Textile dyeing method and device

Abstract

The present invention relates to a method and an apparatus for dyeing textiles, an immobilized enzyme contained in said apparatus required for carrying out the method, and a method for enzymatic production of indigo blue and derivatives thereof.

Description

Technical Field

[0001] The present invention relates to a method and apparatus for dyeing textiles, and to an immobilized enzyme contained in said apparatus required to perform the method. Background of the Invention

[0002] Vat dyes are insoluble dyes that require a reducing agent to dissolve in water. Typically, dyeing with vat dyes involves applying the dye to textiles in its soluble reduced form, and then oxidizing the dye back to its insoluble form, which gives the textiles its color.

[0003] Indigo is a vat dye of formula I:

[0004]

[0005] Substitution of the aromatic ring of indigo with groups such as halogen, alkyl, alkoxy, amino, aryl, aryloxy, and carbonyl provides compounds with a wide variety of colors other than blue, which are part of the so-called indigo derivatives.

[0006] A large portion of indigo and its derivatives are produced synthetically. The Heumann synthesis (scheme 1 below) and the Pfleger synthesis (scheme 2 below) were the original synthetic routes for the industrial-scale preparation of indigo; various variations of these methods are still in use today.

[0007]

[0008] Option 1

[0009]

[0010] Option 2

[0011] The above synthetic route is also used to prepare indigo derivatives; in this case, compounds II', II", III' and III" are substituted with the same groups at the same positions as the desired indigo derivatives to be synthesized.

[0012] The synthesis of indigo and its derivatives, as well as other vat dyes, can also be carried out using enzymes or bacteria that express these enzymes. However, this enzymatic synthesis has not been used in industrial processes.

[0013] The precursors of indigo (such as compounds II', II”, III' and III” of schemes 1 and 2 above) are soluble in aqueous solution, while indigo itself is insoluble and precipitates out after synthesis in aqueous solution. Therefore, as mentioned above, indigo or its derivatives must be reduced (by treatment with a reducing agent, for example as shown in scheme 3).

[0014]

[0015] Option 3

[0016] Compound I is indigo, and compound IV is the water-soluble and reduced form of indigo, called leuco-indigo (or indigo white, because it is colorless).

[0017] Therefore, industrial dyeing methods using indigo or its derivatives as dyes, or typically using vat dyes, involve first synthesizing indigo or its derivatives (or vat dyes) in an aqueous solution in a reactor using known methods (e.g., the synthesis described in Scheme 1 above). Indigo or its derivatives (or vat dyes) are obtained in precipitated form. The aqueous solution containing the 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 reduced vat dyes). This aqueous solution containing dissolved leuco indigo or its derivatives (or reduced vat dyes) is then applied to textiles. Once the solution containing dissolved leuco indigo has wetted the textiles, indigo or its derivatives (or vat dyes) are obtained through oxidation of the leuco indigo or its derivatives (or leuco-vat dyes), thus dyeing the textiles. This oxidation can be carried out, for example, with oxygen from the air. Typically, the indigo dyeing process requires several immersion and oxidation steps to achieve the desired color.

[0018] The reducing agents used to reduce insoluble vat dyes (such as indigo or its derivatives) are irritating chemicals, i.e., chemicals harmful to users and / or the environment, such as sodium hydroxide and sodium dithionite. In fact, large amounts of reducing salts and hydroxides are used in conventional dyeing processes, such as those using indigo or its derivatives as dyes, thus generating significant amounts of wastewater that must be treated before disposal. This step increases the cost of the dyeing process.

[0019] Therefore, there is a need for an improved method for dyeing textiles with vat dyes, particularly indigo and its derivatives, that can reduce the costs of vat dyeing and water treatment processes.

[0020] Another problem with known indigo dyeing methods is that textiles, especially cellulose, can be damaged by prolonged exposure to alkaline process solutions and byproducts present therein. Summary of the Invention

[0021] The purpose of this invention is to solve the above-mentioned problems and to provide a method for dyeing textiles using insoluble dyes, such as vat dyes, particularly indigo and its derivatives, which is safe, cost-effective and environmentally friendly.

[0022] Another object of the present invention is to provide a method for dyeing textiles using indigo and its derivatives as well as insoluble dyes such as vat dyes, which is more sustainable than conventional dyeing methods using indigo and its derivatives or conventional vat dyes.

[0023] This invention provides a method according to claim 1, achieving the above-mentioned and other objectives. The method is a method for dyeing textiles, comprising the enzymatic synthesis of a dye precursor, characterized in that the method includes the following steps:

[0024] a) Contact a solution containing at least one first dye precursor with at least one first immobilized enzyme to convert at least a portion of the first dye precursor into at least one second dye precursor to obtain a solution containing the second dye precursor;

[0025] b) A solution stream containing the second dye precursor is generated, whereby the solution containing the second dye precursor flows from the first immobilized enzyme to the textile.

[0026] c) Contacting the textile with a solution containing the second dye precursor; and

[0027] d) Converting at least a portion of the second dye precursor into at least one dye, thereby dyeing at least a portion of the textiles;

[0028] The first immobilized enzyme is separated from the textile.

[0029] In the following description, "textiles" means any fiber, yarn, rope, fabric, and / or garment that can be dyed, for example, indigo and / or its derivatives. Textile materials can be of natural origin, such as those derived from animals or plants, such as cotton, linen, silk, wool, etc., or can be of synthetic origin, or can be mixtures thereof, such as elastic cotton fabrics or garments. Furthermore, the yarns can be manufactured by any known method, and suitable fabrics can also be manufactured by any known method, such as weaving, knitting, crocheting, knotting, and felting. Additionally, the garments can be any clothing, such as jeans, shirts, casual wear, etc.

[0030] In this invention, "first dye precursor" refers to any soluble compound that can be enzymatically converted into a second dye precursor. In this invention, "second dye precursor" refers to any soluble compound that can be obtained from the first dye precursor and can be converted into an insoluble dye, for example, through dimerization. In this invention, "insoluble dye" refers to any water-insoluble compound conventionally used for dyeing textiles, such as any vat dye, like indigo. Therefore, according to this invention, the first dye precursor, the second dye precursor, and the insoluble dye are interconnected through synthetic pathways, particularly synthetic pathways including a first enzymatic step and a second non-enzymatic step. For example, according to the first dye precursor of this invention, the second dye precursor and the insoluble dye can be 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 reported below. The first dye precursor to be included in the solution of step a) is selected based on the color that the textile to be dyed must have at the end of the dyeing process of this invention, since the color of the textile will be provided by the insoluble dye, which is obtained from the first dye precursor.

[0031] Advantageous examples of the first dye precursor, the second dye precursor, and the insoluble dye are indole and / or its derivatives, indophenol and / or its derivatives, and indigo and / or its derivatives, respectively. Indigo and its derivatives can be synthesized from indole and its derivatives, as shown in Scheme 4 below.

[0032]

[0033] Option 4

[0034] Compound II is indole (a first dye precursor), compound III is indolephenol (a second dye precursor), and compound I is indigo (a dye). Specifically, referring to the reaction of Scheme 4, a hydroxyl group is added to the 3-carbon of indole to obtain indolephenol, which dimers to indigo in aqueous solution. According to the invention, in step a) of the method of the invention, the hydroxyl group is added to indole and / or its derivatives by a first immobilized enzyme. When the first dye precursor is indole and / or its derivative, the second dye precursor is indolephenol and / or its derivative, and the insoluble dye is indigo and / or its derivative, the method of the invention results in the dyeing of textiles with indigo and / or its derivatives without the need for a prior art reduction step. Indigo and / or its derivatives are among the most commonly used dyes in textile dyeing.

[0035] According to the present invention, "indole derivative," "indophenol derivative," and "indigo derivative" refer to the corresponding indole, indophenol, and indigo substituted with one or more substituents, for example, by substitution of one or more carbons at any position selected from the 4, 5, 6, and 7 positions of indole or indophenol by one or more groups, and by substitution of one or more carbons at any position selected from the 4, 4', 5, 5', 6, 6', 7, and 7' positions of indigo by one or more substrates, and / or by substitution of a group on the nitrogen atom of indole, indophenol, or indigo. The one or more groups substituted with one or more carbons can be, for example, but not limited to, the following groups: halogen groups, alkyl groups (e.g., C1-C1), etc. 20 Alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and isobutyl, alkoxy groups (e.g., C1-C...). 20 Alkoxy groups, such as methoxy, ethoxy, butoxy, tert-butoxy, and isobutoxy; aryl groups (e.g., phenyl, substituted phenyl, benzyl, substituted benzyl, naphthyl, anthracene, and heteroaryl); aryloxy groups (e.g., phenoxy and naphthoxy); amino groups (e.g., primary and / or secondary aliphatic and / or aromatic amino groups); nitro and carbonyl groups (e.g., aldehyde groups, such as aromatic and / or aliphatic aldehydes, and ketone groups, such as aromatic and / or aliphatic ketones). Groups substituting for the nitrogen atom can be, for example, but not limited to, the following: alkyl groups (e.g., C1-C1...). 20Alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and isobutyl), aryl groups (e.g., phenyl, substituted phenyl, benzyl, substituted benzyl, naphthyl, anthracene, and heteroaryl), and acyl groups (e.g., formyl, acetyl, propionyl, benzoyl, and acrylyl). Therefore, indole derivatives can be, 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, and indole-6-carboxaldehyde; indolephenol derivatives can be, for example, 4-chloroindolephenol, 5-chloroindolephenol, 6-chloroindolephenol, 7-chloroindolephenol, 5-bromoindolephenol, 6-bromoindolephenol, 5-nitroindolephenol, 5-hydroxyindolephenol, 5-methylindolephenol, 5-methoxyindolephenol, 6-methylindolephenol, 7-methylindolephenol, 5-aminoindolephenol, 1-methylindolephenol, and indolephenol-6-carboxaldehyde. This invention also includes the use of any other indole and indoinol derivatives, as long as these indole derivatives can be reacted and converted into the corresponding indophenol derivatives by enzymatic catalysis. These indophenol derivatives, upon conversion (i.e., dimerization according to step d of the method of this invention), provide corresponding indigo derivatives, each with a different color. According to the invention, "indigo derivative" also refers to asymmetric indigo, i.e., indigo obtained by dimerization of two different indophenol derivatives, or indigo obtained by dimerization of indophenol and indophenol derivatives. The method of this invention can be used to dye textiles with asymmetric indigo when a solution containing two or more different indole derivatives or indole and one or more indole derivatives is contacted with an enzyme in step a). For example, when two different indole derivatives or indole and indole derivatives are contacted with a first immobilized enzyme (step a), two different indophenol derivatives or indophenol and indophenol derivatives are obtained; subsequently, they are contacted with textiles according to step c) of the method. When the two different indolephenol derivatives or indole and indolephenol derivatives are converted into at least one dye according to step d) of the method, three different indigo derivatives are obtained, namely two different symmetrical indigo derivatives and one asymmetrical indigo derivative (e.g., as shown in scheme 5 below), and textiles are thus dyed with more than one dye, one of which is asymmetrical indigo.

