Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21B—FIBROUS RAW MATERIALS OR THEIR MECHANICAL TREATMENT
  • D21B1/00—Fibrous raw materials or their mechanical treatment
  • D21B1/02—Pretreatment of the raw materials by chemical or physical means
  • D21B1/021—Pretreatment of the raw materials by chemical or physical means by chemical means
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
  • C08H8/00—Macromolecular compounds derived from lignocellulosic materials
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
  • C08L1/02—Cellulose; Modified cellulose
  • C08L1/04—Oxycellulose; Hydrocellulose, e.g. microcrystalline cellulose
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0083—Miscellaneous (1.14.99)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
  • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
  • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
  • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
  • D21H11/12—Pulp from non-woody plants or crops, e.g. cotton, flax, straw, bagasse
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
  • D21H11/16—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
  • D21H11/18—Highly hydrated, swollen or fibrillatable fibres
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
  • D21H17/005—Microorganisms or enzymes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

• the present invention generally relates to the field of celluloses, especially nanocelluloses, and more particularly to processes for the manufacture of cellulosic fibers and defibrillation of cellulosic substrates.
• 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.
• nanocelluloses have been identified with a dimension of the order of a nanometer, referred to as the generic name of "nanocelluloses”.
• 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.
• Nanocelluloses are also used in many devices requiring control of nanoscale porosity because of their high surface area.
• 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.
• the nanocelluloses can be classified mainly in two families: cellulose fibrils and cellulose nanocrystals.
• Cellulose nanocrystals also known as “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.
• NCCs have a length from a few tens of nanometers to approximately 1 ⁇ (in particular from 40 nm to 1 ⁇ 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).
• Cellulose fibrils commonly referred to as microfibrillated cellulose (or microfibrillated cellulose) or nano fibrils of cellulose (NFC) are typically isolated from cellulosic mass, by mechanical methods to delaminate the cellulose fibers and release the cellulose fibrils.
• 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 ranging from 25 to 100 nm and a much longer length.
• a first pretreatment strategy consists in pretreating the cellulose fibers with cellulases in order to destructure the fiber before the application of the mechanical homogenization treatment.
• 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 chemical oxidation step of the cellulose fibers (for example Saito et al., Biomacromolecules, Vol.8, No. 8, 2007, pp. 2485-2491).
• 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.
• an oxidant such as sodium hypochlorite catalyzed by the 2,2,6,6-tetramethylpiperidine-1-oxyl radical ("TEMPO")
• the oxidative treatment converts the primary alcohol function at the C 6 position of the glucose unit of the cellulose into a carboxylate function, which leads to the introduction of fillers on the surface of the cellulose fibers. These charges create electrostatic repulsions that facilitate delamination and increase its efficiency.
• nanocellulose production costs remain high, yields uncertain, and quality and properties are variable.
• WO 2016/193617 teaches processes for manufacturing nanocelluloses, comprising a step of enzymatic treatment of said substrates by a cleavage enzyme belonging to the family of lyophilic monooxygenases of polysaccharides (LPMOs), capable of ensuring an oxidative cleavage said cellulosic fibers.
• a cleavage enzyme belonging to the family of lyophilic monooxygenases of polysaccharides (LPMOs), capable of ensuring an oxidative cleavage said cellulosic fibers.
• the present invention aims to provide a method at least partially meeting these expectations.
• the invention relates to a process for preparing a cellulosic substrate for the manufacture of cellulose fibers, which process comprises at least the following steps:
• LPMOs polysaccharides
• said enzyme is a polysaccharide oxidase having an amino acid sequence identity of at least 20% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e- 3 or less.
• the invention relates to the use of a cellulosic substrate obtained according to the preceding method as a cellulosic raw material for preparing cellulose fibers, in particular nanocellulose, in particular cellulose fibrils and / or cellulose nanocrystals via a defibrillation process, especially mechanical.
• the invention relates to a method for defibrillation of a cellulosic substrate, which method comprises at least the following steps:
• the invention relates to a process for producing cellulose fibers, which process comprises at least the following steps:
• LPMOs polysaccharide lyophilic monooxygenases
• said enzyme is a polysaccharide oxidase having an amino acid sequence identity of at least 20% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e "3 or less.
• the invention relates to cellulosic fibers derived from an extraction process, and / or a manufacturing method, as defined above.
• the cellulose fibers which are particularly considered by the invention are cellulose nanofibers, or nanocelluloses.
• Figure 1 Phylogenetic tree of the AAxx family demonstrating that members of the AAxx family strongly cluster together and are very distant from the sequences relating to the families AA9, AA10, AA11 and AA13 respectively.
• Figure 2 Online diagram of the active site of LPMOs of type PcAAxx
• FIG. 3A-3B consensus sequence based on an alignment of 283 sequences belonging to the catalytic module of AAxx families revealing a first histidine conserved in the family
• Figure 4 Morphology of cellulosic fibers of birch (A and B) and resinous (C and D) under the action of polysaccharides monooxygenases.
• Figure 5 Defibrillation state of cellulosic fibers under the action of a polysaccharide monooxygenase (PcAAxxA / SEQ ID No. 1).
• TEM A and C
• AFM B and D
• the object of the invention is to meet these needs.
• 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 lytic monooxygenases.
• polysaccharides commonly referred to as "LPMOs” for “Lytic Polysaccharide Monooxygenases”.
• LPMOs are enzymes whose oxidative function has recently been demonstrated (Vaaje-Kolstad, 2010, Quinlan et al., 2011, Westereng et al., 2011, Horn et al, 2012, Bey et al, 2013). These enzymes, which improve the degradation of lignocellulose (Harris et al., 2010) are found in the latest generation of industrial enzyme cocktails (e.g. Cellic CTec3). These are copper-dependent enzymes that catalyze the oxidative cleavage of cellulose. These enzymes formerly classified in CAZy's GH61 family (“Glycoside Hydrolase”) (www.cazy.org) have been reclassified to the AA9 ("Auxiliary Activity" enzymes) family.
• This new family of enzymes capable of oxidizing polysaccharides is also referred to herein as a "AAxx" protein family.
• the inventors characterized structurally the reference protein PcAAxxB (Genbank # KY769370) of P. coccineus by solving the crystallographic structure of its catalytic module at a resolution of 3 ⁇ . They provided a structural model for the identification of all relevant members of this AAxx family of proteins, in addition to the sequence alignment analysis of more than 300 proteins with significant similarities to PcAAxxB.
• the present invention therefore takes advantage of the particular enzymatic properties of a new class of enzyme with polysaccharide oxidase activity, characterized in that: said enzyme with polysaccharide oxidase activity has an amino acid sequence identity of at least 30% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e -3 or less
• the inventors have identified a crystallographic structure of the polypeptide of sequence SEQ ID NO 2.
