WO2020067546A1 - Composite yarn, method for producing same, and fabric - Google Patents

Composite yarn, method for producing same, and fabric Download PDF

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Publication number
WO2020067546A1
WO2020067546A1 PCT/JP2019/038427 JP2019038427W WO2020067546A1 WO 2020067546 A1 WO2020067546 A1 WO 2020067546A1 JP 2019038427 W JP2019038427 W JP 2019038427W WO 2020067546 A1 WO2020067546 A1 WO 2020067546A1
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amino acid
seq
fibroin
fiber
artificial
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PCT/JP2019/038427
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French (fr)
Japanese (ja)
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明彦 尾関
佑之介 安部
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Ｓｐｉｂｅｒ株式会社
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Publication of WO2020067546A1 publication Critical patent/WO2020067546A1/en

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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02G—CRIMPING OR CURLING FIBRES, FILAMENTS, THREADS, OR YARNS; YARNS OR THREADS
    • D02G3/00—Yarns or threads, e.g. fancy yarns; Processes or apparatus for the production thereof, not otherwise provided for
    • D02G3/02—Yarns or threads characterised by the material or by the materials from which they are made
    • D02G3/04—Blended or other yarns or threads containing components made from different materials
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
    • D06M11/01—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with hydrogen, water or heavy water; with hydrides of metals or complexes thereof; with boranes, diboranes, silanes, disilanes, phosphines, diphosphines, stibines, distibines, arsines, or diarsines or complexes thereof
    • D06M11/05—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with hydrogen, water or heavy water; with hydrides of metals or complexes thereof; with boranes, diboranes, silanes, disilanes, phosphines, diphosphines, stibines, distibines, arsines, or diarsines or complexes thereof with water, e.g. steam; with heavy water

Definitions

  • the present invention relates to a composite yarn, a method for producing the same, and a fabric.
  • Non-Patent Document 1 Since protein fibers have advantages such as biodegradability and low energy for production and processing, demand for various fields is expected to increase in accordance with the growing awareness of environmental conservation in recent years. I have. Animal fiber such as wool among protein fibers has properties such as warm in winter, cool in summer, high moisture absorption and desorption, and elasticity. (For example, Non-Patent Document 1).
  • animal hair fibers are less flammable than cellulose fibers such as cotton, they have the disadvantage that they are more flammable than synthetic fibers such as polyester fibers.
  • synthetic fibers such as polyester fibers.
  • the present inventors have found that the modified fibroin has excellent combustion resistance.
  • the present invention is based on this new finding.
  • the present invention relates to, for example, the following inventions.
  • [1] Contains artificial fibroin fiber and animal hair fiber, The composite yarn, wherein the artificial fibroin fiber contains a modified fibroin and has a contraction history of being irreversibly contracted by contact with water after spinning.
  • [2] The composite yarn according to [1], wherein the artificial fibroin fiber has a wet shrinkage defined by the following formula of 2% or more.
  • wet shrinkage ⁇ 1 ⁇ (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fibril after spinning and before contact with water) ⁇ ⁇ 100 (%) [3]
  • the composite yarn according to [1] or [2], wherein the artificial fibroin fiber has a dry shrinkage defined by the following formula of more than 7%. Drying shrinkage ⁇ 1 ⁇ (length of dried artificial fibroin fiber / length of artificial fibroin raw fiber after spinning and before contact with water) ⁇ ⁇ 100 (%) [4]
  • the composite yarn and the fabric according to the present invention contain the modified fibroin fiber, they are also excellent in moisture absorption and heat generation.
  • the composite yarn according to the present embodiment contains artificial fibroin fiber and animal hair fiber.
  • the composite yarns include, for example, blended yarns, blended yarns, blended yarns, interwoven yarns, plied yarns, covering yarns, and the like.
  • the artificial fibroin fiber according to the present embodiment may contain modified fibroin and have a shrinkage history irreversibly shrunk by contact with water after spinning.
  • the modified fibroin has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Including proteins.
  • the modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence.
  • the N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
  • modified fibroin means artificially produced fibroin (artificial fibroin).
  • the modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin.
  • naturally-derived fibroin as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Is a protein containing the domain sequence to be determined.
  • Modified fibroin may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin).
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have a desired amino acid sequence by chemical synthesis).
  • domain sequence refers to a crystalline region unique to fibroin (typically, corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence).
  • the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues.
  • the number of amino acid residues in the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 .
  • the ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues).
  • At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • a plurality of REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin according to the present embodiment has, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence.
  • an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence can be obtained by performing the following modification.
  • Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods ⁇ in ⁇ Enzymology, 100, 448 (1983).
  • Naturally occurring fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Yes, specifically, for example, fibroin produced by insects or spiders.
  • fibroin produced by insects examples include Bombyx @ mori, Bombyx @ mandarina, natural silkworm (Antheraea @ yamamai), tussah (Anterea @ pernii), and maple silkworm (Erioganyerii). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms such as moga silkworms (Antheraea assama), and silkworms produced by larvae of the hornet beetle Hornet silk protein.
  • fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • Examples of the fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus), such as Orion spider, Elder spider, Red-colored Spider, Blue-colored Spider, etc. Spiders belonging to the genus Argiope genus (Genus Pronus), such as spiders belonging to the genus Procarpus spp., Spiders belonging to the genus Pronus, and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi.
  • Genus Araneus such as Orion spider, Elder spider, Red-colored Spider, Blue-colored Spider, etc.
  • Spiders belonging to the genus Argiope genus such as spiders belonging to the genus Procarpus spp.
  • Spiders belonging to the genus Pronus and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such
  • Spiders belonging to the genus Ordgarius such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Orbalis, and spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Ordgarius.
  • Spiders belonging to the genus Argiope such as Argiope bruennichi, spiders belonging to the genus Argiope sp.
  • Spiders belonging to the genus Spider such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys), spiders belonging to the genus Spiders (genus Cyclosa) such as the spiders belonging to the genus Cyclosa and spiders belonging to the genus Cygnus spp.
  • Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asina such as red-headed spiders, red-backed spiders, blue-backed spiders and urocore-red spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucaegium, such as the spiders Argiope bruennichi and spiders spiders belonging to the genus Leucauge, such as the spiders belonging to the genus Nephila sp.
  • Spiders belonging to the genus Dyschiriognatha such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Latus and the spiders belonging to the genus Lastroconidae belonging to the genus Latus sp.
  • Spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops (Euprosthenops) are produced.
  • Spider silk protein examples include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ph ), U37520 (base sequence)), major ⁇ ampullate ⁇ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number
  • CAJ00428 amino acid sequence
  • AJ97155 base sequence
  • major ⁇ sample ⁇ spidroin ⁇ 2 [Euprosus] ] GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)
  • minor ⁇ silk ⁇ protein ⁇ 1 [Nephila ⁇ clavipes] GenBank Accession No. AAC14589.1 (amino acid sequence)
  • minor ⁇ ampilatte [in] Nephila clavipes] GenBank Accession No. AAC14591.1 (amino acid sequence)
  • minor ampoulate spidroin-like protein [Nephilengys Cruenata] GenBank Accession No. ABR3727.1.
  • fibroin in which sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidroin, ampullate, fibroin, "silk and polypeptide", or “silk and protein” is described as a keyword in DEFINITION from among sequences including INV as DIVISION in the sequence information registered in NCBI @ GenBank. It can be confirmed by extracting a character string of a specific product from a sequence or CDS, and extracting a sequence in which a specific character string is described in TISSUE @ TYPE from SOURCE.
  • the modified fibroin according to this embodiment may be a modified silk (silk) fibroin (an amino acid sequence of a silk protein produced by a silkworm modified), and a modified spider silk fibroin (a spider silk protein produced by an arachnid). Modified amino acid sequence).
  • a modified spider silk fibroin is preferable.
  • the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues.
  • first modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • domain sequence having a reduced content of glycine residues derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • second modified fibroin derived from a large spinal cord marker protein produced in a spider's large ampullate gland
  • a domain sequence having a reduced content of glycine residues derived from a large spinal cord marker protein produced in a spider's large ampul
  • the first modified fibroin includes a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the number of amino acid residues of the (A) n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and an integer of 10 to 20 Is still more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable.
  • the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and more preferably 20 to 100 residues.
  • the first modified fibroin has a total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m
  • the total number is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more.
  • the first modified fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m , and has a C-terminal sequence represented by any of SEQ ID NOS: 1 to 3 or
  • the polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.
  • the amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is
  • the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • the first modified fibroin (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant ⁇ spider ⁇ silk ⁇ protein ⁇ ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% sequence identity.
  • the sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human ⁇ rhinovirus @ 3C protease) recognition site at the N-terminus is added.
  • the 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue.
  • the amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
  • the modified fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.
  • the second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin.
  • the second modified fibroin can be said to have an amino acid sequence corresponding to at least one or more glycine residues in the REP replaced by another amino acid residue, as compared to a naturally occurring fibroin. .
  • the second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been replaced with another amino acid residue. You may.
  • the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
  • the second modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the (A) n motif located at the most C-terminal side to the domain sequence
  • the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • the second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less.
  • the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, and 70% or more. Is still more preferred, and even more preferably 80% or more.
  • the upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
  • the second modified fibroin is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue.
  • a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more.
  • the amino acid sequence can be obtained by designing an amino acid sequence satisfying the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to insertion, addition, and / or addition may be modified.
  • the other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
  • the second modified fibroin examples include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX.
  • the amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted.
  • the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some amino acids on the C-terminal side have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7.
  • the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is repeated four times with a predetermined hinge sequence and His tag sequence added to the C-terminal.
  • the value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%.
  • the values of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%.
  • the value of x / y at the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
  • the modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and contains XGX ( Where X represents an amino acid residue other than glycine.)
  • z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
  • the second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
  • the tag sequence examples include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule.
  • affinity tag is a histidine tag (His tag).
  • His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography).
  • SEQ ID NO: 11 amino acid sequence including a His tag sequence and a hinge sequence.
  • tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • an “epitope tag” utilizing an antigen-antibody reaction can be used.
  • a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
  • the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag.
  • a tag sequence that can be cleaved by a specific protease can be used.
  • protease treatment By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.
  • modified fibroin containing a tag sequence (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799)
  • Modified fibroin comprising a sequence or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
  • amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
  • An amino acid sequence represented by SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) is added to the N-terminal of the amino acid sequence shown.
  • the modified fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX (where X represents an amino acid residue other than glycine.)
  • z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
  • the second modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of the (A) n motif as compared to a naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to naturally occurring fibroin.
  • the third modified fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif from naturally occurring fibroin.
  • the third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) n motif every one (A) n motif May have an amino acid sequence corresponding to the deletion of
  • the third modified fibroin has a domain sequence deletion of at least two (A) n motifs from the N-terminal side to the C-terminal side, and one (A) It may have an amino acid sequence corresponding to the deletion of the n motif repeated in this order.
  • the third modified fibroin may have a domain sequence having an amino acid sequence corresponding to the deletion of the (A) n motif at least every third sequence from the N-terminal side to the C-terminal side. .
  • the third modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side to the C-terminal side] -REP]
  • the number of amino acid residues of REP in the unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units that is 11.3 is x, and the total number of amino acid residues in the domain sequence is y In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more.
  • the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called an n motif.
  • FIG. 1 shows pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP and the second REP, and Pattern 4 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (the comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP with the fifth REP). Second REP comparison). Note that there are other selection methods.
  • the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared.
  • each pattern the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
  • x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
  • x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%.
  • the upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y.
  • / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
  • x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is.
  • the upper limit of x / y is not particularly limited, and may be 100% or less.
  • x / y in naturally occurring fibroin will be described.
  • 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted.
  • x / y was calculated from the amino acid sequence of the naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above calculation method.
  • FIG. 3 shows the results when the indentation ratio is 1: 1.9 to 4.1.
  • the horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency.
  • the x / y of the naturally derived fibroin is less than 64.2% (the highest is 64.14%).
  • the third modified fibroin deletes one or more of the sequence encoding the (A) n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • amino acid residues are further substituted, deleted, inserted and / or added.
  • Amino acid sequence modification corresponding to the above may be performed.
  • the third modified fibroin (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT525) -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.
  • the modified fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) n The motif was deleted, and one [(A) n motif-REP] was inserted before the C-terminal sequence.
  • the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.
  • the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8-11.3.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%.
  • the values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
  • the modified fibroin of (3-i) may be composed of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
  • the modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other Amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (a giza ratio of 1: 1.8 to 11.3).
  • x / y be 64.2% or more, where x is the maximum value of the sum of the base numbers and y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799)
  • Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
  • amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. (Including a His tag sequence and a hinge sequence).
  • the modified fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is N-terminal to C-terminal.
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other
  • the maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 is defined as x.
  • x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of a glycine residue in addition to the content of the (A) n motif reduced as compared to a naturally-derived fibroin.
  • the domain sequence of the fourth modified fibroin is at least one or more (A) n motifs deleted, and further at least one or more glycine residues in the REP, as compared to naturally occurring fibroin. It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects and the like are as described for the second modified fibroin and the third modified fibroin.
  • the fourth modified fibroin (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15
  • a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.
  • Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 are as described above.
  • the fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index therein.
  • a region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
  • the amino acid residue having a large hydrophobicity index is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). More preferably, it is a residue.
  • the fifth modified fibroin may have one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index, and / or one or more amino acids in the REP as compared to a naturally occurring fibroin.
  • one or more amino acid residues may be substituted, deleted, inserted and / or added as compared with naturally occurring fibroin.
  • the fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP.
  • a group for example, an amino acid residue having a positive hydrophobicity index
  • one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP.
  • one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP.
  • the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
  • the fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p,
  • q the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q
  • p / q is 6 .2% or more.
  • hydrophobicity index of amino acid residues
  • a publicly known index Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132).
  • HI hydropathic index
  • sequence A [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”).
  • sequence A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues).
  • the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
  • the fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions.
  • a hydrophobic amino acid residue eg, an amino acid residue having a positive hydrophobicity index
  • inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index.
  • an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin.
  • a modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed. .
  • the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
  • the fifth modified fibroin (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT665), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); Or (5-ii) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the modified fibroin of (5-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410).
  • two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted.
  • the amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there.
  • the amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.
  • the modified fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and is located at the most C-terminal side (A) n
  • amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
  • the fifth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
  • modified fibroin containing a tag sequence (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
  • the modified fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) n
  • all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more
  • P / q is preferably at least 6.2%.
  • the fifth modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.
  • the sixth modified fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
  • the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and preferably 10% or more.
  • the upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
  • the “GPGXX motif content” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence
  • the number obtained by multiplying the total number by 3 ie, the total number of G and P in the GPGXX motif
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented (A)
  • “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) Sequence from n motif to C-terminus of domain sequence (sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated.
  • “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
  • FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin.
  • the method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) n motif-REP] m- (A) n motif” type), all REPs are located at the most C-terminal side.
  • (A) A sequence obtained by removing the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence ”(the sequence shown as“ region A ”in FIG. 5).
  • the sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%. .
  • the “glutamine residue content” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence.
  • the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n
  • the glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif.
  • the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
  • the sixth modified fibroin corresponds to the fact that its domain sequence has one or more glutamine residues in the REP deleted or replaced with other amino acid residues, as compared to the naturally occurring fibroin. It may have an amino acid sequence.
  • the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue.
  • the hydrophobicity index of amino acid residues is as shown in Table 1.
  • amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M )
  • Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can.
  • an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable.
  • the sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. Is still more preferable, and it is particularly preferable that it is 0.4 or more.
  • the upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
  • “REP hydrophobicity” is a value calculated by the following method.
  • Formula 1 Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence.
  • the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence.
  • A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding n motifs.
  • the degree of hydrophobicity of the REP the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
  • the sixth modified fibroin may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin.
  • the sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or remove one or more glutamine residues in the REP. By substituting the amino acid residue with, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • SEQ ID NO: 25 (Met-PRT988), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT889) -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence No.
  • modified fibroin comprising the amino acid sequence represented by (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31
  • An amino acid represented by SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It can be mentioned modified fibroin comprising an amino acid sequence having a sequence at least 90% sequence identity.
  • the modified fibroin of (6-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting VL for all QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410).
  • the amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with TS, and substituting the remaining Q with A.
  • the amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VL, and substituting the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VI and substituting the remaining Q with L.
  • the amino acid sequence represented by SEQ ID NO: 29 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VF and substituting the remaining Q with I.
  • amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL.
  • the amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 32 replaces all QQs in the sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VF, In addition, the remaining Q is replaced with I.
  • the amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI and replacing the remaining Q with V.
  • the amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with IF, and substituting the remaining Q with T.
  • amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 all have glutamine residues.
  • the group content is 9% or less (Table 2).
  • the modified fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It may consist of the amino acid sequence shown.
  • the modified fibroin of (6-ii) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown.
  • the modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
  • modified fibroin containing the tag sequence (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 44 (PRT1028), or ( 6-iv) The amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 and 90 % Modified amino acid sequence having an amino acid sequence having at least Mention may be
  • amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 correspond to SEQ ID NO: 25, respectively.
  • SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin of (6-iii) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.
  • the modified fibroin of (6-iv) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown.
  • the modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
  • the sequence of the secretion signal can be appropriately set according to the type of the host.
  • the modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. Modified fibroin having both of the following characteristics may be used.
  • the modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin.
  • hydrophobic modified fibroin refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. Modified fibroin having an (average HI) greater than 0. The hydrophobicity index is as shown in Table 1. Further, “hydrophilic modified fibroin” is a modified fibroin having an average HI of 0 or less. As the modified fibroin, a hydrophilic modified fibroin is preferred from the viewpoint of excellent combustion resistance, and a hydrophobic modified fibroin is preferred from the viewpoint of excellent moisture absorption and heat generation.
  • hydrophobically modified fibroin examples include the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, SEQ ID NO: 35, Modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.
  • hydrophilic modified fibroin examples include the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: Or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 Modified fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the artificial fibroin fiber according to the present embodiment may contain one kind of modified fibroin alone or may contain two or more kinds of modified fibroin in combination.
  • the modified fibroin may be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
  • the method for producing the nucleic acid encoding the modified fibroin is not particularly limited.
  • the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural fibroin and modifying it by a genetic engineering technique, or a chemical synthesis method. can do.
  • the method for chemically synthesizing nucleic acids is not particularly limited.
  • AKTA oligopilot plus10010 / 100 Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like.
  • a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence comprising an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. May be.
  • the regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host.
  • An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
  • the type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like.
  • a plasmid vector a virus vector
  • a cosmid vector a fosmid vector
  • an artificial chromosome vector an artificial chromosome vector
  • those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are suitably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • microorganisms belonging to the genus Escherichia include, for example, Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
  • Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like.
  • microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis.
  • Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum.
  • Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum.
  • Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold).
  • yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like.
  • filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051).
  • any method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. ⁇ Natl. ⁇ Acad. ⁇ Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
  • a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
  • the modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium.
  • the method of culturing the host in the culture medium can be performed according to a method usually used for culturing the host.
  • the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
  • a prokaryote such as Escherichia coli or a eukaryote such as yeast
  • a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
  • the carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate
  • potassium (I) phosphate potassium (II) phosphate
  • magnesium phosphate magnesium phosphate
  • magnesium sulfate sodium chloride
  • ferrous sulfate manganese sulfate
  • copper sulfate copper sulfate
  • calcium carbonate calcium carbonate
  • ⁇ Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culturing time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside or the like is used.
  • An acid or the like may be added to the medium.
  • the expressed and modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed with a Gaulin homogenizer, Dynomill or the like to obtain a cell-free extract.
  • a method commonly used for isolating and purifying proteins that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei)
  • cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
  • Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.
  • the modified fibroin When the modified fibroin is expressed by forming an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to collect the insoluble form of the modified fibroin as a precipitate fraction.
  • the recovered insoluble form of the modified fibroin can be solubilized with a protein denaturant.
  • a purified sample of the modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the artificial fibroin fibril according to the present embodiment is obtained by spinning the above-mentioned modified fibroin, and contains the above-mentioned modified fibroin as a main component.
  • the artificial fibroin fiber according to the present embodiment is a fiber after spinning and before contact with water.
  • the artificial fibroin fibrils according to the present embodiment can be manufactured by a known spinning method. That is, for example, first, modified fibroin produced according to the above-described method is dissolved in a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), or hexafluoroisopronol (HFIP) as a dissolution promoter. And dissolve it to prepare a dope solution. Next, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain a desired artificial fibroin fibril. Preferred spinning methods include wet spinning and dry-wet spinning.
  • DMSO dimethyl sulfoxide
  • DMF N, N-dimethylformamide
  • HFIP hexafluoroisopronol
  • Preferred spinning methods include wet spinning and dry-wet spinning.
  • FIG. 6 is an explanatory view schematically showing an example of a spinning apparatus for producing artificial fibroin fibrils.
  • the spinning device 10 shown in FIG. 6 is an example of a spinning device for dry-wet spinning, and includes an extruder 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and a drying device 4.
  • a spinning method using the spinning device 10 will be described.
  • the dope solution 6 stored in the storage tank 7 is pushed out of the base 9 by the gear pump 8.
  • a dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope solution 6 is supplied into the coagulation solution 11 of the coagulation solution tank 20 via the air gap 19, the solvent is removed, and the modified fibroin is coagulated to form a fibrous coagulate.
  • the fibrous coagulated material is supplied into the hot water 12 in the stretching bath 21 and stretched.
  • the stretching ratio is determined by the speed ratio between the supply nip roller 13 and the take-off nip roller 14.
  • the drawn fibrous coagulated material is supplied to the drying device 4 and dried in the yarn path 22, and the artificial fibroin raw fiber is obtained as the wound yarn 5.
  • 18a to 18g are yarn guides.
  • the coagulating liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone.
  • the coagulating liquid 11 may appropriately contain water.
  • the temperature of the coagulating liquid 11 is preferably 0 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour. More preferably, it is time.
  • the distance that the coagulated protein passes through the coagulating liquid 11 may be long enough to efficiently remove the solvent, for example, 200 to 500 mm. It is.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and is preferably 1 to 3 m / min.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulation liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as needed.
  • the stretching performed when obtaining the artificial fibroin fibrils includes, for example, dry stretching in addition to wet stretching performed in the coagulation bath 20 and stretching bath 21 described above.
  • Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or steam heating.
  • the temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C.
  • the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
  • Dry heat drawing can be performed using an electric tube furnace, a dry heat plate or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., preferably 160 ° C. to 230 ° C.
  • the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • the wet heat stretching and the dry heat stretching may each be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and further the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be performed in an appropriate combination.
  • the final draw ratio is preferably such that the lower limit is more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, and 6 times of the undrawn yarn (or pre-drawn yarn). Or more, 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, 10 times or less.
  • the artificial fibroin fiber is a fiber spun at a draw ratio of 2 or more, the shrinkage when the artificial fibroin fiber is brought into a wet state by contact with water becomes higher.
  • the artificial fibroin fiber according to the present embodiment can be obtained by a manufacturing method including a shrinking step of shrinking the artificial fibroin raw fiber with water.
  • the shrinking step may include, for example, a step (contact step) of bringing the aforementioned artificial fibroin fibrils (fibers after spinning and before contact with water) into contact with water to shrink irreversibly.
  • the shrinking step may include, after the contacting step, a step of drying and further shrinking the fiber (drying step).
  • FIG. 7 is a diagram showing an example of length changes of artificial fibroin fibrils and artificial fibroin fibers due to contact with water.
  • the artificial fibroin fibrils according to the present embodiment have the property of contracting irreversibly (contact length change in FIG. 7) by contact (wetting) with water. After the primary shrinkage, it is further shrunk when dried (the length change indicated by “secondary shrinkage” in FIG. 7).
  • the artificial fibroin fiber obtained through the primary shrinkage or the secondary shrinkage is brought into contact with water and brought into a wet state, it expands to the same length as that before the secondary shrinkage or to a length similar to that before the secondary shrinkage.
  • the contraction and the elongation are repeated with a width approximately equal to the secondary contraction (the width indicated by “stretch ratio” in FIG. 7).
  • Irreversible shrinkage of the artificial fibroin fibrils in the contact step (“primary shrinkage” in FIG. 7) is considered to occur, for example, for the following reasons. That is, one reason is considered to be due to the secondary structure or tertiary structure of artificial fibroin fibrils, and another reason is, for example, in artificial fibroin fibrils having residual stress due to stretching in the manufacturing process. It is considered that water is infiltrated into the fibers or into the fibers, so that the residual stress is reduced. Therefore, it is considered that the shrinkage rate of the artificial fibroin fibrils in the shrinking step can be arbitrarily controlled in accordance with, for example, the magnitude of the draw ratio in the process of producing the artificial fibroin fibrils described above.
  • the artificial fibroin fibrils before spinning are brought into contact with water after spinning to bring the artificial fibroin fibrils into a wet state.
  • the wet state means a state where at least a part of the artificial fibroin fibrils is wet with water. Thereby, the artificial fibroin fibrils can be shrunk without external force. This contraction is irreversible (corresponding to "primary contraction" in FIG. 7).
  • the temperature of the water contacted with the artificial fibroin fibrils in the contacting step may be lower than the boiling point. Thereby, the handleability, the workability in the shrinking step, etc. are improved.
  • the lower limit of the water temperature is preferably 10 ° C. or higher, more preferably 40 ° C. or higher, and further preferably 70 ° C. or higher.
  • the upper limit of the water temperature is preferably 90 ° C. or less.
  • a method of bringing water into contact with artificial fibroin fibrils is not particularly limited.
  • the method for example, a method of immersing artificial fibroin fibrils in water, a method of spraying water on artificial fibroin fibrils at room temperature or in a state of heated steam, and a method of filling artificial fibroin fibrils with water vapor Exposure to a high humidity environment.
  • the method of immersing the artificial fibroin fibrils in water is preferable because the contraction time can be effectively shortened and the processing equipment can be simplified.
  • the artificial fibroin fibrils when the artificial fibroin fibrils are brought into contact with water in a relaxed state, the artificial fibroin fibrils may not only shrink but also shrink in a wavy manner.
  • the artificial fibroin fibrous fibers are brought into contact with water while being tensioned (pulled) in the fiber axis direction, and the contact step is performed in a state where the artificial fibroin fibrils are not relaxed. Is also good.
  • the method for producing artificial fibroin fibers according to the present embodiment may further include a drying step.
  • the drying step is a step of drying and further shrinking the artificial fibroin fibrils that have passed through the contacting step (or the artificial fibroin fibers obtained through the contacting step) (corresponding to “secondary shrinkage” in FIG. 7). Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known contact-type or non-contact-type drying equipment can be used. Also, the drying temperature is not limited at all, for example, as long as the protein contained in the artificial fibroin fibrils is decomposed or a temperature lower than the temperature at which the artificial fibroin fibrils are thermally damaged.
  • the temperature is in the range of 20 to 150 ° C, and preferably in the range of 50 to 100 ° C. Temperatures in this range allow the fibers to dry more quickly and efficiently without thermal damage to the fibers or degradation of the proteins contained in the fibers.
  • the drying time is appropriately set according to the drying temperature and the like, and, for example, a time that can minimize the influence of the overdrying on the quality and physical properties of the artificial fibroin fiber and the like is adopted.
  • FIG. 8 is an explanatory view schematically showing an example of a production apparatus for producing artificial fibroin fibers.
  • the manufacturing apparatus 40 shown in FIG. 8 includes a feed roller 42 for feeding out artificial fibroin fibers, a winder 44 for winding the artificial fibroin fibers 38, a water bath 46 for performing a contacting step, and a dryer 48 for performing a drying step. , Is configured.
  • the feed roller 42 is configured to be capable of mounting a wound of the artificial fibroin fibril 36, and is rotated from an wound of the artificial fibroin fibril 36 by rotation of an electric motor (not shown). 36 can be continuously and automatically sent out. After being fed from the feed roller 42, the winder 44 can continuously and automatically wind the artificial fibroin fiber 38 manufactured through the contacting step and the drying step by rotation of an electric motor (not shown). .
  • the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.
  • the water bath 46 and the dryer 48 are arranged between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feed direction of the artificial fibroin fibril 36, respectively.
  • the manufacturing apparatus 40 shown in FIG. 8 has relay rollers 50 and 52 for relaying the artificial fibroin raw fibers 36 before and after the contact step running from the feed roller 42 toward the winder 44.
  • the water bath 46 has a heater 54, and the water 47 heated by the heater 54 is stored in the water bath 46.
  • a tension roller 56 is installed in a state of being immersed in water 47.
  • the artificial fibroin fibrils 36 sent out from the feed rollers 42 run toward the winder 44 while being immersed in the water 47 while being wound around the tension rollers 56 in the water bath 46. It has become.
  • the immersion time of the artificial fibroin fibrils 36 in the water 47 is appropriately controlled according to the running speed of the artificial fibroin fibrils 36.
  • the dryer 48 has a pair of hot rollers 58.
  • the pair of hot rollers 58 can be wound around the artificial fibroin fibrils 36 that are separated from the water bath 46 and run toward the winder 44.
  • the artificial fibroin fibrils 36 immersed in water 47 in the water bath 46 are heated by the pair of hot rollers 58 in the dryer 48, dried, and further sent out to the winder 44. It has become.
  • the artificial fibroin fibrils 36 are continuously sent out from the feed roller 42 and immersed in water 47 in a water bath 46.
  • the winding speed of the winder 44 is set lower than the feeding speed of the feed roller 42. Accordingly, the artificial fibroin fibrous material 36 contracts by contact with the water 47 in a state where the artificial fibroin fibrous material 36 is tensioned so as not to relax between the feed roller 42 and the winder 44, so that the occurrence of shrinkage can be prevented.
  • the artificial fibroin fibrils 36 contract irreversibly (corresponding to "primary contraction" in FIG. 7).
  • the artificial fibroin fibrils 36 after contact with the water 47 are heated by the pair of hot rollers 58 of the dryer 48.
  • the artificial fibroin fibrils 36 after contact with the water 47 or the artificial fibroin fibers 38 produced through the contact with the water 47
  • can be dried and further contracted (“secondary contraction” in FIG. 7). ").
