Novel porous carbonaceous material, process for the preparation thereof, and uses thereof

Abstract

The present invention relates to a carbonaceous polymeric material also referred to as porous carbonaceous material complexed with at least one metal ion, preferably a metal cation, a composition comprising a protein immobilized on said material, and methods of preparing the same. The invention further relates to the use of the composition comprising an immobilized protein in particular in heterogeneous biocatalysis applications.

Description

[0001] DESCRIPTION

[0002] New porous carbon material, its preparation process and uses

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a carbonaceous polymeric material also called porous carbonaceous material, or carbon-rich aerogel, comprising at least one complexed metal ion, preferably a metal cation, and a composition comprising at least one protein immobilized on said material and methods of preparing them.

[0005] The invention further relates to the use of the composition comprising at least one immobilized protein in heterogeneous biocatalysis applications in particular.

[0006] The invention also relates to a method for immobilizing proteins carrying an affinity tag on said porous carbon material.

[0007] CONTEXT OF THE INVENTION

[0008] Porous carbon materials or carbon-rich aerogels, known from the state of the art, are conventionally obtained by sol-gel polymerization reactions between carbon-rich organic precursors and drying of the hydrogels thus obtained.

[0009] A porous carbon material of the type prepared and used in the present application is described in the publication “Hydrothermal synthesis of highly porous carbon monoliths from carbohydrates and phloroglucinol”; Nicolas Brun et al.; RSC Advances, 2013, 3.

[0010] This document describes more particularly a novel hydrothermal approach to synthesize highly porous carbon-rich cryogels and aerogels (or carbogels) from carbohydrate-based precursors (e.g. fructose, glucose or xylose) and phenolic compounds (e.g. phloroglucinol). It should be mentioned that these carbohydrates can be isolated from the cellulosic fraction of lignocellulosic biomass, while phloroglucinol is the monomeric unit of phlorotannins and can be isolated from the bark of fruit trees, brown algae or through various biosynthetic pathways. Therefore, the compounds used in the present patent application can be considered renewable and sustainable.

[0011] The carbon-rich aerogels of the state of the art, derived from biomass, are obtained after an additional heat treatment at a temperature above 300°C and offer a wide range of potential applications, such as electrodes for batteries and fuel cells, or adsorbents for the storage of hydrogen and carbon dioxide. The carbon-rich aerogels or porous carbon materials according to the present invention are not subjected to a heat treatment at a temperature > 300°C.

[0012] Furthermore, enzymes are a specific group of proteins that serve as biological catalysts in the metabolism of all living cells. Thus, enzymes are able to specifically transform organic and inorganic molecules into products of interest.

[0013] However, since enzymes are biological molecules developed for a cellular environment, they are often unsuitable for other environments. It is therefore interesting to be able to immobilize enzymes on a solid support and use them as catalysts in this immobilized state.

[0014] Immobilization of enzymes on solid supports has been achieved using different techniques and different solid supports. Adsorption of enzymes on solid surfaces can lead to undesirable interactions between the enzyme and the solid support. For example, it has been shown that adsorption of proteins on silica nanoparticles can lead to changes in the secondary structure of the protein, which can lead to deactivation of the enzyme. It is therefore important that the solid support does not interfere with the structure and activity of the immobilized enzymes.

[0015] Immobilized metal ion affinity chromatography (IMAC) is a protein purification technique based on the affinity of proteins carrying a polyhistidine sequence for metal ions such as Fe 3+ , Cu 2+ , Zn 2+ , Neither 2+ and Co 2+ .

[0016] Thus, for example, metal ions are immobilized on an agarose gel carrying IDA (imido diacetate) and / or NTA (nitrilo triacetate) functional groups and can be selectively complexed by proteins carrying amino acids such as histidine and / or cysteine. An improved version of this technique uses recombinant proteins containing a fused polyhistidine peptide. Although this technique can be successfully applied in chromatographic procedures for protein purification and isolation, gel-immobilized enzymes are less suitable as heterogeneous catalysts in organic synthesis.

[0017] In attempts to prepare heterogeneous catalysts, the principle based on the IMAC affinity tag binding technique was applied to the immobilization of polyhistidine-tagged enzymes on modified silica. Encouraging results were obtained for Candida antarctica lipase B (CalB), but other less stable enzymes were found to be deactivated in the presence of silica, especially in the presence of organic solvents. It is known in the literature that silica nanoparticles have a destabilizing effect on proteins.

[0018] The high costs of enzyme preparation and the frequently observed loss of activity when immobilizing the enzyme on a solid support are obstacles in this development. A standardized and generally usable procedure for enzyme immobilization, which would allow the enzyme to be reused, would be highly desirable. There is still no general and simple method for the preparation of heterogeneous catalysts by enzyme immobilization.

[0019] There is therefore a need for new supports and improved methods for immobilizing enzymes on these supports, and for stable heterogeneous biocatalysts that can be applied in organic synthesis under aqueous or organic reaction conditions.

[0020] It has been surprisingly discovered that a porous carbon material obtained by hydrothermal carbonization of at least one carbohydrate and at least one compound carrying at least one orthodiphenol function, charged with at least one metal ion, makes it possible to immobilize at least one protein preferably via an affinity tag bond, in an improved manner compared to other types of protein immobilization.

[0021] BRIEF OVERVIEW OF THE INVENTION

[0022] In this context of a search for suitable and effective biocatalysis tools, a first aim of the invention is to propose a porous carbon material complexed with at least one metal ion, preferably a metal cation. A second aim of the invention is to propose a composition comprising a protein immobilized on said material or the use of said material for immobilizing a protein. A third aim of the invention is to propose methods for preparing said materials and compositions. Finally, another aim of the invention is to propose particular compositions and their uses.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] According to a first aspect of the invention, the present application relates to a composition comprising: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from at least one carbohydrate, preferably at least one ose, or at least one polysaccharide (biosourced precursor #1), and

[0025] -at least one compound having an orthodiphenol or orthomethoxyphenol function (biosourced precursor #2), and b) at least one metal cation.

[0026] The porous carbon material of the present invention is characterized in 13CP-MAS solid state NMR spectroscopy by a signal at 144 ppm, attributed to the carbons of the orthodiphenol functions.

[0027] The porous carbon material of the present invention is also characterized by bands at 1200, 1280 and 1500 cm' 1 , in FTIR spectroscopy, attributed to the CC and C-0 vibrations of the aromatic nucleus of an orthodiphenol function.

[0028] The porous carbon material of the present invention therefore has at least one orthodiphenol function.

[0029] The porous carbon material of the present invention is obtained by hydrothermal carbonization of at least one carbohydrate, preferably at least one ose, or at least one polysaccharide (biosourced precursor #1) and at least one compound having an orthodiphenol or orthomethoxyphenol function (biosourced precursor #2).

[0030] The biosourced precursor compound #1 that can be used according to the present invention is a carbohydrate, preferably at least one ose, or at least one polysaccharide.

[0031] Carbohydrates are defined by the International Union of Pure and Applied Chemistry (IUPAC) as a class of organic compounds containing a carbonyl group (aldehyde or ketone) and at least two hydroxyl groups (-OH). Included in this class are substances derived from monosaccharides by reduction of the carbonyl group, by oxidation of at least one functional group at the end of the chain to a carboxylic acid, or by replacement of one or more hydroxyl groups by a hydrogen atom, an amino group, a thiol group, or any similar atom. Furthermore, within the scope of the present application, compounds derived from the dehydration of carbohydrates (e.g., furan aldehydes) may also be used.

[0032] A monosaccharide (or sugar) is a carbohydrate monomer. Monosaccharides have at least 3 carbon atoms: they are polyhydroxyaldehydes or polyhydroxyketones.

[0033] Monosaccharides are distinguished by the length of their carbon chain, as follows:

[0034] • trioses: 3-carbon oses, C3H6O3 (glyceraldehyde, dihydroxyacetone);

[0035] • tetroses: 4-carbon oses, C4H8O4 (erythrose, threose, erythrulose);

[0036] • pentoses: 5-carbon oses, C5H10O5 (deoxyribose (C5HI0O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose)

[0037] • hexoses: 6-carbon oses, C6H12O6 (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose);

[0038] • deoxyhexoses: 6-carbon oses, C6H12O5 (fucose, rhamnose);

[0039] • heptoses: 7-carbon oses, C7H14O7 (sedoheptulose, mannoheptulose)

[0040] • octoses: 8-carbon oses, CsHieOs (heptahydroxyoctanal);

[0041] • nonoses: 9-carbon oses, C9H17N1O8 (neuraminic acid or sialic acid).

[0042] An aldose is a monosaccharide consisting of a chain of n carbon atoms with a carbonyl group on the terminal carbon atom, making it an aldehyde, and hydroxyl groups attached to all other carbon atoms. Aldoses have the general chemical formula C n (H2O) n . Since formaldehyde (n=1) and glycolaldehyde (n=2) are not generally considered carbohydrates, the simplest possible aldose is glyceraldehyde (n=3), which contains only three carbon atoms.

