Novel porous carbon-based materials containing immobilized proteins, methods for their preparation and uses

Abstract

The present invention relates to a composition comprising a carbon-based polymer material, also called porous carbon-based material, optionally complexed with at least one metal ion, preferably a metal cation, and a protein immobilized on said material, and a method for preparing the same. The present invention further relates to the use of compositions comprising immobilized proteins as heterogeneous biocatalysts.

Description

[Technical Field]

[0001] The present invention relates to a carbon-based polymer material, also called a porous carbon-based material or a carbon-rich aerogel, comprising a protein immobilized on the material, and a method for preparing the same.

[0002] The invention further relates to the use of compositions comprising immobilized proteins, especially in heterogeneous biocatalysis applications. [Background technology]

[0003] Porous carbon-based materials or carbon-rich aerogels, as known from the state of the art, are conventionally obtained by a sol-gel polymerization reaction between carbon-rich organic precursors and drying of the resulting hydrogel.

[0004] Porous carbon-based materials of the type prepared and used in this application are described in the publication "Hydrothermal synthesis of highly porous carbon monoliths from carbohydrates and phloroglucinol"; Nicolas Brun et al.; RSC Advances, 2013, 3.

[0005] In particular, this research paper describes an original hydrothermal approach for synthesizing highly porous carbon-rich cryogels and aerogels (or carbogels) from carbohydrate precursors (e.g., fructose, glucose, or xylose) and phenolic compounds (e.g., phloroglucinol). It should be noted that these carbohydrates can be isolated from the cellulose fraction of lignocellulosic biomass, while phloroglucinol is the monomer unit of phlorotannins, which can be isolated from fruit trees, brown algae bark, or through various biosynthetic pathways. Therefore, the compounds used in this application can be considered renewable and sustainable.

[0006] State-of-the-art carbon-rich aerogels derived from biomass are obtained after further heat treatment at temperatures above 300°C and offer a wide range of potential applications, e.g., electrodes for batteries and fuel cells or adsorbents for hydrogen and carbon dioxide storage. The carbon-rich aerogel or porous carbon-based material according to the present invention is not subjected to heat treatment at temperatures above 300°C.

[0007] Furthermore, enzymes are a specific group of proteins that serve as biocatalysts in the metabolism of all living cells. Thus, enzymes are capable of specifically converting organic and inorganic molecules into advantageous products.

[0008] However, enzymes are biomolecules developed for the cellular environment and are often unsuitable for other environments. It is therefore interesting to be able to immobilize enzymes on solid supports and use them as catalysts in this immobilized state.

[0009] Solid-state enzyme immobilization has been performed using different techniques and different solid supports. Adsorption of enzymes onto solid surfaces can lead to undesirable interactions between the enzyme and the solid support. For example, adsorption of proteins onto silica nanoparticles can cause changes in the secondary structure of the protein, which can lead to enzyme inactivation. Therefore, it is important that the solid support does not interfere with the structure and activity of the immobilized enzyme.

[0010] The high cost of enzyme preparation and the frequent loss of activity when enzymes are immobilized on solid supports are obstacles to this development. A standardized and versatile procedure for enzyme immobilization that would allow enzyme reuse would be highly desirable. A general and simple method for preparing heterogeneous catalysts by enzyme immobilization is still lacking. Therefore, there is a need for new supports and improved methods for immobilizing enzymes on these supports, as well as stable heterogeneous biocatalysts that can be used in organic synthesis under aqueous or organic reaction conditions. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] "Hydrothermal synthesis of highly porous carbon monoliths from carbohydrates and phloroglucinol"; Nicolas Brun et al.; RSC Advances, 2013, 3. Summary of the Invention [Problem to be solved by the invention]

[0012] In this context of the search for adapted and efficient biocatalysis tools, a first object of the present invention is to propose a porous carbon-based material comprising proteins immobilized thereon. A second object of the present invention is to propose a method for preparing said material. Finally, a final object of the present invention is to propose specific compositions and their uses. [Means for solving the problem]

[0013] According to a first aspect of the present invention, the present application provides: at least one carbohydrate, preferably at least one ose or at least one polyoside, at least one phenolic or polyphenolic compound, a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; at least one immobilized protein, preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), a pyrophosphatase (EC 3.6.1.1), optionally at least one metal cation; The present invention relates to a composition comprising:

[0014] The biologically derived precursor compound #1 that can be used according to the present invention is a carbohydrate, preferably at least one ose or at least one polyoside.

[0015] 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). This class includes substances derived from monosaccharides by reduction of the carbonyl group, oxidation of at least one functional group at the end of the chain to a carboxylic acid, or replacement of one or more hydroxyl groups with a hydrogen atom, an amino group, a thiol group, or any similar atom. Additionally, compounds derived from the dehydration of carbohydrates (e.g., furan aldehyde) may also be used in the context of this application.

[0016] An ose (i.e., a monosaccharide) is a carbohydrate monomer. An ose has at least three carbon atoms; that is, it is a polyhydroxyaldehyde or polyhydroxyketone.

[0017] Oases are distinguished by the length of their carbon chains: · Triose: Three carbon sugar, C3H6O3 (glyceraldehyde, dihydroxyacetone); · Tetrose: tetracarbon sugar, C4H8O4 (erythrose, threose, erythrulose); Pentose: five-carbon sugar, C5H 10 O5 (Deoxyribose (C5H 10 O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose); Hexose: Six carbon sugars, C6H 12 O6 (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose); Deoxyhexose: Six carbon sugar, C6H 12 O5 (fucose, rhamnose); Heptose: seven-carbon sugar, C7H14 O7 (sedoheptulose, mannoheptulose); Octose: eight carbon sugar, C8H 16 O8 (heptahydroxyoctanal); Nonose: nine-carbon sugar, C9H 17 N1O8 (neuraminic acid or sialic acid).

[0018] Aldoses are monosaccharides consisting of a chain of n carbon atoms with a carbonyl group at the terminal carbon atom forming 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 generally not considered carbohydrates, the simplest aldose possible is glyceraldehyde (n=3), which contains only three carbon atoms.

[0019] A ketose is a monosaccharide consisting of a chain of n carbon atoms with a carbonyl group at the non-terminal carbon atom forming a ketone and hydroxyl groups attached to all other carbon atoms.

[0020] According to a preferred embodiment of the invention, at least one ose is: an aldose chosen from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, a ketose selected from among dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose, is selected from among:

[0021] Polysaccharides are a family of polymers of carbohydrates formed by n oses (n≧2) via alpha or beta glycosidic bonds.

[0022] According to a preferred embodiment of the invention, the at least one polysaccharide is: - heterodiholoside (polysaccharides consisting of two different oside units, n=2) selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose and rutinose, or homodiholoside (polysaccharides consisting of two identical oside units, n=2) selected from among inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose, is selected from among:

[0023] According to another aspect of the invention, the bio-derived precursor compound #1 of the porous carbon material consists of a carbohydrate, preferably an ose, or a polysaccharide as described above.

[0024] According to another aspect of the invention, the porous carbon material comprises two bio-derived precursor compounds #1, as described above, consisting of two carbohydrates, preferably two oses, two polysaccharides, or one ose and one polysaccharide. In another aspect of the invention, the porous carbon material comprises three or more bio-derived precursor compounds #1.

[0025] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a precursor compound of biological origin having at least one benzene ring substituted with a hydroxyl group and optionally at least one other functional group selected from among carboxylic acid, aldehyde, ether, ester, alcohol, hydroxyl and ketone groups.

[0026] In this application, the term "bio-based" refers to materials or compounds derived from biomass of plant or animal origin, whether or not chemically modified. These raw materials have the advantage of being renewable.