[0036]

[0037] Option 5

[0038] Reference scheme 5 includes two different indole derivatives, compounds IIa (4-methoxyindole) and IIb (7-chloroindole); two different indolephenol derivatives, compounds IIIa (4-methoxyindolephenol) and IIIb (7-chloroindolephenol); and three different indigo derivatives, compound Ia (4,4'-dimethoxyindole or 4-methoxy-2-(4-methoxy-3-oxo-1,3-dihydro-2H-indole-2-ylidene)-1,2-dihydro-3H-indole- 3-keto; symmetrical indigo derivatives), Ib (7,7'-dichloroindigo or 7-chloro-2-(7-chloro-3-oxo-1,3-dihydro-2H-indole-2-ylidene)-1,2-dihydro-3H-indole-3-one; symmetrical indigo derivatives) and Ic (7-chloro-4'-methoxyindigo or 7-chloro-2-(4-methoxy-1,3-dihydro)-3-oxo-2H-indole-2-ylidene)-1,2-dihydro-3H-indole-3-one; asymmetrical indigo derivatives). Therefore, when at least two indole derivatives, or indole and at least one indole derivative, are contacted with the enzyme, more than one indigo derivative can be obtained in step d) of the method, thereby enabling the dyeing of textiles with more than one dye according to the method of the present invention.

[0039] According to the present invention, "first immobilized enzyme" refers to any enzyme capable of catalyzing the conversion of a first dye precursor to a second dye precursor in step a) of the method according to the present invention. For example, the first immobilized enzyme may be an immobilized enzyme capable of catalyzing the conversion (i.e., oxidation) of indole and / or its derivatives to indophenol and / or its derivatives.

[0040] According to the invention, contacting any compound with an enzyme in step a), for example, contacting a first dye precursor with an immobilized enzyme, means enzymatically converting the compound. This contact can be carried out by contacting a solution stream containing at least this first dye precursor with the enzyme, thereby allowing the dissolved first dye precursor contained in the solution to be contacted with the immobilized enzyme and converted by the enzyme into a second dye precursor.

[0041] According to the present invention, contacting a textile with a solution containing at least a second dye precursor means wetting the textile with the solution, thereby impregnating the textile with the solution containing the second dye precursor.

[0042] According to the present invention, "immobilized" or "immobilized" refers to the process of immobilizing an enzyme. Enzyme immobilization is a conventional method known in the art, involving attaching such an enzyme to a support, preferably covalently, such as to an epoxy-activated resin (e.g., a methacrylate copolymer, as shown in the original text). Relizyme TM , Cellulose, agarose, polystyrene ion exchange resins, aminoacrylate resins, hydrogels (immobilized by occlusion; e.g., agarose, alginate, carrageenan, or gelatin), chelating supports (e.g., Ni- IDA- NTA- IDA-agarose and its derivatives, etc. The type of carrier used for immobilizing enzymes may depend on the type of exposed groups of the enzyme. For example, if the surface amino groups are exposed on the enzyme, an epoxy-activated resin can be used as a carrier: because the amino groups are covalently bound to the epoxy groups of the epoxy-activated resin, the enzyme is immobilized on the epoxy-activated resin. According to the invention, immobilization can be performed by stirring and incubating the enzyme and epoxy-activated resin in a 100 mM potassium phosphate solution containing 0.5 M NaCl at pH 8.0 for 12 hours. Advantageously, the remaining (unreacted) activating groups of the carrier are deactivated after immobilization has been performed; for example, by incubating the remaining activating groups in a solution containing epoxy-activated resin with 10 mM ethanolamine or 10 mM glycine.

[0043] Suitable epoxy-activated resins have particle sizes in the range of 100-1100 micrometers, for example 150 to 300 micrometers, or 200 to 500 micrometers, or 250 to 1000 micrometers, and average pore sizes in the range of 300 to 1800 angstroms, for example 300 to 600 angstroms, or 1200 to 1800 angstroms. In one aspect of the invention, enzyme immobilization can be carried out at a ratio of 5-75 mg (semi-) purified enzyme per gram of wet carrier, more preferably at a ratio of 15-25 mg (semi-) purified enzyme per gram of wet carrier.

[0044] Mutant enzymes (e.g., genetically modified enzymes) can be used in the methods of the present invention, for example, to improve catalytic efficiency or to provide improved binding to the carrier. For example, when the carrier used for enzyme immobilization is an epoxy-activated resin, the enzyme can be modified by introducing a hexalysine (6xLys) or hexahistidine (6xHis) tag sequence (preferably at the N-terminus) to increase its binding to the carrier.

[0045] According to the present invention, "separation" refers to separating the first immobilized enzyme and the textile textile such that the conversion of the second dye precursor to the insoluble dye does not occur at and / or near the first immobilized enzyme, but rather after the solution containing the second dye precursor comes into contact with the textile textile. Thus, the second dye precursor is directly converted to the insoluble dye on the textile textile. Therefore, according to the present invention, the textile textile is downstream of the immobilized enzyme relative to the flow direction of the solution. The enzymes are immobilized, and therefore they are confined and cannot flow with the solution. Such separation can be achieved, for example, by separating the immobilized enzyme (or enzyme system) and the textile textile in different containers or chambers, or by confining the immobilized enzyme (or enzyme system) and said textile textile to different regions of the same container or chamber. Solution parameters, such as flow rate, temperature, and the contact time between the solution and the enzyme and textile textile, can be selected to ensure that the conversion of the second dye precursor to the insoluble dye occurs substantially on the textile textile, i.e., after the solution containing at least the second dye precursor comes into contact with the textile textile.

[0046] Therefore, according to the present invention, the conversion (i.e., precipitation) of the second dye precursor into an insoluble dye at and / or near the first immobilized enzyme is substantially prevented. The applicant has found that precipitation of the insoluble dye at and / or near the enzyme can lead to a loss of enzyme activity; it is believed that the precipitated insoluble dye negatively affects the substrate-enzyme interaction, thereby preventing further contact between the substrate (i.e., the first dye precursor) and the immobilized enzyme.

[0047] Another advantage of this invention is that it avoids the use of reducing agents; therefore, the method of this invention is environmentally friendly, safe, cost-effective and sustainable because it does not produce wastewater containing reducing agents.

[0048] When the method is carried out under standard dyeing conditions and the second dye precursor is indophenol and / or its derivatives, their dimerization into indigo and / or its derivatives is spontaneous. According to the method of the invention, this spontaneous dimerization occurs after a solution containing at least said indophenol and / or its derivatives is contacted with textiles, the textiles being separated from a first immobilized enzyme. The method of the invention allows for the industrial-scale dyeing of textiles using the enzymatic synthesis of indigo and / or its derivatives.

[0049] Advantageously, the method of the present invention can be a continuous method. A continuous method may include adding a first dye precursor upstream of step a) to continuously contact the solution containing the dye precursor with the immobilized enzyme.

[0050] According to one embodiment of the method of the present invention, the solution obtained after step d) is a discharge solution, which may contain unreacted first dye precursor that was not converted into second dye precursor in step a).

[0051] According to one embodiment of the method of the invention, after step d), at least a portion of the discharged solution is returned to the first immobilized enzyme for recycling into the method. This embodiment allows the conversion of unreacted first dye precursor present in the discharged solution into a second dye precursor, which can then be subjected to steps b) to d) of the method of the invention. This embodiment optimizes the use of the first dye precursor because it can be completely converted into the second dye precursor, for example, by repeating the steps of the method, i.e., by guiding the solution flow to contact the first immobilized enzyme several times. Repeating the steps of the method may provide different colors to the dyed textiles; therefore, the method can be carried out until the desired textile color is obtained. Moreover, this embodiment avoids disposing of the discharged solution, which may still contain useful solutes, such as unreacted first dye precursors, buffers, cofactors, etc.

[0052] Furthermore, the first dye precursor can be added to the discharge solution of the immobilized enzyme after step d). This allows the method of the present invention to be carried out continuously.

