Method and apparatus for dyeing fabrics

Abstract

To provide an apparatus for performing a process that dyes a textile comprising enzymatic synthesis of a dye precursor.SOLUTION: An apparatus comprises: a first chamber for contacting indole or a derivative thereof with a first immobilized enzyme to convert it into indoxyl or a derivative thereof; and a second chamber for storing a textile and means to generate a flow, wherein both chambers are in fluid connection with each other, indoxyl or a derivative thereof is flowed from the first chamber to the second chamber for conversion into indigo or a derivative dye thereof, and the textile is dyed.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for dyeing fabrics, and to the immobilized enzymes contained in said apparatus required to carry out said method. [Background technology]

[0002] Vat dyes are insoluble dyes that require a reducing agent to be dissolved in water. Traditionally, dyeing using vat dyes involves applying the dissolved, reduced form of the dye to the fabric, followed by oxidizing the dye back to its insoluble form to impart color to the fabric.

[0003] Indigo is a compound of formula (I) [ka] It is a dye used for dyeing.

[0004] Substitution on the aromatic ring of indigo with groups such as halogen, alkyl, alkoxy, amino, aryl, aryloxy, and carbonyl provides compounds that exhibit a wide range of colors other than blue and are part of the so-called indigo derivatives.

[0005] The majority of indigo and indigo derivatives are produced synthetically. The Heumann synthesis (Scheme 1 below) and the Pfleger synthesis (Scheme 2 below) were the first synthetic routes used to produce indigo on an industrial scale, and variations of these methods are still in use today.

[0006] Scheme 1 [ka] II' III' I

[0007] Scheme 2 [ka] II'' III'' I

[0008] The above synthetic route can also be used to prepare indigo derivatives, where compounds II', II'', III', and III'' are substituted with the same groups at the same positions as in the desired indigo derivative to be synthesized.

[0009] The synthesis of indigo and its derivatives, along with other vat dyes, is also carried out by means of enzymes or bacterially expressed enzymes, but such enzymatic synthesis is not used in industrial processes.

[0010] Although indigo precursors (e.g., compounds II', II'', III', and III'' in the above scheme) are soluble in aqueous solution, indigo is not soluble and precipitates after synthesis in aqueous solution. Therefore, as noted above, indigo and its derivatives must be reduced (e.g., by treatment with a reducing agent, as shown in Scheme 3).

[0011] Scheme 3 [ka] I IV

[0012] Here, compound I is indigo and compound IV is the water-soluble and reduced form of indigo called leuco-indigo (colorless, hence white indigo).

[0013] Therefore, an industrial dyeing process using indigo or its derivatives as the dye, or generally using vat dyes, first involves synthesizing indigo or its derivatives (or vat dyes) in an aqueous solution in a reactor via known methods (e.g., the synthetic route of Scheme 1 above). The indigo or its derivatives (or vat dyes) is obtained as a precipitate. The aqueous solution containing suspended indigo or its derivatives (or vat dyes) is then treated with a reducing agent to obtain an aqueous solution containing dissolved leuco-indigo or its derivatives (or vat dyes). The aqueous solution containing dissolved leuco-indigo or its derivatives (or vat dyes) is then applied to a fabric. After wetting the fabric with the aqueous solution containing dissolved leuco-indigo, oxidation of the leuco-indigo or its derivatives (or vat dyes) yields indigo or its derivatives (or vat dyes), and the fabric is thus dyed. Such oxidation can be carried out, for example, by oxygen in the air. Typically, the indigo dyeing process requires several immersion and oxidation steps to achieve the desired color shade.

[0014] The reducing agents used to reduce insoluble vat dyes, such as indigo or its derivatives, are strong chemicals, i.e., chemicals that are dangerous to the user and / or the environment, such as sodium hydroxide and sodium hydrosulfite. Indeed, in conventional dyeing processes in which, for example, indigo or its derivatives are used as dyes, large amounts of reducing salts and hydroxides are used, which generates large amounts of wastewater that must be treated before disposal. This step increases the cost of the dyeing process. Summary of the Invention [Problem to be solved by the invention]

[0015] Thus, there is a need for an improved method of dyeing fabrics with vat dyes, particularly indigo or its derivatives, that reduces costs for vat dyeing and water treatment processes.

[0016] Another problem with known indigo dyeing methods is that fabrics, particularly cellulose, can be damaged by prolonged exposure to alkaline processing solutions and the by-products present therein. [Means for solving the problem]

[0017] It is an object of the present invention to overcome the above problems and to provide a safe, cost-effective and environmentally friendly method for dyeing fabrics using insoluble dyes, such as vat dyes, in particular using indigo or its derivatives.

[0018] Another object of the present invention is to provide a method for dyeing fabrics using insoluble dyes, e.g., indigo or its derivatives, as well as vat dyes, which is more sustainable than conventional dyeing methods using indigo or its derivatives, or vat dyes in general. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a schematic diagram of the staining method of the present invention. [Figure 2] FIG. 1 is a diagram illustrating an outline of another specific example of the staining method of the present invention. [Figure 3] FIG. 1 is a diagram illustrating an outline of a specific example of a staining method. [Figure 4] 1 is a schematic diagram of an apparatus 10. FIG. [Figure 5] FIG. 2 is another schematic diagram of the device 10. [Figure 6] FIG. 1 is a schematic diagram of an embodiment of a device 10 including a reservoir. [Figure 7] FIG. 1 is a schematic diagram of another embodiment of the apparatus 10 including a recovery tank. [Figure 8] 1 is a diagram illustrating a schematic diagram of an embodiment of the device 10. FIG. [Figure 9] FIG. 2 is a schematic diagram illustrating another embodiment of the device 10. DETAILED DESCRIPTION OF THE INVENTION

[0020] The above object, among other objects, is achieved by a method according to claim 1, i.e. a method for dyeing textiles comprising the enzymatic synthesis of a dye precursor, the method comprising the following steps: a) contacting at least a first dye precursor with at least a first immobilized enzyme to convert at least a portion of said first dye precursor to at least a second dye precursor to obtain a solution comprising said second dye precursor; b) generating a flow of a solution comprising a second dye precursor, thereby causing the solution comprising the second dye precursor to flow from the first immobilized enzyme onto the fabric; c) contacting the fabric with a solution comprising a second dye precursor; and d) converting at least a portion of the second dye precursor to at least one dye, thereby dyeing at least a portion of the fabric. wherein the first immobilized enzyme is spaced from the fabric.

[0021] In the following description, "fabric" refers to various fibers, threads, ropes, woven fabrics, and / or garments, for example, dyed with indigo or its derivatives. The textile materials may be natural materials, such as those of animal or plant origin, such as cotton, linen, silk, wool, etc., or synthetic materials or mixtures thereof, such as stretch cotton fabrics or garments. The threads may be produced by various known methods, and the fabrics may be produced by various known methods, such as weaving, knitting, crocheting, knotting, and felting. The garments may be various garments, such as jeans, shirts, casual wear, etc.

[0022] In the present invention, the term "first dye precursor" refers to any soluble compound that is enzymatically converted into a second dye precursor. In the present invention, the term "second dye precursor" refers to any soluble compound obtained from the first dye precursor and converted into an insoluble dye, for example, via dimerization. In the present invention, the term "insoluble dye" refers to any water-insoluble compound commonly used to dye textiles, such as vat dyes like indigo. Therefore, according to the present invention, the first dye precursor, the second dye precursor, and the insoluble dye are related to each other through a synthetic route, particularly a synthetic route comprising a first enzymatic step and a second non-enzymatic step. For example, the first dye precursor, the second dye precursor, and the insoluble dye according to the present invention are indole and / or its derivatives, indoxyl and / or its derivatives, and indigo and / or its derivatives, respectively, and are related as shown in Scheme 4 below. Which first dye precursor must be included in the solution of step a) is selected depending on the color that the dyed textile must have at the end of the inventive dyeing process, since the color of the fabric is provided by an insoluble dye, which is obtained from the first dye precursor.

[0023] Advantageous examples of the first dye precursor, the second dye precursor, and the insoluble dye are indole and / or a derivative thereof, indoxyl and / or a derivative thereof, and indigo and / or a derivative thereof, respectively. Indigo and / or a derivative thereof are synthesized using indole and a derivative thereof as a raw material, as shown in Scheme 4 below.

[0024] Scheme 4 [ka] II III I

[0025] wherein compound II is indole (first dye precursor), compound II is indoxyl (second dye precursor), and compound I is indigo (dye). In particular, referring to the reaction in Scheme 4, the addition of a hydroxyl group to the 3-carbon of indole produces indoxyl, which dimerizes in aqueous solution to produce indigo. According to the present invention, the addition of a hydroxyl group to indole and / or its derivatives is carried out by an immobilized enzyme in step a) of the method of the present invention. When the first dye precursor is indole and / or its derivatives, the second dye precursor is indoxyl and / or its derivatives, and the insoluble dye is indigo and / or its derivatives, the method of the present invention can dye textiles with indigo and / or its derivatives, which are dyes commonly used in textile dyeing, without going through a reduction step in the prior art.

