Artificial protein fiber, fiber structure, method for producing dyed artificial protein fiber, method for dying artificial protein fiber and dyeing agent for artificial protein fiber

Abstract

To provide an artificial protein fiber excellent in color fastness, a fiber structure obtained from the artificial protein fiber, a method for producing an artificial protein fiber excellent in color fastness, a method for producing an artificial protein fiber excellent in color fastness and an artificial protein fiber dyeing agent excellent in color fastness.SOLUTION: Provided are artificial protein fibers containing artificial proteins, which are dyed with a dye containing bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis, fiber structures obtained from the above artificial protein fibers, methods for producing the above artificial protein fibers, methods for dyeing artificial protein fibers, and dyes for the above artificial protein fibers.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to artificial protein fibers, fiber structures, a method for producing dyed artificial protein fibers, a method for dyeing artificial protein fibers, and a dye for artificial protein fibers. [Background technology]

[0002] In recent years, growing awareness of environmental conservation has led to calls in various fields to move away from petroleum-derived materials. In the field of textile use, for example, natural protein fibers such as silk and wool have come to be used for a variety of purposes, including not only clothing but also daily necessities and industrial applications.

[0003] In these various applications, dyeing natural protein fibers and using them is being considered. For example, Patent Document 1 describes a dye for dyeing keratin fibers, which contains bilirubin oxidase (EC1.3.3.5) derived from Myrothecium verrucaria or Bacillus subtilis. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 099034 Summary of the Invention [Problem to be solved by the invention]

[0005] In recent years, natural protein fibers have the disadvantage that they are difficult to mass-produce industrially, so artificial protein fibers containing artificial proteins have been proposed as alternatives to natural protein fibers (e.g., WO 2013 / 065650), and studies into various applications are underway. Such artificial protein fibers are often dyed to desired colors and used for various purposes, but these applications require color fastness that prevents fading.

[0006] The present invention aims to provide artificial protein fibers with excellent color fastness, fiber structures obtained from the artificial protein fibers, a method for producing artificial protein fibers with excellent color fastness, a method for dyeing artificial protein fibers with excellent color fastness, and a dye for artificial protein fibers with excellent color fastness. [Means for solving the problem]

[0007] Representative embodiments of the present invention are shown below, but the present invention is not limited to these.

[0008] <1> An artificial protein fiber comprising an artificial protein, Stained with an oxidase-containing stain Artificial protein fiber. <2> The oxidase is bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2). <1> The artificial protein fiber according to claim 1. <3> The oxidase is an oxidase derived from a microorganism. <1> The artificial protein fiber according to claim 1. <4> The oxidase is a bilirubin oxidase derived from a microorganism of the genus Myrothecium or the family Bacillus, or a laccase derived from a microorganism of the family Trametes. <3> The artificial protein fiber according to claim 1. <5> The oxidase is bilirubin oxidase derived from Myrothecium verrucaria or Bacillus subtilis, or laccase derived from Trametes sp. or Trametes versicolor. <4> The artificial protein fiber according to claim 1. <6> The dyeing agent further contains an oxidation dye consisting of an indole derivative or an indoline derivative. <1> The artificial protein fiber according to claim 1. <7> The oxidation dye may be 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-butyl-5,6-dihydroxyindoline, 4-hydroxy-5-methoxyindoline, 6-hydroxy-7-methoxyindoline, 6,7-dihydroxyindoline, 4,5-dihydroxyindoline, 4-methoxy-6-hydroxyindoline, N-hexyl-5,6-dihydroxyindoline, 2-methyl-5,6-dihydroxyindoline, 3-methyl-5,6-dihydroxyindoline, Phosphorus, 4-hydroxyindoline, 2,3-dimethyl-5,6-dihydroxyindoline, 2-methyl-5-ethyl-6-hydroxyindoline, 2-methyl-5-hydroxy-6-β-hydroxyethylindoline, 4-hydroxypropylindoline, 2-hydroxy-3-methoxyindoline, 6-hydroxy-5-methoxyindoline, 6-hydroxyindoline, 5-hydroxyindoline, 7-hydroxyindoline, 7-aminoindoline, 5-aminoindoline, 4-aminoindoline, 5,6-dihydroxyindoline-2-carboxamide carboxylic acid, 1-methyl-5,6-dihydroxyindoline, 4,5-dihydroxyindole, 5,6-dihydroxyindole, 6,7-dihydroxyindole, N-methyl-5,6-dihydroxyindole, N-ethyl-5,6-dihydroxyindole, N-hexyl-5,6-dihydroxyindole, 2-methyl-5,6-dihydroxyindole, 3-methyl-5,6-dihydroxyindole, 4-hydroxyindole, 2,3-dimethyl-5,6-dihydroxyindole, 2-methyl-5-ethyl-6-hydroxyindole indole, 2-methyl-5-hydroxy-6-β-hydroxyethyl indole, 4-hydroxypropyl indole, 2-hydroxy-3-methoxy indole, 4-hydroxy-5-methoxy indole, 6-hydroxy-7-methoxy indole, 6-hydroxy-5-methoxy indole, 6-hydroxy indole, 5-hydroxy indole, 7-hydroxy indole, 7-amino indole, 6-amino indole, 5-amino indole, 4-amino indole, 5,6-dihydroxyindole-2-carboxylic acid and 1-methyl-5,6-dihydroxyindole, <6> The artificial protein fiber according to <8> The artificial protein is an artificial structural protein. <1> ~ <7> 10. The artificial protein fiber according to any one of the preceding items. <9> The artificial structural protein has an amino acid sequence containing a repetitive sequence. <8> The artificial protein fiber according to claim 1. <10> the artificially structured protein has an amino acid sequence containing an (A)n motif, which amino acid sequence is different from the amino acid sequence of a naturally occurring protein, the (A)n motif is an amino acid sequence consisting of 2 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 40% or more, and when there are multiple (A)n motifs, these (A)n motifs may be the same amino acid sequence as each other or different amino acid sequences; <9> The artificial protein fiber according to claim 1. <11> The artificial protein is an artificial fibroin. <10> The artificial protein fiber according to claim 1. <12> <1> ~ <11> A fiber structure formed from the artificial protein fiber according to any one of the above. <13> Dyeing artificial protein raw material fibers with a dye containing an oxidase, A method for producing dyed artificial protein fibers. <14> The method comprises treating the fibroin fibers with a dye containing an oxidase. Method for dyeing artificial protein fibers. <15> Contains oxidases, Dyeing agent for artificial protein fibers. [Effects of the Invention]

[0009] According to the present invention, there are provided artificial protein fibers having excellent color fastness, fiber structures obtained from the artificial protein fibers, a method for producing artificial protein fibers having excellent color fastness, a method for dyeing artificial protein fibers having excellent color fastness, and a dye for artificial protein fibers having excellent color fastness. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the evaluation results of color fastness in Example A. [Figure 2] FIG. 1 is a schematic diagram showing an example of a spinning apparatus for producing artificial protein fibers. [Figure 3] 1 is a graph showing the evaluation results of color fastness in Example B. [Figure 4] 10 is a graph showing the evaluation results of uneven dyeing in Example C. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present disclosure will be described in detail, with reference to the drawings as needed. However, the following embodiments are merely examples for explaining the present disclosure, and are not intended to limit the present disclosure to the following contents.

