TECHNICAL FIELD

The present invention relates to the field of dyeing processes. More particularly, it relates to a process for dyeing a cellulosic material, comprising an enzymatic oxidation of a cellulosic material followed by a reductive amination in the presence of an aminated dye.

TECHNICAL BACKGROUND

Reactive dyes have a wide color range and are commonly used for dyeing cellulosic fibers such as cotton and flax. In a reactive dye process, dye molecules are diffused into the fibers and establish chemical bonds with the fibers through a reactive functional group, such as a chlorotriazine, a vinyl sulfone, or a bromoacrylate, attached to the chromophore of the dye. Due to the formation of this permanent bond with the fiber, the reactive dye could not be easily removed by repeated treatment with boiling water under neutral conditions. Thus, the dyes become parts of the fiber, and allows outstanding colour fastness to wash.

However, processes based on reactive dyes have several drawbacks. In particular, the preparation of reactive dyes is not straightforward, since particular syntheses have to be developed depending on the nature of the chromophore and the reactive functional group to be attached thereto. In addition, harsh dyeing conditions, such as highly basic pH’s up to 14, and high temperatures up to 80 °C, have to be used in order to create the bond between the dye and the cellulosic fiber. The use of such basic pH is besides not satisfactory, since it induces a high hydrolysis rate of the reactive dyes during the dyeing process, and consequently significant amounts of reactive dyes can be found in waste water.

In order to overcome the above drawbacks, alternative dyes and dyeing processes have been developed over the last few years. In particular, the use of laccases for converting phenolic and aromatic compounds into colored substances have been studied in dyeing processes. For instance, Hadzhiyska et al. (Biotechnology Letters 2006, 28, 755) have developed a dyeing process, in which cotton is contacted with catechol and 2,5-diaminobenzenesulfonic acid, in the presence of a laccase, at a temperature of 30 to 70 °C and at a pH of 5. However, the weak bonds between the cotton and the dye do not allow to obtain a high colour washfastness. Also, Diaz Blanco et al. (Enzyme and Microbial Technology 2009, 44, 380) have developed a dyeing process, comprising tosylating a cotton material, grafting of an aromatic amine onto the tosylated cotton and then copolymerization of the grafted cotton with a laccase-oxidized catechol. However, such process comprises numerous steps, which makes it difficult to implement, in particular at the industrial scale. In addition, the process uses stoichiometric amounts of tosylating agents and pyridine, which are corrosive, toxic, and fairly expensive.

Thus, there remains a need to provide a dyed cellulosic material obtained by a cost-effective process, which can be implemented under mild and environmentally-friendly conditions.

SUMMARY OF THE INVENTION

In this context, the inventors have developed a two-step process for dyeing a cellulosic material. This process comprises contacting a cellulosic material with an oxidoreductase and a redox mediator, and then contacting the resulting cellulosic material with a dye comprising at least one NH2 group, in the presence of a reducing agent. This two-step process is cost-effective and environmentally-friendly. In particular, the process uses inexpensive and non-toxic reagents, which are advantageously used in catalytic amounts. Also, the process can be implemented under mild conditions, in particular at low temperatures and from moderate acid to moderate basic pH’s. This process allows to create a strong covalent link between the amino dye and the cellulosic material, such that a high colour fastness is obtained.

Thus, the present invention relates to a process for dyeing a cellulosic material comprising the following steps:

(a) contacting a cellulosic material with an oxidoreductase and a redox mediator, so as to obtain an oxidized cellulosic material;

(b) contacting the oxidized cellulosic material of step (a) with a dye comprising at least one NH2 group, in the presence of a reducing agent, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material.

In a particular embodiment, said dye in step (b) is a compound of formula (I):

[H ₂ N-(X-Y)n]m-Z (I), in which:

- n is 0 or 1, - m is an integer from 1 to 4,

- X is an arylene or heteroarylene group, said group being optionally substituted by at least one substituent selected from the group consisting of (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2- Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, heteroaryl, halogen, -CN, - NO2, -C(O)Ri, -C(O)OR2,-C(O)NR ₃ R4, -S(O) ₂ R ₅ , -S(O) ₂ OR ₆ , -NHS(O) ₂ R7, -NHS(O) ₂ OR ₈ , - OR9, -SR10, and -NR11R12,

Ri, R2, R3, R4, Rs, Re, R7, R ₈ , R9, Rio, R11 and R12 being each independently chosen from hydrogen, (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2-Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2- Ci2)heterocycloalkyl, aryl, and heteroaryl,

- Y is a spacer chosen among the following groups: (Ci-Ci2)alkylene, (C2-Ci2)alkenylene, (C2- Ci2)alkynylene, (C3-Ci2)cycloalkylene, (C2-Ci2)heterocycloalkylene, arylene, and heteroarylene, said group being optionally interrupted by at least one group chosen from -O-, - S-, -NH-, -C(O)-, and -S(O) ₂ , and

- Z is a chromophore moiety, or a salt thereof.

In a more particular embodiment, said compound of formula (I) is such that n is 0.

In another more particular embodiment, said compound of formula (I) is such that m is 1 or 2, preferably m is 1.

In a particular embodiment, said compound of formula (I) is such that Z is a chromophore moiety selected from the group consisting of phenyls, rhodamines, anthraquinones, triarylmethanes, phthalocyanines, monoazo dyes, bisazo dyes, triazo dyes, polyazo dyes, biindolylidene diones, indanediones, quinolyl indanediones, acridines, thiazines, thiazoles, oxazines, phenoxazines, xanthenes, chlorines, diketopyrrolopyrroles, quinacridones, anthocyanidins, flavonoids, and derivatives thereof.

In another particular embodiment, said dye is represented by any one of the following formulae:

5 Preferably, said dye is represented by any one of the following formulae:

(I-s).

More preferably, said dye is represented by any one of the following formulae:

In another particular embodiment, said redox mediator is TEMPO.

In another particular embodiment, said oxidoreductase is a laccase. More particularly, the laccase may be a laccase from Bacillus velezensis, Bacillus stratosphericus, Anoxybacillus, Aspergillus, or Trametes versicolor, preferably Trametes versicolor. Preferably, the laccase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and variants thereof, wherein said variants exhibit laccase activity and have at least 60 % of amino acid sequence identity.

In another particular embodiment, step (a) is carried out at a temperature comprised between 10 °C and 60 °C, preferably between 20 °C and 50 °C, and more preferably between 25°C and 35 °C.

In another particular embodiment, step (a) is carried out at a pH comprised between 2.5 and 12, preferably between 4 and 10, more preferably between 4 and 8, even more preferably between 5 and 8.

In another particular embodiment, step (b) is carried out at a temperature comprised between 10 °C and 60 °C, preferably between 30 °C and 50 °C, more preferably between 35°C and 45 °C.

In another particular embodiment, step (b) is carried out in the presence of an acid, such as acetic acid.

In another particular embodiment, step (a) is carried out in water.

In another particular embodiment, step (b) is carried out in water.

In another particular embodiment, said reducing agent is a borane compound, said borane compound being preferably selected from the group consisting of sodium or potassium borohydride, zinc borohydride, sodium cyanoborohydride, borane (BH3), diborane (B2H6), pyridine borane, picoline borane, 5-ethyl-2-methylpyridine borane, morpholine borane, 4- methylmorpholine borane, triethylamine borane, 9-borabicyclo[3.3.1]nonane (9-BBN), monoisopinocampheylborane, dicyclohexylborane, dimesitylborane, disiamylborane, catecholborane, pinacolborane, L-selectride, and a mixture thereof, more preferably picoline borane.

In a particular embodiment, said cellulosic material is chosen from cotton, flax, hemp, jute, viscose, lyocell, rayon, and modal, preferably cotton.

In a preferred embodiment, the process of the present invention comprises the following successive steps: (a) contacting a cellulosic material with a laccase and TEMPO or a derivative thereof, so as to obtain an oxidized cellulosic material;

(b) contacting the oxidized cellulosic material of step (a) with a dye of formula (I) as defined herein, in the presence of a borane compound, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material.

Another object of the present invention is a dyed cellulosic material obtained by a process as defined herein.

