Mise en oeuvre de la pepsine de porc dans un système de solvants biphasiques pour la préparation de peptides opioïdes à partir de l'hémoglobuline bovine

Cinq peptides actifs ont été isolés en une seule étape par CLHP en phase inverse à partir de l'hydrolyse de l'hémoglobine bovine par la pepsine a pH 4,5 : la LVVh-7, la LVV-7 et un peptide potentialisateur de la Bradykinine, qui sont des peptides intermédiaires, ainsi que la néokyotorphine et la VVh-4, qui sont des peptides finaux. L'optimisation de leur production a été obtenue par digestion pepsique de l'hémoglobine dénaturée par l'urée (mécanisme "zipper"). Les couplages de la CLHP en phase inverse avec la SM-IES, pour l'isolement et l'identification rapides des hémorphines, puis avec un détecteur spectral UV, pour la construction d'une librairie spectrale permettant leur quantification dans un hydrolysat peptidique complexe, ont permis de déterminer les conditions optimales de leur production (degré d'hydrolyse). Un plan d'expériences a permis de mettre au point un système biphasique d'extraction de la LVVh-7 et de la VVh-7, composé de butan-2-ol et d'octan-1-ol, capable d'extraire efficacement ces deux hémorphines dans un réacteur à pepsine insolubilisée avec une forte sélectivité (coefficient de partage de 2,6 et de 2,3 pour ces hémorphines). Une méthode d'adsorption sur de l'alumine a permis de récupérer sélectivement les hémorphines pures, tout en retenant l'hème sur le support. La conception d'un réacteur ouvert à solvants biphasiques et à pepsine immobilisée permettra la préparation en continu des deux hémorphines et le recyclage des solvants.LILLE1-BU (590092102) / SudocSudocFranceF

Développement d'un nouveau procédé propre en tannerie pour l'épilage de peaux de veau par traitement protéolytique et oxydatif de surface

Mon travail de thèse a permis de démontrer la faisabilité d un procédé d épilage mixte des peaux de veau, moins polluant que le procédé conventionnel. Des essais d épilage réalisés avec la papaïne ont permis de montrer son implication dans l hydrolyse de la kératine souple au niveau de l épiderme et des gaines épithéliales, contribuant ainsi a la chute du poil. De plus, d après les analyses effectuées en MEB, le collagène n est pas abrase par ce traitement enzymatique. Cependant, l épilage a la papaïne est de 97%, un traitement complémentaire est donc nécessaire pour obtenir un épilage total. Le peroxyde d hydrogène a permis de terminer cet épilage en favorisant la dissolution des fins poils restant sur la peau, tout en conservant le collagène intact. Un procédé mixte chimique-enzymatique a été développé à l échelle semi-industrielle. Un prétraitement enzymatique avec 0,3% de papaïne suivi d un traitement chimique avec 4% de peroxyde d hydrogène a permis l obtention d un épilage total. Compare au procédé conventionnel, ce procédé mixte permet de diminuer la DCO, la DBO et les MEST de respectivement 29,5%, 26,5% et 95%. Cependant, le procédé d épilage mixte est beaucoup plus couteux, 3800EUR contre 60EUR pour traiter 1 tonne de peaux. Ce procédé mixte restant relativement onéreux, une stratégie de recherche de microorganismes possédant des propriétés kératinolytiques a partir de peaux en putréfaction, a permis d isoler un microorganisme identifie comme étant un Bacillus cereus. La mise en culture de ce microorganisme dans un milieu contenant essentiellement des plumes a permis d extraire un surnageant avec une activité kératinolytique et capable d épiler la peau de veau.My thesis has demonstrated the feasibility of a process of unhairing calf skins, which is less polluting than the conventional process. Unhairing tests carried out with papain have shown its impact in the hydrolysis of soft keratin constituting the epidermis and epithelial root sheaths, thus contributing to hair loss. In addition, according to analyses by SEM, the enzymatic treatment does not have an abrasive effect on collagen. However, unhairing with papain was 97% effective, further treatment is also necessary to obtain a total unhairing. Hydrogen peroxide can complete this unhairing, furthering the dissolving of fine hairs remaining on the skin, while keeping collagen intact. A chemical-enzymatic process ( mixed process ) was developed on a semi-industrial scale. An enzymatic pretreatment with 0.3% papain followed by a chemical treatment with 4% hydrogen peroxide resulted in a total unhairing. Compared to conventional process, the mixed process reduces COD, BOD and MEST respectively by 29.5%, 26.5% and 95%. However, the mixed process of unhairing is much more expensive, 3 800 EUR compared with 60 EUR to treat 1 ton of skin. As this mixed process remains relatively expensive, a research strategy for microorganisms possessing keratinolytic properties from rotten skins, enabled the isolation of a microorganism identified as Bacillus cereus. The culture of this microorganism has been established into a medium containing essentially feathers and allowed the extraction of supernatant with a keratinolytic activity and capable of unhairing calf skins.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Development of horseradish peroxidase/layered double hydroxide hybrid catalysis for phenol degradation

