Molded article formed from solidified fibroin and having improved stretchability, method for producing molded article formed from insolubilized fibroin, and molded article formed from insolubilized fibroin

Abstract

Provided is a molded article formed from solidified fibroin, the molded article containing a fibroin protein that contains one or more α-helical structures in each molecule. In the molded article: each of the α-helical structures forms an extended α-helical crystal structure; the extended α-helical crystal structure is constituted by an extended hexagonal unit lattice that is formed by a portion of the fibroin protein; and the lattice spacing d1 between (10-10) planes in the extended hexagonal unit lattice is greater than the lattice spacing d2 between (10-10) planes in a standard hexagonal unit lattice of a standard α-helical crystal structure.

Description

Fibroin coagulated molded article with improved extensibility, method for producing fibroin insolubilized molded article, and fibroin insolubilized molded article

[0001] The present invention relates to a fibroin coagulated molded article, a method for producing a fibroin insolubilized molded article using said fibroin coagulated molded article, and a fibroin insolubilized molded article. The present application claims priority based on Japanese Patent Application No. 2025-031057 filed in Japan on February 28, 2025, and International Application No. PCT / JP2025 / 007053 filed internationally on February 28, 2025, the contents of which are incorporated herein by reference.

[0002] Synthetic resins represented by plastics are used in all areas of human society and have become indispensable materials for modern life. However, on the other hand, problems of global warming caused by large amounts of carbon dioxide generated by the combustion of plastics, and problems of environmental pollution caused by waste plastics such as microplastics resulting from the chemical stability of non-biodegradable plastics have become apparent.

[0003] Therefore, in recent years, toward the realization of a sustainable society, development of environmentally friendly and highly safe alternatives to synthetic resins derived from fossil fuels has been desired. For example, polypeptide insolubilized molded articles made from raw materials such as proteins are one such alternative.

[0004] Fibroin protein has attracted attention as a raw material for polypeptide insolubilized molded articles. Fibroin protein is a fiber component of silkworm silk and spider silk (dragline silk). Since fibroin protein has a β-sheet structure as its crystal structure, it exhibits high toughness with an excellent balance between strength and elongation. Since fibroin protein is composed of polypeptides, it has both biodegradability and high biocompatibility. Therefore, polypeptide insolubilized molded articles using fibroin protein as a raw material are expected as materials in a wide range of fields such as medical materials and structural materials.

[0005] Generally, when processing proteins into polypeptide-insolubilized molded products, liquid protein is used as the raw material. When processing fibroin protein (mainly fibroin H-chain protein), liquid fibroin protein stored in the silk glands of silkworms, etc., before fiber formation, or liquid fibroin protein obtained by expressing it in E. coli, etc., using genetic engineering technology, can be used as the raw material. In addition, when processing fibroin protein, a fibroin protein solution obtained by dissolving already fiber-formed solid fibroin protein in a solvent to make it liquid can also be used as the raw material. Products obtained by re-coagulating and molding a fibroin protein solution are called regenerated fibroin proteins, and due to their ease of processing and high versatility, they are expected to have applications in various fields.

[0006] However, at present, it is not possible to fully control the properties of regenerated fibroin proteins, and the properties of natural fibroin proteins are often lost during the manufacturing process (Non-Patent Literature 1). This poses a major challenge to the practical application of regenerated fibroin materials that utilize the properties of fibroin proteins, such as high toughness and biodegradability.

[0007] Kim HH, et al., 2016, Polymer, 90: 26-33.

[0008] When regenerated fibroin protein contains an α-helix structure, the protein itself is water-soluble and flexible. However, in many cases, regenerated fibroin protein containing an α-helix structure exhibits excellent tensile properties only at high temperatures, and the elongation (strain) when stretched at relatively low temperatures such as room temperature is small, less than 10%. Therefore, regenerated fibroin protein containing an α-helix structure is unsuitable for processing highly oriented insolubilized molded products such as oriented films, oriented rods, and oriented fibers, and the flexible properties of the protein itself have not been fully utilized.

[0009] Furthermore, because high-temperature heating is required for stretching, when using fibroin proteins containing functional regions that are susceptible to heat denaturation, these functional regions can denature, resulting in a failure to obtain the desired function. In addition, the need for heating for stretching is costly and, despite using materials with low environmental impact, ultimately places a burden on the environment.

[0010] Therefore, the object of the present invention is to provide a fibroin solidified molded article that can be stretched under relatively low temperature conditions, a method for producing a fibroin-immobilized molded article using a fibroin solidified molded article, and a fibroin-immobilized molded article.

[0011] In order to solve the above problems, the inventors conducted intensive research and found that when the crystalline region of the fibroin solidified molded product has a specific crystalline structure, the stretchability of the fibroin solidified molded product is dramatically improved.

[0012] The present invention is based on the aforementioned novel findings and includes the following embodiments: [1] A fibroin coagulated molded article comprising a fibroin protein containing one or more α-helix structures in one molecule, wherein the α-helix structures form an extended α-helix crystal structure, the extended α-helix crystal structure is composed of an extended hexagonal unit lattice formed by a portion of the fibroin protein, and the lattice plane spacing d1 between (10-10) faces in the extended hexagonal unit lattice is greater than the lattice plane spacing d2 between (10-10) faces in the standard hexagonal unit lattice of a standard α-helix crystal structure. [2] The fibroin coagulated molded article according to [1], wherein the difference between d1 and d2 is 0.1 Å or more and 0.5 Å or less. [3] The fibroin coagulated molded article according to [1] or [2], wherein d1 is 7.6 Å or more and 8.5 Å or less. [4] A fibroin-coagulated molded article according to any one of [1] to [3], wherein the water content is 7.7% or more and 50% or less. [5] A fibroin-coagulated molded article according to any one of [1] to [4], wherein the fibroin protein further comprises one or more β-sheet structures in one molecule. [6] A fibroin-coagulated molded article according to any one of [1] to [5], wherein the fibroin protein is derived from bagworm silk. [7] A method for producing a fibroin-immobilized molded article, comprising a stretching step of stretching a fibroin-coagulated molded article according to any one of [1] to [6], and a molding step of molding the fibroin-coagulated molded article into a desired shape to produce a fibroin-immobilized molded article. [8] The method for producing the fibroin-immobilized molded article according to [7], wherein the stretching is cold stretching or hot stretching. [9] A fibroin-immobilized molded article produced by the method for producing the fibroin-immobilized molded article according to [7] or [8].

[10] A method for determining the stretchability of a fibroin-coagulated molded article containing fibroin protein by cold stretching or hot stretching, comprising: a structural measurement step of measuring the crystal structure of the fibroin-coagulated molded article; an analysis step of analyzing the size of the lattice plane spacing d between (10-10) planes in the unit hexagonal lattice of the α-helix crystal structure; and a determination step of determining that the fibroin-coagulated molded article is stretchable by cold stretching or hot stretching if the α-helix crystal structure is composed of a unit hexagonal lattice and the lattice plane spacing d between (10-10) planes is greater than the lattice plane spacing d2 between (10-10) planes in the standard unit hexagonal lattice of the standard α-helix crystal structure.

[0013] The fibroin solidified molded article of the present invention provides a fibroin solidified molded article that can be stretched under relatively low temperature conditions.

[0014] According to the method for producing fibroin-insolubilized molded articles of the present invention, insolubilized molded articles can be produced by stretching under relatively low temperature conditions.

[0015] Figure 1 schematically shows the position of the (10-10) plane in a unit hexagonal lattice. In the figure, the hexagonal prism represents the unit hexagonal lattice, the points on the top and bottom surfaces of the unit hexagonal lattice represent the centers of each plane, and the shaded surface represents the (10-10) plane. Figure 2 shows the results of tensile tests at room temperature for the hydrated film and the dried film. Figure 3 shows the X-ray diffraction spectra of the hydrated film and the dried film. In the figure, with respect to the lattice plane spacing of the β-sheet structure, β200 / 210 indicates the position of the peaks indicating the lattice plane spacing of the (200) and (210) planes, β211 indicates the position of the peak indicating the lattice plane spacing of the (211) plane, β202 indicates the position of the peak indicating the lattice plane spacing of the (202) plane, β103 indicates the position of the peak indicating the lattice plane spacing of the (103) plane, and β213 indicates the position of the peak indicating the lattice plane spacing of the (213) plane. Furthermore, in the figure, regarding the lattice plane spacing of the α-helix crystal structure, α10-10 indicates the position of the peak representing the lattice plane spacing of the (10-10) plane, and the asterisk (*) indicates the magnified portion shown in Figure 4. Figure 4 is a magnified view of the portion marked with an asterisk (*) in Figure 3 of the X-ray diffraction spectra of the hydrated film and the dried film. In the figure, α10-10 indicates the position of the peak representing the lattice plane spacing of the (10-10) plane of the α-helix crystal structure. Figure 5 is a diagram showing an example of the measurement results of the water content of the hydrated film and the dried film. In the figure, the horizontal dashed line indicates the level of 0% mass change rate, and the double arrows indicate the mass change rate based on water evaporation due to heating up to 200°C for each film.

[0016] Unless otherwise specified, "a," "an," and "the" are understood to mean "one or more," encompassing both singular and plural forms.

[0017] The term "comprise" means that it may include components other than the component being discussed. The term "consist of" means that it does not include components other than the component being discussed. The term "consistently of" means that it does not include components other than the component being discussed in a manner that performs a special function (such as a manner that completely negates the effect of the invention). In this specification, when "comprise" is used, it includes the "consist of" and "consistently of" manners.

[0018] A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0019] If multiple upper and lower limits are specified for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0020] 1. Fibroin Coagulated Molded Article 1-1. Overview The first aspect of the present invention is a fibroin coagulated molded article. The fibroin coagulated molded article of this aspect contains fibroin protein as an active ingredient and can be stretched under relaxed conditions, in particular, under relatively low temperature conditions that do not involve heat treatment or prolonged heating. The fibroin coagulated molded article of this aspect can be used as a raw material for fibroin immobilized molded articles.

[0021] 1-2. Definitions The terms used herein are defined below.

[0022] "Fibroin protein" refers to the silk protein that constitutes silk threads derived from insects such as silkworms and bagworms, silk threads derived from organisms of the order Araneae, or silk threads derived from organisms of the order Acari. When simply referred to as fibroin protein, it includes both wild-type and mutant forms. In this specification, when referred to as fibroin protein, it means fibroin H-chain protein unless otherwise specified.

[0023] "Fibroin H-chain protein" (hereinafter also referred to as "FibH") is the main protein that constitutes the fibroin complex (silk fibroin elementary unit; SFEU complex), which is the fibrous protein component of silk thread. Generally, FibH refers to the high molecular weight fibrous protein that makes up silk thread derived from insects such as silkworms and bagworms, but in this specification, spidoin protein, which is the high molecular weight fibrous protein that makes up silk thread, especially the dragline thread, derived from organisms of the order Araneae or Acari, is also included in FibH. FibH usually has an amino acid sequence in which clusters of glycine residues (G) and alanine residues (A) are repeated. In this specification, FibH may be natural fibroin H-chain protein (hereinafter referred to as "natural FibH") or artificial fibroin H-chain protein (hereinafter referred to as "artificial FibH").

[0024] Generally, natural fibH consists of an N-terminal region, a central region, and a C-terminal region as its basic constituent elements, in that order from the N-terminus.

[0025] The "N-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the N-terminal side of the central region described below, does not contain repeating units, and consists of 70 to 250 amino acid residues.

[0026] The "central region" is the main region that exhibits the physical properties of natural FibH. This region is responsible for the fibrous properties of FibH and is composed of multiple repeating units consisting of the same and / or different amino acid sequences linked together. A "repeating unit" is the main constituent unit of the central region, and one repeating unit can contain multiple glycine (G) and alanine (A) molecules. Therefore, more than 70% of the amino acid sequence of the central region is composed of glycine and alanine.

[0027] The "C-terminal region" refers to the region in the amino acid sequence that constitutes natural FibH that is located on the C-terminal side of the central region, does not contain repeating units, and consists of 30 to 60 amino acid residues.

[0028] In this specification, "α-βt / s coexisting fibroin protein (α-helix-β-turn / sheet coexisting fibroin protein)" (hereinafter also referred to as "α-βt / s coexisting Fib") refers to a fibroin protein that, when dissolved in water, contains one or more α-helix-forming sequences and β-turn-forming sequences in a single molecule.