FIGURES

Figure 1. FTIR (Fourier-transform infrared spectroscopy) spectra of a cotton oxidized according to step (a) of the process of the invention, and an untreated cotton.

Figure 2. LCUV (Liquid Chromatography-Ultra Violet) chromatograms of the hydrosylate of a cotton dyed according to the process of the invention and the hydrosylate of untreated cotton. Figure 3. LC-MS/UV analysis of the supernatant after enzymatic hydrolysis with cellulases of cotton thread dyed with p-toluidine (A: UV286nm chromatograms - B: m/z detected for each product, Pl, P2 and P3, and corresponding to H ⁺ adducts in positive mode).

Figure 4. LC-MS/UV analysis of the supernatant after enzymatic hydrolysis with cellulases of cotton threads dyed with RHO123 or AR33 (Fig. 4A, Fig. 4C: UV chromatograms obtained from cotton threads grafted with RHO123 or AR33, respectively; Fig. 4B, Fig. 4D: m/z detected for each product and corresponding to H ⁺ adducts in positive mode).

Figure 5. Images of cotton threads dyed according to the process of the invention with RHO123 (Al) or AR33 (Bl), and comparative samples of cotton threads dyes without oxidation (A2, B2) or without reducing agent (A3, B3).

Figure 6. Cotton fabrics dyed with AR33 according to the process of the invention (sample 1), without oxidation (sample 2) and without reducing agent (sample 3).

Figure 7. Images of a cotton fabric dyed with AR33 according to the process of the invention, before washing and after three washing cycles.

DESCRIPTION OF THE INVENTION

Definitions

According to the present invention, the terms below have the following meanings: The terms mentioned herein with prefixes such as for example Ci-Ce, can also be used with lower numbers of carbon atoms such as C1-C2. If, for example, the term C1-C12 is used, it means that the corresponding hydrocarbon chain may comprise from 1 to 12 carbon atoms, especially 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. If, for example, the term Ci-Ce is used, it means that the corresponding hydrocarbon chain may comprise from 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5, or 6 carbon atoms.

The term “alkyl” refers to a saturated, linear or branched aliphatic group. The term “(C1-C12) alkyl” includes for instance methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, or dodecyl.

The term “alkenyl” refers to an unsaturated, linear or branched aliphatic group, having at least one carbon-carbon double bond. The term “(C2-Ci2)alkenyl” includes for instance ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl.

The term “alkynyl” refers to an unsaturated, linear or branched aliphatic group, having at least one carbon-carbon triple bond. The term “(C2-Ci2)alkynyl” includes for instance ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl.

The term “cycloalkyl” corresponds to a saturated or unsaturated mono-, bi- or tri-cyclic alkyl group. It also includes fused, bridged, or spiro-connected cycloalkyl groups. The term “(C3- Ci2)cycloalkyl” includes for instance cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “heterocycloalkyl” corresponds to a saturated or unsaturated cycloalkyl group as above defined further comprising at least one heteroatom such as nitrogen, oxygen, or sulphur atom. It also includes fused, bridged, or spiro-connected heterocycloalkyl groups. The term “(C2-Ci2)heterocycloalkyl includes for instance dioxolanyl, benzo [1,3] dioxolyl, azetidinyl, oxetanyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4- dioxanyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4- dithianyl, pyrrolidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiophenyl.

"Cycloalkyl" and "heterocycloalkyl" also include cycloalkenyl and heterocycloalkenyl which correspond respectively to a cycloalkyl having at least one carbon-carbon double bond and a heterocycloalkyl having at least one carbon-carbon double bond such as cyclohexenyl, and dihydropyranyl. The term “aryl” corresponds to a mono- or bi-cyclic aromatic hydrocarbon having from 6 to 12 carbon atoms. For instance, the term “aryl” includes phenyl, naphtalenyl, or anthracenyl. In a preferred embodiment, the aryl is a phenyl or a naphtalenyl, more preferably a phenyl.

The term “heteroaryl” as used herein corresponds to an aromatic, mono- or poly-cyclic group comprising between 5 and 14 atoms and comprising at least one heteroatom such as nitrogen, oxygen or sulphur atom. As used herein, the term “heteroaryl” further includes the “fused arylheterocycloalkyl” and “fused heteroarylcycloalkyl”. The terms “fused arylheterocycloalkyl” and “fused heteroarylcycloalkyl” correspond to a bicyclic group in which an aryl as above defined or a heteroaryl is respectively bounded to the heterocycloalkyl or the cycloalkyl as above defined by at least two carbons. In other terms, the aryl or the heteroaryl shares a carbon bond with the heterocycloalkyl or the cycloalkyl. Examples of such mono- and poly-cyclic heteroaryl group, fused arylheterocycloalkyl and fused arylcycloalkyl may be: pyridinyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, furazanyl, thiadiazolyl, tetrazolyl, benzofuranyl, thianaphthal enyl, indolyl, indolinyl, indanyl, quinolinyl, isoquinolinyl, benzimidazolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, triazinyl, thianthrenyl, benzofuranyl, dihydrobenzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, chromenyl, xanthenyl, phenoxanthinyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, indazolyl, purinyl, quinolizinyl, phtalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, P-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, indolinyl, isoindolinyl, oxazolidinyl, benzotri azolyl, benzoisoxazolyl, oxindolyl, benzoxazolyl, benzoxazolinyl, benzoxazinyl, benzothienyl, benzothiazolyl, benzodiazepinyl, benzazepinyl, benzoxazepinyl, isatinyl, dihydrobenzodi oxepinyl, dihydropyridyl, pyrimidinyl, s-triazinyl, oxazolyl, or thiofuranyl. A fused arylheterocycloalkyl is for instance an indolinyl (phenyl fused to a pyrrolidinyl) and a dihydrobenzofuranyl (phenyl fused to a dihydrofuranyl).

The term “halogen” corresponds to a fluorine, chlorine, bromine, or iodine atom, preferably a fluorine or a chlorine.

The term “alkylene” refers to a divalent alkyl group, wherein “alkyl” is as defined herein. The term “(Ci-Ci2)alkylene” refers in particular to a group of formula -(CH2) _q - where q is an integer from 1 to 12. “(Ci-Ci2)alkylene” includes for instance methylene, ethylene, propylene, butylene, isobutylene, pentylene, isopentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, or dodecylene. The term “alkenylene” refers to a divalent alkenyl group, wherein “alkenyl” is as defined herein. “(C2-Ci2)alkenylene” includes for instance ethenylene (-CH=CH-), propenylene, butenylene, pentenylene, hexenylene, heptenylene, octenylene, nonenylene, decenylene, undecenylene, or dodecenylene.

The term “alkynylene” refers to a divalent alkynyl group, wherein “alkynyl” is as defined herein. “(C2-Ci2)alkynylene” includes for instance ethynylene (-C=C-), propynylene, butynylene, pentynylene, hexynylene, heptynylene, octynylene, nonynylene, decynylene, undecynylene, or dodecynylene.

The term “cycloalkylene” refers to a divalent cycloalkyl group, wherein “cyclolalkyl” is as defined herein. For instance, a (C3-Ci2)cycloalkylene includes cyclopropylene, cyclopentlyene, or cyclohexylene.

The term “hetercycloalkylene” refers to a divalent hetercycloalkyl group, wherein “hetercyclolalkyl” is as defined herein. For instance, a (C3-Ci2)cycloalkylene includes thiomorpholinylene, pyrazolidinylene, piperidylene, or piperazinylene.

The term “arylene” refers to a divalent aryl group, wherein “aryl” is as defined above. For instance, an arylene includes phenylene.

The term “heteroarylene” refers to a divalent heteroaryl group, wherein “heteroaryl” is as defined herein. For instance, a heteroarylene includes pyridinylene, thiophenylene, or furanyl ene.

The expression “substituted by at least one substituent” means “substituted by one or several substituents” of a given list. The expression “optionally substituted by” means “not substituted or substituted by”.

According to the invention, the term “comprise(s)” or “comprising” (and other comparable terms, e.g., “containing,” and “including”) is “open-ended” and can be generally interpreted such that all of the specifically mentioned features and any optional, additional and unspecified features are included. According to specific embodiments, it can also be interpreted as the phrase “consisting essentially of’ where the specified features and any optional, additional and unspecified features that do not materially affect the basic and novel character! stic(s) of the claimed invention are included or the phrase “consisting of’ where only the specified features are included, unless otherwise stated.