[0053] In the following paragraphs, the method will be disclosed with reference to an embodiment in which the insoluble dye is indigo, the second dye precursor is indophenol, and the first dye precursor is indole. The scope of the invention is not limited to these exemplary compounds. According to one embodiment of the invention, the first immobilized enzyme is an oxidase, whereby, upon contact with the oxidase, indole and / or its derivatives are oxidized to obtain indophenol and / or its derivatives.

[0054] According to the present invention, "oxidase" refers to any enzyme capable of catalyzing the oxidation of its substrate, such as an oxidoreductase (EC1). Suitable oxidoreductases are monooxygenases (EC 1.13); preferably, they are flavin-containing monooxygenases (FMO) (EC1.14.13.8), and more preferably, they are microbial flavin-containing monooxygenases (mFMO). Alternatively, the monooxygenase may be a Baeyer-Villiger monooxygenase (BVMO). Monooxygenases, especially FMO and mFMO, offer excellent conversion rates and binding to many first dye precursors (e.g., indole and / or many of their derivatives), as well as suitable specificity for converting most indole derivatives, and are therefore advantageous to the present invention. Baeyer-Villiger monooxygenase (BVMO) has high homology with FMO and is therefore also advantageous to the present invention. Particularly preferred oxidases for use in the present invention are mFMOs from methylophaga sp., more preferably from strain SK1. This type of mFMO exhibits high solubility in aqueous solutions and can provide high-concentration solutions, thus increasing the amount of the second dye precursor 113 (e.g., indophenol and / or its derivatives) that can be synthesized from this type of mFMO. Furthermore, this type of mFMO is capable of converting most indole derivatives into corresponding indophenol derivatives because it is specific not only to indole or a specific indole derivative. However, the scope of the invention also includes any homologs of the mFMO from the methyl-eating bacteria. According to the invention, genetically modified enzymes, such as genetically modified oxidases, can be used, for example, to improve the oxidation efficiency of the first dye precursor.

[0055] According to one embodiment of the invention, the method further includes using a second immobilized enzyme, preferably an immobilized cofactor regeneration enzyme.

[0056] According to the present invention, the "second immobilized enzyme" can be any enzyme capable of assisting and / or completing the conversion of the first dye precursor to the second dye precursor catalyzed by the first immobilized enzyme. In particular, the second immobilized enzyme can assist this conversion by regenerating the cofactor used by the first immobilized enzyme; in this case, the second immobilized enzyme is an immobilized cofactor regeneration enzyme.

[0057] According to the present invention, "cofactor regenerating enzyme" is any enzyme capable of regenerating (i.e. generating) the cofactor used by the first immobilized enzyme, which is used to catalyze the conversion reaction of the first dye precursor (step a of the method of the present invention).

[0058] Therefore, the second immobilized enzyme can generate the cofactor used by the first immobilized enzyme from a substrate that is cheaper than the cofactor used by the first immobilized enzyme.

[0059] Suitable cofactor regeneration enzymes are known in the art, and are, for example, dehydrogenases such as glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), formate dehydrogenase (FDH), and mutants thereof. Preferably, the cofactor regeneration enzyme is phosphite dehydrogenase (PTDH); an example of a suitable PTDH is disclosed in WO 2004 / 108912 A2. When an oxidase, especially FMO, is used as the first immobilized enzyme, it is advantageous to use a dehydrogenase, such as GDH, FDH, and especially PTDH. GDH, FDH, and PTDH regenerate the cofactors used by these oxidases from glucose, formate, and phosphite, respectively, and are much cheaper and more readily available than the cofactors used by the oxidases. According to the invention, genetically modified cofactor regeneration enzymes can be used, for example, to improve the cofactor regeneration efficiency of the first dye precursor.

[0060] Preferably, both the first and second enzymes are immobilized to provide an enzyme system.

[0061] According to one embodiment of the invention, the immobilized enzyme system is spaced apart from the textile, and the solution flows from the enzyme system to the textile. The method of the present invention also avoids or at least greatly reduces the precipitation of insoluble dyes at or near the immobilized enzyme system.

[0062] According to another embodiment of the 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, and even more preferably a microbial flavin-containing monooxygenase (mFMO). The second enzyme is preferably a cofactor regenerating enzyme, more preferably a dehydrogenase, and even more preferably at least one dehydrogenase selected from glucose dehydrogenase (GDH), phosphorous acid dehydrogenase (PTDH), and formate dehydrogenase (FDH), with phosphorous acid dehydrogenase (PTDH) being the most preferred. Therefore, according to a preferred embodiment, the fusion enzyme is a PTDH-mFMO fusion enzyme.

[0063] Enzyme fusion is a technique known in the art. A suitable fusion enzyme includes regions derived from a first enzyme and regions derived from a second enzyme, each region providing the desired functional properties.

[0064] Immobilization of the fusion enzyme can be achieved by exposing surface groups on any region of the fusion enzyme, such as on the first enzyme region, the second enzyme region, or both. The vector can be selected based on the surface groups exposing any region of the fusion enzyme.

[0065] Mutant fusion enzymes can be used in the methods of the present invention. Such mutant fusion enzymes are genetically modified to, for example, more effectively immobilize or improve catalytic efficiency.

[0066] In one embodiment, the first immobilized enzyme or enzyme system is located in a first container or chamber, and the textile is located in a second container or chamber. Therefore, according to this embodiment, step a) is performed in at least the first chamber, and at least a portion of the dyeing of the textile is performed in at least the second chamber. Thus, according to this embodiment, the conversion of the first dye precursor to the second dye precursor (step a) of the method of the invention occurs in the first chamber, while the conversion of the second dye precursor to the dye occurs in the second chamber, the first chamber being fluidly connected to the second chamber.

[0067] According to one embodiment, the first dye precursor can be prepared enzymatically from one or more starting compounds. For example, indole (i.e., an exemplary first dye precursor) can be obtained enzymatically from tryptophan as a starting compound.

[0068] According to some embodiments, a first dye precursor is produced enzymatically from one or more starting compounds in one or more reactors, different from the first and second chambers. Advantageously, according to some embodiments, such a reactor may be at least fluidly connected to the first chamber.

[0069] According to some embodiments, as described above, a first dye precursor is produced enzymatically in a first chamber starting from one or more starting compounds.

[0070] As used herein, the term "starting compound" refers to a compound that can be converted into a first dye precursor as defined above through one or more enzymatic reactions. Such enzymatic reactions are carried out by one or more starting enzymes. According to some embodiments, the starting enzyme can be immobilized.

[0071] According to some implementation methods, a starting compound (e.g., tryptophan) can be enzymatically converted into its derivatives, such as halogenated derivatives.

[0072] Therefore, as used herein, the term "initiating enzyme" refers to one or more enzymes that can catalyze the conversion of an initiating compound (e.g., tryptophan) into a first dye precursor (e.g., indole).

[0073] Scheme 6 below represents an illustrative reaction scheme in which the first dye precursor (indole(II)) is obtained by enzymatic reaction from the starting compound (tryptophan(IV)):

[0074]

[0075] Option 6

[0076] Referring to Scheme 6, compound IV is tryptophan (starting compound), compound II is indole (first dye precursor), compound III is indophenol (second dye precursor), and compound I is indigo (insoluble dye). According to some embodiments, the conversion of tryptophan to indole can be carried out by a starting enzyme such as tryptophanase or a mutant form thereof (e.g., a mutant form with improved catalytic properties), while the conversion from the first dye precursor to the second dye precursor and from the second dye precursor to the insoluble dye can be carried out as described above. The reaction of Scheme 6 can also be applied to tryptophan, indole, indophenol, and indigo derivatives.

[0077] As used herein, the term "tryptophan derivative" refers to tryptophan substituted with one or more substituents, as disclosed above with respect to indole, indophenol, and indigo derivatives. For example, Scheme 7 represents an exemplary reaction involving tryptophan derivatives and corresponding indole, indophenol, and indigo derivatives. Scheme 7 represents an exemplary reaction in which a starting compound (tryptophan (IV)) is converted to a halogenated derivative (6-bromotryptophan (IVd)). In this case, more than one starting enzyme is required to obtain the desired first dye precursor (6-bromoindole (IId)), namely, tryptophan halogenase and tryptophanase.

[0078]

[0079] Option 7

[0080] Referring to Scheme 7, compound IV is tryptophan (starting compound), compound IVd is 6-bromotryptophan (halogenated derivative of the starting compound), compound IId is 6-bromoindole (first dye precursor), compound IIId is 6-bromoindophenol (second dye precursor), and compound Id is 6,6'-dibromoindigo (also known as Tyrian purple; insoluble dye). As described above, the conversion of tryptophan to 6-bromotryptophan and the conversion of 6-bromotryptophan to 6-bromoindole can be carried out by starting enzymes such as the corresponding tryptophan halogenases and tryptophanases, while the conversion from the first dye precursor to the second dye precursor and the conversion from the second dye precursor to the insoluble dye can be carried out as described above.

[0081] According to some implementations, insoluble dyes are obtained through an enzymatic cascade reaction step (i.e., an enzymatic conversion from the starting compound to the second dye precursor), followed by a non-enzymatic reaction step (i.e., the conversion of the second dye precursor to the insoluble dye occurring on textiles).

[0082] Advantageously, by controlling the parameters of the method of the present invention, the second dye precursor can be directly converted into an insoluble dye and delivered to textiles, and the precipitation of the insoluble dye at and / or near the enzyme is substantially avoided.