International audienceBoth photocatalytic and enzymatic degradation of phenol were studied in order to evaluate and to compare the catalytic potential of both methods. For this, solar-sensitive ZnMe (Me=Al, Cr) layered double hydroxides (LDHs) and their derived mixtures of mixed oxides (MMOs) were synthesized via co-precipitation and calcination at 750 degrees C, while the calcined LDH at 550 degrees C was used for horseradish peroxidase (HRP) immobilization through absorption. The structural, optical and thermal properties of catalysts were investigated by XRD, FTIR, TG/DTA, TEM and UV-Vis analyses. We approached here for the first time the use of LDH-HRP biohybrid for phenol degradation. Further, since HRP operates similarly as photocatalysts, we also investigated the ability of LDH-HRP to generate photo-enzymatic responses, when the biocatalyst is used under solar light. Both degradation methods are able to remove phenol from solution via different pathways, namely step-by-step phenol photodegradation and phenol enzymatic polymerization, when an insoluble product is obtained at the end of reaction. After 7h of reaction, 95% of phenol was removed by a MMOs, while ZnAlLDH+light, ZnAlLDH-HRP and ZnAlLDH-HRP+light removed 20, 25 and 35% of phenol via photo-, enzymatic and photo-enzymatic catalysis, respectively

Lipase catalyzed esterification of formic acid in solvent and solvent-free systems

International audienceEsterification reaction between formic acid and alcohols (C4, C8) catalyzed by lipases was performed in solvent and solvent-free systems. High Performance Liquid Chromatography (HPLC) was used to study the kinetic parameters (Michaelis-Menten) and the reaction conditions by monitoring the ester synthesis. The optimal conditions for formate ester synthesis were found to be: 0.5 M of formic acid, 1.5 M of alcohol, 2% of Novozym 435 at 40 degrees C and 400 rpm in acetonitrile solvent without molecular sieves. In solvent-free system, the optimal conditions were found to be: 1 M of formic acid, 10 M of alcohol, 2% of Novozym 435 at 40 degrees C and 400 rpm without molecular sieves. These optimal conditions resulted in maximum ester yield about 90% for 8 h in solvent system and 5 h in solvent-free system. Among all esterification reactions, the esterification reaction of formic acid with octan-1-ol in solvent-free system was the better regarding the quantity of esterified formic acid, catalytic efficiency of lipase and the required reaction time. Under these optimal conditions, the biocatalyst could be reused for six reaction cycles keeping around 97% of its initial activity after treatment process with n-hexane. Consequently, this enzymatic procedure in solvent-free system could provide formate esters for food and cosmetic applications

High-throughput strain identification and production of fungal enzymatic cocktails for the valorisation of lignocellulosic biomass

International audienc

Innovative microscale workflow from fungi cultures to Cell Wall-Degrading Enzyme screening

International audienceThis study aimed at developing a complete miniaturized high-throughput screening workflow for the evaluation of the Cell Wall-Degrading Enzyme (CWDE) activities produced by any fungal strain directly cultivated on raw feedstock in a submerged manner. In this study, wheat straw was selected as model substrate as it represents an important carbon source but yet poorly valorised to yield high added value products. Fungi were grown in a microbioreactor in a high-throughput (HT) way to replace the fastidious shaking flask cultivations. Both approaches were compared in order to validate our new methodology. The range of CWDE activities produced from the cultures was assayed using AZO-died and pNP-linked substrates in an SBS plate format using a Biomek FXp pipetting platform. As highlighted in this study, it was shown that the CWDE activities gathered from the microbioreactor cultivations were similar or higher to those obtained from shake flasks cultures, with a lower standard deviation on the measured values, making this new method much faster than the traditional one and suitable for HT CWDE production thanks to its pipetting platform compatibility. Also, the results showed that the enzymatic activities measured were the same when doing the assay manually or using the automated method

Transformation Durable du D-glucose en 5-HMF par Catalyse Hybride : du Réacteur Discontinu au Système à Flux Continu

National audienc

Hybrid catalysis: towards an optimal combination of catalysts Application to HMF valorization

National audienc

Valorisation of bio-based furfurals by hybrid catalysis: towards the synthesis of new amine polymers and surfactants

