WO2024153594A1 - Novel porous carbon-based material comprising an immobilized protein, method for preparing same and uses thereof - Google Patents
Novel porous carbon-based material comprising an immobilized protein, method for preparing same and uses thereof Download PDFInfo
- Publication number
- WO2024153594A1 WO2024153594A1 PCT/EP2024/050826 EP2024050826W WO2024153594A1 WO 2024153594 A1 WO2024153594 A1 WO 2024153594A1 EP 2024050826 W EP2024050826 W EP 2024050826W WO 2024153594 A1 WO2024153594 A1 WO 2024153594A1
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- Prior art keywords
- chosen
- immobilized
- protein
- porous carbon
- phenolic
- Prior art date
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- 239000003575 carbonaceous material Substances 0.000 title claims abstract description 132
- 108010058683 Immobilized Proteins Proteins 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title abstract description 46
- 239000000203 mixture Substances 0.000 claims abstract description 71
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y306/01001—Inorganic diphosphatase (3.6.1.1)
Definitions
- New porous carbonaceous material comprising an immobilized protein, its preparation method and its uses
- the present invention relates to a carbonaceous polymeric material also called porous carbonaceous material or carbon-rich airgel, comprising a protein immobilized thereon and methods of preparing the same.
- the invention further relates to the use of the composition comprising an immobilized protein in heterogeneous biocatalysis applications in particular.
- Porous carbonaceous 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.
- 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.
- This document more particularly describes an original 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 ).
- carbohydrate-based precursors e.g. fructose, glucose or xylose
- phenolic compounds e.g. phloroglucinol
- these carbohydrates can be isolated from the cellulosic fraction of lignocellulosic biomass
- 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.
- State-of-the-art carbon-rich aerogels derived from biomass are obtained after further heat treatment at a temperature above 300°C and offer a wide range of potential applications, such as electrodes for batteries and cells. fuel, or adsorbents for storing hydrogen and carbon dioxide.
- the carbon-rich aerogels or porous carbonaceous materials according to the present invention are not subjected to heat treatment at a temperature > 300°C.
- enzymes are a specific group of proteins that serve as biological catalysts in the metabolism of all living cells. Thus, enzymes are capable of specifically transforming organic and inorganic molecules into products of interest.
- 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 to use them as catalysts in this immobilized state.
- the immobilization of enzymes on solid support has been carried out 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, it has been shown that adsorption of proteins onto 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.
- 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 processes making it possible to prepare said materials.
- a final aim of the invention is to propose particular compositions and their uses.
- the present application relates to a composition
- a composition comprising:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- 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)
- At least one metal cation At least one metal cation.
- the biosourced precursor compound #1 usable according to the present invention is a carbohydrate, preferably at least one ose, or at least one polysaccharide.
- Carbohydrates are defined by the International Union of Pure and Applied Chemistry (IUP AC) as a class of organic compounds containing one carbonyl group (aldehyde or ketone) and at least two hydroxyl groups (-OH). Substances derived from monosaccharides by reduction of the carbonyl group, by oxidation of at least one functional group at the end of the chain into a carboxylic acid or by replacement of one or more hydroxyl groups by an atom of hydrogen, an amino group, a thiol group or any similar atom. Furthermore, in the context of the present application, compounds resulting from the dehydration of carbohydrates (e.g. furan aldehydes) can also be used.
- IUP AC International Union of Pure and Applied Chemistry
- a monosaccharide (or monosaccharide) is a carbohydrate monomer.
- the oses have at least 3 carbon atoms: they are polyhydroxyaldehydes or polyhydroxyketones.
- Bones are distinguished by the length of their carbon chain, as follows:
- trioses 3-carbon oses, CsEfcCL (glyceraldehyde, dihydroxyacetone);
- pentoses 5-carbon oses, C5H10O5 (deoxyribose (C5HI0O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose)
- hexoses 6-carbon oses, CôHnOô (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose);
- deoxyhexoses 6-carbon oses, CôHnOs (fucose, rhamnose); • heptoses: 7-carbon oses, C7H14O7 (sedoheptulose, mannoheptulose)
- 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.
- 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.
- the at least one bone is chosen from:
- • -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
- ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose
- a polysaccharide is a polymer from the carbohydrate family formed of a number n of oses (n > 2) by alpha or beta glycosidic bond.
- the at least one polysaccharide is chosen from:
- trehalulose sucrose, turanose, maltulose, leucrose,
- the biosourced precursor compound #1 of the porous carbon material consists of a carbohydrate, preferably a saccharide, or a polysaccharide as described above.
- the porous carbon material comprises two biosourced precursor compounds #1 which consist of two carbohydrates, preferably two oses, two polysaccharides or one ose and a polysaccharide as described above. According to another mode of the invention, the porous carbon material comprises more than two biosourced precursor compounds #1.
- the at least one phenolic or polyphenolic compound is a biosourced precursor compound having at least one benzene ring substituted by a hydroxyl group and optionally at least one other functional group chosen from the groups carboxylic acid, aldehyde, ether, ester, alcohol, hydroxyl and ketone.
- biomass designates 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.
- compound of “natural origin” or biosourced within the meaning of the invention we mean any compound originating from nature (biomass of plant or animal origin).
- This compound can be extracted from renewable terrestrial and marine biomass or from living organisms (animals, micro-organisms), possibly subsequently modified for example chemically, or even obtained following the action of living micro-organisms (for example enzymes or bacteria) on a compound of natural origin according to biofermentation or biosynthesis type processes.
- living micro-organisms for example enzymes or bacteria
- the biosourced precursor compound #2 usable according to the present invention may be a phenol whose benzene ring is further substituted by at least one other functional group chosen from the groups carboxylic acid (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 CH2OH group), hydroxyl and ketone, preferably the groups hydroxyl, carboxylic acid and aldehyde. Any salts of these groups also fall within the scope of the invention.
- the biosourced precursor phenolic or polyphenolic compound usable according to the present invention can be chosen from secondary metabolites of plants.
- Phenolic compounds form the most important group of phytochemicals in plants. They present nearly 8000 molecules divided into around ten chemical classes. Each class is characterized by the presence of a benzoic 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 wood). They are synthesized by plants subjected to difficult conditions (infections, wounds, UV radiation, etc.) and are involved in numerous physiological processes such as cell growth, rhizogenesis, seed germination or fruit ripening.
- 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, made up of one or more benzene rings carrying one or more hydroxyl functions. They can be grouped into numerous classes depending on the complexity of the basic skeleton (C6 core), the degree of modification of this skeleton (oxidation, hydroxylation, etc.) and finally according to the molecules with which they are associated (carbohydrates, lipids, etc.). , proteins, other metabolites).
- the simplest forms are represented by two main groups from which many compounds are derived: hydroxycinnamic acids and flavonoids.
- the complex forms for their part, are mostly the result of the condensation of certain simple forms and contain, among others, tannins and lignins.
- Phenolic acids C ⁇ -CI and C6-C3
- flavonoids C ⁇ -CS-C ⁇
- Lignans C6-C3-C3-C ⁇
- the last group is that of stilbenes (C6-C2-C6); the best known is resveratrol, present in the skin of grapes.
- Flavonoids represent the largest group of polyphenols and the most widely distributed in the plant kingdom. More than 4000 flavonoids different ones 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.
- Condensed tannins also called proanthocyanidins, constitute a particularly interesting, because abundant, source of such phenolic compounds capable of constituting starting products for the formation of epoxy prepolymers.
- condensed tannins are phenolic biopolymers essentially present in soft tissues with rapid growth and renewal, such as leaves, stems, etc. This class of polyphenols are the most abundant after lignins.
- lignins which are constituents of lignocelluloses, structuring 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 agroindustrial residues, for example in fruit marc, and unexploited biomass, in particular in the bark, leaves and needles of trees, vines. , fruits, etc.
- Condensed tannins are non-hydrolysable compounds which can be depolymerized into anthocyanidins when they are heat treated with an acid. They are usually named after the anthocyanidol thus released.
- the monomer (+)-catechol (a flavan-3-ol) is a constituent unit of the dimer catechol -(4a ⁇ 8)-catechol which we will call procyanidol B-3 due to its degradation in an acid medium in cyanidol.
- Many condensed tannins are catechol polymers. This is the reason why condensed tannins are also called “proanthocyanidol” or “proanthocyanidins” (based on the English proanthocyanidins).
- the OH hydroxyls are in the same positions on the monomer and the depolymerization product, only the central ring changes.
- flavanol monomeric units used in the construction of condensed tannins. They can also be substituted by gallic acid or by sugars, generally in position 3 and sometimes in positions 5 and 7.
- Flavanols and anthocyanidols all have three rings: an A ring with generally one or two OH hydroxyls, a C heterocycle with asymmetric carbons 2 and 3 (and a hydroxyl at 3), and a B ring with an OH hydroxyl (afzeléchol, epiafzeléchol ), 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.
- the at least one phenolic or polyphenolic compound is a simple phenol chosen from phenol, catechol, resorcinol, hydroquinone, pyrogallol, or phloroglucinol.
- the at least one phenolic or polyphenolic compound is a compound from the flavonoid family.
- the at least one phenolic or polyphenolic compound is a compound chosen from the subclasses: flavonol, anthocyanin, flavanol, flavanone, isoflavonoid.
- the at least one phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily, 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.
- the at least one phenolic or polyphenolic compound is chosen from: catechin in one of its stereoisomeric forms, gallocatechin, afzéléchol, fisetinidol, guibourtinidol, mesquitol, or robinetinidol.
- the at least one phenolic compound is phloroglucinol.
- the at least one polyphenolic compound is catechin.
- the at least one phenolic or polyphenolic compound is contained in a natural plant extract.
- 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, soya, pea, pine, tomato, garlic or cachou.
- the at least one 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 in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,
- a phlorotannin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
- the porous carbon material comprises two biosourced precursor compounds #2 which consist of two phenolic compounds or polyphenolics as described above. According to another embodiment of the invention, the porous carbon material comprises more than two biosourced precursor compounds #2.
- the porous carbonaceous material obtained by hydrothermal carbonization comprises or is formed from:
- maltose galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, or dihydroxyacetone
- Cachou extract can be obtained from Acacia cachou plant fibers, said extract containing condensed tannins and flavonols.
- 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 this application.
- the porous carbon material can be obtained by a sol-gel polymerization process, known to those skilled in the art, involving at least two biosourced precursors # 1 and 2 such as defined above.
- 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.
- 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.
- Catalyst concentrations can be expressed as reactant-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.
- R/C reactant-catalyst ratio
- the porous carbonaceous material is obtained by hydrothermal carbonization.
- Hydrothermal carbonization is defined as being a process of thermochemical conversion of biomass or in the context of the present invention at least two biosourced precursors # 1 and 2 as defined above with the aim of obtaining a porous carbon monolith in aqueous phase. It is an exothermic process that decreases the oxygen and hydrogen levels of matter 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 biosourced precursors #1 and 2 as defined above and a solvent, preferably water (generally 10% by weight of the two precursors biosourced #1 and 2).
- the porous carbonaceous 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 solution aqueous, at a temperature below 300°C to obtain a solid porous carbon material, b) Wash the solid porous carbon material obtained by successive soaking in a polar solvent.
- the aqueous solution comprises water, and optionally solvents, preferably in quantities miscible with water.
- the reaction mixture comprises only one liquid phase, namely the aqueous solution, preferably water.
- the aqueous solution comprises ethanol, preferably absolute ethanol.
- the mass fraction of water in a water/absolute ethanol mixture is between 1 and 0.3.
- the mass fraction of water in a water/absolute ethanol mixture is between 0.7 and 0.4 when the biosourced precursors #1 and 2 are respectively xylose and phloroglucinol.
- the mass fraction of water in a water/absolute ethanol mixture is between 1 and 0.5 when the biosourced precursors #1 and 2 are respectively xylose and catechin.
- Step (a) of the process can also be called the “hydrothermal treatment step”.
- 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-like material (typically in the absence of oxygen).
- 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 the more preferably from 160 to 200°C or even preferably at 180°C.
- Reaction temperature is intended to refer to the temperature, more specifically the average temperature, inside the reaction mixture, which can be measured with a thermocouple.
- the hydrothermal treatment in the process for preparing the porous carbonaceous material of the present invention is preferably carried out in a pressure-resistant reactor, for example an autoclave.
- step (a) there is no specific limitation as to the duration of step (a) in the process for preparing the porous carbonaceous material of the invention.
- reaction medium can be introduced into a hermetically closed digestion bomb, then placed in an enclosure thermostatically controlled at 180°C for 20 hours.
- Step (a) of the process is carried out until the entire content of the reaction mixture comprising the aqueous solution and the biosourced precursors #1 and 2 has gelled or precipitated into the porous carbonaceous material of the present invention.
- the porous carbonaceous material is prepared by the following process: ajHeating a reaction mixture comprising:
- the porous carbonaceous material is prepared by the following process: ajHeating a reaction mixture comprising:
- 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 compound. or polyphenolic (c2) in a molar ratio (cl/c2) of 10/1 to 1/10, preferably 2/1.
- 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. -say the number of moles of monomeric units (for example flavonoids), in said condensed tannin or said complex natural extract.
- the porous carbon material is prepared by the following process: a) Heating a reaction mixture comprising:
- the porous carbon material is prepared by the following process: a) Heating a reaction mixture comprising:
- the porous carbon material is made up of
- element oxygen preferably 60% by mass of carbon element (C) and 35% by mass of oxygen element (O).
- the porous carbon material consists of 36% O and 60% C for the xylose/phloroglucinol system.
- the porous carbon material is formed from at least one carbohydrate (cl), preferably at least one ose, or at least one polysaccharide (cl) and at least one phenolic compound (c2 ) or polyphenolic in a molar ratio (cl/c2) of 10/1 to 1/10, preferably 2/1.
- the solid porous carbonaceous material is washed by soaking in either ultrapure water, deionized water or a water/polar protic solvent mixture, for example methanol, ethanol or tert-butanol for several hours.
- ultrapure water deionized water
- a water/polar protic solvent mixture for example methanol, ethanol or tert-butanol for several hours.
- 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 step.
- 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, the aqueous solution forming the dispersion phase.
- the carbonaceous material before drying can be called solvogel.
- the carbonaceous material obtained in step (a) can be called hydrogel.
- a hydrogel is considered here as a special type of solvogel, in which the aqueous solution of the dispersion phase is water.
- the solid porous carbonaceous material obtained or obtainable according to the process described above can be dried and transformed into cryogel, airgel or xerogel.
- 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 airgel.
- a supercritical fluid in particular acetone or carbon dioxide
- 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 freeze-drying of said frozen gel to transform it into a cryogel.
- the carbon material i.e. hydrogel or solvogel
- a dry form of hydrogel is obtained by subjecting the carbon material (ie hydrogel or solvogel) to evaporation of the solvent at controlled temperature and pressure to transform it into a xerogel.
- a cryogel can be called an aerogel when the dried gel has largely retained the textural properties after drying.
- Dried gels can be called aerogels regardless of the drying technique used.
- the porous carbonaceous 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 solution aqueous, at a temperature below 300°C, preferably at 180°C to obtain a solid porous carbonaceous material, b) Wash the solid porous carbonaceous material obtained by successive soaking in a polar solvent (ideally in absolute ethanol or in a water/tert-butanol mixture 75/25 in percentages by weight), c) Dry the solid porous carbonaceous material using a supercritical fluid or by freeze-drying.
- a polar solvent ideally in absolute ethanol or in a water/tert-butanol mixture 75/25 in percentages by weight
- hydrogels placed in borosilicate glass beakers are previously placed in immersion in liquid nitrogen (77 K) for 15 minutes, then directly placed in the chamber of the lyophilizer (e.g. a COSMOS lyophilizer 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.
- lyophilizer e.g. a COSMOS lyophilizer from Cryotec
- the solvent in the solvogel is preferably chosen from absolute ethanol or acetone (for example brought by replacement of solvent with absolute ethanol or acetone).
- porous carbon material obtained or obtainable in step (a) is a gelled material, in particular a solidified material composed of aggregated particles forming an interconnected network.
- the porous carbonaceous material obtained or obtainable according to the process described above comprises micropores (pores ⁇ 2 nm), mesopores (pores of 2-50 nm) and macropores (pores > 50 nm).
- the oxygen content is typically greater than 25% for materials directly obtained in the hydrothermal carbonization step (a).
- the surface of the carbon material of the invention as obtained in step (a) is very hydrophilic.
- 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 gaps (or pores) with a diameter 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 again more preferably 40 to 80 nm and preferably around 60 nm.
- the porous carbonaceous material according to the present invention is an aggregated material composed of particles, with a pore size distribution calculated from a nitrogen adsorption/desorption isotherm at 77 K (for the largest pores). narrow; ⁇ 100 nm) and a mercury intrusion porosimetry profile (for the widest pores; > 50 nm).
- the pore size distribution and the pore diameter which corresponds to the peak of the pore size distribution can be provided by the BJH analysis of the nitrogen adsorption/desorption isotherm.
- the pore diameter of the carbon gel according to the present invention 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 carbonaceous material according to the present invention, corresponding to the peak of the pore size distribution, is preferably greater to the average molecular diameter of the diameter of the adsorbed or complexed components.
- the porous carbonaceous 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.
- porous carbonaceous material has a specific surface area of 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 accommodated will be reduced.
- 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.
- the porous carbonaceous 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.
- the so-called total volume was determined at a relative pressure of 0.99 on the nitrogen adsorption/desorption isotherms at -196°C.
- the micropore surface area and volume were determined by applying the t-plot method.
- the 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. Analyzes were performed on a TriStar Micromeritics.
- the total pore volume in the materials is greater 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.
- the specific surface area and the total pore volume of the porous carbon material according to the present invention can be determined by a general volumetric measurement described below. below. Specifically, a porous carbonaceous 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 against each equilibrium pressure. We thus obtain the nitrogen adsorption/desorption isotherm.
- the specific surface area can be determined by the Brunauer Emmett Teller (BET) method, for example by the dinitrogen adsorption technique.
- BET Brunauer Emmett Teller
- the BET equivalent specific surface area was determined by nitrogen adsorption at 77K by carrying out 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).
- This parameter can be determined by mercury intrusion porosimetry indicating the total volume of interconnected pores.
- porous carbon materials as described above can be complexed with metal ions, preferably at least one type of metal cation which is complexed on the external surfaces and the surfaces of the internal pores of the porous carbon material according to the invention.
- the at least one metal cation is chosen from 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 transition metals Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ .
- a chelate is a type of coordination compound in which a single metal ion is attached by coordinated covalent bonds to a molecule or ion called a ligand.
- a coordination compound is formed when groups of atoms, ions, or molecules bond together chemically to each other by donating and accepting pairs of electrons. Groups that donate electron pairs are called ligands. These are generally Lewis bases. The groups accepting electron pairs are often transition metal cations.