[0029] In this specification, "α-βt / s coexisting FibH (α-helix-β-turn / sheet coexisting fibroin H-chain protein)" refers to FibH characterized by containing one or more α-helix-forming sequences and β-turn-forming sequences in a single FibH molecule. Due to this characteristic, α-βt / s coexisting FibH has the property of allowing the α-helix structure and β-turn structure to coexist in a stable molecular form within the peptide chain in aqueous solution, and allowing the α-helix structure and β-sheet structure to coexist in a metastable molecular form within the peptide chain in fibroin coagulated molded articles.

[0030] An "α-helix structure" is a secondary structure in polypeptides, specifically a right-handed helical structure. The pitch per helix is ​​0.54 nm, and hydrogen bonds are formed between the imino group of the first amino acid residue and the carbonyl group of the fourth amino acid residue in a peptide chain, with 3.6 amino acid residues per turn. Some fibroin proteins also form a 31-helix structure containing 3 amino acid residues per turn, in addition to the α-helix structure, but in this specification, the "31-helix structure" is also included under the "α-helix structure".

[0031] In this specification, "β-turn structure" refers to a secondary structure in polypeptides, which is a sheet structure formed by a single molecular chain bending and intramolecular hydrogen bonding.

[0032] A "β-sheet structure" is a secondary structure in polypeptides in which at least two to three parallel β-chains (β-strands) are bonded laterally by intermolecular hydrogen bonds, forming a pleated, sheet-like structure that twists as a whole. Substances containing a β-sheet structure within their molecule may be insoluble in water.

[0033] In this specification, "one molecule" refers to one peptide chain unless otherwise specified. For example, with respect to FibH, one molecule refers to FibH consisting of one peptide chain.

[0034] In this specification, "multiple items" means two or more items, for example, 2 to 1000, 2 to 800, 2 to 600, 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 150, 2 to 100, 2 to 80, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0035] In this specification, "α-helix crystal structure" refers to a crystal structure composed of a unit hexagonal lattice formed by all or part of an α-helix structure. In this specification, "α-helix crystal structure" includes liquid crystal-like structures in which variations are observed in the positional relationships between adjacent molecules along the molecular chain in the c-axis direction of the crystal (Figure 1).

[0036] A "hexagonal unit lattice" is the smallest repeating unit of a spatial lattice formed by the three-dimensional arrangement of lattice points, which are representative points of atoms or groups of atoms. It is a unit lattice that has six-fold symmetry and forms a hexagonal prism with a regular hexagon as its base. Typically, the unit lattice is the unit lattice when a hexagonal close-packed structure is formed. Figure 1 shows the shape of a typical hexagonal unit lattice.

[0037] The term "(10-10) plane" refers to a lattice plane of a unit hexagonal lattice that is expressed as (1,0,-1,0) in notation based on Miller indices. In FIG. 1, the four axes (a1 axis, a2 axis, a3 axis, c axis) of a typical unit hexagonal lattice are indicated by arrows, and the position of the (10-10) plane is indicated by a hatched plane. In FIG. 1, the deeper plane parallel to the hatched plane also corresponds to a (10-10) plane.

[0038] The term "lattice spacing" refers to the distance between adjacent closest planes among mutually parallel lattice planes. The lattice spacing between (10-10) planes refers to the distance between the hatched plane in FIG. 1 and the deeper plane parallel thereto.

[0039] The term "tensile elongation at break" refers to the value of strain (elongation percentage) when a sample breaks, among the mechanical properties measured by a tensile test. In the present specification, unless otherwise specified, tensile elongation at break is used synonymously with nominal tensile strain at break, tensile elongation, and elongation at break. Strain is a value obtained by dividing the displacement between grips in a tensile test by the gripping distance, and is usually expressed as a percentage.

[0040] The term "solidification" means that a substance in a liquid state becomes a solid state, that is, solidification. In particular, a product obtained by solidifying liquid fibroin protein in a liquid state into a solid state is referred to as a solidified fibroin molded article.

[0041] In the present specification, the term "molded article" refers to a solid body having a certain hardness and a specific shape.

[0042] In the present specification, the term "regenerated fibroin" refers to a product obtained by solidifying liquid fibroin dissolved in an aqueous solution and regenerating it as solid fibroin. In the present specification, regenerated fibroin mainly corresponds to regenerated fibroin heavy chain protein regenerated from an aqueous solution of fibroin heavy chain protein.

[0043] As used herein, the term "regenerated fibroin molded article" refers to a fibroin coagulated molded article or an insolubilized fibroin molded article obtained by molding processed regenerated fibroin to have a certain firmness and a specific shape. Examples of the regenerated fibroin molded article include thin film molded articles, fine particle molded articles, and fiber molded articles.

[0044] As used herein, the term "fibroin coagulated molded article" refers to an uninsolubilized treated regenerated fibroin molded article obtained by coagulating a fibroin solution, for example, through solvent removal or the like.

[0045] As used herein, the term "insolubilized fibroin molded article" refers to a regenerated fibroin molded article obtained by coagulating a fibroin solution, for example, through solvent removal or the like, and then subjecting the resulting product to insolubilization treatment. The insolubilized fibroin molded article generally refers to a β-fibroin molded article that becomes insoluble in water because most of the α-helix structures or β-turn structures in fibroin are induced to crystallize into β-sheet structures through the insolubilization treatment. As used herein, fibroin molded articles obtained by subjecting fibroin coagulated molded articles to insolubilization treatment are also included in the insolubilized fibroin molded articles.

[0046] 1-3. Composition The fibroin coagulated molded article of the present embodiment contains fibroin protein as an essential component. A specific description will be given below.

[0047] <Fibroin Protein> The fibroin protein contained in the fibroin coagulated molded article of the present embodiment is not particularly limited in type, as long as it contains one or more α-helix structures in one molecule.

[0048] For example, the fibroin protein may be a natural fibroin protein or an artificial fibroin protein. Further, for example, the fibroin protein may be a wild-type fibroin protein or a mutant fibroin protein.

[0049] In this specification, "natural fibroin protein" refers to fibroin protein that exists in nature. Since natural fibroin protein is a protein encoded by the wild-type genes of various organisms, it is often referred to as "wild-type fibroin protein" in this specification. Specific examples of natural fibroin protein include natural heavy chain proteins.

[0050] In this specification, "natural fibroin H-chain protein" (natural FibH) refers to FibH that exists in nature. Natural FibH is defined as a protein whose full-length amino acid sequence is identical to that of naturally occurring FibH. Regardless of the collection process, any single molecule of FibH whose composition is identical to that of naturally occurring FibH is included in the definition of natural FibH. Examples of natural FibH include FibH directly collected from bagworms, silkworms, or spiders, and FibH obtained by the expression of the FibH gene. Since natural FibH is a protein encoded by the wild-type FibH gene of various organisms, it will also be referred to as "wild-type FibH" below.

[0051] In this specification, "artificial fibroin protein" refers to fibroin proteins that do not exist in nature, and typically includes mutant fibroin proteins.

[0052] In this specification, "artificial fibroin H chain protein" (artificial FibH) refers to FibH that does not exist in nature, and is FibH that has been artificially modified in whole or in part by genetic engineering or other means.

[0053] In this specification, "mutant fibroin H-chain protein" (hereinafter also referred to as "mutant FibH") is FibH composed of an amino acid sequence different from that of wild-type FibH, and is synonymous with artificial FibH in this specification. Mutant FibH does not necessarily need to possess the same physical properties as wild-type FibH. Examples of mutant FibH include mutant FibH obtained by introducing one or more amino acids into the amino acid sequence of FibH, and chimeric FibH (hybrid FibH) obtained by fusing the amino acid sequences of FibH from two or more different insect species.

[0054] In this specification, "wild-type fibroin protein" refers to naturally occurring fibroin protein encoded by the wild-type silk protein gene of various organisms.

[0055] In this specification, "mutant fibroin protein" refers to a silk protein in which all or part of a wild-type fibroin protein has been artificially modified using genetic engineering or other technologies. Mutant fibroin proteins consist of a different amino acid sequence from wild-type fibroin proteins and, in principle, do not exist in nature. However, it is assumed that mutant fibroin proteins include the N-terminal region, central region, and C-terminal region, in order from the N-terminus, which are the basic components of wild-type fibroin proteins. Examples of mutant silk proteins include fibroin proteins in which one or more amino acids have been added, deleted, and / or substituted into the amino acid sequence of a fibroin protein, and chimeric fibroin proteins (hybrid fibroin proteins) in which the amino acid sequences of fibroin proteins from two or more different insects have been fused. Mutant fibroin proteins may have the same physical properties as wild-type fibroin proteins or they may have different physical properties.

[0056] The amino acid sequence of the mutant fibroin protein is not particularly limited, but examples include a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence of the wild-type fibroin protein, or a protein consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the wild-type amino acid sequence.

[0057] In this specification, "amino acid identity" refers to the percentage of identical amino acids in one polypeptide relative to the total number of amino acids in the other polypeptide, after the amino acid sequences of two polypeptides have been aligned and, if necessary, gaps have been introduced into either amino acid sequence to maximize the degree of amino acid agreement between the two polypeptides. This percentage of amino acid identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al, J. Mol. Biol., 215, 403-410, 1990).

[0058] The species from which fibroin proteins are derived in this specification are not particularly limited. For example, species from which fibroin proteins are derived include silkworms, species belonging to the order Araneae, or species belonging to the order Acari.

[0059] In this specification, "silkworm" refers to a general term for insects that possess silk glands and are capable of spinning silk. Specifically, silkworms refer to species from the orders Lepidoptera, Hymenoptera, Neuroptera, Trichoptera, etc., that are primarily capable of spinning silk during the larval stage for nesting, cocooning, or migration. Silkworms only need to be capable of spinning silk, and include those that spin silk at any developmental stage, such as larvae and adults. For example, among the order Lepidoptera, species belonging to families such as Bombycidae, Saturniidae, Psychidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Archtiidae, and Noctuidae are examples of species that can spin large amounts of silk. Specifically, the order Lepidoptera includes, for example, the silkworm (B. mori) and the mulberry silkworm (B. mandarina) belonging to the genus Bombyx, the pearl silkworm (S. cynthia) and the Eri silkworm (S. cynthia ricini) belonging to the genus Samia, the Japanese oak silkworm (A. yamamai) and the Japanese silkworm (A. pernyi) belonging to the genus Antheraea, and the small Japanese oak silkworm (S. yamamai) belonging to the genus Saturnia. Examples include species belonging to the genera Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, which are all members of the Psychidae family. In particular, silkworms, which are the larvae of the silkworm moth, and bagworms, which are the larvae of moths belonging to the Psychidae family, are suitable as silkworms. Specific examples of bagworm moths include the giant bagworm moth (Eumeta japonica), the small bagworm moth (Eumeta minuscula), and the moss bagworm moth (Nipponopsyche fuscescens).

[0060] Examples of organisms belonging to the order Araneae include species belonging to the families Araneidae, Nephilidae, Tetragnathidae, Theridiidae, and Linyphiidae. Specific examples include the Araneus genus species Araneus ventricosus, Araneus uyemurai, Araneus diadematus, and Araneus maccacus; the Argiope genus Araneus amoena; the Caerostris genus Caerostris darwini; and the Nephila genus Nephila species Nephila pilipes, Nephila clavata, and Nephila clavipes.

[0061] In this specification, fibrous proteins such as those derived from silkworm silk, spider silk, and bagworm silk can be suitably used.

[0062] In this specification, "bagworm silk" refers to silk derived from bagworms. In this specification, "silk" refers to a protein-based thread derived from insects, spiders, mites, etc., which is spun by their larvae or adults for purposes such as nesting, movement, anchoring, cocooning, and prey capture. In this specification, silk includes monofilaments, spun fibers, and aggregated fibers.

[0063] In this specification, "spun silk fibers" refer to silk threads in their as-spun state in the case of bagworms and silkworms, and threads in their as-secreted state in the case of spiders. The spinned silk fibers of bagworms and silkworms are composed of difilaments, which are pairs of two single fibers. This form is based on the fact that during spinning, two single fibers extruded from the silk thread filaments located on the left and right sides of the bagworm or silkworm through the spinning opening are bound together by a sericin-like adhesive substance.

[0064] In this specification, "single fiber" refers to the smallest unit of fiber components, a filament, and is also called a monofilament. Single fibers are mainly composed of fibroin-like proteins that make up silk threads. When a spun fiber is composed of multiple single fibers, for example, adhesive substances can be removed by scouring the spun fiber to obtain single fibers.

