METHOD FOR PRODUCING NANOCELLULOSES FROM A CELLULOSE SUBSTRATE

TECHNICAL FIELD TO WHICH THE INVENTION RELATES

The present invention relates, generally, to the field of nanocelluloses, and more particularly the processes for producing these nanocelluloses from a cellulose-based substrate.

TECHNICAL BACKGROUND

Cellulose is one of the most important natural polymers, a virtually inexhaustible raw material, and an important source of materials that are sustainable on the industrial scale.

To date, various forms of cellulose have been identified with a size of about one nanometer, denoted under the generic name of “nanocelluloses”.

The properties of these nanocelluloses, in particular their mechanical properties, their ability to form films and their viscosity, gives them a major advantage in numerous industrial fields.

Nanocelluloses are thus used, for example, as a dispersant or stabilizing additive in the papermaking, pharmaceutical, cosmetics or food-processing industries. They are also part of the composition of paints and varnishes.

Nanocelluloses are also used in many devices that require a control of nanometric porosity, owing to their high specific surface area.

Finally, many nanocomposite materials based on nanocelluloses are currently being developed. This is because the notable mechanical properties of nanocelluloses, their dispersion on the nanometric scale and also their hydrophilic nature, give them excellent gas-barrier properties. These characteristics create in particular a considerable advantage for the production of barrier packagings.

On the basis of their sizes, functions and preparation methods, which themselves depend mainly on the cellulose-based source and on the treatment conditions, nanocelluloses can be classified mainly in two families: cellulose fibrils and cellulose nanocrystals.

Cellulose nanocrystals (also known as NCCs for “nanocrystalline celluloses”) are generally obtained by hydrolysis with a strong acid under strictly controlled temperature, time and stirring conditions. Such a treatment makes it possible to attack the amorphous regions of the fibers while at the same time leaving the more resistant crystalline regions intact. The suspension obtained is then washed by centrifugations and successive dialyses in distilled water. The NCCs most conventionally obtained have a length of a few tens of nanometers to approximately 1 μm (in particular 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 15 nm (typically from 5 to 10 nm).

Cellulose fibrils, commonly denoted cellulose microfibrils (also referred to as MFC for “microfibrilated cellulose”) or cellulose nanofibrils (NFC for “nanofibrilated cellulose”) are typically isolated from cellulose-based materials derived from biomass, by mechanical processes which make it possible to delaminate the cellulose fibers and to release the cellulose fibrils.

For example, document U.S. Pat. No. 4,483,743 describes a process for producing microfibrilated cellulose, which involves passing a liquid suspension of cellulose through a high-pressure Gaulin homogenizer. Repeated passes of the cellulose suspension make it possible to obtain microfibrils which typically have a width ranging from 25 to 100 nm and a much longer length.

In general, the mechanical processes for obtaining cellulose fibrils have the drawback of consuming large amounts of energy. By way of example, it has been evaluated that the use of a homogenizer causes an energy consumption of about 70 000 kWh/t. This high energy consumption, and consequently the high costs of producing nanocelluloses, therefore remain a considerable impediment to their industrial development.

Various cellulose fiber pretreatment strategies have thus been developed in order to reduce the energy consumption required for their mechanical delamination.

A first pretreatment strategy, described for example in application WO 2007/091942, consists in pretreating the cellulose fibers with cellulases so as to destroy the fiber before the application of the mechanical treatment by homogenization.

However, this enzymatic pretreatment is extremely changeable depending on the condition of the fiber and in particular depending on the prior thermochemical history of the fiber.

Furthermore, the quality of the nanocelluloses obtained (in particular the dispersion state and especially the lateral size of the nanofibrils which conditions the wear properties and the energy yields) are very variable.

A second pretreatment strategy is based on a chemical step of oxidation of the cellulose fibers (for example, Saito et al., Biomacromolecules, Vol. 8, No. 8, 2007, pp. 2485-2491).

Typically, the fibers are oxidized with an oxidizing agent such as sodium hypochlorite catalyzed by the 2,2,6,6-tetramethylpiperidine-1-oxyl (“TEMPO”) radical, before undergoing the abovementioned mechanical treatment.

The oxidative treatment converts the primary alcohol function in the 0₆position of the glucose unit of the cellulose into a carboxylate function, which results in the introduction of charges at the surface of the cellulose fibers. These charges create electrostatic repulsions which facilitate the delamination and which increase its efficiency.

However, the removal of the reaction products results in large amounts of highly polluted effluents. In addition, reactant residues persist in the final product and continue to react, altering, in the end, the properties of the nanocelluloses.

Thus, despite the new pretreatment strategies developed, the costs of nanocellulose production remain high, the yields uncertain, and the quality and the properties are variable.

It thus remains necessary to provide new processes for obtaining nanocelluloses, with a lower energy consumption and which are simple and reproducible, according to a route that is not toxic or has low toxicity.

SUBJECT OF THE INVENTION

In order to overcome the abovementioned drawbacks of the prior art, the present invention provides a process for producing nanocelluloses which is based on a step of pretreating cellulose fibers with at least one enzyme belonging to the family of lytic polysaccharide monooxygenases, commonly denoted “LPMOs”.

More particularly, according to the invention, a process is provided for producing nanocelluloses from a cellulose-based substrate comprising cellulose fibers,

said process comprising the following successive steps:

- at least one step of enzymatic treatment of said cellulose-based substrate, by bringing it into contact with at least one cleavage enzyme, then
- at least one step of mechanical treatment of said cellulose-based substrate subjected to said at least one step of enzymatic treatment, in order to delaminate the cellulose fibers and to obtain said nanocelluloses,

characterized in that said at least one cleavage enzyme is chosen from the enzymes belonging to LPMO family.

Typically, LPMOs are capable of performing an oxidative cleavage of cellulose fibers, advantageously of the glucose rings of cellulose fibers, in the presence of an electron donor.

Without being limited by any theory, the action of LPMOs facilitates the production of nanocellulose through two actions:

- the cleavage of the cellulose-based chains causes fragilities within the fibers, facilitating the mechanical delamination,
- the formation of oxidation products makes it possible to introduce charged chemical functions on the surface of the fibers, inducing electrostatic repulsions.

Again without being limited by any theory, the consequence of these combined structural modifications is to promote the separation of the fibers until nanometric dispersion is obtained and to form nanocelluloses (fibrils or nanocrystals) which have new functionalities (degree of charges, chemical functions not currently available).

Other nonlimiting and advantageous characteristics of the production process in accordance with the invention, taken individually or according to all the technically possible combinations, are also described hereinafter and also in the detailed description of the invention.

The electron donor can be chosen from ascorbate, gallate, catechol, reduced glutathione, lignin fragments and fungal carbohydrate dehydrogenases (in particular glucose dehydrogenases and cellobiose dehydrogenases).

Preferably, the LPMOs are chosen from enzymes capable of carrying out a cleavage of the cellulose by oxidation of at least one of the carbon atoms in position(s) C₁, C₄and C₆of the glucose ring. More preferably, the LPMOs are chosen from enzymes capable of carrying out a cleavage of the cellulose by oxidation of at least one of the carbon atoms in position(s) C₁and/or C₄, optionally in combination with C₆, of the glucose ring.

The LPMOs can be chosen from the families of fungal enzymes AA9 (formerly known as GH61) and of bacterial enzymes AA10 (formerly known as CBM33) of the CAZy classification (www.cazy.org). In particular, the LPMOs can be chosen from the LPMOs derived from Podospora anserina and preferably from PaLPMO9A (Genbank CAP68375), PaLPMO9B (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 cellulose-based substrate is obtained from wood, from a cellulose-rich fibrous plant, from beetroot, from citrus fruits, from annual straw plants, from marine animals, from algae, from fungi or from bacteria.

Preferably, the cellulose-based substrate is chosen from chemical papermaking pulps, preferably chemical wood papermaking pulps, more preferably at least one of the following papermaking pulps:

- bleached pulps,
- semi-bleached pulps,
- raw pulps,
- bisulfite pulps,
- sulfate pulps,
- sodium hydroxide pulps,
- kraft pulps.

Said at least one step of mechanical treatment generally comprises at least one of the following mechanical treatments:

- a homogenization treatment,
- a microfluidization treatment,
- an abrasion treatment,
- a cryomilling treatment.

The process can also comprise a post-treatment step, for example an acid treatment, an enzymatic treatment, an oxidation, an acetylation, a silylation, or else a derivatization of certain chemical groups borne by the nanocelluloses.

The invention also relates to the nanocelluloses obtained by carrying out the process of the invention.

Typically, the nanocelluloses obtained consist of cellulose nanofibrils and/or of cellulose nanocrystals.

Preferably, the nanocelluloses comprise glucose rings of which at least one carbon atom is oxidized in position(s) C₁and/or C₄, or even also in position C₆.

FIGURES

FIG. 1: Appearance of kraft fibers of cellulose that have been treated with the PaLPMO9H enzyme according to various enzyme/substrate ratios and subjected to a weak mechanical treatment with a homogenizer-disperser of the Ultra-Turrax type and an ultrasonic treatment. Optical microscopy images of the fibers not treated (control) with the enzyme (A) or treated with enzyme/substrate ratios of 1:50 (B); 1:100 (0); and 1:500 (D).