- the composition is prepared by the following process: a) Heating a reaction mixture comprising an aqueous solution, and at least two biosourced precursors # 1 and 2 as defined above dissolved in the aqueous solution, at a temperature below 300°C to obtain a solid porous carbon material, b) Wash the solid porous carbon material obtained by successive soaking in a polar solvent, c) Dry the solid porous carbon material with using a supercritical fluid or by freeze-drying, d) Impregnate the solid porous carbon material with a solution comprising metal cations to obtain a porous carbon hybrid material complexed with metal cations.
- the adsorption process to allow the metal complex to adsorb on carbon material, and the adsorption conditions are not particularly limited.
- a metal complex can optionally be adsorbed onto a carbonaceous material by placing the porous carbonaceous material in a solution and mixing the solution for a certain period of time at approximately 10 to 100°C.
- the hybrid material is separated from the solution by centrifugation, and the suspension thus obtained is washed and then dried.
- the composition 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 below 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) Dry the solid porous carbon hybrid material using a supercritical fluid or by freeze drying.
- 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.
- the quantity of metal ions adsorbed or complexed on a carbon material is preferably between 0.1 and 40 parts by weight relative to 100 parts by weight of the porous carbon material.
- the composition comprises a quantity of chelated or complexed metal cations, measured by the MEB-EDX method, comprised from 0.2 to 2 mole%, preferably from 0.5 to 1 mole%.
- the composition optionally comprises a quantity of 1 to 5% by weight of metal cation, in particular 2 to 4% by weight.
- the composition comprises a quantity of 20 to 40 mg of iron per gram of porous carbonaceous material.
- the solvent used here 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.
- the concentration of metal complex in the solution is not particularly limited. However, the concentration is preferably about 0.1 to 30 mM.
- metal cation chosen from 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 +
- the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- -the bone is chosen from
- 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
- ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or
- polysaccharide is chosen from:
- a heterodisaccharide 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 and
- -at least one phenolic or polyphenolic compound biosourced precursor #2.
- the composition comprises: A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- -the bone is chosen from
- an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose , galactose, talose, preferably xylose, or
- ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, tagatose, preferably fructose, or
- polysaccharide is chosen from:
- a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, rutinose, or
- a homodiholoside chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobiose , preferably maltose or sucrose,
- a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,
- a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
- a phlorotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
- composition comprises:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- composition comprises:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- composition comprises:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- composition comprises:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- compositions described above can be used to immobilize proteins, in particular at least one enzyme
- 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 with said material and at least one protein (in particular an enzyme) immobilized (on the carbon material porous optionally charged with at least one metal cation), in which 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.
- the protein used here is not particularly limited.
- composition of the present invention thus comprises at least one enzyme chosen from the approved list of the nomenclature and classification of enzymes of the International Union of Biochemistry (IUBMB) and examples thereof include the following proteins:
- 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) .
- 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) .
- the amount of protein adsorbed on the porous carbon material is not particularly limited as long as it exhibits enzymatic activity.
- 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 porous carbonaceous material.
- the method for providing the porous carbon hybrid material of the present invention by causing adsorption of a protein onto 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 buffer at a concentration at which the protein is not precipitated (preferably at a concentration of 0.1 to 1000 mg/ ml).
- a porous carbonaceous 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.)
- 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.
- the carbon hybrid material of the present invention can be provided.
- the composition comprising at least one immobilized protein is prepared by the process comprising the steps consisting of: - Suspend the dried porous carbon material in a solution that may contain metal cations,
- porous carbon hybrid material optionally loaded with metal cations is added to an aqueous solution containing a protein, preferably an enzyme.
- porous carbon hybrid material optionally loaded with metal cations on which at least one protein is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and/or coordination bonds and/or ionic bonds, is washed several times. times with a buffered aqueous solution.
- the protein to be immobilized on the material can be any protein, such as a (recombinant) protein or an enzyme.
- the protein is an enzyme.
- the buffered aqueous solution is preferably a Tris-HCl, or Phosphate, buffer.
- the present application relates to a composition
- a composition comprising: a) A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- -at least one phenolic or polyphenolic compound biosourced precursor #2
- c) at least one protein preferably an enzyme
- the composition comprises a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- -at least one phenolic or polyphenolic compound biosourced precursor #2
- the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- ose preferably at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which the ose is chosen from:
- 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 in which the polysaccharide is chosen from:
- a heterodisaccharide 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, and
- a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol
- a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid
- a phlorotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol, or
- a natural plant extract containing at least one of the compounds above b) optionally at least one metal cation chosen from 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+ , and c) at least one immobilized protein.
- metal cation chosen from Al 3+ , Ag + , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3 + , Cu + , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn
- the composition comprises: a) A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- the binding of the enzyme to the support must be reversible.
- proteins immobilized on the support are enzymes, they contain an active site capable of catalyzing a chemical reaction.
- the immobilized enzyme material is potentially useful as a biocatalyst in organic synthesis.
- the invention relates to the use of an immobilized enzyme material as described herein as a heterogeneous biocatalyst, for example in synthetic organic transformations.
- a preferred mode of the invention concerns the use of the compositions described above as heterogeneous biocatalysts.
- the present invention further relates to a method of catalyzing an enzyme-catalyzed reaction, comprising providing a composition comprising a protein or enzyme immobilized on said porous carbonaceous material according to the invention, and contacting of said composition comprising an enzyme immobilized with at least one substrate on which the enzyme, which is immobilized on said material, is capable of acting.
- the immobilized enzymes were found to tolerate both aqueous conditions as well as a range of organic solvents.
- compositions comprising a protein or an enzyme immobilized on said porous carbon material under reaction conditions in which the free, non-immobilized enzymes would not have been stable.
- composition comprising a protein or enzyme immobilized on said porous carbonaceous material can also be used in a wider pH range than free, non-immobilized enzymes would have tolerated.
- the enzyme which is immobilized on said material may be any enzyme which is useful as a biocatalyst in organic synthetic transformations, including, but not limited to, enzymes acting as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
- 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.
- biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.
- biocatalytic reactions are enantioselective reactions.
- biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.
- two or more different enzymes can be immobilized on the material according to the invention, wherein each of the different enzymes is capable of catalyzing a different reaction.
- the present application relates to a method for catalyzing an enzyme-catalyzed reaction, comprising providing a composition described above, and bringing said composition into contact with at least one substrate on which an enzyme, which is immobilized on said support of the composition, is capable of acting.
- the immobilization yields expressed in mg of immobilized enzyme per mg of enzyme initially present in the solution, are between 90 and 100% for protein loads between 5 and 50% by weight.
- the immobilization speeds expressed in minutes to achieve at least an immobilization rate of at least 75% are between 10 and 120 minutes.
- the process for immobilizing a protein, in particular an enzyme on the porous carbon material is characterized in that the efficiency of the immobilization is between 90% and 100% for protein loads ranging from 5 to 50% by weight.
- the method of immobilizing a protein, in particular an enzyme on the porous carbon material is characterized in that the speed of immobilization to achieve a yield of the immobilization of at least 75% is between 10 and 120 minutes.
- the present application relates to the use of the composition as a heterogeneous biocatalyst.
- the present application concerns:
- a composition comprising:
- a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- 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)
- 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
- ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or
- a heterodisaccharide 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
- a composition 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 in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
- phi orotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
- a composition in which the phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily.
- a composition in which the phenolic or polyphenolic compound is chosen in particular from: catechin or phloroglucinol.
- a composition in which the porous carbonaceous material is obtained by hydrothermal carbonization comprising or formed from:
- a composition in which the at least one metal cation when present is chosen from 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 transition metals Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ .
- a composition comprising: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
- 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 Ni 2+ .
- 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 carbonaceous material.
- Figure 1 is a scanning electron micrograph of a continuously interconnected uniform porous texture for a tannin-xylose airgel prepared in 10 mL of a 15 mass% solution of absolute ethanol in deionized water, treated at 180° C for 20 hours in hydrothermal conditions (autoclave), washed then dried by freeze-drying.
- Figure 2 is a transmission electron micrograph which highlights a macro-mesoporous network for a tannin-xylose airgel prepared in 10 mL of a 15% solution by weight of absolute ethanol in demineralized water, treated at 180 °C for 20 hours in hydrothermal conditions (autoclave), washed then dried by freeze-drying.
- Figures 1 and 2 show primary particles of a few nanometers, aggregated primary particles forming an interconnected network which induces interparticle gaps of a few nanometers to several micrometers.
- Example 1 preparation of a porous carbon material in the form of an airgel
- xylose 0.85 g is dissolved in 10 mL of a mixture of deionized water and absolute ethanol 50/50 m/m.
- the mixture is stirred until a homogeneous solution is obtained.
- Ultrasonic treatment at room temperature for an hour or less can accelerate the homogenization of the solution,
- the resulting solution is treated by hydrothermal carbonization, that is to say introduced into a mineralization bomb (autoclave), then this is closed hermetically and placed in an enclosure thermostatically controlled 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),
- the autoclave is then opened and its contents poured either into ultrapure water (generally 100 mL), or into deionized water, or into a water/polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or in a pure polar protic solvent,
- the solid obtained is washed by successive soaking (generally 3 times 100 mL) with either ultrapure water, deionized water, or a water/polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or in a pure polar protic solvent for several hours (generally 8 hours).
- a hydrogel The hydrogel is then dried by freeze-drying or in a supercritical fluid to obtain an aerogel.
- the following enzyme was prepared: B9L0N2 transaminase (w-TA, EC2 transferase).
- the suspension thus obtained is stirred at 750 rpm for a few hours (generally 2 hours) at 4°C.
- Steps 1 to 4 are repeated once.
- Example 3 Immobilization kinetics 10 mg porous carbonaceous material is brought into contact with 1 mg of enzyme in a total volume of 1 mL.
- the immobilization of the B9L0N2 transaminase occurs in the presence of the cofactor pyridoxal phosphate (PLP).
- the 0, 10, 20 and 30 min samples (10 pL) are diluted to half and the 1h and 2h samples (20 pL) are not diluted.
- the materials are crushed manually with mortar 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.
- Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time.
- the reaction studied is the transamination of pyruvate in the presence of racemic a-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP).
- the protein load used is 10% by mass.
- the reaction mixture is consisting of pyruvate (25mM), racemic a-methylbenzylamine (25mM) PLP (0.lmM) and DMSO (1% wt) in solution in Phosphate buffer 50mM NaCl 300mM pH 7.5.
- the solid consisting of Img of w-TA immobilized on a hydrogel (lOmg) 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.
- the TON of the immobilized enzyme is significantly higher than that of the non-immobilized enzyme.
- the interconnected macroporous structure (pores between 50 nm and 2 microns) therefore facilitates the mass transfer of reactants and products throughout the material.
- the reaction studied is the hydrolysis of / /ra-nitrophenyl butyrate (pNPB) to para-nitrophenol and butanoic acid.
- the reaction is started by adding immobilized CalB (25 mg of airgel loaded with 10% by weight of enzyme). The tests were carried out at 25°C for 1 hour with stirring at 800 rpm. The formation of / /ra-nitrophenol is followed by visible spectrophotometry at 410 nm.
- activity is measured over 5 reaction cycles.
- a reaction cycle proceeds as follows:
- the suspension is centrifuged for 2 to 5 min between 4000 and 6000 rpm.
- Steps 2 to 4 are repeated three times.
- the enzymatic activity measured for immobilized CalB is expressed as moles of p-nitrophenol formed per mass of catalyst (enzyme immobilized on the airgel) for 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:
- Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time.
- the formula is as follows:
- 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.
- 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)
- 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.
- a buffer solution (20 mM Tris pH 7.5 mL) containing 0.250 g of a CalB enzymatic solution (Lypozyme ® STREM CHEMICALS) percolates at a flow rate of 0.1 mL/min in the column by means of a pump. The solution is recovered at the outlet of the column. The airgel 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 outlet of the column.
- a CalB enzymatic solution Lipozyme ® STREM CHEMICALS
- the amount of proteins present in these two solutions are measured by residual activity using the following procedure.
- the reaction studied is the hydrolysis of para-nitrophenyl butyrate (pNPB) into para-nitrophenol and butanoic acid.
- the reaction mixture (6 mL) is introduced into the column prepared as described previously in the “continuous flow immobilization” section.
- the flow is in recirculation, 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 followed by visible spectrophotometry at 410 nm.
- Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time.
- the formula is: 100 • Measurement of flow leaching rate with recirculation using the CalB enzyme immobilized on XPh
- a sample (20 ⁇ L) is taken from the fractions recovered as described previously in the “activity” section.
- the quantity of proteins contained in these samples is determined by residual activity using the following method.
- the leaching rate is obtained by the following formula:
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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
Nouveau matériau carboné poreux comprenant une protéine immobilisée, son procédé de préparation et ses utilisations New porous carbonaceous material comprising an immobilized protein, its preparation method and its uses
DOMAINE DE L’INVENTION FIELD OF INVENTION
La présente invention concerne un matériau polymérique carboné également appelé matériau carboné poreux ou aérogel riche en carbone, comprenant une protéine immobilisée sur ledit matériau et des procédés de préparation de ceux-ci. The present invention relates to a carbonaceous polymeric material also called porous carbonaceous material or carbon-rich airgel, comprising a protein immobilized thereon and methods of preparing the same.
L'invention concerne en outre l'utilisation de la composition comprenant une protéine immobilisée dans des applications de biocatalyse hétérogène notamment. The invention further relates to the use of the composition comprising an immobilized protein in heterogeneous biocatalysis applications in particular.
CONTEXTE DE L’INVENTION BACKGROUND OF THE INVENTION
Les matériaux carbonés poreux ou aérogels riches en carbone, connus de l'état de la technique, sont classiquement obtenus par des réactions de polymérisation sol-gel entre des précurseurs organiques riches en carbone et un séchage des hydrogels ainsi obtenus. Porous carbonaceous 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.
Un matériau carboné poreux du type de celui préparé et utilisé dans la présente demande est décrit dans la publication «Hydrothermal synthesis of highly porous carbon monoliths from carbohydrates and phloroglucinol»; Nicolas Brun et al.; RSC Advances, 2013, 3. 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.
Ce document décrit plus particulièrement une approche hydrothermale originale pour synthétiser des cryogels et des aérogels riches en carbone (ou carbogels) hautement poreux à partir de précurseurs à base de glucides (par exemple fructose, glucose ou xylose) et de composés phénoliques (par exemple phloroglucinol). Il faut mentionner que ces glucides peuvent être isolés de la fraction cellulosique de la biomasse lignocellulosique, tandis que le phloroglucinol est l’unité monomère des phlorotannins et peut être isolé de l’écorce des arbres fruitiers, des algues brunes ou par diverses voies de biosynthèse. Par conséquent, les composés utilisés dans la présente demande de brevet peuvent être considérés comme renouvelables et durables. This document more particularly describes an original 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.
Les aérogels riches en carbone de l’état de la technique, dérivés de la biomasse sont obtenus après un traitement thermique supplémentaire à une température supérieure à 300°C et offrent un large éventail d’applications potentielles, telles que des électrodes pour batteries et piles à combustible, ou des adsorbants pour le stockage de l’hydrogène et du dioxyde de carbone. Les aérogels riches en carbone ou matériaux carbonés poreux selon la présente invention ne sont pas soumis à un traitement thermique à une température > à 300°C.
Par ailleurs, les enzymes sont un groupe spécifique de protéines qui servent de catalyseurs biologiques dans le métabolisme de toutes les cellules vivantes. Ainsi, des enzymes sont capables de transformer spécifiquement des molécules organiques et inorganiques en produits d’intérêt. State-of-the-art carbon-rich aerogels derived from biomass are obtained after further heat treatment at a temperature above 300°C and offer a wide range of potential applications, such as electrodes for batteries and cells. fuel, or adsorbents for storing hydrogen and carbon dioxide. The carbon-rich aerogels or porous carbonaceous materials according to the present invention are not subjected to 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 capable of specifically transforming organic and inorganic molecules into products of interest.
Cependant, puisque les enzymes sont des molécules biologiques développées pour un environnement cellulaire, elles sont souvent inadaptées à d'autres environnements. Il est donc intéressant de pouvoir immobiliser des enzymes sur un support solide et de les utiliser comme catalyseurs dans cet état immobilisé. 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 to use them as catalysts in this immobilized state.
L'immobilisation d'enzymes sur support solide a été réalisée en utilisant différentes techniques et différents supports solides. L'adsorption d'enzymes sur des surfaces solides peut conduire à des interactions indésirables entre l'enzyme et le support solide. Par exemple, il a été montré que l'adsorption de protéines sur des nanoparticules de silice peut conduire à des changements de structure secondaire de la protéine, ce qui peut conduire à une désactivation de l'enzyme. Il est donc important que le support solide n'interfère pas avec la structure et l'activité des enzymes immobilisées. The immobilization of enzymes on solid support has been carried out 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, it has been shown that adsorption of proteins onto 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.
Les coûts élevés concernant la préparation d'enzymes et la perte d'activité fréquemment observée lors de l'immobilisation de l'enzyme sur un support solide sont des obstacles dans ce développement. Une procédure standardisée et généralement utilisable pour l'immobilisation d'enzyme, qui permettrait à l'enzyme d'être réutilisée, serait hautement souhaitable. Il n'existe toujours aucun procédé général et simple pour la préparation de catalyseurs hétérogènes par immobilisation d'enzymes. Il existe donc un besoin pour de nouveaux supports et des procédés améliorés d'immobilisation d'enzymes sur ces supports, et pour des biocatalyseurs hétérogènes stables qui peuvent être appliqués en synthèse organique dans des conditions de réactions aqueuse ou organique. The high costs regarding the preparation of enzymes and the loss of activity frequently observed 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. There is therefore a need for new supports and improved methods of immobilizing enzymes on these supports, and for stable heterogeneous biocatalysts which can be applied in organic synthesis under aqueous or organic reaction conditions.
BREF APERÇU DE L’INVENTION BRIEF OVERVIEW OF THE INVENTION
Dans ce contexte d’une recherche d’outils de biocatalyse adaptés et efficaces, un premier but de l’invention est de proposer un matériau carboné poreux comprenant une protéine immobilisée sur ledit matériau. Un second but de l’invention est de proposer des procédés permettant de préparer lesdits matériaux. Enfin, un dernier but de l’invention est de proposer des compositions particulières et leurs utilisations. In this context of a search for suitable and efficient 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 processes making it possible to prepare said materials. Finally, a final aim of the invention is to propose particular compositions and their uses.
DESCRIPTION DETAILLEE DE L’INVENTION
Selon un premier aspect de l’invention, la présente demande concerne une composition comprenant : DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the invention, the present application relates to a composition comprising:
• Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir: • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside, -d’au moins un composé phénolique ou polyphénolique, -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, -at least one phenolic or polyphenolic compound,
• Au moins une protéine immobilisée, ladite au moins une protéine immobilisée est de préférence une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1) • 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)
• Optionnellement, au moins un cation métallique. • Optionally, at least one metal cation.
