Search

International audience; Oxidative deamination is part of the metabolic pathways of amino acid oxidase and glutamate dehydrogenase enzymes, and enables obtaining α-keto acid from amino compound with ammonia as a by-product. This work presents the utilization of Pt-based catalyst composed of small platinum nanoparticles supported on industrial silica support (Pt/SiO2). This nanozyme was tested in the oxidative deamination reaction of two amino compounds: 5-hydroxymethylfurfurylamine (HMFA) to 5-hydroxymethylfurfural (HMF) and glutamic acid (GA) to α-ketoglutaric acid (KGA). This paper outlines the use of Pt nanozymes with deaminase-like activity. We showed the ability of this nanozymes to be selective and active in liquid phase oxidative deamination of HMFA and glutamic acid in a 6 to 9 pH range at 60°C under atmospheric pressure. Pt/SiO2 nanozyme actually plays as a deaminase mimic with high HMFA conversion (60%) and cumulative yield to the corresponding desaminated keto compounds (67%).

Purpose In this study, a combinatory approach was undertaken to assay the efficiency of fungal enzymatic cocktails from different fermentation conditions to degrade different lignocellulosic biomasses with the aim of finely characterizing fungal enzymatic cocktails. Methods Enzymatic assays (AZO and pNP-linked substrates and ABTS) were used to assess the composition of the fungal enzymatic cocktails for cellulase, xylanase and laccase activities. Comparisons were made with a new range of chromogenic substrates based on complex biomass (CBS substrates). The saccharification efficiency of the cocktails was evaluated as a quantification of the sugar monomers released from the different biomasses after incubation with the enzymatic cocktails. Results The results obtained showed striking differences between the AZO and pNP-linked substrates and the CBS substrates for the same enzymatic cocktails. On AZO and pNP-linked substrates, different hydrolysis profiles were observed between the different fungi species with Aspergillus oryzae being the most efficient. However, the results on CBS substrates were more contrasted depending on the biomass tested. Altogether, the results highlighted that assessing laccase activities and taking into account the complexity of the biomass to degrade were key in order to provide the best enzymatic cocktails. Conclusion The complementary experiments performed in this study showed that different approaches needed to be taken in order to accurately assess the ability of an enzymatic cocktail to be efficient when it comes to lignocellulosic biomass degradation. The saccharification assay proved to be essential to validate the data obtained from both simple and complex substrates.

International audience

International audience; Hybrid catalysis, which combines chemo- and biocatalytic benefits, is an efficient way to address green chemistry principles. 5-Hydroxymethylfurfural (HMF) is a versatile building block in numerous industrial applications. To date, few studies have described the production of its amine derivatives and their polymers. Finding a good methodology to directly transform HMF to 5-aminomethyl-2-furancarboxylic acid (AMFC) therefore represents an important challenge. After selecting the best oxidation catalyst for HMF conversion to 5-aldehyde-2-furancarboxylic acid and immobilizing a transaminase onto a solid carrier, we implemented the first one-pot/two-steps hybrid catalytic process to produce AMFC (77 % yield); this is the most efficient AMFC catalytic production method from HMF reported to date. This process also produced 2,5-furandicarboxylic acid (21 % yield) as a major secondary product that can be applied to polymer syntheses such as polyethylene furanoate. Herein, we report a novel way to access new biosourced polymers based on HMF oxidized and aminated derivatives.

International audience; In the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral amines, we have developed new continuous assays for the screening of amine-transaminases collections. These assays are based on the use of hypotaurine as irreversible amino donor. This β-aminosulfinic acid is converted upon transamination into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily detected by spectrophotometry using Ellman's reagent. Two complementary assays were developed based on this titration method. Firstly, a direct assay allowed detecting various transaminases able to use hypotaurine as amino donor. In a second coupled assay, L-alanine is used as generic donor substrate of aminetransaminases and is regenerated using an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a variety of valuable new biocatalysts for use in synthetic processes.