[0043] A ketose is a monosaccharide consisting of a chain of n carbon atoms having a carbonyl group on the non-terminal carbon atom, making it a ketone, and hydroxyl groups attached to all the other carbon atoms. According to a preferred embodiment of the invention, the at least one ose is chosen from:

[0044] • -an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose, preferably xylose, or

[0045] • -a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose

[0046] A polysaccharide is a polymer of the carbohydrate family formed from a number n of oses (n > 2) by alpha or beta glycosidic bond.

[0047] According to a preferred embodiment of the invention, the at least one polysaccharide is chosen from:

[0048] • -a heterodiholoside (a polysaccharide for which n=2 consisting of 2 different osidic units) notably chosen from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0049] • -a homodiholoside (a polysaccharide for which n=2 consisting of 2 identical osidic units) chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose, preferably maltose or sucrose.

[0050] According to another embodiment of the invention, the biosourced precursor compound #1 of the porous carbon material consists of a carbohydrate, preferably a ose, or a polysaccharide as described above.

[0051] According to another embodiment of the invention, the porous carbon material comprises two biosourced precursor compounds #1 which consist of two carbohydrates, preferably two oses, two polyosides or one ose and one polyoside as described above. According to another embodiment of the invention, the porous carbon material comprises more than two biosourced precursor compounds #1.

[0052] According to a preferred embodiment of the invention, the at least one compound having an orthodiphenol or orthomethoxyphenol function is a biosourced precursor compound having at least one benzene nucleus substituted by two hydroxyl groups in the ortho position or at least one methoxy group in the ortho position.

[0053] In this application, the term "bio-sourced" refers to materials or compounds derived from biomass of plant or animal origin, whether chemically modified or not. These raw materials have the advantage of being renewable.

[0054] For the purposes of the invention, a compound of "natural origin" or biosourced is understood to mean any compound originating from nature (biomass of plant or animal origin). This compound may be extracted from renewable terrestrial and marine biomass or from living organisms (animals, microorganisms), possibly subsequently modified, for example chemically, or obtained following the action of living microorganisms (for example enzymes or bacteria) on a compound of natural origin using biofermentation or biosynthesis-type processes. Compounds derived from petroleum, of fossil origin, do not fall into this category.

[0055] The biosourced precursor compound #2 usable according to the present invention may be a compound having at least one orthodiphenol function.

[0056] The compound having at least one biosourced precursor orthodiphenol or orthomethoxyphenol function that can be used according to the present invention can be chosen from the secondary metabolites of plants. Phenolic compounds form the largest group of phytochemical compounds in plants. They contain nearly 8,000 molecules divided into around ten chemical classes. Each class is characterized by the presence of a benzene ring to which one or more hydroxyl groups are directly linked. These compounds are present in all parts of higher plants (roots, stems, leaves, flowers, pollen, fruits, seeds and wood). They are synthesized by plants subjected to difficult conditions (infections, injuries, UV radiation, etc.) and are involved in numerous physiological processes such as cell growth, rhizogenesis, seed germination or fruit ripening.

[0057] The preferred orthodiphenol or orthomethoxyphenol compounds are shown in the Table below:

[0058] According to a preferred embodiment of the invention, the at least one compound having an orthodiphenol or orthomethoxyphenol function is chosen from: catechol, pyrogallol, caffeic acid, mangiferin, quercetin, cyanidin, catechin, epicatechin, epigallocatechin, anthocyanidol, procyanidol B-3, procyanidol B-4, fuhalols (bifuhahol, trifuhahol), carmalol (diphlorethohydroxycamalol) and tannic acid.

[0059] According to a preferred embodiment of the invention, the at least one compound having at least one orthodiphenol function is catechin.

[0060] According to a preferred embodiment of the invention, the at least one compound having at least one orthodiphenol or orthomethoxyphenol function is contained in a natural plant extract. According to a preferred embodiment of the invention, the natural plant extract containing at least one compound having at least one orthodiphenol or orthomethoxyphenol function is chosen from an extract of strawberry, potato, apple, lemon, walnut, vine, grape, flowers, soybean, pea, pine, tomato, garlic, quebracho wood, mimosa, chestnut or catechu.

[0061] According to another embodiment of the invention, the porous carbon material comprises two biosourced precursor compounds #2 which consist of two compounds having at least one orthodiphenol function as described above. According to another embodiment of the invention, the porous carbon material comprises more than two biosourced precursor compounds #2.

[0062] According to a preferred embodiment of the invention, the porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0063] • maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, or dihydroxyacetone, and

[0064] • catechin, or a catechu extract.

[0065] Cachou extract can be obtained from plant fibers of Acacia cachou, said extract containing condensed tannins and flavonols.

[0066] According to the invention, the term “porous carbon material” represents a polymer material obtained or capable of being obtained by sol-gel polymerization (e.g. by hydrothermal carbonization) from at least two biosourced precursors # 1 and 2 as defined in the present application.

[0067] According to the invention, the molar ratio between the at least one carbohydrate, preferably at least one ose, or at least one polysaccharide and the at least one compound having at least one orthodiphenol or orthomethoxyphenol function is from 10 / 1 to 1 / 10, preferably 1 / 2 or 1 / 1 or 2 / 1 or 3 / 1 or 4 / 1 or 5 / 1.

[0068] In one embodiment of the invention, the porous carbon material is capable of being obtained by a sol-gel polymerization process, known to those skilled in the art, involving at least two biosourced precursors # 1 and 2 as defined above.

[0069] Sol-gel polymerization processes may use a catalyst, for example, acids and bases such as, for example, nitric acid, acetic acid, ascorbic acid, hydrochloric acid, sulfuric acid, boric acid, sodium carbonate, sodium hydroxide, ammonium hydroxide and calcium sulfate.

[0070] Catalyst concentrations can be expressed as reactant-to-catalyst ratio (R / C) which can range, for example, from 10 to 5,000, or from 10 to 2,000, or from 10 to 1,000.

[0071] According to a preferred embodiment of the invention, the porous carbon material is obtained by hydrothermal carbonization.

[0072] Hydrothermal carbonization is defined as a thermochemical conversion process of biomass or, in the context of the present invention, at least two bio-sourced precursors # 1 and 2 as defined above, with the aim of obtaining a porous carbon monolith in aqueous phase. It is an exothermic process which reduces the oxygen and hydrogen levels of the material mainly by dehydration and decarboxylation reactions. Sol-gel polymerization is obtained by applying temperatures ranging from 120°C to 250°C to a mixture of at least two bio-sourced precursors # 1 and 2 as defined above and a solvent, preferably water (generally 10% by mass of the two bio-sourced precursors # 1 and 2).

[0073] In a particular embodiment, the porous carbon material is prepared by the following process: a) Heating a reaction mixture comprising an aqueous solution, and at least two biosourced precursors # 1 and 2 as defined above dissolved in the aqueous solution, at a temperature below 300°C to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a polar solvent.

[0074] The aqueous solution comprises water, and optionally solvents, preferably in amounts miscible with water.

[0075] According to a preferred embodiment, the reaction mixture comprises only one liquid phase, namely the aqueous solution, preferably water.

[0076] For example and without limitation, the aqueous solution comprises ethanol, preferably absolute ethanol.

[0077] According to a preferred embodiment, the mass fraction of water in an absolute water / ethanol mixture is between 1 and 0.3. According to a preferred embodiment, the mass fraction of water in an absolute water / ethanol mixture is between 1 and 0.5 when the biosourced precursors # 1 and 2 are respectively xylose and catechin.

[0078] Step (a) of the process may also be referred to as the “hydrothermal treatment step”.

[0079] Thanks to the presence of water (in the aqueous solution) in step (a), the preparation process of the invention is distinguished for example from pyrolytic processes, for the conversion of biomass or biosourced precursors # 1 and 2, to give a coal-type material (typically in the absence of oxygen).

[0080] The reaction temperature in the hydrothermal treatment step (a) is preferably <300°C, more preferably 100 to 300°C, even more preferably 120 to 250°C and most preferably 160 to 200°C or even more preferably 180°C. The reaction temperature is intended to mean the temperature, more specifically the average temperature, within the reaction mixture, which can be measured with a thermocouple.

[0081] The hydrothermal treatment in the process for preparing the porous carbon material of the present invention is preferably carried out in a pressure-resistant reactor, for example an autoclave.

[0082] There is no specific limitation on the duration of step (a) in the process for preparing the porous carbon material of the invention.

[0083] For example, the reaction medium can be introduced into a hermetically sealed mineralization bomb, then placed in a thermostatically controlled enclosure at 180°C for 20 hours.

[0084] Step (a) of the process is carried out until the entire contents of the reaction mixture comprising the aqueous solution and the bio-sourced precursors #1 and 2 have gelled or precipitated into the porous carbon material of the present invention.

[0085] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0086] -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 0.7 to 0.4, and, -xylose and catechin dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a polar solvent.

[0087] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0088] - an aqueous solution consisting of water and absolute ethanol with a mass fraction of water of 1 to 0.5, and,

[0089] - xylose and catechin or a catechu extract dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a solvent, preferably a polar solvent.

