Fiber-reinforced composite material and method for producing same

Abstract

Provided is a fiber-reinforced composite material that exhibits excellent characteristics in both heat resistance and impact strength regardless of the contained amount of an inorganic filler. The present invention also provides a method for advantageously producing the fiber-reinforced composite material.　This fiber-reinforced composite material comprises a thermoplastic resin, and artificial protein fibers containing an artificial protein. The fiber diameter of the artificial protein fibers is 1-30 μm and the percentage content of the artificial protein fibers is 0.5-30 mass%. This method is for producing said fiber-reinforced composite material.

Description

Fiber-reinforced composite material and its manufacturing method

[0001] The present invention relates to a fiber-reinforced composite material and a method for producing the same.

[0002] As has been well known for some time, thermoplastic resins are used in a variety of applications as general-purpose materials because they have excellent properties such as electrical properties, solvent resistance, and moldability, as well as low specific gravity and low cost.

[0003] Furthermore, resin composites obtained by compounding such thermoplastic resins with inorganic fillers such as glass fiber or wollastonite exhibit high strength characteristics and are therefore used as materials for molded articles that require high strength.

[0004] For example, Patent Document 1 describes an injection-molded article containing a thermoplastic resin and short protein fibers having a fiber length of 24 mm or less dispersed in the thermoplastic resin.

[0005] International Publication No. 2020 / 067518

[0006] On the other hand, due to the recent increase in environmental awareness, the use of inorganic fillers is increasingly being restricted.

[0007] Therefore, the applicant of the present application has proposed a fiber-reinforced composite material (injection molded article) in which bending properties are improved by blending artificial protein short fibers of a specific length into a thermoplastic resin matrix such as polypropylene (for example, Patent Document 1 mentioned above, etc.).

[0008] In such fiber-reinforced composite materials, artificial protein fibers are used instead of inorganic fillers, which can advantageously reduce the environmental load.

[0009] However, the inventors of the present invention have conducted extensive research into such fiber-reinforced composite materials and have found that there is still room for improvement in terms of heat resistance and impact strength.

[0010] An object of the present invention is to provide a fiber-reinforced composite material that exhibits excellent properties in both heat resistance and impact strength, regardless of the content of inorganic filler, and to provide a method for advantageously producing the fiber-reinforced composite material.

[0011] As a result of extensive research to solve the above problems, the inventors have discovered that a fiber-reinforced composite material obtained by blending artificial protein fibers of a specific fiber diameter with a thermoplastic resin in a specific ratio can exhibit a good balance of excellent heat resistance and sufficiently high impact strength.

[0012] The present invention was completed based on these findings, and representative aspects thereof are as follows.

[0013] [1] A fiber-reinforced composite material containing a thermoplastic resin and an artificial protein fiber containing an artificial protein, wherein the fiber diameter of the artificial protein fiber is 1 to 30 μm and the content of the artificial protein fiber is 0.5 to 30 mass%. [2] The fiber-reinforced composite material according to [1], wherein the thermoplastic resin is polypropylene. [3] The fiber-reinforced composite material according to [1] or [2], wherein the artificial protein is an artificial structural protein. [4] The fiber-reinforced composite material according to any one of [1] to [3], wherein the artificial protein is a hydrophobic artificial protein. [5] The fiber-reinforced composite material according to [4], wherein the average hydropathy index (HI) of the hydrophobic artificial protein is 0 or greater. [6] The fiber-reinforced composite material according to any one of [1] to [5], wherein the artificial protein has an amino acid sequence including a repeat sequence. [7] The fiber-reinforced composite material according to [6], wherein the artificial protein has an amino acid sequence including two or more repeats of the domain sequence shown in the following formula 1: Formula 1: REP1-REP2 [In Formula 1, REP1 represents an amino acid sequence consisting of 2 to 27 amino acid residues including at least one alanine, and REP2 represents an amino acid sequence consisting of 10 to 200 amino acid residues. Multiple REP1s may have the same amino acid sequence as each other, or different amino acid sequences. Multiple REP2s may have the same amino acid sequence as each other, or different amino acid sequences.] [8] The fiber-reinforced composite material according to [6], wherein the artificial protein has an amino acid sequence including a domain sequence represented by Formula 2: [(A)n motif-REP3]m or Formula 3: [(A)n motif-REP3]m-(A)n motif. [In Formula 2 and Formula 3, the (A)n motif represents an amino acid sequence consisting of 2 to 27 amino acid residues including at least one alanine, the number of alanine residues relative to the total number of amino acid residues in the (A)n motif being 40% or more, REP3 represents an amino acid sequence consisting of 10 to 200 amino acid residues, and m represents an integer of 2 to 300. Multiple (A)n motifs may have the same amino acid sequence or different amino acid sequences from each other.The plurality of REP3s may have the same amino acid sequence or different amino acid sequences.] [9] The fiber-reinforced composite material according to any one of [6] to [8], wherein the artificial protein has an amino acid sequence in which the content of glutamine residues is reduced compared to naturally-occurring fibroin.

[10] The fiber-reinforced composite material according to [9], wherein the artificial protein has an amino acid sequence in which one or more glutamine residues in REP3 in Formulas 2 and 3 above have been deleted or substituted with other amino acid residues compared to naturally-occurring fibroin.

[11] The fiber-reinforced composite material according to [9] or

[10] , wherein the artificial protein has an amino acid sequence in which the content of glutamine residues relative to the total number of amino acid residues is 9% or less.

[12] The fiber-reinforced composite material according to any one of [1], [6] to

[11] , wherein the artificial protein is artificial fibroin.

[13] The fiber-reinforced composite material according to any one of [1] to

[12] , comprising a kneaded resin of the thermoplastic resin and the artificial protein fiber.

[14] The fiber-reinforced composite material according to any one of [1] to

[13] , wherein the artificial protein fiber has a fiber length of 1 to 24 mm.

[15] The fiber-reinforced composite material according to any one of [1] to

[14] , further comprising maleic anhydride-modified polypropylene.

[16] The fiber-reinforced composite material according to any one of [1] to

[15] , further comprising liquid paraffin.

[17] A method for producing the fiber-reinforced composite material according to any one of [1] to

[16] , comprising compounding the thermoplastic resin and the artificial protein fiber.

[0014] According to the present invention, a fiber-reinforced composite material that exhibits excellent properties in both heat resistance and impact strength regardless of the content of inorganic filler is provided. The present invention also provides a method for advantageously producing the fiber-reinforced composite material.

[0015] 1 is a diagram showing a schematic diagram of a fiber-reinforced composite material according to the present invention; FIG. 2 is a schematic diagram showing the domain arrangement of an artificial protein having a repeating sequence unit; FIG. 3 is a schematic diagram showing an example of a spinning apparatus for producing artificial protein fibers; FIG. 4 is a graph showing the difference in load deflection temperature of fiber-reinforced composite materials depending on the diameter and content of artificial protein fibers; FIG. 5 is a graph showing the difference in impact strength of fiber-reinforced composite materials depending on the diameter and content of artificial protein fibers; FIG. 6 is a graph showing the difference in tensile properties of fiber-reinforced composite materials depending on the diameter and content of artificial protein fibers and the addition of LP or MAPP; FIG. 7 is a graph showing the difference in bending temperature of fiber-reinforced composite materials depending on the content of artificial protein fibers and the addition of LP or MAPP; FIG. 8 is a graph showing the difference in load deflection temperature of fiber-reinforced composite materials depending on the content of artificial protein fibers and the addition of LP or MAPP; 1 is a graph showing the difference in load deflection temperature between a fiber reinforced composite containing artificial protein fiber and a fiber reinforced composite containing natural protein fiber (wool).

[0016] 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.

[0017] (Fiber-reinforced composite material) The fiber-reinforced composite material of this embodiment is a fiber-reinforced composite material containing a thermoplastic resin and an artificial protein fiber containing an artificial protein, wherein the fiber diameter of the artificial protein fiber is 1 to 30 μm, and the content of the artificial protein fiber is 0.5 to 30 mass%.

[0018] Fig. 1 is a diagram schematically illustrating a fiber-reinforced composite material according to this embodiment. As is clear from Fig. 1, the fiber-reinforced composite material 100 according to this embodiment contains artificial protein fibers 102 as a fiber-reinforced resin dispersed within a thermoplastic resin 101 as a matrix resin.

[0019] <Thermoplastic Resin> The thermoplastic resin 1 used as the matrix resin is not particularly limited, and any thermoplastic resin that has conventionally been used as a matrix resin for fiber-reinforced composites can be used. Examples of the thermoplastic resin include hydrophobic thermoplastic resins such as polypropylene, polyethylene, and polystyrene. Among these, polypropylene is particularly preferred.

[0020] <Artificial Protein Fiber> The artificial protein fiber 102 is not particularly limited as long as it contains an artificial protein.

