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    • 专利摘要:
    The invention relates to a process for the production of nanocelluloses from a cellulose substrate comprising cellulose fibers, which process comprises the following successive steps: a step of enzymatic treatment of said cellulosic substrate, by placing it in contact with at least one a cleaving enzyme, then - a step of mechanical treatment of said cellulosic substrate subjected to said enzymatic treatment step, for delaminating said cellulose fibers and obtaining said nanocelluloses, characterized in that said at least one cleavage enzyme is chosen from enzymes belonging to the family of lyophilic monooxygenases polysaccharides (LPMOs) capable of ensuring cleavage in the presence of an electron donor. The invention also relates to nanocelluloses obtained according to this process.
    公开号:FR3037078A1
    申请号:FR1555049
    申请日:2015-06-03
    公开日:2016-12-09
    发明作者:Bernard Cathala；Ana Villares；Jean-Guy Berrin；Celine Moreau
    申请人:Inst Nat de la Rech Agronomique - Inra；Institut National de la Recherche Agronomique INRA；
    IPC主号:
    专利说明:

    [0001] TECHNICAL FIELD TO WHICH THE INVENTION RELATES The present invention relates generally to the field of nanocelluloses, and more particularly to the processes for producing these nanocelluloses from a cellulosic substrate.
    [0002] BACKGROUND TECHNOLOGY Cellulose is one of the most important natural polymers, a virtually inexhaustible raw material, and an important source of sustainable materials on an industrial scale.
    [0003] To date, different forms of cellulose have been identified with a dimension of the order of a nanometer, referred to as the generic name of "nanocelluloses". The properties of these nanocelluloses, in particular their mechanical properties, their ability to form films and their viscosity, give them a major interest in many industrial fields. Nanocelluloses are thus used for example as a dispersant or stabilizer additive in the paper, pharmaceutical, cosmetic or agri-food industries. They are also used in the composition of paints and varnishes.
    [0004] Nanocelluloses are also used in many devices requiring control of nanoscale porosity because of their high surface area. Finally, many nanocomposite materials based on nanocelluloses are currently being developed. Indeed, the remarkable mechanical properties of nanocelluloses, their nanoscale dispersion as well as their hydrophilic nature, give them excellent gas barrier properties. These characteristics are of particular interest for the manufacture of barrier packaging. Based on their dimensions, functions and preparation methods, which themselves mainly depend on the cellulosic source and the treatment conditions, the nanocelluloses can be classified mainly in two families: cellulose fibrils and cellulose nanocrystals. Cellulose nanocrystals (also called "crystalline nanocelluloses" or NCCs for "nanocrystalline cellulose") are generally obtained by hydrolysis with a strong acid under strictly controlled conditions of temperature, duration and agitation. Such a treatment makes it possible to attack the amorphous regions of the fibers while leaving the crystalline regions, which are more resistant and intact. The suspension obtained is then washed by successive centrifugations and dialyses in distilled water. The most conventionally obtained NCCs have a length from a few tens of nanometers to about 1 μm (especially from 40 nm to 1 μm and preferably from 40 nm to 500 nm), and a diameter ranging from 5 to 70 nm, preferably less than at 15 nm (typically 5 to 10 nm).
    [0005] Cellulose fibrils, commonly referred to as cellulose microfibrils (also known as "microfibrillated cellulose" or "microfibrillated cellulose") or cellulose nanofibrils (NFC) are typically isolated from cellulosic materials derived from biomass. by mechanical methods to delaminate the cellulose fibers and release the cellulose fibrils. For example, US 4,483,743 discloses a process for producing microfibrillated cellulose, which involves the passage of a liquid suspension of cellulose through a homogenizer type Gaulin high pressure. Repeated passages of the cellulose suspension make it possible to obtain microfibrils typically having a width of 25 to 100 nm and a much larger length. In general, the mechanical processes for obtaining cellulose fibrils have the disadvantage of consuming large amounts of energy. For example, it has been evaluated that the use of a homogenizer results in an energy consumption of the order of 70000 kWh / t. This significant energy consumption, and consequently the high costs of nanocellulose production, remain a considerable obstacle to their industrial development. Various pre-treatment strategies for cellulose fibers have thus been developed in order to reduce the energy consumption required for their mechanical delamination. A first pretreatment strategy, described for example in the application WO 2007/091942, consists in pretreating the cellulose fibers with cellulases in order to destructure the fiber before the application of the mechanical treatment by homogenization. However, this enzymatic pretreatment is extremely versatile depending on the state of the fiber and in particular according to the prior thermochemical history of the fiber.
    [0006] In addition, the quality of the nanocelluloses obtained (in particular the state of dispersion and in particular the lateral size of the nanofibrils which conditions the properties of use and the energy yields are very variable.) A second pretreatment strategy is based on a step chemical oxidation of cellulose fibers (eg Saito et al., Biomacromolecules, Vol 8, No. 8, 2007, pp. 2485-2491) Typically, the fibers are oxidized with an oxidant such as sodium hypochlorite catalyzed by the 2,2,6,6-tetramethylpiperidine-1-oxyl radical ("TEMPO") before undergoing the aforementioned mechanical treatment, the oxidative treatment converts the primary alcohol function to the C6 position of the glucose unit of the cellulose to a carboxylate function, which leads to the introduction of fillers on the surface of the cellulose fibers.These fillers create electrostatic repulsions which facilitate delamination and increase its efficiency. However, removal of the reaction products leads to large amounts of highly polluted effluents. In addition, reagent residues persist within the final product and continue to react, ultimately altering the properties of the nanocelluloses. Thus, despite the new pretreatment strategies developed, nanocellulose production costs remain high, yields uncertain, and the quality and properties are variable. It therefore remains necessary to propose new processes for obtaining nanocelluloses, with less energy consumption and in a simple and reproducible manner, in a little or no toxic way.
    [0007] OBJECT OF THE INVENTION In order to overcome the aforementioned drawbacks of the state of the art, the present invention proposes a process for manufacturing nanocelluloses based on a pretreatment step of cellulose fibers with at least one enzyme belonging to the family of lyo monooxygenases of 3037078 4 polysaccharides, commonly referred to as "LPM0s" for "Lytic Polysaccharide MonoOxygenases". More particularly, according to the invention, there is provided a process for producing nanocelluloses from a cellulose substrate comprising cellulose fibers, which process comprises the following successive steps: at least one step of enzymatic treatment of said cellulosic substrate, by contacting with at least one cleavage enzyme, and then - at least one mechanical treatment step of said cellulosic substrate subjected to said at least one enzymatic treatment step, to delaminate the cellulose fibers and to obtain said nanocelluloses, characterized in that said at least one cleavage enzyme is selected from enzymes belonging to the LPM0s family. Typically, LPM0s are capable of oxidatively cleaving cellulose fibers, advantageously glucose cycles of cellulose fibers, in the presence of an electron donor. Without being limited by any theory, the action of LPM0s facilitates the manufacture of nanocellulose through two actions: the cleavage of cellulosic chains causes fragilities within the fibers, facilitating the mechanical delamination, the formation of products of Oxidation allows to introduce charged chemical functions on the surface of the fibers by inducing electrostatic repulsions. Without being limited by any theory, these combined structural modifications have the effect of promoting the separation of the fibers to nanometric dispersion and of forming nanocelluloses (fibrils or nanocrystals) having novel functionalities (charge rates, chemical functions at present). not available). Other nonlimiting and advantageous features of the manufacturing method according to the invention, taken individually or in any technically possible combination, are also described hereinafter as well as in the detailed description of the invention. The electron donor may be selected from ascorbate, gallate, cathecol, reduced glutathione, lignin fragments and fungal carbohydrates dehydrogenases (especially glucose dehydrogenases, and cellobiose dehydrogenases). Preferably, the LPM0s are chosen from enzymes capable of cleaving the cellulose by oxidation of at least one carbon atom in position (s) C1, C4 and C6 of the glucose cycle. The LPMOs can be selected from the fungal enzyme families AA9 (formerly known as GH61) and bacterial enzymes AA10 (formerly known as CBM33) of the CAZy classification (www.cazy.org). In particular, the LPM0s may be chosen from LPM0s derived from Podospora anserina and preferably from PaLPMO9A (Genbank CAP68375), PaLPMOB (Genbank CAP73254), PaLPMO9D (Genbank CAP66744) PaLPMO9E (Genbank CAP67740), PaLPMO9F (Genbank CAP71839), PaLPMO9G ( Genbank CAP73072), and PaLPMO9H (Genbank CAP61476) According to the embodiments of the invention, the cellulosic substrate 15 is obtained from wood, a fibrous plant rich in cellulose, beet, citrus, annual plants. straw, marine animals, algae, fungi or bacteria. Preferably, the cellulosic substrate is chosen from chemical papermaking pulps, preferably chemical wood pulp, and more preferably at least one of the following papermaking pulps: bleached pastes, semi-bleached pastes, unbleached pasta, sulphite pastes, sulphate pastes, soda pastes, kraft pastes. Said at least one mechanical treatment step generally comprises at least one of the following mechanical treatments: a homogenization treatment, a microfluidization treatment, an abrasion treatment, a cryomilling treatment. The process may also comprise a post-treatment step, for example an acid treatment, an enzymatic treatment, an oxidation, an acetylation, a silylation, or a derivatization of certain chemical groups carried by the nanocelluloses. The invention also relates to the nanocelluloses obtained by the implementation of the method of the invention. Typically, the nanocelluloses obtained consist of cellulose nanofibrils and / or of cellulose nanocrystals. FIGURES Figure 1: Appearance of cellulose kraft fibers treated with the enzyme PaLPMO9H at various enzyme / substrate ratios and subjected to low mechanical treatment with an Ultra-Turrax type homogenizer-disperser and ultrasonic treatment. Optical microscopy images of untreated (control) fibers with enzyme (A) or treated with 1:50 enzyme / substrate ratios (B); 1: 100 (C); and 1: 500 (D). Figure 2: Appearance of cellulose kraft fibers treated with the enzyme PaLPMO9H at a 1:50 enzyme / substrate ratio and subjected to a weak mechanical treatment with a homogenizer-disperser of the type of Ultra-Turrax and an ultrasonic treatment. Optical microscopy images of the control fibers (A) and the treated fibers (B), and visualization of the nanofibrils obtained by transmission electron microcopy (C) and atomic force microscopy (D). 3: Appearance of the cellulose kraft fibers treated with the enzymes PaLPMO9H and PaLPMO9E according to an enzyme / substrate ratio of 1:50 and then subjected to a weak mechanical treatment with an Ultra-Turrax type homogenizer-disperser and an ultrasound treatment . Optical microscopy images of kraft fibers treated with PaLPMO9H (A) and with PaLPMO9E (B). Atomic force microscopy visualization of nanofibrils obtained from kraft fibers by treatment with LPM0s enzymes PaLPMO9H (C) and PaLPMO9E (D). Figure 4. Appearance of cellulose kraft fibers subjected to two successive treatments with the enzyme PaLPMO9H at different enzyme / substrate ratios then subjected to a weak mechanical treatment with a homogenizer-disperser of the type of Ultra-Turrax and an ultrasonic treatment. Optical microscopy images of fibers treated according to enzyme / substrate ratios of 1:50 (A); 1: 100 (B); 1: 500 (C) and 1: 1000 (D).