  • the artificial fibroin fiber 38 can be further contracted and its length can be kept unchanged.
  • the obtained artificial fibroin fiber 38 is wound by a winder 44 to obtain a roll of the artificial fibroin fiber 38.
  • the artificial fibroin fibrils 36 after being brought into contact with the water 47 by using a drying equipment such as a dry heat plate 64 as shown in FIG. It may be dried. Also in this case, by adjusting the relative speed between the feed speed of the feed roller 42 and the winding speed of the winder 44 in the same manner as in the case where a pair of hot rollers 58 is used as the drying equipment, the artificial fibroin fiber is adjusted. 38 can be further contracted or the length can be unchanged.
  • the drying means is constituted by the dry heat plate 64. Further, the dryer 48 is not essential.
  • the target artificial fibroin fiber 38 can be manufactured automatically, continuously, and extremely easily.
  • FIG. 9 is an explanatory view schematically showing another example of a production apparatus for producing artificial fibroin fibers.
  • FIG. 9A shows a processing apparatus provided in the manufacturing apparatus for performing the contacting step
  • FIG. 9B shows a drying apparatus provided in the manufacturing apparatus for performing the drying step.
  • the manufacturing apparatus shown in FIG. 9 includes a processing apparatus 60 that performs a contacting step on the artificial fibroin fibril 36 and a drying step on the artificial fibroin fibril 36 (or the artificial fibroin fiber 38 manufactured through the contacting step) after the contacting step. And a drying device 62 for making them independent from each other.
  • the processing apparatus 60 shown in FIG. 9A omits the dryer 48 from the manufacturing apparatus 40 shown in FIG. 8, and connects the feed roller 42, the water bath 46, and the winder 44 It has a structure in which the fibroin fibrils 36 are arranged side by side in order from upstream to downstream in the running direction.
  • the artificial fibroin fibrils 36 sent out from the feed roller 42 are immersed in water 47 in a water bath 46 to be contracted. Then, the obtained artificial fibroin fiber 38 is wound up by a winder 44.
  • the drying device 62 shown in FIG. 9B has the feed roller 42 and the winder 44, and a dry heat plate 64.
  • the dry heat plate 64 is disposed between the feed roller 42 and the winder 44 such that the dry heat surface 66 contacts the artificial fibroin fiber 38 and extends in the running direction.
  • the drying device 62 as described above, for example, by controlling the ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 38 can be further contracted, It can be the same.
  • the artificial fibroin fiber 38 can be dried by the drying device 62 after the artificial fibroin fiber 36 is shrunk by the processing device 60 to obtain the artificial fibroin fiber 38. .
  • the processing device may be constituted only by the water bath 46 by omitting the feed roller 42 and the winder 44 from the processing device 60 shown in FIG.
  • a manufacturing apparatus having such a processing apparatus for example, artificial fibroin fibers are manufactured by a so-called batch method.
  • the drying device 62 shown in FIG. 9B is not essential.
  • the artificial fibroin fiber according to the present embodiment may be crimped by contact with water.
  • the degree of crimping is not limited, and the number of crimps may be, for example, 10 to 100 pieces / 40 mm or 20 to 40 pieces / 40 mm.
  • the artificial fibroin fiber crimped by contact with water can be obtained, for example, by contacting the artificial fibroin fibril with an aqueous medium.
  • the aqueous medium is a liquid or gas (steam) medium containing water (including water vapor).
  • the aqueous medium may be water, or a mixture of water and a hydrophilic solvent.
  • the hydrophilic solvent for example, a volatile solvent such as ethanol and methanol or a vapor thereof can be used.
  • the aqueous medium is preferably a mixed solution of water and ethanol.
  • the artificial fibroin fiber dries quickly and has a softer finish.
  • the ratio of water to the volatile solvent or its vapor is not particularly limited.
  • the mass ratio of water: volatile solvent or its vapor may be 10:90 to 90:10.
  • a known oil agent such as an oil for passage through a process (for example, for antistatic) or a finish may be dispersed in the aqueous medium. That is, instead of the aqueous medium, an oil dispersion containing an aqueous medium and an oil dispersed in the aqueous medium can be used. By using such an oil dispersion, the artificial fibroin fibrils can be crimped, and the oil agent can be adhered to the artificial fibroin fibers obtained by crimping the artificial fibroin fibrils. By attaching the oil agent to the artificial fibroin fiber, various properties can be imparted to the artificial fibroin fiber.
  • the amount of the oil agent is not particularly limited, and may be, for example, 1 to 10% by mass or 2 to 5% by mass based on the total amount of the aqueous medium and the oil agent.
  • the temperature of the aqueous medium may be 10 ° C or higher, 25 ° C or higher, 40 ° C or higher, 60 ° C or higher, or 100 ° C or higher, and may be 230 ° C or lower, 120 ° C or lower, or 100 ° C or lower. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230 ° C, more preferably 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, heat denaturation of the artificial fibroin fiber can be prevented. When the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10 ° C.
  • the time for contacting the artificial fibroin fibrils with the aqueous medium is not particularly limited, but may be 30 seconds or more, 1 minute or more, or 2 minutes or more, and is preferably 10 minutes or less from the viewpoint of productivity.
  • the contact of the aqueous medium with the artificial fibroin fibrils may be performed under normal pressure or may be performed under reduced pressure (for example, vacuum).
  • a method of contacting the artificial fibroin fibrils with an aqueous medium a method of immersing the artificial fibroin fibrils in water, a method of spraying the artificial fibroin fibrils with an aqueous medium steam, an environment filled with the aqueous medium steam.
  • Examples include a method of exposing artificial fibroin fibrils.
  • the contact of the aqueous medium with the artificial fibroin fibrils can be performed using a general steam setting device.
  • the steam set device include devices such as a product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and a product name: EPS-400 (manufactured by Tsujii Dyeing Machinery Co., Ltd.).
  • the method of crimping the artificial fibroin fibrils by the steam of the aqueous medium the artificial fibroin fibrils are accommodated in a predetermined accommodation room, while the steam of the aqueous medium is introduced into the accommodation room, and the temperature in the accommodation room is increased. Is adjusted to the above-mentioned predetermined temperature (for example, 100 ° C. to 230 ° C.), and steam is brought into contact with the artificial fibroin fibrils.
  • the step of crimping the artificial fibroin fibrils by contact with the aqueous medium is preferably performed in a state where no tensile force is applied to the artificial fibroin fibrils (no tension is applied in the fiber axis direction), or a predetermined size. (Strained by a predetermined amount in the fiber axis direction). At that time, the degree of crimp can be controlled by adjusting the tensile force applied to the artificial fibroin fibrils.
  • a method of adjusting the tensile force applied to the artificial fibroin fibrils for example, by suspending various weights on the artificial fibroin fibrils
  • a method of adjusting the load applied to the artificial fibroin fibrils A method in which both ends are fixed in a state where the artificial fibroin fibrils are slackened, and the amount of slack is variously changed, and the artificial fibroin fibrils are wound around a wound body such as a paper tube or a bobbin, and the winding force at that time (Tightening force on paper tube or bobbin) may be appropriately changed.
  • the artificial fibroin fibrils may be dried.
  • the drying method is not particularly limited, and may be natural drying, or the artificial fibroin fibrils may be forcibly dried using a drying facility.
  • the crimping with the aqueous medium and the subsequent drying can be performed continuously.
  • the artificial fibroin fibrils can be dried by being immersed in an aqueous medium while being sent from a bobbin, and then blown with hot air or sent over a hot roller.
  • the drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C., preferably 40 to 120 ° C., and more preferably 60 to 100 ° C.
  • the process of shrinking artificial fibroin fibrils with water which is performed to obtain artificial fibroin fibers, includes processes other than simply reducing the length of artificial fibroin fibrils (not shrinking). And crimping the artificial fibroin fibrils (to shorten the length by crimping).
  • the shrinking step contact step, drying step
  • shrinking reducing the length
  • the artificial fibroin fibrils with water and the step of crimping can be performed simultaneously or stepwise.
  • the artificial fibroin fiber according to the present embodiment may have a wet shrinkage defined by the following formula of 2% or more.
  • Wet shrinkage ⁇ 1 ⁇ (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fibril after spinning and before contact with water) ⁇ ⁇ 100 (%)
  • the artificial fibroin fiber according to this embodiment has a wet shrinkage of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, and 5.5% or more. Or 6% or more.
  • the upper limit of the wet shrinkage is not particularly limited, but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3%. It may be:
  • the artificial fibroin fiber according to the present embodiment has a drying shrinkage of 8% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 37% or more, 38% or more. Or 39% or more.
  • the upper limit of the drying shrinkage is not particularly limited, but may be 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less.
  • the artificial fibroin fiber according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, or 24 or more, It may be 26 or more, 28 or more, 29 or more, or 30 or more.
  • LOI limiting oxygen index
  • the LOI value is a value measured in accordance with the Fire and Disaster Management Agency, Dangerous Goods Regulations Section, #Fire and Fire Criterion No. 50, May 31, 1995, based on the test method for synthetic resin having a low granularity or a low melting point.
  • the artificial fibroin fiber according to this embodiment may have a maximum heat of moisture absorption determined by the following formula A of more than 0.025 ° C./g.
  • Maximum heat of moisture absorption ⁇ (Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium, and then moved to high humidity environment) ⁇ (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment) ⁇ (°C) / sample weight (g)
  • the low humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 40%
  • the high humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 90%.
  • the artificial fibroin fiber according to the present embodiment may have a maximum heat of moisture absorption by absorption of not less than 0.026 ° C / g, not less than 0.027 ° C / g, and not less than 0.028 ° C / g. May be 0.029 ° C./g or more, may be 0.030 ° C./g or more, may be 0.035 ° C./g or more, and may be 0.040 ° C./g or more. It may be.
  • the upper limit of the maximum heat of moisture absorption is not particularly limited, but is usually 0.060 ° C./g or less.
  • the artificial fibroin fiber according to the present embodiment may have a heat retention index determined according to the following formula B of more than 0.18.
  • Insulation index Insulation rate (%) / Sample weight (g / m 2 )
  • the heat retention rate (%) means a heat retention rate measured by a dry contact method (temperature: 30 ° C., wind speed: 30 cm / sec), and is calculated by (1 ⁇ a / b) ⁇ 100.
  • a shows the amount of heat dissipated through the test piece
  • b shows the amount of heat dissipated without passing through the test piece.
  • the heat retention can be measured by, for example, a Thermolab II tester or the like.
  • the artificial fibroin fiber according to the present embodiment may have a heat retention index of 0.20 or more, may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, It may be 0.28 or more, may be 0.30 or more, and may be 0.32 or more.
  • the upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.
  • Animal hair fiber in the present specification means animal hair including wool (wool), and fiber derived therefrom.
  • animal hair fibers for example, wool (wool), cashmere (cashmere goat hair), mohair (angora goat hair), angora (angora rabbit hair), alpaca (camelaceae Vicu ⁇ a alpaca hair), vicu ⁇ a ( Camel (Vicuna vicuna hair), Camel (Camelidae camel hair), Lilla (Camelid llama llama hair), and fibers derived therefrom.
  • the composite yarn (blended yarn, mixed fiber yarn, mixed woven yarn, mixed woven yarn, plied yarn, covering yarn, and the like) according to the present embodiment is prepared according to a conventional method except that the above-described artificial fibroin fiber and animal hair fiber are used. Can be manufactured.
  • the composite yarn according to the present embodiment may further contain other fibers other than the artificial fibroin fiber and the animal hair fiber.
  • Other fibers include, for example, synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, hemp, silk, wool, and cashmere. These other fibers are preferably used as long as the effects of the present invention are not impaired.
  • the content ratio of the artificial fibroin fiber and the animal hair fiber in the composite yarn according to the present embodiment is based on the total amount of the artificial fibroin fiber and the animal hair fiber, and the content of the artificial fibroin fiber may be 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more And may be 80% by mass or more.
  • the upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the animal hair fiber. , 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.
  • the content of the artificial fibroin fiber and the animal hair fiber in the composite yarn according to the present embodiment is, based on the total amount of the composite yarn, the total content of the artificial fibroin fiber and the animal hair fiber may be 50% by mass or more, It may be 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more.
  • the upper limit of the total content of the artificial fibroin fiber and the animal hair fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the composite yarn.
  • the fabric according to the present embodiment contains the above-described artificial fibroin fiber and animal hair fiber.
  • the fabric includes woven fabric, knitted fabric (knitted fabric), lace, felt, nonwoven fabric, and the like.
  • the fabric according to the present embodiment may further contain fibers other than the above-described artificial fibroin fiber and animal hair fiber.
  • Other fibers include, for example, synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, hemp, silk, wool, and cashmere. These other fibers are preferably used as long as the effects of the present invention are not impaired.
  • the fabric according to the present embodiment may contain artificial fibroin fiber and animal hair fiber, for example, by being manufactured by mixing the above-mentioned artificial fibroin fiber and animal hair fiber.
  • the fabric according to the present embodiment also contains, for example, a yarn formed from the above-described artificial fibroin fiber, and a yarn formed from animal hair fiber, which is mixed to produce the artificial fibroin fiber and the animal hair fiber. It may be a thing.
  • the fabric according to the present embodiment may also include, for example, an artificial fibroin fiber and an animal hair fiber by being manufactured using the composite yarn according to the present invention.
  • the yarn formed from the above-mentioned artificial fibroin fiber and the yarn formed from animal hair fiber may be a single yarn or a composite yarn with other fibers. Further, in any of the production methods, other fibers and / or yarns formed from other fibers may be used in combination, if necessary.
  • the fabric (woven fabric, knitted fabric, lace, felt, non-woven fabric, etc.) according to the present embodiment can be manufactured according to a conventional method except that the above-described fibers and / or yarns are used.
  • the content ratio of the artificial fibroin fiber and the animal hair fiber in the fabric according to the present embodiment may be such that the content of the artificial fibroin fiber is 10% by mass or more, based on the total amount of the artificial fibroin fiber and the animal hair fiber. At least 30% by mass, at least 40% by mass, at least 50% by mass, at least 60% by mass, at least 70% by mass. And may be 80% by mass or more.
  • the upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the animal hair fiber. , 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.
  • the content of the artificial fibroin fiber and the animal hair fiber in the fabric according to the present embodiment may be such that the total content of the artificial fibroin fiber and the animal hair fiber is 50% by mass or more, and % Or more, 70% or more, 80% or more, and 90% or more.
  • the upper limit of the total content of the artificial fibroin fiber and the animal hair fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the fabric.
  • the cloth according to the present embodiment can be used as a material for clothing, medical supplies, sanitary goods, interior goods, bedding, decorations, bags, accessories, miscellaneous goods, vehicle parts, composite goods with resin and the like, for example. it can.
  • Nucleic acids encoding the designed five types of modified fibroins were respectively synthesized.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • These five types of nucleic acids were respectively cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with the obtained expression vector.
  • the transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005.
  • the temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
  • the seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 5 below) had been added so that the OD 600 was 0.05.
  • the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
  • a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the desired modified fibroin. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band having a size corresponding to the desired modified fibroin depending on the addition of IPTG revealed the presence of the modified fibroin of interest. Expression was confirmed.
  • the precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation. Water was removed from the collected aggregated proteins using a freeze dryer to obtain a freeze-dried powder of modified fibroin.
  • the resulting modified fibroin solution was used as a dope solution (spinning stock solution), and spin-dried and stretched artificial fibroin fibers were manufactured by dry-wet spinning using a spinning device according to the spinning device 10 shown in FIG.
  • the spinning apparatus used in the spinning apparatus 10 shown in FIG. 6 is a second undrawn yarn manufacturing apparatus (between the undrawn yarn manufacturing apparatus 2 (first bath) and the wet heat drawing apparatus 3 (third bath)). (Second bath).
  • the conditions for dry-wet spinning are as follows. Extrusion nozzle diameter: 0.2mm Liquid and temperature in first to third baths: see Table 6 Total draw ratio: see Table 6 Drying temperature: 60 ° C
  • the artificial fibroin raw fibers obtained in Production Examples 1 to 19 are subjected to a contact step of bringing them into contact with water (hereinafter, may be referred to as “primary shrinkage”).
  • a drying step of drying at room temperature is performed (hereinafter, may be referred to as “secondary shrinkage”) to produce artificial fibroin fibers.
  • the length measurement of the artificial fibroin fiber bundle in water was performed with 0.8 g of lead weight attached to the artificial fibroin fiber bundle in order to eliminate the shrinkage of the artificial fibroin fiber bundle.
  • the primary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula I.
  • L0 indicates the length of the artificial fibroin fibril bundle (here, 30 cm) after spinning and before contact with water
  • Lw indicates the length of the artificial fibroin fiber bundle that has undergone primary shrinkage.
  • the primary shrinkage is synonymous with the wet shrinkage.
  • Primary shrinkage ⁇ 1 ⁇ (Lw / L0) ⁇ ⁇ 100 (%)
  • the prepared spinning stock solution was filtered at 60 ° C. with a metal filter having an aperture of 5 ⁇ m, and then allowed to stand in a 30 mL stainless syringe to remove bubbles, and then coagulated with 100% by mass methanol from a solid nozzle having a needle diameter of 0.2 mm. Discharged into the bath. The discharge temperature was 60 ° C. After coagulation, the obtained fibrils were wound and air-dried to obtain artificial fibroin fibrils.
  • the obtained artificial fibroin fiber was subjected to a contact step of bringing it into contact with water, and then subjected to a drying step of drying at room temperature, thereby producing an artificial fibroin fiber (raw fiber).
  • a knitted fabric was manufactured by circular knitting using a circular knitting machine using the obtained raw material fibers.
  • the knitted fabric had a thickness of 180 denier and 18 gauges. 20 g was cut out from the obtained knitted fabric to obtain a test piece.
  • the flammability test was conducted in accordance with the Fire and Disaster Management Agency, Fire and Disaster Criminal Control Division No. 50, May 31, 1995, test method for synthetic resin having a granular or low melting point. The test was performed under the conditions of a temperature of 22 ° C., a relative humidity of 45%, and an air pressure of 1021 hPa. Table 11 shows the measurement results (oxygen concentration (%), burning rate (%), reduced burning rate (%)).
  • the knitted fabric made of artificial fibroin fibers containing the modified fibroin had a limiting oxygen index (LOI) value of 27.2.
  • LOI limiting oxygen index
  • the LOI value is 26 or more, it is considered to be flame retardant. From this result, it can be seen that the artificial fibroin fiber has excellent flame retardancy. Therefore, a composite yarn or fabric excellent in flame retardancy can be obtained by using a composite yarn or fabric in which artificial fibroin fibers and animal hair fibers are mixed.
  • the prepared spinning dope was filtered at 60 ° C. through a 5 ⁇ m-aperture metal filter, then left standing in a 30 mL stainless syringe and defoamed.
  • the mass% methanol was discharged into a coagulation bath.
  • the discharge temperature was 60 ° C., and the discharge pressure was 0.3 MPa.
  • the obtained yarn was wound at a winding speed of 3.00 m / min and air-dried to obtain artificial fibroin fibrils.
  • the obtained artificial fibroin fiber was subjected to a contact step of bringing it into contact with water, and then subjected to a drying step of drying at room temperature, thereby producing an artificial fibroin fiber (raw fiber).
  • a knitted fabric was manufactured by flat knitting using a flat knitting machine.
  • the knitted fabric using PRT918 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 18.
  • the knitted fabric using PRT799 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 16.
  • the thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so as to have almost the same cover factor as that of the knitted fabric using PRT918 fiber and PRT799 fiber. Specifically, it is as shown below.
  • test piece Two pieces of the knitted fabric cut to 10 cm ⁇ 10 cm were aligned, and four sides were sewn to obtain a test piece (sample). After leaving the test specimen in a low humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 40 ⁇ 5%) for 4 hours or more, it was moved to a high humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 90 ⁇ 5%) The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.
  • FIG. 10 is a graph showing an example of the result of the moisture absorption / heating test.
  • the horizontal axis of the graph represents the time (minute) of leaving the sample in the high humidity environment as 0 when the time when the sample was transferred from the low humidity environment to the high humidity environment.
  • the vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor.
  • the point indicated by M corresponds to the maximum value of the sample temperature.
  • Table 12 shows the calculation result of the maximum heat of moisture absorption.
  • the artificial fibroin fiber containing the modified fibroin has a higher maximum heat of moisture absorption and is superior in heat absorption and heat generation as compared with existing materials. Therefore, by mixing artificial fibroin fibers and animal hair fibers to form a composite yarn or fabric, a composite yarn or fabric excellent in heat absorption and heat generation can be obtained.
  • the prepared spinning stock solution was filtered at 60 ° C. with a metal filter having an aperture of 5 ⁇ m, and then allowed to stand in a 30 mL stainless syringe to remove bubbles, and then coagulated with 100% by mass methanol from a solid nozzle having a needle diameter of 0.2 mm. Discharged into the bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain an artificial fibroin fiber (raw material fiber).
  • a knitted fabric was manufactured by flat knitting using a flat knitting machine.
  • the knitted fabric using PRT966 fiber as a raw material fiber had a count of 30 Nm, the number of twists: 1, the number of gauges: 18 GG, and the basis weight: 90.1 g / m 2 .
  • the knitted fabric using PRT799 fiber as the raw material fiber had a count of 30 Nm, a number of twists of 1, a gauge number of GG: 16, and a basis weight of 111.0 g / m 2 .
  • the thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so as to have almost the same cover factor as that of the knitted fabric using PRT966 fiber and PRT799 fiber.
  • Insulation index Insulation rate (%) / Sample weight (g / m 2 )
  • Table 13 shows the calculation results of the heat retention index. The higher the heat retention index, the more the material can be evaluated as having excellent heat retention.
  • the artificial fibroin fiber containing the modified fibroin has a higher heat retention index and is superior in heat retention as compared with existing materials. Therefore, by mixing artificial fibroin fiber and animal hair fiber to form a composite yarn or fabric, a composite yarn or fabric excellent in heat retention can be obtained.

Abstract

The present invention pertains to a composite yarn and a fabric, the yarn containing an artificial fibroin fiber and an animal hair fiber, wherein the artificial fibroin fiber includes a modified fibroin, and has a contraction history of having been irreversibly contracted due to contact with water after being spun.

Description

複合糸、及びその製造方法、並びに布地Composite yarn, method for producing the same, and fabric
　本発明は、複合糸、及びその製造方法、並びに布地に関する。 The present invention relates to a composite yarn, a method for producing the same, and a fabric.
　タンパク質繊維は、生分解性を有すること、生産及び加工のエネルギーが小さいこと等のメリットがあることから、近年の環境保全意識の高まりに応じて、様々な分野への需要の増大が見込まれている。タンパク質繊維の中でもウール等の獣毛繊維は、冬暖かく、夏涼しい、高い吸放湿性を有し、弾力性に富む等の特性を備えているため、衣料用素材を初めとして様々な用途に用いられている（例えば、非特許文献１）。 Since protein fibers have advantages such as biodegradability and low energy for production and processing, demand for various fields is expected to increase in accordance with the growing awareness of environmental conservation in recent years. I have. Animal fiber such as wool among protein fibers has properties such as warm in winter, cool in summer, high moisture absorption and desorption, and elasticity. (For example, Non-Patent Document 1).
　ところが、獣毛繊維は綿等のセルロース繊維よりは燃えにくいものの、ポリエステル繊維等の合成繊維と比べて燃えやすいといった欠点がある。獣毛繊維の耐燃焼性に係る欠点を補うために、獣毛繊維と難燃性の合成繊維を混用することが考えられる。しかしながら、獣毛繊維と合成繊維を混用すると、タンパク質繊維を使用することによって得られるメリットが損なわれてしまう。 However, although animal hair fibers are less flammable than cellulose fibers such as cotton, they have the disadvantage that they are more flammable than synthetic fibers such as polyester fibers. In order to make up for the drawbacks associated with the burning resistance of animal hair fibers, it is conceivable to mix animal hair fibers with flame-retardant synthetic fibers. However, when animal hair fibers and synthetic fibers are mixed, the benefits obtained by using protein fibers are impaired.
　本発明は、タンパク質繊維を使用することによって得られるメリットを損なうことなく、耐燃焼性が向上した、獣毛繊維を含む複合糸及び布地を提供することを目的とする。本発明はまた、当該複合糸の製造方法を提供することを目的とする。 An object of the present invention is to provide a composite yarn and a fabric containing animal hair fibers, which have improved flame resistance without impairing the advantages obtained by using protein fibers. Another object of the present invention is to provide a method for producing the composite yarn.
　本発明者らは、改変フィブロインが優れた耐燃焼性を有することを見い出した。本発明は、この新規な知見に基づくものである。 The present inventors have found that the modified fibroin has excellent combustion resistance. The present invention is based on this new finding.
　本発明は、例えば、以下の各発明に関する。
［１］
　人造フィブロイン繊維と、獣毛繊維とを含有し、
　上記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、複合糸。
［２］
　上記人造フィブロイン繊維は、下記式で定義される湿潤収縮率が２％以上である、［１］に記載の複合糸。
　湿潤収縮率＝｛１－（水に接触させて湿潤状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％）
［３］
　上記人造フィブロイン繊維は、下記式で定義される乾燥収縮率が７％超である、［１］又は［２］に記載の複合糸。
　乾燥収縮率＝｛１－（乾燥状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％）
［４］
　上記人造フィブロイン繊維は、水との接触により捲縮されたものである、［１］～［３］のいずれかに記載の複合糸。
［５］
　上記人造フィブロイン繊維は、限界酸素指数（ＬＯＩ）値が２６．０以上である、［１］～［４］のいずれかに記載の複合糸。
［６］
　上記人造フィブロイン繊維は、下記式Ａに従って求められる最高吸湿発熱度が０．０２５℃／ｇ超である、［１］～［５］のいずれかに記載の複合糸。
　式Ａ：最高吸湿発熱度＝｛（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値）－（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度）｝（℃）／試料重量（ｇ）
［式Ａ中、低湿度環境は、温度２０℃及び相対湿度４０％の環境を意味し、高湿度環境は、温度２０℃及び相対湿度９０％の環境を意味する。］
［７］
　上記人造フィブロイン繊維は、下記式Ｂに従って求められる保温性指数が０．１８超である、［１］～［６］のいずれかに記載の複合糸。
　式Ｂ：保温性指数＝保温率（％）／試料の目付け（ｇ／ｍ２）
［式Ｂ中、保温率（％）は、ドライコンタクト法（温度３０℃、風速３０ｃｍ／秒）で測定された保温率を意味し、（１－ａ／ｂ）×１００で算出される。ａは、試験片を介して放散された熱量を示し、ｂは、試験片を介さないで放散された熱量を示す。］
［８］
　上記改変フィブロインが、改変クモ糸フィブロインである、［１］～［７］のいずれかに記載の複合糸。
［９］
　混紡糸である、［１］～［８］のいずれかに記載の複合糸。
［１０］
　人造フィブロイン繊維と、獣毛繊維とを含有し、
　上記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、布地。
［１１］
　人造フィブロイン繊維と、獣毛繊維とを混合する工程を備え、
　上記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、複合糸の製造方法。 The present invention relates to, for example, the following inventions.
[1]
Contains artificial fibroin fiber and animal hair fiber,
The composite yarn, wherein the artificial fibroin fiber contains a modified fibroin and has a contraction history of being irreversibly contracted by contact with water after spinning.
[2]
The composite yarn according to [1], wherein the artificial fibroin fiber has a wet shrinkage defined by the following formula of 2% or more.
Wet shrinkage = {1− (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fibril after spinning and before contact with water)} × 100 (%)
[3]
The composite yarn according to [1] or [2], wherein the artificial fibroin fiber has a dry shrinkage defined by the following formula of more than 7%.
Drying shrinkage = {1− (length of dried artificial fibroin fiber / length of artificial fibroin raw fiber after spinning and before contact with water)} × 100 (%)
[4]
The composite yarn according to any one of [1] to [3], wherein the artificial fibroin fiber is crimped by contact with water.
[5]
The composite yarn according to any one of [1] to [4], wherein the artificial fibroin fiber has a limiting oxygen index (LOI) value of 26.0 or more.
[6]
The composite yarn according to any one of [1] to [5], wherein the artificial fibroin fiber has a maximum heat of moisture absorption determined by the following formula A of more than 0.025 ° C./g.
Formula A: Maximum heat of moisture absorption = ｛(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium, and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)｝ (℃) / sample weight (g)
[In the formula A, a low humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 40%, and a high humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 90%. ]
[7]
The composite yarn according to any one of [1] to [6], wherein the artificial fibroin fiber has a heat retention index determined according to the following formula B of more than 0.18.
Formula B: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
[In formula B, the heat retention rate (%) means a heat retention rate measured by a dry contact method (temperature: 30 ° C., wind speed: 30 cm / sec), and is calculated by (1−a / b) × 100. a shows the amount of heat dissipated through the test piece, and b shows the amount of heat dissipated without passing through the test piece. ]
[8]
The composite yarn according to any one of [1] to [7], wherein the modified fibroin is a modified spider silk fibroin.
[9]
The composite yarn according to any one of [1] to [8], which is a blended yarn.
[10]
Contains artificial fibroin fiber and animal hair fiber,
The fabric, wherein the artificial fibroin fiber contains a modified fibroin and has a shrinkage history irreversibly shrunk by contact with water after spinning.
[11]
Comprising a step of mixing artificial fibroin fiber and animal hair fiber,
A method for producing a composite yarn, wherein the artificial fibroin fiber contains a modified fibroin and has a contraction history irreversibly contracted by contact with water after spinning.
　本発明によれば、タンパク質繊維を使用することによって得られるメリットを損なうことなく、耐燃焼性が向上した、獣毛繊維を含む複合糸及び布地を提供することが可能となる。 According to the present invention, it is possible to provide a composite yarn and a fabric containing animal hair fibers, which have improved flame resistance without impairing the advantages obtained by using protein fibers.
　本発明に係る複合糸及び布地は、改変フィブロイン繊維を含んでいることから、吸湿発熱性にも優れている。 複合 Since the composite yarn and the fabric according to the present invention contain the modified fibroin fiber, they are also excellent in moisture absorption and heat generation.
