JPWO2019066037A1 - Doping solution, products using it, structural protein fiber and its manufacturing method - Google Patents

Abstract

A doping solution containing a structural protein, a metal compound having a metal atom and an organic group, and a solvent.

Description

The present invention relates to a doping solution and a product using the same. The present invention also relates to structural protein fibers and methods for producing them.

Conventionally, as structural protein fibers, silk fibroin fibers, which are regenerated silk fibers, artificial spider silk fibers, and the like have been known. Some of these manufacturing methods have also been proposed. For example, a method for producing a structural protein fiber by dissolving a structural protein containing silk fibroin in an inorganic salt and a polar solvent having excellent solubility such as dimethylsulfoxide (DMSO) (Patent Document 1), or a natural spider silk structural protein. A method for producing a structural protein fiber having a stress of 350 MPa or more has been proposed by stretching the derived structural protein fiber by the first-stage stretching in moist heat and the second-stage stretching in dry heat (Patent Document 2). Further, a method of reacting structural protein fibers with a cross-linking agent such as carbodiimide or glutaraldehyde to improve stress has also been proposed (Patent Document 3).

International Publication No. 2013/06651 Japanese Unexamined Patent Publication No. 2014-129638 International Publication No. 2012/1654777

Structural protein fibers with even higher stress are required for development in various applications.

An object of the present invention is to provide a doping solution useful for producing structural protein fibers having high stress. Another object of the present invention is to provide a method for producing a structural protein, which can easily produce a structural protein fiber having a high stress. Another object of the present invention is to provide a product formed by using the above-mentioned doping solution, particularly a structural protein fiber having a high stress.

As a result of diligent studies on the above problems, the present inventors can produce structural protein fibers having improved stress by using a dope solution containing a metal compound having an organic group and a structural protein. For the first time, we have found that this is the case, and have completed the present invention. That is, the present invention relates to, for example, the following inventions.

One aspect of the present invention relates to a doping solution containing a structural protein, a metal compound having a metal atom and an organic group, and a solvent.

In one embodiment, the valence of the metal atom may be 2-6 valence.

In one aspect, the metal atom may be at least one selected from the group consisting of titanium, zirconium, chromium, molybdenum, and tungsten.

In one aspect, the viscosity of the doping solution may be 3,000 mPa · sec or more and 60,000 mPa · sec or less.

In one embodiment, the content of the metal compound may be 0.5 equivalents or more and 20 equivalents or less with respect to 1 equivalent of the structural protein.

In one embodiment, the solvent is dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-. It may contain at least one selected from the group consisting of oxide, water, and hexafluoroisopronol.

In one aspect, the doping solution may further contain an inorganic salt.

In one aspect, the inorganic salt may be at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, and alkaline earth metal nitrates.

In one embodiment, the structural protein may be at least one selected from the group consisting of silk fibroin, spider silk fibroin, collagen, resilin, elastin, and keratin.

In one embodiment, the structural protein may be spider silk fibroin.

Another aspect of the present invention relates to a method for producing a structural protein fiber, which comprises a coagulation step of extruding the doping solution into a coagulation solution to coagulate it.

The method for producing a structural protein fiber according to one aspect may further include a dry heat step of drying and heat-treating the structural protein fiber obtained through the coagulation step.

Another aspect of the present invention relates to a product containing a coagulated product of the above-mentioned doping solution. This product may be selected from the group consisting of long fibers, short fibers, yarns, spun yarns, filaments, films, foams, spheres, nanofibrils, hydrogels, resins, papers and their equivalents.

Another aspect of the present invention relates to a structural protein fiber containing spider silk fibroin and a metal atom bonded to the spider silk fibroin.

According to the present invention, it is possible to provide a doping solution useful for producing structural protein fibers having high stress. In addition, the present invention can provide a method for producing a structural protein, which can easily produce a structural protein fiber having a high stress. Further, the present invention can provide a product formed by using the above-mentioned doping solution, particularly a structural protein fiber having a high stress.

It is the schematic which shows an example of the spinning apparatus for manufacturing a structural protein fiber. It is the schematic which shows an example of the heating apparatus for subjecting a structural protein fiber to heat treatment. It is a figure of ^{1 1} H-NMR spectrum of a structural protein in a solution. It is a figure of the ¹³ C-NMR spectrum of the structural protein in a solution. It is a figure of elemental analysis of a structural protein fiber by SEM-EDS energy dispersive X-ray spectroscopy.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. However, the present invention is not limited to the following embodiments.

<Doping liquid>
The doping solution according to this embodiment contains a structural protein, a metal compound having a metal atom and an organic group, and a solvent. The doping solution according to the present embodiment can also be said to be a doping solution in which a structural protein and a metal compound are dissolved in a solvent.

The type of solvent contained in the doping solution is not particularly limited, and may be appropriately selected depending on the type of structural protein and the like. As the solvent, any solvent can be used as long as it can dissolve the structural protein, and examples thereof include an aprotic solvent and a protic solvent. Examples of the aprotonic solvent include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), 1,3-dimethyl-2-imidazolidone (DMI), N-. Methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), etc., or a combination thereof may be used. Examples of protic solvents include water, hexafluoroisoprenaline (HFIP) and the like. In this embodiment, aprotic solvents are preferred.

<Metal compound>
The metal compound according to this embodiment has a metal atom and an organic group. In addition, in this embodiment, an organic group means an atomic group obtained by removing a part of a hydrogen atom from an organic compound, or an organic ligand forming a complex with a metal atom.

As metal atoms, group 1 element alkali metal elements, group 2 elements, group 4 elements, group 5 elements, group 6 elements, group 7 elements, group 8 elements, group 9 elements, and group 1 elements. Examples include group 10 elements, group 11 elements, and metal elements of group 12 elements. The type of metal atom is not particularly limited, but titanium, zirconium, chromium, molybdenum, tungsten and the like are preferable, and titanium, zirconium and chromium are more preferable.

The valence of the metal atom may be 1 to 6 valences, 2 to 6 valences, 3 to 6 valences, 3 to 4 valences, or 4 to 6 valences. Good.