    [0008] DETAILED DESCRIPTION The following description, in combination with the Experimental Results given as non-limiting examples, will make it clear in what the invention is and how it can be achieved. General Definitions The present invention relates to a process for producing nanocelluloses, in particular cellulose fibrils and / or cellulose nanocrystals, from a cellulosic substrate. "Cellulose" means a linear homopolysaccharide derived from biomass (including organic matter of plant origin, including algae, cellulose of animal origin and cellulose of bacterial origin) and consisting of units (or cycles ) glucose (D-Anhydroglucopyranose - AGU for "Anhydro glucose unit") linked together by 6- (1 -4) glycosidic linkages. The repetition pattern is a glucose dimer also called cellobiose dimer. AGUs have 3 hydroxyl functions: 2 secondary alcohols (on carbons in positions 2 and 3 of the glucose cycle) and a primary alcohol (on carbon in position 6 of the glucose cycle). These polymers associate with intermolecular links of the hydrogen bonding type, thus conferring a fibrous structure on the cellulose. In particular, the combination of cellobiose dimers forms an elemental cellulose nanofibril (whose diameter is about 5 nm). The association of 25 elementary nanofibrils forms a nanofibril (whose diameter generally varies from 50 to 500 nm). The arrangement of several of these nanofibrils then forms what is generally called a cellulose fiber. The term "nanocellulose" refers to the various forms of cellulose having a dimension of the order of one nanometer. This term particularly includes, according to the invention, two families of nanocelluloses: cellulose nanocrystals and cellulose fibrils. The terms "cellulose fibrils", "nanofibrils (of cellulose)", "nanofibers (of cellulose)", "nanofibrillated cellulose", "microfibrils (of cellulose)", "microfibrillated cellulose", "microfibrillated cellulose", "cellulose 3037078 8 nanofibrils "are synonymous. In the remainder of the present application, the term "cellulose nanofibrils" (NFCs) will be used generically. Each cellulose nanofibril contains crystalline portions stabilized by a solid network of inter and intra-chain hydrogen bonds. These 5 crystalline regions are separated by amorphous regions. The elimination of the amorphous parts of the cellulose nanofibrils makes it possible to obtain cellulose nanocrystals (NCCs). The NCCs advantageously comprise at least 50% of crystalline part, more preferably at least 55% of crystalline part. They are generally 5 to 70 nm (preferably less than 15 nm) in diameter and 40 to 1 μm in length, preferably 40 to 500 nm in length. The terms "cellulose nanocrystals", "nanocrystalline cellulose", "cellulose whiskers", "microcrystals" or "nanocrystal cellulose" are synonymous. In the remainder of the present application, the term "cellulose nanocrystals" (NCCs) will be used generically. In the case of bacterial cellulose, the nanofibrils, or ribbons, of bacterial cellulose generally have a length of several micrometers and a width ranging from 30 to 60 nm, especially from 45 to 55 nm.
    [0009] Process according to the invention The process for the production of nanocelluloses, according to the invention, comprises the following successive stages: at least one step of enzymatic treatment of a cellulosic substrate comprising cellulose fibers, by bringing it into contact with at least one cleavage enzyme belonging to the family of polysaccharide lyophilic monooxygenases (LPM0s), then - at least one step of mechanical treatment of said cellulosic substrate subjected to said at least one enzymatic treatment step, for delaminating said fibers of cellulose and to obtain said nanocelluloses. One or more, and especially at least two, enzymatic treatment steps can be implemented according to the method of the invention, prior to said at least one mechanical treatment step. For example, at least two enzymatic treatment steps can be carried out successively, prior to said at least one mechanical treatment step. At least one enzymatic treatment step may also be carried out after said at least one mechanical treatment step.
    [0010] When several enzymatic treatment steps are carried out, the treatment conditions (duration, selected LPMO (s), enzyme / cellulose ratio, etc.) may be identical or different from each other. Typically, said at least one enzymatic treatment step, optionally followed by at least one mechanical treatment step, may be repeated, as described above, until complete delamination of the cellulose fibers is achieved. For example, the process of the invention may comprise at least two successive treatment cycles, each treatment cycle comprising at least one step of enzymatic treatment of the cellulosic substrate followed by at least one step of mechanical treatment of said substrate. Without being limited by any theory, the combination according to the invention (i) of an enzymatic treatment with at least one LPMO and (ii) a mechanical delamination treatment, makes it possible to obtain nanocelluloses whose structural characteristics and the mechanical properties are quite different from the existing nanocelluloses in the state of the art. Without being limited by any theory, the process of the invention makes it possible to obtain nanocelluloses in a simple and reproducible manner. Preferably, the size and the mechanical properties of these nanocelluloses are homogeneous.
    [0011] Cellulosic Substrate The cellulosic substrate can be obtained according to the invention from any biomass material (including organic matter of plant origin, including algae, animal or fungal) comprising cellulosic fibers (i.e. cellulose fibers). The cellulosic substrate is advantageously obtained from wood (of which cellulose is the main component), but also from any fibrous plant rich in cellulose, such as, for example, cotton, flax, hemp, bamboo, kapok, coconut fiber (coir), ramie, jute, sisal, raffia, 3037078 10 papyrus and certain reeds, sugarcane bagasse, beet (including beet pulp), citrus fruits, stalks of corn or sorghum, or annual straw plants. Cellulosic substrates can also be obtained from marine animals (such as tunicate for example), algae (such as for example Valonia or Cladophora) or bacteria for bacterial cellulose (for example bacterial strains of the Gluconacetobacter types). ). Depending on the application, cellulose derived from primary walls such as the parenchyma of fruits (for example beets, citrus fruits, etc.) or secondary walls, such as wood, will be chosen. The cellulosic substrate advantageously consists of a cellulosic material prepared by chemical or mechanical means, from any cellulosic source as mentioned above (and in particular from wood).
    [0012] The cellulosic substrate is advantageously in the form of a suspension of cellulose fibers in a liquid medium (preferably an aqueous medium), or a cellulose pulp. The cellulose pulps may be packaged in the "dry" state, typically in a state of dryness greater than or equal to 80%, especially greater than or equal to 90%. The cellulose pulp can then be redispersed in an aqueous medium by mechanical treatment. Preferably, the cellulosic substrate contains at least 90%, especially at least 95% and preferably 100% of cellulosic fibers. Preferably, the cellulosic substrate is suitable for the manufacture of paper or a cellulosic product. The cellulosic substrate is thus preferably chosen from papermaking pulps (or paper pulp), and in particular chemical pulps. In general, the cellulose pulp, and in particular the paper pulp, may contain, in association with the cellulose fibers, hemicellulose and lignin. Preferably, the cellulose pulp contains less than 10% and especially less than 5% of lignin and / or hemicellulose. Preferably, the chemical papermaking pulps contain almost exclusively or exclusively cellulose fibers. The pulp may be chosen from at least one of the following paper pulps: blanched pastes, semi-bleached pastes, unbleached pastes, sulphite pastes (unbleached or bleached), sulphate pastes (unbleached or bleached), soda dough (unbleached or bleached) and kraft dough. It is also possible to use dissolving pastes having a low proportion of hemicellulose, preferably less than 10% and in particular less than or equal to 5%. Preferably, the paper pulps used in a process of the invention are wood pulps, in particular chemical wood pulp.
    [0013] Polysaccharide lysooxygenases - LPMO The cellulosic substrate is thus subjected to at least one pretreatment step with at least one cleavage enzyme belonging to the family of lytic monooxygenases of polysaccharides ("Lytic Polysaccharide Ponooxygenases" or LPMO). . LPM0s are mononuclear enzymes of type II to copper. They have common structural features, including: a flat surface with an active site near its center and a highly conserved binding site for a type II copper ion exposed on the surface of the protein. The interaction between the LPMO enzyme and the surface of the cellulose occurs via the flat face of the LPMO enzyme and involves interactions with polar aromatic residues. The LPMOs that can be used according to the invention are defined by their capacity to catalyze an oxidative cleavage of the cellulose fibers of the cellulosic substrate, by oxidation of at least one of the carbon atoms in the C1, C4 and C6 positions of a glucose cycle. said cellulose fibers. The principle of oxidative cleavage achieved by the LPM0s involves the activation of a CH group followed by a dioxygen-dependent cleavage (02), thus producing oligomers oxidized on at least one of the carbon atoms at the C 1 and C 4 positions. and in C6. The LPMO (s) used are capable of catalyzing a cleavage of the cellulose fibers by oxidation of at least one of the carbon atoms selected from the C1 and / or C4 and / or C6 carbon atoms of a glucose cycle. cellulose. The oxidative cleavage leads to the formation of carboxyl groups on the surface of the cellulose fibers: the oxidative cleavage at the C1 position of a glucose cycle of one unit of cellobiose leads to the formation of a lactone, spontaneously hydrolyzed to acid Aldonic, and - the oxidative cleavage at the C4 position of a glucose cycle of a unit of cellobiose leads to the formation of a ketoalose Oxidation of the alcohol group at the C6 position of a glucose cycle of a unit of cellobiose leads to the formation of a carbonyl group.
