WO2019194245A1 - 高収縮人造フィブロイン紡績糸及びその製造方法、並びに人造フィブロイン紡績糸及びその収縮方法 - Google Patents

高収縮人造フィブロイン紡績糸及びその製造方法、並びに人造フィブロイン紡績糸及びその収縮方法 Download PDF

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WO2019194245A1
WO2019194245A1 PCT/JP2019/014869 JP2019014869W WO2019194245A1 WO 2019194245 A1 WO2019194245 A1 WO 2019194245A1 JP 2019014869 W JP2019014869 W JP 2019014869W WO 2019194245 A1 WO2019194245 A1 WO 2019194245A1
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Prior art keywords
amino acid
fibroin
spun yarn
seq
shrinkage
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PCT/JP2019/014869
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English (en)
French (fr)
Japanese (ja)
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拓海 五十嵐
池田　敦
佑之介 安部
誠仁 原田
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Application filed by Ｓｐｉｂｅｒ株式会社, 小島プレス工業株式会社 filed Critical Ｓｐｉｂｅｒ株式会社
Priority to JP2020512299A priority Critical patent/JP7340262B2/ja
Priority to EP19782354.5A priority patent/EP3800286A4/en
Priority to CN201980023191.4A priority patent/CN112534090B/zh
Publication of WO2019194245A1 publication Critical patent/WO2019194245A1/ja

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    • D—TEXTILES; PAPER
    • D02—YARNS; MECHANICAL FINISHING OF YARNS OR ROPES; WARPING OR BEAMING
    • D02J—FINISHING OR DRESSING OF FILAMENTS, YARNS, THREADS, CORDS, ROPES OR THE LIKE
    • D02J1/00—Modifying the structure or properties resulting from a particular structure; Modifying, retaining, or restoring the physical form or cross-sectional shape, e.g. by use of dies or squeeze rollers
    • D02J1/22—Stretching or tensioning, shrinking or relaxing, e.g. by use of overfeed and underfeed apparatus, or preventing stretch
    • D02J1/223—Stretching in a liquid bath
    • 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
    • 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
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/58—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products
    • D01F6/68—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products from polyaminoacids or polypeptides
    • 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
    • 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
    • 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
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/02—Natural fibres, other than mineral fibres
    • D06M2101/10—Animal fibres
    • D06M2101/12—Keratin fibres or silk
    • D—TEXTILES; PAPER
    • D10—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
    • D10B—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
    • D10B2211/00—Protein-based fibres, e.g. animal fibres
    • D10B2211/20—Protein-derived artificial fibres
    • D10B2211/22—Fibroin

Definitions

  • the present invention relates to a high shrinkage artificial fibroin spun yarn and a production method thereof, and an artificial fibroin spun yarn and a shrinkage method thereof.
  • spun yarns made of synthetic fibers or natural fibers have been used as materials for various knitted fabrics. And in the manufacture of knitted fabrics, spun yarns that can satisfy the requirements for knitted fabrics are selected. For example, when producing clothing and bedding having a high touch and high quality, silk spun yarn or the like is selected as the material.
  • a predetermined processing may be applied to the spun yarn as a material.
  • silk spun yarn that has been shrunk to increase bulkiness is used. May be.
  • salt shrinkage processing As a method for shrinking silk, for example, so-called salt shrinkage processing is known in which silk is immersed in an aqueous solution (salt shrinkage solution) in which an inorganic salt such as calcium nitrate and calcium chloride is dissolved at a high concentration (shrinkage shrinkage).
  • salt shrinkage solution aqueous solution
  • an inorganic salt such as calcium nitrate and calcium chloride
  • synthetic fibers such as polyester fiber, polyamide fiber, and acrylic fiber, which are widely used in clothing and bedding, achieve a shrinkage rate of 40% or more by contacting with boiling water (Patent Document 2).
  • An object of the present invention is to provide a high-shrinkage artificial fibroin spun yarn having a sufficiently high shrinkage ratio, excellent in touch and flexibility, and capable of safe production, and a production method thereof.
  • Another object of the present invention is to provide an artificial fibroin spun yarn and a method for shrinking the same, which can safely produce a high shrinkage artificial fibroin spun yarn with a sufficiently high shrinkage rate.
  • the present invention relates to the following inventions, for example.
  • [1] Shrinked artificial fibroin spun yarn containing modified fibroin, A high shrinkage artificial fibroin spun yarn having a shrinkage rate defined by the following formula I of more than 1%.
  • Shrinkage rate ⁇ 1 ⁇ (length of the spun artificial fibroin spun yarn / length of spun man-made fibroin spun yarn before contacting with water after spinning) ⁇ ⁇ 100 (%) (Formula I)
  • Shrinkage rate ⁇ 1 ⁇ (length of the spun artificial fibroin spun yarn / length of spun man-made fibroin spun yarn before contacting with water after spinning) ⁇ ⁇ 100 (%) (Formula I)
  • Shrinkage rate ⁇ 1 ⁇ (length of the spun artificial fibroin spun yarn / length of spun man-made fibroin spun yarn before contacting with water after spinning) ⁇ ⁇ 100 (%) (Formula I)
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has an amino acid sequence with a reduced content of n motif (A) corresponding to deletion of at least one or more (A) n motifs compared to naturally occurring fibroin, [1] to [5]
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , From the N-terminal side to the C-terminal side, compare the number of REP amino acid residues of two adjacent [(A) n motif-REP] units in order, and the number of REP amino acid residues with a small number of amino acid residues. Is the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of the other REP of 1.8 to 11.3.
  • n motif represents an amino acid sequence composed of 2 to 27 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , Compared with naturally-occurring fibroin, the content of glycine residues corresponding to at least one or more glycine residues in REP was replaced with another amino acid residue in the domain sequence was reduced.
  • n motif represents an amino acid sequence composed of 2 to 27 amino acid residues
  • (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • n represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the domain sequence is at least one or more of at least one motif sequence selected from GGX and GPGXX in REP (where X represents an amino acid residue other than glycine).
  • the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue is 10% or more with respect to the entire motif sequence.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , XGX contained in all REPs in the sequence excluding the sequence from the domain sequence to the most C-terminal side (A) n motif to the C-terminus of the domain sequence (where X is an amino acid residue other than glycine)
  • the total number of amino acid residues in the amino acid sequence consisting of z) is z, and the domain sequence is located at the most C-terminal side (A) in the sequence excluding the sequence from the n motif to the C-terminus of the domain sequence
  • the high shrinkage artificial fibroin spun yarn according to any one of [1] to [5], wherein z / w is 50.9% or more, where w is the total number of amino acid residues of [6] to [6] 9]
  • the method for producing a high shrinkage artificial fibroin spun yarn according to any one of [9] the method for shrinking a man-made fibroin
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is equivalent to the substitution of one or more glycine residues in REP with another amino acid residue as compared with naturally-occurring fibroin, and further includes one or more amino acid residues.
  • the high-shrinkage artificial fibroin spun yarn according to any one of [19] to [22], which has an amino acid sequence corresponding to substitution, deletion, insertion and / or addition of , Shrinkage method of artificial fibroin spun yarn, or artificial fibroin spun yarn.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m ,
  • the domain sequence has one or more amino acid residues in REP substituted with amino acid residues having a higher hydrophobicity index and / or one or more hydrophobicity in REP compared to naturally occurring fibroin.
  • the high-shrinkage artificial fibroin spinning according to any one of [1] to [5], which has an amino acid sequence including a region having a locally large hydrophobic index, corresponding to insertion of an amino acid residue having a large sex index A method for producing a high-shrinkage artificial fibroin spun yarn according to any one of [6] to [9], a method for shrinking an artificial fibroin spun yarn according to any of [10] to [13], or [14] Man-made fibroin spun yarn described in 1.
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more. .
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • 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).
  • I isoleucine
  • V valine
  • L leucine
  • F phenylalanine
  • C cysteine
  • M methionine
  • A alanine
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , (A) The average of the hydrophobicity index of four consecutive amino acid residues in all REPs included in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence from the domain sequence. The total number of amino acid residues contained in a region having a value of 2.6 or more is p, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence.
  • n motif represents an amino acid sequence composed of 2 to 27 amino acid residues
  • A the number of alanine residues relative to the total number of amino acid residues in n motif is 83% or more.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin is obtained by replacing one or more amino acid residues in REP with an amino acid residue having a large hydrophobicity index and / or one or more hydrophobic groups in REP as compared to naturally occurring fibroin.