[0021] <Artificial Proteins> Artificial proteins include recombinant proteins and synthetic proteins. In other words, as used herein, "artificial protein" refers to a protein that has been artificially produced. An artificial protein may have a domain sequence that is different from the amino acid sequence of a naturally occurring protein, or it may have the same amino acid sequence as a naturally occurring protein. Furthermore, an "artificial protein" may use the amino acid sequence of a naturally occurring protein as is, or may be a protein whose amino acid sequence has been modified based on the amino acid sequence of a naturally occurring protein (for example, a protein whose amino acid sequence has been modified by modifying the gene sequence of a cloned naturally occurring protein), or may be a protein that has been artificially designed and synthesized without relying on a naturally occurring protein (for example, a protein having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding a designed amino acid sequence). Unlike naturally occurring proteins, the amino acid sequence of an artificial protein can be freely designed. Therefore, in artificial protein fibers containing such artificial proteins, the functions, characteristics, physical properties, etc. of the artificial protein can be controlled as desired by appropriately designing the amino acid sequence of the artificial protein. Furthermore, because uniform molecular design is always possible, proteins with high homology to the target protein and suited to the purpose can be stably obtained, which advantageously stabilizes the quality of artificial protein fibers containing artificial proteins.

[0022] The number of amino acid residues in the artificial protein is not particularly limited, and may be, for example, 50 or more. The number of amino acid residues may also be, for example, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more. The number of amino acid residues may be, for example, 5000 or less, 4500 or less, 4000 or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, or 1000 or less. The fewer the number of amino acid residues, the higher the solubility in solvents tends to be. The preferred number of amino acid residues in the protein is, for example, 100 to 5000, 150 to 4500, 200 to 4000, 250 to 3500, 300 to 3000, 350 to 2500, 400 to 2000, 450 to 1500, or 500 to 1000.

[0023] The molecular weight of the artificial protein is not particularly limited, and may be, for example, 2 kDa to 500 kDa. Furthermore, the molecular weight 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 may be 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The molecular weight may be, for example, 2 kDa to 500 kDa, 3 kDa to 500 kDa, 4 kDa to 500 kDa, 5 kDa to 500 kDa, 6 kDa to 500 kDa, 7 kDa to 500 kDa or more, 8 kDa to 500 kDa, 9 kDa to 500 kDa, 10 kDa to 500 kDa, 20 kDa to 400 kDa, 30 kDa to 360 kDa, 40 kDa to 360 kDa, 50 kDa to 360 kDa, 60 kDa to 300 kDa, 70 kDa to 300 kDa, 80 kDa to 300 kDa, 90 kDa to 200 kDa, or 100 kDa to 200 kDa.

[0024] The artificial protein may have a glycine residue content of 10 to 55% based on the number of amino acid residues. The glycine residue content may be, for example, 13% to 55%, 15% to 55%, 18% to 55%, 20% to 55%, 22% to 55%, or 25% to 55%. In this specification, the "glycine residue content" is a value represented by the following formula: Glycine residue content = (number of glycine residues contained in the artificial protein / total number of amino acid residues in the artificial protein) x 100 (%)

[0025] Furthermore, in this specification, the alanine residue content, serine residue content, threonine residue content, proline residue content, and tyrosine residue content described below have the same meanings as those obtained by replacing glycine residue with alanine residue, serine residue, threonine residue, proline residue, and tyrosine residue, respectively, in the above formula.

[0026] The artificial protein may have a total content (total content) of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any of the serine residue content, threonine residue content, tyrosine residue content, the sum of the serine residue content and threonine residue content, the sum of the serine residue content and tyrosine residue content, the sum of the threonine residue content and tyrosine residue content, or the sum of the serine residue content, threonine residue content, and tyrosine residue content), alanine residue content, and glycine residue content, based on the number of amino acid residues. This total content may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. There is no particular upper limit to the total content, but it may be, for example, 90% or less, 85% or less, or 80% or less.

[0027] The artificial protein may have a total serine residue content, threonine residue content, and tyrosine residue content, based on the number of amino acid residues, of 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 15% or more, or 16% or more. The total serine residue content, threonine residue content, and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0028] The artificial protein has an even distribution of serine, threonine, or tyrosine residues, and the total content of serine, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 4% or more, 5% or more, 10% or more, or 15% or more, or may be 50% or less, 40% or less, 30% or less, or 20% or less.

[0029] [Artificial structural protein] Examples of the artificial protein according to this embodiment include proteins that can be used for industrial purposes. "Usable for industrial purposes" means that the protein can be used for various general-purpose materials that are used indoors or outdoors. A specific example of an artificial protein that can be used for industrial purposes is an artificial structural protein.

[0030] 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.

[0031] When forming artificial structural proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds and thus produce 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, 10-40%, and may be 12-40%, 15-40%, 18-40%, 20-40%, or 22-40%. From the viewpoint of obtaining molded products with superior strength, the glycine residue content may be, for example, 10-55%, and may be 11-55%, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%.

[0032] It is preferable that 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 a structural protein is likely to inhibit the formation of strong intermolecular hydrogen bonds during processing of the molded article obtained by molding, 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, more than 5.5%, 6.0% or more, more than 6.5%, 7.0% or more, more than 7.5%, 8.0% or more, more 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.

[0033] [Hydrophobic Artificial Protein] The artificial protein according to this embodiment may be a hydrophobic artificial protein. When the artificial protein is a hydrophobic artificial protein, the water resistance of the artificial protein fiber (hydrophobic artificial protein fiber) obtained using the hydrophobic artificial protein can be advantageously improved. Furthermore, a fiber-reinforced composite material containing a hydrophobic artificial protein fiber and a thermoplastic resin exhibits superior properties in both heat resistance and impact strength compared to a fiber-reinforced composite material containing a hydrophilic artificial protein fiber and a thermoplastic resin, and can reliably exhibit even more excellent properties in impact strength in particular. Furthermore, a fiber-reinforced resin containing a hydrophobic artificial protein fiber and a hydrophobic thermoplastic resin can exhibit even more excellent properties in both heat resistance and impact strength in particular.

[0034] The hydrophobicity of an artificial protein can be estimated using the average HI (hydropathy index) value of each amino acid constituting the artificial protein as an index. Herein, the average HI value of a hydrophobic artificial protein may sometimes be greater than 0, for example, over the entire length of the amino acid sequence of the hydrophobic artificial protein. Furthermore, the average HI may be, for example, 0.00 or greater, greater than 0.00 and 0.10 or greater, 0.20 or greater, 0.22 or greater, 0.25 or greater, 0.30 or greater, 0.35 or greater, 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. Furthermore, the upper limit of the average HI is not particularly limited, but may be, for example, 1.00 or less or 0.7 or less.

[0035] The average HI value of the hydrophobic artificial protein and the hydrophobicity of the repeat sequence unit described below are determined according to known methods using known hydrophobicity indices of amino acid residues. Known hydrophobicity indices of amino acid residues are shown in Table 1. For example, the hydrophobicity may be calculated according to the method described in Kyte J, Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein," J. Mol. Biol., 157, pp. 105-132.

[0036]

[0037] Furthermore, the hydrophobic artificial protein may have low solubility in an aqueous lithium bromide solution (concentration: 9 M) at 60°C. That is, the hydrophobic artificial protein may have a maximum concentration, when dissolved in an aqueous lithium bromide solution (concentration: 9 M) at 60°C, of, for example, less than 30% by mass, less than 25% by mass, less than 20% by mass, less than 15% by mass, less than 10% by mass, less than 5% by mass, or less than 1% by mass. The hydrophobic artificial protein may also be completely insoluble in an aqueous lithium bromide solution (concentration: 9 M) at 60°C. When the hydrophobic artificial protein has low solubility in an aqueous lithium bromide solution at 60°C, the above-described effects obtained when the hydrophobic artificial protein is used as a material for forming various molded articles, including artificial protein fibers, can be more advantageously and reliably obtained.

[0038] The hydrophobic artificial protein may have a water contact angle of 55° or more. Furthermore, the water contact angle may be 60° or more, 65° or more, or 70° or more. The water contact angle can be evaluated by forming a film made of the hydrophobic artificial protein on a substrate, dropping water onto the film, and measuring the contact angle after 5 seconds. When the hydrophobic artificial protein has a water contact angle of 55° or more, the above-described effects obtained when using the hydrophobic artificial protein as a material for forming various molded articles, including artificial protein fibers, can be more advantageously and reliably obtained.

[0039] The hydrophobic artificial protein may have excellent hot hydrolysis resistance. For example, the hot hydrolysis resistance may be such that, when a 5 w / v % aqueous dispersion of the hydrophobic artificial protein is prepared and the dispersion is heated to 100°C, the protein does not decompose for at least 5 hours. When the hydrophobic artificial protein has excellent hot hydrolysis resistance, the above-described effects obtained when the hydrophobic artificial protein is used as a material for forming various molded articles, including artificial protein fibers, can be more advantageously and reliably obtained.

[0040] [Artificial Protein Having a Repeat Sequence] The artificial protein according to this embodiment may have a repeat sequence. That is, the artificial protein according to this embodiment may have multiple amino acid sequences (repeat sequence units) with high sequence identity within the artificial protein. The number of amino acid residues in the repeat sequence unit may be 6 to 200. The total number of glycine residues, serine residues, glutamine residues, and alanine residues relative to the total number of amino acid residues in the repeat sequence unit may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. Furthermore, the sequence identity between the repeat sequence units may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The hydrophobicity of the repeat sequence unit (i.e., the sum of the HIs of the amino acids contained in the repeat sequence) 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, more than 0.00, 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 of the repeat sequence unit is not particularly limited, but may be, for example, 1.0 or less or 0.7 or less.