International audience; The search for the optimal coupling of several types of catalysts has inspired the development of multicatalytic hybrid materials (MCHMs) featuring chemical and biological catalysts co-immobilised on the same support. This complex interdisciplinary strategy, located at the crossroads of chemistry, biology, and materials science, calls for a wide range of skills and offers access to MCHMs with diverse catalytic properties and applications. In particular, numerous organic and inorganic supports, both rigid or flexible, have been used to develop remarkable hybrid catalysts that exhibit synergetic effects and achieve yields and enantiomeric excesses unattainable through the separate use of catalyst constituents even in onepot/one step processes. As the spearhead of hybrid catalysis, MCHMs concentrate the very essence of this field, requiring seamless communication and collaboration between chemists, biologists, materials scientists, and modelling specialists. Future developments in this area are expected to revolutionise catalysis and make it an inter-or even transdisciplinary research area. † This article is dedicated to P. H. Dixneuf for his outstanding contribution to organometallic chemistry and catalysis.

International audience; Direct methanol oxidation is expected to play a central role in low-polluting future power sources. However, the sluggish and complex electro-oxidation of methanol is one of the limiting factors for any practical application. To solve this issue, the use of plasmonic is considered as a promising way to accelerate the methanol oxidation reaction. In this study, we report on a novel approach for achieving enhanced methanol oxidation currents. Perforated gold thin film anodes were decorated with Pt/Ru via electrochemical deposition and investigated for their ability for plasmon-enhanced electrocatalytic methanol oxidation in alkaline media. The novel methanol oxidation anode (AuNHs/PtRu), combining the strong light absorption properties of a gold nanoholes array-based electrode (AuNHs) with surface-anchored bimetallic Pt/Ru nanostructures, known for their high activity toward methanol oxidation, proved to be highly efficient in converting methanol via the hot holes generated in the plasmonic electrode. Without light illumination, AuNHs/PtRu displayed a maximal current density of 13.7 mA/cm2 at -0.11 V vs Ag/AgCl. Enhancement to 17.2 mA/cm2 was achieved under 980 nm laser light illumination at a power density of 2 W/cm2. The thermal effect was negligible in this system, underlining a dominant plasmon process. Fast generation and injection of charge carriers were also evidenced by the abrupt change in the current density upon laser irradiation. The good stability of the interface over several cycles makes this system interesting for methanol electro-oxidation.

International audience; The various levels of integration of chemo-and bio-catalysis towards hybrid catalysis Q1 Egon Heuson * a and Franck Dumeignil b Combining catalysts is not a recent concept. For several decades, it has enabled the development of processes that are more economical in terms of solvents, energy, and carbon emissions. This strategy leads the way in current catalytic research aiming to reduce the impact of the chemical industry on the environment and replace synthetic routes based on petroleum with those based on biomass. In particular, hybrid catalysis, consisting of the integrated combination of several catalysts of different types, often a chemical and bio-catalyst, represents one of the most promising innovations in the field, especially when the two catalysts are combined in a single multicatalytic material. Several examples of such achievements have already been reported; however, these are rare compared to single-type catalyst combinations. It is important to understand the issues that govern hybrid catalysis to overcome the obstacles affecting its implementation, starting with the difficulties of communication between scientists of different fields. To surmount this barrier, this article proposes a new naming system for multicatalytic processes and reactions to unify common terms describing the same concepts. This system allows for the comparison of multicatalytic systems developed in both chemistry and biology and highlights their differences, similarities, and limitations. Hybrid catalysis is a rapidly expanding interdisciplinary field that builds on the developments from both the field of catalysis and materials science. Although the number of synthetic examples is limited, they are growing exponentially using the latest innovative materials for the production of multicatalytic materials for use in optimized "one-pot/one-step" processes.