    [0014] In certain embodiments, the LPMO (s) used catalyze cleavage of the cellulose fibers by oxidation of at least one of the carbon atoms selected from the C1 and / or C4 carbon atoms of a ring. glucose of cellulose. LPM0s catalyze the oxidative cleavage of a cellobiose unit in the presence of an external electron donor. This electron donor, generally a low molecular weight molecule, is selected from ascorbate, reduced glutathione, gallate, cathecol, lignin fragments, or an enzyme of the carbohydrate dehydrogenase family.
    [0015] Preferably, the carbohydrate dehydrogenases are chosen from fungal enzymes, in particular cellobiose dehydrogenase (CDHs). CDHs (or Cellobiose oxidoreductases - EC 1.1.99.18) catalyze the reaction [Cellobiose + electron acceptor <=> cellobiono-1,5-lactone + reduced acceptor]. These are fungal hemoflavoenzymes belonging to the superfamily of glucose-methanol-choline (GMC) oxidoreductases. CDHs are monomeric enzymes bearing two prosthetic groups, a heme group b and a flavin adenine dinucleotide. The flavoprotein domain of CDHs catalyzes the two-electron oxidation of cellobiose to lactone using an electron acceptor. This electron acceptor may for example be chosen from dioxygen, quinones and phenoxy radicals or LPMOs. The activity of a CDH enzyme can be determined by following the reduction of the 2,6-dichlorophenol indophenol reagent (DCPIP) in a sodium acetate buffer containing cellobiose (Bey et al., 2011, Microb Cell Fact 10: 113 ).
    [0016] Examples of CDHs usable in combination with at least one LPMO enzyme and also acting as an electron donor may be selected from CDHs derived from Pycnoporus cinnabarinus, Humicola insolens, Podospora anserina, or Myceliophthora thermophila.
    [0017] More preferably, an LPMO enzyme is used for which cellulolytic activity (i.e., catalyzing the oxidative cleavage of cellulose) has been identified. The oxidative cleavage activity of LPM0s on a cellulosic substrate can be tested in cleavage assays as described in the Example portion of the present application.
    [0018] More specifically, the LPM0s used in the invention are advantageously chosen from the so-called "auxiliary activity" (or "Auxiliary Activity" - AA) enzymes according to the classification established in the CAZy database, relating to the enzymes active on carbohydrates (CAZy - Carbohydrate Active enZyme database - http://www.cazy.org/ - See also 15 Levasseur et al., Biotechnology for Biofuels 2013, 6:41). More preferably, the enzymatic treatment step is carried out with at least one enzyme chosen from the LPM0s enzymes of the so-called AA9, AA10, AA11 and AA13 families, according to the classification established in the CAZy database.
    [0019] The LPMO enzyme according to the invention may contain a carbohydrate binding module carbohydrate binding module which is specific for CBM1 type cellulose according to the CAZy classification. The enzymes listed in this application are identified by references Genbank (identifying a genetic sequence) and Uniprot when the latter is available (identifying a protein sequence - see Table 1). By default, the reference indicated in parentheses for each enzyme corresponds to the reference "Genbank". Preferably, at least one enzyme of the AA9 family and / or at least one enzyme of the AA10 family of the CAZy classification is used (advantageously exclusively). The enzymes of the AA9 family, listed in Table 1 below, are fungal enzymes widely distributed in the genome of most ascomycetes and some basidiomycetes (fungi). In general, the enzymes of the AA9 family were initially classified in the family of glycoside hydrolases 61 (GH61) of the CAZy classification. Specific analyzes have since shown that the endoglucanase activity of AA9 enzymes was low or non-existent (Morgenstern I et al., Briefings in Functional Genomics vol.3 (6P): 471-481).
    [0020] Preferably, LPM0s are used whose endoglucanase activity is insignificant or non-existent. The copper ion of LPM0s of the AA9 family is bound to the protein according to a hexacoordination model involving at least 2 conserved histidine residues and water molecules.
    [0021] Enzymes of the AA9 family catalyze an oxidative cleavage of the cellobiose unit on carbon in the C 1 and / or C 4 positions, preferably on the C 1 or C 4 carbon. Certain enzymes (T. aurantiacus TaGH61A (G3XAP7) and Podospora anserina PaGH61 B (B2AVF1)) could catalyze an oxidative cleavage of cellobiose on a C6 carbon.
    [0022] LPM0s of the AA9 family expressed in fungi generally show a post-translational modification consisting of methylation of the N-terminal histidine residue. Preferably, LPMOs of the AA9 family comprising at least one CBM1 or CBM18 (CBM for "carbohydrate binding module") domain are used in the N-terminal position. These enzymes then comprise a flat surface formed of several polar aromatic residues forming a CBM1 or CBM18 type domain. More preferably, said at least one LPMO of the AA9 family is derived from Podospora anserina and / or Neurospora crassa.
    [0023] The AA9 family of enzymes derived from Podospora anserina are typically selected from the group consisting of PaLPMO9A (CAP68375), PaLPMO9B (CAP73254), PaLPMO9D (CAP66744), PaLPMO9E (CAP67740), PaLPMO9F (CAP71839), PaLPMO9G (CAP73072), PaLPMO9H (CAP61476). Preferably, the enzymes PaLPMO9E (CAP67740) and / or PaLPMO9H (CAP61476) are used. The enzymes of the AA9 family derived from Neurospora crassa are typically selected from the group consisting of NcLPMO9C (EAA36362), NcLPMO9D (EAA32426 / CAD21296), NcLPMO9E (EAA26873), NcLPMO9F (EAA26656 / CAD70347), NcLPMO9M (EAA33178), NcU00836 (EAA34466). ), 3037078 NcU02240 (EAA30263), NcU07760 (EAA29018). The enzymes of the AA10 family (CAZy classification) were formerly classified in the CBM33 family (or "carbohydrate binding module family 33") of the CAZy classification.
    [0024] The AA10 family of LPM0s comprises to date more than a thousand enzymes, identified particularly in bacteria, but also in some eukaryotes as well as in a few viruses. The LPMOs of the AA10 family have a structure similar to that of the AA9 family of enzymes and in particular at least one N-terminal tyrosine residue which is involved in binding with the copper ion. However, in most LPM0s of the AA10 family, one of the other tyrosine residues involved in the axial bonding of the copper ion is replaced by a phenylalanine residue. For these enzymes, oxidative activity has been demonstrated on chitin and cellulose.
    [0025] Preferably, the enzymes of the AA10 family are multimodular and comprise a CBM domain in the N-terminal position. These domains are typically CBM2, CBM5, CBM10, CBM12 domains as well as fibronectin type III modules. The AA11 family is characterized by enzymes that perform C1-cleavage cleavage on chitin. The enzyme Aspergillus oryzae will preferably be selected (see also Hemsworth et al., Nature Chemical Biology 2014 (10): 122-126 - Discovery and characterization of a new family of lytic polysaccharide monooxygenases). The AA13 family is characterized by enzymes that effect C1-Cl oxidative cleavage on starch. The Aspergillus nidulans enzyme will preferably be chosen (Lo Leggio et al., Nat. Commun. 2015 (22) 6: 5961 -Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase). In general, the enzymatic treatment step is carried out using at least one LPMO enzyme listed in Table 2 below.
    [0026] In certain embodiments of the method of the invention, said at least one enzyme of the family of LPM0s (advantageously of the AA9 family and / or of the AA10 family) is used in combination with at least one cellulase. The cellulase is advantageously chosen from at least one endoglucanase (for example an endoglucanase) and / or at least one carbohydrate dehydrogenase (advantageously a cellobiose dehydrogenase (CDHs)). Carbohydrate dehydrogenases can act as electron donors for LPM0s. In practice, said at least one LPMO enzyme used is advantageously purified from a culture supernatant of a fungus and / or produced in a heterologous system, in particular in a bacterium, a fungus or a yeast, for example in the yeast Pichia pastoris. Said at least one LPMO enzyme is mixed with the cellulosic substrate, so as to allow contact between said at least one enzyme and the cellulose fibers. The enzymatic treatment step is preferably carried out with gentle stirring, so as to ensure good dispersion of the enzymes within the fibers. This enzymatic treatment step is for example carried out for a period ranging from 24 to 72 hours (preferably 48 hours).
    [0027] Preferably, the enzymatic treatment step is carried out at a temperature of from 30 to 45 ° C. According to the invention, said at least one LPMO enzyme can be added to the cellulosic substrate in a ratio (or ratio) enzyme / cellulose ranging from 1: 1000 to 1: 50, in particular from 1: 500 to 1: 50 or 1: 100. at 1: 50 or else from 1: 1000 to 1: 500, from 1: 500 to 1: 100. Preferably, said at least one LPMO enzyme is used at a concentration ranging from 0.001 to 10 g / l, especially from 0.1 to 5 g / L, and more preferably 0.5 to 5 g / L. According to a particular embodiment, the cellulosic substrate is subjected to at least two (or only two) successive enzymatic treatment steps (in series, advantageously separated by a rinsing step). The LPM0s used in each of these enzymatic treatment steps are identical or different; the conditions (in particular the enzyme / substrate ratio) are identical or different between these successive steps. In this case, the Examples demonstrate that the fibers are fully destructured, including at low enzyme / cellulose ratios.
    [0028] Mechanical treatment step (s) The pretreated cellulosic substrate is then subjected to at least one mechanical treatment step which is intended to delaminate the cellulose fibers to obtain the nanocelluloses.