[0041] An artificial protein having a repeat sequence may have an amino acid sequence containing a domain sequence (repeat sequence unit) represented by Formula 1: REP1-REP2. Such an artificial protein may contain, for example, 2 or more, 10 or more, 100 or more, or 200 or more of the domain sequence. The upper limit may be 300 or less. Further, further amino acid sequences (N-terminal sequence and C-terminal sequence) may be added to either or both of the N-terminal and C-terminal ends of multiple consecutive domain sequences. The N-terminal sequence and C-terminal sequence are regions that do not contain repeat sequences and consist of approximately 100 amino acid residues.

[0042] In addition, REP1 in the above formula 1 represents, for example, an amino acid sequence composed of 2 to 27 amino acid residues. The number of amino acid residues in REP1 may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Furthermore, REP2 represents, for example, an amino acid sequence composed of 10 to 200 amino acid residues. Multiple REP1s may have the same amino acid sequence as each other, or different amino acid sequences. Multiple REP2s may have the same amino acid sequence as each other, or different amino acid sequences.

[0043] The domain sequence of an artificial protein having the above-described repeat sequence unit may be, for example, an amino acid sequence that generates a crystalline region and an amorphous region.

[0044] A domain sequence consisting of an amino acid sequence that generates a crystalline region and an amorphous region may be, for example, an amino acid sequence represented by formula 2: [(A)n motif-REP3]m or formula 3: [(A)n motif-REP3]m-(A)n motif. In an artificial protein having such an amino acid sequence, the crystalline region corresponds, for example, to the (A)n motif in the amino acid sequence, and the amorphous region corresponds, for example, to the REP3 in the amino acid sequence. Note that even in an artificial protein having an amino acid sequence that includes a domain sequence represented by formula 2 or formula 3, the above-mentioned N-terminal sequence and C-terminal sequence may be further added to either or both of the N-terminal and C-terminal ends of the domain sequence.

[0045] Here, the (A)n motif represents an amino acid sequence having at least one alanine residue, and the number of amino acid residues may be 2 to 27. The number of amino acid residues in the (A)n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Furthermore, the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n 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 (A)n motif is composed only of alanine residues). The (A)n motif may have a total number of alanine residues, serine residues, threonine residues, and valine residues relative to the total number of amino acid residues in the (A)n motif of 80% or more, but may also be 85% or more, more preferably 90% or more, or may be 95% or more, or may be 100% (meaning that the motif is composed of only one or more amino acid residues selected from alanine residues, serine residues, threonine residues, and valine residues). REP3 represents an amino acid sequence composed of 2 to 200 amino acid residues. REP3 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. The (A)n motifs may have the same amino acid sequence as each other or different amino acid sequences. REP3s may also have the same amino acid sequence as each other or different amino acid sequences.

[0046] The (A)n motif primarily contains alanine residues and therefore tends to form an α-helix structure or a β-sheet structure. The inclusion of the (A)n motif in the repeating sequence unit results in the artificial protein of this embodiment having these secondary structures repeatedly. Therefore, as described below, when the artificial protein is formed into a fiber, these secondary structures are expected to provide high strength.

[0047] An example of an artificial protein having the above-described repeating sequence unit is artificial fibroin (hereinafter also referred to as modified fibroin). An example of the fibroin is naturally-occurring fibroin. An example of the naturally-occurring fibroin is 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.

[0048] More specific examples of naturally occurring fibroin include fibroins whose sequence information is registered in NCBIGenBank. For example, among the sequences registered in NCBIGenBank that contain INV as a division, the fibroin can be identified by extracting sequences in which spidroin, ampullate, fibroin, "silk and polypeptide," or "silk and protein" are described as keywords in the DEFINITION, a specific character string for "product" from the CDS, and a specific character string for the TISSUETYPE from the SOURCE.

[0049] As used herein, "artificial fibroin" refers to artificially produced fibroin (artificial fibroin). The artificial fibroin may be a fibroin with an amino acid sequence different from that of naturally occurring fibroin, or may be a fibroin with the same amino acid sequence as that of naturally occurring fibroin. Artificial fibroin can be produced by known methods, for example, by the method described in International Publication No. 2019 / 194263. Furthermore, when the artificial fibroin is expressed by forming an insoluble body within cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the artificial fibroin as a precipitate fraction. The recovered insoluble body of the artificial 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 sample.

[0050] 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.

[0051] "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, it is preferable for the molding material to contain, and more preferably consist of, artificial spider silk fibroin.

[0052] As described above, the artificial fibroin may be a protein containing a domain sequence represented by formula 2: [(A)n motif-REP3]m or formula 3: [(A)n motif-REP3]m-(A)n 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 are 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.

[0053] Specific examples of artificial fibroins include artificial fibroins derived from the major spinal dragline silk protein produced in the major ampullate gland of spiders (as described in WO 2019 / 194263 (first artificial fibroin)), artificial fibroins having a domain sequence with a reduced content of glycine residues (second artificial fibroin), artificial fibroins having a domain sequence with a reduced content of (A)n motifs (third artificial fibroin), artificial fibroins having a reduced content of glycine residues and a reduced content of (A)n motifs (fourth artificial fibroin), artificial fibroins having a domain sequence containing a region with a locally high hydrophobic index (fifth artificial fibroin), and artificial fibroins having a domain sequence with a reduced content of glutamine residues (sixth artificial fibroin). The definitions of each of the first to sixth artificial fibroins are incorporated herein by reference in the contents of WO 2019 / 194263.

[0054] 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.

[0055] Examples of tag sequences include affinity tags that utilize specific affinity (binding ability, affinity) with other molecules. Specific examples of affinity tags include histidine tags (His tags). His tags are short peptides consisting of approximately 4 to 10 histidine residues, and have the property of specifically binding to metal ions such as nickel, so they can be used to isolate artificial fibroin by chelating metal chromatography. Specific examples of tag sequences include the amino acid sequence shown in SEQ ID NO: 8 (an amino acid sequence including a His tag sequence and a hinge sequence).

[0056] 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.

[0057] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody specific to the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, artificial fibroin can be easily purified with high specificity.

[0058] 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.

[0059] 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 2 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 NO: 4 corresponds to the aforementioned first artificial fibroin.

[0060]

[0061] The artificial fibroin may be an artificial fibroin having at least two or more 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.

[0062] The molecular weight of the artificial fibroin 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.

[0063] Incidentally, the artificial protein having a repeating sequence unit according to this embodiment (e.g., artificial fibroin) may have an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

[0064] Furthermore, an artificial protein having a repeat sequence unit may contain at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP3.

[0065] When an artificial protein having a repeat sequence unit contains a GPGXX motif in REP3, the GPGXX motif content is usually 1% or more, or may be 5% or more, or may be 10% or more. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

[0066] As used herein, the term "GPGXX motif content" refers to a value calculated by the following method. In an artificial protein containing a domain sequence represented by Formula 2: [(A)n motif-REP3]m or Formula 3: [(A)n motif-REP3]m-(A)n motif, the GPGXX motif content is calculated as s / t, where s is the sum of three times the number of GPGXX motifs contained in all REP3 contained in the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence (i.e., equivalent to the total number of Gs and Ps in the GPGXX motifs), and t is the total number of amino acid residues in all REP3 contained in the domain sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence and further excluding the (A)n motif.

[0067] The reason why the calculation of the GPGXX motif content targets "the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" is that the "sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence corresponding to REP3) may contain a sequence that has low correlation with the sequence characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation result of the GPGXX motif content, and this influence is to be excluded. Note that when a "GPGXX motif" is located at the C-terminus of REP3, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

[0068] FIG. 2 is a schematic diagram showing the domain sequence of an artificial protein having repeat sequence units (for example, artificial fibroin). A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 2. First, in the domain sequence of the artificial protein shown in FIG. 2 (which is of the "[(A)n motif-REP3]m-(A)n motif" type), all REP3 is contained in "the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown as "Region A" in FIG. 2). Therefore, the number of GPGXX motifs required to calculate s is 7, and s is 7×3=21. Similarly, all of REP3 is contained in "the sequence obtained by excluding from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown as "Region A" in Figure 2), and therefore the total number of amino acid residues t of all REP3, further excluding the (A)n motif from this sequence, is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, which is 21 / 150 = 14.0% in the case of the artificial protein in Figure 2.

[0069] An artificial protein having repeating sequence units may have a glutamine residue content of 9% or less, 7% or less, 4% or less, or 0%.

[0070] In this specification, the "glutamine residue content" is a value calculated by the following method. In an artificial protein containing a domain sequence represented by Formula 2: [(A)n motif-REP3]m or Formula 3: [(A)n motif-REP3]m-(A)n motif, in all REP3 contained in a sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence (a sequence corresponding to "region A" in Figure 2), the total number of glutamine residues contained in that region is defined as u, and the total number of amino acid residues in all REP3 obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence and further excluding the (A)n motif is defined as t. The reason for targeting "the sequence obtained by excluding from the domain sequence the sequence from the most C-terminal (A)n motif to the C-terminus of the domain sequence" in calculating the glutamine residue content is the same as that described above.