• the enzymes with polysaccharide oxidase activity according to the invention are characterized by the presence of a conserved active site fixing the copper, also referred to herein as an active site fixing the copper of the "histidine brace” type, formed by two residues Histidine and a Tyrosine one of the two histidine residues being N-terminal histidine after cleavage of the signal peptide.
• the inventors have shown here that the plurality of proteins belonging to this new AAxx family can be differentiated from other lytic oxygenases of the polysaccharides in that this new AAxx family does not, necessarily, have a module which fixes the carbohydrates (CBM).
• CBM carbohydrates
• a polysaccharide oxidizing enzyme such as those identified in the present application can act in synergy with other previously known polysaccharide oxidizing enzymes such as the lytic oxygenases of polysaccharides (generally called LPMOs). to improve the hydrolysis of these polysaccharides.
• LPMOs polysaccharide oxidizing enzymes
• the experimental data described here show that the polysaccharide oxidizing enzymes identified by the inventors can target distinct sugar units constituting a polysaccharide (for example cellulose, hemicellulose or lignocellulose), or else can target distinct sugar unit chemical groups. from these targeted by already known LPMOs, such as AA9 (also called GH61), AA10, AAl l and AA13.
• a polysaccharide for example cellulose, hemicellulose or lignocellulose
• LPMOs such as AA9 (also called GH61), AA10, AAl l and AA13.
• the inventors have unexpectedly discovered that the polysaccharide oxidizing enzymes considered according to the invention can act synergistically with cellulases to degrade polysaccharide-containing materials, such as cellulose-containing and lignocellulose-like materials.
• polysaccharide oxidizing enzymes identified herein can also act synergistically with LPMOs to degrade polysaccharide-containing materials, such as cellulose-containing and lignocellulose-like materials.
• the polysaccharide oxidizing enzymes of the present invention can be considered either separately or in combination with other enzymes capable of oxidizing or degrading polysaccharides, as well as mixtures thereof.
• the inventors have identified a new class of polysaccharide oxidizing enzymes that can be used in a variety of processes to degrade polysaccharide-containing materials, and more particularly in a wide variety of processes to degrade lignocellulosic materials.
• the inventors are of the opinion that the AAxx enzymes of the invention are capable of acting preferentially on the xylans bound to cellulose and more specifically on the xylans having a rigidity and a conformation. similar to the underlying cellulosic chains.
• the invention thus relates to (i) an original process for preparing a cellulosic substrate; (ii) using the cellulosic substrate thus obtained as a raw material for forming cellulose fibers, especially nanocelluloses; (iii) a method for defibrillating a cellulosic substrate; and (iv) a process for making cellulose fibers.
• a process for producing cellulose fibers as defined herein may thus consist of the combined implementation of a process for preparing a cellulosic substrate and then a defibrillation process on said prepared substrate.
• nanocelluloses are aimed at applications in 3 major areas:
• the nanocrystals are usually stabilized in colloidal suspensions by electrostatic repulsions induced by the presence of surface charges. They can serve as stabilizer of hydrophobic phases and thus enter the composition of paints, varnishes, cosmetics, etc. They can also make it possible to disperse functional agents such as opacifiers for applications (titanium dioxide, for example in paper mills), or reinforcers (clays).
• Porous materials (foams, aerogels, membranes, etc.):
• the very large surface area of the nanocelluloses makes it possible to access a control of the porosity of the materials at the nanometric level after drying. This control gives access to various types of applications where porosity or a large specific surface area are key features: filtration membrane, catalytic support, thermal superinsurance.
• Nanocelluloses have quite remarkable mechanical properties. On the other hand, dispersion at the nanoscale and their hydrophilic nature gives them gas barrier properties. These characteristics pave the way for their use particularly in the field of packaging.
• the present invention relates to a method 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 ) of glucose (D-Anhydroglucopyranose - AGU for "Anhydro glucose unit”) interconnected by glycoside bonds ⁇ - (1-4).
• 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).
• the combination of the cellobiose dimeric chains forms a nano elemental fibrillar of cellulose (the diameter of which is about 5 nm).
• the combination of elemental nanofibrials forms a nano-fibril (generally ranging in diameter from 50 to 500 nm, which includes 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 and 500 nm).
• the arrangement of several of these nanofibrials then forms what is generally called a cellulose fiber.
• cellulose fiber refers to all of the forms of cellulose that can be obtained after a defibrillation process, or delamination of a cellulosic substrate; which includes cellulose forms having a nanometer dimension, as well as cellulose forms having a larger dimension.
• nanocellulose refers to the various forms of cellulose having a dimension of the order of one nanometer. This term particularly encompasses according to the invention, two families of nanocelluloses: cellulose nanocrystals and cellulose fibrils.
• cellulose fibrils are synonymous.
• Each nano fibril of cellulose contains crystalline parts stabilized by a solid network of inter- and intra-chain hydrogen bonds. These crystalline regions are separated by amorphous regions.
• NCCs cellulose nanocrystals
• the NCCs advantageously comprise at least 50% of crystalline part, more preferably at least 55% of crystalline part. They generally have a diameter ranging from 5 to 70 nm (preferably less than 15 nm) and a length ranging from 40 nm to approximately 1 ⁇ m, preferably ranging from 40 nm to 500 nm; which includes 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 and 500 nm.
• cellulose nanocrystals are synonymous.
• cellulose whiskers are synonymous.
• microcrystals or “nanocrystal cellulose” are synonymous.
• NCCs cellulose nanocrystals
• polysaccharide-containing material includes a substance or composition comprising polysaccharides.
• polysaccharide is used in its conventional sense and refers to polymeric carbohydrates composed of long chains of monosaccharides held together by glycosidic linkages. During their hydrolysis, the polysaccharides release the monosaccharides or oligosaccharides constituting them.
• lignocellulose-containing material refers to a material initially consisting of cellulose, hemicellulose and lignin. This term is synonymous with “lignocellulosic material”. Such material is often referred to as “biomass”.
• a material containing lignocellulose is an example of a cellulosic substrate capable of forming cellulose fibers, within the meaning of the invention.
• a "polysaccharide oxidizing enzyme” encompasses polypeptides with the following properties:
• said polypeptide produces hydrogen peroxide in the presence of oxygen and an electron donor compound, such as ascorbates,
• said polypeptide increases in a dose-dependent manner, in the absence or in the presence of an electron donor compound, the degradation of a material containing polysaccharides such as lignocellulose, caused by cellulases and / or xylanases,
• said polypeptide increases in a dose-dependent manner, in the absence or in the presence of an electron donor compound, the degradation of a material containing polysaccharides such as lignocellulose, caused by cellulases, in the presence of a or more LPMOs, such as LPMOs separated into a group comprising AA9, AA10, AA1 1 and AA13.