La catalyse hybride a émergé il y a une vingtaine d’années. Cette discipline, qui vise à combiner un catalyseur chimique avec un catalyseur biologique afin de produire un système multi-catalytique possédant des propriétés supplémentaires par rapport à celles des catalyseurs unitaires, est longtemps restée peu étudiée car faisant appel à des compétences très diversifiées rarement rassemblées au sein de travaux communs. Ces dernières années, l’accroissement de l’intérêt pour les recherches inter- voire transdisciplinaires a largement bénéficié à son développement et le nombre d’exemples d’applications de la catalyse hybride croît aujourd’hui de manière exponentielle. Historiquement mise au point pour les procédés de résolution cinétique et de déracémisation, elle s’illustre désormais dans de très nombreux domaines, avec des finalités très différentes, depuis la régénération de cosubstrats enzymatiques coûteux, jusqu’à la mise en place de tandems catalytiques intégrés permettant la synthèse de composés jusque-là inaccessibles. Elle bénéficie à ce titre de nombreuses avancées en chimie et biologie/biologie moléculaire, qu’il s’agisse des nouvelles techniques de modélisation ou analytiques, de la possibilité de concevoir de nouveaux catalyseurs plus efficaces et tolérants, ou encore de l’élaboration de dispositifs réactionnels plus performants grâce aux génies des réacteurs et des procédés. Cette importante diversité de catalyseurs et de techniques représente un espoir prometteur pour le renouveau d’une catalyse qui peine souvent à répondre aux défis environnementaux de demain. Hybrid catalysis emerged about twenty years ago. This discipline aims at combining a chemical catalyst with a biological catalyst to produce a multi-catalytic system, with additional properties compared to those of single catalysts, has long remained little studied because it calls on a wide range of skills that are rarely brought together in joint work. In recent years, however, the growing interest in inter- or even transdisciplinary research has greatly benefited its development, and the number of examples of hybrid catalysis applications is now growing exponentially. Historically developed for kinetic resolution and deracemisation processes, hybrid catalysis is now illustrated in many fields, with very different purposes, from the regeneration of costly enzyme co-substrates to the implementation of integrated catalytic tandems allowing the synthesis of previously inaccessible compounds. In this respect, it benefits from numerous advances in chemistry and biology/molecular biology, whether in terms of new modelling or analytical techniques, the possibility of designing new, more efficient and tolerant catalysts, or the development of more efficient reaction devices thanks to reactor and process engineering. This important diversity of catalysts and techniques now represents a promising hope for the renewal of catalysis that is often struggling to meet tomorrow’s environmental challenges. 454

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. 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. 454

L’efficacité des catalyseurs chimiques et biologiques pour la réalisation de réactions chimiques au niveau industriel n’est plus à démontrer. Leur spécificité et sélectivité en font des outils essentiels à de nombreux procédés, et ils représentent à ce titre un outil majeur dans l’établissement de l’industrie de demain. Historiquement, les catalyseurs ont été très majoritairement utilisés de manière unitaire, les procédés en faisant intervenir plusieurs s’inscrivant le plus souvent dans des stratégies multi-étapes. Cependant, les deux dernières décennies ont vu un essor de la recherche autour des réactions multi-catalytiques qui combinent deux catalyseurs ou plus afin de bénéficier d’effets collaboratifs. Si des catalyseurs de natures différentes ne sont que très rarement mis en jeu ensemble, la combinaison d’un catalyseur chimique et d’un biocatalyseur, appelée « catalyse hybride », représente très probablement le type de réaction multi-catalytique le plus prometteur de par la diversité et l’efficacité inédites permises. Elle n’en demeure pas moins la plus complexe à mettre en œuvre, faisant appel à des compétences de domaines différents (chimie, biologie, matériaux, etc.). Les perspectives offertes donnent lieu à des travaux pionniers qui poussent les chercheurs à mettre au point de nouvelles stratégies permettant de contourner les limitations et inhibitions qui ne manquent pas d’apparaitre lorsque l’on force ces deux types de catalyseurs à travailler de concert. The effectiveness of chemical as well as biological catalysts for carrying out chemical reactions on an industrial level is no longer to be demonstrated. Their specificity and selectivity make them essential tools for many processes, and as such, they represent a major key in the establishment of tomorrow's industry. Historically, catalysts have mostly been used in a unitary manner, with processes involving several catalysts most often forming part of multi-stage strategies. However, the last two decades have seen a significant increase in research into multi-catalytic reactions that combine two or more catalysts in order to benefit from collaborative effects. While catalysts of different natures are very rarely used together, the combination of a chemical catalyst and a biocatalyst, known as "hybrid catalysis", is most likely the most promising type of multi-catalytic reaction because of the unprecedented diversity and efficiency allowed. Nevertheless, it remains the most complex to implement, calling on skills from different fields (chemistry, biology, materials, etc.). The prospects it offers are giving riseto pioneering work that is pushing researchers to develop new strategies for overcoming the limitations and inhibitions that inevitably arise when these two types of catalyst are forced to work together. 454