    [0029] Delamination (also called "fibrillation" or "defibrillation") consists in separating, by a mechanical phenomenon, the cellulose fibers into nanocelluloses. As demonstrated by the Examples below, the oxidative cleavage of the cellulose fibers catalyzed by the at least one LPMO facilitates the delamination of these cellulose fibers during the mechanical treatment step. This mechanical delamination step of the cellulose fibers can then be carried out under less stringent conditions and therefore less expensive in terms of energy. Furthermore, the use of LPM0s according to the invention makes it possible to introduce into the cellulose fibers charged groups inducing electrostatic repulsions, without contamination with treatment reagents, as when using TEMPO reagents. The mechanical treatments for delaminating the cellulose fibers are known to those skilled in the art and can be used in the process of the invention.
    [0030] In general, mention may be made of weak mechanical treatments with a homogenizer-disperser (for example of the type of Ultra-Turrax) and / or ultrasonic treatments. For example, it is still possible to refer to the document by Lavoine N et al (Carbohydrate Polymers, 2012, (92): 735-64) which describes in particular (pages 740 to 25 744) mechanical treatments for the preparation of microfibrillated cellulose (for example cellulose nanofibrils). Typically, a mechanical treatment may be chosen from mechanical treatments for homogenization, microfluidization, abrasion, or cryomilling.
    [0031] The homogenization treatment involves passing the pretreated cellulosic substrate, typically a cellulose pulp or a liquid cellulose slurry, through a narrow, high pressure space (as described, for example, in US Patent 4,486,743). This homogenization treatment is preferably carried out by means of a Gaulin homogenizer. In such a device, the pretreated cellulosic substrate, typically in the form of a cellulose suspension, is pumped at high pressure and dispensed through a small orifice automatic valve. A rapid succession of valve openings and closures subject the fibers to a large pressure drop (typically at least 20 MPa) and high speed shear action followed by high speed deceleration impact. . The passage of the substrate into the orifice is repeated (generally 8 to 10 times) until the cellulose suspension becomes stable. In order to maintain a temperature of the product in a range of 70 to 80 ° C during the homogenization treatment, cooling water is generally used. This homogenization treatment can also be carried out using a microfluidizer type device (see, for example, Sisqueira et al., Polymer 2010 2 (4): 728-65). In such a device, the cellulose suspension 15 passes through a typical "z" -shaped thin chamber (the channel dimensions of which are generally between 200 and 400 μm) under high pressure (about 2070 bar). The high shear rate that is applied (generally greater than 107.s-1) makes it possible to obtain very fine nanofibrils. A variable number of passages (for example from 2 to 30, in particular from 10 to 30 or from 5 to 25, and in particular from 5 to 20) with chambers of different sizes may be used to increase the degree of fibrillation. The abrasion or grinding treatment (see for example Iwamoto Set al., 2007 Applied Physics A89 (2): 461-66) is based on the use of a grinding device capable of exerting shear forces provided by grinding stones. The pretreated cellulosic substrate, generally in the form of a cellulose pulp, is passed between a static grinding stone and a rotating grinding stone, typically at a rate in the order of 1500 rotations per minute (rpm). Several passes (usually between 2 and 5) may be necessary to obtain nano-sized fibrils. A mixer-type device (for example as described in Unetani K et al., Biomacromolecules 2011, 12 (2), pp.348-53) can also be used to produce microfibrils from the pretreated cellulosic substrate, for example from a suspension of wood fibers.
    [0032] The cryomilling (or cryoconcassage) treatment (Dufresne et al., 1997, Journal of Applied Polymer Science, 64 (6): 1185-94) consists in grinding a suspension of pretreated cellulosic substrate previously frozen with liquid nitrogen. . The ice crystals formed inside the cells explode cell membranes and release wall fragments. These processes are generally used for the production of cellulose microfibrils from products, or residues, of agriculture. Cellulosic substrate post-treatment step (s) In some embodiments, the manufacturing method comprises at least one post-treatment step of the cellulosic substrate, carried out after said substrate has been mechanically processed. Generally, said at least one post-treatment step aims at increasing the degree of fibrillation of the obtained nanocelluloses and / or at imparting to said nanocelluloses new mechanical properties, depending on the applications envisaged. Said at least one post-treatment step can in particular be chosen from an acid treatment, an enzymatic treatment, an oxidation, an acetylation, a silylation, or a derivatisation of certain chemical groups carried by the microfibrils. Reference may also be made, for example, to the document by Lavoine N et al (Carbohydrate Polymers, 2012, (92): 735-64) which describes in particular (point 2.3, pages 747 to 748) post-treatments which can be combined with different pretreatments and mechanical treatments of cellulosic fibers.
    [0033] Nanocelluloses according to the invention The process according to the invention thus makes it possible to obtain nanocelluloses, in particular cellulose nanocrystals and / or cellulose nanofibrils. In contrast to the NFCs obtained after oxidation with TEMPO type chemical reagents, the nanocelluloses obtained by the process of the invention are free of oxidation reaction residues.
    [0034] EXPERIMENTAL RESULTS 1. Tests for the cleavage of cellulose with an LPMO enzyme can be carried out according to the following protocol: The cleavage test is carried out in a volume of 300 μl of liquid containing 4.4 μM of LPMO enzyme and 1 mM ascorbate and 0.1% (w / v) phosphoric acid swollen cellulose powder (PASC phosphoric acidswoller cellulose - prepared as described in Wood TM, Methods Enzym 1988, 160: 19-25) in 50 mM sodium acetate buffer at pH 4.8 or 5 μM cello-oligosaccharides (Megazyme, Wicklow, Ireland) in 10 mM sodium acetate buffer pH 4.8. The enzymatic reaction is carried out in a 2 ml tube incubated in a thermomixer (Eppendorf, Montesson, France) at 50 ° C. and 580 rpm (rotation per minute). After 16 hours of incubation, the sample is heated to 100 ° C. for 10 minutes in order to stop the enzymatic reaction and then centrifuged at 16,000 rpm for 15 minutes at 4 ° C. in order to separate the solution fraction. of the remaining insoluble fraction. The cleaved products obtained can be analyzed by ion exchange chromatography and / or by mass spectrometry (MALDI-TOF). 2. Preliminary tests were conducted to demonstrate the effectiveness of the nanocellulose manufacturing process according to the invention. These tests were carried out on a papermaking fiber (cellulose kraft fiber) using LMPO enzyme from the AA9 family from Podospora anserina (PaLPMO9E (Genbank CAP67740) and / or PaLPMO9H (Genbank CAP61476)) and produced in a heterologous system. in yeast (Pichia pastoris). The fibers are contacted with enzymes (at a concentration of 1 g / L and in enzyme / cellulose ratios of 1: 50, 1: 100, 1: 500 and 1: 1000) and ascorbate (2 mM) and subjected to gentle stirring for 48 hours at 40 ° C.
    [0035] The treated fibers are then subjected to mechanical action with a homogenizer-disperser (Ultra-Turrax power 500 W, maximum speed for 3 minutes), followed by sonication for 3 minutes. Compared with substrates not treated with the enzyme, it is observed that defibrillation is facilitated for all the enzyme / cellulose ratios used (FIG. 1B-D - the photos show, qualitatively, defibrillation). In the absence of enzyme, the fibers remain intact and no defibrillation is observed (see untreated control fibers, Figure 1A). The dispersions were then analyzed by TEM (Transmission Electron Microscopy) and AFM (Atomic Force Microscopy). In the absence of LPM0s enzymes (FIG. 2A), it is found that very few structures are visible at the nanoscale. On the other hand, for fibers treated with the LPMO enzyme, structures of nanometric dimensions are easily identified both in the supernatant and in the experiment pellets. The fibers are completely unstructured, revealing the crystalline zones of the fiber (FIG. 2C-D). Treatment of the fibers with the enzyme PaLPMO9E combined with subsequent mechanical treatment (FIGS. 3B and D) produces a cellulose defibrillation similar to that obtained with an identical method involving the enzyme PaLPMO9H (see FIG. 3A and C). In general, FIGS. 2C, 2D, 3C and 3D demonstrate indisputably that nanocelluloses are obtained. If the fibers having undergone a first treatment with the LPMO enzyme are again subjected to a second successive treatment with an LPMO enzyme under the conditions described above, followed by the mechanical treatment, the fibers are completely destructured, including the enzyme ratios. cellulose (ie, the ratios 1/500 and 1/1000) (Figure 4).