[0026] According to the present invention, "indole derivatives," "indoxyl derivatives," and "indigo derivatives" refer to indole, indoxyl, and indigo, respectively, substituted with one or more substituents, for example, one or more groups at one or more carbon atoms at various positions selected from the 4-, 5-, 6-, and 7-positions of indole or indoxyl, and the 4-, 4'-, 5-, 5'-, 6-, 6'-, and 7-, 7'-positions of indigo, and / or a group at the nitrogen atom of indole, indoxyl, or indigo. The one or more groups substituting one or more carbon atoms can be, for example, a halogen, an alkyl group (e.g., C1-C 20 alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, and isobutyl), alkoxy groups (e.g., C-C 20The nitrogen-substituting groups include, but are not limited to, alkoxy groups (e.g., methoxy, ethoxy, butoxy, tertiary-butoxy, and isobutoxy), aryl groups (e.g., phenyl, substituted phenyl, benzyl, substituted benzyl, naphthyl, anthracenyl, and heteroaryl), aryloxy groups (e.g., phenoxy and naphthoxy), amine groups (e.g., primary and / or secondary aliphatic and / or aromatic amine groups), nitro groups, and carbonyl groups (e.g., aldehyde groups, e.g., aromatic and / or aliphatic aldehydes, and ketones, e.g., aromatic and / or aliphatic ketones). Groups substituting the nitrogen include alkyl groups (e.g., C-C 20Examples of such groups include, but are not limited to, alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tertiary-butyl, and isobutyl), aryl groups (e.g., phenyl, substituted phenyl, benzyl, substituted benzyl, naphthyl, anthracenyl, and heteroaryl), and acetyl groups (e.g., formyl, acetyl, propionyl, benzoyl, and acryloyl). Thus, indole derivatives are, for example, 4-chloroindole, 5-chloroindole, 6-chloroindole, 7-chloroindole, 5-bromoindole, 6-bromoindole, 5-nitroindole, 5-hydroxyindole, 5-methylindole, 5-methoxyindole, 6-methylindole, 7-methylindole, 5-aminoindole, 1-methylindole, indole-6-carboxaldehyde; and indoxyl derivatives are, for example, 4-chloroindoxyl, 5-chloroindoxyl, 6-chloroindoxyl, 7-chloroindoxyl, 5-bromoindoxyl, 6-bromoindoxyl, 5-nitroindoxyl, 5-hydroxyindoxyl, 5-methylindoxyl, 5-methoxyindoxyl, 6-methylindoxyl, 7-methylindoxyl, 5-aminoindoxyl, 1-methylindoxyl, indoxyl-6-carboxaldehyde. The present invention also encompasses the use of various other indole and indoxyl derivatives, provided that the indole derivatives react and are converted by enzymatic catalysis to the corresponding indoxyl derivatives. When these indoxyl derivatives are converted (i.e., dimerized according to step d) of the method of the present invention), they each produce a corresponding indigo derivative having a different color. According to the present invention, "indigo derivative" also refers to an asymmetric indigo, i.e., an indigo derived from the dimerization of two different indoxyl derivatives, or an indoxyl or indoxyl derivative. Dyeing of fabric with an asymmetric indigo is achieved according to the present invention when a solution comprising two or more different indole derivatives, or indole and one or more indoxyl derivatives, is contacted with an enzyme in step a).For example, when two different indole derivatives, or an indole and an indole derivative, are contacted with the first immobilized enzyme (step a), two different indoxyl derivatives, or an indoxyl and an indoxyl derivative, are obtained, which are then contacted with a fabric according to process step c). When such two different indoxyl derivatives, or an indoxyl and an indoxyl derivative, are converted to at least one dye according to process step d), three different indigo derivatives, i.e., two different asymmetric indigo derivatives and one asymmetric indigo derivative (e.g., as shown in the scheme below), are obtained, and thus the fabric is dyed with one or more dyes, one of which is an asymmetric indigo.

[0027] Scheme 5 [ka]

[0028] Referring to Scheme 5, the two different indole derivatives are compounds IIa (4-methoxyindole) and IIb (7-chloroindole), the two different indoxyl derivatives are compounds IIIa (4-methoxyindoxyl) and IIIb (7-chloroindoxyl), and the three different indigo derivatives are compounds Ia (4,4'-dimethoxyindigo or 4-methoxy-2-(4-methoxy-3-oxo-1,3-dihydro-2H-indol-2-ylindene)-1,2-dihydro-3H-indole). Indole derivatives Ib (7,7'-dichloroindigo or 7-chloro-2-(7-chloro-3-oxo-1,3-dihydro-2H-indol-2-ylindene)-1,2-dihydro-3H-indol-3-one; symmetrical indigo derivatives), Ib (7,7'-dichloroindigo or 7-chloro-2-(7-chloro-3-oxo-1,3-dihydro-2H-indol-2-ylindene)-1,2-dihydro-3H-indol-3-one; symmetrical indigo derivatives), and Ic (7-chloro-4'-methoxyindigo or 7-chloro-2-(4-methoxy-1,3-dihydro-3-oxo-2H-indol-2-ylindene)-1,2-dihydro-3H-indol-3-one; asymmetrical indigo derivatives). Thus, in step d) of the process, when at least two indole derivatives, or indole and at least one indole derivative, are contacted with the enzyme, one or more indigo derivatives are obtained, thereby achieving dyeing of a textile with one or more dyes according to the method of the present invention.

[0029] According to the present invention, a "first immobilized enzyme" refers to any enzyme capable of catalyzing the conversion of a first dye precursor to a second dye precursor according to step a) of the method of the present invention. For example, the first immobilized enzyme is an immobilized enzyme capable of catalyzing the conversion (i.e., oxidation) of indole and / or its derivatives to indoxyl and / or its derivatives.

[0030] According to the present invention, in step a), contacting various compounds with an enzyme, e.g., contacting a first dye precursor with an immobilized enzyme, means that such compounds are enzymatically converted by contacting a stream of a solution comprising at least such a first dye precursor with the enzyme, whereby the dissolved first dye precursor contained in such solution is sequentially contacted with the immobilized enzyme and enzymatically converted to a second dye precursor.

[0031] According to the present invention, contacting a fabric with a solution comprising at least a second dye precursor means wetting the fabric with such solution, whereby the fabric is impregnated with the solution in which the second dye precursor is dissolved.

[0032] According to the present invention, "immobilized" or "immobilization" refers to the method of immobilizing an enzyme. Immobilization of an enzyme is a common method known in the art, including binding, preferably covalent binding, of such enzymes to supports, such as epoxy-activated resins (e.g., methacrylate copolymers, e.g., Eupergit®, SepaBeads®, Relizyme®, Purolite®), cellulose, agarose, polystyrene-based ion exchange resins, aminoacrylate resins, hydrogels (immobilization by occlusion; e.g., agarose, alginate, carrageenan, or gelatin), chelating supports (e.g., Ni-Sepharose®, IDA-Sepharose®, NTA-Sepharose®, IDA-Agarose, and derivatives thereof), etc. The type of support used to immobilize the enzyme depends on the exposed groups of the enzyme. For example, if surface amino groups are exposed on the enzyme, an epoxy-activated resin is used as the support: the amino groups covalently bond to the epoxy groups of the epoxy-activated resin, and the enzyme is thus immobilized on the epoxy-activated resin. According to the present invention, immobilization is carried out by incubating the enzyme and the epoxy-activated resin in a 100 mM potassium phosphate solution at pH 8.0 containing 0.5 M NaCl for 12 hours with stirring. Advantageously, after immobilization, the remaining (unreacted) activated groups on the support are deactivated: for example, the deactivation of the remaining activated groups is carried out by incubating 10 mM ethanolamine or 10 mM glycine in a solution comprising the epoxy-activated resin.

[0033] Suitable epoxy-activated resins have a particle size in the range of 100-1100μ, e.g., 150-300μ, or 200-500μ, or 250-1000μ, with an average pore size in the range of 300-1800 Å, e.g., 300-600 Å, or 1200-1800 Å. In one embodiment of the invention, the enzyme is immobilized at a ratio in the range of 5-75 mg (semi-)purified enzyme per gram of wet support, more preferably in the range of 15-25 mg (semi-)purified enzyme per gram of wet support.

[0034] In the methods of the present invention, mutant enzymes (e.g., genetically engineered enzymes) are used, for example, to increase catalytic efficiency or to provide improved binding to a support. For example, if the support used to immobilize the enzyme is an epoxy-activated resin, the enzyme can be modified, preferably through the introduction of a hexalysine (6xLys) or hexahistidine (6xHis) tag sequence at the N-terminus, to increase binding to the support.

[0035] According to the present invention, "spaced apart" refers to positioning the first immobilized enzyme and the textile such that conversion of the second dye precursor to an insoluble dye does not occur at and / or near the first immobilized enzyme, but instead occurs after the solution containing the second dye precursor comes into contact with the textile. Therefore, conversion of the second dye precursor to an insoluble dye is obtained directly on the textile. According to the present invention, the textile is thus positioned downstream of the immobilized enzyme in the direction of solution flow. The enzyme is immobilized so that it is confined and cannot flow with the solution flow. Such spacing can be achieved, for example, by placing the immobilized enzyme (or enzyme system) and the textile in different containers or chambers, or by confining the immobilized enzyme (or enzyme system) and the textile to different regions in the same container or chamber. The solution parameters, such as flow rate, temperature, and contact time of the solution with the enzyme and fabric, are selected to ensure that conversion of the second dye precursor to an insoluble dye occurs substantially on the fabric, i.e., after the solution comprising at least the second dye precursor contacts the fabric.