Dans la recherche de couplages optimaux de catalyseurs, les chercheurs pionniers en catalyse hybride se sont tout naturellement tournés vers l’élaboration de matériaux multi-catalytiques hybrides (MMCH) constitués à partir de co-immobilisation sur un même support de catalyseurs chimiques et biologiques. Cette exécution disciplinaire innovante, située au carrefour de la chimie, la biologie et la science des matériaux, fait appel à de très nombreuses compétences, et sa complexité n’a d’égale que la diversité des propriétés catalytiques que peuvent offrir ces MMCH. En particulier, l’utilisation d’une large variété de supports organiques et inorganiques, rigides et flexibles, a d’ores et déjà permis l’élaboration de quelques déclinaisons remarquables de catalyseurs hybrides démontrant de véritables effets de synergie, avec des rendements et des excès énantiomériques finaux inatteignables en utilisant des catalyseurs de manière isolée, et ce même dans des procédés en 1P1S. Véritable fer de lance de la catalyse hybride, les MMCH concentrent l’essence même de ce domaine, nécessitant communication et collaboration extrêmement étroites entre chimistes, biologistes, chercheurs en science des matériaux, ou encore en modélisation. Son développement sera sans aucun doute dans un futur proche le témoin d’une véritable (r)évolution de la recherche en catalyse vers des thématiques inter-, voire transdisciplinaires. In the search for optimal coupling of catalysts, it is quite naturally that pioneering researchers in hybrid catalysis have turned toward the development of hybrid multi-catalytic materials (HMCMs) constituted from the co-immobilization on the same carrier of chemical and biological catalysts. Such an innovative disciplinary variant, located at the crossroads of chemistry, biology and materials sciences, calls for a wide range of skills ; its complexity is only equaled by the diversity of catalytic properties that HMCMs can offer. In particular, the use of a wide variety of organic and inorganic supports, either rigid or flexible, has already led to the development of some remarkable examples of hybrid catalysts exhibiting real synergy effects, with yields and final enantiomeric excesses that are unreachable when using each catalyst separately, even in 1P1S processes. As the spearhead of hybrid catalysis, HMCMs concentrate the very essence of this field, requiring seamless communication and collaboration between chemists, biologists, materials science researchers and modeling specialists. Its development will in the near future undoubtedly witness a real (r)evolution of research in catalysis towards inter-, or even trans-disciplinary themes. 454

International audience; This study presents a miniaturized 3-methyl-2-benzothiazolinone hydrazone hydrochloride-assay (MBTHassay) 1 , optimized for high-throughput measurement of shrimp phenol oxidase activities and the screening of potential inhibitors of post-mortem melanosis. To validate this approach, assay parameters, including limit of detection and dynamic range, were compared to values obtained for DOPAchrome-assay, used here as a reference method. A reliable high-throughput automatized analysis was also performed, under standardized parameters, in order to compare and characterize 40 potential inhibitors of phenol oxidase activities. Among the selected molecules, twenty showed inhibitory action. Enzyme-inhibitor dissociation constant (K i), median inhibitor concentration (IC 50) and inhibition modes could be determined for nine of them. 4-Hexylresorcinol, Nphenylthiourea L-cystein and glutathione displayed K i values lower than sulfite salts (Na 2 S 2 O 5 and Na 2 SO 3) used as positive controls.