Le composé précurseur biosourcé #1 utilisable selon la présente invention est un glucide, de préférence au moins un ose, ou au moins un polyoside. The biosourced precursor compound #1 usable according to the present invention is a carbohydrate, preferably at least one ose, or at least one polysaccharide.
Les glucides sont définis par l'Union internationale de chimie pure et appliquée (IUP AC) comme une classe de composés organiques contenant un groupe carbonyle (aldéhyde ou cétone) et au moins deux groupes hydroxyle (-OH). On inclut dans cette classe les substances issues des monosaccharides par réduction du groupe carbonyle, par oxydation d'au moins un groupe fonctionnel à l'extrémité de la chaîne en acide carboxylique ou par remplacement d'un ou plusieurs groupes hydroxyle par un atome d'hydrogène, un groupe amino, un groupe thiol ou par tout atome similaire. Par ailleurs, dans le cadre de la présente demande, les composés issus de la déshydratation des glucides, (p.e les aldéhydes furaniques) peuvent également être utilisés. Carbohydrates are defined by the International Union of Pure and Applied Chemistry (IUP AC) as a class of organic compounds containing one carbonyl group (aldehyde or ketone) and at least two hydroxyl groups (-OH). Substances derived from monosaccharides by reduction of the carbonyl group, by oxidation of at least one functional group at the end of the chain into a carboxylic acid or by replacement of one or more hydroxyl groups by an atom of hydrogen, an amino group, a thiol group or any similar atom. Furthermore, in the context of the present application, compounds resulting from the dehydration of carbohydrates (e.g. furan aldehydes) can also be used.
Un ose (ou monosaccharide) est un monomère de glucide. Les oses possèdent au moins 3 atomes de carbone : ce sont des polyhydroxyaldéhydes ou des polyhydroxycétones. A monosaccharide (or monosaccharide) is a carbohydrate monomer. The oses have at least 3 carbon atoms: they are polyhydroxyaldehydes or polyhydroxyketones.
On distingue les oses par la longueur de leur chaîne carbonée, comme suit : Bones are distinguished by the length of their carbon chain, as follows:
• les trioses : oses à 3 carbones, CsEfcCL (glycéraldéhyde, dihydroxyacétone) ;• trioses: 3-carbon oses, CsEfcCL (glyceraldehyde, dihydroxyacetone);
• les tétroses : oses à 4 carbones, C4H8O4 (érythrose, thréose, érythrulose) ; • tetroses: 4-carbon oses, C4H8O4 (erythrose, threose, erythrulose);
• les pentoses : oses à 5 carbones, C5H10O5 (désoxyribose (C5HI0O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose) • pentoses: 5-carbon oses, C5H10O5 (deoxyribose (C5HI0O4), ribose, arabinose, xylose, lyxose, ribulose, xylulose)
• les hexoses : oses à 6 carbones, CôHnOô (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose) ; • hexoses: 6-carbon oses, CôHnOô (allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose);
• les désoxyhexoses : oses à 6 carbones, CôHnOs (fucose, rhamnose) ;
• les heptoses : oses à 7 carbones, C7H14O7 (sédoheptulose, mannoheptulose)• deoxyhexoses: 6-carbon oses, CôHnOs (fucose, rhamnose); • heptoses: 7-carbon oses, C7H14O7 (sedoheptulose, mannoheptulose)
• les octoses : oses à 8 carbones, CSHIÔOS (heptahydroxyoctanal) ; • octoses: 8-carbon oses, CSHIÔOS (heptahydroxyoctanal);
• les nonoses : oses à 9 carbones, C9H17N1O8 (acide neuraminique ou acide sialique). • nonoses: 9-carbon oses, C9H17N1O8 (neuraminic acid or sialic acid).
Un aldose est un monosaccharide constitué d’une chaîne de n atomes de carbone comportant un groupe carbonyle sur l'atome de carbone terminal, ce qui en fait un aldéhyde, et des groupes hydroxyle reliés à tous les autres atomes de carbone. Les aldoses ont la formule chimique générale Cn(H2O)n. Étant donné que le formaldéhyde (n=l) et le glycolaldéhyde (n=2) ne sont généralement pas considérés comme des glucides, l'aldose le plus simple possible est le glycéraldéhyde (n=3), qui ne contient que trois atomes de carbone. 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 Cn (H2O) n . Since formaldehyde (n=l) and glycolaldehyde (n=2) are not generally considered carbohydrates, the simplest possible aldose is glyceraldehyde (n=3), which contains only three atoms of carbon.
Un cétose est un monosaccharide constitué d’une chaîne de n atomes de carbone comportant un groupe carbonyle sur l'atome de carbone non terminal, ce qui en fait une cétone, et des groupes hydroxyle reliés à tous les autres atomes de carbone. 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.
Selon un mode préféré de l’invention, le au moins un ose est choisi parmi : According to a preferred mode of the invention, the at least one bone is chosen from:
• -un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, 1’ arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose, l’idose, le galactose, et le talose, de préférence le xylose, ou • -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
• -un cétose notamment choisi parmi : le dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, et le tagatose, de préférence le fructose • -a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose
Un polyoside est un polymère de la famille des glucides formés d'un nombre n d'oses (n > 2) par liaison glycosidique alpha ou beta. A polysaccharide is a polymer from the carbohydrate family formed of a number n of oses (n > 2) by alpha or beta glycosidic bond.
Selon un mode préféré de l’invention, le au moins un polyoside est choisi parmi : According to a preferred embodiment of the invention, the at least one polysaccharide is chosen from:
• -un hétérodiholoside (un polyoside pour lequel n=2 constitué de 2 unités osidiques différentes) notamment choisi parmi : le tréhalulose, le saccharose, le turanose, le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, et le rutinose, ou
• -un homodiholoside (un polyoside pour lequel n=2 constitué de 2 unités osidiques identiques) notamment choisi parmi : l’inulobiose, l’alpha2-mannobiose, l’alpha3-mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, et le gentiobiose, de préférence le maltose ou le saccharose. • -a heterodiholoside (a polysaccharide for which n=2 consisting of 2 different saccharide 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 saccharide units) chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-mannobiose, trehalose, kojibiose, nigerose, maltose , isomaltose, sophorose, laminaribiose, cellobiose, and gentiobiose, preferably maltose or sucrose.
Selon un autre mode de l’invention, le composé précurseur biosourcé #1 du matériau carboné poreux consiste en un glucide, de préférence un ose, ou un polyoside tels que décrits ci-dessus. According to another mode of the invention, the biosourced precursor compound #1 of the porous carbon material consists of a carbohydrate, preferably a saccharide, or a polysaccharide as described above.
Selon un autre mode de l’invention, le matériau carboné poreux comprend deux composés précurseurs biosourcés #1 qui consistent en deux glucides, de préférence deux oses, deux polyosides ou un ose et un polyoside tels que décrits ci-dessus. Selon un autre mode de l’invention, le matériau carboné poreux comprend plus de deux composés précurseurs biosourcés #1. According to another mode of the invention, the porous carbon material comprises two biosourced precursor compounds #1 which consist of two carbohydrates, preferably two oses, two polysaccharides or one ose and a polysaccharide as described above. According to another mode of the invention, the porous carbon material comprises more than two biosourced precursor compounds #1.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est un composé précurseur biosourcé ayant au moins un noyau benzénique substitué par un groupe hydroxyle et optionnellement au moins un autre groupe fonctionnel choisi parmi les groupes acide carboxylique, aldéhyde, éther, ester, alcool, hydroxyle et cétone. 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 ring substituted by a hydroxyl group and optionally at least one other functional group chosen from the groups carboxylic acid, aldehyde, ether, ester, alcohol, hydroxyl and ketone.
Dans la présente demande, le terme "biosourcé" désigne des matériaux ou composés issus de la biomasse d’origine végétale ou animale, modifiés chimiquement ou non. Ces matières premières présentent l’avantage d’être renouvelables. In this application, the term "biosourced" designates 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.
On entend par composé "d’origine naturelle" ou biosourcé au sens de l’invention, tout composé en provenance de la nature (biomasse d’origine végétale ou animale). Ce composé peut être extrait de la biomasse terrestre et marine renouvelable ou d’organismes vivants (animaux, micro-organismes), éventuellement ultérieurement modifié par exemple chimiquement, ou encore obtenu suite à l’action de micro-organismes vivants (par exemple des enzymes ou bactéries) sur un composé d’origine naturelle selon des procédés de type biofermentation ou biosynthèse. Les composés issus du pétrole, d’origine fossile, n’entrent pas dans cette catégorie. By compound of "natural origin" or biosourced within the meaning of the invention, we mean any compound originating from nature (biomass of plant or animal origin). This compound can be extracted from renewable terrestrial and marine biomass or from living organisms (animals, micro-organisms), possibly subsequently modified for example chemically, or even obtained following the action of living micro-organisms (for example enzymes or bacteria) on a compound of natural origin according to biofermentation or biosynthesis type processes. Compounds from petroleum, of fossil origin, do not fall into this category.
Le composé précurseur biosourcé #2 utilisable selon la présente invention peut être un phénol dont le noyau benzénique est en outre substitué par au moins un autre groupe fonctionnel
choisi parmi les groupes acide carboxylique (notamment le groupe carboxy COOH), aldéhyde (notamment le groupe formyle CHO), éther (notamment le groupe méthoxy CH3O), ester, alcool (notamment le groupe CH2OH), hydroxyle et cétone, de préférence les groupes hydroxyle, acide carboxylique et aldéhyde. Les éventuels sels de ces groupes entrent également dans le cadre de l’invention. The biosourced precursor compound #2 usable according to the present invention may be a phenol whose benzene ring is further substituted by at least one other functional group chosen from the groups carboxylic acid (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 CH2OH group), hydroxyl and ketone, preferably the groups hydroxyl, carboxylic acid and aldehyde. Any salts of these groups also fall within the scope of the invention.
Le composé phénolique ou polyphénolique précurseur biosourcé utilisable selon la présente invention peut être choisi parmi les métabolites secondaires des végétaux. Les composés phénoliques forment le groupe des composés phytochimiques le plus important des plantes. Ils présentent près de 8000 molécules divisées en une dizaine de classes chimiques. Chaque classe est caractérisée par la présence d'un noyau benzoïque auquel un ou plusieurs groupes hydroxyles sont directement liés. Ces composés sont présents dans toutes les parties des végétaux supérieurs (racines, tiges, feuilles, fleurs, pollen, fruits, graines et bois). Ils sont synthétisés par les plantes soumises à des conditions difficiles (infections, blessures, radiation UV, ...) et sont impliqués dans de nombreux processus physiologiques comme la croissance cellulaire, la rhizogenèse, la germination des graines ou la maturation des fruits. The biosourced precursor phenolic or polyphenolic compound usable according to the present invention can be chosen from secondary metabolites of plants. Phenolic compounds form the most important group of phytochemicals in plants. They present nearly 8000 molecules divided into around ten chemical classes. Each class is characterized by the presence of a benzoic 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 wood). They are synthesized by plants subjected to difficult conditions (infections, wounds, UV radiation, etc.) and are involved in numerous physiological processes such as cell growth, rhizogenesis, seed germination or fruit ripening.
Les composés phénoliques comprennent des molécules simples, comme les acides phénoliques, ou des composés hautement polymérisés, comme les tanins et les lignines. Les composés phénoliques peuvent être classés selon la complexité, le degré et les liaisons possibles du squelette de base avec d'autres molécules. Les polyphénols sont des molécules très diversifiées, constituées d’un ou plusieurs cycles benzéniques portant une ou plusieurs fonctions hydroxyles. Ils peuvent être regroupés en de nombreuses classes suivant la complexité du squelette de base (noyau C6), le degré de modification de ce squelette (oxydation, hydroxylation, . . . .) et enfin suivant les molécules auxquelles ils sont associés (glucides, lipides, protéines, autres métabolites). Les formes les plus simples sont représentées par deux principaux groupes dont dérivent de nombreux composés : les acides hydroxycinnamiques et les flavonoïdes. Les formes complexes quant à elles, sont pour la plupart issues de la condensation de certaines formes simples et renferment, entre autres, les tannins et les lignines. 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, made up of one or more benzene rings carrying one or more hydroxyl functions. They can be grouped into numerous classes depending on the complexity of the basic skeleton (C6 core), the degree of modification of this skeleton (oxidation, hydroxylation, etc.) and finally according to the molecules with which they are associated (carbohydrates, lipids, etc.). , proteins, other metabolites). The simplest forms are represented by two main groups from which many compounds are derived: hydroxycinnamic acids and flavonoids. The complex forms, for their part, are mostly the result of the condensation of certain simple forms and contain, among others, tannins and lignins.
Les polyphénols sont répartis en différents groupes, définis en fonction de la structure de leur squelette carboné. Les acides phénoliques (CÔ-CI et C6-C3) et surtout les flavonoïdes (CÔ-CS-CÔ) sont les plus fréquemment retrouvés dans le règne végétal. Les lignanes (C6-C3-C3- CÔ), moins répandus. Le dernier groupe est celui des stilbènes (C6-C2-C6) ; le plus connu est le resvératrol, présent dans la peau du raisin. Les flavonoïdes représentent le groupe le plus vaste des polyphénols et le plus largement distribué dans le règne végétal. Plus de 4000 flavonoïdes
différents ont été identifiés. Elles diffèrent par le degré d’oxydation du noyau hétérocyclique oxygéné. Le tableau 1 ci-dessous résume les principales classes des composés phénoliques. [Table 1]
Les tanins condensés, également nommés proanthocyanidines, constituent une source particulièrement intéressante, car abondante, de tels composés phénoliques susceptibles de constituer des produits de départ pour la formation de prépolymères époxydes. En effet, les tanins condensés sont des biopolymères phénoliques essentiellement présents dans les tissus mous à croissance et renouvellement rapides, tels que les feuilles, les tiges, etc. Cette classe de
polyphénols est la plus abondante après celle des lignines. Contrairement aux lignines qui sont des constituants des lignocelluloses, éléments structurants des parois secondaires des cellules végétales, les tanins condensés sont stockés dans les vacuoles des cellules sous formes d'organites, et sont donc facilement extractibles. Ces composés se retrouvent notamment dans de nombreuses ressources naturelles disponibles et variées telles que les résidus agroindustriels, par exemple dans les marcs fruitiers, et la biomasse non exploitée, en particulier dans l'écorce, les feuilles et les aiguilles d'arbres, la vigne, les fruits, etc. Polyphenols are divided into different groups, defined according to the structure of their carbon skeleton. Phenolic acids (CÔ-CI and C6-C3) and especially flavonoids (CÔ-CS-CÔ) are the most frequently found in the plant kingdom. Lignans (C6-C3-C3-CÔ), less common. The last group is that of stilbenes (C6-C2-C6); the best known is resveratrol, present in the skin of grapes. Flavonoids represent the largest group of polyphenols and the most widely distributed in the plant kingdom. More than 4000 flavonoids different ones 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, because abundant, source of such phenolic compounds capable of constituting starting products for the formation of epoxy 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 are the most abundant after lignins. Unlike lignins which are constituents of lignocelluloses, structuring 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 agroindustrial residues, for example in fruit marc, and unexploited biomass, in particular in the bark, leaves and needles of trees, vines. , fruits, etc.
Les tanins condensés sont des composés non hydrolysables mais qui peuvent être dépolymérisés en anthocyanidols lorsqu'ils sont traités à chaud par un acide. Ils sont habituellement dénommés d'après l'anthocyanidol ainsi libéré. Par exemple, le monomère (+)- catéchol (un flavan-3-ol) est une unité constitutive du dimère catéchol -(4a^8)-catéchol que l'on nommera procyanidol B-3 en raison de sa dégradation en milieu acide en cyanidol. De nombreux tanins condensés sont des polymères de catéchol. C'est la raison pour laquelle on dénomme aussi les tanins condensés «proanthocyanidol» ou «proanthocyanidines» (sur le modèle de l'anglais proanthocyanidins). Les hydroxyles OH sont dans les mêmes positions sur le monomère et le produit de dépolymérisation, seul change le cycle central. Condensed tannins are non-hydrolysable compounds which can be depolymerized into anthocyanidins when they are heat treated with an 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 we will call procyanidol B-3 due to its degradation in an acid medium in cyanidol. Many condensed tannins are catechol polymers. This is the reason why condensed tannins are also called “proanthocyanidol” or “proanthocyanidins” (based on the English proanthocyanidins). The OH hydroxyls are in the same positions on the monomer and the depolymerization product, only the central ring changes.
Il existe une douzaine d'unités monomères flavanols entrant dans la construction des tanins condensés. Elles peuvent en outre être substituées par l'acide gallique ou par des sucres, généralement en position 3 et parfois en position 5 et 7. There are a dozen flavanol monomeric units used in the construction of condensed tannins. They can also be substituted by gallic acid or by sugars, generally in position 3 and sometimes in positions 5 and 7.
Flavanols et anthocyanidols comportent tous trois cycles : un cycle A avec en général un ou deux hydroxyles OH, un hétérocycle C avec des carbones asymétriques 2 et 3 (et un hydroxyle en 3), et un cycle B avec un hydroxyle OH (afzeléchol, épiafzeléchol), deux OH (catéchol et épicatéchol), ou trois OH (gallocatéchol, épigallocatéchol). Les composés avec la configuration cis- (2R,3R) ont un nom préfixé par épi-, les autres sont dans la configuration traw -(2R,3 S). Flavanols and anthocyanidols all have three rings: an A ring with generally one or two OH hydroxyls, a C heterocycle with asymmetric carbons 2 and 3 (and a hydroxyl at 3), and a B ring with an OH hydroxyl (afzeléchol, epiafzeléchol ), 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.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est un phénol simple choisi parmi le phénol, catéchol, résorcinol, hydroquinone, pyrogallol, ou phloroglucinol. 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.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est un composé de la famille des flavonoïdes. According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is a compound from the flavonoid family.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est un composé choisi parmi les sous-classes : flavonol, anthocyane, flavanol, flavanone, isoflavonoïde.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est un composé de la sous-famille des flavanols ou catéchines, dont la structure est basée sur le 2-phényl-3-chromanol. Les structures oligomères et polymères des flavanols constituent la classe des proanthocyanidols ou tanins condensés. 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 flavanol or catechin subfamily, 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.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est choisi parmi : la catéchine sous une de ses formes stéréo-isomériques, gallocatéchine, afzéléchol, fisétinidol, guibourtinidol, mesquitol, ou robinétinidol. 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, afzéléchol, fisetinidol, guibourtinidol, mesquitol, or robinetinidol.