FIG. 2: Appearance of kraft fibers of cellulose that have been treated with the PaLPMO9H enzyme at an enzyme/substrate ratio of 1:50 and subjected to a weak mechanical treatment with a homogenizer-disperser of the Ultra-Turrax type and an ultrasonic treatment. Optical microscopy images of the control fibers (A) and of the treated fibers (B), and visualization of the nanofibrils obtained, by transmission electron microscopy (C) and atomic force microscopy (D).

FIG. 3: Appearance of kraft fibers of cellulose that have been treated with the PaLPMO9H and PaLPMO9E enzymes according to an enzyme/substrate ratio of 1:50 and then subjected to a weak mechanical treatment with a homogenizer-disperser of the Ultra-Turrax type and an ultrasonic treatment. Optical microscopy images of the kraft fibers treated with PaLPMO9H (A) and with PaLPMO9E (B). Visualization by atomic force microscopy of the nanofibrils obtained from the kraft fibers by treatment with the LPMO enzymes PaLPMO9H (C) and PaLPMO9E (D).

FIG. 4: Appearance of kraft fibers of cellulose that have been subjected to two successive treatments with the PaLPMO9H enzyme at various enzyme/substrate ratios and then subjected to a weak mechanical treatment with a homogenizer/disperser of the Ultra-Turrax type and an ultrasonic treatment. Optical microscopy images of the fibers treated according to the enzyme/substrate ratios of 1:50 (A); 1:100 (B); 1:500 (C) and 1:1000 (D).

FIG. 5: Analysis of the AFM photos for the PaLPMO9H enzyme, so as to characterize the height profile (FIG. 5A) and the size distribution (FIG. 5B) of the nanocelluloses. Legend to FIG. 5A: example of a height profile obtained on the surface, width (x, μm) versus height (y, nm); legend to FIG. 5B: histogram of height distribution with height (x, nm) versus number (y).

DETAILED DESCRIPTION

The description which follows, in combination with the Experimental Results given by way of nonlimiting examples, will provide a clear understanding of what the invention consists of and how it can be carried out.

General Definitions

The present invention relates to a process for producing nanocelluloses, in particular cellulose fibrils and/or cellulose nanocrystals, from a cellulose-based substrate.

The term “cellulose” is intended to mean a linear homopolysaccharide derived from biomass (encompassing organic matter of plant origin, algae included, cellulose of animal origin and also cellulose of bacterial origin) and consisting of units (or rings) of glucose (D-anhydroglucopyranose—AGU for “anhydro glucose unit”) which are linked to one another by β-(1-4) glycosidic bonds. The repeat unit is a glucose dimer also known as cellobiose dimer.

AGUs have 3 hydroxyl functions: 2 secondary alcohols (on the carbons in positions 2 and 3 of the glucose ring) and a primary alcohol (on the carbon in position 6 of the glucose ring).

These polymers link together via intermolecular bonds of hydrogen bond type, thus conferring a fibrous structure on the cellulose. In particular, the linking of cellobiose dimers forms an elementary cellulose nanofibril (the diameter of which is approximately 5 nm). The linking of elementary nanofibrils forms a nanofibril (the diameter of which generally ranges from 50 to 500 nm). The arranging of several of these nanofibrils then forms what is generally referred to as a cellulose fiber.

The term “nanocelluloses” denotes the various forms of cellulose having a size of about one nanometer. This encompasses in particular, according to the invention, two nanocellulose families: cellulose nanocrystals and cellulose fibrils.

The terms “cellulose fibrils”, “(cellulose) nanofibrils”, “(cellulose) nanofibers”, “nanofibrilated cellulose”, “(cellulose) microfibrils”, “microfibrilated cellulose”, and “cellulose nanofibrils” are synonymous. In the remainder of the present application, the term “cellulose nanofibrils” (NFCs) will be used generically.

Each cellulose nanofibril contains crystalline parts stabilized by a solid network of inter-chain and intra-chain hydrogen bonds. These crystalline regions are separated by amorphous regions.

Elimination of the amorphous parts of cellulose nanofibrils makes it possible to obtain cellulose nanocrystals (NCCs).

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 μm, preferably ranging from 40 nm to 500 nm.

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, nanofibrils, or ribbons, of bacterial cellulose generally have a length of several micrometers and a width ranging from 30 to 60 nm, in particular from 45 to 55 nm.

Process According to the Invention

The process for producing nanocelluloses, according to the invention, comprises the following successive steps:

- at least one step of enzymatic treatment of a cellulose-based substrate comprising cellulose fibers, by bringing it into contact with at least one cleavage enzyme belonging to the family of lytic polysaccharide monooxygenases (LPMOs), then
- at least one step of mechanical treatment of said cellulose-based substrate subjected to said at least one step of enzymatic treatment, in order to delaminate said cellulose fibers and to obtain said nanocelluloses.

One or more, and in particular at least two, steps of enzymatic treatment can be carried out according to the process of the invention, prior to said at least one step of mechanical treatment. For example, at least two steps of enzymatic treatment can be carried out successively, prior to said at least one step of mechanical treatment.

At least one step of enzymatic treatment can also be carried out after said at least one step of mechanical treatment.

When several steps of enzymatic treatment are carried out, the treatment conditions (time, LPMO(s) chosen, enzyme/cellulose ratio, etc.) can be identical to or different than one another.

Typically, said at least one step of enzymatic treatment, optionally followed by at least one step of mechanical treatment, can be repeated, as described above, until complete delamination of the cellulose fibers is obtained.

For example, the process of the invention can comprise at least two successive treatment cycles, each treatment cycle comprising at least one step of enzymatic treatment of the cellulose-based 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) of a mechanical delamination treatment makes it possible to obtain nanocelluloses of which the structural characteristics and the mechanical properties are entirely different than the nanocelluloses that exist in the prior art.

Again without being limited by any theory, the process of the invention makes it possible to obtain nanocelluloses simply and reproducibly. Preferably, the size and the mechanical properties of these nanocelluloses are uniform.

Cellulose-Based Substrate

The cellulose-based substrate can be obtained according to the invention from any matter of the biomass (encompassing organic matter of plant origin, algae included, animal origin or fungal origin) comprising cellulose-based fibers (that is to say cellulose fibers).

The cellulose-based substrate is advantageously obtained from wood (of which cellulose is the main component), but also from any cellulose-rich fibrous plant, for instance cotton, flax, hemp, bamboo, kapok, coconut fibers (coir), ramie, jute, sisal, raffia, papyrus and certain reeds, sugarcane bagasse, beetroot (and in particular beetroot pulp), citrus fruits, corn stalks or sorghum stalks, or else annual straw plants.

The cellulose-based substrates can also be obtained from marine animals (such as tunicates for example), algae (for instance Valonia or Cladophora) or bacteria for bacterial cellulose (for instance bacterial strains of Gluconacetobacter types).

Depending on the applications, cellulose from primary walls, for instance the parenchyma of fruits (for example beetroots, citrus fruits, etc.), or from secondary walls, for instance wood, will be chosen.

The cellulose-based substrate advantageously consists of a cellulose- based material prepared by chemical or mechanical means from any cellulose- based source as mentioned above (and in particular from wood).

The cellulose-based substrate is advantageously in the form of a suspension of cellulose fibers in a liquid medium (preferably an aqueous medium), or of a cellulose pulp.

The cellulose pulps can be conditioned in the “dry” state, that is to say typically in a state of dryness greater than or equal to 80%, in particular greater than or equal to 90%. The cellulose pulp can subsequently be redispersed in an aqueous medium by mechanical treatment.

Preferably, the cellulose-based substrate contains at least 90%, in particular at least 95% and preferably 100% of cellulose fibers.

Preferably, the cellulose-based substrate is suitable for the production of paper or of a cellulose-based product. The cellulose-based substrate is thus preferably chosen from papermaking pulps (or paper pulp), and in particular chemical papermaking pulps.

Generally, the cellulose pulp and in particular the papermaking pulp can contain, in combination with the cellulose fibers, hemicellulose and lignin. Preferably, the cellulose pulp contains less than 10% and in particular less than 5% of lignin and/or of hemicellulose.

Preferably, the chemical papermaking pulps contain virtually exclusively, or even exclusively, cellulose fibers.

The papermaking pulp can be chosen from at least one of the following papermaking pulps: bleached pulps, semi-bleached pulps, raw pulps, (raw or bleached) bisulfite pulps, (raw or bleached) sulfate pulps, (raw or bleached) sodium hydroxide pulps and kraft pulps.

It is also possible to use pulps to be dissolved, that have a low proportion of hemicellulose, preferably less than 10% and in particular less than or equal to 5%.

Preferably, the papermaking pulps used in a process of the invention are wood pulps, in particular chemical papermaking pulps of wood.

Lytic Polysaccharide Monooxydenases—LPMOs

The cellulose-based substrate is thus subjected to at least one step of pretreatment with at least one cleavage enzyme belonging to the lytic polysaccharide monooxygenase (LPMO) family.

LPMOs are mononuclear type II copper enzymes. They have common structural characteristics, in particular:

- a planar surface with an active site located close to its center and
- a highly conserved binding site for a type II copper ion exposed at the surface of the protein.

The interaction between the LPMO enzyme and the surface of the cellulose occurs by means of the planar 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 cellulose-based substrate, by oxidation of at least one of the carbon atoms in positions C₁, C₄and C₆of a glucose ring of said cellulose fibers.