The “salts” include inorganic as well as organic acids salts. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, maleic, methanesulfonic and the like. The “salts” also include inorganic as well as organic base salts. Representative examples of suitable inorganic bases include sodium or potassium salt, an alkaline earth metal salt, such as a calcium or magnesium salt, or an ammonium salt.

As used herein, the term “sequence identity” or “identity” refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, for purposes herein, % amino acid sequence identity values refers to values generated using a local alignment algorithm, preferably the Basic Local Alignment Search Tool (BLAST) that finds regions of local similarity between sequences and calculates the statistical significance of matches, wherein all search parameters are set to default values, i.e. blastp algorithm, Expect threshold= 0.05, word size= 3, Scoring matrix = BLOSUM62, Gap costs: existence=l l, extension = 1, Conditional compositional score matrix adjustment.

The present invention provides a simple and environmentally-friendly two-step process for dyeing a cellulosic material. The process of the present invention comprises the following steps:

(a) contacting a cellulosic material with an oxidoreductase and a redox mediator, so as to obtain an oxidized cellulosic material;

(b) contacting the oxidized cellulosic material of step (a) with a dye comprising at least one NEL group, in the presence of a reducing agent, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material. As used herein, a “cellulosic material” refers to any material comprising or consisting of cellulose. The cellulosic material may have any size and any form, and may be from any source. For instance, the cellulose may be from a natural or synthetic source. In a particular embodiment, the cellulosic material comprises at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, more preferably at least 80%, 90%, 95%, 98%, 99%, or 100% by weight of cellulose.

In another particular embodiment, the cellulosic material comprises or consists of fibers comprising or consisting of cellulose. More particularly, the cellulosic material may be a textile, for instance a non-woven or woven fabric.

In a preferred embodiment, the cellulosic material is chosen from cotton, flax, hemp, jute, viscose, lyocell, rayon, and modal. More preferably, the cellulosic material is cotton.

In step (a) of the process of the invention, the cellulosic material is contacted with an oxidoreductase and a redox mediator, such that an oxidized cellulosic material is obtained.

As used herein, an “oxidoreductase” refers to an enzyme which is able to trigger or catalyze an oxidoreduction reaction. The oxidoreductase may in particular be able to oxidize the redox mediator. The oxidoreductase may be from any source, such as bacteria, fungi, lichen, insects, or plants. The oxidoreductase may in particular be any known oxidoreductase, and variants thereof. More particularly, the oxidoreductase may be selected from known bacteria, fungi, lichen, insect, or plant oxidoreductases, and variants thereof. Preferably, said variants exhibit oxidoreductase activity and have at least 60 %, preferably at least 70% or 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to any of said oxidoreductase.

Given that the process of the invention can be carried out under mild temperatures, the oxidoreductase may be thermostable or not.

In a particular embodiment, the oxidoreductase is selected from the group consisting of a laccase, a p-diphenol: oxygen oxidoreductase, a ferroxidase (such as EC 1.10.3.2), a laccaselike multicopper oxidase (such as EC 1.10.3.-), a cellobiose dehydrogenase (such as EC 1.1.99.18), a glucose 1-oxidase (such as EC 1.1.3.4), an aryl alcohol oxidase (such as EC 1.1.3.7) , an alcohol oxidase (such as EC 1.1.3.13), a pyranose oxidase (such as EC 1.1.3.10), an acceptor-oxygen oxidase (such as EC 1.1.3.-) , a galactose oxidase (such as EC 1.1.3.9), a glyoxal oxidase (such as EC 1.2.3.15), an alcohol oxidase (such as EC 1.1.3.13), a raffinose oxidase (such as EC 1.1.3.-) and a lytic polysaccharide monooxygenase (LPMO) (such as EC 1.14.99.-), and combinations thereof.

As used herein, the EC number refers to the Enzyme Commission number, which is a numerical classification scheme for enzymes based on chemical reactions such enzymes catalyze.

In a preferred embodiment, the oxidoreductase is a laccase. In such embodiment, the laccase may be from any source, such as bacteria, fungi, lichen, insects, or plants. Said laccase may be any known laccase, in particular may be selected from known bacteria, fungi, lichen, insect, or plant laccases, and variants thereof. Preferably, said variants exhibit laccase activity and have at least 60 %, preferably at least 70 % or 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to any of said laccase. The laccase activity can be determined by photometrical test using phenolic substrates and by monitoring the oxidized products. Examples of phenolic substrates for the laccase assay include, but are not limited to, guaiacol, 2.6-dimethoxyphenol, syringaldazine, 2,2-azino-bis-(3-ethylbenzothiazoline-6- sulphonic acid, benzenediol, and 3,4-dimethoxybenzyl alcohol. A method for the determination of laccase activity is in particular described in the following articles: Johannes & Maicherczyk, Journal of Biotechnology, 2000, 78, 193-199; Harkin & Obst, Ex-perientia 1973, 37, 381-387; Prillinger & Esser Molec. Gen. Genet. 1975, 156, 333-345.

In a particular embodiment, the laccase is a laccase from a bacterium or a fungus.

Examples of bacteria producing an oxidoreductase (such as a laccase) include, but are not limited to:

- Bacillus, such as Bacillus velezensis (for instance Bacillus velezensis TCCC 111904), Bacillus stratosphericus (for instance Bacillus stratosphericus BCMC2), Bacillus subtilis, Bacillus pumilus, Bacillus clausii, Bacillus halodurans, or Bacillus tequilensis (for instance Bacillus tequilensis SN4),

- Anoxybacillus, for instance Anoxybacillus sp. UARK-01,

- Pseudomonas, such as Pseudomonas extremorientalis,

- Streptomyces, such as Streptomyces viridochromogenes, Streptomyces ipomoea, or Streptomyces cyaneus, - Marinomonas, such as Marinomonas mediterranea,

- Escherichia, such as Escherichia coli,

- Klebsiella, such as Klebsiella pneumoniae, and

- Yersinia, such as Yersinia enter ocolitica.

An example of laccase from Bacillus pumilus is Bacillus pumilus CotA. An example of laccase from Bacillus subtilis is Bacillus subtilis CotA. An example of laccase from Bacillus clausii is Bacillus clausii CotA. An example of oxidoreductase from Escherichia coli is Escherichia coli CueO.

Examples of fungi producing an oxidoreductase (such as a laccase) include, but are not limited to:

- Trametes, such as Trametes versicolor (for instance Trametes versicolor 52J or Trametes versicolor ATCC 32745), Trametes pubescens, Trametes villosa, Trametes hirsuta, o Trametes trogii,

- Antrodiella, such as Antrodiella faginea,

- Aspergillus, such as Aspergillus oryzae or Aspergillus niger (for instance Aspergillus niger CBS 513.88),

- Cerrena, such as Cerrena maxima or Cerrena sp. RSD1,

- Pycnoporus, such as Pycnoporus cinnabarinus or Pycnoporus sanguineus

- Lentinus, such as Lentinus tigrinus, or Lentinus sp. WR2,

- Coriolopsis, such as Coriolopsis gallica (for instance Coriolopsis gallica SAH-12), Coriolopsis cinerea (for instance Coriolopsis cinerea A3387), or Coriolopsis caperata,

- Coriolus, such as Coriolus zonatus,

- Phlebia, such as Phlebia brevispora,

- Steccherinum, such as Steccherinum ochraceum or Steccherinum murashkinski,

- Rigidoporus such as Rigidoporous microporus,

- Myceliophtora such as Myceliophtora thermophila,

- Melanocarpus such as Melanocarpus albomyces,

- Laccaria such as Laccaria bicolor,

- Coprinopsis, such as Coprinopsis cinerea,

- Abortiporus, such as Abortiporus biennis,

- Thielavia, such as Thielavia arenaria,

- Deconica, such as Deconica castanella, and - Trichoderma, such as Trichoderma asperellum (Tor instance Trichoderma asperellum BPI.MBT1).

