WO2020067514A1 - Modified spider silk fibroin fiber and method for producing same - Google Patents
Modified spider silk fibroin fiber and method for producing same Download PDFInfo
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- WO2020067514A1 WO2020067514A1 PCT/JP2019/038375 JP2019038375W WO2020067514A1 WO 2020067514 A1 WO2020067514 A1 WO 2020067514A1 JP 2019038375 W JP2019038375 W JP 2019038375W WO 2020067514 A1 WO2020067514 A1 WO 2020067514A1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/02—Elements
- C08K3/04—Carbon
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K7/00—Use of ingredients characterised by shape
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
- D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
Definitions
- the present invention relates to a modified spider silk fibroin fiber and a method for producing the same.
- the present invention also relates to a dispersion that can be used for producing the modified spider silk fibroin fiber.
- the present invention also relates to products comprising the modified fibroin fibers described above.
- fibroin fiber is attracting attention as a new material with high utility value in the future.
- Conventionally, regenerated silk fibroin fibers and spider silk fibroin fibers are known as fibroin fibers.
- Many methods for producing small-diameter fibroin fibers have been reported.
- Non-Patent Document 2 a method of obtaining fibers having a diameter of 2 ⁇ m by forming fibers from the above-mentioned dope solution using a microfluidic device, and then drawing and impregnating the fibers in an aqueous alcohol solution. Have been.
- Patent Document 1 a method has been reported in which a fiber having an average diameter of 1 ⁇ m or less is obtained by discharging a spider silk fibroin dope solution from a spinneret to which a voltage is applied by electrospinning.
- an object of the present invention is to provide a small-diameter fibroin fiber by a simple method.
- the present invention relates to, for example, the following inventions.
- the polar solvent is hexafluoroisopropanol, hexafluoroacetone, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile
- the dispersion according to [1] which is at least one selected from the group consisting of N-methylmorpholine N-oxide, formic acid, ethylene glycol, tetrahydrofuran, and water.
- planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide.
- liquid [4] A modified spider silk fibroin, a carbon material, and a polar solvent, The carbon material is carbon black nanoparticles, The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran.
- a carbon material dispersion which is at least one selected from the group consisting of: [5] The dispersion according to any one of [1] to [4], wherein the content of the carbon material is 25 parts by mass or less based on 100 parts by mass of the modified spider silk fibroin. [6] The dispersion according to any one of [1] to [5], which is a dope. [7] A modified spider yarn comprising a modified spider yarn fibroin and at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles, and having an average fiber diameter of 3 ⁇ m or less. Fibroin fiber.
- Spider silk fibroin fiber [10] A product comprising the modified spider silk fibroin fiber according to any one of [7] to [9].
- Modified spider silk fibroin a carbon material, comprising a step of forming fibrils by a dry spinning method from a dispersion containing a polar solvent, wherein the carbon material is carbon black nanoparticles, production of modified spider silk fibroin fibers Method.
- [16] Modified spider silk fibroin, a carbon material, and a polar solvent, a step of forming fibrils from a dispersion liquid, Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils, A method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is one or more layers of planar graphene.
- Modified spider silk fibroin a carbon material, a polar solvent, a step of forming fibrils by a dry spinning method from a dispersion containing, Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils, And a method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is carbon black nanoparticles.
- Modified spider silk fibroin comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent, A method for dispersing a carbon material, wherein the carbon material is one or two or more layers of planar graphene.
- Modified spider silk fibroin comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent
- the carbon material is carbon black nanoparticles
- the polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran.
- a method for dispersing a carbon material which is at least one selected from the group.
- a dispersion aid for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin, wherein the carbon material is one or more layers of planar graphene.
- a modified auxiliary agent for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin, The carbon material is carbon black nanoparticles,
- the polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. At least one selected from dispersing aids.
- a thinning agent for producing a modified spider silk fibroin fiber having a reduced diameter comprising at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles.
- the carbon material dispersion liquid of the present embodiment includes a modified spider silk fibroin, a carbon material, and a polar solvent, and the carbon material is one or two or more layers of planar graphene.
- the term “dispersion” refers to a precipitate, an aggregate, or a precipitate and an aggregate that is visually free from a precipitate, an aggregate, or a precipitate and an aggregate even after standing still for at least 7 days. Refers to a solution without any objects.
- the carbon material dispersion of the present invention is excellent in dispersibility of the carbon material, can sufficiently suppress aggregation and precipitation of the carbon material, and is excellent in operability.
- the dispersion of this embodiment can be used as it is, or appropriately concentrated or diluted, as a spinning stock solution (dope solution) for producing a modified spider silk fibroin fiber.
- the carbon material dispersion liquid of the present embodiment has good dispersibility and excellent operability, it can be suitably used for uses other than the raw material of the modified spider silk fibroin fiber of the present invention. For example, it can be suitably used for producing a composite material containing a carbon material.
- planar graphene refers to graphene and graphene analogs that form a two-dimensional sheet-like molecular structure with contained carbon atoms. Therefore, carbon nanotubes that form a cylindrical structure with carbon atoms and fullerenes that form a spherical structure are excluded from “planar graphene”.
- the “planar graphene” may have a two-dimensional sheet-like structure in a part of its molecular structure, and the entire molecular structure may not be planar.
- the planar graphene may have a two-dimensional sheet-like molecular structure of one layer or two or more layers.
- planar graphene examples include graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, reduced functionalized graphene oxide, and the like. Among these, graphene oxide is preferable.
- the degree of oxidation of graphene oxide is not particularly limited, and known graphene oxide can be used.
- the degree of oxidation of graphene oxide may be, for example, from 4 to 55%, from 4 to 50%, from 4 to 45%, from 4 to 40%, from 4 to 35%. May be 4 to 30%, may be 4 to 25%, may be 4 to 20%, may be 4 to 15%, may be 4 to 10%.
- the functionalized graphene oxide has a structure in which a part of the oxygen-containing functional group of the graphene oxide is chemically modified with another functional group or the like.
- Known functionalized graphene oxide can be used.
- the functionalized graphene oxide include alkylamine-functionalized graphene oxide, ammonia-functionalized graphene oxide, amine-functionalized graphene oxide, thiol-functionalized graphene oxide, alkyne-functionalized graphene oxide, and glucose-functionalized graphene oxide.
- the carbon / oxygen ratio of the reduced graphene oxide is not particularly limited, and a known reduced graphene oxide can be used.
- the carbon / oxygen ratio of the reduced graphene oxide may be, for example, 90/10 or 75/35.
- the functionalized reduced graphene oxide has a structure in which a part of the oxygen-containing functional group of reduced graphene oxide is chemically modified with another functional group or the like.
- Known functionalized reduced graphene oxide can be used.
- Examples of the functionalized reduced graphene oxide include amine-functionalized reduced graphene oxide, octadecylamine-functionalized reduced graphene oxide, piperazine-functionalized reduced graphene oxide, tetraethylenepentamine-functionalized reduced graphene oxide, and glucose-modified reduction.
- Graphene oxide and the like are examples of the functionalized reduced graphene oxide.
- planar graphene in the form of powder, sheet, liquid or the like can be used.
- planar graphene in a powder or liquid form it is preferable to use planar graphene in a powder or liquid form.
- graphene it is preferable to use planar graphene in a liquid form.
- planar graphene in a liquid form a graphene ink in which planar graphene is dissolved or dispersed in a solvent or a dispersion medium can be used.
- any polar solvent that can disperse or dissolve the modified spider silk fibroin can be used.
- the polar solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), and 1,3.
- Organic solvents such as -dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), formic acid, ethylene glycol and tetrahydrofuran (THF); Water and the like.
- hexafluoroisopropanol, dimethyl sulfoxide and formic acid are more preferred, and dimethyl sulfoxide and formic acid are even more preferred.
- These organic solvents may include water. These solvents may be used alone or as a mixture of two or more.
- Carbon black nanoparticles In the carbon material dispersion liquid of the present embodiment, as the carbon material, carbon black nanoparticles can be used instead of or together with the planar graphene. Carbon black nanoparticles are fine particles formed of carbon. In the present embodiment, as the carbon black nanoparticles, it is preferable to use fine particles having an average particle diameter of less than 3 ⁇ m, more preferably to use fine particles of less than 2 ⁇ m, and still more preferably to use fine particles of less than 1 ⁇ m.
- the method for producing carbon black nanoparticles is not particularly limited, and for example, using carbon black nanoparticles produced by a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method. Can be.
- a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method.
- carbon black nanoparticles are used as the carbon material
- N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, and the like are used as polar solvents for the carbon material dispersion. Examples thereof include N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. These solvents may be used alone or as a mixture of two or more.
- the carbon material dispersion of the present embodiment is prepared by using a modified spider silk fibroin, a carbon material, and a polar solvent.
- the dispersion is, for example, a mixing and dispersing machine (for example, a ball mill, a bead mill, a sand mill, a roll mill, a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, an ultrasonic device, an attritor, a dissolver, a paint shaker, etc.) in which a carbon material and a polar solvent are mixed. And then adding the modified spider silk fibroin.
- a mixing and dispersing machine for example, a ball mill, a bead mill, a sand mill, a roll mill, a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, an ultrasonic device, an attritor, a dissolver, a paint shaker, etc.
- the dispersion may be prepared by adding a carbon material to a solution obtained by mixing a modified spider silk fibroin and a polar solvent using a mixing disperser, and mixing the carbon material and the polar solvent using a mixing disperser. And a solution in which the modified spider silk fibroin is dissolved in a different polar solvent.
- the carbon material dispersion liquid of the present embodiment may be in a liquid form or a semi-solid form such as a paste or a gel, but is preferably in a liquid form.
- the upper limit of the content of the carbon material in the carbon material dispersion of the present embodiment may be 25 parts by mass or less, 20 parts by mass or less, and 100 parts by mass of the modified spider silk fibroin. Not more than 10 parts by mass, not more than 5 parts by mass, not more than 4 parts by mass, not more than 3 parts by mass, not more than 1 part by mass, Good.
- the lower limit of the content of the carbon material in the carbon material dispersion liquid of the present embodiment may be not less than 0.01 part by mass, and not less than 0.03 part by mass, based on 100 parts by mass of the modified spider silk fibroin.
- it may be 0.05 parts by mass or more, may be 0.06 parts by mass or more, may be 0.07 parts by mass or more, may be 0.08 parts by mass or more, and may be 0.09 parts by mass. Parts or more.
- the content of the carbon material in the carbon material dispersion liquid of the present embodiment may be 0.01 to 25 parts by mass, 0.05 parts by mass or more 25 parts by mass or less, may be 0.01 parts by mass or more and 20 parts by mass or less, may be 0.05 parts by mass or more and 20 parts by mass or less, 0.01 parts by mass or more and 15 parts by mass or less May be 0.05 to 15 parts by mass, 0.01 to 10 parts by mass, 0.05 to 10 parts by mass, 0.01 to 5 parts by mass, 0.05 to 5 parts by mass, 0.01 to 4 parts by mass, 0.05 or more parts by mass 4 parts by mass or less, and 0.01 parts by mass or less.
- the amount may be 0.01 to 1 part by mass, and may be 0.05 to 1 part by mass.
- the upper limit of the content of the carbon material in the dope solution is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin. Is preferably 0.95 parts by mass or less, 0.9 parts by mass or less, 0.8 parts by mass or less, 0.7 parts by mass or less, and 0.1 part by mass or less. 6 parts by mass or less, 0.5 parts by mass or less, 0.4 parts by mass or less, 0.3 parts by mass or less, and 0.2 parts by mass or less. May be.
- the lower limit of the content of the carbon material in the dope solution may be 0.01 parts by mass or more, 0.03 parts by mass or more with respect to 100 parts by mass of the modified spider silk fibroin. It may be at least 05 parts by mass, at least 0.06 parts by mass, at least 0.07 parts by mass, at least 0.08 parts by mass, at least 0.09 parts by mass. May be.
- the content of the carbon material in the dope solution is preferably, for example, 0.01 part by mass or more and 1 part by mass or less, and preferably 0.01 part by mass or more and 1 part by mass with respect to 100 parts by mass of the modified spider silk fibroin.
- the amount is more preferably less than 0.03 parts by mass and less than 1 part by mass, particularly preferably from 0.05 parts by mass to less than 1 part by mass, and more preferably from 0.05 parts by mass to 0.1 part by mass.