    [0036] 3037078 Table 1: Listed fungal enzymes of the AA9 family (CAZy classification) Name Organism Ref. GenBank Ref. Uniprot Cell Agaricus bisporus D649 AAA53434.1 Q00023 AfA5C5.025 Aspergillus fumigatus CAF31975.1 Q6MYM8 endoglucanase / CMCase (Eng61) Aspergillus fumigatus MKU1 AFJ54163.1 endo-p-1,4-glucanase B (EgIB; AkCeI61A) (CeI61A) Aspergillus kawachii NBRC4308 BAB62318.1 Q96WQ9 AN1041.2 Aspergillus EAA65609.1 C8VTW9 FGSC A4 nidulans Q5BEI9 AN3511.2 Aspergillus EAA59072.1 Q5B7G9 FGSC A4 nidulans AN9524.2 Aspergillus EAA66740.1 CBF83171.1 C8VI93 FGSC A4 nidulans Q5AQA6 AN7891.2 Aspergillus EAA59545.1 Q5AUY9 nidulans FGSC A4 AN6428.2 Aspergillus EAA58450.1 C8V0F9 nidulans FGSC A4 Q5AZ52 AN3046.2 Aspergillus EAA63617.1 C8VIS7 nidulans FGSC A4 Q5B8T4 AN3860.2 (EgIF) Aspergillus EAA59125.1 C8V6H2 nidulans FGSC A4 Q5B6H0 endo-p-1,4 Aspergillus EAA64722.1 ABF50850.1 Q5BCX8 Ascargillus Ascargillus Ascargillus EAA64722.1 ABF50850.1 Ascargillus Ascargillus Ascargillus FGSC A4 A2388.2 Aspergillus EAA64499.1 C8VN P4 nidulans FGSC A4 Q5BAP2 An04g08550 Aspergillus niger CBS 513.88 CAK38942.1 A2QJX0 An08g05230 Aspergillus niger CBS 513.88 CAK45495.1 A2QR94 An12g02540 ace pergillus niger CBS 513.88 CAK41095.1 A2QYU6 Anl 2g04610 Aspergillus niger CBS 513.88 CAK97151.1 A2QZE1 An14g02670 Aspergillus niger CBS 513.88 CAK46515.1 A2R313 3037078 23 Name Organism Ref. GenBank Ref. Uniprot An15g04570 Aspergillus dip CBS 513.88 CAK97324.1 A2R5J9 An15g04900 Aspergillus dip CBS 513.88 CAK42466.1 A2R5NO A0090005000531 Aspergillus oryzae RIB40 BAE55582.1 Q2US83 A0090001000221 Aspergillus oryzae RIB40 BAE56764.1 Q2UNV1 A0090023000056 Aspergillus oryzae RIB40 BAE58643.1 Q2UIH2 A0090023000159 Aspergillus oryzae RIB40 BAE58735.1 Q2U180 A0090023000787 Aspergillus oryzae RIB40 BAE59290.1 Q2UGM5 A0090012000090 Aspergillus oryzae RIB40 BAE60320.1 Q2UDP5 A0090138000004 Aspergillus oryzae RIB40 BAE64395.1 Q2U220 A0090103000087 Aspergillus oryzae RIB40 BAE65561.1 Q2TYW2 EC16 (E6) Bipolaris maydis C4 AAM76663.1 Q8JOH7 glycoside hydrolase family 61 protein ( Bofut4_p103280.1) Botryotinia fuckeliana T4 CCD34368.1 blood glycoside family 61 protein (Bofut4_p003870.1) Botryotinia fuckeliana T4 CCD47228.1 glycoside hydrolase family 61 protein (Bofut4_p109330.1) Botryotinia fuckeliana T4 CCD48549.1 glycoside hydrolase family 61 protein (Bofut4_p025380. 1) Botryotinia fuckeliana T4 CCD50139.1 blood glycoside family 61 protein (Bofut4_p025430.1) Botryotinia fuckeliana T4 CCD50144.1 family glycoside hydrolase 61 protein (Bofut4_p018100.1) Botryotinia fuckeliana T4 CCD51504.1 family glycoside hydrolase 61 protein (Bofut4_p031660.1) Botryotinia fuckeliana T4 CCD49290. 1 glycoside hydrolase family 61 protein (Bofut4_p000920.1) Botryotinia fuckeliana T4 CCD52645.1 BofuT4P143000045001 Botryotinia fuckeliana T4 CCD50451.2 CCD50451.1 3037078 24 Name Organism Ref. GenBank Ref. Uniprot ORF Chaetomium thermophilum CT2 AGY80102.1 ORF (fragment) Chaetomium thermophilum CT2 AGY80103.1 ORF (fragment) Chaetomium thermophilum CT2 AGY80104.1 ORF (fragment) Chaetomium thermophilum CT2 AGY80105.1 cellobiohydrolase family protein 61, partial (Cbh61-2) ( fragment) Chaetomium thermophilum CT2 AGY80103.1 cellobiohydrolase family protein 61, partial (Cbh61-3) (fragment) Chaetomium thermophilum CT2 AGY80104.1 cellobiohydrolase family protein 61, partial (Cbh61-4) (fragment) Chaetomium thermophilum CT2 AGY80105.1 ORF ( possible fragment) Colletotrichum graminicola M2 CAQ16278.1 B5WYD8 ORF Colletotrichum graminicola M2 CAQ16206.1 B5WY66 ORF Colletotrichum graminicola M2 CAQ16208.1 B5WY68 ORF Colletotrichum graminicola M2 CAQ16217.1 B5WY77 unnamed protein product Coprinopsis cinerea CAG27578.1 CG B_A6300C Cryptococcus bacillisporus WM276 ADV19810.1 CNAG_00601 Cryptococcus neoformans var. grubii H99 (Cryne H99 1) AFR92731.1 AFR92731.2 Cell Cryptococcus neoformans var. neoformans AAC39449.1 059899 CNA05840 (Cell) Cryptococcus neoformans var. neoformans JEC21 (Cryne JEC21_1) AAW41121.1 F5HH24 ORF (fragment) Flammulina velutipes KACC 42777 ADX07320.1 3037078 25 Name Organism Ref. GenBank Ref. Uniprot FFUJ 12340 Fusarium fujikuroi 1M! 58289 (Fusfu1) CCT72465.1 FFUJ 13305 Fusarium fujikuroi 1M! 58289 (Fusfu1) CCT67119.1 FFUJ 07829 Fusarium fujikuroi 1M! 58289 (Fusful) CCT69268.1 FFUJ 12621 Fusarium fujikuroi 1M! 58289 (Fusful) CCT72729.1 FFUJ 12840 Fusarium fujikuroi 1M! 58289 (Fusful) CCT72942.1 FFUJ 09373 Fusarium fujikuroi 1M! 58289 (Fusful) CCT73805.1 FFUJ 10599 Fusarium fujikuroi 1M! 58289 (Fusful) CCT74544.1 FFUJ 10643 Fusarium fujikuroi 1M! 58289 (Fusful) CCT74587.1 FFUJ 14514 Fusarium fujikuroi 1M! 58289 (Fusful) CCT67584.1 FFUJ 11399 Fusarium fujikuroi 1M! 58289 (Fusful) CCT75380.1 FFUJ 14514 Fusarium fujikuroi 1M! 58289 CCT67584.1 FFUJ 11399 Fusarium fujikuroi 1M! 58289 CCT75380.1 FFUJ 04652 Fusarium fujikuroi 1M! 58289 (Fusful) CCT64153.1 FFUJ 03777 Fusarium fujikuroi 1M! 58289 (Fusful) CCT64954.1 FFUJ 04940 Fusarium fujikuroi 1M! 58289 (Fusful) CCT63889.1 Sequence 122805 from patent US 7214786 Fusarium graminearum ABT35335.1 FG03695.1 (CeI61E) Fusarium XP 383871.1 graminearum PH-1 unnamed protein product Fusarium CEF78545.1 graminearum PH-1 unnamed protein product Fusarium CEF74901.1 graminearum PH-1 unnamed protein product Fusarium CEF78472.1 graminearum PH-1 303 70 78 26 Name Organism Ref. GenBank Ref. Uniprot unnamed protein product Fusarium CEF86346.1 graminearum PH-1 unnamed protein product Fusarium CEF87450.1 graminearum PH-1 unnamed protein product Fusarium CEF85876.1 graminearum PH-1 unnamed protein product Fusarium CEF86254.1 graminearum PH-1 unnamed protein product Fusarium CEF87657 .1 graminearum PH-1 unnamed protein product Fusarium CEF76256.1 graminearum PH-1 unnamed protein product Fusarium CEF78876.1 graminearum PH-1 unnamed protein product Fusarium CEF79735.1 graminearum PH-1 unnamed protein product Fusarium CEF74460.1 graminearum PH-1 unamed protein product Fusarium CEF84640.1 graminearum PH-1 endo-p-1,4-glucanase (CeI61G) Gloeophyllum trabeum AEJ35168.1 GH61D Heterobasidion parviporum AF072234.1 GH61B Heterobasidion parviporum AF072233.1 GH61A Heterobasidion parviporum AF072232.1 GH61F Heterobasidion parviporum AF072235 .1 GH61G Heterobasidion parviporum AF072236.1 GH61H Heterobasidion parviporum AF072237.1 GH61I Heterobasidion parviporum AF072238.1 GH61J Heterobasidio n parviporum AF072239.1 unnamed protein product Humicola insolens CAG27577.1 303 70 78 27 Name Organism Ref. GenBank Ref. Uniprot endoglucanase IV (EgiV) Hypocrea orientalis EU7-22 AFD50197.1 GH61A (GH61A) Lasiodiplodia theobromae CBS 247.96 CAJ81215.1 GH61 B (GH61B) Lasiodiplodia theobromae CBS 247.96 CAJ81216.1 GH61C (GH61C) Lasiodiplodia theobromae CBS 247.96 CAJ81217.1 GH61D (GH61D) Lasiodiplodia theobromae CBS 247.96 CAJ81218.1 ORF Leptosphaeria maculans v23.1.3 CBX91313.1 E4ZJM8 ORF Leptosphaeria maculans v23.1.3 CBX93546.1 E4ZQ11 ORF Leptosphaeria maculans v23.1.3 CBX94224.1 E4ZS44 ORF Leptosphaeria maculans v23.1.3 CBX94532.1 E4ZSU4 ORF Leptosphaeria maculans v23.1.3 CBX94572.1 E4ZSY4 ORF Leptosphaeria maculans v23.1.3 CBX95655.1 E4ZVM9 ORF Leptosphaeria maculans v23.1.3 CBX96476.1 E4ZZ41 ORF Leptosphaeria maculans v23.1.3 CBX96550.1 E4ZYM4 ORF Leptosphaeria maculans v23.1.3 CBX96949.1 E5A089 ORF Leptosphaeria maculans v23.1.3 CBX97718.1 E5A201 ORF Leptosphaeria maculans v23.1.3 CBX98126.1 E5A3B3 ORF Leptosphaeria maculans v23.1.3 CBY01974.1 E5AFI5 ORF Leptosphaeria maculans v23.1.3 CBY02242.1 E5 ACP0 3037078 28 Name Body Ref. GenBank Ref. Uniprot ORF Leptosphaeria maculans v23.1.3 CBX91667.1 E4ZK72 ORF Leptosphaeria maculans v23.1.3 CBX93965.1 E4ZQA3 ORF Leptosphaeria maculans v23.1.3 CBX98254.1 E5A3P1 ORF (fragment) Leptosphaeria maculans v23.1.3 CBY00196.1 E5A955 ORF Leptosphaeria maculans v23.1.3 CBY01204.1 E5AC13 predicted protein Leptosphaeria maculans v23.1.3 CBY01256.1 E5ADG7 (Lema_p000430.1) (fragment) ORF (fragment) Leptosphaeria maculans v23.1.3 CBY01257.1 E5ADG8 lytic polysaccharide monooxygenase Leucoagaricus gongylophorus Ae322 CDJ79823.1 MG05364.4 Magnaporthe grisea 70-15 (Maggrl) EAA54572.1 XP 359989.1 MG07686.4 Gray Magnaporthe 70-15 (Maggrl) EAA53409.1 XP 367775.1 G4N3E5 MG07300.4 Gray Magnaporthe 70-15 (Maggrl) EAA56945.1 XP 367375.1 G4MUY8 MG08020.4 Gray Magnaporthe 70-15 (Maggrl) EAA57051.1 XP 362437.1 MG08254.4 Magnaporthe grayea 70-15 (Maggrl) EAA57285.1 XP 362794.1 G4MXC7 MG08066.4 (fragment) Magnaporthe grayea 70-15 (Maggrl) EAA57097.1 XP 362483.1 G4MXS5 MG04547. 