[0012] (artificial protein fiber) The artificial protein fiber of this embodiment is an artificial protein fiber containing an artificial protein, and is dyed with a dye containing an oxidase, specifically, for example, a dye containing bilirubin oxidase (EC1.3.3.5) or laccase (EC1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis.

[0013] The inventors discovered that dyeing artificial protein fibers with the dyestuff provides superior color fastness compared to dyeing natural protein fibers with the dyestuff, leading to the completion of the present invention. The dyeing mechanism is presumed to be as follows: A water-soluble, pre-enzyme reaction dye precursor is dissolved in an aqueous solution, and the dye precursor is allowed to penetrate into the artificial protein fiber. Next, a certain amount of enzyme is added to the dissolved dye precursor and artificial protein fiber to oxidize the dye precursor. As a result, the dye precursor that has penetrated into the surface or interior of the artificial protein fiber is polymerized by an oxidation reaction and insolubilized to form a dye. The insolubilized dye accumulates on the surface or interior of the artificial protein fiber, making it impossible for the dye to be removed from the artificial protein fiber. All of these processes can be carried out at room temperature, eliminating the need for heat treatment or the addition of chemicals. This allows the artificial protein fiber to be dyed without denaturing it with heat or chemicals. Although oxidation reactions can occur in air, the addition of enzymes makes the oxidation of the dye precursor highly efficient. It is presumed that this mechanism makes it possible to dye artificial protein fibers. Furthermore, the present inventors have found that when artificial protein fibers are dyed with the dyeing agent, the occurrence of dyeing unevenness is suppressed compared to when natural protein fibers are dyed with the dyeing agent.

[0014] <Artificial protein fiber> Artificial protein fibers are not particularly limited as long as they contain an artificial protein. Here, artificial proteins include recombinant proteins and synthetic proteins. In other words, in this specification, "artificial protein" refers to a protein that has been artificially produced. An artificial protein may be a protein whose domain sequence is different from the amino acid sequence of a naturally occurring protein, or it may be a protein whose domain sequence is identical to the amino acid sequence of a naturally occurring protein. Furthermore, an "artificial protein" may be one that uses the amino acid sequence of a naturally occurring protein as is, one whose amino acid sequence has been modified based on the amino acid sequence of a naturally occurring protein (for example, one whose amino acid sequence has been modified by modifying the gene sequence of a cloned naturally occurring protein), or one that has been artificially designed and synthesized without relying on a naturally occurring protein (for example, one having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence).

[0015] Furthermore, examples of artificial proteins include proteins that can be used for industrial purposes. "Usable for industrial purposes" means that the proteins can be used, for example, in various general-purpose materials for indoor and outdoor use. Specific examples of artificial proteins that can be used for industrial purposes include artificial structural proteins.

[0016] [Artificial structural proteins] A structural protein refers to a protein involved in the structure of a living organism, a protein that constitutes a structure produced by a living organism, or a protein derived from such a protein. An artificial structural protein also refers to a protein that self-aggregates under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. Furthermore, an artificial structural protein can also be said to be a protein that contains repeated motifs consisting of a characteristic amino acid sequence or a specific number of amino acid residues and forms the skeleton of an organism or material. An artificial structural protein is an artificially produced version of such a structural protein. Examples of such artificial structural proteins include artificial fibroin, artificial keratin, artificial collagen, artificial elastin, and artificial resilin.

[0017] When forming artificial structural proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds and thus yield stronger molded products. Furthermore, alanine and glycine residues are amino acids with nonpolar side chains, and therefore are arranged so that they face inward during the folding process in polypeptide production, making them more likely to form α-helix or β-sheet structures. Therefore, a high proportion of amino acids such as glycine and alanine residues is desirable. From the viewpoint of obtaining molded products with superior strength, the alanine residue content may be, for example, 10 to 40%, and may be 12 to 40%, 15 to 40%, 18 to 40%, 20 to 40%, or 22 to 40%. From the viewpoint of obtaining molded products with superior strength, the glycine residue content may be, for example, 10 to 55%, and may be 11 to 55%, 13 to 55%, 15 to 55%, 18 to 55%, 20 to 55%, 22 to 55%, or 25 to 55%.

[0018] As used herein, the term "alanine residue content" refers to the ratio of the number of alanine residues to the total number of amino acid residues constituting a protein, and is a value expressed by the following formula:

[0019] Alanine residue content = (number of alanine residues in protein / total number of amino acid residues in protein) x 100 (%)

[0020] Furthermore, the glycine residue content, serine residue content, threonine residue content, proline residue content, and tyrosine residue content have the same meaning as those obtained by replacing the alanine residue in the above formula with glycine residue, serine residue, threonine residue, proline residue, and tyrosine residue, respectively.

[0021] Preferably, the artificial structural protein contains amino acids with relatively large side chains or flexible amino acids uniformly throughout its entire sequence to a certain extent. Specifically, the structural protein may contain a motif containing tyrosine, threonine, and proline residues in a repeated cycle. Such structural proteins are more likely to inhibit the formation of strong intermolecular hydrogen bonds during processing of molded articles, thereby improving processability. For example, the total content of proline, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 5% or more, greater than 5.5%, 6.0% or more, greater than 6.5%, 7.0% or more, greater than 7.5%, 8.0% or more, greater than 8.5%, 9.0% or more, 10.0% or more, or 15.0% or more. Furthermore, for example, the total content of proline, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0022] The artificial structural protein may have an amino acid sequence that includes a repeat sequence. That is, the artificial structural protein according to this embodiment may have multiple amino acid sequences (repeat sequence units) with high sequence identity within the artificial structural protein. The number of amino acid residues in the repeat sequence unit is preferably 6 to 200. Furthermore, the sequence identity between the repeat sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The hydrophobicity index (hydropathy index) of the repeating sequence unit may be, for example, -0.80 or more, -0.70 or more, -0.60 or more, -0.50 or more, -0.40 or more, -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more. The upper limit of the hydrophobicity index of the repeating sequence unit is not particularly limited, but may be, for example, 1.0 or less or 0.7 or less.

[0023] Artificial structural proteins are (A) n The amino acid sequence may include an amino acid sequence containing the motif. n The motif refers to an amino acid sequence consisting mainly of alanine residues. (A) n The number of amino acid residues in the motif may be 2 to 27, or may be an integer of 2 to 20, 2 to 16, or 2 to 12. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed only of alanine residues). The artificial structural protein is (A) n Preferably, the artificial structural protein contains a plurality of motifs. n If there are multiple motifs, their (A) n The motifs may be identical to each other in amino acid sequence or may be different in amino acid sequence. Also, (A) n Preferably, the amino acid sequence containing the motif is different from the amino acid sequence of a naturally occurring protein. Among these, the artificial protein is (A) n (A) having an amino acid sequence containing a motif, the amino acid sequence being different from the amino acid sequence of a naturally occurring protein; n The motif represents an amino acid sequence consisting of 2 to 27 amino acid residues, and (A) n A preferred embodiment of this invention is one in which the number of alanine residues relative to the total number of amino acid residues in the motif is 40% or more. n Preferred embodiments of the motif are as described above.