[0027] "Naturally derived" or biologically derived compounds within the meaning of the present invention refer to any compound derived from nature (plant or animal biomass). The compound may be extracted from renewable terrestrial and marine biomass or living organisms (animals, microorganisms), possibly subsequently modified, for example chemically, or may be obtained by the action of living microorganisms (e.g., enzymes or bacteria) on naturally occurring compounds using methods such as biofermentation or biosynthesis. Petroleum-based compounds of fossil origin do not fall into this category.

[0028] The bio-derived precursor compounds #2 that can be used according to the present invention can be phenols whose benzene ring is further substituted with at least one other functional group selected from the group consisting of carboxylic acid (especially COOH), aldehyde (especially CHO), ether (especially CH3O), ester, alcohol (especially CH2OH), hydroxyl, and ketone, preferably hydroxyl, carboxylic acid, and aldehyde. Any salts within these groups are also part of the present invention.

[0029] Biogenic phenolic or polyphenolic precursor compounds that can be used in accordance with the present invention can be selected from plant secondary metabolites. Phenolic compounds form the most important group of phytochemicals in plants. They consist of approximately 8,000 molecules divided into over a dozen chemical classes. Each class is characterized by the presence of a benzoic acid ring to which one or more hydroxyl groups are directly attached. These compounds are present in all parts of higher plants (roots, stems, leaves, flowers, pollen, fruits, seeds, and xylem). These compounds are synthesized by plants exposed to harsh conditions (infection, wounding, ultraviolet radiation, etc.) and are involved in many physiological processes, such as cell growth, rooting, seed germination, or fruit ripening.

[0030] Phenolic compounds include simple molecules such as phenolic acids or compounds with a high degree of polymerization, such as tannins and lignins. Phenolic compounds can be classified according to the complexity, degree, and possible linkages between the basic skeleton and other molecules. Polyphenols are highly diverse molecules consisting of one or more benzene rings carrying one or more hydroxyl functional groups. These polyphenols can be divided into many classes depending on the complexity of the basic skeleton (C6 ring), the degree of modification of this skeleton (oxidation, hydroxylation, etc.), and finally, the molecules to which they are associated (carbohydrates, lipids, proteins, other metabolites). The simplest forms are represented by two major groups, from which numerous compounds are derived: hydroxycinnamic acids and flavonoids. On the other hand, complex forms are mostly the result of condensation of several simpler forms, including tannins and lignins, among others.

[0031] Polyphenols are divided into different groups defined by the structure of their carbon skeleton. Phenolic acids (C6-C1 and C6-C3), especially flavonoids (C6-C3-C6), are the most frequently found in the plant kingdom. Lignans (C6-C3-C3-C6) are less common. The last group is the stilbene (C6-C2-C6) group, the best known of which is resveratrol, found in grape skins. Flavonoids represent the largest group of polyphenols and are the most widely distributed in the plant kingdom. Over 4,000 different flavonoids have been identified. They differ in the degree of oxidation of the oxygenated heterocycle. Table 1 below summarizes the major classes of phenolic compounds.

[0032] [Table 1]

[0033] Condensed tannins, also known as proanthocyanidins, are a particularly interesting source of such phenolic compounds that can serve as starting materials for the formation of epoxy prepolymers. Condensed tannins are phenolic biopolymers primarily present in rapidly growing and regenerating soft tissues, such as leaves and stems. This class of polyphenols is the second most abundant after the lignin class. Unlike lignin, which is a component of lignocellulose, the structural element 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 a variety of available natural resources, including agro-industrial residues, such as fruit pomace, and in untapped biomass, particularly bark, leaves and needles, vines, and fruits.

[0034] Condensed tannins are compounds that cannot be hydrolyzed but can be depolymerized to anthocyanidols when treated with acid at high temperatures. They are usually named after the anthocyanidol thus released. For example, the monomer (+)-catechol (flavan-3-ol) is the building block of the catechol-(4α→8)-catechol dimer, which is called proanthocyanidol B-3 due to its decomposition to cyanidol in an acidic environment. Many condensed tannins are catechol polymers. For this reason, condensed tannins are also called "proanthocyanidols" or "proanthocyanidins" (based on the English word "proanthocyanidins"). The OH hydroxyl is in the same position on the monomer and the depolymerized product; only the central ring changes.

[0035] There are a dozen or so flavanol monomer units used in the construction of condensed tannins, which may be substituted by gallic acid or sugars, usually at position 3, and occasionally at positions 5 and 7.

[0036] All flavanols and anthocyanidols have three rings: an A ring, usually with one or two OH hydroxyls; a C heterocycle with asymmetric carbons 2 and 3 (and 3 hydroxyls); and a B ring with one OH hydroxyl (afzelecol, epiafzelecol), two OH (catechol and epicatechol), or three OH (gallocatechol, epigallocatechol). Compounds with a cis- (2R,3R) configuration have names prefixed with epi-; others have a trans- (2R,3S) configuration.

[0037] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a simple phenol selected from among phenol, catechol, resorcinol, hydroquinone, pyrogallol or phloroglucinol.

[0038] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound of the flavonoid family.

[0039] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound selected from the subclasses of flavonols, anthocyanins, flavanols, flavanones and isoflavonoids.

[0040] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily, which has a structure based on 2-phenyl-3-chromanol. The oligomeric and polymeric structures of flavanols constitute the class of proanthocyanidols or condensed tannins.

[0041] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is chosen from among catechin, gallocatechin, afzelicol, fisetinidol, giburtinidol, mesquitol or robinetinidol in one of its stereoisomers.

[0042] According to a preferred embodiment of the invention, the at least one phenolic compound is phloroglucinol.

[0043] According to a preferred embodiment of the invention, the at least one polyphenolic compound is a catechin.

[0044] According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is contained in a natural plant extract.

[0045] According to a preferred embodiment of the invention, the natural vegetable extract containing at least one phenolic or polyphenolic compound is selected from among extracts of strawberry, potato, apple, lemon, walnut, liana, grape, flower, soybean, pea, pine, tomato, garlic or catechu.

[0046] According to a preferred embodiment of the invention, the at least one phenol or polyphenol compound is: simple phenols selected from phenol, catechol, resorcinol, hydroquinone, pyrogallol and phloroglucinol; phenolic compounds of the flavonoid family selected from the subclasses: flavonols, anthocyanidols, anthocyanins, flavanols, flavanones, stilbenoids, isoflavonoids, Condensed tannins, phlorotannins, chosen in particular from fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, equol and diphlorethydroxychamanol; or natural plant extracts containing at least one of these compounds, is.

[0047] According to another aspect of the invention, the porous carbon material comprises two bio-derived precursor compounds #2 consisting of two phenolic or polyphenolic compounds as described above. According to another aspect of the invention, the porous carbon material comprises three or more bio-derived precursor compounds #2.

[0048] According to a preferred embodiment of the present invention, the porous carbon-based material obtained by hydrothermal carbonization comprises: maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose or dihydroxyacetone, and - phloroglucinol, catechin or catechu extract, It comprises or is formed from:

[0049] Catechu extract can be obtained from the plant fiber of Acacia catechu, said extract containing condensed tannins and flavonols.

[0050] According to the present invention, a "porous carbon-based material" refers to a polymeric material obtained or obtainable by sol-gel polymerization (e.g., hydrothermal carbonization) from at least two bio-derived precursors #1 and #2 as defined in the present application.

[0051] In one embodiment of the present invention, the porous carbon-based material is obtainable by a sol-gel polymerization method known to those skilled in the art, involving at least two bio-derived precursors #1 and #2 as defined above.

[0052] The sol-gel polymerization process can use catalysts, such as acids and bases, including nitric acid, acetic acid, ascorbic acid, hydrochloric acid, sulfuric acid, boric acid, sodium carbonate, sodium hydroxide, ammonium hydroxide, and calcium sulfate.