[0090] According to a preferred embodiment, the carbon material is formed from at least one carbohydrate, preferably an aldose or ketose, even more preferably at least one heterodiholoside, or homodiholoside (cl) and at least one compound having at least one orthodiphenol or orthomethoxyphenol function (c2) in a molar ratio (cl / c2) of from 10 / 1 to 1 / 10, preferably 2 / 1. For complex natural extracts and condensed tannins, the molar ratio (cl / c2) is defined by the ratio between the number of moles of carbohydrates (cl) and the number of moles of compounds having at least one orthodiphenol or orthomethoxyphenol function.

[0091] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0092] -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 0.7 to 0.4, and, -xylose and catechin or a catechu extract dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a solvent, preferably a polar solvent.

[0093] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0094] - an aqueous solution consisting of water and absolute ethanol with a mass fraction of water of 1 to 0.5, and,

[0095] -xylose and catechin (molar ratio 2 / 1) dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbon material, b) Wash the solid porous carbon material obtained by successive soaking in a solvent, preferably a polar solvent.

[0096] According to a preferred embodiment, the porous carbon material consists of

[0097] • 40-70% by mass in carbon element (C)

[0098] • 30-40% by mass in oxygen element (O) preferably 60% by mass in carbon element (C) and 35% by mass in oxygen element (O).

[0099] According to a preferred embodiment, the porous carbon material consists of 36% O and 60% C for the xylose / catechol system or catechu extract.

[0100] According to a preferred embodiment, the porous carbon material is formed from at least one carbohydrate (cl), preferably at least one ose, or at least one polysaccharide (cl) and at least one compound having at least one orthodiphenol or orthomethoxyphenol function (c2) or in a molar ratio (cl / c2) of from 10 / 1 to 1 / 10, preferably 2 / 1. The solid porous carbon material is washed by soaking either in ultrapure or deionized water or in a water / polar protic solvent mixture, for example methanol, ethanol or tert-butanol for several hours.

[0101] The purpose of washing is to extract soluble compounds not incorporated into the structure of the porous carbon material and to prepare the gel for the drying stage.

[0102] The carbonaceous material obtained or obtainable in the hydrothermal carbonization step (a) is typically composed of primary nanoparticles aggregated into an interconnected network forming the dispersed phase, with the aqueous solution forming the dispersion phase.

[0103] The carbonaceous material before drying can be called a solvogel. When the aqueous solution is water, the carbonaceous material obtained in step (a) can be called a hydrogel.

[0104] Therefore, a hydrogel is considered here as a special type of solvogel, in which the aqueous solution of the dispersion phase is water.

[0105] The solid porous carbon material obtained or obtainable according to the process described above can be dried and transformed into cryogel, aerogel or xerogel.

[0106] According to a first variant, a dry form of a hydrogel or solvogel is obtained by subjecting the carbon material (i.e. hydrogel or solvogel) to contact with a supercritical fluid, in particular acetone or carbon dioxide to transform it into an aerogel.

[0107] According to a second variant, a dry form of hydrogel is obtained by subjecting the carbon material (i.e. hydrogel or solvogel) to freezing of the gel thus isolated and lyophilization of said frozen gel to transform it into a cryogel.

[0108] According to a third variant, a dry form of hydrogel is obtained by subjecting the carbon material (i.e. hydrogel or solvogel) to evaporation of the solvent at controlled temperature and pressure to transform it into a xerogel.

[0109] Furthermore, a cryogel can be called an aerogel when the dried gel has largely retained the textural properties after drying.

[0110] Dried gels can be called aerogels regardless of the drying technique used.

[0111] According to the gel drying method, it is possible to control the pore size and the structure of the porous carbon materials obtained according to the invention. In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising an aqueous solution, and at least two bio-sourced precursors # 1 and 2 as defined above dissolved in the aqueous solution, at a temperature below 300°C, preferably at 180°C to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a polar solvent (ideally in absolute ethanol or in a water / tert-butanol mixture 75 / 25 in mass percentages), c) Drying the solid porous carbon material using a supercritical fluid or by freeze-drying.

[0112] The hydrogels placed in borosilicate glass beakers are first immersed in liquid nitrogen (77 K) for 15 minutes, then directly placed in the freeze-dryer chamber (e.g. a COSMOS freeze-dryer from Cryotec). Drying is carried out under vacuum < 50 mTorr for 48 hours with a cold trap at -80°C which ensures the condensation of water or solvent vapors.

[0113] Supercritical drying and freeze-drying are preferred to maintain the pore system. In a particularly preferred embodiment, the solvent is removed by extraction with supercritical CO2 for this process, the solvent in the solvogel is preferably selected from absolute ethanol or acetone (e.g., provided by solvent replacement with absolute ethanol or acetone).

[0114] Texture of porous carbon material

[0115] The porous carbon material obtained or obtainable in step (a) is a gelled material, in particular a solidified material composed of aggregated particles forming an interconnected network. The porous carbon material obtained or obtainable according to the method described above comprises micropores (pores < 2 nm), mesopores (pores of 2-50 nm) and macropores (pores > 50 nm).

[0116] Furthermore, as opposed to conventional activated carbons (e.g. those produced by pyrolysis), the oxygen content is typically higher than 25% for materials directly obtained in the hydrothermal carbonization step (a).

[0117] The surface of the carbon material of the invention as obtained in step (a) is very hydrophilic.

[0118] Textural properties of porous carbon material

[0119] Pore ​​diameter

[0120] The porous carbon material according to the present invention is composed of aggregated particles forming an interconnected network. This interconnected network of aggregated particles induces interparticle interstices (or pores) with a diameter of between 10 pm and 5 nm, more preferably with an average diameter of 5 to 200 nm, more preferably with an average diameter of 25 to 100 nm and even more preferably of 40 to 80 nm and preferably around 60 nm.

[0121] Furthermore, the porous carbon material according to the present invention is an aggregated material composed of the particles, with a pore size distribution calculated from a nitrogen adsorption / desorption isotherm at 77 K (for the narrowest pores; < 100 nm) and a mercury intrusion porosimetry profile (for the widest pores; > 50 nm).

[0122] In addition, the pore size distribution and the pore diameter corresponding to the peak of the pore size distribution can be provided by BJH analysis of the nitrogen adsorption / desorption isotherm.

[0123] The pore diameter of the carbon gel according to the present invention, corresponding to the peak of the pore size distribution, is preferably in a range of 40 to 80 nm.

[0124] It should be noted that, in order to prevent the reduction of the amount of adsorbed components, the pore diameter of the porous carbon material according to the present invention, corresponding to the peak of the pore size distribution, is preferably larger than the average molecular diameter of the diameter of the adsorbed or complexed components.

[0125] Specific surface area

[0126] The porous carbon material according to the present invention preferably has a specific surface area of ​​300 to 1000 m 2 / g, more preferably has a specific surface area of ​​600 to 800 m 2 / g.

[0127] If the porous carbon material has a specific surface area less than 100 m 2 / g, the surface area with which the adsorbed components are in contact will be reduced, and the number of pores in which the adsorbed components are housed will be reduced.

[0128] Porous volume

[0129] In addition, the total pore volume of the porous carbon material according to the present invention is not particularly limited because it also varies depending on the specific surface area and the pore diameter which corresponds to the peak of the pore size distribution. However, the porous carbon material according to the present invention preferably has a total pore volume of 0.1 to 5 cm 3 / g, more preferably has a total pore volume of 0.2 to 2.5 cm 3 / g.

[0130] The so-called total volume was determined at a relative pressure of 0.99 on nitrogen adsorption / desorption isotherms at -196°C. The surface area and micropore volume were determined by applying the t-plot method. Pore diameter distributions were evaluated by applying the BJH method on the desorption branch of the isotherms. The aerogels were degassed for at least 6 h at 0.05 mbar on a Micromeritics VacPrep before analyses. The analyses were performed on a Micromeritics TriStar. The total pore volume in the materials is higher than that obtained by nitrogen sorption volumetry at -196°C, since the largest pores (> 100 nm in diameter) cannot be analyzed by this method. The largest pores (> 100 nm in diameter) were analyzed by mercury intrusion porosimetry. The volume of these large pores is greater than 3 mL / g for catechin / xylose aerogels.The specific surface area and total pore volume of the porous carbon material according to the present invention can be determined by a general volumetric measurement described below. Specifically, a porous carbon material is placed in a container and cooled to the temperature of liquid nitrogen (-196°C). Then, nitrogen gas is introduced into the container and the amount of nitrogen gas adsorbed on the carbon gel is determined based on the volumetric method. Then, the pressure of nitrogen gas introduced into the container is gradually changed and the amount of nitrogen gas adsorbed on the carbon gel is plotted as a function of each equilibrium pressure. Thus, the nitrogen adsorption / desorption isotherm is obtained.

[0131] Alternatively, the specific surface area can be determined by the Brunauer Emmett Teller (BET) method, for example by the nitrogen adsorption technique.

[0132] The BET equivalent specific surface area was determined by nitrogen adsorption at 77K by performing isotherms at relative pressures P / PO between 0.01 and 0.99. The BET method was applied over a range of relative pressures between 0.1 and 0.3 (based on the Rouquerol transform).

[0133] According to the present invention, an aerogel prepared from mixtures of catechin or a natural extract of catechu and a sugar (see Figures 3 and 4) has a total volume of 0.5 to 0.8 cm 3 / g, an external volume of 0.5 to 0.8 cm 3 / g and a micropore volume of 0.01 to 0.05 cm 3 / g.