[0024] The artificial structural protein may be artificial fibroin. Examples of fibroin include naturally occurring fibroin. Examples of naturally occurring fibroin include fibroin produced by insects or spiders. Natural fibroin is a fibrous protein with a molecular weight of approximately 370,000, composed of two subunits, and has a high content of glycine, alanine, serine, and tyrosine residues, with these amino acid residues accounting for nearly 90% of the total number of amino acid residues. Natural fibroin has a crystalline region rich in amino acid residues with relatively small side chains such as glycine, alanine, and serine, and an amorphous region containing amino acid residues with relatively large side chains such as tyrosine.

[0025] More specific examples of naturally occurring fibroin include fibroins whose sequence information is registered in NCBI GenBank. For example, among the sequences registered in NCBI GenBank that contain INV as their division, they can be identified by extracting sequences with keywords spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" in their definition, sequences with a specific product character string in their CDS, and sequences with a specific character string in their TISSUE TYPE SOURCE.

[0026] As used herein, "artificial fibroin" refers to artificially produced fibroin (artificial fibroin). The artificial fibroin may be a fibroin having an amino acid sequence different from that of naturally occurring fibroin, or may be a fibroin having an amino acid sequence identical to that of naturally occurring fibroin. The artificial fibroin can be produced by known methods, for example, by the method described in International Publication No. 2019 / 194263. When the modified fibroin is expressed as an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble modified fibroin as a precipitate fraction. The recovered insoluble modified fibroin may be inactivated by adding citric acid and water (e.g., at 80°C for 2 hours), washed with water by filter press filtration, granulated, and dried to obtain a purified preparation.

[0027] Artificial fibroin may be a fibrous protein having a structure similar to that of naturally occurring fibroin, or may be a fibroin having a sequence similar to the repetitive sequence of naturally occurring fibroin. The "similar sequence to the repetitive sequence of fibroin" may be a sequence actually found in naturally occurring fibroin, or a sequence similar thereto.

[0028] "Artificial fibroin" may be a naturally occurring fibroin whose amino acid sequence has been modified (e.g., an amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), as long as it has the amino acid sequence specified in this disclosure. Alternatively, it may be an artificially designed amino acid sequence independent of naturally occurring fibroin (e.g., an artificial fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence). Artificial fibroins whose amino acid sequence has been modified are also included in the category of artificial fibroin, provided that the amino acid sequence differs from that of naturally occurring fibroin. Examples of artificial fibroins include artificial silk fibroin (a silk protein produced by silkworms) and artificial spider silk fibroin (a spider silk protein produced by spiders) whose amino acid sequence has been modified. Because artificial fibroin is relatively easy to fibrillate and has high fiber-forming ability, the molding material preferably contains artificial spider silk fibroin, and more preferably consists of artificial spider silk fibroin.

[0029] Artificial fibroin has the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m -(A) n The artificial fibroin may be a protein containing a domain sequence represented by a motif. The artificial fibroin may have further amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeats of the amino acid motif characteristic of fibroin and consist of about 100 amino acid residues.

[0030] As used herein, a "domain sequence" refers to a sequence of a molecule having the formula 1: [(A) n Motif-REP] m , or Formula 2: [(A) n Motif-REP] m-(A) n The amino acid sequence represented by the motif (A) n The motif represents an amino acid sequence mainly consisting of alanine residues, and the number of amino acid residues is an integer between 2 and 27. (A) n The number of amino acid residues in the motif may be an integer of 2 to 27, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. n The ratio of the number of alanine residues to the total number of amino acid residues in the motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that the motif is composed of only alanine residues). n At least seven of the motifs may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may also be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. (A) n The motifs may have the same or different amino acid sequences, and the REPs present in multiple instances may have the same or different amino acid sequences.

[0031] Specific examples of artificial fibroins include, for example, an artificial fibroin derived from the major spinal dragline silk protein produced in the major ampullate gland of spiders (as described in International Publication No. 2019 / 194263) (first artificial fibroin), an artificial fibroin having a domain sequence with a reduced content of glycine residues (second artificial fibroin), (A) n An artificial fibroin having a domain sequence with a reduced motif content (third artificial fibroin), the content of glycine residues, and (A) nThese include an artificial fibroin with a reduced motif content (fourth artificial fibroin), an artificial fibroin with a domain sequence containing a region with a locally high hydrophobic index (fifth artificial fibroin), and an artificial fibroin with a domain sequence with a reduced glutamine residue content (sixth artificial fibroin). The definitions of the first to sixth artificial fibroins are set forth in WO 2019 / 194263, the contents of which are incorporated herein by reference.

[0032] The artificial fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus, which allows the artificial fibroin to be isolated, immobilized, detected, visualized, and the like.

[0033] An example of a tag sequence is an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide consisting of approximately 4 to 10 histidine residues, and has the property of specifically binding to metal ions such as nickel, so it can be used to isolate artificial fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 8 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0034] Furthermore, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione, and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

[0035] Furthermore, "epitope tags" that utilize antigen-antibody reactions can also be used. By adding an antigenic peptide (epitope) as a tag sequence, antibodies specific to the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin), myc, and FLAG tags. By using epitope tags, artificial fibroin can be easily purified with high specificity.

[0036] Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with the protease, the artificial fibroin from which the tag sequence has been cleaved can be recovered.

[0037] Specific examples of artificial fibroins include those represented by SEQ ID NOS: 1 to 7. The artificial fibroin may be an artificial fibroin represented by SEQ ID NOS: 1 to 7 or an artificial fibroin containing an amino acid sequence having 90% or more sequence identity with these amino acid sequences. The respective contents of alanine residues, glycine residues, serine residues, threonine residues, tyrosine residues, glutamine residues, and lysine residues in the artificial fibroins represented by SEQ ID NOS: 1 to 7 are shown in Table 1 below. The artificial fibroins represented by SEQ ID NOS: 1 and 7 correspond to the aforementioned fourth artificial fibroin, the artificial fibroins represented by SEQ ID NOS: 2, 3, 5, and 6 correspond to the aforementioned sixth artificial fibroin, and the artificial fibroin represented by SEQ ID NOS: 4 corresponds to the aforementioned first artificial fibroin.

[0038] [Table 1]

[0039] The artificial fibroin may be an artificial fibroin that combines at least two or more of the characteristics of the first artificial fibroin, the second artificial fibroin, the third artificial fibroin, the fourth artificial fibroin, the fifth artificial fibroin, and the sixth artificial fibroin.

[0040] The molecular weight of the artificial fibroin according to this embodiment is not particularly limited, and may be, for example, 2 kDa or more and 700 kDa or less. The molecular weight of the artificial fibroin according to this embodiment may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, or 700 kDa or less, 600 kDa or less, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less.

[0041] The fiber diameter of the artificial protein fiber is not particularly limited and may be selected depending on the application, but the lower limit may be, for example, 1 μm or more, 2 μm or more, 3 μm or more, 5 μm or more, 7 μm or more, 10 μm or more, 16 μm or more, 20 μm or more, or 25 μm or more. The upper limit of the diameter of the artificial protein fiber is not limited to any specific numerical value, but may be, for example, 30 μm or less, 25 μm or less, 20 μm or less, 16 μm or less, or 10 μm or less. A suitable range for the fiber diameter of the artificial protein is determined, for example, by appropriately combining any of the above-mentioned lower and upper limits.