Group 1 elements include lithium (I) (Li ⁺ ), sodium (I) (Na ⁺ ), potassium (I) (K ⁺ ), rubidium (I) (Rb ⁺ ), cesium (I) (Cs ⁺ ). ), Francium (I) (Fr ⁺ ) and the like. Group 2 elements include beryllium (I) (Be ⁺ ), beryllium (II) (Be ²⁺ ), beryllium (III) (Be ³⁺ ), magnesium (I) (Mg ⁺ ), magnesium (II) (Mg ²⁺ ). ), Calcium (I) (Ca ⁺ ), Calcium (II) (Ca ²⁺ ), Strontium (I) (Sr ⁺ ), Strontium (II) (Sr ²⁺ ), Barium (II) (Ba ²⁺ ) and the like. .. Group 4 elements include titanium (I) (Ti ⁺ ), titanium (II) (Ti ²⁺ ), titanium (III) (Ti ³⁺ ), titanium (IV) (Ti ⁴⁺ ), zirconium (IV) (Zr ⁴⁺ ). ), Hafnium (IV) (Hf ⁴⁺ ) and the like. Group 5 elements include vanadium (I) (V ⁺ ), vanadium (II) (V ²⁺ ), vanadium (V) (V ⁵⁺ ), niobium (V) (Nb ⁵⁺ ), tantalum (V) (Ta ⁵⁺ ). ) Etc. can be mentioned. Group 6 elements include chromium (III) (Cr ³⁺ ), chromium (VI) (Cr ⁶⁺ ), molybdenum (I) (Mo ⁺ ), molybdenum (II) (Mo ²⁺ ), molybdenum (III) (Mo ³⁺ ). ), Molybdenum (IV) (Mo ⁴⁺ ), molybdenum (V) (Mo ⁵⁺ ), molybdenum (VI) (Mo ⁶⁺ ), tungsten (VI) (W ⁶⁺ ) and the like. Examples of Group 7 elements include manganese (II) (Mn ²⁺ ) and manganese (IV) (Mn ⁴⁺ ). Group 8 elements include iron (II) (Fe ²⁺ ), iron (III) (Fe ³⁺ ), ruthenium (II) (Ru ²⁺ ), ruthenium (III) (Ru ³⁺ ), ruthenium (IV) (Ru ⁴⁺ ). ) Etc. can be mentioned. Examples of Group 9 elements include cobalt (III) (Co ³⁺ ) and the like. Examples of Group 10 elements include nickel (II) (Ni ²⁺ ), palladium (II) (Pd ²⁺ ), platinum (II) (Pt ²⁺ ) and the like. Group 11 elements include copper (I) (Cu ⁺ ), copper (II) (Cu ²⁺ ), silver (I) (Ag ⁺ ), gold (I) (Au ⁺ ), gold (III) (Au ³⁺ ). ) Etc. can be mentioned. Examples of the Group 12 element include zinc (II) (Zn ²⁺ ) and the like. Group 13 elements include aluminum (III) (Al ³⁺ ), gallium (III) (Ga ³⁺ ), indium (III) (In ³⁺ ), thallium (I) (Tl ⁺ ), thallium (III) (Tl ³⁺ ). ) Etc. can be mentioned. Examples of Group 14 elements include tin (IV) (Sn ⁴⁺ ), lead (II) (Pb ²⁺ ) and the like. Examples of Group 15 elements include bismuth (III) (Bi ³⁺ ) and the like.

When the metal compound is a compound having a metal atom and an organic ligand, examples of the organic ligand include a monodentate ligand and a polydentate ligand. Examples of the polydentate ligand include a bidentate ligand, a tridentate ligand, and a hexadentate ligand. Examples of the ligand include cyclopentadienyl anion, acetylacetonate (hereinafter referred to as "acac"), ethylenediamine, glycine, oxalic acid, 2,2'-bipyridine, 1,2- (bisphosphino) ethane. , Glycinato, diethylenetriamine, ethylenediaminetetraacetate and the like.

The metal compound may be, for example, a metal chelate, a metal alkoxide, a metallocene, or the like. The ligand of the metal chelate is not particularly limited, and may be appropriately selected according to the solvent used.

Examples of the metal chelate include metal acetylacetonates and the like. Examples of metallic acetylacetonate include lithium acetylacetonate (Li (acac)), potassium acetylacetonate (K (acac)), sodium acetylacetonate (Na (acac)), and rubidium acetylacetonate (Rb (acac)). )), Cesium Acetylacetone (Cs (acac)), Berylium (II) Acetylacetone (Be (acac) ₂ ), Magnesium (II) Acetylacetone (Mg (acac) ₂ ), Calcium (II) Acetylacetone inert (Ca _(acac) 2), bis (acetylacetonato) di Aqua strontium _{(II) (Sr (acac)} 2 (H 2 O) 2), bis (acetylacetonato) di Aqua barium (II) (Ba ( acac) ₂ (H ₂ O) ₂ ), barium (II) hexafluoroacetylacetonate, titanium (IV) acetylacetonate (Ti (acac) ₄ ), titanium (IV) oxyacetylacetonate, titanium (IV) ( Diisopropoxide) bis (acetylacetoneate), bishexachlorotitanate [tris (acetylacetonate) titanium (IV)], zirconium (IV) acetylacetonate (Zr (acac) ₄ ), zirconium (IV) tributoxy Monoacetylacetonate, zirconium (IV) dibutoxybis (ethylacetoneacetate), vanadium (IV) bis (acetylacetonetate) oxide, vanadium (IV) oxyacetylacetonate, niobtriacetylacetonate, tantalum (I) acetylacetonate (Ta (acac)), Chromium (III) Acetylacetone (Cr (acac) ₄ ), Bis (2,4-Pentandionato) Molybdenum (VI) Dioxide, Tungsten (II) Bis (Acetylacetone), Manganese (III) Acetylacetonate (Mn (acac) ₃ ), Iron (III) Acetylacetonate (Fe (acac) ₃ ), Luthenium (III) Acetylacetonate (Ru (acac) ₃ ), Cobalt (II) Acetylacetone Nate (Co (acac) ₂ ), rhodium (III) acetylacetoneate, iridium (III) acetylacetonate, nickel (II) acetylacetonate (Ni (acac) ₂ ), palladium (II) acetylacetonate (Pd (acac) ₂ ), Platinum (II) Acetylacetone (Pt (acac) ₂ ), Copper (II) Acetylacetone (Cu (acac) ₂ ), Vinyltriethylsilane (Hexafluoroacetylacetonate) Silver ( I), Dimethyl acetylacetonate gold (III), dimethyl (trifluoroacetylacetonate) gold (III), zinc (II) acetylacetonate (Zn (acac) ₂ ), aluminum (III) acetylacetonate (Al ( acac) ₃ ), gallium (III) acetylacetonate, indium (III) acetylacetonate, tallium (I) acetylacetonate, tin (IV) acetylacetonate dichloride, lead (II) acetylacetonate, and bismuth (III). ) -2,4-Pentandionate and the like.