- 95 parts by mass or less may be 0.05 parts by mass or more and 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, and may be 0.05 parts by mass or more and 0 parts by mass or less.
- the concentration of the modified spider silk fibroin in the carbon material dispersion of the present embodiment may be 1 to 40% by mass, 1 to 35% by mass, and 1 to 40% by mass when the total amount of the dispersion is 100% by mass. 30% by mass, 1 to 25% by mass, 1 to 20% by mass, 1 to 15% by mass, 1 to 10% by mass, 1 to 1% by mass It may be 5% by mass, 1-3% by mass, 1-2% by mass.
- concentration of the modified spider silk fibroin is 1% by mass or more, the dispersibility of the carbon material can be sufficiently improved.
- the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to avoid a decrease in the dispersibility of the carbon material due to a significant increase in viscosity.
- the concentration of the modified spider silk fibroin may be 10 to 40% by mass when the total amount of the dope solution is 100% by mass. More preferably, it is 10 to 35% by mass, more preferably 12 to 35% by mass, more preferably 15 to 35% by mass, more preferably 15 to 30% by mass, The content is more preferably 20 to 35% by mass, and particularly preferably 20 to 30% by mass.
- the concentration of the modified spider silk fibroin is 10% by mass or more, the dope solution can be more stably discharged from the spinneret, and the productivity is improved.
- the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to prevent the holes of the spinneret from being closed when the dope is discharged from the spinneret, thereby improving the productivity.
- the dispersion may be stirred or shaken for a certain period of time to promote dissolution. At that time, if necessary, the dispersion may be heated to a temperature at which it can be dissolved depending on the modified spider silk fibroin and the polar solvent used. The dispersion may be heated to, for example, 30C or higher, 40C or higher, 50C or higher, 60C or higher, 70C or higher, 80C or higher, or 90C or higher. The upper limit of the heating temperature is, for example, equal to or lower than the boiling point of the polar solvent.
- the viscosity of the dispersion of this embodiment may be set as appropriate.
- the viscosity is not particularly limited as long as it allows dry spinning. From the viewpoint of productivity, it may be 3000 to 50,000 mPa ⁇ sec at 25 ° C. 5000 to 50000 mPa ⁇ sec, 5000 to 40000 mPa ⁇ sec, 5000 to 30000 mPa ⁇ sec, 5000 to 20000 mPa ⁇ sec, and 5000 to 15000 mPa ⁇ sec. 5000 to 12000 mPa ⁇ sec.
- the viscosity of the spinning solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
- the dispersion of the present embodiment may further contain an inorganic salt.
- the inorganic salt can be used as a promoter for dissolving the modified spider silk fibroin in a polar solvent.
- the inorganic salt may be an inorganic salt composed of the following Lewis acid and Lewis base.
- the Lewis base include a halide ion and the like.
- Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions.
- Examples of the inorganic salt include an alkali metal halide and an alkaline earth metal halide.
- Specific examples of the alkali metal halide include lithium chloride and lithium bromide.
- Specific examples of the alkaline earth metal halide include magnesium chloride and calcium chloride. Among these inorganic salts, lithium chloride and calcium chloride are particularly preferred.
- the preparation of the dispersion may be easier.
- the content of the inorganic salt is 0.1% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more based on the total amount of the dispersion. Or 15% by mass or more, and may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
- the dispersion may further contain various additives as necessary.
- the additives include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin.
- the content of the additive may be 50 parts by mass or less based on 100 parts by mass of the total modified fibroin in the spinning solution.
- the modified spider silk fibroin used as a raw material is not particularly limited, and may be fibroin produced by a microorganism or the like by genetic recombination technology, or fibroin produced by synthesis. However, naturally derived spider silk fibroin is excluded from the modified spider silk fibroin.
- modified spider silk fibroin refers to a spider silk fibroin having an amino acid sequence different from that of a naturally occurring spider silk fibroin. Spider silk fibroin having the same amino acid sequence as the spider silk fibroin.
- spider silk fibroin of natural origin examples include spider silk fibroin produced by spiders such as large tubule wicking silk protein, weft silk protein, and small ampullate gland protein. Since the large spinneret thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity.
- the weft of spider silk has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft has a lower stress than the large spinneret and has a high elasticity.
- the large spinal cord marker thread protein is produced by the large ampullate gland of spiders, and has the characteristic of excellent toughness.
- Examples of the large spinal cord marker thread protein include the large ampullate spidroins MaSp1 and MaSp2 derived from the American spider (Nephila laclavipes), and ADF3 and ADF4 derived from Araneus diadematus.
- ADF3 is one of the two major bookmarker thread proteins of the Japanese spider.
- the spider silk protein derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and elongation and toughness.
- weft protein is produced in the flagellar gland of spiders.
- a flagellated silk protein (flagelliform @ silk @ protein) derived from the American spider (Nephila @ clavipes) can be mentioned.
- spider silk fibroins produced by spiders include, for example, spiders belonging to the genus Araneus (Araneus sp.), Such as Orion spiders, Japanese spiders, A. spiders and A. spiders, and the spiders of the Japanese spiders Spiders belonging to the genus (Neoscona), spiders belonging to the genus Argiope (Pronus) such as Argiope serrata, spiders, spiders belonging to the genus Cyrarchachne such as the Torinofundamashi and Otorinofundamashi, and Spiders belonging to the genus Spider spiders (Genus Gasteracantha) such as Tibato spiders, spiders belonging to the genus Ordgarius, such as spiders belonging to the genus Orthodox spiders, such as the spider spider Spider spiders and the spiders spiders spiders, etc.
- spiders belonging to the genus Araneus Such as Orion spiders, Japanese spiders, A. spiders and A. spiders,
- Spiders belonging to the genus Argiope such as Argiope bruennichi and Argiope bruennichi, spiders belonging to the genus Arachnura (genus Arachnura) such as the arachnid spider, spiders such as the spiders belonging to the genus Acusilas and the spiders of the spider spiders belonging to the genus Acusilas such as the spider Spiders belonging to the genus Cytophora, spiders belonging to the spider spider belonging to the genus Cytophora (genus Poltys), spiders belonging to the genus Spiders belonging to the genus Spiders belonging to the genus Poltys, spiders belonging to the genus Spiders, spiders belonging to the spider, spiders belonging to the genus Cyclos sp.
- Argiope Argiope
- genus Arachnura genus Arachnura
- spiders belonging to the genus Acusilas such as the arachnid
- Spider silk proteins produced by spiders belonging to the genus Chorizopes, and spider silk spiders, Asagata spiders, Harabiroashida spiders, and urocore spiders The spiders belonging to the genus Tetragnatha (genus Tetragnatha), the spiders belonging to the genus Tetragnatha, the spiders belonging to the genus Leucauge, the spiders belonging to the genus Leucauge, and the genus E belonging to the spiders sp.
- the spiders belonging to the genus L such as spiders belonging to the spiders belonging to the genus Menosira, such as the spider spider, the spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Menosira, the spiders belonging to the spiders belonging to the spiders spiders belonging to the spiders, the black widow spider, the red widow spider, and the black spiders And spiders belonging to the genus Euprostenops (Tetragnathidae), such as spiders belonging to the genus Euprostenops Spider silk proteins produced by spiders.
- spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ph ), U37520 (base sequence)), major ⁇ ampullate ⁇ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number
- the modified spider silk fibroin according to the present embodiment is represented by, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It may be a protein containing a domain sequence.
- an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to one or both of the N-terminal side and the C-terminal side of the domain sequence.
- the N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
- a modified spider silk fibroin is preferably used as the modified fibroin because it is excellent in heat retention, moisture absorption and heat generation and / or flame retardancy.
- domain sequence refers to a crystalline region unique to fibroin (typically, corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence).
- the amino acid represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Means an array.
- the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues.
- the number of amino acid residues in the n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16.
- the ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues).
- At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues.
- REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
- the REP may be an amino acid sequence composed of 10 to 200 amino acid residues, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or The amino acid sequence may be composed of 10 to 180 amino acid residues.
- m represents an integer of 2 to 300, and 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 of 20 to 140 or 20 to 120.
- the plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
- a plurality of REPs may have the same amino acid sequence or different amino acid sequences.
- the modified spider silk fibroin is, for example, a modified spider silk fibroin whose amino acid sequence is modified based on the amino acid sequence of the spider silk fibroin (for example, by changing the gene sequence of a cloned naturally occurring spider silk fibroin,
- the amino acid sequence may be modified or artificially designed and synthesized without using spider silk fibroin of natural origin (for example, a desired amino acid sequence can be synthesized by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). May be included).
- the modified spider silk fibroin is, for example, an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by making modifications. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods ⁇ in ⁇ Enzymology, 100, 448 (1983).
- modified spider silk fibroin examples include a modified spider silk fibroin (first modified spider silk fibroin) derived from a large spinal canal thread protein produced in the large ampullate gland of a spider, and the content of glycine residues Spider silk fibroin (second modified spider silk fibroin), (A) modified spider silk fibroin with reduced n- motif content (third modified spider silk fibroin), glycine residue content And (A) a modified spider silk fibroin having a reduced content of the n motif (fourth modified spider silk fibroin), and a modified spider silk fibroin (fifth modified spider silk fibrin having a domain sequence including a region having a locally large hydrophobicity index). Modified spider silk fibroin) and a modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified silk fibroin). Yarn fibroin), and the like.
- first modified spider silk fibroin derived from a large spinal canal thread protein produced in the large ampullate gland of
- the modified spider silk fibroin (first modified spider silk fibroin) derived from the large spinal cord marker silk protein produced in the large ampullate of the spider is represented by Formula 1: [(A) n motif-REP] m And proteins containing the domain sequence to be performed.
- n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and still more preferably an integer of 10 to 20.
- An integer of 4 to 16 is still more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred.
- the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. It is more preferably a residue, and even more preferably 20 to 75 residues.
- the first modified spider silk fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m It is preferably at least 40%, more preferably at least 60%, even more preferably at least 70%, based on the total number of residues.
- the first modified spider silk fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m and has a C-terminal sequence represented by any one of SEQ ID NOs: 1 to 3.
- the protein may be a sequence or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOS: 1 to 3.
- the amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is
- the amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
- the first modified spider silk fibroin (1-i) the amino acid sequence represented by SEQ ID NO: 4 or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 having 90% or more sequence identity Modified spider silk fibroin containing an amino acid sequence having sex properties.
- the sequence identity is preferably 95% or more.
- the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human ⁇ rhinovirus @ 3C protease) recognition site at the N-terminus is added.
- the 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue.
- the amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
- the modified spider silk fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.
- the modified spider silk fibroin having a reduced content of glycine residues has a domain sequence in which the content of glycine residues is reduced as compared with a naturally occurring spider silk fibroin.
- Having an amino acid sequence of The second modified spider silk fibroin has an amino acid sequence corresponding to at least one or more glycine residues in the REP has been replaced with another amino acid residue, as compared to a naturally occurring spider silk fibroin. It can be said.
- the second modified spider silk fibroin has GGX and GPGXX in the REP (where G is a glycine residue, P is a proline residue, and X is other than glycine, as compared with the naturally occurring spider silk fibroin.
- G is a glycine residue
- P is a proline residue
- X is other than glycine, as compared with the naturally occurring spider silk fibroin.
- an amino acid sequence corresponding to the substitution of at least one or a plurality of glycine residues in the motif sequence with another amino acid residue is determined. You may have.
- the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
- the second modified spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the above domain sequence, the (A) n motif located at the most C-terminal side to the above
- the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence is represented by z
- z / w is 30%
- it may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more.
- the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
- the second modified spider silk fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue.
- the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less.
- the content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
- z / w (%) can be calculated by dividing z by w.
- z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70/70. %, Still more preferably 80% or more.
- the upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
- the second modified spider silk fibroin is obtained, for example, by replacing at least a part of a nucleotide sequence encoding a glycine residue from a cloned naturally occurring spider silk fibroin gene sequence to encode another amino acid residue. It can be obtained by modification.
- a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more.
- an amino acid sequence satisfying the above aspect from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
- one or more amino acid residues are further substituted,
- the amino acid sequence corresponding to the deletion, 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 includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
- a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
- the modified spider silk fibroin of (2-i) will be described.