[0134] Interconnected porosity

[0135] This parameter can be determined by mercury intrusion porosimetry indicating the total volume of interconnected pores.

[0136] Ion complexation

[0137] The porous carbon materials as described above are complexed with metal ions, preferably at least one type of metal cation, which is complexed on the outer surfaces and the inner pore surfaces of the porous carbon material according to the invention.

[0138] According to a preferred embodiment of the invention, the at least one metal cation is chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0139] The metal cation is complexed or chelated to the porous carbon material. A chelate is a type of coordination compound in which a single metal ion is attached by coordinate covalent bonds to a molecule or ion called a ligand. A coordination compound is formed when groups of atoms, ions, or molecules chemically bond to each other by donating and accepting pairs of electrons. Groups that donate pairs of electrons are called ligands. These are usually Lewis bases. Groups that accept pairs of electrons are often transition metal cations.

[0140] According to the present invention, the term "hybrid material" refers to the porous carbon material on which the metal ions are complexed. This is the composition within the meaning of the present invention.

[0141] In a particular embodiment, the composition (porous carbon hybrid material) is prepared by the following method: a) Heating a reaction mixture comprising an aqueous solution, and at least two biosourced precursors # 1 and 2 as defined above dissolved in the aqueous solution, at a temperature below 300°C to obtain a solid porous carbon material, b) Washing the solid porous carbon material obtained by successive soaking in a polar solvent, c) Drying the solid porous carbon material using a supercritical fluid or by freeze-drying, d) Impregnating the solid porous carbon material with a solution comprising metal cations to obtain a porous carbon hybrid material complexed with metal cations.

[0142] The adsorption process to allow the metal complex to adsorb on a carbon material, the adsorption conditions are not particularly limited. For example, a metal complex can be adsorbed on a carbon material by placing the porous carbon material in a solution and mixing the solution for a certain period of time at about 10 to 100°C. The hybrid material is separated from the solution by centrifugation, and the resulting suspension is washed and then dried.

[0143] According to another variant of the invention, the composition (porous carbon hybrid material) is prepared by the following process: a) Heating a reaction mixture comprising an aqueous solution, and at least two biosourced precursors # 1 and 2 as defined above dissolved in the aqueous solution, with a solution comprising metal cations, at a temperature below 300°C to obtain a solid porous carbon material, b) Washing the solid porous carbon hybrid material obtained by successive soaking in a polar solvent, c) Drying the solid porous carbon hybrid material using a supercritical fluid or by freeze-drying.

[0144] The amount of metal ions complexed with the porous carbon material to form the hybrid material or composition of the present invention is not particularly limited. However, the amount of metal ions adsorbed or complexed on a carbon material is preferably 0.1 to 40 parts by weight relative to 100 parts by weight of the porous carbon material.

[0145] In a particular embodiment, the composition comprises an amount of chelated or complexed metal cations, measured by the SEM-EDX method, of from 0.2 to 2 mol%, preferably from 0.5 to 1 mol%.

[0146] In a particular embodiment, the composition comprises an amount of 1 to 5% by mass of metal cation, in particular 2 to 4% by mass.

[0147] In a particular embodiment, the composition comprises an amount of 20 to 40 mg of Iron per gram of porous carbon material. The solvent used herein is not particularly limited as long as it can dissolve and / or disperse metal complexes. Examples thereof include: acetic acid, water, ethylene glycol, DMSO and DMF.

[0148] The concentration of metal complex in the solution is not particularly limited. However, the concentration is preferably about 0.1 to 500 mM.

[0149] According to a preferred embodiment of the invention, the composition (solid porous carbon hybrid material) comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0150] • -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0151] • -at least one compound having at least one orthodiphenol or orthomethoxyphenol function (biosourced precursor #2), and b) at least one metal cation chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0152] According to a preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0153] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which

[0154] -the dare is chosen from

[0155] • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose idose, galactose, and talose, preferably xylose, or

[0156] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or - the polysaccharide is chosen from: • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0157] • a homodiholoside chosen in particular from inulobiose, alphaZ-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose, preferably maltose or sucrose and

[0158] -at least one compound having at least one orthodiphenol or orthomethoxyphenol function (biosourced precursor #2), and b) at least one metal cation chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0159] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0160] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which

[0161] -the dare is chosen from

[0162] • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose idose, galactose, talose, preferably xylose, or

[0163] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, tagatose, preferably fructose, or

[0164] -the polysaccharide is chosen from:

[0165] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, rutinose, or • a homodiholoside chosen in particular from: inulobiose, alphaZ-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobiose, preferably maltose or sucrose,

[0166] • -at least one compound having an orthodiphenol or orthomethoxyphenol function chosen from: catechol, pyrogallol, caffeic acid, mangiferin, quercetin, cyanidin, catechin, epicatechin, epigallocatechin, anthocyanidol, procyanidol B-3, procyanidol B-4, fuhalols (bifuhahol, trifuhahol), carmalol (diphlorethohydroxycamalol) and tannic acid or a natural plant extract containing at least one of the above compounds and b) at least one metal cation chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither2+ .

[0167] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0168] -xylose, or dihydroxyacetone,

[0169] - catechin, and b) at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0170] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0171] -xylose,

[0172] - catechin, and b) at least one metal cation which is Fe 3+

[0173] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0174] -xylose,

[0175] - catechin, and b) at least one metal cation which is Co 2+

[0176] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0177] -dihydroxyacetone,

[0178] - catechin, and b) at least one metal cation which is Fe 3+

[0179] According to the present invention, all the compositions described above can be used to immobilize proteins, in particular at least one enzyme

[0180] Immobilization of a protein

[0181] A second object of the invention is to provide a composition comprising a protein or an enzyme immobilized on said solid porous carbon hybrid material or composition. The solid porous carbon hybrid material of the present invention comprises the solid porous carbon material described above as a support, at least one metal ion complexed to said material and at least one protein (in particular an enzyme) immobilized (on the porous carbon material loaded with at least one metal cation), wherein the at least one immobilized protein preferably contains an affinity tag and is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and / or coordination bonds and / or ionic bonds.

[0182] The protein used here is not particularly limited.

[0183] The composition of the present invention thus comprises at least one enzyme selected from the approved list of the International Union of Biochemistry Nomenclature and Classification of Enzymes (IUBMB) and examples thereof include the following proteins:

[0184] EC 1 Oxido-reductases, EC 2 Transferases, EC 3 Hydrolases, EC 4 Lyases, EC 5 Isomerases, EC 6 Ligases and EC 7 Translocases.

[0185] Among these proteins, at least one protein chosen from the group consisting in particular of, α-arabinosidase, α-galactosidase, α-rhamnosidase, β-galactosidase, β-glucanase, β-glucosidase, β-glucanase, β-glucanase, β-mannanase, γ-lactamase, acetolactate decarboxylase, activase, adenosine deaminase, aminoacylase, aminopeptidase, amylase, amyloglucosidase, asparginase, aspartase, bromelain, carbonic anhydrase, catalase, cellulase, chitinase, chymosin, collagenase, cyclodextrinase, deoxyribonuclease I, dextranase, enoate reductase, epimerase, esterase, formate dehydrogenase, galactinol synthase, glucanotransferase, glucoamylase, glucose isomerase, glucose oxidase, glutenase, hemicellulase, hexose oxidase, inulinase, invertase, laccase, lactase, lactate dehydrogenase, leucine dehydrogenase, levanase, lipase, lipoxygenase, lysozyme, methane monooxygenase,monoamine oxidase, muramidase, naphthalene dioxygenase, naphthalene monooxygenase, naringinase, nattokinase, nitrile hydratase, papain, pectinase, pectinesterase, penicillin G, acylase, pentosanase, phenoloxidase, phenylalanine dehydrogenase, phytase, polyethylesterase, polygalacturonase, protease, protopectinase, pullulanase, pyrophosphatase, pyruvate transaminase, raffinose synthase, raffinose synthase, rennet, sacrosidase, serratiopeptidase, sphingosine kinase, stachyose synthase, tannase, taxolase, thermolysin, transaminase, transglutimases, trypsin, urease, xylanase, xylose isomerase.,

[0186] According to another preferred embodiment of the invention, the composition comprises a protein chosen from the group consisting in particular of an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), a pyrophosphatase (EC3.6.1.1).

[0187] Protein fillers

[0188] With regard to the porous carbon hybrid material of the present invention, the amount of protein adsorbed on the porous carbon material is not particularly limited as long as it has enzymatic activity. However, the amount of protein adsorbed on the porous carbon material is preferably 10 to 80 parts by weight relative to 100 parts by weight of the porous carbon material, preferably 10 to 50 parts by weight relative to 100 parts by weight of the porous carbon material.

[0189] In addition, the method for providing the porous carbon hybrid material of the present invention by causing a protein to adsorb onto a porous carbon material is not particularly limited. It is possible to use, for example, the impregnation method. The impregnation method described below is more preferable. That is, the protein is first dissolved in water or a buffer at a concentration at which the protein is not precipitated (preferably at a concentration of 0.1 to 1000 mg / ml). A porous carbon material is then suspended in the resulting solution at a temperature at which the solution does not freeze and at which the protein does not denature (preferably 0 to 50°C.) Thus, the protein is brought into contact with the porous carbon material for at least 5 minutes or more, preferably for 30 minutes or more, and thus the protein is immobilized in the pores of the carbon gel.In this way, the carbon hybrid material of the present invention can be provided.