Examples of metal alkoxides include lithium methoxyd, lithium isopropoxide, lithium-tert-butoxide, lithium-tert-pentoxide, sodium ethoxide, sodium ethoxide, potassium-tert-butoxide, cesium ethoxide, magnesium ethoxide, Calcium methoxyd, strontium (IV) oxyisopropoxide, barium ethoxide, titanium (IV) isopropoxide, titanium (IV) tetrapropoxide, zirconium (IV) tetranormal propoxide, zirconium tetranormalbutoxide, hafnium-tert -Butoxide, vanadium (IV) oxyisopropoxide, niobium (V) ethoxide, tantalum (V) ethoxide, tantalum (V) butoxide, chromium (III) tetrabutoxide, tungsten (VI) ethoxide, manganese methoxyd, iron (III ) Ethoxide, cobalt (II) isopropoxide, nickel 2-methoxyethoxydo, platinum ethoxide, copper (II) ethoxide, trimethoxyaluminum, aluminum ethoxide, gallium ethoxide, tin (II) ethoxide, tetranormal butoxystin ( IV), tetra-tert-butoxystin (IV), bismuth (IV) normal butoxide and the like.

Examples of metallocenes are titanosendichloride (Ti (C ₆ H ₅ ) ₂ Cl ₂ ), zirconosen dichloride (Zr (C ₆ H ₅ ) ₂ Cl ₂ ), trichloro (cyclopentadienyl) zirconium (IV), bis ( Pentamethylcyclopentadienyl) zirconium (IV) dichloride, zirconocene chloride hydride, trichloro (cyclopentadienyl) titanium (IV), bis (cyclopentadienyl) dimethylzirconium (IV), vanadinosen dichloride, chromocene, ferrocene , 1,1'-bis (di-tert-butylphosphino) ferrocene, 1,1'-bis (diisopropylphosphino) ferrocene, cyclopentadienylferrocene, ruthenocene, cyclopentadienylbis (triphenylphosphine) ruthenium (II) ) Chloride, cyclopentadienyl (dimethylfumarate) (triethylphosphite) cobalt (I), cyclopentadienyltalium, cobaltsen (III) hexafluorophosphate, cyclopentadienyl cobaltdicarbonyl, nickelocene, etc. Be done.

The method for preparing the doping solution is not particularly limited, but the doping solution is usually prepared by a method including dissolving a structural protein, a metal compound, and if necessary, other components in a solvent. The dope solution may be agitated or shaken for some time. At that time, the doping solution may be heated if necessary. For example, the doping solution may be heated to 50 ° C. or higher, 60 ° C. or higher, 70 ° C. or higher, 80 ° C. or higher, 90 ° C. or higher, or 120 ° C. or higher. The upper limit of the heating temperature is not particularly limited, but usually 130 ° C. or lower or about 85 ° C. is sufficient.

The viscosity of the doping solution is not particularly limited as long as it can be spun, but in consideration of industrial productivity, it is preferably 3,000 to 70,000 mPa · sec and 3,000 to 60,000 mPa · sec. , 3,000 to 55,000 mPa · sec is more preferable, and 3,000 to 50,000 mPa · sec is even more preferable.

The amount of the metal compound contained in the doping solution is not particularly limited, and may be appropriately determined according to the molecular weight of the structural protein, the concentration of the structural protein, and the like. For example, the content of the metal compound is 0.5 equivalent or more, 1 equivalent or more, 2 equivalent or more, 3 equivalent or more, 4 equivalent or more, 5 equivalent or more, 6 equivalent or more, 7 equivalent or more, relative to 1 equivalent of the structural protein. It may be 10 equivalents or more, 13 equivalents or more, 15 equivalents or more, 17 equivalents or more, 20 equivalents or more, and the like. The content of the metal compound may be any concentration as long as it does not cause gelation, and can be appropriately determined according to the molecular weight of the structural protein to be used. The content of the metal compound may be, for example, 28 equivalents or less, 26 equivalents or less, 24 equivalents or less, or 20 equivalents or less with respect to 1 equivalent of the structural protein. Here, the equivalent of the metal compound means the ratio of the amount (mol) of the metal atom contained in the metal compound to 1 equivalent (mol) of the structural protein.

The concentration of the structural protein in the doping solution is not particularly limited, and is, for example, 10% by mass or more, 15% by mass or more, 17% by mass or more, 20% by mass or more, 22% by mass or more, etc., based on the mass of the doping solution. It may be there. The concentration of the structural protein in the doping solution may be, for example, 35% by mass or less, 30% by mass or less, 28% by mass or less, 26% by mass or less, etc., based on the mass of the doping solution.

The doping solution may further contain an inorganic salt. Inorganic salts can function as solubilizers for structural proteins. Examples of the inorganic salt include alkali metal halides, alkaline earth metal halides, and alkaline earth metal nitrates. Specific examples of the inorganic salt include lithium carbonate, lithium chloride, calcium chloride, calcium nitrate, lithium bromide, barium bromide, calcium bromide, barium chlorate, sodium perchlorate, lithium perchlorate, and barium perchlorate. , Calcium perchlorate, magnesium perchlorate. At least one of these inorganic salts may be added to the solvent.

The amount of the inorganic salt contained in the doping solution is not particularly limited, and is appropriately determined according to the type of the inorganic salt, the amount of the structural protein, and the like. The content of the inorganic salt is, for example, 1.0 part by mass or more, 2.0 part by mass or more, 4.0 part by mass or more, 5.0 part by mass or more, and 9. It may be 0 parts by mass or more, 15 parts by mass or more, and 20 parts by mass or more. Further, the amount of the inorganic salt may be, for example, 40 parts by mass or less, 35 parts by mass or less, and 30 parts by mass or less with respect to 100 parts by mass of the total amount of the structural protein.

<Structural protein>

The structural protein according to the present embodiment is not particularly limited, and may be produced by a microorganism or the like by a gene recombination technique, or may be produced by synthesis. Alternatively, the structural protein may be a purified naturally occurring structural protein.