[0036] Therefore, in accordance with the present invention, conversion of the second dye precursor to an insoluble dye (i.e., precipitation) at and / or near the first immobilized enzyme is substantially prevented. The inventors have discovered that precipitation of the insoluble dye at and / or near the enzyme results from a loss of enzyme activity; the precipitated insoluble dye negatively impacts substrate-enzyme interactions and furthermore appears to prevent contact of the substrate (i.e., the first dye precursor) with the immobilized enzyme.

[0037] Another advantage of the present invention is that the use of reducing agents is avoided, and therefore no wastewater containing reducing agents is produced, making the process environmentally friendly, safe, cost-effective and sustainable.

[0038] When the method is carried out under standard dyeing conditions and the second dye precursor is indoxyl and / or its derivatives, their dimerization to indigo and / or its derivatives is spontaneous. According to the method of the present invention, such spontaneous dimerization occurs after a solution comprising at least said indoxyl and / or its derivatives is contacted with a textile (spaced apart from the first immobilized enzyme). The method of the present invention makes it possible to use the enzymatic synthesis of indigo and / or its derivatives to dye textiles in an industrial-scale process.

[0039] Advantageously, the process of the invention is a continuous process, which allows the addition of the first dye precursor upstream of step a), so that the solution comprising the dye precursors is continuously contacted with the immobilized enzyme.

[0040] According to one embodiment of the method of the present invention, the solution obtained after step d) is a waste solution comprising unreacted first dye precursor that was not converted to the second dye precursor in step a).

[0041] According to one embodiment of the method of the present invention, at least a portion of the waste solution is recycled to the first immobilized enzyme after step d) and reused in the method. In this embodiment, unreacted first dye precursor present in the waste solution can be converted to a second dye precursor, which can then be subjected to steps b) to d) of the method of the present invention. This embodiment optimizes the use of the first dye precursor, for example, by repeating the method steps, i.e., by directing the solution flow to contact the first immobilized enzyme several times, thereby completely converting the first dye precursor to the second dye precursor. By repeating the method steps, different color shades can be provided for the dyed textile; in this way, the method is carried out until the desired color shade for the textile is obtained. Furthermore, this embodiment avoids the disposal of waste solution that still contains useful solutes, such as unreacted first dye precursor, buffers, cofactors, etc.

[0042] Additionally, the first dye precursor can be added to the waste solution that is returned to the immobilized enzyme after step d), thereby allowing the method of the invention to be carried out continuously.

[0043] In the following paragraphs, the method will be described in detail with reference to an embodiment in which the insoluble dye is indigo, the second dye precursor is indoxyl, and the first dye precursor is indole. The scope of the invention is not limited to these exemplified compounds. According to an embodiment of the present invention, the first immobilized enzyme is an oxidase, whereby indole and / or its derivatives are oxidized to indoxyl and / or its derivatives upon contact with the oxidase.

[0044] According to the present invention, "oxidase" refers to any enzyme capable of catalyzing the oxidation of its substrate, e.g., oxidoreductase (EC 1). Suitable oxidoreductases are monooxygenases (EC 1.13); preferably, flavin-containing monooxygenases (FMOs) (EC 1.14.13.8), and more preferably, microbial flavin-containing monooxygenases (mFMOs). Alternatively, the monooxygenases are Baeyer-Villiger monooxygenases (BVMOs). Monooxygenases, particularly FMOs and mFMOs, provide good conversion and conjugation of many first dye precursors, e.g., indole and / or its derivatives, with favorable specificity for converting many indole derivatives, and are thus useful in the present invention. Baeyer-Villiger monooxygenases (BVMOs) share similar homology to FMOs and are thus also useful in the present invention. A particularly suitable oxidase for use in the present invention is mFMO from Methylophaga sp., more preferably strain SK1. This type of mFMO is highly soluble in aqueous solution, providing highly concentrated solutions, thus increasing the amount of second dye precursor 113, e.g., indole and / or its derivatives (synthesized by this type of mFMO). Furthermore, this type of mFMO is not specific for indole or one particular indole derivative, and therefore can convert many indole derivatives to the corresponding indoxyl derivatives. However, various analogs of the Methylophaga mFMO are also encompassed within the scope of the present invention. Generally, mutant enzymes, e.g., recombinant oxidases, are used in accordance with the present invention, for example, to improve the oxidation efficiency of the first dye precursor.

[0045] According to one embodiment of the invention, the method also comprises using a second immobilized enzyme, preferably an immobilized cofactor-regenerating enzyme.

[0046] According to the present invention, a "second immobilized enzyme" is any enzyme that assists and / or completes the conversion of a first dye precursor to a second dye precursor catalyzed by a first immobilized enzyme. In particular, the second immobilized enzyme can support such conversion by regenerating a cofactor used by the first immobilized enzyme; in this case, the second immobilized enzyme is an immobilized cofactor-regenerating enzyme.

[0047] According to the present invention, a "cofactor-regenerating enzyme" is any enzyme capable of regenerating (i.e., generating) the cofactor used by the first immobilized enzyme catalyzing the conversion reaction of the first dye precursor (step a) of the method of the present invention).

[0048] The second immobilized enzyme can thus produce the cofactor used by the first immobilized enzyme from a substrate that is less expensive than the cofactor used by said first immobilized enzyme.

[0049] Suitable cofactor-regenerating enzymes are known in the art, such as glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), formate dehydrogenase (FDH), and variants thereof. Preferably, the cofactor-regenerating enzyme is phosphite dehydrogenase (PTDH); examples of suitable PTDHs are disclosed in WO 2004 / 108912. In particular, when an oxidase, particularly an FMO, is used as the first immobilized enzyme, the use of dehydrogenases, such as GDH, FDH, and especially PTDH, is advantageous. GDH, FDH, and PTDH regenerate the cofactors used by such oxidases using glucose, formate, and phosphite, respectively, which are significantly cheaper than the cofactors used by the oxidases and are widely commercially available. A recombinant cofactor-regenerating enzyme is used in accordance with the present invention, for example, to improve the cofactor regeneration efficiency of the first dye precursor.

[0050] Preferably, the first and second enzymes are both immobilized to provide an enzyme system.

[0051] According to one embodiment of the invention, the immobilized enzyme system is spaced from the fabric and the solution flows from the enzyme system to the fabric. The method of the invention avoids or at least greatly reduces precipitation of insoluble dyes at or near the immobilized enzyme system.

[0052] According to another embodiment of the present invention, the first immobilized enzyme and the second immobilized enzyme are provided as immobilized fusion enzymes, thus providing a fusion system. Preferably, the first immobilized enzyme is an oxidase, more preferably a monooxygenase, even more preferably a microbial flavin-containing monooxygenase (mFMO). The second enzyme is preferably a cofactor-regenerating enzyme, more preferably at least one dehydrogenase selected from the group consisting of glucose dehydrogenase (GDH), phosphate dehydrogenase (PTDH), and formate dehydrogenase (FDH), most preferably phosphate dehydrogenase (PTDH). Thus, according to a preferred embodiment, the fusion system is a PTDH-mFMO fusion enzyme.

[0053] Fusion of enzymes is a technique known in the art. A suitable fusion enzyme comprises a region from a first enzyme and a region from a second enzyme, each region providing an essential functional property.

[0054] The fusion enzymes are immobilized via surface-exposed groups in each region of the fusion enzyme, for example, the first enzyme region, the second enzyme region, or both regions. The carrier is selected depending on the surface-exposed groups in either region of the fusion enzyme.

[0055] The methods of the invention can use mutant fusion enzymes that have been genetically modified, for example, to be more effectively immobilized or to improve catalytic efficiency.

[0056] In one embodiment, a first immobilized enzyme or enzyme system is placed in a first container or chamber, and a textile is placed in a second container or chamber. Thus, according to this embodiment, step a) is carried out in at least a first chamber, and dyeing of at least a portion of the textile is carried out in at least a second chamber. Thus, according to said embodiment, conversion of a first dye precursor to a second dye precursor (step a) of the method of the present invention) occurs in a first chamber, while conversion of the second dye precursor to the dye occurs in the second chamber (fluidly connected to the first chamber).

[0057] According to one embodiment, the first dye precursor is enzymatically produced from one or more feedstock compounds. For example, indole (i.e., a representative first dye precursor) is enzymatically obtained using tryptophan as a feedstock compound.

[0058] According to an embodiment, the first dye precursor is enzymatically produced using one or more raw materials in one or more reactors different from the first and second chambers, and advantageously, according to an embodiment, such reactors are in fluid communication with at least the first chamber.

[0059] According to a specific embodiment, as described above, a first dye precursor is enzymatically generated in the first chamber from one or more source compounds.

[0060] As used herein, the term "source compound," as defined above, refers to a compound that is converted into a first dye precursor by one or more enzymatic reactions. Such enzymatic reactions are carried out by one or more source enzymes. According to a specific example, the source enzymes are immobilized.

[0061] According to a specific example, a starting compound (eg, tryptophan) is enzymatically converted to a derivative thereof, eg, a halogenated derivative.

