Novel porous carbon-based material comprising an immobilized protein, method for preparing same and uses thereof

Abstract

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

Description

[0001] Novel porous carbon material comprising immobilized protein, its preparation method and uses

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a carbonaceous polymeric material also called porous carbonaceous material or carbon-rich aerogel, comprising a protein immobilized on said material and methods of preparing the same.

[0004] The invention further relates to the use of the composition comprising an immobilized protein in heterogeneous biocatalysis applications in particular.

[0005] CONTEXT OF THE INVENTION

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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. 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.

[0010] 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.

[0011] 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.

[0012] 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. Therefore, there is 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.

[0013] BRIEF OVERVIEW OF THE INVENTION

[0014] 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 comprising a protein immobilized on said material. A second aim of the invention is to propose methods for preparing said materials. Finally, a final aim of the invention is to propose particular compositions and their uses.

[0015] DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the invention, the present application relates to a composition comprising:

[0016] • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0017] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide, -at least one phenolic or polyphenolic compound,

[0018] • At least one immobilized protein, 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)

[0019] • Optionally, at least one metal cation.

[0020] 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.

[0021] Carbohydrates are defined by the International Union of Pure and Applied Chemistry (IUP AC) 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.

[0022] A monosaccharide (or sugar) is a carbohydrate monomer. Monosaccharides have at least 3 carbon atoms: they are polyhydroxyaldehydes or polyhydroxyketones.

[0023] Monosaccharides are distinguished by the length of their carbon chain, as follows:

[0024] • trioses: 3-carbon oses, CsEfcCL (glyceraldehyde, dihydroxyacetone);

[0025] • tetroses: 4-carbon oses, C4H8O4 (erythrose, threose, erythrulose);

[0026] • pentoses: 5-carbon oses, C5H10O5 (deoxyribose (C5HI0O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose)

[0027] • hexoses: 6-carbon oses, CôHnOô (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose);

[0028] • deoxyhexoses: 6-carbon oses, CôHnOs (fucose, rhamnose); • heptoses: 7-carbon oses, C7H14O7 (sedoheptulose, mannoheptulose)

[0029] • octoses: 8-carbon oses, CSHIÔOS (heptahydroxyoctanal);

[0030] • nonoses: 9-carbon oses, C9H17N1O8 (neuraminic acid or sialic acid).

[0031] 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.

[0032] A ketose is a monosaccharide consisting of a chain of n carbon atoms with a carbonyl group on the non-terminal carbon atom, making it a ketone, and hydroxyl groups attached to all other carbon atoms.

[0033] According to a preferred embodiment of the invention, the at least one ose is chosen from:

[0034] • -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

[0035] • -a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose

[0036] A polysaccharide is a polymer of the carbohydrate family formed from a number n of oses (n > 2) by alpha or beta glycosidic bond.

[0037] According to a preferred embodiment of the invention, the at least one polysaccharide is chosen from:

[0038] • -a heterodiholoside (a polysaccharide for which n=2 consisting of 2 different osidic units) chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or • -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.

[0039] 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.

[0040] 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.

[0041] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a biosourced precursor compound having at least one benzene nucleus substituted by a hydroxyl group and optionally at least one other functional group chosen from carboxylic acid, aldehyde, ether, ester, alcohol, hydroxyl and ketone groups.

[0042] 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.

[0043] 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.

[0044] The biosourced precursor compound #2 that can be used according to the present invention may be a phenol whose benzene nucleus is further substituted by at least one other functional group chosen from carboxylic acid groups (in particular the carboxy group COOH), aldehyde (in particular the formyl group CHO), ether (in particular the methoxy group CH3O), ester, alcohol (in particular the group CH2OH), hydroxyl and ketone, preferably hydroxyl, carboxylic acid and aldehyde groups. Any salts of these groups also fall within the scope of the invention.

[0045] The biosourced precursor phenolic or polyphenolic compound 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 benzoic nucleus 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.

[0046] Phenolic compounds include simple molecules, such as phenolic acids, or highly polymerized compounds, such as tannins and lignins. Phenolic compounds can be classified according to the complexity, degree and possible bonds of the basic skeleton with other molecules. Polyphenols are very diverse molecules, consisting of one or more benzene rings carrying one or more hydroxyl functions. They can be grouped into numerous classes according to the complexity of the basic skeleton (C6 ring), the degree of modification of this skeleton (oxidation, hydroxylation, . . . .) and finally according to the molecules with which they are associated (carbohydrates, lipids, proteins, other metabolites). The simplest forms are represented by two main groups from which many compounds are derived: hydroxycinnamic acids and flavonoids.Complex forms, on the other hand, are mostly produced by the condensation of certain simple forms and contain, among other things, tannins and lignins.