• a "polysaccharide oxidizing enzyme” according to the invention has been demonstrated to be particularly effective for oxidizing xylan, and especially xylan absorbed on cellulose.
• the term "electron donor compound” is used herein in its usual sense for a person skilled in the art.
• an electron donor compound is a chemical entity capable of donating electrons to another compound.
• An electron donor compound is a reducing agent because of its ability to donate electrons and is itself oxidized when it gives electrons to another chemical entity.
• An electron donor compound as specified above for the oxidation properties of polysaccharides includes, but is not limited to, ascorbates and cellobiose dehydrogenases (CDHs).
• the reducing agent may advantageously be provided by biomass (lignin) which can act as an electron donor.
• BLAST-P method also called Protein Basic Local Alignment Search Tool method
• the BLAST-P method has in particular been described in Altschul et al. (1990, Mol Mol, Vol 215 (No. 3): 403-410), Altschul et al. (1997, Nucleic Acids Res, Vol 25: 3389-3402) and Altschul et al. (2005, FEBS J. Vol 272: 5101-5109).
• the BLAST-P method is preferably used according to the following parameters: (i) Expected threshold: 10; (ii) Word Size: 6; (iii) Max Matches in a Query range: 0; (iv) Matrix: BLOSSUM62; (v) Gap costs: Existence 11, Extension 1; (vi) Compositional Adjustments: Conditional compositional score matrix adjustment, (vii) No f ⁇ lter; (viii) No mask.
• the score of an alignment, S is calculated as the sum of the substitution and gap scores. Substitution scores are given in a table (see PAM, BLOSUM below).
• the gap scores are usually calculated as the sum of the G, the opening gap penalty and L, the extension penalty of a gap. For a gap of length n, the cost of a gap would be G + Ln.
• the choice of gap costs, G and L are empirical, but it is usual to choose a high value for G (10-15) and a low value for L (1-2).
• Optimal alignment means aligning two sequences with the highest score possible.
• Amino acid identity represents the extent to which two amino acid sequences have the same residues at the same positions in an alignment, and is often expressed as a percentage.
• BLOSUM (Blocks Substitution Matrix) matrices are surrogate scoring matrices in which the scores for each position are derived from the observation of the substitution frequency of blocks in local alignments for related proteins. Each matrix is drawn for a particular evolution distance.
• the alignment from which the scores were derived was created from sequences sharing no more than 62% identity. Sequences that have more than 62% identity are represented by a single sequence in alignment so as not to overrepresent members close to the same family.
• an E-value (also called Expect Value) is a calculated parameter when the BLAST-P method is used, said parameter representing the number of different alignments with equivalent scores or with better than S of which the appearance is expected during a search in the database by chance.
• E value also called Expect Value
• the "percentage identity" between two polypeptides means that the percentage of identical amino acids between the two polypeptide sequences to be compared, obtained after optimal alignment, this percentage being entirely statistical and the differences between the two polypeptide sequences being distributed randomly along their length.
• the comparison of two polypeptide sequences is traditionally carried out by comparing the sequences after optimally aligning them, the said comparison must be able to be conducted by segment or by using an "alignment window".
• the optimal alignment of the sequences for their comparison is achieved using the BLAST-P comparison software.
• the percentage identity between two amino acid sequences is determined by comparing the two optimally aligned sequences within which the nucleic acid sequences to be compared may contain additions or deletions with respect to the sequence of amino acids. reference of the optimal alignment between the two polypeptide sequences.
• the percentage of identity is calculated by determining the number of positions in which an amino acid is identical between the two sequences, preferably between two complete sequences, and then dividing this number of identical positions by the total number of positions in the window. alignment and multiplying the result by 100 to obtain the percentage identity between the two sequences.
• polypeptide sequences having at least 20% amino acid identity with a reference sequence include those having at least 21%, 22%, 23%, 24%, 25%, 26 %, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 28, 59% , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% of amino acid identity with said reference sequence.
• the "percentage identity" between two nucleic acid sequences represents the percentage of identical nucleotide residues between two nucleic acid sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly over the length of the sequences.
• the comparison of two nucleic acid sequences is traditionally performed by comparing the sequences after optimally aligning them, said comparison being segmentable or using an "alignment window”. Optimal alignment of the sequences for comparison is performed using the BLAST-N comparison software.
• the percentage identity between two nucleic acid sequences is determined by comparing the two optimally aligned sequences in which the nucleic acid sequences to be compared may contain additions or deletions with respect to the reference sequence. the optimal alignment between the two sequences
• the percentage of identity is calculated according to the number of positions at which the nucleotide residues are identical between the two sequences, preferably between the two complete sequences, and then by dividing this number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
• nucleotide sequences having at least 20% identity with the reference sequence include those having at least 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28 %, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 28%, 59%, 60%, 61% 62% 63% 64% 65% 66% 67% 68% 69% 70% 71% 72% 73% 74% 75% 76% 77% 78 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with the reference sequence.
• an E value of 10 e “3 or less includes the E-value of 1 e “ 3 or less, 1 e “4 or less, 1 e “ 5 or less, 1 e “6 or less, 1 e “7 or less, 1 " 8 or less, 1 "9 or less, 1 " 10 or less, 1 "20 or less, 1 " 30 or less, 1 "40 or less, 1 " 50 or less, 1 "60 or less, 1 " 70 or less, 1 "80 or less, 1 " 90 or less and 1 "100 or less.
• chemical treatment refers to all chemical pretreatments that allow the separation and / or release of cellulose, hemicellulose and / or lignin.
• suitable chemical pretreatments include, for example, dilute acids, lime, bases, organic solvents, ammonia, sulfur dioxide, carbon dioxide.
• wet oxidation and controlled pH hydrothermolysis are also considered chemical pretreatments.
• the pretreatment methods using ammonia are in particular described in PCT applications WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901.
• mechanical pretreatment refers to all mechanical (or physical) treatments that allow the separation and / or release of cellulose, hemicellulose and / or lignin from a material containing lignocellulose .
• mechanical pretreatments include different types of grinding, irradiation, steam explosion and hydrothermolysis.
• Mechanical pretreatment includes fragmentation of a solid (comminution or mechanical size reduction). Solid fragmentation includes dry milling, wet milling and vibratory milling techniques. Mechanical pretreatment may also include high pressures and / or high temperatures (steam explosion). In some representations of the pretreatment step, said step may combine mechanical and chemical pretreatment.
• biological pretreatment refers to all biological treatments that allow the separation and / or release of cellulose, hemicellulose and / or lignin from a material containing lignocellulose.