International audience; For powder catalyst characterization, Fourier Transform Infrared (FTIR), Raman, and X-Ray Fluorescence (XRF) spectrometers and X-Ray Diffraction (XRD) are available in high-throughput (HT) configurations, for example at the REALCAT platform to sequentially analyse multiple sets of samples. To remove the bottleneck resulting from the use of different sample holders for each equipment, a unique multi-well plate was developed. This paper details the design of such a plate including the selection of the fabrication material and the plate dimensioning based on the study of the 4 different physical interactions between matter and electromagnetic radiations for the aforementioned techniques. This new plate consists of a holder for removable wells enabling the avoidance of cross-contamination between samples. Raman, a focusing technique, has no strict constraint on the plate design. The number of wells, their geometry, spacing and dimensions were adjusted to deal with the constraints of IR optics. The well depth was set according to the XRF maximum penetration depth in the sample. The well diameter was optimized in order to obtain from the X-ray spot size the maximum achievable intensity. Poly-methyl-methacrylate (PMMA) was chosen as the material for the new plate due to its amorphous structure (no peak in XRD analysis) and ease with which it can be cut by a laser. Finally, the flatness of the multi-well plate was validated on the most challenging instrument: XRD. This new plate allows fast sample filling/preparation, requires small quantities of catalyst (50 to 80 mg) in each well and is compatible and convenient for HT experimentation.

International audience; Enzymatic transamination is a useful method for the green and highly enantioselective synthesis of chiral amines and non‐canonical amino acids which are of major importance as intermediates in medicinal chemistry. However, transamination reactions are usually reversible and synthetic applications of transaminases often require the implementation of an equilibrium shift strategy. Herein, we report a highly effective approach using glutamine as smart amine donor. This amino acid is converted upon transamination into 2‐oxoglutaramate which undergoes a fast cyclisation displacing the transamination equilibrium. We have developed a new activity assay in order to identify transaminases from biodiversity able to convert various α‐keto acids into valuable amino acids of l‐ or d‐series in the presence of glutamine as amine donor. Discovered transaminases were then used to prepare in high yield and with high enantioselectivity three amino acids of pharmaceutical importance, homophenylalanine, homoalanine and tert‐leucine by simply using a nearly stoichiometric amount of glutamine as amine donor.

International audience; Esterification 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.

International audience; Lipopeptides, such as surfactins are important biosurfactants produced by Bacillus sp. that find applications in many areas (environment, medicine, and food industries). Giving their importance, the use of simple detection methods will facilitate screening and quantification. In the present work, the authors describe a completely automated workflow for the screening of lipopeptide-producing strains, including quantification. First, isolated colonies from environmental samples are automatically picked and inoculated in 96 wells growth plate. After overnight incubation, surfactin produced in the broth is quantified, using a new sensitive fluorescent method. The method uses fluorescein (FL), which is an anionic dye at neutral to alkaline pH and forms a stable complex with the cationic surfactant cetylpiridinium chloride (CPC), quenching fluorescence. Upon addition of surfactin or other lipopeptides, fluorescein is released from the CPC-FL complex and quantified. The robustness of this method is assessed by comparing the quantification results to those conventionally measured by RP-UPLC and the results of strain screening are confirmed by MALDI-ToF analysis. The authors report for the first time the successful application of this analytical method for high-throughput screening of novel lipopeptide-producing strains.; Les lipopeptides, tels que les surfactines, sont des biosurfactants importants produits par Bacillus sp. qui trouvent des applications dans de nombreux domaines (environnement, médecine et industries alimentaires). Compte tenu de leur importance, l'utilisation de méthodes de détection simples facilitera leur criblage et leur quantification. Dans le présent travail, les auteurs décrivent un workflow de travail entièrement automatisé pour l'identification des souches productrices de lipopeptides, ainsi que la quantification de ces derniers. Tout d'abord, les colonies isolées des échantillons environnementaux sont automatiquement prélevées et inoculées dans une plaque 96 puits. Après une incubation d'une nuit, la surfactine produite dans le milieu de culture est quantifiée à l'aide d'une nouvelle méthode de lecture basée sur la fluorescence. La méthode utilise la fluorescéine (FL), qui est un colorant anionique au pH neutre à alcalin et qui forme un complexe stable avec le tensioactif cationique chlorure de cétylpiridinium (CPC), éteignant la fluorescence. Lors de l'ajout de surfactine ou d'autres lipopeptides, la fluorescéine est libérée du complexe CPC-FL et quantifiée. La robustesse de cette méthode est évaluée en comparant les résultats de quantification à ceux mesurés conventionnellement par RP-UPLC et les résultats du criblage de souches sont confirmés par analyse MALDI-ToF. Les auteurs rapportent ainsi ici pour la première fois l'application réussie de cette méthode analytique pour le criblage à haut débit de nouvelles souches productrices de lipopeptides.