[0042] The length of the artificial protein fiber according to this embodiment is not particularly limited and may be a value within the range of 1 to 24 mm. Furthermore, the fiber length may be a value within the range of 5 to 20 mm, a value within the range of 8 to 17 mm, or a value within the range of 10 to 15 mm.

[0043] <Dyeing agent> The artificial protein fiber according to this embodiment is dyed with a dye containing an oxidase, specifically, for example, a dye containing bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2). Hereinafter, bilirubin oxidase will also be referred to as "BO." In this embodiment, bilirubin oxidase is an enzyme classified into EC1.3.3.5 according to the Enzyme Commission number determined by the International Union of Biochemistry and Molecular Biology, and laccase is an enzyme classified into EC1.10.3.2. Dyeing methods using these enzymes allow dyeing to be carried out at relatively low temperatures, and denaturation of the artificial protein fibers is suppressed, resulting in excellent safety during dyeing.

[0044] Whether or not the artificial protein fiber according to this embodiment has been dyed with a dye containing an oxidase, specifically, for example, a dye containing bilirubin oxidase (EC1.3.3.5) or laccase (EC1.10.3.2), can be confirmed, for example, by the following method. This can be confirmed by carrying out dyeing under the conditions described in the examples and under comparative conditions in which only the enzyme is omitted (substrate (dye precursor) and buffer solution were added to various fabrics and reacted for the same time under the same conditions), and comparing the color difference between each dyed fabric and the fabric before dyeing. These enzymes are described in detail below.

[0045] 〔enzyme〕 -Oxidase- Oxidase is an enzyme that electron-reduces oxygen to produce water molecules. Examples of oxidases include glutathione peroxidase, ascorbate peroxidase, heme peroxidase, lactoperoxidase, myeloperoxidase, horseradish peroxidase, copper oxidase, iron oxidase, lignin peroxidase, manganese peroxidase, and P450. Among these, copper oxidase is preferred. The copper oxidase may be a copper-containing monooxidase or a copper-containing multicopper oxidase (hereinafter also referred to as a multicopper oxidase). Examples of copper-containing monooxidases include dopamine-β-monooxygenase, peptidylglycine α-hydroxylating monooxygenase, tyrosinase, and methane monooxygenase. The copper oxidase is preferably a multicopper oxidase. Multicopper oxidases are enzymes containing 2 to 8 copper atoms in the molecule, which are necessary for enzymatic activity, and which generate water molecules by electron reduction of oxygen. The multicopper oxidase is preferably at least one selected from phenol oxidase and bilirubin oxidase, more preferably phenol oxidase, and even more preferably polyphenol oxidase. Examples of multicopper oxidases include laccase, bilirubin oxidase, ascorbate oxidase, ceruloplasmin, Fet3p, CueO, CotA, stellacyanin, tyrosinase, catechol oxidase, and nitrite reductase. Among these, laccase or bilirubin oxidase is particularly preferred.

[0046] -Bilirubin oxidase (BO)- The BO according to this embodiment is a BO derived from a microorganism, preferably a BO derived from a microorganism of the genus Myrothecium or Bacillus. Furthermore, the original producer of the BO used in this embodiment is preferably Myrothecium verrucaria or Bacillus subtilis. The producer may be a wild-type strain (a strain isolated from nature that has not been subjected to mutation or modification treatment such as genetic engineering) or a mutant strain. BOs produced by a transformant obtained by introducing the BO gene of Myrothecium verrucaria into a host microorganism also fall under the category of Myrothecium verrucaria-derived BOs. The same applies to BOs derived from Bacillus subtilis. In this way, BOs produced by genetic engineering (recombinant BOs) may be used.

[0047] Specific examples of BO-producing bacteria used in this embodiment include Myrothecium verrucaria MT-1 (FERM-BP 653 (Agricultural and Biological Chemistry, Vol. 45, pp. 2383-2384 (1981))), Myrothecium verrucaria IFO 6113, Myrothecium verrucaria IFO 6133, Myrothecium verrucaria IFO 6351, Myrothecium verrucaria IFO 9056, and Bacillus subtilis ATCC 23857. These strains can be cultured in liquid or solid form by conventional methods, and purified bilirubin oxidase preparations can be obtained from the culture broth by extraction, salting out, dialysis, ion exchange, gel filtration, and the like.

[0048] For details of BO derived from Myrothecium verrucaria, please refer to International Publication No. 2013 / 099034.

[0049] For details of BO derived from Bacillus subtilis, refer to Japanese Patent No. 4743854.

[0050] BO derived from Myrothecium verrucaria is commercially available from Amano Enzyme Inc., and BO derived from Bacillus subtilis is commercially available from Asahi Kasei Pharma Corporation under the product name Bilirubin Oxidase, abbreviated as BODII, and these commercially available BOs can also be used in this embodiment.

[0051] -Laccase- Laccase is an oxidase capable of oxidizing phenols and is found in plants, fungi, bacteria, animals, and the like. For example, laccases derived from fungi and bacteria are preferred. Examples of fungi and bacteria that produce laccase include Alphaproteobacteria, Aspergillus, Neurospora, Podospora, Botrytis, Coryvia, Phomes, Lentinus, Pleurotus, Trametes, Rhizoctonia, Coprinus, Psatilla, Myceliophthora, Scytharia, Polyporus, Phlebia, Coriolus, Bacillus, Pseudomonas, and Escherichia. Laccases derived from microorganisms of the genus Trametes are preferred, and laccases derived from Trametes sp. or Trametes versicolor are more preferred.

[0052] For example, these fungi or bacteria can be cultured in liquid or solid form by conventional methods, and purified laccase preparations can be obtained from the culture solution by extraction, salting out, dialysis, ion exchange, gel filtration, etc. These fungi or bacteria may be wild-type strains or mutant strains, or may be produced from transformants obtained by introducing genes into host microorganisms.

[0053] Laccase is commercially available as laccase (derived from Trametes sp.; manufactured by Amano Enzyme Inc.) or laccase (derived from Trametes versicolor; manufactured by SIGMA-ALDRICH), and these commercially available laccases can also be used in this embodiment.

[0054] 〔dye〕 The dyeing agent is preferably used in combination with a dye for dyeing. Specifically, it is preferable to use a dye and an enzyme in combination. In one aspect, the staining agent of this embodiment is provided as a composition in which the dye and the enzyme are mixed. The staining agent of this embodiment is called a single-component staining agent. On the other hand, the staining agent of this embodiment can also be provided in the form of a kit consisting of a first component containing the dye and a second component containing the enzyme. The staining agent of this embodiment is called a two-component staining agent. In the case of a two-component staining agent, each component is provided in a separate container or compartment, and the two components are mixed upon use. It can be said that single-component staining agents are superior to two-component staining agents in that they not only have a simpler configuration but also enable a more convenient method of use.

[0055] The dye is preferably an oxidation dye, and the enzyme contained in the dyeing agent in this embodiment acts on the oxidation dye to achieve the desired dyeing effect.