The structural protein may be, for example, a structural protein and an artificial structural protein derived from the structural protein. The structural protein means a structural protein that forms or retains a structure, morphology, etc. in a living body. That is, the structural protein may be a naturally occurring structural protein, and is a modified protein in which a part of the amino acid sequence (for example, 10% or less of the amino acid sequence) is modified depending on the amino acid sequence of the naturally occurring structural protein. It may be. Examples of the structural protein include fibroin, keratin, collagen, elastin, resilin and the like.

The structural protein may be fibroin. The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. In particular, the structural protein may be silk fibroin, spider silk fibroin, or a combination thereof. When silk fibroin and spider silk fibroin are used in combination, the ratio of silk fibroin may be, for example, 40 parts by mass or less, 30 parts by mass or less, or 10 parts by mass or less with respect to 100 parts by mass of spider fibroin.

Silk thread is a fiber (cocoon thread) obtained from a cocoon made by silkworm, which is a larva of Bombyx mori. Generally, one cocoon thread is composed of two silk fibroins and a sericin that covers them from the outside. Silk fibroin is composed of a large number of fibrils. Silk fibroin is covered with four layers of sericin. Practically, silk filaments obtained by dissolving and removing outer sericin by refining are used for clothing applications. A general silk thread has a specific gravity of 1.33, a fineness of 3.3 decitex on average, and a fiber length of about 1300 to 1500 m. Silk fibroin is obtained from natural or domestic silkworm cocoons, or used or discarded silk fabrics.

The silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, or a combination thereof. Sericin-removed silk fibroin is purified by removing sericin that covers silk fibroin and other fats. The silk fibroin purified in this way is preferably used as a lyophilized powder. Sericin-unremoved silk fibroin is unrefined silk fibroin from which sericin and the like have not been removed.

The spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of a natural spider silk structure protein and a polypeptide derived from the natural spider silk structure protein (artificial spider silk structure protein).

Examples of the natural spider silk structure protein include a large spider canal bookmark thread structure protein, a weft thread protein, and a vial-shaped gland structure protein. Since the large spit tube bookmark thread has a repeating region consisting of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of the spider silk has a characteristic that it does not have a crystalline region but has a repeating region consisting of an amorphous region. The weft yarn has a lower stress than the large discharge pipe bookmark yarn, but has high elasticity.

The large spider canal bookmark thread structure protein is produced in the large bottle-shaped gland of a spider and has a characteristic of being excellent in toughness. Examples of the large spider thread structure protein include large bottle-shaped gland spiders MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Angulate orbweaver spider (Araneus diadematus). ADF3 is one of the two major bookmark thread proteins of the European garden spider. The polypeptide derived from the natural spider silk structure protein may be a polypeptide derived from these bookmark thread structure proteins. The polypeptide derived from ADF3 is relatively easy to synthesize, and has excellent properties in terms of strength and elongation and toughness.

Weft structural proteins are produced in the flagelliform gland of spiders. Examples of the weft structure protein include flagelliform silk protein derived from the American silk spider (Nephila clavipes).

The polypeptide derived from the natural spider silk structure protein may be a recombinant spider silk structure protein. Examples of the recombinant spider silk structure protein include variants, analogs and derivatives of the natural spider silk structure protein. A preferred example of such a polypeptide is a recombinant spider silk structure protein of a large spit tube bookmark thread protein (also referred to as a "polypeptide derived from a large spit tube bookmark thread structure protein").

Examples of the fibroin-like structural protein, a structural protein derived from a large spit tube bookmark thread and a structural protein derived from silkworm silk, include a protein containing a domain sequence represented by the formula 1: [(A) n motif-REP1] m. Can be mentioned. Here, in the formula 1, in the (A) n motif, A represents an alanine residue, and n is preferably an integer of 2 to 27, preferably 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to. It may be an integer of 16, 10 to 16, and the number of alanine residues with respect to the total number of amino acid residues in the (A) n motif may be 40% or more, 60% or more, 70% or more, 80% or more. , 90% or more, 100% (meaning that it is composed only of alanine residues). REP1 shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REP1s may have the same amino acid sequence or different amino acid sequences.

In the above, a structural protein in which the industrial productivity is improved while maintaining the strength and elongation by deleting the n motif (A) in the formula 1 may be used. As the frequency of deletion, for example, the number of amino acid residues of REP of two adjacent [(A) n motif-REP1] units is sequentially compared from the N-terminal side to the C-terminal side, and the amino acid residues are deleted. When the number of amino acid residues of the REP having a small number is 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3. The above two adjacent [(A) n motif-REP1] units. When x is the maximum value of the total value obtained by adding the number of amino acid residues in the above domain sequence and y is the total number of amino acid residues in the domain sequence, a structural protein having x / y of 50% or more can be mentioned.

Further, in the REP in the formula 1, a structural protein having an amino acid sequence having a reduced content of the glycine residue, which corresponds to replacing at least one or a plurality of glycine residues in the REP with another amino acid residue. It may be. Examples of such a structural protein include a structural protein in which the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue is 10% or more of the total motif sequence.

Specific examples of the structural protein derived from the large spit tube bookmark thread include a structural protein containing the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

Examples of the structural protein derived from the weft structural protein include a structural protein containing a domain sequence represented by the formula 2: [REP2] _o (here, in the formula 2, REP2 is derived from Gly-Pro-Gly-Gly-X. The constituent amino acid sequence is shown, where X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr) and valine (Val). O represents an integer of 8 to 300. .) Can be mentioned. Specifically, a structural protein containing the amino acid sequence shown in SEQ ID NO: 2 can be mentioned. The amino acid sequence shown in SEQ ID NO: 3 is the N-terminal corresponding to the repeat portion and motif of the partial sequence (NCBI accession number: AAF36090, GI: 7106224) of the woolen silk structure protein of the American jellyfish obtained from the NCBI database. Amino acid sequence from residues 1220 to 1659 (referred to as PR1 sequence) and partial sequence of fluffy silk structure protein of American jellyfish obtained from NCBI database (NCBI accession number: AAC38847, GI: 2833649). ) C-terminal amino acid sequences from the 816th residue to the 907th residue were bound, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 4 was added to the N-terminal of the bound sequence. It is a thing.