[0053] The catalyst concentration can be expressed as a reagent to catalyst (R / C) ratio, which can range, for example, from 10 to 5000, or from 10 to 2000, or from 10 to 1000.

[0054] According to a preferred embodiment of the present invention, the porous carbon-based material is obtained by hydrothermal carbonization.

[0055] Hydrothermal carbonization is defined as a thermochemical conversion process of biomass, or at least two bio-based precursors #1 and #2, as defined above, aimed at obtaining porous carbon monoliths in an aqueous phase. It is an exothermic process that reduces the oxygen and hydrogen levels of the material primarily through dehydration and decarboxylation reactions. Sol-gel polymerization is achieved by applying temperatures between 120 °C and 250 °C to a mixture of at least two bio-based precursors #1 and #2, as defined above, and a solvent, preferably water (usually 10% by weight of the two bio-based precursors #1 and #2).

[0056] In certain embodiments, the porous carbon material is prepared by the following method: (a) heating a reaction mixture comprising an aqueous solution and at least two bio-derived precursors #1 and #2 as defined above dissolved therein to a temperature below 300°C to obtain a solid porous carbon material; (b) washing the resulting solid porous carbon-based material by successive immersions in a polar solvent;

[0057] An aqueous solution may contain water and, preferably, an amount of a solvent that is miscible with water.

[0058] According to a preferred embodiment, the reaction mixture consists of only one liquid phase, namely an aqueous solution, preferably water.

[0059] For example, and without limitation, the aqueous solution includes ethanol, preferably absolute ethanol.

[0060] According to a preferred embodiment, the mass fraction of water in the water / absolute ethanol mixture is between 1 and 0.3.

[0061] According to a preferred embodiment, the mass fraction of water in the water / absolute ethanol mixture is 0.7-0.4 when bio-derived precursors #1 and #2 are xylose and phloroglucinol, respectively.

[0062] According to a preferred embodiment, the mass fraction of water in the water / absolute ethanol mixture is between 1 and 0.5 when bio-derived precursors #1 and #2 are xylose and catechin, respectively.

[0063] Step (a) of the method may also be referred to as the "hydrothermal treatment step."

[0064] Due to the presence of water (in aqueous solution) in step (a), the preparation method of the present invention differs from, for example, biomass conversion or pyrolysis processes for bio-derived precursors #1 and #2 with the goal of providing charcoal-like materials (typically in the absence of oxygen).

[0065] The reaction temperature in the hydrothermal treatment step (a) is preferably below 300° C., preferably between 100 and 300° C., even more preferably between 120 and 250° C., and most preferably between 160 and 200° C. or preferably 180° C. Reaction temperature is intended to mean the temperature inside the reaction mixture, more particularly the average temperature, which can be measured with a thermocouple.

[0066] The hydrothermal treatment in the method for preparing the porous carbon-based material of the present invention is preferably carried out in a pressure-resistant reactor, such as an autoclave.

[0067] There is no specific limit on the duration of step (a) in the method for preparing the porous carbon-based material of the present invention.

[0068] For example, the reaction medium can be introduced into a sealed mineralization bomb, which is then placed in a thermostatic bath at 180° C. for 20 hours.

[0069] Step (a) of the method is carried out until the entire contents of the reaction mixture, including the aqueous solution and bio-derived precursors #1 and #2, have gelled or precipitated into the porous carbon-based material of the present invention.

[0070] In certain embodiments, the porous carbon material is prepared by the following method: (a) - an aqueous solution of water and absolute ethanol having a mass moisture content of 0.7 to 0.4; - xylose and phloroglucinol dissolved in an aqueous solution, heating the reaction mixture comprising the compound at a temperature of less than 300°C (preferably 180°C) to obtain a solid porous carbon material; (b) Washing the obtained solid porous carbon-based material by successive immersions in polar solvents.

[0071] In certain embodiments, the porous carbon material is prepared by the following method: (a)- An aqueous solution of water and absolute ethanol having a mass moisture content of 1 to 0.5; - xylose and catechin dissolved in an aqueous solution, heating the reaction mixture comprising the compound at a temperature of less than 300°C (preferably 180°C) to obtain a solid porous carbon material; (b) Washing the obtained solid porous carbon-based material by successive immersions in a solvent, preferably a polar solvent.

[0072] According to a preferred embodiment, the carbon-based material is formed from at least one carbohydrate, preferably an aldose or ketose, even more preferably at least one heterodiholoside or homodiholoside (c1) and at least one phenolic or polyphenolic compound (c2), in a molar ratio (c1 / c2) of 10:1 to 1:10, preferably 2:1. For complex natural extracts and condensed tannins, the molar ratio (c1 / c2) is defined as the ratio between the number of moles of carbohydrate (c1) 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 complex natural extract.

[0073] In certain embodiments, the porous carbon material is prepared by the following method: (a) - an aqueous solution of water and absolute ethanol having a mass moisture content of 0.7 to 0.4; - xylose and phloroglucinol dissolved in an aqueous solution, heating the reaction mixture comprising the compound at a temperature of less than 300°C (preferably 180°C) to obtain a solid porous carbon material; (b) Washing the obtained solid porous carbon-based material by successive immersions in a solvent, preferably a polar solvent.

[0074] In certain embodiments, the porous carbon material is prepared by the following method: (a)- An aqueous solution of water and absolute ethanol having a mass moisture content of 1 to 0.5; - xylose and catechin dissolved in an aqueous solution (molar ratio 2:1), heating the reaction mixture comprising the compound at a temperature of less than 300°C (preferably 180°C) to obtain a solid porous carbon material; (b) Washing the obtained solid porous carbon-based material by successive immersions in a solvent, preferably a polar solvent.

[0075] According to a preferred embodiment, the porous carbon material comprises: 40 to 70% by weight of elemental carbon (C), 30-40% by weight of elemental oxygen (O), Preferably 60% by weight of carbon element (C) and 35% by weight of oxygen element (O), It consists of:

[0076] According to a preferred embodiment, the porous carbon material consists of 36% O and 60% C for the xylose / phloroglucinol system.

[0077] According to a preferred embodiment, the porous carbon-based material is formed from at least one carbohydrate (c1), preferably at least one ose or at least one polysaccharide (c1), and at least one phenolic compound (C2) or polyphenolic compound, in a molar ratio (c1 / c2) of 10:1 to 1:10, preferably 2:1.

[0078] The solid porous carbon material is washed by immersion for several hours in either ultrapure water, deionized water or a polar protic water / solvent mixture such as methanol, ethanol or tert-butanol.

[0079] The purpose of washing is to extract soluble compounds that are not incorporated within the structure of the porous carbon-based material and to prepare the gel for the drying step.

[0080] The carbonaceous material obtained or obtainable in the hydrothermal carbonization step (a) is typically composed of primary nanoparticles aggregated in the form of an interconnected network forming the dispersed phase, together with the aqueous solution forming the dispersed phase.

[0081] The carbon-based material before drying can be called a solvogel. If the aqueous solution is water, the carbon-based material obtained in step (a) can be called a hydrogel.

[0082] Thus, hydrogels are considered herein as a special type of solvogel in which the aqueous solution of the dispersed phase is water.

[0083] The solid porous carbon material obtained or obtainable by the methods described above can be dried and converted into a cryogel, aerogel or xerogel.

[0084] According to a first variant, the hydrogel or solvogel in dry form is obtained by contacting a carbon-based material (i.e. a hydrogel or solvogel) with a supercritical fluid, in particular acetone or carbon dioxide, to convert it into an aerogel.

[0085] According to a second variant, the hydrogel in dry form is obtained by subjecting the carbon-based material (i.e. hydrogel or solvogel) to freezing of the isolated gel and freeze-drying of the frozen gel to convert it into a cryogel.

[0086] According to a third variant, the hydrogel in dry form is obtained by subjecting a carbon-based material (i.e. a hydrogel or solvogel) to evaporation of the solvent at controlled temperature and pressure to convert it into a xerogel.