- the amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin with GQX.
- the amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted.
- the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7.
- the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
- the value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%.
- the values of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%.
- the value of x / y at the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
- the modified spider silk fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
- the modified spider silk fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
- the modified spider silk 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 spider silk 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.
- the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
- the second modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
- the tag sequence examples include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule.
- affinity tag is a histidine tag (His tag).
- His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography).
- SEQ ID NO: 12 amino acid sequence including a His tag sequence and a hinge sequence.
- tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
- GST glutathione-S-transferase
- MBP maltose binding protein
- an “epitope tag” utilizing an antigen-antibody reaction can be used.
- a peptide (epitope) showing antigenicity as a tag sequence an antibody against the epitope can be bound.
- the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag.
- a tag sequence that can be cleaved by a specific protease can be used.
- protease treatment By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified spider silk fibroin from which the tag sequence has been separated can also be recovered.
- the second modified fibroin containing a tag sequence (2-iii) the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, or (2-iv) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned.
- amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
- an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
- the modified spider silk fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
- the modified spider silk fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
- the modified spider silk 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 spider silk fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and is contained in REP.
- the total number of amino acid residues in the amino acid sequence consisting of XGX is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
- the second modified spider silk 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 the host.
- (A) n motifs modified spider silk fibroin content is reduced (Third modified spider silk fibroin), the domain sequence is compared to the naturally occurring silk fibroin, containing (A) n motif It has a reduced amino acid sequence. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to a naturally occurring spider silk fibroin.
- the third modified spider silk fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif from a naturally occurring spider silk fibroin.
- the third modified spider silk fibroin has a domain sequence of at least one for every 1-3 (A) n motifs from the N-terminus to the C-terminus compared to naturally occurring spider silk fibroin. A) It may have an amino acid sequence corresponding to the deletion of the n motif.
- the third modified spider silk fibroin has a deletion of the (A) n motif whose domain sequence is at least two contiguous from the N-terminal side to the C-terminal side, as compared with the naturally-derived spider silk fibroin, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
- the third modified spider silk fibroin has an amino acid sequence whose domain sequence corresponds to the deletion of the (A) n motif at least every other region from the N-terminal to the C-terminal. Is also good.
- the third modified spider silk fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) The number of amino acid residues of the REP of the [ n motif-REP] unit is sequentially compared, and when the number of amino acid residues of the REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.
- the maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units of 8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is y.
- the number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
- FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from spider silk fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called an n motif.
- the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared.
- each pattern the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
- x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
- x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and more preferably at least 70%. Even more preferably, it is even more preferably 75% or more, and particularly preferably 80% or more.
- the upper limit of x / y is not particularly limited, and may be, for example, 100% or less.
- x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y.
- / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
- the third modified spider silk fibroin is a modified spider silk fibroin in which at least seven of the (A) n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y is 46.4% Or more, more preferably 50% or more, still more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, It is particularly preferred that it is 80% or more.
- the upper limit of x / y is not particularly limited, and may be 100% or less.
- the third modified spider silk fibroin is, for example, one or more of the sequences encoding the (A) n motif such that x / y is 64.2% or more from the cloned spider silk fibroin gene sequence. Can be obtained by deleting Further, for example, 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 spider silk fibroin derived from nature. Alternatively, it can be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
- one or more amino acid residues are further substituted, deleted, inserted and / or substituted.
- the amino acid sequence corresponding to the addition may be modified.
- the third modified spider silk fibroin (3-i) the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18 , A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
- the modified spider silk fibroin (3-i) will be described.
- the amino acid sequence represented by SEQ ID NO: 18 is different from the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin in that every two (A) n motifs are deleted from the N-terminal side to the C-terminal side. And one [(A) n motif-REP] was inserted in front of the C-terminal sequence.
- the amino acid sequence represented by SEQ ID NO: 7 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 18 with GQX.
- the amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7.
- the amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
- the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8 to 11.3.
- the value of x / y in the amino acid sequence represented by SEQ ID NO: 18 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%.
- the value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%.
- the value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.3%.
- the values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
- the modified spider silk fibroin of (3-i) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
- the modified spider silk fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
- the modified spider silk 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 spider silk fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has C-terminal sequence from the N-terminal side.
- the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared toward the terminal side, when the number of amino acid residues of REP having a small number of amino acid residues is 1, Of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of the other REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3).
- x / y be 64.2% or more, where x is the maximum value of the sum of the number of amino acid residues and y is the total number of amino acid residues in the domain sequence.
- the third modified spider silk fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
- the third modified spider silk fibroin including the tag sequence, (3-iii) the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (3-iv) ) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
- amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively.
- an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
- the modified spider silk fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
- the modified spider silk fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
- the modified spider silk 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 spider silk fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and has C-terminal sequence from the N-terminal side.
- the third modified spider silk 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 the host.
- the modified spider silk fibroin (the fourth modified spider silk fibroin) in which the content of the glycine residue and the content of the (A) n motif are reduced has a domain sequence that is lower than that of a naturally derived spider silk fibroin.
- (A) having an amino acid sequence in which the content of glycine residues is reduced in addition to the reduced content of the n motif.
- the domain sequence of the fourth modified spider silk fibroin differs from the naturally occurring spider silk fibroin in that at least one or more (A) n motifs have been deleted, and at least one or more of the It can be said that the glycine residue has an amino acid sequence corresponding to the substitution of another amino acid residue.
- the modified spider silk fibroin (the second modified spider silk fibroin) in which the content of the glycine residue was reduced and the content of the (A) n motif were reduced.
- a modified spider silk fibroin having the characteristics of the modified spider silk fibroin (third modified spider silk fibroin). Specific embodiments and the like are as described for the second modified spider silk fibroin and the third modified spider silk fibroin.
- the fourth modified spider silk fibroin (4-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9 can be mentioned.
- Specific embodiments of the modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 are as described above.
- the modified spider silk fibroin having a domain sequence including a region having a locally large hydrophobicity index has a domain sequence of 1 in REP compared to naturally occurring spider silk fibroin.
- local substitution corresponding to substitution of a plurality of amino acid residues with amino acid residues having a large hydrophobicity index and / or insertion of one or more amino acid residues having a large hydrophobicity index into REP. May have an amino acid sequence containing a region having a large hydrophobicity index.
- a region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
- the fifth modified fibroin is that one or more amino acid residues in the REP have been replaced with amino acid residues having a large hydrophobicity index, and / or Or, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, further, compared with naturally occurring spider silk fibroin, substitution or deletion of one or more amino acid residues, insertion, And / or there may be an amino acid sequence modification corresponding to the addition.
- the fifth modified spider silk fibroin is obtained by, for example, removing one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. Further, for example, one or more hydrophilic amino acid residues in REP are replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids are contained in REP.
- one or more hydrophilic amino acid residues in REP are replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids are contained in REP.
- amino acid sequence corresponding to insertion of a residue can also be obtained by designing an amino acid sequence corresponding to insertion of a residue and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
- one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of the naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acid residues in the REP.
- the amino acid sequence may further be modified corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues.
- the fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence.
- the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p,
- q the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q
- p / q is 6 .2% or more.
- hydrophobicity index of amino acid residues
- a publicly known index Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132).
- HI hydropathic index
- sequence A [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”).
- sequence A the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues).
- the average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
- p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Is still more preferred, and even more preferably 30% or more.
- the upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
- the fifth modified spider silk fibroin is, for example, one or more hydrophilic amino acid residues in REP (for example, the amino acid sequence of the cloned naturally-derived spider silk fibroin is changed so as to satisfy the above-mentioned p / q conditions).
- Replacing an amino acid residue with a negative hydrophobicity index with a hydrophobic amino acid residue eg, an amino acid residue with a positive hydrophobicity index
- a hydrophobic amino acids in the REP It can be obtained by inserting a residue to locally modify the amino acid sequence to include a region having a large hydrophobicity index.
- an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
- one or more amino acid residues in the REP were replaced with amino acid residues having a higher hydrophobicity index as compared to naturally occurring spider silk fibroin, and / or one or more amino acid residues in the REP.
- the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues was performed. Is also good.
- the amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
- the fifth modified spider silk fibroin examples include (5-i) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21 can be mentioned.
- the modified spider silk fibroin of (5-i) will be described.
- the amino acid sequence represented by SEQ ID NO: 22 is obtained by deleting the amino acid sequence having consecutive alanine residues in the (A) n motif of a spider silk fibroin of natural origin so that the number of consecutive alanine residues becomes five. It is.
- the amino acid sequence represented by SEQ ID NO: 19 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues at every other REP into the amino acid sequence represented by SEQ ID NO: 22; A part of the amino acids at the C-terminal side are deleted so that the molecular weight of the amino acid sequence to be obtained is almost the same.
- the amino acid sequence represented by SEQ ID NO: 23 is different from the amino acid sequence represented by SEQ ID NO: 22 by inserting two alanine residues at the C-terminal side of each (A) n motif, and further including a part of glutamine (Q) residue.
- a group is substituted with a serine (S) residue, and some amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 22 is almost the same.
- the amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23.
- the amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23.
- the modified spider silk fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
- the modified spider silk 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 spider silk fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most C-terminal (A )
- the REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more.
- the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
- the fifth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
- a modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 26.
- amino acid sequences represented by SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 correspond to the amino acid sequence represented by SEQ ID NO: 12 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
- the modified spider silk fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
- the modified spider silk fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
- the modified spider silk 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 spider silk fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most C-terminal (A )
- the REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more.
- the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
- the fifth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
- the sequence of the secretion signal can be appropriately set according to the type of the host.
- the modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues has a reduced content of glutamine residues as compared to naturally occurring spider silk fibroin Has an amino acid sequence.
- the sixth modified spider silk fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
- the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and preferably 10% or more.
- the upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
- the “GPGXX motif content” is a value calculated by the following method.
- Formula 1 [(A) n motif -rep] m
- Formula 2 [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal (A) In all REPs included in the sequence except for the sequence from the n motif to the C-terminus of the domain sequence from the n motif to the domain sequence, the total number of GPGXX motifs contained in the region was tripled ( That is, s is defined as s (corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n Assuming that the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is
- “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) sequence from the n motif to the C-terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of spider silk fibroin. Is small (that is, when the domain sequence is short), which affects the calculation result of the GPGXX motif content, so that this effect is eliminated.
- “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
- FIG. 3 is a schematic diagram showing the domain sequence of spider silk fibroin.
- the method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of spider silk fibroin ("[(A) n motif-REP] m- (A) n motif" type) shown in FIG. 3, all REPs are located at the "most C-terminal position".
- all REPs are “sequences in which the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence shown as “region A” in FIG. 3). ),
- the sixth modified spider silk fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and more preferably 0%. Particularly preferred.
- 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) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs included in the sequence (sequence corresponding to “region A” in FIG.
- the sixth modified spider silk fibroin had one or more glutamine residues in the REP deleted or substituted with other amino acid residues in the domain sequence compared to the naturally occurring spider silk fibroin. It may have an amino acid sequence corresponding to this.
- the “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue.
- the hydrophobicity index of amino acid residues is as shown in Table 1.
- an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable.
- the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more. It is still more preferably 3 or more, and particularly preferably 0.4 or more.
- the upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
- REP hydrophobicity is a value calculated by the following method.
- Formula 1 [(A) n motif -rep] m
- Formula 2 [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs contained in the sequence (sequence corresponding to “region A” in FIG.
- the sixth modified spider silk fibroin may have a domain sequence that lacks one or more glutamine residues in the REP, and / or one or more glutamine residues in the REP, as compared to the naturally occurring spider silk fibroin.
- the modification corresponding to the substitution of a glutamine residue with another amino acid residue the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is also included. There may be.
- the sixth modified spider silk fibroin may be, for example, by deleting one or more glutamine residues in the REP from the cloned naturally occurring spider silk fibroin gene sequence, and / or one or more glutamine in the REP. It can be obtained by replacing a residue with another amino acid residue.
- one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more glutamine residues in REP have been replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the substitution and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
- the sixth modified spider silk fibroin (6-i) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: A modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 43, or (6-ii) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: Modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 43 can be mentioned.
- the modified spider silk fibroin of (6-i) will be described.