• Biological pretreatments may involve the application of microorganisms capable of solubilizing lignin (see, for example, Hsu, 1996, Pretreatment of Biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, ed., Taylor & Francis, Washington, DC, 179-212, Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic / microbial conversion of lignocellulosic biomass, Adv ApplMicrobiol 39: 295-333, McMillan, 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, Baker, and Overend, eds., ACS Symposium Series 566, American Chemical Society, Washington,
• the invention relates to a process for preparing a cellulosic substrate for the manufacture of cellulose fibers, which process comprises at least the following steps:
• LPMOs polysaccharides
• said enzyme has an amino acid sequence identity of at least 20% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e "3 or less.
• the invention relates to the use of a cellulosic substrate prepared according to the preceding method, as cellulosic raw material for preparing cellulose fibers, in particular nanocellulose, in particular cellulose fibrils and / or of cellulose nanocrystals via a defibrillation process, especially mechanical.
• the invention relates to a process for extracting cellulose fibers, which process comprises at least the following steps:
• the invention relates to a process for manufacturing cellulose fibers, which process comprises at least the following steps:
• LPMOs polysaccharide lyophilic monooxygenases
• polysaccharide oxidase active enzyme has an amino acid sequence identity of at least 20% with a SEQ reference polypeptide
• Said enzyme with polysaccharide oxidase activity 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).
• the enzymatic treatment step is carried out at a temperature ranging from 30 to 50 ° C, in particular from 30 to 45 ° C.
• the pH of the reaction conditions of the enzyme in contact with the cellulosic substrate is generally between 3 and 7, which includes between 4 and 7, and especially between 4 and 6.
• 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 from 1: 1000 to 1: 500, from 1: 500 at 1: 100.
• said at least one LPMO enzyme is used at a concentration ranging from 0.001 to 10 g / l, in particular from 0.1 to 5 g / l, and more preferably from 0.5 to 5 g / l.
• 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 LPMO or LPMOs implemented during 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.
• tests for cleavage of the cellulose with an LPMO enzyme according to the invention can be carried out according to the following protocol:
• a cleavage test can be performed in a volume of 300 ⁇ l of liquid containing 4.4 ⁇ l of LPMO enzyme and 1 mM of ascorbate and 0.1% (weight / volume) of cellulose powder swollen with phosphoric acid (PASC phosphoric acid-swollen cellulose - prepared as described in Wood TM, Methods Enzym 1988, 160: 19-25) in 50 mM of sodium acetate buffer at pH 4.8 or 5 ⁇ l of cello-oligosaccharides ( Megazyme, Wicklow, Ireland) in 10 mM sodium acetate buffer at pH 4.8.
• PASC phosphoric acid-swollen cellulose - prepared as described in Wood TM, Methods Enzym 1988, 160: 19-25 in 50 mM of sodium acetate buffer at pH 4.8 or 5 ⁇ l of cello-oligosaccharides ( Megazyme, Wicklow, Ireland) in 10 mM sodium acetate buffer at pH 4.8.
• the enzymatic reaction can be carried out in a 2 ml tube incubated in a thermomixer (Eppendorf, Montesson, France) at 50 ° C. and 580 rpm (rotation per minute).
• the fibers are contacted with enzymes (at a concentration of between 1 and 5 g / L and at ratios of enzyme / cellulose of 1: 50, 1: 100, 1: 500 and 1: 1000) and ascorbate (2 mM) and then gently stirred for 48 hours at 40 ° C.
• enzymes at a concentration of between 1 and 5 g / L and at ratios of enzyme / cellulose of 1: 50, 1: 100, 1: 500 and 1: 1000
• ascorbate 2 mM
• the sample After 16 hours of incubation, the sample is heated at 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 treated fibers are then subjected to a mechanical action with a homogenizer-disperser (Ultra-Turrax power 500 W, maximum speed during 3 minutes), followed by sonication for 3 minutes.
• a homogenizer-disperser Ultra-Turrax power 500 W, maximum speed during 3 minutes
• the said processes are characterized in that the cellulose fibers obtained at the end of the process are cellulose nanofibers.
• said methods are characterized in that the electron donor is selected from ascorbate, gallate, catechol, reduced glutathione, lignin fragments and fungal carbohydrate dehydrogenases; and preferably ascorbate.
• the said processes are characterized in that the cellulosic substrate is obtained from wood, a fibrous plant rich in cellulose, beetroot, citrus fruits, annual straw plants, animals marine, algae, fungi or bacteria.
• the said processes are characterized in that the cellulosic substrate is chosen from chemical papermaking pastes, preferably chemical wood papermaking pastes, and preferably at least one of the following papermaking pastes: pasta bleached, semi-bleached pasta, unbleached pasta, sulphite pulp, sulphate dough, soda dough, kraft pulp.
• chemical papermaking pastes preferably chemical wood papermaking pastes, and preferably at least one of the following papermaking pastes: pasta bleached, semi-bleached pasta, unbleached pasta, sulphite pulp, sulphate dough, soda dough, kraft pulp.
• the said processes are characterized in that the cellulosic substrate is a paper pulp derived from wood, annual plants or fiber plants.
• the said methods are characterized in that the said at least one mechanical treatment step comprises at least one of the following mechanical treatments:
• the said methods are characterized in that, following said mechanical treatment step, said method comprises a post-treatment step chosen from: an acid treatment, an enzymatic treatment, an oxidation, an acetylation, a silylation or a derivatization of chemical groups carried by said cellulose fibers.
• a post-treatment step chosen from: an acid treatment, an enzymatic treatment, an oxidation, an acetylation, a silylation or a derivatization of chemical groups carried by said cellulose fibers.
• the invention relates to cellulosic fibers resulting from a defibrillation process and / or a process for producing cellulose fibers as defined above.
• Said cellulosic fibers can be characterized in that said cellulose fibers, preferably nanocellulose, comprise glucose rings of which at least one carbon atom is oxidized in position (s) Ci and / or C 4 , or even C 6 .
• the cellulosic fibers are nano fibrils of cellulose. Mechanical treatment step (s)
• the cellulosic substrate brought into contact with said enzyme is then subjected to at least one mechanical treatment step which is intended to delaminate the cellulose fibers to obtain the nanocelluloses.
• Delamination also called “fibrillation” or “defibrillation” consists in separating, by a mechanical phenomenon, the cellulose fibers within the cellulosic substrate, in particular for the manufacture of nanocelluloses.
• 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.
• the use of LPMOs 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.
• a mechanical treatment may be chosen from mechanical treatments for homogenization, microfluidization, abrasion, or cryomilling.