As a collagen-derived structural protein, for example, a structural protein containing a domain sequence represented by the formula 3: [REP3] _p (where, in formula 3, p represents an integer of 5 to 300. REP3 is Gly-X. An amino acid sequence composed of one Y is shown, and X and Y indicate arbitrary amino acid residues other than Gly. A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) Can be done. Specifically, a structural protein containing the amino acid sequence shown in SEQ ID NO: 5 can be mentioned. The amino acid sequence shown in SEQ ID NO: 5 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 4 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a structural protein derived from resilin, for example, a structural protein containing a domain sequence represented by the formula 4: [REP4] _q (here, in formula 4, q represents an integer of 4 to 300. REP4 is Ser-J1. The amino acid sequence composed of J-Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. Indicates an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. Good.) Can be mentioned. Specifically, a structural protein containing the amino acid sequence shown in SEQ ID NO: 6 can be mentioned. The amino acid sequence shown in SEQ ID NO: 6 replaces Thr at residue 87 with Ser and Asn at residue 95 in the amino acid sequence of recillin (Axion No. NP 61157, Gl: 24654243 of NCBI GenBank). The amino acid sequence (tag sequence) shown by SEQ ID NO: 9 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.

Examples of the structural protein derived from elastin include structural proteins having an amino acid sequence such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a structural protein containing the amino acid sequence shown in SEQ ID NO: 7 can be mentioned. The amino acid sequence shown by SEQ ID NO: 7 is the amino acid sequence shown by SEQ ID NO: 4 at the N-terminal of the amino acid sequence from residue 121 to 390 of the amino acid sequence of accession number AAC98395 of NCBI GenBank. And the hinge arrangement) are added.

Examples of the keratin-derived structural protein include Type I keratin of Capra hilcus. Specifically, a structural protein containing the amino acid sequence shown in SEQ ID NO: 8 (amino acid sequence of accession number ACY30466 of GenBank of NCBI) can be mentioned.

As the above-mentioned structural protein and the structural protein derived from the structural protein, one type can be used alone, or two or more types can be used in combination.

The structural protein contained in the dope solution is, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the structural protein 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 structural protein is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural structural protein, or a method of chemically synthesizing the nucleic acid. The chemical synthesis method of nucleic acid is also not particularly limited. For example, based on the amino acid sequence information of the structural protein obtained from the NCBI web database or the like, AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized in (1) by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the structural protein, a nucleic acid encoding a structural protein 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-terminal of the above amino acid sequence is synthesized. You may.

The regulatory sequence is a sequence that controls the expression of the recombinant structural protein in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the protein of interest may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the 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 depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, one that can be autonomously replicated in the host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding a target structural protein can be transcribed is preferably used. Be done.

As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Philum and the like. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, examples of the vector into which the nucleic acid encoding the target structural protein is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110 , PNCO2 (Japanese Unexamined Patent Publication No. 2002-238569) and the like.

Eukaryotic hosts include, for example, yeast and filamentous fungi (molds, etc.). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

When a eukaryote is used as a host, examples of the vector into which the nucleic acid encoding the target structural protein is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.

As a method for expressing 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. ..

The structural protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the structural protein in the culture medium, and collecting the structural protein from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

The carbon source may be any assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Examples of the nitrogen source include ammonium salts of inorganic 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 and corn steep liquor. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests can be used. As the inorganic salts, for example, primary potassium phosphate, secondary potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culturing 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.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture, if necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as needed. For example, isopropyl-β-D-thiogalactopyranoside and the like are used when culturing microorganisms transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing microorganisms transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

Isolation and purification of the expressed structural protein can be carried out by a commonly used method. For example, when the structural protein is expressed in a lysed state in cells, the host cells are collected by centrifugation after the completion of culture, suspended in an aqueous buffer, and then an ultrasonic crusher, a French press, or a manton. Crush the host cells with a gaulin homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, methods usually used for isolating and purifying structural proteins, that is, a solvent extraction method, a salting-out method using sulfite, a desalting method, an organic solvent, etc. Precipitation method according to, diethylaminoethyl (DEAE) -Sepharose, anion exchange chromatography method using a resin such as DIAION HPA-75 (manufactured by Mitsubishi Kasei), and a resin such as S-Sepharose FF (manufactured by Pharmacia). Cation exchange chromatography method, hydrophobic chromatography method using resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis such as isoelectric point electrophoresis Purified preparations can be obtained by using methods such as the law alone or in combination.

When the structural protein is expressed by forming an insoluble matter in the cell, the host cell is similarly recovered, crushed, and centrifuged to recover the insoluble matter of the structural protein as a precipitation fraction. The insoluble form of the recovered structural protein can be solubilized with a structural protein denaturing agent. After the operation, a purified preparation of the structural protein can be obtained by the same isolation and purification method as described above. When the protein is secreted extracellularly, the structural protein can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

<Manufacturing method of structural protein fiber>
An example of a method for producing structural protein fibers will be described below.

The method for producing a structural protein fiber according to the present embodiment includes a coagulation step. The coagulation step may be a step of extruding the doping solution according to the above embodiment into the coagulating solution to coagulate it, and is a step of extruding the doping solution according to the above embodiment into the air to heat the solvent to vaporize and solidify it. It may be. FIG. 1 is a schematic view showing an example of a spinning apparatus for producing structural protein fibers. The spinning device 10 shown in FIG. 1 is an example of a spinning device for dry / wet spinning, and includes an extrusion device 1, a coagulation device 2 having a coagulation bath 20, a cleaning device 3 having a washing bath 21, and a heating device 17. The drying apparatus 4 to be provided is provided in order from the upstream side.

The extrusion device 1 has a storage tank 7, in which the spinning stock solution 6 is stored. As the spinning stock solution 6, the doping solution according to the above-described embodiment is used. The coagulation liquid 11 (for example, methanol) is stored in the coagulation bath 20. The spinning stock solution 6 is pushed out from a nozzle 9 provided with an air gap 19 between the spinning stock solution 6 and the coagulating solution 11 by a gear pump 8 attached to the lower end of the storage tank 7. The extruded spinning stock solution 6 is supplied into the coagulating solution 11 through the air gap 19. In the coagulation liquid 11, the solvent is removed from the spinning stock solution 6 to coagulate the structural protein, and structural protein fibers are formed. The formed structural protein fibers are guided to the washing tub 21 via the thread guides 18a, 18b, 18c and 18d, and are washed by the washing liquid 12 in the washing tub 21. The washed structural protein fibers are sent by the first nip roller 13 and the second nip roller 14 installed in the washing bath 21, and are introduced into the heating device 17 via the thread guides 18e, 18f and 18g. At this time, for example, if the rotation speed of the second nip roller 14 is set to be faster than the rotation speed of the first nip roller 13, the structural protein fiber 36 stretched at a magnification corresponding to the rotation speed ratio can be obtained. The structural protein fibers 36 stretched in the washing liquid 12 are dried when passing through the path 22 in the heating device 17 after leaving the washing bath 21, and then wound up by the winder 23. In this way, the structural protein fiber 36 is finally obtained as a wound product 5 wound around the winder 23 by the spinning device 10.