改変フィブロインのドメイン配列の一例を示す模式図である。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 天然由来のフィブロインのｚ／ｗ（％）の値の分布を示す図である。It is a figure which shows the distribution of the value of z / w (%) of the fibroin derived from nature. 天然由来のフィブロインのｘ／ｙ（％）の値の分布を示す図である。It is a figure which shows the distribution of the value of x / y (%) of fibroin derived from nature. 改変フィブロインのドメイン配列の一例を示す模式図である。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 改変フィブロインのドメイン配列の一例を示す模式図である。It is a schematic diagram which shows an example of the domain sequence of a modified fibroin. 人造フィブロイン原繊維を製造するための紡糸装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing artificial fibroin fibrils. 水との接触による人造フィブロイン原繊維及び人造フィブロイン繊維の長さ変化の例を示す図である。It is a figure showing an example of length change of artificial fibroin fibrils and artificial fibroin fibers by contact with water. 人造フィブロイン繊維を製造するための製造装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the manufacturing apparatus for manufacturing an artificial fibroin fiber. 人造フィブロイン繊維を製造するための製造装置の一例を概略的に示す説明図である。It is explanatory drawing which shows roughly an example of the manufacturing apparatus for manufacturing an artificial fibroin fiber. 吸湿発熱性試験の結果の一例を示すグラフである。It is a graph which shows an example of the result of a moisture absorption exothermic test.
　以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔複合糸〕
　本実施形態に係る複合糸は、人造フィブロイン繊維と、獣毛繊維とを含有する。複合糸には、例えば、混紡糸、混繊糸、混織糸、交織糸、合撚糸及びカバーリング糸等が含まれる。 (Composite yarn)
The composite yarn according to the present embodiment contains artificial fibroin fiber and animal hair fiber. The composite yarns include, for example, blended yarns, blended yarns, blended yarns, interwoven yarns, plied yarns, covering yarns, and the like.
（人造フィブロイン繊維）
　本実施形態に係る人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有するものであってよい。 (Artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment may contain modified fibroin and have a shrinkage history irreversibly shrunk by contact with water after spinning.
（改変フィブロイン）
　本実施形態に係る改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。改変フィブロインは、ドメイン配列のＮ末端側及びＣ末端側のいずれか一方又は両方に更にアミノ酸配列（Ｎ末端配列及びＣ末端配列）が付加されていてもよい。Ｎ末端配列及びＣ末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、１００残基程度のアミノ酸からなる。 (Modified fibroin)
The modified fibroin according to this embodiment has a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Including proteins. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
　本明細書において「改変フィブロイン」とは、人為的に製造されたフィブロイン（人造フィブロイン）を意味する。改変フィブロインは、そのドメイン配列が、天然由来のフィブロインのアミノ酸配列とは異なるフィブロインであってもよく、天然由来のフィブロインのアミノ酸配列と同一であるフィブロインであってもよい。本明細書でいう「天然由来のフィブロイン」もまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。 As used herein, the term “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be the same as the amino acid sequence of naturally occurring fibroin. The term “naturally-derived fibroin” as used herein is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Is a protein containing the domain sequence to be determined.
　「改変フィブロイン」は、天然由来のフィブロインのアミノ酸配列をそのまま利用したものであってもよく、天然由来のフィブロインのアミノ酸配列に依拠してそのアミノ酸配列を改変したもの（例えば、クローニングした天然由来のフィブロインの遺伝子配列を改変することによりアミノ酸配列を改変したもの）であってもよく、また天然由来のフィブロインに依らず人工的に設計及び合成したもの（例えば、設計したアミノ酸配列をコードする核酸を化学合成することにより所望のアミノ酸配列を有するもの）であってもよい。 "Modified fibroin" may be a directly used amino acid sequence of a naturally occurring fibroin, or a modified amino acid sequence based on the amino acid sequence of a naturally occurring fibroin (for example, cloned naturally occurring fibroin). The amino acid sequence may be modified by modifying the gene sequence of fibroin), or may be artificially designed and synthesized without using naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence may be used). Which have a desired amino acid sequence by chemical synthesis).
　本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域（典型的には、アミノ酸配列の（Ａ）ｎモチーフに相当する。）と非晶領域（典型的には、アミノ酸配列のＲＥＰに相当する。）を生じるアミノ酸配列であり、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるアミノ酸配列を意味する。ここで、（Ａ）ｎモチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は２～２７である。（Ａ）ｎモチーフのアミノ酸残基数は、２～２０、４～２７、４～２０、８～２０、１０～２０、４～１６、８～１６、又は１０～１６の整数であってよい。また、（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は４０％以上であればよく、６０％以上、７０％以上、８０％以上、８３％以上、８５％以上、８６％以上、９０％以上、９５％以上、又は１００％（アラニン残基のみで構成されることを意味する。）であってもよい。ドメイン配列中に複数存在する（Ａ）ｎモチーフは、少なくとも７つがアラニン残基のみで構成されてもよい。ＲＥＰは２～２００アミノ酸残基から構成されるアミノ酸配列を示す。ＲＥＰは、１０～２００アミノ酸残基から構成されるアミノ酸配列であってもよい。ｍは２～３００の整数を示し、１０～３００の整数であってもよい。複数存在する（Ａ）ｎモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するＲＥＰは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。 As used herein, the term “domain sequence” refers to a crystalline region unique to fibroin (typically, corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence). The amino acid represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Means an array. Here, the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 . (A) The ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.
　本実施形態に係る改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列に対し、例えば、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行うことで得ることができる。アミノ酸残基の置換、欠失、挿入及び／又は付加は、部分特異的突然変異誘発法等の当業者に周知の方法により行うことができる。具体的には、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．１０，６４８７（１９８２）、Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１００，４４８（１９８３）等の文献に記載されている方法に準じて行うことができる。 The modified fibroin according to the present embodiment has, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to a cloned natural fibroin gene sequence. Can be obtained by performing the following modification. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods {in} Enzymology, 100, 448 (1983).
　天然由来のフィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質であり、具体的には、例えば、昆虫又はクモ類が産生するフィブロインが挙げられる。 Naturally occurring fibroin is a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. Yes, specifically, for example, fibroin produced by insects or spiders.
　昆虫が産生するフィブロインとしては、例えば、ボンビックス・モリ（Ｂｏｍｂｙｘ　ｍｏｒｉ）、クワコ（Ｂｏｍｂｙｘ　ｍａｎｄａｒｉｎａ）、天蚕（Ａｎｔｈｅｒａｅａ　ｙａｍａｍａｉ）、柞蚕（Ａｎｔｅｒａｅａ　ｐｅｒｎｙｉ）、楓蚕（Ｅｒｉｏｇｙｎａ　ｐｙｒｅｔｏｒｕｍ）、蓖蚕（Ｐｉｌｏｓａｍｉａ　Ｃｙｎｔｈｉａ　ｒｉｃｉｎｉ）、樗蚕（Ｓａｍｉａ　ｃｙｎｔｈｉａ）、栗虫（Ｃａｌｉｇｕｒａ　ｊａｐｏｎｉｃａ）、チュッサー蚕（Ａｎｔｈｅｒａｅａ　ｍｙｌｉｔｔａ）、ムガ蚕（Ａｎｔｈｅｒａｅａ　ａｓｓａｍａ）等のカイコが産生する絹タンパク質、及びスズメバチ（Ｖｅｓｐａ　ｓｉｍｉｌｌｉｍａ　ｘａｎｔｈｏｐｔｅｒａ）の幼虫が吐出するホーネットシルクタンパク質が挙げられる。 Examples of the fibroin produced by insects include Bombyx @ mori, Bombyx @ mandarina, natural silkworm (Antheraea @ yamamai), tussah (Anterea @ pernii), and maple silkworm (Erioganyerii). ), Silkworms produced by silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antheraea mylitta), silkworms such as moga silkworms (Antheraea assama), and silkworms produced by larvae of the hornet beetle Hornet silk protein.
　昆虫が産生するフィブロインのより具体的な例としては、例えば、カイコ・フィブロインＬ鎖（ＧｅｎＢａｎｋアクセッション番号Ｍ７６４３０（塩基配列）、及びＡＡＡ２７８４０．１（アミノ酸配列））が挙げられる。 よ り More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
　クモ類が産生するフィブロインとしては、例えば、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属（Ａｒａｎｅｕｓ属）に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属（Ｎｅｏｓｃｏｎａ属）に属するクモ、コオニグモモドキ等のコオニグモモドキ属（Ｐｒｏｎｕｓ属）に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属（Ｃｙｒｔａｒａｃｈｎｅ属）に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属（Ｇａｓｔｅｒａｃａｎｔｈａ属）に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属（Ｏｒｄｇａｒｉｕｓ属）に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属（Ａｒｇｉｏｐｅ属）に属するクモ、キジロオヒキグモ等のオヒキグモ属（Ａｒａｃｈｎｕｒａ属）に属するクモ、ハツリグモ等のハツリグモ属（Ａｃｕｓｉｌａｓ属）に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属（Ｃｙｔｏｐｈｏｒａ属）に属するクモ、ゲホウグモ等のゲホウグモ属（Ｐｏｌｔｙｓ属）に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属（Ｃｙｃｌｏｓａ属）に属するクモ、及びヤマトカナエグモ等のカナエグモ属（Ｃｈｏｒｉｚｏｐｅｓ属）に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属（Ｔｅｔｒａｇｎａｔｈａ属）に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属（Ｌｅｕｃａｕｇｅ属）に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属（Ｎｅｐｈｉｌａ属）に属するクモ、キンヨウグモ等のアズミグモ属（Ｍｅｎｏｓｉｒａ属）に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属（Ｄｙｓｃｈｉｒｉｏｇｎａｔｈａ属）に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属（Ｌａｔｒｏｄｅｃｔｕｓ属）に属するクモ、及びユープロステノプス属（Ｅｕｐｒｏｓｔｈｅｎｏｐｓ属）に属するクモ等のアシナガグモ科（Ｔｅｔｒａｇｎａｔｈｉｄａｅ科）に属するクモが産生するスパイダーシルクタンパク質が挙げられる。スパイダーシルクタンパク質としては、例えば、ＭａＳｐ（ＭａＳｐ１及びＭａＳｐ２）、ＡＤＦ（ＡＤＦ３及びＡＤＦ４）等の牽引糸タンパク質、ＭｉＳｐ（ＭｉＳｐ１及びＭｉＳｐ２）等が挙げられる。 Examples of the fibroin produced by spiders include spiders belonging to the genus Araneus (genus Araneus), such as Orion spider, Elder spider, Red-colored Spider, Blue-colored Spider, etc. Spiders belonging to the genus Argiope genus (Genus Pronus), such as spiders belonging to the genus Procarpus spp., Spiders belonging to the genus Pronus, and spider spiders belonging to the genus Cynotarachne, such as the genus Cyrtarachne, such as Torinofundamashi and Otorinofundamashi. Spiders belonging to the genus Ordgarius, such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Orbalis, and spiders belonging to the genus Ordgarius, such as the spiders belonging to the genus Ordgarius. Spiders belonging to the genus Argiope such as Argiope bruennichi, spiders belonging to the genus Argiope sp. Spiders belonging to the genus Spider (Genus Poltys) such as spiders belonging to the genus (Cytophora) and spiders belonging to the genus (Potys), spiders belonging to the genus Spiders (genus Cyclosa) such as the spiders belonging to the genus Cyclosa and spiders belonging to the genus Cygnus spp. Spider silk proteins produced by spiders belonging to the genus Chorizopes), and asina such as red-headed spiders, red-backed spiders, blue-backed spiders and urocore-red spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Leucaegium, such as the spiders Argiope bruennichi and spiders spiders belonging to the genus Leucauge, such as the spiders belonging to the genus Nephila sp. Spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Latus and the spiders belonging to the genus Lastroconidae belonging to the genus Latus sp. Spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops (Euprosthenops) are produced. Spider silk protein. Examples of the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).
　クモ類が産生するスパイダーシルクタンパク質のより具体的な例としては、例えば、ｆｉｂｒｏｉｎ－３（ａｄｆ－３）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１０（アミノ酸配列）、Ｕ４７８５５（塩基配列））、ｆｉｂｒｏｉｎ－４（ａｄｆ－４）［Ａｒａｎｅｕｓ　ｄｉａｄｅｍａｔｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ４７０１１（アミノ酸配列）、Ｕ４７８５６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ０４５０４（アミノ酸配列）、Ｕ３７５２０（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｌａｔｒｏｄｅｃｔｕｓ　ｈｅｓｐｅｒｕｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ６８８５６（アミノ酸配列）、ＥＦ５９５２４６（塩基配列））、ｄｒａｇｌｉｎｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ｓｐｉｄｒｏｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖａｔａ由来］（ＧｅｎＢａｎｋアクセッション番号ＡＡＬ３２４７２（アミノ酸配列）、ＡＦ４４１２４５（塩基配列））、ｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　１［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ由来］（ＧｅｎＢａｎｋアクセッション番号ＣＡＪ００４２８（アミノ酸配列）、ＡＪ９７３１５５（塩基配列））、及びｍａｊｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ　２［Ｅｕｐｒｏｓｔｈｅｎｏｐｓ　ａｕｓｔｒａｌｉｓ］（ＧｅｎＢａｎｋアクセッション番号ＣＡＭ３２２４９．１（アミノ酸配列）、ＡＭ４９０１６９（塩基配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　１［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５８９．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　２［Ｎｅｐｈｉｌａ　ｃｌａｖｉｐｅｓ］（ＧｅｎＢａｎｋアクセッション番号ＡＡＣ１４５９１．１（アミノ酸配列））、ｍｉｎｏｒ　ａｍｐｕｌｌａｔｅ　ｓｐｉｄｒｏｉｎ－ｌｉｋｅ　ｐｒｏｔｅｉｎ［Ｎｅｐｈｉｌｅｎｇｙｓ　ｃｒｕｅｎｔａｔａ］（ＧｅｎＢａｎｋアクセッション番号ＡＢＲ３７２７８．１（アミノ酸配列）等が挙げられる。 More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ｐｈ ), U37520 (base sequence)), major \ ampullate \ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number ABA45, amino acid sequence of A23) )), Major \ sample \ spidroin \ 1 [from Euprosthenops \ australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ97155 (base sequence)), and major \ sample \ spidroin \ 2 [Euprosus] ] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor \ silk \ protein \ 1 [Nephila \ clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor \ ampilatte [in] Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampoulate spidroin-like protein [Nephilengys Cruenata] (GenBank Accession No. ABR3727.1.
　天然由来のフィブロインのより具体的な例としては、更に、ＮＣＢＩ　ＧｅｎＢａｎｋに配列情報が登録されているフィブロインを挙げることができる。例えば、ＮＣＢＩ　ＧｅｎＢａｎｋに登録されている配列情報のうちＤＩＶＩＳＩＯＮとしてＩＮＶを含む配列の中から、ＤＥＦＩＮＩＴＩＯＮにｓｐｉｄｒｏｉｎ、ａｍｐｕｌｌａｔｅ、ｆｉｂｒｏｉｎ、「ｓｉｌｋ及びｐｏｌｙｐｅｐｔｉｄｅ」、又は「ｓｉｌｋ及びｐｒｏｔｅｉｎ」がキーワードとして記載されている配列、ＣＤＳから特定のｐｒｏｄｕｃｔの文字列、ＳＯＵＲＣＥからＴＩＳＳＵＥ　ＴＹＰＥに特定の文字列の記載された配列を抽出することにより確認することができる。 よ り More specific examples of naturally occurring fibroin include fibroin in which sequence information is registered in NCBI GenBank. For example, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" is described as a keyword in DEFINITION from among sequences including INV as DIVISION in the sequence information registered in NCBI @ GenBank. It can be confirmed by extracting a character string of a specific product from a sequence or CDS, and extracting a sequence in which a specific character string is described in TISSUE @ TYPE from SOURCE.
　本実施形態に係る改変フィブロインは、改変絹（シルク）フィブロイン（カイコが産生する絹タンパク質のアミノ酸配列を改変したもの）であってもよく、改変クモ糸フィブロイン（クモ類が産生するスパイダーシルクタンパク質のアミノ酸配列を改変したもの）であってもよい。改変フィブロインとしては、改変クモ糸フィブロインが好ましい。 The modified fibroin according to this embodiment may be a modified silk (silk) fibroin (an amino acid sequence of a silk protein produced by a silkworm modified), and a modified spider silk fibroin (a spider silk protein produced by an arachnid). Modified amino acid sequence). As the modified fibroin, a modified spider silk fibroin is preferable.
　改変フィブロインの具体的な例として、クモの大瓶状腺で産生される大吐糸管しおり糸タンパク質に由来する改変フィブロイン（第１の改変フィブロイン）、グリシン残基の含有量が低減されたドメイン配列を有する改変フィブロイン（第２の改変フィブロイン）、（Ａ）ｎモチーフの含有量が低減されたドメイン配列を有する改変フィブロイン（第３の改変フィブロイン）、グリシン残基の含有量、及び（Ａ）ｎモチーフの含有量が低減された改変フィブロイン（第４の改変フィブロイン）、局所的に疎水性指標の大きい領域を含むドメイン配列を有する改変フィブロイン（第５の改変フィブロイン）、並びにグルタミン残基の含有量が低減されたドメイン配列を有する改変フィブロイン（第６の改変フィブロイン）が挙げられる。 Specific examples of the modified fibroin include a modified fibroin (first modified fibroin) derived from a large spinal cord marker protein produced in a spider's large ampullate gland, and a domain sequence having a reduced content of glycine residues. (A second modified fibroin), (A) a modified fibroin having a domain sequence with a reduced content of the n motif (a third modified fibroin), a glycine residue content, and (A) n Modified fibroin having a reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence containing a region having a large hydrophobicity index (fifth modified fibroin), and glutamine residue content Modified fibroin having a reduced domain sequence (sixth modified fibroin).
　第１の改変フィブロインとしては、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質が挙げられる。第１の改変フィブロインにおいて、（Ａ）ｎモチーフのアミノ酸残基数は、３～２０の整数が好ましく、４～２０の整数がより好ましく、８～２０の整数が更に好ましく、１０～２０の整数が更により好ましく、４～１６の整数が更によりまた好ましく、８～１６の整数が特に好ましく、１０～１６の整数が最も好ましい。第１の改変フィブロインは、式１中、ＲＥＰを構成するアミノ酸残基の数は、１０～２００残基であることが好ましく、１０～１５０残基であることがより好ましく、２０～１００残基であることが更に好ましく、２０～７５残基であることが更により好ましい。第１の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるアミノ酸配列中に含まれるグリシン残基、セリン残基及びアラニン残基の合計残基数がアミノ酸残基数全体に対して、４０％以上であることが好ましく、６０％以上であることがより好ましく、７０％以上であることが更に好ましい。 The first modified fibroin includes a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . In the first modified fibroin, the number of amino acid residues of the (A) n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, and an integer of 10 to 20 Is still more preferable, an integer of 4 to 16 is still more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and more preferably 20 to 100 residues. Is even more preferable, and even more preferably 20 to 75 residues. The first modified fibroin has a total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m The total number is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more.
　第１の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるアミノ酸配列の単位を含み、かつＣ末端配列が配列番号１～３のいずれかに示されるアミノ酸配列又は配列番号１～３のいずれかに示されるアミノ酸配列と９０％以上の相同性を有するアミノ酸配列であるポリペプチドであってもよい。 The first modified fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m , and has a C-terminal sequence represented by any of SEQ ID NOS: 1 to 3 or The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3.
　配列番号１に示されるアミノ酸配列は、ＡＤＦ３（ＧＩ：１２６３２８７、ＮＣＢＩ）のアミノ酸配列のＣ末端の５０残基のアミノ酸からなるアミノ酸配列と同一であり、配列番号２に示されるアミノ酸配列は、配列番号１に示されるアミノ酸配列のＣ末端から２０残基取り除いたアミノ酸配列と同一であり、配列番号３に示されるアミノ酸配列は、配列番号１に示されるアミノ酸配列のＣ末端から２９残基取り除いたアミノ酸配列と同一である。 The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
　第１の改変フィブロインのより具体的な例として、（１－ｉ）配列番号４（ｒｅｃｏｍｂｉｎａｎｔ　ｓｐｉｄｅｒ　ｓｉｌｋ　ｐｒｏｔｅｉｎ　ＡＤＦ３ＫａｉＬａｒｇｅＮＲＳＨ１）で示されるアミノ酸配列、又は（１－ｉｉ）配列番号４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。配列同一性は、９５％以上であることが好ましい。 As more specific examples of the first modified fibroin, (1-i) an amino acid sequence represented by SEQ ID NO: 4 (recombinant \ spider \ silk \ protein \ ADF3KaiLargeNRSH1) or (1-ii) an amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroin comprising an amino acid sequence having at least% sequence identity. The sequence identity is preferably 95% or more.
　配列番号４で示されるアミノ酸配列は、Ｎ末端に開始コドン、Ｈｉｓ１０タグ及びＨＲＶ３Ｃプロテアーゼ（Ｈｕｍａｎ　ｒｈｉｎｏｖｉｒｕｓ　３Ｃプロテアーゼ）認識サイトからなるアミノ酸配列（配列番号５）を付加したＡＤＦ３のアミノ酸配列において、第１～１３番目の反復領域をおよそ２倍になるように増やすとともに、翻訳が第１１５４番目アミノ酸残基で終止するように変異させたものである。配列番号４で示されるアミノ酸配列のＣ末端のアミノ酸配列は、配列番号３で示されるアミノ酸配列と同一である。 The amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human \ rhinovirus @ 3C protease) recognition site at the N-terminus is added. The 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
　（１－ｉ）の改変フィブロインは、配列番号４で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.
　第２の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、グリシン残基の含有量が低減されたアミノ酸配列を有する。第２の改変フィブロインは、天然由来のフィブロインと比較して、少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。 The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. The second modified fibroin can be said to have an amino acid sequence corresponding to at least one or more glycine residues in the REP replaced by another amino acid residue, as compared to a naturally occurring fibroin. .
　第２の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中のＧＧＸ及びＧＰＧＸＸ（但し、Ｇはグリシン残基、Ｐはプロリン残基、Ｘはグリシン以外のアミノ酸残基を示す。）から選ばれる少なくとも一つのモチーフ配列において、少なくとも１又は複数の当該モチーフ配列中の１つのグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものであってもよい。 The second modified fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine, as compared with a naturally-derived fibroin. At least one motif sequence selected from the group consisting of at least one glycine residue in one or more of the motif sequences has been replaced with another amino acid residue. You may.
　第２の改変フィブロインは、上述のグリシン残基が別のアミノ酸残基に置換されたモチーフ配列の割合が、全モチーフ配列に対して、１０％以上であってもよい。 は In the second modified fibroin, the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
　第２の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の全ＲＥＰに含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を除いた配列中の総アミノ酸残基数をｗとしたときに、ｚ／ｗが３０％以上、４０％以上、５０％以上又は５０．９％以上であるアミノ酸配列を有するものであってもよい。（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数は８３％以上であってよいが、８６％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることが更に好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が更により好ましい。 The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the (A) n motif located at the most C-terminal side to the domain sequence The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus is represented by z, From, when the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is defined as w, z / w is 30% or more; It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
　第２の改変フィブロインは、ＧＧＸモチーフの１つのグリシン残基を別のアミノ酸残基に置換することにより、ＸＧＸからなるアミノ酸配列の含有割合を高めたものであることが好ましい。第２の改変フィブロインは、ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合が３０％以下であることが好ましく、２０％以下であることがより好ましく、１０％以下であることが更に好ましく、６％以下であることが更により好ましく、４％以下であることが更によりまた好ましく、２％以下であることが特に好ましい。ドメイン配列中のＧＧＸからなるアミノ酸配列の含有割合は、下記ＸＧＸからなるアミノ酸配列の含有割合（ｚ／ｗ）の算出方法と同様の方法で算出することができる。 ２ The second modified fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
　ｚ／ｗの算出方法を更に詳細に説明する。まず、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる全てのＲＥＰから、ＸＧＸからなるアミノ酸配列を抽出する。ＸＧＸを構成するアミノ酸残基の総数がｚである。例えば、ＸＧＸからなるアミノ酸配列が５０個抽出された場合（重複はなし）、ｚは５０×３＝１５０である。また、例えば、ＸＧＸＧＸからなるアミノ酸配列の場合のように２つのＸＧＸに含まれるＸ（中央のＸ）が存在する場合は、重複分を控除して計算する（ＸＧＸＧＸの場合は５アミノ酸残基である）。ｗは、ドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列に含まれる総アミノ酸残基数である。例えば、図１に示したドメイン配列の場合、ｗは４＋５０＋４＋１００＋４＋１０＋４＋２０＋４＋３０＝２３０である（最もＣ末端側に位置する（Ａ）ｎモチーフは除いている。）。次に、ｚをｗで除すことによって、ｚ／ｗ（％）を算出することができる。 The method of calculating z / w will be described in more detail. First, in a fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m (modified fibroin or naturally occurring fibroin), (A) n located closest to the C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (there is no duplication), z is 50 × 3 = 150. Further, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues are used. is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located closest to the C-terminus to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A) n motif located at the most C-terminal side is excluded). Next, z / w (%) can be calculated by dividing z by w.
　ここで、天然由来のフィブロインにおけるｚ／ｗについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、フィブロイン中のＧＧＸからなるアミノ酸配列の含有割合が６％以下である天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｚ／ｗを算出した。その結果を図２に示す。図２の横軸はｚ／ｗ（％）を示し、縦軸は頻度を示す。図２から明らかなとおり、天然由来のフィブロインにおけるｚ／ｗは、いずれも５０．９％未満である（最も高いもので、５０．８６％）。 Here, z / w in naturally occurring fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Of all the extracted fibroins, a naturally-derived one containing a domain sequence represented by Formula 1: [(A) n motif-REP] m and containing 6% or less of a GGX amino acid sequence in the fibroin Z / w was calculated from the amino acid sequence of fibroin by the above-described calculation method. The result is shown in FIG. The horizontal axis in FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w of the naturally derived fibroin is less than 50.9% (the highest one is 50.86%).
　第２の改変フィブロインにおいて、ｚ／ｗは、５０．９％以上であることが好ましく、５６．１％以上であることがより好ましく、５８．７％以上であることが更に好ましく、７０％以上であることが更により好ましく、８０％以上であることが更によりまた好ましい。ｚ／ｗの上限に特に制限はないが、例えば、９５％以下であってもよい。 In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, still more preferably 58.7% or more, and 70% or more. Is still more preferred, and even more preferably 80% or more. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
　第２の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、グリシン残基をコードする塩基配列の少なくとも一部を置換して別のアミノ酸残基をコードするように改変することにより得ることができる。このとき、改変するグリシン残基として、ＧＧＸモチーフ及びＧＰＧＸＸモチーフにおける１つのグリシン残基を選択してもよいし、またｚ／ｗが５０．９％以上になるように置換してもよい。また、例えば、天然由来のフィブロインのアミノ酸配列から上記態様を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中のグリシン残基を別のアミノ酸残基に置換したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The second modified fibroin is modified, for example, by replacing at least a part of the base sequence encoding a glycine residue from the cloned natural fibroin gene sequence to encode another amino acid residue. Obtainable. At this time, as the glycine residue to be modified, a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more. Alternatively, for example, the amino acid sequence can be obtained by designing an amino acid sequence satisfying the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of another amino acid residue for the glycine residue in the REP from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. The amino acid sequence corresponding to insertion, addition, and / or addition may be modified.
　上記の別のアミノ酸残基としては、グリシン残基以外のアミノ酸残基であれば特に制限はないが、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基、メチオニン（Ｍ）残基、プロリン（Ｐ）残基、フェニルアラニン（Ｆ）残基及びトリプトファン（Ｗ）残基等の疎水性アミノ酸残基、グルタミン（Ｑ）残基、アスパラギン（Ｎ）残基、セリン（Ｓ）残基、リシン（Ｋ）残基及びグルタミン酸（Ｅ）残基等の親水性アミノ酸残基が好ましく、バリン（Ｖ）残基、ロイシン（Ｌ）残基、イソロイシン（Ｉ）残基、フェニルアラニン（Ｆ）残基及びグルタミン（Ｑ）残基がより好ましく、グルタミン（Ｑ）残基が更に好ましい。 The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
　第２の改変フィブロインのより具体的な例として、（２－ｉ）配列番号６（Ｍｅｔ－ＰＲＴ３８０）、配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）若しくは配列番号９（Ｍｅｔ－ＰＲＴ７９９）で示されるアミノ酸配列、又は（２－ｉｉ）配列番号６、配列番号７、配列番号８若しくは配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 More specific examples of the second modified fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.
　（２－ｉ）の改変フィブロインについて説明する。配列番号６で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１０（Ｍｅｔ－ＰＲＴ３１３）で示されるアミノ酸配列のＲＥＰ中の全てのＧＧＸをＧＱＸに置換したものである。配列番号７で示されるアミノ酸配列は、配列番号６で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号８で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列の各（Ａ）ｎモチーフのＣ末端側に２つのアラニン残基を挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、配列番号７の分子量とほぼ同じとなるようにＣ末端側の一部のアミノ酸を欠失させたものである。配列番号９で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中に存在する２０個のドメイン配列の領域（但し、当該領域のＣ末端側の数アミノ酸残基が置換されている。）を４回繰り返した配列のＣ末端に所定のヒンジ配列とＨｉｓタグ配列が付加されたものである。 The modified fibroin (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some amino acids on the C-terminal side have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). Is repeated four times with a predetermined hinge sequence and His tag sequence added to the C-terminal.