Selon un mode préféré de l’invention, le au moins un composé phénolique est le phloroglucinol. According to a preferred embodiment of the invention, the at least one phenolic compound is phloroglucinol.
Selon un mode préféré de l’invention, le au moins un composé polyphénolique est la catéchine. According to a preferred embodiment of the invention, the at least one polyphenolic compound is catechin.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est contenu dans un extrait naturel végétal. According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is contained in a natural plant extract.
Selon un mode préféré de l’invention, l’extrait naturel végétal contenant au moins un composé phénolique ou polyphénolique est choisi parmi un extrait de fraise, pomme de terre, pomme, citron, noix, vigne, raisin, fleurs, soja, pois, pin, tomate, ail ou cachou. According to a preferred mode 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, soya, pea, pine, tomato, garlic or cachou.
Selon un mode préféré de l’invention, le au moins un composé phénolique ou polyphénolique est According to a preferred embodiment of the invention, the at least one phenolic or polyphenolic compound is
• un phénol simple notamment choisi parmi: le phénol, le catéchol, le résorcinol, 1’ hydroquinone, le pyrogallol, et le phloroglucinol, • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,
• un composé phénolique de la famille des flavonoïdes notamment choisi parmi les sous-classes: flavonol, anthocyanidols, anthocyane, flavanol, flavanone, stilbénoïdes, isoflavonoïde, • a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isoflavonoid,
• un tanin condensé • a condensed tannin
• un phlorotanin notamment choisi parmi: le fucol, le difucol, le trifucol, le phloréthol, le diphloréthol, le bifuhalol, le trifuhalol, lefucophloréthol, le fuhalol, l’eckol, et le diphloréthohydroxycamanol • a phlorotannin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
• ou un extrait naturel végétal contenant au moins un des composés ci- dessus. • or a natural plant extract containing at least one of the compounds above.
Selon un autre mode de l’invention, le matériau carboné poreux comprend deux composés précurseurs biosourcés #2 qui consistent en deux composés phénoliques ou
polyphénoliques tels que décrits ci-dessus. Selon un autre mode de l’invention, le matériau carboné poreux comprend plus de deux composés précurseurs biosourcés #2. According to another mode of the invention, the porous carbon material comprises two biosourced precursor compounds #2 which consist of two phenolic compounds or polyphenolics as described above. According to another embodiment of the invention, the porous carbon material comprises more than two biosourced precursor compounds #2.
Selon un mode préféré de l’invention, le matériau carboné poreux obtenu par carbonisation hydrothermale comprend ou est formé à partir de : According to a preferred embodiment of the invention, the porous carbonaceous material obtained by hydrothermal carbonization comprises or is formed from:
• maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, érythrose, ou dihydroxyacétone, et • maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, or dihydroxyacetone, and
• phloroglucinol, catéchine, ou d’un extrait de cachou. • phloroglucinol, catechin, or cashew extract.
L’extrait de cachou peut être obtenu à partir de fibres végétales d’ Acacia à cachou, ledit extrait contenant des tanins condensés et des flavonols. Cachou extract can be obtained from Acacia cachou plant fibers, said extract containing condensed tannins and flavonols.
Selon l’invention, le terme «matériau carboné poreux» représente un matériau polymère obtenu ou susceptible d’être obtenu par polymérisation sol-gel (p.e une carbonisation hydrothermale) à partir d’au moins deux précurseurs biosourcé # 1 et 2 tels que définis dans la présente demande. 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 this application.
Dans un mode de réalisation de l’invention, le matériau carboné poreux est susceptible d'être obtenu par un procédé de polymérisation sol-gel, connu de l’homme du métier, faisant intervenir au moins deux précurseurs biosourcés # 1 et 2 tels que définis ci-dessus. In one embodiment of the invention, the porous carbon material can be obtained by a sol-gel polymerization process, known to those skilled in the art, involving at least two biosourced precursors # 1 and 2 such as defined above.
Les procédés de polymérisation sol-gel peuvent utiliser un catalyseur, par exemple, des acides et des bases tels que, par exemple, l'acide nitrique, l'acide acétique, l'acide ascorbique, l'acide chlorhydrique, l'acide sulfurique, l’acide borique, le carbonate de sodium, l'hydroxyde de sodium, l'hydroxyde d'ammonium et le sulfate de calcium. 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.
Les concentrations de catalyseur peuvent être exprimées en rapport réactif-catalyseur (R/C) qui peut aller, par exemple, de 10 à 5 000, ou de 10 à 2 000, ou de 10 à 1 000. Catalyst concentrations can be expressed as reactant-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.
Selon un mode préféré de l’invention, le matériau carboné poreux est obtenu par carbonisation hydrothermale. According to a preferred embodiment of the invention, the porous carbonaceous material is obtained by hydrothermal carbonization.
La carbonisation hydrothermale est définie comme étant un processus de conversion thermochimique de la biomasse ou dans le cadre de la présente invention au moins deux précurseurs biosourcés # 1 et 2 tels que définis ci-dessus dans le but d’obtenir un monolithe de carbone poreux en phase aqueuse. Il s’agit d’un processus exothermique qui diminue les taux d’oxygène et d’hydrogène de la matière principalement par des réactions de déshydratation et de décarboxylation. La polymérisation sol-gel est obtenue en appliquant des températures
comprises de 120°C à 250°C à un mélange d’au moins deux précurseurs biosourcé # 1 et 2 tels que définis ci -dessus et d’un solvant, de préférence d’eau (en général 10% en masse des deux précurseurs biosourcé # 1 et 2). Hydrothermal carbonization is defined as being a process of thermochemical conversion of biomass or in the context of the present invention at least two biosourced precursors # 1 and 2 as defined above with the aim of obtaining a porous carbon monolith in aqueous phase. It is an exothermic process that decreases the oxygen and hydrogen levels of matter 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 biosourced precursors #1 and 2 as defined above and a solvent, preferably water (generally 10% by weight of the two precursors biosourced #1 and 2).
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : a)Chauffer un mélange réactionnel comprenant une solution aqueuse, et, au moins deux précurseurs biosourcés # 1 et 2 tels que définis ci-dessus dissous dans la solution aqueuse, à une température inférieure à 300°C pour obtenir un matériau carboné poreux solide, b)Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant polaire. In a particular embodiment, the porous carbonaceous 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 solution aqueous, at a temperature below 300°C to obtain a solid porous carbon material, b) Wash the solid porous carbon material obtained by successive soaking in a polar solvent.
La solution aqueuse comprend de l'eau, et éventuellement des solvants, de préférence en quantités miscibles avec l'eau. The aqueous solution comprises water, and optionally solvents, preferably in quantities miscible with water.
Selon un mode de réalisation préféré, le mélange réactionnel ne comprend qu'une seule phase liquide, à savoir la solution aqueuse, de préférence de l’eau. According to a preferred embodiment, the reaction mixture comprises only one liquid phase, namely the aqueous solution, preferably water.
Par exemple et sans limitation, la solution aqueuse comprend de l'éthanol, de préférence de l’éthanol absolu. For example and without limitation, the aqueous solution comprises ethanol, preferably absolute ethanol.
Selon un mode de réalisation préféré la fraction massique en eau dans un mélange eau/éthanol absolu est comprise de 1 à 0,3. According to a preferred embodiment, the mass fraction of water in a water/absolute ethanol mixture is between 1 and 0.3.
Selon un mode de réalisation préféré la fraction massique en eau dans un mélange eau/éthanol absolu est comprise de 0,7 à 0.4 lorsque les précurseurs biosourcés # 1 et 2 sont respectivement le xylose et le phloroglucinol. According to a preferred embodiment, the mass fraction of water in a water/absolute ethanol mixture is between 0.7 and 0.4 when the biosourced precursors #1 and 2 are respectively xylose and phloroglucinol.
Selon un mode de réalisation préféré la fraction massique en eau dans un mélange eau/éthanol absolu est comprise de 1 à 0.5 lorsque les précurseurs biosourcés # 1 et 2 sont respectivement le xylose et la catéchine. According to a preferred embodiment, the mass fraction of water in a water/absolute ethanol mixture is between 1 and 0.5 when the biosourced precursors #1 and 2 are respectively xylose and catechin.
L'étape (a) du procédé peut également être appelée « étape de traitement hydrothermal ». Step (a) of the process can also be called the “hydrothermal treatment step”.
Grâce à la présence d'eau (dans la solution aqueuse) dans l'étape (a), le procédé de préparation de l'invention se distingue par exemple des procédés pyrolytiques, pour la conversion de biomasse ou des précurseurs biosourcés # 1 et 2, pour donner un matériau de type charbon (typiquement en l'absence d'oxygène). 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-like material (typically in the absence of oxygen).
La température de réaction dans l'étape de traitement hydrothermal (a) est de préférence <300°C, plus préférablement de 100 à 300°C, encore plus préférablement de 120 à 250°C et le
plus préférablement de 160 à 200°C ou encore de préférence à 180°C. La température de réaction est destinée à désigner la température, plus spécifiquement la température moyenne, à l'intérieur du mélange réactionnel, qui peut être mesurée avec un thermocouple. 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 the more preferably from 160 to 200°C or even preferably at 180°C. Reaction temperature is intended to refer to the temperature, more specifically the average temperature, inside the reaction mixture, which can be measured with a thermocouple.
Le traitement hydrothermal dans le procédé de préparation du matériau carboné poreux de la présente invention est de préférence effectué dans un réacteur résistant à la pression, par exemple un autoclave. The hydrothermal treatment in the process for preparing the porous carbonaceous material of the present invention is preferably carried out in a pressure-resistant reactor, for example an autoclave.
Il n'y a pas de limitation spécifique quant à la durée de l'étape (a) dans le procédé de préparation du matériau carboné poreux de l'invention. There is no specific limitation as to the duration of step (a) in the process for preparing the porous carbonaceous material of the invention.
Par exemple, le milieu réactionnel peut être introduit dans une bombe de minéralisation fermée hermétiquement, puis placé dans une enceinte thermostatée à 180°C pendant 20 heures. For example, the reaction medium can be introduced into a hermetically closed digestion bomb, then placed in an enclosure thermostatically controlled at 180°C for 20 hours.
L'étape (a) du procédé est mise en œuvre jusqu'à ce que la totalité du contenu du mélange réactionnel comprenant la solution aqueuse et les précurseurs biosourcés # 1 et 2 se soit gélifié ou précipité en matériau carboné poreux de la présente invention. Step (a) of the process is carried out until the entire content of the reaction mixture comprising the aqueous solution and the biosourced precursors #1 and 2 has gelled or precipitated into the porous carbonaceous material of the present invention.
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : ajChauffer un mélange réactionnel comprenant : In a particular embodiment, the porous carbonaceous material is prepared by the following process: ajHeating a reaction mixture comprising:
-une solution aqueuse constituée d’eau et d’éthanol absolu avec une fraction massique en eau comprise de 0,7 à 0,4, et, -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 0.7 to 0.4, and,
-du xylose et du phloroglucinol dissous dans la solution aqueuse, à une température inférieure à 300°C (de préférence à 180°C) pour obtenir un matériau carboné poreux solide, b)Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant polaire. -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) Wash the solid porous carbon material obtained by successive soaking in a polar solvent.
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : ajChauffer un mélange réactionnel comprenant : In a particular embodiment, the porous carbonaceous material is prepared by the following process: ajHeating a reaction mixture comprising:
-une solution aqueuse constituée d’eau et d’éthanol absolu avec une fraction massique en eau comprise de 1 à 0,5, et, -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 1 to 0.5, and,
-du xylose et de la catéchine dissous dans la solution aqueuse, à une température inférieure à 300°C (de préférence à 180°C) pour obtenir un matériau carboné poreux solide, b)Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant, de préférence un solvant polaire.
Selon un mode de réalisation préférée, le matériau carboné est formé à partir d’au moins un glucide, de préférence un aldose ou cétose, encore plus de préférence au moins un hétérodiholoside, ou homodiholoside (cl) et d’au moins un composé phénolique ou polyphénolique (c2) dans un rapport molaire (cl/c2) compris de 10/1 à 1/10, de préférence 2/1. Pour les extraits naturels complexes et tanins condensés, le rapport molaire (cl/c2) est défini par le rapport entre le nombre de moles de glucides (cl) et le nombre de moles de composés phénoliques et/ou polyphénoliques, c’est-à-dire le nombre de moles d’unités monomériques (par exemple flavonoïdes), dans ledit tanin condensé ou ledit extrait naturel complexe. -xylose and catechin dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbonaceous material, b) Wash the solid porous carbonaceous 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 compound. or polyphenolic (c2) in a molar ratio (cl/c2) of 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. -say the number of moles of monomeric units (for example flavonoids), in said condensed tannin or said complex natural extract.
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : a)Chauffer un mélange réactionnel comprenant : In a particular embodiment, the porous carbon material is prepared by the following process: a) Heating a reaction mixture comprising:
-une solution aqueuse constituée d’eau et d’éthanol absolu avec une fraction massique en eau comprise de 0,7 à 0,4, et, -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 0.7 to 0.4, and,
-du xylose et du phloroglucinol dissous dans la solution aqueuse, à une température inférieure à 300°C (de préférence à 180°C) pour obtenir un matériau carboné poreux solide, b)Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant, de préférence un solvant polaire. -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) Wash the solid porous carbon material obtained by successive soaking in a solvent, preferably a polar solvent.
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : a)Chauffer un mélange réactionnel comprenant : In a particular embodiment, the porous carbon material is prepared by the following process: a) Heating a reaction mixture comprising:
-une solution aqueuse constituée d’eau et d’éthanol absolu avec une fraction massique en eau comprise de 1 à 0,5, et, -an aqueous solution consisting of water and absolute ethanol with a water mass fraction of 1 to 0.5, and,
-du xylose et de la catéchine (ratio molaire 2/1) dissous dans la solution aqueuse, à une température inférieure à 300°C (de préférence à 180°C) pour obtenir un matériau carboné poreux solide, b)Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant, de préférence un solvant polaire. -xylose and catechin (molar ratio 2/1) dissolved in the aqueous solution, at a temperature below 300°C (preferably 180°C) to obtain a solid porous carbon material, b) Wash the carbon material porous solid obtained by successive soaking in a solvent, preferably a polar solvent.
Selon un mode de réalisation préférée, le matériau carboné poreux est constitué deAccording to a preferred embodiment, the porous carbon material is made up of
• 40-70% massique en élément carbone (C) • 40-70% by mass of carbon element (C)
• 30-40% massique en élément oxygène (O)
de préférence 60% massique en élément carbone (C) et 35% massique en élément oxygène (O). • 30-40% by mass of element oxygen (O) preferably 60% by mass of carbon element (C) and 35% by mass of oxygen element (O).
Selon un mode de réalisation préférée, le matériau carboné poreux est constitué de 36% O et 60% C pour le système xylose/phloroglucinol. According to a preferred embodiment, the porous carbon material consists of 36% O and 60% C for the xylose/phloroglucinol system.
Selon un mode de réalisation préférée, le matériau carboné poreux est formé à partir d’au moins un glucide (cl), de préférence au moins un ose, ou au moins un polyoside (cl) et d’au moins un composé phénolique (c2) ou polyphénolique dans un rapport molaire (cl/c2) compris de 10/1 à 1/10, de préférence 2/1. According to a preferred embodiment, the porous carbon material is formed from at least one carbohydrate (cl), preferably at least one ose, or at least one polysaccharide (cl) and at least one phenolic compound (c2 ) or polyphenolic in a molar ratio (cl/c2) of 10/1 to 1/10, preferably 2/1.
Le matériau carboné poreux solide est lavé par trempage soit dans de l’eau ultrapure, soit désionisée soit dans un mélange eau/solvant protique polaire, par exemple le méthanol, l’éthanol ou le tert-butanol pendant plusieurs heures. The solid porous carbonaceous material is washed by soaking in either ultrapure water, deionized water or a water/polar protic solvent mixture, for example methanol, ethanol or tert-butanol for several hours.
Le lavage a pour but d'extraire des composés solubles non incorporés dans la structure du matériau carboné poreux et de préparer le gel à l’étape de séchage. 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 step.
Le matériau carboné obtenu ou pouvant être obtenu dans l'étape de carbonisation hydrothermale (a) est typiquement composé de nanoparticules primaires agrégées en un réseau interconnecté formant la phase dispersée, la solution aqueuse formant la phase de dispersion. 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, the aqueous solution forming the dispersion phase.
Le matériau carboné avant séchage peut être appelé solvogel. Lorsque la solution aqueuse est de l'eau, le matériau carboné obtenu à l'étape (a) peut être appelé hydrogel. The carbonaceous material before drying can be called solvogel. When the aqueous solution is water, the carbonaceous material obtained in step (a) can be called hydrogel.
Par conséquent, un hydrogel est considéré ici comme un type spécial de solvogel, dans lequel la solution aqueuse de la phase de dispersion est de l'eau. Therefore, a hydrogel is considered here as a special type of solvogel, in which the aqueous solution of the dispersion phase is water.
Le matériau carboné poreux solide obtenu ou pouvant être obtenu selon le procédé décrit ci-dessus peut être séché est transformé en cryogel, en aérogel ou en xérogel. The solid porous carbonaceous material obtained or obtainable according to the process described above can be dried and transformed into cryogel, airgel or xerogel.
Selon une première variante, une forme sèche d’un hydrogel ou solvogel est obtenue en soumettant le matériau carboné (i.e hydrogel ou solvogel) au contact d’un fluide-supercritique, notamment de l’acétone ou du dioxyde de carbone pour le transformer en un aérogel. 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 airgel.
Selon une seconde variante, une forme sèche d’hydrogel est obtenue en soumettant le matériau carboné (i.e hydrogel ou solvogel) à une congélation du gel ainsi isolé et une lyophilisation dudit gel congelé pour le transformer en un cryogel. 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 freeze-drying of said frozen gel to transform it into a cryogel.
Selon une troisième variante, une forme sèche d’hydrogel est obtenue en soumettant le matériau carboné (i.e hydrogel ou solvogel) à une évaporation du solvant à température et pression contrôlées pour le transformer en un xérogel.
Par ailleurs, un cryogel peut être nommé aérogel lorsque le gel séché a largement conservé les propriétés texturales après séchage. According to a third variant, a dry form of hydrogel is obtained by subjecting the carbon material (ie 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.
Les gels séchés peuvent être nommés aérogels indépendamment de la technique de séchage utilisée. Dried gels can be called aerogels regardless of the drying technique used.
Selon le procédé de séchage du gel, il est possible de contrôler la taille des pores et la structure des matériaux carbonés poreux obtenus selon l’invention. Depending on the gel drying process, it is possible to control the size of the pores and the structure of the porous carbon materials obtained according to the invention.