The principle of the oxidative cleavage carried out by the LPMOs involves the activation of a C-H group followed by a dioxygen (O₂)-dependent cleavage, thus producing oligomers that are oxidized on at least one of the carbons in positions C₁, C₄and C₆.

The LPMO(s) used are capable of catalyzing a cleavage of the cellulose fibers by oxidation of at least one of the carbons chosen from the carbons in positions C₁and/or C₄and/or C₆of a glucose ring of the cellulose. The oxidative cleavage results in the formation of carboxyl groups at the surface of the cellulose fibers:

- the oxidative cleavage in position C₁of a glucose ring of a cellobiose unit leads to the formation of a lactone, which is spontaneously hydrolyzed to aldonic acid, and
- the oxidative cleavage in position C₄of a glucose ring of a cellobiose unit results in the formation of a ketoaldose.

The oxidation of the alcohol group in position C₆of a glucose ring of a cellobiose unit results in the formation of a carbonyl group.

In some embodiments, the LPMO(s) used catalyze(s) a cleavage of the cellulose fibers by oxidation of at least one of the carbons chosen from the carbons in position(s) C₁and/or C₄of a glucose ring of the cellulose, optionally in combination with the carbon in position C₆.

LPMOs catalyze the oxidative cleavage of a cellobiose unit in the presence of an external electron donor.

This electron donor, generally a molecule of low molecular weight, is chosen from ascorbate, reduced glutathione, gallate, catechol, lignin fragments, or else an enzyme of the carbohydrate dehydrogenase family.

Preferably, the carbohydrate dehydrogenases are chosen from fungal enzymes, in particular cellobiose dehydrogenases (CDHs).

CDHs (or cellobiose oxidoreductases—EC 1.1.99.18) catalyze the [cellobiose+electron acceptor<=>cellobiono-1,5-lactone+reduced acceptor] reaction. They are fungal hemoflavoenzymes belonging to the glucose-methanol-choline (GMC) oxidoreductase superfamily. 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 can for example be chosen from dioxygen, quinones and phenoxy radicals or LPMOs.

The activity of a CDH enzyme can be determined according to the reduction of the reagent 2,6-dichlorophenol indophenol (DCPIP) in a sodium acetate buffer containing cellobiose (Bey et al., 2011, Microb. Cell Fact. 10:113).

Examples of CDHs that can be used in combination with at least one LPMO enzyme and which also act as an electron donor can be chosen from the CDHs originating from Pycnoporus cinnabarinus, Humicola insolens, Podospora anserina, or Myceliophthora thermophila.

More preferably, an LPMO enzyme for which a cellulolytic activity (that is to say an activity that catalyzes the oxidative cleavage of the cellulose) has been identified is used. The oxidative cleavage activity of LPMOs on a cellulose-based substrate can be tested in cleavage tests as described in the Example section of the present application.

More specifically, the LPMOs used in the invention are advantageously chosen from enzymes said to have “auxiliary activity” (AA) according to the classification established in the CAZy database, relating to enzymes that are active on carbohydrates (CAZy—Carbohydrate Active enZyme database—http://www.cazy.org/—see also Levasseur et al., Biotechnology for Biofuels 2013, 6: 41).

More preferably, the step of enzymatic treatment is carried out with at least one enzyme chosen from the LPMO enzymes of the families referred to as AA9, AA10, AA11 and AA13, according to the classification established in the CAZy database.

The LPMO enzyme according to the invention can contain a carbohydrate binding protein module specific for cellulose of CBM1 type according to the CAZy classification.

The enzymes listed in the present application are identified by the Genbank reference (identifying a genetic sequence) and the Uniprot reference when the latter is available (identifying a protein sequence—see table 1). By default, the reference indicated between parentheses for each enzyme corresponds to the “Genbank” reference.

Preferably, at least one enzyme of the AA9 family and/or at least one enzyme of the AA10 family of the CAZy classification is (advantageously exclusively) used.

The enzymes of the AA9 family, listed in table 1 hereinafter, are fungal enzymes widely distributed in the genome of most ascomycetes and in some basidiomycetes (fungi).

Generally, the enzymes of the AA9 family were initially classified in the family of glycoside hydrolases 61 (GH61) of the CAZy classification. Specific analyses have since shown that the endoglucanase activity of the AA9 enzymes is weak, or even nonexistent (Morgenstern I et al., Briefings in Functional Genomics vol. 3(6P): 471-481).

Preferably, LPMOs of which the endoglucanase activity is not significant or is inexistent are used.

The copper ion of the LPMOs of the AA9 family is bound to the protein according to a hexacoordination model involving at least 2 conserved histidine residues and water molecules.

The enzymes of the AA9 family catalyze an oxidative cleavage of the cellobiose unit on the carbon in position C₁and/or C₄, preferably on the carbon in position C₁or C₄. Some enzymes (T. aurantiacus TaGH61A (G3XAP7) and Podospora anserina PaGH61 B (B2AVF1)) could catalyze an oxidative cleavage of the cellobiose on a carbon in position C₆.

The LPMOs of the AA9 family that is expressed in fungi generally exhibits a post-translational modification consisting of a methylation of the N-terminal histidine residue.

Preferably, LPMOs of the AA9 family comprising at least one CBM1 or CBM18 domain (CBM for “carbohydrate binding module”) in the N-terminal position are used. These enzymes then comprise a planar surface made up of several polar aromatic residues forming a domain of CBM1 or CBM18 type.

More preferably, said at least one LPMO of the AA9 family is derived from Podospora anserina and/or from Neurospora crassa.

The enzymes of the AA9 family derived from Podospora anserina are typically chosen from the group consisting of PaLPMO9A (CAP68375), PaLPMO9B (CAP73254), PaLPMO9D (CAP66744), PaLPMO9E (CAP67740), PaLPMO9F (CAP71839), PaLPMO9G (CAP73072) and PaLPMO9H (CAP61476).

Preferably, the PaLPMO9E (CAP67740) and/or PaLPMO9H (CAP61476) enzymes are used.

The enzymes of the AA9 family derived from Neurospora crassa are typically chosen from the group consisting of NcLPM09C (EAA36362), NcLPMO9D (EAA32426 /CAD21296), NcLPMO9E (EAA26873), NcLPMO9F (EAA26656/CAD70347), NcLPMO9M (EAA33178), NcU00836 (EAA34466), NcU02240 (EAA30263) and 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.

The LPMO family AA10 comprises, at the current time, more than about a thousand enzymes, identified particularly in bacteria, but also in some eukaryotes and also in some viruses.

The LPMOs of the AA10 family have a structure similar to that of the enzymes of the AA9 family and in particular at least one conserved tyrosine residue in the N-terminal position, which is involved in the binding with the copper ion. However, in most LPMOs of the AA10 family, one of the other tyrosine residues involved in the axial binding of the copper ion is replaced with a phenylalanine residue. For these enzymes, an oxidative activity has been demonstrated on chitin and on cellulose.

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 and CBM12 domains and also fibronectin type III modules.

The AA11 family is characterized by enzymes which carry out an oxidative cleavage in position C₁on chitin. The enzyme of Aspergillus oryzae will preferably be chosen (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 which carry out an oxidative cleavage in position C₁on starch. The enzyme of Aspergillus nidulans 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).

Generally, the step of enzymatic treatment is carried out by means of at least one LPMO enzyme listed in table 2 hereinafter.

In certain embodiments of the process of the invention, said at least one enzyme of the LPMO family (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 one endoglucanase) and/or at least one carbohydrate dehydrogenase (advantageously one cellobiose dehydrogenase (CDH)). The carbohydrate dehydrogenases can act as an electron donor for the LPMOs.

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 Pichia pastoris yeast.

Said at least one LPMO enzyme is mixed with the cellulose-based substrate, so as to allow said at least one enzyme to be brought into contact with the cellulose fibers.

The step of enzymatic treatment is preferably carried out with gentle stirring, so as to ensure good dispersion of the enzymes within the fibers. This step of enzymatic treatment is for example carried out for a period ranging from 24 h to 72 h (preferably for 48 h).

Preferably, the step of enzymatic treatment is carried out at a temperature ranging from 30 to 45° C.

According to the invention, said at least one LPMO enzyme can be added to the cellulose-based substrate according to an enzyme/cellulose ratio ranging from 1:1000 to 1:50, in particular from 1:500 to 1:50 or from 1:100 to 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, in particular from 0.1 to 5 g/l, and more preferably from 0.5 to 5 g/l.

According to one particular embodiment, the cellulose-based substrate is subjected to at least two (or even only to two) successive steps of enzymatic treatment (in series, advantageously separated by a rinsing step).

The LPMO(s) used during each of these steps of enzymatic treatment is (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 entirely destructured, including at low enzyme/cellulose ratios.

Step(s) of Mechanical Treatment(s)

The pretreated cellulose-based substrate is then subjected to at least one step of mechanical treatment which is intended to delaminate the cellulose fibers in order to obtain nanocelluloses.

The delamination (also referred to as “fibrillation” or “defibrillation”) consists in separating the cellulose fibers into nanocelluloses, via a mechanical phenomenon.

As demonstrated through the examples below, the oxidative cleavage of the cellulose fibers, catalyzed by said at least one LPMO, facilitates the delamination of these cellulose fibers during the step of mechanical treatment.