[0083] According to some embodiments, the method of the present invention may further include the following steps: prior to step a), contacting a solution containing at least one starting compound with at least one starting enzyme to convert at least a portion of such starting compound into a first dye precursor, to obtain a solution containing at least one of the first dye precursors.

[0084] Another object of the present invention is the apparatus according to claim 11, for performing a method of dyeing textiles, i.e., an apparatus for dyeing textiles comprising: a first chamber containing at least a first immobilized enzyme and at least a solution containing a dye precursor, at least one second chamber containing textiles, and means for generating a solution flow. The first chamber is fluidly connected to the second chamber, whereby the solution containing at least the dye precursor can flow from the first chamber to the second chamber, wherein at least a portion of the dye precursor is converted into dye for dyeing at least a portion of the textiles. The second chamber optionally includes an outlet means for removing the solution from the second chamber.

[0085] According to another embodiment of the present invention, the device further includes:

[0086] - at least one or more reservoirs fluidly connected to the first chamber, such that a solution containing the dye precursor can flow from the reservoir to the first chamber; and / or

[0087] - One or more collection tanks that are fluidly connected to the outlet device of the second chamber.

[0088] A reservoir according to this embodiment can be provided to the device of the invention, particularly the first chamber containing the first immobilized enzyme, to supply a solution containing at least the first dye precursor, such as the first dye precursor. In practice, the reservoir can be intentionally configured to allow a user to easily feed solutions and / or solutes necessary for carrying out the method of the invention using the device. Thus, a solution, for example, containing at least the first dye precursor, can be added to the reservoir, and this solution can then be fed into the first chamber containing the first immobilized enzyme (and ultimately the second enzyme) via a fluid connection between the reservoir and the first chamber. The collection tank according to this embodiment allows for the collection of the drain solution obtained once the textile has been dyed (e.g., after step d of the method of the invention).

[0089] According to another embodiment, the apparatus of the present invention further includes means for generating a solution flow. The means for generating the solution flow, such as one or more pumps, allows the solution contained in the apparatus of the present invention to flow.

[0090] According to some embodiments, the device may further include one or more reactors in fluid connection with the device, preferably with the first chamber. The one or more reactors may contain one or more initiating enzymes and solutions containing one or more initiating compounds.

[0091] According to some embodiments, the first chamber may contain one or more initiating enzymes and a solution containing one or more initiating compounds to enzymatically generate a first dye precursor.

[0092] Another object of the present invention is an immobilized fusion enzyme according to claim 19, namely an immobilized fusion enzyme comprising a carrier and at least one fusion enzyme immobilized on the carrier, wherein the fusion enzyme is a PTDH-mFMO fusion enzyme.

[0093] The PTDH-mFMO fusion enzyme has been found to be particularly useful for carrying out the methods of the present invention (i.e., dyeing of textiles), especially when the dyeing must be performed with indigo and / or its derivatives.

[0094] Another object of the present invention is the use of the immobilized fusion enzyme according to claim 20, namely the use of the immobilized fusion enzyme in a textile dyeing method, wherein the immobilized fusion enzyme is a regenerase-oxidase fusion enzyme, preferably a PTDH-mFMO fusion enzyme, i.e., the immobilized fusion enzyme according to the prior object of the present invention.

[0095] It has been revealed that the immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, is particularly useful in staining methods (e.g., the staining method of the present invention) using insoluble dyes, especially indigo and / or its derivatives. In fact, the immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, provides excellent reaction rates and yields in the oxidation of first dye precursors, particularly indole and / or its derivatives. Therefore, the immobilized fusion enzyme, particularly the immobilized PTDH-mFMO fusion enzyme, is optimal for the synthesis of insoluble dyes, particularly indigo and / or its derivatives, for the staining method according to the present invention.

[0096] Another object of the present invention is the method according to claim 21, namely, a method for producing indigo or indigo derivatives by enzymatic synthesis, comprising the following steps:

[0097] a') Converting tryptophan or a tryptophan derivative in the presence of at least one tryptophanase to obtain indole or an indole derivative.

[0098] b') Hydroxylating the indole or indole derivative obtained in step a') in the presence of at least one oxidase to obtain indolephenol or indolephenol derivative; and

[0099] c') The indophenol or indophenol derivative obtained in step b') is converted into indigo or indigo derivative.

[0100] The reaction scheme of the method of the present invention is shown in Scheme 6 above.

[0101] The method of the present invention provides the synthesis of indigo or indigo derivatives from tryptophan or tryptophan derivatives 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 the cost-effective production of indigo and / or indigo derivatives, such as Thiol violet.

[0102] Similarly, advantageously, the method of the present invention allows for the industrial-scale production of indigo and / or indigo derivatives.

[0103] As used herein, "tryptophan derivative" refers to tryptophan substituted with groups and positions as defined above for indole and indolephenol derivatives.

[0104] The tryptophan derivative in step a') is preferably a tryptophan halogenated derivative, which can be obtained by halogenating tryptophan in the presence of at least a tryptophan halogenase and a halogen source. The reaction scheme of this embodiment is represented by Scheme 7 above (wherein the halogenated derivative is a 6-bromine derivative).

[0105] According to some implementation methods, tryptophan can be used as a starting compound for the enzymatic production of indigo and indigo derivatives. Advantageously, using tryptophan as a starting compound allows for the cost-effective production of indigo and / or indigo derivatives.

[0106] As used herein, “halogenated derivatives” refers to any tryptophan, indole, indophenol, and indole whose one or more carbons at the 5, 6, 7, and 8 positions (and for indole, the 5', 6', 7', and 8' positions) are substituted with a halogen group, particularly a fluorine, chlorine, bromine, or iodine group. For example, tryptophan halogenated derivatives are 6-bromotryptophan (compound IVd of Scheme 7 above) and 7-chlorotryptophan; indole halogenated derivatives are 6-bromoindole (compound IId of Scheme 7 above) and 7-chloroindole; indophenol halogenated derivatives are 6-bromoindophenol (compound IIId of Scheme 7 above) and 7-chloroindophenol; and indole halogenated derivatives are Thiol violet (6,6'-dibromoindole, compound Id of Scheme 7 above) and 7,7'-dichloroindole.

[0107] According to some embodiments, the enzymes used in the methods and processes of the present invention can be isolated enzymes, preferably purified or semi-purified, thereby allowing the methods of the present invention to be carried out without expressing them in bacteria. Preferably, the isolated enzymes are immobilized enzymes.

[0108] The method of the present invention can be carried out in a reactor, thus providing a one-pot reaction. This provides indigo or its derivatives as a solid precipitate after step c'), which can be separated from the reaction mixture (e.g., by filtration). In other words, when the method of the present invention is carried out according to a one-pot reaction, the obtained indigo or its derivatives can be separated and purified from the reaction mixture.

[0109] According to some embodiments, step c') can be performed in the presence of textiles, such that at least a portion of the obtained indigo or indigo derivative is deposited on the textiles.

[0110] According to some embodiments, steps a') to c') are carried out in an aqueous medium and generate an aqueous medium flow, thereby allowing steps a') to c') to be carried out in different reactors or at different locations within a single reactor. This embodiment is advantageous for carrying out each step according to its optimal parameters, such as temperature, pH, amount of enzyme substrate, etc.

[0111] According to some exemplary embodiments, the method of the present invention can be applied in a two-stage packed bed reactor, such as... It is carried out in a rotating bed reactor (RBR).

[0112] The method of the present invention is preferably carried out in an aqueous medium. This aqueous medium preferably has a neutral or weakly alkaline pH, for example, 7.0 to 10, preferably 7.4 or 8. Therefore, this aqueous medium may contain a buffer, such as a potassium phosphate buffer. Some tryptophan derivatives, such as 6-bromotryptophan, are poorly soluble in aqueous media, and the method of the present invention can be carried out using these tryptophan derivatives suspended in the aqueous medium.

[0113] Step a') involves cleaving the carbon-carbon bonds on tryptophan or its derivatives in the presence of tryptophanase. This tryptophan derivative is preferably synthesized by performing step a').

[0114] Scheme 8 represents the reaction step a'):

[0115]

[0116] Option 8

[0117] Compound IV is tryptophan, compound II is indole, compound V is pyruvate, and TRPase is tryptophanase. Tryptophanase has been observed to catalyze the conversion of tryptophan derivatives to indole derivatives, such as the conversion of 6-bromotryptophan to 6-bromoindole.

[0118] Tryptophanases (systematic name: L-tryptophan indole lyase (deamination; formation of pyruvate)) are known enzymes that break the carbon-carbon bond of tryptophan and release indole. They can use pyridoxal phosphate (PLP) as a cofactor. The tryptophanase suitable for the method of this invention is *Escherichia coli*. 10β tryptophanase.

[0119] PLP can be optionally added to the reaction mixture of step a') to improve the conversion of tryptophan or its derivatives.

[0120] Step b') of the present invention involves hydroxylation of at least the 3-carbon of the indole or a derivative thereof obtained in step a') in the presence of an oxidase and O2. Therefore, step b') provides indophenol or a derivative thereof.

[0121] Suitable oxidases are those mentioned above, such as methyl-eating bacteria strain SK1 and the microbial FMO of Bayer-Villiger monooxygenase.

[0122] The oxidase requires O2, or oxygen, in the reaction mixture to catalyze the hydroxylation of indole or its derivatives. The O2 required for step b') can be oxygen normally dissolved in the aqueous reaction mixture, or the reaction mixture can be saturated with O2 to achieve the maximum conversion of indole or its derivatives.