Dans un mode de réalisation particulier, le matériau carboné poreux est préparé par le procédé suivant : a) Chauffer un mélange réactionnel comprenant une solution aqueuse, et, au moins deux précurseurs biosourcé # 1 et 2 tels que définis ci-dessus dissous dans la solution aqueuse, à une température inférieure à 300°C, de préférence à 180°C pour obtenir un matériau carboné poreux solide, b) Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant polaire (idéalement dans de l’éthanol absolu ou dans un mélange eau/tert-butanol 75/25 en pourcentages massiques), c) Sécher le matériau carboné poreux solide à l’aide d’un fluide supercritique ou par lyophilisation. In a particular embodiment, the porous carbonaceous 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 solution aqueous, at a temperature below 300°C, preferably at 180°C to obtain a solid porous carbonaceous material, b) Wash the solid porous carbonaceous material obtained by successive soaking in a polar solvent (ideally in absolute ethanol or in a water/tert-butanol mixture 75/25 in percentages by weight), c) Dry the solid porous carbonaceous material using a supercritical fluid or by freeze-drying.
Les hydrogels disposés dans des béchers en verre borosilicaté sont préalablement placés en immersion dans de l’azote liquide (77 K) pendant 15 minutes, puis directement placé dans la chambre du lyophilisateur (p.e un lyophilisateur COSMOS de Cryotec). Le séchage est effectué sous vide < 50 mTorr pendant 48h avec un piège de froid à -80°C qui assure la condensation des vapeurs d'eau ou de solvants. The hydrogels placed in borosilicate glass beakers are previously placed in immersion in liquid nitrogen (77 K) for 15 minutes, then directly placed in the chamber of the lyophilizer (e.g. a COSMOS lyophilizer 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.
Le séchage supercritique et la lyophilisation sont préférés pour maintenir le système de pores. Selon un mode de réalisation particulièrement préféré, le solvant est éliminée par extraction avec du CO2 supercritique pour ce procédé, le solvant dans le solvogel est de préférence choisi parmi l'éthanol absolu ou l'acétone (par exemple amené par remplacement de solvant par l'éthanol absolu ou l'acétone). Supercritical drying and freeze drying are preferred to maintain the pore system. According to a particularly preferred embodiment, the solvent is eliminated by extraction with supercritical CO2 for this process, the solvent in the solvogel is preferably chosen from absolute ethanol or acetone (for example brought by replacement of solvent with absolute ethanol or acetone).
Texture du matériau carboné poreux
Le matériau carboné poreux obtenu ou pouvant être obtenu dans l'étape (a), est un matériau gélifié, en particulier une matière solidifiée composée de particules agrégées formant un réseau interconnecté. Texture of 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.
Le matériau carboné poreux obtenu ou pouvant être obtenu selon le procédé décrit ci- dessus comprend des micropores (pores < 2 nm), mésopores (pores de 2-50 nm) et macropores (pores > 50 nm). The porous carbonaceous material obtained or obtainable according to the process described above comprises micropores (pores < 2 nm), mesopores (pores of 2-50 nm) and macropores (pores > 50 nm).
En outre, par opposition aux charbons actifs classiques (par exemple ceux produits par pyrolyse), la teneur en oxygène est typiquement supérieure à 25 % pour les matériaux directement obtenus dans l'étape de carbonisation hydrothermale (a). Furthermore, as opposed to conventional activated carbons (for example those produced by pyrolysis), the oxygen content is typically greater than 25% for materials directly obtained in the hydrothermal carbonization step (a).
La surface du matériau carboné de l'invention telle qu'obtenue à l'étape (a) est très hydrophile. The surface of the carbon material of the invention as obtained in step (a) is very hydrophilic.
Propriétés texturales du matériau carboné poreux Textural properties of porous carbonaceous material
Diamètre des pores Pore diameter
Le matériau carboné poreux selon la présente invention est composé de particules agrégées formant un réseau interconnecté. Ce réseau interconnecté de particules agrégées induit des interstices interparticulaires (ou pores) avec un diamètre compris entre 10 pm et 5 nm, plus préférablement avec un diamètre moyen de 5 à 200 nm, plus préférablement avec un diamètre moyen de 25 à 100 nm et encore plus préférable de 40 à 80 nm et de préférence autour de 60 nm. 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 gaps (or pores) with a diameter 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 again more preferably 40 to 80 nm and preferably around 60 nm.
De plus, le matériau carboné poreux selon la présente invention est une matière agrégée composée des particules, avec une distribution de taille de pore calculée à partir d'une isotherme d'adsorption/désorption d’azote à 77 K (pour les pores les plus étroits ; < 100 nm) et un profil de porosimétrie par intrusion de mercure (pour les pores les plus larges ; > 50 nm). In addition, the porous carbonaceous material according to the present invention is an aggregated material composed of particles, with a pore size distribution calculated from a nitrogen adsorption/desorption isotherm at 77 K (for the largest pores). narrow; < 100 nm) and a mercury intrusion porosimetry profile (for the widest pores; > 50 nm).
De plus, la distribution de la taille des pores et le diamètre des pores qui correspond au pic de la distribution de la taille des pores peuvent être fournis par l'analyse BJH de l'isotherme d'adsorption/désorption d'azote. In addition, the pore size distribution and the pore diameter which corresponds to the peak of the pore size distribution can be provided by the BJH analysis of the nitrogen adsorption/desorption isotherm.
Le diamètre des pores du gel de carbone selon la présente invention, correspondant au pic de la distribution de taille des pores, est de préférence dans une gamme de 40 à 80 nm.
Il convient de noter que, dans le but d'empêcher la réduction de la quantité de composants adsorbés, le diamètre des pores du matériau carboné poreux selon la présente invention, correspondant au pic de la distribution de la taille des pores, est de préférence supérieur au diamètre moléculaire moyen du diamètre des composants adsorbés ou complexés. 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 carbonaceous material according to the present invention, corresponding to the peak of the pore size distribution, is preferably greater to the average molecular diameter of the diameter of the adsorbed or complexed components.
Surface spécifique Specific surface
Le matériau carboné poreux selon la présente invention présente de préférence une surface spécifique de 300 à 1000 m2/g, plus préférablement présente une surface spécifique de 600 à 800 m2/g. The porous carbonaceous 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.
Si le matériau carboné poreux a une surface spécifique inférieure à 100 m2/g, la surface avec laquelle les composants adsorbés sont en contact sera réduite, et le nombre de pores dans lesquels les composants adsorbés sont logés sera réduit. If the porous carbonaceous material has a specific surface area of 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 accommodated will be reduced.
Volume poreux Porous volume
De plus, le volume poreux total du matériau carboné poreux selon la présente invention n'est pas particulièrement limité car il varie également en fonction de la surface spécifique et du diamètre des pores qui correspond au pic de la distribution de taille des pores. Cependant, le matériau carboné poreux selon la présente invention a de préférence un volume total de pores de 0,1 à 5 cm3/g, plus préférablement a un volume total de pores de 0,2 à 2,5 cm3/g. Furthermore, 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 carbonaceous 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.
Le volume dit total a été déterminé à une pression relative de 0,99 sur les isothermes d'adsorption/désorption d’azote à -196°C. La surface et le volume de micropore ont été déterminés en appliquant la méthode t-plot. Les distributions des diamètres de pores ont été évaluées en appliquant la méthode BJH sur la branche de désorption des isothermes. Les aérogels ont été dégazés pendant au moins 6 h à 0,05 mbar sur un VacPrep Micromeritics avant analyses. Les analyses ont été réalisées sur un TriStar Micromeritics. Le volume total de pores dans les matériaux est supérieur à celui obtenu par volumétrie de sorption d’azote à -196°C, puisque les pores les plus larges (> 100 nm de diamètre) ne peuvent pas être analysés par cette méthode. Les pores les plus larges (> 100 nm de diamètre) ont été analysés par porosimétrie par intrusion de mercure. Le volume de ces pores larges est supérieur à 3 mL/g pour des aérogels phloroglucinol/xylose. The so-called total volume was determined at a relative pressure of 0.99 on the nitrogen adsorption/desorption isotherms at -196°C. The micropore surface area and volume were determined by applying the t-plot method. The 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. Analyzes were performed on a TriStar Micromeritics. The total pore volume in the materials is greater 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.
La surface spécifique et le volume poreux total du matériau carboné poreux selon la présente invention peuvent être déterminés par une mesure volumétrique générale décrite ci-
dessous. Plus précisément, un matériau carboné poreux est placé dans un récipient et refroidi à la température de l'azote liquide (-196°C). Ensuite, de l'azote gazeux est introduit dans le récipient et la quantité d'azote gazeux adsorbée sur le gel de carbone est déterminée sur la base de la méthode volumétrique. Ensuite, la pression d'azote gazeux introduite dans le récipient est progressivement modifiée et la quantité d'azote gazeux adsorbée sur le gel de carbone est tracée en fonction de chaque pression d'équilibre. On obtient ainsi l'isotherme d'adsorption/désorption d'azote. The specific surface area and the total pore volume of the porous carbon material according to the present invention can be determined by a general volumetric measurement described below. below. Specifically, a porous carbonaceous 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 against each equilibrium pressure. We thus obtain the nitrogen adsorption/desorption isotherm.
Par ailleurs, la surface spécifique peut être déterminée par la méthode de Brunauer Emmett Teller (BET), par exemple par la technique d’adsorption de diazote. Furthermore, the specific surface area can be determined by the Brunauer Emmett Teller (BET) method, for example by the dinitrogen adsorption technique.
La surface spécifique équivalent BET a été déterminée par adsorption d’azote à 77K en réalisant des isothermes à des pressions relatives P/PO comprises entre 0,01 et 0,99. La méthode BET a été appliquée sur une gamme de pressions relatives comprises entre 0,1 et 0,3 (en se basant sur la transformée de Rouquerol). The BET equivalent specific surface area was determined by nitrogen adsorption at 77K by carrying out 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).
Porosité interconnectée Interconnected porosity
Ce paramètre peut être déterminé par porosimétrie par intrusion de mercure indiquant le volume total de pores interconnectés. This parameter can be determined by mercury intrusion porosimetry indicating the total volume of interconnected pores.
Complexation d’ions Ion complexation
Les matériaux carbonés poreux tels que décrits ci -dessus peuvent être complexés avec des ions métalliques de préférence au moins un type de cation métallique qui est complexé sur les surfaces externes et les surfaces des pores internes du matériau carboné poreux selon l’invention. The porous carbon materials as described above can be complexed with metal ions, preferably at least one type of metal cation which is complexed on the external surfaces and the surfaces of the internal pores of the porous carbon material according to the invention.
Selon un mode particulier de l’invention, le au moins un cation métallique est choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. According to a particular embodiment of the invention, the at least one metal cation is chosen from 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 transition metals Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ .
Le cation métallique est complexé ou chélaté au matériau carboné poreux. Un chélate est un type de composé de coordination dans lequel un ion métallique unique est fixé par des liaisons covalentes coordonnées à une molécule ou à un ion appelé ligand. Un composé de coordination est formé lorsque des groupes d'atomes, d'ions ou de molécules se lient
chimiquement les uns aux autres en donnant et en acceptant des paires d'électrons. Les groupes qui donnent des paires d'électrons sont appelés ligands. Ce sont généralement des bases de Lewis. Les groupes acceptant des paires d'électrons sont souvent des cations de métaux de transition. 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 coordinated covalent bonds to a molecule or ion called a ligand. A coordination compound is formed when groups of atoms, ions, or molecules bond together chemically to each other by donating and accepting pairs of electrons. Groups that donate electron pairs are called ligands. These are generally Lewis bases. The groups accepting electron pairs are often transition metal cations.
Selon la présente invention, le terme « matériau hybride » fait référence au matériau carboné poreux sur lequel est complexé les ions métalliques. Il s’agit d’une composition au sens de la présente invention. 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.
Dans un mode de réalisation particulier, la composition (matériau hybride carboné poreux) est préparée par le procédé suivant : a) Chauffer un mélange réactionnel comprenant une solution aqueuse, et, au moins deux précurseurs biosourcé # 1 et 2 tels que définis ci-dessus dissous dans la solution aqueuse, à une température inférieure à 300°C pour obtenir un matériau carboné poreux solide, b) Laver le matériau carboné poreux solide obtenu par trempage successifs dans un solvant polaire, c) Sécher le matériau carboné poreux solide à l’aide d’un fluide supercritique ou par lyophilisation, d) Imprégner le matériau carboné poreux solide avec une solution comprenant des cations métalliques pour obtenir un matériau hybride carboné poreux complexé avec des cations métalliques. In a particular embodiment, 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, at a temperature below 300°C to obtain a solid porous carbon material, b) Wash the solid porous carbon material obtained by successive soaking in a polar solvent, c) Dry the solid porous carbon material with using a supercritical fluid or by freeze-drying, d) Impregnate the solid porous carbon material with a solution comprising metal cations to obtain a porous carbon hybrid material complexed with metal cations.
Le processus d'adsorption pour permettre au complexe métallique de s'adsorber sur un matériau carboné, et les conditions d'adsorption ne sont pas particulièrement limités. The adsorption process to allow the metal complex to adsorb on carbon material, and the adsorption conditions are not particularly limited.
Par exemple, un complexe métallique peut être optionnellement adsorbé sur un matériau carboné en plaçant le matériau carboné poreux dans une solution et en mélangeant la solution pendant une certaine période de temps à environ 10 à 100°C. Le matériau hybride est séparé de la solution par centrifugation, et la suspension ainsi obtenue est lavée puis séchée. For example, a metal complex can optionally be adsorbed onto a carbonaceous material by placing the porous carbonaceous material in a solution and mixing the solution for a certain period of time at approximately 10 to 100°C. The hybrid material is separated from the solution by centrifugation, and the suspension thus obtained is washed and then dried.
Selon une autre variante de l’invention la composition (matériau hybride carboné poreux) est préparée par le procédé suivant : a) Chauffer un mélange réactionnel comprenant une solution aqueuse, et, au moins deux précurseurs biosourcé # 1 et 2 tels que définis ci-dessus dissous dans la solution aqueuse, avec une solution comprenant des cations métalliques, à une température inférieure à 300°C pour obtenir un matériau carboné poreux solide, b) Laver le matériau hybride carboné poreux solide obtenu par trempage successifs dans un solvant polaire,
c) Sécher le matériau hybride carboné poreux solide à l’aide d’un fluide supercritique ou par lyophilisation. 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 below 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) Dry the solid porous carbon hybrid material using a supercritical fluid or by freeze drying.
La quantité d’ions métalliques complexé au matériau carboné poreux pour former le matériau hybride ou la composition de la présente invention n'est pas particulièrement limitée. Cependant, la quantité d’ions métalliques adsorbés ou complexés sur un matériau carboné est de préférence comprise de 0,1 à 40 parties en poids par rapport à 100 parties en poids du matériau carboné poreux. 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 quantity of metal ions adsorbed or complexed on a carbon material is preferably between 0.1 and 40 parts by weight relative to 100 parts by weight of the porous carbon material.
Dans un mode de réalisation particulier, la composition comprend une quantité de cations métalliques chélatés ou complexés, mesurée par la méthode MEB-EDX, comprise de 0,2 à 2% molaire, de préférence de 0,5 à 1% molaire. In a particular embodiment, the composition comprises a quantity of chelated or complexed metal cations, measured by the MEB-EDX method, comprised from 0.2 to 2 mole%, preferably from 0.5 to 1 mole%.
Dans un mode de réalisation particulier, la composition comprend optionnellement une quantité de 1 à 5 % massique de cation métallique en particulier de 2 à 4% massique. In a particular embodiment, the composition optionally comprises a quantity of 1 to 5% by weight of metal cation, in particular 2 to 4% by weight.
Dans un mode de réalisation particulier, la composition comprend une quantité de 20 à 40 mg de Fer par gramme de matériau carboné poreux. In a particular embodiment, the composition comprises a quantity of 20 to 40 mg of iron per gram of porous carbonaceous material.
Le solvant utilisé ici n'est pas particulièrement limité tant qu'il peut dissoudre et/ou disperser des complexes métalliques. Des exemples de ceux-ci comprennent : l'acide acétique, l'eau, l'éthylène glycol, le DMSO et le DMF. The solvent used here 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.
La concentration de complexe métallique dans la solution n'est pas particulièrement limitée. Cependant, la concentration est de préférence d'environ 0,1 à 30 mM. The concentration of metal complex in the solution is not particularly limited. However, the concentration is preferably about 0.1 to 30 mM.
Selon un mode particulier, la composition (matériau hybride carboné poreux solide) comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to a particular mode, the composition (solid porous carbon hybrid material) comprises: a) a porous carbon material obtained by hydrothermal carbonization comprising or formed from:
• -d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside (précurseur biosourcé #1), et • -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide (biosourced precursor #1), and
• -d’au moins un composé phénolique ou polyphénolique (précurseur biosourcé #2), et
b) optionnellement au moins un cation métallique choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. • -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+ , 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+ .
Selon un mode particulier, la composition comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to a particular embodiment, the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside, dans laquelle -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide, in which
-l’ose est choisi parmi -the bone is chosen from
• un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, l’arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose, l’idose, le galactose, et le talose, de préférence le xylose, ou • 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
• un cétose notamment choisi parmi : la dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, et le tagatose, de préférence le fructose, ou • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or
-le polyoside est choisi parmi : -the polysaccharide is chosen from:
• un hétérodiholoside notamment choisi parmi : le tréhalulose, le saccharose , le turanose , le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, et le rutinose, ou • a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or
• un homodiholoside notamment choisi parmi l’inulobiose, l’alpha2- mannobiose, I’alpha3-Mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, et le gentiobiose, de préférence maltose ou saccharose et • 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
-d’au moins un composé phénolique ou polyphénolique (précurseur biosourcé #2). b) optionnellement au moins un cation métallique choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. -at least one phenolic or polyphenolic compound (biosourced precursor #2). b) optionally at least one metal cation chosen from 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+ .