[0071] An artificial protein having a repeat sequence unit may have an amino acid sequence in which the domain sequence is such that, compared to naturally occurring fibroin, one or more glutamine residues in REP3 in Formulas 2 and 3 above have been deleted or substituted with other amino acid residues (thereby reducing the content of glutamine residues).

[0072] The "other amino acid residue" may be any amino acid residue other than glutamine, but is preferably an amino acid residue with a higher hydrophobicity index than glutamine. The hydrophobicity indexes of amino acid residues are as shown in Table 1 above.

[0073] As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include those selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine ​​(C), methionine (M), and alanine (A) are more preferred, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferred.

[0074] In an artificial protein comprising a domain sequence represented by formula 2 or 3, the hydrophobicity of REP3 is preferably -0.8 or higher, more preferably -0.7 or higher, even more preferably 0 or higher, even more preferably 0.3 or higher, and particularly preferably 0.4 or higher. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or lower, or 0.7 or lower.

[0075] As used herein, the "hydrophobicity of REP3" is a value calculated by the following method. In an artificial protein (e.g., artificial fibroin) containing a domain sequence represented by Formula 2: [(A)n motif-REP3]m or Formula 3: [(A)n motif-REP3]m-(A)n motif, the hydrophobicity of REP3 is calculated as v / t, where v is the sum of the hydrophobicity indices of each amino acid residue in all REP3 contained in the sequence obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence (the sequence corresponding to "region A" in Figure 2), and t is the total number of amino acid residues in all REP3 obtained by removing from the domain sequence the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence and further removing the (A)n motif. The reason for using "the sequence obtained by excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminus of the domain sequence" in calculating the hydrophobicity of REP3 is the same as that described above.

[0076] An artificial protein having a repeat sequence unit may have a domain sequence that, compared to naturally occurring fibroin, corresponds to the deletion of one or more glutamine residues in REP3 and / or the substitution of one or more glutamine residues in REP3 with other amino acid residues, and may also have a further amino acid sequence modification that corresponds to the substitution, deletion, insertion and / or addition of one or more amino acid residues.

[0077] An artificial protein having a repeat sequence unit can be obtained, for example, by deleting one or more glutamine residues in REP3 from the cloned gene sequence of naturally occurring fibroin and / or substituting one or more glutamine residues in REP3 with other amino acid residues. Alternatively, it can be obtained, for example, by designing an amino acid sequence corresponding to the deletion of one or more glutamine residues in REP3 from the amino acid sequence of naturally occurring fibroin and / or the substitution of one or more glutamine residues in REP3 with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0078] More specific examples of artificial proteins having repeat sequence units include (i) modified fibroins containing the amino acid sequence shown in SEQ ID NO:9 (Met-PRT888), SEQ ID NO:10 (Met-PRT965), SEQ ID NO:11 (Met-PRT889), SEQ ID NO:12 (Met-PRT916), SEQ ID NO:13 (Met-PRT918), SEQ ID NO:14 (Met-PRT699), SEQ ID NO:15 (Met-PRT698), SEQ ID NO:16 (Met-PRT966), SEQ ID NO:17 (Met-PRT917), or SEQ ID NO:18 (Met-PRT1028); or (ii) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0079] The artificial protein (i) will now be described. The amino acid sequence shown in SEQ ID NO: 9 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO: 10 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with TS, and substituting the remaining Q with A. The amino acid sequence shown in SEQ ID NO: 11 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with VL, and substituting the remaining Q with I. The amino acid sequence shown in SEQ ID NO: 12 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with VI, and substituting the remaining Q with L. The amino acid sequence shown in SEQ ID NO: 13 is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with VF, and substituting the remaining Q with I.

[0080] The amino acid sequence shown in SEQ ID NO: 14 is obtained by substituting all QQs with VLs in the amino acid sequence shown in SEQ ID NO: 20 (Met-PRT525). The amino acid sequence shown in SEQ ID NO: 15 is obtained by substituting all QQs with VLs in the amino acid sequence shown in SEQ ID NO: 20 and substituting the remaining Qs with I.

[0081] The amino acid sequence shown in SEQ ID NO: 16 is obtained by substituting all QQ with VF and the remaining Q with I in a sequence in which the 20 domain sequence region present in the amino acid sequence shown in SEQ ID NO: 19 (Met-PRT410) is repeated twice.

[0082] The amino acid sequence shown in SEQ ID NO: 17 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with LI and substituting the remaining Q with V. The amino acid sequence shown in SEQ ID NO: 18 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence shown in SEQ ID NO: 19 with IF and substituting the remaining Q with T.

[0083] The amino acid sequences represented by SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18 all have a glutamine residue content of 9% or less (Table 3).

[0084]

[0085] The artificial protein (i) may consist of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0086] The artificial protein (ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18. The artificial protein (ii) is also a protein comprising a domain sequence represented by formula 2: [(A)n motif-REP3]m or formula 3: [(A)n motif-REP3]m-(A)n motif. The sequence identity is preferably 95% or more.

[0087] The artificial protein (ii) preferably has a glutamine residue content of 9% or less, and the artificial protein (ii) preferably has a GPGXX motif content of 10% or more.

[0088] Artificial proteins having repeating sequence units may contain tag sequences at either or both of the N-terminus and C-terminus, which allows for isolation, immobilization, detection, visualization, etc. of the artificial protein.

[0089] More specific examples of artificial proteins containing tag sequences include (iii) modified fibroins containing the amino acid sequence shown in SEQ ID NO:21 (PRT888), SEQ ID NO:22 (PRT965), SEQ ID NO:23 (PRT889), SEQ ID NO:24 (PRT916), SEQ ID NO:3 (PRT918), SEQ ID NO:25 (PRT699), SEQ ID NO:26 (PRT698), SEQ ID NO:2 (PRT966), SEQ ID NO:27 (PRT917) or SEQ ID NO:28 (PRT1028), or (iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:2, SEQ ID NO:27 or SEQ ID NO:28.

[0090] The amino acid sequences represented by SEQ ID NOs:21, 22, 23, 24, 3, 25, 26, 2, 27, and 28 are obtained by adding the amino acid sequence represented by SEQ ID NO:8 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences represented by SEQ ID NOs:9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, respectively. Because only a tag sequence has been added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences represented by SEQ ID NOs:21, 22, 23, 24, 3, 25, 26, 2, 27, and 28 all have a glutamine residue content of 9% or less (Table 4).

[0091]

[0092] The artificial protein (iii) may consist of the amino acid sequence shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:3, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:2, SEQ ID NO:27 or SEQ ID NO:28.

[0093] The artificial protein (iv) comprises an amino acid sequence that has 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 2, SEQ ID NO: 27, or SEQ ID NO: 28. The artificial protein (iv) is also a protein that comprises a domain sequence represented by formula 2: [(A)n motif-REP3]m or formula 3: [(A)n motif-REP3]m-(A)n motif. The sequence identity is preferably 95% or more.

[0094] The artificial protein (iv) preferably has a glutamine residue content of 9% or less, and the artificial protein (iv) preferably has a GPGXX motif content of 10% or more.

[0095] In one embodiment, an artificial protein having a repeat sequence unit may contain a secretion signal for releasing the protein produced in a recombinant protein production system outside the host. The sequence of the secretion signal can be appropriately designed depending on the type of host.

[0096] [Artificial Protein Derivative] The artificial protein according to this embodiment may be an artificial protein derivative such as a block copolymer. For example, the artificial protein derivative may be a block copolymer including a first segment including a polypeptide chain and a second segment bound to the first segment.

[0097] The polypeptide chain constituting the first segment may be, for example, an artificial protein as described in detail above in the sections [Artificial Protein], [Hydrophobic Artificial Protein], and [Artificial Protein Having Repetitive Sequences].

[0098] The second segment may be a molecular chain having a plasticizing function for polypeptide chains. A molecular chain having a plasticizing function for polypeptide chains means a molecular chain having a function of increasing the flexibility of a fiber compared to a fiber containing only polypeptide chains. Fibers containing a block copolymer having a second segment containing a molecular chain having a plasticizing function for polypeptide chains can have high flexibility suitable for fabrics used in clothing, etc. In addition, block copolymers containing a second segment containing a molecular chain having a plasticizing function for polypeptide chains can be biodegradable.

[0099] The second segment may be a molecular chain comprising a polyoxyalkylene, polyester, polycarbonate, polyamide, polyol, or modified polysaccharide chain, which may have a plasticizing function for the polypeptide chain.