[0123] Step c') is non-enzymatic and involves the oxidation and dimerization of indophenol or its derivatives to indigo or its derivatives.

[0124] According to some embodiments, step c') of the method of the present invention can be carried out in the presence of a textile at least separated from the oxidase, such that the generation of indigo or indigo derivatives occurs directly on the textile, thereby dyeing at least a portion of the textile. In this case, it is advantageous to substantially avoid the precipitation of indigo or its derivatives at and / or near the enzyme.

[0125] Step c') can occur spontaneously after step b') (provided that the concentration of O2 is sufficient to oxidize indophenol or its derivatives), or it can be driven to occur (e.g., by adding O2 to the reaction mixture).

[0126] The O2 required for step c') can be oxygen that is normally dissolved in the aqueous reaction mixture, or the reaction mixture can be saturated with O2 to achieve the maximum conversion of indole or its derivatives.

[0127] In one embodiment, the tryptophan derivative of step a') is a tryptophan halogenated derivative, which is obtained by another step: i) halogenating tryptophan in the presence of at least one tryptophan halogenase.

[0128] Tryptophan halogenases are known enzymes capable of catalyzing the halogenation of tryptophan at various sites. Tryptophan halogenases are typically flavin-dependent halogenases, meaning they use FAD or FADH2 as a cofactor. Suitable tryptophan halogenases according to the method of the present invention are thermophilic tryptophan halogenases, such as the thermophilic tryptophan halogenase of *Streptomyces violaceusniger*.

[0129] According to some implementation methods, the tryptophan halogenase is a thermophilic tryptophan halogenase of strain SPC6 of Streptomyces violaceum.

[0130] For example, thermophilic tryptophan halogenases can have the following sequence: LNNVVIVGGGTAGWMTASYLKAAFGDRIDITLVESGHIGAVGVGEATFSDIRHFFEFLGLKEKDWMPACNATYKLAVRFENWREKGHYFYHPFEQMRSVNGFPLTDWWLKQGPTDRFDKDCFVMASVIDAGLSPRHQDGTLIDQPFDEGADEMQGLTMSEHQGKTQFPYAYQFEAALLAKYLTKYSVERGVKHIVDDVREVSLDDRGWITGVRTGEHGDLTGDLFIDCTGFRGLLLNQALEEPFISYQDT LPNDSAVALQVPMDMERRGILPCTTATAQDAGWIWTIPLTGRVGTGYVYAKDYLSPEEAERTLREFVGPAAADVEANHIRMRIGRSRNSWVKNCVAIGLSSGFVEPLESTGIFFIHHAIEQLVKNFPAADWNSMH RDLYNSAVSHVMDGVREFLVLHYVAAKRNDTQYWRDTKTRKIPDSLAERIEKWKVQLPDSETVYPYYHGLPPYSYMCILLGMGGIELKPSPALALADGGAAQREFEQIRNKTQRLTEVLPKAYDYFTQ(SEQ.ID NO.1).

[0131] This type of tryptophan halogenase preferably catalyzes the halogenation of the carbon at the 6-position of tryptophan, thus making it suitable for producing Thial violet (6,6'-dibromoindigo) according to the method of the present invention.

[0132] Another tryptophan halogenase suitable for the method of the present invention is tryptophan halogenase PrnA, preferably PrnA of Pseudomonas fluorescens, which preferably catalyzes the halogenation of tryptophan at its 5th or 7th carbon.

[0133] For example, tryptophan halogenase (PrnA) may have the following sequence: (SEQ.ID.NO.2).

[0134] According to some implementations, tryptophan halogenase can be a genetically modified enzyme; in other words, tryptophan halogenase can be a mutant form. For example, tryptophan halogenase can be a mutant form of tryptophan halogenase from the *Streptomyces violaceum* strain SPC6, or a mutant form of tryptophan halogenase PrnA.

[0135] Since tryptophan must react with halogens in the presence of tryptophan halogenase to convert into indigo halogenated derivatives, this embodiment requires a halogen source in the reaction mixture. A suitable halogen source according to the method of the present invention can be a halide salt, i.e., a salt in which the anion is a halide ion. Suitable halide salts can be magnesium, silver, sodium, potassium, lithium, and calcium halide salts, such as NaCl, KCl, KI, LiCl, CuCl2, CuBr2, AgCl, CaCl2, CaBr2, ClF, MgCl2, MgBr2, etc.

[0136] This embodiment is advantageously carried out at a temperature of 20°C to 60°C, preferably 25°C to 40°C, more preferably about 30°C, for 30 minutes to 4 hours, preferably 1 hour to 3 hours, more preferably about 2 hours.

[0137] According to some embodiments, cofactor regenerating enzymes can be used to regenerate cofactors that may be required by the enzymes used in the methods of the present invention.

[0138] According to some embodiments, step b') can be performed in the presence of at least one enzyme suitable for NADPH cofactor regeneration. Preferably, the enzyme suitable for NADPH cofactor regeneration is selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH) as described below, more preferably PTDH as described below, thereby providing an FMO-NADPH regenerating enzyme system. Advantageously, this embodiment provides an enzyme system in which an expensive cofactor (i.e., NADPH) is regenerated by consuming an inexpensive cofactor (e.g., glucose, phosphite, or formate). For example, an oxidase (e.g., FMO) can use NADPH as a cofactor, which can be produced by an NADPH regenerating enzyme using inexpensive cofactors such as glucose, phosphite, or formate.

[0139] In another embodiment, tryptophan is halogenated in the presence of flavin reductase and NAD(P)H regenerase to obtain its halogenated derivative, wherein the NAD(P)H regenerase is preferably selected from the group consisting of glucose dehydrogenase (GDH), phosphorous acid dehydrogenase (PTDH), and formate dehydrogenase (FDH), more preferably PTDH, thereby providing a tryptophan halogenase-flavin reductase-NAD(P)H regenerase system.

[0140] Flavin reductase (EC 1.5.1.30) is an enzyme that catalyzes the following reactions:

[0141] Riboflavin + NADPH + H + → Reduced riboflavin + NADP + H +

[0142] NAD(P)H regenerators are enzymes that produce NADH or NADPH, such as GDH, PTDH, and FDH. Advantageously, this embodiment provides an enzyme system in which expensive cofactors (i.e., FAD and NADH or NADPH) are regenerated by consuming cheaper cofactors (e.g., glucose, phosphite, or formate), thereby improving the industrial feasibility of the method of the present invention. For example, tryptophan halogenases can use FAD as a cofactor, which can be produced by flavin reductases that can use NADH or NADPH as cofactors, which can be produced by NAD(P)H regenerators using inexpensive cofactors (e.g., glucose, phosphite, and formate).

[0143] The flavin reductase suitable for the method of the present invention can be a flavin reductase of Bacillus subtilis, especially a flavin reductase of Bacillus subtilis strain WU-S2B.

[0144] For example, flavin reductase can have the following sequence:

[0145] MKVLVLAFHPNMEQSVVNRAFADTLKDAPGITLRDLYQEYPDEAIDVEKEQKLCEEHDRIVFQFPLYWYSSPPLLKKWLDHVLLYGWAYGTNGTALRGKEFMVAVSAGAPEEAYQAGGSNHYAISELLRPFQATSNFIGTTYLPPYVFYQAGTAGKSELAEGATQYREHVLKSF (SEQ.ID NO.3).

[0146] Mutant forms of any enzyme used in the method of this invention can be used to improve the yield and industrial feasibility of the method of this invention.

[0147] For example, FMO mutants selected from W319A, C78I, C78IY207W, and C78IY207W W319F were found to improve the catalytic activity of FMO for 6-bromoindole. Furthermore, NADPH regenerators can be mutants that improve NADPH production, such as PTDH disclosed in WO2004 / 108912 A2.

[0148] According to some implementation methods, when the enzyme requires a cofactor, it can be provided as a fusion enzyme with a cofactor regenerating enzyme.

[0149] For example, tryptophan halogenase and flavin reductase can be provided as fusion enzymes, and FMO and NADPH regenerator can be provided as fusion enzymes, preferably as PTDH-FMO. According to this embodiment, only three individual enzymes can be used in the method of the invention (when performing optional step i), namely, the tryptophan halogenase-flavin reductase fusion enzyme, the tryptophanase, and the FMO-NADPH regeneration fusion enzyme. The NADPH regeneration portion of the latter fusion enzyme is capable of regenerating the required NADPH from its inexpensive substrate, phosphite, both the FMO and flavin reductase regions of the fusion enzyme.

[0150] Another object of the present invention is dyed textiles obtainable by the method of the present invention.

[0151] Another object of the present invention is to obtain dyed textiles by the method of the present invention when step c') of the method is performed in the presence of textiles. Attached Figure Description

[0152] Figure 1 This is a schematic diagram of the method of the present invention.

[0153] Figure 2 Another embodiment of the method of the present invention is illustrated schematically.

[0154] Figure 3 An embodiment of the method is illustrated schematically;

[0155] Figure 4 This is a schematic diagram of device 10.

[0156] Figure 5 This is another schematic diagram of device 10.

[0157] Figure 6 An embodiment of the device 10 including a storage unit is illustrated schematically;

[0158] Figure 7 Another embodiment of the device 10, including a collection tank, is illustrated schematically;

[0159] Figure 8 One embodiment of device 10 is illustrated schematically; and

[0160] Figure 9 Another embodiment of the device 10 is illustrated schematically. Detailed Implementation

[0161] The objects and embodiments of the present invention will now be disclosed in more detail with reference to the accompanying drawings.