[0047] Polyphenols are divided into different groups, defined according to the structure of their carbon skeleton. Phenolic acids (C6-C1 and C6-C3) and especially flavonoids (C6-C8-C10) are the most frequently found in the plant kingdom. Lignans (C6-C3-C3-C6) are less common. The last group is that of stilbenes (C6-C2-C6); the best known is resveratrol, present in grape skin. Flavonoids represent the largest group of polyphenols and the most widely distributed in the plant kingdom. More than 4000 different flavonoids have been identified. They differ in the degree of oxidation of the oxygenated heterocyclic ring. Table 1 below summarizes the main classes of phenolic compounds. [Table 1] Condensed tannins, also called proanthocyanidins, constitute a particularly interesting source, because they are abundant, of such phenolic compounds likely to constitute starting products for the formation of epoxide prepolymers. Indeed, condensed tannins are phenolic biopolymers essentially present in soft tissues with rapid growth and renewal, such as leaves, stems, etc. This class of polyphenols is the most abundant after lignins. Unlike lignins which are constituents of lignocelluloses, structural elements of the secondary walls of plant cells, condensed tannins are stored in the vacuoles of cells in the form of organelles, and are therefore easily extractable.These compounds are found in particular in many available and varied natural resources such as agro-industrial residues, for example in fruit pomace, and unexploited biomass, in particular in the bark, leaves and needles of trees, vines, fruits, etc.

[0048] Condensed tannins are non-hydrolyzable compounds that can be depolymerized into anthocyanidols when treated with heat and acid. They are usually named after the anthocyanidol thus released. For example, the monomer (+)-catechol (a flavan-3-ol) is a constituent unit of the dimer catechol -(4a^8)-catechol, which will be called procyanidol B-3 because of its degradation in acidic conditions to cyanidol. Many condensed tannins are polymers of catechol. This is why condensed tannins are also called "proanthocyanidol" or "proanthocyanidins" (after the English proanthocyanidins). The OH hydroxyls are in the same positions on the monomer and the depolymerization product; only the central ring changes.

[0049] There are about a dozen flavanol monomer units used in the construction of condensed tannins. They can also be substituted by gallic acid or sugars, usually in position 3 and sometimes in positions 5 and 7.

[0050] Flavanols and anthocyanidols all have three rings: an A ring with usually one or two OH hydroxyls, a C heterocycle with asymmetric carbons 2 and 3 (and a hydroxyl at 3), and a B ring with one OH hydroxyl (afzelechol, epiafzelechol), two OH (catechol and epicatechol), or three OH (gallocatechol, epigallocatechol). Compounds with the cis- (2R,3R) configuration have a name prefixed with epi-, the others are in the traw - (2R,3S) configuration.

[0051] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a simple phenol chosen from phenol, catechol, resorcinol, hydroquinone, pyrogallol, or phloroglucinol.

[0052] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound from the flavonoid family.

[0053] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound chosen from the subclasses: flavonol, anthocyanin, flavanol, flavanone, isoflavonoid. According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound of the subfamily of flavanols or catechins, the structure of which is based on 2-phenyl-3-chromanol. The oligomeric and polymeric structures of flavanols constitute the class of proanthocyanidols or condensed tannins.

[0054] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is chosen from: catechin in one of its stereoisomeric forms, gallocatechin, afzelechol, fisetinidol, guibourtinidol, mesquitol, or robinetinidol.

[0055] According to a preferred embodiment of the invention, the at least one phenolic compound is phloroglucinol.

[0056] According to a preferred embodiment of the invention, the at least one polyphenolic compound is catechin.

[0057] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is contained in a natural plant extract.

[0058] According to a preferred embodiment of the invention, the natural plant extract containing at least one phenolic or polyphenolic compound is chosen from an extract of strawberry, potato, apple, lemon, walnut, vine, grape, flowers, soy, pea, pine, tomato, garlic or catechu.

[0059] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is

[0060] • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,

[0061] • a phenolic compound from the flavonoid family, notably chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,

[0062] • a condensed tannin

[0063] • a phlorotannin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol

[0064] • or a natural plant extract containing at least one of the above compounds.

[0065] According to another embodiment of the invention, the porous carbon material comprises two biosourced precursor compounds #2 which consist of two phenolic or polyphenolic compounds as described above. According to another embodiment of the invention, the porous carbon material comprises more than two biosourced precursor compounds #2.