The coagulating solution may be a solution capable of desolving the spinning stock solution. Examples of the coagulating liquid include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulant may contain water. The temperature of the coagulating liquid is preferably 0 to 30 ° C. The length of the coagulation liquid tank may be any length as long as the solvent can be efficiently removed, and is, for example, 200 to 500 mm. The residence time of the structural protein fibers formed by coagulation in the coagulation liquid may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Structural protein fibers may be stretched (or pre-stretched) in the coagulation fluid. Pre-drawing yarn is formed by pre-drawing. In the pre-drawing, the undrawn yarn can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.

The structural protein fibers are stretched while heating the washing liquid in the washing bath 21. The cleaning liquid may be, for example, water or a mixed solvent of water and an organic solvent. Stretching performed in a heated cleaning solution (or solvent) may be referred to by those skilled in the art as moist heat stretching. The temperature of moist heat stretching (temperature of the washing liquid) may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In moist heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, 2 to 8 times, 5 to 10 times, 6 to 10 times, 5 to 8 times, or. It is preferably stretched 6 to 8 times.

The lower limit of the final draw ratio of the structural protein fiber is preferably 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, and 7 times with respect to the undrawn yarn (or the pre-drawn yarn). , 8 times, or 9 times. The upper limit of the final draw ratio of the structural protein fiber is preferably any one of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. ..

The method for producing a structural protein fiber according to the present embodiment may further include a dry heat step after the coagulation step. The dry heat step is a step of dry heat treating the structural protein fibers obtained through the coagulation step. FIG. 2 is a schematic view showing an example of a heating device for subjecting the structural protein fiber obtained as described above to a heating (dry heat) treatment as a post-treatment. The heating device 62 shown in FIG. 2 has a feed roller 42 and a winder 44, and a drying heat plate 64 provided between them. The dry heat plate 64 has a dry heat surface 66 extending in the direction from the feed roller 42 toward the winder 44.

The structural protein fibers 36 are continuously fed from the feed roller 42, and the fed structural protein fibers 36 are heated while moving along the dry heat surface 66. The conditions of this dry heat treatment are not particularly limited, and are appropriately determined according to the type of metal compound, the type of structural protein, and the like. For example, the dry heat temperature may be 150 ° C., 180 ° C., 200 ° C., 220 ° C., 240 ° C., and the dry heat time is 10 seconds or longer, 20 seconds or longer, 30 seconds or longer, 1 minute or longer, 2 minutes or longer. It may be 3 minutes or more. It is also possible to continuously produce the heat-treated structural protein fibers from the doping solution by combining the spinning device 10 of FIG. 1 and the heating device 62 of FIG. In this case, the structural protein fiber after spinning may be subjected to heating (dry heat) treatment as it is without being wound around the winder, or the structural protein fiber 36 after spinning may be heated as it is without undergoing drying. Post-treatment by (dry heat) may be performed.

<Product>
The structural protein fiber formed from the doping solution according to the present embodiment can be applied to woven fabrics, knitted fabrics, braids, non-woven fabrics and the like as fibers or threads. It can also be applied to high-strength applications such as ropes, surgical sutures, flexible fasteners for electrical components, and bioactive materials for transplantation (eg, artificial ligaments and aortic bands). These can be produced according to the method described in Japanese Patent No. 5427322 and the like.

The structural protein fibers according to the present embodiment can also be applied to long fibers, short fibers, spun yarns, filaments, films, papers, foams, spheres, nanofibrils, hydrogels, resins and their equivalents. It can be produced according to the methods described in JP-A-2009-505668, JP-A-2009-505668, Patent No. 5678283, Patent No. 4638735 and the like.

These products can be said to be products containing a coagulated product of the doping solution according to the present embodiment, and can also be said to be products containing a structural protein and a metal atom bonded to the structural protein.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to the above embodiment.

1. 1. Production of spider silk structure protein (spider silk fibroin: PRT799) (synthesis of gene encoding spider silk structure protein and construction of expression vector)
Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes, a modified fibroin having the amino acid sequence shown in SEQ ID NO: 1 and having a molecular weight of about 200 kDa ( Hereinafter, it is also referred to as “PRT799”).

The amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, and its N-terminal. It has the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 4 added to.

Nucleic acid encoding the designed PRT799 was synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the structural protein expression vector pET-22b (+) to obtain an expression vector.

Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 1) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was carried out for about 15 hours until the OD ₆₀₀ reached 5, to obtain a seed culture solution.

The seed culture solution was added to the jar fermenter to which 500 ml of the production medium (Table 2 below) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Moreover, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. The culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of PRT799. When 20 hours had passed after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture broths before and after the addition of IPTG, and the expression of PRT799 was confirmed by the appearance of a band having a size corresponding to PRT799 depending on the addition of IPTG.

(Purification of spider silk fibroin)
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 crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. 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 at 60 ° C. Stir with a stirrer for 30 minutes 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 structure protein (PRT799) obtained after dialysis was recovered by centrifugation. Moisture was removed from the recovered aggregated structural protein with a lyophilizer to obtain a lyophilized powder of PRT799.

The degree of purification of PRT799 in the obtained lyophilized powder was confirmed by image analysis of the results of polyacrylamide gel electrophoresis of the powder using Totallab (nonliner dynamics ltd.). As a result, the degree of purification of PRT799 was about 85%.

2. Production of structural protein fibers (Example 1)
<Preparation of doping solution>
Dimethyl sulfoxide (DMSO) was used as the solvent. Zirconium (IV) acetylacetonate Zr (acac) ₄ was added to DMSO as a metal compound, and the mixture was dissolved by heating to 80 ° C. with stirring using a mechanical stirrer. Further, a dry powder of spider silk fibroin (PRT799) was added so as to have a concentration of 11% by mass, and the mixture was heated to 120 ° C. to dissolve the spider silk fibroin. After filtering with a metal filter having a mesh size of 3 μm, defoaming was performed to obtain a Zr ⁴⁺ -containing doping solution (Zr ⁴⁺ -containing doping solution). The amount of Zr (acac) ₄ added was 5 equivalents (5 mol) with respect to 1 equivalent (1 mol) of spider silk fibroin (PRT799). Moreover, the viscosity of the prepared doping solution at 90 ° C. was measured. The results are shown in Table 3. The viscosity of the spinning stock solution was measured using an electromagnetic spinning viscometer (Kyoto Denshi Kogyo Co., Ltd.) under the condition of a rotation speed of 1000 rpm under sealing. The doping solution was not colored by the addition of Zr ⁴⁺ .