　配列番号１０で示されるアミノ酸配列（天然由来のフィブロインに相当）におけるｚ／ｗの値は、４６．８％である。配列番号６で示されるアミノ酸配列、配列番号７で示されるアミノ酸配列、配列番号８で示されるアミノ酸配列、及び配列番号９で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ５８．７％、７０．１％、６６．１％及び７０．０％である。また、配列番号１０、配列番号６、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のギザ比率（後述する）１：１．８～１１．３におけるｘ／ｙの値は、それぞれ１５．０％、１５．０％、９３．４％、９２．７％及び８９．８％である。 Ｚ The value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The values of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%. In addition, the value of x / y at the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
　（２－ｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
　（２－ｉｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（２－ｉｉ）の改変フィブロインは、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが５０．９％以上であることが好ましい。 The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
　第２の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変フィブロインの単離、固定化、検出及び可視化等が可能となる。 ２The second modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
　タグ配列として、例えば、他の分子との特異的親和性（結合性、アフィニティ）を利用したアフィニティタグを挙げることができる。アフィニティタグの具体例として、ヒスチジンタグ（Ｈｉｓタグ）を挙げることができる。Ｈｉｓタグは、ヒスチジン残基が４から１０個程度並んだ短いペプチドで、ニッケル等の金属イオンと特異的に結合する性質があるため、金属キレートクロマトグラフィー（ｃｈｅｌａｔｉｎｇ　ｍｅｔａｌ　ｃｈｒｏｍａｔｏｇｒａｐｈｙ）による改変フィブロインの単離に利用することができる。タグ配列の具体例として、例えば、配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含むアミノ酸配列）が挙げられる。 Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including a His tag sequence and a hinge sequence).
　また、グルタチオンに特異的に結合するグルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）、マルトースに特異的に結合するマルトース結合タンパク質（ＭＢＰ）等のタグ配列を利用することもできる。 タ グ Alternatively, tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
　さらに、抗原抗体反応を利用した「エピトープタグ」を利用することもできる。抗原性を示すペプチド（エピトープ）をタグ配列として付加することにより、当該エピトープに対する抗体を結合させることができる。エピトープタグとして、ＨＡ（インフルエンザウイルスのヘマグルチニンのペプチド配列）タグ、ｍｙｃタグ、ＦＬＡＧタグ等を挙げることができる。エピトープタグを利用することにより、高い特異性で容易に改変フィブロインを精製することができる。 Furthermore, an “epitope tag” utilizing an antigen-antibody reaction can be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag. By using the epitope tag, the modified fibroin can be easily purified with high specificity.
　さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも使用することができる。当該タグ配列を介して吸着したタンパク質をプロテアーゼ処理することにより、タグ配列を切り離した改変フィブロインを回収することもできる。 Further, a tag sequence that can be cleaved by a specific protease can be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified fibroin from which the tag sequence has been separated can also be recovered.
　タグ配列を含む改変フィブロインのより具体的な例として、（２－ｉｉｉ）配列番号１２（ＰＲＴ３８０）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しくは配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（２－ｉｖ）配列番号１２、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the modified fibroin containing a tag sequence, (2-iii) the amino acid represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
　配列番号１６（ＰＲＴ３１３）、配列番号１２、配列番号１３、配列番号１４及び配列番号１５で示されるアミノ酸配列は、それぞれ配列番号１０、配列番号６、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. An amino acid sequence represented by SEQ ID NO: 11 (including a His tag sequence and a hinge sequence) is added to the N-terminal of the amino acid sequence shown.
　（２－ｉｉｉ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
　（２－ｉｖ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（２－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（２－ｉｖ）の改変フィブロインは、配列番号１２、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＲＥＰ中に含まれるＸＧＸ（但し、Ｘはグリシン以外のアミノ酸残基を示す。）からなるアミノ酸配列の総アミノ酸残基数をｚとし、上記ドメイン配列中のＲＥＰの総アミノ酸残基数をｗとしたときに、ｚ／ｗが５０．９％以上であることが好ましい。 The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, and contains XGX ( Where X represents an amino acid residue other than glycine.) When z is the total number of amino acid residues in the amino acid sequence consisting of Is preferably 50.9% or more.
　第２の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 {Circle around (2)} The second modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたアミノ酸配列を有する。第３の改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものということができる。 The third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of the (A) n motif as compared to a naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to naturally occurring fibroin.
　第３の改変フィブロインは、天然由来のフィブロインから（Ａ）ｎモチーフを１０～４０％欠失させたことに相当するアミノ酸配列を有するものであってもよい。 The third modified fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif from naturally occurring fibroin.
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって１～３つの（Ａ）ｎモチーフ毎に１つの（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 The third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal side toward the C-terminal one to three (A) n motif every one (A) n motif May have an amino acid sequence corresponding to the deletion of
　第３の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、少なくともＮ末端側からＣ末端側に向かって２つ連続した（Ａ）ｎモチーフの欠失、及び１つの（Ａ）ｎモチーフの欠失がこの順に繰り返されたことに相当するアミノ酸配列を有するものであってもよい。 The third modified fibroin has a domain sequence deletion of at least two (A) n motifs from the N-terminal side to the C-terminal side, and one (A) It may have an amino acid sequence corresponding to the deletion of the n motif repeated in this order.
　第３の改変フィブロインは、そのドメイン配列が、少なくともＮ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。 The third modified fibroin may have a domain sequence having an amino acid sequence corresponding to the deletion of the (A) n motif at least every third sequence from the N-terminal side to the C-terminal side. .
　第３の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、Ｎ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが２０％以上、３０％以上、４０％以上又は５０％以上であるアミノ酸配列を有するものであってもよい。（Ａ）ｎモチーフ中の全アミノ酸残基数に対するアラニン残基数は８３％以上であってよいが、８６％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることが更に好ましく、１００％であること（アラニン残基のみで構成されることを意味する）が更により好ましい。 The third modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side to the C-terminal side] -REP] The number of amino acid residues of REP in the unit is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to When the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units that is 11.3 is x, and the total number of amino acid residues in the domain sequence is y In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
　ｘ／ｙの算出方法を図１を参照しながら更に詳細に説明する。図１には、改変フィブロインからＮ末端配列及びＣ末端配列を除いたドメイン配列を示す。当該ドメイン配列は、Ｎ末端側（左側）から（Ａ）ｎモチーフ－第１のＲＥＰ（５０アミノ酸残基）－（Ａ）ｎモチーフ－第２のＲＥＰ（１００アミノ酸残基）－（Ａ）ｎモチーフ－第３のＲＥＰ（１０アミノ酸残基）－（Ａ）ｎモチーフ－第４のＲＥＰ（２０アミノ酸残基）－（Ａ）ｎモチーフ－第５のＲＥＰ（３０アミノ酸残基）－（Ａ）ｎモチーフという配列を有する。 The method of calculating x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called an n motif.
　隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットは、重複がないように、Ｎ末端側からＣ末端側に向かって、順次選択する。このとき、選択されない［（Ａ）ｎモチーフ－ＲＥＰ］ユニットが存在してもよい。図１には、パターン１（第１のＲＥＰと第２のＲＥＰの比較、及び第３のＲＥＰと第４のＲＥＰの比較）、パターン２（第１のＲＥＰと第２のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン３（第２のＲＥＰと第３のＲＥＰの比較、及び第４のＲＥＰと第５のＲＥＰの比較）、パターン４（第１のＲＥＰと第２のＲＥＰの比較）を示した。なお、これ以外にも選択方法は存在する。 Two adjacent [(A) n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so that there is no overlap. At this time, there may be an unselected [(A) n motif-REP] unit. FIG. 1 shows pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP and the second REP, and Pattern 4 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (the comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP with the fifth REP). Second REP comparison). Note that there are other selection methods.
　次に各パターンについて、選択した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニット中の各ＲＥＰのアミノ酸残基数を比較する。比較は、よりアミノ酸残基数の少ない方を１としたときの、他方のアミノ酸残基数の比を求めることによって行う。例えば、第１のＲＥＰ（５０アミノ酸残基）と第２のＲＥＰ（１００アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第１のＲＥＰを１としたとき、第２のＲＥＰのアミノ酸残基数の比は、１００／５０＝２である。同様に、第４のＲＥＰ（２０アミノ酸残基）と第５のＲＥＰ（３０アミノ酸残基）の比較の場合、よりアミノ酸残基数の少ない第４のＲＥＰを１としたとき、第５のＲＥＰのアミノ酸残基数の比は、３０／２０＝１．５である。 Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of the other amino acid residues when the smaller number of the amino acid residues is set to 1. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP is The ratio of the number of amino acid residues is 100/50 = 2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is set to 1, the fifth REP Is 30/20 = 1.5.
　図１中、よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８～１１．３となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組を実線で示した。本明細書中、この比をギザ比率と呼ぶ。よりアミノ酸残基数の少ない方を１としたときに、他方のアミノ酸残基数の比が１．８未満又は１１．３超となる［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの組は破線で示した。 In FIG. 1, when the smaller number of amino acid residues is set to 1, the [(A) n motif-REP] unit pair in which the ratio of the other amino acid residues is 1.8 to 11.3 is set. Shown by solid line. In the present specification, this ratio is called a jagged ratio. Assuming that the smaller number of amino acid residues is 1, the pair of [(A) n motif-REP] units in which the ratio of the other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line. Indicated.
　各パターンにおいて、実線で示した隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットの全てのアミノ酸残基数を足し合わせる（ＲＥＰのみではなく、（Ａ）ｎモチーフのアミノ酸残基数もである。）。そして、足し合わせた合計値を比較して、当該合計値が最大となるパターンの合計値（合計値の最大値）をｘとする。図１に示した例では、パターン１の合計値が最大である。 In each pattern, the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
　次に、ｘをドメイン配列の総アミノ酸残基数ｙで除すことによって、ｘ／ｙ（％）を算出することができる。 Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
　第３の改変フィブロインにおいて、ｘ／ｙは、５０％以上であることが好ましく、６０％以上であることがより好ましく、６５％以上であることが更に好ましく、７０％以上であることが更により好ましく、７５％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、例えば、１００％以下であってよい。ギザ比率が１：１．９～１１．３の場合には、ｘ／ｙは８９．６％以上であることが好ましく、ギザ比率が１：１．８～３．４の場合には、ｘ／ｙは７７．１％以上であることが好ましく、ギザ比率が１：１．９～８．４の場合には、ｘ／ｙは７５．９％以上であることが好ましく、ギザ比率が１：１．９～４．１の場合には、ｘ／ｙは６４．２％以上であることが好ましい。 In the third modified fibroin, x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and even more preferably at least 70%. Preferably, it is still more preferably at least 75%, particularly preferably at least 80%. The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y. / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
　第３の改変フィブロインが、ドメイン配列中に複数存在する（Ａ）ｎモチーフの少なくとも７つがアラニン残基のみで構成される改変フィブロインである場合、ｘ／ｙは、４６．４％以上であることが好ましく、５０％以上であることがより好ましく、５５％以上であることが更に好ましく、６０％以上であることが更により好ましく、７０％以上であることが更によりまた好ましく、８０％以上であることが特に好ましい。ｘ／ｙの上限に特に制限はなく、１００％以下であればよい。 When the third modified fibroin is a modified fibroin in which at least seven of the (A) n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y should be 46.4% or more. Is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, and even more preferably 80% or more. It is particularly preferred that there is. The upper limit of x / y is not particularly limited, and may be 100% or less.
　ここで、天然由来のフィブロインにおけるｘ／ｙについて説明する。まず、上述のように、ＮＣＢＩ　ＧｅｎＢａｎｋにアミノ酸配列情報が登録されているフィブロインを例示した方法により確認したところ、６６３種類のフィブロイン（このうち、クモ類由来のフィブロインは４１５種類）が抽出された。抽出された全てのフィブロインのうち、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列で構成される天然由来のフィブロインのアミノ酸配列から、上述の算出方法により、ｘ／ｙを算出した。ギザ比率が１：１．９～４．１の場合の結果を図３に示す。 Here, x / y in naturally occurring fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the exemplified method, 663 types of fibroins (among them, 415 types of spider-derived fibroins) were extracted. Among all the extracted fibroins, x / y was calculated from the amino acid sequence of the naturally occurring fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above calculation method. Was calculated. FIG. 3 shows the results when the indentation ratio is 1: 1.9 to 4.1.
　図３の横軸はｘ／ｙ（％）を示し、縦軸は頻度を示す。図３から明らかなとおり、天然由来のフィブロインにおけるｘ／ｙは、いずれも６４．２％未満である（最も高いもので、６４．１４％）。 横 The horizontal axis in FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is evident from FIG. 3, the x / y of the naturally derived fibroin is less than 64.2% (the highest is 64.14%).
　第３の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列から、ｘ／ｙが６４．２％以上になるように（Ａ）ｎモチーフをコードする配列の１又は複数を欠失させることにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から、ｘ／ｙが６４．２％以上になるように１又は複数の（Ａ）ｎモチーフが欠失したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列から（Ａ）ｎモチーフが欠失したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The third modified fibroin, for example, deletes one or more of the sequence encoding the (A) n motif from the cloned natural fibroin gene sequence such that x / y is 64.2% or more. Can be obtained. Further, for example, an amino acid sequence corresponding to deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more based on the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. Amino acid sequence modification corresponding to the above may be performed.
　第３の改変フィブロインのより具体的な例として、（３－ｉ）配列番号１７（Ｍｅｔ－ＰＲＴ３９９）、配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）若しくは配列番号９（Ｍｅｔ－ＰＲＴ７９９）で示されるアミノ酸配列、又は（３－ｉｉ）配列番号１７、配列番号７、配列番号８若しくは配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), or SEQ ID NO: 9 (Met-PRT525) -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, Modified fibroin can be mentioned.
　（３－ｉ）の改変フィブロインについて説明する。配列番号１７で示されるアミノ酸配列は、天然由来のフィブロインに相当する配列番号１０（Ｍｅｔ－ＰＲＴ３１３）で示されるアミノ酸配列から、Ｎ末端側からＣ末端側に向かって２つおきに（Ａ）ｎモチーフを欠失させ、更にＣ末端配列の手前に［（Ａ）ｎモチーフ－ＲＥＰ］を１つ挿入したものである。配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列は、第２の改変フィブロインで説明したとおりである。 The modified fibroin (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 17 differs from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin in that every two amino acids from the N-terminal side to the C-terminal side (A) n The motif was deleted, and one [(A) n motif-REP] was inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 is as described for the second modified fibroin.
　配列番号１０で示されるアミノ酸配列（天然由来のフィブロインに相当）のギザ比率１：１．８～１１．３におけるｘ／ｙの値は１５．０％である。配列番号１７で示されるアミノ酸配列、及び配列番号７で示されるアミノ酸配列におけるｘ／ｙの値は、いずれも９３．４％である。配列番号８で示されるアミノ酸配列におけるｘ／ｙの値は、９２．７％である。配列番号９で示されるアミノ酸配列におけるｘ／ｙの値は、８９．８％である。配列番号１０、配列番号１７、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列におけるｚ／ｗの値は、それぞれ４６．８％、５６．２％、７０．１％、６６．１％及び７０．０％である。 Ｘ The amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8-11.3. The value of x / y in the amino acid sequence represented by SEQ ID NO: 17 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
　（３－ｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (3-i) may be composed of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
　（３－ｉｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（３－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（３－ｉｉ）の改変フィブロインは、配列番号１７、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３（ギザ比率が１：１．８～１１．３）となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 The modified fibroin (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, and is N-terminal to C-terminal. , The number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other Amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (a giza ratio of 1: 1.8 to 11.3). It is preferable that x / y be 64.2% or more, where x is the maximum value of the sum of the base numbers and y is the total number of amino acid residues in the domain sequence.
　第３の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方に上述したタグ配列を含んでいてもよい。 The third modified fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
　タグ配列を含む改変フィブロインのより具体的な例として、（３－ｉｉｉ）配列番号１８（ＰＲＴ３９９）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しくは配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（３－ｉｖ）配列番号１８、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the modified fibroin containing a tag sequence, (3-iii) the amino acid represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525), or SEQ ID NO: 15 (PRT799) Modified fibroin comprising a sequence or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned. .
　配列番号１８、配列番号１３、配列番号１４及び配列番号１５で示されるアミノ酸配列は、それぞれ配列番号１７、配列番号７、配列番号８及び配列番号９で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are obtained by adding SEQ ID NO: 11 to the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. (Including a His tag sequence and a hinge sequence).
　（３－ｉｉｉ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
　（３－ｉｖ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（３－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (3-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（３－ｉｖ）の改変フィブロインは、配列番号１８、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつＮ末端側からＣ末端側に向かって、隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのＲＥＰのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないＲＥＰのアミノ酸残基数を１としたとき、他方のＲＥＰのアミノ酸残基数の比が１．８～１１．３となる隣合う２つの［（Ａ）ｎモチーフ－ＲＥＰ］ユニットのアミノ酸残基数を足し合わせた合計値の最大値をｘとし、ドメイン配列の総アミノ酸残基数をｙとしたときに、ｘ／ｙが６４．２％以上であることが好ましい。 The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is N-terminal to C-terminal. , The number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and when the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 is defined as x. Preferably, x / y is 64.2% or more, where y is the total number of amino acid residues in the domain sequence.
　第３の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 The third modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
　第４の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、（Ａ）ｎモチーフの含有量が低減されたことに加え、グリシン残基の含有量が低減されたアミノ酸配列を有するものである。第４の改変フィブロインのドメイン配列は、天然由来のフィブロインと比較して、少なくとも１又は複数の（Ａ）ｎモチーフが欠失したことに加え、更に少なくともＲＥＰ中の１又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。すなわち、第４の改変フィブロインは、上述した第２の改変フィブロインと、第３の改変フィブロインの特徴を併せ持つ改変フィブロインである。具体的な態様等は、第２の改変フィブロイン、及び第３の改変フィブロインで説明したとおりである。 The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of a glycine residue in addition to the content of the (A) n motif reduced as compared to a naturally-derived fibroin. Have The domain sequence of the fourth modified fibroin is at least one or more (A) n motifs deleted, and further at least one or more glycine residues in the REP, as compared to naturally occurring fibroin. It can be said that it has an amino acid sequence equivalent to being replaced with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects and the like are as described for the second modified fibroin and the third modified fibroin.
　第４の改変フィブロインのより具体的な例として、（４－ｉ）配列番号７（Ｍｅｔ－ＰＲＴ４１０）、配列番号８（Ｍｅｔ－ＰＲＴ５２５）、配列番号９（Ｍｅｔ－ＰＲＴ７９９）、配列番号１３（ＰＲＴ４１０）、配列番号１４（ＰＲＴ５２５）若しくは配列番号１５（ＰＲＴ７９９）で示されるアミノ酸配列、又は（４－ｉｉ）配列番号７、配列番号８、配列番号９、配列番号１３、配列番号１４若しくは配列番号１５で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。配列番号７、配列番号８、配列番号９、配列番号１３、配列番号１４又は配列番号１５で示されるアミノ酸配列を含む改変フィブロインの具体的な態様は上述のとおりである。 As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 And a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 are as described above.
　第５の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する、局所的に疎水性指標の大きい領域を含むアミノ酸配列を有するものであってよい。 The fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP have been replaced by amino acid residues having a large hydrophobicity index as compared to naturally occurring fibroin, and / or It may have an amino acid sequence locally including a region having a large hydrophobicity index, corresponding to insertion of one or more amino acid residues having a large hydrophobicity index therein.
　局所的に疎水性指標の大きい領域は、連続する２～４アミノ酸残基で構成されていることが好ましい。 領域 A region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
　上述の疎水性指標の大きいアミノ酸残基は、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましい。 The amino acid residue having a large hydrophobicity index is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). More preferably, it is a residue.
　第５の改変フィブロインは、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に、天然由来のフィブロインと比較して、１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変があってもよい。 The fifth modified fibroin may have one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index, and / or one or more amino acids in the REP as compared to a naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, one or more amino acid residues may be substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be an amino acid sequence modification corresponding to the above.
　第５の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列からＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変を行ってもよい。 The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned natural fibroin gene sequence, It can be obtained by substituting a group (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. In addition, for example, one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP. Can be obtained by designing an amino acid sequence corresponding to the insertion of the amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more hydrophilic amino acid residues in REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.
　第５の改変フィブロインは、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含み、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフから上記ドメイン配列のＣ末端までの配列を上記ドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であるアミノ酸配列を有してもよい。 The fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence. In all REPs included in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p, When the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q, p / q is 6 .2% or more.
　アミノ酸残基の疎水性指標については、公知の指標（Ｈｙｄｒｏｐａｔｈｙ　ｉｎｄｅｘ：Ｋｙｔｅ　Ｊ，＆Ｄｏｏｌｉｔｔｌｅ　Ｒ（１９８２）“Ａ　ｓｉｍｐｌｅ　ｍｅｔｈｏｄ　ｆｏｒ　ｄｉｓｐｌａｙｉｎｇ　ｔｈｅ　ｈｙｄｒｏｐａｔｈｉｃ　ｃｈａｒａｃｔｅｒ　ｏｆ　ａ　ｐｒｏｔｅｉｎ”，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，１５７，ｐｐ．１０５－１３２）を使用する。具体的には、各アミノ酸の疎水性指標（ハイドロパシー・インデックス、以下「ＨＩ」とも記す。）は、下記表１に示すとおりである。 Regarding the hydrophobicity index of amino acid residues, a publicly known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132). Specifically, the hydropathic index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ｐ／ｑの算出方法を更に詳細に説明する。算出には、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列から、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列を除いた配列（以下、「配列Ａ」とする）を用いる。まず、配列Ａに含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値を算出する。疎水性指標の平均値は、連続する４アミノ酸残基に含まれる各アミノ酸残基のＨＩの総和を４（アミノ酸残基数）で除して求める。疎水性指標の平均値は、全ての連続する４アミノ酸残基について求める（各アミノ酸残基は、１～４回平均値の算出に用いられる。）。次いで、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域を特定する。あるアミノ酸残基が、複数の「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」に該当する場合であっても、領域中には１アミノ酸残基として含まれることになる。そして、当該領域に含まれるアミノ酸残基の総数がｐである。また、配列Ａに含まれるアミノ酸残基の総数がｑである。 The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”). First, in all REPs included in sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
　例えば、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が２０カ所抽出された場合（重複はなし）、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、連続する４アミノ酸残基（重複はなし）が２０含まれることになり、ｐは２０×４＝８０である。また、例えば、２つの「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が１アミノ酸残基だけ重複して存在する場合、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域には、７アミノ酸残基含まれることになる（ｐ＝２×４－１＝７。「－１」は重複分の控除である。）。例えば、図４に示したドメイン配列の場合、「疎水性指標の平均値が２．６以上となる連続する４アミノ酸残基」が重複せずに７つ存在するため、ｐは７×４＝２８となる。また、例えば、図４に示したドメイン配列の場合、ｑは４＋５０＋４＋４０＋４＋１０＋４＋２０＋４＋３０＝１７０である（Ｃ末端側の最後に存在する（Ａ）ｎモチーフは含めない）。次に、ｐをｑで除すことによって、ｐ／ｑ（％）を算出することができる。図４の場合２８／１７０＝１６．４７％となる。 For example, when “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a deduction for the overlap). For example, in the case of the domain sequence shown in FIG. 4, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping. 28. Also, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (the last (A) n motif on the C-terminal side is not included). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.
　第５の改変フィブロインにおいて、ｐ／ｑは、６．２％以上であることが好ましく、７％以上であることがより好ましく、１０％以上であることが更に好ましく、２０％以上であることが更により好ましく、３０％以上であることが更によりまた好ましい。ｐ／ｑの上限は、特に制限されないが、例えば、４５％以下であってもよい。 In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
　第５の改変フィブロインは、例えば、クローニングした天然由来のフィブロインのアミノ酸配列を、上記のｐ／ｑの条件を満たすように、ＲＥＰ中の１又は複数の親水性アミノ酸残基（例えば、疎水性指標がマイナスであるアミノ酸残基）を疎水性アミノ酸残基（例えば、疎水性指標がプラスであるアミノ酸残基）に置換すること、及び／又はＲＥＰ中に１又は複数の疎水性アミノ酸残基を挿入することにより、局所的に疎水性指標の大きい領域を含むアミノ酸配列に改変することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列から上記のｐ／ｑの条件を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び／又はＲＥＰ中に１又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当する改変を行ってもよい。 The fifth modified fibroin is, for example, one or a plurality of hydrophilic amino acid residues (for example, a hydrophobicity index) in the REP so that the amino acid sequence of the cloned natural fibroin is satisfied so as to satisfy the above-mentioned p / q conditions. Substituting a negative amino acid residue with a hydrophobic amino acid residue (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into the REP By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can also be obtained by designing an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP have been replaced by amino acid residues with a higher hydrophobicity index and / or one or more amino acid residues in the REP as compared to naturally occurring fibroin. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, a modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues may be performed. .
　疎水性指標の大きいアミノ酸残基としては、特に制限はないが、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）が好ましく、バリン（Ｖ）、ロイシン（Ｌ）及びイソロイシン（Ｉ）がより好ましい。 The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
　第５の改変フィブロインのより具体的な例として、（５－ｉ）配列番号１９（Ｍｅｔ－ＰＲＴ７２０）、配列番号２０（Ｍｅｔ－ＰＲＴ６６５）若しくは配列番号２１（Ｍｅｔ－ＰＲＴ６６６）で示されるアミノ酸配列、又は（５－ｉｉ）配列番号１９、配列番号２０若しくは配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As a more specific example of the fifth modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT665), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); Or (5-ii) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
　（５－ｉ）の改変フィブロインについて説明する。配列番号１９で示されるアミノ酸配列は、配列番号７（Ｍｅｔ－ＰＲＴ４１０）で示されるアミノ酸配列に対し、Ｃ末端側の端末のドメイン配列を除いて、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入し、更に一部のグルタミン（Ｑ）残基をセリン（Ｓ）残基に置換し、かつＣ末端側の一部のアミノ酸を欠失させたものである。配列番号２０で示されるアミノ酸配列は、配列番号８（Ｍｅｔ－ＰＲＴ５２５）で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を１カ所挿入したものである。配列番号２１で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列に対し、ＲＥＰ一つ置きにそれぞれ３アミノ酸残基からなるアミノ酸配列（ＶＬＩ）を２カ所挿入したものである。 The modified fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 19 consists of three amino acid residues every other REP, except for the domain sequence of the terminal at the C-terminal side, with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). In this amino acid sequence, two amino acid sequences (VLI) were inserted, some glutamine (Q) residues were further substituted with serine (S) residues, and some C-terminal amino acids were deleted. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525). is there. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 8.
　（５－ｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
　（５－ｉｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（５－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（５－ｉｉ）の改変フィブロインは、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and is located at the most C-terminal side (A) n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
　第５の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。 The fifth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
　タグ配列を含む改変フィブロインのより具体的な例として、（５－ｉｉｉ）配列番号２２（ＰＲＴ７２０）、配列番号２３（ＰＲＴ６６５）若しくは配列番号２４（ＰＲＴ６６６）で示されるアミノ酸配列、又は（５－ｉｖ）配列番号２２、配列番号２３若しくは配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。 As more specific examples of the modified fibroin containing a tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv) ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
　配列番号２２、配列番号２３及び配列番号２４で示されるアミノ酸配列は、それぞれ配列番号１９、配列番号２０及び配列番号２１で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。 The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 correspond to the amino acid sequence represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
　（５－ｉｉｉ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
　（５－ｉｖ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（５－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
　（５－ｉｖ）の改変フィブロインは、配列番号２２、配列番号２３又は配列番号２４で示されるアミノ酸配列と９０％以上の配列同一性を有し、かつ最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、連続する４アミノ酸残基の疎水性指標の平均値が２．６以上となる領域に含まれるアミノ酸残基の総数をｐとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をｑとしたときに、ｐ／ｑが６．２％以上であることが好ましい。 The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located at the most C-terminal side (A) n In all REPs contained in the sequence excluding the sequence from the motif to the C-terminus of the domain sequence from the domain sequence, amino acids contained in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more When the total number of residues is p, and the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is q , P / q is preferably at least 6.2%.
　第５の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 (5) The fifth modified fibroin may include a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
　第６の改変フィブロインは、天然由来のフィブロインと比較して、グルタミン残基の含有量が低減されたアミノ酸配列を有する。 The sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced as compared with naturally occurring fibroin.
　第６の改変フィブロインは、ＲＥＰのアミノ酸配列中に、ＧＧＸモチーフ及びＧＰＧＸＸモチーフから選ばれる少なくとも一つのモチーフが含まれていることが好ましい。 ６ The sixth modified fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
　第６の改変フィブロインが、ＲＥＰ中にＧＰＧＸＸモチーフを含む場合、ＧＰＧＸＸモチーフ含有率は、通常１％以上であり、５％以上であってもよく、１０％以上であるのが好ましい。ＧＰＧＸＸモチーフ含有率の上限に特に制限はなく、５０％以下であってよく、３０％以下であってもよい。 場合 When the sixth modified fibroin contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
　本明細書において、「ＧＰＧＸＸモチーフ含有率」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列に含まれる全てのＲＥＰにおいて、その領域に含まれるＧＰＧＸＸモチーフの個数の総数を３倍した数（即ち、ＧＰＧＸＸモチーフ中のＧ及びＰの総数に相当）をｓとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、ＧＰＧＸＸモチーフ含有率はｓ／ｔとして算出される。 In the present specification, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the number of GPGXX motifs contained in the region of all REPs contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total number by 3 (ie, the total number of G and P in the GPGXX motif) is represented by s, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is represented (A) When the total number of amino acid residues of all REPs excluding the (A) n motif is represented by t, the content of the GPGXX motif is calculated as s / t. It is.