[0100] Examples of polyoxyalkylenes that can constitute the second segment include polyoxyethylene, polyoxypropylene, and polyoxytetramethylene. Examples of polyesters that can constitute the second segment include polylactic acid, poly(3-hydroxybutanoic acid), polyhydroxybutanoic acid / hydroxyvaleric acid copolymer, polyhydroxybutanoic acid / 4-hydroxybutanoic acid copolymer, polyhydroxybutanoic acid / hydroxyhexanoic acid copolymer, polytrimethylene terephthalate, butanediol / long-chain dicarboxylic acid copolymer, polyethylene terephthalate, polybutylene succinate, polybutylene succinate-adipate copolymer, polybutylene adipate-terephthalate copolymer, polycaprolactone, and polytrimethylene furan dicarboxylate. Polycarbonates that can constitute the second segment may contain structural units derived from an aliphatic diol, and examples thereof include 1,6-hexanediol polycarbonate, 1,5-pentanediol polycarbonate, and 1,10-decanediol carbonate. Examples of polyamides that can constitute the second segment include nylon 3, nylon 4, nylon 5, nylon 6, nylon 11, and nylon 610. Examples of polyols that can constitute the second segment include polyvinyl alcohol and ethylene-vinyl alcohol copolymers. Examples of modified polysaccharide chains that can constitute the second segment include chemically modified polysaccharide chains of cellulose, starch, chitin, chitosan, etc. Specific examples of modified polysaccharide chains include cellulose acetate, ethyl cellulose, starch acetate, hydroxypropylated starch, carboxymethyl chitin, and carboxymethyl chitosan.

[0101] [Modified Artificial Protein] The artificial protein according to this embodiment may be a chemically modified artificial protein, that is, an artificial protein in which, for example, at least some or all of the lysine residues, serine residues, threonine residues, tyrosine residues, or cysteine ​​residues in the artificial protein have been modified to impart a specific function, property, or the like.

[0102] [Production of proteins by microbiological techniques] The artificial protein according to this embodiment may be produced by microbiological techniques. The artificial protein may be produced with reference to the descriptions in WO 2017 / 188430, WO 2017 / 188434, WO 2017 / 222034, WO 2018 / 025886, WO 2019 / 022163, etc.

[0103] Artificial proteins can be produced, for example, by a method including a step of expressing a nucleic acid in a host transformed with an expression vector. Expression methods include direct expression, as well as secretory production and fusion protein expression, in accordance with the methods described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, the protein can be obtained as a polypeptide to which sugars or sugar chains have been added.

[0104] The impact failure mechanism of fiber-reinforced composite materials is primarily a mechanism in which the fracture energy is dissipated by the fracture propagating from the impact fracture initiation point by bypassing the three-dimensional arrangement of dispersed fibers. In the fiber-reinforced composite material according to this embodiment, the inclusion of artificial protein fibers having a specific fiber diameter at a specific content sufficiently exhibits the effect of bypassing fracture propagation from the fracture initiation point, which is thought to result in improved impact strength.

[0105] Furthermore, one of the mechanisms of impact failure of fiber-reinforced composites is that the interfacial adhesion between the artificial protein fiber and the thermoplastic resin (matrix resin) transmits the fracture stress to the artificial protein fiber, and the fracture energy is dissipated by the deformation of the fiber itself. The artificial protein fiber contained in the fiber-reinforced composite of this embodiment can be made to have higher toughness than natural protein fibers by arbitrarily designing the protein molecules (i.e., the artificial protein fiber contained in the fiber-reinforced composite of this embodiment may have higher toughness than natural protein fibers). Fiber-reinforced composites containing such artificial protein fibers exhibit a higher level of fracture energy dissipation effect due to the deformation of the fiber itself, based on the high toughness of the artificial protein fiber, which is also expected to improve impact strength. Furthermore, the addition of a modified polyolefin such as maleic acid-modified polypropylene to the thermoplastic resin may provide more sufficient interfacial adhesion between the artificial protein fiber and the thermoplastic resin, thereby more reliably transmitting the fracture stress to the artificial protein fiber and advantageously dissipating the fracture energy by the deformation of the fiber itself. The lower limit of the toughness value of the artificial protein fiber is not particularly limited, but is, for example, 71 MJ / m ３ or more, and 80 MJ / m ３ or more, and 90 MJ / m ３ or more, and may be 100 MJ / m ３ or more, and 110 MJ / m ３ or more, and 120 MJ / m ３ The upper limit of the toughness value of the artificial protein fiber is not particularly limited, but it is preferably 150 MJ / m or more. ３ or less, 140 MJ / m ３ or less, and 130 MJ / m ３ or less, and 120 MJ / m ３ It may be 110 MJ / m or less, and may be 100 MJ / m ３ or less, and 90 MJ / m ３ or less, and ３or less. A suitable range for the toughness value is, for example, a range that appropriately combines the various suitable upper and lower limit values ​​described above. The toughness value of an artificial protein fiber can be determined, for example, as follows. The strength, initial modulus of elasticity (measured at the maximum slope of 20 points. Measurements were made at 50 msec intervals, and the maximum slope when the slope was calculated at 20-point intervals was taken as the initial modulus of elasticity), and elongation of the artificial protein fiber are measured using a tensile tester (Shimadzu EZ-S small benchtop tester) at an ambient temperature of 25°C and a relative humidity of 60%, and the toughness is calculated. The sample is attached to a cardboard mold, and the distance between the grippers is 20 mm and the pulling speed is 10 mm / min. The load cell capacity is 1 N, and the gripper is a clip-type. The measured value is the average value of the number of samples (n = 5). The toughness is calculated using the following formula: [E / (r2 × π × L) × 1000] (unit: MJ / m ３ ) where, E is the breaking energy (unit: J), r is the radius of the fiber (unit: mm), π is the circular constant, and L is the distance between the grips during the tensile test: 20 mm.

[0106] Additionally, in fiber-reinforced composites, the artificial protein fibers dispersed in the thermoplastic resin reinforce and support the thermoplastic resin, which is thought to improve the load-heat distortion characteristics (load deflection temperature). In the fiber-reinforced composites according to the embodiments, the artificial protein fibers having a specific fiber diameter are contained at a specific content, which is thought to sufficiently reinforce the thermoplastic resin, thereby improving the load-heat distortion characteristics. Furthermore, by arbitrarily designing the protein, artificial protein fibers can be made to have higher heat resistance than natural protein fibers (i.e., the artificial protein fibers contained in the fiber-reinforced composites according to the present embodiments may have higher heat resistance than natural protein fibers), thereby enabling them to be combined with thermoplastic resins while suppressing thermal degradation. In fiber-reinforced composites containing such artificial protein fibers, the high heat resistance of the artificial protein fibers is expected to further improve the load-heat distortion characteristics (load deflection temperature).

[0107] In the fiber-reinforced composite material of this embodiment, in order to enhance the effect of fracture propagation detouring from the fracture initiation point and improve impact strength, it is preferable that the fiber diameter of the artificial protein fiber be small and that the content of the artificial protein fiber in the entire fiber-reinforced composite material be low. On the other hand, in order to reinforce the thermoplastic resin and improve the load-heat deformation characteristics, it is desirable that the content of the artificial protein fiber be high. However, in a fiber-reinforced composite material, if the content of the artificial protein fiber is less than 0.5% by mass, the number of artificial protein fibers present in the thermoplastic resin will be small, which may result in the effect of fracture propagation detouring from the fracture initiation point being insufficient, making it difficult to obtain sufficient impact strength. Furthermore, even if the content of the artificial protein fiber exceeds 30% by mass or the fiber diameter exceeds 30 μm, there is a risk that the improvement in impact strength obtained by the inclusion of the artificial protein fiber will be insufficient. Furthermore, it is not easy to obtain artificial protein fibers with a fiber diameter of less than 1 μm using known wet spinning, dry spinning, or dry-wet spinning methods, which may increase the cost of fiber production.

[0108] Therefore, in the fiber-reinforced composite material according to this embodiment, the fiber diameter of the artificial protein fiber is advantageously set to a value within the range of 1 to 30 μm, and the content of the artificial protein fiber is set to a value within the range of 0.5 to 30 mass %. In such a fiber-reinforced composite material, excellent properties are exhibited in both heat resistance and impact strength, regardless of the content of the inorganic filler.

[0109] As mentioned above, the lower limit of the fiber diameter of the artificial protein fiber may be 1 μm or more, and is not particularly limited to a specific numerical value, but may be, for example, 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. Similarly, as mentioned above, the upper limit of the diameter of the artificial protein fiber may be 30 μm or less, and is not particularly limited to a specific numerical value, but may be, for example, 25 μm or less, 20 μm or less, 16 μm or less, or 10 μm or less. A suitable range of the fiber diameter of the artificial protein is determined, for example, by appropriately combining any of the above-mentioned lower and upper limits. For example, the fiber diameter of the artificial protein fiber may be 1 to 25 μm, 5 to 25 μm, 10 to 25 μm, 1 to 20 μm, 5 to 20 μm, 10 to 20 μm, 16 to 20 μm, 5 to 30 μm, 10 to 30 μm, 16 to 30 μm, or 20 to 30 μm.

[0110] Furthermore, as mentioned above, the lower limit of the artificial protein fiber content in the fiber-reinforced composite material may be 0.5% by mass or more, and although the specific numerical value is not particularly limited, it may be, for example, 1% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, or 25% by mass or more. Furthermore, as mentioned above, the upper limit of the artificial protein fiber content may be 30% by mass or less, and although the specific numerical value is not particularly limited, it may be, for example, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, or 1% by mass or less. A suitable range of the artificial protein fiber content is determined, for example, by appropriately combining any of the above-mentioned lower and upper limits. For example, the content of artificial protein fibers may be 1 to 25% by mass, 1 to 20% by mass, 1 to 15% by mass, 5 to 25% by mass, 5 to 20% by mass, 5 to 15% by mass, 7 to 25% by mass or more, 7 to 20% by mass, 7 to 15% by mass, 10 to 25% by mass, 10 to 20% by mass or more, or 10 to 15% by mass.