  • the modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif; Glutamine residue content corresponding to the domain sequence having one or more glutamine residues in REP deleted or substituted with other amino acid residues compared to naturally occurring fibroin
  • Glutamine residue content corresponding to the domain sequence having one or more glutamine residues in REP deleted or substituted with other amino acid residues compared to naturally occurring fibroin
  • the high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5] and the high-shrinkage artificial fibroin spun yarn according to any of [6] to [9] having a reduced amino acid sequence A method, a shrinkage method of an artificial fibroin spun yarn according to any one of [10] to [13], or an artificial fibroin spun yarn according to [14].
  • (A) n motif represents an amino acid sequence composed of 2 to 27 amino acid residues, and (A) the number of alanine residues is 80% with respect to the total number of amino acid residues in n motif. That's it.
  • REP indicates an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the above-mentioned modified fibroin contains a GPGXX (wherein X represents an amino acid residue other than a glycine residue) motif in REP, and has a GPGXX motif content of 10% or more, as described in [29] Fibroin spun yarn, high shrinkage artificial fibroin spun yarn production method, artificial fibroin spun yarn shrinkage method, or artificial fibroin spun yarn.
  • the modified fibroin has a glutamine residue content of 9% or less, the high shrinkage artificial fibroin spun yarn according to [29] or [30], the method for producing a high shrinkage artificial fibroin spun yarn, the shrinkage of the artificial fibroin spun yarn Method or artificial fibroin spun yarn.
  • the other amino acid residues are isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine ( Any one of [29] to [31], which is an amino acid residue selected from the group consisting of T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H)
  • a high-shrinkage artificial fibroin spun yarn a method for producing a high-shrinkage artificial fibroin spun yarn, a method for shrinking a man-made fibroin spun yarn, or a man-made fibroin spun yarn.
  • the modified fibroin is a high shrinkage artificial fibroin spun yarn according to any one of [29] to [32], wherein the hydrophobicity of REP is ⁇ 0.8 or more, a method for producing a high shrinkage artificial fibroin spun yarn, Artificial fibroin spun yarn shrinkage method or artificial fibroin spun yarn.
  • the modified fibroin corresponds to the deletion of one or more glutamine residues in REP or substitution with other amino acid residues as compared to naturally occurring fibroin,
  • the high-shrinkage artificial fibroin yarn or high-shrinkage artificial fibroin according to any one of [29] to [33], which has an amino acid sequence corresponding to substitution, deletion, insertion and / or addition of a plurality of amino acid residues A method for producing spun yarn, a shrinking method for artificial fibroin spun yarn, or a artificial fibroin spun yarn.
  • the modified fibroin is a high shrinkage artificial fibroin spun yarn according to any one of [1] to [5], which has a limiting oxygen index (LOI) value of 26.0 or more, and any one of [6] to [9]
  • the method for producing a high shrinkage artificial fibroin spun yarn according to [10] the method for shrinking a man-made fibroin spun yarn according to any one of [10] to [13], or the artificial fibroin spun yarn according to [14].
  • the modified fibroin is a high-shrinkage artificial fibroin spun yarn according to any one of [1] to [5], which has a maximum moisture absorption exotherm calculated according to the following formula A of more than 0.025 ° C./g, [6] to [9]
  • a method for producing a high shrinkage artificial fibroin spun yarn according to any one of [9] a method for shrinking an artificial fibroin spun yarn according to any one of [10] to [13], or a man-made fibroin spin according to [14] yarn.
  • the present invention it is possible to provide a high-shrinkage artificial fibroin spun yarn having a sufficiently high shrinkage ratio, excellent in touch and flexibility, and capable of safe production, and a production method thereof. According to the present invention, it is also possible to provide an artificial fibroin spun yarn that can be produced safely with a sufficiently high shrinkage rate and a shrinkage method thereof.
  • the high-shrinkage artificial fibroin spun yarn according to this embodiment is a contracted artificial fibroin fiber containing modified fibroin.
  • the high shrinkage artificial fibroin spun yarn according to this embodiment has a shrinkage rate defined by the following formula I of more than 1%.
  • Shrinkage rate ⁇ 1 ⁇ (length of the spun artificial fibroin spun yarn / length of spun man-made fibroin spun yarn before contacting with water after spinning) ⁇ ⁇ 100 (%) (Formula I)
  • the modified fibroin according to the present 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. It is a protein containing.
  • an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to either 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 not limited to these, but are typically regions having no amino acid motif repeat characteristic of fibroin and consisting of about 100 amino acids.
  • modified fibroin means an 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.
  • Natural 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.
  • a protein comprising a domain sequence to be processed.
  • the “modified fibroin” may be one that uses the amino acid sequence of naturally-occurring fibroin as it is, or a modified amino acid sequence based on the amino acid sequence of naturally-occurring fibroin (for example, a cloned naturally-derived fibroin).
  • the amino acid sequence may be modified by modifying the gene sequence of fibroin), or artificially designed and synthesized without relying on natural fibroin (for example, a nucleic acid encoding the designed amino acid sequence). It may be one having a desired amino acid sequence by chemical synthesis.
  • the modified fibroin according to the present embodiment is preferably a modified spider silk fibroin because it is excellent in heat retention, moisture absorption heat generation and / or flame retardancy.
  • domain sequence refers to a fibroin-specific crystal region (typically corresponding to the (A) n motif in the amino acid sequence) and an amorphous region (typically in the REP of the amino acid sequence).
  • (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues of 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 (A) n motif may be 40% or more, such as 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 only of alanine residues).
  • a plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues alone.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP is an amino acid sequence composed of 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, or 10-40 amino acid residues. May be. m represents an integer of 2 to 300, 8 to 300, 10 to 300, 20 to 300, 40 to 300, 60 to 300, 80 to 300, 10 to 200, 20 to 200, 20 to 180, 20 to 160, It may be an integer from 20 to 140, or 20 to 120. A plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • the modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to, for example, substitution, deletion, insertion and / or addition of one or a plurality of amino acid residues to the cloned gene sequence of naturally derived fibroin. It can be obtained by modifying the above. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial-directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983), and the like.
  • Naturally-derived 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.
  • Specific examples include fibroin produced by insects or spiders.
  • fibroin produced by insects include, for example, Bombyx mori, Kwako (Bombyx mandarina), Tenaea (Antheraea palaniii), and ⁇ ⁇ (Eriothyraminey).
  • Silkworms produced by silkworms such as Samia cythia, chestnut worms (Caligula japonica), Chuser moth (Antherea mylitta), Antheraea assama, and vespax (Vespaxia spp.) Hornet silk protein.
  • fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).
  • Fibroin produced by spiders includes, for example, spiders belonging to the genus spider (Araneus spp.) Such as the spider spider, the spider spider, the red spider spider, and the bean spider, the genus spiders of the genus Araneus, the spider spider spider, the spider spider genus e Spiders, spiders such as spiders, spiders belonging to the genus Spider, spiders belonging to the genus Pronos, spiders belonging to the genus Trinofunda, such as Torinofundamas (genus Cyrtarachne) Spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Spider (Ordgarius genus), such as the spiders, the spiders, and the spiders belonging to the genus Ordgarius Spiders belonging to the genus Argiope, such as the genus Argiope, spiders belonging to the genus Arachnura, such as the white-tailed spider, spiders belonging to the
  • Spiders belonging to the genus Azumigumi (Menosira)
  • spiders belonging to the genus Dyschiriognatha (genus Dyschiriognatha) such as the common spider spider, the black spider spider, the genus Spider genus belonging to the genus Spider belonging to the genus and the genus Spider belonging to the genus Spider belonging to the genus
  • Produced by spiders belonging to the family Tetragnathidae such as spiders belonging to the genus Prostenops
  • Examples include spider silk protein.
  • the spider silk protein include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.
  • spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers 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 spiroin 1 [derived from Nephila clavipes] (GenBank amino acid sequence 4) ), U37520 (base sequence)), major ampulate spidro n 1 [derived from Latroductus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidrin 2 [derived from Nephila clavata (GenBank accession number AAL32 base sequence 45 AAL32 base sequence amino acid 44, amino acid sequence 44 AAL47)
  • Naturally derived fibroin include fibroin whose sequence information is registered in NCBI GenBank.
  • sequence information is registered in NCBI GenBank.
  • spidin, sample, fibroin, “silk and polypeptide”, or “silk and protein” is described as a keyword in DEFINITION from sequences including INV as DIVISION among the sequence information registered in NCBI GenBank. It can be confirmed by extracting a character string of a specific product from the sequence, CDS, and a sequence in which the specific character string is described from SOURCE to TISSUE TYPE.
  • the modified fibroin according to the present embodiment may be a modified silk fibroin (a modified amino acid sequence of a silk protein produced by a silkworm), or a modified spider silk fibroin (a spider silk protein produced by a spider). It may be a modified amino acid sequence).