This step of mechanical delamination of the cellulose fibers can then be carried out under conditions that are less drastic and therefore less costly in terms of energy. Moreover, the use of LPMOs according to the invention makes it possible to introduce into the cellulose fibers charged groups which create electrostatic repulsions, without contamination with treatment reagents, such as when TEMPO reagents are used.

The mechanical treatments intended to delaminate cellulose fibers are known to those skilled in the art and can be implemented in the process of the invention.

Generally, mention may be made of weak mechanical treatments with a homogenizer-disperser (for example of the Ultra-Turrax type) and/or ultrasonic treatments.

Reference may also for example be made to the document of Lavoine N et al. (Carbohydrate Polymers, 2012, (92): 735-64) which describes in particular (pages 740 to 744) mechanical treatments for preparing microfibrilated cellulose (for example cellulose nanofibrils).

Typically, a mechanical treatment can be chosen from mechanical homogenization, microfluidization, abrasion or cryomilling treatments.

The homogenization treatment involves passing the pretreated cellulose- based substrate, typically a cellulose pulp or a liquid suspension of cellulose, through a narrow space under high pressure (as described for example in patent U.S. Pat. No. 4,486,743).

This homogenization treatment is preferably carried out by means of a homogenizer of Gaulin type. In such a device, the pretreated cellulose-based substrate, typically in the form of a cellulose suspension, is pumped at high pressure and distributed through an automatic valve with a small orifice. A rapid succession of openings and closings of the valve subjects the fibers to a considerable drop in pressure (generally of at least 20 MPa) and to a high-speed shear action followed by a high-speed deceleration impact. The passing of the substrate through the orifice is repeated (generally from 8 to 10 times) until the cellulose suspension becomes stable. In order to maintain a product temperature in a range of from 70 to 80° C. during the homogenization treatment, cooling water is generally used.

This homogenization treatment can also be carried out by means of a device of the microfluidizer type (see for example Sisqueira et al. Polymer 2010 2(4): 728-65). In such a device, the cellulose suspension passes through a typically “z”-shaped thin chamber (the dimensions of the channel of which are generally between 200 and 400 μm) under high pressure (approximately 2070 bar). The high shear rate which is applied (generally greater than 10⁷.s⁻¹) makes it possible to obtain very fine nanofibrils. A variable number of passes (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 can be used, in order to increase the degree of fibrillation.

The abrasion or milling treatment (see for example Iwamoto S et al., 2007 Applied Physics A89(2): 461-66) is based on the use of a milling device capable of exerting shear forces provided by milling stones.

The pretreated cellulose-based substrate, generally in the form of a cellulose pulp, is passed between a static milling stone and a rotating milling stone, typically at a speed of about 1500 revolutions per minute (rpm). Several passes (generally between 2 and 5) may be required in order to obtain fibrils of nanometric size.

A device of mixer type (for example as described in Unetani K et al., Biomacromolecules 2011, 12(2), pp. 348-53) can also be used to produce microfibrils from pretreated cellulose-based substrate, for example from a suspension of wood fibers.

The cryomilling (or cryocrushing) treatment (Dufresne et al., 1997, Journal of Applied Polymer Science, 64(6): 1185-94) consists in milling a suspension of pretreated cellulose-based substrate frozen beforehand with liquid nitrogen. The ice crystals formed inside the cells cause the cell membranes to explode and release wall fragments. These processes are generally used for the production of cellulose microfibrils from agricultural products or residues.

Step(s) of Post-Treatment of the Cellulose-Based Substrate

In certain embodiments, the production process comprises at least one step of post-treatment of the cellulose-based substrate, carried out after said substrate has been subjected to the mechanical treatment.

Generally, said at least one post-treatment step aims to increase the degree of fibrillation of the nanocelluloses obtained and/or to confer new mechanical properties on said nanocelluloses, as a function of the applications envisioned.

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 else a derivatization of certain chemical groups borne 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 that can be combined with various pretreatments and mechanical treatments of the cellulose fibers.

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.

Contrary to the NFCs obtained after oxidation by chemical reagents of TEMPO type, the nanocelluloses obtained by means of the process of the invention are devoid of oxidation reagent residues (namely, for example, sodium bromide, sodium hypochlorite, sodium chlorite, the (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical (TEMPO), derivatives or analogs).

Preferably, at the end of the process according to the invention, the nanocelluloses comprise at least one glucose ring (typically several glucose rings) of which at least one of the carbon atoms in positions C₁and/or C₄, or even also C₆, is oxidized by an oxidative cleavage phenomenon.

The nanocelluloses according to the invention thus comprise glucose rings which are:

- monooxidized on the carbon atom in position C₁, and/or
- monooxidized on the carbon atom in position C₄, and/or
- doubly oxidized on the atoms in positions C₁and C₄.

These glucose rings, oxidized on the atoms in positions C₁and/or C₄, can also comprise an oxidized carbon atom in position C₆.

The term “oxidized carbon atom” is intended to mean in particular a carbon atom which comprises a carbonyl function, and advantageously also a carboxyl function.

The nanocelluloses according to the invention are thus advantageously negatively charged, because of the presence of various surface functions including carboxylate functions on the carbons in positions C₁and/or C₄(contrary to the TEMPO process which results in a specific oxidation of the carbon in position C₆).

Experimental Results

1. Tests for cleavage of the cellulose by an LPMO enzyme can be carried out according to the following protocol:

The cleavage test is carried out at a volume of 300 μl of liquid containing 4.4 μM of LPMO enzyme and 1 mM of ascorbate and 0.1% (weight/volume) of powder of phosphoric acid-swollen cellulose (PASO—prepared as described in Wood T M, Methods Enzym 1988, 160: 19-25) in 50 mM of a sodium acetate buffer at pH 4.8 or 5 μM of cellooligosaccharides (Megazyme, Wicklow, Ireland) in 10 mM of sodium acetate buffer at 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 (revolutions per minute).

After incubation for 16 h, the sample is brought to 100° C. for 10 minutes in order to stop the enzymatic reaction, then centrifuged at 16 000 revolutions per minute (rpm) for 15 minutes at 4° C. in order to separate the solution fraction from remainder insoluble fraction.

The cleaved products obtained can be analyzed by ion exchange chromatography and/or by mass spectrometry (MALDI-TOF).

2. Preliminary tests were carried out in order to demonstrate the efficiency of the process for producing nanocellulose according to the invention.

These tests were carried out on a papermaking fiber (cellulose kraft fibers) by means of LPMO enzyme of the AA9 family derived from Podospora anserina (PaLPMO9E (Genbank CAP67740) and/or PaLPMO9H (Genbank CAP61476)) and produced in a heterologous system in yeast (Pichia pastoris).

The fibers are brought into contact with the enzymes (at a concentration of 1 g/l and according to enzyme/cellulose ratios of 1:50, 1:100, 1:500 and 1:1000) and with ascorbate (2 mM) and then subjected to gentle stirring for 48 hours at 40° C.

The treated fibers are then subjected to a mechanical action with a homogenizer-disperser (Ultra-Turrax power 500 W, maximum speed for 3 minutes), followed by an ultrasonic treatment for 3 minutes.

Compared with the substrates not treated with the enzyme, it is observed that the defibrillation is facilitated for all of the enzyme/cellulose ratios used (FIG. 1 B-D—the photos show, qualitatively, the defibrillation).

In the absence of enzymes, the fibers remain intact and no defibrillation is noted (see the non-treated control fibers, FIG. 1A).

The dispersions were then analyzed by TEM (Transmission Electron Microscopy) and AFM (Atomic Force Microscopy).

In the absence of LPMO enzymes (FIG. 2A), it is noted that very few structures are visible on the nanometric scale.

On the other hand, for the fibers treated with the LPMO enzyme, structures of nanometric sizes are easily pinpointed, both in the supernatant and in the pellets of the experiment. The fibers are entirely destructured, allowing crystalline zones of the fiber to appear (FIG. 2C-D).

The treatment of the fibers with the PaLPMO9E enzyme combined with the subsequent mechanical treatment (FIGS. 3B and D) produces a defibrillation of the cellulose that is similar to that obtained with an identical process involving the PaLPMO9H enzyme (see FIG. 3A and C).

Generally, FIGS. 2C, 2D, 3C and 3D unquestionably demonstrate that nanocelluloses are obtained.

If the fibers that have 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 entirely destructured, including at the low enzyme/cellulose ratios (that is to say the 1/500 and 1/1000 ratios) (FIG. 4).

The AFM photos for the PaLPMO9H enzyme were analyzed using the WSxM software in order to characterize the height profile (FIG. 5A) and the size distribution (FIG. 5B) of the nanocelluloses.

This analysis shows that, at the 1:50 enzyme/cellulose ratio, the nanocelluloses have a diameter of less than 100 nm.

This result confirms that the products obtained by means of the process according to the invention are nanocelluloses.

TABLE 1

Listed fungal enzymes of the AA9 family (CAZy classification)

Name
Organism
GenBank ref.
Uniprot ref.