[0087] Alternatively, if the dried gel retains most of its textural properties after drying, the cryogel can be called an aerogel.

[0088] A dried gel can be referred to as an aerogel regardless of the drying technique used.

[0089] Depending on the gel drying method, it is possible to control the pore size and structure of the porous carbon material obtained according to the present invention.

[0090] In certain embodiments, the porous carbon material is prepared by the following method: a) heating a reaction mixture comprising an aqueous solution and at least two bio-derived precursors #1 and #2 as defined above dissolved therein to a temperature of less than 300°C, preferably 180°C, to obtain a solid porous carbon-based material; b) washing the obtained solid porous carbon material by successive immersions in a polar solvent (ideally in absolute ethanol or in a 75 / 25 by weight mixture of water / tert-butanol); c) Drying the solid porous carbon-based material using a supercritical fluid or by freeze-drying.

[0091] The hydrogel, placed in a borosilicate glass beaker, is pre-immersed in liquid nitrogen (77 K) for 15 minutes and then placed directly into the freeze-dryer chamber (e.g., a Cryotec COSMOS freeze-dryer). Drying is carried out under a vacuum of less than 50 mTorr for 48 hours using a cold trap at -80°C to ensure condensation of water vapor or solvent.

[0092] To maintain the pore system, supercritical drying and freeze-drying are preferred. According to a particularly preferred embodiment, the solvent is removed by extraction with supercritical CO2 for this method, and the solvent in the solvogel is preferably selected from absolute ethanol or acetone (e.g., provided by solvent displacement with absolute ethanol or acetone).

[0093] Porous carbon material texture The porous carbon-based material obtained or obtainable in step (a) is a gelled material, in particular a solidified material made up of agglomerated particles forming an interconnected network.

[0094] The porous carbon-based materials obtained or obtainable by the above-mentioned methods include micropores (pores less than 2 nm), mesopores (pores between 2 and 50 nm) and macropores (pores greater than 50 nm).

[0095] Furthermore, in contrast to conventional activated carbons (eg those produced by pyrolysis), the oxygen content is typically greater than 25% for the material directly obtained in the hydrothermal carbonization step (a).

[0096] The surface of the carbon-based material of the present invention obtained in step (a) is highly hydrophilic.

[0097] [Texture properties of porous carbon materials] Pore ​​diameter The porous carbon material according to the present invention is composed of agglomerated particles that form an interconnected network, which induces gaps (or pores) between the particles having a diameter of 10 μm to 5 nm, preferably an average diameter of 5 to 200 nm, more preferably an average diameter of 25 to 100 nm, and even more preferably an average diameter of 40 to 80 nm, preferably around 60 nm.

[0098] Furthermore, the porous carbon material of the present invention is an aggregate material composed of particles having a pore size distribution calculated from a nitrogen adsorption / desorption isotherm at 77 K (narrowest pores less than 100 nm) and a mercury intrusion porosimetry profile (widest pores greater than 50 nm).

[0099] Furthermore, 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.

[0100] The pore diameter of the carbon gel according to the present invention, which corresponds to the peak of the pore size distribution, is preferably within the range of 40 to 80 nm.

[0101] It should be pointed out that the pore diameter of the porous carbon-based 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 adsorbed or complexed component, in order to prevent a decrease in the amount of adsorbed component.

[0102] specific surface area The porous carbon material according to the present invention preferably has a particle size of 300 to 1000 m 2 / g, and more preferably 600 to 800 m 2 / g specific surface area.

[0103] Porous carbon material is 100m 2If the specific surface area is less than 1 / 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 accommodated will also be reduced.

[0104] porous volume Furthermore, the total pore volume of the porous carbon material according to the present invention is not particularly limited, since it also varies with the pore diameter corresponding to the peak of the pore size distribution and the specific surface area. However, the porous carbon material according to the present invention preferably has a pore volume of 0.1 to 5 cm. 3 / g, more preferably 0.2 to 2.5 cm 3 / g total pore volume.

[0105] The so-called total volume was determined at a relative pressure of 0.99 on the nitrogen adsorption / desorption isotherm at -196 °C. The surface area and volume of the micropores were determined by applying the t-plot method. The pore size distribution was evaluated by applying the BJH method to the isothermal desorption branch. The aerogels were degassed for at least 6 h at 0.05 mbar in a VacPrep Micromeritics system before analysis. The analysis was performed in a TriStar Micromeritics system. The total pore volume in the material is larger than that obtained by nitrogen adsorption volume at -196 °C because this method cannot analyze the largest pores (diameters >100 nm). The largest pores (diameters >100 nm) were analyzed by mercury intrusion porosimetry. The volume of these large pores is >3 mL / g for the phloroglucinol / xylose aerogels.

[0106] The specific surface area and total porous volume of the porous carbon material according to the present invention can be determined by a general volumetric measurement as described below. Specifically, the porous carbon material is placed in a container and cooled to liquid nitrogen temperature (-196°C). Nitrogen gas is then introduced into the container, and the amount of nitrogen gas adsorbed onto the carbon gel is determined based on a volumetric method. The pressure of the nitrogen gas introduced into the container is then gradually changed, and the amount of nitrogen gas adsorbed onto the carbon gel is plotted according to each equilibrium pressure. This results in a nitrogen adsorption / desorption isotherm.

[0107] Additionally, the specific surface area can be determined by the Brunauer Emmett Teller method (BET), for example, by nitrogen adsorption techniques.

[0108] The BET equivalent specific surface area was determined by nitrogen adsorption at 77 K by constructing isotherms at relative pressures P / P0 between 0.01 and 0.99. The BET method was applied over the relative pressure range of 0.1 to 0.3 (based on the Rouquerol transformation).

[0109] Interconnected porosity This parameter can be determined by mercury intrusion porosimetry, which indicates the total volume of interconnected pores.

[0110] Ion complexation The porous carbon-based material as described above is capable of complexing with metal ions, preferably at least one type of metal cation, which is complexed on the outer surface and the inner pore surfaces of the porous carbon-based material according to the present invention.

[0111] According to a particular embodiment of the invention, at least one metal cation is Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+, Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ , preferably one or more cations of a transition metal Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ is selected from among:

[0112] Metal cations are complexed or chelated with porous carbon-based materials. A chelate is a type of coordination compound in which a single metal ion is bound by a coordinate covalent bond to a molecule or ion called a ligand. A coordination compound is formed when groups of atoms, ions, or molecules chemically bond with each other by donating and accepting electron pairs. The electron-donating groups are called ligands. They are usually Lewis bases. The electron-accepting groups are often transition metal cations.

[0113] According to the present invention, "hybrid material" means a porous carbon-based material in which metal ions are complexed, which is a composition within the meaning of the present invention.

[0114] In certain embodiments, the composition (porous carbon-based hybrid material) is prepared by the following method: a) heating a reaction mixture comprising an aqueous solution and at least two bio-derived precursors #1 and #2 as defined above dissolved therein to a temperature below 300°C to obtain a solid porous carbon-based material; b) washing the obtained solid porous carbon material by successive immersions in a polar solvent; c) Drying the solid porous carbon-based material using a supercritical fluid or by freeze-drying. d) Impregnating a solid porous carbon material with a solution containing metal cations to obtain a porous carbon hybrid material complexed with the metal cations.

[0115] The adsorption method and adsorption conditions for allowing the metal complex to be adsorbed onto the carbon-based material are not particularly limited.

[0116] For example, metal complexes can be optionally adsorbed onto the porous carbon-based material by placing the material in a solution and mixing the solution for a period of time at about 10-100° C. The hybrid material is separated from the solution by centrifugation, and the resulting suspension is washed and dried.