4 Magnaporthe grisea 70-15 ( Maggrl) EAA50788.1 XP 362102.1 G4MS66 MG08409.4 Magnaporthe grayea 70-15 (Maggrl) EAA57439.1 XP 362640.1 G4MVX4 3037078 29 Name Body Ref. GenBank Ref. Uniapot MG09709.4 Magnaporthe EAA49718.1 G4NAI5 grayea 70-15 XP 364864.1 (Maggrl) MG06069.4 Magnaporthe EAA52941.1 G4N560 grayea 70-15 XP 369395.1 (Maggrl) MG09439.4 Magnaporthe EAA51422.1 G4NHT8 grayea 70-15 XP 364487.1 Maggrl) MG06229.4 Magnaporthe EAA56258.1 grayea 70-15 XP 369714.1 (Maggrl) MG07631.4 Magnaporthe EAA53354.1 G4N2Z0 grayea 70-15 XP 367720.1 (Maggrl) MGG 06621 Magnaporthe XP 003716906.1 grayea 70-15 XP 370106.1 (Maggrl) MGG 12696 Magnaporthe grayea 70-15 XP 003721313.1 (Maggrl) MGG 02502 Magnaporthe XP_003709306.1 grayea 70-15 EAA54517.1 (Maggrl) XP 365800.1 MGG 04057 Magnaporthe XP_003719782.1 grayea 70-15 EAA50298.1 (Maggrl) XP 361583.1 MGG 13241 Magnaporthe grayea 70-15 XP 003711808.1 (Maggrl) MGG 13622 Magnaporthe grayea 70-15 XP 003717521.1 (Maggrl) MGG 07575 Magnaporthe XP_003711490.1 grayea 70-15 EAA53298.1 (Maggrl) XP 367664.1 MGG 11948 Magnaporthe grayea 70-15 XP 003709110.1 (Maggrl ) MGG 16080 (fragment) Magnaporthe grayea 70-15 XP 003709033.1 (Maggrl) 303707 8 30 Name Body Ref. GenBank Ref. Uniprot MGG 16043 (fragment) Magnaporthe grisea 70-15 (Maggrl) XP 003708922.1 MGG 12733 (probable fragment) Magnaporthe grisea 70-15 (Maggrl) XP 003716689.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Malbranchea cinnamomea CBS 115.68 CCP37674.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Melanocarpus albomyces CBS 638.94 CCP37668.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Myceliophthora fergusii CBS 406.69 CCP37667.1 MYCTH 2112799 Myceliophthora thermophila ATCC 42464 AE061257.1 MYCTH 79765 Myceliophthora thermophila ATCC Myceliophthora thermophila ATCC 42464 MYCTH Mycophosphorophosphates Myceliophthora ATCC 42464 AE055082.1 MYCTH Mycophosphorophosphates Myceliophthora thermophila ATCC 42464 AE055502.1 MYCTH Myceliophthora thermophila ATCC 42464 AE055652.1 MYCTH lycan -cl e avi ng enzyme (StCeI61a; MYCTH_46583) (CeI61A) Myceliopht hora thermophila ATCC 42464 AE056542.1 3037078 31 Name Body Ref. GenBank Ref. Uniprot MYCTH_2301632 Myceliophthora thermophila ATCC 42464 AE056547.1 MYCTH_100518 Myceliophthora thermophila ATCC 42464 AE056642.1 lytic polysaccharide Myceliophthora thermophila ATCC 42464 AE056665.1 monooxygenases (active on cellulose) (MYCTH_92668) MYCTH_2060403 Myceliophthora thermophila ATCC 42464 AE058412.1 MYCTH_2306673 Myceliophthora thermophila ATCC 42464 AE058921. Myceliophthora thermophila ATCC 42464 MYCTH_103537 MYCCI-1_55803 MYCCTI-1_55803 Myceliophthora thermophila ATCC 42464 Mycophosphoric acid ATCC 42464 AE059955.1 Mycophosphorophosphate on cellulose) Mycophthora thermophila ATCC 42464 MYCTH_2311323 Myceliophthora thermophila ATCC 42464 AE061305.1 MYCTH_47093 (fragment) Myceliophthora thermophila ATCC 42464 AE056498.1 MYCTH_80312 Myceliophthora thermophila ATCC 42464 42464 AE058169.1 3037078 32 Name Body Ref. GenBank Ref. Uniprot lytic polysaccharide Neurospora crassa OR74A CAD21296.1 01 K8B6 monooxygenase (active on cellulose) (PMO-2; NcLPM09D; GH61-4; NCU01050) (LPMO9D) EAA32426.1 XP 326543.1 Q8WZQ2 lytic polysaccharide Neurospora crassa OR74A CAD70347.1 01K401 monooxygenase ( active on cellulose) (PMO-03328; NcLPM09F; GH61-6; NCU03328) (LPMO9F) EAA26656.1 XP 322586.1 0873G1 lytic polysaccharide Neurospora crassa OR74A CAE81966.1 Q7SHD9 monooxygenase (PM0-01867; NcLPMO9J; GH61-10; NCU01867; EAA36262 .1 B13N4.070) (LPMO9J) XP 329057.1 NCU02344.1 (B23N11.050) Neurospora crassa OR74A CAF05857.1 07S411 EAA30230.1 XP 331120.1 lytic polysaccharide monooxygenase (active on cellulose) (PM0-3; NcLPM09M; GH61-13; NcPM0- Neurospora crassa OR74A EAA33178.1 XP 328604.1 07SA19 3; NCU07898) (LPMO9M) NCU05969.1 Neurospora crassa OR74A EAA29347.1 XP 325824.1 07S1V2 lytic polysaccharide monooxygenase (active on cellulose and cellooligosaccharides) (PM0-02916; NcLPM09C; GH61-3 NCU02916) (LPMO9C) Neurospora crassa OR74A EA A36362.1 XP 330104.1 07SH18 lytic polysaccharide monooxygenase (active on cellulose) (GH61-2; NCU07760) Neurospora crassa OR74A EAA29018.1 XP 328466.1 07S111 NCU07520.1 Neurospora crassa OR74A EAA29132.1 XP 327806.1 07S1A0 lytic polysaccharide monooxygenase (active on cellulose) (GH61-1; NCU02240) Neurospora crassa OR74A EAA30263.1 XP 331016.1 Q7S439 lytic polysaccharide Neurospora crassa OR74A EAA34466.1 XP 325016.1 Q7SCJ5 monooxygenase (active on cellulose) (NCU00836) 3037078 33 Name Organism Ref. GenBank Ref. Uniospot lytic polysaccharide Neurospora crassa OR74A EAA26873.1 XP 330877.1 Q7RWN7 monooxygenase (active on cellulose) (PM0-083760; NcLPM09E; GH61-5; NCU08760) (LPMO9E) NCU07974.1 Neurospora crassa OR74A EAA33408.1 XP 328680.1 Q7SAR4 NCU03000.1 B24P7.180) Neurospora crassa OR74A EAA36150.1 Q7RV41 CAB97283.2 XP 330187.1 Q9P3R7 monooxygenase cellulose Penicillium oxalicum GZ-2 A1006742.1 Pc12g13610 Penicillium chrysogenum Wisconsin 54-1255 (PenchWisc1_1) CAP80988.1 B6H016 Pc13g07400 Penicillium chrysogenum Wisconsin 54-1255 (PenchWisc1_1 ) CAP91809.1 B6H3U0 Pc13g13110 Penicillium chrysogenum Wisconsin 54-1255 (PenchWisc1_1) CAP92380.1 B6H3A3 Pc20g11100 Penicillium chrysogenum Wisconsin 54-1255 (PenchWisc1_1) CAP86439.1 B6HG02 Ce161 (CeI61A) Phanerochaete chrysosporium BKM-F-1767 AAM22493.1 Q8NJI9 Lytic polysaccharide monohydrogenase active on cellulose (Gh61D; PcGH61D) Phanerochaete chrysosporium K-3 BAL43430.1 PIIN 01487 Piriformospora indica (Pirinl) CCA67659.1 PIIN 02110 Piriformospora i ndica (Pirinl) CCA68244.1 PIIN 03975 Piriformospora indica (Pirinl) CCA70035.1 PIIN 04357 Piriformospora indica (Pirinl) CCA70418.1 3037078 34 Name Organism Ref. GenBank Ref. English PIIN 06117 Piriformospora indica (Pirinl) CCA72182.1 PIIN 06118 Piriformospora indica (Pirinl) CCA72183.1 PIIN 06127 Piriformospora indica (Pirinl) CCA72192.1 PIIN 06155 Piriformospora indica (Pirinl) CCA72220 .1 PI IN 07098 Piriformospora indica (Pirinl) CCA73144.1 PIIN 07105 Piriformospora indica (Pirinl) CCA73151.1 PIIN 08199 Piriformospora indica (Pirinl) CCA74246.1 PI IN 08783 Piriformospora indica (Pirin1) CCA74814.1 PI IN 09022 Piriformospora indica ( Pirinl) CCA75037.1 PIIN 00566 (fragment) Piriformospora indica (Pirinl) CCA66803.1 PIIN 01484 Piriformospora indica (Pirinl) CCA67656.1 PIIN 01485 (fragment) Piriformospora indica (Pirinl) CCA67657.1 PIIN 01486 (fragment) Piriformospora indica (Pirinl) ) PTH 05699 (fragment) Piriformospora indica (Pirinl) CCA71764.1 PIIN 06156 Piriformospora indica (Pirinl) CCA72221.1 PI IN 08402 Piriformospora indica (Pirinl) CCIN74449.1 PIIN 1 0315 (fragment) Piriformospora indica (Pirinl) CCA76320.1 PIIN 1 0660 (fragment) Piriformospora indica (Pirinl) CCA76671.1 3037078 Name Organism Ref. GenBank Ref. Uniprot PIIN 00523 (fragment) Piriformospora indica (Pirinl) CCA77877.1 Putative Glycoside Hydrolase Family 61 Podospora anserina S mat + (Podan2) CDP30131.1 CAP64732.1 B2AL94 Putative Glycoside Hydrolase Family 61 Podospora anserina S mat + (Podan2) CDP30928.1 CAP71532. 