- the amino acid sequence represented by SEQ ID NO: 7 is based on the nucleotide sequence and amino acid sequence of a naturally occurring fibroin, Nephila clavipes (GenBank Accession Number: P468804.1, GI: 1174415), and (A) n
- the amino acid sequence in which the alanine residues in the motif are consecutive has been modified such that the number of consecutive alanine residues is changed to 5, for example, to improve productivity.
- the glutamine residue (Q) is not modified, so that the glutamine residue content is almost the same as that of naturally occurring fibroin.
- M_PRT965 represented by SEQ ID NO: 28 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with TS and replacing the remaining Qs with A.
- the amino acid sequence represented by SEQ ID NO: 29 (M_PRT889) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and replacing the remaining Qs with I.
- the amino acid sequence (M_PRT525) represented by SEQ ID NO: 34 is obtained by inserting two alanine residues into a region (A 5 ) in which alanine residues are continuous with Met-PRT410 (SEQ ID NO: 7), In this example, two C-terminal domain sequences were deleted, and 13 glutamine residues (Q) were replaced with a serine residue (S) or a proline residue (P).
- the amino acid sequence represented by SEQ ID NO: 43 (Met-PRT966) is obtained by converting all the QQs in the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence before the amino acid sequence represented by SEQ ID NO: 42 is added to the C-terminus) to VF , And the remaining Q is replaced by I.
- amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 all have a glutamine residue content of 9% or less. (Table 2).
- the modified spider silk fibroin of (6-i) consists of the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 It may be something.
- the modified spider silk fibroin of (6-ii) has an amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 and 90 % Or more.
- the modified spider silk fibroin of (6-ii) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence.
- the sequence identity is preferably 95% or more.
- the modified spider silk fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified spider silk fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
- the sixth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
- the sixth modified spider silk fibroin including the tag sequence, (6-iii) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: A modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 41 or SEQ ID NO: 44, or (6-iv) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 44 can be mentioned.
- amino acid sequences represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, respectively.
- the amino acid sequence represented by SEQ ID NO: 12 (including the His tag sequence and the hinge sequence) was added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 Things.
- amino acid sequence represented by SEQ ID NO: 44 has a glutamine residue content of 9% or less (Table 3).
- the modified spider silk fibroin of (6-iii) consists of the amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44 It may be something.
- the modified spider silk fibroin of (6-iv) has an amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44, and 90 % Or more.
- the modified spider silk fibroin of (6-iv) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence.
- the sequence identity is preferably 95% or more.
- the modified spider silk fibroin of (6-iv) preferably has a glutamine residue content of 9% or less.
- the modified spider silk fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
- the sixth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host.
- the sequence of the secretion signal can be appropriately set according to the type of the host.
- the modified spider silk fibroin comprises a first modified spider silk fibroin, a second modified spider silk fibroin, a third modified spider silk fibroin, a fourth modified spider silk fibroin, a fifth modified spider silk fibroin, and a sixth modified spider silk fibroin.
- the modified spider silk fibroin having at least two or more characteristics may be used.
- the modified spider silk fibroin may be a hydrophilic modified spider silk fibroin or a hydrophobic modified spider silk fibroin.
- Hydrophobic modified spider silk fibroin refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified spider silk fibroin, and then dividing the sum by the total number of amino acid residues (average HI). ) Is 0 or more.
- the hydrophobicity index is as shown in Table 1.
- the hydrophilic modified spider silk fibroin is a modified spider silk fibroin having the above average HI of less than 0.
- hydrophobic modified spider silk fibroin examples include the sixth modified fibroin described above. More specific examples of the hydrophobically modified spider silk fibroin include amino acids represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 Modified spider silk fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.
- hydrophilic modified spider silk fibroin examples include the above-mentioned first modified fibroin, second modified fibroin, third modified fibroin, fourth modified fibroin, and fifth modified fibroin.
- More specific examples of the hydrophilic spider silk protein include an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: Modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
- the above-mentioned modified spider silk fibroin can be used alone or in combination of two or more.
- the modified spider silk fibroin is, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified spider silk fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing a nucleic acid.
- the method for producing the nucleic acid encoding the modified spider silk fibroin is not particularly limited.
- the nucleic acid can be produced by a method of amplifying and cloning by polymerase chain reaction (PCR) using a gene encoding a modified spider silk fibroin, or a method of chemically synthesizing.
- the method for chemically synthesizing nucleic acids is not particularly limited. For example, based on amino acid sequence information of spider silk proteins obtained from the NCBI web database or the like, AKTA oligopilot plus 10/100 (GE Healthcare Japan KK) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like by PCR or the like.
- a nucleic acid encoding the modified spider silk fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus was synthesized. You may.
- the regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host.
- An inducible promoter that functions in a host cell and is capable of inducing the expression of the desired modified spider silk fibroin may be used as the promoter.
- An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
- the type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like.
- a plasmid vector a virus vector
- a cosmid vector a fosmid vector
- an artificial chromosome vector an artificial chromosome vector
- those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified spider silk fibroin can be transcribed are suitably used. .
- any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
- the expression vector is capable of autonomous replication in the prokaryote and contains a promoter, a ribosome binding sequence, a nucleic acid encoding a modified spider silk fibroin, and a transcription termination sequence. It is preferably a vector. A gene that controls the promoter may be included.
- Prokaryotes include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like.
- Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli.
- Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
- Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like.
- microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis.
- Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum.
- Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum.
- Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
- Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
- a prokaryote when used as a host, as a vector for introducing a nucleic acid encoding a modified spider silk fibroin, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
- pBTrp2 manufactured by Boehringer Mannheim
- pGEX manufactured by Pharmacia
- pUC18 pBluescriptII, pSuex, pET22b, pCold
- pUB110 pNCO2
- Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold).
- yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like.
- filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
- examples of a vector into which a nucleic acid encoding a modified spider silk fibroin is introduced include YEp13 (ATCC 37115) and YEp24 (ATCC 37051).
- any method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
- a method using calcium ions [Proc. ⁇ Natl. ⁇ Acad. ⁇ Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
- a method for expressing a nucleic acid by a host transformed with an expression vector in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
- the modified spider silk fibroin can be produced, for example, by culturing the transformed host in a culture medium, producing and accumulating the modified spider silk fibroin in the culture medium, and collecting the modified spider silk fibroin from the culture medium.
- the method of culturing the transformed host in a culture medium can be performed according to a method generally used for culturing a host.
- the culture medium contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, thereby efficiently culturing the host.
- a natural medium or a synthetic medium may be used as long as the medium can be used.
- the carbon source may be any as long as the host can assimilate it.
- examples include glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysates, and organic acids such as acetic acid and propionic acid. And alcohols such as ethanol and propanol.
- the nitrogen source for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
- ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
- potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate
- potassium (I) phosphate potassium (II) phosphate
- magnesium phosphate magnesium phosphate
- magnesium sulfate sodium chloride
- ferrous sulfate manganese sulfate
- copper sulfate copper sulfate
- calcium carbonate calcium carbonate
- ⁇ Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
- the culture temperature is, for example, 15 to 40 ° C.
- the culturing time is usually 16 hours to 7 days.
- the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
- the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
- antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture.
- an inducer may be added to the medium as necessary.
- isopropyl- ⁇ -D-thiogalactopyranoside or the like is used.
- An acid or the like may be added to the medium.
- the modified spider silk fibroin produced by the transformed host can be isolated and purified by a method usually used for protein isolation and purification.
- a method usually used for protein isolation and purification For example, when the modified spider silk fibroin is expressed in a dissolved state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The host cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract.
- a method commonly used for isolating and purifying proteins that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
- a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei)
- cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia).
- the modified spider silk fibroin when expressed by forming an insoluble body in the cells, the host cells are similarly recovered, crushed, and centrifuged to obtain an insoluble body of the modified spider silk fibroin as a precipitate fraction. Collect.
- the recovered insoluble form of the modified spider silk fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified spider silk fibroin can be obtained by the same isolation and purification method as described above.
- the modified spider silk fibroin When the modified spider silk fibroin is secreted extracellularly, the modified spider silk fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
- the method for producing a modified spider silk fibroin fiber includes a step of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming a raw fiber from the dispersion. And at least a step.
- fibrils refers to fibrous solids formed from a dispersion used as a spinning dope (dope) by cooling the dispersion, evaporating a solvent (vaporizing), a chemical reaction, or the like.
- the step of forming fibrils from the dispersion may include discharging the above-described dispersion, or may include drawing the fibrils from the above-described dispersion, discharging the dispersion, and It may include both drawing fibrils from the dispersion.
- the fibrils formed from the dispersion may be used as modified spider silk fibroin fibers as they are, or modified spider silk fibroin fibers obtained by subjecting the fibrils to processing such as stretching described below. Good.
- the method for producing a modified spider silk fibroin fiber according to the present embodiment may further include a step of defoaming a dispersion used as a spinning stock solution (dope solution) (defoaming step).
- the liquid When performed in a liquid, the liquid is preferably a liquid having a low affinity for the dispersion, for example, a non-polar solvent.
- the point of contact between the dispersion and the device When drawing fibrils is performed in a gas, the point of contact between the dispersion and the device may be at the gas / dispersion interface, and when drawing fibrils in a liquid, The point of contact between the dispersion and the device may be at the liquid / dispersion interface. By drawing the fibrils, the fiber diameter of the fibrils can be made smaller.
- the device to be brought into contact with the dispersion is preferably one having a sharp tip in order to form a small point of contact with the dispersion, and examples thereof include a needle, a pipette tip, tweezers, a skewer, a toothpick, and a thin stick.
- examples of the material of the device include, but are not limited to, plastic, metal, glass, and wood.
- the speed at which the device is withdrawn in the direction away from the point of contact with the dispersion is adjusted by the viscosity of the dispersion. Further, it is preferable that the drawing speed is constant. By keeping the pulling speed constant, it is possible to prevent the fiber from being twisted, sagged or cut, and to suppress the variation in the fiber diameter.
- the drawing speed for example, 0.1 cm / sec or more, 0.1 cm / sec to 15 m / sec, 1 cm / sec to 5 m / sec, 3 cm / sec to 1 m / sec, 5 cm / sec to 50 cm / sec, 5 cm / sec 3535 cm / sec.
- the tip of the fibril and the tip of the instrument are adhered, but once the fibril is pulled out, the tip of the instrument and the Does not have to be bonded to the tip.
- the fibrils continue to be drawn out of the dispersion liquid even if the tip of the device and the tips of the fibrils are not bonded.
- discharge of dispersion liquid means discharging the dispersion liquid from the nozzle with or without applying pressure to the dispersion liquid.
- the fine fibers are formed by the dispersion liquid finely discharged through the nozzle becoming solid by evaporation (vaporization) of the solvent, chemical reaction, or the like.
- the destination where the dispersion liquid is discharged may be discharged into the air or may be discharged into a liquid (coagulating liquid).
- the method of discharging the dispersion liquid from the nozzle is not particularly limited.
- a method using a metering pump as a means for sending the spinning solution can be used.
- the discharge amount can be appropriately adjusted according to the production speed.
- a spinneret may be used as a nozzle for discharging the dispersion.
- the spinneret shape, hole shape, number of holes, and the like of the spinneret are not particularly limited, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like.
- the hole diameter of the spinneret can be, for example, 0.01 mm or more and 0.6 mm or less.
- the hole diameter is 0.01 mm or more, the pressure loss can be reduced and the equipment cost can be reduced.
- the pore diameter is 0.6 mm or less, it is possible to reduce the necessity of a stretching operation for reducing the fiber diameter, and it is possible to reduce the possibility of stretching breakage from discharge to winding.
- either the discharging of the dispersion or the extraction of the fibrils may be performed to form the fibrils, or both may be performed.
- the dispersion is discharged from a nozzle, the tip of the device is brought into contact with the discharged dispersion, and the device is dispersed. By pulling out the fiber in a direction away from the fiber and pulling out the fiber from the contact point, the fiber can be formed.
- the dry spinning method is a method of spinning (forming fibrils) by evaporating (vaporizing) a solvent in a spinning solution (dope solution).
- a spinning solution a spinning solution
- the drawing of the fibrils from the dispersion may be performed in a gas such as air.
- the discharge may be performed in a gas such as air.
- the fibrils are formed by evaporating the solvent in the dispersion. That is, in the present embodiment, in the step of forming the fibrils, the fibrils may be formed by a dry spinning method.