  • modified spider silk fibroin is preferable because it is excellent in heat retention, moisture absorption heat generation and / or flame retardancy.
  • modified fibroin examples include a modified fibroin derived from a large sphincter bookmarker protein produced in a large spider gland (first modified fibroin), a domain sequence with a reduced content of glycine residues Modified fibroin (second modified fibroin) having (A) modified fibroin (third modified fibroin) having a domain sequence with reduced content of n motif, content of glycine residue, and (A) n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin having a domain sequence that includes a region with a large hydrophobic index locally (fifth modified fibroin), and glutamine residue content Are modified fibroins having a reduced domain sequence (sixth modified fibroin).
  • Examples of the first modified fibroin include a protein comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m .
  • (A) the number of amino acid residues of the 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 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 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues.
  • the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) n motif-REP] m is an amino acid residue.
  • 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 an amino acid sequence unit represented by Formula 1: [(A) n motif-REP] m , and the C-terminal sequence is represented by any one of SEQ ID NOs: 1 to 3 or It may be a polypeptide that is 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 identical to 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 sequence
  • the amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminal, and the amino acid sequence shown in SEQ ID NO: 3 has 29 residues removed from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
  • modified fibroin As a more specific example 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 Mention may be made of modified fibroin comprising an amino acid sequence having a sequence identity of at least%. The sequence identity is preferably 95% or more.
  • the amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) consisting of a start codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminus.
  • the 13th repeat region was increased to approximately double, and the translation was mutated to terminate at the 1154th amino acid residue.
  • the C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4 is identical to the amino acid sequence shown in SEQ ID NO: 3.
  • the modified fibroin (1-i) may be composed of the 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 compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with 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 to naturally occurring fibroin.
  • G is a glycine residue
  • P is a proline residue
  • X is an amino acid residue other than glycine
  • at least one glycine residue in at least one or more of the motif sequences is substituted with another amino acid residue. May be.
  • the ratio of the motif sequence in which the above glycine residue is replaced with another amino acid residue may be 10% or more with respect to the entire motif sequence.
  • the second modified fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side from the domain sequence (A) from the n motif to the domain sequence.
  • 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 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • the second modified fibroin is preferably one in which the content ratio 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, still more preferably 10% or less, % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable.
  • 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 XGX below.
  • a fibroin modified fibroin or naturally-occurring fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m , (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 included in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence.
  • z / w (%) can be calculated by dividing z by w.
  • z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. It is still more preferable that it is 80% or more. Although there is no restriction
  • the second modified fibroin is obtained by, for example, modifying a cloned natural fibroin gene sequence so as to encode another amino acid residue by substituting at least a part of a base sequence encoding a glycine residue.
  • a glycine residue in GGX motif and GPGXX motif may be selected as a glycine residue to be modified, or substitution may be performed so that z / w is 50.9% or more.
  • an amino acid sequence satisfying the above-described aspect can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues are further substituted or deleted.
  • the amino acid sequence corresponding to the insertion 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 a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, M) hydrophobic amino acid residues such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, hydrophilic amino acid residues such as lysine (K) residues and glutamic acid (E) residues are preferred, and valine (V) residues, leucine (L) residues, isoleucine (I) residues, F) residue and glutamine (Q) residue are more preferred, and glutamine (Q) residue is still 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 shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin (2-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin.
  • the amino acid sequence represented by SEQ ID NO: 7 is the amino acid sequence represented by SEQ ID NO: 6, wherein every two (A) n motifs are deleted from the N-terminal side to the C-terminal side, and further before the C-terminal sequence.
  • One [(A) n motif-REP] is inserted into the.
  • 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 a part of glutamine (Q) residues. Substituted with a serine (S) residue and a part of the amino acid at the C-terminal side was deleted so as to be almost the same as the molecular weight of SEQ ID NO: 7.
  • the amino acid sequence represented by SEQ ID NO: 9 is 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 obtained by adding a predetermined hinge sequence and a His tag sequence to the C-terminus of the sequence repeated four times.
  • 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 z / w values of the amino acid sequence shown by SEQ ID NO: 6, the amino acid sequence shown by SEQ ID NO: 7, the amino acid sequence shown by SEQ ID NO: 8, and the amino acid sequence shown by SEQ ID NO: 9 are 58.7%, 70.1%, 66.1% and 70.0%.
  • the value of x / y at the ratio of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 (described later) 1: 1.8 to 11.3 is: 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.
  • the modified fibroin (2-i) may be composed of 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 (2-ii) includes 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 is contained in REP (XGX ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the second modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of binding specifically to metal ions such as nickel. Therefore, the isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography) Can be used.
  • Specific examples of the tag sequence include the amino acid sequence represented by SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).
  • GST glutathione-S-transferase
  • MBP maltose-binding protein
  • an “epitope tag” using an antigen-antibody reaction can also be used.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence, an antibody against the epitope can be bound.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of hemagglutinin of influenza virus
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • a tag sequence that can be separated with a specific protease can also be used.
  • 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)
  • Examples include (2-iv) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .
  • amino acid sequences represented by SEQ ID NO: 16 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.
  • amino acid sequence shown in SEQ ID NO: 11 is added to the N-terminus of the amino acid sequence shown.
  • the modified fibroin may be composed of 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 (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 (2-iv) has an XGX (which 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 is contained in REP ( Where X is an amino acid residue other than glycine.) Z / w where z is the total number of amino acid residues of the amino acid sequence consisting of z and w is the total number of amino acid residues of REP in the domain sequence. Is preferably 50.9% or more.
  • the second modified fibroin may contain a secretion signal for releasing the 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 host.
  • the third modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) n motif compared to 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 more (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 deletion of.
  • the third modified fibroin has a domain sequence that is at least two consecutive from the N-terminal side to the C-terminal side compared to the naturally occurring fibroin (A) deletion of the n motif, 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 an amino acid sequence whose domain sequence corresponds to that at least every two (A) n motifs are deleted from the N-terminal side to the C-terminal side. .
  • the third modified fibroin includes a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) n motifs from the N-terminal side toward the C-terminal side. -REP]
  • the ratio of the number of amino acid residues in the other REP is 1.8 to
  • x the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units that becomes 11.3
  • x the total number of amino acid residues in the domain sequence is y
  • 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 95% or more. More preferably, it is 100% (meaning that it is composed only of alanine residues).
  • FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin.
  • the domain sequence is from the N-terminal side (left side): (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 n motif.
  • FIG. 1 includes pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and 4th REP and 5th REP), pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison), pattern 4 (first REP and Comparison of the second REP).
  • pattern 1 compare between the first REP and the second REP, and comparison between the third REP and the fourth REP
  • pattern 2 comparison between the first REP and the second REP, and 4th REP and 5th REP
  • pattern 3 (2nd REP and 3rd REP comparison, 4th REP and 5th REP comparison
  • pattern 4 first REP and Comparison of the second REP
  • the number of amino acid residues of each REP in the two adjacent [(A) n motif-REP] units selected is compared.
  • each pattern the number of all amino acid residues of two adjacent [(A) n motif-REP] units indicated by solid lines is added (not only REP but also (A) the number of amino acid residues of the n motif. is there.). Then, the total value added is compared, and the total value (maximum value of the total value) of the pattern having the maximum total value is set as 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 of the domain sequence.
  • x / y is preferably 50% or more, more preferably 60% or more, still more preferably 65% or more, and even more preferably 70% or more. Preferably, it is still more preferably 75% or more, and particularly preferably 80% or more. There is no restriction
  • x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x / y / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.
  • a plurality of third modified fibroins are present in the domain sequence (A)
  • x / y is 46.4% or more It is preferably 50% or more, more preferably 55% or more, still more preferably 60% or more, still more preferably 70% or more, and more preferably 80% or more. It is particularly preferred.
  • x / y in naturally derived fibroin will be described.
  • 663 types of fibroin (of which 415 types were derived from spiders) were extracted.
  • x / y is calculated from the amino acid sequence of naturally derived fibroin composed of the domain sequence represented by Formula 1: [(A) n motif-REP] m by the above-described calculation method.
  • FIG. 3 shows the results when the jagged ratio is 1: 1.9 to 4.1.
  • the horizontal axis indicates x / y (%), and the vertical axis indicates frequency.
  • x / y in naturally derived fibroin is less than 64.2% (the highest, 64.14%).
  • one or a plurality of sequences encoding the n motif is deleted so that x / y is 64.2% or more from the cloned gene sequence of naturally occurring fibroin.