[0117] According to another variant of the invention, the composition (porous carbon-based hybrid material) is prepared by the following method: a) heating a reaction mixture comprising an aqueous solution and at least two bio-derived precursors #1 and #2 as defined above dissolved therein, together with a solution containing metal cations, to a temperature below 300°C to obtain a solid porous carbon-based material; b) washing the resulting solid porous carbon hybrid material by successive immersions in polar solvents; c) drying the solid porous carbon hybrid material using supercritical fluids or by freeze-drying.

[0118] The amount of metal ions complexed with the porous carbon-based 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 the carbon-based material is preferably 0.1 to 40 parts by weight per 100 parts by weight of the porous carbon-based material.

[0119] In certain embodiments, the composition comprises chelated or complexed metal cations in an amount ranging from 0.2 to 2 mole %, preferably 0.5 to 1 mole %, as determined by SEM-EDX techniques.

[0120] In certain embodiments, the composition optionally comprises metal cations in an amount of 1 to 5% by weight, especially 2 to 4% by weight.

[0121] In certain embodiments, the composition comprises iron in an amount of 20-40 mg per gram of porous carbon-based material.

[0122] The solvent used here is not particularly limited as long as it can dissolve and / or disperse the metal complex. Examples of the solvent include acetic acid, water, ethylene glycol, DMSO, and DMF.

[0123] There is no particular limitation on the concentration of the metal complex in the solution, however, the concentration is preferably about 0.1 to 30 mM.

[0124] In particular, the composition (solid porous carbon hybrid material) comprises: (a) at least one carbohydrate, preferably at least one ose, or at least one polysaccharide (biological precursor #1); and At least one phenolic or polyphenolic compound (biogenic precursor #2) A porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization, (b) Optionally, Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ at least one metal cation selected from among, preferably one or more transition metal cations Fe3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ .

[0125] In a particular embodiment, the composition comprises: (a) - at least one carbohydrate, preferably at least one ose or at least one polyoside, - Oath: an aldose chosen from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, or a ketose chosen from among dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose, or - Polysaccharides: heterodiholoside selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose and rutinose, or homodiphorosides selected from inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose, and - at least one phenolic or polyphenolic compound (biogenic precursor #2), a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; (b) Optionally, Al 3+ , Ag + , Ba 2+ , Ca 2+, Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+ at least one metal cation selected from among, preferably one or more transition metal cations Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ .

[0126] In another particular embodiment, the composition comprises: (a) - at least one carbohydrate, preferably at least one ose or at least one polyoside, - Oath: an aldose chosen from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, or a ketose chosen from among dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose, or - Polysaccharides: a heterodiholoside selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, rutinose, or homodiphorosides selected from inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose, - at least one phenolic or polyphenolic compound selected from the following: simple phenols selected from phenol, catechol, resorcinol, hydroquinone, pyrogallol and phloroglucinol; phenolic compounds of the flavonoid family selected from the subclasses: flavonols, anthocyanidols, anthocyanins, flavanols, flavanones, stilbenoids, isoflavonoids, Condensed tannins, phlorotannins, chosen in particular from fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, equol and diphlorethydroxychamanol; or natural plant extracts containing at least one of these compounds, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) Optionally, Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2at least one metal cation selected from among 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ .

[0127] In another particular embodiment, the composition comprises: (a)-xylose or dihydroxyacetone, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; (b) optionally, Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ and at least one metal cation selected from:

[0128] In another particular embodiment, the composition comprises: (a)-xylose, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; (b) optionally, Fe 3+ and at least one metal cation which is

[0129] In another particular embodiment, the composition comprises: (a)-xylose, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) Optionally, Co 2+ and at least one metal cation which is

[0130] In another particular embodiment, the composition comprises: (a)-Dihydroxyacetone, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) optionally, Fe 3+ and at least one metal cation which is

[0131] According to the invention, all of the above-mentioned compositions can be used to immobilize proteins, in particular at least one enzyme.

[0132] [Protein immobilization] A second object of the present invention is to propose a composition comprising a protein or enzyme immobilized on the solid porous carbon hybrid material or composition. The solid porous carbon hybrid material of the present invention comprises the above-mentioned solid porous carbon material as a support, at least one metal ion complexed to the material, and at least one protein (in particular an enzyme) immobilized (optionally on the porous carbon material loaded 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.

[0133] The protein used here is not particularly limited.

[0134] Thus, the compositions of the present invention comprise at least one enzyme selected from the International Union of Biochemistry (IUBMB) approved list of enzyme nomenclature and classification, examples of which include the following proteins: EC1 oxidoreductases, EC2 transferases, EC3 hydrolases, EC4 lyases, EC5 isomerases, EC6 synthases and EC7 transportases.

[0135] Among these proteins, at least one protein is, in particular, α-acetolactate, α-arabinosidase, α-galactosidase, α-rhamnosidase, β-galactosidase, β-glucanase, β-glucosidase, β-glucanase, β-mannanase, γ-lactamase, acetolactate decarboxylase, activase, adenosine deaminase, aminoacylase, aminopeptidase, amylase, amyloglucosidase, asparginase, aspartase, bromelain, carbonic anhydrase enzymes, 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 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 The enzyme is selected from the group consisting of enzymes such as sucrose, polygalacturonase, protease, protopectinase, pullulanase, pyrophosphatase, pyruvate transaminase, raffinose synthase, rennet, saccharosidase, serratiopeptidase, sphingosine kinase, stachyose synthase, tannase, taxolase, thermolysin, transaminase, transglutaminase, trypsin, urease, xylanase, and xylose isomerase.

[0136] According to another preferred embodiment of the invention, the composition comprises a protein selected in particular from the group consisting of enoate reductases (EC 1.3.1.31), transaminases (EC 2.6.1), pyrophosphatases (EC 3.6.1.1).

[0137] Protein loading Regarding the porous carbon hybrid material of the present invention, the amount of protein adsorbed onto the porous carbon hybrid material is not particularly limited as long as it exhibits enzymatic activity, but the amount of protein adsorbed onto the porous carbon hybrid material is preferably 10 to 80 parts by weight per 100 parts by weight of the porous carbon hybrid material, and more preferably 10 to 50 parts by weight per 100 parts by weight of the porous carbon hybrid material.

[0138] Furthermore, the method for providing the porous carbon-based hybrid material of the present invention by adsorbing the protein onto the porous carbon-based material is not particularly limited. For example, sublimation and impregnation methods can be used. The impregnation method described below is more preferred. Specifically, the protein is first dissolved in water or a buffer solution at a concentration at which the protein does not precipitate (preferably 0.1 to 1000 mg / ml). Next, the porous carbon-based material is suspended in the resulting solution at a temperature at which the solution does not freeze and the protein does not denature (preferably 0 to 50°C). The protein is then contacted with the porous carbon-based material for at least 5 minutes, preferably 30 minutes or more, and thereby immobilized within the pores of the carbon gel. In this way, the carbon-based hybrid material of the present invention can be provided.

[0139] In certain embodiments, a composition comprising at least one immobilized protein is prepared by a method comprising the following steps: - suspending the dried porous carbon material in a solution that may contain metal cations; - optionally washing the porous carbon hybrid material if metal cations are loaded; - immobilizing a protein (enzyme) on the porous carbon hybrid material optionally loaded with metal cations by reacting said protein with the porous carbon material optionally loaded with metal cations; - washing the suspension to recover the immobilized proteins on the porous carbon hybrid material, optionally loaded with metal cations.

[0140] More specifically, the porous carbon hybrid material, optionally loaded with metal cations, is added to an aqueous solution containing a protein, preferably an enzyme.

[0141] Optionally, the metal cation-loaded porous carbon hybrid material on which at least one protein is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and / or coordinate bonds and / or ionic bonds is washed several times with a buffered aqueous solution.

[0142] The protein to be immobilized on the material can be any protein, for example a (recombinant) protein or an enzyme.