[0062] As used herein, the term "starting material enzyme" thus refers to one or more enzymes capable of catalyzing the conversion of a starting material compound, e.g., tryptophan, to a first dye precursor, e.g., indole.

[0063] Scheme 6 shows a reaction scheme in which the first dye precursor (indole (II)) is enzymatically obtained from a starting compound (tryptophan (IV)). Reaction Scheme 6 [ka]

[0064] Referring to Scheme 6, compound IV is tryptophan (starting compound), compound II is indole (first dye precursor), compound III is indoxyl (second dye precursor), and compound I is indigo (insoluble dye). According to a specific example, the conversion of tryptophan to indole is carried out by a starting enzyme, such as tryptophanase, or a mutant thereof (e.g., a mutant having improved catalytic properties), and the conversion of the first dye precursor to the second dye precursor and the second dye precursor to the insoluble dye is carried out as described above. The reaction of Scheme 6 also applies to tryptophan, indole, indoxyl, and indigo derivatives.

[0065] As used herein, the term "tryptophan derivative" refers to tryptophan substituted with one or more substituents, as disclosed for indole, indoxyl, and indigo derivatives. For example, illustrative reactions involving tryptophan derivatives and the corresponding indole, indoxyl, and indigo derivatives are shown in Scheme 7. Scheme 7 shows illustrative reactions in which a starting compound (tryptophan (IV)) is converted to a halogenated derivative (6-bromotryptophan (IVd)). In this case, one or more starting enzymes, i.e., tryptophan halogenase and tryptophanase, are required to obtain the desired first dye precursor (6-bromoindole (IId)).

[0066] Scheme 7 [ka]

[0067] Referring to Scheme 7, compound IV is tryptophan (the starting compound), compound IVd is 6-bromotryptophan (a halogenated derivative of the starting compound), and compound IId is Compound IIId is 6-bromoindole (first dye precursor), compound IIId is 6-bromoindoxyl (second dye precursor), and compound Id is 6,6'-dibromoindigo (also known as Tyrian purple; an insoluble dye). As described above, the conversion of tryptophan to 6-bromotryptophan and the conversion of 6-bromotryptophan to 6-bromoindole are carried out by source enzymes, such as tryptophan halogenase and tryptophanase, respectively, and the conversion of the first dye precursor to the second dye precursor and the second dye precursor to the insoluble dye are carried out as described above.

[0068] According to a specific example, the insoluble dye is obtained by an enzymatic cascade reaction process (i.e., enzymatic conversion starting from a starting compound to a second dye precursor), followed by a non-enzymatic reaction process (i.e., conversion of the second dye precursor to an insoluble dye occurring on the fabric).

[0069] Advantageously, by controlling the parameters of the process of the present invention, conversion of the second dye precursor to an insoluble dye directly on the fabric can be obtained, thereby avoiding precipitation of the insoluble dye at and / or near the enzyme.

[0070] According to a specific embodiment, the method of the present invention may further comprise the step of contacting a solution comprising at least source compounds with at least source enzymes to convert at least a portion of such source compounds into a first dye precursor, to obtain a solution comprising at least said first dye precursor, before carrying out step a).

[0071] Another object of the present invention is an apparatus according to claim 11 for carrying out a method for dyeing fabrics, i.e., an apparatus for dyeing fabrics, comprising a first chamber containing a solution comprising at least a first immobilized enzyme and at least a dye precursor, at least a second chamber containing a fabric, and means for generating a flow of the solution. The first chamber is in fluid communication with the second chamber, whereby the solution comprising at least the dye precursors can flow from the first chamber to the second chamber, at least a portion of the dye precursors being converted to dye to dye at least a portion of the fabric. The second chamber optionally comprises outlet means for removing the solution from the second chamber.

[0072] According to another embodiment of the present invention, the apparatus further comprises:

[0073] one or more reservoirs in fluid communication with at least the first chamber, such that a solution comprising the dye precursors can flow from the reservoirs to the first chamber; and / or one or more collection tanks in fluid communication with the outlet means of the second chamber; The compound comprises:

[0074] The reservoir according to this embodiment, when installed, is set to provide a solution comprising at least a first dye precursor, e.g., the first dye precursor, to the device of the present invention, in particular to the first chamber containing the first immobilized enzyme. Indeed, the reservoir is installed for the purpose of allowing a user to easily supply solutions and / or solutes (such solutions and / or solutes required for carrying out the method of the present invention by the device of the present invention) to the device of the present invention. Thus, for example, a solution comprising at least the first dye precursor is added to the reservoir, and then such solution is supplied to the first chamber containing the first immobilized enzyme (and ultimately the second enzyme) by means of a fluid connection between the reservoir and the first chamber. The recovery tank according to this embodiment allows for the collection of waste solution obtained after the textile has been dyed (e.g., after step d) of the method of the present invention).

[0075] According to another embodiment, the device of the present invention also comprises a means for generating a flow of the solution, said means for generating a flow of the solution, e.g., one or more pumps, allowing the flow of the solution contained in the device of the present invention.

[0076] According to a specific embodiment, the device can include one or more reactors in fluid communication with the device, preferably with the first chamber, which can contain a solution comprising one or more source enzymes and one or more source compounds.

[0077] According to a specific example, a solution comprising one or more source enzymes and one or more source compounds can be contained within the first chamber for enzymatically producing a first dye precursor.

[0078] Another object of the present invention is an immobilized fusion enzyme according to claim 19, i.e. an immobilized fusion enzyme comprising a carrier and at least a fusion enzyme immobilized on said carrier, said fusion enzyme being a PTDH-mFMO fusion enzyme.

[0079] The PTDH-mFMO fusion enzyme has been found to be particularly useful in the method of the present invention, namely dyeing of textiles, especially when said dyeing is carried out using indigo and / or its derivatives.

[0080] Another object of the present invention is the use according to claim 20, i.e. the use of an immobilized fusion enzyme in a method for dyeing textiles, said immobilized fusion enzyme being a regenerating enzyme-oxidase fusion enzyme, such as a PTDH-mFMO fusion enzyme, i.e. an immobilized fusion enzyme according to the above object of the present invention.

[0081] The immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, has been found to be particularly useful in dyeing methods (e.g., the dyeing method of the present invention) in which insoluble dyes, particularly indigo and / or its derivatives, are used as the dye. Indeed, the immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, provides good reaction rates and yields in the oxygenation of the first dye precursor, particularly indole and / or its derivatives. Therefore, the immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, is optimal for synthesizing insoluble dyes, particularly indigo and / or its derivatives, and for performing the dyeing method of the present invention. Another object of the present invention is a method according to claim 21, namely the following steps: a') converting tryptophan or a derivative thereof in the presence of at least tryptophanase to obtain indole and / or a derivative thereof; b') hydroxylating the indole and / or derivative thereof obtained in step a') in the presence of at least an oxidase to obtain indoxyl or a derivative thereof; and c') converting the indoxyl or its derivative obtained in step b') into indigo and / or an indigo derivative. The present invention relates to a method for producing indigo and / or its derivatives by enzymatic synthesis, comprising:

[0082] The reaction scheme for the process of the present invention is shown in Scheme 6 above.

[0083] The method of the present invention provides a synthesis of indigo and / or indigo derivatives from tryptophan or a tryptophan derivative as a starting material through enzymatic cascade reaction steps (steps a') and b') and a non-enzymatic step (step c')). The method of the present invention is particularly advantageous for producing indigo and / or indigo derivatives, such as Tyrian purple, in a cost-effective manner.

[0084] The method of the present invention also makes it possible to produce indigo and / or indigo derivatives on an industrial scale.

[0085] As used herein, "tryptophan derivative" refers to tryptophan substituted with groups and positions as defined above for indole and indoxyl derivatives.

[0086] The tryptophan derivative in step a') is preferably a halogenated tryptophan derivative obtained by halogenating tryptophan in the presence of at least a tryptophan halogenase and a halogen source. The reaction scheme of this specific example is shown in Scheme 7 above (where the halogenated derivative is a 6-bromo derivative).

[0087] According to a specific example, tryptophan is used as a raw material compound for the enzymatic production of indigo and indigo derivatives. Advantageously, the use of tryptophan as a raw material compound allows for cost-effective production of indigo and / or indigo derivatives.

[0088] As used herein, "halogenated derivative" refers to tryptophan, indole, indoxyl, and indigo substituted with a halogen, particularly fluorine, chlorine, bromine, or iodine, on one or more carbons at the 5, 6, 7, and 8 positions (for indigo, the 5', 6', 7', and 8' positions). For example, tryptophan halogenated derivatives are 6-bromotryptophan (compound IVd in Scheme 7 above) and 7-chlorotryptophan, indole halogenated derivatives are 6-bromoindole (compound IId in Scheme 7 above) and 7-chloroindole, indoxyl halogenated derivatives are 6-bromoindoxyl (compound IIId in Scheme 7 above) and 7-chloroindoxyl, and indigo halogenated derivatives are Tyrian purple (6,6'-dibromoindigo; compound Id in Scheme 7 above) and 7,7'-dichloroindigo.

[0089] According to a specific embodiment, the enzymes used in the methods of the invention, as well as in the staining methods of the invention, are isolated enzymes (preferably purified or semi-purified), so that the methods of the invention are carried out without expression in bacteria. Preferably, said isolated enzymes are immobilized enzymes.