  • an amino acid sequence corresponding to the deletion of one or more (A) n motifs is designed so that x / y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence.
  • one or more amino acid residues are further substituted, deleted, inserted and / or added.
  • the amino acid sequence corresponding to this may be modified.
  • third modified fibroin examples include (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 -PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, Mention may be made of modified fibroin.
  • the modified fibroin (3-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 17 is derived from the amino acid sequence represented by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally-occurring fibroin from the N-terminal side to the C-terminal side every two (A) n
  • the motif is deleted, and one [(A) n motif-REP] is 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 in the second modified fibroin.
  • the value of x / y in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%.
  • the value of x / y in the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 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 z / w values in the amino acid sequences shown in 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% and 66. respectively. 1% and 70.0%.
  • the modified fibroin (3-i) may consist 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 (3-ii) includes 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 of (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 from the N-terminal side to the C-terminal side
  • the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared, and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other
  • x / y is 64.2% or more, where x is the maximum total value of the total number of bases and y is the total number of amino acid residues in the domain sequence.
  • the third modified fibroin may contain the tag sequence described above at one or both of the N-terminal and C-terminal.
  • modified fibroin containing a tag sequence 3-iii) amino acids represented by SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799)
  • a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the sequence or (3-iv) the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .
  • amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 11 at the N-terminus 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. Are added to the amino acid sequence (including His tag sequence and hinge sequence).
  • the modified fibroin may be composed of 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 (3-iv) comprises 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 (3-iv) has a sequence identity of 90% or more 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 from the N-terminal side to the C-terminal side.
  • the other X is the maximum total value of the total number of amino acid residues of two adjacent [(A) n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3.
  • x / y is preferably 64.2% or more.
  • the third modified fibroin may contain a secretion signal for releasing the 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 host.
  • the fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced glycine residue content in addition to (A) a reduced content of the n motif compared to naturally occurring fibroin. It is what you have.
  • the domain sequence of the fourth modified fibroin has at least one or more (A) n motifs deleted as compared to naturally occurring fibroin, and at least one or more glycine residues in the REP. It can be said to have an amino acid sequence corresponding to the substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the second modified fibroin described above and the third modified fibroin. Specific embodiments and the like are as described in the second modified fibroin and the third modified fibroin.
  • SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), 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 are replaced with amino acid residues having a higher hydrophobicity index and / or REP compared to naturally occurring fibroin. It may have an amino acid sequence including a region having a large hydrophobicity index corresponding to the insertion of one or more amino acid residues having a large hydrophobicity index.
  • the region where the hydrophobic index is locally large is preferably composed of 2 to 4 amino acid residues.
  • the amino acid residue having a large hydrophobicity index is an amino acid 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 has one or more amino acid residues in REP substituted with amino acid residues having a higher hydrophobicity index and / or one or more in REP compared to naturally occurring fibroin.
  • substitution, deletion, insertion and / or addition of one or more amino acid residues as compared with naturally occurring fibroin There may be amino acid sequence modifications corresponding to the above.
  • the fifth modified fibroin is obtained by removing 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 in REP.
  • hydrophilic amino acid residues for example, amino acid residues having a negative hydrophobicity index
  • a group for example, an amino acid residue having a positive hydrophobicity index
  • one or more hydrophilic amino acid residues in REP are substituted 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 It can also be obtained by designing an amino acid sequence corresponding to insertion of, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • one or more hydrophilic amino acid residues in REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin and / or one or more hydrophobic amino acids 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 comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and is located on the most C-terminal side (A) from the n motif to the C terminus of the domain sequence.
  • p is the total number of amino acid residues included in the region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more
  • (A) When the total number of amino acid residues contained in the sequence excluding the sequence from the n motif to the C terminus of the domain sequence, which is located at the most C-terminal side, from the domain sequence is q, p / q is 6 It may have an amino acid sequence that is 2% or more.
  • hydrophobicity index of amino acid residues As for the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) “A simple method for displaying the hydropathic character of bio.p. 7”. 105-132). Specifically, the hydrophobicity index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
  • a sequence obtained by removing the sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m to the most C-terminal side from the domain (A) n motif to the C terminus of the domain sequence. (Hereinafter referred to as “array A”).
  • array A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated.
  • the average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in the four consecutive amino acid residues by 4 (number of amino acid residues).
  • the average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 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 if a certain amino acid residue corresponds to a plurality of “four consecutive amino acid residues whose average value of hydrophobicity index is 2.6 or more”, it should be included as one amino acid residue in the region. become.
  • 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.
  • the average value of the hydrophobicity index of four consecutive amino acid residues is 2
  • p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and preferably 20% or more. Even more preferably, it is still more preferably 30% or more.
  • the upper limit of p / q is not particularly limited, but may be 45% or less, for example.
  • 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 naturally-derived fibroin thus cloned satisfies the above p / q condition. Is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index) and / or one or more hydrophobic amino acid residues are inserted in 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.
  • hydrophilic amino acid residues for example, a hydrophobicity index
  • an amino acid sequence satisfying the above p / q conditions can be designed from the amino acid sequence of naturally derived fibroin, and a nucleic acid encoding the designed amino acid sequence can be obtained by chemical synthesis.
  • one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index and / or one or more amino acid residues in REP.
  • modifications 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 ) are 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-PRT720), 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 (5-i) will be described.
  • the amino acid sequence represented by SEQ ID NO: 19 consists of 3 amino acid residues for every other REP with respect to the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), excluding the domain sequence of the terminal on the C-terminal side.
  • An amino acid sequence (VLI) is inserted at two positions, a part of glutamine (Q) residues is substituted with a serine (S) residue, and a part of amino acids on the C-terminal side is deleted.
  • the amino acid sequence represented by SEQ ID NO: 20 is an amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there.
  • the amino acid sequence shown in SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues into the amino acid sequence shown in SEQ ID NO: 8 every other REP.
  • the modified fibroin (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 (5-ii) comprises 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 a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal.
  • modified fibroin containing the tag sequence examples include (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 And a modified fibroin comprising an amino acid sequence having 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.
  • amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminus of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Including a sequence and a hinge sequence).
  • the modified fibroin may be composed of the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24.
  • the modified fibroin (5-iv) comprises an amino acid sequence having 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.
  • 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 a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) n
  • the amino acids included in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more P is the total number of residues
  • P / q is preferably 6.2% or more.
  • the fifth modified fibroin may contain a secretion signal for releasing the 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 host.
  • the sixth modified fibroin has an amino acid sequence in which the content of glutamine residues is reduced compared to 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 ratio of the GPGXX motif is usually 1% or more, may be 5% or more, and is preferably 10% or more.
  • the upper limit of GPGXX motif content rate 50% or less may be sufficient and 30% or less may be sufficient.
  • GPGXX motif content is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m- (A) fibroin (modified fibroin or naturally derived) containing a domain sequence represented by the n motif In fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the domain sequence (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
  • the number obtained by multiplying the total number by three is s, and is located at the most C-terminal side.
  • the sequence from the n motif to the C-terminal of the domain sequence is determined from the domain sequence.
  • the content ratio of the GPGXX motif is calculated as s / t, where t is the total number of amino acid residues of all REPs excluding the n motif. It is.
  • “A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminal of the domain sequence from the domain sequence” (A)
  • the sequence from the n motif to the C terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence that is not highly correlated with the sequence characteristic of fibroin, and m is small In this case (that is, when the domain sequence is short), the calculation result of the content ratio of the GPGXX motif is affected, so this influence is excluded.
  • the “GPGXX motif” is located at the C-terminus of REP, even if “XX” is, for example, “AA”, it is treated as “GPGXX motif”.
  • FIG. 5 is a schematic diagram showing the domain sequence of the modified fibroin.
  • all REPs are “a sequence located at the most C-terminal side (A)
  • the sequence from the n motif to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence indicated by “region A” in FIG. ))
  • 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 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • A) the sequence from the n- motif to the C-terminal of the domain sequence located on the most C-terminal side is included.
  • the total number of glutamine residues contained in the region is u
  • the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence
  • (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 "A sequence located at the most C-terminal side (A) excluding the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.
  • the sixth modified fibroin corresponds to its domain sequence having one or more glutamine residues in REP deleted or replaced with other amino acid residues compared to 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 the glutamine residue. Table 1 shows the hydrophobicity index of amino acid residues.
  • amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), 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. More preferably, it is an amino acid residue selected from isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
  • the hydrophobicity of REP is preferably ⁇ 0.8 or more, more preferably ⁇ 0.7 or more, still more preferably 0 or more, and 0.3 or more. It is still more preferable that it is and it is especially preferable that it is 0.4 or more.