[0111] Furthermore, as mentioned above, the fiber-reinforced composite material according to this embodiment can more reliably exhibit excellent properties in both heat resistance and impact strength, particularly impact strength, when the artificial protein fibers contained therein are hydrophobic artificial protein fibers rather than hydrophilic artificial protein fibers. This is thought to be because hydrophobic artificial protein fibers containing hydrophobic artificial proteins (especially those with an average HI of greater than 0 over the entire length of the amino acid sequence) disperse more easily in thermoplastic resins than hydrophilic artificial protein fibers, facilitating the formation of an interface between the thermoplastic resin and the fiber, and as a result, the impact fracture mechanism described above can be more reliably implemented. Furthermore, particularly when both the artificial protein fibers and the thermoplastic resin are hydrophobic, the dispersibility of the artificial protein fibers in the thermoplastic resin is further improved, further facilitating the formation of an interface between the thermoplastic resin and the fiber, thereby improving the impact strength of the fiber-reinforced composite material.

[0112] The length of the artificial protein fibers contained in the fiber-reinforced composite material 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. By setting the fiber length to 24 mm or less, the bending properties of the fiber-reinforced composite material are improved. Furthermore, by setting the fiber length to 1 mm or more, the mechanical strength of the fiber-reinforced composite material is improved.

[0113] [Other Components] In addition to the artificial protein fiber, the fiber-reinforced composite material according to this embodiment may further contain maleic anhydride-modified polypropylene or liquid paraffin. By including these components, the bending properties and tensile properties of the fiber-reinforced composite material can be expected to be improved. In addition, the fiber-reinforced composite material may contain general additives and the like that are included in conventional fiber-reinforced composite materials.

[0114] -Maleic anhydride-modified polypropylene- The inclusion of maleic anhydride-modified polypropylene in a fiber-reinforced composite material can increase the yield stress. Furthermore, the inclusion of maleic anhydride-modified polypropylene can increase the flexural strength and flexural modulus due to, for example, improved adhesion at the interface between the thermoplastic resin and the artificial protein fiber. The maleic anhydride-modified polypropylene is not particularly limited, and known modifiers can be used. The degree of modification in the maleic anhydride-modified polypropylene is not particularly limited. For example, the content of maleic acid-modified groups (M value) per 100 parts by mass of the maleic anhydride-modified polypropylene may be 0.5 to 5.0 parts by mass, or 0.8 to 3.0 parts by mass. Specific examples of commercially available maleic anhydride-modified polypropylene include Mitsui Chemicals Admer, Sanyo Chemicals Umex, DuPont's MZ series, and Exxelor from Exxon.

[0115] The content of maleic anhydride-modified polypropylene may be 1.0 to 20.0 parts by mass, 2.0 to 18.0 parts by mass, or 5.0 to 15.0 parts by mass relative to 100 parts by mass of the artificial protein fiber. When the content is 1 part by mass or more, the yield stress, flexural strength, and flexural modulus of the fiber-reinforced composite material can be increased, and when the content is 20.0 parts by mass or less, the fiber-reinforced composite material also has an excellent Young's modulus.

[0116] Liquid paraffin: When the fiber-reinforced composite material contains liquid paraffin, the breakage of the artificial protein fiber is suppressed, and therefore the yield stress and Young's modulus can be increased. There are no particular limitations on the liquid paraffin, and any liquid paraffin known as a modifier can be used. There are no particular limitations on the number of carbon atoms in the liquid paraffin, and it is preferably 5 to 35, and more preferably 10 to 25.

[0117] The content of liquid paraffin may be 1.0 to 20.0 parts by mass, 2.0 to 18.0 parts by mass, or 5.0 to 15.0 parts by mass relative to 100 parts by mass of the above-mentioned artificial protein fiber. If the content is 1 part by mass or more, the yield stress and Young's modulus of the fiber-reinforced composite material can be increased, and if the content is 20.0 parts by mass or less, the fiber-reinforced composite material will also have excellent bending properties.

[0118] Other Additives Examples of other additives include antioxidants and lubricants.

[0119] In addition, in one preferred aspect of the present embodiment, the fiber-reinforced composite material has an inorganic filler content of 5% by mass or less relative to the total mass of the fiber-reinforced composite material. The content is preferably 1% by mass or less, and more preferably 0.1% by mass or less. The lower limit of the content is not particularly limited, and may be 0% by mass. Examples of inorganic fillers include wollastonite and glass fiber.

[0120] In addition, in one preferred aspect of the present embodiment, the fiber-reinforced composite material has a natural protein fiber content of 5% by mass or less relative to the total mass of the fiber-reinforced composite material. The content is preferably 1% by mass or less, and more preferably 0.1% by mass or less. The lower limit of the content is not particularly limited, and may be 0% by mass. Examples of natural protein fibers include cotton fiber and wool fiber.

[0121] The fiber-reinforced composite material according to this embodiment preferably contains a mixed resin of the thermoplastic resin and the artificial protein fiber, and more preferably contains a mixed resin. As the kneading method, a known kneading method can be used.

[0122] <Method for producing fiber-reinforced composite material> The method for producing a fiber-reinforced composite material according to this embodiment includes compounding the thermoplastic resin and the artificial protein fiber.

[0123] The method for forming the composite is not particularly limited, and any method can be used as long as the thermoplastic resin and the artificial protein fiber are mixed, and a method in which they are kneaded is preferred.

[0124] The timing of adding the other components is not particularly limited. For example, they may be added in advance to the resin component in the above-mentioned composite method, or may be added simultaneously with composite formation.

[0125] One example of a production method is to add the artificial protein fiber to the thermoplastic resin and then knead it using a known kneader. Another example of a case in which other components are further used is to mix the thermoplastic resin with an antioxidant and a lubricant to obtain mixture A, and then add the artificial protein fiber, liquid paraffin, and maleic acid-modified polypropylene to mixture A and knead them. In the above-mentioned embodiment, a preferred embodiment is to separately mix the artificial protein fiber and liquid paraffin to obtain mixture B, mix mixture A and mixture B, and then add the maleic acid-modified polypropylene and knead them. Another preferred embodiment is to feed the thermoplastic resin, the artificial protein fiber, and the other components into a known kneader and knead them. A known extruder or injection molding machine can be used as the kneader, and the thermoplastic resin, the artificial protein fiber, and other components added as needed can be kneaded in the molding machine and extruded or injected through a nozzle to form a fiber-reinforced composite. Alternatively, the thermoplastic resin, the artificial protein fiber, and other components added as needed can be kneaded in a known kneader to produce pellets, and the pellets can be molded into a fiber-reinforced composite material using a known extrusion molding machine or injection molding.

[0126] <Applications> The application of the fiber-reinforced composite material according to the present embodiment is not particularly limited. For example, the fiber-reinforced composite material can be suitably used for components such as frames, inner panels, and outer panels of structures such as automobiles, railway vehicles, ships, aircraft, rockets, artificial satellites, and robots.

[0127] Fiber-reinforced composites can be suitably used for automobile brake discs, wheels, fuel tanks, hoods, roofs, side doors, back doors, luggage inner and outer panels, exterior parts, structural parts, undercarriage parts, engine-related parts, fuel cell and electric vehicle parts, and other parts. Examples of exterior parts include bumpers, rocker moldings, pillars, roof moldings, and fuel lids. Examples of structural parts include bumper reinforcements, rear floor pans, crash boxes, dash panels, rear partitions, seatback frames, and braces. Examples of undercarriage parts include lower absorbers and undercovers. Examples of engine-related parts include engine covers, engine undercovers, cylinder head covers, oil pans, radiator supports, and timing belt and chain covers. Examples of fuel cell and electric vehicle parts include stack frames, stack end plates, fuel cell cells, inverter covers, inverter cases, rotor motors, stator motors, and reactors. Decorative parts include interior and exterior parts such as ornaments, switch bases, registers, console boxes, cup holders, cluster panels, emblems, etc. Other parts include brackets, anchors, pedals, sheet metal parts, etc.

[0128] Furthermore, the fiber reinforced composite material can be suitably used for aircraft nose landing gear doors, main landing gear doors, floor beams, engine covers, ailerons, wing-body fairings, horizontal stabilizers, vertical stabilizers, rudders, elevators, etc.

[0129] Fiber reinforced composite materials are also used in ship bodies, ship masts, drone frames, suitcases, cargo containers, housings for electronic and electrical devices (for example, personal computers, displays, projectors, cameras, mobile phones, smartphones, tablets, etc.), watch bezels, watch bodies, fishing tackle (for example, fishing rods and reels), golf club shafts, golf club heads, rackets (for example, for tennis, badminton, squash, and table tennis), bicycle frames, front forks, rims, baseball bats, skis, snowboards, skateboards, etc. The material can also be suitably used for applications such as ski boards, surfboards, wakeboards, various bindings, canoe hulls, canoe paddles, sleds in general (for example, bobsleigh sleds), hockey sticks, ski poles, kendo bamboo swords, Japanese bows, Western bows, table tennis tables, billiard cues, gateball sticks, construction and civil engineering materials, wind power generation blades (windmills), flywheels, pipes, parabolic antennas, prosthetic limbs, wheelchairs, beds, portable ramps, crutches, artificial bones, and protective and bulletproof goods (for example, helmets, shields, bulletproof vests).