In a particular embodiment, the laccase is a laccase from Bacillus velezensis, Bacillus stratosphericus, Anoxybacillus, Aspergillus (such as Aspergillus niger), or Trametes versicolor, preferably from Trametes versicolor.

In a particular embodiment, the laccase comprises, or consists of, an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5, and variants thereof, said variants exhibiting laccase activity and having at least 60 %, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to any of SEQ ID NO: 1 to 5.

In a more particular embodiment, the laccase is a laccase from Bacillus velezensis, and more particularly from Bacillus velezensis TCCC 111904. More particularly, the laccase is preferably a laccase comprising, or consisting of, the following amino acid sequence: SEQ ID NO: 1, or a variant thereof exhibiting laccase activity and having at least 60 %, preferably at least 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to SEQ ID NO: 1. A laccase having the amino acid sequence SEQ ID NO: 1 is described in Li et al. Sci. Total. Environ. 2020, 713, 136713.

In another more particular embodiment, the laccase is a laccase from Anoxybacillus and more particularly from Anoxybacillus sp. UARK-01. More particularly, the laccase is preferably a laccase comprising, or consisting of, the following amino acid sequence: SEQ ID NO: 2, or a variant thereof exhibiting laccase activity and having at least 60 %, preferably at least 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to SEQ ID NO: 2. A laccase having the amino acid sequence SEQ ID NO: 2 is described in Al-Kahem Al-balawi et al. Curr Microbiol. 2017,74, 762-771.

In another more particular embodiment, the laccase is a laccase from Bacillus stratosphericus, and more particularly from Bacillus stratosphericus BCMC2. More particularly, the laccase is preferably a laccase comprising, or consisting of, the following amino acid sequence: SEQ ID NO: 3, or a variant thereof exhibiting laccase activity and having at least 60 %, preferably at least 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to SEQ ID NO: 3. A laccase having the amino acid sequence SEQ ID NO: 3 is described in Xiao et al. Int J Biol Macromol. 2021, 179, 270-278.

In another particular embodiment, the laccase is a laccase from Aspergillus, such as Aspergillus niger. More particularly, the laccase is preferably a laccase comprising, or consisting of, the following amino acid sequence: SEQ ID NO: 5, or a variant thereof exhibiting laccase activity and having at least 60 %, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to SEQ ID NO: 5.

In a preferred embodiment, the laccase is a laccase from Trametes versicolor. More particularly, the laccase is preferably a laccase comprising, or consisting of, the following amino acid sequence: SEQ ID NO: 4, or a variant thereof exhibiting laccase activity and having at least 60 %, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 75%, more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, and even more preferably at least 90%, 95%, 96%, 97%, 98% or 99%, amino acid sequence identity to SEQ ID NO: 4.

As used herein, a “redox mediator” refers to a compound that is able to exchange (in particular, provide and then recover) at least one electron. In particular, the redox mediator may be able to be oxidized by the oxidoreductase to form an oxidized redox mediator, and said oxidized redox mediator may be able to oxidize the cellulose.

In a particular embodiment, the redox mediator is an oxyl radical, preferably a nitroxyl radical. As used herein, an “oxyl radical” denotes an organic compound having one free radical (i.e. one single unpaired electron) on an oxygen atom. As used herein, a “nitroxyl radical” denotes an oxyl radical as defined herein, wherein said oxygen atom having one free radical is bound to a nitrogen atom. In a more particular embodiment, the redox mediator is an nitroxyl radical selected from the group consisting of 2,2,6,6-tetramethylpiperidin-l-yl)oxyl (TEMPO), 2,2,5,5-tetramethyl-l- pyrrolidinyloxyl (PROXYL), aminyloxyl, imino nitroxyl, nitronyl nitroxyl, iminoxyl, derivatives thereof, and a mixture thereof.

In a particular embodiment, an aminyloxyl is a compound of formula (Il-a): wherein R13 and R14 are each independently chosen from (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2- Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, and heteroaryl.

In another particular embodiment, an imino nitroxyl is a compound of formula (Il-b) : wherein R15, Ri6, and R17 are each independently chosen from (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2-Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, and heteroaryl, or R15 and Ri6 form together with the atoms to which they are attached a C5-C12 nitrogencontaining cycle.

In another particular embodiment, a nitronyl nitroxyl is a compound of formula (II-c): wherein Ris, R19, and R20 are each independently chosen from (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2-Cn)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, and heteroaryl, or Ris and R19 form together with the atoms to which they are attached a C5-C12 nitrogencontaining cycle.

In a particular embodiment, an iminoxyl is a compound of formula (Il-d): wherein R21 and R22 are each independently chosen from (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2- Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, and heteroaryl.

Preferably, the redox mediator is TEMPO or a derivative thereof. TEMPO can be represented as follows:

As used herein, a “derivative of TEMPO” refers to a molecule of TEMPO which is substituted by at least one (for instance, one, two or three, preferably one) substituent. For instance, a derivative of TEMPO can be represented by the following formula (II- 1): wherein R23 is selected from the group consisting from OH, NH2, CN, C(O)OH, oxo, (Ci- C ₆ )alkyl, -O-(Ci-C ₆ )alkyl.

More preferably, the redox mediator is TEMPO.

In step (a), contacting may be carried out by mixing the cellulosic material, the oxidoreductase and the redox mediator in a suitable solvent, preferably water. More particularly, such contacting may be carried out by dipping the cellulosic material into a solvent (preferably water) comprising the oxidoreductase and the redox mediator.

Step (a) of the process of the invention can be carried out under mild conditions, in particular mild temperatures and pH’s. In a particular embodiment, step (a) is carried out at a temperature comprised between 10 °C and 60 °C, preferably between 20 °C and 50 °C, more preferably between 25°C and 35 °C. In another particular embodiment, step (a) is carried out at a pH comprised between 2.5 and 12, preferably between 4 and 10, more preferably between 4 and 8, even more preferably between 5 and 8. Step (a) may be carried out in aqueous medium. The pH of step (a) can in particular be controlled by using a pHstat system and/or a buffer. Examples of buffer include, but are not limited to, a sodium phosphate buffer, an acetate buffer, a citrate buffer or Tri-CuCh buffer.

In another particular embodiment, step (a) is carried out for a duration comprised between 1 hour and 30 hours, for instance between 5 hours and 15 hours. Step (a) may be carried out under air.

The oxidoreductase and the redox mediator may be used in catalytic amounts. The concentration of the redox mediator in step (a) may for instance be comprised between 0.1 mM and 100 mM. The concentration of the oxidoreductase in step (a) may for instance be comprised between 0.001 U/mL and 100 U/mL.

Step (a) of the process of the invention allows to obtain an oxidized cellulosic material. An “oxidized cellulosic material” may in particular refer to a cellulosic material in which all or part of the alcohol functions of the cellulose are in oxidized forms, preferably in aldehyde and optionally ketone and/or carboxylic acid. In a particular embodiment, the solvent (preferably water) comprising the oxidoreductase and the redox mediator may be re-used after recovering of the oxidized cellulosic material.

Step (b) of the process of the invention comprises contacting the oxidized cellulosic material of step (a) with a dye comprising at least one NH2 group, in the presence of a reducing agent, so as to obtain a dyed cellulosic material.

As used herein, the “dye comprising at least one NH2 group” refers to any inorganic or organic compound comprising a chromophore moiety and at least one NH2 group. As used herein, the term “chromophore moiety” denotes a chemical moiety comprising conjugated double bonds (for instance, aromatic bonds) and imparting a color to the dye, typically by absorbing a part of a visible light and by reflecting, transmitting, or diffusing a complementary part of said visible light. The chromophore moiety and the at least one NH2 group may be covalently linked to each other directly or indirectly (i.e. through a spacer), preferably directly.

The chromophore moiety may in particular be chosen among chromophore moieties that are usually used in dyeing processes, and derivatives thereof. Such chromophore moieties are in particular described in the following articles: Klaus Hunger, Industrial dyes, chemistry, properties, applications, 2003, Wiley; Benkhaya et al. Inorganic Chemistry Communications, 2020, 115, 107891. “Derivatives” of chromophore moieties include in particular derivatives substituted by one or more substituents (such as -CF3, -CN, -NO2, -OH, Ci-Ce alkyl, halogen, - S(O)2ONa, or -C(O)O(Ci-Ce alkyl)) and having their chromophore activity preserved.