[0056] As used herein, the term "oxidation dye" refers to a dye that requires an oxidation reaction for color development or dyeing. Oxidation dyes include "dye precursors" that undergo oxidative polymerization themselves to develop color, and "dye auxiliaries (couplers)" that exhibit specific color tones by polymerizing with the dye precursor. In this embodiment, it is preferable to use an indole analog as the oxidation dye. Various indole analogs can be used. More preferably, an appropriate oxidation dye is selected from compounds that correspond to indole derivatives or indoline derivatives, or their salts (e.g., acid addition salts such as hydrochlorides, hydrobromides, sulfates, tartrates, lactates, or acetates). Specific examples of indole derivatives and indoline derivatives include 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-butyl-5,6-dihydroxyindoline, 4-hydroxy-5-methoxyindoline, 6-hydroxy-7-methoxyindoline, 6,7-dihydroxyindoline, 4,5-dihydroxyindoline, 4-methoxy-6-hydroxyindoline, N-hexyl-5,6-dihydroxyindoline, 2-methyl-5,6-dihydroxyindoline, 3-methyl-5,6-dihydroxyindoline, 4-hydroxyindoline, 2,3-dimethyl-5,6-dihydroxyindoline, 2-methyl-5-ethyl-6-hydroxyindoline, and 2-methyl-5-hydroxy-6-β-hydroxyethylindoline. , 4-hydroxypropylindoline, 2-hydroxy-3-methoxyindoline, 6-hydroxy-5-methoxyindoline, 6-hydroxyindoline, 5-hydroxyindoline, 7-hydroxyindoline, 7-aminoindoline, 5-aminoindoline, 4-aminoindoline, 5,6-dihydroxyindoline-2-carboxylic acid, 1-methyl-5,6-dihydroxyindoline, 4,5-dihydroxyindole, 5,6-dihydroxyindole, 6,7-dihydroxyindole, N-methyl-5,6-dihydroxyindole, N-ethyl-5,6-dihydroxyindole, N-hexyl-5,6-dihydroxyindole, 2-methyl-5,6-dihydroxyindole, 3-methyl-5,6-dihydroxyindole, 4-hydroxyindole, 2,3-dimethyl-5,The oxidation dye is selected from the group consisting of 6-dihydroxyindole, 2-methyl-5-ethyl-6-hydroxyindole, 2-methyl-5-hydroxy-6-β-hydroxyethylindole, 4-hydroxypropylindole, 2-hydroxy-3-methoxyindole, 4-hydroxy-5-methoxyindole, 6-hydroxy-7-methoxyindole, 6-hydroxy-5-methoxyindole, 6-hydroxyindole, 5-hydroxyindole, 7-hydroxyindole, 7-aminoindole, 6-aminoindole, 5-aminoindole, 4-aminoindole, 5,6-dihydroxyindole-2-carboxylic acid, and 1-methyl-5,6-dihydroxyindole. In a preferred embodiment, one or more oxidation dyes selected from the group consisting of 5,6-dihydroxyindole, 5,6-dihydroxyindoline, 5,6-dihydroxyindole-2-carboxylic acid, 5,6-dihydroxyindoline-2-carboxylic acid, 4-aminoindole, 5-hydroxyindole, and 5-aminoindole are used.

[0057] The dyeing agent of this embodiment uses one or more oxidation dyes. The combined use of two or more oxidation dyes is effective, for example, for improving the dyeing effect and adjusting the color (tone). In particular, it is preferable to combine an oxidation dye classified as a dye precursor with an oxidation dye classified as a dye auxiliary.

[0058] The amount of the oxidation dye to be blended is not particularly limited, and may be determined taking into consideration the properties and intended use of the oxidation dye to be used. In the case of a one-component dye, the oxidation dye may be blended in an amount of, for example, 0.01 to 20% by mass, preferably 0.05 to 10% by mass, and more preferably 0.05 to 1% by mass, based on the total amount of the dye. In the case of a two-component dye, the oxidation dye may be blended in an amount of, for example, 0.01 to 20% by mass, preferably 0.05 to 10% by mass, and more preferably 0.05 to 1% by mass, based on the total amount of the first component that will contain the oxidation dye.

[0059] In addition to oxidative dyes, direct dyes can be used to enhance the dyeing effect and adjust the color tone. Examples of direct dyes include 2-amino-4-nitrophenol, 2-amino-5-nitrophenol, 1-amino-4-methylaminoanthraquinone, nitro-paraphenylenediamine hydrochloride, 1,4-diaminoanthraquinone, nitro-paraphenylenediamine, picramic acid, sodium picramate, 2-amino-5-nitrophenol sulfate, resorcinol, nitro-paraphenylenediamine sulfate, paranitro-o-phenylenediamine sulfate, paranitro-meta-phenylenediamine sulfate, Natural Orange 6 (2-hydroxy-1,4-naphthoquinone), Acid Orange 8, Acid Violet 17, Remazol Brilliant Blue, Evans Blue, and Acid Blue 80.

[0060] The amount of the direct dye to be blended is not particularly limited. In the case of a one-component dye, the direct dye can be blended in an amount of, for example, 0.01 to 20% by mass relative to the total amount of the dye. In the case of a two-component dye, the direct dye is blended in the first component containing the oxidative dye and / or the second component containing the enzyme. In this case, the blending amount is, for example, 0.01 to 20% by mass relative to the total amount of the first component, and 0.01 to 20% by mass relative to the total amount of the second component. As with oxidative dyes, two or more types of direct dyes can be used in combination.

[0061] [Other ingredients] In addition to the above components, the dye in this embodiment may contain additional components (optional components). Examples of optional components include acidic compounds, alkaline compounds, reducing agents such as thiolactic acid, sodium sulfite, and N-acetyl-L-cysteine, surfactants, oily components, silicones, thickeners, solvents, water, chelating agents, amino acids, various salts, moisturizers, preservatives, UV protection agents, alcohols, polyhydric alcohols, and fragrances.

[0062] The dyeing agent of this embodiment may contain an alkaline compound. When a dyeing agent containing an alkaline compound is applied, it promotes swelling of the artificial protein fiber, improving the dyeing effect. The amount of alkaline compound is set so as to achieve a desired pH (e.g., pH 4.0 to 10.0) during use. An example of the amount of alkaline compound to be added in the case of a one-component dyeing agent is 0.01 to 20% by mass relative to the total amount of the dyeing agent. On the other hand, in the case of a two-component dyeing agent, the alkaline compound is generally added to the first component, which will contain the oxidation dye, and the amount to be added is, for example, 0.01 to 20% by mass relative to the total amount of the first component (however, the alkaline compound may be added to the second component instead of the first component, or may be added to both the first and second components). Specific examples of alkaline compounds include amine compounds such as monoethanolamine, monoisopropanolamine, triethanolamine, and diethanolamine, and inorganic compounds such as ammonia, sodium hydroxide, potassium hydroxide, ammonium carbonate, sodium carbonate, sodium hydrogencarbonate, potassium carbonate, and potassium hydrogencarbonate.

[0063] The dye of this embodiment is prepared in the form of a paste (cream), aerosol, gel, liquid, foam, etc. In the case of a two-component formulation consisting of a first component (containing an oxidation dye) and a second component (containing an enzyme), the forms of the first component and the second component do not need to be the same.