[0066] According to a preferred embodiment of the invention, the porous carbon material obtained by hydrothermal carbonization comprises or is formed from:

[0067] • maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, or dihydroxyacetone, and

[0068] • phloroglucinol, catechin, or catechu extract.

[0069] Cachou extract can be obtained from plant fibers of Acacia cachou, said extract containing condensed tannins and flavonols.

[0070] 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. hydrothermal carbonization) from at least two biosourced precursors # 1 and 2 as defined in the present application.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] According to a preferred embodiment of the invention, the porous carbon material is obtained by hydrothermal carbonization.

[0075] 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).

[0076] 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 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.

[0077] The aqueous solution comprises water, and optionally solvents, preferably in amounts miscible with water.

[0078] According to a preferred embodiment, the reaction mixture comprises only one liquid phase, namely the aqueous solution, preferably water.

[0079] For example and without limitation, the aqueous solution comprises ethanol, preferably absolute ethanol.

[0080] According to a preferred embodiment, the mass fraction of water in an absolute water / ethanol mixture is between 1 and 0.3.

[0081] According to a preferred embodiment, the mass fraction of water in an absolute water / ethanol mixture is between 0.7 and 0.4 when the biosourced precursors #1 and 2 are respectively xylose and phloroglucinol.

[0082] 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.

[0083] Step (a) of the process may also be referred to as the “hydrothermal treatment step”.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] There is no specific limitation on the duration of step (a) in the process for preparing the porous carbon material of the invention.

[0088] 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.

[0089] 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.

[0090] In a particular embodiment, the porous carbon material is prepared by the following method: ajHeating a reaction mixture comprising:

[0091] - an aqueous solution consisting of water and absolute ethanol with a mass fraction of water of 0.7 to 0.4, and,

[0092] -xylose and phloroglucinol 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.

[0093] In a particular embodiment, the porous carbon material is prepared by the following method: ajHeating 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 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. 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 phenolic or polyphenolic compound (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 phenolic and / or polyphenolic compounds, i.e. the number of moles of monomeric units (e.g. flavonoids), in said condensed tannin or said complex natural extract.

[0096] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0097] - an aqueous solution consisting of water and absolute ethanol with a mass fraction of water of 0.7 to 0.4, and,

[0098] -xylose and phloroglucinol 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.

[0099] In a particular embodiment, the porous carbon material is prepared by the following method: a) Heating a reaction mixture comprising:

[0100] - an aqueous solution consisting of water and absolute ethanol with a mass fraction of water of 1 to 0.5, and,

[0101] -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)Washing the solid porous carbon material obtained by successive soaking in a solvent, preferably a polar solvent.

[0102] According to a preferred embodiment, the porous carbon material consists of

[0103] • 40-70% by mass in carbon element (C)

[0104] • 30-40% by mass in oxygen element (O) preferably 60% by mass in carbon element (C) and 35% by mass in oxygen element (O).

[0105] According to a preferred embodiment, the porous carbon material consists of 36% O and 60% C for the xylose / phloroglucinol system.

[0106] 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 polyoside (cl) and at least one phenolic (c2) or polyphenolic compound in a molar ratio (cl / c2) of from 10 / 1 to 1 / 10, preferably 2 / 1.

[0107] The solid porous carbon material is washed by soaking either in ultrapure or deionized water or in a water / polar protic solvent mixture, e.g., methanol, ethanol, or tert-butanol, for several hours.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] Therefore, a hydrogel is considered here as a special type of solvogel, in which the aqueous solution of the dispersion phase is water.

[0112] The solid porous carbon material obtained or obtainable according to the process described above can be dried and transformed into cryogel, aerogel or xerogel.

[0113] 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.

[0114] 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.

[0115] In 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. Furthermore, a cryogel can be called an aerogel when the dried gel has largely retained the textural properties after drying.

[0116] Dried gels can be called aerogels regardless of the drying technique used.

[0117] Depending on the gel drying process, it is possible to control the pore size and structure of the porous carbon materials obtained according to the invention.

[0118] 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 biosourced precursors # 1 and 2 as defined above 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 (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.

[0119] 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.

[0120] 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).

[0121] Texture of the porous carbon material 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.

[0122] 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).

[0123] 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).

[0124] The surface of the carbon material of the invention as obtained in step (a) is very hydrophilic.

[0125] Textural properties of porous carbon material

[0126] Pore ​​diameter

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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. 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.

[0131] Specific surface area

[0132] 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.