International audienceThe new and highly innovative field of research called "hybrid catalysis", that combines chemo and biocatalysts, has emerged over the last two decades.[1] It is still in its infancy but already reveals numerous advantages compared to traditional processes, particularly in terms of energy savings, atom savings, but also eco-friendliness. Therefore, one of the application fields in which hybrid catalysis seems particularly attractive is that of biomass valorisation. In particular, hybrid catalysis could help achieving the production of numerous new highly valuable building blocks from complex feedstocks. Among them, 5-hydroxymethylfurfural (HMF) is described as one of the most important biobased platform molecules, with various production processes from lignocellulose. Still, its valorisation in the form of furfurylamines has been little studied, and the routes for their synthesis remain generally complex and sustainable. Nevertheless, several of these furfurylamines are very promising molecules for numerous applications, such as 5-aminomethyl-2-furancarboxilic acid (AMFC) which, thanks to its free amine and carboxylic acid moieties, can be used as a precursor of new polyamides or surfactants. Recently, several works have described the synthesis of the latter by biocatalysis, but none of them were able to directly synthesise it from HMF. Thus, we recently proposed the synthesis of AMFC directly from HMF using a hybrid one-pot/two-step process combining a platinum nanoparticle and a transaminase, both immobilised on silica beads (Fig. 1).[2] This process resulted in a 77% yield of the desired product, with 100% conversion of HMF and only one by-product, FDCA, another high value compound. A one-pot/one-step process is currently being developed using a newly discovered thermostable transaminase. In addition, this first hybrid step has since been complemented by a second step to obtain new amphiphilic molecules from AMFC, to target the production of innovative surfactants. The idea was to be able to graft aliphatic chains onto the amine function through the formation of an amide bond using sustainable catalysts and common alcohols. We therefore developed a second hybrid route, this time combining a gold nanoparticle immobilised on different supports to carry out the oxidation of the aliphatic alcohols into fatty acids, the latter then being coupled to the AMFC with the help of a CoA ligase, also thermostable. Here, 71% and 44% of amide formation could be obtained after 64h with butanol and pentanol as starting material respectively, in a one-pot/two-step process. Noteworthy, the limiting step remains the enzymatic one with 100% of alcohols to acids conversion. In addition to the overcoming of this bottleneck, a fully integrated one-pot/one-step process is also currently being developed

La catalyse, la diversité au service de l’efficacité

It is estimated today that 90% of chemical processes use less than one catalyst, and that 30-40% of the world economy depends directly or indirectly on the use of catalysts. These chemical or biological species are extremely widespread at the industrial level; they make it possible to better control the selectivity and specificity of a wide variety of chemical reactions for the customised formation of very varied target compounds, by playing on the reaction pathways and thus the activation energies, while most often favouring kinetics. In general, catalysts are the site of physicochemical environments with properties different from those of conventional reaction media. They promote or allow specific reaction events (catalytic act) implementing their properties, and even allow the creation of new reaction intermediates facilitating and/or directing reactions. The diversity of catalysts provides access to an extremely wide range of more or less complex molecular mechanisms, the understanding of which allowing for a better control of reactions and optimisation of catalytic processes, as well as their reasoned coupling according to so-called "multi-catalytic" reactions that benefit from the additional novel properties of these catalytic systems assembled in this way.On estime aujourd’hui que 90 % des procédés chimiques font appel à au moins un catalyseur, et que 30 à 40 % de l’économie mondiale dépendent directement ou indirectement de l’utilisation de catalyseurs. Ces espèces chimiques ou biologiques sont extrêmement répandues au niveau industriel ; elles permettent de mieux maitriser la sélectivité et la spécificité d’une grande diversité de réactions chimiques pour la formation à façon de composés cibles très variés, en jouant sur les chemins réactionnels et donc les énergies d’activations, tout en favorisant le plus souvent la cinétique.De manière générale, les catalyseurs sont le lieu d’environnements physicochimiques aux propriétés différentes de celles des milieux réactionnels conventionnels. Ils favorisent ou permettent des évènements réactionnels spécifiques (acte catalytique) mettant en œuvre leurs propriétés, et permettent même la création de nouveaux intermédiaires réactionnels facilitant et/ou orientant les réactions.La diversité des catalyseurs permet d’accéder à un panel extrêmement large de mécanismes moléculaires plus ou moins complexes dont la compréhension permet en retour un meilleur contrôle des réactions et l’optimisation de procédés catalytiques, ainsi que leur couplage raisonné selon des réactions dites « multi-catalytiques » qui tirent bénéfice des propriétés supplémentaires inédites de ces systèmes catalytiques ainsi assemblés