[0064] (fiber structure) The fiber structure according to this embodiment is a fiber structure formed from the artificial protein fiber according to this embodiment. In this specification, the term "fibrous structure" refers to an aggregate of processed fibers, and examples thereof include spun yarn, woven fabric, knitted fabric, braided fabric, and nonwoven fabric. The fiber structure may be a structure consisting only of the artificial protein fibers according to this embodiment, or may further contain other fibers such as other artificial protein fibers, other natural fibers, and other synthetic fibers in addition to the artificial protein fibers according to this embodiment.

[0065] <Application> The uses of the artificial protein fiber according to this embodiment and the fiber structure according to this embodiment are not particularly limited, and they can be used without particular limitation in applications where fiber structures are used, such as clothing, packaging materials, rugs, interior materials, furniture, bedding, building materials, wall materials, and filter materials.

[0066] (Manufacturing method of artificial protein fiber) The method for producing artificial protein fibers of the present invention includes a step (dyeing step) of dyeing artificial protein raw material fibers using a dye containing an oxidase, specifically, for example, a dye containing bilirubin oxidase (EC1.3.3.5) or laccase (EC1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis.

[0067] <Dyeing process> [Artificial protein fiber] The artificial protein raw fiber is a fiber before it is dyed with a dye. Preferred aspects of the artificial protein raw fiber are the same as the preferred aspects of the artificial protein fiber according to the present embodiment described above, except that it is not dyed.

[0068] [Dyeing agent] Preferred aspects of the dyeing agent used in the dyeing step are the same as the preferred aspects of the dyeing agent described above for the artificial protein fiber according to this embodiment.

[0069] [Dyeing method] In the dyeing step, it is preferable to treat the artificial protein raw material fiber with the dyeing agent described above in the presence of oxygen (i.e., in an oxygen atmosphere). In the case of a one-component dye, for example, the dye is diluted as necessary (dilution is not essential), and then the dye is brought into contact with the artificial protein raw material fiber by coating, immersion, or the like. In the case of a two-component dye, for example, the first and second components are mixed (they may be diluted or dissolved in a solvent as necessary), and then the dye is brought into contact with the artificial protein raw fiber by coating, immersion, etc. One component (the first or second component) may be brought into contact with the artificial protein raw fiber, and then the other component may be brought into contact with the artificial protein raw fiber, so that both components are mixed on the surface of the fiber, etc.

[0070] By the above-mentioned operation, the dye and the artificial protein raw fiber are brought into contact with each other. This contact state is then maintained for the time required for the desired dyeing. At this time, the temperature may be maintained or increased to enhance the dyeing effect or to dye more quickly or efficiently. The maintenance time is, for example, about 10 minutes to 3 hours, preferably about 20 minutes to 2 hours.

[0071] The pH conditions during treatment depend on the staining agent used. However, it is also possible to adjust the pH using a separate pH adjuster. The pH during treatment is, for example, 4.0 to 10.0, or may be 5.0 to 9.0, 6.0 to 9.0, 5.0 to 8.0, or 5.0 to 7.0, or may be 5.0 or higher and less than 7.0.

[0072] The temperature during treatment depends on the dye used, but is, for example, 5°C to 50°C, may be 10°C to 50°C, or may be 15°C to 40°C.

[0073] After the above treatment, the resulting artificial protein fibers may be washed and finally dried. For washing, for example, water, detergent, etc., can be used. Examples of drying methods include air drying (natural drying), hot air drying, spin drying, suction drying, and barrel drying.

[0074] In the method for producing dyed artificial protein fibers of the present invention, the oxidation and polymerization of the oxidation dye occur due to the action of an enzyme, and the artificial protein raw fiber is dyed. In this embodiment, because an enzyme is used in the oxidation reaction, there is less damage to the dyed material during the dyeing process. The dyeing agent in this embodiment enables effective dyeing while reducing the impact on the artificial protein raw fiber.

[0075] (Method for dyeing artificial protein fibers) The method for dyeing artificial protein fibers according to this embodiment includes a step (dyeing step) of treating fibroin fibers with a dye containing an oxidase, specifically, for example, a dye containing bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis. A preferred aspect of the dyeing step in the method for dyeing artificial protein fibers according to this embodiment is the same as a preferred aspect of the dyeing step in the method for producing artificial protein fibers according to this embodiment, except that the artificial protein raw fiber is limited to fibroin fiber.

[0076] (dyeing agent for artificial protein fibers) The dye for artificial protein fibers according to this embodiment contains an oxidase, specifically, for example, bilirubin oxidase (EC1.3.3.5) or laccase (EC1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis. Preferred aspects of the dye for artificial protein fibers according to this embodiment are the same as the preferred aspects of the dye explained for the artificial protein fibers according to this embodiment above. [Example]

[0077] The present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, processing details, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

[0078] Example A (Fabric manufacturing) <Manufacturing artificial protein fibers> [Production of artificial fibroin] Based on the base sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), an artificial fibroin (hereinafter also referred to as "PRT966") having the amino acid sequence shown in SEQ ID NO: 2 was designed.

[0079] Next, a nucleic acid encoding PRT966 was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. The nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme digestion with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

[0080] Escherichia coli BLR(DE3) was transformed with the pET22b(+) expression vector containing a nucleic acid encoding PRT966. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture was added to 100 mL of seed culture medium (see table below) containing ampicillin so that the OD600 became 0.005. The culture temperature was maintained at 30°C, and flask culture was continued until the OD600 reached 5 (approximately 15 hours), yielding a seed culture.

[0081] [Table 2]

[0082] The seed culture was added to a jar fermenter containing 500 ml of production medium (see table below) so that the OD600 was 0.05. The culture temperature was maintained at 37°C, and the pH was controlled to a constant 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.

[0083] [Table 3]

[0084] Immediately after complete consumption of glucose in the production medium, a feed solution (455 g glucose / L, 120 g yeast extract / L) was added at a rate of 1 mL / min. The culture temperature was maintained at 37°C and pH was controlled at 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration for 20 hours. Subsequently, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM to induce PRT966 expression. Twenty hours after IPTG addition, the culture was centrifuged and the cells were harvested. SDS-PAGE was performed using cells prepared from the cultures before and after IPTG addition. The IPTG-dependent expression of PRT966 was confirmed by the appearance of a band of the size corresponding to PRT966.

[0085] -Purification of PRT966- Two hours after the addition of IPTG, the cells were harvested and washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF and disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until highly purified. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL and dissolved by stirring at 60°C for 30 minutes. After dissolution, the cells were dialyzed against water using a dialysis tube (Cellulose tube 36 / 32, Sanko Junyaku Co., Ltd.). The white aggregated protein (PRT966) obtained after dialysis was collected by centrifugation, and the water was removed using a freeze-dryer to collect a freeze-dried powder.

[0086] The purity of PRT966 in the resulting freeze-dried powder was confirmed by image analysis of polyacrylamide gel electrophoresis using Totallab (Nonlinear Dynamics Ltd.), which revealed that the purity of PRT966 was approximately 85%.

[0087] (2) Production of artificial fibroin fibers The artificial fibroin (PRT966) was added to dimethyl sulfoxide (DMSO) to a concentration of 24% by mass, and then LiCl was added as a dissolution promoter to a concentration of 4.0% by mass. The mixture was then dissolved using a shaker for 3 hours. Dust and bubbles were then removed to obtain a dope solution. The solution viscosity of the dope solution was 5000 cP (centipoise) at 90°C.