[0133] 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.

[0134] Porous volume

[0135] 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.

[0136] 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 phloroglucinol / xylose aerogels.

[0137] 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.

[0138] Alternatively, the specific surface area can be determined by the Brunauer Emmett Teller (BET) method, for example by the nitrogen adsorption technique.

[0139] 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).

[0140] Interconnected porosity

[0141] This parameter can be determined by mercury intrusion porosimetry indicating the total volume of interconnected pores.

[0142] Ion complexation

[0143] The porous carbon materials as described above may be 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.

[0144] According to a particular 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+ .

[0145] 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.

[0146] According to the present invention, the term "hybrid material" refers to the porous carbon material on which the metal ions are complexed. This is a composition within the meaning of the present invention.

[0147] 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.

[0148] The adsorption process to enable the metal complex to adsorb on a carbon material, and the adsorption conditions are not particularly limited.

[0149] For example, a metal complex can be optionally adsorbed onto a carbonaceous material by placing the porous carbonaceous material in a solution and mixing the solution for a 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.

[0150] 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.

[0151] 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.

[0152] 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%.

[0153] In a particular embodiment, the composition optionally comprises an amount of 1 to 5% by mass of metal cation, in particular 2 to 4% by mass.

[0154] In a particular embodiment, the composition comprises an amount of 20 to 40 mg of Iron per gram of porous carbon material.

[0155] The solvent used herein is not particularly limited as long as it can dissolve and / or disperse metal complexes. Examples of these include: acetic acid, water, ethylene glycol, DMSO, and DMF.

[0156] The concentration of metal complex in the solution is not particularly limited. However, the concentration is preferably about 0.1 to 30 mM.

[0157] According to a particular embodiment, the composition (solid porous carbon hybrid material) comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0158] • -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0159] • -at least one phenolic or polyphenolic compound (biosourced precursor #2), and b) optionally 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+ .

[0160] According to a particular embodiment, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0161] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which

[0162] -the dare is chosen from

[0163] • 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

[0164] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or

[0165] -the polysaccharide is chosen from:

[0166] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0167] • 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 and

[0168] -at least one phenolic or polyphenolic compound (biosourced precursor #2). b) optionally 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+ .

[0169] According to another particular embodiment, the composition comprises: A porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0170] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which

[0171] -the dare is chosen from

[0172] • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, preferably xylose, or

[0173] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, tagatose, preferably fructose, or

[0174] -the polysaccharide is chosen from:

[0175] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, rutinose, or

[0176] • a homodiholoside chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobiose, preferably maltose or sucrose,

[0177] -at least one phenolic or polyphenolic compound chosen from:

[0178] • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,

[0179] • a phenolic compound from the flavonoid family, notably chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,

[0180] • a condensed tannin

[0181] • a phlorotannin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol

[0182] • or a natural plant extract containing at least one of the above compounds. and b) optionally 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+ .

[0183] According to another particular mode, the composition comprises:

[0184] A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0185] -xylose, or dihydroxyacetone,

[0186] - phloroglucinol or catechin. b) optionally at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0187] According to another particular mode, the composition comprises:

[0188] A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0189] -xylose,

[0190] -phloroglucinol or catechin. b) optionally at least one metal cation which is Fe 3+

[0191] According to another particular mode, the composition comprises:

[0192] A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0193] -xylose,

[0194] -phloroglucinol or catechin, and b) optionally at least one metal cation which is Co 2+

[0195] According to another particular mode, the composition comprises:

[0196] A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0197] -dihydroxyacetone,

[0198] -phloroglucinol or catechin, and b) optionally at least one metal cation which is Fe 3+ According to the present invention, all the compositions described above can be used to immobilize proteins, in particular at least one enzyme

[0199] Immobilization of a protein

[0200] A second aim 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 optionally charged with at least one metal cation), wherein the at least one immobilized protein is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and / or coordination bonds and / or ionic bonds.

[0201] The protein used here is not particularly limited.

[0202] 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:

[0203] EC 1 Oxido-reductases, EC 2 Transferases, EC 3 Hydrolases, EC 4 Lyases, EC 5 Isomerases, EC 6 Ligases and EC 7 Translocases.

[0204] Among these proteins, at least one protein selected from the group consisting in particular of α-acetolactate, 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, β-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, phenol oxidase, phenylalanine dehydrogenase, phytase, polyethylesterase, polygalacturonase, protease, protopectinase, pullulanase, pyrophosphatase, pyruvate transaminase, raffinose synthase, rennet, sacrosidase, serratiopeptidase, sphingosine kinase, stachyose synthase, tannase, taxolase, thermolysin, transaminase, transglutimases, trypsin, urease, xylanase, xylose isomerase.