1 B2B346 Pa_1_500 Podospora anserina S mat + (Podan2) CAP59702.1 CDP22345.1 B2A9F5 Pa_4_350 Podospora anserina S mat + (Podan2) CAP61395.1 CDP27750.1 B2AD80 Pa_4_1020 Podospora anserina S mat + (Podan2) CAP61476.1 CDP27830.1 B2ADG1 Pa_0_270 Podospora anserina S mat + (Podan2) CAP61650.1 CDP28001.1 B2ADY5 Pa_5_8940 Podospora anserina S mat + (Podan2) CAP64619.1 CDP30017.1 B2AKU6 Pa_5_4100 (fragment) Podospora anserina S mat + (Podan2) CAP64865.1 CDP29378.1 B2ALM7 Pa_5_6950 Podospora anserina S mat + (Podan2) CAP65111.1 CDP29800.1 B2AMI8 Pa_5_10660 Podospora anserina S mat + (Podan2) CAP65855.1 CDP30283.1 B2APD8 Pa_5_10760 Podospora anserina S mat + (Podan2) CAP65866.1 CDP30272.1 B2APE9 Pa_5_11630 (fragment) Podospora anserina S mat + (Podan2) CAP65971.1 CDP30166.1 B2API9 Pa_4_7570 Podospora anserina S mat + (Podan2) CAP66744.1 CDP28479.1 B2ARG6 3037078 36 Name Organism Ref. GenBank Ref. Uniprot Pa_1_21900 (fragment) Podospora anserina S mat + (Podan2) CAP67176.1 CDP24589.1 B2AS05 Pa_1_22040 Podospora anserina S mat + (Podan2) CAP67190.1 CDP24603.1 B2AS19 Pa_1_22150 (fragment) Podospora anserina S mat + (Podan2) CAP67201.1 CDP24614. 1 B2AS30 Pa_6_11220 Podospora anserina S mat + (Podan2) CAP67466.1 CDP30332.1 B2ASU3 Pa_6_11370 Podospora anserina S mat + (Podan2) CAP67481.1 CDP30347.1 B2ASV8 Pa_6_11470 Podospora anserina S mat + (Podan2) CAP67493.1 CDP30359.1 B2ASX0 Pa_1_16300 Podospora anserina S mat + (Podan2) CAP67740.1 CDP23998.1 B2ATL7 Pa_7_5030 Podospora anserina S mat + (Podan2) CAP68173.1 CDP31642.1 B2AUVO Pa_7_3770 Podospora anserina S mat + (Podan2) CAP68309.1 CDP31780.1 B2AV86 Pa_7_3390 Podospora anserina S mat + (Podan2) CAP68352.1 CDP31823.1 B2AVC8 lytic polysaccharide mono-oxygenase active on cellulose (Gh61B; Pa_7_3160) (Gh61B) Podospora anserina S mat + (Podan2) CAP68375.1 CDP31846.1 B2AVF1 Pa_6_7780 Podospora anserina S mat + (Podan2) CAP71839.1 CDP31230. 1 B2B403 Pa_ 2_1700 Podospora anserina S mat + (Podan2) CAP72740.1 CDP25137.1 B2B4L5 Pa_2_4860 Podospora anserina S mat + (Podan2) CAP73072.1 CDP25472.1 B2B5J7 303 70 78 37 Name Organism Ref. GenBank Ref. Uniprot lytic polysaccharide mono-oxygenase active on cellulose (Gh61A; Pa_2_6530) (Gh61A) Podospora anserina S mat + (Podan2) CAP73254.1 CDP25655.1 B2B629 Pa_2_7040 Podospora anserina S mat + (Podan2) CAP73311.1 CDP25714.1 B2B686 Pa_2_7120 Podospora anserina S mat + (Podan2) CAP73320.1 CDP25723.1 B2B695 Pa_3_190 Podospora anserina S mat + (Podan2) CAP61048.1 CDP26500.1 B2AC83 Pa_3_2580 Podospora anserina S mat + (Podan2) CAP70156.1 CDP26748.1 B2AZV6 Pa_3_3310 Podospora anserina S mat + (Podan2) CAP70248 .1 CDP26841.1 B2AZD4 endo-p-1,4-glucanase (Eg11; PIEGL1) Pyrenochaeta lycopersici ISPaVe ER 1211 AEV53599.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Rasamsonia CCP37669.1 byssochlamydoides CBS 151.75 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Remersonia thermophila CBS 540.69 CCP37675.1 RHTOOS 28e01816g Rhodosporidium toruloides CECT1137 CDR49619.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Scytalidium CCP37676.1 indonesiacum CBS 259.81 SMU2916 (fragment) Sordaria CAQ58424.1 C1KU36 macrospora k-hell lytic polysaccharide active mono-oxygenase on cellulose Thermoascus aurantiacus ABW56451.1 ACS05720.1 3037078 38 Name Organism Ref. GenBank Ref. Uniprot copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Thermoascus aurantiacus CBS 891.70 CCP37673.1 ORF Thermoascus aurantiacus var. levisporus AG068294.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Thermomyces dupontii CBS 236.58 CCP37672.1 copper-dependent polysaccharide monooxygenases (Gh61) (fragment) Thermomyces lanuginosus CBS 632.91 CCP37678.1 unnamed protein product Thielavia terrestris CAG27576.1 THITE 2106556 Thielavia terrestris NRRL 8126 AE062422.1 THITE 2116536 Thielavia terrestris NRRL 8126 AE067662.1 THITE 2040127 Thielavia terrestris NRRL 8126 AE064605.1 THITE 2119040 Thielavia terrestris NRRL 8126 AE069044.1 THITE 115795 Thielavia terrestris NRRL 8126 AE064177.1 THITE 2110890 Thielavia terrestris NRRL 8126 AE064593 .1 THITE 2112626 Thielavia terrestris NRRL 8126 AE065532.1 THITE 2076863 Thielavia terrestris NRRL 8126 AE065580.1 THITE 170174 Thielavia terrestris NRRL 8126 AE066274.1 THITE 2044372 Thielavia terrestris NRRL 8126 AE067396.1 THITE 2170662 Thielavia terrestris NRRL 8126 AE068023.1 TH ITE 128130 Thielavia terrestris NRRL 8126 AE068157.1 3037078 39 Name Body Ref. GenBank Ref. Uniprot THITE 2145386 Thielavia terrestris NRRL 8126 AE068577.1 THITE 2054543 Thielavia terrestris NRRL 8126 AE068763.1 THITE 2059487 Thielavia terrestris NRRL 8126 AE071031.1 THITE 2142696 Thielavia terrestris NRRL 8126 AE067395.1 THITE 43665 Thielavia terrestris NRRL 8126 AE069043.1 THITE 2085430 (fragment ) Thielavia terrestris NRRL 8126 AE063926.1 THITE 2122979 Thielavia terrestris NRRL 8126 XP 003657366.1 Cellulase-enhancing factor (GH61 B) Thielavia terrestris NRRL 8126 ACE10231.1 Sequence 4 from patent US 7361495 (GH61C) Thielavia terrestris NRRL 8126 ACE10232.1 Sequence 4 from Sequence 6 from US 7361495 (GH61D) Thielavia terrestris NRRL 8126 ACE10233.1 active on cellulose (131562; TtGH61E) (GH61E) Thielavia terrestris NRRL 8126 AE071030.1 ACE10234.1 Sequence 10 from patent US 7361495 (GH6 1G) Thielavia terrestris NRRL 8126 ACE10235.1 Sequence 10 from patent US 7361495 (GH61G) Thielavia terrestris NRRL 8126 ACE10235.1 Lytic polysaccharide active monohydrogenase on cellulose (EG7; HjGH61 B) (Ce161B = GH61B) Trichoderma reesei QM6A AAP57753 .Beta.1-endo-p-1,4-glucanase IV (EGIV; Eg14; EG4) (CeI61A) Trichoderma reesei RUTC-30 CAA71999.1 014405 endoglucanase Trichoderma saturnisporum ADB89217.1 D3JTC4 (EnGluIV) EndoGluIV) 3037078 Name Organism Ref. GenBank Ref. Uniprot endoglucanase IV (IVIV, EG IV) Trichoderma sp. SSL ACH92573.1 B5TYI4 endoglucanase VII (EgvII) Trichoderma viride AS 3.3711 ACD36971.1 B4YEW1 endoglucanase IV (EgIV) Trichoderma viride AS 3.3711 ADJ57703.1 ACD36973.1 B4YEW3 D9IXC6 AAA12YMO5FL uncultured eukaryote CCA94933.1 AAA2YGO1 FL uncultured eukaryote CCA94930.1 AAA15Y110FL uncultured eukaryote CCA94931.1 AAA21YH11FL uncultured eukaryote CCA94932.1 ABA3YPO5FL eukaryote eukaryote CCA94934.1 endoglucanase II (Eg11) Volvariella volvacea AFP23133.1 endoglucanase II (Eg11) Volvariella volvacea V14 AAT64005.1 Q6E5B4 unknown Zea mays B73 ACF86151.1 unknown (ZM_BFc0036G02) Zea mays B73 ACF78974.1 ACR36748.1 B4FA31 3037078 41 Table 2: LPMOs (families AA9, AA10 and AA11 of the CAZy classification). By "substrate specificity" is meant the type of cleaved substrate (oxidative cleavage) by the corresponding LPMO enzyme.