[0088] (spinning) The dope solution obtained as described above was used in a spinning apparatus 10 shown in Fig. 2 to carry out a known dry-wet spinning process to obtain an artificial fibroin monofilament. The dry-wet spinning process was carried out under the following conditions. Extrusion nozzle diameter: 0.1 mm Extrusion speed: 327.6 ml / h Temperature of coagulation liquid (methanol): 2°C Winding speed: 99.5 m / min Stretching ratio: 4.52x Drying temperature: 80℃ Air gap length: 5mm Fig. 2 is a schematic diagram showing an example of a spinning apparatus for producing protein fibers. The spinning apparatus 10 shown in Fig. 2 is an example of a spinning apparatus for dry-wet spinning, and includes, in order from the upstream side, an extrusion apparatus 1, a coagulation bath 20, a washing bath 21, and a drying apparatus 4. The extrusion device 1 has a storage tank 7, in which a dope solution (spinning solution) 6 is stored. A coagulation liquid 11 (e.g., methanol) is stored in a coagulation bath 20. The dope solution 6 is extruded from a nozzle 9 provided with an air gap 19 between the nozzle and the coagulation liquid 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded dope solution 6 is supplied into the coagulation liquid 11 through the air gap 19. In the coagulation liquid 11, the solvent is removed from the dope solution 6, and the protein is coagulated. The coagulated protein is introduced into a washing bath 21, where it is washed with a washing liquid 12 in the washing bath 21, and then sent to the drying device 4 by a first nip roller 13 and a second nip roller 14 installed in the washing bath 21. At this time, for example, by setting the rotation speed of the second nip roller 14 faster than the rotation speed of the first nip roller 13, protein fibers 36 drawn at a ratio corresponding to the ratio of the rotation speeds are obtained. The protein fibers 36 stretched in the washing solution 12 are dried as they pass through the drying device 4 after leaving the washing bath 21, and then wound up by a winder. In this way, the protein fibers 36 are obtained by the spinning device 10 as a wound product 5 that is finally wound up by the winder. Note that 18a to 18g are yarn guides.

[0089] <Manufacturing artificial protein fiber fabric> (1) Manufacture of spun yarn A plurality of artificial fibroin fibers obtained as described above were bundled and cut to a length of 38 mm using a tabletop fiber cutter to produce artificial fibroin staples. The produced artificial fibroin staples were immersed in water at 40°C for 1 minute to cause crimping, and then dried at 40°C for 18 hours to obtain crimped artificial fibroin staples. Artificial fibroin spun yarn was produced using this crimped artificial fibroin staple and known cotton spinning equipment.

[0090] (2) Fabric manufacturing An artificial protein fiber spun yarn knitted fabric was produced using the artificial fibroin spun yarn (20 count (cotton count) two-ply yarn) obtained as described above and a Marusan 18-gauge test circular knitting machine manufactured by Marui Sen-i Kikai Co., Ltd. For comparison, a wool spun yarn knitted fabric and a silk spun yarn knitted fabric were produced using a commercially available wool spun yarn (32 count (wool count) two-ply yarn) and a silk spun yarn (34 count (wool count) two-ply yarn) and the above circular knitting machine, respectively.

[0091] <Fabric dyeing> All of the following steps were carried out at room temperature (23°C). The fabrics of each example (artificial protein spun yarn knitted fabric) or comparative examples (wool spun yarn knitted fabric and silk spun yarn knitted fabric) were soaked in water and dried overnight. Next, 100 mg of the substrate (dye precursor) shown in Table 4 below was weighed into a 50 mL Falcon tube, and 44 mL of Milli-Q water and 5 mL of the buffer solution shown in Table 4 below were added, followed by stirring with a rotator to obtain a mixed solution. The dried dough and the mixed liquid after stirring were placed in a beaker and stirred with a stirrer for 15 minutes, and the dough was turned over and stirred for another 15 minutes. 1 mL of the enzyme listed in Table 4 below was added to the beaker and stirred for 60 minutes. The fabric was then added to the soaping solution and stirred for 30 minutes. The soaping solution used was 0.25 mL of Liquid Monogen New (manufactured by Aikuma Dye Co., Ltd.) plus 50 mL of Milli-Q water. Thereafter, the fabric was washed with water and dried to obtain a dyed fabric.

[0092] [Table 4]

[0093] (Evaluation of color fastness) In each example or comparative example, 40 mL of the laundry solution was weighed into a 50 mL Falcon tube, and the dyed fabric was placed in. The laundry solution used was a mixture of 0.2 g of Top (a weak alkaline detergent, manufactured by Lion Corporation) and 39.8 g of Milli-Q water. The Falcon tube was agitated by end-over-end stirring at room temperature (23°C) and 60 rpm for 2 hours, after which the dyed fabric was taken out and dried at room temperature (23°C). The fabric before being put into the Falcon tube and the fabric after being dried were subjected to CIELAB 1976 L according to the method described in JIS Z 8781-4:2013. * a * b * Color difference (ΔE * ab) was calculated. ΔE * The smaller the ab, the less color fading occurs when washed, and the higher the color fastness. The evaluation results are shown in Figure 1. FIG. 1 shows that the fabrics according to Examples 1 to 3 of the present invention, which use artificial protein fibers, are superior in color fastness compared to Comparative Examples 1 to 6, which use natural protein fibers such as wool and silk.

[0094] Example B In the same manner as in Example A, artificial protein fibers were obtained.

[0095] <Fabric dyeing> All of the following steps were carried out at room temperature (23°C). The fabrics of each example (artificial protein spun yarn knitted fabric) or comparative examples (wool spun yarn knitted fabric and silk spun yarn knitted fabric) were soaked in water and dried overnight. Next, 100 mg of the substrate (dye precursor) shown in Table 6 below was weighed into a 50 mL Falcon tube, and 44 mL of Milli-Q water and 5 mL of the buffer solution shown in Table 5 below were added, followed by stirring with a rotator to obtain a mixed solution. The dried dough and the mixed liquid after stirring were placed in a beaker and stirred with a stirrer for 15 minutes, and the dough was turned over and stirred for another 15 minutes. 1 mL of the enzyme listed in Table 5 below was added to the beaker and stirred for 60 minutes. The fabric was then added to the soaping solution and stirred for 30 minutes. The soaping solution used was 0.25 mL of Liquid Monogen New (manufactured by Aikuma Dye Co., Ltd.) plus 50 mL of Milli-Q water. Thereafter, the fabric was washed with water and dried to obtain a dyed fabric.