[0143] Preferably, the protein is an enzyme.

[0144] The aqueous buffer solution is preferably a Tris-HCl or phosphate buffer.

[0145] According to a second aspect of the present invention, the present application relates to a composition comprising: a) - at least one carbohydrate, preferably at least one ose or at least one polysaccharide (biological precursor #1), and - at least one phenolic or polyphenolic compound (biogenic precursor #2), a porous carbon-based material comprising or formed from the porous carbon-based material and obtained by hydrothermal carbonization; b) optionally at least one metal cation, and c) At least one protein, preferably an enzyme.

[0146] According to a preferred embodiment of the invention, the composition comprises: a) - at least one carbohydrate, preferably at least one ose or at least one polysaccharide (biological precursor #1), and - at least one phenolic or polyphenolic compound (biogenic precursor #2), a porous carbon-based material comprising or formed from the porous carbon-based material and obtained by hydrothermal carbonization; b) optionally at least one metal cation, and c) At least one immobilized protein, preferably an enzyme.

[0147] In another preferred form of the invention, the composition comprises: (a) - at least one carbohydrate, preferably at least one ose or at least one polyoside, - Oath: an aldose chosen from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, or a ketose chosen from among dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose, or - Polysaccharides: heterodiholoside selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose and rutinose, or a homodiphoroside selected from among inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose, is selected from among - at least one phenolic or polyphenolic compound selected from the following: simple phenols selected from phenol, catechol, resorcinol, hydroquinone, pyrogallol and phloroglucinol; phenolic compounds of the flavonoid family selected from the subclasses: flavonols, anthocyanidols, anthocyanins, flavanols, flavanones, stilbenoids, isoflavonoids, Condensed tannins, Phlorotannins selected from fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, equol, and diphlorethydroxychamanol, or natural plant extracts containing at least one of these compounds; a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; (b) Optionally, Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Pb 2+ , Sn 2+ , Sn 4+ , Zn 2+at least one metal cation selected from among 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ , and (c) at least one immobilized protein;

[0148] In another preferred form of the invention, the composition comprises: (a)-xylose or dihydroxyacetone, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) optionally, Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ at least one metal cation selected from (c) at least one immobilized protein;

[0149] In another preferred form of the invention, the composition comprises: (a)-xylose, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) optionally, Fe 3+ at least one metal cation which is (c) at least one immobilized protein;

[0150] In another preferred form of the invention, the composition comprises: (a)-xylose, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) Optionally, Co 2+at least one metal cation which is (c) at least one immobilized protein;

[0151] In another preferred form of the invention, the composition comprises: (a)-Dihydroxyacetone, - phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) optionally, Fe 3+ at least one metal cation which is (c) at least one immobilized protein;

[0152] For example, when the solid material according to the invention is used for the purification and isolation of proteins, the binding of the enzyme to the support must be reversible.

[0153] For the use of immobilized enzymes in heterogeneous biocatalysis, strong binding of the enzyme to the support is desirable.

[0154] When the proteins immobilized on the supports as described above are enzymes, they contain active sites capable of catalyzing chemical reactions.

[0155] Thus, immobilized enzyme materials are potentially useful as biocatalysts in organic synthesis.

[0156] Thus, in another aspect, the present invention relates to the use of the immobilized enzyme material described herein as a heterogeneous biocatalyst, for example in synthetic organic transformations.

[0157] A preferred embodiment of the present invention relates to the use of the above-described composition as a heterogeneous biocatalyst.

[0158] The present invention further relates to a method for catalyzing an enzyme-catalyzed reaction, comprising providing a composition comprising a protein or enzyme immobilized on the porous carbon-based material of the present invention, and contacting the composition comprising the immobilized enzyme with at least one substrate with which the enzyme immobilized on the material is capable of reacting.

[0159] Immobilized enzymes have been shown to tolerate both aqueous conditions as well as a range of organic solvents.

[0160] This allows compositions comprising proteins or enzymes immobilized on the porous carbon material to be used under reaction conditions where free, non-immobilized enzymes would not be stable.

[0161] It is conceivable that compositions comprising proteins or enzymes immobilized on the porous carbon material can also be used over a wider pH range than the free, non-immobilized enzyme would tolerate.

[0162] The enzymes immobilized on the material include oxidoreductases, transferases, hydrolases, and lyases. The enzyme may be any enzyme that is useful as a biocatalyst in synthetic organic transformations, including but not limited to enzymes that react as isomerases, isomerases, and synthases.

[0163] Thus, compositions comprising enzymes immobilized on the materials can be used as heterogeneous biocatalysts in any organic reaction in which the immobilized enzyme can specifically catalyze the reaction.

[0164] Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation-reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.

[0165] A highly useful class of biocatalytic reactions is the enantioselective reaction.

[0166] Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation-reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.

[0167] In one embodiment, two or more different enzymes may be immobilized on the material of the present invention, where each different enzyme has the ability to catalyze a different reaction.

[0168] It may then be possible to use materials containing two or more different immobilized enzymes as heterogeneous biocatalysts in multi-step or cascade reactions.

[0169] According to another aspect of the present invention, the present application relates to a method for catalyzing an enzyme-catalyzed reaction, comprising providing the composition described above and contacting said composition with at least one substrate on which the enzyme immobilized on said support of the composition is capable of acting.

[0170] Immobilization yield and rate The immobilization yield, expressed as mg of immobilized enzyme per mg of enzyme originally present in solution, is 90-100% for protein loadings of 5-50% by mass.

[0171] The immobilization rate, expressed in minutes, to achieve at least 75% immobilization is 10 to 120 minutes.

[0172] According to a preferred embodiment of the present invention, the method for immobilizing proteins, in particular enzymes, on porous carbon-based materials is characterized by an immobilization yield of 90% to 100% for protein loadings in the range of 5 to 50 wt.%.

[0173] According to a preferred embodiment of the present invention, the method for immobilizing proteins, particularly enzymes, on porous carbon-based materials is characterized by an immobilization rate of 10 to 120 minutes to achieve an immobilization yield of at least 75%.

[0174] According to another aspect of the invention, the present application relates to the use of the composition as a heterogeneous biocatalyst.

[0175] According to a final aspect of the invention, the present application relates to: at least one carbohydrate, preferably at least one ose or at least one polyoside, at least one phenolic or polyphenolic compound, a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; at least one protein, preferably an immobilized enzyme, said at least one immobilized protein being preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), a pyrophosphatase (EC 3.6.1.1), optionally at least one metal cation; A composition comprising:

[0176] - Oath: an aldose chosen from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, or a ketose chosen from among dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose, or - Polyosides: heterodiholoside selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose and rutinose, or a homodiphoroside selected from among inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose, composition.

[0177] Phenol or polyphenol compounds: simple phenols selected from phenol, catechol, resorcinol, hydroquinone, pyrogallol and phloroglucinol; phenolic compounds of the flavonoid family selected from the subclasses: flavonols, anthocyanidols, anthocyanins, flavanols, flavanones, stilbenoids, isoflavonoids, Condensed tannins, phlorotannins, chosen in particular from fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, equol and diphlorethydroxychamanol; or natural plant extracts containing at least one of these compounds, The composition.

[0178] A composition wherein the phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily.

[0179] A composition wherein the phenolic or polyphenolic compound is selected from catechin or phloroglucinol.

[0180] Porous carbon-based materials: Maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and - Phloroglucinol and catechin from catechu extract 1. A composition comprising or formed from:

[0181] If present, at least one metal ion is Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K. + , Mg 2+ , Mn 2+ , Na + , Ni 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 Ni 2+ The composition is selected from:

[0182] (a) - an ose selected from xylose or dihydroxyacetone; a phenolic or polyphenolic compound selected from phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) at least one immobilized protein, preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), or a pyrophosphatase (EC 3.6.1.1); (c) optionally, Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ or Ni 2+ at least one metal cation selected from A composition comprising:

[0183] A composition in which the amount of protein adsorbed on the porous carbon-based material is preferably 10 to 80 parts by weight per 100 parts by weight of the porous carbon-based material, and even more preferably 10 to 50 parts by weight per 100 parts by weight of the porous carbon-based material.