Selon un autre mode particulier, la composition comprend :
Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to another particular mode, the composition comprises: A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside, dans laquelle -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide, in which
-l’ose est choisi parmi -the bone is chosen from
• un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, l’arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose,l’idose, le galactose, le talose, de préférence le xylose, ou • an aldose chosen in particular from: glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose , galactose, talose, preferably xylose, or
• un cétose notamment choisi parmi : la dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, le tagatose, de préférence le fructose, ou • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, tagatose, preferably fructose, or
-le polyoside est choisi parmi : -the polysaccharide is chosen from:
• un hétérodiholoside notamment choisi parmi: le tréhalulose, le saccharose , le turanose , le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, le rutinose, ou • a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, rutinose, or
• un homodiholoside notamment choisi parmi: l’inulobiose, l’alpha2-mannobiose, I’alpha3-Mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, le gentiobiose, de préférence maltose ou saccharose, • a homodiholoside chosen in particular from: inulobiose, alpha2-mannobiose, alpha3-Mannobiose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobiose , preferably maltose or sucrose,
-d’au moins un composé phénolique ou polyphénolique choisi parmi : -at least one phenolic or polyphenolic compound chosen from:
• un phénol simple notamment choisi parmi: le phénol, le catéchol, le résorcinol, 1’ hydroquinone, le pyrogallol, et le phloroglucinol, • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,
• un composé phénolique de la famille des flavonoïdes notamment choisi parmi les sous-classes: flavonol, anthocyanidols, anthocyane, flavanol, flavanone, stilbénoïdes, isofl avonoïde, • a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
• un tanin condensé • a condensed tannin
• un phlorotanin notamment choisi parmi: le fucol, le difucol, le trifucol, le phloréthol, le diphloréthol, le bifuhalol, le trifuhalol, lefucophloréthol, le fuhalol, l’eckol, et le diphloréthohydroxycamanol • a phlorotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
• ou un extrait naturel végétal contenant au moins un des composés ci-dessus. et
b) optionnellement au moins un cation métallique choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. • 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+ , 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+ .
Selon un autre mode particulier, la composition comprend : According to another particular mode, the composition comprises:
Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, ou de dihydroxyacétone, -xylose, or dihydroxyacetone,
-de phloroglucinol ou de catéchine. b) optionnellement au moins un cation métallique choisi parmi Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. -phloroglucinol or catechin. b) optionally at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ .
Selon un autre mode particulier, la composition comprend : According to another particular mode, the composition comprises:
Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, -xylose,
-de phloroglucinol ou de catéchine. b) optionnellement au moins un cation métallique qui est Fe3+ -phloroglucinol or catechin. b) optionally at least one metal cation which is Fe 3+
Selon un autre mode particulier, la composition comprend : According to another particular mode, the composition comprises:
Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, -xylose,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique qui est Co2+ -phloroglucinol or catechin, and b) optionally at least one metal cation which is Co 2+
Selon un autre mode particulier, la composition comprend : According to another particular mode, the composition comprises:
Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de dihydroxyacétone, -dihydroxyacetone,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique qui est Fe3+
Selon la présente l’invention, toutes les compositions décrites ci-dessus peuvent être utilisées pour immobiliser des protéines, en particulier au moins une enzyme -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
Immobilisation d’une protéine Immobilization of a protein
Un second but de l’invention est de proposer une composition comprenant une protéine ou une enzyme immobilisée sur ledit matériau hybride carboné poreux solide ou composition. Le matériau hybride carboné poreux solide de la présente invention comprend le matériau carboné poreux solide décrit ci-dessus en tant que support, au moins un ion métallique complexé audit matériau et au moins une protéine (en particulier une enzyme) immobilisée (sur le matériau carboné poreux optionnellement chargé en au moins un cation métallique), dans laquelle la au moins une protéine immobilisée est immobilisée par des liaisons dites faibles (liaisons de van der Waals, liaisons hydrogène) et/ou des liaisons de coordination et/ou des liaisons ioniques. 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 with said material and at least one protein (in particular an enzyme) immobilized (on the carbon material porous optionally charged with at least one metal cation), in which 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.
La protéine utilisée ici n'est pas particulièrement limitée. The protein used here is not particularly limited.
La composition de la présente invention comprend ainsi au moins une enzyme choisie dans la liste approuvée de la nomenclature et de la classification des enzymes de l'Union internationale de biochimie (IUBMB) et des exemples de celle-ci comprennent les protéines suivantes : The composition of the present invention thus comprises at least one enzyme chosen from the approved list of the nomenclature and classification of enzymes of the International Union of Biochemistry (IUBMB) and examples thereof include the following proteins:
EC 1 Oxydo-réductases, EC 2 Transférases, EC 3 Hydrolases, EC 4 Lyases, EC 5 Isomérases, EC 6 Ligases et EC 7 Translocases. EC 1 Oxido-reductases, EC 2 Transferases, EC 3 Hydrolases, EC 4 Lyases, EC 5 Isomerases, EC 6 Ligases and EC 7 Translocases.
Parmi ces protéines, au moins une protéine choisie dans le groupe notamment constitué par l'a-acétolactate, l'a-arabinosidase, l'a-galactosidase, l'a-rhamnosidase, la P-galactosidase, la P-glucanase, la P-glucosidase, la P-glucanase, la P-mannanase, T y-lactamase, l’acétolactate décarboxylase, l’activase, l’adénosine désaminase, l’aminoacylase, T aminopeptidase, l’amylase, l’amyloglucosidase, l’asparginase, l’aspartase, labromélaïne, anhydrase carbonique, la catalase, la cellulase, la chitinase, la chymosine, la collagénase, la cyclodextrinase, la désoxyribonucléase I, la dextranase, la enoate-reductase, l’épimérase, l’estérase, la formiate déshydrogénase, la galactinol synthase, la glucanotransférase, la glucoamylase, la glucose isomérase, la glucose oxydase, la glutenase, l’hémicellulase, l’hexose oxydase, l’inulinase, l’invertase, la laccase, la lactase, la lactate déshydrogénase, la leucine déshydrogénase, la levanase, la lipase, la lipoxygénase, le lysozyme, la méthane monooxygénase, la monoamine oxydase, la muramidase, la naphtalène dioxygénase, la naphtalène monooxygénase, la
naringinase, la nattokinase, la nitrile hydratase, la papaïne, la pectinase, la pectinestérase, la pénicilline G, l’acylase, la pentosanase, la phénol oxydase, la phénylalanine déshydrogénase, la phytase, la polyéthylestérase, la polygalacturonase, la protéase, la protopectinase, la pullulanase, la pyrophosphatase, la pyruvate transaminase, la raffinose synthase, la raffinose synthase, la présure, la sacrosidase, la serratiopeptidase, la sphingosine kinase, la stachyose synthase, la tannase, la taxolase, la thermolysine, la transaminase, la transglutimases, la trypsine, l’uréase, la xylanase, la xylose isomérase. Among these proteins, at least one protein chosen from the group notably consisting of a-acetolactate, a-arabinosidase, a-galactosidase, a-rhamnosidase, P-galactosidase, P-glucanase, P-glucosidase, P-glucanase, P-mannanase, T y-lactamase, acetolactate decarboxylase, activase, adenosine deaminase, aminoacylase, T aminopeptidase, amylase, amyloglucosidase, asparginase, aspartase, abromelain, 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, raffinose synthase, rennet, sacrosidase, serratiopeptidase, sphingosine kinase, stachyose synthase, tannase, taxolase, thermolysin, transaminase, transglutimases, trypsin, urease, xylanase, xylose isomerase.
Selon un autre mode préféré de l’invention, la composition comprend une protéine choisie dans le groupe notamment constitué par une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1). 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) .
Charges protéiques Protein fillers
En ce qui concerne le matériau hybride carboné poreux de la présente invention, la quantité de protéine adsorbée sur le matériau carboné poreux n'est pas particulièrement limitée tant qu'elle présente une activité enzymatique. Cependant, la quantité de protéine adsorbée sur le matériau carboné poreux est de préférence de 10 à 80 parties en poids par rapport à 100 parties en poids du matériau carboné poreux, de préférence de 10 à 50 parties en poids par rapport à 100 parties en poids du matériau carboné poreux. As for 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 exhibits 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 porous carbonaceous material.
De plus, le procédé pour fournir le matériau hybride carboné poreux de la présente invention en provoquant l'adsorption d'une protéine sur matériau carboné poreux n'est pas particulièrement limité. Il est possible d'utiliser, par exemple, la méthode de sublimation et la méthode d'imprégnation. Le procédé d'imprégnation décrit ci-dessous est plus préférable. C'est- à-dire que la protéine est d'abord dissoute dans de l'eau ou un tampon à une concentration à laquelle la protéine n'est pas précipitée (de préférence à une concentration de 0,1 à 1 000 mg/ml). Un matériau carboné poreux est ensuite mis en suspension dans la solution résultante à une température à laquelle la solution ne gèle pas et à laquelle la protéine ne se dénature pas (de préférence 0 à 50°C.) Ainsi, la protéine est mise en contact avec le matériau carboné poreux pendant au moins 5 minutes ou plus, de préférence pendant 30 minutes ou plus et ainsi la protéine est immobilisée dans les pores du gel de carbone. De cette manière, le matériau hybride carboné de la présente invention peut être fourni. Furthermore, the method for providing the porous carbon hybrid material of the present invention by causing adsorption of a protein onto 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 buffer at a concentration at which the protein is not precipitated (preferably at a concentration of 0.1 to 1000 mg/ ml). A porous carbonaceous 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.
Dans un mode de réalisation particulier, la composition comprenant au moins une protéine immobilisée est préparée par le procédé comprenant les étapes consistant à :
- Mettre en suspension le matériau carboné poreux séché dans une solution pouvant contenir des cations métalliques, In a particular embodiment, the composition comprising at least one immobilized protein is prepared by the process comprising the steps consisting of: - Suspend the dried porous carbon material in a solution that may contain metal cations,
- Optionnellement laver le matériau hybride carboné poreux lorsqu’il est chargé en cations métalliques - Optionally wash the porous carbon hybrid material when it is loaded with metal cations
- Immobiliser une protéine (enzyme) sur le matériau hybride carboné poreux optionnellement chargé en cations métalliques en faisant réagir le matériau carboné poreux optionnellement chargé en cations métalliques avec ladite protéine; - Immobilize 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;
-laver la suspension et récupérer la protéine immobilisée sur le matériau hybride carboné poreux optionnellement chargé en cations métalliques. -wash the suspension and recover the protein immobilized on the porous carbon hybrid material optionally loaded with metal cations.
Plus spécifiquement, le matériau hybride carboné poreux optionnellement chargé en cations métalliques est ajouté à une solution aqueuse contenant une protéine de préférence une enzyme. More specifically, the porous carbon hybrid material optionally loaded with metal cations is added to an aqueous solution containing a protein, preferably an enzyme.
Le matériau hybride carboné poreux optionnellement chargé en cations métalliques sur lequel est immobilisé au moins une protéine par des liaisons dites faibles (liaisons de van der Waals, liaisons hydrogène) et/ou des liaisons de coordination et/ou des liaisons ioniques, est lavé plusieurs fois avec une solution aqueuse tamponnée. The porous carbon hybrid material optionally loaded with metal cations on which at least one protein is immobilized by so-called weak bonds (van der Waals bonds, hydrogen bonds) and/or coordination bonds and/or ionic bonds, is washed several times. times with a buffered aqueous solution.
La protéine à immobiliser sur le matériau peut être n'importe quelle protéine, telle qu'une protéine (recombinante) ou une enzyme. The protein to be immobilized on the material can be any protein, such as a (recombinant) protein or an enzyme.
De préférence, la protéine est une enzyme. Preferably, the protein is an enzyme.
La solution aqueuse tamponnée est de préférence un tampon Tris-HCl, ou Phosphate. The buffered aqueous solution is preferably a Tris-HCl, or Phosphate, buffer.
Selon un deuxième aspect de l’invention, la présente demande concerne une composition comprenant : a) Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to a second aspect of the invention, the present application relates to a composition comprising: a) A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside (précurseur biosourcé #1), et -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide (biosourced precursor #1), and
-d’au moins un composé phénolique ou polyphénolique (précurseur biosourcé #2), b) optionnellement au moins un cation métallique, et c) au moins une protéine, de préférence une enzyme. -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.
Selon un mode préféré de l’invention, la composition comprend
a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to a preferred mode of the invention, the composition comprises a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside (précurseur biosourcé #1), et -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide (biosourced precursor #1), and
-d’au moins un composé phénolique ou polyphénolique (précurseur biosourcé #2), b) optionnellement au moins un cation métallique, et c) au moins une protéine immobilisée, de préférence une enzyme. -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.
Selon un autre mode préféré de l’invention, la composition comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to another preferred embodiment of the invention, the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide de préférence au moins un ose, ou au moins un polyoside, dans laquelle l’ose est choisi parmi : -at least one carbohydrate, preferably at least one ose, or at least one polysaccharide, in which the ose is chosen from:
• un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, l’arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose,l’idose, le galactose, et le talose, de préférence le xylose, ou • 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
• un cétose notamment choisie parmi: la dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, et le tagatose, de préférence le fructose, ou dans laquelle le polyoside est choisi parmi : • 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:
• un hétérodiholoside notamment choisi parmi : le tréhalulose, le saccharose , le turanose , le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, et le rutinose, ou • a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or
• un homodiholoside notamment choisi parmi : l’inulobiose, l’alpha2- mannobiose, I’alpha3-Mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, et le gentiobiose, de préférence maltose ou saccharose, et • 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
-d’au moins un composé phénolique ou polyphénolique choisi parmi : -at least one phenolic or polyphenolic compound chosen from:
• un phénol simple notamment choisi parmi: le phénol, le catéchol, le résorcinol, 1’ hydroquinone, le pyrogallol, et le phloroglucinol,,
• un composé phénolique de la famille des flavonoïdes notamment choisi parmi les sous-classes: flavonol, anthocyanidols, anthocyane, flavanol, flavanone, stilbénoïdes, isofl avonoïde, • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol, • a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
• un tanin condensé • a condensed tannin
• un phlorotanin notamment choisi parmi: le fucol, le difucol, le trifucol, le phloréthol, le diphloréthol, le bifuhalol, le trifuhalol, lefucophloréthol, le fuhalol, l’eckol, et le diphloréthohydroxycamanol, ou • a phlorotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol, or
• un extrait naturel végétal contenant au moins un des composés ci- dessus, b) optionnellement au moins un cation métallique choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+, et c) au moins une protéine immobilisée. • a natural plant extract containing at least one of the compounds above, b) optionally at least one metal cation chosen from 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+ , and c) at least one immobilized protein.
Selon un autre mode préféré de l’invention, la composition comprend : a) Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to another preferred embodiment of the invention, the composition comprises: a) A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, ou de dihydroxyacétone, -xylose, or dihydroxyacetone,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique choisi parmi Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+ et c) au moins une protéine immobilisée. -phloroglucinol or catechin, and b) optionally at least one metal cation chosen from Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ and c) at least one immobilized protein.
Selon un autre mode préféré de l’invention, la composition comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to another preferred embodiment of the invention, the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, -xylose,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique qui est Fe3+, et c) au moins une protéine immobilisée.
Selon un autre mode préféré de l’invention, la composition comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : -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 carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de xylose, -xylose,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique qui est Co2+, et c) au moins une protéine immobilisée. -phloroglucinol or catechin, and b) optionally at least one metal cation which is Co 2+ , and c) at least one immobilized protein.
Selon un autre mode préféré de l’invention, la composition comprend : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : According to another preferred embodiment of the invention, the composition comprises: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-de dihydroxyacétone, -dihydroxyacetone,
-de phloroglucinol ou de catéchine, et b) optionnellement au moins un cation métallique qui est Fe3+, et c) au moins une protéine immobilisée. -phloroglucinol or catechin, and b) optionally at least one metal cation which is Fe 3+ , and c) at least one immobilized protein.
Par exemple, si le matériau solide selon l’invention est utilisé pour la purification et l'isolement d'une protéine, la liaison de l'enzyme au support doit être réversible. 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.
Pour l'utilisation d'enzymes immobilisées dans une biocatalyse hétérogène, une forte liaison de l'enzyme au support est souhaitable. For the use of immobilized enzymes in heterogeneous biocatalysis, strong binding of the enzyme to the support is desirable.
Si les protéines immobilisées sur le support, telles que décrites ci-dessus, sont des enzymes, elles contiennent un site actif capable de catalyser une réaction chimique. If the proteins immobilized on the support, as described above, are enzymes, they contain an active site capable of catalyzing a chemical reaction.
Ainsi, la matière d'enzyme immobilisée est potentiellement utile comme biocatalyseur dans la synthèse organique. Thus, the immobilized enzyme material is potentially useful as a biocatalyst in organic synthesis.
Par conséquent, dans un autre aspect, l'invention concerne l'utilisation d'un matériau d'enzyme immobilisé tel que décrit ici en tant que biocatalyseur hétérogène, par exemple dans des transformations organiques synthétiques. 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.
Un mode préféré de l’invention, concerne l’utilisation des compositions décrites ci- dessus en tant que biocatalyseurs hétérogènes. A preferred mode of the invention concerns the use of the compositions described above as heterogeneous biocatalysts.
La présente invention concerne en outre un procédé de catalyse d'une réaction catalysée par une enzyme, comprenant la fourniture d'une composition comprenant une protéine ou une enzyme immobilisée sur ledit matériau carboné poreux selon l'invention, et la mise en contact
de ladite composition comprenant une enzyme immobilisée avec au moins un substrat sur lequel l’enzyme, qui est immobilisée sur ledit matériau, est capable d'agir. The present invention further relates to a method of catalyzing an enzyme-catalyzed reaction, comprising providing a composition comprising a protein or enzyme immobilized on said porous carbonaceous material according to the invention, and contacting of said composition comprising an enzyme immobilized with at least one substrate on which the enzyme, which is immobilized on said material, is capable of acting.
On a trouvé que les enzymes immobilisées tolèrent à la fois des conditions aqueuses ainsi qu'une gamme de solvants organiques. The immobilized enzymes were found to tolerate both aqueous conditions as well as a range of organic solvents.
Ceci permet d'utiliser les compositions comprenant une protéine ou une enzyme immobilisée sur ledit matériau carboné poreux dans des conditions réactionnelles dans lesquelles les enzymes libres, non immobilisées n'auraient pas été stables. This makes it possible to use compositions comprising a protein or an enzyme immobilized on said porous carbon material under reaction conditions in which the free, non-immobilized enzymes would not have been stable.
Il est possible que la composition comprenant une protéine ou une enzyme immobilisée sur ledit matériau carboné poreux puisse également être utilisée dans une plage de pH plus large que les enzymes libres, non immobilisées n'auraient tolérée. It is possible that the composition comprising a protein or enzyme immobilized on said porous carbonaceous material can also be used in a wider pH range than free, non-immobilized enzymes would have tolerated.
L'enzyme qui est immobilisée sur ledit matériau peut être n'importe quelle enzyme qui est utile comme biocatalyseur dans des transformations synthétiques organiques, y compris, mais sans s'y limiter, des enzymes agissant comme des oxydoréductases, des transférases, des hydrolases, des lyases, des isomérases et des ligases. The enzyme which is immobilized on said material may be any enzyme which is useful as a biocatalyst in organic synthetic transformations, including, but not limited to, enzymes acting as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
Ainsi, les compositions comprenant une enzyme immobilisée sur ledit matériau peuvent être utilisés comme biocatalyseurs hétérogènes dans toute réaction organique dans laquelle l'enzyme immobilisée est capable de catalyser spécifiquement la réaction. 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.