Cel1

Agaricus bisporus

AAA53434.1
Q00023

D649

AfA5C5.025

Aspergillus

CAF31975.1
Q6MYM8

fumigatus

endoglucanase/CMCase

Aspergillus

AFJ54163.1

(Eng61)

fumigatus MKU1

endo-β-1,4-glucanase B (EgIB;

Aspergillus

BAB62318.1
Q96WQ9

AkCel61A) (Cel61A)

kawachii

NBRC4308

AN1041.2

Aspergillus

EAA65609.1
C8VTW9

nidulans FGSC A4

Q5BEI9

AN3511.2

Aspergillus

EAA59072.1
Q5B7G9

nidulans FGSC A4

AN9524.2

Aspergillus

EAA66740.1
C8VI93

nidulans FGSC A4
CBF83171.1
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-β-1,4-glucanase

Aspergillus

EAA64722.1
Q5BCX8

(AN1602.2)

nidulans FGSC A4
ABF50850.1

AN2388.2

Aspergillus

EAA64499.1
C8VNP4

nidulans FGSC A4

Q5BAP2

An04g08550

Aspergillus niger

CAK38942.1
A2QJX0

CBS 513.88

An08g05230

Aspergillus niger

CAK45495.1
A2QR94

CBS 513.88

An12g02540

Aspergillus niger

CAK41095.1
A2QYU6

CBS 513.88

An12g04610

Aspergillus niger

CAK97151.1
A2QZE1

CBS 513.88

An14g02670

Aspergillus niger

CAK46515.1
A2R313

CBS 513.88

An15g04570

Aspergillus niger

CAK97324.1
A2R5J9

CBS 513.88

An15g04900

Aspergillus niger

CAK42466.1
A2R5N0

CBS 513.88

AO090005000531

Aspergillus oryzae

BAE55582.1
Q2US83

RIB40

AO090001000221

Aspergillus oryzae

BAE56764.1
Q2UNV1

RIB40

AO090023000056

Aspergillus oryzae

BAE58643.1
Q2UIH2

RIB40

AO090023000159

Aspergillus oryzae

BAE58735.1
Q2UI80

RIB40

AO090023000787

Aspergillus oryzae

BAE59290.1
Q2UGM5

RIB40

AO090012000090

Aspergillus oryzae

BAE60320.1
Q2UDP5

RIB40

AO090138000004

Aspergillus oryzae

BAE64395.1
Q2U220

RIB40

AO090103000087

Aspergillus oryzae

BAE65561.1
Q2TYW2

RIB40

Cel6 (E6)

Bipolaris maydis

AAM76663.1
Q8J0H7

C4

glycoside hydrolase family 61

Botryotinia

CCD34368.1

protein (Bofut4_p103280.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD47228.1

protein (Bofut4_p003870.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD48549.1

protein (Bofut4_p109330.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD50139.1

protein (Bofut4_p025380.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD50144.1

protein (Bofut4_p025430.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD51504.1

protein (Bofut4_p018100.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD49290.1

protein (Bofut4_p031660.1)

fuckeliana T4

glycoside hydrolase family 61

Botryotinia

CCD52645.1

protein (Bofut4_p000920.1)

fuckeliana T4

BofuT4P143000045001

Botryotinia

CCD50451.2

fuckeliana T4
CCD50451.1

ORF

Chaetomium

AGY80102.1

thermophilum CT2

ORF (fragment)

Chaetomium

AGY80103.1

thermophilum CT2

ORF (fragment)

Chaetomium

AGY80104.1

thermophilum CT2

ORF (fragment)

Chaetomium

AGY80105.1

thermophilum CT2

cellobiohydrolase family protein

Chaetomium

AGY80103.1

61, partial (Cbh61-2) (fragment)

thermophilum CT2

cellobiohydrolase family protein

Chaetomium

AGY80104.1

61, partial (Cbh61-3) (fragment)

thermophilum CT2

cellobiohydrolase family protein

Chaetomium

AGY80105.1

61, partial (Cbh61-4) (fragment)

thermophilum CT2

ORF (possible fragment)

Colletotrichum

CAQ16278.1
B5WYD8

graminicola M2

ORF

Colletotrichum

CAQ16206.1
B5WY66

graminicola M2

ORF

Colletotrichum

CAQ16208.1
B5WY68

graminicola M2

ORF

Colletotrichum

CAQ16217.1
B5WY77

graminicola M2

unnamed protein product

Coprinopsis

CAG27578.1

cinerea

CGB_A6300C

Cryptococcus

ADV19810.1

bacillisporus

WM276

CNAG_00601

Cryptococcus

AFR92731.1

neoformans var.
AFR92731.2

grubii H99

(Cryne_H99_1)

Cel1

Cryptococcus

AAC39449.1
O59899

neoformans var.

neoformans

CNA05840 (Cel1)

Cryptococcus

AAW41121.1
F5HH24

neoformans var.

neoformans JEC21

(Cryne_JEC21_1)

ORF (fragment)

Flammulina

ADX07320.1

velutipes KACC

42777

FFUJ_12340

Fusarium fujikuroi

CCT72465.1

IMI 58289 (Fusfu1)

FFUJ_13305

Fusarium fujikuroi

CCT67119.1

IMI 58289 (Fusfu1)

FFUJ_07829

Fusarium fujikuroi

CCT69268.1

IMI 58289 (Fusfu1)

FFUJ_12621

Fusarium fujikuroi

CCT72729.1

IMI 58289 (Fusfu1)

FFUJ_12840

Fusarium fujikuroi

CCT72942.1

IMI 58289 (Fusfu1)

FFUJ_09373

Fusarium fujikuroi

CCT73805.1

IMI 58289 (Fusfu1)

FFUJ_10599

Fusarium fujikuroi

CCT74544.1

IMI 58289 (Fusfu1)

FFUJ_10643

Fusarium fujikuroi

CCT74587.1

IMI 58289 (Fusfu1)

FFUJ_14514

Fusarium fujikuroi

CCT67584.1

IMI 58289 (Fusfu1)

FFUJ_11399

Fusarium fujikuroi

CCT75380.1

IMI 58289 (Fusfu1)

FFUJ_14514

Fusarium fujikuroi

CCT67584.1

IMI 58289

FFUJ_11399

Fusarium fujikuroi

CCT75380.1

IMI 58289

FFUJ_04652

Fusarium fujikuroi

CCT64153.1

IMI 58289 (Fusfu1)

FFUJ_03777

Fusarium fujikuroi

CCT64954.1

IMI 58289 (Fusfu1)

FFUJ_04940

Fusarium fujikuroi

CCT63889.1

IMI 58289 (Fusfu1)

Sequence 122805 from patent

Fusarium

ABT35335.1

U.S. Pat. No. 7,214,786

graminearum

FG03695.1 (Cel61E)

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

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

unnamed protein product

Fusarium

CEF84640.1

graminearum PH-1

endo-β-1,4-glucanase (Cel61G)

Gloeophyllum

AEJ35168.1

trabeum

GH61D

Heterobasidion

AFO72234.1

parviporum

GH61B

Heterobasidion

AFO72233.1

parviporum

GH61A

Heterobasidion

AFO72232.1

parviporum

GH61F

Heterobasidion

AFO72235.1

parviporum

GH61G

Heterobasidion

AFO72236.1

parviporum

GH61H

Heterobasidion

AFO72237.1

parviporum

GH61I

Heterobasidion

AFO72238.1

parviporum

GH61J

Heterobasidion

AFO72239.1

parviporum

unnamed protein product

Humicola insolens

CAG27577.1

endoglucanase IV (EgiV)

Hypocrea orientalis

AFD50197.1

EU7-22

GH61A (GH61A)

Lasiodiplodia

CAJ81215.1

theobromae CBS

247.96

GH61B (GH61B)

Lasiodiplodia

CAJ81216.1

theobromae CBS

247.96

GH61C (GH61C)

Lasiodiplodia

CAJ81217.1

theobromae CBS

247.96

GH61D (GH61D)

Lasiodiplodia

CAJ81218.1

theobromae CBS

247.96

ORF

Leptosphaeria

CBX91313.1
E4ZJM8

maculans v23.1.3

ORF

Leptosphaeria

CBX93546.1
E4ZQ11

maculans v23.1.3

ORF

Leptosphaeria

CBX94224.1
E4ZS44

maculans v23.1.3

ORF

Leptosphaeria

CBX94532.1
E4ZSU4

maculans v23.1.3

ORF

Leptosphaeria

CBX94572.1
E4ZSY4

maculans v23.1.3

ORF

Leptosphaeria

CBX95655.1
E4ZVM9

maculans v23.1.3

ORF

Leptosphaeria

CBX96476.1
E4ZZ41

maculans v23.1.3

ORF

Leptosphaeria

CBX96550.1
E4ZYM4

maculans v23.1.3

ORF

Leptosphaeria

CBX96949.1
E5A089

maculans v23.1.3

ORF

Leptosphaeria

CBX97718.1
E5A201

maculans v23.1.3

ORF

Leptosphaeria

CBX98126.1
E5A3B3

maculans v23.1.3

ORF

Leptosphaeria

CBY01974.1
E5AFI5

maculans v23.1.3

ORF

Leptosphaeria

CBY02242.1
E5ACP0

maculans v23.1.3

ORF

Leptosphaeria

CBX91667.1
E4ZK72

maculans v23.1.3

ORF

Leptosphaeria

CBX93965.1
E4ZQA3

maculans v23.1.3

ORF

Leptosphaeria

CBX98254.1
E5A3P1

maculans v23.1.3

ORF (fragment)