[0190] In a particular embodiment, the composition comprising at least one immobilized protein is prepared by the method comprising the steps of:

[0191] - Suspend the dried porous carbon material in a solution containing metal cations, to obtain a porous carbon hybrid material loaded with metal cations

[0192] - Wash the porous carbon hybrid material loaded with metal cations

[0193] - Immobilizing a protein (enzyme) on the porous carbon hybrid material loaded with metal cations by reacting the porous carbon material loaded with metal cations with said protein preferably carrying an affinity marker, preferably a polyhistidine tag;

[0194] -wash the suspension and recover the protein immobilized on the porous carbon hybrid material loaded with metal cations.

[0195] More specifically, the porous carbon hybrid material loaded with metal cations is added to an aqueous solution containing a protein preferably carrying a polyhistidine tag at its N-terminus or C-terminus. Tl

[0196] As used herein, the term "affinity-tagged protein" refers to a recombinant protein in which an affinity tag, as defined above, has been added to the target protein.

[0197] Affinity-tagged proteins may be prepared by recombinant DNA technology using methods known in the art, such as by ligation of DNA fragments or by PCR techniques. Affinity-tagged proteins may also be referred to as "fusion-tagged proteins" or "fusion proteins."

[0198] The term "polyhistidine tag" refers to a chain of at least two histidine residues, which is attached to the C or N terminus of a protein.

[0199] The polyhistidine tag is preferably a chain of at least six histidine residues.

[0200] The term "polyhistidine-tagged enzyme" refers to a recombinant enzyme in which the target enzyme is fused with a polyhistidine tag as defined above.

[0201] The term "affinity tag" refers to a defined group, such as an organic or organometallic molecule, a protein fragment, or the like, that is attached to a recombinant protein and is capable of binding to a specific group immobilized on a matrix.

[0202] The porous carbon hybrid material loaded with metal cations on which at least one protein is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and / or coordination bonds and / or ionic bonds, is washed several times with a buffered aqueous solution.

[0203] The affinity tag used in the invention may be any tag capable of specifically binding to the porous carbon material for which it has an affinity.

[0204] Affinity binding can be the result of, for example, van der Waals interaction, hydrogen bonding, ionic bonding, or hydrophobic interaction.

[0205] In any case, the affinity binding must be strong enough to allow the affinity tag and the porous carbon material to remain tightly bound to each other at least until certain specific conditions are applied to dissociate the affinity tag from the porous carbon material. The protein to be immobilized on the material can be any protein containing an affinity tag, such as a (recombinant) protein or an enzyme containing an affinity tag.

[0206] Preferably, the protein is an enzyme containing an affinity tag.

[0207] A number of affinity tags and corresponding matrices are known in the art.

[0208] In a preferred embodiment, the affinity tag on the protein is a polyhistidine tag and the solid material contains a chelated metal ion.

[0209] The buffered aqueous solution is preferably a Tris-HCl, or Phosphate buffer.

[0210] For the purposes of the present invention, an affinity label is a label which has a specific affinity for metal cations.

[0211] According to a second aspect of the invention, the present application relates to a composition comprising: a) a porous carbon material formed from:

[0212] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0213] -at least one compound having an orthodiphenol or orthomethoxyphenol function (biosourced precursor #2), b) at least one metal cation, and c) at least one protein preferably comprising a polyhistidine tag

[0214] According to a preferred embodiment of the invention, the composition comprises a) a porous carbon material formed from:

[0215] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0216] -at least one compound having an orthodiphenol or orthomethoxyphenol function (biosourced precursor #2), b) at least one metal cation, and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag. According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material formed from:

[0217] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which the ose is chosen from:

[0218] • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose idose, galactose, and talose, preferably xylose, or

[0219] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or in which the polysaccharide is chosen from:

[0220] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0221] • a homodiholoside chosen in particular from: inulobiose, alphaZ-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose, preferably maltose or sucrose, and

[0222] -at least one compound having an orthodiphenol or orthomethoxyphenol function chosen from: catechol, pyrogallol, caffeic acid, mangiferin, quercetin, cyanidin, catechin, epicatechin, epigallocatechin, anthocyanidol, procyanidol B-3, procyanidol B-4, fuhalols (bifuhahol, trifuhahol), carmalol (diphlorethohydroxycamalol) and tannic acid,

[0223] • Or

[0224] • a natural plant extract containing at least one of the above compounds, b) at least one metal cation chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+, Zn 2+ , Cu 2+ , or Neither 2+ , and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag.

[0225] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material formed from:

[0226] -xylose, or dihydroxyacetone,

[0227] - catechin, and b) at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag.

[0228] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material formed from:

[0229] -xylose,

[0230] - catechin, and b) at least one metal cation which is Fe 3+, and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag.

[0231] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material formed from:

[0232] -xylose,

[0233] - catechin, and b) at least one metal cation which is Co 2+ , and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag.

[0234] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material formed from:

[0235] -dihydroxyacetone,

[0236] - catechin, and b) at least one metal cation which is Fe 3+ , and c) at least one immobilized protein, preferably said at least one immobilized protein comprising a polyhistidine tag.

[0237] For example, if the solid material according to the invention is used for the purification and isolation of an affinity-labeled protein, the binding of the enzyme to the support must be reversible. In such cases, it is preferred that the chelated metal ion is Fe 3+ , Neither 2+ or Co 2+ These metal ions bind strongly enough to immobilize a polyhistidine-tagged enzyme, but are also capable of releasing the immobilized enzyme when specific conditions are applied, such as treatment with a buffered solution containing imidazole or ethylenediaminetetraacetate (EDTA).

[0238] For the use of immobilized enzymes in heterogeneous biocatalysis, strong binding of the enzyme to the support is desirable.

[0239] In such cases, it is preferred that the chelated metal ion is Co 2+ or Fe 3+ , and most preferably Fe 3+, since this results in particularly strong binding of the polyhistidine tag to said material. As demonstrated in the examples, leaching of the enzyme or metal ion from a material comprising an immobilized protein in the presence of Fe 3+ as a chelated metal ion is almost negligible. The absence of leaching allows the immobilized protein material (the biocatalyst) to be used in catalytic quantities. The use of catalytic quantities is particularly important in continuous flow reactions.

[0240] Another advantage of Fe 3+ as a chelated metal ion is that this metal is non-toxic.

[0241] The high affinity of the polyhistidine tag for metal ions such as Co 2+ or Fe 3+allows immobilization of polyhistidine-tagged proteins to be performed from crude solutions containing the proteins without the need for extensive purification of the solution prior to the immobilization step. Organic matter that does not contain a polyhistidine tag binds to chelated metal ions only weakly, or not at all, and is readily removed from the material comprising at least one polyhistidine-tagged protein by washing with, for example, water or buffered aqueous solutions. Thus, if the polyhistidine-tagged protein is prepared by intracellular overexpression, protein immobilization can be performed directly from the cell lysate. Alternatively, if the polyhistidine-tagged protein is secreted from the host organism, protein immobilization can be performed directly from the cell culture supernatant.

[0242] Dissociation of bound protein can be achieved using standard IMAC methods.

[0243] The bound protein can, for example, be released from the support by lowering the pH or by adding a competitive molecule with equal or greater affinity for the chelated metal ions than the polyhistidine group, for example, by applying a buffered solution containing imidazole or ethylenediaminetetraacetate (EDTA).

[0244] After dissociation of the purified proteins from the support, the polyhistidine tag can be removed from the proteins, if necessary, by techniques known in the art, for example by cleaving the affinity tag with a suitable enzyme such as a specific protease, thereby obtaining the pure, tag-free protein.

[0245] If the proteins immobilized on the support, as described above, are enzymes, they contain an active site capable of catalyzing a chemical reaction.

[0246] Thus, the immobilized enzyme material is potentially useful as a biocatalyst in organic synthesis.

[0247] Therefore, in another aspect, the invention relates to the use of an immobilized enzyme material as described herein as a heterogeneous biocatalyst, for example in synthetic organic transformations.

[0248] A preferred embodiment of the invention relates to the use of the compositions described above as heterogeneous biocatalysts.

[0249] The present invention further relates to a method of catalyzing an enzyme-catalyzed reaction, comprising providing a composition comprising a protein or an enzyme immobilized on said porous carbon material according to the invention, and contacting said composition comprising an immobilized enzyme with at least one substrate on which the enzyme, which is immobilized on said material, is capable of acting.

[0250] Immobilization of enzymes on said material by affinity tag binding, as described herein, improves the stability of the enzymes used. The immobilized enzymes have been found to tolerate both aqueous conditions as well as a range of organic solvents.

[0251] This allows compositions comprising a protein or enzyme immobilized on said porous carbon material to be used under reaction conditions in which free, non-immobilized enzymes would not have been stable.