    [0037] By "known selectivity" is meant the carbon of the glucose cycle oxidized by the corresponding LPMO enzyme. By "Modularity" is meant the CAZy class (AA9, 10 or 11) of the enzyme and the known presence of a conserved domain (CBM or X278). Body Ref. Ref. Other names Substrate specificity Known selectivity Modulahte Uniprot GenBank fungi A. oryzae Q2UA85 BAE61530 AoAAll chitin Cl AA11-X278 A. nidulans C8VG F8 EAA AnAA13 starch Cl AA13- CBM20 62623.1 M. G2QI82 AE060271 MYCTH 11208 9 cellulose Cl AA9 thermophila M. thermophila G2QAB5 AE056665 MYCTH 92668 cellulose Cl AA9 N. crassa Q7RWN7 EAA26873 NcLPMO9E cellulose Cl AA9-CBM1 N. crassa Q1K8B6 Q8WZQ2 EAA32426 CAD21296 NcLPMO9D cellulose C4 AA9 N. crassa Q7SA19 EAA33178 NcLPMO9M cellulose C1, C4 AA9 N. crassa Q7SHI8 EAA36362 NcLPMO9C cellulose hemicellulose C4 AA9- CBM1 N. crassa Q1K4Q1 EAA26656 CAD70347 NcLPMO9F cellulose Cl AA9 N. crassa Q7SCJ5 EAA34466 NcU00836 cellulose Cl AA9-CBM1 N. crassa Q7SCE9 EAA NcAA13 starch Cl AA13-CBM20 34371.2 N. crassa Q7S439 EAA30263 NcU02240 cellulose C4 AA9-CBM1 N. crassa Q7S111 EAA29018 NcU07760 cellulose C1, C4 AA9-CBM1 P.H1AE14 BAL43430 PcLPMO9D cellulose Cl AA9 chrysosporium P. anserina B2B629 CAP73254 PaGH61A PaLMPOB cellulose Cl a, C4a AA9-CBM1 P. anserina B2AVF1 CAP68375 PaGH61 B PaLMPO9A cellulose C1, C4 AA9-CBM1 P. anserina B2ARG6 CAP66744 PaLPMO9D cellulose Cl AA9 CBM1 P. anserina B2ATL7 CAP67740 PaLPMO9E cellulose Cl AA9 CBM1 P. anserina B2B403 CAP71839 PaLPMO9F cellulose nd AA9 CBM1 3037078 42 Organization Ref. Ref. Other names Substrate specificity Known selectivity Modularity Uniprot GenBank P. anserina B2B5J7 CAP73072 PaLPMO9G cellulose nd AA9 CBM1 P. anserina B2ADG1 CAP64476 PaLPMO9H cellulose Cl, C4 AA9 CBM1 T. aurantiacus G3XAP7 ABW56451 TaGH61A cellulose Cl AA9 T. terrestris G2RGE5 AE071030 cellulose nd AA9 T . reesei Q7Z9M7 AAP57753 cellulose na AA9 T. reesei 014405 CAA71999 CeI61A cellulose na AA9 Bacteria Bacillus El UUV3 CB142985 nana AA10 amyloliquefaciens Burkholderia pseudomallei 1710b Q3JY22 ABA49030 BURPS1710b 0114 nana AA10 (BpAA10A) Bacillus Q62YN7 AAU22121 chitin Cl AA10 licheniformes Caldibacillus cellulovorans Q9RFX5 AAF22274 [3- 1, 4- ndnd AA10 mannanase (ManA) Enterococcus faecalis Q838S1 AA080225 ELPM010A chitin Cl AA10 Hahella chefuensis Q2SNS3 ABC27701 LPMO (HcAA10-2, HCH 00807) cellulose nd AA10 Serratia 083009 AAU88202 SmLPM010A chitin Cl AA10 marcescens Streptomyces coelicolor Q9RJC1 CAB61160 ScLPM010B cellulose chitin Cl , C4 AA10 Strep tomyces coelicolor Q9RJY2 CAB61600 ScLPM010C cellulose Cl AA10-CBM2 Thermobifida fusca Q47QG3 AAZ55306 TILPM010A cellulose chitin Cl, C4 AA10 Thermobifida fusca Q47PB9 AAZ55700 TILPM010B cellulose Cl AA10-CBM2 V. Q9KLD5 AAF96709 VcLPM010B n.e. no. AA10 cholerae 01
    权利要求:
    Claims (11)
    [0001]
    REVENDICATIONS1. A process for the manufacture of nanocelluloses from a cellulosic substrate comprising cellulose fibers, which process comprises the following successive steps: at least one step of enzymatic treatment of said cellulosic substrate, by contacting with at least one enzyme of cleaving, then - at least one step of mechanical treatment of said cellulosic substrate subjected to said at least one enzymatic treatment step, to delaminate said cellulose fibers and to obtain said nanocelluloses, characterized in that said at least one cleavage enzyme is chosen among the enzymes belonging to the family of lyophilic monooxygenases of polysaccharides (LPM0s) capable of ensuring an oxidative cleavage of said cellulose fibers in the presence of an electron donor.
    [0002]
    2. Process for the production of nanocelluloses according to claim 1, characterized in that the LPM0s are chosen from enzymes capable of cleaving the cellulose by oxidation of at least one of the carbon atoms in positions C1, C4 and C6 of the glucose cycle.
    [0003]
    3. Process for the manufacture of nanocelluloses according to claim 2, characterized in that the LPM0s are chosen from the AA9 and AA10 families of the CAZy classification.
    [0004]
    4. Process for the production of nanocelluloses according to any one of claims 1 to 3, characterized in that the LMPOs are chosen from LPM0s from Podospora anserina, preferably from PaLPMO9A (Genbank CAP68375), PaLPMOB (Genbank CAP73254) , PaLPMO9D (Genbank CAP66744) PaLPMO9E (Genbank CAP67740), PaLPMO9F (Genbank CAP71839), PaLPMO9G (Genbank CAP73072), PaLPMO9H (Genbank CAP61476).
    [0005]
    5. Process for the production of nanocelluloses according to any one of Claims 1 to 4, characterized in that the electron donor is chosen from ascorbate, gallate, catechol, reduced glutathione and lignin fragments. and fungal carbohydrate dehydrogenases.
    [0006]
    6. Process for the production of nanocelluloses according to any one of claims 1 to 5, characterized in that the cellulosic substrate is obtained from wood, a fibrous plant rich in cellulose, beetroot, citrus fruit. , annual straw plants, marine animals, algae, fungi or bacteria.
    [0007]
    7. Process for the production of nanocelluloses according to any one of Claims 1 to 6, characterized in that the cellulosic substrate is chosen from chemical papermaking pastes, preferably chemical wood pulps, more preferably one of at least the following paper pulps: - bleached doughs, - semi-bleached doughs, - unbleached doughs, - sulphite doughs, - sulphate doughs, - soda doughs, - kraft doughs. 15
    [0008]
    8. Process for the manufacture of nanocelluloses according to any one of claims 1 to 7, characterized in that said at least one mechanical treatment step comprises at least one of the following mechanical treatments: a homogenization treatment, a microfluidization treatment, an abrasion treatment, a cryomilling treatment.
    [0009]
    9. Process for the production of nanocelluloses according to any one of claims 1 to 8, characterized in that, following said at least one mechanical treatment step, said method comprises a post-treatment step, for example an acid treatment , an enzymatic treatment, an oxidation, an acetylation, a silylation, or a derivatization of certain chemical groups carried by the nanocelluloses.
    [0010]
    10. Process for the manufacture of nanocelluloses according to any one of claims 1 to 9, characterized in that the nanocelluloses obtained consist of cellulose nanofibrils and / or cellulose nanocrystals.
    [0011]
    11. Nanocellulose resulting from a manufacturing process according to any one of claims 1 to 10.
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    同族专利:
    公开号 | 公开日
    EP3303689A1|2018-04-11|
    US20180142084A1|2018-05-24|
    FR3037078B1|2018-07-27|
    WO2016193617A1|2016-12-08|
    CA2988109A1|2016-12-08|
    引用文献:
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    CN111116762B|2020-01-10|2022-02-18|天津科技大学|Preparation method of hydrophobic cellulose nanocrystals|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1555049|2015-06-03|
    FR1555049A|FR3037078B1|2015-06-03|2015-06-03|PROCESS FOR THE PRODUCTION OF NANOCELLULOSES FROM A CELLULOSIC SUBSTRATE|FR1555049A| FR3037078B1|2015-06-03|2015-06-03|PROCESS FOR THE PRODUCTION OF NANOCELLULOSES FROM A CELLULOSIC SUBSTRATE|
    CA2988109A| CA2988109A1|2015-06-03|2016-06-01|Method for producing nanocelluloses from a cellulose substrate|
    US15/577,964| US20180142084A1|2015-06-03|2016-06-01|Method for producing nanocelluloses from a cellulose substrate|
    PCT/FR2016/051306| WO2016193617A1|2015-06-03|2016-06-01|Method for producing nanocelluloses from a cellulose substrate|
    EP16734421.7A| EP3303689A1|2015-06-03|2016-06-01|Method for producing nanocelluloses from a cellulose substrate|
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