Examples of chromophore moieties include, but are not limited to, phenyls, rhodamines, anthraquinones, triarylmethanes, phthalocyanines, monoazo dyes, bisazo dyes, triazo dyes, polyazo dyes, biindolylidene diones, indanediones, quinolyl indanediones, acridines, thiazines, thiazoles, oxazines, phenoxazines, xanthenes, chlorines, diketopyrrolopyrroles, quinacridones, anthocyanidins, flavonoids, and derivatives thereof.

In a particular embodiment, said dye is a dye comprising at least one NH2 group having a pKa equal to or less than 11, preferably equal to or less than 9, more preferably equal to or less than 7, even more preferably equal to or less than 5. For instance, said pKa may be comprised between -5 and 11, between -5 and 9, between -5 and 7, between 0 and 7, between 3 and 7, between -5 and 5, between 0 and 5, or between 3 and 5.

As used herein, the “pKa of said at least one NH2 group” refers to -log(Ka), where Ka is the acid dissociation constant between said at least one NH2 group and the corresponding acidic form NHC. The pKa can be determined by any techniques known to the skilled artisan, for instance by potentiometric titration or conductometry.

In another particular embodiment, said NH2 group is a NH2 group directly linked to an aromatic group such as an arylene or heteroarylene. In such embodiment, the dye is thus an aromatic amine.

In a more particular embodiment, said dye is a compound of formula (I): [H ₂ N-(X-Y)n]m-Z (I), in which: - n is 0 or 1,

- m is an integer from 1 to 4,

- X is an arylene or heteroarylene group, said group being optionally substituted by at least one substituent selected from the group consisting of (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2- Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, heteroaryl, halogen, -CN, - NO2, -C(O)Ri, -C(O)OR2,-C(O)NR ₃ R4, -S(O) ₂ R ₅ , -S(O) ₂ OR ₆ , -NHS(O) ₂ R7, -NHS(O) ₂ OR ₈ , - OR9, -SR10, and -NR11R12,

Ri, R2, R3, R4, Rs, Re, R7, R ₈ , R9, Rio, R11 and R12 being each independently chosen from hydrogen, (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2-Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2- Ci2)heterocycloalkyl, aryl, and heteroaryl,

- Y is a spacer chosen among the following groups: (Ci-Ci2)alkylene, (C2-Ci2)alkenylene, (C2- Ci2)alkynylene, (C3-Ci2)cycloalkylene, (C2-Ci2)heterocycloalkylene, arylene, and heteroarylene, said group being optionally interrupted by at least one group chosen from -O-, - S-, -NH-, -C(O)-, and -S(O) ₂ , and

- Z is a chromophore moiety, or a salt thereof.

A compound of formula (I) according to the invention thus comprises a chromophore moiety Z linked to a number m of H2N-(X-Y) _n - groups.

As used herein, m represents the number of [H2N-(X-Y) _n ]- groups covalently linked to the chromophore moiety Z. According to the present invention, m is an integer from 1 to 4, preferably m is 1 or 2, more preferably m is 1.

As used herein, n represents the number of (X-Y) group in a [H2N-(X-Y) _n ]- group, where the (X-Y) group is covalently linked to NH2 and to the chromophore moiety. According to the present invention, n is 0 or 1. In other words, according to the present invention, each NH2 of the compound of formula (I) may be linked to the chromophore, directly (when n is 0) or through a (X-Y) group (when n is 1).

When m is higher than 1, namely 2, 3, or 4, it is understood that each n is independently 0 or 1.

In a particular embodiment, said compound of formula (I) is such that n is 1. In such an embodiment, when m is higher than 1, it is understood that each n is 1. In a particular embodiment, X is an arylene group, preferably a phenylene, said group being optionally substituted by at least one substituent selected from the group consisting of (Ci- Cn)alkyl, (C2-Ci2)alkenyl, (C2-Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2-Ci2)heterocycloalkyl, aryl, heteroaryl, halogen, -CN, -NO2, -C(O)Ri, -C(O)OR2,-C(O)NR ₃ R4, -S(O) ₂ R ₅ , -S(O) ₂ OR ₆ , - NHS(O) ₂ R ₇ , -NHS(O) ₂ OR ₈ , -OR9, -SR10, and -NR11R12,

Ri, R2, R3, R4, Rs, Re, R7, R ₈ , R9, Rio, R11 and R12 being each independently chosen from hydrogen, (Ci-Ci2)alkyl, (C2-Ci2)alkenyl, (C2-Ci2)alkynyl, (C3-Ci2)cycloalkyl, (C2- Ci2)heterocycloalkyl, aryl, and heteroaryl.

In another particular embodiment, Y is a spacer chosen among the following groups: (Ci- Ci2)alkylene and arylene, said group being optionally interrupted by at least one group chosen from -O-, -S-, -NH-, -C(O)-, and -S(O) ₂ .

In a preferred embodiment, said compound of formula (I) is such that n is 0. In such an embodiment, when m is higher than 1, it is understood that each n is 0. A compound of formula (I) wherein n is 0 can be represented by the following formula (1-0):

(H ₂ N) _m -Z (1-0), wherein m and Z are as defined herein.

In a particular embodiment, said compound of formula (I) (or (1-0)) is such that Z is a chromophore moiety selected from the group consisting of phenyls, rhodamines, anthraquinones, triarylmethanes, phthalocyanines, monoazo dyes, bisazo dyes, triazo dyes, polyazo dyes, biindolylidene diones, indanediones, quinolyl indanediones, acridines, thiazines, thiazoles, oxazines, phenoxazines, xanthenes, chlorines, diketopyrrolopyrroles, quinacridones, anthocyanidins, and flavonoids.

In a more particular embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

In another particular embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

In a preferred embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

In another preferred embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

(I-s). In a more preferred embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

In an even more preferred embodiment, said dye (or the compound of formula (I)) is represented by any one of the following formulae:

The compound of formula (I-a) as represented above is also called herein “/?ara-toluidine”. The compound of formula (I-b) as represented above is also called herein “rhodamine 123”. The compound of formula (I-f) as represented above is also called herein “acid red 266”. The compound of formula (I-h) as represented above is also called herein “acid red 33”. The compound of formula (I-p) as represented above is also called herein “acid violet 19”. The compound of formula (I-r) as represented above is also called herein “acid blue 25”. The compound of formula (I-s) as represented above is also called herein “acid black 41”.

As used herein, the “reducing agent” relates to one or more compounds that are able to reduce an imine group into an amine group. The reducing agent may in particular be any reducing agent usually used in a reductive amination reaction. More particularly, said reducing agent may be a hydride-type reducing agent. As used herein, a hydride-type reducing agent is a compound having one or more hydrides (formally: “H ”) that can be transferred to another compound, resulting in the reduction of said other compound.

In a particular embodiment, said reducing agent is a borane compound. As used herein, a “borane compound” refers to any organic compound having at least one B-H bond. In a more particular embodiment, the reducing agent is a borane compound selected from the group consisting of sodium or potassium borohydride, zinc borohydride, sodium cyanoborohydride, borane (BH3), diborane (B2H6), pyridine borane, picoline borane, 5-ethyl-2-methylpyridine borane, morpholine borane, 4-methylmorpholine borane, triethylamine borane, 9- borabicyclo[3.3.1]nonane (9-BBN), monoisopinocampheylborane, dicyclohexylborane, dimesitylborane, di si amylborane, catecholborane, pinacolborane, L-selectride, and a mixture thereof.

In a preferred embodiment, said reducing agent is picoline borane.

In step (b), contacting may be carried out by mixing the oxidized cellulosic material obtained in step (a), said dye comprising at least one NH2 group, and said reducing agent in a suitable solvent, preferably water. More particularly, such contacting may be carried out by dipping said oxidized cellulosic material into a solvent (preferably water) comprising the dye and the reducing agent.

Step (b) of the process of the invention can be carried out under mild conditions, in particular mild temperatures. In a particular embodiment, step (b) is carried out at a temperature comprised between 10 °C and 60 °C, preferably between 30 °C and 50 °C, more preferably between 35°C and 45 °C. In another particular embodiment, step (b) is carried out for a duration comprised between 30 minutes and 30 hours, for instance between 2 hours and 5 hours. Step (b) may be carried out under air.