[0130] 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.

[0131] (Relationship between fiber diameter and amount of artificial protein fiber and load deflection temperature) The relationship between fiber diameter and amount of artificial protein fiber and load deflection temperature was investigated for fiber-reinforced composites containing polypropylene (PP) and artificial protein fiber.

[0132] <Production of artificial protein fiber> [Production of modified fibroin] A modified fibroin (hereinafter also referred to as "PRT966") having the amino acid sequence shown in SEQ ID NO: 2 was designed based on the base sequence and amino acid sequence of fibroin derived from Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415).

[0133] The amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which amino acid residues have been substituted, inserted, and deleted from the amino acid sequence of fibroin derived from Nephila clavipes in order to improve productivity, and further has the amino acid sequence shown in SEQ ID NO: 8 (tag sequence and hinge sequence) added to the N-terminus.

[0134] 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 treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

[0135] 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 reached 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.

[0136]

[0137] The seed culture was added to a jar fermenter containing 500 ml of production medium (see the 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.

[0138]

[0139] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, 120 g / 1 L of yeast extract) was added at a rate of 1 mL / min. The culture temperature was maintained at 37°C, and the culture was controlled at a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM to induce the expression of PRT966. 20 hours after the addition of IPTG, the culture was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the cultures before and after the addition of IPTG. Expression of PRT966 was confirmed by the appearance of a band of a size corresponding to PRT966, which was dependent on the addition of IPTG.

[0140] -Purification of PRT966- The bacterial cells harvested 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed bacterial cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing approximately 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (GEANiroSoavi). 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 with a stirrer at 60°C for 30 minutes. After dissolution, the solution was dialyzed against water using a dialysis tube (Cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.) The white aggregated protein (PRT966) obtained after dialysis was recovered by centrifugation, and the water was removed using a freeze-dryer to recover a freeze-dried powder.

[0141] The purity of PRT966 in the resulting lyophilized powder was confirmed by image analysis of polyacrylamide gel electrophoresis using Totallab (nonlinear dynamics Ltd.). The result showed that the purity of PRT966 was approximately 85%. The HI (HI for the full-length amino acid sequence) of PRT966 was 0.47.

[0142] (2) Production of modified fibroin fiber: The modified fibroin (PRT966) was added to formic acid to a concentration of 30% by mass, and then dissolved for 3 hours using a shaker. Dust and bubbles were then removed to obtain a dope solution. The solution viscosity of the dope solution was 15,000 cP (centipoise) at 90°C.

[0143] (Spinning) Using the dope solution obtained as described above and the spinning apparatus 10 shown in Figure 3, known wet spinning was performed to obtain three types of modified fibroin monofilaments with fiber diameters of 10 µm, 16 µm, and 30 µm. In this case, to obtain the three types of modified fibroin monofilaments with different fiber diameters, wet spinning was performed under the following conditions. The coagulation liquid used was a sodium sulfate aqueous solution with a concentration of 14.4% by mass. <Monofilament with a fiber diameter of 10 μm> Extrusion nozzle diameter: 0.08 mm Linear discharge speed: 3.7 m / min Coagulation liquid temperature: 40°C Take-up speed: 71.3 m / min Draw ratio in coagulation bath: 4.0 times Draw ratio in washing bath: 4.82 times Drying temperature: 140°C <Monofilament with a fiber diameter of 16 μm> Extrusion nozzle diameter: 0.1 mm Linear discharge speed: 3.7 m / min Coagulation liquid temperature: 40°C Take-up speed: 33.0 m / min Draw ratio in coagulation bath: 2.66 times Draw ratio in washing bath: 4.17 times Drying temperature: 100°C <Monofilament with a fiber diameter of 30 μm> Extrusion nozzle diameter: 0.1 m Linear discharge speed: 22.3 m / min Coagulation liquid temperature: 3.4°C Take-up speed: 78.2 m / min Draw ratio in coagulation bath: 1.3 times Draw ratio in washing bath: 2.61 times Drying temperature: 70°C Figure 3 is a schematic diagram showing an example of a spinning apparatus for producing protein fibers. The spinning apparatus 10 shown in Figure 3 is an example of a spinning apparatus for wet spinning, and includes, in order from upstream, an extruder 1, a coagulation bath 20, a washing bath 21, and a drying apparatus 4. The extruder 1 has a storage tank 7 in which a dope solution (spinning stock solution) 6 is stored. A coagulation bath 20 stores a coagulation liquid 11. The dope solution 6 is extruded by a gear pump 8 attached to the lower end of the storage tank 7 from a nozzle 9 installed so that its tip is immersed in the coagulation liquid 11. The extruded dope solution 6 is supplied into the coagulation liquid 11. The solvent is removed from the dope solution 6 in the coagulation liquid 11, and the protein is coagulated. The coagulated protein is introduced into a washing bath 21, washed with a washing solution 12 in the washing bath 21, and then sent to a 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, if the rotation speed of the second nip roller 14 is set faster than the rotation speed of the first nip roller 13, the protein fibers 36 are obtained that are drawn at a ratio corresponding to the rotation speed ratio. After being drawn in the washing solution 12, the protein fibers 36 are dried while passing through the drying device 4 after leaving the washing bath 21, and then wound up by the 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.

[0144] (2) Additive for fiber-reinforced composites Three types of modified fibroin monofilaments with different fiber diameters obtained by the above-mentioned method were bundled together in a bundle of 48 monofilaments each, and then cut to an average length of 1 mm using a fiber cutter to obtain short fibers of three types of artificial protein fibers with different fiber diameters. The artificial protein fibers are artificial structural protein fibers and are artificial fibroins.

[0145] Example 1 Production of Fiber-Reinforced Composite Material Using a blender (7012BU, Waring), PP (polypropylene), AO (antioxidant), and CaSt (lubricant, calcium stearate) shown in the table below were mixed for 1 minute (room temperature (23°C, same below), rotation speed 3400 rpm) to obtain a mixture. In examples containing artificial protein fibers, artificial protein fibers having fiber diameters shown in the table below were added to the above mixture in amounts of 5, 10, 15, or 20% by mass of the resulting material, and the mixture was mixed by hand. In examples containing MAPP (maleic anhydride-modified polypropylene), MAPP was added to the PP mixture containing the artificial protein fibers in an amount of 10% by mass of the artificial protein fibers, and the mixture was mixed by hand. In examples not containing artificial protein fibers, MAPP in the amount shown in the table below was added to a mixture not containing the artificial protein fibers and mixed. In examples containing LP (liquid paraffin), the artificial protein fiber added was a mixture prepared by previously mixing artificial protein fiber and LP in an amount of 10% by mass of the artificial protein fiber in a mortar. In examples not containing artificial protein fiber, LP was added to a mixture containing no artificial protein fiber in the amount shown in the table below and mixed. Next, the mixture was melted and kneaded for 10 minutes under a nitrogen atmosphere using a small twin-screw kneader (MC15, Xplore Instruments) (kneading temperature: 190°C, screw rotation speed: 150 rpm), to produce fiber-reinforced composites 1 to 3 or comparative materials 1 to 3.

[0146]

[0147] In the table, the materials used are as follows: PP: Polypropylene (PX600N, SunAllomer Co., Ltd.) Artificial protein fiber: Product manufactured by the above-mentioned method LP: Liquid paraffin (Fujifilm Wako Pure Chemical Industries, Ltd.) MAPP: Maleic anhydride-modified polypropylene (UMEX1010, Sanyo Chemical Industries, Ltd.) AO: Antioxidant (Irganox B225, BASF Japan Ltd.) CaSt: Lubricant (Calcium stearate, Taihei Chemical Industry Co., Ltd.)

[0148] (Example 2) <Evaluation of Load Deflection Temperature> The obtained fiber reinforced composite materials 1 to 3 or comparative materials 1 to 3 were subjected to load deflection temperature measurement in accordance with JIS K7191: 2015 standard to evaluate heat resistance. For the fiber reinforced composite materials 1 to 3 or comparative materials 1 to 3, an injection molding machine (IM12, Xplore Instruments) was used to mold rectangular test pieces in accordance with JIS K7139: 2009 standard Type B1 (injection pressure 1.6 MPa, mold temperature 40 ° C). For each molded product, the load deflection temperature was measured using a heat distortion temperature measuring device (HD-500, Yasuda Seiki Seisakusho Co., Ltd.) at a test start temperature of 50 ° C, a temperature rise temperature of 120 ° C / h, and a load of 0.45 MPa. The higher the load deflection temperature, the better the heat resistance.