[0184] A composition, wherein the specific activity of an enzyme immobilized on a porous carbon-based material is greater than the specific activity of the same enzyme in free form. [Brief explanation of the drawings]

[0185] [Figure 1] Scanning electron micrograph of a continuously interconnected, uniformly porous structure for tannin-xylose aerogels prepared in 10 mL of a 15 wt % dry solution of absolute ethanol in demineralized water, treated under hydrothermal conditions (autoclave) at 180 °C for 20 h, washed, and then dried by freeze-drying. [Figure 2] Transmission electron micrograph showing macro- and mesoporous networks for tannin-xylose aerogels prepared in 10 mL of a 15% by weight solution of absolute ethanol in demineralized water, treated under hydrothermal conditions (autoclave) at 180 °C for 20 h, washed, and then dried by freeze-drying.

[0186] Figures 1 and 2 show primary particles of a few nanometers that are aggregated primary particles that form an interconnected network that induces interparticle gaps of a few nanometers to a few micrometers. DETAILED DESCRIPTION OF THE INVENTION

[0187] Experimental Department Example 1: Preparation of porous carbon-based materials in the form of aerogels Dissolve 0.85 g of xylose in 10 mL of a mixture of deionized water and 50 / 50 m / m absolute ethanol.

[0188] To the above solution, add 0.35 g of phloroglucinol.

[0189] - The mixture is stirred until a homogenous solution is obtained. Sonication at room temperature for up to 1 hour accelerates the homogenization of the solution. The resulting solution is treated by hydrothermal carbonization, i.e. introduced into a mineralization bomb (autoclave), which is then sealed and left in a thermostatic bath at 180°C for 20 hours. At the end of this period, the autoclave and its contents are cooled to room temperature over several hours (usually 2 hours). The autoclave is then opened and its contents poured into either ultrapure water (typically 100 mL), deionized water, a polar protic water / solvent mixture (methanol, ethanol, propanol, butanol or their isomers) or a pure polar protic solvent. The resulting solid is washed by successive immersions (usually three times 100 mL) for several hours (usually 8 hours) in either ultrapure water, or deionized water, or polar protic water / solvent mixtures (methanol, ethanol, propanol, butanol or their isomers), or pure polar protic solvents. This results in a hydrogel. The hydrogel is then dried by freeze-drying or in a supercritical fluid to obtain an aerogel.

[0190] Example 2: Protein immobilization 10 mg of aerogel obtained under the conditions described above are added to an aqueous solution containing (1 mg) of protein.

[0191] The following enzymes were prepared: Transaminase B9L0N2 (w-TA, EC2 transferase).

[0192] 25 mg of aerogel obtained under the above conditions is added to a buffer solution (Tris 20 mM pH = 7, 1 mL) containing 2.5 mg of the enzyme CalB (Candida antartica lipase B).

[0193] The suspension thus obtained is stirred at 750 rpm at 4° C. for several hours (usually 2 hours).

[0194] After stirring, the suspension is centrifuged (4000-6000 rpm for 2-5 minutes). After centrifugation, the supernatant is removed. The solid thus obtained is washed twice with an aqueous buffer solution: 1) Add buffer (1 mL); 2) The resulting suspension is stirred at room temperature for a few minutes (usually 5 minutes). 3) Centrifuge the suspension (4000-6000 rpm for 2-5 minutes). 4) After centrifugation, remove the supernatant.

[0195] Repeat steps 1 to 4 once.

[0196] This results in proteins immobilized on the porous carbon-based material.

[0197] Example 3: Immobilization Kinetics 10 mg of porous carbon-based material is combined with 1 mg of enzyme to give a total volume of 1 mL.

[0198] Immobilization of transaminase B9L0N2 (w-TA, EC2 transferase) occurs in the presence of pyridoxal phosphate cofactor (PLP).

[0199] The 0, 10, 20 and 30 min samples (10 μL) are diluted in half, while the 1 h and 2 h samples (20 μL) are left undiluted.

[0200] Mix 20 μL of sample (Ech1-2) with 1 mL of Bradford reagent in the dark for 15 min.

[0201] result Kinetics of immobilization of B9L0N2 (1 mg) on ​​xylose / catechin (10 mg) aerogels prepared under the conditions described above.

[0202] [Table 2]

[0203] The results in Table 2 above show that on average, at least 80% immobilization is achieved after 10 minutes.

[0204] These results demonstrate that the immobilization of enzymes onto porous carbon-based materials is simple, rapid, and effective.

[0205] Kinetics of CalB immobilization (2.5 mg) on ​​different aerogels prepared under the above-mentioned conditions.

[0206] [Table 3]

[0207] The results in Table 3 above show that an average immobilization rate of at least 80% is achieved after 10 minutes for XPh medium.

[0208] These results show that for CalB, the XPh material allows for a relatively better immobilization (99.47% at 120 min) than the XCat material (89.825% at 120 min), although the two performances are still quite similar. This difference can be explained by the more pronounced hydrophobic character on the surface of the XPh material than the XCat material. Indeed, it has been shown in the literature that CalB lipase has a higher affinity for immobilization materials with relatively hydrophobic surfaces.

[0209] The material is manually crushed using a mortar and pestle and then sieved by passing it through a first sieve of 300 microns and a second sieve of 100 microns to retain only the fraction between 100 and 300 microns.

[0210] Example 4: Performance Conversion is expressed as moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time.

[0211] For w-TA, the reaction studied is the transamination of pyruvate to alanine in the presence of racemic α-methylbenzylamine and to acetophenone in the presence of pyridoxal phosphate cofactor (PLP). The protein loading used is 10% by weight. The reaction mixture consists of pyruvate (25 mM), racemic α-methylbenzylamine (25 mM), PLP (0.1 mM), and DMSO (1 wt%) dissolved in 50 mM NaCl phosphate buffer, 300 mM pH 7.5. 1 mg of w-TA solid immobilized on a hydrogel (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 4.

[0212] [Table 4]

[0213] Table 4: Catalytic performance (catalytic turnover number, TON) of the studied free or immobilized enzymes (B9L0N2 / w-TA).

[0214] The TON of the immobilized enzyme is significantly higher than that of the non-immobilized enzyme.

[0215] These results are superior to those described to date in the literature, where in most cases it has been observed that the activity of the enzyme is significantly reduced after immobilization.

[0216] Thus, the interconnected macroporous structure (pores between 50 nm and 2 microns) facilitates mass transport of reactants and products throughout the material.

[0217] For CalB, the reaction studied is the hydrolysis of paranitrophenyl butyrate (pNPB) to paranitrophenol and butanoic acid. The reaction mixture consists of pNPB (15 mM) as a solution in 20 mM Tris-HCl buffer, pH = 7.0, containing 1 wt% Genapol X-100. The reaction is initiated by adding immobilized CalB (25 mg of aerogel loaded with 10 wt% enzyme). The test was carried out at 25 °C for 1 h with 800 rpm agitation. The formation of paranitrophenol is monitored by visible spectrophotometry at 410 nm.

[0218] For the immobilized enzyme, the activity is measured over five reaction cycles, which are carried out as follows: 1) Carry out the enzymatic reaction as described above. 2) At the end of the reaction, the suspension is centrifuged at 4000-6000 rpm for 2-5 minutes. 3) The supernatant is collected and 1 mL of buffer solution (20 mM Tris-HCl, pH=7) is added to the solid. 4) Shake the new suspension at 800 rpm for 3 minutes. 5) Repeat steps 2-4 three times.