<Spinning>
The obtained doping solution was used as a spinning stock solution, and spinning and stretched structural protein fibers were formed by dry-wet spinning using the spinning device 10 shown in FIG. The formed structural protein fibers were dried and then wound up. The conditions for dry-wet spinning are as follows.
Extruded nozzle diameter: 0.2 mm
Coagulant (methanol) temperature: 5 ° C
Coagulation bath stretching ratio: 1.06 times Water washing bath stretching ratio: 5 times Total stretching ratio: 6.56 times Drying temperature: 60 ° C

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was allowed to stand in a constant temperature and humidity chamber (LHL-113 type manufactured by ESPEC) at 20 ° C. and a relative humidity of 65% for 24 hours, and then INSTRON (registered trademark) FORCE TRANSDUCER 2519- It was measured using 101.

(Comparative Example 1)
A doping solution was prepared in the same manner as in Example 1 except that Zr (acac) ₄ was not added, and the viscosity of the obtained doping solution at 90 ° C. was measured. Further, using the obtained doping solution, structural protein fibers were formed in the same manner as in Example 1, and the mechanical strength of the obtained structural protein fibers was measured. Table 3 shows the relative stress values when the maximum stress value of the structural protein fiber in Example 1 is 100%.

The viscosity of the doping solution of Comparative Example 1 at 90 ° C. was 1,000 mPa · sec, and the viscosity of the doping solution of Example 1 at 90 ° C. was 5,000 mPa · sec. The dope solution of Comparative Example 1 had significantly improved spinnability as compared with the dope solution of Comparative Example 1.

Comparing Example 1 and Comparative Example 1, the relative stress value of the structural protein fiber of Comparative Example 1 was lower than 100%. In other words, it was confirmed that the maximum stress value was improved in the structural protein fiber formed by using the doping solution to which the metal compound was added, as compared with the structural protein fiber formed by using the doping solution to which the metal compound was not added.

(Example 2)
<Preparation of doping solution>
A dope solution was prepared in the same manner as in Example 1 except that dimethyl sulfoxide (LiCl / DMSO) in which LiCl was dissolved was used as a solvent. The amount of LiCl added was 630 equivalents (630 mol) with respect to 1 equivalent (1 mol) of spider silk fibroin. Further, the viscosity of the obtained doping solution at 90 ° C. was measured in the same manner as in Example 1. The viscosity of the obtained doping solution at 90 ° C. was 4,940 mPa · sec.

<Spinning>
The obtained doping solution was used as a spinning stock solution, and structural protein fibers were formed in the same manner as in Example 1.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. Table 4 shows the relative stress values when the maximum stress value of the structural protein fiber in Example 1 is 100%.

As shown in Table 4, when the doping solution contained LiCl, the maximum stress value of the structural protein fiber was further improved as compared with the case where the doping solution did not contain LiCl.

(Example 3)
<Preparation of doping solution>
A dope solution was prepared in the same manner as in Example 2 except that the concentration of spider silk fibroin (PRT799) was 24% by mass, and the viscosity of the obtained dope solution at 90 ° C. was measured. The measurement results are shown in Table 5.

<Spinning>
The obtained doping solution was used as a spinning stock solution, and structural protein fibers were formed in the same manner as in Example 1.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. Table 5 shows the relative stress values when the maximum stress value of the structural protein fiber in Comparative Example 2 described later is 100%.

(Example 4)
<Preparation of doping solution>
A dope solution was prepared in the same manner as in Example 3 except that titanocene dichloride (Ti (C ₅ H ₅ ) ₂ Cl ₂ ) was used as the metal compound, and the viscosity of the obtained dope solution at 90 ° C. was measured. .. The results are shown in Table 5.

<Spinning>
The obtained doping solution was used as a spinning stock solution, and structural protein fibers were formed in the same manner as in Example 1.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. Table 5 shows the relative stress values when the maximum stress value of the structural protein fiber in Comparative Example 2 described later is 100%.

(Example 5)
<Preparation of doping solution>
A dope solution was prepared in the same manner as in Example 3 except that chromium acetylacetonate (III) (Cr (acac) ₃ ) was used as the metal compound, and the viscosity of the obtained dope solution at 90 ° C. was measured. did. The results are shown in Table 5.

<Spinning>
The obtained doping solution was used as a spinning stock solution to form structural protein fibers in the same manner as in Example 1.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. Table 5 shows the relative stress values when the maximum stress value of the structural protein fiber in Comparative Example 2 described later is 100%.

(Comparative Example 2)
<Preparation of doping solution>
A doping solution was prepared in the same manner as in Example 3 except that no metal compound was added to the doping solution, and the viscosity of the obtained doping solution at 90 ° C. was measured. The results are shown in Table 5.

<Spinning>
The obtained doping solution was used as a spinning stock solution to form structural protein fibers in the same manner as in Example 1.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1.

In the doping solutions of Examples 3 to 6, the spinnability was significantly improved as compared with the doping solution of Comparative Example 2. Further, as shown in Table 5, in Comparative Example 2 to which the metal compound was not added, stretching up to 5 times was possible, but in Examples 3 to 5 to which the metal compound was added, stretching was possible up to 7 times. I was able to increase the magnification. In addition, an improvement in the maximum stress value was observed in Examples 3 to 5 as compared with Comparative Example 2.

(Examples 6 to 18)
<Preparation of doping solution>
A doping solution was prepared in the same manner as in Example 2 except that the concentration of spider silk fibroin (PRT799) and the amount of Zr (acac) ₄ added were changed to the concentrations shown in Table 6.

<Spinning>
Structural protein fibers were formed in the same manner as in Example 1 except that the obtained doping solution was used as a spinning stock solution and the drawing ratio in a water washing bath was set to the ratio shown in Table 6.