- Examples of the method for evaporating the solvent include drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying.
- drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying.
- the solvent evaporates spontaneously without performing aggressive drying such as hot-air drying, heat-drying, and air-drying, and the fibrils are removed. It can be formed and is efficient.
- carbon black nanoparticles are used as the carbon material contained in the dispersion, it is preferable to form the fibrils by a dry spinning method.
- the formed fibrils may be wound by a winding device such as a winder.
- a winding device By using a winding device, fibers can be continuously produced. Further, after the raw fiber is drawn out, the raw fiber can be continuously drawn out by winding the raw fiber with a winding device.
- a winder may be used as the winding device. In the winder, winding can be performed by appropriately adjusting winding conditions such as tension and contact pressure. As the winder, a known winder may be used.
- the fiber diameter of the fibrils can be controlled. Specifically, the fiber diameter can be made thinner by increasing the winding speed, and a fiber having a large fiber diameter can be manufactured by reducing the winding speed.
- the method for producing the modified spider silk fibroin fiber may further include a step of drawing the formed raw fiber (drawing step).
- the original fiber is drawn at an arbitrary draw ratio, and the drawn fiber can be used as a modified spider yarn fibroin fiber of the present embodiment for any use.
- Examples of the stretching method include wet heat stretching and dry heat stretching.
- Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or steam heating.
- the wet heat stretching temperature is preferably from 50 to 90 ° C, more preferably from 75 to 85 ° C.
- the temperature is 50 ° C. or more, the pore diameter in the fiber can be made small and stable.
- the temperature is 90 ° C. or lower, the temperature can be easily set and spinning stability is improved.
- the draw ratio in the wet heat drawing is, for example, 1 to 30 times, 1 to 25 times, 1 to 20 times, or 1 to 30 times with respect to the undrawn yarn (or pre-drawn yarn). 1515 times, 1 ⁇ 10 times, 2 ⁇ 10 times, 2 ⁇ 8 times, 2 ⁇ 6 times, 2 ⁇ 24 times And may be 2-3 times.
- Dry heat drawing can be performed by drawing in the air using a device equipped with a heat source such as a contact hot plate and a non-contact furnace, but the device is not particularly limited. Any device can be used as long as it can raise the temperature of the fiber to a predetermined temperature and can draw the fiber at a predetermined magnification.
- the temperature at which the dry heat stretching is performed may be, for example, 100 ° C to 270 ° C, 140 ° C to 230 ° C, 140 ° C to 200 ° C, or 160 ° C to 200 ° C. , 160 ° C to 180 ° C.
- the draw ratio in the dry heat drawing step may be, for example, 1 to 30 times, 1 to 25 times, or 1 to 20 times with respect to the undrawn yarn (or pre-drawn yarn). 1 to 15 times, 1 to 10 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, 2 to 4 times And may be 2-3 times.
- the wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, as the stretching step, 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, and the second-stage stretching is performed by wet-heat stretching. Stretching can be performed by appropriately combining wet heat stretching and dry heat stretching, such as by performing dry heat stretching.
- the lower limit of the final draw ratio of the fiber after the drawing step is, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or more with respect to the undrawn yarn (or pre-drawn yarn). It may be any of double, eight, or nine times.
- the upper limit of the final draw ratio of the fiber after the drawing step is, for example, any one of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. It may be.
- the final stretching magnification may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, or 5 to 15 times. And may be 5 to 13 times.
- the draw ratio is not limited as long as the desired properties such as fiber thickness and mechanical properties can be obtained.
- an oil agent may be added to the fiber, if necessary, for the purpose of imparting antistatic properties, convergence properties, lubricity and the like.
- the type of oil agent to be applied, the amount to be applied, and the like are not particularly limited, and can be appropriately adjusted in consideration of the use of the fiber, the handleability of the fiber, and the like.
- the modified spider silk fibroin fiber according to the present embodiment can be manufactured.
- the above-described method is an example, and the modified spider silk fibroin fiber according to the present embodiment may be manufactured by a method other than the above-described method.
- the modified spider silk fibroin fiber according to the present embodiment is a fiber containing the modified spider silk fibroin and one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
- the content of the carbon material may be from 0.01 part by mass to 1 part by mass, from 0.01 part by mass to less than 1 part by mass, based on the content of the modified spider silk fibroin.
- 03 parts by mass or more and less than 1 part by mass may be 0.05 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and 0.95 parts by mass or less, and may be 0.05 parts by mass.
- It may be 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, may be 0.05 parts by mass or more and 0.7 parts by mass or less, and may be 0.05 parts by mass or less. It may be not less than 0.6 part by mass and not less than 0.05 part by mass, and may be not less than 0.05 part by mass and not more than 0.3 part by mass.
- the method for producing a modified spider silk fibroin nonwoven fabric includes the steps of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming fibrils from the dispersion. And at least a step of intertwining the fibrils or modified spider silk fibroin fibers produced from the fibrils.
- the manufactured modified spider silk fibroin nonwoven fabric may be used for any purpose as it is.
- the step of forming the fibrils may include discharging the dispersion described above, and may include extracting the fibrils from the dispersion described above, and discharging the dispersion and from the dispersion. It may include both drawing fibrils.
- the modified spider silk fibroin nonwoven fabric according to the present embodiment may be manufactured by a known nonwoven fabric manufacturing method using raw fibers or modified spider silk fibroin fibers manufactured from raw fibers.
- a web including a single-layer web and a laminated web
- a chemical bond method The fibers of the web can be bonded together by a dipping method, a spraying method, etc.
- a needle punching method to obtain a nonwoven fabric.
- the manufacturing process of the nonwoven fabric may further include a process of adjusting the fiber density of the nonwoven fabric (adjustment process).
- the fiber density (basis weight) is a value defined by the weight per unit area of the nonwoven fabric.
- the fiber density of the nonwoven fabric can be adjusted by, for example, increasing or decreasing the amount of fibers constituting the web, and in the case of a laminated web, can be adjusted by increasing or decreasing the number of layers.
- Various products including the modified spider silk fibroin fiber according to the present embodiment can be manufactured.
- Such products include, for example, fabrics such as fibers, yarns, and fabrics, knits, braids, non-woven fabrics, paper, and cotton.
- the fiber include a long fiber, a short fiber, a monofilament, and a multifilament
- examples of the yarn include a spun yarn, a twisted yarn, a false twisted yarn, a processed yarn, a mixed fiber, and a mixed spun yarn.
- fabrics such as woven fabrics, knits, braids, non-woven fabrics, and the like, paper, cotton, and the like can be manufactured. These products can be manufactured by a known method.
- the present invention also provides a method for dispersing a carbon material, comprising a step of mixing a modified spider silk fibroin, a carbon material of 25 parts by mass or less based on 100 parts by weight of the modified spider silk fibroin, and a polar solvent.
- the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles.
- the present invention also provides a dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin.
- a dispersion aid for dispersing a carbon material in a polar solvent comprising a modified spider silk fibroin.
- the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles.
- the modified spider silk fibroin improves the dispersibility of the carbon material in the polar solvent. That is, the modified spider silk fibroin can be used as a dispersion aid for the carbon material.
- the present invention also provides a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
- a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles.
- the specific carbon material can have an extremely small fiber diameter. That is, the specific carbon material can be used as a diameter reducing agent for producing a modified spider silk fibroin fiber having a reduced diameter.
- the amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 12 (tag sequence and hinge sequence) is added to the N-terminus.
- nucleic acids encoding the designed spider silk fibroins PRT799, PRT918, and PRT966 having the designed amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 39, and SEQ ID NO: 44 were synthesized.
- An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid.
- the nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
- the seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) had been added so that the OD 600 was 0.05.
- the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the concentration of dissolved oxygen in the culture solution was maintained at 20% of the saturated concentration of dissolved oxygen.
- a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
- the temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, 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. Thereafter, 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified spider silk fibroin.
- IPTG isopropyl- ⁇ -thiogalactopyranoside
- the precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain modified spider silk fibroin (PRT799, PRT918, PRT966).
- 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
- Example 1 (Preparation of dispersion liquid) (Example 1) 3.16 g of formic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide (manufactured by Sigma-Aldrich, product number: 796034) were mixed, and the temperature was 60 ° C. using an ultrasonic cleaning device (manufactured by BRANSON). For 100 minutes by ultrasonic vibration. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-described production step was added, and the mixture was heated and dissolved with an aluminum block heater at 40 ° C. for 1 hour while stirring to prepare a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
- Example 2 A dispersion was prepared in the same manner as in Example 1, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 0.5% by mass. did. Specifically, the amount of formic acid added was 1.9 g, the amount of modified spider silk fibroin was 0.1 g, and the amount of graphene oxide was 0.5 mg.
- Example 3 A dispersion was prepared in the same manner as in Example 1, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 1% by mass.
- Example 4 3.16 g of dimethyl sulfoxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide were mixed, and stirred by ultrasonic vibration at a temperature of 60 ° C. for 100 minutes using an ultrasonic cleaning device. Immediately, 1 g of the modified fibroin (PRT799) obtained in the process of producing the modified fibroin was added, and the mixture was heated with a 90 ° C. aluminum block heater for 3 hours to dissolve with stirring, thereby preparing a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
- Example 5 A dispersion was prepared in the same manner as in Example 4, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 4% by mass.
- Example 6 A dispersion was prepared in the same manner as in Example 4, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 20% by mass.
- Comparative Example 1 As a comparative example, the dispersibility of graphene oxide in a formic acid single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 1, except that the modified spider silk fibroin was not added.
- Comparative Example 2 As a comparative example, the dispersibility of graphene oxide in a DMSO single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 4, except that the modified spider silk fibroin was not added.
- the dispersibility of graphene oxide in the dispersion was evaluated.
- the dispersibility was evaluated by visually confirming the precipitate of graphene oxide in the dispersion liquid after stirring.
- Table 6 shows the evaluation results of the dispersibility.
- the evaluation criteria for dispersibility are as follows. :: After the stirring, the dispersion state is maintained even after being left for one month or more. :: After stirring, the dispersion state is maintained even if the mixture is allowed to stand for 14 days or more. ⁇ : sedimented within 48 hours after stirring. ⁇ : sedimentation within 24 hours after stirring.
- Example 7 Preparation of dope solution (spinning stock solution) 3.16 g of formic acid and 1 mg of graphene oxide were mixed, and the mixture was stirred by ultrasonic vibration at a temperature of 60 ° C. for 99 minutes using an ultrasonic cleaning device, and a graphene oxide / formic acid dispersion was obtained.
- a graphene oxide / formic acid dispersion was obtained.
- 1 g of the modified spider silk fibroin (PRT799) obtained in the above-mentioned modified fibroin production step was added to the graphene oxide / formic acid dispersion, and heated with a 40 ° C aluminum block heater for 2 hours with stirring to dissolve. And a dope liquid (dispersion liquid) was prepared.
- the obtained dope liquid was a viscous black liquid and had a viscosity at 25 ° C. of 6,230 [mPa ⁇ sec].
- the added amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dope solution.
- the addition amount of graphene oxide was 0.1% by mass based on the modified fibroin.
- (2) Dry spinning Dry spinning was performed at room temperature using a spinning apparatus.
- the prepared graphene oxide-containing dope liquid (dispersion liquid) was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle.
- the tip of the needle was brought into contact with the end of the droplet to draw out the dope solution into a fibrous form.
- the drawn fiber was wound up by a winder to form a fiber continuously while drafting, thereby obtaining a modified spider silk fibroin fiber.
- the winding speed of the winder was set to the maximum speed of 20.82 m / min (34.7 cm / sec).
- FIG. 4 shows an SEM image of the modified spider silk fibroin fiber obtained in Example 7.
- Table 7 shows the analysis result (fiber diameter) of the SEM image of FIG.
- the ImageJ software is described in the following document. Schindelin, J .; Arganda-Carreras, I .; & France, E.C. et al.
- DiameterJ plug-in is described in the following document. Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: Validated open source nanofiber diameter measurement tool. Biomaterials 2015; 61: p. 327-38. doi: 10.1016 / j. biomaterials. 2015.05.015.
- a dope solution containing no graphene oxide was prepared.
- a dope solution was prepared in the same procedure as in Example 7 except that graphene oxide was not added.