  • the “hydrophobicity of REP” is a value calculated by the following method.
  • Formula 1 [(A) n motif-REP] m
  • Formula 2 [(A) n motif-REP] m-
  • A) the sequence from the n- motif to the C-terminal of the domain sequence located on the most C-terminal side is included.
  • the sum of the hydrophobicity index of each amino acid residue in the region is represented by v, and the sequence from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence is excluded from the domain sequence, and ( A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the n motif.
  • t is the total number of amino acid residues of all REPs excluding the n motif.
  • the sixth modified fibroin has its domain sequence deleted one or more glutamine residues in REP and / or one or more glutamine residues in REP compared to naturally occurring fibroin.
  • modifications corresponding to substitution of other amino acid residues there may also be amino acid sequence modifications corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues. .
  • the sixth modified fibroin is, for example, deleting one or more glutamine residues in REP from the cloned gene sequence of naturally occurring fibroin and / or other one or more glutamine residues in REP. It can obtain by substituting to the amino acid residue.
  • one or more glutamine residues in REP are deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP are replaced with other amino acid residues.
  • it can also be obtained by designing a corresponding amino acid sequence and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
  • sixth modified fibroin examples include (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met -PRT916), a modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698) or SEQ ID NO: 32 (Met-PRT966), or (6-ii) 90% or more sequence identity with the amino acid sequence 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 or SEQ ID NO: 32 Mention may be made of modified fibroin comprising an amino acid sequence having.
  • the (6-i) modified fibroin will be described.
  • the amino acid sequence represented by SEQ ID NO: 25 is obtained by substituting VL for 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 replacing 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 replacing the remaining Q with I.
  • the amino acid sequence represented by SEQ ID NO: 28 is obtained by replacing all QQs in the amino acid sequence represented by SEQ ID NO: 7 with VI and replacing the remaining Q with L.
  • the amino acid sequence represented by SEQ ID NO: 29 is one in which all QQs in the amino acid sequence represented by SEQ ID NO: 7 are substituted with VF, and the remaining Q is substituted with I.
  • the amino acid sequence represented by SEQ ID NO: 30 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT468) in which all QQs are substituted with VL.
  • the amino acid sequence shown in SEQ ID NO: 31 is obtained by substituting all QQs in the amino acid sequence shown in SEQ ID NO: 8 with VL and replacing the remaining Q with I.
  • 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 and SEQ ID NO: 32 all have a glutamine residue content of 9% or less. Yes (Table 2).
  • the modified fibroin (6-i) comprises the amino acid sequence 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 or SEQ ID NO: 32. There may be.
  • the modified fibroin of (6-ii) is 90% or more of the amino acid sequence 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 or SEQ ID NO: 32 An amino acid sequence having the following sequence identity is included.
  • the modified fibroin of (6-ii) is also represented by the formula 1: [(A) n motif-REP] m or the formula 2: [(A) n motif-REP] m- (A) n motif.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a tag sequence at one or both of the N-terminal and C-terminal. This makes it possible to isolate, immobilize, detect and visualize the modified fibroin.
  • modified fibroin containing a 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) or modified fibroin comprising the amino acid sequence shown by SEQ ID NO: 40 (PRT966), or (6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • 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, and SEQ ID NO: 40 are SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, respectively.
  • the amino acid sequence represented by SEQ ID NO: 11 was added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32. Is.
  • 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.
  • amino acid sequence represented by SEQ ID NO: 40 has a glutamine residue content of 9% or less (Table 3).
  • the modified fibroin (6-iii) comprises 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, or SEQ ID NO: 40. There may be.
  • the modified fibroin of (6-iv) has 90% or more of 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 or SEQ ID NO: 40
  • An amino acid sequence having the following sequence identity is included.
  • the modified fibroin of (6-iv) is also a domain represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif.
  • the sequence identity is preferably 95% or more.
  • the modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less.
  • the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.
  • the sixth modified fibroin may contain a secretion signal for releasing the 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 host.
  • the modified fibroin has 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. It may be a modified fibroin having the following characteristics.
  • the modified fibroin according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, 26 It may be above, 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 test method for powdery granular or low melting point synthetic resin on May 31, 1995, Fire Department 50, Hazardous Materials Regulation Division Director.
  • the modified fibroin according to the present embodiment may have a maximum moisture absorption exotherm calculated according to the following formula A of more than 0.025 ° C./g.
  • a low humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 40%
  • a high humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 90%.
  • the modified fibroin according to the present embodiment may have a maximum moisture absorption exotherm of 0.026 ° C./g or more, 0.027 ° C./g or more, or 0.028 ° C./g or more. May be 0.029 ° C./g or more, 0.030 ° C./g or more, 0.035 ° C./g or more, and 0.040 ° C./g or more. There may be. Although there is no restriction
  • the modified fibroin according to the present embodiment may have a heat retention index determined according to the following formula B of 0.20 or more.
  • Thermal insulation index Heat retention ratio (%) / Sample weight (g / m 2 )
  • a heat retention rate means the heat retention rate measured by the dry contact method using a thermolab II type tester (under a wind of 30 cm / sec), and is determined by a method described in an example column described later. The value to be measured.
  • the heat retention index of the modified fibroin according to this embodiment may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, may be 0.28 or more, and may be 0. .30 or more and 0.32 or more.
  • exponent there is no restriction
  • exponent For example, it may be 0.60 or less or 0.40 or less.
  • the modified fibroin according to the present embodiment includes, 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. It 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 such as amplification by polymerase chain reaction (PCR), cloning, modification by genetic engineering techniques, or chemical synthesis. be able to.
  • the method for chemically synthesizing nucleic acids is not particularly limited. For example, based on the amino acid sequence information of fibroin obtained from the NCBI web database, etc., AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) A gene can be chemically synthesized by a method of linking oligonucleotides that are synthesized automatically by PCR or the like.
  • a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. May be.
  • Regulatory sequences are sequences that control the expression of modified fibroin in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in the host cell and can induce expression of the modified fibroin may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • vectors for introducing a nucleic acid encoding a modified fibroin include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of a vector into which a nucleic acid encoding a modified fibroin is introduced include YEP13 (ATCC 37115) and YEp24 (ATCC 37051).
  • 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.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method, 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, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
  • 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 from the culture medium.
  • the method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture 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 as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed modified fibroin can be performed by a commonly used method.
  • the host cells are recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton.
  • the host cells are disrupted with a Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • a method usually used for isolation and purification of modified fibroin that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method by DE, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), or a resin such as S-Sepharose FF (manufactured by Pharmacia) Electrophoresis such as cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Use methods such as law alone or in combination It is possible to obtain a purified product.
  • a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA
  • the modified fibroin when expressed by forming an insoluble substance in the cell, the host cell is similarly collected, crushed, and centrifuged to collect the modified fibroin insoluble substance as a precipitate fraction.
  • the recovered insoluble form of modified fibroin can be solubilized with a protein denaturant.
  • a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above.
  • the modified fibroin when secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture with a technique such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.
  • the modified fibroin fiber (filament) according to the present embodiment is obtained by spinning the above-described modified fibroin and contains the above-described modified fibroin as a main component.
  • the modified fibroin fiber can be produced by a known spinning method.
  • the modified fibroin produced according to the above-described method is converted into dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF),
  • a dope solution is prepared by adding to a solvent such as formic acid or hexafluoroisopropanol (HFIP) together with an inorganic salt as a dissolution accelerator, if necessary, and dissolving.
  • the modified fibroin fiber can be obtained by spinning using this dope solution by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning.
  • Preferred spinning methods include wet spinning or dry wet spinning.
  • FIG. 6 is an explanatory view schematically showing an example of a spinning device for producing a modified fibroin fiber.
  • a spinning device 10 illustrated in FIG. 6 is an example of a spinning device for dry and wet spinning, and includes an extrusion device 1, an undrawn yarn production device 2, a wet heat drawing device 3, and a drying device 4.
  • the dope solution 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8.
  • the dope solution may be filled into a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded dope liquid 6 is supplied into the coagulating liquid 11 in the coagulating liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulated body is formed.
  • the fibrous solidified body is supplied into the hot water 12 in the drawing bath 21 and drawn.
  • the draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14.
  • the stretched fibrous solidified body is supplied to the drying device 4 and dried in the yarn path 22, and the modified fibroin fiber 36 is obtained as the wound body 5.
  • Reference numerals 18a to 18g denote thread guides.
  • the coagulation liquid 11 may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone.
  • the coagulation liquid 11 may appropriately contain water.