　ＧＰＧＸＸモチーフ含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としているのは、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列」（ＲＥＰに相当する配列）には、フィブロインに特徴的な配列と相関性の低い配列が含まれることがあり、ｍが小さい場合（つまり、ドメイン配列が短い場合）、ＧＰＧＸＸモチーフ含有率の算出結果に影響するので、この影響を排除するためである。なお、ＲＥＰのＣ末端に「ＧＰＧＸＸモチーフ」が位置する場合、「ＸＸ」が例えば「ＡＡ」の場合であっても、「ＧＰＧＸＸモチーフ」として扱う。 In the calculation of the content ratio of the GPGXX motif, “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) Sequence from n motif to C-terminus of domain sequence (sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of fibroin, and m may be small. In this case (that is, when the domain sequence is short), the calculation result of the GPGXX motif content is affected, so that this effect is eliminated. When the “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
　図５は、改変フィブロインのドメイン配列を示す模式図である。図５を参照しながらＧＰＧＸＸモチーフ含有率の算出方法を具体的に説明する。まず、図５に示した改変フィブロインのドメイン配列（「［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフ」タイプである。）では、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図５中、「領域Ａ」で示した配列。）に含まれているため、ｓを算出するためのＧＰＧＸＸモチーフの個数は７であり、ｓは７×３＝２１となる。同様に、全てのＲＥＰが「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」（図５中、「領域Ａ」で示した配列。）に含まれているため、当該配列から更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数ｔは５０＋４０＋１０＋２０＋３０＝１５０である。次に、ｓをｔで除すことによって、ｓ／ｔ（％）を算出することができ、図５の改変フィブロインの場合２１／１５０＝１４．０％となる。 FIG. 5 is a schematic diagram showing the domain sequence of a modified fibroin. The method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) n motif-REP] m- (A) n motif” type), all REPs are located at the most C-terminal side. (A) A sequence obtained by removing the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence ”(the sequence shown as“ region A ”in FIG. 5). The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are “sequences obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” (the sequence shown as “region A” in FIG. 5). ), The total number t of amino acid residues of all REPs excluding the (A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t (%) can be calculated. In the case of the modified fibroin of FIG. 5, 21/150 = 14.0%.
　第６の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましく、７％以下であることがより好ましく、４％以下であることが更に好ましく、０％であることが特に好ましい。 The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%. .
　本明細書において、「グルタミン残基含有率」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図５の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域に含まれるグルタミン残基の総数をｕとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、グルタミン残基含有率はｕ／ｔとして算出される。グルタミン残基含有率の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, the “glutamine residue content” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the total number of glutamine residues contained in the region is defined as u, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
　第６の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、又は他のアミノ酸残基に置換したことに相当するアミノ酸配列を有するものであってよい。 The sixth modified fibroin corresponds to the fact that its domain sequence has one or more glutamine residues in the REP deleted or replaced with other amino acid residues, as compared to the naturally occurring fibroin. It may have an amino acid sequence.
　「他のアミノ酸残基」は、グルタミン残基以外のアミノ酸残基であればよいが、グルタミン残基よりも疎水性指標の大きいアミノ酸残基であることが好ましい。アミノ酸残基の疎水性指標は表１に示すとおりである。 The “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.
　表１に示すとおり、グルタミン残基よりも疎水性指標の大きいアミノ酸残基としては、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）アラニン（Ａ）、グリシン（Ｇ）、スレオニン（Ｔ）、セリン（Ｓ）、トリプトファン（Ｗ）、チロシン（Ｙ）、プロリン（Ｐ）及びヒスチジン（Ｈ）から選ばれるアミノ酸残基を挙げることができる。これらの中でも、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）、フェニルアラニン（Ｆ）、システイン（Ｃ）、メチオニン（Ｍ）及びアラニン（Ａ）から選ばれるアミノ酸残基であることがより好ましく、イソロイシン（Ｉ）、バリン（Ｖ）、ロイシン（Ｌ）及びフェニルアラニン（Ｆ）から選ばれるアミノ酸残基であることが更に好ましい。 As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can. Among them, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
　第６の改変フィブロインは、ＲＥＰの疎水性度が、－０．８以上であることが好ましく、－０．７以上であることがより好ましく、０以上であることが更に好ましく、０．３以上であることが更により好ましく、０．４以上であることが特に好ましい。ＲＥＰの疎水性度の上限に特に制限はなく、１．０以下であってよく、０．７以下であってもよい。 The sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more, and 0.3 or more. Is still more preferable, and it is particularly preferable that it is 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
　本明細書において、「ＲＥＰの疎水性度」は、以下の方法により算出される値である。
　式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むフィブロイン（改変フィブロイン又は天然由来のフィブロイン）において、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列（図５の「領域Ａ」に相当する配列。）に含まれる全てのＲＥＰにおいて、その領域の各アミノ酸残基の疎水性指標の総和をｖとし、最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除き、更に（Ａ）ｎモチーフを除いた全ＲＥＰのアミノ酸残基の総数をｔとしたときに、ＲＥＰの疎水性度はｖ／ｔとして算出される。ＲＥＰの疎水性度の算出において、「最もＣ末端側に位置する（Ａ）ｎモチーフからドメイン配列のＣ末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, “REP hydrophobicity” is a value calculated by the following method.
Formula 1: Fibroin containing a domain sequence represented by [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif (modified fibroin or naturally occurring fibroin) Fibroin), the sequence (the sequence corresponding to “region A” in FIG. 5) in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence. In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is defined as v, and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence. A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding n motifs. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
　第６の改変フィブロインは、そのドメイン配列が、天然由来のフィブロインと比較して、ＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当する改変に加え、更に１又は複数のアミノ酸残基を置換、欠失、挿入及び／又は付加したことに相当するアミノ酸配列の改変があってもよい。 The sixth modified fibroin may have a domain sequence that is missing one or more glutamine residues in the REP and / or one or more glutamine residues in the REP, as compared to the naturally occurring fibroin. In addition to the modification corresponding to the substitution of with another amino acid residue, there may be an amino acid sequence modification equivalent to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .
　第６の改変フィブロインは、例えば、クローニングした天然由来のフィブロインの遺伝子配列からＲＥＰ中の１又は複数のグルタミン残基を欠失させること、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換することにより得ることができる。また、例えば、天然由来のフィブロインのアミノ酸配列からＲＥＰ中の１又は複数のグルタミン残基を欠失したこと、及び／又はＲＥＰ中の１又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。 The sixth modified fibroin may, for example, delete one or more glutamine residues in the REP from the cloned naturally occurring fibroin gene sequence and / or remove one or more glutamine residues in the REP. By substituting the amino acid residue with In addition, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
　第６の改変フィブロインのより具体的な例として、（６－ｉ）配列番号２５（Ｍｅｔ－ＰＲＴ８８８）、配列番号２６（Ｍｅｔ－ＰＲＴ９６５）、配列番号２７（Ｍｅｔ－ＰＲＴ８８９）、配列番号２８（Ｍｅｔ－ＰＲＴ９１６）、配列番号２９（Ｍｅｔ－ＰＲＴ９１８）、配列番号３０（Ｍｅｔ－ＰＲＴ６９９）、配列番号３１（Ｍｅｔ－ＰＲＴ６９８）、配列番号３２（Ｍｅｔ－ＰＲＴ９６６）、配列番号４１（Ｍｅｔ－ＰＲＴ９１７）若しくは配列番号４２（Ｍｅｔ－ＰＲＴ１０２８）で示されるアミノ酸配列を含む改変フィブロイン、又は（６－ｉｉ）配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号４１若しくは配列番号４２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む改変フィブロインを挙げることができる。 As more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT988), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met-PRT889) -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence No. 42 (Met-PRT1028), or a modified fibroin comprising the amino acid sequence represented by (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 An amino acid represented by SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It can be mentioned modified fibroin comprising an amino acid sequence having a sequence at least 90% sequence identity.
　（６－ｉ）の改変フィブロインについて説明する。配列番号２５で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）中のＱＱを全てＶＬに置換したものである。配列番号２６で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＴＳに置換し、かつ残りのＱをＡに置換したものである。配列番号２７で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。配列番号２８で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＩに置換し、かつ残りのＱをＬに置換したものである。配列番号２９で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列中のＱＱを全てＶＦに置換し、かつ残りのＱをＩに置換したものである。 The modified fibroin of (6-i) will be described. The amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting VL for all QQ in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410). The amino acid sequence represented by SEQ ID NO: 26 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with TS, and substituting the remaining Q with A. The amino acid sequence represented by SEQ ID NO: 27 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VL, and substituting the remaining Q with I. The amino acid sequence represented by SEQ ID NO: 28 is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with VI and substituting the remaining Q with L. The amino acid sequence represented by SEQ ID NO: 29 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VF and substituting the remaining Q with I.
　配列番号３０で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ５２５）中のＱＱを全てＶＬに置換したものである。配列番号３１で示されるアミノ酸配列は、配列番号８で示されるアミノ酸配列中のＱＱを全てＶＬに置換し、かつ残りのＱをＩに置換したものである。 ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 30 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with VL. The amino acid sequence represented by SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence represented by SEQ ID NO: 8 with VL and substituting the remaining Q with I.
　配列番号３２で示されるアミノ酸配列は、配列番号７で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ４１０）中に存在する２０個のドメイン配列の領域を２回繰り返した配列中のＱＱを全てＶＦに置換し、かつ残りのＱをＩに置換したものである。 The amino acid sequence represented by SEQ ID NO: 32 replaces all QQs in the sequence obtained by repeating twice the domain of the 20 domain sequences present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) with VF, In addition, the remaining Q is replaced with I.
　配列番号４１で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ９１７）は、配列番号７で示されるアミノ酸配列中のＱＱを全てＬＩに置換し、かつ残りのＱをＶに置換したものである。配列番号４２で示されるアミノ酸配列（Ｍｅｔ－ＰＲＴ１０２８）は、配列番号７で示されるアミノ酸配列中のＱＱを全てＩＦに置換し、かつ残りのＱをＴに置換したものである。 ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 41 (Met-PRT917) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with LI and replacing the remaining Q with V. The amino acid sequence represented by SEQ ID NO: 42 (Met-PRT1028) is obtained by substituting all QQ in the amino acid sequence represented by SEQ ID NO: 7 with IF, and substituting the remaining Q with T.
　配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号４１及び配列番号４２で示されるアミノ酸配列は、いずれもグルタミン残基含有率は９％以下である（表２）。 The amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 all have glutamine residues. The group content is 9% or less (Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　（６－ｉ）の改変フィブロインは、配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号４１又は配列番号４２で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It may consist of the amino acid sequence shown.
　（６－ｉｉ）の改変フィブロインは、配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号４１又は配列番号４２で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（６－ｉｉ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (6-ii) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence. The sequence identity is preferably 95% or more.
　（６－ｉｉ）の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましい。また、（６－ｉｉ）の改変フィブロインは、ＧＰＧＸＸモチーフ含有率が１０％以上であることが好ましい。 The modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
　第６の改変フィブロインは、Ｎ末端及びＣ末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変フィブロインの単離、固定化、検出及び可視化等が可能となる。 ６The sixth modified fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified fibroin can be isolated, immobilized, detected, visualized, and the like.
　タグ配列を含む改変フィブロインのより具体的な例として、（６－ｉｉｉ）配列番号３３（ＰＲＴ８８８）、配列番号３４（ＰＲＴ９６５）、配列番号３５（ＰＲＴ８８９）、配列番号３６（ＰＲＴ９１６）、配列番号３７（ＰＲＴ９１８）、配列番号３８（ＰＲＴ６９９）、配列番号３９（ＰＲＴ６９８）、配列番号４０（ＰＲＴ９６６）、配列番号４３（ＰＲＴ９１７）若しくは配列番号４４（ＰＲＴ１０２８）で示されるアミノ酸配列を含む改変フィブロイン、又は（６－ｉｖ）配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４３若しくは配列番号４４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含む改変フィブロインを挙げることができる。 As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 44 (PRT1028), or ( 6-iv) The amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 and 90 % Modified amino acid sequence having an amino acid sequence having at least Mention may be made of the emissions.
　配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４３及び配列番号４４で示されるアミノ酸配列は、それぞれ配列番号２５、配列番号２６、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号４１及び配列番号４２で示されるアミノ酸配列のＮ末端に配列番号１１で示されるアミノ酸配列（Ｈｉｓタグ配列及びヒンジ配列を含む）を付加したものである。Ｎ末端にタグ配列を付加しただけであるため、グルタミン残基含有率に変化はなく、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４３及び配列番号４４で示されるアミノ酸配列は、いずれもグルタミン残基含有率が９％以下である（表３）。 The amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, and SEQ ID NO: 44 correspond to SEQ ID NO: 25, respectively. SEQ ID NO: 11 at the N-terminal of the amino acid sequence represented by SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: Amino acid sequence (including a His tag sequence and a hinge sequence). Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　（６－ｉｉｉ）の改変フィブロインは、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４３又は配列番号４４で示されるアミノ酸配列からなるものであってもよい。 The modified fibroin of (6-iii) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.
　（６－ｉｖ）の改変フィブロインは、配列番号３３、配列番号３４、配列番号３５、配列番号３６、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４３又は配列番号４４で示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列を含むものである。（６－ｉｖ）の改変フィブロインもまた、式１：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ、又は式２：［（Ａ）ｎモチーフ－ＲＥＰ］ｍ－（Ａ）ｎモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、９５％以上であることが好ましい。 The modified fibroin of (6-iv) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif A protein containing a sequence. The sequence identity is preferably 95% or more.
　（６－ｉｖ）の改変フィブロインは、グルタミン残基含有率が９％以下であることが好ましい。また、（６－ｉｖ）の改変フィブロインは、ＧＰＧＸＸモチーフ含有率が１０％以上であることが好ましい。 The modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
　第６の改変フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。 The sixth modified fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
　改変フィブロインは、第１の改変フィブロイン、第２の改変フィブロイン、第３の改変フィブロイン、第４の改変フィブロイン、第５の改変フィブロイン、及び第６の改変フィブロインが有する特徴のうち、少なくとも２つ以上の特徴を併せ持つ改変フィブロインであってもよい。 The modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. Modified fibroin having both of the following characteristics may be used.
　改変フィブロインは、親水性改変フィブロインであってもよく、疎水性改変フィブロインであってもよい。本明細書において、「疎水性改変フィブロイン」とは、改変フィブロインを構成する全てのアミノ酸残基の疎水性指標（ＨＩ）の総和を求め、次にその総和を全アミノ酸残基数で除した値（平均ＨＩ）が０超である改変フィブロインである。疎水性指標は表１に示したとおりである。また、「親水性改変フィブロイン」とは、平均ＨＩが０以下である改変フィブロインである。改変フィブロインとしては、耐燃焼性に優れるという観点から、親水性改変フィブロインが好ましく、吸湿発熱性に優れるという観点からは、疎水性改変フィブロインが好ましい。 The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. As used herein, the term "hydrophobic modified fibroin" refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. Modified fibroin having an (average HI) greater than 0. The hydrophobicity index is as shown in Table 1. Further, “hydrophilic modified fibroin” is a modified fibroin having an average HI of 0 or less. As the modified fibroin, a hydrophilic modified fibroin is preferred from the viewpoint of excellent combustion resistance, and a hydrophobic modified fibroin is preferred from the viewpoint of excellent moisture absorption and heat generation.
　疎水性改変フィブロインとしては、例えば、配列番号２７、配列番号２８、配列番号２９、配列番号３０、配列番号３１、配列番号３２、配列番号３３又は配列番号４３で示されるアミノ酸配列、配列番号３５、配列番号３７、配列番号３８、配列番号３９、配列番号４０、配列番号４１又は配列番号４４で示されるアミノ酸配列を含む改変フィブロインが挙げられる。 Examples of the hydrophobically modified fibroin include the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, SEQ ID NO: 35, Modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.
　親水性改変フィブロインとしては、例えば、配列番号４で示されるアミノ酸配列、配列番号６、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列、配列番号１３、配列番号１１、配列番号１４又は配列番号１５で示されるアミノ酸配列、配列番号１８、配列番号７、配列番号８又は配列番号９で示されるアミノ酸配列、配列番号１７、配列番号１１、配列番号１４又は配列番号１５で示されるアミノ酸配列、配列番号１９、配列番号２０又は配列番号２１で示されるアミノ酸配列を含む改変フィブロインが挙げられる。 Examples of the hydrophilic modified fibroin include the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: Or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 Modified fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
　本実施形態に係る人造フィブロイン繊維は、改変フィブロインを１種単独で含有するものであってもよく、改変フィブロイン２種以上を組み合わせて含有するものであってもよい。 人 The artificial fibroin fiber according to the present embodiment may contain one kind of modified fibroin alone or may contain two or more kinds of modified fibroin in combination.
（改変フィブロインの製造方法）
　上記いずれの実施形態に係る改変フィブロインも、例えば、当該改変フィブロインをコードする核酸配列と、当該核酸配列に作動可能に連結された１又は複数の調節配列とを有する発現ベクターで形質転換された宿主により、当該核酸を発現させることにより生産することができる。 (Method for producing modified fibroin)
The modified fibroin according to any of the above embodiments may be, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
　改変フィブロインをコードする核酸の製造方法は、特に制限されない。例えば、天然のフィブロインをコードする遺伝子を利用して、ポリメラーゼ連鎖反応（ＰＣＲ）などで増幅しクローニングし、遺伝子工学的手法により改変する方法、又は、化学的に合成する方法によって、当該核酸を製造することができる。核酸の化学的な合成方法も特に制限されず、例えば、ＮＣＢＩのウェブデータベースなどより入手したフィブロインのアミノ酸配列情報をもとに、ＡＫＴＡ　ｏｌｉｇｏｐｉｌｏｔ　ｐｌｕｓ　１０／１００（ＧＥヘルスケア・ジャパン株式会社）などで自動合成したオリゴヌクレオチドをＰＣＲなどで連結する方法によって遺伝子を化学的に合成することができる。この際に、改変フィブロインの精製及び／又は確認を容易にするため、上記のアミノ酸配列のＮ末端に開始コドン及びＨｉｓ１０タグからなるアミノ酸配列を付加したアミノ酸配列からなる改変フィブロインをコードする核酸を合成してもよい。 方法 The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural fibroin and modifying it by a genetic engineering technique, or a chemical synthesis method. can do. The method for chemically synthesizing nucleic acids is not particularly limited. For example, based on amino acid sequence information of fibroin obtained from the NCBI web database or the like, AKTA oligopilot plus１００10 / 100 (GE Healthcare Japan Co., Ltd.) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like. At this time, to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence comprising an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. May be.
　調節配列は、宿主における改変フィブロインの発現を制御する配列（例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等）であり、宿主の種類に応じて適宜選択することができる。プロモーターとして、宿主細胞中で機能し、改変フィブロインを発現誘導可能な誘導性プロモーターを用いてもよい。誘導性プロモーターは、誘導物質（発現誘導剤）の存在、リプレッサー分子の非存在、又は温度、浸透圧若しくはｐＨ値の上昇若しくは低下等の物理的要因により、転写を制御できるプロモーターである。 The regulatory sequence is a sequence that controls the expression of the modified fibroin in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and is capable of inducing the expression of a modified fibroin may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
　発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等、宿主の種類に応じて適宜選択することができる。発現ベクターとしては、宿主細胞において自立複製が可能、又は宿主の染色体中への組込みが可能で、改変フィブロインをコードする核酸を転写できる位置にプロモーターを含有しているものが好適に用いられる。 種類 The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed are suitably used.
　宿主として、原核生物、並びに酵母、糸状真菌、昆虫細胞、動物細胞及び植物細胞等の真核生物のいずれも好適に用いることができる。 As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
　原核生物の宿主の好ましい例として、エシェリヒア属、ブレビバチルス属、セラチア属、バチルス属、ミクロバクテリウム属、ブレビバクテリウム属、コリネバクテリウム属及びシュードモナス属等に属する細菌を挙げることができる。エシェリヒア属に属する微生物として、例えば、エシェリヒア・コリ等を挙げることができる。ブレビバチルス属に属する微生物として、例えば、ブレビバチルス・アグリ等を挙げることができる。セラチア属に属する微生物として、例えば、セラチア・リクエファシエンス等を挙げることができる。バチルス属に属する微生物として、例えば、バチルス・サチラス等を挙げることができる。ミクロバクテリウム属に属する微生物として、例えば、ミクロバクテリウム・アンモニアフィラム等を挙げることができる。ブレビバクテリウム属に属する微生物として、例えば、ブレビバクテリウム・ディバリカタム等を挙げることができる。コリネバクテリウム属に属する微生物として、例えば、コリネバクテリウム・アンモニアゲネス等を挙げることができる。シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属に属する微生物として、例えば、シュードモナス・プチダ等を挙げることができる。 好 ま し い Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like. Examples of microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis. Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
　原核生物を宿主とする場合、改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ｐＢＴｒｐ２（ベーリンガーマンハイム社製）、ｐＧＥＸ（Ｐｈａｒｍａｃｉａ社製）、ｐＵＣ１８、ｐＢｌｕｅｓｃｒｉｐｔＩＩ、ｐＳｕｐｅｘ、ｐＥＴ２２ｂ、ｐＣｏｌｄ、ｐＵＢ１１０、ｐＮＣＯ２（特開２００２－２３８５６９号公報）等を挙げることができる。 When a prokaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
　真核生物の宿主としては、例えば、酵母及び糸状真菌（カビ等）を挙げることができる。酵母としては、例えば、サッカロマイセス属、ピキア属、シゾサッカロマイセス属等に属する酵母を挙げることができる。糸状真菌としては、例えば、アスペルギルス属、ペニシリウム属、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属等に属する糸状真菌を挙げることができる。 Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold). Examples of the yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
　真核生物を宿主とする場合、改変フィブロインをコードする核酸を導入するベクターとしては、例えば、ＹＥＰ１３（ＡＴＣＣ３７１１５）、ＹＥｐ２４（ＡＴＣＣ３７０５１）等を挙げることができる。上記宿主細胞への発現ベクターの導入方法としては、上記宿主細胞へＤＮＡを導入する方法であればいずれも用いることができる。例えば、カルシウムイオンを用いる方法〔Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．　ＵＳＡ，６９，２１１０（１９７２）〕、エレクトロポレーション法、スフェロプラスト法、プロトプラスト法、酢酸リチウム法、コンピテント法等を挙げることができる。 場合 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. {Natl. {Acad. {Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
　発現ベクターで形質転換された宿主による核酸の発現方法としては、直接発現のほか、モレキュラー・クローニング第２版に記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができる。 As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
　改変フィブロインは、例えば、発現ベクターで形質転換された宿主を培養培地中で培養し、培養培地中に当該改変フィブロインを生成及び蓄積させ、該培養培地から採取することにより製造することができる。宿主を培養培地中で培養する方法は、宿主の培養に通常用いられる方法に従って行うことができる。 The modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing the host in the culture medium can be performed according to a method usually used for culturing the host.
　宿主が、大腸菌等の原核生物又は酵母等の真核生物である場合、培養培地として、宿主が資化し得る炭素源、窒素源及び無機塩類等を含有し、宿主の培養を効率的に行える培地であれば天然培地及び合成培地のいずれを用いてもよい。 When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, a culture medium containing a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, so that the host can be cultured efficiently. If so, either a natural medium or a synthetic medium may be used.
　炭素源としては、上記形質転換微生物が資化し得るものであればよく、例えば、グルコース、フラクトース、スクロース、及びこれらを含有する糖蜜、デンプン及びデンプン加水分解物等の炭水化物、酢酸及びプロピオン酸等の有機酸、並びにエタノール及びプロパノール等のアルコール類を用いることができる。窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物を用いることができる。無機塩類としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムを用いることができる。 The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid Organic acids and alcohols such as ethanol and propanol can be used. As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
　大腸菌等の原核生物又は酵母等の真核生物の培養は、例えば、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことができる。培養温度は、例えば、１５～４０℃である。培養時間は、通常１６時間～７日間である。培養中の培養培地のｐＨは３．０～９．０に保持することが好ましい。培養培地のｐＨの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。 培養 Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is usually 16 hours to 7 days. The pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
　また、培養中、必要に応じて、アンピシリン及びテトラサイクリン等の抗生物質を培養培地に添加してもよい。プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、ｌａｃプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはイソプロピル－β－Ｄ－チオガラクトピラノシド等を、ｔｒｐプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。 抗 生 During the culture, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. An acid or the like may be added to the medium.
　発現させた改変フィブロインの単離及び精製は通常用いられている方法で行うことができる。例えば、当該改変フィブロインが、細胞内に溶解状態で発現した場合には、培養終了後、宿主細胞を遠心分離により回収し、水系緩衝液に懸濁した後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー及びダイノミル等により宿主細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、タンパク質の単離精製に通常用いられている方法、すなわち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル（ＤＥＡＥ）－セファロース、ＤＩＡＩＯＮ　ＨＰＡ－７５（三菱化成社製）等のレジンを用いた陰イオン交換クロマトグラフィー法、Ｓ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（Ｐｈａｒｍａｃｉａ社製）等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の方法を単独又は組み合わせて使用し、精製標品を得ることができる。 単 離 The expressed and modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The host cells are crushed with a Gaulin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia). Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.
　また、改変フィブロインが細胞内に不溶体を形成して発現した場合は、同様に宿主細胞を回収後、破砕し、遠心分離を行うことにより、沈殿画分として改変フィブロインの不溶体を回収する。回収した改変フィブロインの不溶体はタンパク質変性剤で可溶化することができる。該操作の後、上記と同様の単離精製法により改変フィブロインの精製標品を得ることができる。当該改変フィブロインが細胞外に分泌された場合には、培養上清から当該改変フィブロインを回収することができる。すなわち、培養物を遠心分離等の手法により処理することにより培養上清を取得し、その培養上清から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。 When the modified fibroin is expressed by forming an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to collect the insoluble form of the modified fibroin as a precipitate fraction. The recovered insoluble form of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
（人造フィブロイン原繊維の製造方法）
　本実施形態に係る人造フィブロイン原繊維は、上述した改変フィブロインを紡糸したものであり、上述した改変フィブロインを主成分として含む。本実施形態に係る人造フィブロイン原繊維は、紡糸後、水と接触する前の繊維である。 (Method of producing artificial fibroin fibrils)
The artificial fibroin fibril according to the present embodiment is obtained by spinning the above-mentioned modified fibroin, and contains the above-mentioned modified fibroin as a main component. The artificial fibroin fiber according to the present embodiment is a fiber after spinning and before contact with water.
　本実施形態に係る人造フィブロイン原繊維は、公知の紡糸方法によって製造することができる。すなわち、例えば、まず、上述した方法に準じて製造した改変フィブロインをジメチルスルホキシド（ＤＭＳＯ）、Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）、又はヘキサフルオロイソプロノール（ＨＦＩＰ）等の溶媒に、溶解促進剤としての無機塩と共に添加し、溶解してドープ液を作製する。次いで、このドープ液を用いて、湿式紡糸、乾式紡糸、乾湿式紡糸又は溶融紡糸等の公知の紡糸方法により紡糸して、目的とする人造フィブロイン原繊維を得ることができる。好ましい紡糸方法としては、湿式紡糸又は乾湿式紡糸を挙げることができる。 人 The artificial fibroin fibrils according to the present embodiment can be manufactured by a known spinning method. That is, for example, first, modified fibroin produced according to the above-described method is dissolved in a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), or hexafluoroisopronol (HFIP) as a dissolution promoter. And dissolve it to prepare a dope solution. Next, using this dope solution, spinning is performed by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning to obtain a desired artificial fibroin fibril. Preferred spinning methods include wet spinning and dry-wet spinning.
　図６は、人造フィブロイン原繊維を製造するための紡糸装置の一例を概略的に示す説明図である。図６に示す紡糸装置１０は、乾湿式紡糸用の紡糸装置の一例であり、押出し装置１と、未延伸糸製造装置２と、湿熱延伸装置３と、乾燥装置４とを有している。 FIG. 6 is an explanatory view schematically showing an example of a spinning apparatus for producing artificial fibroin fibrils. The spinning device 10 shown in FIG. 6 is an example of a spinning device for dry-wet spinning, and includes an extruder 1, an undrawn yarn manufacturing device 2, a wet heat drawing device 3, and a drying device 4.
　紡糸装置１０を使用した紡糸方法を説明する。まず、貯槽７に貯蔵されたドープ液６が、ギアポンプ８により口金９から押し出される。ラボスケールにおいては、ドープ液をシリンダーに充填し、シリンジポンプを用いてノズルから押し出してもよい。次いで、押し出されたドープ液６は、エアギャップ１９を経て、凝固液槽２０の凝固液１１内に供給され、溶媒が除去されて、改変フィブロインが凝固し、繊維状凝固体が形成される。次いで、繊維状凝固体が、延伸浴槽２１内の温水１２中に供給されて、延伸される。延伸倍率は供給ニップローラ１３と引き取りニップローラ１４との速度比によって決まる。その後、延伸された繊維状凝固体が、乾燥装置４に供給され、糸道２２内で乾燥されて、人造フィブロイン原繊維が、巻糸体５として得られる。１８ａ～１８ｇは糸ガイドである。 A spinning method using the spinning device 10 will be described. First, the dope solution 6 stored in the storage tank 7 is pushed out of the base 9 by the gear pump 8. In a laboratory scale, a dope solution may be filled in a cylinder and extruded from a nozzle using a syringe pump. Next, the extruded dope solution 6 is supplied into the coagulation solution 11 of the coagulation solution tank 20 via the air gap 19, the solvent is removed, and the modified fibroin is coagulated to form a fibrous coagulate. Next, the fibrous coagulated material is supplied into the hot water 12 in the stretching bath 21 and stretched. The stretching ratio is determined by the speed ratio between the supply nip roller 13 and the take-off nip roller 14. After that, the drawn fibrous coagulated material is supplied to the drying device 4 and dried in the yarn path 22, and the artificial fibroin raw fiber is obtained as the wound yarn 5. 18a to 18g are yarn guides.