- (2) Dry spinning Dry spinning was performed at room temperature using a tabletop spinning apparatus in the same manner as in Example 7.
- the prepared dope solution was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle.
- the tip of the needle When a droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet, and an attempt was made to draw out the dope solution in a fibrous form. In addition, the dope solution could not be drawn out into a fibrous form, and fibers could not be formed.
- Example 8 (Production and evaluation of nonwoven fabric) (Example 8) (1) Preparation of Dope Solution (Spinning Stock Solution) In the same manner as in Example 3, a dope solution of graphene oxide / formic acid / modified spider silk fibroin (PRT799) was prepared. (2) Dry spinning Dry spinning was performed using a commercially available cotton candy manufacturing apparatus (manufactured by Azuma Co., Ltd.). The apparatus was turned on, and the disk-shaped rotating section inside the pan of the cotton candy manufacturing apparatus was rotated. The disk-shaped rotating part is a rotating part around an inlet for feeding a rough or the like when a cotton candy is manufactured using a cotton candy confectionery apparatus as usual.
- the prepared graphene oxide-containing dope solution was filled in a syringe, and defoamed by centrifugation.
- a dope solution is discharged into the air from a 0.2-mm-diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet contacts the disk-shaped rotating part of the cotton candy manufacturing device.
- the fibers were continuously formed by utilizing the rotation of the disk-shaped rotating portion to obtain a modified spider silk fibroin nonwoven fabric.
- FIG. 5 shows an SEM image of the nonwoven fabric obtained in Example 8.
- Table 8 shows the analysis result of the SEM image of FIG. (Comparative Example 4)
- (1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 8, except that graphene oxide was not added. (2) Dry spinning The prepared dope solution was filled in a syringe and defoamed by centrifugation.
- the dry spinning was performed using a commercially available cotton candy manufacturing apparatus in the same manner as in Example 2.
- the apparatus was turned on and the rotating pan was rotated.
- the dope liquid is discharged into the air from a 0.2 mm diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet is brought into contact with a disk-shaped rotating portion of a cotton candy manufacturing apparatus. An attempt was made to form fibers, but no fibers could be formed.
- the dope solution to which no graphene oxide was added failed to form a nonwoven fabric by dry spinning, whereas the dope solution to which graphene oxide was added (Example 8).
- a nonwoven fabric could be formed by dry spinning.
- the average fiber diameter of the obtained modified spider silk fibroin nonwoven fabric was as small as 1 to 2 ⁇ m, and unexpectedly excellent results could be obtained.
- the nonwoven fabric had a sufficiently small fiber diameter, a heating step for removing (vaporizing) the solvent from the dope solution was unnecessary.
- Reference Example 1 Flammability test of modified fibroin A freeze-dried powder of modified fibroin (PRT799) was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was added. Used and mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
- the obtained spinning stock solution was heated to 90 ° C., filtered through a metal filter having a mesh size of 5 ⁇ m, and allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 90 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
- a knitted fabric (thickness: 180 denier, gauge number: 18) was manufactured by circular knitting using a circular knitting machine by using a twisted yarn obtained by twisting raw fibers. 20 g of the obtained knitted fabric was cut out and used as a test piece.
- the flammability test was based on the “Test method for synthetic resin with powdery or low melting point” described in “Fire Danger No. 50 (May 31, 1995)”. The test was performed under the conditions of a temperature of 22 ° C., a relative humidity of 45%, and an air pressure of 1021 hPa. Table 9 shows the measurement results (oxygen concentration (%), burning rate (%), reduced burning rate (%)).
- the knitted fabric made of the modified fibroin (PRT799) fiber had a limiting oxygen index (LOI) value of 27.2.
- LOI limiting oxygen index
- the LOI value is 26 or more, it is known to be flame retardant. It can be seen that the modified fibroin has excellent flame retardancy.
- Reference Example 2 Evaluation of heat generation by moisture absorption of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethyl sulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was used. And mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
- the obtained spinning dope was heated to 60 ° C., filtered with a metal filter having a mesh size of 5 ⁇ m, and then allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
- Table 10 shows the thickness and gauge number of the knitted fabric using PRT918 fiber or PRT799 fiber.
- the thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so that the same cover factor as the knitted fabric of the modified fibroin fiber was obtained. Specifically, it is as follows.
- test piece Two knitted fabrics cut to 10 cm ⁇ 10 cm were aligned, and four sides were sewn to obtain a test piece (sample). After leaving the test specimen in a low humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 40 ⁇ 5%) for 4 hours or more, it was moved to a high humidity environment (temperature 20 ⁇ 2 ° C, relative humidity 90 ⁇ 5%) The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.
- FIG. 6 is a graph showing an example of the result of the moisture absorption / heating test.
- the horizontal axis of the graph represents the time (minute) of leaving the sample in the high humidity environment as 0 when the time when the sample was transferred from the low humidity environment to the high humidity environment.
- the vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor.
- the point indicated by M corresponds to the maximum value of the sample temperature.
- Table 11 shows the calculation results of the maximum heat of moisture absorption of each knitted fabric.
- the modified fibroin (PRT918 and PRT799) has a higher maximum heat of moisture absorption and excellent heat generation by moisture absorption than the existing materials.
- Reference Example 3 Evaluation of heat retention of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was shaken using a shaker. The mixture was dissolved by mixing for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
- the obtained spinning dope was heated to 60 ° C., filtered through a metal filter having a mesh size of 5 ⁇ m, and then left standing in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
- a knitted fabric was produced by flat knitting using a flat knitting machine.
- Table 12 shows the count, the number of twists, the number of gauges, and the basis weight of the knitted fabric using the PRT966 fiber or the PRT799 fiber.
- the knitted fabric using other raw material fibers was adjusted to have almost the same cover factor as the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.
- the heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothing directly touched in a dry state).
- a dry contact method a method assuming that the skin and clothing directly touched in a dry state.
- One knitted fabric cut into a rectangle of 20 cm ⁇ 20 cm was used as a test piece (sample).
- the test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) radiated through the test piece was determined under the condition of a wind speed in the wind tunnel of 30 cm / sec. Without setting the test piece, the amount of heat (b) radiated under the same conditions as above was determined, and the heat retention (%) was calculated according to the following formula B.
- Insulation index Insulation rate (%) / Sample weight (g / m 2 )
- Table 11 shows the calculation results of the heat retention index. The higher the heat retention index, the more the material can be evaluated as having excellent heat retention.
- the modified fibroin (PRT966 and PRT799) has a high heat retention index and is excellent in heat retention as compared with existing materials.
- the modified fibroin when the modified fibroin is modified spider silk fibroin, the heat retention, the moisture absorption and heat generation and / or the flame retardancy can be more excellent.
- the modified spider silk fibroin By using the modified spider silk fibroin to produce the fiber of the present invention, a fiber having excellent heat retention, moisture absorption and heat generation and / or flame retardancy, and a small diameter can be obtained.
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Abstract
The present invention provides a carbon material dispersion containing a modified spider silk fibroin, a carbon material, and a polar solvent, wherein the carbon material is a planar graphene composed of one or more layers.
Description
本発明は、改変クモ糸フィブロイン繊維及びその製造方法に関する。本発明はまた、上記改変クモ糸フィブロイン繊維の製造に用いることのできる分散液にも関する。本発明はまた、上記改変フィブロイン繊維を含む製品にも関する。
The present invention relates to a modified spider silk fibroin fiber and a method for producing the same. The present invention also relates to a dispersion that can be used for producing the modified spider silk fibroin fiber. The present invention also relates to products comprising the modified fibroin fibers described above.
各種産業分野において、将来的に利用価値の高い新素材としてフィブロイン繊維に関心が寄せられている。フィブロイン繊維として、従来から再生絹フィブロイン繊維やクモ糸フィブロイン繊維が知られている。細径のフィブロイン繊維の製造方法も多数報告されている。
フ ィ In various industrial fields, fibroin fiber is attracting attention as a new material with high utility value in the future. Conventionally, regenerated silk fibroin fibers and spider silk fibroin fibers are known as fibroin fibers. Many methods for producing small-diameter fibroin fibers have been reported.
細径のフィブロイン繊維の製造方法としては、例えば、精錬後の蚕絹フィブロインを透析して濃縮した蚕絹フィブロイン水溶液に、2-モルホリノエタンスルホン酸-トリスヒドロキシメチルアミノメタン緩衝液と塩化カルシウム水溶液を添加して作製した紡糸原液(ドープ液)を、シリンジに接続した毛細管から気中に吐出させて、繊維を形成させた後、さらにアルコール水溶液中で繊維を延伸及び含浸処理することで、直径5.7μmの繊維を得る方法(非特許文献1)が報告されている。
As a method for producing a fine-diameter fibroin fiber, for example, a 2-morpholinoethanesulfonic acid-trishydroxymethylaminomethane buffer solution and a calcium chloride aqueous solution are added to a silkworm silk fibroin aqueous solution obtained by dialyzing and concentrating refined silkworm silk fibroin. The spun stock solution (dope solution) produced by the addition is discharged into the air from a capillary tube connected to a syringe to form fibers, and the fibers are further stretched and impregnated in an alcohol aqueous solution to obtain a fiber having a diameter of 5%. A method for obtaining a 0.7 μm fiber (Non-Patent Document 1) has been reported.
また、例えば、上述のドープ液を、マイクロ流体装置を用いて繊維形成させ、さらにアルコール水溶液中で繊維を延伸及び含浸処理することで、直径2μmの繊維を得る方法(非特許文献2)が報告されている。
In addition, for example, a method of obtaining fibers having a diameter of 2 μm by forming fibers from the above-mentioned dope solution using a microfluidic device, and then drawing and impregnating the fibers in an aqueous alcohol solution (Non-Patent Document 2) is reported. Have been.
また、例えば、クモ糸フィブロインのドープ液を、電圧をかけた口金からエレクトロスピニングにより吐出させることで、平均直径が1μm以下の繊維を得る方法(特許文献1)が報告されている。
方法 Also, for example, a method has been reported in which a fiber having an average diameter of 1 μm or less is obtained by discharging a spider silk fibroin dope solution from a spinneret to which a voltage is applied by electrospinning (Patent Document 1).
しかしながら、上記先行技術文献に開示される製造方法は、十分に細径のフィブロイン繊維を得るために特殊な器具又は装置を必要としたり、多くの工程を必要としたりする。
However, the production methods disclosed in the above-mentioned prior art documents require special tools or equipment or many steps to obtain fibroin fibers having a sufficiently small diameter.
したがって、本発明は、簡便な方法によって、細径のフィブロイン繊維を提供することを目的とする。
Therefore, an object of the present invention is to provide a small-diameter fibroin fiber by a simple method.
本発明者は、上記課題を解決すべく、鋭意検討を重ねた。その結果、改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される炭素材料と、極性溶媒とを含む分散液を用いることで、特殊な器具又は装置、及び多くの工程を必要とせず簡便に、細径を有するフィブロイン繊維が得られることを見出した。
The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, by using a dispersion containing a modified spider silk fibroin, a carbon material selected from one or more layers of planar graphene and carbon black nanoparticles, and a polar solvent, a special instrument or device, and It has been found that a fibroin fiber having a small diameter can be easily obtained without requiring many steps.