[0162] The present invention provides a method for dyeing textiles 22, comprising the enzymatic synthesis of a dye precursor, characterized in that the method includes the following steps:

[0163] a) Contact a solution containing at least one first dye precursor 112 with at least one first immobilized enzyme 12 to convert at least a portion of the at least one first dye precursor 112 into at least one second dye precursor 113 to obtain a solution containing the at least one second dye precursor 113.

[0164] b) Generate a flow of the solution containing at least one second dye precursor 113, whereby the solution containing at least one second dye precursor 113 flows from the first immobilized enzyme 12 to the textile.

[0165] c) Contact the solution containing at least the second dye precursor 113 with the textile 22; and

[0166] d) Convert at least a portion of the second dye precursor 113 into at least one dye 111, thereby dyeing at least a portion of the textile 22;

[0167] The first immobilized enzyme 12 is spaced apart from the textile 22, for example, as shown in the figure. Figure 1 As shown.

[0168] Figure 1 The diagram illustrates the method of the present invention, specifically showing the conversion of the first dye precursor 112 into the second dye precursor 113 by the first immobilized enzyme 12, followed by the flow of a solution containing at least the second dye precursor 113 onto the textile 22 (separated from the first immobilized enzyme 12), and finally the conversion of the second dye precursor 113 directly onto the textile 22 into the insoluble dye 111.

[0169] More specifically, see reference Figure 1 The method of the present invention will be disclosed with reference to the indole-to-indigo pathway. The method dyes textile 22 by means of the following steps: contacting a solution stream containing an indole dye precursor 112 with at least one first immobilized enzyme 12 or enzyme system (step a) thereby converting at least a portion of the indole to indolephenol 113 due to enzyme catalysis. The solution at this point contains indolephenol 113. The flow of the solution brings the solution containing indolephenol 113 into contact with textile 22; advantageously, parameters such as flow rate are controlled so that the solution reaches the textile 22 immediately or shortly thereafter after the synthesis of indolephenol 113. Thus, the conversion of indolephenol 113 to indigo 111 can be achieved on the textile 22, and the dyeing of the textile 22 is realized. In particular, when the solution containing indolephenol 113 wets the textile 22, at least a portion of the indolephenol 113 is directly converted to indigo 111 on the textile 22.

[0170] According to the present invention, the method generates a solution flow that first flows to enzyme 12 (or enzyme system) and then from enzyme 12 (or enzyme system) to textile 22, for example, as... Figure 1As shown, the straight arrows represent the direction of solution flow. Any direction of solution flow is possible, provided that the solution is first supplied to the enzyme 12 and then from the enzyme 12 to the textile 22. For example, when the first immobilized enzyme 12 is below the textile 22, another direction of solution flow could be from bottom to top; or when the first immobilized enzyme 12 is above the textile 22, another direction of solution flow could be from top to bottom; or when the first immobilized enzyme 12 and the textile 22 are located in different regions of an annular chamber, the other direction of solution flow could be annular, such as a helical tubular chamber, for example, when the first immobilized enzyme 12 and the textile 22 are held in radially opposite regions within such a helical tubular chamber.

[0171] The use of flow in the method of the present invention can prevent indigo 111 from precipitating at and / or near the immobilized enzyme 12, because the solution containing at least the second dye precursor 113 flows to the textile 22 spaced apart from the first immobilized enzyme 12, and then the conversion of the second dye precursor 113 to the insoluble dye 111 occurs.

[0172] Specifically, when the second dye precursor 113 is spontaneously converted into the insoluble dye 111, for example when the second dye precursor 113 is indophenol and / or its derivatives and the insoluble dye 111 is indigo and / or its derivatives, a solution stream containing at least the second dye precursor 113 is generated, thereby the solution has flowed to the textile 22 before the second dye precursor 113 is spontaneously converted at and / or near a fixed oxidase and precipitated as the insoluble dye 111.

[0173] When the second dye precursor 113 does not spontaneously convert to the insoluble dye 111, or when it does not spontaneously convert in a suitable amount, conditions can be altered, such as pH and / or temperature, and / or reagents can be added, and / or a gas such as oxygen can be supplied to the textile 22 and the location of the second dye precursor 113 to induce the second dye precursor 113 to convert to the insoluble dye 111 on the textile 22. Conversely, in step a) of the method, solution conditions can also be controlled to prevent conversion until the solution reaches the textile.

[0174] Furthermore, according to step a) of the method, two or more different first dye precursors 112 can be contacted with enzyme 12 to obtain two or more different second dye precursors 113. Then, according to step d) of the method, such two or more different second dye precursors 113 can be converted to obtain one or more different dyes 111 on textile 22; finally, if the conversion of such two or more different second dye precursors 113 in step d) does not occur spontaneously, or if the conversion does not occur in a suitable amount, such conversion can be achieved by adding other reactants and / or by changing the parameters of the solution containing them. An illustrative example of contacting two or more different dye precursors with enzyme 12 is shown in embodiment 5 above. When two or more different first dye precursors 112 are used in the method according to the invention, two or more different enzymes 12 may be required; for example, when two or more different first dye precursors 112 require different enzymatic reactions to be converted into the corresponding two or more different second dye precursors 113, or when two or more different first dye precursors 112 cannot be substrates of the same enzyme 12.

[0175] The solution containing at least one first dye precursor 112 may also contain other solutes and is the solution that has been contacted with the first immobilized enzyme 12 or enzyme system in step a) of the method. The solution containing at least one second dye precursor 113 is the solution obtained after step a) by converting at least a portion of the first dye precursor 112 into the second dye precursor 113, and may also contain other solutes, such as some unreacted first dye precursor 112.

[0176] As described above, the solution according to the invention may contain other functional solutes, such as salts, buffers, cofactors, and oxygen and / or peroxide scavengers (e.g., catalase). Preferably, the concentration of the substrate contained in the aqueous solution saturates the catalytic enzyme, such 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, the exemplary solution according to the invention may contain 100 mM potassium phosphate buffer (pH 8.0), 0.5 M NaCl, 100 μM NADPH, 20 mM sodium phosphite, and 1 nM bovine liver catalase, with water as the solvent.

[0177] The method of the present invention can provide dyeing of textile 22 intermittently or continuously. For the latter, the first dye precursor 112 needs to be added to the solution, for example before step a), so that the solution containing at least the first dye precursor 112 is continuously contacted with the first immobilized enzyme 12 to continuously synthesize the second dye precursor 113. The first dye precursor 112 is advantageously added to keep the first immobilized enzyme 12 saturated. It may also be necessary to add other solutes, such as cofactors, buffers, and oxygen, for example before step a), to carry out the method of the present invention in a continuous manner.

[0178] The temperature and pH value of the method according to the present invention can be varied, and can be the temperature and pH value conventionally used in the enzymatic synthesis of insoluble dyes.

[0179] The temperature of the solution according to the method of the present invention can be, for example, in the range of 20 to 40, preferably 25 to 30. The pH of the solution according to the method of the present invention can be, for example, in the range of 7.0 to 10.0, preferably 7.5 to 9.0, even more preferably 7.5 to 8.5, and most preferably 8.0.

[0180] In step a), the contact time between the first immobilized enzyme 12 and the solution containing indole 112 can be varied to achieve different colors in the dyed textiles, and can be varied, for example, by changing the flow rate of the solution.

[0181] The oxygen concentration in the solution can be a relevant parameter for the overall dyeing yield because oxygen is involved in the conversion of the first dye precursor to the second dye precursor and / or the conversion of the second dye precursor to the insoluble dye (e.g., when the first dye precursor is indole and / or its derivatives, the second dye precursor is indophenol and / or its derivatives, and the insoluble dye is indigo and / or its derivatives). Therefore, the oxygen concentration in the solution can vary, for example, based on the amount of insoluble dye to be synthesized or the amount of textile to be dyed. To achieve, for example, maximum conversion rates of indole and / or its derivatives and indophenol and / or its derivatives, the solution is advantageously saturated with oxygen. It is also advantageous to monitor and control the oxygen concentration, and oxygen can be added as needed to maintain solution saturation.

[0182] Other parameters of the method of the present invention can be selected based on, for example, the type of textile that must be dyed and which dye to use as the final dye.

[0183] According to one embodiment of the method of the invention, the solution stream generated in step d), the so-called "discharge solution," is guided back to the chamber or region containing the immobilized enzyme system. The discharge solution is the solution obtained after at least a portion of indophenol 113 has been converted to indigo 111, which is immobilized on the textile 22, and may contain unreacted first dye precursor 112, such as unreacted indole, particularly if the first dye precursor 112 did not completely react with the enzyme 12 in step a) of the method of the invention. This embodiment... Figure 2 As shown in the diagram, the effluent containing indole is continuously or in batches, directly or indirectly, returned to the enzyme system to allow the remaining indole to react.

[0184] Preferably, the method includes the step of adding a portion of the first dye precursor 112 to the discharge solution. Advantageously, the first dye precursor 112 is added to keep the first immobilized enzyme 12 saturated. This provides a continuously operating method of the invention.

[0185] In one embodiment of the invention, the first immobilized enzyme 12 is an oxidase as previously defined. The use of an oxygenase is particularly useful when the first dye precursor 112 must be oxidized to convert into the second dye precursor 113, for example when the first dye precursor 112 is indole and / or a derivative thereof, the second dye precursor 113 is indophenol and / or a derivative thereof, and the insoluble dye 111 is indigo and / or a derivative thereof.