Leptosphaeria

CBY00196.1
E5A955

maculans v23.1.3

ORF

Leptosphaeria

CBY01204.1
E5AC13

maculans v23.1.3

predicted protein

Leptosphaeria

CBY01256.1
E5ADG7

(Lema_p000430.1) (fragment)

maculans v23.1.3

ORF (fragment)

Leptosphaeria

CBY01257.1
E5ADG8

maculans v23.1.3

lytic polysaccharide

Leucoagaricus

CDJ79823.1

monooxygenase

gongylophorus

Ae322

MG05364.4

Magnaporthe

EAA54572.1

grisea 70-15
XP_359989.1

(Maggr1)

MG07686.4

Magnaporthe

EAA53409.1
G4N3E5

grisea 70-15
XP_367775.1

(Maggr1)

MG07300.4

Magnaporthe

EAA56945.1
G4MUY8

grisea 70-15
XP_367375.1

(Maggr1)

MG08020.4

Magnaporthe

EAA57051.1

grisea 70-15
XP_362437.1

(Maggr1)

MG08254.4

Magnaporthe

EAA57285.1
G4MXC7

grisea 70-15
XP_362794.1

(Maggr1)

MG08066.4 (fragment)

Magnaporthe

EAA57097.1
G4MXS5

grisea 70-15
XP_362483.1

(Maggr1)

MG04547.4

Magnaporthe

EAA50788.1
G4MS66

grisea 70-15
XP_362102.1

(Maggr1)

MG08409.4

Magnaporthe

EAA57439.1
G4MVX4

grisea 70-15
XP_362640.1

(Maggr1)

MG09709.4

Magnaporthe

EAA49718.1
G4NAI5

grisea 70-15
XP_364864.1

(Maggr1)

MG06069.4

Magnaporthe

EAA52941.1
G4N560

grisea 70-15
XP_369395.1

(Maggr1)

MG09439.4

Magnaporthe

EAA51422.1
G4NHT8

grisea 70-15
XP_364487.1

(Maggr1)

MG06229.4

Magnaporthe

EAA56258.1

grisea 70-15
XP_369714.1

(Maggr1)

MG07631.4

Magnaporthe

EAA53354.1
G4N2Z0

grisea 70-15
XP_367720.1

(Maggr1)

MGG_06621

Magnaporthe

XP_003716906.1

grisea 70-15
XP_370106.1

(Maggr1)

MGG_12696

Magnaporthe

XP_003721313.1

grisea 70-15

(Maggr1)

MGG_02502

Magnaporthe

XP_003709306.1

grisea 70-15
EAA54517.1

(Maggr1)
XP_365800.1

MGG_04057

Magnaporthe

XP_003719782.1

grisea 70-15
EAA50298.1

(Maggr1)
XP_361583.1

MGG_13241

Magnaporthe

XP_003711808.1

grisea 70-15

(Maggr1)

MGG_13622

Magnaporthe

XP_003717521.1

grisea 70-15

(Maggr1)

MGG_07575

Magnaporthe

XP_003711490.1

grisea 70-15
EAA53298.1

(Maggr1)
XP_367664.1

MGG_11948

Magnaporthe

XP_003709110.1

grisea 70-15

(Maggr1)

MGG_16080 (fragment)

Magnaporthe

XP_003709033.1

grisea 70-15

(Maggr1)

MGG_16043 (fragment)

Magnaporthe

XP_003708922.1

grisea 70-15

(Maggr1)

MGG_12733 (probable fragment)

Magnaporthe

XP_003716689.1

grisea 70-15

(Maggr1)

copper-dependent

Malbranchea

CCP37674.1

polysaccharide monooxygenases

cinnamomea CBS

(Gh61) (fragment)
115.68

copper-dependent

Melanocarpus

CCP37668.1

polysaccharide monooxygenases

albomyces CBS

(Gh61) (fragment)
638.94

copper-dependent

Myceliophthora

CCP37667.1

polysaccharide monooxygenases

fergusii CBS

(Gh61) (fragment)
406.69

MYCTH_2112799

Myceliophthora

AEO61257.1

thermophila ATCC

42464

MYCTH_79765

Myceliophthora

AEO56016.1

thermophila ATCC

42464

MYCTH_110651

Myceliophthora

AEO54509.1

thermophila ATCC

42464

MYCTH_2298502

Myceliophthora

AEO55082.1

thermophila ATCC

42464

MYCTH_2299721

Myceliophthora

AEO55652.1

thermophila ATCC

42464

MYCTH_2054500

Myceliophthora

AEO55776.1

thermophila ATCC

42464

MYCTH_111088

Myceliophthora

AEO56416.1

thermophila ATCC

42464

β-glycan-cleaving enzyme

Myceliophthora

AEO56542.1

(StCel61a; MYCTH_46583)

thermophila ATCC

(Cel61A)
42464

MYCTH_2301632

Myceliophthora

AEO56547.1

thermophila ATCC

42464

MYCTH_100518

Myceliophthora

AEO56642.1

thermophila ATCC

42464

lytic polysaccharide

Myceliophthora

AEO56665.1

monooxygenases (active on

thermophila ATCC

cellulose) (MYCTH_92668)
42464

MYCTH_2060403

Myceliophthora

AEO58412.1

thermophila ATCC

42464

MYCTH_2306673

Myceliophthora

AEO58921.1

thermophila ATCC

42464

MYCTH_116175 (fragment)

Myceliophthora

AEO59482.1

thermophila ATCC

42464

MYCTH_96032

Myceliophthora

AEO59823.1

thermophila ATCC

42464

MYCTH_103537

Myceliophthora

AEO59836.1

thermophila ATCC

42464

MYCTH_55803

Myceliophthora

AEO59955.1

thermophila ATCC

42464

lytic polysaccharide

Myceliophthora

AEO60271.1

monooxygenases (active on

thermophila ATCC

cellulose) (MYCTH_112089)
42464

MYCTH_85556

Myceliophthora

AEO61304.1

thermophila ATCC

42464

MYCTH_2311323

Myceliophthora

AEO61305.1

thermophila ATCC

42464

MYCTH_47093 (fragment)

Myceliophthora

AEO56498.1

thermophila ATCC

42464

MYCTH_80312

Myceliophthora

AEO58169.1

thermophila ATCC

42464

lytic polysaccharide

Neurospora crassa

CAD21296.1
Q1K8B6

monooxygenase (active on
OR74A
EAA32426.1
Q8WZQ2

cellulose) (PMO-

XP_326543.1

2; NcLPMO9D; GH61-

4; NCU01050) (LPMO9D)

lytic polysaccharide

Neurospora crassa

CAD70347.1
Q1K4Q1

monooxygenase (active on
OR74A
EAA26656.1
Q873G1

cellulose) (PMO-

XP_322586.1

03328; NcLPMO9F; GH61-

6; NCU03328) (LPMO9F)

lytic polysaccharide

Neurospora crassa

CAE81966.1
Q7SHD9

monooxygenase (PMO-01867;
OR74A
EAA36262.1

NcLPMO9J; GH61-10;

XP_329057.1

NCU01867;

B13N4.070) (LPMO9J)

NCU02344.1 (B23N11.050)

Neurospora crassa

CAF05857.1
Q7S411

OR74A
EAA30230.1

XP_331120.1

lytic polysaccharide

Neurospora crassa

EAA33178.1
Q7SA19

monooxygenase (active on
OR74A
XP_328604.1

cellulose) (PMO-

3; NcLPMO9M; GH61-13; NcPMO-

3; NCU07898) (LPMO9M)

NCU05969.1

Neurospora crassa

EAA29347.1
Q7S1V2

OR74A
XP_325824.1

lytic polysaccharide

Neurospora crassa

EAA36362.1
Q7SHI8

monooxygenase (active on
OR74A
XP_330104.1

cellulose and

cellooligosaccharides) (PMO-

02916; NcLPMO9C; GH61-

3; NCU02916) (LPMO9C)

lytic polysaccharide

Neurospora crassa

EAA29018.1
Q7S111

monooxygenase (active on
OR74A
XP_328466.1

cellulose) (GH61-2; NCU07760)

NCU07520.1

Neurospora crassa

EAA29132.1
Q7S1A0

OR74A
XP_327806.1

lytic polysaccharide

Neurospora crassa

EAA30263.1
Q7S439

monooxygenase (active on
OR74A
XP_331016.1

cellulose) (GH61-1; NCU02240)

lytic polysaccharide

Neurospora crassa

EAA34466.1
Q7SCJ5

monooxygenase (active on
OR74A
XP_325016.1

cellulose) (NCU00836)

lytic polysaccharide

Neurospora crassa

EAA26873.1
Q7RWN7

monooxygenase (active on
OR74A
XP_330877.1

cellulose) (PMO-

08760; NcLPMO9E; GH61-

5; NCU08760) (LPMO9E)

NCU07974.1

Neurospora crassa

EAA33408.1
Q7SAR4

OR74A
XP_328680.1

NCU03000.1 (B24P7.180)

Neurospora crassa

EAA36150.1
Q7RV41

OR74A
CAB97283.2
Q9P3R7

XP_330187.1

cellulose monooxygenase

Penicillium

AIO06742.1

oxalicum GZ-2

Pc12g13610

Penicillium

CAP80988.1
B6H016

chrysogenum

Wisconsin 54-1255

(PenchWisc1_1)