  • the temperature of the coagulation 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 through which the coagulated modified fibroin passes through the coagulating liquid 11 may be long enough to efficiently remove the solvent. 500 mm.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time in the coagulating liquid 11 may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulating liquid tank 20 may be provided in multiple stages, and the stretching may be performed in each stage or a specific stage as necessary.
  • the stretching performed when obtaining the modified fibroin fiber for example, dry stretching is employed in addition to the above-described pre-stretching in the coagulating liquid tank 20 and the wet heat stretching performed in the stretching bath 21.
  • Wet and hot stretching can be performed in warm water, in a solution obtained by adding an organic solvent or the like to warm water, and in steam heating.
  • the temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • undrawn yarn or predrawn yarn
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, 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 the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and 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, and 10 times or less.
  • the artificial fibroin spun yarn is obtained by a method including a cutting step of cutting the above-described modified fibroin fiber (filament) into an appropriate length to obtain a modified fibroin staple, and a spinning step of spinning the obtained modified fibroin staple. be able to.
  • the cutting step can be performed using any apparatus that can cut the modified fibroin fiber.
  • An example of such an apparatus is a table type fiber cutting machine (s / NO. IT-160201-NP-300).
  • the length of the modified fibroin staple is not particularly limited, but is, for example, 20 mm or more, 20 to 140 mm, 70 to 140 mm, or 20 to 70 mm.
  • the spinning process can be performed by a known spinning method.
  • the spinning method include cotton spinning, lashing, and spinning methods.
  • the apparatus used for these spinning methods is not particularly limited, and a commonly used apparatus can be used.
  • the modified fibroin staple may be opened or defibrated by a hair opener (opener) or a breaker (breaker).
  • the method for producing the artificial fibroin spun yarn may have a crimping step before or after the cutting step.
  • the crimping step may be, for example, a step of performing a mechanical crimping method such as an indentation method, and the modified fibroin fiber (filament) or the modified fibroin staple is brought into contact with an aqueous medium to be crimped (hereinafter, referred to as a crimping method).
  • the process may be referred to as “water crimp”.
  • the aqueous medium is a liquid or gas (steam) medium containing water (including water vapor).
  • the aqueous medium may be water or a mixed solution of water and a hydrophilic solvent.
  • a hydrophilic solvent it is also possible to use volatile solvents, such as ethanol and methanol, or its vapor
  • the aqueous medium may be a mixed liquid of water and a volatile solvent such as ethanol and methanol, and is preferably water or a mixed liquid of water and ethanol.
  • the ratio of water to the volatile solvent or the vapor thereof is not particularly limited.
  • the water: volatile solvent or the vapor thereof may be 10:90 to 90:10 by mass ratio.
  • the proportion of water is preferably 30% by mass or more, and may be 40% by mass or 50% by mass or more.
  • the aqueous medium is preferably a liquid or gas at 10 to 230 ° C. containing water (including water vapor).
  • 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 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 from 100 to 230 ° C, more preferably from 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, thermal modification of the modified fibroin filament can be prevented.
  • 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 modified fibroin filament high. 60 ° C. or less is preferable.
  • the time of contact with the aqueous medium is not particularly limited, but may be 30 seconds or more, may be 1 minute or more, or 2 minutes or more, and is preferably 10 minutes or less from the viewpoint of productivity. In the case of steam, it is considered that a large contraction rate can be obtained in a shorter time than liquid.
  • the contact with the aqueous medium may be performed under normal pressure or under reduced pressure (for example, vacuum).
  • Examples of the method of contacting with an aqueous medium include a method of immersing the modified fibroin filament or the modified fibroin staple in the aqueous medium, a method of spraying the modified fibroin filament or the modified fibroin staple on the aqueous medium, and a steam of the aqueous medium filled. And a method of exposing the modified fibroin filament or the modified fibroin staple to the environment.
  • the aqueous medium is steam
  • the contact of the aqueous medium with the modified fibroin filament or the modified fibroin staple can be performed using a general steam setting apparatus.
  • the steam setting device include devices such as product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and product name: EPS-400 (manufactured by Sakurai Dyeing Machinery Co., Ltd.).
  • the modified fibroin filament or the modified fibroin staple is accommodated in a predetermined accommodating chamber, while the aqueous medium steam is introduced into the accommodating chamber. Then, the steam is brought into contact with the modified fibroin filament or the modified fibroin staple while adjusting the temperature in the accommodation chamber to the predetermined temperature (for example, 100 ° C. to 230 ° C.).
  • the crimping step of the modified fibroin filament or modified fibroin staple by contact with an aqueous medium preferably no tensile force is applied to the single fiber and bundle (such as wrinkles) of the modified fibroin filament and modified fibroin staple ( It is carried out in a state in which no tension is applied in the fiber axis direction, or in a state where a predetermined amount is added (a tension is applied in a predetermined amount in the fiber axis direction).
  • the degree of crimping can be controlled by adjusting the tensile force applied to the modified fibroin filament or the modified fibroin staple.
  • the filament and the staple are loaded.
  • a method of adjusting the load a method of fixing both ends in a state where the filament and the staple are loosened, a method of changing the amount of looseness, and winding the filament around a wound body such as a paper tube or a bobbin. Examples thereof include a method of appropriately changing the winding force (clamping force to the paper tube or bobbin).
  • the modified fibroin filament or modified fibroin staple may be further dried after contacting with an aqueous medium.
  • the drying method is not particularly limited, and the drying may be natural drying or hot air or 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 shrinkage rate of the artificial fibroin spun yarn can be further increased by performing spinning using a staple in which the crimped staple and the uncrimped staple are mixed in the spinning process. .
  • the artificial fibroin spun yarn may be a single yarn or a mixed yarn such as a twin yarn.
  • a spun yarn of 100% modified fibroin a blended yarn of at least one selected from a staple of 100% modified fibroin and other protein staples and chemical fiber staples, modified fibroin and others
  • an oil agent may be attached in advance before the spinning process. Oil adhesion can be carried out at any stage in the manufacturing process.
  • the oil agent may be attached before the cutting process, simultaneously with the cutting process, or after the cutting process.
  • the oil agent is not particularly limited, and may be a known oil agent used for general purposes such as process passability and functionality imparting such as antistatic, friction reducing, flexibility imparting, or water repellency imparting. Any of them can be used.
  • the artificial fibroin spun yarn according to the present embodiment includes the above-described modified fibroin and can be highly contracted only by contacting with moisture (water, water vapor, etc.).
  • the possibility of high shrinkage means that the shrinkage rate defined by the following formula II is more than 1%.
  • Shrinkage rate ⁇ 1 ⁇ (length of artificial fibroin spun yarn subjected to shrinkage processing including contact with water having less than boiling point / length of artificial fibroin spun yarn before performing the shrinkage processing) ⁇ ⁇ 100 (% (Formula II)
  • the artificial fibroin spun yarn according to the present embodiment includes the above-described modified fibroin, and the shrinkage rate defined by the formula II is more than 1%, so that the shrinkage is higher than that of a conventionally known protein spun yarn. It is possible to shrink at a rate, and it is possible to shrink at a higher shrinkage rate under milder conditions than a conventionally known synthetic yarn.
  • the shrinkage of the artificial fibroin spun yarn according to this embodiment is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, It may be 15% or more, 20% or more, 25% or more, 30% or more.
  • the upper limit of the shrinkage rate is not particularly limited, but may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less.
  • the artificial fibroin spun yarn according to the present embodiment includes a modified fibroin fiber (meaning a filament after spinning) as a raw material, and when there is no contact history with moisture (water, water vapor, etc.), High shrinkage is possible. That is, when there is no history of contact with moisture, the shrinkage of the artificial fibroin spun yarn according to this embodiment may be, for example, more than 7%, preferably 15% or more, and more than 25%. Is more preferably 32% or more, still more preferably 40% or more, still more preferably 48% or more, particularly preferably 56% or more, and 64% or more. It is especially more preferable that it is 72% or more.
  • the upper limit of shrinkage is usually 80% or less.
  • the artificial fibroin spun yarn according to this embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, or 24 or more. 26 or more, 28 or more, 29 or more, or 30 or more.
  • LOI limiting oxygen index
  • the artificial fibroin spun yarn according to this embodiment may have a maximum moisture absorption exotherm calculated according to the following formula A of more than 0.025 ° C./g.
  • a low humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 40%
  • a high humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 90%.
  • the artificial fibroin spun yarn according to this embodiment may have a maximum moisture absorption exotherm of 0.026 ° C / g or more, 0.027 ° C / g or more, and 0.028 ° C / g or more. May be 0.029 ° C / g or more, 0.030 ° C / g or more, 0.035 ° C / g or more, and 0.040 ° C / g. It may be the above. Although there is no restriction
  • the artificial fibroin spun yarn according to this embodiment may have a heat retention index determined according to the following formula B of 0.20 or more.