• the homogenization treatment involves the passage of the pretreated cellulosic substrate, typically a cellulose pulp or a liquid suspension of cellulose, through a narrow space under high pressure (as described for example in US Pat. No. 4,486,743).
• the pretreated cellulosic substrate typically a cellulose pulp or a liquid suspension of cellulose
• This homogenization treatment is preferably carried out using a Gaulin type homogenizer.
• the pretreated cellulosic substrate typically in the form of a cellulose suspension
• the pretreated cellulosic substrate 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 velocity deceleration impact.
• the passage of the substrate into the orifice is repeated (generally 8 to 10 times) until the cellulose suspension becomes stable.
• cooling water is generally used.
• This homogenization treatment can also be implemented using a micro-fluidizer type device (see, for example, Sisqueira et al., Polymer 2010 2 (4): 728-65).
• a micro-fluidizer type device see, for example, Sisqueira et al., Polymer 2010 2 (4): 728-65.
• the cellulose suspension passes through a thin chamber typically shaped "z" (whose channel dimensions are generally between 200 and 400 ⁇ ) under high pressure (about 2070 bar).
• the high shear that is applied generally greater than 10 7 .s _1) allows 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
• chambers of different sizes may be used to increase the degree of fibrillation.
• 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., Bio Macromolecules 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.
• the method of defibrillating and / or fabricating cellulose fibers comprises at least one post-treatment step of the cellulosic substrate, carried out after said substrate has been mechanically processed.
• said at least one post-treatment step aims to increase the degree of fibrillation of the celluloses (in particular nanocelluloses) obtained and / or to confer on said nanocelluloses new mechanical properties, depending on the intended applications.
• 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 derivatization of certain chemical groups carried by the micro-fibrils.
• 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 (ie cellulose fibers). ).
• biomass material including organic matter of plant origin, including algae, animal or fungal
• cellulosic fibers ie 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, papyrus and certain reeds, sugarcane bagasse, beetroot (including beet pulp), citrus fruits, stems maize or sorghum, or annual straw plants.
• wood of which cellulose is the main component
• any fibrous plant rich in cellulose such as, for example, cotton, flax, hemp, bamboo, kapok, coconut fiber (coir), ramie, jute, sisal, raffia, papyrus and certain reeds, sugarcane bagasse, beetroot (including beet pulp), citrus fruits, stems maize or sorghum, or annual straw plants.
• the 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 Gluconacetobacter types). .
• marine animals such as tunicate for example
• algae such as for example Valonia or Cladophora
• bacteria for bacterial cellulose for example bacterial strains of Gluconacetobacter types.
• cellulose from primary walls such as the parenchyma of fruits (for example beetroot, 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).
• 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, ie 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.
• the cellulosic substrate contains at least 90%, especially at least 95% and preferably 100% of cellulosic fibers.
• the cellulosic substrate is suitable for the manufacture of paper or of a cellulosic product.
• the cellulosic substrate is thus preferably chosen from papermaking pulps (or paper pulp), and in particular chemical pulps.
• the cellulose pulp may contain, in association with the cellulose fibers, hemicellulose and lignin.
• the cellulose pulp contains less than 10% and especially less than 5% of lignin and / or hemicellulose.
• the chemical papermaking pulps contain almost exclusively or exclusively cellulose fibers.
• the paper 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) , pasta with soda (unbleached or blanched) and kraft pasta.
• dissolving pastes having a low proportion of hemicellulose, preferably less than 10% and in particular less than or equal to 5%.
• the paper pulps used in a process of the invention are wood pulps, in particular chemical wood pulp.
• the cellulosic substrate is a paper pulp derived from wood, annual plants or fiber plants.
• a material containing lignocellulose, as defined below, is an example of a cellulosic substrate particularly considered according to the invention.
• the material containing lignocellulose contains at least 30%> wt. %, preferably at least 50 wt. %, even more preferably, at least 70% wt. %, and even more preferably at least 90%) wt. % lignocellulose. It is noted that lignocellulose-containing material may also contain other components such as protein material, starch, sugars, such as fermentable sugars and / or non-fermentable sugars.
• Materials containing lignocellulose may be any material containing lignocellulose.
• the material containing lignocellulose contains at least 30% wt. %, preferably at least 50 wt. %, even more preferably, at least 70% wt. %, and even more preferably at least 90% wt. % lignocellulose. It is important to understand that material containing the lignocellulose may also contain other components such as protein material, starch, sugars, such as fermentable sugars and / or sugars that can not ferment.
• the material containing lignocellulose is usually present, for example, in stems, leaves, bran, envelopes and spines of plants or leaves, branches, and wood of trees.
• the material containing the lignocellulose may also be herbaceous material, agricultural and forestry residues, municipal solid waste, paper waste, and pulp and paper mill residues.
• lignocellulose-containing material may be in the form of plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.
• the lignocellulose-containing material is a lignocellulosic biomass selected from the group consisting of: grass, switchgrass, spartin, ryegrass, false bald beech, miscanthus, moss residues. sugar processing, sugarcane bagasse, agricultural waste, rice straw, rice husk, barley straw, corn cob, cereal straw, wheat straw, straw, canola, oat straw, oat hull, corn stover, soy flour, corn flour, forestry waste, recycled wood pulp fiber, paper sludge, sawdust , hardwood, softwood, agave and its combinations.
• the lignocellulose-containing material is selected from the group consisting of: corn meal, corn fiber, rice straw, pine wood, wood chips, poplar, bagasse , paper and waste processing pulp.
• the material containing the lignocellulose is a lignocellulosic biomass based on wood.
• material containing lignocellulose include hardwood such as poplar and birch, softwood, cereal straw such as wheat straw, perennial millet, municipal solid waste, organic industrial waste, office papers and a mixture of these.
• the material containing lignocellulose is selected from pine, poplar and wheat straw.
• LPMO type enzyme according to the invention (AAxx family)
• An enzyme according to the invention is defined as an enzyme with polysaccharide oxidase activity, said enzyme having an amino acid sequence identity of at least 20% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e ⁇ 3 or less.
• said polysaccharide oxidase activity enzyme has an amino acid sequence identity of at least 30% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e- 3 or less.
• said polysaccharide oxidase active enzyme has an amino acid sequence identity of at least 60% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e- 3 or less.
• said polysaccharide oxidase active enzyme has an amino acid sequence identity of at least 90% with a reference polypeptide of SEQ ID NO. 1, 2 or 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e- 3 or less.
• said polysaccharide oxidase-active enzyme has an amino acid sequence identity of at least 90%> with a reference polypeptide of SEQ ID NO 1, from the BLAST-P comparison method. , said BLAST-P comparison method resulting in an E-value (E-value) of 10 e "3 or less.