[0149] Figure 4 shows the results of measuring the deflection temperature under load for fiber reinforced composites 1 to 3 and comparative materials 1 to 3. Figure 4 shows that the deflection temperature under load is higher for fiber reinforced composites 1 to 3 compared to comparative materials 1 to 3, which do not contain artificial protein fibers. Furthermore, it can be seen that the deflection temperature under load increases with the increase in the artificial protein fiber content in fiber reinforced composites 1 to 3. It can also be seen that the fiber diameter of the artificial protein fiber does not have a significant effect on the deflection temperature under load in fiber reinforced composites 1 to 3.

[0150] Example 3 Evaluation of Impact Strength The obtained fiber reinforced composite materials 1 to 3 or comparative materials 1 to 3 were subjected to a Charpy impact test in accordance with JIS K 7111:2012 to evaluate their impact strength. For each material, a rectangular test piece in accordance with JIS K 7139:2009 Type B1 was molded (injection pressure 1.6 MPa, mold temperature 40°C) using an injection molding machine (IM12, Xplore Instruments) and notched. A Charpy impact test was performed on the rectangular test piece after notching using a digital impact tester (Toyo Seiki Seisakusho, Ltd.) to measure its impact strength.

[0151] Figure 5 shows the results of measuring the impact strength of fiber-reinforced composites 1 to 3 and comparative materials 1 to 3. Figure 5 shows that the impact strength of fiber-reinforced composites 1 to 3 is increased compared to comparative materials 1 to 3, which do not contain artificial protein fibers. Furthermore, it can be seen that the impact strength of fiber-reinforced composite 1 decreases when the artificial protein fiber content is 15% by mass or more. This is presumably because, when the fiber diameter of the artificial protein fiber is small, the entanglement and aggregation of the artificial protein fibers increases as the artificial protein fiber concentration increases, and the effect of bypassing fracture propagation from the fracture initiation point is not fully exerted. Furthermore, it can be seen that, in fiber-reinforced composites 2 to 3, when the fiber diameter of the artificial protein fiber is large, such as 16 μm or more, the artificial protein fiber content does not significantly affect the impact strength.

[0152] Example 4 <Evaluation of Tensile Properties> Fiber reinforced composite materials 4 and 5 having the compositions shown in the table below were prepared in the same manner as in Example 1.

[0153]

[0154] Tensile tests were conducted in accordance with ASTM D 638 for Fiber Reinforced Composite Materials 1, 4, and 5, and Comparative Materials 1 and 3, respectively, to evaluate the tensile properties of PP alone and the composites. For each material, an injection molding machine (IM12, Xplore Instruments) was used to fabricate dumbbell-shaped test specimens in accordance with ASTM D 638 Type-1 (injection pressure 1.6 MPa, mold temperature 40°C). Using a precision universal testing machine (AG-Xplus, Shimadzu Corporation), tensile tests were conducted on the dumbbell-shaped test specimens at room temperature at a test speed of 20 mm / min, and the yield stress and Young's modulus were measured.

[0155] Figure 6 shows the results of measuring the yield stress and Young's modulus for fiber-reinforced composites 1, 4, and 5 or comparative materials 1 and 3. Figure 6 shows that the yield stress and Young's modulus are higher in fiber-reinforced composites 1, 4, and 5 compared to comparative materials 1 and 3, which do not contain artificial protein fibers. It can also be seen that both the yield stress and Young's modulus increase with an increase in the artificial protein fiber content. In addition, it can be seen that the addition of LP increases the yield stress and Young's modulus, and that the addition of MAPP increases the yield stress.

[0156] Example 5 <Evaluation of Flexural Properties> A fiber reinforced composite material 6 having the composition shown in the table below was produced in the same manner as in Example 1.

[0157]

[0158] Fiber-reinforced composites 1, 4, 5, and 6, and comparative materials 1 to 3, were each subjected to a bending test in accordance with JIS K 7171:2016 to evaluate the bending properties of the PP alone and the composites. For each material, rectangular test specimens conforming to JIS K7139:2009 Type B1 were molded using an injection molding machine (IM12, Xplore Instruments) at an injection pressure of 1.6 MPa and a mold temperature of 40°C. Bending tests were performed on the rectangular test specimens at room temperature at a test speed of 2 mm / min using a precision universal testing machine (AG-Xplus, Shimadzu Corporation). Figure 7 shows the bending strength and bending modulus obtained from the bending test. Average values ​​are plotted, with maximum and minimum values ​​indicated by error bars. The bending strength and bending modulus of the fiber-reinforced composites tended to increase with increasing artificial protein fiber content. The flexural strength and flexural modulus of the test specimen of fiber-reinforced composite 6 were the highest compared to the other fiber-reinforced composites, which is thought to be due to the improved adhesion at the interface between the artificial protein fiber and PP caused by MAPP.

[0159] Example 6 Evaluation of Deflection Temperature Under Load The deflection temperatures under load of the fiber reinforced composite materials 1, 4, 5, and 6 and the comparative materials 1 to 3 were measured in the same manner as in Example 2.

[0160] Figure 8 shows the results of measuring the deflection temperature under load for fiber-reinforced composite materials 1, 4, 5, and 6, and comparative materials 1 to 3. Figure 8 shows that the deflection temperature under load is higher for fiber-reinforced composite materials 1, 4, 5, and 6, compared to comparative materials 1 to 3, which do not contain artificial protein fibers. Furthermore, it can be seen that the deflection temperature under load increases with the increase in the artificial protein fiber content in fiber-reinforced composite materials 1, 4, 5, and 6.

[0161] Example 7 Evaluation of impact strength and load deflection temperature Fiber-reinforced composite material 7 was prepared using the same method as for fiber-reinforced composite material 1, except that the amount of artificial protein fiber was 3.8, 7.5, 11.0, or 15.0 mass% of the resulting material. Comparative material 4, having the composition shown in the table below, was prepared using the same method as for comparative material 1. Furthermore, comparative materials 5 to 12 were prepared by replacing the artificial protein fiber in fiber-reinforced composite material 7 with the compounds shown in the table below.

[0162]

[0163]

[0164] Details of the compounds in the table are as follows: Artificial protein fiber (60°C, vacuum dried for 4 hours): Product manufactured by the method described above PA6-F10 SD 10 μm: Commercially available product PA6 filament yarn - white: Commercially available product PA6 filament yarn - dope-dyed: Commercially available product PA6 - flocculent 18 μm: Commercially available product PA66 - flocculent 12 μm: Commercially available product Wool 10-30 μm: Commercially available product Glass fiber: Commercially available product

[0165] For fiber-reinforced composite material 7 and comparative materials 1, 4 to 12, the deflection temperature under load was measured using the same method as in Example 2, and the results are shown in Figure 9. Furthermore, for fiber-reinforced composite material 7 and comparative materials 1, 4 to 12, the impact strength was measured using the same method as in Example 3, and the results are shown in Figure 10. From Figures 9 and 10, it can be seen that when synthetic resin fibers such as nylon are used, both the impact strength and the deflection temperature under load are inferior to when artificial protein fibers are used. Furthermore, from Figure 9, it can be seen that when natural protein fibers such as wool are used, both the impact strength and the deflection temperature under load are inferior to when artificial protein fibers are used.

[0166] 1...extrusion device, 4...drying device, 6...dope solution, 10...spinning device, 20...coagulation bath, 21...washing bath, 36...protein fiber, 100...fiber-reinforced composite material, 101...thermoplastic resin, 102...artificial protein fiber

Claims

1. A fiber-reinforced composite material containing a thermoplastic resin and an artificial protein fiber containing an artificial protein, wherein the fiber diameter of the artificial protein fiber is 1 to 30 μm, and the content of the artificial protein fiber is 0.5 to 30 mass%.

2. The fiber reinforced composite of claim 1, wherein said thermoplastic resin is polypropylene.

3. The fiber-reinforced composite material of claim 1, wherein the artificial protein is an artificial structural protein.

4. The fiber-reinforced composite material of claim 1, wherein the artificial protein is a hydrophobic artificial protein.

5. The fiber-reinforced composite material according to claim 4, wherein the average hydropathic index of said hydrophobic artificial protein is 0 or greater.

6. A fiber-reinforced composite material according to any one of claims 1 to 5, comprising a kneaded resin of the thermoplastic resin and the artificial protein fiber.

7. The fiber-reinforced composite material of claim 1, wherein the artificial protein has an amino acid sequence that includes a repeat sequence.

8. The fiber-reinforced composite material according to claim 7, wherein the artificial protein has an amino acid sequence including an (A)n motif, the amino acid sequence being different from that of a naturally occurring protein, the (A)n motif being an amino acid sequence consisting of 2 to 27 amino acid residues, the number of alanine residues relative to the total number of amino acid residues in the (A)n motif being 40% or more, and when there are multiple (A)n motifs, the (A)n motifs may be the same or different amino acid sequences.

9. The fiber-reinforced composite material of claim 1, wherein the artificial protein is artificial fibroin.

10. The fiber-reinforced composite of claim 1, wherein the artificial protein fibers have a fiber length of 1 to 24 mm.

11. The fiber-reinforced composite of claim 1, further comprising maleic anhydride-modified polypropylene.

12. The fiber-reinforced composite material according to claim 1 or 11, further comprising liquid paraffin.

13. A method for producing the fiber-reinforced composite material of claim 1, comprising compounding the thermoplastic resin with the artificial protein fiber.

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