation, which was in agreement with the prediction of the model. The maximum vanillin yield of 384.40 mg/L was predicted when corn steep liquor, ferulic acid concentration and pH were 7.72 g/L, 2.33 g/L, and 9.34 respectively. Fermentation system in a bioreactor has been proven to be an efficient system for the study of controlled fermentation variables when compared to a shake flask study. The influence of agitation, aeration, time and pH were analysed by Taguchi orthogonal array design for the upscale of acetoin in a bioreactor. The optimized parameters in 1.3L of fermentation vessel were as follows: 300 rpm agitation, 1.5 slpm aeration; 2 days’ fermentation time and 6.5 pH value. Agitation with above 70% was the most contributing factor and other variables were less than 30% in the percentage analysis of variance of each fermentation variables in the batch study of acetoin. A fourfold gain in acetoin titre (42.30 g/L) was obtained with the same substrate concentration in a lab-scale bioreactor on scaling up when compared with the shake flask batch study. The validated acetoin concentration of 41.72 g/L was obtained after a triplicate experiment to confirm the possibility of reproducing acetoin using the optimized conditions. Many separation techniques have been proven to recover value-added products from fermentation broth with a preference for several methods above other and new techniques that are emerging. Supercritical fluids separation using CO2 is one such technique. The feasibility of acetoin concentration and recovery was studied in supercritical CO2 pilot plant with pressure ranges of 100 to 300 bar, CO2 feed rate of 5 to 15 kg/h, at a process temperature of 37 and 80 °C in simulated and fermentation broth, respectively. The validated conditions for the fractionation of acetoin by supercritical fluid extraction (SFE) were determined as follows: extraction pressure, 300 bar; CO2 feed rate, 15 kg/h; extraction temperature 37 °C; and fractionation time of 30 minutes. At these operating conditions, the percentage recovery of acetoin with respect to the feed solution at the raffinate for the simulated and actual ermentation broth was 77.8% (0.20 g/L) and 77% (0.15 g/L) respectively. A two-fold extract increase was obtained after 30 minutes of fractionation. The study provides the technical feasibility and the base case data which are critical to the development and design of processes for production and recovery of acetoin. The lesson gleaned from this study may be extended to develop processes for the production and recovery of other bioproducts (ethanol and vanillin).