[0252] It is possible that the composition comprising a protein or enzyme immobilized on said porous carbon material may also be used in a wider pH range than the free, non-immobilized enzymes would have tolerated.

[0253] Since the native activity of the enzyme is retained, the composition comprising a protein or an enzyme immobilized on said porous carbon material has a higher enzymatic activity per protein mass than the initial non-immobilized protein material. For example, the turnover number (TON), defined as the number of moles of product formed per mole of protein over a given time, is higher for an enzyme immobilized according to the invention compared to a free enzyme.

[0254] The enzyme that is immobilized on said material may be any enzyme that is useful as a biocatalyst in organic synthetic transformations, including, but not limited to, enzymes acting as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.

[0255] Thus, compositions comprising an enzyme immobilized on said material can be used as heterogeneous biocatalysts in any organic reaction in which the immobilized enzyme is capable of specifically catalyzing the reaction.

[0256] Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.

[0257] Particularly useful biocatalytic reactions are enantioselective reactions.

[0258] Specific examples of biocatalytic reactions include the synthesis of chiral amines from prochiral ketones, the synthesis of esters from carboxylic acids and alcohols or from esters and alcohols, the synthesis of hydroxylated molecules, or the synthesis of terpenes or terpenoids. In one embodiment, two or more different enzymes may be immobilized on the material of the invention, wherein each of the different enzymes is capable of catalyzing a different reaction.

[0259] It may then be possible to use the material containing two or more different immobilized enzymes as a heterogeneous biocatalyst in a multi-step or cascade reaction.

[0260] According to another aspect of the invention, the present application relates to a method for catalyzing an enzyme-catalyzed reaction, comprising providing a composition described above, and contacting said composition with at least one substrate on which an enzyme, which is immobilized on said support of the composition, is capable of acting.

[0261] Yield and downtime speed

[0262] The immobilization yields, expressed in mg of enzyme immobilized per mg of enzyme initially present in the solution, are between 90 and 100% for protein loads between 5 and 50% by mass.

[0263] The immobilization speeds expressed in minutes to reach at least an immobilization rate of at least 75% are between 10 and 120 minutes.

[0264] According to a preferred embodiment of the invention, the method for immobilizing a protein, in particular an enzyme on the porous carbon material, is characterized in that the immobilization yield is between 90% and 100% for protein loads between 5 and 50% by mass.

[0265] According to a preferred embodiment of the invention, the method for immobilizing a protein, in particular an enzyme on the porous carbon material, is characterized in that the immobilization speed to achieve an immobilization yield of at least 75% is between 10 and 120 minutes.

[0266] According to a final aspect of the invention, the present application relates to the use of the composition as a heterogeneous biocatalyst. Enzymatic performances

[0267] The measured enzymatic performances are expressed as the number of reactions catalyzed by enzyme (Turnover number, TON) over a given time. The TON represents the number of moles of substrate converted (or product formed) by the number of moles of enzyme immobilized in a given volume.

[0268] According to a preferred embodiment of the invention, the specific activity (which is the quantity of substrate transformed per unit of time and per mass of enzyme) of an enzyme immobilized on the porous carbon material is greater than that of the same free enzyme, i.e. not immobilized on said porous carbon material.

[0269] In Example 3, measurements of the enzymatic performance of several immobilized enzymes show a significantly higher TON than that of the non-immobilized enzyme.

[0270] These results are better than those described to date in the literature where it is observed that in most cases the activity of the enzyme is strongly reduced after immobilization.

[0271] List of figures

[0272] Figure 1 is a scanning electron micrograph of a uniform, continuously interconnected porous texture for a tannin-xylose aerogel prepared in 10 mL of a 15% by mass solution of absolute ethanol in deionized water, treated at 180°C for 20 hours under hydrothermal conditions (autoclave), washed and then dried by freeze-drying.

[0273] Figure 2 is a transmission electron micrograph that highlights a macro-mesoporous network for a tannin-xylose aerogel prepared in 10 mL of a 15% mass solution of absolute ethanol in demineralized water, treated at 180°C for 20 hours under hydrothermal conditions (autoclave), washed and then dried by lyophilization.

[0274] Figures 1 and 2 show primary particles of a few nanometers, aggregated primary particles forming an interconnected network that induces interparticle interstices of a few nanometers to several micrometers. Figure 3 represents the textural properties determined by nitrogen sorption volumetry at 77K of aerogel prepared from a mixture of a sugar and natural catechu extract.

[0275] Figure 4 represents the textural properties determined by nitrogen sorption volumetry at 77K of aerogel prepared from a mixture of a ose and catechin.

[0276] Figure 5A represents an NMR spectrum 13C CP-MAS, of an aerogel formed by hydrothermal carbonization of a xylose-catechin mixture (top, dark) and xylose-phloroglucinol (bottom, light) and Figure 5B an FTIR spectrum of an aerogel formed by hydrothermal carbonization of a xylose-catechin mixture (top, dark) and xylose-phloroglucinol (bottom, light).

[0277] Experimental part

[0278] Example 1: Preparation of a porous carbon material in the form of an aerogel (based on xylose / catechin / Fe 3+ (FeXCat))

[0279] 0.85 g of xylose is dissolved in 10 mL of a mixture of deionized water and absolute ethanol 50 / 50 m / m.

[0280] 0.35 g of catechin is added to the previous solution.

[0281] The mixture is stirred until a homogeneous solution is obtained. Ultrasonic treatment at room temperature for one hour or less can accelerate the homogenization of the solution,

[0282] The resulting solution is treated by hydrothermal carbonization, i.e. introduced into a mineralization bomb (autoclave), then this is hermetically sealed and placed in a thermostatically controlled enclosure at 180°C for 20 hours. At the end of this period, the autoclave and its contents are cooled to room temperature for a few hours (generally 2 hours).

[0283] The autoclave is then opened and its contents poured either into ultrapure water (usually 100 mL), or into deionized water, or into a water / polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or into a pure polar protic solvent,

[0284] The solid obtained is washed by successive soaking (usually 3 times 100 mL) with either ultrapure water, deionized water, a water / polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or a pure polar protic solvent for several hours (usually 8 hours). This produces a hydrogel. The hydrogel is then dried by lyophilization or in a supercritical fluid to obtain an aerogel.

[0285] The aerogels obtained under the conditions described above (200 mg) are added to 10 mL of an aqueous solution containing 400 mmol / L of FeCH

[0286] The suspension thus obtained is stirred for 4 hours at 20 rpm at room temperature.

[0287] After stirring, the suspension is centrifuged (5 min at 5000 rpm). After centrifugation, the supernatant is removed.

[0288] The solid thus obtained is washed with water 3 times:

[0289] 1) Water is added (10 mL),

[0290] 2) The suspension thus obtained is stirred for a few minutes (generally 5 minutes) at room temperature,

[0291] 3) The suspension is centrifuged (5 min at 5000 rpm),

[0292] 4) After centrifugation, the supernatant is removed.

[0293] Steps 1 to 4 are repeated 2 times.

[0294] Example 2: Immobilization of a protein

[0295] 10 mg aerogel complexed with Fe ions 3+ obtained under the conditions described above is added to an aqueous solution containing (1 mg) of protein carrying a polyhistidine tag at its N-terminus.

[0296] The following three enzymes were prepared: Trypanosoma brucei pyrophosphatase (TbVSPl, EC3 Hydrolase), B9L0N2 transaminase (w-TA, EC2 transferase) and Galdieria sulphuraria old Yellow Enzyme (GsOYE, EC1 Oxydoreductase).

[0297] The suspension thus obtained is stirred at 750rpm for a few hours

[0298] (usually 2 hours) at 4°C. After stirring, the suspension is centrifuged (between 4000 and 6000rpm for 2 to 5 min). After centrifugation, the supernatant is removed. The solid thus obtained is washed with a buffered aqueous solution twice:

[0299] 1) Buffer is added (lmL),

[0300] 2) The suspension thus obtained is stirred for a few minutes (usually 5 minutes) at room temperature,

[0301] 3) The suspension is centrifuged (between 4000 and 6000rpm for 2 to 5 min),

[0302] 4) After centrifugation, the supernatant is removed.

[0303] Steps 1 to 4 are repeated once.

[0304] This gives a protein carrying a polyhistidine sequence immobilized on the porous carbon material complexed with Fe ions. 3+ .

[0305] Variable protein loads

[0306] The aerogels obtained under the conditions described above (10 mg) are added to a buffer solution (Tris HCl 50 mM NaCl 150 mM pH=7.5, 1 mL) containing 1 / 2.5 / 5 mg of GsOYE enzyme according to the desired protein load (10 / 25 / 50% by mass) or 1 / 2.5 mg of B9L0N2 enzyme for a protein load of 10 and 25% by mass.

[0307] The suspension thus obtained is stirred at 800 rpm for 2 hours at 4°C.

[0308] For the kinetic monitoring of immobilization, samples are taken at 10, 20, 30, 60, 90 and 120 min. To do this, the suspension is centrifuged between 4000 and 6000 rpm for 2 to 5 min and then a sample of 20 pL is taken. Stirring of the reaction mixture is resumed immediately after taking the sample.

[0309] After the last sample, all of the supernatant is discarded.