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Transaminases are efficient tools for the stereoselective conversion of prochiral ketones into valuable chiral amines. Notably, the diversity of naturally occurring α-transaminases offers access to a wide range of l- and d-α-amino acids. We describe here two continuous colorimetric assays for the quantification of transamination activities between a keto acid and a standard donor substrate (l- or d-Glutamic acid or cysteine sulfinic acid). These assays are helpful for kinetic studies as well as for high-throughput screening of enzyme collections., Les transaminases sont des outils efficaces pour la conversion stéréosélective de cétones prochirales en amines chirales de haute valeur ajoutée. Notamment, la diversité des α-transaminases naturelles offre un accès à une large gamme de l- et d-α-aminoacides. Nous décrivons ici deux tests colorimétriques continus pour la quantification des activités de transamination entre un acide cétonique et un substrat donneur standard (acide l- ou d-glutamique ou acide cystéine sulfinique). Ces tests sont utiles pour les études cinétiques ainsi que pour le criblage à haut débit de collections d'enzymes.

Transaminases are efficient tools for the stereoselective conversion of prochiral ketones into valuable chiral amines. Notably, the diversity of naturally occurring α-transaminases offers access to a wide range of L- and D-α-amino acids. We describe here two continuous colorimetric assays for the quantification of transamination activities between a keto acid and a standard donor substrate (L- or D-Glutamic acid or cysteine sulfinic acid). These assays are helpful for kinetic studies as well as for high-throughput screening of enzyme collections.

International audience

The effects of yeast ICluyveromyces marxianus S-2-05, of cheese origin, were assessed on the intestine anaerobe symbiont Bacteroides thetaiotaomicron ATCC 29741 to unveil any changes in its antioxidant properties. To this end, these microorganisms were grown and incubated either separately, or co-incubated, under anaerobic atmosphere. Afterwards, the microbial cells were recovered and washed, and extracts were prepared using a sterile detergent solution to mimic the intestine detergent content. The extracts prepared from K. marxianus S-2-05 and reference strain K. marxianus MUCL 29917, grown under different conditions, were assessed for their antioxidant properties against superoxide anion and hydrogen peroxide. Extracts from both yeasts showed antioxidative effects, which were particularly important for K. marxianus S-02-5 after anaerobic incubation. Moreover, K. marxianus S-02-5 displayed a high level of activity against the aforementioned reactive oxygen species, enhancing that of B. thetaiotaomicron ATCC 29741, after the co-incubation process. Two-dimensional polyacrylamide gel electrophoresis was used to separate the proteins extracted. Superoxide dismutase, thiol peroxidase, rubrerythrin-intensively produced by B. thetaiotaomicron induced by the yeast-were identified by mass spectrometry. The antioxidative potential evidenced for K. marxianus S-02-5 is another advantage which could justify the utilization of this strain as a probiotic for countering intestinal inflammatory processes.

International audience; An efficient simultaneous cascade of two enzymatic steps catalyzed by a thermostable transaminase and transketolase was performed at elevated temperatures allowing the synthesis of naturally rare L-erythro (3S,4S) ketoses. L-ribulose, 5-deoxy-L-ribulose, D-tagatose and L-psicose, which are highly valuable chiral building blocks and display prominent biological properties, were obtained on a preparative scale with excellent stereoselectivities and good yields. A thermostable transketolase from Geobacillus stearothermophilus catalyzed at high temperatures the stereospecific synthesis of L-erythro (3S,4S)-configured ketoses from (2S)-hydroxylated aldehydes and β-hydroxypyruvate in which the latter is generated in an unprecedented manner in situ from natural L-serine and pyruvate using a novel thermostable L-α-transaminase from the thermophilic bacterium Thermosinus carboxydivorans. Overall, this cascade synthesis prevents the thermal decomposition of the labile β-hydroxypyruvate and offers an efficient and environmentally friendly procedure.