In another particular embodiment, step (b) is carried out in the presence of an acid. Examples of acid include, but are not limited to, hydrochloric acid, sulfuric acid, a carboxylic acid such as formic acid, acetic acid, propionic acid, or benzoic acid. Preferably, said acid is acetic acid. The concentration of said acid in step (b) may advantageously be comprised between 2 % and 30 % (v/v).

The dye and the reducing agent may be used in excess with respect to the oxidized cellulosic material. The concentration of the dye (for instance a compound of formula (I)) in step (b) may for instance be comprised between 0.01 mM and 10 M. The concentration of the reducing agent in step (b) may for instance be comprised between 0.1 mM and 1 M.

In a particular embodiment, the molar ratio of the carbonyl groups of the oxidized cellulosic material to the dye is comprised between 1/20 and 1/1, preferably between 1/10 and 1/1. In a particular embodiment, the molar ratio of the carbonyl groups of the oxidized cellulosic material to the reducing agent is comprised between 1/2 and 1/1, preferably between 1/1.5 and 1/1. The molar amount of carbonyl groups of the oxidized cellulosic material can be determined by spectrophotometry, in particular according to the method as described below in the examples.

In an embodiment where the oxidized cellulosic material is dipped into a solvent (preferably water) comprising the dye and the reducing agent, said solvent comprising the dye and the reducing agent may be re-used after recovering of the oxidized cellulosic material.

Step (b) of the process of the invention allows to obtain a dyed cellulosic material. In step (c) of the process of the invention, the dyed cellulosic material can be recovered by any suitable technique known to the skilled artisan, for instance by collecting the dyed cellulosic material from the solvent comprising the dye and the reducing agent.

Advantageously, steps (a), (b) and (c) of the process of the invention are carried out successively.

In a particular embodiment, the process comprises the following successive steps:

(a) contacting a cellulosic material with a laccase and TEMPO or a derivative thereof, so as to obtain an oxidized cellulosic material;

(b) contacting the oxidized cellulosic material of step (a) with a dye of formula (I) as defined herein (preferably a dye of formula (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I- k), (1-1), (I-m), (I-n), (I-o), (I-p), (I-q), (I-r), or (I-s) as represented above, more preferably of formula (I-a), (I-b) or (I-h)), in the presence of a borane compound, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material.

In a particular embodiment, the process comprises the following successive steps:

(a) contacting a cellulosic material with a laccase and TEMPO or a derivative thereof, so as to obtain an oxidized cellulosic material; (b) contacting the oxidized cellulosic material of step (a) with a dye of formula (I) as defined herein (preferably a dye of formula (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I- k), (1-1), (I-m), (I-n), (I-o), (I-p), (I-q), (I-r), or (I-s) as represented above, more preferably of formula (I-a), (I-b) or (I-h)), in the presence of a borane compound, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material, wherein step (a) is carried out at a temperature comprised between 10 °C and 60 °C (preferably between 20 and 50 °C), and step (b) is carried out at a temperature comprised between 10 °C and 60 °C (preferably between 30 and 50 °C).

In another particular embodiment, the process comprises the following successive steps:

(a) contacting a cellulosic material chosen from cotton, flax, hemp, jute, viscose, lyocell, rayon or modal (preferably cotton) with a laccase from Bacillus velezensis, Bacillus stratosphericus, Anoxybacillus, Aspergillus, or Trametes versicolor, and TEMPO or a derivative thereof, so as to obtain an oxidized cellulosic material;

(b) contacting the oxidized cellulosic material of step (a) with a dye of formula (I) as defined herein (preferably a dye of formula (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (I-g), (I-h), (I-i), (I-j), (I- k), (1-1), (I-m), (I-n), (I-o), (I-p), (I-q), (I-r), or (I-s) as represented above, more preferably of formula (I-a), (I-b) or (I-h)), in the presence of picoline borane, so as to obtain a dyed cellulosic material; and

(c) recovering a dyed cellulosic material, wherein step (a) is carried out at a temperature comprised between 10 °C and 60 °C (preferably between 20 and 50 °C), and step (b) is carried out at a temperature comprised between 10 °C and 60 °C (preferably between 30 and 50 °C).

Another object of the present invention is a dyed cellulosic material obtained by a process as defined herein.

The invention will also be described in further detail in the following examples, which are not intended to limit the scope of this invention, as defined by the attached claims. EXAMPLES

Materials and Methods

The laccase from Trametes versicolor was purchased from Sigma Aldrich.

Purification of the laccase from Trametes versicolor Discontinuous diafiltration was performed using a Labscale™ system (Millipore, Merck) with a Pellicon XL filter PXC030C50 (30 KDa) (Merck) regenerated cellulose membrane (filter area 1.14 m ² ) at a fixed transmembrane pressure of 2.1 bar at 4°C. The feeding chamber was filled with 50 mL of a solution of TvL at 30 g/L and diafiltrated twice with 500 mL of water or 20 mM acetate buffer pH 6.0 under and then concentrated to 15 mL. Before diafiltration, the membrane was previously equilibrated with the corresponding buffer for 15 min. After each trial, the membrane was flushed with distilled water and stored in 50 mM NaOH. The diafiltrated and concentrated enzymatic preparation was analyzed by HPAEC-PAD analysis.

Determination of laccase activity. The laccase activity was determined using a colorimetric assay with 50 pL of 1 mM ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 10 pL of laccase solution, and 440 pL of acetate buffer (20 mM, pH 3.5). Oxidation of ABTS was monitored by measuring the absorbance increase at 420 nm (8420= 3.6 x 10 ⁴ M7 ¹ cm ¹ ) at 30°C using a UV-vis spectrophotometer (Cary 100 Bio). A unit (U) of laccase activity is defined as the amount of laccase that oxidizes 1 pmol of ABTS per minute.

IC-HRMS of the oxidized Glc5 The mass of Glc5 oxidation products were determined using liquid anion exchange chromatography on a Dionex ICS-5000+ reagent-free HPIC (Thermo Fisher Scientific, Sunnyvale, CA, USA) system coupled with a Thermo Scientific linear trap quadrupole (LTQ) Orbitrap Velos hybrid Fourier-transform (FT) mass spectrometer (FTMS; Thermo Fisher Scientific, San Jose, CA, USA). Samples were separated within 50 min using a linear gradient elution of 7 mM to 100 mM of KOH applied to an IC Dionex™ lonPac™ AS11- HC column (250 x 2 mm) equipped with an ASH Thermo Scientific™ Dionex™ lonPac™ guard column (50 x 2 mm) at a flow rate of 0.38 mL/min. The column and autosampler temperature were 25°C and 4°C, respectively. The injected sample volume was 5 pL. Conditions for the electrospray ionization (ESI) at negative mode was as follows: spray voltage was at 2.7 kV, capillary and desolvation temperatures were 350 and 350°C, respectively, and the maximum injection time was 50 ms. Nitrogen was used as the sheath gas (pressure, 50 units) and auxiliary gas (pressure, 5 units). The automatic gain control (AGC) was set at le6 for fullscan mode, with a mass resolution of 60.000 (at 400 m/z). For the full scan MS analysis, the spectra were recorded in the range of m/z 80.0-1000.0. Finally, data acquisition was performed using Thermo Scientific Xcalibur software.

LC-HRMS/UV analysis of the grafted Glc5 with ■toluidine or Rhodamine Analyses were carried out on a vanquish™ system coupled to a Thermo Sceintific Q Exactive™ Plus hydrid quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher). Samples were separated within 95 min using an isocratic elution with a mixture containing 10% ACN/ 40% H2O/ 50% 20 mM ammonium acetate applied to a Shodex™ AsahipakNH2P-504E (4,6mm x 250 mm) equipped with a Shodex™ Asahipak NH2P-50 4A guard column (4.6 x 10 mm) at a flow rate of 0.7 mL/min. The column and autosampler temperature were 40°C and 4°C, respectively. The injected sample volume was 10 pL. The LC eluates was monitored at different wavelength according to the grafted compound: 286 nm for /?-toluidine and 540 nm for rhodamine 123. Conditions for the electrospray ionization (ESI) at negative mode was as follows: Conditions for the electrospray ionization (ESI) at negative mode was as follows: spray voltage was at 2.75 kV, capillary and desolvation temperatures were 400 and 400°C, respectively, and the maximum injection time was 100 ms. Nitrogen was used as the sheath gas (pressure, 75 units) and auxiliary gas (pressure, 20 units). The automatic gain control (AGC) was set at le6 for fullscan mode, with a mass resolution of 70.000 (at 800 m/z). For the full scan MS analysis, the spectra were recorded in the range of m/z 80.0-1000.0. Finally, data acquisition was performed using Thermo Scientific Xcalibur software.