Des exemples de telles réactions biocatalytiques comprennent, mais sans s'y limiter, des réactions d'oxydation et de réduction enzymatiques, des réactions d'hydrolyse enzymatique et des réactions d'isomérisation enzymatique. Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.
Des réactions biocatalytiques particulièrement utiles sont des réactions énantiosélectives. Particularly useful biocatalytic reactions are enantioselective reactions.
Des exemples de telles réactions biocatalytiques comprennent, mais sans s'y limiter, des réactions d'oxydation et de réduction enzymatiques, des réactions d'hydrolyse enzymatique et des réactions d'isomérisation enzymatique. Examples of such biocatalytic reactions include, but are not limited to, enzymatic oxidation and reduction reactions, enzymatic hydrolysis reactions, and enzymatic isomerization reactions.
Dans un mode de réalisation, deux enzymes différentes ou plus peuvent être immobilisées sur le matériau selon l’invention, dans lequel chacune des enzymes différentes est capable de catalyser une réaction différente. In one embodiment, two or more different enzymes can be immobilized on the material according to the invention, wherein each of the different enzymes is capable of catalyzing a different reaction.
Il peut alors être possible d'utiliser le matériau contenant deux enzymes immobilisées différentes ou plus en tant que biocatalyseur hétérogène dans une réaction en plusieurs étapes ou en cascade.
Selon un autre aspect de l’invention, la présente demande concerne un procédé pour catalyser une réaction catalysée par une enzyme, comprenant la fourniture d'une composition décrite ci -dessus, et mise en contact de ladite composition avec au moins un substrat sur lequel une enzyme, qui est immobilisée sur ledit support de la composition, est susceptible d'agir. It may then be possible to use the material containing two or more different immobilized enzymes as a heterogeneous biocatalyst in a multistep 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 bringing said composition into contact with at least one substrate on which an enzyme, which is immobilized on said support of the composition, is capable of acting.
Rendement et vitesse d’immobilisation Yield and speed of immobilization
Les rendements d’immobilisation, exprimés en mg d’enzyme immobilisée par mg d’enzyme initialement présente dans la solution sont compris de 90 à 100% pour des charges protéiques comprises de 5 à 50% massique. The immobilization yields, expressed in mg of immobilized enzyme per mg of enzyme initially present in the solution, are between 90 and 100% for protein loads between 5 and 50% by weight.
Les vitesses d’immobilisation exprimée en minutes pour atteindre au moins un taux d’immobilisation d’au moins 75% sont comprises de 10 à 120 minutes. The immobilization speeds expressed in minutes to achieve at least an immobilization rate of at least 75% are between 10 and 120 minutes.
Selon un mode préféré de l’invention, le procédé d’immobilisation d’une protéine, en particulier d’une enzyme sur le matériau carboné poreux est caractérisé en ce que le rendement de l'immobilisation est compris de 90% à 100% pour des charges protéiques comprises de 5 à 50% massique. According to a preferred embodiment of the invention, the process for immobilizing a protein, in particular an enzyme on the porous carbon material is characterized in that the efficiency of the immobilization is between 90% and 100% for protein loads ranging from 5 to 50% by weight.
Selon un mode préféré de l’invention, le procédé d’immobilisation d’une protéine, en particulier d’une enzyme sur le matériau carboné poreux est caractérisé en ce que la vitesse d’immobilisation pour atteindre un rendement de l'immobilisation d’au moins 75% est comprise de 10 à 120 minutes. According to a preferred embodiment of the invention, the method of immobilizing a protein, in particular an enzyme on the porous carbon material is characterized in that the speed of immobilization to achieve a yield of the immobilization of at least 75% is between 10 and 120 minutes.
Selon un autre aspect de l’invention, la présente demande concerne l’utilisation de la composition comme biocatalyseur hétérogène. According to another aspect of the invention, the present application relates to the use of the composition as a heterogeneous biocatalyst.
Selon un dernier aspect de l’invention, la présente demande concerne : According to a final aspect of the invention, the present application concerns:
Une composition comprenant : A composition comprising:
• Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir: • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside,-at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide,
-d’au moins un composé phénolique ou polyphénolique, -at least one phenolic or polyphenolic compound,
• Au moins une protéine, de préférence une enzyme immobilisée, ladite au moins une protéine immobilisée est de préférence une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1)• 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)
• Optionnellement, au moins un cation métallique.
Une composition dans laquelle • Optionally, at least one metal cation. A composition in which
-l’ose est: -the dare is:
• un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, 1’ arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose, l’idose, le galactose, et le talose, de préférence le xylose, ou • 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
• un cétose notamment choisi parmi : le dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, et le tagatose, de préférence le fructose, ou • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or
-le polyoside est : -the polysaccharide is:
• un hétérodiholoside notamment choisi parmi : le tréhalulose, le saccharose , le turanose , le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, et le rutinose, ou • a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or
• un homodiholoside notamment choisi parmi : l’inulobiose, l’alpha2-mannobiose, l’alpha3-mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, et le gentiobiose, de préférence le maltose ou le saccharose • 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
Une composition dans laquelle le composé phénolique ou polyphénolique est : A composition in which the phenolic or polyphenolic compound is:
• un phénol simple notamment choisi parmi: le phénol, le catéchol, le résorcinol, 1’ hydroquinone, le pyrogallol, et le phloroglucinol, • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,
• un composé phénolique de la famille des flavonoïdes notamment choisi parmi les sous-classes: flavonol, anthocyanidols, anthocyane, flavanol, flavanone, stilbénoïdes, isofl avonoïde, • a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
• un tanin condensé, • a condensed tannin,
• un phi orotanin notamment choisi parmi: le fucol, le difucol, le trifucol, le phloréthol, le diphloréthol, le bifuhalol, le trifuhalol, lefucophloréthol, le fuhalol, l’eckol, et le diphloréthohydroxycamanol • a phi orotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
• ou un extrait naturel végétal contenant au moins un de ces composés.
Une composition dans laquelle le composé phénolique ou polyphénolique est un composé de la sous-famille des flavanols ou catéchines. • or a natural plant extract containing at least one of these compounds. A composition in which the phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily.
Une composition dans laquelle le composé phénolique ou polyphénolique est notamment choisi parmi : la catéchine ou le phloroglucinol. A composition in which the phenolic or polyphenolic compound is chosen in particular from: catechin or phloroglucinol.
Une composition dans laquelle le matériau carboné poreux est obtenu par carbonisation hydrothermale comprenant ou formé à partir de : A composition in which the porous carbonaceous material is obtained by hydrothermal carbonization comprising or formed from:
-maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, érythrose, dihydroxyacétone, et -maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and
-phloroglucinol, catéchine, d’un extrait de cachou. -phloroglucinol, catechin, from a cashew extract.
Une composition dans laquelle le au moins un cation métallique lorsqu‘il est présent est choisi parmi Al3+, Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Cu+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Sn2+, Sn4+, Zn2+, de préférence un ou des cations de métaux de transition Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. A composition in which the at least one metal cation when present is chosen from 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 transition metals Fe 3+ , Co 2+ , Zn 2+ , Cu 2+ , or Ni 2+ .
Une composition comprenant : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : A composition comprising: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’un ose choisi parmi le xylose, ou la dihydroxyacétone -an ose chosen from xylose, or dihydroxyacetone
-d’un composé phénolique ou polyphénolique choisi parmi le phloroglucinol ou la catéchine, et b) au moins une protéine immobilisée, ladite au moins une protéine immobilisée est de préférence une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1), et c) optionnellement, au moins un cation métallique choisi parmi Fe3+, Co2+, Zn2+, Cu2+, ou Ni2+. -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 Ni 2+ .
Une composition dans laquelle la quantité de protéine adsorbée sur le matériau carboné poreux est de préférence de 10 à 80 parties en poids par rapport à 100 parties en poids du matériau carboné poreux, encore plus de préférence de 10 à 50 parties en poids par rapport à 100 parties en poids du matériau carboné poreux.
Une composition dans laquelle l’activité spécifique de l’enzyme immobilisée sur le matériau carboné poreux est supérieure à celle de la même enzyme libre. 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 carbonaceous material. A composition in which the specific activity of the enzyme immobilized on the porous carbon material is greater than that of the same free enzyme.
Liste des figures List of Figures
La figure 1 est une micrographie électronique à balayage d’une texture poreuse uniforme continuellement interconnectée pour un aérogel tannin-xylose préparé dans 10 mL d’une solution à 15% massique d’éthanol absolu dans de l’eau déminéralisée, traité à 180°C pendant 20 heures en conditions hydrothermales (autoclave), lavé puis séché par lyophilisation. Figure 1 is a scanning electron micrograph of a continuously interconnected uniform porous texture for a tannin-xylose airgel prepared in 10 mL of a 15 mass% solution of absolute ethanol in deionized water, treated at 180° C for 20 hours in hydrothermal conditions (autoclave), washed then dried by freeze-drying.
La figure 2 est une micrographie électronique à transmission qui met en évidence un réseau macro-mésoporeux pour un aérogel tannin-xylose préparé dans 10 mL d’une solution à 15% massique d’éthanol absolu dans de l’eau déminéralisée, traité à 180°C pendant 20 heures en conditions hydrothermales (autoclave), lavé puis séché par lyophilisation. Figure 2 is a transmission electron micrograph which highlights a macro-mesoporous network for a tannin-xylose airgel prepared in 10 mL of a 15% solution by weight of absolute ethanol in demineralized water, treated at 180 °C for 20 hours in hydrothermal conditions (autoclave), washed then dried by freeze-drying.
Les figures 1 et 2 montrent des particules primaires de quelques nanomètres, particules primaires agrégées formant un réseau interconnecté qui induit des interstices interparticulaires de quelques nanomètres à plusieurs micromètres. Figures 1 and 2 show primary particles of a few nanometers, aggregated primary particles forming an interconnected network which induces interparticle gaps of a few nanometers to several micrometers.
Partie expérimentale Experimental part
Exemple 1 : préparation d’un matériau carboné poreux sous la forme d’un aérogel Example 1: preparation of a porous carbon material in the form of an airgel
0,85 g de xylose est dissout dans 10 mL d’un mélange d’eau désionisée et d’éthanol absolu 50/50 m/m. 0.85 g of xylose is dissolved in 10 mL of a mixture of deionized water and absolute ethanol 50/50 m/m.
0,35 g de phloroglucinol est ajoutée à la solution précédente. 0.35 g of phloroglucinol is added to the previous solution.
Le mélange est agité jusqu’à obtenir une solution homogène. Un traitement aux ultrasons à température ambiante pendant une heure ou moins peut permettre d’accélérer l’homogénéisation de la solution, The mixture is stirred until a homogeneous solution is obtained. Ultrasonic treatment at room temperature for an hour or less can accelerate the homogenization of the solution,
La solution résultante est traitée par carbonisation hydrothermale c’est-à-dire introduite dans une bombe de minéralisation (autoclave), puis celle-ci est fermée hermétiquement et placée dans une enceinte thermostatée à 180°C pendant 20h,
A l’issue de ce délai, l’autoclave et son contenu sont refroidis à température ambiante pendant quelques heures (généralement 2h), The resulting solution is treated by hydrothermal carbonization, that is to say introduced into a mineralization bomb (autoclave), then this is closed hermetically and placed in an enclosure thermostatically controlled 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),
L’autoclave est ensuite ouvert et son contenu versé soit dans de l’eau ultrapure (généralement 100 mL), soit dans de l’eau désionisée, soit dans un mélange eau/ solvant protique polaire (méthanol, éthanol, propanol, butanol ou leurs isomères), soit dans un solvant protique polaire pur, The autoclave is then opened and its contents poured either into ultrapure water (generally 100 mL), or into deionized water, or into a water/polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or in a pure polar protic solvent,
Le solide obtenu est lavé par trempage successifs (généralement 3 fois 100 mL) avec soit dans de l’eau ultrapure, soit dans de l’eau désionisée, soit dans un mélange eau/solvant protique polaire (méthanol, éthanol, propanol, butanol ou leurs isomères), soit dans un solvant protique polaire pur pendant plusieurs heures (généralement 8 heures). On obtient ainsi un hydrogel. L’hydrogel est ensuite séché par lyophilisation ou dans un fluide supercritique pour obtenir un aérogel. The solid obtained is washed by successive soaking (generally 3 times 100 mL) with either ultrapure water, deionized water, or a water/polar protic solvent mixture (methanol, ethanol, propanol, butanol or their isomers), or in a pure polar protic solvent for several hours (generally 8 hours). We thus obtain a hydrogel. The hydrogel is then dried by freeze-drying or in a supercritical fluid to obtain an aerogel.
Exemple 2 : Immobilisation d’une protéine Example 2: Immobilization of a protein
10 mg aérogel obtenus dans les conditions décrites ci-dessus sont ajoutés à une solution aqueuse contenant (Img) de protéine. 10 mg airgel obtained under the conditions described above are added to an aqueous solution containing (Img) of protein.
L’enzyme suivante a été préparée : la transaminase B9L0N2 (w-TA, EC2 transférase). The following enzyme was prepared: B9L0N2 transaminase (w-TA, EC2 transferase).
25 mg aérogel obtenus dans les conditions décrites ci-dessus sont ajoutés à une solution tampon (Tris 20 mM pH=7, 1 mL) contenant 2,5 mg d’enzyme CalB (Candida antartica lipase B). 25 mg airgel obtained under the conditions described above are added to a buffer solution (20 mM Tris pH=7.1 mL) containing 2.5 mg of CalB enzyme (Candida antarctica lipase B).
La suspension ainsi obtenue est agitée à 750rpm pendant quelques heures (généralement 2 heures) à 4°C. The suspension thus obtained is stirred at 750 rpm for a few hours (generally 2 hours) at 4°C.
Après agitation, la suspension est centrifugée (entre 4000 et ôOOOrpm pendant 2 à 5 min). Après centrifugation, la surnageant est éliminé. Le solide ainsi obtenu est lavé avec une solution aqueuse tamponnée 2 fois : After stirring, the suspension is centrifuged (between 4000 and 6OOOrpm for 2 to 5 min). After centrifugation, the supernatant is removed. The solid thus obtained is washed with a buffered aqueous solution twice:
1 ) Du tampon est aj outé ( 1 mL), 1) Buffer is added (1 mL),
2) La suspension ainsi obtenue est agitée pendant quelques minutes (généralement 5 minutes) à température ambiante, 2) The suspension thus obtained is stirred for a few minutes (generally 5 minutes) at room temperature,
3) La suspension est centrifugée (entre 4000 et ôOOOrpm pendant 2 à 5 min), 3) The suspension is centrifuged (between 4000 and ôOOOrpm for 2 to 5 min),
4) Après centrifugation, le surnageant est éliminé. 4) After centrifugation, the supernatant is removed.
Les étapes 1 à 4 sont recommencées 1 fois. Steps 1 to 4 are repeated once.
On obtient ainsi une protéine immobilisée sur le matériau carboné poreux. We thus obtain a protein immobilized on the porous carbon material.
Exemple 3 Cinétiques d’immobilisation
10 mg matériau carboné poreux sont mis en présence avec 1 mg d’enzyme dans un volume total de 1 mL. Example 3 Immobilization kinetics 10 mg porous carbonaceous material is brought into contact with 1 mg of enzyme in a total volume of 1 mL.
L'immobilisation de la transaminase B9L0N2 (w-TA, EC2 transférase) se fait en présence du cofacteur pyridoxal phosphate (PLP). The immobilization of the B9L0N2 transaminase (w-TA, EC2 transferase) occurs in the presence of the cofactor pyridoxal phosphate (PLP).
Les échantillons 0, 10, 20 et 30 min (10 pL) sont dilués au demi et les échantillons Ih et 2h (20 pL) ne sont pas dilués. The 0, 10, 20 and 30 min samples (10 pL) are diluted to half and the 1h and 2h samples (20 pL) are not diluted.
20 pL d'échantillon (Echl à 2) mélangé à 1 mL de réactif de Bradford, à l'obscurité pendant 15 min. 20 pL of sample (Echl at 2) mixed with 1 mL of Bradford reagent, in the dark for 15 min.
Résultats Results
Cinétique d’immobilisation de B9L0N2 (Img) sur un aérogel Xylose/Catéchine(10mg) préparé dans les conditions décrites ci -dessus.
Kinetics of immobilization of B9L0N2 (Img) on a Xylose/Catechin aerogel (10mg) prepared under the conditions described above.
Les résultats du tableau 2 ci-dessus montrent qu’un taux d’immobilisation d’au moins 80% est atteint en moyenne après 10 minutes. The results in Table 2 above show that an immobilization rate of at least 80% is reached on average after 10 minutes.
Ces résultats montrent que l’immobilisation d’une enzyme sur le matériau carboné poreux est simple, rapide et efficace. These results show that the immobilization of an enzyme on the porous carbon material is simple, rapid and effective.
Cinétique d’immobilisation de CalB (2,5mg) sur différents aérogels préparés dans les conditions décrites ci-dessus.
Kinetics of immobilization of CalB (2.5 mg) on different aerogels prepared under the conditions described above.
Les résultats du tableau 3 ci-dessus montrent qu’un taux d’immobilisation d’au moins 80% est atteint en moyenne après 10 minutes pour des supports XPh. The results in Table 3 above show that an immobilization rate of at least 80% is reached on average after 10 minutes for XPh supports.
Ces résultats montrent que dans le cas de CalB, le matériau XPh permet une relativement meilleure immobilisation (99,47% en 120 min) que le matériau XCat (89,825% en 120 min) bien que les 2 performances soient tout de même assez proches. Cette différence pourrait s’expliquer par un caractère hydrophobe plus marqué en surface du matériau XPh que du matériau XCat. En effet, il a été démontré dans la littérature que la lipase CalB présentait une plus forte affinité pour les matériaux d’immobilisation dont les surfaces sont relativement hydrophobes. These results show that in the case of CalB, the XPh material allows relatively better immobilization (99.47% in 120 min) than the XCat material (89.825% in 120 min) although the two performances are still quite close. This difference could be explained by a more marked hydrophobic nature on the surface of the XPh material than of the XCat material. Indeed, it has been demonstrated in the literature that CalB lipase has a stronger affinity for immobilization materials whose surfaces are relatively hydrophobic.
Les matériaux sont broyés manuellement au mortier puis tamisé par passage sur un premier tamis de 300 microns et un second de 100 microns pour ne retenir que la fraction comprise 100 et 300 microns. The materials are crushed manually with mortar 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.
Exemple 4 : Performances Example 4: Performance
Les taux de conversion sont exprimés en moles de produit formé par moles de substrat initialement présent, mesurés et calculés après un temps de réaction donné. Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time.