　凝固液１１としては、脱溶媒できる溶媒であればよく、例えば、メタノール、エタノール及び２－プロパノール等の炭素数１～５の低級アルコール、並びにアセトン等を挙げることができる。凝固液１１は、適宜水を含んでいてもよい。凝固液１１の温度は、０～３０℃であることが好ましい。口金９として、直径０．１～０．６ｍｍのノズルを有するシリンジポンプを使用する場合、押出し速度は１ホール当たり、０．２～６．０ｍｌ／時間が好ましく、１．４～４．０ｍｌ／時間であることがより好ましい。凝固したタンパク質が凝固液１１中を通過する距離（実質的には、糸ガイド１８ａから糸ガイド１８ｂまでの距離）は、脱溶媒が効率的に行える長さがあればよく、例えば、２００～５００ｍｍである。未延伸糸の引き取り速度は、例えば、１～２０ｍ／分であってよく、１～３ｍ／分であることが好ましい。凝固液１１中での滞留時間は、例えば、０．０１～３分であってよく、０．０５～０．１５分であることが好ましい。また、凝固液１１中で延伸（前延伸）をしてもよい。凝固液槽２０は多段設けてもよく、また延伸は必要に応じて、各段、又は特定の段で行ってもよい。 The coagulating liquid 11 may be any solvent that can remove the solvent, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulating liquid 11 may appropriately contain water. The temperature of the coagulating liquid 11 is preferably 0 to 30 ° C. When a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm is used as the base 9, the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole, and 1.4 to 4.0 ml / hour. More preferably, it is time. The distance that the coagulated protein passes through the coagulating liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) may be long enough to efficiently remove the solvent, for example, 200 to 500 mm. It is. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and is preferably 1 to 3 m / min. The residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and is preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulating liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as needed.
　なお、人造フィブロイン原繊維を得る際に実施される延伸は、例えば、上記した凝固液槽２０内で行う前延伸、及び延伸浴槽２１内で行う湿熱延伸の他、乾熱延伸も採用される。 Note that the stretching performed when obtaining the artificial fibroin fibrils includes, for example, dry stretching in addition to wet stretching performed in the coagulation bath 20 and stretching bath 21 described above.
　湿熱延伸は、温水中、温水に有機溶剤等を加えた溶液中、又はスチーム加熱中で行うことができる。温度としては、例えば、５０～９０℃であってよく、７５～８５℃が好ましい。湿熱延伸では、未延伸糸（又は前延伸糸）を、例えば、１～１０倍延伸することができ、２～８倍延伸することが好ましい。 Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In the wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
　乾熱延伸は、電気管状炉、乾熱板等を使用して行うことができる。温度としては、例えば、１４０℃～２７０℃であってよく、１６０℃～２３０℃が好ましい。乾熱延伸では、未延伸糸（又は前延伸糸）を、例えば、０．５～８倍延伸することができ、１～４倍延伸することが好ましい。 Dry heat drawing can be performed using an electric tube furnace, a dry heat plate or the like. The temperature may be, for example, 140 ° C. to 270 ° C., preferably 160 ° C. to 230 ° C. In dry heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
　湿熱延伸及び乾熱延伸はそれぞれ単独で行ってもよく、またこれらを多段で、又は組み合わせて行ってもよい。すなわち、一段目延伸を湿熱延伸で行い、二段目延伸を乾熱延伸で行う、又は一段目延伸を湿熱延伸行い、二段目延伸を湿熱延伸行い、更に三段目延伸を乾熱延伸で行う等、湿熱延伸及び乾熱延伸を適宜組み合わせて行うことができる。 (4) The wet heat stretching and the dry heat stretching may each be performed alone, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and further the third stage stretching is performed by dry heat stretching. For example, wet heat stretching and dry heat stretching can be performed in an appropriate combination.
　最終的な延伸倍率は、その下限値が、未延伸糸（又は前延伸糸）に対して、好ましくは、１倍超、２倍以上、３倍以上、４倍以上、５倍以上、６倍以上、７倍以上、８倍以上、９倍以上のうちのいずれかであり、上限値が、好ましくは４０倍以下、３０倍以下、２０倍以下、１５倍以下、１４倍以下、１３倍以下、１２倍以下、１１倍以下、１０倍以下である。人造フィブロイン原繊維が２倍以上の延伸倍率で紡糸された繊維であると、人造フィブロイン原繊維を水に接触させて湿潤状態にした際の収縮率は、より高くなる。 The final draw ratio is preferably such that the lower limit is more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, and 6 times of the undrawn yarn (or pre-drawn yarn). Or more, 7 times or more, 8 times or more, 9 times or more, and the upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less. , 12 times or less, 11 times or less, 10 times or less. When the artificial fibroin fiber is a fiber spun at a draw ratio of 2 or more, the shrinkage when the artificial fibroin fiber is brought into a wet state by contact with water becomes higher.
（人造フィブロイン繊維の製造方法）
　本実施形態に係る人造フィブロイン繊維は、人造フィブロイン原繊維を水により収縮させる収縮工程を備える製造方法により得ることができる。収縮工程は、例えば、上述した人造フィブロイン原繊維（紡糸後、水と接触する前の繊維）を、水と接触させて不可逆的に収縮させるステップ（接触ステップ）を備えるものであってよい。収縮工程は、接触ステップの後、繊維を乾燥させて更に収縮させるステップ（乾燥ステップ）を備えるものであってもよい。 (Manufacturing method of artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment can be obtained by a manufacturing method including a shrinking step of shrinking the artificial fibroin raw fiber with water. The shrinking step may include, for example, a step (contact step) of bringing the aforementioned artificial fibroin fibrils (fibers after spinning and before contact with water) into contact with water to shrink irreversibly. The shrinking step may include, after the contacting step, a step of drying and further shrinking the fiber (drying step).
　図７は、水との接触による人造フィブロイン原繊維及び人造フィブロイン繊維の長さ変化の例を示す図である。本実施形態に係る人造フィブロイン原繊維は、水に接触（湿潤）させることにより不可逆的に収縮する（図７中、「一次収縮」で示した長さ変化）特性を有する。一次収縮後、乾燥させると更に収縮する（図７中、「二次収縮」で示した長さ変化）。一次収縮又は二次収縮を経て得られた人造フィブロイン繊維は、水に接触させて湿潤状態にすると二次収縮前と同一又はそれに近似した長さにまで伸長し、以後乾燥と湿潤を繰り返すと、二次収縮と同程度の幅（図７中、「伸縮率」で示した幅）で、収縮と伸長を繰り返す。 FIG. 7 is a diagram showing an example of length changes of artificial fibroin fibrils and artificial fibroin fibers due to contact with water. The artificial fibroin fibrils according to the present embodiment have the property of contracting irreversibly (contact length change in FIG. 7) by contact (wetting) with water. After the primary shrinkage, it is further shrunk when dried (the length change indicated by “secondary shrinkage” in FIG. 7). When the artificial fibroin fiber obtained through the primary shrinkage or the secondary shrinkage is brought into contact with water and brought into a wet state, it expands to the same length as that before the secondary shrinkage or to a length similar to that before the secondary shrinkage. The contraction and the elongation are repeated with a width approximately equal to the secondary contraction (the width indicated by “stretch ratio” in FIG. 7).
　接触ステップでの人造フィブロイン原繊維の不可逆的な収縮（図７中の「一次収縮」）は、例えば、以下の理由により生ずると考えられる。すなわち、一つの理由は、人造フィブロイン原繊維の二次構造又は三次構造に起因すると考えられ、また別の一つの理由は、例えば、製造工程での延伸等によって残留応力を有する人造フィブロイン原繊維において、水が繊維間又は繊維内へ浸入することにより、残留応力が緩和されることで生ずると考えられる。したがって、収縮工程での人造フィブロイン原繊維の収縮率は、例えば、上記した人造フィブロイン原繊維の製造過程での延伸倍率の大きさに応じて任意にコントロールすることもできると考えられる。 不可 Irreversible shrinkage of the artificial fibroin fibrils in the contact step (“primary shrinkage” in FIG. 7) is considered to occur, for example, for the following reasons. That is, one reason is considered to be due to the secondary structure or tertiary structure of artificial fibroin fibrils, and another reason is, for example, in artificial fibroin fibrils having residual stress due to stretching in the manufacturing process. It is considered that water is infiltrated into the fibers or into the fibers, so that the residual stress is reduced. Therefore, it is considered that the shrinkage rate of the artificial fibroin fibrils in the shrinking step can be arbitrarily controlled in accordance with, for example, the magnitude of the draw ratio in the process of producing the artificial fibroin fibrils described above.
　接触ステップでは、紡糸後、水と接触する前の人造フィブロイン原繊維を水と接触させて、人造フィブロイン原繊維を湿潤状態にする。湿潤状態とは、人造フィブロイン原繊維の少なくとも一部が水で濡れた状態を意味する。これにより、外力によらずに人造フィブロイン原繊維を収縮させることができる。この収縮は不可逆的なものである（図７の「一次収縮」に相当する）。 In the contacting step, the artificial fibroin fibrils before spinning are brought into contact with water after spinning to bring the artificial fibroin fibrils into a wet state. The wet state means a state where at least a part of the artificial fibroin fibrils is wet with water. Thereby, the artificial fibroin fibrils can be shrunk without external force. This contraction is irreversible (corresponding to "primary contraction" in FIG. 7).
　接触ステップで人造フィブロイン原繊維に接触させる水の温度は、沸点未満であってよい。これにより、取扱い性及び収縮工程の作業性等が向上する。また、収縮時間を充分に短縮するという観点からは、水の温度の下限値が、１０℃以上であることが好ましく、４０℃以上であることがより好ましく、７０℃以上であることが更に好ましい。水の温度の上限値は９０℃以下であることが好ましい。 温度 The temperature of the water contacted with the artificial fibroin fibrils in the contacting step may be lower than the boiling point. Thereby, the handleability, the workability in the shrinking step, etc. are improved. In addition, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit of the water temperature is preferably 10 ° C. or higher, more preferably 40 ° C. or higher, and further preferably 70 ° C. or higher. . The upper limit of the water temperature is preferably 90 ° C. or less.
　接触ステップにおいて、水を人造フィブロイン原繊維に接触させる方法は、特に限定されない。当該方法として、例えば、人造フィブロイン原繊維を水中に浸漬する方法、人造フィブロイン原繊維に対して水を常温で又は加温したスチーム等の状態で噴霧する方法、及び人造フィブロイン原繊維を水蒸気が充満した高湿度環境下に暴露する方法等が挙げられる。これらの方法の中でも、接触ステップにおいては、収縮時間の短縮化が効果的に図れるとともに、加工設備の簡素化等が実現できることから、人造フィブロイン原繊維を水中に浸漬する方法が好ましい。 方法 In the contacting step, a method of bringing water into contact with artificial fibroin fibrils is not particularly limited. As the method, for example, a method of immersing artificial fibroin fibrils in water, a method of spraying water on artificial fibroin fibrils at room temperature or in a state of heated steam, and a method of filling artificial fibroin fibrils with water vapor Exposure to a high humidity environment. Among these methods, in the contacting step, the method of immersing the artificial fibroin fibrils in water is preferable because the contraction time can be effectively shortened and the processing equipment can be simplified.
　接触ステップにおいて、人造フィブロイン原繊維を弛緩させた状態で水に接触させると、人造フィブロイン原繊維が、単に収縮するだけでなく、波打つように縮れてしまうことがある。このような縮れの発生を防止するために、例えば、人造フィブロイン原繊維を繊維軸方向に緊張させ（引っ張り）ながら水と接触させるなど、人造フィブロイン原繊維を弛緩させない状態で接触ステップを実施してもよい。 In the contacting step, when the artificial fibroin fibrils are brought into contact with water in a relaxed state, the artificial fibroin fibrils may not only shrink but also shrink in a wavy manner. In order to prevent the occurrence of such shrinkage, for example, the artificial fibroin fibrous fibers are brought into contact with water while being tensioned (pulled) in the fiber axis direction, and the contact step is performed in a state where the artificial fibroin fibrils are not relaxed. Is also good.
　本実施形態に係る人造フィブロイン繊維の製造方法は、乾燥ステップを更に備えるものであってもよい。乾燥ステップは、接触ステップを経た人造フィブロイン原繊維（又は接触ステップを経て得られた人造フィブロイン繊維）を乾燥させて更に収縮させる工程である（図７の「二次収縮」に相当する）。乾燥は、例えば、自然乾燥でもよく、乾燥設備を使用して強制的に乾燥させてもよい。乾燥設備としては、接触型又は非接触型の公知の乾燥設備がいずれも使用可能である。また、乾燥温度も、例えば、人造フィブロイン原繊維に含まれるタンパク質が分解したり、人造フィブロイン原繊維が熱的損傷を受けたりする温度よりも低い温度であれば何ら限定されるものではないが、一般には、２０～１５０℃の範囲内の温度であり、５０～１００℃の範囲内の温度であることが好ましい。温度がこの範囲にあることにより、繊維の熱的損傷、又は繊維に含まれるタンパク質の分解が生ずることなく、繊維が、より迅速且つ効率的に乾燥される。乾燥時間は、乾燥温度等に応じて適宜に設定され、例えば、過乾燥による人造フィブロイン繊維の品質及び物性等への影響が可及的に排除され得る時間等が採用される。 方法 The method for producing artificial fibroin fibers according to the present embodiment may further include a drying step. The drying step is a step of drying and further shrinking the artificial fibroin fibrils that have passed through the contacting step (or the artificial fibroin fibers obtained through the contacting step) (corresponding to “secondary shrinkage” in FIG. 7). Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known contact-type or non-contact-type drying equipment can be used. Also, the drying temperature is not limited at all, for example, as long as the protein contained in the artificial fibroin fibrils is decomposed or a temperature lower than the temperature at which the artificial fibroin fibrils are thermally damaged. Generally, the temperature is in the range of 20 to 150 ° C, and preferably in the range of 50 to 100 ° C. Temperatures in this range allow the fibers to dry more quickly and efficiently without thermal damage to the fibers or degradation of the proteins contained in the fibers. The drying time is appropriately set according to the drying temperature and the like, and, for example, a time that can minimize the influence of the overdrying on the quality and physical properties of the artificial fibroin fiber and the like is adopted.
　図８は、人造フィブロイン繊維を製造するための製造装置の一例を概略的に示す説明図である。図８に示す製造装置４０は、人造フィブロイン原繊維を送り出すフィードローラ４２と、人造フィブロイン繊維３８を巻き取るワインダー４４と、接触ステップを実施するウォーターバス４６と、乾燥ステップを実施する乾燥機４８と、を有して構成されている。 FIG. 8 is an explanatory view schematically showing an example of a production apparatus for producing artificial fibroin fibers. The manufacturing apparatus 40 shown in FIG. 8 includes a feed roller 42 for feeding out artificial fibroin fibers, a winder 44 for winding the artificial fibroin fibers 38, a water bath 46 for performing a contacting step, and a dryer 48 for performing a drying step. , Is configured.
　より詳細には、フィードローラ４２は、人造フィブロイン原繊維３６の巻回物が装着可能とされており、図示しない電動モータ等の回転によって、人造フィブロイン原繊維３６の巻回物から人造フィブロイン原繊維３６を連続的且つ自動的に送り出し得るようになっている。ワインダー４４は、フィードローラ４２から送り出された後、接触ステップと乾燥ステップを経て製造された人造フィブロイン繊維３８を、図示しない電動モータの回転によって連続的且つ自動的に巻き取り得るようになっている。なお、ここでは、フィードローラ４２による人造フィブロイン原繊維３６の送出し速度と、ワインダー４４による人造フィブロイン繊維３８の巻取り速度とが、互いに独立して制御可能とされている。 More specifically, the feed roller 42 is configured to be capable of mounting a wound of the artificial fibroin fibril 36, and is rotated from an wound of the artificial fibroin fibril 36 by rotation of an electric motor (not shown). 36 can be continuously and automatically sent out. After being fed from the feed roller 42, the winder 44 can continuously and automatically wind the artificial fibroin fiber 38 manufactured through the contacting step and the drying step by rotation of an electric motor (not shown). . Here, the feeding speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.
　ウォーターバス４６と乾燥機４８は、フィードローラ４２とワインダー４４との間に、人造フィブロイン原繊維３６の送り方向の上流側と下流側にそれぞれ並んで配置されている。なお、図８に示す製造装置４０は、フィードローラ４２からワインダー４４に向かって走行する接触ステップ前及び後の人造フィブロイン原繊維３６を中継するリレーローラ５０及び５２を有している。 The water bath 46 and the dryer 48 are arranged between the feed roller 42 and the winder 44 on the upstream side and the downstream side in the feed direction of the artificial fibroin fibril 36, respectively. The manufacturing apparatus 40 shown in FIG. 8 has relay rollers 50 and 52 for relaying the artificial fibroin raw fibers 36 before and after the contact step running from the feed roller 42 toward the winder 44.
　ウォーターバス４６はヒータ５４を有し、このヒータ５４にて加温された水４７が、ウォーターバス４６内に収容されている。また、ウォーターバス４６内には、テンションローラ５６が、水４７中に浸漬された状態で設置されている。これにより、フィードローラ４２から送り出された人造フィブロイン原繊維３６が、ウォーターバス４６内を、テンションローラ５６に巻き掛けられた状態で水４７中に浸漬されつつ、ワインダー４４側に向かって走行するようになっている。なお、人造フィブロイン原繊維３６の水４７中への浸漬時間は、人造フィブロイン原繊維３６の走行速度に応じて適宜にコントロールされる。 The water bath 46 has a heater 54, and the water 47 heated by the heater 54 is stored in the water bath 46. In the water bath 46, a tension roller 56 is installed in a state of being immersed in water 47. As a result, the artificial fibroin fibrils 36 sent out from the feed rollers 42 run toward the winder 44 while being immersed in the water 47 while being wound around the tension rollers 56 in the water bath 46. It has become. The immersion time of the artificial fibroin fibrils 36 in the water 47 is appropriately controlled according to the running speed of the artificial fibroin fibrils 36.
　乾燥機４８は、一対のホットローラ５８を有している。一対のホットローラ５８は、ウォーターバス４６内から離脱してワインダー４４側に向かって走行する人造フィブロイン原繊維３６が巻き掛け可能とされている。これにより、ウォーターバス４６内で水４７に浸漬された人造フィブロイン原繊維３６が、乾燥機４８内で一対のホットローラ５８にて加熱され、乾燥させられた後、ワインダー４４に向かって更に送り出されるようになっている。 The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 can be wound around the artificial fibroin fibrils 36 that are separated from the water bath 46 and run toward the winder 44. Thus, the artificial fibroin fibrils 36 immersed in water 47 in the water bath 46 are heated by the pair of hot rollers 58 in the dryer 48, dried, and further sent out to the winder 44. It has become.
　このような構造を有する製造装置４０を用いて、人造フィブロイン繊維３８を製造する際には、先ず、例えば、図６に示された紡糸装置１０を用いて紡糸された人造フィブロイン原繊維３６の巻回物をフィードローラ４２に装着する。次に、フィードローラ４２から人造フィブロイン原繊維３６を連続的に送り出して、ウォーターバス４６内で水４７に浸漬させる。このとき、例えば、ワインダー４４の巻き取り速度をフィードローラ４２の送り出し速度よりも遅くしておく。これにより、人造フィブロイン原繊維３６が、フィードローラ４２とワインダー４４との間で弛緩しないように緊張された状態で、水４７との接触により収縮するため、縮れの発生を防止することができる。水４７との接触により人造フィブロイン原繊維３６は不可逆的に収縮する（図７の「一次収縮」に相当する）。 When manufacturing the artificial fibroin fiber 38 using the manufacturing apparatus 40 having such a structure, first, for example, the winding of the artificial fibroin raw fiber 36 spun using the spinning apparatus 10 shown in FIG. The food is mounted on the feed roller 42. Next, the artificial fibroin fibrils 36 are continuously sent out from the feed roller 42 and immersed in water 47 in a water bath 46. At this time, for example, the winding speed of the winder 44 is set lower than the feeding speed of the feed roller 42. Accordingly, the artificial fibroin fibrous material 36 contracts by contact with the water 47 in a state where the artificial fibroin fibrous material 36 is tensioned so as not to relax between the feed roller 42 and the winder 44, so that the occurrence of shrinkage can be prevented. Upon contact with the water 47, the artificial fibroin fibrils 36 contract irreversibly (corresponding to "primary contraction" in FIG. 7).
　次に、水４７と接触した後の人造フィブロイン原繊維３６（又は水４７との接触を経て製造された人造フィブロイン繊維３８）を、乾燥機４８の一対のホットローラ５８により加熱する。これにより、水４７と接触した後の人造フィブロイン原繊維３６（又は水４７との接触を経て製造された人造フィブロイン繊維３８）を乾燥させ、更に収縮させることができる（図７の「二次収縮」に相当する）。このとき、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との比率をコントロールすることで、人造フィブロイン繊維３８を更に収縮させることもできるし、長さを変化させないこともできる。そして、得られた人造フィブロイン繊維３８をワインダー４４にて巻き取って、人造フィブロイン繊維３８の巻回物を得る。 Next, the artificial fibroin fibrils 36 after contact with the water 47 (or the artificial fibroin fibers 38 produced through the contact with the water 47) are heated by the pair of hot rollers 58 of the dryer 48. Thereby, the artificial fibroin fibrils 36 after contact with the water 47 (or the artificial fibroin fibers 38 produced through the contact with the water 47) can be dried and further contracted (“secondary contraction” in FIG. 7). "). At this time, by controlling the ratio between the feed speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 38 can be further contracted and its length can be kept unchanged. Then, the obtained artificial fibroin fiber 38 is wound by a winder 44 to obtain a roll of the artificial fibroin fiber 38.
　なお、一対のホットローラ５８に代えて、図９（ｂ）に示されるような乾熱板６４等、単なる熱源のみからなる乾燥設備を用いて水４７と接触した後の人造フィブロイン原繊維３６を乾燥させてもよい。この場合にも、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との互いの相対速度を、乾燥設備として一対のホットローラ５８を使用する場合と同様に調節することにより、人造フィブロイン繊維３８を更に収縮させることもできるし、長さを変化させないこともできる。ここでは、乾燥手段が乾熱板６４にて構成されることとなる。また、乾燥機４８は必須ではない。 Note that, instead of the pair of hot rollers 58, the artificial fibroin fibrils 36 after being brought into contact with the water 47 by using a drying equipment such as a dry heat plate 64 as shown in FIG. It may be dried. Also in this case, by adjusting the relative speed between the feed speed of the feed roller 42 and the winding speed of the winder 44 in the same manner as in the case where a pair of hot rollers 58 is used as the drying equipment, the artificial fibroin fiber is adjusted. 38 can be further contracted or the length can be unchanged. Here, the drying means is constituted by the dry heat plate 64. Further, the dryer 48 is not essential.
　上述のように、製造装置４０を用いることによって、目的とする人造フィブロイン繊維３８を自動的且つ連続的に、しかも極めて容易に製造することができる。 As described above, by using the manufacturing apparatus 40, the target artificial fibroin fiber 38 can be manufactured automatically, continuously, and extremely easily.
　図９は、人造フィブロイン繊維を製造するための製造装置の別の例を概略的に示す説明図である。図９（ａ）は、当該製造装置に備わる、接触ステップを実施する加工装置を示し、図９（ｂ）は、当該製造装置に備わる、乾燥ステップを実施する乾燥装置を示す。図９に示される製造装置は、人造フィブロイン原繊維３６に対する接触ステップを実施する加工装置６０と、接触ステップ後の人造フィブロイン原繊維３６（又は接触ステップを経て製造された人造フィブロイン繊維３８）を乾燥させる乾燥装置６２とを有し、それらが互いに独立した構造とされている。 FIG. 9 is an explanatory view schematically showing another example of a production apparatus for producing artificial fibroin fibers. FIG. 9A shows a processing apparatus provided in the manufacturing apparatus for performing the contacting step, and FIG. 9B shows a drying apparatus provided in the manufacturing apparatus for performing the drying step. The manufacturing apparatus shown in FIG. 9 includes a processing apparatus 60 that performs a contacting step on the artificial fibroin fibril 36 and a drying step on the artificial fibroin fibril 36 (or the artificial fibroin fiber 38 manufactured through the contacting step) after the contacting step. And a drying device 62 for making them independent from each other.
　より具体的には、図９（ａ）に示す加工装置６０は、図８に示された製造装置４０から乾燥機４８を省略して、フィードローラ４２とウォーターバス４６とワインダー４４とを、人造フィブロイン原繊維３６の走行方向の上流から下流側に向かって順に並べて配置してなる構造を有している。このような加工装置６０は、フィードローラ４２から送り出された人造フィブロイン原繊維３６を、ウォーターバス４６内の水４７中に浸漬させて、収縮させるようになっている。そして、得られた人造フィブロイン繊維３８をワインダー４４にて巻き取るように構成されている。 More specifically, the processing apparatus 60 shown in FIG. 9A omits the dryer 48 from the manufacturing apparatus 40 shown in FIG. 8, and connects the feed roller 42, the water bath 46, and the winder 44 It has a structure in which the fibroin fibrils 36 are arranged side by side in order from upstream to downstream in the running direction. In such a processing device 60, the artificial fibroin fibrils 36 sent out from the feed roller 42 are immersed in water 47 in a water bath 46 to be contracted. Then, the obtained artificial fibroin fiber 38 is wound up by a winder 44.
　図９（ｂ）に示す乾燥装置６２は、フィードローラ４２及びワインダー４４と、乾熱板６４とを有している。乾熱板６４は、フィードローラ４２とワインダー４４との間に、乾熱面６６が、人造フィブロイン繊維３８に接触し、且つその走行方向に沿って伸びるように配置されている。この乾燥装置６２では、前述したように、例えば、フィードローラ４２の送出し速度とワインダー４４の巻取り速度との比率をコントロールすることで、人造フィブロイン繊維３８を更に収縮させることもできるし、長さを変化させないこともできる。 乾燥 The drying device 62 shown in FIG. 9B has the feed roller 42 and the winder 44, and a dry heat plate 64. The dry heat plate 64 is disposed between the feed roller 42 and the winder 44 such that the dry heat surface 66 contacts the artificial fibroin fiber 38 and extends in the running direction. In the drying device 62, as described above, for example, by controlling the ratio of the feed speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 38 can be further contracted, It can be the same.
　このような構造を有する製造装置を用いることによって、人造フィブロイン原繊維３６を加工装置６０により収縮させて人造フィブロイン繊維３８を得た後、乾燥装置６２にて人造フィブロイン繊維３８を乾燥させることができる。 By using the manufacturing device having such a structure, the artificial fibroin fiber 38 can be dried by the drying device 62 after the artificial fibroin fiber 36 is shrunk by the processing device 60 to obtain the artificial fibroin fiber 38. .
　なお、図９（ａ）に示された加工装置６０からフィードローラ４２とワインダー４４とを省略して、ウォーターバス４６のみで加工装置を構成してもよい。このような加工装置を有する製造装置を用いる場合には、例えば、人造フィブロイン繊維が、いわゆるバッチ式で製造されることとなる。また、図９（ｂ）に示す乾燥装置６２は必須ではない。 Note that the processing device may be constituted only by the water bath 46 by omitting the feed roller 42 and the winder 44 from the processing device 60 shown in FIG. When a manufacturing apparatus having such a processing apparatus is used, for example, artificial fibroin fibers are manufactured by a so-called batch method. The drying device 62 shown in FIG. 9B is not essential.
（捲縮）
　本実施形態に係る人造フィブロイン繊維は、水との接触により捲縮されたものであってもよい。捲縮の度合いは制限されず、捲縮数は、例えば、１０～１００個／４０ｍｍであってよく、２０～４０個／４０ｍｍであってもよい。水との接触により捲縮された人造フィブロイン繊維は、例えば、人造フィブロイン原繊維を水性媒体と接触させることにより得ることができる。水性媒体とは、水（水蒸気を含む。）を含む液体又は気体（スチーム）の媒体である。水性媒体は水であってもよいし、水と親水性溶媒との混合液であってもよい。また、親水性溶媒としては、例えば、エタノール及びメタノール等の揮発性溶媒又はその蒸気を用いることも可能である。水性媒体は、水とエタノールとの混合溶液であることが好ましい。揮発性溶媒又はその蒸気を含む水性媒体を使用することで、人造フィブロイン繊維が早く乾燥し、よりソフト感のある仕上がりになる。水と揮発性溶媒又はその蒸気との比率は、特に限定されず、例えば、水：揮発性溶媒又はその蒸気は、質量比で１０：９０～９０：１０であってもよい。 (Crimp)
The artificial fibroin fiber according to the present embodiment may be crimped by contact with water. The degree of crimping is not limited, and the number of crimps may be, for example, 10 to 100 pieces / 40 mm or 20 to 40 pieces / 40 mm. The artificial fibroin fiber crimped by contact with water can be obtained, for example, by contacting the artificial fibroin fibril with an aqueous medium. The aqueous medium is a liquid or gas (steam) medium containing water (including water vapor). The aqueous medium may be water, or a mixture of water and a hydrophilic solvent. Further, as the hydrophilic solvent, for example, a volatile solvent such as ethanol and methanol or a vapor thereof can be used. The aqueous medium is preferably a mixed solution of water and ethanol. By using an aqueous medium containing a volatile solvent or its vapor, the artificial fibroin fiber dries quickly and has a softer finish. The ratio of water to the volatile solvent or its vapor is not particularly limited. For example, the mass ratio of water: volatile solvent or its vapor may be 10:90 to 90:10.
　水性媒体が液体である場合、水性媒体には、例えば、工程通過用（例えば、帯電防止用等）又は仕上げ用の油剤等、公知の油剤を分散させてもよい。すなわち、水性媒体の代わりに、水性媒体と、水性媒体に分散された油剤とを含む油剤分散液を使用することもできる。このような油剤分散液を用いることによって、人造フィブロイン原繊維を捲縮させるとともに、人造フィブロイン原繊維の捲縮により得られた人造フィブロイン繊維に油剤を付着させることができる。人造フィブロイン繊維に油剤を付着させることで、人造フィブロイン繊維に種々の特性を付与することができる。なお、油剤の量は、特に限定されず、例えば、水性媒体と油剤の全量に対して１～１０質量％であってもよく、２～５質量％であってよい。 In the case where the aqueous medium is a liquid, a known oil agent such as an oil for passage through a process (for example, for antistatic) or a finish may be dispersed in the aqueous medium. That is, instead of the aqueous medium, an oil dispersion containing an aqueous medium and an oil dispersed in the aqueous medium can be used. By using such an oil dispersion, the artificial fibroin fibrils can be crimped, and the oil agent can be adhered to the artificial fibroin fibers obtained by crimping the artificial fibroin fibrils. By attaching the oil agent to the artificial fibroin fiber, various properties can be imparted to the artificial fibroin fiber. The amount of the oil agent is not particularly limited, and may be, for example, 1 to 10% by mass or 2 to 5% by mass based on the total amount of the aqueous medium and the oil agent.