[0205] 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).

[0206] Protein fillers

[0207] 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.

[0208] In addition, the method for providing the porous carbon hybrid material of the present invention by causing a protein to adsorb onto the porous carbon material is not particularly limited. It is possible to use, for example, the sublimation method and 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 contacted 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.

[0209] In a particular embodiment, the composition comprising at least one immobilized protein is prepared by the method comprising the steps of: - Suspending the dried porous carbon material in a solution which may contain metal cations,

[0210] - Optionally wash the porous carbon hybrid material when loaded with metal cations

[0211] - Immobilizing a protein (enzyme) on the porous carbon hybrid material optionally loaded with metal cations by reacting the porous carbon material optionally loaded with metal cations with said protein;

[0212] -wash the suspension and recover the protein immobilized on the porous carbon hybrid material optionally loaded with metal cations.

[0213] More specifically, the porous carbon hybrid material optionally loaded with metal cations is added to an aqueous solution containing a protein, preferably an enzyme.

[0214] The porous carbon hybrid material optionally charged 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.

[0215] The protein to be immobilized on the material can be any protein, such as a (recombinant) protein or an enzyme.

[0216] Preferably, the protein is an enzyme.

[0217] The buffered aqueous solution is preferably a Tris-HCl, or Phosphate buffer.

[0218] According to a second 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:

[0219] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0220] -at least one phenolic or polyphenolic compound (biosourced precursor #2), b) optionally at least one metal cation, and c) at least one protein, preferably an enzyme.

[0221] According to a preferred embodiment of the invention, the composition comprises a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0222] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide (biosourced precursor #1), and

[0223] -at least one phenolic or polyphenolic compound (biosourced precursor #2), b) optionally at least one metal cation, and c) at least one immobilized protein, preferably an enzyme.

[0224] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0225] -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which the ose is chosen from:

[0226] • 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

[0227] • 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:

[0228] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0229] • 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, and

[0230] -at least one phenolic or polyphenolic compound chosen from:

[0231] • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol, • a phenolic compound from the flavonoid family chosen in particular from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,

[0232] • a condensed tannin

[0233] • a phlorotannin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol, or

[0234] • a natural plant extract containing at least one of the above compounds, b) optionally 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.

[0235] According to another preferred embodiment of the invention, the composition comprises: a) A porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0236] -xylose, or dihydroxyacetone,

[0237] - phloroglucinol or catechin, and b) optionally 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.

[0238] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0239] -xylose,

[0240] -phloroglucinol or catechin, and b) optionally at least one metal cation which is Fe 3+ , and c) at least one immobilized protein. According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0241] -xylose,

[0242] -phloroglucinol or catechin, and b) optionally at least one metal cation which is Co 2+ , and c) at least one immobilized protein.

[0243] According to another preferred embodiment of the invention, the composition comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:

[0244] -dihydroxyacetone,

[0245] -phloroglucinol or catechin, and b) optionally at least one metal cation which is Fe 3+, and c) at least one immobilized protein.

[0246] For example, if the solid material according to the invention is used for the purification and isolation of a protein, the binding of the enzyme to the support must be reversible.

[0247] For the use of immobilized enzymes in heterogeneous biocatalysis, strong binding of the enzyme to the support is desirable.

[0248] If the proteins immobilized on the support, as described above, are enzymes, they contain an active site capable of catalyzing a chemical reaction.

[0249] Thus, the immobilized enzyme material is potentially useful as a biocatalyst in organic synthesis.

[0250] 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.

[0251] A preferred embodiment of the invention relates to the use of the compositions described above as heterogeneous biocatalysts.

[0252] 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.

[0253] Immobilized enzymes were found to tolerate both aqueous conditions as well as a range of organic solvents.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.

[0259] Particularly useful biocatalytic reactions are enantioselective reactions.

[0260] Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.

[0261] 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.

[0262] 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. 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.

[0263] Yield and downtime speed

[0264] 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.

[0265] The immobilization speeds expressed in minutes to reach at least an immobilization rate of at least 75% are between 10 and 120 minutes.

[0266] 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.

[0267] 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.

[0268] According to another aspect of the invention, the present application relates to the use of the composition as a heterogeneous biocatalyst.