Pour w-TA, la réaction étudiée est la transamination du pyruvate en présence de a- methylbenzylamine racémique en alanine et acétophénone en présence du cofacteur pyridoxal phosphate (PLP). La charge protéique utilisée est de 10% en masse. Le mélange réactionnel est
constitué de pyruvate (25mM), d’a-methylbenzylamine racémique (25mM) de PLP (0,lmM) et de DMSO (1% wt) en solution dans du tampon Phosphate 50mM NaCI 300mM pH 7,5. Le solide constitué de Img de w-TA immobilisée sur un hydrogel (lOmg) est ajouté au mélange réactionnel. La formation de l’acétophénone est suivie en continu en spectroscopie UV à 245nm. Les résultats sont résumés dans le Tableau 4.
For w-TA, the reaction studied is the transamination of pyruvate in the presence of racemic a-methylbenzylamine to alanine and acetophenone in the presence of the cofactor pyridoxal phosphate (PLP). The protein load used is 10% by mass. The reaction mixture is consisting of pyruvate (25mM), racemic a-methylbenzylamine (25mM) PLP (0.lmM) and DMSO (1% wt) in solution in Phosphate buffer 50mM NaCl 300mM pH 7.5. The solid consisting of Img of w-TA immobilized on a hydrogel (lOmg) 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.
Tableau 4 : Performances catalytique (Turnover numbers, TON des enzymes libres ou immobilisées étudiées (B9L0N2/w-TA). Table 4: Catalytic performances (Turnover numbers, TON of the free or immobilized enzymes studied (B9L0N2/w-TA).
Le TON de l’enzyme immobilisée est significativement supérieur à celui de l’enzyme non immobilisée. The TON of the immobilized enzyme is significantly higher than that of the non-immobilized enzyme.
Ces résultats sont meilleurs que ceux décrits à ce jour dans la littérature où Ton observe que dans la plupart des cas l’activité de l’enzyme est fortement réduite après immobilisation. 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 greatly reduced after immobilization.
La structure macroporeuse interconnectée (pores entre 50 nm et 2 microns) facilite donc bien le transfert de masse des réactifs et des produits dans tout le matériau. The interconnected macroporous structure (pores between 50 nm and 2 microns) therefore facilitates the mass transfer of reactants and products throughout the material.
Pour CalB, la réaction étudiée est l’hydrolyse du / /ra-nitrophényl butyrate (pNPB) en para- nitrophénol et acide butanoïque. Le mélange réactionnel est constitué de pNPB (15 mM) en solution dans du tampon Tris-HCl 20 mM pH=7,0 contenant du Genapol X-100 (1% massique). La réaction est démarrée par ajout de la CalB immobilisée (25 mg d’aérogel chargé avec 10 % massique d’enzyme). Les essais ont été réalisé à 25°C pendant 1 h avec une agitation de 800 rpm. La formation du / /ra-nitrophénol est suivie par spectrophotométrie visible à 410 nm.
Pour les enzymes immobilisées, l’activité est mesurée sur 5 cycles réactionnels. Un cycle réactionnel se déroule comme suit : For CalB, the reaction studied is the hydrolysis of / /ra-nitrophenyl butyrate (pNPB) to 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 weight). The reaction is started by adding immobilized CalB (25 mg of airgel loaded with 10% by weight of enzyme). The tests were carried out at 25°C for 1 hour with stirring at 800 rpm. The formation of / /ra-nitrophenol is followed by visible spectrophotometry at 410 nm. For immobilized enzymes, activity is measured over 5 reaction cycles. A reaction cycle proceeds as follows:
1) La réaction enzymatique est réalisée comme décrit ci-dessus. 1) The enzymatic reaction is carried out as described above.
2) A la fin de la réaction la suspension est centrifugée 2 à 5 min entre 4000 et 6000 rpm. 2) At the end of the reaction, the suspension is centrifuged for 2 to 5 min between 4000 and 6000 rpm.
3) Le surnageant est récupéré et 1 mL de solution tampon (Tris-HCl à 20 mM pH=7) est ajoutée au solide. 3) The supernatant is recovered and 1 mL of buffer solution (Tris-HCl at 20 mM pH=7) is added to the solid.
4) La nouvelle suspension est agitée 3 min à 800 rpm. 4) The new suspension is stirred for 3 min at 800 rpm.
5) Les étapes 2 à 4 sont répétées trois fois. 5) Steps 2 to 4 are repeated three times.
L’activité enzymatique mesurée pour la CalB immobilisée est exprimée en mole de p- nitrophénol formée par masse de catalyseur (enzyme immobilisée sur l’aérogel) durant un temps donné. Pour ce faire la quantité de p-nitrophénol dosée dans chacun des surnageants récupérés à la suite des 5 étapes précédentes est additionnée. La formule est la suivante :
The enzymatic activity measured for immobilized CalB is expressed as moles of p-nitrophenol formed per mass of catalyst (enzyme immobilized on the airgel) for 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:
Ces activités sont mesurées pour les enzymes non immobilisées (libres) et pour les enzymes immobilisées par la technique décrite ci-dessus. These activities are measured for non-immobilized (free) enzymes and for immobilized enzymes by the technique described above.
Les taux de conversion sont exprimés en moles de produit formé par moles de substrat initialement présent, mesurés et calculés après un temps de réaction donné. La formule est la suivante :
Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time. The formula is as follows:
Les résultats sont résumés dans le Tableau 5.
Tableau 5 : Taux de conversion mesurés pour CalB immobilisée sur XPh et sur XCat
The results are summarized in Table 5. Table 5: Conversion rates measured for CalB immobilized on XPh and on XCat
Tableau 6 : Activité enzymatique mesurée pour CalB immobilisée sur XPh et sur XCat Table 6: Enzymatic activity measured for CalB immobilized on XPh and on XCat
Exemple 5: Lixiviation Example 5: Leaching
Pour chaque cycle réactionnel, les quatre surnageants issus des étapes 1 à 3 du protocole d’activité sont conservés. La quantité de protéines contenue dans ces solutions est dosée par mesure de l’activité enzymatique résiduelle, en procédant comme suit. For each reaction cycle, the four supernatants from steps 1 to 3 of the activity protocol are kept. The quantity of proteins contained in these solutions is determined by measuring the residual enzymatic activity, using the following procedure.
Un prélèvement de chaque surnageant des cycles réactionnels (20 pL) est ajouté à une solution de réaction contenant 1,5 mM de substrat (pNPB), 1% de Genapol et 20 mM de tampon Tris HCl pH=7. La solution est agitée pendant 5 min à 25°C. La quantité de /?-nitrophénol produite lors de cette réaction est comparée à une gamme étalon pour déterminer à la masse de protéine contenue dans le prélèvement. Elle est ainsi rapportée à la quantité initiale de protéine immobilisée sur le solide pour obtenir le taux de lixiviation. A sample of each supernatant from 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.
On définit ainsi le taux de lixiviation cumulée :
We define the cumulative leaching rate as follows:
Tableau 7 : Taux de lixiviation mesurée à chaque cycle d’utilisation de l’enzyme immobilisée sur 5 cycles réactionnels sur 2 supports différents (XPh : xylose/Phloroglucinol et XCat : xylose/ Cathéchine) 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)
Comme pour les expériences d’immobilisation, ces expériences montrent que la lixiviation (par cycle et cumulée sur 5 cycles) de CalB lorsqu’elle est immobilisée sur XPh est plus faible que sur XCat. Ceci pourrait également s’expliquer par un caractère hydrophobe plus marqué en surface du matériau XPh que du matériau XCat. En effet, il a été démontré dans la littérature que la lipase CalB présentait une plus forte affinité pour les matériaux d’immobilisation dont les surfaces sont relativement hydrophobes. 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 nature on the surface of the XPh material than the XCat material. Indeed, it has been demonstrated in the literature that CalB lipase has a stronger affinity for immobilization materials whose surfaces are relatively hydrophobic.
EXPERIENCES EN FLUX (avec et sans recirculation ; sans recirculation = flux continu) FLOW EXPERIMENTS (with and without recirculation; without recirculation = continuous flow)
Immobilisation de CalB sur XPh en flux continu : Immobilization of CalB on XPh in continuous flow:
Les aérogels XPh obtenus dans les conditions décrites précédemment (65 mg) sont introduits dans une colonne thermostatée dont la température est maintenue à 4°C.L’extrémité de la colonne est constituée dans un fritté permettant de retenir l’aérogel. 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.
Une solution tampon (Tris 20 mM pH=7, 5 mL) contenant 0,250g d’une solution enzymatique CalB (Lypozyme ® STREM CHEMICALS) percole à un débit 0, 1 mL/min dans
la colonne par le biais d’une pompe. La solution est récupérée à la sortie de la colonne. L’aérogel contenu dans la colonne est ensuite lavée par percolation de solution tampon à un débit de 0,1 mL/min pendant 50 min. La solution est aussi récupérée à la sortie de la colonne. A buffer solution (20 mM Tris pH=7.5 mL) containing 0.250 g of a CalB enzymatic solution (Lypozyme ® STREM CHEMICALS) percolates at a flow rate of 0.1 mL/min in the column by means of a pump. The solution is recovered at the outlet of the column. The airgel 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 outlet of the column.
La quantité de protéines présentes dans ces deux solutions sont mesurée par activité résiduelle en procédant comme suit. The amount of proteins present in these two solutions are measured by residual activity using the following procedure.
Un prélèvement des solutions récupérées (20 pL) est ajouté à une solution de réaction contenant 1,5 mM de substrat (pNPB), 1% de Genapol et 20 mM de tampon Tris HCl pH=7. La solution est agitée pendant 5 min à 25°C. La quantité de p-nitrophénol produite lors de cette réaction est comparée à une gamme étalon pour déterminer à la masse de protéine contenue dans le prélèvement. 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.
On obtient ainsi la protéine CalB immobilisée sur un hydrogel XPh (avec une charge protéique de 7,7 % en masse) dans une colonne pour être utilisée en recirculation. We thus obtain the CalB protein immobilized on an XPh hydrogel (with a protein loading of 7.7% by mass) in a column to be used in recirculation.
• Mesure de taux de conversion en flux avec recirculation en utilisant l’enzyme CalB immobilisée sur XPh • Measurement of conversion rate in flow with recirculation using the CalB enzyme immobilized on XPh
La réaction étudiée est l'hydrolyse du para-nitrophényl butyrate (pNPB) en para- nitrophénol et acide butanoïque. Le mélange réactionnel est constitué de pNPB (5 mM) en solution dans du tampon Tris-HCI 20 mM pH=7,0 contenant du Genapol X-100 (1% massique). 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 weight).
Le mélange réactionnel (6 mL) est introduit dans la colonne préparée comme décrit précédemment dans la partie « immobilisation en flux continu ». Le flux est en recirculation, la solution récupérée en sortie de colonne est directement réinjectée dans la colonne à un débit de 0,1 mL/min. La colonne est thermostatée, la température est maintenue à 35°C. Un prélèvement (100 pL) est réalisé toutes les heures pendant 5h. La formation du para- nitrophénol est suivie par spectrophotométrie visible à 410 nm. The reaction mixture (6 mL) is introduced into the column prepared as described previously in the “continuous flow immobilization” section. The flow is in recirculation, 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 followed by visible spectrophotometry at 410 nm.
Les taux de conversion sont exprimés en moles de produit formé par moles de substrat initialement présent, mesurés et calculés après un temps de réaction donné. La formule est la suivante : 100
• Mesure de taux de Lixiviation en flux avec recirculation en utilisant l’enzyme CalB immobilisée sur XPh Conversion rates are expressed in moles of product formed per moles of substrate initially present, measured and calculated after a given reaction time. The formula is: 100 • Measurement of flow leaching rate with recirculation using the CalB enzyme immobilized on XPh
Un prélèvement (20 pL) est réalisé dans les fractions récupérées comme décrit précédemment dans la partie « activité ». La quantité de protéines contenue dans ces prélèvements est dosée par activité résiduelle en procédé comme suit. A sample (20 μL) is taken from the fractions recovered as described previously in the “activity” section. The quantity of proteins contained in these samples is determined by residual activity using the following method.
Le prélèvement est ajouté à une solution de réaction contenant 1,5 mM de substrat (pNPB), 1% de Genapol et 20 mM de tampon Tris HCl pH=7. La solution est agitée pendant 5 min à 25°C. La quantité de /?-nitrophénol produite lors de cette réaction est comparée à une gamme étalon pour déterminer à la masse de protéine contenue dans le prélèvement. Elle est ainsi rapportée à la quantité initiale de protéine immobilisée sur le solide pour obtenir le taux de lixiviation. 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.
Le taux de lixiviation est obtenu par la formule suivante :
The leaching rate is obtained by the following formula:
Ces expériences montrent que l’immobilisation en flux d’une protéine est possible sur nos matériaux et qu’elle est rapide et efficace. Le caractère hydrophobe semble être plus marqué en surface des matériaux XPh que XCat puisque CalB s’immobilise mieux et se décroche moins sur le premier que sur le second.
Les taux de conversion observés en flux sont les mêmes que ceux mesurés pour l’enzyme libre et en batch à temps de contact égal démontrant des transferts de matière très efficaces et une très bonne accessibilité du substrat à l’enzyme.
These experiments show that 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 first than on the second. 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 material transfers and very good accessibility of the substrate to the enzyme.
Claims
1. Composition comprenant : 1. Composition comprising:
• Un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : • A porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’au moins un glucide, de préférence au moins un ose, ou au moins un polyoside, -at least one carbohydrate, preferably at least one carbohydrate, or at least one polysaccharide,
-d’au moins un composé phénolique ou polyphénolique,-at least one phenolic or polyphenolic compound,
• Au moins une protéine, de préférence une enzyme immobilisée, ladite au moins une protéine immobilisée est de préférence une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1) • 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 selon la revendication 1 dans laquelle 2. Composition according to claim 1 in which
-l’ose est: -the dare is:
• un aldose notamment choisi parmi : le glycéraldéhyde, l’érythrose, le thréose, le ribose, 1’ arabinose, le xylose, le lyxose, l’allose, l’altrose, le glucose, le mannose, le gulose, l’idose, le galactose, et le talose, de préférence le xylose, ou • 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
• un cétose notamment choisi parmi : le dihydroxyacétone, l’érythrulose, le ribulose, le xylulose, le fructose, le psicose, le sorbose, et le tagatose, de préférence le fructose, ou • a ketose chosen in particular from: dihydroxyacetone, erythrulose, ribulose, xylulose, fructose, psicose, sorbose, and tagatose, preferably fructose, or
-le polyoside est : -the polysaccharide is:
• un hétérodiholoside notamment choisi parmi : le tréhalulose, le saccharose , le turanose , le maltulose, le leucrose, l’isomaltulose, le gentiobiulose, le mélibiose, le lactulose, le lactose, et le rutinose, ou • a heterodisaccharide chosen in particular from: trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiulose, melibiose, lactulose, lactose, and rutinose, or
• un homodiholoside notamment choisi parmi : l’inulobiose, l’alpha2-mannobiose, l’alpha3-mannobiose, le tréhalose, le kojibiose, le nigerose, le maltose, l’isomaltose, le sophorose, le laminaribiose, le cellobiose, et le gentiobiose, de préférence le maltose ou le saccharose
• 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 selon la revendication 1 dans laquelle le composé phénolique ou polyphénolique est : 3. Composition according to claim 1 in which the phenolic or polyphenolic compound is:
• un phénol simple notamment choisi parmi: le phénol, le catéchol, le résorcinol, 1’ hydroquinone, le pyrogallol, et le phloroglucinol, • a simple phenol chosen in particular from: phenol, catechol, resorcinol, hydroquinone, pyrogallol, and phloroglucinol,
• un composé phénolique de la famille des flavonoïdes notamment choisi parmi les sous-classes: flavonol, anthocyanidols, anthocyane, flavanol, flavanone, stilbénoïdes, isofl avonoïde, • a phenolic compound from the flavonoid family in particular chosen from the subclasses: flavonol, anthocyanidols, anthocyanin, flavanol, flavanone, stilbenoids, isofl avonoid,
• un tanin condensé, • a condensed tannin,
• un phi orotanin notamment choisi parmi: le fucol, le difucol, le trifucol, le phloréthol, le diphloréthol, le bifuhalol, le trifuhalol, lefucophloréthol, le fuhalol, l’eckol, et le diphloréthohydroxycamanol • a phi orotanin chosen in particular from: fucol, difucol, trifucol, phlorethol, diphlorethol, bifuhalol, trifuhalol, lefucophlorethol, fuhalol, eckol, and diphlorethohydroxycamanol
• ou un extrait naturel végétal contenant au moins un de ces composés. • or a natural plant extract containing at least one of these compounds.
4. Composition selon la revendication 3, dans laquelle le composé phénolique ou polyphénolique est un composé de la sous-famille des flavanols ou catéchines. 4. Composition according to claim 3, in which the phenolic or polyphenolic compound is a compound of the flavanol or catechin subfamily.
5. Composition selon la revendication 3 ou 4, dans laquelle le composé phénolique ou polyphénolique est notamment choisi parmi : la catéchine ou le phloroglucinol. 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 selon la revendication 1, dans laquelle le matériau carboné poreux est formé à partir de : 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, érythrose, dihydroxyacétone, et -maltose, galactose, lactose, glucose, mannose, sucrose, arabinose, ribose, fructose, xylose, erythrose, dihydroxyacetone, and
-phloroglucinol, catéchine, d’un extrait de cachou. -phloroglucinol, catechin, from a cashew extract.
7. Composition selon la revendication 1, comprenant : a) un matériau carboné poreux obtenu par carbonisation hydrothermale comprenant ou formé à partir : 7. Composition according to claim 1, comprising: a) a porous carbonaceous material obtained by hydrothermal carbonization comprising or formed from:
-d’un ose choisi parmi le xylose, ou la dihydroxyacétone -an ose chosen from xylose, or dihydroxyacetone
-d’un composé phénolique ou polyphénolique choisi parmi le phloroglucinol ou la catéchine, et
b) au moins une protéine immobilisée, ladite au moins une protéine immobilisée est de préférence une enoate reductase (EC 1.3.1.31), une transaminase (EC 2.6.1), une pyrophosphatase (EC3.6.1.1), et 8. Composition selon l’une des revendications 1 à 7, dans laquelle la quantité de protéine adsorbée sur le matériau carboné poreux est de 10 à 80 parties en poids par rapport à 100 parties en poids du matériau carboné poreux, encore plus de préférence de 10 à 50 parties en poids par rapport à 100 parties en poids du matériau carboné poreux. 9. Composition selon l’une des revendications 1 à 8, dans laquelle l’activité spécifique de l’enzyme immobilisée sur le matériau carboné poreux est supérieure à celle de la même enzyme libre.
-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, in which the quantity of protein adsorbed on the porous carbon material is 10 to 80 parts by weight relative to 100 parts by weight of the porous carbon material, even more preferably 10 to 50 parts by weight relative to 100 parts by weight of the porous carbonaceous material. 9. Composition according to one of claims 1 to 8, in which 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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