　水性媒体の温度は、１０℃以上、２５℃以上、４０℃以上、６０℃以上、又は１００℃以上であってよく、２３０℃以下、１２０℃以下、又は１００℃以下であってよい。より具体的には、水性媒体が気体（スチーム）である場合、水性媒体の温度は１００～２３０℃が好ましく、１００～１２０℃がより好ましい。水性媒体のスチームが２３０℃以下であると、人造フィブロイン繊維の熱変性を防ぐことができる。水性媒体が液体である場合、水性媒体の温度は、効率良く捲縮を付与する観点から、１０℃以上、２５℃以上、又は４０℃以上が好ましく、人造フィブロイン繊維の繊維強度を高く保つ観点から、６０℃以下が好ましい。水性媒体に人造フィブロイン原繊維を接触させる時間は、特に制限されないが、３０秒以上、１分以上、又は２分以上であってよく、生産性の観点から１０分以下であることが好ましい。人造フィブロイン原繊維への水性媒体の接触は、常圧下で行ってもよく、減圧下（例えば、真空）で行ってもよい。 温度 The temperature of the aqueous medium may be 10 ° C or higher, 25 ° C or higher, 40 ° C or higher, 60 ° C or higher, or 100 ° C or higher, and may be 230 ° C or lower, 120 ° C or lower, or 100 ° C or lower. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230 ° C, more preferably 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, heat denaturation of the artificial fibroin fiber can be prevented. When the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10 ° C. or higher, 25 ° C. or higher, or 40 ° C. or higher from the viewpoint of efficiently imparting crimp, and from the viewpoint of keeping the fiber strength of the artificial fibroin fiber high. , 60 ° C or lower. The time for contacting the artificial fibroin fibrils with the aqueous medium is not particularly limited, but may be 30 seconds or more, 1 minute or more, or 2 minutes or more, and is preferably 10 minutes or less from the viewpoint of productivity. The contact of the aqueous medium with the artificial fibroin fibrils may be performed under normal pressure or may be performed under reduced pressure (for example, vacuum).
　人造フィブロイン原繊維を水性媒体に接触させる方法としては、人造フィブロイン原繊維を水中に浸漬する方法、人造フィブロイン原繊維に対して水性媒体のスチームを噴霧する方法、水性媒体のスチームが充満した環境に人造フィブロイン原繊維を暴露する方法等が挙げられる。水性媒体がスチームである場合、人造フィブロイン原繊維への水性媒体の接触は、一般的なスチームセット装置を使用して行うことができる。スチームセット装置の具体例としては、製品名：ＦＭＳＡ型スチームセッター（福伸工業株式会社製）、製品名：ＥＰＳ－４００（辻井染機工業株式会社製）等の装置を挙げることができる。水性媒体のスチームにより人造フィブロイン原繊維を捲縮させる方法の具体例としては、所定の収容室内に人造フィブロイン原繊維を収容する一方、収容室内に水性媒体のスチームを導入して、収容室内の温度を上記所定温度（例えば、１００℃～２３０℃）に調整しつつ、人造フィブロイン原繊維にスチームを接触させることが挙げられる。 As a method of contacting the artificial fibroin fibrils with an aqueous medium, a method of immersing the artificial fibroin fibrils in water, a method of spraying the artificial fibroin fibrils with an aqueous medium steam, an environment filled with the aqueous medium steam. Examples include a method of exposing artificial fibroin fibrils. When the aqueous medium is steam, the contact of the aqueous medium with the artificial fibroin fibrils can be performed using a general steam setting device. Specific examples of the steam set device include devices such as a product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and a product name: EPS-400 (manufactured by Tsujii Dyeing Machinery Co., Ltd.). As a specific example of the method of crimping the artificial fibroin fibrils by the steam of the aqueous medium, the artificial fibroin fibrils are accommodated in a predetermined accommodation room, while the steam of the aqueous medium is introduced into the accommodation room, and the temperature in the accommodation room is increased. Is adjusted to the above-mentioned predetermined temperature (for example, 100 ° C. to 230 ° C.), and steam is brought into contact with the artificial fibroin fibrils.
　なお、水性媒体との接触による人造フィブロイン原繊維の捲縮工程は、好ましくは人造フィブロイン原繊維に対して引張力が何ら加えられない（繊維軸方向に何ら緊張されない）状態、又は所定の大きさだけ加えられた（繊維軸方向に所定量だけ緊張させられた）状態で実施される。その際に、人造フィブロイン原繊維に加えられる引張力を調整することで、捲縮の程度をコントロールすることが可能となる。人造フィブロイン原繊維に加えられる引張力の調整方法としては、例えば、人造フィブロイン原繊維に様々な重さの重りを吊す等して、人造フィブロイン原繊維に対して負荷される荷重を調整する方法、人造フィブロイン原繊維を弛ませた状態で両末端を固定するとともに、その弛み量を種々変更する方法、人造フィブロイン原繊維を紙管又はボビン等の被巻回体に巻き付けるとともに、その際の巻き付け力（紙管又はボビンへの締付力）を適宜に変更する方法等が挙げられる。 The step of crimping the artificial fibroin fibrils by contact with the aqueous medium is preferably performed in a state where no tensile force is applied to the artificial fibroin fibrils (no tension is applied in the fiber axis direction), or a predetermined size. (Strained by a predetermined amount in the fiber axis direction). At that time, the degree of crimp can be controlled by adjusting the tensile force applied to the artificial fibroin fibrils. As a method of adjusting the tensile force applied to the artificial fibroin fibrils, for example, by suspending various weights on the artificial fibroin fibrils, a method of adjusting the load applied to the artificial fibroin fibrils, A method in which both ends are fixed in a state where the artificial fibroin fibrils are slackened, and the amount of slack is variously changed, and the artificial fibroin fibrils are wound around a wound body such as a paper tube or a bobbin, and the winding force at that time (Tightening force on paper tube or bobbin) may be appropriately changed.
　人造フィブロイン原繊維を水性媒体に接触させた後、人造フィブロイン原繊維を乾燥してもよい。乾燥方法は、特に限定されず、自然乾燥でもよく、乾燥設備を使用して強制的に人造フィブロイン原繊維を乾燥させてもよい。水性媒体による捲縮とその後の乾燥は、連続的に行うことができる。具体的には、例えば、ボビンから人造フィブロイン原繊維を送り出しながら、水性媒体中に浸漬した後、熱風を吹き付けるか、又はホットローラー上に送り出すことで乾燥することができる。乾燥温度としては、特に限定されず、例えば、２０～１５０℃であってよく、４０～１２０℃であることが好ましく、６０～１００℃であることがより好ましい。 (4) After the artificial fibroin fibrils are brought into contact with the aqueous medium, the artificial fibroin fibrils may be dried. The drying method is not particularly limited, and may be natural drying, or the artificial fibroin fibrils may be forcibly dried using a drying facility. The crimping with the aqueous medium and the subsequent drying can be performed continuously. Specifically, for example, the artificial fibroin fibrils can be dried by being immersed in an aqueous medium while being sent from a bobbin, and then blown with hot air or sent over a hot roller. The drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C., preferably 40 to 120 ° C., and more preferably 60 to 100 ° C.
　上記からも明らかなように、人造フィブロイン繊維を得るために実施される、人造フィブロイン原繊維の水による収縮工程には、人造フィブロイン原繊維の長さを短縮するだけの（縮れさせない）工程以外に、人造フィブロイン原繊維を捲縮させる（縮れさせて長さを短縮させる）工程も含まれる。なお、条件の設定次第では、人造フィブロイン原繊維を水により収縮（長さを短縮）させる収縮工程（接触ステップ、乾燥ステップ）と捲縮させる工程を同時に又は段階的に実施することもできる。 As is clear from the above, the process of shrinking artificial fibroin fibrils with water, which is performed to obtain artificial fibroin fibers, includes processes other than simply reducing the length of artificial fibroin fibrils (not shrinking). And crimping the artificial fibroin fibrils (to shorten the length by crimping). Depending on the setting of the conditions, the shrinking step (contact step, drying step) of shrinking (reducing the length) the artificial fibroin fibrils with water and the step of crimping can be performed simultaneously or stepwise.
（人造フィブロイン繊維の実施形態）
　本実施形態に係る人造フィブロイン繊維は、下記式で定義される湿潤収縮率が２％以上であってよい。
　湿潤収縮率＝｛１－（水に接触させて湿潤状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％） (Embodiment of artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment may have a wet shrinkage defined by the following formula of 2% or more.
Wet shrinkage = {1− (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fibril after spinning and before contact with water)} × 100 (%)
　本実施形態に係る人造フィブロイン繊維は、湿潤収縮率が、２．５％以上、３％以上、３．５％以上、４％以上、４．５％以上、５％以上、５．５％以上、又は６％以上であってよい。湿潤収縮率の上限は特に限定されないが、８０％以下、６０％以下、４０％以下、２０％以下、１０％以下、７％以下、６％以下、５％以下、４％以下、又は３％以下であってよい。 The artificial fibroin fiber according to this embodiment has a wet shrinkage of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, and 5.5% or more. Or 6% or more. The upper limit of the wet shrinkage is not particularly limited, but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3%. It may be:
　本実施形態に係る人造フィブロイン繊維は、下記式で定義される乾燥収縮率が７％超であってよい。
　乾燥収縮率＝｛１－（乾燥状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％） The artificial fibroin fiber according to the present embodiment may have a drying shrinkage defined by the following formula of more than 7%.
Drying shrinkage = {1− (length of dried artificial fibroin fiber / length of artificial fibroin raw fiber after spinning and before contact with water)} × 100 (%)
　本実施形態に係る人造フィブロイン繊維は、乾燥収縮率が、８％以上、１０％以上、１５％以上、２０％以上、２５％以上、３０％以上、３５％以上、３７％以上、３８％以上、又は３９％以上であってよい。乾燥収縮率の上限は特に限定されないが、８０％以下、７０％以下、６０％以下、５０％以下、又は４０％以下であってよい。 The artificial fibroin fiber according to the present embodiment has a drying shrinkage of 8% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 37% or more, 38% or more. Or 39% or more. The upper limit of the drying shrinkage is not particularly limited, but may be 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less.
　本実施形態に係る人造フィブロイン繊維は、限界酸素指数（ＬＯＩ）値が、１８以上であってよく、２０以上であってもよく、２２以上であってもよく、２４以上であってもよく、２６以上であってもよく、２８以上であってもよく、２９以上であってもよく、３０以上であってもよい。本明細書において、ＬＯＩ値は、消防庁危険物規制課長　消防危５０号平成７年５月３１日の粉粒状又は融点の低い合成樹脂の試験方法に準拠して測定される値である。 The artificial fibroin fiber according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, or 24 or more, It may be 26 or more, 28 or more, 29 or more, or 30 or more. In the present specification, the LOI value is a value measured in accordance with the Fire and Disaster Management Agency, Dangerous Goods Regulations Section, #Fire and Fire Criterion No. 50, May 31, 1995, based on the test method for synthetic resin having a low granularity or a low melting point.
　本実施形態に係る人造フィブロイン繊維は、下記式Ａに従って求められる最高吸湿発熱度が０．０２５℃／ｇ超であってよい。
　式Ａ：最高吸湿発熱度＝｛（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値）－（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度）｝（℃）／試料重量（ｇ）
　なお、式Ａ中、低湿度環境は、温度２０℃及び相対湿度４０％の環境を意味し、高湿度環境は、温度２０℃及び相対湿度９０％の環境を意味する。 The artificial fibroin fiber according to this embodiment may have a maximum heat of moisture absorption determined by the following formula A of more than 0.025 ° C./g.
Formula A: Maximum heat of moisture absorption = ｛(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium, and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)｝ (℃) / sample weight (g)
In Formula A, the low humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 40%, and the high humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 90%.
　本実施形態に係る人造フィブロイン繊維は、最高吸湿発熱度が０．０２６℃／ｇ以上であってもよく、０．０２７℃／ｇ以上であってもよく、０．０２８℃／ｇ以上であってもよく、０．０２９℃／ｇ以上であってもよく、０．０３０℃／ｇ以上であってもよく、０．０３５℃／ｇ以上であってもよく、０．０４０℃／ｇ以上であってもよい。最高吸湿発熱度の上限に特に制限はないが、通常、０．０６０℃／ｇ以下である。 The artificial fibroin fiber according to the present embodiment may have a maximum heat of moisture absorption by absorption of not less than 0.026 ° C / g, not less than 0.027 ° C / g, and not less than 0.028 ° C / g. May be 0.029 ° C./g or more, may be 0.030 ° C./g or more, may be 0.035 ° C./g or more, and may be 0.040 ° C./g or more. It may be. The upper limit of the maximum heat of moisture absorption is not particularly limited, but is usually 0.060 ° C./g or less.
　本実施形態に係る人造フィブロイン繊維は、下記式Ｂに従って求められる保温性指数が０．１８超であってよい。
　式Ｂ：保温性指数＝保温率（％）／試料の目付け（ｇ／ｍ２）
　なお、式Ｂ中、保温率（％）は、ドライコンタクト法（温度３０℃、風速３０ｃｍ／秒）で測定された保温率を意味し、（１－ａ／ｂ）×１００で算出される。ａは、試験片を介して放散された熱量を示し、ｂは、試験片を介さないで放散された熱量を示す。保温率は、例えば、サーモラボＩＩ型試験機等によって測定できる。 The artificial fibroin fiber according to the present embodiment may have a heat retention index determined according to the following formula B of more than 0.18.
Formula B: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
In Formula B, the heat retention rate (%) means a heat retention rate measured by a dry contact method (temperature: 30 ° C., wind speed: 30 cm / sec), and is calculated by (1−a / b) × 100. a shows the amount of heat dissipated through the test piece, and b shows the amount of heat dissipated without passing through the test piece. The heat retention can be measured by, for example, a Thermolab II tester or the like.
　本実施形態に係る人造フィブロイン繊維は、保温性指数が０．２０以上であってよく、０．２２以上であってよく、０．２４以上であってよく、０．２６以上であってよく、０．２８以上であってよく、０．３０以上であってよく、０．３２以上であってよい。保温性指数の上限に特に制限はないが、例えば、０．６０以下、又は０．４０以下であってよい。 The artificial fibroin fiber according to the present embodiment may have a heat retention index of 0.20 or more, may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, It may be 0.28 or more, may be 0.30 or more, and may be 0.32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.
（獣毛繊維）
　本明細書における獣毛繊維は、ウール（羊毛）を含む動物の毛、及びそれら由来の繊維を意味する。獣毛繊維としては、例えば、ウール（羊毛）、カシミヤ（カシミヤ山羊の毛）、モヘア（アンゴラ山羊の毛）、アンゴラ（アンゴラ兎の毛）、アルパカ（ラクダ科ビキューナ属アルパカの毛）、ビキューナ（ラクダ科ビキューナ属ビキューナの毛）、キャメル（ラクダ科ラクダ属ラクダの毛）、及びリャマ（ラクダ科ラマ属リャマの毛）等、並びにそれら由来の繊維が挙げられる。 (Animal fiber)
Animal hair fiber in the present specification means animal hair including wool (wool), and fiber derived therefrom. As animal hair fibers, for example, wool (wool), cashmere (cashmere goat hair), mohair (angora goat hair), angora (angora rabbit hair), alpaca (camelaceae Vicuña alpaca hair), vicuña ( Camel (Vicuna vicuna hair), Camel (Camelidae camel hair), Lilla (Camelid llama llama hair), and fibers derived therefrom.
　本実施形態に係る複合糸（混紡糸、混繊糸、混織糸、交織糸、合撚糸及びカバーリング糸等）は、上述した人造フィブロイン繊維及び獣毛繊維を使用する他は、常法に従って製造することができる。本実施形態に係る複合糸は、人造フィブロイン繊維及び獣毛繊維以外の他の繊維を更に含有していてもよい。他の繊維としては、例えば、ポリエステル、ポリアミド繊維等の合成繊維、及び綿、麻、シルク、ウール、カシミア等の天然繊維が挙げられる。これら他の繊維は、本発明による効果を損なわない範囲で使用するのが好ましい。 The composite yarn (blended yarn, mixed fiber yarn, mixed woven yarn, mixed woven yarn, plied yarn, covering yarn, and the like) according to the present embodiment is prepared according to a conventional method except that the above-described artificial fibroin fiber and animal hair fiber are used. Can be manufactured. The composite yarn according to the present embodiment may further contain other fibers other than the artificial fibroin fiber and the animal hair fiber. Other fibers include, for example, synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, hemp, silk, wool, and cashmere. These other fibers are preferably used as long as the effects of the present invention are not impaired.
　本実施形態に係る複合糸における人造フィブロイン繊維と獣毛繊維の含有比率は、人造フィブロイン繊維と獣毛繊維の合計量を基準として、人造フィブロイン繊維の含有量が１０質量％以上であってよく、２０質量％以上であってよく、３０質量％以上であってよく、４０質量％以上であってよく、５０質量％以上であってよく、６０質量％以上であってよく、７０質量％以上であってよく、８０質量％以上であってよい。人造フィブロイン繊維の含有量の上限は、人造フィブロイン繊維と獣毛繊維の合計量を基準として、９０質量％以下であってよく、８０質量％以下であってよく、７０質量％以下であってよく、６０質量％以下であってよく、５０質量％以下であってよく、４０質量％以下であってよく、３０質量％以下であってよく、２０質量％以下であってよい。 The content ratio of the artificial fibroin fiber and the animal hair fiber in the composite yarn according to the present embodiment is based on the total amount of the artificial fibroin fiber and the animal hair fiber, and the content of the artificial fibroin fiber may be 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more And may be 80% by mass or more. The upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the animal hair fiber. , 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.
　本実施形態に係る複合糸における人造フィブロイン繊維と獣毛繊維の含有量は、複合糸全量を基準として、人造フィブロイン繊維と獣毛繊維との合計含有量が、５０質量％以上であってよく、６０質量％以上であってよく、７０質量％以上であってよく、８０質量％以上であってよく、９０質量％以上であってよい。人造フィブロイン繊維と獣毛繊維との合計含有量の上限に特に制限はなく、複合糸全量を基準として、１００質量％であってよく、９５質量％以下であってよい。 The content of the artificial fibroin fiber and the animal hair fiber in the composite yarn according to the present embodiment is, based on the total amount of the composite yarn, the total content of the artificial fibroin fiber and the animal hair fiber may be 50% by mass or more, It may be 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more. The upper limit of the total content of the artificial fibroin fiber and the animal hair fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the composite yarn.
〔布地〕
　本実施形態に係る布地は、上述した人造フィブロイン繊維及び獣毛繊維を含有する。布地には、織物、編物（メリヤス生地）、レース、フェルト及び不織布等が含まれる。 (Cloth)
The fabric according to the present embodiment contains the above-described artificial fibroin fiber and animal hair fiber. The fabric includes woven fabric, knitted fabric (knitted fabric), lace, felt, nonwoven fabric, and the like.
　本実施形態に係る布地は、上述した人造フィブロイン繊維及び獣毛繊維以外の他の繊維を更に含有するものであってもよい。他の繊維としては、例えば、ポリエステル、ポリアミド繊維等の合成繊維、及び綿、麻、シルク、ウール、カシミア等の天然繊維が挙げられる。これら他の繊維は、本発明による効果を損なわない範囲で使用するのが好ましい。 布 The fabric according to the present embodiment may further contain fibers other than the above-described artificial fibroin fiber and animal hair fiber. Other fibers include, for example, synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, hemp, silk, wool, and cashmere. These other fibers are preferably used as long as the effects of the present invention are not impaired.
　本実施形態に係る布地は、例えば、上述した人造フィブロイン繊維及び獣毛繊維を混用して製造することで、人造フィブロイン繊維及び獣毛繊維を含有するものとしてもよい。本実施形態に係る布地はまた、例えば、上述した人造フィブロイン繊維から形成される糸、及び獣毛繊維から形成される糸を混用して製造することで、人造フィブロイン繊維及び獣毛繊維を含有するものとしてもよい。本実施形態に係る布地はまた、例えば、本発明に係る複合糸を使用して製造することで、人造フィブロイン繊維及び獣毛繊維を含有するものとしてもよい。また、上述した人造フィブロイン繊維から形成される糸、及び獣毛繊維から形成される糸は、単一糸であってもよく、他の繊維との複合糸であってもよい。さらに、いずれの製造方法においても、必要に応じて、他の繊維、及び／又は他の繊維から形成される糸を併用してもよい。 布 The fabric according to the present embodiment may contain artificial fibroin fiber and animal hair fiber, for example, by being manufactured by mixing the above-mentioned artificial fibroin fiber and animal hair fiber. The fabric according to the present embodiment also contains, for example, a yarn formed from the above-described artificial fibroin fiber, and a yarn formed from animal hair fiber, which is mixed to produce the artificial fibroin fiber and the animal hair fiber. It may be a thing. The fabric according to the present embodiment may also include, for example, an artificial fibroin fiber and an animal hair fiber by being manufactured using the composite yarn according to the present invention. Further, the yarn formed from the above-mentioned artificial fibroin fiber and the yarn formed from animal hair fiber may be a single yarn or a composite yarn with other fibers. Further, in any of the production methods, other fibers and / or yarns formed from other fibers may be used in combination, if necessary.
　本実施形態に係る布地（織物、編物、レース、フェルト及び不織布等）は、上述した繊維及び／又は糸を使用する他は、常法に従って製造することができる。 布 The fabric (woven fabric, knitted fabric, lace, felt, non-woven fabric, etc.) according to the present embodiment can be manufactured according to a conventional method except that the above-described fibers and / or yarns are used.
　本実施形態に係る布地における人造フィブロイン繊維と獣毛繊維の含有比率は、人造フィブロイン繊維と獣毛繊維の合計量を基準として、人造フィブロイン繊維の含有量が１０質量％以上であってよく、２０質量％以上であってよく、３０質量％以上であってよく、４０質量％以上であってよく、５０質量％以上であってよく、６０質量％以上であってよく、７０質量％以上であってよく、８０質量％以上であってよい。人造フィブロイン繊維の含有量の上限は、人造フィブロイン繊維と獣毛繊維の合計量を基準として、９０質量％以下であってよく、８０質量％以下であってよく、７０質量％以下であってよく、６０質量％以下であってよく、５０質量％以下であってよく、４０質量％以下であってよく、３０質量％以下であってよく、２０質量％以下であってよい。 The content ratio of the artificial fibroin fiber and the animal hair fiber in the fabric according to the present embodiment may be such that the content of the artificial fibroin fiber is 10% by mass or more, based on the total amount of the artificial fibroin fiber and the animal hair fiber. At least 30% by mass, at least 40% by mass, at least 50% by mass, at least 60% by mass, at least 70% by mass. And may be 80% by mass or more. The upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the animal hair fiber. , 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.
　本実施形態に係る布地における人造フィブロイン繊維と獣毛繊維の含有量は、布地全量を基準として、人造フィブロイン繊維と獣毛繊維との合計含有量が、５０質量％以上であってよく、６０質量％以上であってよく、７０質量％以上であってよく、８０質量％以上であってよく、９０質量％以上であってよい。人造フィブロイン繊維と獣毛繊維との合計含有量の上限に特に制限はなく、布地全量を基準として、１００質量％であってよく、９５質量％以下であってよい。 The content of the artificial fibroin fiber and the animal hair fiber in the fabric according to the present embodiment may be such that the total content of the artificial fibroin fiber and the animal hair fiber is 50% by mass or more, and % Or more, 70% or more, 80% or more, and 90% or more. The upper limit of the total content of the artificial fibroin fiber and the animal hair fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the fabric.
　本実施形態に係る布地は、例えば、衣料、医療品、衛生用品、インテリア用品、寝具、装飾品、バッグ、小物、雑貨、車両用部品、樹脂等との複合品等の材料として使用することができる。 The cloth according to the present embodiment can be used as a material for clothing, medical supplies, sanitary goods, interior goods, bedding, decorations, bags, accessories, miscellaneous goods, vehicle parts, composite goods with resin and the like, for example. it can.
　以下、実施例等に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and the like. However, the present invention is not limited to the following examples.
〔試験例１：人造フィブロイン繊維の製造及び収縮性評価〕
（１）改変フィブロインの製造
（改変フィブロインをコードする核酸の合成、及び発現ベクターの構築）
　配列番号１８で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ３９９）、配列番号１２で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ３８０）、配列番号１３で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ４１０）、配列番号１５で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ７９９）、及び配列番号３７で示されるアミノ酸配列を有する改変フィブロイン（ＰＲＴ９１８）を設計した。 [Test Example 1: Manufacture of artificial fibroin fiber and evaluation of shrinkage]
(1) Production of modified fibroin (synthesis of nucleic acid encoding modified fibroin and construction of expression vector)
Modified fibroin having the amino acid sequence represented by SEQ ID NO: 18 (PRT399), modified fibroin having the amino acid sequence represented by SEQ ID NO: 12 (PRT380), modified fibroin having the amino acid sequence represented by SEQ ID NO: 13 (PRT410), SEQ ID NO: A modified fibroin having the amino acid sequence represented by No. 15 (PRT799) and a modified fibroin having the amino acid sequence represented by SEQ ID NO: 37 (PRT918) were designed.
　設計した５種類の改変フィブロインをコードする核酸をそれぞれ合成した。当該核酸には、５’末端にＮｄｅＩサイト、終止コドン下流にＥｃｏＲＩサイトを付加した。これら５種類の核酸をクローニングベクター（ｐＵＣ１１８）にそれぞれクローニングした。その後、同核酸をＮｄｅＩ及びＥｃｏＲＩで制限酵素処理して切り出した後、タンパク質発現ベクターｐＥＴ－２２ｂ（＋）に組換えてそれぞれ発現ベクターを得た。 (4) Nucleic acids encoding the designed five types of modified fibroins were respectively synthesized. The nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon. These five types of nucleic acids were respectively cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
（改変フィブロインの発現）
　得られた発現ベクターで、大腸菌ＢＬＲ（ＤＥ３）を形質転換した。当該形質転換大腸菌を、アンピシリンを含む２ｍＬのＬＢ培地で１５時間培養した。当該培養液を、アンピシリンを含む１００ｍＬのシード培養用培地（表４）にＯＤ６００が０．００５となるように添加した。培養液温度を３０℃に保ち、ＯＤ６００が５になるまでフラスコ培養を行い（約１５時間）、シード培養液を得た。 (Expression of modified fibroin)
Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　当該シード培養液を５００ｍｌの生産培地（下記表５）を添加したジャーファーメンターにＯＤ６００が０．０５となるように添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持した。 The seed culture solution was added to a jar fermenter to which 500 ml of a production medium (Table 5 below) had been added so that the OD 600 was 0.05. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. The concentration of dissolved oxygen in the culture was maintained at 20% of the saturated concentration of dissolved oxygen.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　生産培地中のグルコースが完全に消費された直後に、フィード液（グルコース４５５ｇ／１Ｌ、Ｙｅａｓｔ　Ｅｘｔｒａｃｔ　１２０ｇ／１Ｌ）を１ｍＬ／分の速度で添加した。培養液温度を３７℃に保ち、ｐＨ６．９で一定に制御して培養した。培養液中の溶存酸素濃度を、溶存酸素飽和濃度の２０％に維持しながら、２０時間培養を行った。その後、１Ｍのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を培養液に対して終濃度１ｍＭになるよう添加し、目的とする改変フィブロインを発現誘導させた。ＩＰＴＧ添加後２０時間経過した時点で、培養液を遠心分離し、菌体を回収した。ＩＰＴＧ添加前とＩＰＴＧ添加後の培養液から調製した菌体を用いてＳＤＳ－ＰＡＧＥを行い、ＩＰＴＧ添加に依存した目的とする改変フィブロインに相当するサイズのバンドの出現により、目的とする改変フィブロインの発現を確認した。 フ ィ ー ド Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the desired modified fibroin. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of a band having a size corresponding to the desired modified fibroin depending on the addition of IPTG revealed the presence of the modified fibroin of interest. Expression was confirmed.
（改変フィブロインの精製）
　ＩＰＴＧを添加してから２時間後に回収した菌体を２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｂｕｆｆｅｒ（ｐＨ７．４）で洗浄した。洗浄後の菌体を約１ｍＭのＰＭＳＦを含む２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）に懸濁させ、高圧ホモジナイザー（ＧＥＡ　Ｎｉｒｏ　Ｓｏａｖｉ社）で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４）で洗浄した。洗浄後の沈殿物を１００ｍｇ／ｍＬの濃度になるように８Ｍ　グアニジン緩衝液（８Ｍ　グアニジン塩酸塩、１０ｍＭ　リン酸二水素ナトリウム、２０ｍＭ　ＮａＣｌ、１ｍＭ　Ｔｒｉｓ－ＨＣｌ、ｐＨ７．０）で懸濁し、６０℃で３０分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ（三光純薬株式会社製のセルロースチューブ３６／３２）を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収した。回収した凝集タンパク質から凍結乾燥機で水分を除き、改変フィブロインの凍結乾燥粉末を得た。 (Purification of modified fibroin)
Two hours after the addition of IPTG, the collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation. Water was removed from the collected aggregated proteins using a freeze dryer to obtain a freeze-dried powder of modified fibroin.
（２）人造フィブロイン原繊維の製造
（ドープ液の調製）
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに改変フィブロインの凍結乾燥粉末を、濃度１８質量％又は２４質量％となるよう添加し（表６参照）、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液を得た。 (2) Manufacture of artificial fibroin fibrils (preparation of dope solution)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0% by mass was prepared as a solvent, and a lyophilized powder of modified fibroin was added thereto to a concentration of 18% by mass or 24% by mass (see Table 1). 6), and dissolved for 3 hours using a shaker. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution.