[0219] The enzyme activity measured for immobilized CalB is expressed as the number of moles of p-nitrophenol formed per mass of catalyst (enzyme immobilized on aerogel) in a given time. To do this, the amount of p-nitrophenol measured in each of the supernatants collected after the previous five steps is summed. The formula is as follows:

number

[0220] These activities are measured for non-immobilized (free) and immobilized enzymes by the techniques described above.

[0221] Conversion is expressed as the number of moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time, according to the following formula:

number

[0222] The results are summarized in Table 5.

[0223] [Table 5]

[0224] Table 5: Conversion rates measured for CalB immobilized on XPh and XCat

[0225] [Table 6]

[0226] Table 6: Enzyme activity measured for CalB immobilized on XPh and XCat

[0227] Example 5: Leaching For each reaction cycle, retain the four supernatants from steps 1-3 of the activity protocol. The amount of protein contained in these solutions is determined by measuring the residual enzyme activity as follows:

[0228] A sample (20 μL) of the supernatant from each reaction cycle was added to a reaction solution containing 1.5 mM substrate (pNPB), 1% genapol, and 20 mM Tris-HCl buffer, pH 7. The solution was stirred at 25°C for 5 minutes. The amount of p-nitrophenol produced during the reaction was compared with a standard range to determine the mass of protein contained in the sample. This yielded the leaching rate, which can be related to the initial amount of protein immobilized on the solid.

number

[0229] The cumulative leaching rate is defined as:

number

[0230] The results are summarized in Table 7.

[0231] [Table 7]

[0232] Table 7: Leaching rates measured at each cycle of use of immobilized enzyme over five reaction cycles on two different supports (XPh: xylose / phloroglucinol and XCat: xylose / catechin).

[0233] As with the immobilization experiments, these experiments show that leaching of CalB (cycle by cycle and cumulative over 5 cycles) is lower when immobilized on XPh than on XCat. This can also be explained by the more hydrophobic nature of the surface of the XPh material than on the surface of the XCat material. Indeed, literature has shown that CalB lipase has a higher affinity for immobilization materials with relatively hydrophobic surfaces.

[0234] Flow experiments (with and without recirculation; no recirculation = continuous flow) Immobilization of CalB on XPh in continuous flow The XPh aerogel (65 mg) obtained under the above conditions is introduced into a thermostatic column whose temperature is maintained at 4° C. The end of the column is made of sinter to retain the aerosol.

[0235] A buffer solution (Tris 20 mM pH = 7, 5 mL) containing 0.250 g of CalB enzyme solution (Lypozyme® STREM CHEMICALS) is pumped into the column at a rate of 0.1 mL / min. The solution is collected at the column outlet. The aerogel stored in the column is then washed by permeating the buffer solution at a rate of 0.1 mL / min for 50 minutes. The solution is also collected at the column outlet.

[0236] The amount of protein present in these two solutions is measured by residual activity by proceeding as follows.

[0237] A sample (20 μL) of the collected solution 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 at 25 °C for 5 min. The amount of p-nitrophenol produced during this reaction is compared with a standard range to determine the mass of protein contained in the sample.

[0238] CalB protein is thus immobilized on XPh hydrogel in the column (at a protein loading of 7.7% by mass) for use in recycling.

[0239] Measurement of conversion rate in a flow-through system with recirculation using CalB enzyme immobilized on XPh The reaction studied is the hydrolysis of paranitrophenyl butyrate (pNPB) to paranitrophenol and butanoic acid. The reaction mixture consisted of pNPB (5 mM) as a solution in 20 mM Tris-HCl buffer, pH 7.0, containing Genapol X-100 (1% by weight).

[0240] The reaction mixture (6 mL) is introduced into the column prepared as described above in the "Continuous Flow Immobilization" section. The flow is recycled, and the solution collected at the column outlet is directly reinjected into the column at a rate of 0.1 mL / min. The column is thermostatically controlled, maintaining the temperature at 35°C. Samples (100 μL) are taken every hour for 5 hours. The formation of paranitrophenol is monitored by visible spectrophotometry at 410 nm.

[0241] Conversion is expressed as the number of moles of product formed per mole of substrate initially present, measured and calculated after a given reaction time, according to the following formula:

number

[0242] Measurement of leaching rate in a flow with recirculation using CalB enzyme immobilized on XPh

[0243] Samples (20 μL) are taken from the collected fractions as described above in the "Activity" section. The amount of protein contained in these samples is determined by the residual activity in the following process.

[0244] 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 minutes at 25°C. The amount of p-nitrophenol produced during the reaction is compared with a standard range to determine the mass of protein contained in the sample. This gives the leaching rate, which can be related to the initial amount of protein immobilized on the solid.

[0245] The leaching rate is given by the following formula:

number

[0246] The cumulative leaching rate is defined as:

number

[0247] The results are summarized in Table 8.

[0248] [Table 8]

[0249] These experiments demonstrate that protein immobilization during flow is possible on our material and is both fast and efficient. CalB immobilized better and released less on XPh than on XCat, suggesting that the hydrophobic properties are more pronounced on the surface of the XPh material than on XCat.

[0250] The conversion observed during flow was the same as that measured for the free enzyme in batch at the same contact time, demonstrating very efficient material transfer and very good accessibility of the substrate to the enzyme.

Claims

1. - at least one carbohydrate, preferably at least one ose or at least one polyoside, at least one phenolic or polyphenolic compound, a porous carbon-based material comprising or formed from these and obtained by hydrothermal carbonization; at least one protein, preferably an immobilized enzyme, said at least one immobilized protein being preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), a pyrophosphatase (EC 3.6.1.1), A composition comprising:

2. - Oath: an aldose selected from among glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, preferably xylose, or a ketose selected from dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose and tagatose, preferably fructose; or - Polyosides: heterodiholoside selected from trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose and rutinose, or a homodiphoroside selected from among inulobiose, alpha 2-mannobiose, alpha 3-mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose and gentiobiose, preferably maltose or sucrose; The composition of claim 1.

3. Phenol or polyphenol compounds: simple phenols selected from phenol, catechol, resorcinol, hydroquinone, pyrogallol and phloroglucinol; phenolic compounds of the flavonoid family selected from the subclasses: flavonols, anthocyanidols, anthocyanins, flavanols, flavanones, stilbenoids, isoflavonoids; Condensed tannins, phlorotannins, chosen in particular from fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, fucophlorethol, fuhalol, equol and diphlorethydroxychamanol; or natural plant extracts containing at least one of these compounds, The composition of claim 1 ,

4. 4. The composition of claim 3, wherein the phenol or polyphenol compound is a compound of the flavanol or catechin subfamily.

5. 5. A composition according to claim 3 or 4, wherein the phenolic or polyphenolic compound is chosen in particular from among catechin or phloroglucinol.

6. The porous carbon material: maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and - Phloroglucinol and catechin derived from catechu extract, The composition of claim 1 formed from

7. (a) an ose selected from among xylose or dihydroxyacetone; - phenolic or polyphenolic compounds selected from phloroglucinol or catechin, a porous carbon-based material comprising or formed from the above and obtained by hydrothermal carbonization, and (b) at least one immobilized protein, preferably an enoate reductase (EC 1.3.1.31), a transaminase (EC 2.6.1), or a pyrophosphatase (EC 3.6.1.1); The composition of claim 1 comprising:

8. 8. The composition according to claim 1, wherein the amount of protein adsorbed on the porous carbon-based material is 10 to 80 parts by weight per 100 parts by weight of the porous carbon-based material, and even more preferably 10 to 50 parts by weight per 100 parts by weight of the porous carbon-based material.

9. 9. The composition of claim 1, wherein the specific activity of the immobilized enzyme on the porous carbon-based material is greater than the specific activity of the same enzyme in free form.

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