Pc13g07400

Penicillium

CAP91809.1
B6H3U0

chrysogenum

Wisconsin 54-1255

(PenchWisc1_1)

Pc13g13110

Penicillium

CAP92380.1
B6H3A3

chrysogenum

Wisconsin 54-1255

(PenchWisc1_1)

Pc20g11100

Penicillium

CAP86439.1
B6HG02

chrysogenum

Wisconsin 54-1255

(PenchWisc1_1)

Cel61 (Cel61A)

Phanerochaete

AAM22493.1
Q8NJI9

chrysosporium

BKM-F-1767

Lytic polysaccharide mono-

Phanerochaete

BAL43430.1

oxygenase active on cellulose

chrysosporium K-3

(Gh61D; PcGH61D)

PIIN_01487

Piriformospora

CCA67659.1

indica (Pirin1)

PIIN_02110

Piriformospora

CCA68244.1

indica (Pirin1)

PIIN_03975

Piriformospora

CCA70035.1

indica (Pirin1)

PIIN_04357

Piriformospora

CCA70418.1

indica (Pirin1)

PIIN_04637

Piriformospora

CCA70703.1

indica (Pirin1)

PIIN_06117

Piriformospora

CCA72182.1

indica (Pirin1)

PIIN_06118

Piriformospora

CCA72183.1

indica (Pirin1)

PIIN_06127

Piriformospora

CCA72192.1

indica (Pirin1)

PIIN_06155

Piriformospora

CCA72220.1

indica (Pirin1)

PIIN_07098

Piriformospora

CCA73144.1

indica (Pirin1)

PIIN_07105

Piriformospora

CCA73151.1

indica (Pirin1)

PIIN_08199

Piriformospora

CCA74246.1

indica (Pirin1)

PIIN_08783

Piriformospora

CCA74814.1

indica (Pirin1)

PIIN_09022

Piriformospora

CCA75037.1

indica (Pirin1)

PIIN_00566 (fragment)

Piriformospora

CCA66803.1

indica (Pirin1)

PIIN_01484

Piriformospora

CCA67656.1

indica (Pirin1)

PIIN_01485 (fragment)

Piriformospora

CCA67657.1

indica (Pirin1)

PIIN_01486 (fragment)

Piriformospora

CCA67658.1

indica (Pirin1)

PIIN_04356

Piriformospora

CCA70417.1

indica (Pirin1)

PIIN_05699 (fragment)

Piriformospora

CCA71764.1

indica (Pirin1)

PIIN_06156

Piriformospora

CCA72221.1

indica (Pirin1)

PIIN_08402

Piriformospora

CCA74449.1

indica (Pirin1)

PIIN_10315 (fragment)

Piriformospora

CCA76320.1

indica (Pirin1)

PIIN_10660 (fragment)

Piriformospora

CCA76671.1

indica (Pirin1)

PIIN_00523 (fragment)

Piriformospora

CCA77877.1

indica (Pirin1)

Putative Glycoside Hydrolase

Podospora

CDP30131.1
B2AL94

Family 61

anserina S mat+
CAP64732.1

(Podan2)

Putative Glycoside Hydrolase

Podospora

CDP30928.1
B2B346

Family 61

anserina S mat+
CAP71532.1

(Podan2)

Pa_1_500

Podospora

CAP59702.1
B2A9F5

anserina S mat+
CDP22345.1

(Podan2)

Pa_4_350

Podospora

CAP61395.1
B2AD80

anserina S mat+
CDP27750.1

(Podan2)

Pa_4_1020

Podospora

CAP61476.1
B2ADG1

anserina S mat+
CDP27830.1

(Podan2)

Pa_0_270

Podospora

CAP61650.1
B2ADY5

anserina S mat+
CDP28001.1

(Podan2)

Pa_5_8940

Podospora

CAP64619.1
B2AKU6

anserina S mat+
CDP30017.1

(Podan2)

Pa_5_4100 (fragment)

Podospora

CAP64865.1
B2ALM7

anserina S mat+
CDP29378.1

(Podan2)

Pa_5_6950

Podospora

CAP65111.1
B2AMI8

anserina S mat+
CDP29800.1

(Podan2)

Pa_5_10660

Podospora

CAP65855.1
B2APD8

anserina S mat+
CDP30283.1

(Podan2)

Pa_5_10760

Podospora

CAP65866.1
B2APE9

anserina S mat+
CDP30272.1

(Podan2)

Pa_5_11630 (fragment)

Podospora

CAP65971.1
B2API9

anserina S mat+
CDP30166.1

(Podan2)

Pa_4_7570

Podospora

CAP66744.1
B2ARG6

anserina S mat+
CDP28479.1

(Podan2)

Pa_1_21900 (fragment)

Podospora

CAP67176.1
B2AS05

anserina S mat+
CDP24589.1

(Podan2)

Pa_1_22040

Podospora

CAP67190.1
B2AS19

anserina S mat+
CDP24603.1

(Podan2)

Pa_1_22150 (fragment)

Podospora

CAP67201.1
B2AS30

anserina S mat+
CDP24614.1

(Podan2)

Pa_6_11220

Podospora

CAP67466.1
B2ASU3

anserina S mat+
CDP30332.1

(Podan2)

Pa_6_11370

Podospora

CAP67481.1
B2ASV8

anserina S mat+
CDP30347.1

(Podan2)

Pa_6_11470

Podospora

CAP67493.1
B2ASX0

anserina S mat+
CDP30359.1

(Podan2)

Pa_1_16300

Podospora

CAP67740.1
B2ATL7

anserina S mat+
CDP23998.1

(Podan2)

Pa_7_5030

Podospora

CAP68173.1
B2AUV0

anserina S mat+
CDP31642.1

(Podan2)

Pa_7_3770

Podospora

CAP68309.1
B2AV86

anserina S mat+
CDP31780.1

(Podan2)

Pa_7_3390

Podospora

CAP68352.1
B2AVC8

anserina S mat+
CDP31823.1

(Podan2)

lytic polysaccharide mono-

Podospora

CAP68375.1
B2AVF1

oxygenase active on cellulose

anserina S mat+
CDP31846.1

(Gh61B; Pa_7_3160)(Gh61B)
(Podan2)

Pa_6_7780

Podospora

CAP71839.1
B2B403

anserina S mat+
CDP31230.1

(Podan2)

Pa_2_1700

Podospora

CAP72740.1
B2B4L5

anserina S mat+
CDP25137.1

(Podan2)

Pa_2_4860

Podospora

CAP73072.1
B2B5J7

anserina S mat+
CDP25472.1

(Podan2)

lytic polysaccharide mono-

Podospora

CAP73254.1
B2B629

oxygenase active on cellulose

anserina S mat+
CDP25655.1

(Gh61A; Pa_2_6530)(Gh61A)
(Podan2)

Pa_2_7040

Podospora

CAP73311.1
B2B686

anserina S mat+
CDP25714.1

(Podan2)

Pa_2_7120

Podospora

CAP73320.1
B2B695

anserina S mat+
CDP25723.1

(Podan2)

Pa_3_190

Podospora

CAP61048.1
B2AC83

anserina S mat+
CDP26500.1

(Podan2)

Pa_3_2580

Podospora

CAP70156.1
B2AZV6

anserina S mat+
CDP26748.1

(Podan2)

Pa_3_3310

Podospora

CAP70248.1
B2AZD4

anserina S mat+
CDP26841.1

(Podan2)

endo-β-1,4-glucanase (Egl1;

Pyrenochaeta

AEV53599.1

PIEGL1)

lycopersici ISPaVe

ER 1211

copper-dependent

Rasamsonia

CCP37669.1

polysaccharide monooxygenases

byssochlamydoides

(Gh61) (fragment)
CBS 151.75

copper-dependent

Remersonia

CCP37675.1

polysaccharide monooxygenases

thermophila CBS

(Gh61) (fragment)
540.69

RHTO0S_28e01816g

Rhodosporidium

CDR49619.1

toruloides

CECT1137

copper-dependent

Scytalidium

CCP37676.1

polysaccharide monooxygenases

indonesiacum CBS

(Gh61) (fragment)
259.81

SMU2916 (fragment)

Sordaria

CAQ58424.1
C1KU36

macrospora k-hell

lytic polysaccharide mono-

Thermoascus

ABW56451.1

oxygenase active on cellulose

aurantiacus

ACS05720.1

copper-dependent

Thermoascus

CCP37673.1

polysaccharide monooxygenases

aurantiacus CBS

(Gh61) (fragment)
891.70

ORF

Thermoascus

AGO68294.1

aurantiacus var.