すなわち、本発明は、例えば、以下の各発明に関する。
[1]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
上記炭素材料が一層又は二層以上の平面状グラフェンである、炭素材料分散液。
[2]
上記極性溶媒が、ヘキサフルオロイソプロパノール、ヘキサフルオロアセトン、ジメチルスルホキシド、N,N-ジメチルホルムアミド、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフラン及び水からなる群から選ばれる少なくとも1種である、[1]に記載の分散液。
[3]
上記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、[1]又は[2]に記載の分散液。
[4]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
上記炭素材料がカーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、炭素材料分散液。
[5]
上記炭素材料の含有量が、上記改変クモ糸フィブロイン100質量部に対して25質量部以下である、[1]~[4]のいずれか一に記載の分散液。
[6]
ドープ液である、[1]~[5]のいずれか一に記載の分散液。
[7]
改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料と、を含み、平均繊維径が3μm以下である、改変クモ糸フィブロイン繊維。
[8]
上記炭素材料の含有量が、上記改変クモ糸フィブロイン100質量部に対して1質量部以下である、[7]に記載の改変クモ糸フィブロイン繊維。
[9]
上記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、[7]又は[8]に記載の改変クモ糸フィブロイン繊維。
[10]
[7]~[9]のいずれか一に記載の改変クモ糸フィブロイン繊維を含む、製品。
[11]
上記製品が、繊維、糸、布帛、編み物、組み物、不織布、紙、及び綿からなる群から選択される少なくとも1種である、[10]に記載の製品。
[12]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程を備え、上記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン繊維の製造方法。
[13]
上記原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させる、[12]に記載の改変クモ糸フィブロイン繊維の製造方法。
[14]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程を備え、上記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン繊維の製造方法。
[15]
上記原繊維を形成させる工程が、上記分散液から原繊維を引き出すことを含む、[12]~[14]のいずれか一に記載の改変クモ糸フィブロイン繊維の製造方法。
[16]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程と、
上記原繊維又は上記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、上記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン不織布の製造方法。
[17]
上記原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させる、[16]に記載の改変クモ糸フィブロイン不織布の製造方法。
[18]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程と、
上記原繊維又は上記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、上記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン不織布の製造方法。
[19]
改変クモ糸フィブロインと、上記改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含み、
上記炭素材料が、一層又は二層以上の平面状グラフェンである、炭素材料の分散方法。
[20]
改変クモ糸フィブロインと、上記改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含み、
上記炭素材料が、カーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、炭素材料の分散方法。
[21]
改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤であって、上記炭素材料が一層又は二層以上の平面状グラフェンである、分散補助剤。
[22]
改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤であって、
上記炭素材料がカーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、分散補助剤。
[23]
一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料を含む、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤。 That is, the present invention relates to, for example, the following inventions.
[1]
A modified spider silk fibroin, a carbon material, and a polar solvent,
A carbon material dispersion, wherein the carbon material is one or two or more layers of planar graphene.
[2]
When the polar solvent is hexafluoroisopropanol, hexafluoroacetone, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, The dispersion according to [1], which is at least one selected from the group consisting of N-methylmorpholine N-oxide, formic acid, ethylene glycol, tetrahydrofuran, and water.
[3]
The dispersion according to [1] or [2], wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide. liquid.
[4]
A modified spider silk fibroin, a carbon material, and a polar solvent,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. A carbon material dispersion which is at least one selected from the group consisting of:
[5]
The dispersion according to any one of [1] to [4], wherein the content of the carbon material is 25 parts by mass or less based on 100 parts by mass of the modified spider silk fibroin.
[6]
The dispersion according to any one of [1] to [5], which is a dope.
[7]
A modified spider yarn comprising a modified spider yarn fibroin and at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles, and having an average fiber diameter of 3 μm or less. Fibroin fiber.
[8]
The modified spider silk fibroin fiber according to [7], wherein the content of the carbon material is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin.
[9]
The modification according to [7] or [8], wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide. Spider silk fibroin fiber.
[10]
A product comprising the modified spider silk fibroin fiber according to any one of [7] to [9].
[11]
The product according to [10], wherein the product is at least one selected from the group consisting of fibers, yarns, fabrics, knits, braids, nonwoven fabrics, paper, and cotton.
[12]
Modified spider silk fibroin, a carbon material, a polar solvent, comprising the step of forming fibrils from a dispersion containing, the carbon material is a single or two or more layers of planar graphene, modified spider silk fibroin fiber Production method.
[13]
The method for producing a modified spider silk fibroin fiber according to [12], wherein in the step of forming the raw fiber, the raw fiber is formed by a dry spinning method.
[14]
Modified spider silk fibroin, a carbon material, comprising a step of forming fibrils by a dry spinning method from a dispersion containing a polar solvent, wherein the carbon material is carbon black nanoparticles, production of modified spider silk fibroin fibers Method.
[15]
The method for producing a modified spider silk fibroin fiber according to any one of [12] to [14], wherein the step of forming the fibril includes extracting the fibril from the dispersion.
[16]
Modified spider silk fibroin, a carbon material, and a polar solvent, a step of forming fibrils from a dispersion liquid,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
A method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is one or more layers of planar graphene.
[17]
The method for producing a modified spider silk fibroin nonwoven fabric according to [16], wherein in the step of forming the raw fibers, the raw fibers are formed by a dry spinning method.
[18]
Modified spider silk fibroin, a carbon material, a polar solvent, a step of forming fibrils by a dry spinning method from a dispersion containing,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
And a method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is carbon black nanoparticles.
[19]
Modified spider silk fibroin, comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent,
A method for dispersing a carbon material, wherein the carbon material is one or two or more layers of planar graphene.
[20]
Modified spider silk fibroin, comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. A method for dispersing a carbon material, which is at least one selected from the group.
[21]
A dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin, wherein the carbon material is one or more layers of planar graphene.
[22]
A modified auxiliary agent for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. At least one selected from dispersing aids.
[23]
A thinning agent for producing a modified spider silk fibroin fiber having a reduced diameter, comprising at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles.
[1]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
上記炭素材料が一層又は二層以上の平面状グラフェンである、炭素材料分散液。
[2]
上記極性溶媒が、ヘキサフルオロイソプロパノール、ヘキサフルオロアセトン、ジメチルスルホキシド、N,N-ジメチルホルムアミド、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフラン及び水からなる群から選ばれる少なくとも1種である、[1]に記載の分散液。
[3]
上記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、[1]又は[2]に記載の分散液。
[4]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
上記炭素材料がカーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、炭素材料分散液。
[5]
上記炭素材料の含有量が、上記改変クモ糸フィブロイン100質量部に対して25質量部以下である、[1]~[4]のいずれか一に記載の分散液。
[6]
ドープ液である、[1]~[5]のいずれか一に記載の分散液。
[7]
改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料と、を含み、平均繊維径が3μm以下である、改変クモ糸フィブロイン繊維。
[8]
上記炭素材料の含有量が、上記改変クモ糸フィブロイン100質量部に対して1質量部以下である、[7]に記載の改変クモ糸フィブロイン繊維。
[9]
上記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、[7]又は[8]に記載の改変クモ糸フィブロイン繊維。
[10]
[7]~[9]のいずれか一に記載の改変クモ糸フィブロイン繊維を含む、製品。
[11]
上記製品が、繊維、糸、布帛、編み物、組み物、不織布、紙、及び綿からなる群から選択される少なくとも1種である、[10]に記載の製品。
[12]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程を備え、上記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン繊維の製造方法。
[13]
上記原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させる、[12]に記載の改変クモ糸フィブロイン繊維の製造方法。
[14]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程を備え、上記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン繊維の製造方法。
[15]
上記原繊維を形成させる工程が、上記分散液から原繊維を引き出すことを含む、[12]~[14]のいずれか一に記載の改変クモ糸フィブロイン繊維の製造方法。
[16]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程と、
上記原繊維又は上記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、上記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン不織布の製造方法。
[17]
上記原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させる、[16]に記載の改変クモ糸フィブロイン不織布の製造方法。
[18]
改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程と、
上記原繊維又は上記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、上記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン不織布の製造方法。
[19]
改変クモ糸フィブロインと、上記改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含み、
上記炭素材料が、一層又は二層以上の平面状グラフェンである、炭素材料の分散方法。
[20]
改変クモ糸フィブロインと、上記改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含み、
上記炭素材料が、カーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、炭素材料の分散方法。
[21]
改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤であって、上記炭素材料が一層又は二層以上の平面状グラフェンである、分散補助剤。
[22]
改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤であって、
上記炭素材料がカーボンブラックナノパーティクルであり、
上記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、分散補助剤。
[23]
一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料を含む、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤。 That is, the present invention relates to, for example, the following inventions.
[1]
A modified spider silk fibroin, a carbon material, and a polar solvent,
A carbon material dispersion, wherein the carbon material is one or two or more layers of planar graphene.
[2]
When the polar solvent is hexafluoroisopropanol, hexafluoroacetone, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, The dispersion according to [1], which is at least one selected from the group consisting of N-methylmorpholine N-oxide, formic acid, ethylene glycol, tetrahydrofuran, and water.
[3]
The dispersion according to [1] or [2], wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide. liquid.
[4]
A modified spider silk fibroin, a carbon material, and a polar solvent,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. A carbon material dispersion which is at least one selected from the group consisting of:
[5]
The dispersion according to any one of [1] to [4], wherein the content of the carbon material is 25 parts by mass or less based on 100 parts by mass of the modified spider silk fibroin.
[6]
The dispersion according to any one of [1] to [5], which is a dope.
[7]
A modified spider yarn comprising a modified spider yarn fibroin and at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles, and having an average fiber diameter of 3 μm or less. Fibroin fiber.
[8]
The modified spider silk fibroin fiber according to [7], wherein the content of the carbon material is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin.
[9]
The modification according to [7] or [8], wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide. Spider silk fibroin fiber.
[10]
A product comprising the modified spider silk fibroin fiber according to any one of [7] to [9].
[11]
The product according to [10], wherein the product is at least one selected from the group consisting of fibers, yarns, fabrics, knits, braids, nonwoven fabrics, paper, and cotton.
[12]
Modified spider silk fibroin, a carbon material, a polar solvent, comprising the step of forming fibrils from a dispersion containing, the carbon material is a single or two or more layers of planar graphene, modified spider silk fibroin fiber Production method.
[13]
The method for producing a modified spider silk fibroin fiber according to [12], wherein in the step of forming the raw fiber, the raw fiber is formed by a dry spinning method.
[14]
Modified spider silk fibroin, a carbon material, comprising a step of forming fibrils by a dry spinning method from a dispersion containing a polar solvent, wherein the carbon material is carbon black nanoparticles, production of modified spider silk fibroin fibers Method.
[15]
The method for producing a modified spider silk fibroin fiber according to any one of [12] to [14], wherein the step of forming the fibril includes extracting the fibril from the dispersion.
[16]
Modified spider silk fibroin, a carbon material, and a polar solvent, a step of forming fibrils from a dispersion liquid,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
A method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is one or more layers of planar graphene.
[17]
The method for producing a modified spider silk fibroin nonwoven fabric according to [16], wherein in the step of forming the raw fibers, the raw fibers are formed by a dry spinning method.
[18]
Modified spider silk fibroin, a carbon material, a polar solvent, a step of forming fibrils by a dry spinning method from a dispersion containing,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
And a method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is carbon black nanoparticles.
[19]
Modified spider silk fibroin, comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent,
A method for dispersing a carbon material, wherein the carbon material is one or two or more layers of planar graphene.
[20]
Modified spider silk fibroin, comprising the step of mixing 25 parts by mass or less of a carbon material with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. A method for dispersing a carbon material, which is at least one selected from the group.
[21]
A dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin, wherein the carbon material is one or more layers of planar graphene.
[22]
A modified auxiliary agent for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. At least one selected from dispersing aids.
[23]
A thinning agent for producing a modified spider silk fibroin fiber having a reduced diameter, comprising at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles.
本発明の分散液を用いることによって、特殊な器具又は装置、及び多くの工程を必要とせず、細径のフィブロイン繊維を簡便に製造することが可能となる。
用 い る By using the dispersion of the present invention, it is possible to easily produce a small-diameter fibroin fiber without requiring a special instrument or device and many steps.
以下、本発明の実施形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。
Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
〔炭素材料分散液〕
本実施形態の炭素材料分散液は、改変クモ糸フィブロインと、炭素材料と、極性溶媒とを含み、炭素材料は、一層又は二層以上の平面状グラフェンである。本明細書において、「分散液」とは、目視において沈降物、凝集物、又は沈降物及び凝集物がなく、少なくとも7日間静置後においても目視において沈降物、凝集物、又は沈降物及び凝集物がない状態の溶液をいう。本発明の炭素材料分散液は、炭素材料の分散性に優れ、炭素材料の凝集及び沈殿を充分に抑制することができ、操作性に優れている。本実施形態の分散液は、そのまま、あるいは適宜濃縮又は希釈して、改変クモ糸フィブロイン繊維を製造するための、紡糸原液(ドープ液)として用いることができる。また、本実施形態の炭素材料分散液は分散性がよく、操作性に優れていることから、本発明の改変クモ糸フィブロイン繊維の原料以外の用途にも、好適に用いることが可能である。例えば、炭素材料を含む複合材料の製造にも好適に用いることができる。 (Carbon material dispersion)
The carbon material dispersion liquid of the present embodiment includes a modified spider silk fibroin, a carbon material, and a polar solvent, and the carbon material is one or two or more layers of planar graphene. As used herein, the term “dispersion” refers to a precipitate, an aggregate, or a precipitate and an aggregate that is visually free from a precipitate, an aggregate, or a precipitate and an aggregate even after standing still for at least 7 days. Refers to a solution without any objects. The carbon material dispersion of the present invention is excellent in dispersibility of the carbon material, can sufficiently suppress aggregation and precipitation of the carbon material, and is excellent in operability. The dispersion of this embodiment can be used as it is, or appropriately concentrated or diluted, as a spinning stock solution (dope solution) for producing a modified spider silk fibroin fiber. In addition, since the carbon material dispersion liquid of the present embodiment has good dispersibility and excellent operability, it can be suitably used for uses other than the raw material of the modified spider silk fibroin fiber of the present invention. For example, it can be suitably used for producing a composite material containing a carbon material.