HPLC-UV-MS analysis of hydrolyzed cotton fibers Analyses were performed with an Ultimate

3000 series chromatograph equipped with a Dionex 340 UV/VIS detector and coupled with a simple quadruple mass spectrometer (MSQ Plus, Thermo Scientific) and separated by a Phenomenex PFP C18 column (Luna 5 pm, 100 A, 250 x 4.6 mm, USA) maintained at 40 °C.

Oligosaccharides grafted with p-toluidine (“pT”), Rhodamine 123 (“RHO123”) or Acid Red 33 (“AR33”) were respectively detected by UV at 286 nm, 500 nm and 532 nm depending on the maximum absorption wavelength of the dyes. Injected samples (50 pL) were eluted using A= ELO/formic acid 0.05 %, (v/v) and B=ACN/formic acid 0.05 % (v/v) as eluents. The gradient used for samples grafted with RHO123 was as following: 0-20 min from 10% B to 30% B for samples grafted with RHO123 or 0-50 min from 10% B to 30% B for sample grafted with AR33, then for all sample 30% B was increased to 40% B within 10 minutes, then 40% B was increased to 100% B within 5 min and kept with 100% B for 5 min finally return to the initial condition and 5 min equilibration of the column. In both cases, a flow rate of 0.5 mL per min was used. The mass spectrometer was used in positive mode with a voltage cone at 75 V, the temperature of the electrospray ionization (ESI) ion source was 450 °C and the gas carrier was nitrogen. The spray voltage was set at 3.5 kV. The mass spectrometer scanned from m/z 100 to 1500. The data acquisition and processing were performed using Chromeleon™ 7.2 data systems. of the freeze-dried oxidized cotton fibers were recorded using a Nicolet

IS 10 infrared spectrophotometer with a Golden Gate attenuated total reflection (ATR) attachment having a diamond crystal (Thermo Nicolet, USA). Absorbance measurements were performed over a wavelength range from 400 to 3900 cm’ ¹ with a resolution of 16 cm’ ¹ and 32 scans per sample were recorded.

Elemental analyses were performed on untreated and treated cotton fibers to determine their carbon, hydrogen, nitrogen content. Elemental analyses were performed in the facility available in Laboratoire de Chimie de Coordination du CNRS using PerkinElmer 2400 Series II Analyzer, Toulouse. The detection range for nitrogen was between 0.001 mg and 6.0 mg.

Color washing-fastness test the color resistance test of fabric colored samples consisted of three sequential washing cycles with laundry detergent. Each step was performed at 60 °C for 60 min. After the last step, the fabrics samples were dried overnight at room temperature for color strength evaluation.

Determination of carbonyl groups into oxidized cotton thread

The content of carbonyl groups in the cotton threads was determined using a spectrophotometric method published by Szabolcs (1961) and optimized by Strlic and Pihlar (1997). The “TTC reagent” was prepared by mixing 20 mL of Triphenyltetrazolium chloride (TTC) at 0.5 M with 20 mL of KOH at 0.3 M. The solution was mixed well and left to stand for 10 min at room temperature before use. 10 mg of dry cotton thread was inserted in a glass tube with 2 mL of water and 2 mL of freshly prepared TTC reagent, and then incubated for 6 min at 80 °C in a water bath. The suspension (4 mL) was mixed with MeOH (41 mL) to solubilize the reddish-crystals of TTF (triphenyltetrazolium formazan) produced. The absorbance of the solution was measured at 482 nm. The amount of carbonyl groups was calculated from the calibration curve determined with D-glucose. The mean values were calculated from at least three individual assays. A calibration curve was established using anhydrous D-glucose as standard. A solution of D-glucose at 5.5 mM was prepared, left 30 min at room temperature and then dissolved in water to obtain Absorbances were measured at 482 nm using a UV-Vis Cary 3500 spectrophotometer (Agilent Technologies, Santa Clara, CA, United States) with the CaryWinUV Scan software (Vers.5.0.0.999).

Example 1. Process for dyeing a cellulosic material

1) Glc5

Enzymatic Oxidation of cellopentaose (Glc5). Oxidation reaction was performed in the presence of 27 mM of Glc5, 6 mM of TEMPO and 5.4 U.mL' ¹ of laccase from Trametes versicolor in 5 mL of acetate buffer (20 mM, pH 6.0) at 30 °C. The reaction was stirred at 500 rpm in open flask for 8h and monitoring by sampling 250 pL of the reaction mixture for subsequent analyses.

LC-HRMS analysis showed that aldehyde and carboxylic acid functions are obtained:

- tr = 2.45 min, [M-H]' : m/z 827.27, [(M-H+Na)-H] : m/z 863.24,

- tr = 2.90 min, [M-H]' : m/z 843.26,

- tr = 4.30 min, [M-H]' : m/z 841.24.

Reaction with the dye (p-toluidine (I-a)). After 8h of enzymatic oxidation of Glc5 (as described above), 500 pL of the reaction medium was mixed for 3h with 2.4 mM of /?-toluidine, 10 mM of 2-picoline-borane and 10% of acetic acid at room temperature.

LC-HRMS analysis showed the grafting of -toluidine on Glc5:

- tr = 4.76 min, [M-H]': m/z 916.33,

- tr = 4.89 min, [M-H]’: m/z 918.34, - tr = 41.69 min, [M-H]': m/z 932.32,

- tr = 44.19 min, [M-H]’: m/z 932.32.

Reaction with the dye (Rhodamine 123 (I-b)). A similar procedure was applied to Glc5 using Rhodamine 123 as a dye.

LC-HRMS analysis showed the grafting of Rhodamine 123 on Glc5: - tr = 2.63 min, [M-H]': m/z 1154.37.

2) Cotton

Enzymatic Oxidation of Cotton. 215 mg of cotton thread (SAS Bruneel textiles, Perenchies, 59 , ready to dye) was washed 6 times with 10 mL of distilled water and dried overnight at air. The resulting cotton was then dipped during 8 hours in an open flask containing 10 mL of a reactional medium containing:

- Medium: Trametes Versicolor Laccase: 5.4 U/mL (54U), TEMPO: 6 mM, Sodium phosphate buffer (50 mM pH 6.0)

- Conditions: Stirring 500 rpm; Open flasks; 30 °C; 8h.

- Analytics. The resulting cotton was washed 6 times with 10 mL of water, dried overnight and analyzed by FTIR where a new absorption band at 1605-1610 cm' ¹ (such as 1608 cm' ¹ ) appeared compared to untreated cotton showing the appearance of a new C=O bond (Figure 1). Solid state NMR revealed that approximately 2% of the cotton had been oxidized.

The initial amount of carbonyl group (i.e., before oxidation with laccase/TEMPO) was 59 ± 8 pmol per gram of cotton. After 7 hours of reaction, the amount is 178±9 pmol of carbonyl groups per gram of cotton threads, corresponding to 6173 pmoles of equivalent glucose.

Reaction with the dye p-Toluidine (I-a). 213 mg of the oxidized cotton was then placed in an open vessel at 40 °C containing 10 mL of an aqueous solution of 10% acetic acid with the following components:

- Medium: p-Toluidine:0.1 M ; 2-picoline borane (Pic-BH3):48 mM (molar ratio of 1 :5:1.2 for carbonyl groups, amine groups and Pic-BH?).

- Conditions: T°C=40°C; Time=3h; 500 rpm The resulting cotton was washed with 8x 10 mL of 50:50 JfcChEtOH and 2 x 10 mL H2O. Between each wash, samples were stirred for 30 s and finally dried at room temperature, for two days.