（紡糸）
　得られた改変フィブロイン溶液をドープ液（紡糸原液）とし、図６に示す紡糸装置１０に準じた紡糸装置を用いた乾湿式紡糸によって、紡糸及び延伸された人造フィブロイン原繊維を製造した。用いた紡糸装置は、図６に示す紡糸装置１０において、未延伸糸製造装置２（第１浴）及び湿熱延伸装置３（第３浴）の間に、更に第２の未延伸糸製造装置（第２浴）を備えるものである。乾湿式紡糸の条件は以下のとおりである。
　押出しノズル直径：０．２ｍｍ
　第１浴～第３浴中の液体及び温度：表６参照
　総延伸倍率：表６参照
　乾燥温度：６０℃ (spinning)
The resulting modified fibroin solution was used as a dope solution (spinning stock solution), and spin-dried and stretched artificial fibroin fibers were manufactured by dry-wet spinning using a spinning device according to the spinning device 10 shown in FIG. The spinning apparatus used in the spinning apparatus 10 shown in FIG. 6 is a second undrawn yarn manufacturing apparatus (between the undrawn yarn manufacturing apparatus 2 (first bath) and the wet heat drawing apparatus 3 (third bath)). (Second bath). The conditions for dry-wet spinning are as follows.
Extrusion nozzle diameter: 0.2mm
Liquid and temperature in first to third baths: see Table 6 Total draw ratio: see Table 6 Drying temperature: 60 ° C
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（３）人造フィブロイン繊維の製造及び評価
　製造例１～１９で得た各人造フィブロイン原繊維に対して、水に接触させる接触ステップを施すこと（以下、「一次収縮」ということがある。）、又は当該接触ステップを施した後、室温で乾燥させる乾燥ステップを施すこと（以下、「二次収縮」ということがある。）により、人造フィブロイン繊維を製造した。 (3) Production and Evaluation of Artificial Fibroin Fibers The artificial fibroin raw fibers obtained in Production Examples 1 to 19 are subjected to a contact step of bringing them into contact with water (hereinafter, may be referred to as “primary shrinkage”). Alternatively, after performing the contacting step, a drying step of drying at room temperature is performed (hereinafter, may be referred to as “secondary shrinkage”) to produce artificial fibroin fibers.
＜一次収縮＞
　製造例１～１９で得た人造フィブロイン原繊維の巻回物から、それぞれ、長さ３０ｃｍの複数本の人造フィブロイン原繊維を切り出した。それら複数本の人造フィブロイン原繊維を束ねて、繊度１５０デニールの人造フィブロイン原繊維束を得た。各人造フィブロイン原繊維束に０．８ｇの鉛錘を取り付け、その状態で各人造フィブロイン原繊維束を表７～１０に示す温度の水に１０分間浸漬した（接触ステップ）。その後、水中で各人造フィブロイン繊維束の長さを測定した。水中での人造フィブロイン繊維束の長さ測定は、人造フィブロイン繊維束の縮れを無くすために、人造フィブロイン繊維束に０．８ｇの鉛錘を取り付けたまま実施した。次いで、各人造フィブロイン繊維の一次収縮率（％）を、下記式Ｉに従って算出した。式Ｉ中、Ｌ０は紡糸後、水と接触する前の人造フィブロイン原繊維束の長さ（ここでは３０ｃｍ）を示し、Ｌｗは一次収縮を経た人造フィブロイン繊維束の長さを示す。なお、一次収縮率は、湿潤収縮率と同義である。
　式Ｉ：一次収縮率＝｛１－（Ｌｗ／Ｌ０）｝×１００（％） <Primary shrinkage>
From artificial wound fibroin fibrils obtained in Production Examples 1 to 19, a plurality of artificial fibroin fibrils each having a length of 30 cm were cut out. The plurality of artificial fibroin fibrils were bundled to obtain an artificial fibroin fibril bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each artificial fibroin fibril bundle, and in this state, each artificial fibroin fibril bundle was immersed in water at a temperature shown in Tables 7 to 10 for 10 minutes (contact step). Thereafter, the length of each artificial fibroin fiber bundle was measured in water. The length measurement of the artificial fibroin fiber bundle in water was performed with 0.8 g of lead weight attached to the artificial fibroin fiber bundle in order to eliminate the shrinkage of the artificial fibroin fiber bundle. Next, the primary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula I. In Formula I, L0 indicates the length of the artificial fibroin fibril bundle (here, 30 cm) after spinning and before contact with water, and Lw indicates the length of the artificial fibroin fiber bundle that has undergone primary shrinkage. The primary shrinkage is synonymous with the wet shrinkage.
Formula I: Primary shrinkage = {1− (Lw / L0)} × 100 (%)
＜二次収縮＞
　一次収縮での水への浸漬（接触ステップ）の後、人造フィブロイン繊維束を水中から取り出した。取り出した人造フィブロイン繊維束を、０．８ｇの鉛錘を取り付けたまま、室温で２時間おいて乾燥させた（乾燥ステップ）。乾燥後、各人造フィブロイン繊維束の長さを測定した。次いで、各人造フィブロイン繊維の二次収縮率（％）を、下記式ＩＩに従って算出した。式ＩＩ中、Ｌ０は紡糸後、水と接触する前の人造フィブロイン原繊維束の長さ（ここでは３０ｃｍ）を示し、Ｌｗｄは二次収縮を経た人造フィブロイン繊維束の長さを示す。なお、二次収縮率は、乾燥収縮率と同義である。
　式ＩＩ：二次収縮率＝｛１－（Ｌｗｄ／Ｌ０）｝×１００（％） <Secondary shrinkage>
After immersion in water with primary shrinkage (contact step), the artificial fibroin fiber bundle was removed from the water. The removed artificial fibroin fiber bundle was dried at room temperature for 2 hours with a 0.8 g lead weight attached (drying step). After drying, the length of each artificial fibroin fiber bundle was measured. Next, the secondary shrinkage (%) of each artificial fibroin fiber was calculated according to the following formula II. In Formula II, L0 represents the length of the artificial fibroin fiber bundle (here, 30 cm) after spinning and before contact with water, and Lwd represents the length of the artificial fibroin fiber bundle that has undergone secondary shrinkage. The secondary shrinkage is synonymous with the dry shrinkage.
Formula II: Secondary shrinkage = {1− (Lwd / L0)} × 100 (%)
　結果を表７～１０に示す。
Figure JPOXMLDOC01-appb-T000007
 The results are shown in Tables 7 to 10.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
〔試験例２：人造フィブロイン繊維の難燃性評価〕
（人造フィブロイン繊維の製造）
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに試験例１の「（１）改変フィブロインの製造」で製造した改変フィブロイン（ＰＲＴ７９９）の凍結乾燥粉末を、濃度２４質量％となるよう添加し、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液（紡糸原液）を得た。 [Test Example 2: Evaluation of flame retardancy of artificial fibroin fiber]
(Manufacture of artificial fibroin fiber)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0% by mass was prepared as a solvent, and the modified fibroin (PRT799) produced in “(1) Production of modified fibroin” in Test Example 1 was frozen there. The dry powder was added to a concentration of 24% by mass and dissolved for 3 hours using a shaker. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
　調製した紡糸原液を６０℃にて目開き５μｍの金属フィルターで濾過し、次いで３０ｍＬのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径０．２ｍｍのソリッドノズルから１００質量％メタノール凝固浴槽中へ吐出させた。吐出温度は６０℃であった。凝固後、得られた原糸を巻き取り、自然乾燥させて人造フィブロイン原繊維を得た。 The prepared spinning stock solution was filtered at 60 ° C. with a metal filter having an aperture of 5 μm, and then allowed to stand in a 30 mL stainless syringe to remove bubbles, and then coagulated with 100% by mass methanol from a solid nozzle having a needle diameter of 0.2 mm. Discharged into the bath. The discharge temperature was 60 ° C. After coagulation, the obtained fibrils were wound and air-dried to obtain artificial fibroin fibrils.
　得られた人造フィブロイン原繊維に対して、水に接触させる接触ステップを施した後、室温で乾燥させる乾燥ステップを施すことにより、人造フィブロイン繊維（原料繊維）を製造した。 (4) The obtained artificial fibroin fiber was subjected to a contact step of bringing it into contact with water, and then subjected to a drying step of drying at room temperature, thereby producing an artificial fibroin fiber (raw fiber).
（編地の製造）
　得られた原料繊維を使用して、丸編機を使用した丸編みで編地を製造した。編地は、太さ１８０デニール、ゲージ数１８とした。得られた編地から２０ｇ切り出して試験片とした。 (Manufacture of knitted fabric)
A knitted fabric was manufactured by circular knitting using a circular knitting machine using the obtained raw material fibers. The knitted fabric had a thickness of 180 denier and 18 gauges. 20 g was cut out from the obtained knitted fabric to obtain a test piece.
（燃焼性試験）
　燃焼性試験は、消防庁危険物規制課長　消防危５０号平成７年５月３１日の粉粒状又は融点の低い合成樹脂の試験方法に準拠した。試験は、温度２２℃、相対湿度４５％、気圧１０２１ｈＰａの条件下で実施した。測定結果（酸素濃度（％）、燃焼率（％）、換算燃焼率（％））を表１１に示す。
Figure JPOXMLDOC01-appb-T000011
 (Flammability test)
The flammability test was conducted in accordance with the Fire and Disaster Management Agency, Fire and Disaster Criminal Control Division No. 50, May 31, 1995, test method for synthetic resin having a granular or low melting point. The test was performed under the conditions of a temperature of 22 ° C., a relative humidity of 45%, and an air pressure of 1021 hPa. Table 11 shows the measurement results (oxygen concentration (%), burning rate (%), reduced burning rate (%)).
Figure JPOXMLDOC01-appb-T000011
　難燃性試験の結果、改変フィブロイン（ＰＲＴ７９９）を含む人造フィブロイン繊維で編んだ編地の限界酸素指数（ＬＯＩ）値は２７．２であった。一般にＬＯＩ値が２６以上あれば難燃性があるとされる。この結果から、人造フィブロイン繊維は、難燃性に優れていることが分かる。したがって、人造フィブロイン繊維と獣毛繊維を混用した複合糸又は布地とすることにより、難燃性に優れた複合糸又は布地を得ることができる。 結果 As a result of the flame retardancy test, the knitted fabric made of artificial fibroin fibers containing the modified fibroin (PRT799) had a limiting oxygen index (LOI) value of 27.2. Generally, if the LOI value is 26 or more, it is considered to be flame retardant. From this result, it can be seen that the artificial fibroin fiber has excellent flame retardancy. Therefore, a composite yarn or fabric excellent in flame retardancy can be obtained by using a composite yarn or fabric in which artificial fibroin fibers and animal hair fibers are mixed.
〔試験例３：人造フィブロイン繊維の吸湿発熱性評価〕
（人造フィブロイン繊維の製造）
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに試験例１の「（１）改変フィブロインの製造」で製造した改変フィブロイン（ＰＲＴ９１８又はＰＲＴ７９９）の凍結乾燥粉末を、濃度２４質量％質量％となるよう添加し、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液（紡糸原液）を得た。 [Test Example 3: Evaluation of heat generation and moisture absorption of artificial fibroin fiber]
(Manufacture of artificial fibroin fiber)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0% by mass was prepared as a solvent, and the modified fibroin (PRT918 or PRT799) produced in “(1) Production of modified fibroin” in Test Example 1 was prepared there. Was added to a concentration of 24% by mass and dissolved using a shaker for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
　調製した紡糸原液を６０℃にて目開き５μｍの金属フィルターで濾過し、次いで３０ｍＬのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径０．２ｍｍのソリッドノズルから窒素ガスを用い１００質量％メタノール凝固浴槽中へ吐出させた。吐出温度は６０℃であり、吐出圧は０．３ＭＰａであった。凝固後、得られた原糸を巻き取り速度３．００ｍ／分で巻き取り、自然乾燥させて人造フィブロイン原繊維を得た。 The prepared spinning dope was filtered at 60 ° C. through a 5 μm-aperture metal filter, then left standing in a 30 mL stainless syringe and defoamed. The mass% methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C., and the discharge pressure was 0.3 MPa. After coagulation, the obtained yarn was wound at a winding speed of 3.00 m / min and air-dried to obtain artificial fibroin fibrils.
　得られた人造フィブロイン原繊維に対して、水に接触させる接触ステップを施した後、室温で乾燥させる乾燥ステップを施すことにより、人造フィブロイン繊維（原料繊維）を製造した。 (4) The obtained artificial fibroin fiber was subjected to a contact step of bringing it into contact with water, and then subjected to a drying step of drying at room temperature, thereby producing an artificial fibroin fiber (raw fiber).
　比較のため、原料繊維として、市販されているウール繊維、コットン繊維、テンセル繊維、レーヨン繊維及びポリエステル繊維を用意した。 市 販 For comparison, commercially available wool fibers, cotton fibers, Tencel fibers, rayon fibers, and polyester fibers were prepared as raw fibers.
（編地の製造）
　各原料繊維を使用して、横編機を使用した横編みで編地を製造した。原料繊維としてＰＲＴ９１８繊維を使用した編地は、太さ：１／３０Ｎ（毛番手単糸）、ゲージ数：１８とした。原料繊維としてＰＲＴ７９９繊維を使用した編地は、太さ：１／３０Ｎ（毛番手単糸）、ゲージ数：１６とした。その他の原料繊維を使用した編地は、ＰＲＴ９１８繊維及びＰＲＴ７９９繊維を使用した編地とほぼ同一のカバーファクターとなるように太さ及びゲージ数を調整した。具体的には、以下に示すとおりである。
　ウール　太さ：２／３０Ｎ（双糸）、ゲージ数：１４
　コットン　太さ：２／３４Ｎ（双糸）、ゲージ数：１４
　テンセル　太さ：２／３０Ｎ（双糸）、ゲージ数：１５
　レーヨン　太さ：１／３８Ｎ（単糸）、ゲージ数：１４
　ポリエステル　太さ：１／６０Ｎ（単糸）、ゲージ数：１４ (Manufacture of knitted fabric)
Using each raw material fiber, a knitted fabric was manufactured by flat knitting using a flat knitting machine. The knitted fabric using PRT918 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 18. The knitted fabric using PRT799 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 16. The thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so as to have almost the same cover factor as that of the knitted fabric using PRT918 fiber and PRT799 fiber. Specifically, it is as shown below.
Wool Thickness: 2 / 30N (double thread), Number of gauges: 14
Cotton Thickness: 2 / 34N (double yarn), Number of gauges: 14
Tencel Thickness: 2 / 30N (double yarn), Number of gauges: 15
Rayon Thickness: 1 / 38N (single yarn), Number of gauges: 14
Polyester Thickness: 1 / 60N (single yarn), Number of gauges: 14
（吸湿発熱性試験）
　１０ｃｍ×１０ｃｍに裁断した編地を２枚合わせにし、四辺を縫い合わせて試験片（試料）とした。試験片を低湿度環境（温度２０±２℃、相対湿度４０±５％）で４時間以上放置した後、高湿度環境（温度２０±２℃、相対湿度９０±５％）に移し、試験片内部中央に取り付けた温度センサーにより３０分間、１分間隔で温度の測定を行った。 (Hygroscopic heat generation test)
Two pieces of the knitted fabric cut to 10 cm × 10 cm were aligned, and four sides were sewn to obtain a test piece (sample). After leaving the test specimen in a low humidity environment (temperature 20 ± 2 ° C, relative humidity 40 ± 5%) for 4 hours or more, it was moved to a high humidity environment (temperature 20 ± 2 ° C, relative humidity 90 ± 5%) The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.
　測定結果から、下記式Ａに従って、最高吸湿発熱度を求めた。
　式Ａ：最高吸湿発熱度＝｛（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値）－（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度）｝（℃）／試料重量（ｇ） From the measurement results, the maximum heat of moisture absorption was calculated according to the following formula A.
Formula A: Maximum heat of moisture absorption = ｛(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium, and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)｝ (℃) / sample weight (g)
　図１０は、吸湿発熱性試験の結果の一例を示すグラフである。グラフの横軸は、試料を低湿度環境から高湿度環境に移した時点を０とし、高湿度環境での放置時間（分）を示す。グラフの縦軸は、温度センサーで測定した温度（試料温度）を示す。図１０に示したグラフ中、Ｍで示した点が、試料温度の最高値に対応している。 FIG. 10 is a graph showing an example of the result of the moisture absorption / heating test. The horizontal axis of the graph represents the time (minute) of leaving the sample in the high humidity environment as 0 when the time when the sample was transferred from the low humidity environment to the high humidity environment. The vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor. In the graph shown in FIG. 10, the point indicated by M corresponds to the maximum value of the sample temperature.
　最高吸湿発熱度の算出結果を表１２に示す。
Figure JPOXMLDOC01-appb-T000012
 Table 12 shows the calculation result of the maximum heat of moisture absorption.
Figure JPOXMLDOC01-appb-T000012
　改変フィブロイン（ＰＲＴ９１８又はＰＲＴ７９９）を含む人造フィブロイン繊維は、既存の材料と比べて、最高吸湿発熱度が高く、吸湿発熱性に優れていることが分かる。したがって、人造フィブロイン繊維と獣毛繊維を混用して複合糸又は布地とすることにより、吸湿発熱性に優れた複合糸又は布地を得ることができる。 人 It can be seen that the artificial fibroin fiber containing the modified fibroin (PRT918 or PRT799) has a higher maximum heat of moisture absorption and is superior in heat absorption and heat generation as compared with existing materials. Therefore, by mixing artificial fibroin fibers and animal hair fibers to form a composite yarn or fabric, a composite yarn or fabric excellent in heat absorption and heat generation can be obtained.
〔試験例４：人造フィブロイン繊維の保温性評価〕
　４．０質量％になるようにＬｉＣｌを溶解させたジメチルスルホキシド（ＤＭＳＯ）を溶媒として用意し、そこに改変フィブロインの凍結乾燥粉末を、濃度２４質量％となるよう添加し、シェーカーを使用して３時間溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液（紡糸原液）を得た。 [Test Example 4: Evaluation of heat retention of artificial fibroin fiber]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to have a concentration of 4.0% by mass was prepared as a solvent, and a freeze-dried powder of the modified fibroin was added thereto so as to have a concentration of 24% by mass. Dissolved for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
　調製した紡糸原液を６０℃にて目開き５μｍの金属フィルターで濾過し、次いで３０ｍＬのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径０．２ｍｍのソリッドノズルから１００質量％メタノール凝固浴槽中へ吐出させた。吐出温度は６０℃であった。凝固後、得られた原糸を巻き取り、自然乾燥させて人造フィブロイン繊維（原料繊維）を得た。 The prepared spinning stock solution was filtered at 60 ° C. with a metal filter having an aperture of 5 μm, and then allowed to stand in a 30 mL stainless syringe to remove bubbles, and then coagulated with 100% by mass methanol from a solid nozzle having a needle diameter of 0.2 mm. Discharged into the bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain an artificial fibroin fiber (raw material fiber).
　比較のため、原料繊維として、市販されているウール繊維、シルク繊維、綿繊維、レーヨン繊維及びポリエステル繊維を用意した。 市 販 For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers and polyester fibers were prepared as raw material fibers.
　各原料繊維を使用して、横編機を使用した横編みで編地を製造した。原料繊維としてＰＲＴ９６６繊維を使用した編地は、番手：３０Ｎｍ、撚り本数：１、ゲージ数：１８ＧＧ、目付け：９０．１ｇ／ｍ２とした。原料繊維としてＰＲＴ７９９繊維を使用した編地は、番手：３０Ｎｍ、撚り本数：１、ゲージ数ＧＧ：１６、目付け：１１１．０ｇ／ｍ２とした。その他の原料繊維を使用した編地は、ＰＲＴ９６６繊維及びＰＲＴ７９９繊維を使用した編地とほぼ同一のカバーファクターとなるように太さ及びゲージ数を調整した。具体的には、以下のとおりである。
　ウール　番手：３０Ｎｍ、撚り本数：２、ゲージ数：１４ＧＧ、目付け：２４２．６ｇ／ｍ２
　シルク　番手：６０Ｎｍ、撚り本数：２、ゲージ数：１４ＧＧ、目付け：２２５．２ｇ／ｍ２
　綿　番手：３４Ｎｍ、撚り本数：２、ゲージ数：１４ＧＧ、目付け：１９４．１ｇ／ｍ２
　レーヨン　番手：３８Ｎｍ、撚り本数：１、ゲージ数：１４ＧＧ、目付け：１８１．８ｇ／ｍ２
　ポリエステル　番手：６０Ｎｍ、撚り本数：１、ゲージ数：１４ＧＧ、目付け：１８４．７ｇ／ｍ２ Using each raw material fiber, a knitted fabric was manufactured by flat knitting using a flat knitting machine. The knitted fabric using PRT966 fiber as a raw material fiber had a count of 30 Nm, the number of twists: 1, the number of gauges: 18 GG, and the basis weight: 90.1 g / m 2 . The knitted fabric using PRT799 fiber as the raw material fiber had a count of 30 Nm, a number of twists of 1, a gauge number of GG: 16, and a basis weight of 111.0 g / m 2 . The thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so as to have almost the same cover factor as that of the knitted fabric using PRT966 fiber and PRT799 fiber. Specifically, it is as follows.
Wool count: 30 Nm, number of twists: 2, gauge: 14 GG, basis weight: 242.6 g / m 2
Silk count: 60 Nm, number of twists: 2, gauge: 14 GG, basis weight: 225.2 g / m 2
Cotton count: 34 Nm, number of twists: 2, gauge: 14 GG, weight: 194.1 g / m 2
Rayon count: 38 Nm, number of twists: 1, number of gauges: 14 GG, basis weight: 181.8 g / m 2
Polyester count: 60 Nm, number of twists: 1, gauge: 14 GG, basis weight: 184.7 g / m 2
（保温性試験）
　保温性は、カトーテック株式会社製のＫＥＳ－Ｆ７サーモラボＩＩ試験機を使用し、ドライコンタクト法（皮膚と衣服が乾燥状態で直接触れた時を想定した方法）を用いて評価した。２０ｃｍ×２０ｃｍに裁断した編地１枚を試験片（試料）とした。試験片を、一定温度（３０℃）に設定した熱板にセットし、風洞内風速３０ｃｍ／秒の条件で、試験片を介して放散された熱量（ａ）を求めた。試験片をセットしない状態で、上記同様の条件で放散された熱量（ｂ）を求め、下記の式に従い保温率（％）を算出した。
保温率（％）＝（１－ａ／ｂ）×１００ (Insulation test)
The heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothing directly touched in a dry state). One piece of the knitted fabric cut to 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) radiated through the test piece was determined under the condition of a wind speed in the wind tunnel of 30 cm / sec. Without setting the test piece, the amount of heat dissipated (b) under the same conditions as above was determined, and the heat retention (%) was calculated according to the following equation.
Heat retention rate (%) = (1-a / b) × 100
　測定結果から、下記式Ｂに従って、保温性指数を求めた。
　式Ｂ：保温性指数＝保温率（％）／試料の目付け（ｇ／ｍ２） From the measurement results, the heat retention index was determined according to the following formula B.
Formula B: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
　保温性指数の算出結果を表１３に示す。保温性指数が高いほど、保温性に優れる材料と評価することができる。 Table 13 shows the calculation results of the heat retention index. The higher the heat retention index, the more the material can be evaluated as having excellent heat retention.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　表１３に示すとおり、改変フィブロイン（ＰＲＴ９６６及びＰＲＴ７９９）を含む人造フィブロイン繊維は、既存の材料と比べて、保温性指数が高く、保温性に優れていることが分かる。したがって、人造フィブロイン繊維と獣毛繊維を混用して複合糸又は布地とすることにより、保温性に優れた複合糸又は布地を得ることができる。 と お り As shown in Table 13, the artificial fibroin fiber containing the modified fibroin (PRT966 and PRT799) has a higher heat retention index and is superior in heat retention as compared with existing materials. Therefore, by mixing artificial fibroin fiber and animal hair fiber to form a composite yarn or fabric, a composite yarn or fabric excellent in heat retention can be obtained.
　１…押出し装置、２…未延伸糸製造装置、３…湿熱延伸装置、４…乾燥装置、６…ドープ液、１０…紡糸装置、２０…凝固液槽、２１…延伸浴槽、３６…人造フィブロイン原繊維、３８…人造フィブロイン繊維、４０…製造装置、４２…フィードローラ、４４…ワインダー、４６…ウォーターバス、４８…乾燥機、５４…ヒータ、５６…テンションローラ、５８…ホットローラ、６０…加工装置、６２…乾燥装置、６４…乾熱板。 DESCRIPTION OF SYMBOLS 1 ... Extrusion apparatus, 2 ... Unstretched yarn manufacturing apparatus, 3 ... Wet heat stretching apparatus, 4 ... Drying apparatus, 6 ... Dope liquid, 10 ... Spinning apparatus, 20 ... Coagulation liquid tank, 21 ... Stretching bath tank, 36 ... Artificial fibroin raw material Fiber: 38: artificial fibroin fiber, 40: manufacturing device, 42: feed roller, 44: winder, 46: water bath, 48: dryer, 54: heater, 56: tension roller, 58: hot roller, 60: processing device , 62: drying device, 64: dry heat plate.

Claims (11)

  1. 　人造フィブロイン繊維と、獣毛繊維とを含有し、
    　前記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、複合糸。     Contains artificial fibroin fiber and animal hair fiber,
    The composite yarn, wherein the artificial fibroin fiber contains a modified fibroin, and has a contraction history irreversibly contracted by contact with water after spinning.
  2. 　前記人造フィブロイン繊維は、下記式で定義される湿潤収縮率が２％以上である、請求項１に記載の複合糸。
    　湿潤収縮率＝｛１－（水に接触させて湿潤状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％）     The composite yarn according to claim 1, wherein the artificial fibroin fiber has a wet shrinkage defined by the following formula of 2% or more.
    Wet shrinkage = {1− (length of artificial fibroin fiber wetted by contact with water / length of artificial fibroin fibril after spinning and before contact with water)} × 100 (%)
  3. 　前記人造フィブロイン繊維は、下記式で定義される乾燥収縮率が７％超である、請求項１又は２に記載の複合糸。
    　乾燥収縮率＝｛１－（乾燥状態にした人造フィブロイン繊維の長さ／紡糸後、水と接触する前の人造フィブロイン原繊維の長さ）｝×１００（％）     3. The composite yarn according to claim 1, wherein the artificial fibroin fiber has a dry shrinkage defined by the following formula of more than 7%. 4.
    Drying shrinkage = {1− (length of dried artificial fibroin fiber / length of artificial fibroin raw fiber after spinning and before contact with water)} × 100 (%)
  4. 　前記人造フィブロイン繊維は、水との接触により捲縮されたものである、請求項１～３のいずれか一項に記載の複合糸。 The composite yarn according to any one of claims 1 to 3, wherein the artificial fibroin fiber is crimped by contact with water.
  5. 　前記人造フィブロイン繊維は、限界酸素指数（ＬＯＩ）値が２６．０以上である、請求項１～４のいずれか一項に記載の複合糸。 The composite yarn according to any one of claims 1 to 4, wherein the artificial fibroin fiber has a limiting oxygen index (LOI) value of 26.0 or more.
  6. 　前記人造フィブロイン繊維は、下記式Ａに従って求められる最高吸湿発熱度が０．０２５℃／ｇ超である、請求項１～５のいずれか一項に記載の複合糸。
    　式Ａ：最高吸湿発熱度＝｛（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値）－（試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度）｝（℃）／試料重量（ｇ）
    ［式Ａ中、低湿度環境は、温度２０℃及び相対湿度４０％の環境を意味し、高湿度環境は、温度２０℃及び相対湿度９０％の環境を意味する。］     The composite yarn according to any one of claims 1 to 5, wherein the artificial fibroin fiber has a maximum heat of moisture absorption determined according to the following formula A of more than 0.025 ° C / g.
    Formula A: Maximum heat of moisture absorption = ｛(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium, and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)｝ (℃) / sample weight (g)
    [In the formula A, a low humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 40%, and a high humidity environment means an environment at a temperature of 20 ° C. and a relative humidity of 90%. ]
  7. 　前記人造フィブロイン繊維は、下記式Ｂに従って求められる保温性指数が０．１８超である、請求項１～６のいずれか一項に記載の複合糸。
    　式Ｂ：保温性指数＝保温率（％）／試料の目付け（ｇ／ｍ２）
    ［式Ｂ中、保温率（％）は、ドライコンタクト法（温度３０℃、風速３０ｃｍ／秒）で測定された保温率を意味し、（１－ａ／ｂ）×１００で算出される。ａは、試験片を介して放散された熱量を示し、ｂは、試験片を介さないで放散された熱量を示す。］     The composite yarn according to any one of claims 1 to 6, wherein the artificial fibroin fiber has a heat retention index determined according to the following formula B of more than 0.18.
    Formula B: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
    [In formula B, the heat retention rate (%) means a heat retention rate measured by a dry contact method (temperature: 30 ° C., wind speed: 30 cm / sec), and is calculated by (1−a / b) × 100. a shows the amount of heat dissipated through the test piece, and b shows the amount of heat dissipated without passing through the test piece. ]
  8. 　前記改変フィブロインが、改変クモ糸フィブロインである、請求項１～７のいずれか一項に記載の複合糸。 複合 The composite yarn according to any one of claims 1 to 7, wherein the modified fibroin is a modified spider silk fibroin.
  9. 　混紡糸である、請求項１～８のいずれか一項に記載の複合糸。 The composite yarn according to any one of claims 1 to 8, which is a blended yarn.
  10. 　人造フィブロイン繊維と、獣毛繊維とを含有し、
    　前記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、布地。     Contains artificial fibroin fiber and animal hair fiber,
    A fabric, wherein the artificial fibroin fibers comprise modified fibroin and have a shrinkage history irreversibly shrunk by contact with water after spinning.
  11. 　人造フィブロイン繊維と、獣毛繊維とを混合する工程を備え、
    　前記人造フィブロイン繊維は、改変フィブロインを含み、かつ紡糸後に水との接触により不可逆的に収縮された収縮履歴を有する、複合糸の製造方法。     Comprising a step of mixing artificial fibroin fiber and animal hair fiber,
    A method for producing a composite yarn, wherein the artificial fibroin fiber contains a modified fibroin and has a shrinkage history irreversibly shrunk by contact with water after spinning.
PCT/JP2019/038427 2018-09-28 2019-09-27 Composite yarn, method for producing same, and fabric WO2020067546A1 (en)

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