[0310] The solid thus obtained is washed twice with a buffer solution:

[0311] 1) 1 mL of buffer is added.

[0312] 2) The suspension thus obtained is stirred for a few minutes (usually 5 minutes) at room temperature.

[0313] 3) The suspension is centrifuged (between 4000 and 6000 rpm for 2 to 5 min).

[0314] 4) After centrifugation, the supernatant is discarded. Steps 1 to 4 are repeated once.

[0315] The amount of protein present in the samples is measured by Bradford assay. This results in the GsOYE protein immobilized on a hydrogel (aerogel) with a protein load varying from 10 to 50% by mass and the B9L0N2 protein immobilized on a hydrogel (aerogel) with a protein load varying from 10% to 25% by mass.

[0316] Continuous flow immobilization:

[0317] • B9L0N2

[0318] The FeXCat aerogels obtained under the conditions described above (50 mg) are introduced into a non-thermostatic column whose end is made of a frit to retain the aerogel.

[0319] A buffer solution (50 mM Phosphate, 300 mM NaCl, pH=7.5, 5 mL) containing 5 mg of B9L0N2 enzyme percolates at a flow rate of 0.1 mL / min through the column via a pump. The solution is recovered at the column outlet. The aerogel contained in the column is then washed by percolation of buffer solution at a flow rate of 0.1 mL / min for 50 min. The solution is also recovered at the column outlet.

[0320] The amount of protein present in these two solutions is measured by Bradford assay.

[0321] This results in the B9L0N2 protein immobilized on a FeXCat hydrogel (with a protein load of 10% by mass) in a column for use in continuous flow and recirculation.

[0322] • GsOYE

[0323] The FeXCat aerogels obtained under the conditions described above (50 mg) are introduced into a non-thermostatic column whose end is made of a frit to retain the aerogel.

[0324] A buffer solution (Tris HCl 50 mM NaCl 150 mM pH=7.5, 5 mL) containing 5 mg of GsOYE4 enzyme percolates at a flow rate of 0.1 mL / min into the column via a pump. The solution is recovered at the column outlet. The aerogel contained in the column is then washed by percolation of buffer solution at a flow rate of 0.1 mL / min for 50 min. The solution is also recovered at the column outlet.

[0325] The amount of protein present in these two solutions is measured by Bradford assay.

[0326] This gives the GsOYE protein immobilized on a FeXCat hydrogel (with a protein load of 10% by mass) in a column to be used in recirculation.

[0327] Example 3 Immobilization kinetics 10 mg porous carbon material complexed with Fe ions 3+ are brought together with 1 mg of enzyme bearing a polyhistidine tag in a total volume of 1 mL.

[0328] Immobilization of B9L0N2 transaminase occurs in the presence of the cofactor pyridoxal phosphate (PLP).

[0329] Immobilization of pyrophosphatase TbVSPl occurs in the presence of the cofactor Mg2+.

[0330] The 0, 10, 20 and 30 min samples (10 pL) are diluted half-way and the 1h and 2h samples (20 pL) are not diluted. 20 pL of sample (Echl at 4) mixed with 1 mL of Bradford reagent, in the dark for 15 min.

[0331] Results Table 2: Immobilization kinetics of TbVSPl (lmg) on ​​an aerogel

[0332] Xylose / Catechin complexed with Fe ions 3+ (10 mg) prepared under the conditions described above. Table 3: Immobilization kinetics of B9L0N2 (lmg) on ​​a Xylose / Catechin aerogel complexed with Fe ions 3+ (10 mg) prepared under the conditions described above.

[0333] Table 4: Immobilization kinetics of GsOYE (lmg) on ​​a Xylose / Catechin aerogel complexed with Fe ions 3+ (10 mg) prepared under the conditions described above.

[0334] The results in Tables 2-4 show that the immobilization rates to achieve an immobilization rate of at least 75% are between 10 and 120 minutes. These results show that the immobilization of an enzyme on the porous carbon material complexed with Fe ions 3+ is simple, fast and efficient.

[0335] Example 4: Performance

[0336] Conversion rates are expressed in moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time.

[0337] For TbVSPl, the reaction studied is the hydrolysis of pyrophosphate to phosphate in the presence of the cofactor Mg 2+ . The protein charge used is 10% by mass. The reaction mixture consists of sodium pyrophosphate (5mM), MgCl (ImM) in solution in Tris-HCl buffer 50mM NaCl 200 mM pH 8.5. The solid consists of 1mg of TbVSPl carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions 3+ (10 mg) is added to the reaction mixture. The formation of phosphate is monitored by determination of phosphmolybdate / malachite green in visible spectroscopy at 630 nm. The results are summarized in Table 5.

[0338] For w-TA, the reaction studied is the transamination of pyruvate in the presence of racemic α-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP). The protein charge used is 10% by mass. The reaction mixture consists of pyruvate (25mM), racemic α-methylbenzylamine (25mM), PLP (0.1mM) and DMSO (1% wt) in solution in 50mM NaCl 300mM pH 7.5 Phosphate buffer. The solid consists of 1mg of w-TA carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ (10 mg) is added to the reaction mixture. The formation of acetophenone is continuously monitored by UV spectroscopy at 245 nm. The results are summarized in Table 5.

[0339] For GsOYE, the reaction studied is the reduction of cyclohexenone to cyclohexanone in the presence of the cofactor NADH which oxidizes to NAD+. The protein charge used is 10% by mass. The reaction mixture consists of cyclohexenone (50mM), NADH (75mM) and DMSO (1% wt) in solution in Tris-HCl 50mM NaCl 150mM pH 7.5 buffer. The solid consists of 1mg of GsOYE carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ (10 mg) is added to the reaction mixture. The disappearance of NADH is monitored by UV spectroscopy at 340 nm. The results are summarized in Table 5.

[0340] Table 5: Catalytic performances (Turnover numbers, TON and Conversion rate) of the 3 free or immobilized enzymes in the presence of Fe ions 3+(Trypanosoma brucei pyrophosphatase (TbVSPl, EC3 Hydrolase), B9L0N2 transaminase (w-TA, EC2 Transferase) and G. sulphuraria Old Yellow Enzyme (GsOYE, EC1 Oxidoreductase).

[0341] In all 3 cases, the TON of the immobilized enzyme is significantly higher than that of the non-immobilized enzyme. The conversions obtained with the immobilized enzymes are always equal to or higher than those obtained with the respective free enzymes.

[0342] An aerogel complexed with Co ions 2+ obtained under the same conditions as those described above in the presence of Fe ions 3+ and a B9L0N2 transaminase (w-TA, EC2 transferase) carrying a polyhistidine tag at its N-terminus was prepared. The ratio of immobilized TON enzyme vs. non-immobilized (free) TON enzyme is 764 / 520.

[0343] These results are better than those described to date in the literature where it is observed that in most cases the activity of the enzyme is strongly reduced after immobilization. The interconnected macroporous structure (pores between 50 nm and 2 microns) therefore facilitates the mass transfer of reactants and products throughout the material.

[0344] Example 5: Reuse

[0345] The measured enzymatic performances are expressed as the number of reactions catalyzed per enzyme (Turnover numbers, TON) over a given time. These activities are measured for non-immobilized (free) enzymes and for enzymes immobilized under the reaction conditions below.

[0346] For TbVSPl, the reaction studied is the hydrolysis of pyrophosphate to phosphate in the presence of the cofactor Mg 2+. The protein charge used is 10% by mass. The reaction mixture consists of sodium pyrophosphate (5mM), MgCh (ImM) in solution in Tris-HCl buffer 50mM NaCl 200mM pH 8.5. The solid consists of 1mg of TbVSPl carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions 3+ (10 mg) is added to the reaction mixture. The formation of phosphate is monitored by determination of phosphomolybdate / malachite green in visible spectroscopy at 630 nm.

[0347] For w-TA, the reaction studied is the transamination of pyruvate in the presence of racemic α-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP). The protein charge used is 10% by mass. The reaction mixture consists of pyruvate (25mM), racemic α-methylbenzylamine (25mM) and PLP (0.1mM) and DMSO (1% wt) in solution of 50mM NaCl 300mM pH 7.5 Phosphate buffer. The solid consists of 1mg of w-TA carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ (10 mg) is added to the reaction mixture. The formation of acetophenone is continuously monitored by UV spectroscopy at 245 nm.

[0348] For GsOYE, the reaction studied is the reduction of cyclohexenone to cyclohexanone in the presence of the cofactor NADH which oxidizes to NAD+. The protein charge used is 10% by mass. The reaction mixture consists of cyclohexenone (50mM), NADH (75mM) and DMSO (1% wt) in solution in Tris-HCl 50mM NaCl 150mM pH 7.5 buffer. The solid consists of 1mg of GsOYE carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ (10 mg) is added to the reaction mixture. The disappearance of NADH is monitored by UV spectroscopy at 340 nm. Step 1: The solid consists of a protein carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions 3+ is added to the reaction mixture.

[0349] Step 2: After 10 minutes of reaction at 25°C for TbVSPl and w-TA or after 1 hour of reaction at 30°C for GsOYE, the suspension is centrifuged. The supernatant and the pellet are separated.