[0090] The process of the present invention is carried out in one reactor, thereby providing a one-pot reaction. Thus, after step c'), indigo or its derivatives are obtained as a solid precipitate, which is isolated (filtered) from the reaction mixture. In other words, when the process of the present invention is carried out according to a one-pot reaction, the obtained indigo or its derivatives are isolated and purified from the reaction mixture.

[0091] According to a particular embodiment, step c') is carried out in the presence of a fabric, so that at least a portion of the resulting indigo or indigo derivative is deposited on the fabric.

[0092] According to a specific embodiment, steps a') to c') are carried out in an aqueous medium, and a flow of aqueous medium is generated, whereby steps a') to c') are carried out in different reactors or at different locations in one reactor. This embodiment is advantageous for carrying out each step according to its optimum parameters, such as temperature, pH, amount of enzyme substrate, etc.

[0093] According to an exemplary embodiment, the process of the present invention is carried out in a two-stage packed bed reactor, such as a Spinchem® rotating bed reactor (RBR).

[0094] The process of the present invention is preferably carried out in an aqueous medium. Such an aqueous medium preferably has a neutral or slightly alkaline pH, for example, 7.0 to 10, preferably 7.4 or 8. Such an aqueous medium may thus contain a buffer, for example, a potassium phosphate buffer. Some tryptophan derivatives, such as 6-bromotryptophan, are poorly soluble in aqueous media, and the process of the present invention is carried out using the tryptophan derivative suspended in an aqueous medium.

[0095] Step a') comprises cleaving a carbon-carbon bond on tryptophan or a tryptophan derivative in the presence of tryptophanase. Such a tryptophan derivative is preferably synthesized by carrying out step a').

[0096] The reaction of step a') is shown in Scheme 8. Scheme 8 [ka]

[0097] where compound IV is tryptophan, compound II is indole, compound V is pyruvate, and TRPase is tryptophanase. It has been observed that tryptophanase is used to catalyze the conversion of tryptophan derivatives to indole derivatives, e.g., the conversion of 6-bromotryptophan to 6-bromoindole.

[0098] Tryptophanase (systematic name: L-tryptophan indole lyase (deaminating; pyruvate forming)) is a known enzyme that cleaves the carbon-carbon bond of tryptophan to release indole. They can use pyridoxal phosphate (PLP) as a cofactor. A suitable tryptophanase for use in the process of the present invention is tryptophanase from Escherichia coli NEB® 10β.

[0099] PLP is optionally added to the reaction mixture of step a') to improve the yield of conversion of tryptophan or its derivatives.

[0100] Step b') of the present invention comprises hydroxylating the indole or its derivative obtained from step a') at least at carbon 3 in the presence of an oxidase and O. Step b') thus provides indoxyl or its derivative.

[0101] Suitable oxidases are those described above, such as the bacterial FMO and Baeyer-Villiger monooxygenase from Methylophaga sp. strain SK1. The oxidase requires O2, i.e., oxygen, in the reaction mixture to catalyze the hydroxylation of indole or its derivatives. The O2 required to carry out step b') is typically dissolved oxygen in the aqueous reaction mixture, or the reaction mixture may be saturated with O2 to achieve maximum conversion of indole or its derivatives.

[0102] Step c') is non-enzymatic and involves the oxidation and dimerization of indoxyl or its derivatives to indigo and / or its derivatives.

[0103] According to a particular embodiment, step c') of the process of the invention is carried out in the presence of a textile, the textile being at least spaced from said oxidase, so that the production of indigo or an indigo derivative is carried out directly on the textile and at least a portion of said textile is dyed, advantageously in such a case that precipitation of indigo or an indigo derivative in the vicinity of the enzyme and / or in the vicinity is substantially avoided.

[0104] Step c') can occur spontaneously after step b') (provided that the O2 concentration is adequate to oxidize indoxyl or a derivative thereof) or can be induced (e.g., by adding O2 to the reaction mixture).

[0105] The O required to carry out step c') is typically dissolved oxygen in the aqueous reaction mixture, or the reaction mixture may be saturated with O to achieve maximum conversion of the indole or its derivative.

[0106] In one embodiment, the tryptophan derivative of step a') is a tryptophan halogenated derivative obtained by a further step i) of halogenating tryptophan in the presence of at least a tryptophan halogenase.

[0107] Tryptophan halogenases are known enzymes that can catalyze the halogenation of tryptophan at various positions. Tryptophan halogenases are typically flavin-dependent halogenases, i.e., they use FAD or FADH2 as a cofactor. A preferred tryptophan halogenase for the process of the present invention is a thermophilic tryptophan halogenase, such as the thermophilic tryptophan halogenase from Streptomyces violaceus niger.

[0108] According to a specific example, the tryptophan halogenase is the thermophilic tryptophan halogenase of Streptomyces violaceus niger strain SPC6.

[0109] For example, a thermophilic tryptophan halogenase has the following sequence: LNNVVIVGGGTAGWMTASYLKAAFGDRIDITLVESGHIGAVGVGEATFSDIRHFFEFLGLKEKDWMPACNATYKLAVRFENWREKGHYFYHPFEQMRSVNGFPLTDWWLKQGPTDRFDKDCFVMASVIDA GLSPRHQDGTLIDQPFDEGADEMQGLTMSEHQGKTQFPYAYQFEAALLAKYLTKYSVERGVKHIVDDVREVSLDDRGWITGVRTGEHGDLTGDLFIDCTGFRGLLLNQALEEPFISYQDTLPNDSAVALQ VPMDMERRGILPCTTATAQDAGWIWTIPLTGRVGTGYVYAKDYLSPEEAERTLREFVGPAAADVEANHIRMRIGRSRNSWVKNCVAIGLSSGFVEPLESTGIFFIHHAIEQLVKNFPAADWNSMHRDLYNSAVSHVMDGVREFLVLHYVAAKRNDTQYWRDTKTRKIPDSLAERIEKWKVQLPDSETVYPYYHGLPPYSYMCILLGMGGIELKPSPALALADGGAAQREFEQIRNKTQRLTEVLPKAYDYFTQ (SEQ ID NO: 1).

[0110] This type of tryptophan halogenase preferably catalyzes the halogenation of the carbon at the 6-position of tryptophan, and is therefore suitable for producing Tyrian purple (6,6'-dibromoindigo) according to the process of the present invention.

[0111] Another tryptophan halogenase suitable for the process of the present invention is tryptophan halogenase PrnA, preferably Pseudomonas fluorescens tryptophan halogenase PrnA, which preferably catalyzes the halogenation of tryptophan at the 5- or 7-carbon position of tryptophan.

[0112] For example, tryptophan halogenase (PrnA) has the following sequence: (SEQ ID NO: 2).

[0113] In some embodiments, the tryptophan halogenase is a recombinant enzyme, i.e., a mutant tryptophan halogenase, such as a mutant thermophilic tryptophan halogenase from Streptomyces violaceus niger strain SPC6 or a mutant tryptophan halogenase PrnA.

[0114] To be converted to the indigo halogen derivative, tryptophan must be reacted with a halogen in the presence of a tryptophan halogenase, and thus, in practice, this embodiment requires a halogen source in the reaction mixture. A preferred halogen source for the process of the present invention is a halogen salt, i.e., a salt whose anion is a halogen ion. Suitable halogen salts include magnesium, silver, sodium, potassium, lithium, and calcium halogen salts, such as NaCl, KCl, KI, LiCl, CuCl, CuBr, AgCl, CaCl, CaBr, ClF, MgCl, MgBr, and the like.

[0115] This specific example is advantageously carried out at a temperature of 20 to 60°C, preferably 25 to 40°C, and more preferably 30°C, for a period of 30 minutes to 4 hours, preferably 1 to 3 hours, and even more preferably about 2 hours.

[0116] According to a specific embodiment, a cofactor-regenerating enzyme is used to regenerate the cofactor required by the enzyme used in the process of the invention.

[0117] According to a specific example, step b') is carried out in the presence of at least an enzyme suitable for regenerating the cofactor NADPH. Preferably, the enzymes suitable for regenerating the cofactor NADPH are glucose dehydrogenase (GDH), phosphate dehydrogenase (PTDH), and formate dehydrogenase (FDH), as described below, and more preferably PTDH, as described below, thereby providing an FMO-NADPH-regenerating enzyme system. Advantageously, this example provides an enzyme system in which an expensive cofactor (i.e., NADPH) is regenerated by consuming a cheaper cofactor (e.g., glucose, phosphite, or formate). For example, an oxidase, such as an FMO, can use NADPH (generated by an NADPH-regenerating enzyme using cheaper cofactors, such as glucose, phosphite, and formate) as a cofactor.

[0118] In another embodiment, halogenation of tryptophan to obtain its halogenated derivative is carried out in the presence of flavin reductase and an NAD(P)H-regenerating enzyme selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH) (more preferably, PTDH), thereby providing a tryptophan halogenase-flavin reductase-NAD(P)H-regenerating enzyme system.