[0065] In this specification, "composite fiber" refers to a fiber composed of multiple fiber bundles, also known as a multifilament. This is so-called raw silk, and in principle is composed of multiple single fibers, but in this specification, it also includes cases where it is composed of multiple single fibers and spinning fibers, or multiple spinning fibers. Composite fibers in this specification also include mixed fibers made by mixing multiple types of silk yarn (for example, multiple types of bagworm silk; multiple types of silkworm silk; multiple types of spider silk; silk composed of two or more of bagworm silk, silkworm silk, and spider silk). Composite fibers in this specification include not only twisted yarn fibers but also untwisted yarn fibers.

[0066] Silk threads can include scaffolding silks, dragging silks, and nesting silks, but in this specification, silk threads include all of them. "Scaffolding silks" are silk threads spun or secreted by insects or spiders for movement, while dragging silks are silk threads spun or secreted to hang from branches, leaves, etc. "Nesting silks," on the other hand, are silk threads that make up nests, and are spun or secreted to bind leaf fragments or branch fragments together, or to create the nest or the inner wall of the nest, which is the living area.

[0067] The fibroin proteins used herein may be produced by the organism from which they originate, synthesized by other means, or a combination thereof. For example, methods other than those used by the organism from which the protein originates include chemical synthesis and synthesis by organisms other than the organism from which the protein originates (including cells and microorganisms).

[0068] In this specification, "microorganism" refers to a single-celled organism, encompassing both eukaryotic single-celled organisms and prokaryotes, typically referring to prokaryotes. The type of microorganism is not particularly limited and may be any microorganism commonly used in the field of genetic engineering. For example, yeast and Escherichia coli are suitable microorganisms. Unless otherwise specified, in this specification, "microorganism" refers to a transformed organism (genetically modified microorganism) that has been made capable of expressing the target protein through genetic engineering.

[0069] The fibroin coagulated molded article of this embodiment may contain only one type of fibroin protein, or it may contain a combination of multiple types of fibroin proteins. When the fibroin coagulated molded article contains multiple types of fibroin proteins, for example, it may contain a combination of fibroin proteins derived from multiple species, or it may contain a combination of fibroin proteins composed of multiple amino acid sequences derived from the same species.

[0070] The fibroin protein contained in the fibroin coagulated molded article of this embodiment contains one or more α-helix structures in one molecule. Therefore, the amino acid sequence of the fibroin protein of this embodiment includes an α-helix-forming sequence.

[0071] In this specification, "α-helix-forming sequence" refers to an amino acid sequence that, in whole or in part, forms an α-helix in an aqueous solution or a fibroin protein coagulated molded article. For example, an α-helix-forming sequence is the amino acid sequence represented by (A)n (where n is an integer ≥ 6), specifically the amino acid sequence shown in Sequence ID No. 1. Another example of an α-helix-forming sequence is the amino acid sequence represented by (GGX)n (where X is an alanine residue (A) or a tyrosine residue (Y), and n is an integer ≥ 1), specifically the amino acid sequence shown in Sequence ID No. 2. The helix structure formed by (GGX)n corresponds to a "31-helix structure" in which the helix pitch is slightly longer than that of an α-helix structure, but in this specification, the "31-helix structure" is also included as one of the structures of an α-helix structure.

[0072] The fibroin protein contained in the fibroin coagulated molded article of this embodiment may further include one or more β-sheet structures. In this case, the amino acid sequence of the fibroin protein of this embodiment includes a β-turn forming sequence.

[0073] In this specification, "β-turn-forming sequence" refers to an amino acid sequence that, in whole or in part, forms a β-turn structure in an aqueous solution, and in whole or in part, forms a β-sheet structure in a fibroin-coated molded article. Thus, this β-turn-forming sequence can form different secondary structures in an aqueous solution and in a fibroin-coated molded article. Examples of β-turn-forming sequences include the amino acid sequence represented by (GX)n (where X is an alanine residue or a serine residue, and n is an integer ≥ 3), specifically the amino acid sequence shown in Sequence ID No. 3. Another example of a β-turn-forming sequence is the amino acid sequence represented by (GAGAGX)n (where X is a serine residue or a tyrosine residue, and n is an integer ≥ 1), specifically the amino acid sequence shown in Sequence ID No. 4. However, exceptionally, in the wild-type silkworm fibroin H chain protein, (GX)n and (GAGAGX)n do not form a β-sheet structure in the fibroin protein coagulation molded product, but instead form a water-soluble crystalline structure consisting of an aggregate of β-turn structures called Silk-I type.

[0074] For example, fibroin proteins contain one or more repeating units in their amino acid sequence. For example, repeating units from natural FibH can be suitably used as these repeating units.

[0075] The fibroin protein preferably contains two or more core sequences within the repeating unit. A "core sequence" refers to a sequence consisting of more than ten amino acid residues that appears repeatedly multiple times within the repeating unit.

[0076] For example, mutant bagworm FibH contains one or more repeat units in its amino acid sequence. Examples of specific amino acid sequences that constitute one repeat unit in bagworm FibH include the amino acid sequences shown in SEQ ID NOs. 5 to 13, amino acid sequences in which one or more amino acids are added, deleted, or substituted in the amino acid sequences shown in SEQ ID NOs. 5 to 13, or amino acid sequences that have 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity with the amino acid sequences shown in SEQ ID NOs. 5 to 13 are amino acid sequences of repeat units unique to wild-type bagworm FibH of the giant bagworm moth, which has the α-βt / s coexisting FibH configuration.

[0077] Specific examples of mutant bagworm FibH include the amino acid sequence shown in SEQ ID NO: 14, which contains four repeat units consisting of the amino acid sequences shown in SEQ ID NOs: 5 to 13; the amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 14; or the amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 14. The amino acid sequence shown in SEQ ID NO: 14 is an amino acid sequence consisting of a part of the wild-type bagworm FibH of the giant bagworm moth.

[0078] An example of a specific base sequence that encodes the amino acid sequence shown in Sequence ID No. 14 is the base sequence shown in Sequence ID No. 15.

[0079] Specific examples of mutant or artificial fibroin proteins include, for instance, artificial FibH having the α-βt / s coexisting FibH structure.

[0080] As mutant or artificial fibroin proteins, chimeric (hybrid) fibroin proteins can be used, which are a combination of fibroin protein from one species and fibroin protein from another species. For example, as mutant or artificial fibroin proteins, chimeric FibH can be used, which is a combination of FibH from bagworms and FibH from other insects. A specific example of such chimeric FibH is a combination of bagworm FibH and silkworm FibH. A concrete example of such a chimeric FibH is the bagworm FibH of the giant bagworm moth and silkworm FibH, which consists of the amino acid sequence shown in Sequence ID No. 22. In this chimeric FibH, positions 1 to 153 and 466 to 524 are amino acid sequences derived from silkworm FibH, and positions 156 to 463 contain amino acid sequences derived from bagworm FibH of the giant bagworm moth. In the case of chimeric FibH, it may possess the physical properties of the FibH of each of the species from which it is derived. Chimeric FibH may be a FibH consisting of an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 14, or a FibH consisting of an amino acid sequence having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 22. A specific nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO: 22 is, for example, the nucleotide sequence shown in SEQ ID NO: 23.

[0081] Another example of a mutant bagworm FibH is terminal mutant bagworm FibH, which contains mutations in either the N-terminal region, the C-terminal region, or both of the wild-type bagworm FibH.

[0082] The mutation, if it is a deletion, may be a complete deletion of either the N-terminal region or the C-terminal region, or both, or a partial deletion. Here, "partial" refers to one or two or more consecutive or discontinuous amino acids from the amino acid sequence constituting a specific amino acid region (here, the N-terminal region and / or C-terminal region), and less than the total number of amino acids. Preferably, the mutation is a deletion of a number of amino acids that can cause loss of function in the terminal region. For example, the mutation may be a deletion of five or more, eight or more, ten or more, twelve or more, fifteen or more, eighteen or more, or twenty or more consecutive or discontinuous amino acids. In terminal mutant bagworm FibH, either the N-terminal region or the C-terminal region, or both, may be deleted.

[0083] In addition to deletions, mutations may also include the addition or substitution of one or more amino acids. The amino acids to be added or substituted are not particularly limited, but amino acids that may lose the function of their terminal region are preferred.

[0084] Specific examples of terminal region mutant bagworm FibH include the amino acid sequences shown in SEQ ID NOs: 16-18. SEQ ID NO: 16 is the amino acid sequence of terminal region mutant bagworm FibHΔC, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with the entire C-terminal region deleted. SEQ ID NO: 17 is the amino acid sequence of terminal region mutant bagworm FibHΔN, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with the entire N-terminal region deleted. SEQ ID NO: 18 is the amino acid sequence of terminal region mutant bagworm FibHΔN / C, which is a mutant bagworm FibH having the amino acid sequence shown in SEQ ID NO: 14, but with both the N-terminal and C-terminal regions completely deleted.

[0085] Examples of specific base sequences of genes encoding terminal region mutant bagworm FibH include the terminal region mutant bagworm FibHΔC gene shown in SEQ ID NO: 19, the terminal region mutant bagworm FibHΔN gene shown in SEQ ID NO: 20, and the terminal region mutant bagworm FibHΔN / C gene shown in SEQ ID NO: 21.

[0086] The fibroin protein contains one or more α-helix-forming sequences within the amino acid sequence of the repeating unit or other regions, and optionally further contains one or more β-turn-forming sequences.

[0087] The α-helix-forming sequence and the β-turn-forming sequence may be amino acid sequences present in natural FibH, or they may be amino acid sequences modified from or artificially designed from those sequences.

[0088] The ratio of bases in the α-helix-forming sequence and the β-turn-forming sequence contained in the fibroin protein is not particularly limited.

[0089] For example, the β-turn-forming sequence may contain more or fewer base pairs than the α-helix-forming sequence. Specifically, for example, the number of base pairs in the β-turn-forming sequence may be 0.1 to 20 times, 0.1 to 10 times, 0.1 to 5 times, 0.1 to 4 times, 0.1 to 3 times, 0.1 to 2.5 times, 0.1 to 2.4 times, 0.1 to 2.3 times, 0.5 to 20 times, 0.5 to 10 times, 0.5 to 5 times, 0.5 to 4 times, 0.5 to 3 times, 0.5 to 2.5 times, 0.5 to 2.4 times, 0.5 to 2.3 times, 0.9 x - 20x, 0.9x - 10x, 0.9x - 5x, 0.9x - 4x, 0.9x - 3x, 0.9x - 2.5x, 0.9x - 2.4x, 0.9x - 2.3x, 1x - 20x, 1x - 10x, 1x - 5x, 1x - 4x , 1x - 3x, 1x - 2.5x, 1x - 2.4x, 1x - 2.3x, 1.1x - 20x, 1.1x - 10x, 1.1x - 5x, 1.1x - 4x, 1.1x - 3x, 1.1x - 2.5x, 1.1x - 2.4x, 1. 1x - 2.3x, 1.5x - 20x, 1.5x - 10x, 1.5x - 5x, 1.5x - 4x, 1.5x - 3x, 1.5x - 2.5x, 1.5x - 2.4x, 1.5x - 2.3x, 1.9x - 20x, 1.9x - 10x, 1.9x - 5x, 1.9x - 4x, 1.9x - 3x, 1.9x - 2.5x, 1.9x - 2.4x, 1.9x - 2.3x, 2.0x - 20x, 2.0x - 10x, 2.0x - 5x, 2.0x - 4x, 2.0 It can be set to 2x to 3x, 2.0x to 2.5x, 2.0x to 2.4x, 2.0x to 2.3x, 2.1x to 20x, 2.1x to 10x, 2.1x to 5x, 2.1x to 4x, 2.1x to 3x, 2.1x to 2.5x, 2.1x to 2.4x, 2.1x to 2.3x, 2.2x to 20x, 2.2x to 10x, 2.2x to 5x, 2.2x to 4x, 2.2x to 3x, 2.2x to 2.5x, 2.2x to 2.4x, 2.2x to 2.3x, etc.

[0090] α-βt / s coexisting FibH is defined as having at least one α-helix-forming sequence and one β-turn-forming sequence in its amino acid sequence, regardless of their position or number. For example, in the amino acid sequence of α-βt / s coexisting FibH, the α-helix-forming sequences and β-turn-forming sequences may form separate clusters in multiple consecutive sequences, or they may alternate consecutively. Furthermore, the α-helix-forming sequences and β-turn-forming sequences may be randomly distributed within the amino acid sequence of α-βt / s coexisting FibH.