• said polysaccharide oxidase active enzyme has an amino acid sequence identity of at least 90%> with a reference polypeptide of SEQ ID NO 2, from the BLAST-P comparison method. , said BLAST-P comparison method resulting in an E-value (E-value) of 10 e "3 or less.
• said enzyme with polysaccharide oxidase activity has an amino acid sequence identity of at least 90% with a reference polypeptide of SEQ ID NO 3, from the BLAST-P comparison method, said BLAST-P comparison method resulting in an E-value (E-value) of 10 e- 3 or less.
• said polysaccharide oxidase active enzyme is encoded by a nucleic acid having at least 90% sequence identity with a nucleic acid selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. And SEQ ID NO. 6.
• said enzyme with polysaccharide oxidase activity is chosen from the group comprising polypeptides having one of the following GenBank references: ALO60293.1; CCA68158.1; CCA68159.1; CCA68161.1; CCA71530.1; CCA72554.1; CCA72555.1; CCO30796.1; CCT73728.1; CDM26384.1; CD076981.1; CD076983.1; CDO76990.1; CDR41535.1; CDZ98469.1; CDZ98532.1; CDZ98792.1; CDZ98793.1; CEJ62913.1; CEJ80690.1; CEL55274.1; CEL55761.1; CEN61973.1; CEQ41736.1; CRL20539.1; CUA74138.1; CUA75968.1; EEB87294.1; EEB88604.1; EEB93106.1; EGU12035.1; EGU79270.1; EJU02917.1; EJU04796.1; EJU04797.1
• KIJ93961.1 KIKO 1335.1; KIKO 1364.1; KIK03019.1; KIK24220.1; KIK24223.1; KIK45012.1; KIK47453.1; KIK58046.1; KIK60325.1; KIK64405.1; KIK64418.1;
• said enzyme with polysaccharide oxidase activity consists of a recombinant protein.
• said recombinant protein is produced in a yeast which has been genetically transformed to produce said recombinant enzyme with polysaccharide oxidase activity.
• said polysaccharide oxidase active enzyme having an amino acid sequence comprising, or even consisting of in, SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO. 3, and / or at least one of the enzymes identified by their GenBank number above.
• said polysaccharide oxidase active enzyme is encoded by a nucleic acid having a nucleic acid sequence selected from a group comprising SEQ ID NO. 4, SEQ ID NO. And SEQ ID NO. 6.
• the cellulosic support in question can be put into contact with a plurality of enzymes with polysaccharide oxidase activity according to the invention, and more particularly a plurality of enzymes with polysaccharide oxidase activity comprising a sequence amino acid comprising, or even consisting of, SEQ ID NO. 1, SEQ ID NO.2, SEQ ID NO. 3, and / or at least one of the enzymes identified by their GenBank number above.
• the cellulosic support considered can be put into contact with a plurality of enzymes with polysaccharide oxidase activity according to the invention, and more particularly a plurality of enzymes with polysaccharide oxidase activity coded by a nucleic acid selected from a group of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, and / or at least one of the enzymes identified by their GenBank number above.
• said enzyme with polysaccharide oxidase activity is used in a composition with polysaccharide oxidase activity.
• said plurality of polysaccharide-oxidase active enzymes comprises between 2 and 10 distinct polysaccharide oxidase enzymes, which includes 2, 3, 4, 5, 6, 7, 8, 9 , 10, enzymes with distinct polysaccharide oxidase activity.
• a polysaccharide oxidase-active composition is capable of comprising one or more other polysaccharide oxidase activity enzymes selected from LPMOs, including enzymes selected from a group consisting of: AA9, AA10, AA11 and AA13 LPMOs Methods for identifying polypeptides belonging to the AAxx family
• the inventors describe here the crystallographic structure of the catalytic modulus PcAAxxB (JGI ID 1372210, GenBank ID # KY769370) belonging to an enzyme with polysaccharide oxidase activity. Also disclosed are nearly 300 enzymes with polysaccharide oxidase activity according to the invention, which belong to the same enzymatic family, including the polypeptides of sequence SEQ ID No. 1, SEQ ID NO.
• sequence SEQ ID No. 3 corresponds to the polypeptide SEQ ID No. 3
• bioinformatic methods exist for identifying new variants forming part of the so-called new family of LPMOs, by implementing, on the one hand, in silico modeling based on the said crystallographic structure, and on the other hand apart from said sequence alignments.
• programs capable of producing such three-dimensional models include:
• SWISS-MODEL homology-modeling server see Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information (Nucleic Acids Research 2014 (1 July 2014) 42 (W1): W252-W258).
• enzymes with polysaccharide oxidase activity according to a method comprising the following steps: al) identifying one or more polypeptides that may have polysaccharide oxidase activity;
• a2 identifying experimental coordinates of a main chain of a reference polypeptide, including or not the positions of the side chains, said main chain and / or said side chains being included in a polypeptide of sequence SEQ ID NO. 2 or SEQ ID NO. 7, or a fragment thereof with polysaccharide oxidase activity;
• a3) identifying a sequence alignment of one or more of said candidate polypeptides with a reference polypeptide having a sequence identity of 20% or more with SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO 3 or SEQ ID No. 7 according to the BLAST-P comparison method, characterized in that said one or more candidate polypeptides have an E-value of 10 e -3 or less;
• the nucleotide sequence was synthesized by codon optimization from P. pastoris (GenScript, Piscataway, USA, and ensuie inserted with the native signal sequence into a pPICZaA vector (Invitrogen, Cergy-Pontoise, France) using the BstBl and Xbal restriction, in phase with the C-terminal (His) 6 tag sequence
• P. pastoris X33 strain and the pPICZaA vector are components of the Easy Select Expression System (Invitrogen), and all media and protocols are described in the manufacturer's manual (Invitrogen).
• the cells are then transferred to 400 ml of BMMY containing 1 ml.l -1 of PTM4 salts at 20 ° C. with shaking (200 rpm) for 3 days, supplemented with 3% (v / v) of methanol each day.
• the transformant producing the greatest amount of protein was selected for the bioreactor culture. This is carried out in fermenters of the New type Brunswick BioFlo 115 containing 1.3L (Eppendorf, Hamburg, Germany) according to the fermentation protocol of P. pastoris (Invitrogen) containing some modifications.
• P. pastoris is cultivated in solid YPD agar medium (20 ⁇ l-1 peptone, 10 ⁇ l-1 yeast extract, 20 ⁇ l-1 glucose, 20 ⁇ l-1 agar). 100 ml of BMGY medium contained in a flask of 500 ml were inoculated from an isolated colony of P. pastoris and incubated at 30 ° C.