[0119] Flavin reductase (EC 1.5.1.30) is an enzyme that catalyzes the following reaction: Riboflavin + NADPH + H + → Reduced riboflavin + NADP + H + On the other hand, NAD(P)H-regenerating enzymes are enzymes that generate NADH or NADPH, such as GDH, PTDH, and FDH. Advantageously, this embodiment provides an enzyme system in which an expensive cofactor (i.e., FAD, NADH, or NADPH) is regenerated by consuming a cheaper cofactor (e.g., glucose, phosphite, or formate), improving the industrial feasibility of the process of the present invention. For example, tryptophan halogenase can use FAD as a cofactor (FAD is generated by flavin reductase using NADH or NADPH as a cofactor; NADH or NADPH is generated by NAD(P)H-regenerating enzymes using a cheaper cofactor, e.g., glucose, phosphite, or formate).

[0120] A preferred flavin reductase useful in the process of the present invention is the flavin reductase of Bacillus subtilis, particularly the flavin reductase of Bacillus subtilis strain WU-S2B.

[0121] For example, flavin reductase has the following sequence: MKVLVLAFHPNMEQSVVNRAFADTLKDAPGITLRDLYQEYPDEAIDVEKEQKLCEEHDRIVFQFPLYWYSSPPLLKKWLDHVLLYGWAYGTNGTALRGKEFMVAVSAGAPEEAYQAGGSNHYAISELLRPFQATSNFIGTTYLPPYVFYQAGTAGKSELAEGATQYREHVLKSF (SEQ ID NO: 3).

[0122] In order to improve the yield and industrial feasibility of the process of the present invention, variants of the various enzymes used in the process of the present invention may be used.

[0123] To improve the catalytic activity of FMOs toward 6-bromoindole, FMO mutants selected from the group consisting of W319A, C78I, C78I Y207W, and C78I Y207W W319F were discovered. Additionally, NADPH-regenerating enzymes, such as PTDH as disclosed in WO 2004 / 108912, are mutants that improve NADPH production.

[0124] According to a specific example, when an enzyme requires a cofactor, such enzyme is provided as a fusion enzyme with a cofactor-regenerating enzyme.

[0125] For example, tryptophan halogenase and flavin reductase are provided as fusion enzymes, and the FMO and NADPH-regenerating enzyme are provided as fusion enzymes, preferably as a PTDH-FMO. According to this embodiment, only three individual enzymes are used in the process of the present invention (if optional step i) is performed: tryptophan halogenase-flavin reductase fusion enzyme, tryptophanase, and FMO-NADPH-regenerating fusion enzyme. The NADPH-regenerating portion of the final fusion enzyme can regenerate NADPH, which is required for both the FMO and flavin reductase domains of the fusion enzyme, using their inexpensive substrate, i.e., phosphite, as a starting material.

[0126] Yet another object of the present invention is the dyed fabric obtained via the dyeing process of the present invention.

[0127] Furthermore, the object of the present invention is the dyed fabric obtained via the process of the present invention, when step c') of the process is carried out in the presence of a fabric.

[0128] Next, the object and embodiments of the present invention will be described in detail with reference to the drawings. The object of the present invention is a method for dyeing a fabric 22 comprising the enzymatic synthesis of a dye precursor, said method comprising the following steps: a) contacting a solution comprising at least a first dye precursor 112 with at least a first immobilized enzyme 12 to convert at least a portion of said at least first dye precursor 112 into at least a second dye precursor 113, to obtain a solution comprising at least a second dye precursor 113; b) generating a flow of the solution comprising the at least second dye precursor 113, thereby causing the solution comprising the at least second dye precursor 113 to flow from the first immobilized enzyme 12 onto the fabric; c) contacting the solution comprising the at least second dye precursor 113 with the fabric; and d) converting at least a portion of the at least second dye precursor 113 to at least a dye 111, thereby dyeing at least a portion of the fabric 22. For example, as shown in FIG. 1, the at least first immobilized enzyme 12 is characterized by being spaced apart from the fabric 22.

[0129] FIG. 1 shows a schematic diagram of the dyeing method of the present invention, in particular showing the conversion of the first dye precursor 112 to the second dye precursor 113 by the first immobilized enzyme 12, followed by the flow of a solution comprising at least the second dye precursor 113 onto a fabric 22 (spaced apart from the first immobilized enzyme 12), and finally the conversion of the second dye precursor 113 to the insoluble dye 111 directly on the fabric 22.

[0130] More specifically, with reference to FIG. 1 , the dyeing method of the present invention is disclosed with reference to the indole to indigo route. The dyeing method provides dyeing of a fabric 22 as a result of the following steps: a flow of a solution comprising an indole dye precursor 112 is brought into contact with at least an immobilized enzyme 12 or enzyme system (step a)), thus obtaining the conversion of at least a portion of the indole to indoxyl 113 by an enzyme-catalyzed reaction. The solution then comprises indoxyl 113. The flow of the solution allows the solution comprising indoxyl 113 to come into contact with the fabric 22; advantageously, parameters such as the flow rate are controlled so that the solution reaches the fabric 22 immediately or shortly after the synthesis of indoxyl 113. In this way, the conversion of indoxyl 113 to indigo 111 is obtained in the fabric 22, and dyeing of the fabric 22 is achieved. In particular, when the fabric 22 is wetted with the solution comprising indoxyl 113, at least a portion of the indoxyl 113 is converted to indigo 111 directly on the fabric 22.

[0131] The dyeing method according to the present invention generates a flow of solution first to the enzyme 12 (or enzyme system) and then from the enzyme 12 (or enzyme system) to the fabric 22, as shown in FIG. 1 (arrows indicate the direction of solution flow). The flow of solution can be in any direction, as long as the solution is supplied first to the enzyme 12 and then from the enzyme 22 to the fabric 22. For example, other directions of solution flow are bottom-to-top when the first immobilized enzyme 12 is below the fabric 22, or top-to-bottom when the first immobilized enzyme 12 is above the fabric 22, and may be circular when the first immobilized enzyme 12 and the fabric 22 are contained in different zones of a single annular, e.g., donut-shaped, chamber, e.g., when they are held in diametrically opposed zones within such a donut-shaped chamber.

[0132] The use of flow in the dyeing method of the present invention allows a solution comprising at least a second dye precursor 113 to contact the fabric 22 (spaced apart from the first immobilized enzyme 12) and then conversion of the second dye precursor 113 to the insoluble dye 111 occurs, thereby preventing precipitation of indigo 111 at and / or near the immobilized enzyme 12.

[0133] In particular, if the second dye precursor 113 spontaneously converts to the insoluble dye 111, for example, if the second dye precursor 113 is indoxyl and / or its derivatives and the insoluble dye 111 is indigo and / or its derivatives, a flow of a solution comprising at least the second dye precursor 113 is generated, whereby the solution flows onto the fabric 22 before the second dye precursor 113 spontaneously converts and precipitates as the insoluble dye 111 at and / or near the immobilized oxidase.

[0134] If the second dye precursor 113 does not spontaneously convert to the insoluble dye 111, or does not spontaneously convert in a suitable amount, it is possible to change conditions, such as pH and / or temperature, and / or add reagents and / or supply gas, such as oxygen, in the presence of the textile 22 and the second dye precursor 113 to promote the conversion of the second dye precursor 113 to the insoluble dye 111. Conversely, in step a) of the dyeing process, the solution conditions can be controlled to prevent conversion until the solution reaches the textile.

[0135] Furthermore, according to step a) of the dyeing process, two or more first dye precursors 112 can be contacted with the enzyme 12, thereby obtaining two or more different second dye precursors 113. These two or more different second dye precursors 113 are then converted according to step d) of the dyeing process to obtain one or more different dyes 111 on the textile 22; finally, the conversion of these two or more different second dye precursors 113 in step d) is achieved by adding further reagents and / or modifying the parameters of the solution comprising them, if such conversion does not occur spontaneously or in suitable amounts. An example in which two or more different dye precursors are contacted with the enzyme 12 is shown in Scheme 5 above. Two or more different enzymes 12 are required when two or more different first dye precursors 112 are used in accordance with the dyeing method of the present invention; for example, when two or more different first dye precursors 112 require different enzymatic reactions to be converted to different second dye precursors 113, or when two or more different first dye precursors 112 are not substrates for the same enzyme 12.

[0136] The solution comprising at least a first dye precursor 112, which may contain other solutes, is the solution that is contacted with the first immobilized enzyme 12 or enzyme system in step a) of the dyeing method. The solution comprising at least a second dye precursor 113, which is the solution obtained after step a) after at least a portion of said first dye precursor 112 has been converted to said second dye precursor 113, may also contain other solutes, for example some unreacted first dye precursor 112.

[0137] As mentioned above, solutions according to the present invention can contain other functional solutes, such as buffers, cofactors, and oxygen and / or peroxide scavengers (e.g., catalase). Preferably, the concentration of substrate contained in the aqueous solution saturates the catalytic enzyme, so that the first immobilized enzyme can effectively catalyze the conversion of the first dye precursor 112 to the second dye precursor 113. When the first dye precursor 112 is indole, the first immobilized enzyme 12 is mFMO, and the second immobilized enzyme is PTDH, an illustrative solution according to the present invention comprises 100 mM potassium phosphate buffer (pH 8.0), 0.5 M NaCl, 100 μM NADPH, 20 mM sodium phosphite, and 1 nM beef liver catalase, along with water as the solvent.