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. Tables 6 and 7 show the relative stress values when the maximum stress value of the structural protein fiber in Comparative Example 2 is 100%.

As shown in Tables 6 and 7, the largest structural protein fiber formed using a dope solution in which the amount of Zr (acac) ₄ added was 5 equivalents and the concentration of spider silk fibroin (PRT799) was 24% by mass. The stress value was obtained.

(Examples 19 to 21)
<Dry heat treatment>
The structural protein fiber formed by using the doping solution to which Zr ⁴⁺ obtained in Example 2 was added was subjected to dry heat treatment as a post-treatment using the heating device 62 shown in FIG. 2, and the structural protein after dry heat treatment was performed. The fiber was wound up. The conditions of the dry heat treatment are as follows. The processing temperature (dry-bulb plate temperature) was the temperature shown in Table 8, respectively.
Delivery speed: 100 cm / min
Winding speed: 100 cm / min
Dry heat plate length: 100 cm

<Measurement of physical properties of fibers>
The mechanical strength of the obtained structural protein fiber was measured in the same manner as in Example 1. The results are shown in Table 8. The relative stress values in Table 8 are represented by relative values [%] when the stress values of the structural protein fibers obtained in Example 2 are taken as 100%.

As shown in Table 8, the maximum stress value of the structural protein fiber was improved by the dry heat treatment. The maximum stress value of the structural protein fiber subjected to dry heat treatment at 150 ° C. was improved 1.5 times.

(Example 22)
In order to analyze the structural protein and Zr ⁴⁺ , spider fibroin (PRT799) was dissolved in DMSO-d ₆ containing Zr (acac) ₄ so as to be 2 wt%, and ¹ H-NMR was measured at 400 MHz. Although there was no significant change in the chemical shift of each amino acid in the protein, a characteristic signal attributed to acetylacetone liberated from Zr (acac) ₄ was observed around 5.65 ppm (indicated by a star in FIG. 3). Signal). In addition, signals derived from the same compound were observed at 1.99, 2.10 and 3.65 ppm on the high magnetic field side. Signals appeared at 204, 190, 58, and 31 ppm in ¹³ C-NMR of the same sample, and these chemical shifts were almost in agreement with the literature values of acetylacetone (Fig. 4). From the above, it was confirmed that Zr ⁴⁺ was coordinated with respect to the structural protein due to the presence of acetylacetone in the solution.

<Elemental analysis of fibers (SEM-EDS energy dispersive X-ray spectroscopy)>
Elemental analysis of the structural protein fibers obtained in Example 2 was performed using an energy dispersive X-ray spectroscope built into Pro-X manufactured by Phoenix-world. After fixing the structural protein fiber to the pin stub holder and confirming a reflected electron image (SEM image) of 4000 to 6000 times, an electron beam is irradiated with an accelerating voltage of 15 kV, and EDS (energy dispersive X-ray spectrometer: Energy). Elemental analysis was performed by measuring with a dispersive spectrometer). A semi-quantitative analysis of Zr was performed from the intensity of the Zr Lα ray (2.04 kV) emitted from the sample.

As shown in FIG. 5, it was confirmed that the structural protein fiber contained Zr.

<Inorganic analysis of fibers (ICP emission spectroscopy)>
The residual amount of Li in the structural protein fibers obtained in Example 2 was measured using an ICP (high frequency inductively coupled plasma) emission spectroscopic analyzer manufactured by Hitachi High-Tech Science Corporation.
Specifically, a structural protein fiber (sample) was pretreated using a microwave sample pretreatment apparatus manufactured by Milestone General Co., Ltd. 7 ml of nitric acid and 1 ml of hydrogen peroxide were added to 0.3 g of the pretreated sample and dissolved to prepare a solution, and ICP emission spectroscopic analysis was performed (detection limit: 5 mg / kg).

Li was not detected in the structural protein fibers, and it was confirmed that LiCl added as an inorganic salt did not remain.

According to the present invention, it is possible to provide a doping solution useful for producing structural protein fibers having high stress.

1 ... Extruder, 2 ... Coagulant, 3 ... Cleaning device, 4 ... Drying device, 6 ... Spinning stock solution, 10 ... Spinning device, 12 ... Cleaning solution, 13 ... First nip roller, 14 ... Second nip roller, 19 ... Air gap , 20 ... coagulation bathtub, 64 ... drying plate, 36 ... structural protein fiber, 42 ... feed roller, 44 ... winder, 62 ... heating device.

Claims (15)

A doping solution containing a structural protein, a metal compound having a metal atom and an organic group, and a solvent. The doping solution according to claim 1, wherein the metal atom has a valence of 2 to 6 valences. The dope solution according to claim 1 or 2, wherein the metal atom is at least one selected from the group consisting of titanium, zirconium, chromium, molybdenum, and tungsten. The doping solution according to any one of claims 1 to 3, wherein the doping solution has a viscosity of 3,000 mPa · sec or more and 60,000 mPa · sec or less. The doping solution according to any one of claims 1 to 4, wherein the content of the metal compound is 0.5 equivalents or more and 20 equivalents or less with respect to 1 equivalent of the structural protein. The solvent is dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, water. The dope solution according to any one of claims 1 to 5, which comprises at least one selected from the group consisting of hexafluoroisopronol and. The doping solution according to any one of claims 1 to 6, wherein the doping solution further contains an inorganic salt. The dope solution according to claim 7, wherein the inorganic salt is at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, and alkaline earth metal nitrates. The dope solution according to any one of claims 1 to 8, wherein the structural protein is at least one selected from the group consisting of silk fibroin, spider silk fibroin, collagen, resilin, elastin, and keratin. The doping solution according to any one of claims 1 to 9, wherein the structural protein is spider silk fibroin. A method for producing a structural protein fiber using the doping solution according to any one of claims 1 to 10.
A method for producing a structural protein fiber, which comprises a coagulation step of extruding the dope solution into a coagulation solution to coagulate it. The method for producing a structural protein fiber according to claim 11, further comprising a dry heat step of drying and heat-treating the structural protein fiber obtained through the coagulation step. A product containing a coagulated product of the doping solution according to any one of claims 1 to 10.
A product selected from the group consisting of long fibers, short fibers, yarns, spun yarns, filaments, films, foams, spheres, nanofibrils, hydrogels, resins, paper and their equivalents. Structural protein fibers containing spider silk fibroin and metal atoms. The structural protein fiber according to claim 14, wherein the metal atom is bonded to the spider silk fibroin.