[0091] In natural FibH, the α-helix-forming sequence and the β-turn-forming sequence may be contained within the repeating unit of the central region, or they may be contained outside the repeating unit, such as the N-terminal region and / or the C-terminal region.

[0092] The mutant bagworm FibH may optionally have an exogenous signal peptide at its N-terminus. A "signal peptide" is an extracellular translocation signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space. After translation, signal peptides are cleaved and removed by signal peptidases before being secreted into the extracellular space. Signal peptides have a positively charged amino acid such as Lys or Arg at their N-terminus, followed by a sequence of highly hydrophobic amino acids such as Ala, Leu, Val, Ile, and Phe.

[0093] In the case of secreted proteins, they typically have an endogenous signal peptide at their N-terminus. For example, in the chimeric FibH of bagworm FibH and silkworm FibH, which is composed of the amino acid sequence shown in Sequence ID No. 22, the amino acids from positions 1 to 21 correspond to the endogenous signal peptide derived from silkworm FibH, so there is no need to add an exogenous signal peptide.

[0094] The C-terminal side of the signal peptide may also have an amino acid sequence containing a post-insertion signal sequence that promotes cleavage of the signal peptide and secretion of the protein, and / or a signal peptidase recognition site that cleaves the signal peptide from the fusion protein.

[0095] The fibroin protein may also include functional protein regions, etc. By using the fibroin coagulation molded product of this embodiment, an insolubilized molded product can be manufactured under relaxed conditions, in particular conditions that do not involve heat treatment or prolonged heating. Therefore, by including a functional region based on proteins that are easily denatured, especially by heat (such as enzymes or fluorescent proteins), the insolubilized molded product can also exhibit the function based on that region.

[0096] <Structure of fibroin coagulated molded product> The α-helix structure formed by the fibroin protein forms an extended α-helix crystal structure in the fibroin coagulated molded product of this embodiment.

[0097] Here, "extended α-helix crystal structure" refers to a crystal structure composed of an extended hexagonal unit lattice, in which all or part of the fibroin protein forms a hexagonal close-packed structure.

[0098] In this specification, "standard α-helix crystal structure" refers to a crystal structure in a dried fibroin coagulated molded article that is composed of a unit hexagonal lattice formed by all or part of the α-helix structures that constitute a part of the fibroin protein. In this specification, the unit hexagonal lattice in this crystal structure is referred to as the "standard unit hexagonal lattice."

[0099] In this specification, "extended unit hexagonal lattice" refers to a unit hexagonal lattice having a lattice plane spacing d1 between (10-10) faces that is greater than the lattice plane spacing d2 between (10-10) faces in a standard unit hexagonal lattice.

[0100] In this specification, an extended α-helix crystal structure is a crystal structure in a fibroin coagulated molded article that is composed of an extended unit hexagonal lattice formed by all or part of the α-helix structures that constitute a part of the fibroin protein.

[0101] The degree of drying of a fibroin solidified molded article having a standard α-helix crystal structure is sufficient as long as the moisture content of the fibroin solidified molded article is reduced, and the specific moisture content is not particularly limited. For example, a fibroin solidified molded article having a standard α-helix crystal structure includes both fibroin solidified molded articles obtained without actively increasing the moisture content and fibroin solidified molded articles obtained by performing a treatment to reduce the moisture content.

[0102] The specific moisture content of a dry fibroin solidified molded product can be, for example, 8.2% or less, 8.1% or less, 8% or less, 7.9% or less, 7.8% or less, 7.7% or less, 7.6% or less, etc., based on the rate of change in mass when the temperature is raised from room temperature (approximately 25°C) to 200°C at a heating rate of 5°C / min.

[0103] The specific value of d2 is not particularly limited. For example, in the case of fibroin-coated molded products derived from the silk of the wild-type bagworm moth, d2 is typically 7.5 Å.

[0104] d1 only needs to be greater than d2, and the difference in lattice plane spacing is not particularly limited. For example, the difference in lattice plane spacing can be 0.1 Å or more, 0.15 Å or more, 0.175 Å or more, 0.2 Å or more, 0.225 Å or more, 0.25 Å or more, 0.3 Å or more, 0.35 Å or more, 0.375 Å or more, or 0.4 Å or more. The upper limit of the difference in lattice plane spacing is not particularly limited as long as it is distinguishable from other lattice plane spacings, for example, it can be 2 Å or less, 1.5 Å or less, 1 Å or less, 0.9 Å or less, 0.8 Å or less, 0.7 Å or less, 0.6 Å or less, 0.5 Å or less, 0.45 Å or less, or 0.4 Å or less. Specific ranges for the difference in lattice plane spacing include, for example, 0.05 Å to 2 Å, 0.1 Å to 2 Å, 0.1 Å to 1.5 Å, 0.1 Å to 1 Å, 0.1 Å to 0.5 Å, 0.15 Å to 1 Å, 0.15 Å to 0.8 Å, 0.15 Å to 0.6 Å, 0.15 Å to 0.5 Å, 0.175 Å to 1 Å, 0.175 Å to 0.8 Å, 0.175 Å to 0.6 Å, 0.175 Å to 0.5 Å, 0.175 Å to 0.45 Å, 0.2 Å to 1 Å, 0.2 Å to 0.8 Å, 0.2 Å to 0.6 Å, and 0.2 Å to 0.5 Å. Examples include 0.2Å~0.45Å, 0.225Å~0.8Å, 0.225Å~0.6Å, 0.225Å~0.5Å, 0.225Å~0.45Å, 0.3Å~0.8Å, 0.3Å~0.6Å, 0.3Å~0.5Å, 0.3Å~0.45Å, 0.35Å~0.8Å, 0.35Å~0.6Å, 0.35Å~0.5Å, 0.35Å~0.45Å, 0.35Å~0.4Å, 0.4Å~0.8Å, 0.4Å~0.6Å, 0.4Å~0.5Å, 0.4Å~0.45Å, etc.

[0105] The specific value of d1 is not particularly limited. For example, the lower limit of d1 may be 7.6 Å or greater, 7.65 Å or greater, 7.675 Å or greater, 7.7 Å or greater, 7.725 Å or greater, 7.75 Å or greater, 7.8 Å or greater, 7.85 Å or greater, 7.875 Å or greater, or 7.9 Å or greater. The upper limit of d1 is not particularly limited as long as it is distinguishable from other lattice plane spacings, for example, 9 Å or less, 8.8 Å or less, 8.6 Å or less, 8.5 Å or less, 8.4 Å or less, 8.3 Å or less, 8.2 Å or less, 8.1 Å or less, 8 Å or less, 7.95 Å or less, or 7.9 Å or less.The specific range of d1 is, for example, 7.55 Å to 9 Å, 7.55 Å to 8.5 Å, 7.55 Å to 8.4 Å, 7.55 Å to 8.3 Å, 7.55 Å to 8.2 Å, 7.55 Å to 8.1 Å, 7.55 Å to 8 Å, 7.55 Å to 7.95 Å, 7.55 Å to 7.9 Å, 7.6 Å to 8.5 Å, 7.6 Å to 8.4 Å, 7.6 Å to 8.3 Å, 7.6 Å to 8.2 Å, 7.6 Å to 8.1 Å, 7.6 Å to 8 Å, 7.6 Å to 7.95 Å, 7.6 Å to 7.9 Å, and 7.65 Å to 8.5 Å. Å, 7.65Å to 8.4Å, 7.65Å to 8.3Å, 7.65Å to 8.2Å, 7.65Å to 8.1Å, 7.65Å to 8Å, 7.65Å to 7.95Å, 7.65Å to 7.9Å, 7.675Å to 8.5Å, 7.675Å to 8.4Å , 7.675Å to 8.3Å, 7.675Å to 8.2Å, 7.675Å to 8.1Å, 7.675Å to 8Å, 7.675Å to 7.95Å, 7.675Å to 7.9Å, 7.7Å to 8.5Å, 7.7Å to 8.4Å, 7.7Å to 8.3Å , 7.7Å to 8.2Å, 7.7Å to 8.1Å, 7.7Å to 8Å, 7.7Å to 7.95Å, 7.7Å to 7.9Å, 7.725Å to 8.5Å, 7.725Å to 8.4Å, 7.725Å to 8.3Å, 7.725Å to 8.2Å, 7.7 25Å to 8.1Å, 7.725Å to 8Å, 7.725Å to 7.95Å, 7.725Å to 7.9Å, 7.75Å to 8.5Å, 7.75Å to 8.4Å, 7.75Å to 8.3Å, 7.75Å to 8.2Å, 7.75Å to 8.1Å, 7. Examples include 75Å–8Å, 7.75Å–7.95Å, 7.75Å–7.9Å, 7.8Å–8.5Å, 7.8Å–8.4Å, 7.8Å–8.3Å, 7.8Å–8.2Å, 7.8Å–8.1Å, 7.8Å–8Å, 7.8Å–7.95Å, 7.8Å–7.9Å, 7.85Å–8.5Å, 7.85Å–8.4Å, 7.85Å–8.3Å, 7.85Å–8.2Å, 7.85Å–8.1Å, 7.85Å–8Å, 7.85Å–7.95Å, 7.85Å–7.9Å, etc.

[0106] The spacing of other grid planes in an extended unit hexagonal grid is not particularly limited. The spacing of other grid planes in an extended unit hexagonal grid may be smaller than, the same as, or larger than, a standard unit hexagonal grid.

[0107] The measurement of the crystal structure can be performed using any measurement method known in the art and is not particularly limited. For example, the method exemplified in the structural measurement step of the fifth embodiment can be used to measure the crystal structure. For example, it can be confirmed whether the lattice plane spacing falls within the above range by analysis based on the results of a wide-angle X-ray diffraction measurement performed under the conditions of CuKα line (40kV, 30mA; wavelength 1.5418Å) and camera length 50mm.

[0108] The lattice plane spacing of the β-sheet crystal structure formed by the β-sheet structure is not particularly limited, but preferably, one or more lattice plane spacings of the extended β-sheet crystal structure do not change from those of the standard unit hexagonal lattice. For example, the lattice plane spacing of one or more or all of the faces selected from the group consisting of (200), (210), (211), (202), (103), and (213) faces of the β-sheet crystal structure does not change from those of the standard unit hexagonal lattice. For example, in the fibroin solidified molded product of this embodiment, the lattice plane spacing of the (210) face of the β-sheet crystal structure does not change whether or not it is extended. Here, "lattice plane spacing does not change" means that the lattice plane spacing does not change by 0.05 Å or more, for example, 0.04 Å or more, 0.03 Å or more, or 0.02 Å or more.

[0109] The factors that cause the lattice plane spacing d1 to expand are not particularly limited. For example, the lattice plane spacing d1 can be expanded by the presence of many other molecules, such as a solvent.

[0110] The type of solvent in this case is not particularly limited, but may include, for example, polar or nonpolar organic solvents, water or aqueous solutions, or combinations thereof. Polar solvents, such as aqueous solvents, can be suitably used. Specific examples of solvents include, for example, the solvents exemplified in the dissolution step of the third embodiment.