[0138] The dyeing method of the present invention can provide for dyeing of fabric 22 in a batch or continuous manner. To be carried out in a continuous manner, it is required to add the first dye precursor 112, for example, to a solution before step a), so that a solution comprising at least the first dye precursor 112 is continuously contacted with the first immobilized enzyme 12, and the second dye precursor 113 is continuously synthesized. Advantageously, the first dye precursor 112 is added to maintain the first immobilized enzyme 12 in a saturated state. To carry out the dyeing method of the present invention in a continuous manner, it is also required to add other solutes, for example, cofactors, buffers, and oxygen, for example, before step a).

[0139] The temperature and pH values ​​for the dyeing method of the present invention can be varied and are those commonly used in the enzymatic synthesis of insoluble dyes.

[0140] The temperature of the solution used in the dyeing method of the present invention is, for example, 20 to 40° C., preferably 25 to 30° C. The pH of the solution used in the dyeing method of the present invention is 7.0 to 10.0, preferably 7.5 to 9.0, more preferably 7.5 to 8.5, and most preferably 8.0.

[0141] The contact time of the first immobilized enzyme 12 with the solution comprising indole 112 can be varied to achieve different shadings of the dyed fabric, for example, by changing the flow rate of the solution.

[0142] The oxygen concentration in the solution is an important parameter for the overall dyeing yield because oxygen participates in the conversion of the first dye precursor to the second dye precursor and / or the conversion of the second dye precursor to an insoluble dye (e.g., when the first dye precursor is indole and / or its derivatives, the second dye precursor is indoxyl and / or its derivatives, and the insoluble dye is indigo and / or its derivatives). Thus, the oxygen concentration in the solution is varied, for example, based on the amount of insoluble dye synthesized or the amount of fabric to be dyed. For example, to achieve maximum conversion of indole and / or its derivatives and indoxyl and / or its derivatives, the solution is preferably saturated with oxygen. The oxygen concentration is preferably monitored and controlled, and oxygen is added as required to maintain the solution saturated.

[0143] Other parameters of the dyeing process of the present invention are selected depending, for example, on what type of fabric is being dyed and what dye is selected as the final dye.

[0144] According to one embodiment of the dyeing method of the present invention, the resulting solution stream from step d), the so-called "waste solution," is directed back to the chamber or zone containing the immobilized enzyme system. The waste solution is the solution obtained after at least a portion of the indoxyl 113 has been converted to indigo 111 (fixed to the fabric 22), and in particular comprises unreacted first dye precursor 112, e.g., indole, if the first dye precursor 112 was not fully reacted by the enzyme 12 in step a) of the dyeing method of the present invention. This embodiment is shown in Figure 2. The waste solution comprising indole is fed back to the enzyme system, where the remaining indole is reacted, directly or indirectly, continuously or batchwise.

[0145] Preferably, the dyeing method provides a step of adding some first dye precursor 112 to the waste solution. Advantageously, said first dye precursor 112 is added to maintain said first immobilized enzyme 12 in a saturated state. This allows the dyeing method of the present invention to be carried out in a continuous manner.

[0146] In one embodiment of the present invention, the first immobilized enzyme 12 is an oxidase, as defined above. The use of an oxygenase is particularly beneficial when oxidizing the first dye precursor 112 to convert it to the second dye precursor 113, for example when the first dye precursor 112 is indoxyl and / or its derivatives, the second dye precursor 113 is indoxyl and / or its derivatives, and the insoluble dye 111 is indigo and / or its derivatives.

[0147] The dyeing method of the present invention further comprises the presence in the dyeing plant of a second immobilized enzyme, preferably an immobilized cofactor-regenerating enzyme as defined above. This provides an enzyme system in which the first immobilized enzyme catalyzes the conversion of the first dye precursor 112, and the immobilized cofactor-regenerating enzyme regenerates the cofactor required by the first dye precursor 112. The support used to immobilize the second immobilized enzyme may be the same as or different from the support used to immobilize the first immobilized enzyme, depending on the surface-exposed groups of the second enzyme. If possible, the same support is used to immobilize both the first and second enzymes.

[0148] The type of immobilized cofactor-regenerating enzyme depends on the cofactor used by the first immobilized enzyme 12. For example, if the first immobilized enzyme 12 is a flavin-containing monooxygenase (FMO; using NADPH as a cofactor), the immobilized cofactor-regenerating enzyme is at least a dehydrogenase that generates NADPH, such as a dehydrogenase selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH). PTDH is a soluble NADPH-regenerating enzyme obtained, for example, from Pseudomonas stutzeri, that uses phosphite as a substrate to catalyze the production of NADPH. Using an FMO (preferably mFMO) together with PTDH provides good oxidation rates and efficient regeneration of NADPH, making it effective in the synthesis of many second dye precursors 113, particularly indoxyl and / or its derivatives.

[0149] The solution used in the staining method and device of the present invention can contain a cofactor and / or a substrate for the second immobilized enzyme. For example, when GDH, PTDH, or FDH is used as the immobilized cofactor-regenerating enzyme, the solution can contain glucose, phosphite, or formate (i.e., the substrates for GDH, PTDH, and FDH, respectively).

[0150] As with the first enzyme, variants of the second enzyme (e.g., genetically engineered second enzymes) can be used in the staining methods of the invention, for example, to improve the regeneration of desired cofactors or to improve their binding properties to carriers.

[0151] In a specific embodiment of the present invention, the first enzyme and the second enzyme are provided as a fusion enzyme, as previously defined. This provides an enzyme system.

[0152] Referring to FIG. 3, another embodiment of the dyeing method of the present invention is shown. This embodiment provides for the production of a first dye precursor 112, such as indole, starting from one raw compound 114, such as tryptophan. According to this embodiment, the dyeing method of the present invention further comprises a step of contacting at least the raw compound 114 with at least a starting enzyme 14 to convert at least a portion of the raw compound 114 into the first dye precursor 112 and obtain a solution containing the first dye precursor 112, thereby performing steps a) to d) of the dyeing method of the present invention as described above with reference to FIGS. 1 and 2. When the first immobilized enzyme 12 and the starting enzyme 14 are spaced apart, a flow of the solution containing the first dye precursor 112 (obtained by converting the raw compound 114) is generated to bring the solution into contact with the enzyme 12, thereby causing the solution to flow from the starting enzyme 14 to the first immobilized enzyme 12, as shown in FIG. 3, for example. Since the waste solution resulting from step d) may contain unreacted starting compound 114, recycling of the waste solution to the reactor or area containing the starting enzyme 14 and / or the first chamber or area containing the first immobilized enzyme 12 is advantageous in order to optimize the conversion of unreacted starting compound 114 and / or first dye precursor 112.

[0153] In a further embodiment of the dyeing method of the present invention, step a) is carried out in the first chamber 11, and dyeing of the textile 22 is carried out in the second chamber 21. A solution comprising a first dye precursor 112 is provided in the first chamber 11, whereby the solution comes into contact with the first enzyme 12 contained in the first chamber 11, and the first dye precursor 112 is enzymatically converted to a second dye precursor 113. A solution flow is then generated, and the solution, now containing at least the second dye precursor 113, flows into the second chamber 21 containing the textile 22, whereby the solution and the second dye precursor 113 come into contact and impregnate the textile 22. Finally, the second dye precursor 113 is converted to a dye 111 directly on the textile 22.

[0154] The flow of the solution can be generated by any suitable means, such as a pump 50 or gravity (if the first chamber 11 is located above the second chamber 21).

[0155] The first chamber 11 is a container suitable for containing an enzyme system containing the enzyme 12 or a second immobilized enzyme and an aqueous solution. The first chamber 11 may include means for containing and retaining the first immobilized enzyme 12 (and ultimately the enzyme system) therein, such as one or more filters. The first chamber 11 may advantageously include one or more means or sensors for monitoring solution parameters, such as pH, solution temperature, oxygen concentration, flow rate, etc. The dimensions, shape, and material of the first chamber 11 are arbitrarily selected depending on many factors, such as the amount of fabric 22 to be dyed and the dimensions and shape of the second chamber 21.

[0156] The second chamber 21 is a container suitable for containing at least the fabric 22 and an aqueous solution, in which conversion of the second dye precursor 113 to the insoluble dye 111 takes place. The second chamber 21 may also include means for holding the fabric 22 in place to allow for a faster or more complete dyeing process. The second chamber 21 may advantageously include one or more means or sensors for monitoring solution parameters, such as pH, solution temperature, oxygen concentration, flow rate, etc. The size, shape and material of the second chamber 21 are selected depending on many factors, such as the amount of fabric to be dyed and the size and shape of the first chamber 11.

[0157] The first chamber 11 and the second chamber 21 are individually temperature controlled, for example by means of a jacket, for example a water jacket.

[0158] In Figure 4, the first chamber 11 is fluidly connected to the second chamber 21. The fluid connection is made by a fluid connector 11a, e.g., a tube or pipe, the dimensions, shape and material of which are selected by a person skilled in the art to ensure a fluid connection between the first and second chambers and to ensure that they can effectively contain the aqueous solutions circulating in the first chamber 11 and the second chamber 21 and are inert to the aqueous solutions. Such dimensions, shape and material are optionally varied according to, e.g., the amount of fabric to be dyed and the required flow rate for the solution. The fluid connector (e.g., a tube or pipe) can advantageously include probes for measuring parameters such as flow rate, temperature, solution pH and oxygen concentration, as well as portholes, portholes and / or doors for monitoring the dyeing method of the present invention and for taking, e.g., solution samples.