  • the heat retention index of the artificial fibroin spun yarn according to this embodiment may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, and may be 0.28 or more. 0.30 or more and 0.32 or more.
  • exponent there is no restriction
  • exponent For example, it may be 0.60 or less or 0.40 or less.
  • the shrinking process includes bringing the artificial fibroin spun yarn into contact with water having a boiling point (hereinafter also referred to as “contacting step”).
  • the shrinkage processing may further include drying the artificial fibroin spun yarn after contact with water (after the contact step) (hereinafter also referred to as “drying step”).
  • the temperature of the water to be brought into contact with the artificial fibroin spun yarn in the contact step may be less than the boiling point. Thereby, the handleability, the workability of shrinkage processing, and the like are improved. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit value 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 lower.
  • the method for bringing water into contact with the artificial fibroin spun yarn is not particularly limited.
  • the method include, for example, a method of immersing a man-made fibroin spun yarn in water, a method of spraying water on a man-made fibroin spun yarn at room temperature or in a heated steam state, and the man-made fibroin spun yarn filled with water vapor. And a method of exposing to a high humidity environment.
  • the shrinkage time can be shortened effectively, and simplification of the processing equipment can be realized. Therefore, the method of immersing the artificial fibroin spun yarn in water is preferable.
  • the drying step is a step of drying the artificial fibroin spun yarn that has undergone the contact step. Drying may be, for example, natural drying or forced drying using a drying facility. As the drying equipment, any known drying equipment of contact type or non-contact type can be used. Also, the drying temperature is not limited as long as it is lower than the temperature at which the protein (modified fibroin, etc.) contained in the artificial fibroin spun yarn is decomposed or the artificial fibroin spun yarn is thermally damaged. Although it is not, it is generally a temperature within the range of 20 to 150 ° C., preferably a temperature within the range of 50 to 100 ° C.
  • the artificial fibroin spun yarn is dried more quickly and efficiently without thermal damage of the artificial fibroin spun yarn or decomposition of the protein contained in the artificial fibroin spun yarn.
  • the drying time is appropriately set according to the drying temperature or the like, and for example, a time that can eliminate the influence on the quality and physical properties of the artificial fibroin spun yarn by overdrying as much as possible is adopted.
  • the shrinkage of the artificial fibroin spun yarn according to the present embodiment can be controlled by controlling, for example, the temperature of water to be contacted, the contact time with water, and the tensile force when contacting with water.
  • the artificial fibroin spun yarn according to the present embodiment is capable of high shrinkage, for example, the high shrinkage artificial fibroin spun yarn can be obtained through the above-described shrinkage processing (contacting step and drying step as necessary). .
  • the shrinkage rate defined by the following formula I is more than 1%.
  • Shrinkage rate ⁇ 1 ⁇ (length of the spun artificial fibroin spun yarn / length of spun man-made fibroin spun yarn before contacting with water after spinning) ⁇ ⁇ 100 (%) (Formula I)
  • the shrinkage of the high shrinkage artificial fibroin spun yarn according to this embodiment is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% Above, it may be 15% or more, 20% or more, 25% or more, 30% or more.
  • the upper limit of the shrinkage rate is not particularly limited, but may be 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less.
  • the artificial fibroin spun yarn according to the present embodiment includes a modified fibroin fiber (meaning a filament after spinning) as a raw material, and when there is no contact history with moisture (water, water vapor, etc.), High shrinkage is possible. That is, when there is no history of contact with moisture, the shrinkage rate of the high shrinkage artificial fibroin spun yarn according to the present embodiment may be, for example, more than 7%, preferably 15% or more, and more than 25%. More preferably, it is more preferably 32% or more, still more preferably 40% or more, still more preferably 48% or more, particularly preferably 56% or more, 64 % Or more is particularly preferable, and 72% or more is most preferable.
  • the upper limit of shrinkage is usually 80% or less.
  • the high-shrinkage artificial fibroin spun yarn 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. May be 26 or more, 28 or more, 29 or more, or 30 or more.
  • LOI limiting oxygen index
  • the high-shrinkage artificial fibroin spun yarn according to this embodiment may have a maximum moisture absorption exotherm calculated according to the following formula A of more than 0.025 ° C./g.
  • Maximum moisture absorption exotherm ⁇ (maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then transferred to a high humidity environment) ⁇ (sample Sample temperature when placed in a low-humidity environment until the temperature reaches equilibrium and then moved to a high-humidity environment) ⁇ (° C.) / Sample weight (g)
  • a low humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 40%
  • a high humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 90%.
  • the high-shrinkage artificial fibroin spun yarn according to the present embodiment may have a maximum moisture absorption exotherm of 0.026 ° C./g or more, 0.027 ° C./g or more, and 0.028 ° C./g. It may be 0.029 ° C./g or more, 0.030 ° C./g or more, 0.035 ° C./g or more, 0.040 ° C. / G or more.
  • limiting in particular in the upper limit of the maximum moisture absorption heat generation degree Usually, it is 0.060 degrees C / g or less.
  • the high shrinkage artificial fibroin spun yarn according to the present embodiment may have a heat retention index determined according to the following formula B of 0.20 or more.
  • the heat retention index of the high shrinkage artificial fibroin spun yarn according to this embodiment may be 0.22 or more, may be 0.24 or more, may be 0.26 or more, and may be 0.28 or more. It may be 0.30 or more, and may be 0.32 or more. Although there is no restriction
  • the method for shrinking an artificial fibroin spun yarn according to the present embodiment includes a step of shrinking an artificial fibroin spun yarn containing modified fibroin by bringing it into contact with water having a boiling point below.
  • the shrinking step may further include drying the artificial fibroin spun yarn after contact with water.
  • the artificial fibroin spun yarn shrinking method according to the present embodiment can be carried out in the same manner as the shrinking process described above.
  • Each nucleic acid encoding a protein having the designed amino acid sequence was 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 cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was digested with NdeI and EcoRI, respectively, and then excised, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.
  • the seed culture was added to a jar fermenter to which 500 ml of production medium (Table 5) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. SDS-PAGE was performed using cells prepared from the culture solution before and after IPTG addition, and the appearance of a band of a size corresponding to the target modified fibroin depending on the addition of IPTG Expression was confirmed.
  • the washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 60 ° C. And stirred 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.). White aggregated protein obtained after dialysis was collected by centrifugation. Water was removed from the recovered aggregated protein with a freeze dryer to obtain freeze-dried powders of the desired modified fibroin (PRT399, PRT380, PRT410, PRT799, and PRT918).
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl,
  • spun and drawn modified fibroin fibers were produced by dry and wet spinning using a spinning device similar to the spinning device 10 shown in FIG.
  • the spinning device used is a spinning device 10 shown in FIG. 6, and a second undrawn yarn production device (a first bath) and a wet heat drawing device 3 (third bath) are further provided between the undrawn yarn production device 2 (first bath) and the wet heat drawing device 3 (third bath).
  • a second bath The dry and wet spinning conditions are as follows. Extrusion nozzle diameter: 0.2 mm Liquid and temperature in the first to third baths: see Table 6 Total draw ratio: see Table 6 Drying temperature: 60 ° C.
  • a plurality of modified fibroin fibers for testing each having a length of 30 cm were cut from the wound product of modified fibroin fibers obtained in Production Examples 1 to 19.
  • the plurality of modified fibroin fibers were bundled to obtain a modified fibroin fiber bundle having a fineness of 150 denier.
  • 0.8 g of lead weight was attached to each modified fibroin fiber bundle, and in this state, each modified fibroin fiber bundle was immersed in water at a temperature shown in Tables 7 to 10 (shrinkage processing 1). Thereafter, the length of each modified fibroin fiber bundle was measured in water.
  • the modified fibroin fiber (filament) according to the present invention has a sufficiently high shrinkage rate. Therefore, it is recognized that the artificial fibroin spun yarn spun from the modified fibroin fiber (filament) according to the present invention also has a sufficiently high shrinkage rate. Further, the shrinkage rate of the modified fibroin fiber (filament) and the artificial fibroin spun yarn according to the present invention is controlled by controlling, for example, the temperature of water to be contacted, the contact time with water, and the tensile force when contacting with water. I can understand what I can do.
  • Example 1 Production and evaluation of artificial fibroin spun yarn
  • DMSO Dimethyl sulfoxide
  • PRT799 modified fibroin obtained in Test Example 1 was prepared there.
  • the lyophilized powder was added to a concentration of 24% by mass and dissolved using a shaker for 3 hours. Thereafter, insoluble matters and bubbles were removed to obtain a modified fibroin solution.