[0111] For example, if the lattice plane spacing d1 is expanded by increasing the content of the aqueous solvent, the water content of the fibroin solidified molded product in this embodiment should be higher than the water content of the dried fibroin solidified molded product described above. For example, the lower limit of the water content of the fibroin solidified molded product in this embodiment should be 7.7% or more, greater than 7.7%, 7.8% or more, 7.9% or more, 8% or more, 8.1% or more, 8.2% or more, 8.3% or more, 8.4% or more, 8.5% or more, 8.6% or more, 8.7% or more, 8.9% or more, 9% or more, 9.5% or more, 10% or more, 10.5% or more, 11% or more, 11.1% or more, 11.2% or more, 11.3% or more, 11.4% or more, 11.5% or more, 11.6% or more, or 11.7% or more. Furthermore, for example, the upper limit of the moisture content of the fibroin solidified molded product according to this embodiment may be 50% or less, 45% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 30% or less, 25% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14.5% or less, 14% or less, 13.5% or less, 13% or less, 12.5% ​​or less, 12% or less, 11.9% or less, 11.8% or less, or 11.7% or less. Specific ranges of moisture content include, for example, 7.7% to 50%, 7.7% to 36%, 7.7% to 35%, 7.7% to 30%, 7.7% to 25%, 7.7% to 20%, 7.7% to 15%, 7.7% to 13%, 7.7% to 12%, 7.7% to 11.7%, over 7.7% and up to 50%, over 7.7% and up to 36%, over 7.7% and up to 35%, over 7.7% and up to 30%, over 7.7% and up to 25%, over 7.7% and up to 20%, over 7.7% and up to 15%, over 7.7% and up to 13%, over 7.7% and up to 12%, over 7.7% and up to 11.7%, 8% to 50%, 8% to 36%, 8% % to 35%, 8% to 30%, 8% to 25%, 8% to 20%, 8% to 15%, 8% to 13%, 8% to 12%, 8% to 11.7%, 8.5% to 50%, 8.5% to 36%, 8.5% to 35%, 8.5% to 30%, 8.5% to 25%, 8.5% to 20%, 8.5% to 15%, 8.5% to 13%, 8.5% to 12%, 8.5% to 11.7%, 8.7% to 50%, 8.7% to 36%, 8.7% to 35%, 8.7% to 30%, 8.7% to 25%, 8.7% to 20%, 8.7% to 15%, 8.7% to 13%, 8.7% to 12%, 8.7% to 11.7%, 9%–50%, 9%–36%, 9%–35%, 9%–30%, 9%–25%, 9%–20%, 9%–15%, 9%–13%, 9%–12%, 9%–11.7%, 10%–50%, 10%–36%, 10%–35%, 10%–30%, 10%–25%, 10%–20%, 10%–15%, 10%– 13%, 10%–12%, 10%–11.7%, 10.5%–50%, 10.5%–36%, 10.5%–35%, 10.5%–30%, 10.5%–25%, 10.5%–20%, 10.5%–15%, 10.5%–13%, 10.5%–12%, 10.5%–11.7%, 11%–50%, 11% ~36%, 11%~35%, 11%~30%, 11%~25%, 11%~20%, 11%~15%, 11%~13%, 11%~12%, 11%~11.7%, 11.5%~50%, 11.5%~36%, 11.5%~35%, 11.5%~30%, 11.5%~25%, 11.5%~20%, 11.5 Examples include % to 15%, 11.5% to 13%, 11.5% to 12%, 11.5% to 11.7%, 11.7% to 50%, 11.7% to 36%, 11.7% to 35%, 11.7% to 30%, 11.7% to 25%, 11.7% to 20%, 11.7% to 15%, 11.7% to 13%, 11.7% to 12%, etc.

[0112] The moisture content should be within the range described above, measured by the rate of change in mass when the temperature is raised from room temperature (approximately 25°C) to 200°C at a heating rate of 5°C / min.

[0113] Whether a fibroin protein forms an α-helix structure and whether a fibroin coagulated molded product has an extended α-helix crystal structure composed of an extended hexagonal unit lattice can be determined based on whether the amino acid sequence of the fibroin protein has an α-helix-forming sequence and on the water content of the fibroin coagulated molded product, without examining the three-dimensional structure of the fibroin protein and the crystal structure of the fibroin coagulated molded product.

[0114] The shape of the fibroin-coated molded article in this embodiment is not particularly limited, and may be any shape commonly used for resin before stretching. For example, the shape of the fibroin-coated molded article may be rod-shaped, fibrous, flat (film-shaped (including sheet-shaped), plate-shaped, etc.), other granular (convex, polyhedral, spherical, columnar, conical, pellet-shaped or granular, etc.), or a combination thereof.

[0115] The size (dimensions) of the fibroin-coated molded product, such as length and thickness, or particle size, or fiber cross-sectional diameter, is not particularly limited.

[0116] The content of crystalline regions in the fibroin solidified molded product is not particularly limited, as long as the lattice plane spacing d1 can be detected by the measurement method described in the fifth embodiment. For example, the fibroin solidified molded product of this embodiment may contain amorphous regions.

[0117] 1-4. Physical Properties The physical properties of the fibroin solidified molded article of this embodiment are not particularly limited, as long as it is a ductile material that can be easily stretched at a relatively low temperature. Preferably, the fibroin solidified molded article of this embodiment is a ductile material having a yield point.

[0118] Typically, the tensile yield stress (tensile strength) of the fibroin solidified molded product of this embodiment is lower than that of the dry fibroin solidified molded product having a standard α-helix crystal structure composed of a standard unit hexagonal lattice. For example, the tensile yield stress of the fibroin solidified molded product of this embodiment is 1 MPa or more, 2 MPa or more, 3 MPa or more, 4 MPa or more, 5 MPa or more, 6 MPa or more, 7 MPa or more, 8 MPa or more, 8.1 MPa or more, 8.2 MPa or more, 8.3 MPa or more, or 8.4 MPa or more lower than that of the dry fibroin solidified molded product. Also, for example, the difference between the tensile yield stress of the fibroin solidified molded product of this embodiment and the tensile yield stress of the dry fibroin solidified molded product may be 30 MPa or less, 25 MPa or less, 20 MPa or less, 15 MPa or less, 10 MPa or less, 9 MPa or less, or 8.5 MPa or less. The range of the difference between the tensile yield stress of the fibroin solidified molded product and the tensile yield stress of the dried fibroin solidified molded product in this embodiment is, for example, 1 MPa to 30 MPa, 1 MPa to 20 MPa, 1 MPa to 15 MPa, 1 MPa to 10 MPa, 1 MPa to 9 MPa, 1 MPa to 8.5 MPa, 4 MPa to 30 MPa, 4 MPa to 20 MPa, 4 MPa to 15 MPa, 4 MPa to 10 MPa, 4 MPa to 9 MPa, 4 MPa to 8.5 MPa, 6 MPa to 30 MPa, 6 MPa to 20 MPa, 6 MPa to 15 MPa, 6 MPa to 10 MPa, and 6 MPa to 9 MPa. Examples include 6MPa to 8.5MPa, 8MPa to 30MPa, 8MPa to 20MPa, 8MPa to 15MPa, 8MPa to 10MPa, 8MPa to 9MPa, 8MPa to 8.5MPa, 8.2MPa to 30MPa, 8.2MPa to 20MPa, 8.2MPa to 15MPa, 8.2MPa to 10MPa, 8.2MPa to 9MPa, 8.2MPa to 8.5MPa, 8.4MPa to 30MPa, 8.4MPa to 20MPa, 8.4MPa to 15MPa, 8.4MPa to 10MPa, 8.4MPa to 9MPa, 8.4MPa to 8.5MPa, etc.

[0119] The specific tensile yield stress of the fibroin solidified molded article in this embodiment is not particularly limited. For example, the lower limit of the tensile yield stress may be 64.5 MPa or less, 64 MPa or less, 63 MPa or less, 62 MPa or less, 61 MPa or less, 60 MPa or less, 59 MPa or less, 58 MPa or less, 57 MPa or less, or 56.5 MPa or less. Also, for example, the upper limit of the tensile yield stress may be 40 MPa or more, 45 MPa or more, 50 MPa or more, 51 MPa or more, 52 MPa or more, 53 MPa or more, 54 MPa or more, 55 MPa or more, 55.5 MPa or more, or 56 MPa or more. Furthermore, for example, the range of tensile yield stress can be found to be 40 MPa to 64.5 MPa, 40 MPa to 60 MPa, 40 MPa to 57 MPa, 40 MPa to 56.5 MPa, 50 MPa to 64.5 MPa, 55 MPa to 60 MPa, 55 MPa to 57 MPa, 55 MPa to 56.5 MPa, 56 MPa to 64.5 MPa, 56 MPa to 60 MPa, 56 MPa to 57 MPa, 56 MPa to 56.5 MPa, etc.

[0120] The tensile elongation at break of the fibroin solidified molded product of this embodiment is greater than that of the dried fibroin solidified molded product having a standard α-helix crystal structure composed of a standard unit hexagonal lattice. For example, the tensile elongation at break of the fibroin solidified molded product of this embodiment may be 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 25 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 81 times or more, 82 times or more, 83 times or more, 84 times or more, or 85 times or more than the tensile elongation at break of the dried fibroin solidified molded product. Furthermore, for example, the tensile elongation at break of the fibroin solidified molded product according to this embodiment may be 5% or more, 10% or more, 15% or more, 20% or more, 40% or more, 50% or more, 75% or more, 100% or more, 150% or more, 175% or more, 200% or more, 210% or more, 220% or more, 225% or more, or 230% or more greater than the tensile elongation at break of the dried fibroin solidified molded product.

[0121] For example, the tensile elongation at break of the fibroin solidified molded product according to this embodiment may be 20% or more, 30% or more, 50% or more, 70% or more, 90% or more, 100% or more, 110% or more, 120% or more, 150% or more, 175% or more, 200% or more, 210% or more, 215% or more, 220% or more, 230% or more, 231% or more, 232% or more, or 233% or more.

[0122] 1-5. Effects The fibroin solidified molded article of this embodiment provides a fibroin solidified molded article that is extremely tough and has extremely high impact strength. This solidified molded article can be easily stretched to a high stretch ratio under relaxed conditions, especially under conditions that do not involve heat treatment or prolonged heating, and has excellent moldability.

[0123] 2. Method for Improving the Stretchability of Fibroin Coagulated Molded Articles 2-1. Overview The second aspect of the present invention is a method for improving the tensile elongation at break of fibroin coagulated molded articles. The method of this aspect includes an expansion step as an essential step. According to the method of this aspect, the stretchability of fibroin coagulated molded articles, including tensile properties such as tensile elongation at break, can be improved.

[0124] 2-2. Process The method of this embodiment includes an expansion step as an essential step, and includes a dissolution step, a solidification step, and a pre-molding step as optional steps. Each step will be described below.

[0125] 2-2-1. Dissolution Step This step is an optional step of the method according to this embodiment, and is a step of dissolving fibroin protein in a solvent to prepare a dope solution.

[0126] This step can be performed, for example, when returning a fibroin solidified molded product to the method of this embodiment back into the doping solution and then re-preparing it as a fibroin solidified molded product. The specific details of this step are the same as those described in the dissolution step in the third embodiment.

[0127] 2-2-2. Coagulation Step This step is an optional step of the method according to this embodiment, and is a step of coagulating the fibroin protein dissolved in the dope solution to prepare a fibroin coagulated molded product. If the dissolution step is performed, this step can be performed simultaneously or afterward.

[0128] The specific details of this process are the same as those described in the solidification process in the third aspect.

[0129] 2-2-3. Pre-molding process The "pre-molding process" is an optional step of the method according to this embodiment, and is a process of molding the fibroin solidified molded product into a desired shape. Typically, this process is performed simultaneously with the solidification process.

[0130] This process can be carried out, for example, when it is required to change the shape of the fibroin solidified molded product, which is the raw material of this embodiment. The specific details of this process are the same as those described in the pre-molding process in the third embodiment.

[0131] 2-2-4. Expansion Step This step is an essential step of the method according to this embodiment, and is a step of expanding the α-helix crystal structure of the fibroin solidified molded product to an expanded α-helix crystal structure. This step can be performed simultaneously with or after the solidification step. Furthermore, if a pre-molding step is performed, this step can be performed simultaneously with or after it.

[0132] The expansion method used in this process is not particularly limited, as long as it can change the α-helix crystal structure of the fibroin-coagulated molded product into an expanded α-helix crystal structure. For example, one expansion method is a treatment that increases the content of molecules other than fibroin protein, such as solvents, in the fibroin-coagulated molded product.

[0133] In this case, the process may involve contacting the fibroin-coated molded product with the target molecule whose content is to be increased, relaxing the removal conditions for the target molecule, or a combination thereof.

[0134] When the target molecule is a solvent, specific examples of solvents include polar solvents such as aqueous solvents like water, as exemplified in the dissolution step of the third embodiment.

[0135] In this case, the expansion in this process includes increasing the solvent content of the fibroin solidified molded product.

[0136] Any method that allows for direct contact between a solid and a liquid can be used to bring the fibroin-coated molded product into contact with a solvent. Specifically, for example, the fibroin-coated molded product can be brought into contact with the solvent by immersing it in the solvent, exposing it to the solvent vapor, spraying, spraying, or coating the fibroin-coated molded product with the solvent, or a combination thereof.

[0137] The method used in this process causes the solvent to come into contact with all or part of the fibroin-coated molded product (including the entire surface).

[0138] The contact time is not particularly limited as long as the solvent can penetrate the fibroin-coated molded product. For example, the contact time can be determined by considering the method used, the type of solvent, etc. For example, the lower limit of the contact time can be 1 second or more, 2 seconds or more, 3 seconds or more, 5 seconds or more, 10 seconds or more, 15 seconds or more, 20 seconds or more, 25 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 15 minutes or more, 30 minutes or more, 45 minutes or more, 60 minutes or more, 90 minutes or more, 2 hours or more, 3 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, or 16 hours or more. The upper limit of the contact time is not particularly limited, but for example, it can be 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, 1 day or less, 20 hours or less, 18 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less.