[0159] Another object of the present invention is an apparatus 10 for dyeing a fabric 22, said apparatus comprising a first chamber 11 containing a solution comprising at least a first immobilized enzyme 12 and at least a dye precursor, at least a second chamber 21 containing a fabric 22, and means 50 for generating a solution flow, said first chamber 11 being in fluid communication with said second chamber 21, so that a solution comprising at least a dye precursor flows from said first chamber 11 to said second chamber 21, and at least a portion of said dye precursor is converted to a dye 111 to dye at least a portion of said fabric 22, as shown, for example, in Figure 4. said second chamber 21 optionally comprises outlet means for removing said solution from said second chamber 21.

[0160] 4 shows the first chamber 11 containing the first immobilized enzyme 12 and the second chamber 21 containing the fabric 22. Arrows indicate the flow of a solution comprising at least a dye precursor from the first chamber 11 to the second chamber 21. Within the second chamber 21, at least a portion of the second dye precursor 113 is converted to an insoluble dye 111 to dye at least a portion of the fabric 22 contained in the second chamber 21.

[0161] 5 is a schematic diagram of one embodiment of the apparatus 10, in which the second chamber 21 has an outlet means 21a that is fluidly connected to the first chamber 11. Thus, waste solution can be discharged from the second chamber 21 and subsequently fed to the first chamber 11 (as indicated by the arrow in FIG. 5). The outlet means 21a can be, for example, any of the various tubes or pipes described above.

[0162] In order to carry out the dyeing method of the present invention in a continuous manner using the apparatus 10, it is advantageous to maintain a supply of the cofactor and substrate and the first immobilized enzyme 12 to the first chamber 11, thereby allowing the continuous production of the second dye precursor 113. For this reason, compounds (e.g., cofactor and substrate) are added to the first chamber 11 and / or to the waste solution (which is returned to the first chamber 11). The apparatus 10 of Figure 5 is therefore provided with means for such addition, comprising a supply means 11b connected to the first chamber 11 and / or a supply means 21b connected to the outlet means 21a.

[0163] One embodiment of the device 10 of the present invention further includes a means 50 for generating a solution flow, which allows the solution contained within the device 10 of the present invention to flow.

[0164] If the first immobilized enzyme 12 is not specific for only one substrate and is thus capable of converting different first dye precursors 112, different colors can be obtained for the dyed fabric 22 simply by changing the reagents provided to the apparatus 10. By changing the first dye precursor 112, without changing the apparatus 10 and / or the enzyme 12 contained therein, different dyes 111 can be obtained: these dyes 111 are suitable for dyeing the fabric 22 in the second chamber 21. For example, if the enzyme 12 contained in the first chamber 11 is the fusion enzyme PTDH-mFMO (which, together with its derivatives, can convert indole), providing a solution comprising indole to the apparatus 10 will provide a blue fabric. If, in the same dyeing method, a solution comprising 5-hydroxyindole is used and provided to the apparatus 10 instead of a solution comprising indole, a brown dye and a brown-dyed fabric will be obtained.

[0165] Figure 6 shows another embodiment of the device 10, further comprising a reservoir 31 in fluid communication with at least the first chamber 11, so that a solution comprising a dye precursor can flow from the reservoir 31 to the first chamber 11. Figure 7 shows another embodiment of the device 10, further comprising a collection tank 41 in fluid communication with the outlet means 21a of the second chamber 21.

[0166] The reservoir 31 may be any container capable of containing an aqueous solution, e.g., a solution comprising at least the first dye precursor 112, e.g., indole and / or its derivatives. The reservoir 31 may be a container from which a solution comprising at least the first dye precursor 112 is supplied to the apparatus 10 for carrying out the dyeing method of the present invention. Therefore, the reservoir 31 is advantageously configured to allow an operator to easily supply the solution and / or solutes therein, and its shape and dimensions are appropriately selected. The apparatus 10 of FIG. 6 comprises a means 50 for generating a solution flow, e.g., a pump, for causing the solution to flow from the reservoir 31 to the first chamber 11, as indicated by the arrow in FIG. 6, thus supplying the solution comprising the first dye precursor 112 to the first chamber 11.

[0167] FIG. 8 shows an apparatus 10 including both a reservoir 31 and a recovery tank 41. The apparatus of FIG. 8 is useful for practicing the dyeing method of the present invention. A solution comprising at least a first dye precursor is provided to the reservoir 31. A pump 50 then generates a solution flow from the reservoir 31 to the first chamber 11, where the solution is brought into contact with an enzyme. The solution, now containing a second dye precursor 113, flows from the first chamber 11 to the second chamber 21, where a fabric 22 is placed. At least a portion of the second dye precursor 113 is converted to dye 111, which dyes at least a portion of the fabric 22 in the second chamber 21, and a waste solution is obtained. The waste solution is removed from the second chamber 21 via outlet means 1a and collected in the recovery tank 41. Collection of such waste solutions is advantageous if they must be treated, for example, to remove any precipitates (e.g., insoluble dye 111) that may be present; such treatment is carried out in a recovery tank 41. The waste solution is returned to the first immobilized enzyme 12 present in the first chamber 11 (flowing through reservoir 31), so that any unreacted first compound 112 in the solution comes into contact with the first immobilized enzyme 12. Solutes, for example, the first dye precursor 112 and cofactors, may also be added to the solution via reservoir 31. The device 10 shown in FIG. 8 may be provided with other means for adding solutes, for example, a supply means 11b connected to the first chamber 11 and / or a supply means 21b connected to the outlet means 21a (not shown in FIG. 8).

[0168] In the device 10 of the present invention, there can be one or more reservoirs 31 and / or one or more collection tanks 41, configured in series and / or in parallel.

[0169] In another embodiment, as shown in FIG. 9, the apparatus 10 of the present invention further includes a reactor 51 for producing a first dye precursor 112, such as indole, from one or more starting compounds 114, such as tryptophan. The reactor 51 contains at least a starting enzyme 14. A solution containing the starting compounds 114 is supplied to the reactor 51 by a supply means 51b, whereby the solution comes into contact with the starting enzyme 14 and the starting compound 114 is converted into the first dye precursor 112. The solution containing the first dye precursor 112 then flows into the first chamber 11 by generating a flow of the solution using a second pump 50b. Once the solution containing the first dye precursor 112 has flowed into the first chamber 11, steps a) to d) of the dyeing method of the present invention are carried out. FIG. 9 shows an embodiment in which the reactor 51 is set up in parallel with the flow of solution flowing from the first chamber 11 to the second chamber 21; however, embodiments in which one or more reactors 51 are set up in series with the flow of solution, preferably upstream of the first chamber 11, are also encompassed by the present invention.

Claims

1. An apparatus (10) for carrying out a method for dyeing a fabric (22), said apparatus comprising: a first chamber (11) containing a solution comprising at least a first immobilized enzyme and at least a dye precursor; at least a second chamber (21) containing a fabric (22) and a means (50) for generating a solution flow, said first chamber (11) being in fluid communication with said second chamber (21), whereby a solution comprising at least indoxyl and / or its derivatives flows through said first chamber (11). the indoxyl and / or its derivatives can flow from a chamber (11) to the second chamber (21), wherein at least a portion of the indoxyl and / or its derivatives is converted to an indigo and / or its derivative dye (111) in the presence of the fabric (22) to dye at least a portion of the fabric (22), and the second chamber (21) optionally comprises an outlet means (21a) for removing the solution from the second chamber (21), and the first immobilized enzyme (12) is an oxidase.

2. The device (10) described in claim 1, wherein the first immobilized enzyme (12) is an oxygenase.

3. The apparatus (10) of claim 1, wherein the first immobilized enzyme (12) is a monooxygenase.

4. The device (10) described in claim 1, wherein the first immobilized enzyme (12) is mFMO.

5. 5. The device (10) according to any one of claims 1 to 4, wherein the first chamber (11) further contains at least a second enzyme.

6. The apparatus (10) described in claim 5, wherein the second enzyme is at least a cofactor-regenerating enzyme.

7. The device (10) described in claim 5, wherein the second enzyme is at least one dehydrogenase.

8. The device (10) described in claim 5, wherein the second enzyme is a dehydrogenase selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH).

9. The device (10) described in claim 5, wherein the second enzyme is phosphite dehydrogenase (PTDH).

10. 10. The device (10) according to any one of claims 5 to 9, wherein the first immobilized enzyme (12) and the second enzyme are provided as immobilized fusion enzymes.

11. 11. The device (10) of claim 10, wherein the immobilized fusion enzyme is PTDH-mFMO.

12. moreover, one or more reservoirs (31) in fluid communication with the first chamber (11), whereby a solution comprising a dye precursor can flow from the reservoir (31) to said first chamber (11); and / or one or more recovery tanks (41) in fluid communication with the outlet means of the second chamber (21); The device (10) according to any one of claims 1 to 11, comprising:

13. An apparatus (10) according to any one of the preceding claims, wherein the outlet means (21a) of the second chamber (21) is in fluid connection with the first chamber (11).

Images