[0186] The method of the present invention can further provide a second immobilized enzyme present in plants for staining, preferably an immobilized cofactor regeneration enzyme as previously defined. This provides an enzyme system in which a first immobilized enzyme catalyzes the conversion of a first dye precursor 111, while the immobilized cofactor regeneration enzyme regenerates the cofactor required for the first dye precursor 111. The carrier used to immobilize the second immobilized enzyme may be the same as or different from the carrier used to immobilize the first immobilized enzyme, depending on the surface-exposed groups of the second enzyme. If possible, the same carrier is used to immobilize both the first and second enzymes.

[0187] The type of immobilized cofactor regenerating enzyme depends on which cofactor is used by the first immobilized enzyme 12. For example, when the first immobilized enzyme 12 is a flavin-containing monooxygenase (FMO) (which uses NADPH as a cofactor), the immobilized cofactor regenerating enzyme can be at least one dehydrogenase that produces NADPH, such as dehydrogenases selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH). PTDH is a soluble NADPH regenerating enzyme obtainable from, for example, Pseudomonas stutzeri, which uses phosphite as a substrate to catalyze the production of NADPH. The use of FMO (preferably mFMO) in conjunction with PTDH has shown effectiveness in the synthesis of many second dye precursors 113, particularly indophenol and / or its derivatives, because it provides a good oxidation rate and efficient regeneration of NADPH.

[0188] The solution used in the method and apparatus of the present invention may also contain a cofactor and / or substrate of the second immobilized enzyme. For example, if GDH, PTDH, or FDH is used as the immobilized cofactor regeneration enzyme, the solution may also contain glucose, phosphite, or formate (i.e., substrates of GDH, PTDH, and FDH, respectively).

[0189] Similar to the first enzyme, a mutated second enzyme (e.g., a genetically modified second enzyme) can be used in the methods of the present invention, for example, to improve the regeneration of the desired cofactor or to improve its binding properties with the vector.

[0190] In one embodiment of the invention, the first enzyme 12 and the second enzyme are provided as fusion enzymes as previously defined. This provides an enzyme system.

[0191] refer to Figure 3 This illustrates another embodiment of the method of the present invention. This embodiment provides the production of a first dye precursor 112 (e.g., indole) starting from a starting compound 114 (e.g., tryptophan). According to this embodiment, the method of the present invention includes the additional step of contacting a solution containing at least one starting compound 114 with at least one starting enzyme 14 to convert at least a portion of the starting compound 114 into the first dye precursor 112, thereby obtaining a solution containing the first dye precursor 112, such that steps a) to d) of the method of the present invention can be performed as already referred to Figure 1 and 2 The process is carried out as disclosed. When the first immobilized enzyme 12 and the initiating enzyme 14 are separated, a flow of solution can be generated to bring the solution containing the first dye precursor 112 (obtained by converting the starting compound 114) into contact with the enzyme 12, thereby allowing the solution to flow from the initiating enzyme 14 to the first immobilized enzyme 12, for example as... Figure 3As shown. Since the effluent obtained from step d) may also contain unreacted starting compound 114, it is advantageous to recycle the effluent back to the reactor or region containing the starting enzyme 14 and / or the first chamber or region containing the first immobilized enzyme 11 to optimize the conversion of unreacted starting compound 114 and / or the first dye precursor 112.

[0192] In another embodiment of the method of the present invention, step a) is performed in the first chamber 11, and the dyeing of the textile 22 is performed in the second chamber 21. A solution containing a first dye precursor 112 is provided into the interior of the first chamber 11, whereby the solution contacts a first enzyme 12 contained in the first chamber 11, and the first dye precursor 112 is enzymatically converted into a second dye precursor 113. A solution flow is then generated, at which point a solution 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 can contact and impregnate the textile 22. Finally, the second dye precursor 113 is directly converted into dye 111 on the textile 22.

[0193] The flow of the solution is generated by any suitable means, such as pump 50 or gravity (if the first chamber 11 is located above the second chamber 21).

[0194] The first chamber 11 is a container suitable for containing enzyme 12 or an enzyme system including a second immobilized enzyme, as well as an aqueous solution. The first chamber 11 may also include means therein for containing and confining the first immobilized catalytic enzyme 12 (and the final enzyme system), such as one or more filters. The first chamber 11 may advantageously include one or more devices or sensors to allow monitoring of solution parameters, such as pH, solution temperature, oxygen concentration, flow rate, etc. The size, shape, and material of the first chamber 11 can be arbitrarily chosen based on many factors, such as the number of textiles 22 to be dyed and the size and shape of the second chamber 21.

[0195] The second chamber 21 is a container suitable for containing at least the textile 22 and an aqueous solution, and wherein the conversion of the second dye precursor 113 to the insoluble dye 111 can occur. The second chamber 21 may also include means for holding the textile 22 in a predetermined position to allow for a faster or more complete dyeing process. The second chamber 21 may advantageously include one or more devices or sensors that allow monitoring of solution parameters, such as pH, solution temperature, oxygen concentration, flow rate, etc. The size, shape, and material of the second chamber 21 can be selected based on many factors, such as the number of textiles to be dyed and the size and shape of the first chamber 11.

[0196] The first chamber 11 and the second chamber 21 can be thermally regulated independently, for example by means of a jacket device (e.g., a water jacket).

[0197] exist Figure 4 In this invention, the first chamber 11 is fluidly connected to the second chamber 21. This fluid connection can be made via a fluid connector 11a (e.g., a tube or pipe), the size, shape, and material of which can be selected by those skilled in the art to ensure the fluid connection between the first and second chambers, and which are capable of effectively accommodating the aqueous solution circulating in the first chamber 11 and the second chamber 21, and are advantageously inert to these aqueous solutions. The size, shape, and material can be arbitrarily varied depending on, for example, the amount of textile to be dyed and the required flow rate of the solution. The fluid connector (e.g., a tube or pipe) may advantageously include probes for measuring parameters such as flow rate, temperature, solution pH, and oxygen concentration, as well as observation holes, round windows, and / or doors for monitoring the process of the invention and, for example, sampling the solution.

[0198] Another object of the present invention is an apparatus 10 for dyeing textiles 22, comprising: a first chamber 11 containing at least one first immobilized enzyme 12 and a solution containing at least one dye precursor; at least one second chamber 21 containing the textile 22; and means 50 for generating a solution flow, wherein the first chamber 11 is fluidly connected to the second chamber 21, whereby the solution containing at least one dye precursor can flow from the first chamber 11 to the second chamber 21, wherein at least a portion of the dye precursor is converted into dye 111 for dyeing at least a portion of the textile 22, for example as... Figure 4 As shown. The second chamber 21 optionally also includes an outlet device for removing the solution from the second chamber 21.

[0199] In particular, Figure 4 The diagram illustrates a first chamber 11 containing the first immobilized enzyme 12 and a second chamber 21 containing the textile 22. Straight arrows indicate the flow of a solution containing at least one dye precursor from the first chamber 11 to the second chamber 21. In the second chamber, at least a portion of the second dye precursor 113 is converted into an insoluble dye 111 to dye at least a portion of the textile 22 contained in the second chamber 21.

[0200] Figure 5 A schematic diagram of an embodiment of the device 10 is shown, wherein the second chamber 21 has an outlet device 21a, and wherein the outlet device 21a is fluidly connected to the first chamber 11. In this way, the discharged solution can be removed from the second chamber 21 and subsequently fed into the first chamber 11 (e.g., Figure 5(As shown by the straight arrow in the diagram). The outlet device 21a can be any pipe or conduit, such as those described above.

[0201] In order to continuously perform the method of the invention while using the device 10, it is useful to maintain the supply of cofactors and substrates to the first chamber 11 and the first immobilized enzyme 12, so that they can continuously generate the second dye precursor 113. Therefore, compounds (e.g., cofactors and substrates) are added to the first chamber 11 and / or to the effluent solution flowing back to the first chamber 11. Figure 5 The device 10 accordingly has means for making such additions and includes a feed device 11b connected to the first chamber 11 and / or a feed device 21b connected to the outlet device 21a.

[0202] In one embodiment of the device 10 of the present invention, a means 50 for generating a solution flow, such as a pump, is also included to cause the solution contained in the device 10 of the present invention to flow.

[0203] When the first immobilized enzyme 12 is not only specific to a single substrate and is therefore capable of converting different first dye precursors 112, different colors can be provided to the textile 12 to be dyed simply by changing the reagents supplied to the apparatus 10. Different dyes 111 can be obtained by changing the first dye precursor 112 without changing the apparatus 10 and / or the enzyme 12 contained therein; these dyes 111 are suitable for dyeing the textile 22 in the second chamber 21. For example, when the enzyme 12 contained in the first chamber 11 is a fusion enzyme PTDH-mFMO (which is capable of converting indole and its derivatives), feeding a solution containing indole to the apparatus 10 provides blue textiles. If, in the same dyeing process, a solution containing 5-hydroxyindole is fed instead of a solution containing indole to the apparatus 10, brown dye and brown-dyed textiles are obtained.

[0204] Figure 6 Another embodiment of the device 10 is shown, which further includes a reservoir 31 in fluid connection with at least the first chamber 11, such that a solution containing the dye precursor can flow from the reservoir 31 to the first chamber 11. Figure 7 Another embodiment of the device 10 is shown, which also includes a collection tank 41 in fluid connection with the outlet device 21a of the second chamber 21.