[0139] The expansion method can be specifically carried out by, for example, immersing the fibroin-coated molded product in a solvent (such as water) for 30 minutes or more, 45 minutes or more, or 60 minutes or more. Alternatively, the expansion method can be carried out by, for example, exposing the fibroin-coated molded product to a vapor atmosphere environment (for example, under the saturated vapor pressure of a solvent (such as water)) for 60 minutes or more, 90 minutes or more, or 2 hours or more.

[0140] The contact temperature is not particularly limited as long as the solvent can penetrate the fibroin solidified molded product. For example, the contact temperature can be determined by considering the method used, the type of solvent, etc. For example, the contact temperature can be room temperature (1°C to 30°C), ambient temperature (15°C to 25°C), or low temperature (30°C to 40°C), and specifically, it can be carried out under the temperature conditions exemplified for the stretching temperature in the fourth embodiment. The stretching method may involve contact at temperatures exceeding these conditions, but in the case of the method in this embodiment, it is not necessary to perform contact at high temperatures.

[0141] The expansion method by relaxing the removal conditions can typically be implemented when the solvent whose content is increased by the expansion method in this step is the same solvent that is removed in the solidification step.

[0142] Specific mitigation methods vary depending on the solidification method used and are not particularly limited, but examples include lowering the processing temperature, shortening the processing period, increasing the solvent concentration during processing, or a combination thereof. The degree to which each condition is changed is not particularly limited, as long as it differs from the conditions normally used.

[0143] Increasing the solvent concentration during processing can be achieved, for example, by increasing the solvent concentration in an atmosphere containing the target solvent when the coagulation process is carried out in that atmosphere. Alternatively, for example, when exposing a coagulation solution to a doping solution, this can be achieved by increasing the solvent concentration in the coagulation solution, i.e., by decreasing the concentration of the coagulation solution.

[0144] The following description uses, but is not limited to, a case where water is the solvent and solidification occurs under a water vapor atmosphere.

[0145] For example, the ambient temperature can be lowered by 1°C, 2°C, 3°C, 4°C, or 5°C or more from the normally used temperature. Furthermore, the ambient temperature can be within ranges such as 0°C to 30°C, 4°C to 30°C, 10°C to 30°C, 0°C to 25°C, 4°C to 25°C, 10°C to 25°C, 0°C to 20°C, 4°C to 20°C, or 10°C to 20°C.

[0146] For example, the processing period can be shortened by 1% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 55% or more compared to the time normally used. Also, if solidification is performed by air drying, the processing period can be set to 1 to 13 days, 2 to 10 days, 3 to 9 days, 4 to 8 days, 4 to 7 days, 3 to 6 days, 5 to 6 days, 6 to 13 days, 6 to 10 days, or 6 to 9 days, etc.

[0147] For example, the relative humidity as the concentration of the aqueous solvent can be increased from the relative humidity normally used. Specifically, for example, the difference in relative humidity can be increased by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 27% or more, 28% or more, 29% or more, or 30% or more. Also, for example, the relative humidity can be 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 85% to 100%, or 90% to 100%, etc. When processing under a vapor atmosphere of a solvent other than aqueous solvent, the relative pressure to the saturated vapor pressure of that solvent can be set as described above.

[0148] More specifically, the expansion method can be carried out, for example, by using relaxed removal (drying) conditions in a water vapor atmosphere at a temperature of 4°C to 30°C (10°C to 25°C, etc.) and a relative humidity of 70% to 100% (85% to 100%, etc.) for a period of 2 to 10 days (4 to 8 days, etc.).

[0149] The extension process can be performed multiple times. In this case, the method and conditions of each process may be the same or may differ in one or more conditions. For example, both relaxation of the removal condition and contact may be performed.

[0150] Specifically, for example, the aforementioned exposure to a vapor atmosphere environment can be carried out in combination with the aforementioned mitigated drying treatment under a water vapor atmosphere.

[0151] 2-3. Effects According to the method of this embodiment, when the fibroin protein used contains one or more α-helix structures in one molecule, the stretchability, such as tensile properties, bending properties, elasticity, flexibility, etc., and especially tensile properties such as tensile elongation at break, can be easily improved. In particular, the improvement of stretchability by the method of this embodiment does not require melting and re-solidification or changes in raw materials, and is achieved simply by applying a simple treatment to the fibroin solidified molded product. Therefore, it is possible to improve stretchability without making major changes to the manufacturing process of existing fibroin recycled materials. The method of this embodiment can be used as a method for improving tensile properties, bending properties, elasticity, flexibility, tensile elongation at break, etc., depending on the purpose.

[0152] 3. Method for Manufacturing a Fibroin Coagulated Molded Article 3-1. Overview The third aspect of the present invention is a method for manufacturing a fibroin coagulated molded article. The method of this aspect includes a coagulation step and an expansion step as essential steps. According to the method of this aspect, the fibroin coagulated molded article described in the first aspect can be manufactured.

[0153] 3-2. Process The method of this embodiment includes a solidification step and a dilation step as essential steps, and includes a fibroin synthesis step, a purification step, a dissolution step, and a pre-molding step as optional steps. Each step will be described below.

[0154] 3-2-1. Fibroin Synthesis Step This step is an optional step of the method of this embodiment and is a step of synthesizing a crude fibroin protein containing a fibroin protein that contains one or more α-helix structures in one molecule.

[0155] In this process, any protein synthesis method known in the art can be used, and the specific method is not particularly limited. For example, crude fibroin protein can be synthesized by silkworms or spiders or other silkworms, or by microorganisms, that are capable of synthesizing fibroin proteins containing one or more α-helix structures in a single molecule, as described in the first embodiment.

[0156] In particular, when using silkworms, the silkworms used may be natural organisms or organisms that have undergone genetic modification or other treatments. When using silkworms, crude fibroin protein may be recovered by collecting the silk spun or secreted from the organism, or it may be recovered directly from the lumen of the silk gland.

[0157] When using microorganisms, genetically modified microorganisms are typically used, and crude fibroin protein is recovered from the culture medium in which the microorganisms grow and / or from within the microbial cells.

[0158] 3-2-2. Purification Step This step is an optional step of the method according to this embodiment, and is a step of purifying the fibroin protein that will be used as a raw material for the coagulated molded product from the synthesized crude fibroin protein. This step can be performed simultaneously with or after the fibroin synthesis step.

[0159] This process can be carried out, for example, when the crude fibroin protein contains a large amount of impurities.

[0160] In this process, any protein purification method known in the art can be used, and the specific method is not particularly limited. If crude fibroin protein is recovered as silk thread in the fibroin synthesis process, it can be purified, for example, by using a method commonly used for scouring silk thread.

[0161] Furthermore, for example, when crude fibroin protein is recovered from the lumen of a silk gland or from microorganisms, chromatography methods such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reversed-phase column chromatography, hydrophobic chromatography, chromatographic focusing, and HPLC, electrophoresis methods such as ammonium sulfate fractionation, ultrafiltration, immunoadsorption, solvent extraction, desalting, precipitation with organic solvents, isoelectric focusing, or combinations thereof can be used.

[0162] 3-2-3. Dissolution Step This step is an optional step of the method according to this embodiment, and is a step of dissolving fibroin protein in a solvent to prepare a dope solution. When the fibroin synthesis step is performed, this step can be performed simultaneously with or after it.

[0163] This process can be carried out, for example, when fibroin proteins insoluble in water are used. Such fibroin proteins also include fibroin coagulated molded products.

[0164] If the crude fibroin protein does not contain many impurities or if fibroin protein of sufficient purity is available, the crude fibroin protein can be used directly in this process. Furthermore, if the impurities in the crude fibroin protein are insoluble in the solvent used in this process, and are removed by this process, the purification process can be performed simultaneously with this process. Alternatively, for example, the purification process may be performed after preparing the dope solution in this process.

[0165] "Doping solution" refers to a solution in which the raw material, fibroin protein, is dissolved. In this specification, the doping solution specifically refers to a solution in which fibroin protein containing one or more α-helix structures in one molecule is dissolved as a raw material, and serves as the raw material for the fibroin coagulated molded article described in the first embodiment.

[0166] The solvent for the doping solution can be any solvent commonly used in the art and is not particularly limited. For example, the solvent can be a polar or nonpolar organic solvent, an aqueous solvent such as water or an aqueous solution, or a combination thereof, preferably a polar solvent.

[0167] Specific examples of polar organic solvents include dimethyl sulfoxide, N-methylmorpholine N-oxide (NMMO), N-methylmorpholine (NMM), 1-butyl-3-methylimidazolium chloride (BMIMCl), 1,5-diazabicyclo[4.3.0]non-5-enium acetate, dioxane, hydrazine, N,N-dimethylformamide, N,N-dimethylacetamide, 1,1,1,3,3,3-hexafluoro-2-propanol, methanol, ethanol, propanol, formic acid, or combinations thereof.

[0168] The doping solution may contain inorganic salts, and the specific type of inorganic salt is not particularly limited. The concentration of inorganic salt in the aqueous solution of inorganic salt is not particularly limited. For example, the aqueous solution of inorganic salt may be a saturated solution or an unsaturated solution.

[0169] Other dissolution conditions such as temperature and time vary depending on the solvent and inorganic salt used, and are not particularly limited. Examples of temperatures include freezing temperatures (below 0°C), refrigeration temperatures (0°C to 10°C), low temperatures (10°C to 20°C), cool places (1°C to 15°C), room temperature (15°C to 25°C), ambient temperature (1°C to 30°C), lukewarm temperatures (30°C to 40°C), 40°C to 100°C, 60°C to 100°C, 80°C to 100°C, 40°C to 80°C, and 60°C to 80°C. Examples of times include 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 50 minutes or more, 60 minutes or more, 2 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 2 days or more, 5 days or more, and 1 week or more.

[0170] 3-2-4. Coagulation Process This process is an essential step of the method according to this embodiment, and involves coagulating the fibroin protein dissolved in the dope solution to prepare a coagulated molded product. If a dissolution process is performed, this process can be performed simultaneously or afterward.

[0171] The coagulation method is not particularly limited, as long as it can make the fibroin protein visible as a solid. For example, any coagulation method known in the art can be used, such as desolvation treatment, exposure to a coagulation solution, or changes in solvent composition (ionic strength, salt concentration, pH, etc.).

[0172] In this specification, "solvent removal treatment" refers to a treatment that removes the solvent (e.g., water) from a dope solution in order to coagulate fibroin protein. The specific solvent removal treatment is not particularly limited, but drying is one example. "Drying" refers to reducing the solvent by evaporation or sublimation.

[0173] The drying method is not particularly limited, as long as it can reduce the amount of solvent in the doping solution. For example, drying methods include natural drying by exposure to the outside air, dehumidification drying by sealing in a sealed container with a desiccant, sun drying, ventilation drying by blowing hot air, heat drying, far-infrared drying, low-temperature drying, moist drying by maintaining humidity, vacuum drying by degassing, freeze-drying, or a combination thereof. All of these techniques are well known and can be carried out according to conventional methods. For example, natural drying and moist drying are preferred drying methods, and drying can also be carried out under wind-blocking conditions as needed.

[0174] The drying conditions vary depending on the method used and are not particularly limited. For example, if the expansion step is performed simultaneously with this step by relaxing the solvent removal conditions, the drying conditions can be those exemplified in the expansion step of the second embodiment.

[0175] On the other hand, if the extension process is performed separately from the main process, the main process can be carried out under the conditions normally used for each method.

[0176] For example, when moisture drying is performed under windproof conditions, the ambient temperature can be the temperature conditions exemplified for the stretching temperature in the fourth embodiment. Also, for example, the relative humidity can be 20% to 100%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 60% to 100%, etc. The processing period is not particularly limited, but for example, it can be 1 to 20 days, 5 to 20 days, 10 to 20 days, 10 to 17 days, 10 to 15 days, 10 to 14 days, 12 to 20 days, 12 to 17 days, 12 to 16 days, 14 to 20 days, etc.