Spectrometrical analysis revealed that the amount of aldehyde bond had diminished by a 10- fold between ungrafted cotton after oxidation and grafted cotton. Acid hydrolysis of the grafted cotton (H2SO4 72% (13.5 M), Volume ~2 mL, T°C=30°C, Time=lh) showed the appearance of two news peaks that absorb at 286 nm (Figure 2). This shows the grafting of p-toluidine at Cl and C6.

Enzymatic hydrolysis of the dyed cotton using a cellulase cocktail from Trichoderma reesei was carried out and analyzed the hydrolysis products by LC-MS/UV: after washing, 50 mg of the dyed cotton were incubated with 500 pL of cellulase in sodium acetate buffer (50 mM, pH 4.5) at 40°C for 24h. The untreated cotton fibers were also hydrolyzed in the same conditions to serve as control, and they were more rapidly degraded than the dyed threads. The reactions were stopped after 24 h. The HPLC-UV286nm analysis of the hydrolysis products recovered in the reaction supernatant after centrifugation revealed the presence of two main peaks corresponding to products Pl (tr= 9.34 min) and P2 (tr= 9.62min), which both absorb at 286 nm and were not present in the control experiment. LC-UV/MS analysis in positive mode further demonstrated that Pl (m/z 432 [M+H] ⁺ ) corresponds to a cellobiose molecule grafted with p- Toluidine at one of the C-6 position of the molecule, P2 (m/z 270 [M+H] ⁺ ) is a glucose molecule grafted with a />-Toluidine at C-6 position and P3 (tr= 24.7 min) (not detected in UV with m/z 521 [M+H] ⁺ ) is a cellobiose with two />-Toluidine molecules grafted at each of its C- 6 position (Figures 3 A and 3B).

Reaction with the dye Rhodamine 123 (I-b). A similar procedure was applied to the oxidized cotton using Rhodamine 123 (0.1 M) as a dye.

Cellulase-hydrolysis of cotton threads dyed with Rhodamine 123 was carried out under the same conditions as described above. HPLC-UVsoonm/MS analysis showed the presence of the product P4 (tr= 22.29 min) with m/z 507 [M+H] ⁺ corresponding to a glucose covalently grafted with a Rhodamine 123 at C-6 position. Another experiment was conducted with 1 g of threads. Hydrolyates of the oxidized threads over 72 h with an excess of cellulases were centrifuged and the supernatant was analyzed by LC-MS/UV. In addition to the already identified product P4, another product P5 (m/z 669 [M+H] ⁺ ) was identified, which corresponds to cellobiose grafted with a Rhodamine 123 on one of the two possible C-6 position (Figures 4A and 4B). Reaction with the dye Acid Red 33 (I-f). A similar procedure was applied to the oxidized cotton using Acid Red 33 (0.1 M) as a dye.

Cellulase-hydrolysis of cotton threads dyed with Acid Red 33 was carried out under the same conditions as described above.

HPLC-UV532nm/MS analysis highlighted two products P6 (tr= 18.69 min) and P’6 (tr= 19.54 min) with the same m/z 745 [M+H] ⁺ value corresponding to cellobiose with Acid Red 33 grafted at one of the two possible C-6 position (either on the reducing or non-reducing unit) and that elute at different retention times (Figures 4C and 4D).

Comparative tests:

Figures 5 A and 5B show cotton threads dyed with Rhodamine 123 and Acid Red 33 dyes respectively for assays according to the invention (Al, Bl) and the controls (A2, A3, B2, B3), after reaction and washing steps (Table 1).

Table 1

“+” means that an intense coloration of the material was obtained after dyeing and washing, means that a low coloration of the material was obtained after dyeing and washing.

Remarkably, the oxidized cotton threads incubated with Rhodamine 123 (sample Al) or Acid Red 33 (sample Bl) and pic-BHs retain an intense coloration after washing in comparison to the controls, indicating an improved wet fastness. A much lower coloration was obtained with the unoxidized fibers treated in similar conditions (samples A2 and B2) and the oxidized cotton incubated only with the aminated dyes before washing (samples A3 and B3). This demonstrates that both the oxidation and the reductive amination steps are crucial to obtain a high coloration and wet fastness. More particularly, the result obtained in absence of reducing agent shows that the coloration and wet fastness obtained with an amine bond (Al, Bl) are improved with respect to those obtained with an imine bond (A3, B3). Table 2 shows the elemental composition obtained for each series of experiments (Series A and B). The amount of nitrogen found in oxidized threads treated with Rhodamine 123 and Acid Red 33, and pic-BEE was of 0.30% and 0.08%, respectively. No traces of nitrogen could be detected in the control showing that the persistent color remaining after washing of samples A2/B2 and A3/B3 is due to very low amounts of residual dyes.

Table 2:

Series A N (%) C (%) H (%)

Unoxidized cotton 0.00±0.00 42.29±0.01 6.79±0.05

Cotton oxidized using TvL/TEMPO 0.00±0.00 41.49±0.06 6.55±0.01

Oxidized Cotton +Rhodaminel23+ pic-BHs (Al) 0.30±0.00 42.82±0.12 6.32±0.01

Unoxidized cotton + Rhodamine 123 + Pic -BHs (A2) 0.00±0.00 42.10±0.06 6.48±0.04

Oxidized Cotton + Rhodamine 123 (A3) 0.00±0.00 41.93±0.13 6.31±0.01

Series B N (%) C (%) H (%)

Oxidized cotton + Acid Red33+ with pic-BH ₃ (Bl) 0.08±0.01 41.93±0.04 6.32±0.06

Unoxidized cotton + Acid Red33 + Pic-BHs (B2) 0.00±0.00 42.47±0.08 6.49±0.10

Oxidized cotton + Acid Red33 (B3) 0.00±0.00 42.04±0.13 6.31±0.04

3) Cotton fabric a) A 5.8 cm x 4 cm piece of cotton fabric was immersed in a solution containing 5.4 U/mL of laccase fiber and 0.6 mM of TEMPO. The solution was stirred at 140 rpm at 30°C during 7 h. After oxidation, the fabric was rinsed with 125 mL of water and then washed with 100 mL of EtOH/EEO 70/30 before a final rinse with 125 mL distilled water. The washed fabric was dried overnight at room temperature.

The reductive amination was carried out at 40°C in a solution containing 10% of acid acetic using the molar ratio of 1 : 1.5: 1.2 for carbonyl groups, amine groups and pic-BEE. As pic-BEE has a low solubility in water, it was previously prepared in 80% MeOH (v/v). After reductive amination, the dyed cotton material was rinsed three times with 150 mL of water, then washed twice with 50 mL of 50 mM NaOH solution before three successive final rinses with 150 mL of distilled water each. The washed fibers were dried overnight at room temperature.

Figure 6 shows that the cotton fabric dyed with AR33 according to the process of the invention retain an intense coloration after washing in comparison to the controls (sample 2: no oxidation, sample 3: no reducing agent) indicating an improved wet fastness. b) At the industrial scale: 1 kg of weaved cotton fabric was dipped in a 50 kg bath of water containing 54 mg of Trametes Versicolor Laccase (270 kUnits, 50 Units/mg), 47 g of TEMPO (6mM, 0.93 g/L) and 6 kg of phosphate buffer (50 mM, 120 g/L) for 8 hours at 30 °C. The resulting fabric was then rinsed in 50 kg of water and in another tank containing 45 kg of water, 5 kg of acetic acid (10%), 0,1 mM of a dyestuff, 250 g of picoline borane (48mM, 5g/L) at 40

°C during 3 hours. The resulting cotton was washed with water and dried at air.

Fastness study of grafted cotton fabric:

A cotton fabric dyed with AR33 was utilized for the study of the dye washing-fastness. For that propose, a piece of 23,2 cm ² of colored cotton fabric with AR33 has been subjected to three washing cycles (in a washing machine) of one hour each at 60°C with a laundry detergent.

After three washing cycles at 60°C, the staining of the fabric has persisted being close to that of the starting material (Figure 7). This result demonstrates the strength and the good washing fastness of the dyed material.