International audience; Efficient bi-enzymatic cascades combining aldolases and α-transaminases were designed for the synthesis of γ-hydroxy-α-amino acids. These recycling cascades provide high stereoselectivity, atom economy, and an equilibrium shift of the transamination. L-syn or anti-4-hydroxyglutamic acid and D-anti-4,5-dihydroxynorvaline were thus prepared in 83–95% yield in one step from simple substrates.; Des cascades bi-enzymatiques efficaces combinant aldolases et α-transaminases ont été conçues pour la synthèse de γ-hydroxy-α-aminoacides. Ces cascades de recyclage permettent une stéréosélectivité élevée, une économie d'atomes et un déplacement d'équilibre de la transamination. L'acide L-syn ou anti-4-hydroxyglutamique et la D-anti-4,5-dihydroxynorvaline ont ainsi été préparés avec un rendement de 83-95% en une étape à partir de substrats simples.

This multidisciplinary project was devoted to the discovery of new biocatalysts for the synthesis of chiral amines, through the screening of libraries of enzymes selected from the biodiversity. It involved the achievement of various tasks.The design and construction of three collections of transaminases, with a total of around 800 enzymes, was carried out within a strong collaboration with the Genoscope institute of Evry. We have developed novel efficient enzymatic assays allowing the high- throughput screening of the transaminase libraries and the detection, with high sensitivity, of new biocatalysts for a variety of transamination reactions. A rigorous statistical data analysis allowed highlighting a large set of hits, in which 24 transaminases were selected for further studies. After confirming the screening results, we have explored the substrate spectrum of the selected enzymes and studied the specific properties of some of them. Finally, we have demonstrated the usefulness of the new identified biocatalysts through their implementation for several applications. Some enzymes were used in an enzymatic cascade, whereas others allowed the synthesis of L- or D-homophénylalanine through an efficient innovative process.In addition to the multiple perspectives of application, this PhD work constitutes a valuable contribution to the fundamental knowledge about enzymatic transaminations.; e travail de thèse pluridisciplinaire a eu pour objectif la découverte de nouveaux outils biocatalytiques pour la synthèse d’amines chirales, par criblage de banques d’enzymes issues de la biodiversité. Il a impliqué un ensemble de tâches diverses.Trois collections de transaminases, comptant un total d’environ 800 enzymes, ont été construites dans le cadre d’une collaboration étroite avec le Génoscope d’Evry. Nous avons mis au point différents tests d’activité originaux, qui ont permis un criblage haut- débit de ces banques de transaminases et la détection, très sensible, de nouveaux catalyseurs pour une grande variété de réactions de transamination. Un traitement statistique rigoureux des données a permis de révéler un ensemble de hits, au sein duquel 24 TA ont été sélectionnées pour des études plus approfondies. Après avoir confirmé les résultats du criblage, nous avons exploré plus en détail le spectre de substrats de ces enzymes et les propriétés spécifiques de certaines d’entre elles. Nous avons enfin démontré l’intérêt de ces nouveaux biocatalyseurs à travers des premières applications. Certaines TA ont ainsi pu être engagées dans une cascade enzymatique ou mises en œuvre dans un procédé innovant pour la synthèse des deux énantiomères de l’homophénylalanine.En plus d’offrir de nombreuses perspectives d’applications, ce travail de thèse représente une contribution notable à l’avancée des connaissances fondamentales sur les réactions de transamination enzymatique.

International audience; In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine.

International audience; The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B (MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians. Despite their importance and being a prevalent target for the development of inhibitors as drugs, no systematic study of substrate specificity has been reported. In this study we present a systematic study of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs. Km and kcat values were determined for four N-alkyl analogs 2–5 and four aryl halide analogs 6–9 at MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be relevant for future drug design within the field of monoamines.