[0177] In this specification, "coagulation solution" refers to a liquid that has the effect of dehydrating and coagulating fibroin proteins dissolved in the dope solution. The coagulation solution is not limited to any liquid that can rapidly dehydrate the dope solution and coagulate the fibroin proteins. Examples of coagulation solutions include lower alcohols (methanol, ethanol, propanol, etc.), acetone, dimethyl sulfoxide, tetrahydrofuran, diethyl ether, acetonitrile, N,N-dimethylformamide, N-methyl-2-pyrrolidone, 1,4-dioxane, or solutions containing these. If the coagulation solution is a solution containing a lower alcohol or acetone, its concentration may be, for example, 10% to 98%, 20% to 97%, 30% to 96%, 40% to 95%, 50% to 90%, 60% to 85%, or 70% to 80%.

[0178] Additional optional processing may be performed during this process. Specific processing is not limited to dilution, mixing, or concentration of the dope solution, or addition or combination thereof.

[0179] As an optional treatment, for example, the doping solution can be adjusted to a predetermined concentration before coagulation. The concentration of the doping solution used for coagulation is not particularly limited, but for example, in terms of mass % of regenerated fibroin protein relative to the mass of the aqueous solution (hereinafter the same applies in this specification), it can be 0.1% to 40%, 0.5% to 40%, 1% to 40%, 1.1% to 40%, 1.2% to 40%, 1.5% to 40%, 3% to 40%, 3.5% to 40%, 4% to 40% Examples include %, 0.1% to 20%, 0.5% to 20%, 1% to 20%, 1.1% to 20%, 1.2% to 20%, 1.5% to 20%, 3% to 20%, 3.5% to 20%, 4% to 20%, 0.5% to 10%, 1% to 10%, 1.1% to 10%, 1.2% to 10%, 1.5% to 10%, 3% to 10%, 3.5% to 10%, 4% to 10%, etc.

[0180] 3-2-5. Pre-molding process The "pre-molding process" is an optional step of the method according to this embodiment, and is a process of molding the fibroin solidified molded product into a desired shape. Typically, this process is performed simultaneously with the solidification process.

[0181] This process can be carried out, for example, when the shape of the fibroin solidified molded product according to this embodiment is fixed.

[0182] The molding method used in this process can be any method known in the manufacturing fields of synthetic resins, chemical fibers, and metal molding, which is used to obtain solids of a desired shape. When obtaining a solidified molded product of a desired shape during solidification from the doping liquid, this process is performed simultaneously with the solidification process. Furthermore, by using, for example, the method exemplified in the molding process of the fourth embodiment, the solidified molded product obtained in the solidification process can be molded without dissolving it.

[0183] The specific molding method varies depending on the desired shape and is not particularly limited, but a method that does not involve stretching is preferably used.

[0184] If the desired shape is fibrous, for example, a fiber molding method can be used as the molding method. Known fiber molding methods include dry fiber molding, wet fiber molding, molten fiber molding, emulsion fiber molding, and gel fiber molding. For example, dry fiber molding or wet fiber molding can be suitably used as the fiber molding method.

[0185] The "dry fiber forming method" is a method of forming fibers by extruding a doping solution into a gas through a nozzle and evaporating the solvent.

[0186] The "wet fiber forming method" is a method of forming fibers by extruding a doping solution into a suitable coagulation solution.

[0187] Methods for forming the film include, for example, solution casting, wet film formation, compression molding, and extrusion molding, as well as methods used for coating.

[0188] The "solution casting method" is a method of forming a film by pouring a doping solution onto a smooth surface, allowing it to adhere to the surface, and then evaporating the solvent by heating or other means. The smooth surface can be the surface of a petri dish, a drum (casting drum), or a smooth stainless steel belt. Furthermore, a mold can be placed on the smooth surface to achieve the desired shape if necessary.

[0189] "Wet film formation" is a method of forming a film by extruding a doping solution into a suitable solidifying solution.

[0190] "Compression molding" is a molding method in which a dope liquid is poured into a female mold and then compressed and solidified in a male mold.

[0191] The coating method typically involves bringing a coating liquid into contact with the surface of an object. A mold can be used as the object and a doping solution as the coating liquid in this process. Methods for bringing the coating liquid into contact with the surface include, for example, nozzle flow, spray, dip, roll, and spin methods.

[0192] In addition to the various methods described above, injection molding and other methods can be used to form the material into a plate shape.

[0193] "Injection molding" is a molding method in which a dope liquid is poured into a mold and allowed to solidify.

[0194] As for the molding method, for example, molding may be performed by fabrication using a 3D printer or the like. In this case, the fabrication method is not particularly limited, but fused deposition modeling (FDM) or the like can be used. Furthermore, in addition to these methods, the various molding methods described in the molding process of the fourth embodiment can also be used.

[0195] 3-2-6. Expansion Step This step is an essential step of the method of this embodiment, and is a step of expanding the α-helix crystal structure of the fibroin solidified molded product to an expanded α-helix crystal structure, thereby providing the fibroin solidified molded product described in the first embodiment. This step can be performed simultaneously with or after the solidification step. Furthermore, if a pre-molding step is performed, this step can be performed simultaneously with or after it. The specific details of this step are the same as those described for the expansion step in the second embodiment.

[0196] 4. Method for Manufacturing Fibroin-Isolated Molded Articles 4-1. Overview The fourth aspect of the present invention is a method for manufacturing fibroin-immobilized molded articles. The method of this aspect includes a stretching step and a molding step as essential steps. According to the method of this aspect, immobilized molded articles can be manufactured by stretching under relaxed conditions, particularly at a high stretching ratio under relatively low temperature conditions.

[0197] 4-2. Process The method of this embodiment includes a stretching process and a molding process as essential processes, and includes a preparation process and a determination process as optional processes. Each process will be described below.

[0198] 4-2-1. Preparation Process The "preparation process" is an optional process and is a process for preparing the fibroin solidified molded product described in the first embodiment.

[0199] In this process, the method described in the third embodiment is carried out. This process includes a solidification step and an expansion step as essential steps, and includes a fibroin synthesis step, a purification step, a dissolution step, and a pre-shaping step as optional steps. The solidification step is carried out in accordance with the description of the solidification process, the fibroin synthesis step in accordance with the fibroin synthesis process, the purification step in accordance with the purification process, and the dissolution step in accordance with the description of the dissolution process in the third embodiment. Furthermore, the expansion step is carried out in accordance with the description of the expansion process in the second embodiment.

[0200] 4-2-2. Step for Determining Stretchability The "step for determining stretchability" is an optional step and is a step for determining whether the fibroin solidified molded product can be stretched by cold stretching or hot stretching. If the preparation step is performed, this step can be performed simultaneously with or after the preparation step.

[0201] In this process, the method described in the fifth embodiment is carried out. This process includes a structural measurement step, an analysis step, and a determination step as essential steps, and the structural measurement step is in accordance with the description of the structural measurement process, the analysis step is in accordance with the analysis process, and the determination step is in accordance with the description of the determination process in the fifth embodiment.

[0202] 4-2-3. Stretching Process The "stretching process" is an essential process and involves stretching the fibroin solidified molded product described in the first embodiment. This process can be performed after the preparation process if one is performing it. It can also be performed after the pre-molding process if one is performing it, and can be performed simultaneously with or after the structural measurement process if one is performing it.

[0203] Any stretching method known in the art can be used for this process. The stretching in this process may be performed by wet stretching or dry stretching.

[0204] Furthermore, conditions such as temperature and stretching speed are not particularly limited. For example, in the case of wet heat stretching, it is typically carried out at an average temperature of about 50 to 90°C in liquid or vapor water, organic solvent, or a combination thereof. In the case of dry heat stretching, it is typically carried out at an average temperature of about 140 to 270°C using an electric tubular furnace, dry heat plate, etc.

[0205] The fibroin solidified molded product used in this embodiment exhibits significantly improved tensile properties at relatively low temperatures. Therefore, this process can be performed not only by the hot stretching described above, but also by cold stretching or warm stretching.

[0206] In this specification, "heat stretching" refers to a stretching method in which heat treatment is applied throughout the stretching process. In contrast, "cold stretching" and "warm stretching" refer to stretching methods in which no heat treatment is applied during stretching, or in which heat treatment is limited to a short period of time. Specifically, "cold stretching" refers to stretching performed at an average temperature of less than 30°C. "Warm stretching" refers to stretching performed at an average temperature of 30 to 50°C. The average temperature is the weighted average of the ambient temperatures through which the yarn passes during stretching, weighted by the time spent in each environment.

[0207] The average temperature when performing stretching by cold stretching or hot stretching is not particularly limited, but for example, it can be done at temperatures of 0°C or higher, 4°C or higher, 5°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 27°C or higher, or 30°C or higher. Alternatively, for example, it can be done at temperatures of 50°C or lower, 45°C or lower, 40°C or lower, 35°C or lower, 30°C or lower, 27°C or lower, 25°C or lower, 20°C or lower, 15°C or lower, 10°C or lower, or 5°C or lower. Specific average temperature ranges include, for example, 0-50°C, 4-45°C, 0-20°C, 4-20°C, 10-40°C, 15-40°C, 20-35°C, 25-35°C, etc.

[0208] "Cold stretching" and "hot stretching" may include a short period of heating during stretching. The heating time in this case is not particularly limited. Specifically, the heating time can be, for example, 0.1 seconds to 30 minutes, 1 second to 20 minutes, 2 seconds to 10 minutes, 5 seconds to 5 minutes, 10 seconds to 2 minutes, 15 seconds to 1 minute, 20 seconds to 50 seconds, 30 seconds to 40 seconds, 0.1 seconds to 30 seconds, 0.1 seconds to 20 seconds, 0.1 seconds to 15 seconds, 0.1 seconds to 10 seconds, 0.1 seconds to 5 seconds, 0.1 seconds to 2 seconds, 0.1 seconds to 1 second, etc.

[0209] The stretching ratio can be appropriately selected depending on the purpose and is not particularly limited. Since the fibroin solidified molded product described in the first embodiment has excellent tensile properties, stretching can be performed at the same stretching ratio as for hot stretching, even in the cold stretching and / or hot stretching described above.

[0210] The extension may be carried out in multiple steps, or along multiple axes. In this case, the conditions for each extension may be the same, or they may differ for one or more extensions.

[0211] 4-2-4. Molding Process The "molding process" is an essential process in which a fibroin solidified molded product or a fibroin immobilized molded product is molded into a desired shape to produce a fibroin immobilized molded product. This process can be performed simultaneously with or after the stretching process.

[0212] The method used in this process is widely known in fields such as the manufacturing of synthetic resins and chemical fibers, and can be appropriately selected according to the type of insolubilized molded product to be manufactured. The type of insolubilized molded product is not particularly limited, but examples include the shape exemplified for the fibroin solidified molded product in the first embodiment.

[0213] If the desired shape is one obtained as a result of stretching a fibroin solidified molded product, this process is performed simultaneously with the stretching process through a stretching treatment.

[0214] When this process is performed separately from the stretching process, it is preferable that the molding is carried out without dissolving the fibroin solidified molded product.

[0215] Any molding method known in the fields of resin processing or metal processing can be used. Examples of molding methods include, but are not limited to, vacuum forming, bonding / welding, lamination, cutting, punching, bending, or combinations thereof.

[0216] "Vacuum forming" is a method of forming a softened sheet-like solidified product into the shape of the mold by bringing it into contact with a mold and then reducing the pressure in the gap between the mold and the solidified product.

[0217] "Adhesion and welding" refers to a method of forming by joining multiple solidified molded products together using an adhesive or by softening the bonding area.

[0218] "Laminate molding" is a method of forming by stacking solidified molded products in sheet-like or other shapes and then softening them by applying pressure, heating, etc.

[0219] "Cutting" refers to a method of shaping solidified molded products by cutting and removing material. This includes methods such as water jet machining.

[0220] "Die-cutting" is a method of shaping by pressing a die against a solidified molded product.

[0221] "Bending" is a molding process in which a solidified molded product is softened by applying pressure or heating, then bent, and finally solidified.

[0222] 5. Method for Determining Stretchability 5-1. Overview The fifth aspect of the present invention is a method for determining stretchability. The method of this aspect includes a structural measurement step, an analysis step, and a determination step as essential steps. According to the method of this aspect, it is possible to determine whether the fibroin solidified molded product in question can be stretched by cold stretching or hot stretching.

[0223] 5-2. Process 5-2-1. Structural Measurement Process The "structural measurement process" is a process for measuring the crystal structure of the fibroin solidified molded product.

[0224] The method used in this process can be any known method available for analyzing the crystalline structure of resins, etc. Examples of methods that can be used in this process include X-ray diffraction, electron diffraction, nuclear magnetic resonance analysis, and Fourier transform IR spectroscopy. Specifically, examples of methods that can be used in this process include wide-angle X-ray diffraction and small-angle X-ray scattering, with wide-angle X-ray diffraction being particularly suitable.