[0269] According to a final aspect of the invention, the present application relates to:

[0270] A composition comprising:

[0271] • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0272] -at least one carbohydrate, preferably at least one sugar, or at least one polysaccharide,

[0273] -at least one phenolic or polyphenolic compound,

[0274] • At least one protein, preferably an immobilized enzyme, 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)

[0275] • Optionally, at least one metal cation. A composition in which

[0276] -the dare is:

[0277] • 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

[0278] • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or

[0279] -the polysaccharide is:

[0280] • a heterodiholoside chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or

[0281] • 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

[0282] A composition in which the phenolic or polyphenolic compound is:

[0283] • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,

[0284] • a phenolic compound from the flavonoid family, notably chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,

[0285] • a condensed tannin,

[0286] • a phi orotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol

[0287] • or a natural plant extract containing at least one of these compounds. A composition in which the phenolic or polyphenolic compound is a compound from the flavanol or catechin subfamily.

[0288] A composition in which the phenolic or polyphenolic compound is chosen in particular from: catechin or phloroglucinol.

[0289] A composition wherein the porous carbonaceous material is obtained by hydrothermal carbonization comprising or formed from:

[0290] -maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and

[0291] -phloroglucinol, catechin, from a catechu extract.

[0292] A composition in which the at least one metal cation when present is selected 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+ .

[0293] A composition comprising: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:

[0294] -a sugar chosen from xylose or dihydroxyacetone

[0295] -a phenolic or polyphenolic compound chosen from phloroglucinol or catechin, and b) at least one immobilized protein, 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), and c) optionally, at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Neither 2+ .

[0296] A composition in which 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, even more preferably 10 to 50 parts by weight relative to 100 parts by weight of the porous carbon material. A composition in which the specific activity of the enzyme immobilized on the porous carbon material is higher than that of the same enzyme free.

[0297] List of figures

[0298] 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.

[0299] 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.

[0300] Figures 1 and 2 show primary particles of a few nanometers, aggregated primary particles forming an interconnected network which induces interparticle interstices of a few nanometers to several micrometers.

[0301] Experimental part

[0302] Example 1: Preparation of a porous carbon material in the form of an aerogel

[0303] 0.85 g of xylose is dissolved in 10 mL of a mixture of deionized water and absolute ethanol 50 / 50 m / m.

[0304] 0.35 g of phloroglucinol is added to the previous solution.

[0305] 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,

[0306] 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).

[0307] 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,

[0308] 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.

[0309] Example 2: Immobilization of a protein

[0310] 10 mg of aerogel obtained under the conditions described above are added to an aqueous solution containing (1 mg) of protein.

[0311] The following enzyme was prepared: B9L0N2 transaminase (w-TA, EC2 transferase).

[0312] 25 mg of aerogel obtained under the conditions described above are added to a buffer solution (Tris 20 mM pH=7, 1 mL) containing 2.5 mg of CalB enzyme (Candida antartica lipase B).

[0313] The suspension thus obtained is stirred at 750rpm for a few hours (generally 2 hours) at 4°C.

[0314] After stirring, the suspension is centrifuged (between 4000 and 6000 rpm for 2 to 5 min). After centrifugation, the supernatant is removed. The solid thus obtained is washed with a buffered aqueous solution twice:

[0315] 1) Buffer is added (1 mL),

[0316] 2) The suspension thus obtained is stirred for a few minutes (usually 5 minutes) at room temperature,

[0317] 3) The suspension is centrifuged (between 4000 and 6000 rpm for 2 to 5 min),

[0318] 4) After centrifugation, the supernatant is removed.

[0319] Steps 1 to 4 are repeated once.

[0320] This produces a protein immobilized on the porous carbon material.

[0321] Example 3 Immobilization kinetics 10 mg of porous carbon material are placed in the presence of 1 mg of enzyme in a total volume of 1 mL.

[0322] Immobilization of B9L0N2 transaminase (w-TA, EC2 transferase) occurs in the presence of the cofactor pyridoxal phosphate (PLP).

[0323] The 0, 10, 20 and 30 min samples (10 pL) are diluted halfway and the 1 h and 2 h samples (20 pL) are not diluted.

[0324] 20 pL of sample (Echl at 2) mixed with 1 mL of Bradford reagent, in the dark for 15 min.

[0325] Results

[0326] Immobilization kinetics of B9L0N2 (Img) on ​​a Xylose / Catechin aerogel (10mg) prepared under the conditions described above.

[0327] The results in Table 2 above show that an immobilization rate of at least 80% is achieved on average after 10 minutes.

[0328] These results show that immobilizing an enzyme on the porous carbon material is simple, rapid and efficient.