[0350] Step 3: The solid thus obtained is washed twice with a buffered aqueous solution: 1) Buffer is added (lmL). 2) The suspension thus obtained is stirred for a few minutes (usually 5 minutes) at room temperature. 3) The suspension is centrifuged. 4) After centrifugation, the supernatant is removed. Steps 1 to 4 are repeated once.

[0351] Steps 1, 2 and 3 are repeated successively 4 times, which corresponds to 5 cycles of use of the material consisting of a protein carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ .

[0352] The results obtained are presented in Table 6.

[0353] Table 6: Catalytic performances (Turnover numbers, TON and Conversion rate) of the 3 immobilized enzymes studied (Trypanosoma brucei pyrophosphatase (TbVSPl, EC3 Hydrolase), B9L0N2 transaminase (w-TA, EC2 Transferase) and G. sulphuraria Old Yellow Enzyme (GsOYE, EC1 Oxydoreductase) over 5 reaction cycles.

[0354] These results demonstrate that it is possible to use proteins carrying a polyhistidine sequence immobilized on a hydrogel complexed with Fe ions. 3+ at least 5 times in a row without significant loss of activity.

[0355] Example 6: Leaching For each reaction cycle, the three supernatants from steps 2 and 3 of the reuse protocol are kept (i.e. the reaction mixture after 10 min or 1 h of reaction and the buffer solutions used for the two washes of the solid). The quantity of proteins contained in these solutions is measured using the Bradford method. The mass of protein contained in the solutions is thus obtained and related to the initial quantity of protein immobilized on the solid to obtain the leaching rate. The leaching rate for each cycle represents the quantity of proteins contained in the supernatant and in the successive washes compared to the total quantity of protein immobilized on the material. This rate can be expressed as a %.

[0356] The results are summarized in Table 7.

[0357] Table 7: Leaching rate measured at each cycle of use of the 3 immobilized enzymes studied (Trypanosoma brucei pyrophosphatase (TbSVPl, EC3 Hydrolase), B9L0N2 transaminase (w-TA, EC2 Transferase) and G. sulphuraria Old Yellow Enzyme (GsOYE, EC1 Oxidoreductase) over 5 reaction cycles.

[0358] The observed leaching rates are very low. This means that the proteins adhere strongly to the surface of the materials studied. This observation is consistent with the strong affinity between the polyhistidine tag and the immobilized metal.

[0359] The observed leaching rates are very low: much lower than for other protein immobilization methods such as adsorption or encapsulation and of the same order of magnitude as for methods where a covalent bond to the support is formed via the use of a chemical grafting agent. However, we do not need the latter since the bond between the immobilized protein and the support is obtained thanks to the very strong affinity between the immobilized metal and the protein carrying a polyhistidine tag. The metal itself is in very strong interaction with the support, notably via chemical functions on its surface (carboxylic acid, phenol, catechol in particular).

[0360] FLOW EXPERIMENTS (with and without recirculation; without recirculation = continuous flow)

[0361] • Recirculating flow using the B9L0N2 enzyme immobilized on Fe-XCat

[0362] The reaction studied for the B9L0N2 enzyme is the transamination of pyruvate in the presence of racemic α-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP). The reaction mixture consists of pyruvate (50 mM), racemic α-methylbenzylamine (50 mM), PLP (0.1 mM) and DMSO (1% wt) in solution in 50 mM NaCl 300 mM Phosphate buffer pH=7.5.

[0363] The reaction mixture (10 mL) is introduced into the column prepared as previously described in the “immobilization” section. The flow is recirculated, the solution recovered at the column outlet is directly reinjected into the column at a flow rate of 0.1 mL / min. A sample (100 pL) is taken every 10 min for 1 h. The formation of acetophenone is monitored by UV spectrophotometry at 245 nm.

[0364] The enzymatic activity measured at time t for immobilized B9L0N2 is expressed in moles of acetophenone formed per mole of enzyme. To do this, the quantity of acetophenone is measured in each sample.

[0365] Conversion rates are expressed in moles of product formed (acetophenone) per mole of substrate (a-methylbenzylamine) initially present. 100 • Continuous flow using B9L0N2 enzyme immobilized on Fe-XCat

[0366] The reaction studied for the enzyme B9L0N2 is the transamination of pyruvate in the presence of racemic α-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP). The reaction mixture consists of pyruvate (25 mM), racemic α-methylbenzylamine (25 mM), PLP (0.1 mM) and DMSO (1% wt) in solution in 50 mM NaCl 300 mM Phosphate buffer pH=7.5.

[0367] The reaction mixture is introduced into the column prepared as previously described in the “immobilization” section. The flow is continuous with a flow rate of 0.1 mL / min, the solution at the column outlet is recovered in the form of a 1 mL fraction for 5 hours. The formation of acetophenone is monitored by UV spectrophotometry at 245 nm.

[0368] The enzymatic activity measured at time t for immobilized B9L0N2 is expressed in moles of acetophenone formed per mole of enzyme. To do this, the quantity of acetophenone is measured in each sample.

[0369] Conversion rates are expressed in moles of product formed (acetophenone) per mole of substrate (a-methylbenzylamine) initially present. 100

[0370] • Recirculating flow using GsOYE enzyme immobilized on Fe-XCat

[0371] The reaction studied is the reduction of cyclohexenone to cyclohexanone in the presence of the cofactor NADH which oxidizes to NAD+. The reaction mixture consists of cyclohexenone (50 mM), NADH (50 mM) and DMSO (1% wt) in solution in Tris-HCl 50 mM NaCl 150 mM buffer pH=7.5.

[0372] The reaction mixture (10 mL) is introduced into the column prepared as previously described in the “immobilization” section. The flow is recirculated, the solution recovered at the column outlet is directly reinjected into the column at a flow rate of 1 mL / min. A sample (100 pL) is taken every 10 min for 2 h (Day 1), then the column is stored overnight at 4°C and a new reaction mixture (10 mL) is prepared to be reintroduced into the column (Day 2). The disappearance of NADH is monitored by UV spectrophotometry at 340 nm.

[0373] The enzymatic activity measured for immobilized GsOYE is expressed in moles of NADH consumed per mole of GsOYE enzyme. To do this, the quantity of residual NADH is measured in each sample.

[0374] Conversion rates are expressed in moles of cofactor (NADH) consumed per moles of initial cofactor (NADH).

[0375] Results :

[0376] Table 8: Flow performance with recirculation using B9L0N2 enzyme immobilized on Fe-XCat Table 9: Performance in non-recirculating flow (continuous flow) using the enzyme

[0377] B9L0N2 immobilized on Fe-XCat Table 10: Performance in recirculating flow using the GsOYE enzyme immobilized on Fe-XCat The experimental results with B9L0N2 in recirculating flow, in continuous flow (without recirculation) and with GsOYE in recirculating flow demonstrate the possibility of using the Fe-XCat material to immobilize different enzymes and use them in flow. The low leaching rates (<5%) indicate that the enzymes remain largely in interaction with the immobilization support material.

Claims

Claims 1. Composition comprising: • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from: -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide, -at least one compound having an orthodiphenol or orthomethoxyphenol function, and • At least one metal cation, said porous carbon material has at least one orthodiphenol function.

2. Composition according to claim 1 in which -the dare is: • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose, preferably xylose, or • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or -the polysaccharide is: • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or • a homodiholoside chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose, preferably maltose or sucrose 3. Composition according to claim 1 in which the compound having an orthodiphenol or orthomethoxyphenol function is chosen from: catechol, pyrogallol, caffeic acid, mangiferin, quercetin, cyanidin, catechin, epicatechin, epigallocatechin, anthocyanidol, procyanidol B-3, procyanidol B-4, fuhalols (bifuhahol, trifuhahol), carmalol (diphlorethohydroxycamalol) and tannic acid, or a natural plant extract containing at least one of these compounds.

4. Composition according to claim 3, in which the compound having an orthodiphenol function is catechin.

5. Composition according to claim 1, in which the porous carbon material is formed from: -maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and - catechin, or a catechu extract.

6. Composition according to claim 1, in which the porous carbon material has a molar ratio between the at least one carbohydrate, preferably at least one ose, or at least one polysaccharide and the at least one compound having at least one orthodiphenol or orthomethoxyphenol function of 1 / 2 or 1 / 1 or 2 / 1 or 3 / 1 or 4 / 1 or 5 / 1.

7. Composition according to claim 1, in which the at least one metal cation is chosen from Al 3+ , Ag + , Ba 2+ , That 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , N / A + , Neither 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

8. Composition according to one of claims 1 to 7, further comprising at least one immobilized protein, said at least one immobilized protein preferably comprising a polyhistidine tag and said at least one immobilized protein is preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), a pyrophosphatase (EC3.6.1.1).

9. Composition according to one of claims 1 to 8, in which the quantity of protein adsorbed on the porous carbon material is 10 to 80 parts by weight relative to 100 parts by weight of the porous carbon material, more preferably 10 to 50 parts by weight relative to 100 parts by weight of the porous carbon material, more preferably 25 to 50 parts by weight relative to 100 parts by weight of the porous carbon material.

10. Composition according to claim 8 or 9, wherein the specific activity of the enzyme immobilized on the porous carbon material is greater than that of the same free enzyme.

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