[0225] The measurement conditions are not particularly limited and can be selected as appropriate depending on the method used, as long as they allow for the measurement of the lattice plane spacing of the α-helix crystal structure. For example, when performing X-ray diffraction measurements, the wavelength of the irradiated X-rays is not particularly limited. For example, X-ray diffraction measurements can use X-rays with wavelengths such as 0.1 Å to 10 Å, 0.1 Å to 5 Å, 0.1 Å to 2 Å, 0.1 Å to 1.6 Å, 0.5 Å to 1.6 Å, 0.6 Å to 1.6 Å, 0.8 Å to 1.6 Å, 1.0 Å to 1.6 Å, 1.3 Å to 1.6 Å, and 1.4 Å to 1.6 Å.

[0226] If necessary, the fibroin-coated molded product can be subjected to any desired processing. Examples include shredding into measurable sizes or preparing thin film samples.

[0227] 5-2-2. Analysis Process The "analysis process" is a process of analyzing the size of the lattice plane spacing d between (10-10) planes in the unit hexagonal lattice of the α-helix crystal structure. This process can be performed simultaneously with or after the structural measurement process.

[0228] The analysis performed in this process is not particularly limited, as long as it can quantitatively identify the magnitude of the lattice plane spacing d between (10-10) planes.

[0229] For example, the lattice plane spacing can be calculated from the peak reading (2θ) based on Bragg's formula. The peak of the (10-10) plane (d) in a unit hexagonal lattice can be determined by, for example, using CuKα rays (wavelength λ (Å): 1.5418 Å), where the peak of the (10-10) plane (d=7.5 Å) in a standard unit hexagonal lattice is observed around 2θ=11.8°. Furthermore, if measurements are taken using any wavelength other than CuKα rays (e.g., molybdenum source X-rays, tungsten source X-rays, etc.), the scattering vector q(Å) does not depend on the wavelength λ (Å) of the X-rays used. -1 It is preferable to evaluate using ). Here, q is calculated as 4πsinθ / λ. In this case, the peak of the (10-10) plane is q = 0.84 Å. -1 It is observed in the vicinity.

[0230] 5-2-3. Determination Step The "determination step" is a step in which the fibroin solidified molded product is determined to be stretchable by cold stretching or hot stretching if the α-helix crystal structure is composed of a unit hexagonal lattice and the lattice plane spacing d between the (10-10) planes is greater than the lattice plane spacing d2 between the (10-10) planes in the standard unit hexagonal lattice of the standard α-helix crystal structure. This step can be performed simultaneously with or after the analysis step.

[0231] The criteria for judgment in this case are not particularly limited, but for example, if the analysis result falls within the range of the difference in grid plane spacing or the value of d1 exemplified in the first embodiment, it can be determined that stretching at a relatively low temperature (cold stretching or hot stretching) is possible, and if it does not fall within that range, it can be determined that stretching at a relatively low temperature is not possible. The temperature conditions for stretching at a relatively low temperature in this case are in accordance with the description in the fourth embodiment.

[0232] <Example 1. Preparation of solidified molded product and tensile test> (Objective) To investigate the relationship between tensile properties and microstructure of fibroin solidified molded products.

[0233] (Methods) 1. Preparation of fibroin protein aqueous solution First, an aqueous solution of fibroin protein derived from bagworm silk was prepared. As the bagworm silk, silk spun by bagworms (Larva of the giant bagworm moth: final instar larvae collected in Tsukuba City, Ibaraki Prefecture) was used. To remove components other than fibroin protein, the silk was treated with an aqueous sodium carbonate solution and then scouring. After dissolving the scouring silk in an aqueous lithium thiocyanate solution, it was placed in a cellulose dialysis tube and desalted by dialysis with ultrapure water to obtain a regenerated aqueous fibroin protein solution.

[0234] The concentration of the prepared fibroin protein aqueous solution was calculated to be 1.2%, using the dry weight as the solute weight. The fibroin protein aqueous solution was further concentrated by air-drying in a cellulose dialysis tube to prepare a 4.0% concentrate.

[0235] 2. Preparation of the cast film: 0.75 mL of the concentrated solution was poured into a petri dish and allowed to air dry to obtain a 25 mm x 12 mm cast film.

[0236] Air drying was carried out under wind-free conditions using a constant temperature and humidity chamber set to 20°C and 90% relative humidity. Films air-dried for 6 days were classified as hydrated films, and those air-dried for 14 days were classified as dry films.

[0237] 3. Tensile Test A 20mm x 2mm test piece was cut from each prepared film, and the film thickness, width, and grip distance of the test piece were measured. A Digimicro MF-501 (Nikon) was used to measure the film thickness. The tensile test was performed using a desktop precision universal testing machine AUTOGRAPH AGS-X 5kN (Shimadzu Corporation) and its attached 50N load cell at a tensile speed of 1.0mm / min. Measurements and tests were performed at room temperature (approximately 23-25°C).

[0238] Stress was calculated by dividing the force value obtained from the tensile test by the cross-sectional area of ​​the sample before tensioning (product of film thickness and width). Strain was calculated by dividing the displacement obtained from the tensile test by the working length (distance between grips) before tensioning.

[0239] 4. Wide-angle X-ray diffraction measurement: 5 mm × 1 mm test pieces were further cut from each prepared film and subjected to wide-angle X-ray diffraction measurement. A wide-angle X-ray diffractometer T-WAXS (Rigaku) ​​was used for the measurement, and measurements were performed using CuKα rays (40 kV, 30 mA; wavelength 1.5418 Å) with a camera length of 50 mm, and the measurement results were obtained as a two-dimensional pattern. A hybrid multi-dimensional pixel detector HyPix-6000 (Rigaku) ​​was used as the detector, and silicon (Si) 111 reflection was used for camera length calibration.

[0240] A 2θ profile was extracted from the obtained 2D pattern, and the 2θ value was obtained for each observed peak. The lattice plane spacing was calculated from the obtained 2θ value based on Bragg's equation below. λ was set to 1.5418 Å. 2d sinθ = λ (where d: lattice plane spacing, λ: X-ray wavelength)

[0241] In particular, for the diffraction peaks corresponding to the lattice plane spacing of the (10-10) plane in the hexagonal close-packed structure of the α-helix, we used the peak value observed around 2θ, which corresponds to d=7.5Å.

[0242] (Results) The results are shown in Figures 2 to 4. Figure 2 shows the results of tensile tests at room temperature for the hydrated film and the dry film. The dry film reached a yield point of approximately 64.8 MPa at a strain of approximately 2.7%, and then fractured at a strain of approximately 11% (solid line in Figure 2). On the other hand, the hydrated film reached a yield point of approximately 56.3 MPa at a strain of approximately 4.4%, and then stretched without fracture up to a strain of over 230% (dashed line in Figure 2). The tensile elongation at break of the dry film was 11%, while the tensile elongation at break of the hydrated film was an extremely large value of 234%.

[0243] This indicates that the hydration film dramatically improves the tensile properties, changing them to a slightly softer and extremely tougher material, while also increasing the area under the stress-strain curve and resulting in extremely high impact strength.

[0244] Figures 3 and 4 show the X-ray diffraction spectra of the hydrated and dried films. Both films showed substantially similar spectra, indicating the presence of α-helix and β-sheet structures (Figure 3). In particular, for the β-sheet structure, no significant differences were observed between the hydrated and dried films in any of the (200) / (210) plane (β200 / 210 in Figure 3: 4.3 Å), (211) plane (β211 in Figure 3: 3.6 Å), (202) plane (β202 in Figure 3: 2.8 Å), (103) plane (β103 in Figure 3: 2.2 Å), and (213) plane (β213 in Figure 3: 2.1 Å).

[0245] On the other hand, for the α-helix structure, a significant difference was observed in the lattice plane spacing of the (10-10) plane (α10-10 in Figure 3 and Figure 4), with the spacing being 0.4 Å larger in the hydrated film compared to the dry film.

[0246] The change in the elongation properties at break of both films was suggested to be due to a change in the spacing of the lattice planes in this α-helix structure.

[0247] <Example 2. Relationship between water content and structure> (Objective) To investigate the relationship between water content and lattice plane spacing in fibroin solidified molded products.

[0248] (Method) 1. Film Preparation Films were prepared from the concentrated solution prepared in the same manner as in Example 1. The air-drying conditions were the same as in Example 1, except that the dry film was treated in a constant temperature and humidity chamber at 25°C and 35% relative humidity for 7 days, and the hydrated film was treated at 20°C and 90% relative humidity for 7 days.

[0249] The hydrated film was further subjected to a high-humidity treatment, in which it was exposed to saturated water vapor at room temperature (approximately 23°C) for 2 hours.

[0250] 2. For each wide-angle X-ray diffraction film, the lattice plane spacing of the (10-10) plane was measured in the same manner as in Example 1.

[0251] 3. Thermogravimetric Analysis and Evaluation of Moisture Content Approximately 5 mg of test material was cut from each prepared film and subjected to thermogravimetric analysis. A Thermo plus EV02 TG-DTA / S thermogravimetric analyzer (Rigaku) ​​was used for the measurement, and the temperature was raised from room temperature (approximately 25°C) to 250°C at a heating rate of 5°C / min, and the mass loss that occurred during this process was measured. To prevent oxidation of the sample during measurement, all measurements were performed under nitrogen gas inflow.

[0252] It has been reported that all mass loss due to heating up to 200°C is attributable to water loss (Yazawa K. et al., Biomacromolcules 2016, 17, 1057-1066). Therefore, the initial mass at room temperature is W RT W is the mass after heating up to 200℃. 200 The initial moisture content (%) of each film was calculated using the following formula: Initial moisture content (%) = 100 × (W RT -W 200 ) / W RT

[0253] The moisture content of each film was determined as the average of the measurement results of three samples cut from different locations on the same film.

[0254] (Results) The results are shown in Figure 5. Figure 5 shows an example of the measurement results for the water content of dry film and hydrated film.

[0255] The lattice plane spacings between the (10-10) planes of the dry film and the hydrated film were 7.5 Å and 7.9 Å, respectively, as in Example 1.

[0256] As shown in Figure 5, the hydrated film experienced greater mass loss upon heating up to 200°C compared to the dry film. The average moisture content of the dry film was 7.6%, while the average moisture content of the hydrated film was 11.7%.

[0257] In the semi-hydrated film that was not subjected to high-humidity treatment, the average moisture content was 8.5%, and the lattice plane spacing between (10-10) planes was 7.7 Å.

[0258] From the above, it was suggested that the structural changes observed in Example 1 were due to differences in water content.

[0259] While preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Additions, omissions, substitutions, and other modifications are possible without departing from the spirit of the invention. The present invention is not limited by the foregoing description, but only by the scope of the appended claims.

Claims

1. A fibroin coagulated molded article comprising a fibroin protein containing one or more α-helix structures in one molecule, wherein the α-helix structures form an extended α-helix crystal structure, the extended α-helix crystal structure is composed of an extended hexagonal unit lattice formed by a portion of the fibroin protein, and the lattice plane spacing d1 between (10-10) faces in the extended hexagonal unit lattice is greater than the lattice plane spacing d2 between (10-10) faces in the standard hexagonal unit lattice of a standard α-helix crystal structure.

2. The fibroin solidified molded article according to claim 1, wherein the difference between d1 and d2 is 0.1 Å or more and 0.5 Å or less.

3. The fibroin solidified molded article according to claim 1 or 2, wherein d1 is 7.6 Å or more and 8.5 Å or less.

4. A fibroin solidified molded article according to any one of claims 1 to 3, wherein the water content is 7.7% or more and 50% or less.

5. The fibroin coagulated molded article according to any one of claims 1 to 4, wherein the fibroin protein further comprises one or more β-sheet structures in one molecule.

6. The fibroin coagulated molded article according to any one of claims 1 to 5, wherein the fibroin protein is derived from bagworm silk.

7. A method for producing a fibroin-insolubilized molded article, comprising: a stretching step of stretching a fibroin-solidified molded article according to any one of claims 1 to 6; and a molding step of molding the fibroin-solidified molded article into a desired shape to produce a fibroin-insolubilized molded article.

8. The manufacturing method according to claim 7, wherein the stretching is cold stretching or hot stretching.

9. A fibroin-insolubilized molded article produced by the manufacturing method described in claim 7 or 8.

Images