[0329] Immobilization kinetics of CalB (2.5 mg) on ​​different aerogels prepared under the conditions described above.

[0330] The results in Table 3 above show that an immobilization rate of at least 80% is achieved on average after 10 minutes for XPh supports.

[0331] These results show that in the case of CalB, the XPh material allows a relatively better immobilization (99.47% in 120 min) than the XCat material (89.825% in 120 min) although the 2 performances are still quite close. This difference could be explained by a more marked hydrophobic character on the surface of the XPh material than of the XCat material. Indeed, it has been demonstrated in the literature that the CalB lipase has a stronger affinity for immobilization materials whose surfaces are relatively hydrophobic.

[0332] The materials are ground manually in a mortar and then sieved by passing through a first sieve of 300 microns and a second of 100 microns to retain only the fraction between 100 and 300 microns.

[0333] Example 4: Performance

[0334] Conversion rates are expressed in moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time.

[0335] 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 loading 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 consisting of 1mg of w-TA immobilized on a hydrogel (10mg) is added to the reaction mixture. The formation of acetophenone is continuously monitored by UV spectroscopy at 245nm. The results are summarized in Table 4.

[0336] Table 4: Catalytic performances (Turnover numbers, TON of the free or immobilized enzymes studied (B9L0N2 / w-TA).

[0337] The TON of the immobilized enzyme is significantly higher than that of the non-immobilized enzyme.

[0338] 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.

[0339] The interconnected macroporous structure (pores between 50 nm and 2 microns) therefore facilitates the mass transfer of reactants and products throughout the material.

[0340] For CalB, the reaction studied is the hydrolysis of / / ra-nitrophenyl butyrate (pNPB) into para-nitrophenol and butanoic acid. The reaction mixture consists of pNPB (15 mM) dissolved in 20 mM Tris-HCl buffer pH=7.0 containing Genapol X-100 (1% by mass). The reaction is started by adding immobilized CalB (25 mg of aerogel loaded with 10% by mass of enzyme). The tests were carried out at 25°C for 1 h with stirring at 800 rpm. The formation of / / ra-nitrophenol is monitored by visible spectrophotometry at 410 nm. For immobilized enzymes, the activity is measured over 5 reaction cycles. A reaction cycle proceeds as follows:

[0341] 1) The enzymatic reaction is carried out as described above.

[0342] 2) At the end of the reaction the suspension is centrifuged for 2 to 5 min between 4000 and 6000 rpm.

[0343] 3) The supernatant is collected and 1 mL of buffer solution (Tris-HCl at 20 mM pH=7) is added to the solid.

[0344] 4) The new suspension is stirred for 3 min at 800 rpm.

[0345] 5) Steps 2 to 4 are repeated three times.

[0346] The enzymatic activity measured for immobilized CalB is expressed in moles of p-nitrophenol formed per mass of catalyst (enzyme immobilized on the aerogel) during a given time. To do this, the quantity of p-nitrophenol measured in each of the supernatants recovered following the 5 previous steps is added. The formula is as follows:

[0347] These activities are measured for non-immobilized (free) enzymes and for immobilized enzymes by the technique described above.

[0348] Conversion rates are expressed in moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time. The formula is as follows:

[0349] The results are summarized in Table 5. Table 5: Conversion rates measured for CalB immobilized on XPh and on XCat

[0350] Table 6: Enzymatic activity measured for CalB immobilized on XPh and on XCat

[0351] Example 5: Leaching

[0352] For each reaction cycle, the four supernatants from steps 1 to 3 of the activity protocol are retained. The amount of protein contained in these solutions is measured by measuring the residual enzymatic activity, proceeding as follows.

[0353] A sample of each supernatant of the reaction cycles (20 pL) is added to a reaction solution containing 1.5 mM substrate (pNPB), 1% Genapol and 20 mM Tris HCl buffer pH=7. The solution is stirred for 5 min at 25°C. The quantity of / ?-nitrophenol produced during this reaction is compared to a standard range to determine the mass of protein contained in the sample. It is thus related to the initial quantity of protein immobilized on the solid to obtain the leaching rate.

[0354] Leaching rate at cycle N (%)

[0355] The cumulative leaching rate is defined as follows:

[0356] The results are summarized in Table 7.