• the first step of the batch culture is in 400 mL of minimum medium containing 40 ⁇ L-1 glycerol; 26.7 ml.L-1 H 3 P04; 14.9 ⁇ L-1 MgSO 4 .7H 2 O; 0.93 ⁇ L-1 CaSO 4 .2H 2 O; 7.7 gL-1 KCl; 4.13 ⁇ L-1 KOH; 4.35 mL.L-1 TMP 1 solution (6 gL-1 CuSO 4 .5H 2 O, 0.08 gL-1 Nal, 3 gL-1MnSO 4 .H 2 O, 0.2 gL-1 Na 2 MoO 4 .2H 2 O, 0.02 gL-1 H 3 BO 3 0.5 ⁇ L-1 CoCl 2, 20 ⁇ L-1 ZnCl 2 .7H 2
• the second phase begins with the simultaneous addition of 50 g of sorbitol and 0.5%) of methanol (v / v) to the bioreactor so that the yeasts switch on metabolism of methanol (about 5 hours).
• stirring is increased to 500 rpm and the pH is gradually increased to pH6 by adding ammonium hydroxide (28% ov / v).
• the induction phase begins with the addition of a methanol solution containing 12 ml.L-1 of PTM1 salts (containing copper) in a "fed-batch" mode.
• the initial flow rate is 1.47 mL.h-1 and is increased after about 14 hours of incubation at 2.94mL.hl.
• the induction phase is carried out at 20 ° C. and the dissolved oxygen concentration is maintained at 20% by stirring (800 rpm), gas flow (0.2-1 vvm) and the oxigen (0-50%) cascade. ). The induction phase is continued for 144h.
• the supernatants of the culture are recovered after centrifugation at 2700 g for 5 min at 4 ° C. and are passed through 0.45 ⁇ filters (Millipore, Molsheim, France) in order to remove the remaining cells.
• the pH is adjusted to 7.8 and the supernatants are filtered a second time through filters of 0.2 ⁇ before being deposited in an HP His Trap column of 5 mL (GE healthcare, Bue, France) connected to the Akta system Xpress (GE Healthcare).
• the column is equilibrated in a buffer containing 50 mM Tris HCl pH 7.8 and 150 mM NaCl (buffer A) before charging.
• the column is washed with 5 column volumes (CV) of buffer A containing 10 mM of imidazole. The elution is then carried out by the passage of 5 CV of buffer A containing 150 mM of imidazole.
• the fractions containing the purified protein are assembled and concentrated using a 3kDa vivaspin concentrator (Sartorius, Palaiseau, France) before being loaded into a HiLoad 16/600 Superdex 75 Prep Grade column (GE Helthcare) for separation in 50 mM buffer acetate at pH 5.2. Gel filtration analysis showed that PcAAxx proteins are monomeric in solution even after addition of copper.
• the salts contained in the culture medium are diluted 10 times in 20 mM Tris-HCl pH 8 and the proteins are then concentrated using a Pellicon-2 cassette with an off of lOkDa ( Millipore) to a volume of approximately 200 mL that will be loaded into a DEAE High Prep 20 mL column (GE Healthcare).
• the proteins are then eluted from the column in a linear gradient of 1M NaCl (0 to 700mM in 200mL).
• the fractions are then analyzed by SDS-PAGE for the presence of the recombinant protein and the fractions containing the protein are collected in the same sample and concentrated.
• the concentrated proteins are finally incubated overnight in a solution containing their molar equivalent of copper before another separation step using a HiLoad 16/600 Superdex 75 Prep Grade column in 50 mM acetate buffer pH 5.2. Finally the fractions containing the purified proteins are collected in the same sample and concentrated with a 3kDa vivaspin concentration column (Sartorius).
• the purified PcAAxxB proteins (JGI ID 1372210, GenBank ID # KY769370) are concentrated with a Vivaspin 10kDa concentrator in polyethersulfone (Sartorius). Protein concentration is determined by measuring ⁇ 280 nm of the solution with a Nanodrop ND-2000 (Wilmington, Delaware, USA). All the crystallization experiments were carried out at 20 ° C. by the seated droplet technique (diffusion by steam) in a 96-well crystallization plate (Swissci) with a crystallization robot mosquito® Crystal (TTP labtech).
• the reservoirs contain 40 ⁇ l of commercial screening solution and the crystallization drops are prepared by mixing 100 ⁇ l of reservoir solution with 100, 200 and 300 ⁇ l of solution containing the protein to be crystallized.
• a first result was obtained after one week from one of the conditions prepared with Screen AmSO 4 solution (Qiagen) containing 2.4 M (NH 4) 2 SO 4 and 0.1 M citric acid pH 4.
• this crystallization condition was optimized by mixing the protein solution at 28 mg.mL-1 with a precipitation solution composed of 2.4 M (NH 4) 2 S 04 and 0.1 M citric acid at pH 4.4 in a volume ratio of 3: 1.
• the crystals of PcAAxxB are generated in one week at the dimension of 0.15x0.15x0.05 mm.
• the crystals belong to the P41212 space group and have 204x204x 110 ⁇ axes and 2 molecules per asymmetric unit.
• AT 5 Data collection, structure determination and refinement
• the crystals of PcAAxx are incubated for 5 min in a solution of 2.4M Li 2 SO 4 to replace the 2.4M of (NH 4) 2 SO 4 in the stock solution prior to instant cooling in liquid nitrogen.
• a derived crystal is obtained by introducing a heavy atom by incubating the crystals in the reservoir solution supplemented with 55 mM gadoteridol-gadolinium complex (Gd) before cooling.
• the initial diffraction data were collected with a beamline light line ID23-1, while the MAD data was collected with a beamline ID30B light line at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.
• ESRF European Synchrotron Radiation Facility
• the data was then indexed and integrated into the P41212 space group using XDS.
• the necessary data processing was carried out with the CCP4 program suite.
• the determination of the GD3 + substructure and the subsequent phasing combined with solvent flattening was done with SHELXC / D / E, and using the S AD data collected on the edge of the Gd which allowed obtain a correlation coefficient of 71.8%.
• the P. coccineus AAxx sequences (Genbank ID KY769369 and KY769370) were compared to the non-redundant sequence database of NCBI with BlastP (29) in February 2016. Blast analyzes from AAxx have not been performed. allowed to find AA9s, AAlOs, AAl ls, or AA13s with significant scores, and vice versa. MUSCLE has been used to achieve multiple alignments. In order to avoid interference due to the presence or absence of additional residues, signal peptides and C-terminal extensions have been removed. Bioinformatic analyzes were performed from 286 fungal genomes previously sequenced and shared by JGI collaborators.