  • the obtained modified fibroin solution was used as a dope solution (spinning stock solution), and dry-wet spinning was performed using a spinning device according to the spinning device 10 shown in FIG. 6 to obtain modified fibroin filaments (24 multifilaments).
  • the obtained modified fibroin filament was wound on a bobbin.
  • the dry and wet spinning conditions are as follows. Coagulation liquid (methanol) temperature: 5-10 ° C Stretch ratio: 5 times Drying temperature: 80 ° C
  • Modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm with a table-type fiber cutter to obtain modified fibroin staples. A part of the obtained staple was crimped by being immersed in water at 40 ° C. for 1 minute to be crimped, and then dried at 40 ° C. for 18 hours to obtain a crimped staple. Next, staples containing crimped staples and uncrimped staples at a ratio of 7: 3 (weight ratio) were spun by a known spinning device to obtain an artificial fibroin spun yarn 1. The fineness (count) of the artificial fibroin spun yarn 1 was 30 Nm.
  • Example 2 Production and evaluation of artificial fibroin spun yarn
  • a modified fibroin filament was obtained in the same manner as in Example 1.
  • Modified fibroin filaments (24 multifilaments) were cut into a length of 40 mm with a table-type fiber cutter to obtain modified fibroin staples.
  • the obtained staple was crimped by being immersed in water at 40 ° C. for 1 minute to be crimped, and then dried at 40 ° C. for 18 hours to obtain a crimped staple.
  • the crimped staple was spun by a known spinning device to obtain an artificial fibroin spun yarn 3.
  • the fineness (count) of the artificial fibroin spun yarn 3 was 3 Nm.
  • the artificial fibroin spun yarn of the present invention had a sufficiently high shrinkage rate, and the highly contracted artificial fibroin spun yarn after shrinkage was excellent in touch and flexibility.
  • the high shrinkage artificial fibroin spun yarn of the present invention can be produced by bringing the artificial fibroin spun yarn into contact with water having a boiling point below and, if necessary, drying after contact with water. Can be manufactured.
  • Escherichia coli BLR (DE3) was transformed with the obtained expression vector.
  • the transformed Escherichia 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 (Table 12) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
  • the seed culture was added to a jar fermenter to which 500 mL of production medium (Table 13) was added so that the OD 600 was 0.05.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and cultured for 20 hours.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin.
  • the culture solution was centrifuged, and the cells were collected. Perform SDS-PAGE using cells prepared from the culture before and after adding IPTG, and confirm the expression of the desired modified fibroin by the appearance of the desired modified fibroin size band depending on the addition of IPTG. did.
  • the cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4).
  • the washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 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 resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure.
  • the washed precipitate 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) to a concentration of 100 mg / mL, and 60 ° C. And stirred 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 collected by centrifugation, water was removed with a freeze dryer, and lyophilized powder was collected to obtain modified fibroin (PRT918, PRT966 and PRT799).
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • PRT918 and PRT966 are hydrophobically modified fibroin with an average HI greater than 0.
  • PRT799 is a hydrophilic modified fibroin with an average HI of 0 or less.
  • the prepared spinning dope is filtered through a metal filter with an opening of 5 ⁇ m at 90 ° C., then left to stand in a 30 mL stainless syringe, defoamed, and then solidified with 100 mass% methanol from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 90 ° C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fiber (raw fiber).
  • the knitted fabric was manufactured by circular knitting using a circular knitting machine using the twisted yarn obtained by twisting the obtained raw material fibers.
  • the knitted fabric had a thickness of 180 denier and a gauge number of 18. 20 g was cut out from the obtained knitted fabric to obtain a test piece.
  • the flammability test was in accordance with the test method for powdered or low melting point synthetic resin on May 31, 1995, Fire Department 50, Hazardous Materials Regulation Section Manager. The test was performed under conditions of a temperature of 22 ° C., a relative humidity of 45%, and an atmospheric pressure of 1021 hPa. Table 14 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).
  • the critical oxygen index (LOI) value of the knitted fabric knitted with the modified fibroin (PRT799) fiber was 27.2.
  • the LOI value is 26 or more, it is said that it has flame retardancy. It can be seen that the modified fibroin is excellent in flame retardancy.
  • the prepared spinning dope is filtered through a metal filter having a mesh size of 5 ⁇ m at 60 ° C., then left to stand in a 30 mL stainless syringe and defoamed, and then 100 mass% methanol coagulated from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fiber (raw fiber).
  • a knitted fabric was produced by flat knitting using a flat knitting machine.
  • the knitted fabric using PRT918 fiber as the raw 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 1/30 N (hair count single yarn) and a gauge number of 16.
  • the thickness and the number of gauges were adjusted so that the knitted fabric using other raw material fibers had almost the same cover factor as the knitted fabric using PRT918 fiber and PRT799 fiber. Specifically, it is as follows.
  • test piece 2 pieces of knitted fabric cut to 10 cm ⁇ 10 cm were put together, and four sides were sewn together to obtain a test piece (sample). After leaving the test piece in a low humidity environment (temperature 20 ⁇ 2 ° C., relative humidity 40 ⁇ 5%) for 4 hours or more, it is transferred to a high humidity environment (temperature 20 ⁇ 2 ° C., relative humidity 90 ⁇ 5%). The temperature was measured at intervals of 1 minute by a temperature sensor attached to the center of the interior for 30 minutes.
  • FIG. 7 is a graph showing an example of the results of the hygroscopic exothermic test.
  • the horizontal axis of the graph represents the time (minutes) left in the high humidity environment, with the time when the sample was transferred from the low humidity environment to the high humidity environment as 0.
  • 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 15 shows the calculation results of the maximum moisture absorption exotherm.
  • modified fibroin PRT918 and PRT799
  • PRT918 and PRT799 has a higher maximum moisture absorption exotherm and superior moisture absorption exothermicity as compared with existing materials.
  • the prepared spinning dope is filtered through a metal filter having a mesh size of 5 ⁇ m at 60 ° C., then left to stand in a 30 mL stainless syringe and defoamed, and then 100 mass% methanol coagulated from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound up and naturally dried to obtain modified fibroin fiber (raw fiber).
  • a knitted fabric was produced by flat knitting using a flat knitting machine.
  • the knitted fabric using PRT966 fiber as the raw material fiber had a count of 30 Nm, a twist number of 1, a gauge number of 18 GG, and a basis weight of 90.1 g / m 2 .
  • the knitted fabric using PRT799 fiber as the raw material fiber had a count: 30 Nm, a twist number: 1, a gauge number GG: 16, and a basis weight: 111.0 g / m 2 .
  • the knitted fabric using other raw material fibers was adjusted in thickness and gauge number so as to have almost the same cover factor as the knitted fabric using PRT966 fiber and PRT799 fiber. Specifically, it is as follows.
  • the heat retention was evaluated by 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 clothes are directly touched in a dry state).
  • a dry contact method a method assuming that the skin and clothes are directly touched in a dry state.
  • One 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) dissipated through the test piece was determined under the condition of the wind speed in the wind tunnel of 30 cm / second. With no test piece set, the amount of heat (b) dissipated under the same conditions as above was determined, and the heat retention rate (%) was calculated according to the following formula.
  • Thermal insulation rate (%) (1 ⁇ a / b) ⁇ 100
  • Table 16 shows the calculation results of the heat retention index. It can be evaluated that the higher the heat retention index, the better the heat retention material.
  • the modified fibroin (PRT966 and PRT799) has a higher heat retention index and is superior in heat retention compared to existing materials.

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  • Engineering & Computer Science (AREA)
  • Textile Engineering (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Mechanical Engineering (AREA)
  • Peptides Or Proteins (AREA)
  • Yarns And Mechanical Finishing Of Yarns Or Ropes (AREA)
  • Chemical Or Physical Treatment Of Fibers (AREA)
  • Artificial Filaments (AREA)
  • Knitting Of Fabric (AREA)
PCT/JP2019/014869 2018-04-03 2019-04-03 高収縮人造フィブロイン紡績糸及びその製造方法、並びに人造フィブロイン紡績糸及びその収縮方法 WO2019194245A1 (ja)

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EP19782354.5A EP3800286A4 (en) 2018-04-03 2019-04-03 SPUN SYNTHETIC YARN WITH HIGH SHRINKAGE, METHOD OF PRODUCTION, SPUN SYNTHETIC YARN AND METHOD OF SHRINKING THE SAME
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CN113737521A (zh) * 2021-09-27 2021-12-03 溧阳市天目湖农业发展有限公司 一种长效抗菌的天然蚕丝纤维及其加工工艺

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