[0205] The reservoir 31 is any container capable of holding an aqueous solution, such as a solution containing at least the first dye precursor 112 (e.g., indole and / or its derivatives). The reservoir 31 can be a container from which a solution containing at least the first dye precursor 112 is fed into the apparatus 10 for the dyeing process of the present invention. Therefore, the reservoir 31 is advantageously positioned so that the operator can easily feed the solution and / or solutes therein, and can accordingly select its shape and size. Figure 6 The device 10 also includes a means 50 for generating a solution flow, such as a pump, to allow the solution to flow from the reservoir 31 to the first chamber 11, such as... Figure 6 As indicated by the straight arrow, the solution containing the first dye precursor 112 is thus fed into the first chamber 11.

[0206] Figure 8 The device 10, which includes both a storage container 31 and a collection tank 41, is shown. Figure 8 The apparatus can be used to perform the dyeing process of the present invention. A solution containing at least one first dye precursor is provided in a reservoir 31. A flow of this solution is then generated by a pump 50, and the solution flows from the reservoir 31 to a first chamber 11. Contact between this solution and enzyme 12 is achieved within the first chamber 11. At this time, a solution containing a second dye precursor 113 flows from the first chamber 11 to a second chamber 21, in which textile 22 is located. At least a portion of the second dye precursor 113 is converted into dye 111 to dye at least a portion of the textile 22 in the second chamber 21, and an excrement solution is obtained. The excrement solution is removed from the second chamber 21 through an outlet device 21a and collected in a collection tank 41. Collecting the excrement solution may be advantageous, for example, when such excrement solution must be treated to remove any precipitates (e.g., insoluble dye 111); such treatment can be carried out in the collection tank 41. The drained solution flows back into the first immobilized enzyme 12 contained in the first chamber 11 (through the reservoir 31), allowing any unreacted first compound 112 in the solution to contact the first immobilized enzyme 12. Solutes such as the first dye precursor 111 and cofactors can also be added to the solution through the reservoir 31. Figure 8 The device 10 shown may also have other means for feeding the solute, such as a feed device 11b connected to the first chamber 11 and / or a feed device 21b connected to the outlet device 21a. Figure 8 (Not shown in the image).

[0207] In the device 10 of the present invention, there may be more than one storage tank 31 and / or more than one collection tank 41, and they may be arranged in series and / or in parallel.

[0208] In another embodiment, the apparatus 10 of the present invention further includes a reactor 51 for producing a first dye precursor 112 (e.g., indole) from one or more starting compounds 114 (e.g., tryptophan), for example, as shown in the figure. Figure 9 As shown. This reactor 51 contains at least a starting enzyme 14. A solution containing the starting compound 114 can be fed into the reactor 51 via a feed device 51b, where the solution is contacted with the starting enzyme 14, causing the starting compound 114 to be converted into a first dye precursor 112. Then, a flow of the solution is generated by a second pump 50b, allowing the solution containing the first dye precursor 112 to flow into the first chamber 11. Once the solution containing the first dye precursor 112 has flowed into the first chamber 11, steps a} to d} of the method of the present invention can be performed. Figure 9 One embodiment is shown in which reactor 51 is arranged parallel to the flow of solution from first chamber 11 to second chamber 21; however, the invention includes embodiments in which one or more reactors 51 are arranged in series with respect to the flow of the solution, preferably upstream of first chamber 11.

Claims

1. A method for dyeing textiles (22), comprising the enzymatic synthesis of a dye precursor, characterized in that, The method includes the following steps: a) Contact a solution containing at least one first dye precursor (112) with at least one first immobilized enzyme (12) to convert at least a portion of the first dye precursor (112) into at least one second dye precursor (113) to obtain a solution containing the at least one second dye precursor (113). b) A flow of the solution containing the second dye precursor (113) is generated, whereby the solution containing the second dye precursor (113) flows from the first immobilized enzyme (12) to the textile (22). c) Contact the solution containing the second dye precursor (113) with the textile (22); and d) Convert at least a portion of the second dye precursor (113) into at least one dye (111) thereby dyeing at least a portion of the textile (22); The first immobilized enzyme (12) is spaced apart from the textile (22).

2. The method as claimed in the preceding claims, wherein, In step d), an excretion solution is obtained, generating a flow of the excretion solution, thereby allowing the excretion solution to flow to the at least one first immobilized enzyme (12).

3. The method as described in claim 1 or 2, wherein, The at least one first dye precursor (112) is indole and / or a derivative thereof, the at least one second dye precursor (113) is indophenol and / or a derivative thereof, and the at least one dye (111) is indigo and / or a derivative thereof.

4. The method as described in any of the preceding claims, wherein, The first immobilized enzyme is an oxidase, preferably an oxygenase, more preferably a monooxygenase, whereby the first dye precursor is oxidized when it comes into contact with the oxidase to obtain the second dye precursor.

5. The method as described in any of the preceding claims, further comprising a second immobilized enzyme, preferably an immobilized cofactor regeneration enzyme.

6. The method of claim 5, wherein, The cofactor regenerating enzyme is a dehydrogenase; preferably at least one dehydrogenase selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH), and formate dehydrogenase (FDH); more preferably phosphite dehydrogenase (PTDH).

7. The method of claim 5 or 6, wherein, The first immobilized enzyme and the second immobilized enzyme are provided as immobilized fusion enzymes, preferably PTDH-mFMO.

8. The method according to any one of claims 4 to 7, wherein, Oxygen is added to the solution.

9. The method as described in any of the preceding claims, wherein, Step a) is carried out in at least one first chamber (11), and the dyeing of at least part of the textile (22) is carried out in at least one second chamber (21).

10. The method of claim 9, wherein, The first dye precursor (112) is produced enzymatically from one or more starting compounds (114) in one or more reactors (51) different from the first chamber (11) and the second chamber (21).

11. The method of claim 9, wherein, In the first chamber (11), the first dye precursor (112) is produced enzymatically from one or more starting compounds (114).

12. An apparatus (10) for dyeing textiles (22), comprising: A first chamber (11) contains at least one first immobilized enzyme (12) and a solution containing at least one dye precursor; At least one second chamber (21) containing textile (22); and means (50) for generating a solution flow, wherein the first chamber (11) is fluidly connected to the second chamber (21), whereby the solution containing at least one dye precursor can flow from the first chamber (11) to the second chamber (21), whereby at least a portion of the dye precursor is converted into dye (111) to dye at least a portion of the textile (22), and wherein the second chamber (21) optionally includes an outlet means (21a) for removing the solution from the second chamber (21).

13. The device (10) as claimed in claim 12, wherein, The first immobilized enzyme (12) is an oxidase, preferably an oxygenase, more preferably a monooxygenase, and most preferably mFMO.

14. The device (10) as claimed in claim 12 or 13, wherein, The first chamber (11) further contains at least one second enzyme, preferably at least one cofactor regenerating enzyme, more preferably at least one dehydrogenase, and even more preferably a dehydrogenase selected from the group consisting of glucose dehydrogenase (GDH), phosphite dehydrogenase (PTDH) and formate dehydrogenase (FDH); most preferably phosphite dehydrogenase (PTDH).

15. The device (10) as claimed in claim 14, wherein, The first immobilized enzyme (12) and the second enzyme are provided as immobilized fusion enzymes.

16. The device (10) as claimed in claim 15, wherein, The immobilized fusion enzyme is PTDH-mFMO.

17. The device (10) as claimed in any one of claims 12-16, further comprising: - One or more reservoirs (31) fluidly connected to the at least one first chamber (11) such that a solution containing the dye precursor can flow from the reservoir (31) to the first chamber (11); and / or - One or more collection tanks (41) that are fluidly connected to the outlet device of the second chamber (21).

18. The device (10) as claimed in any one of claims 12 to 17, wherein, The outlet device (21a) of the second chamber (21) is fluidly connected to the first chamber (11).

19. An immobilized fusion enzyme comprising a carrier and at least one fusion enzyme immobilized on the carrier, wherein the fusion enzyme is a PTDH-mFMO fusion enzyme.

20. Use of the immobilized fusion enzyme as described in claim 19 in a textile dyeing method.

21. A method for producing indigo or indigo derivatives by enzymatic synthesis, the method comprising the following steps: a') Converting tryptophan or a tryptophan derivative in the presence of at least one tryptophanase to obtain indole or an indole derivative. b') Hydroxylating the indole or indole derivative obtained in step a') in the presence of at least one oxidase to obtain indolephenol or indolephenol derivative; and c') The indophenol or indophenol derivative obtained in step b') is converted into indigo or indigo derivative.

22. The method as described in any of the preceding claims, wherein, The tryptophan derivative in step a') is a tryptophan halogenated derivative, and the method further includes the following steps: i) halogenating tryptophan in the presence of at least one tryptophan halogenating enzyme and a halogen source to obtain the tryptophan halogenated derivative.

23. The method of claim 21 or 22, wherein, The tryptophanase, the oxidase, and the tryptophan halogenase are separate enzymes, preferably immobilized enzymes.

24. The method according to any one of claims 21-23, wherein, Steps a') to c') are carried out in an aqueous medium and generate an aqueous medium flow, thereby steps a') to c') being carried out in different reactors or at different locations within a single reactor.

25. The method according to any one of claims 21 to 24, wherein, The halogen is bromine, the tryptophan derivative is 6-bromotryptophan, and the indigo derivative is Thiol violet.

26. The method according to any one of claims 21 to 25, wherein, Step c') is performed in the presence of textiles at least separated from the oxidase, thereby dyeing at least a portion of the textiles.

27. Dyed textiles obtained by the method of any one of claims 1 to 11 or 26.

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