• Protein clusters are available through JGI (https://goo.gl/ZAa2NX) for each variety of fungi. 100 protein alignments from selected protein clusters were previously cleaned and merged to generate a phylogenetic tree. These clusters are present in one copy, as far as possible, in one copy in all varieties of fungi to improve the score / l / n (n represents the number of copies in the genome). Sequences from the clusters were mafft aligned and cleaned with Gblocks, and the phylogenetic tree was generated by concatenating alignments with Fasttree. The tree is visualized with Dendroscope and Bio :: phylo.
• AAxx enzymes were loaded with copper from copper salts (sulfate or acetate) during or after purification. Proteins were incubated with 10 molar equivalents of copper salts between 2h and overnight at 4 ° C. Excess copper was then removed by diafiltration with 3kDa centricons or by gel filtration chromatography. The presence of copper can be determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), as described below.
• ICP-MS Inductively Coupled Plasma Mass Spectrometry
• the samples Prior to analysis, the samples are mineralized in a mixture containing 2/3 nitric acid (Sigma-Aldrich, 65% purissime) and 1/3 hydrochloric acid (Fluka, 37%, Trace Select) at 120 ° C. .
• Residues are diluted in ultrapure water (2 mL) prior to ICP-MS analysis.
• the instrument for ICP-MS analysis is ICAP Q (ThermoElectron, Les Ullis, France), equipped with a collision chamber.
• the calibration curve is obtained by diluting a certified multi-element solution (Sigma-Aldrich).
• Aqueous dispersions of Kraft cellulose fibers are adjusted to pH 5.2 in acetate buffer (50 mM) in a final reaction volume of 5 mL.
• the polypeptide of sequence SEQ ID No. 1 (LPMO AAxx) is added to the fibers at a final concentration of 20 mg.g- 1 in the presence of 1 mM ascorbic acid.
• Enzyme is performed at 40 ° C with gentle stirring for 48 hours.
• the samples are then dispersed by a Polytron PT2100 Kinematica AG homogenizer, Germany) for 3 minutes, and then sonicated with a QSonica Q700-type sonicator (20 kHz, QSonica LLC, Newtown, USA) at 350 W ultrasonic power.
• the reference sample is subjected to the same treatment, in the absence of enzyme.
• the cellulose fibers (reference and treated with PcAAxx) are deposited on a glass slide and observed by polarizing microscope BX51 (Olympus France SAS) with a 4x objective. Images are taken with a U-CMAD3 type camera (Olympus Japan).
• AFM atomic force microscopy
• TEM transmission electron microscopy
• Example 1 Crystallographic Structure of the Catalytic Module of PcAAxxB (JGI ID 1372210; GenBank ID # KY769370) to 3.0 A
• the crystallographic structure of the catalytic modulus of PcAAxxB (JGI ID 1372210, GenBank ID # KY769370), refined to a resolution of 3.0 ⁇ , reveals a structured core and a catalytic site formed according to a canonical coordination mode of histidine brace ", exposed on the surface.
• PcAAxxB (# KY769370) was produced in large quantities in Pichia pastoris and purified to homogeneity.
• PcAAxxB The structure of PcAAxxB was resolved by the MAD technique (multiple wave length anomalous dispersion) from the gadolinium signal, then refined to 3.0 ⁇ resolution.
• the core of the protein is structured into a large antiparallel ⁇ sandwich, a folding that is broadly similar to that already identified for other families.
• the active site of PcAAxxB consists of Hisl, His99 and Tyrl76, which form a canonical coordination mode of the "histidine brace" type (see FIG. 2).
• the surface of PcAAxxB has a rippled shape with a clamp formed by two protruding loops, visible from the pdb format (Protein Data Bank). Five N-glycans are attached to the crystallographic structure of PcAAxxB through the following Asparagine residues: Asn13, Asn76, Asn133, Asn183 and Asn217.
• the crystallographic structure further indicates the presence of 10 Cysteine residues involved in five disulfide bridges at the following positions: Cys67 &Cys90; Cys109 &Cys136; Cys133 &Cys158; Cysl60 &Cysl82; Cys202 & Cys218.
• the crystallized structure includes two molecules per asymmetric unit.
• the coordinates of the channel A are here presented in pdb format.
• Chain B which is also part of the asymmetric unit, is not shown here.
• sequence SEQ ID No. 7 corresponds to the minimal fragment of sequence SEQ ID No. 2 comprising the three amino acids involved in the catalytic triad fixing the copper, including the residue Histidine in the N-terminal position, and further comprising the sandwich- ⁇ antiparallel to the Thr185 residue.
• SEQ ID No. 2 may be furthermore positioned relative to a consensus sequence derived from FIG. 3, as follows: Table 1A: Consensus Sequence and Position on SEQ ID No. 2
• the fibers are brought into contact with an enzyme with polysaccharide oxidase activity according to the invention, of sequence SEQ ID No. 1, and ascorbate then subjected to gentle stirring for 48 hours. In the absence of enzyme, the fibers remain intact and no modification is observed.
• the dispersions were then analyzed by light microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM).
• TEM transmission electron microscopy
• AFM atomic force microscopy
• the degradation of ligocellulosic biomass by polypeptides of SEQ ID NO. 1, SEQ ID NO. 2 and a T.reesei cellulase cocktail was tested by sequential reactions.
• Cellulosic carriers of the "poplar" type were first incubated with 2.2 ⁇ l (equivalent to 70 ⁇ g) of polypeptide of sequence SEQ ID NO. 1 or 2.2 ⁇ (equivalent to 70 ⁇ g) of polypeptide of sequence SEQ ID NO.2 for 48 hours; following which 10 ⁇ g of T. reesei TR3012 cellulase cocktail were added. The reactions were incubated for 24 hours.
• Example 5 Polysaccharide Degradation Activity of Polypeptides of Sequence SEQ ID NO. 1, and SEQ ID NO. 2 in a sequential degradation test of lignocellulose, in the presence of LPMO type AA9
• SEQ ID NO. 1 in combination with LPMOs type AA9, was tested by sequential reactions.
• Cellulosic carriers of the "poplar" type were first incubated with (i) a control medium, (ii) 2.2 ⁇ (equivalent to 35 ⁇ g) of polypeptide SEQ ID NO. 1, (iii) 2.2. ⁇ of LPMO type AA9 and (iv) 1.1 ⁇ of polypeptide SEQ ID NO. 1 and 1.1. ⁇ of LPMO type AA9, for 48 hours; following which 10 ⁇ g of T. reesei TR3012 cellulase cocktail were added. The reactions were incubated for 24 hours.
• the analysis of the soluble sugars released was done according to several methodologies (DNS assay, RTU assay and HPAEC), which showed an improvement in the release of glucose and cello-oligosaccharides.

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