[0096] [Table 5]

[0097] (Evaluation of color fastness) In each example or comparative example, 40 mL of the laundry solution was weighed into a 50 mL Falcon tube, and the dyed fabric was placed in. The laundry solution used was a mixture of 0.2 g of Top (a weak alkaline detergent, manufactured by Lion Corporation) and 39.8 g of Milli-Q water. The Falcon tube was agitated by end-over-end stirring at room temperature (23°C) and 60 rpm for 2 hours, after which the dyed fabric was taken out and dried at room temperature (23°C). The fabric before being put into the Falcon tube (before dyeing) and the fabric after drying (after dyeing) were subjected to CIELAB 1976 L according to the method described in JIS Z 8781-4:2013. * a * b * Color difference (ΔE * ab) was calculated. ΔE * The ab values ​​were measured at five points, the four corners and the center of the fabric, and the average value and standard deviation were calculated. * The smaller the ab, the less color fading occurs when washed, and the higher the color fastness. The evaluation results are shown in Figure 3. The graph shows the average value, and the error bars show the standard deviation. From Figure 3, it can be seen that even when the substrate was changed to an indoline derivative, the fabrics in Examples 4 to 6 of the present application, which used artificial protein fibers, had superior color fastness compared to Comparative Examples 7 to 9, which used silk, a natural protein fiber.

[0098] Example C In the same manner as in Example A, artificial protein fibers were obtained.

[0099] <Fabric dyeing> All of the following steps were carried out at room temperature (23°C). The fabric of each example (artificial protein spun yarn knitted fabric) or comparative example (silk spun yarn knitted fabric) was soaked in water and dried overnight. Next, 100 mg of the substrate (dye precursor) shown in Table 6 below was weighed into a 50 mL Falcon tube, and 44 mL of Milli-Q water and 5 mL of the buffer solution shown in Table 5 below were added, followed by stirring with a rotator to obtain a mixed solution. The dried dough and the mixed liquid after stirring were placed in a beaker and stirred with a stirrer for 15 minutes, and the dough was turned over and stirred for another 15 minutes. 1 mL of the enzyme listed in Table 6 below was added to the beaker and stirred for 60 minutes. The fabric was then added to the soaping solution and stirred for 30 minutes. The soaping solution used was 0.25 mL of Liquid Monogen New (manufactured by Aikuma Dye Co., Ltd.) plus 50 mL of Milli-Q water. Thereafter, the fabric was washed with water and dried to obtain a dyed fabric.

[0100] [Table 6]

[0101] (Evaluation of dyeing unevenness) The dyed fabric was subjected to CIELAB 1976 L according to the method described in JIS Z 8781-4:2013. * a * b * Color difference (ΔE * ab) was calculated. ΔE * Measurements were taken at five points (the four corners and the center) of the fabric, and the standard deviation was calculated. The smaller the standard deviation, the less uneven the dyeing at that position on the fabric. The evaluation results are shown in Figure 4. FIG. 4 shows that the occurrence of dyeing unevenness is suppressed in the fabrics of Examples 7 and 8 of the present application, which use artificial protein fibers, compared to Comparative Examples 10 to 13, which use natural protein fibers such as wool or silk. [Explanation of symbols]

[0102] 1...extrusion device, 4...drying device, 6...dope liquid, 10...spinning device, 20...coagulation bath, 21...washing bath, 36...protein fiber.

Claims

1. An artificial protein fiber comprising an artificial protein, Stained with an oxidase-containing stain Artificial protein fiber.

2. 2. The artificial protein fiber of claim 1, wherein the oxidase is bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2).

3. 10. The artificial protein fiber of claim 1, wherein the oxidase is an oxidase derived from a microorganism.

4. 4. The artificial protein fiber according to claim 3, wherein the oxidase is a bilirubin oxidase derived from a microorganism of the genus Myrothecium or the family Bacillus, or a laccase derived from a microorganism of the family Trametes.

5. 5. The artificial protein fiber of claim 4, wherein the oxidase is bilirubin oxidase derived from Myrothecium verrucaria or Bacillus subtilis, or laccase derived from Trametes sp. or Trametes versicolor.

6. The artificial protein fiber of claim 1 , wherein the dyeing agent further comprises an oxidation dye consisting of an indole derivative or an indoline derivative.

7. The oxidation dye may be 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-butyl-5,6-dihydroxyindoline, 4-hydroxy-5-methoxyindoline, 6-hydroxy-7-methoxyindoline, 6,7-dihydroxyindoline, 4,5-dihydroxyindoline, 4-methoxy-6-hydroxyindoline, N-hexyl-5,6-dihydroxyindoline, 2-methyl-5,6-dihydroxyindoline, 3-methyl-5,6-dihydroxyindoline, Phosphorus, 4-hydroxyindoline, 2,3-dimethyl-5,6-dihydroxyindoline, 2-methyl-5-ethyl-6-hydroxyindoline, 2-methyl-5-hydroxy-6-β-hydroxyethylindoline, 4-hydroxypropylindoline, 2-hydroxy-3-methoxyindoline, 6-hydroxy-5-methoxyindoline, 6-hydroxyindoline, 5-hydroxyindoline, 7-hydroxyindoline, 7-aminoindoline, 5-aminoindoline, 4-aminoindoline, 5,6-dihydroxyindoline-2-carboxamide carboxylic acid, 1-methyl-5,6-dihydroxyindoline, 4,5-dihydroxyindole, 5,6-dihydroxyindole, 6,7-dihydroxyindole, N-methyl-5,6-dihydroxyindole, N-ethyl-5,6-dihydroxyindole, N-hexyl-5,6-dihydroxyindole, 2-methyl-5,6-dihydroxyindole, 3-methyl-5,6-dihydroxyindole, 4-hydroxyindole, 2,3-dimethyl-5,6-dihydroxyindole, 2-methyl-5-ethyl-6-hydroxyindole indole, 2-methyl-5-hydroxy-6-β-hydroxyethyl indole, 4-hydroxypropyl indole, 2-hydroxy-3-methoxy indole, 4-hydroxy-5-methoxy indole, 6-hydroxy-7-methoxy indole, 6-hydroxy-5-methoxy indole, 6-hydroxy indole, 5-hydroxy indole, 7-hydroxy indole, 7-amino indole, 6-amino indole, 5-amino indole, 4-amino indole, 5,6-dihydroxyindole-2-carboxylic acid and 1-methyl-5,The artificial protein fiber according to claim 6, wherein the oxidation dye is selected from the group consisting of 6-dihydroxyindole.

8. The artificial protein fiber according to any one of claims 1 to 7, wherein the artificial protein is an artificial structural protein.

9. The artificial protein fiber according to claim 8 , wherein the artificial structural protein has an amino acid sequence that includes a repeat sequence.

10. 10. The artificial protein fiber according to claim 9, wherein the artificial structural protein has an amino acid sequence including an (A)n motif, the amino acid sequence being different from the amino acid sequence of a naturally occurring protein, the (A)n motif exhibits an amino acid sequence consisting of 2 to 27 amino acid residues, and the number of alanine residues relative to the total number of amino acid residues in the (A)n motif is 40% or more, and when multiple (A)n motifs are present, the (A)n motifs may have the same amino acid sequence or different amino acid sequences.

11. The artificial protein fiber of claim 10 , wherein the artificial protein is artificial fibroin.

12. A fiber structure formed from the artificial protein fiber according to any one of claims 1 to 7.

13. Dyeing artificial protein raw material fibers with a dye containing an oxidase, A method for producing dyed artificial protein fibers.

14. The method comprises treating fibroin fibers with a dye containing bilirubin oxidase (EC 1.3.3.5) or laccase (EC 1.10.3.2) derived from Myrothecium verrucaria or Bacillus subtilis, Method for dyeing artificial protein fibers.

15. Contains oxidases, Dyeing agent for artificial protein fibers.

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