[0357] Table 7: Leaching rate measured at each cycle of use of the immobilized enzyme over 5 reaction cycles on 2 different supports (XPh: xylose / Phloroglucinol and XCat: xylose / Cathechin)

[0358] As with the immobilization experiments, these experiments show that the leaching (per cycle and cumulative over 5 cycles) of CalB when immobilized on XPh is lower than on XCat. This could also be explained by a more marked hydrophobic character on the surface of the XPh material than of the XCat material. Indeed, it has been demonstrated in the literature that the CalB lipase has a stronger affinity for immobilization materials whose surfaces are relatively hydrophobic.

[0359] FLOW EXPERIMENTS (with and without recirculation; without recirculation = continuous flow)

[0360] Immobilization of CalB on XPh in continuous flow:

[0361] The XPh aerogels obtained under the conditions described above (65 mg) are introduced into a thermostatically controlled column whose temperature is maintained at 4°C. The end of the column is made of a frit to retain the aerogel.

[0362] A buffer solution (Tris 20 mM pH=7.5 mL) containing 0.250 g of CalB enzyme solution (Lypozyme ® STREM CHEMICALS) 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.

[0363] The amount of protein present in these two solutions is measured by residual activity by proceeding as follows.

[0364] A sample of the recovered solutions (20 pL) is added to a reaction solution containing 1.5 mM substrate (pNPB), 1% Genapol and 20 mM Tris HCl buffer pH=7. The solution is stirred for 5 min at 25°C. The quantity of p-nitrophenol produced during this reaction is compared to a standard range to determine the mass of protein contained in the sample.

[0365] This gives the CalB protein immobilized on an XPh hydrogel (with a protein load of 7.7% by mass) in a column to be used in recirculation.

[0366] • Measurement of conversion rates in flow with recirculation using the CalB enzyme immobilized on XPh

[0367] The reaction studied is the hydrolysis of para-nitrophenyl butyrate (pNPB) into para-nitrophenol and butanoic acid. The reaction mixture consists of pNPB (5 mM) dissolved in 20 mM Tris-HCl buffer pH=7.0 containing Genapol X-100 (1% by mass).

[0368] The reaction mixture (6 mL) is introduced into the column prepared as previously described in the “continuous flow 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. The column is thermostatically controlled, the temperature is maintained at 35°C. A sample (100 pL) is taken every hour for 5 hours. The formation of para-nitrophenol is monitored by visible spectrophotometry at 410 nm.

[0369] Conversion rates are expressed in moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time. The formula is: 100 • Measurement of flow leaching rates with recirculation using the CalB enzyme immobilized on XPh

[0370] A sample (20 pL) is taken from the recovered fractions as described previously in the “activity” section. The quantity of proteins contained in these samples is measured by residual activity using the following procedure.

[0371] The sample is added to a reaction solution containing 1.5 mM substrate (pNPB), 1% Genapol and 20 mM Tris HCl buffer pH=7. The solution is stirred for 5 min at 25°C. The quantity of / ?-nitrophenol produced during this reaction is compared to a standard range to determine the mass of protein contained in the sample. It is thus related to the initial quantity of protein immobilized on the solid to obtain the leaching rate.

[0372] The leaching rate is obtained by the following formula:

[0373] The results are summarized in Table 8.

[0374] These experiments show that the flow immobilization of a protein is possible on our materials and that it is rapid and efficient. The hydrophobic character seems to be more marked on the surface of XPh materials than XCat since CalB immobilizes better and detaches less on the former than on the latter. The conversion rates observed in flow are the same as those measured for the free enzyme and in batch at equal contact time demonstrating very efficient mass transfers and very good accessibility of the substrate to the enzyme.

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 phenolic or polyphenolic compound, • At least one protein, preferably an immobilized enzyme, 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) 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 phenolic or polyphenolic compound is: • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol, • a phenolic compound from the flavonoid family, notably chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid, • a condensed tannin, • a phi orotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol • or a natural plant extract containing at least one of these compounds.

4. Composition according to claim 3, in which the phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily.

5. Composition according to claim 3 or 4, in which the phenolic or polyphenolic compound is in particular chosen from: catechin or phloroglucinol.

6. 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 -phloroglucinol, catechin, from a catechu extract.

7. Composition according to claim 1, comprising: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from: -a sugar chosen from xylose or dihydroxyacetone -a phenolic or polyphenolic compound chosen from phloroglucinol or catechin, and b) at least one immobilized protein, 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), and 8. Composition according to one of claims 1 to 7, wherein the amount of protein adsorbed on the porous carbon material is from 10 to 80 parts by weight relative to 100 parts by weight of the porous carbon material, even more preferably from 10 to 50 parts by weight relative to 100 parts by weight of the porous carbon material.

9. Composition according to one of claims 1 to 8, 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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