levisporus

copper-dependent

Thermomyces

CCP37672.1

polysaccharide monooxygenases

dupontii CBS

(Gh61) (fragment)
236.58

copper-dependent

Thermomyces

CCP37678.1

polysaccharide monooxygenases

lanuginosus CBS

(Gh61) (fragment)
632.91

unnamed protein product

Thielavia terrestris

CAG27576.1

THITE_2106556

Thielavia terrestris

AEO62422.1

NRRL 8126

THITE_2116536

Thielavia terrestris

AEO67662.1

NRRL 8126

THITE_2040127

Thielavia terrestris

AEO64605.1

NRRL 8126

THITE_2119040

Thielavia terrestris

AEO69044.1

NRRL 8126

THITE_115795

Thielavia terrestris

AEO64177.1

NRRL 8126

THITE_2110890

Thielavia terrestris

AEO64593.1

NRRL 8126

THITE_2112626

Thielavia terrestris

AEO65532.1

NRRL 8126

THITE_2076863

Thielavia terrestris

AEO65580.1

NRRL 8126

THITE_170174

Thielavia terrestris

AEO66274.1

NRRL 8126

THITE_2044372

Thielavia terrestris

AEO67396.1

NRRL 8126

THITE_2170662

Thielavia terrestris

AEO68023.1

NRRL 8126

THITE_128130

Thielavia terrestris

AEO68157.1

NRRL 8126

THITE_2145386

Thielavia terrestris

AEO68577.1

NRRL 8126

THITE_2054543

Thielavia terrestris

AEO68763.1

NRRL 8126

THITE_2059487

Thielavia terrestris

AEO71031.1

NRRL 8126

THITE_2142696

Thielavia terrestris

AEO67395.1

NRRL 8126

THITE_43665

Thielavia terrestris

AEO69043.1

NRRL 8126

THITE_2085430 (fragment)

Thielavia terrestris

AEO63926.1

NRRL 8126

THITE_2122979

Thielavia terrestris

XP_003657366.1

NRRL 8126

cellulase-enhancing

Thielavia terrestris

ACE10231.1

factor (GH61B)
NRRL 8126

Sequence 4 from patent U.S. Pat. No.

Thielavia terrestris

ACE10232.1

7,361,495 (GH61C)
NRRL 8126

Sequence 4 from patent U.S. Pat. No.

Thielavia terrestris

ACE10232.1

7,361,495 (GH61C)
NRRL 8126

Sequence 6 from patent U.S. Pat. No.

Thielavia terrestris

ACE10233.1

7,361,495 (GH61D)
NRRL 8126

Sequence 6 from patent U.S. Pat. No.

Thielavia terrestris

ACE10233.1

7,361,495 (GH61D)
NRRL 8126

lytic polysaccharide mono-

Thielavia terrestris

AEO71030.1

oxygenase active on cellulose
NRRL 8126
ACE10234.1

(131562; TtGH61E)(GH61E)

Sequence 10 from patent U.S. Pat. No.

Thielavia terrestris

ACE10235.1

7,361,495 (GH61G)
NRRL 8126

Sequence 10 from patent U.S. Pat. No.

Thielavia terrestris

ACE10235.1

7,361,495 (GH61G)
NRRL 8126

Lytic polysaccharide mono-

Trichoderma reesei

AAP57753.1
Q7Z9M7

oxygenase active on cellulose
QM6A
ABH82048.1

(EG7; HjGH61B)(Cel61B = GH61B)

ACK19226.1

ACR92640.1

endo-γ-1,4-glucanase IV

Trichoderma reesei

CAA71999.1
O14405

(EGIV; Egl4; EG4) (Cel61A)
RUTC-30

endoglucanase

Trichoderma

ADB89217.1
D3JTC4

(EnGluIV; EndoGluIV)

saturnisporum

endoglucanase IV (EgIV; EG IV)

Trichoderma sp.
ACH92573.1
B5TYI4

SSL

endoglucanase VII (EgvII)

Trichoderma viride

ACD36971.1
B4YEW1

AS 3.3711

endoglucanase IV (EgIV)

Trichoderma viride

ADJ57703.1
B4YEW3

AS 3.3711
ACD36973.1
D9IXC6

AAA12YM05FL
uncultured
CCA94933.1

eukaryote

AAA2YG01FL
uncultured
CCA94930.1

eukaryote

AAA15YI10FL
uncultured
CCA94931.1

eukaryote

AAA21YH11FL
uncultured
CCA94932.1

eukaryote

ABA3YP05FL
uncultured
CCA94934.1

eukaryote

endoglucanase II (EgII)

Volvariella

AFP23133.1

volvacea

endoglucanase II (EgII)

Volvariella

AAT64005.1
Q6E5B4

volvacea V14

Unknown

Zea mays B73
ACF86151.1

unknown (ZM_BFc0036G02)

Zea mays B73
ACF78974.1
B4FA31

ACR36748.1

TABLE 2

LPMOs (AA9, AA10 and AA11 families of the CAZy classification)

Uniprot
GenBank

Substrate
Known

Organism
ref.
ref.
Other names
specificity
selectivity
Modularity

fungi

A. oryzae

Q2UA85
BAE61530
AoAA11
chitin
C1
AA11-

X278

A. nidulans

C8VGF8
EAA62623.1
AnAA13
starch
C1
AA13-

CBM20

M. thermophila

G2QI82
AEO60271
MYCTH_112089
cellulose
C1
AA9

M. thermophila

G2QAB5
AEO56665
MYCTH_92668
cellulose
C1
AA9

N. crassa

Q7RWN7
EAA26873
NcLPMO9E
cellulose
C1
AA9-

CBM1

N. crassa

Q1K8B6
EAA32426
NcLPMO9D
cellulose
C4
AA9

Q8WZQ2
CAD21296

N. crassa

Q7SA19
EAA33178
NcLPMO9M
cellulose
C1, C4
AA9

N. crassa

Q7SHI8
EAA36362
NcLPMO9C
cellulose
C4
AA9-

hemi-

CBM1

cellulose

N. crassa

Q1K4Q1
EAA26656
NcLPMO9F
cellulose
C1
AA9

CAD70347

N. crassa

Q7SCJ5
EAA34466
NcU00836
cellulose
C1
AA9-

CBM1

N. crassa

Q7SCE9
EAA34371.2
NcAA13
starch
C1
AA13-

CBM20

N. crassa

Q7S439
EAA30263
NcU02240
cellulose
C4
AA9-

CBM1

N. crassa

Q7S111
EAA29018
NcU07760
cellulose
C1, C4
AA9-

CBM1

P. chrysosporium

H1AE14
BAL43430
PcLPMO9D
cellulose
C1
AA9

P. anserina

B2B629
CAP73254
PaGH61A
cellulose
C1^a, C4^a
AA9-

PaLMPOB

CBM1

P. anserina

B2AVF1
CAP68375
PaGH61B
cellulose
C1, C4
AA9-

PaLMPO9A

CBM1

P. anserina

B2ARG6
CAP66744
PaLPMO9D
cellulose
C1
AA9

CBM1

P. anserina

B2ATL7
CAP67740
PaLPMO9E
cellulose
C1
AA9

CBM1

P. anserina

B2B403
CAP71839
PaLPMO9F
cellulose
n.d
AA9

CBM1

P. anserina

B2B5J7
CAP73072
PaLPMO9G
cellulose
n.d
AA9

CBM1

P. anserina

B2ADG1
CAP64476
PaLPMO9H
cellulose
C1, C4
AA9

CBM1

T. aurantiacus

G3XAP7
ABW56451
TaGH61A
cellulose
C1
AA9

T. terrestris

G2RGE5
AEO71030

cellulose
n.d.
AA9

T. reesei

Q7Z9M7
AAP57753

cellulose
n.d.
AA9

T. reesei

O14405
CAA71999
Cel61A
cellulose
n.d.
AA9

Bacteria

Bacillus

E1UUV3
CBI42985

n.d.
n.d.
AA10

amyloliquefaciens

Burkholderia

Q3JY22
ABA49030
BURPS1710b_0114
n.d.
n.d.
AA10

pseudomallei

(BpAA10A)

1710b

Bacillus

Q62YN7
AAU22121

chitin
C1
AA10

licheniformes

Caldibacillus

Q9RFX5
AAF22274
β-1,4-
n.d.
n.d.
AA10

cellulovorans

mannanase

(ManA)

Enterococcus

Q838S1
AAO80225
EfLPMO10A
chitin
C1
AA10

faecalis

Hahella

Q2SNS3
ABC27701
LPMO
cellulose
nd
AA10

chejuensis

(HcAA10-

2; HCH_00807)

Serratia

O83009
AAU88202
SmLPMO10A
chitin
C1
AA10

marcescens

Streptomyces

Q9RJC1
CAB61160
ScLPMO10B
cellulose
C1, C4
AA10

coelicolor

chitin

Streptomyces

Q9RJY2
CAB61600
ScLPMO10C
cellulose
C1
AA10-

coelicolor

CBM2

Thermobifida

Q47QG3
AAZ55306
TfLPMO10A
cellulose
C1, C4
AA10

fusca

chitin

Thermobifida

Q47PB9
AAZ55700
TfLPMO10B
cellulose
C1
AA10-

fusca

CBM2

V. cholerae

Q9KLD5
AAF96709
VcLPMO10B
n.d.
n.d.
AA10

O1

The term “substrate specificity” is intended to mean the type of substrate cleaved (oxidative cleavage) by the corresponding LPMO enzyme.

The term “known selectivity” is intended to mean the carbon of the glucose ring oxidized by the corresponding LPMO enzyme.

The term “modularity” is intended to mean the CAZy class (AA9, 10 or 11) of the enzyme and the known presence of a conserved domain (CBM or X278).

METHOD FOR PRODUCING NANOCELLULOSES FROM A CELLULOSE SUBSTRATE

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