本実施形態の炭素材料分散液は、改変クモ糸フィブロインと、炭素材料と、極性溶媒とを含み、炭素材料は、一層又は二層以上の平面状グラフェンである。本明細書において、「分散液」とは、目視において沈降物、凝集物、又は沈降物及び凝集物がなく、少なくとも7日間静置後においても目視において沈降物、凝集物、又は沈降物及び凝集物がない状態の溶液をいう。本発明の炭素材料分散液は、炭素材料の分散性に優れ、炭素材料の凝集及び沈殿を充分に抑制することができ、操作性に優れている。本実施形態の分散液は、そのまま、あるいは適宜濃縮又は希釈して、改変クモ糸フィブロイン繊維を製造するための、紡糸原液(ドープ液)として用いることができる。また、本実施形態の炭素材料分散液は分散性がよく、操作性に優れていることから、本発明の改変クモ糸フィブロイン繊維の原料以外の用途にも、好適に用いることが可能である。例えば、炭素材料を含む複合材料の製造にも好適に用いることができる。 (Carbon material dispersion)
The carbon material dispersion liquid of the present embodiment includes a modified spider silk fibroin, a carbon material, and a polar solvent, and the carbon material is one or two or more layers of planar graphene. As used herein, the term “dispersion” refers to a precipitate, an aggregate, or a precipitate and an aggregate that is visually free from a precipitate, an aggregate, or a precipitate and an aggregate even after standing still for at least 7 days. Refers to a solution without any objects. The carbon material dispersion of the present invention is excellent in dispersibility of the carbon material, can sufficiently suppress aggregation and precipitation of the carbon material, and is excellent in operability. The dispersion of this embodiment can be used as it is, or appropriately concentrated or diluted, as a spinning stock solution (dope solution) for producing a modified spider silk fibroin fiber. In addition, since the carbon material dispersion liquid of the present embodiment has good dispersibility and excellent operability, it can be suitably used for uses other than the raw material of the modified spider silk fibroin fiber of the present invention. For example, it can be suitably used for producing a composite material containing a carbon material.
〔平面状グラフェン〕
本実施形態において、「平面状グラフェン」とは、含まれる炭素原子同士で2次元シート状の分子構造を形成するグラフェン及びグラフェン類縁体を意味する。したがって、炭素原子同士で筒状構造を形成するカーボンナノチューブ類及び球状構造を形成するフラーレン類は、「平面状グラフェン」から除かれる。「平面状グラフェン」は、その分子構造の一部に2次元シート状構造を有していればよく、その分子構造の全部が平面状でなくてもよい。平面状グラフェンは、2次元シート状の分子構造が一層であってもよいし、二層以上であってもよい。 [Flat graphene]
In the present embodiment, “planar graphene” refers to graphene and graphene analogs that form a two-dimensional sheet-like molecular structure with contained carbon atoms. Therefore, carbon nanotubes that form a cylindrical structure with carbon atoms and fullerenes that form a spherical structure are excluded from “planar graphene”. The “planar graphene” may have a two-dimensional sheet-like structure in a part of its molecular structure, and the entire molecular structure may not be planar. The planar graphene may have a two-dimensional sheet-like molecular structure of one layer or two or more layers.
本実施形態において、「平面状グラフェン」とは、含まれる炭素原子同士で2次元シート状の分子構造を形成するグラフェン及びグラフェン類縁体を意味する。したがって、炭素原子同士で筒状構造を形成するカーボンナノチューブ類及び球状構造を形成するフラーレン類は、「平面状グラフェン」から除かれる。「平面状グラフェン」は、その分子構造の一部に2次元シート状構造を有していればよく、その分子構造の全部が平面状でなくてもよい。平面状グラフェンは、2次元シート状の分子構造が一層であってもよいし、二層以上であってもよい。 [Flat graphene]
In the present embodiment, “planar graphene” refers to graphene and graphene analogs that form a two-dimensional sheet-like molecular structure with contained carbon atoms. Therefore, carbon nanotubes that form a cylindrical structure with carbon atoms and fullerenes that form a spherical structure are excluded from “planar graphene”. The “planar graphene” may have a two-dimensional sheet-like structure in a part of its molecular structure, and the entire molecular structure may not be planar. The planar graphene may have a two-dimensional sheet-like molecular structure of one layer or two or more layers.
平面状グラフェンとしては、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン、還元型機能化酸化グラフェン等が挙げられる。これらの中でも酸化グラフェンが好ましい。
Examples of planar graphene include graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, reduced functionalized graphene oxide, and the like. Among these, graphene oxide is preferable.
酸化グラフェンの酸化度には、特に制限はなく、公知の酸化グラフェンを用いることができる。酸化グラフェンの酸化度は、例えば、4~55%であってよく、4~50%であってよく、4~45%であってよく、4~40%であってよく、4~35%であってよく、4~30%であってよく、4~25%であってよく、4~20%であってよく、4~15%であってよく、4~10%であってよい。
酸化 The degree of oxidation of graphene oxide is not particularly limited, and known graphene oxide can be used. The degree of oxidation of graphene oxide may be, for example, from 4 to 55%, from 4 to 50%, from 4 to 45%, from 4 to 40%, from 4 to 35%. May be 4 to 30%, may be 4 to 25%, may be 4 to 20%, may be 4 to 15%, may be 4 to 10%.
機能化酸化グラフェンは、酸化グラフェンの酸素含有官能基の一部が、他の官能基等によって化学修飾された構造を有する。公知の機能化酸化グラフェンを用いることができる。機能化酸化グラフェンとしては、例えば、アルキルアミン機能化酸化グラフェン、アンモニア機能化酸化グラフェン、アミン機能化酸化グラフェン、チオール機能化酸化グラフェン、アルキン機能化酸化グラフェン、グルコース機能化酸化グラフェン等が挙げられる。
The functionalized graphene oxide has a structure in which a part of the oxygen-containing functional group of the graphene oxide is chemically modified with another functional group or the like. Known functionalized graphene oxide can be used. Examples of the functionalized graphene oxide include alkylamine-functionalized graphene oxide, ammonia-functionalized graphene oxide, amine-functionalized graphene oxide, thiol-functionalized graphene oxide, alkyne-functionalized graphene oxide, and glucose-functionalized graphene oxide.
還元型酸化グラフェンの炭素/酸素比には、特に制限はなく、公知の還元型酸化グラフェンを用いることができる。還元型酸化グラフェンの炭素/酸素比は、例えば、90/10であってよく、75/35であってよい。
炭素 The carbon / oxygen ratio of the reduced graphene oxide is not particularly limited, and a known reduced graphene oxide can be used. The carbon / oxygen ratio of the reduced graphene oxide may be, for example, 90/10 or 75/35.
機能化還元型酸化グラフェンは、還元型酸化グラフェンの酸素含有官能基の一部が、他の官能基等によって化学修飾された構造を有する。公知の機能化還元型酸化グラフェンを用いることができる。機能化還元型酸化グラフェンとしては、例えば、アミン機能化還元型酸化グラフェン、オクタデシルアミン機能化還元型酸化グラフェン、ピペラジン機能化還元型酸化グラフェン、テトラエチレンペンタアミン機能化還元型酸化グラフェン、グルコース修飾還元型酸化グラフェン等が挙げられる。
The functionalized reduced graphene oxide has a structure in which a part of the oxygen-containing functional group of reduced graphene oxide is chemically modified with another functional group or the like. Known functionalized reduced graphene oxide can be used. Examples of the functionalized reduced graphene oxide include amine-functionalized reduced graphene oxide, octadecylamine-functionalized reduced graphene oxide, piperazine-functionalized reduced graphene oxide, tetraethylenepentamine-functionalized reduced graphene oxide, and glucose-modified reduction. Graphene oxide and the like.
本実施形態には、粉末、シート、液体等の形態の平面状グラフェンを用いることができる。これらの中でも粉末又は液体の形態の平面状グラフェンを用いることが好ましい。また、グラフェンを用いる際は、液体の形態の平面状グラフェンを用いることが好ましい。液体の形態の平面状グラフェンとして、溶媒又は分散媒に平面状グラフェンを溶解又は分散させたグラフェンインクを用いることができる。
に は In the present embodiment, planar graphene in the form of powder, sheet, liquid or the like can be used. Among these, it is preferable to use planar graphene in a powder or liquid form. When using graphene, it is preferable to use planar graphene in a liquid form. As the planar graphene in a liquid form, a graphene ink in which planar graphene is dissolved or dispersed in a solvent or a dispersion medium can be used.
〔極性溶媒〕
本実施形態の炭素材料分散液に使用する極性溶媒は、改変クモ糸フィブロインを分散又は溶解し得るものであれば、いずれも使用することができる。極性溶媒としては、例えば、ヘキサフルオロイソプロパノール(HFIP)、ヘキサフルオロアセトン(HFA)、ジメチルスルホキシド(DMSO)、N,N-ジメチルホルムアミド(DMF)、N,N-ジメチルアセトアミド(DMA)、1,3-ジメチル-2-イミダゾリドン(DMI)、N-メチル-2-ピロリドン(NMP)、アセトニトリル、N-メチルモルホリンN-オキシド(NMO)、ギ酸、エチレングリコール、及びテトラヒドロフラン(THF)等の有機溶媒、及び水等が挙げられる。 (Polar solvent)
As the polar solvent used in the carbon material dispersion liquid of the present embodiment, any polar solvent that can disperse or dissolve the modified spider silk fibroin can be used. Examples of the polar solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), and 1,3. Organic solvents such as -dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), formic acid, ethylene glycol and tetrahydrofuran (THF); Water and the like.
本実施形態の炭素材料分散液に使用する極性溶媒は、改変クモ糸フィブロインを分散又は溶解し得るものであれば、いずれも使用することができる。極性溶媒としては、例えば、ヘキサフルオロイソプロパノール(HFIP)、ヘキサフルオロアセトン(HFA)、ジメチルスルホキシド(DMSO)、N,N-ジメチルホルムアミド(DMF)、N,N-ジメチルアセトアミド(DMA)、1,3-ジメチル-2-イミダゾリドン(DMI)、N-メチル-2-ピロリドン(NMP)、アセトニトリル、N-メチルモルホリンN-オキシド(NMO)、ギ酸、エチレングリコール、及びテトラヒドロフラン(THF)等の有機溶媒、及び水等が挙げられる。 (Polar solvent)
As the polar solvent used in the carbon material dispersion liquid of the present embodiment, any polar solvent that can disperse or dissolve the modified spider silk fibroin can be used. Examples of the polar solvent include hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), and 1,3. Organic solvents such as -dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), acetonitrile, N-methylmorpholine N-oxide (NMO), formic acid, ethylene glycol and tetrahydrofuran (THF); Water and the like.
改変クモ糸フィブロインの溶解性をより良好にする観点からは、ヘキサフルオロイソプロパノール、ジメチルスルホキシド及びギ酸がより好ましく、ジメチルスルホキシド及びギ酸がさらに好ましい。これらの有機溶媒は、水を含んでいてもよい。これらの溶媒は、1種単独で使用してもよく、2種以上を混合して使用してもよい。
か ら From the viewpoint of improving the solubility of the modified spider silk fibroin, hexafluoroisopropanol, dimethyl sulfoxide and formic acid are more preferred, and dimethyl sulfoxide and formic acid are even more preferred. These organic solvents may include water. These solvents may be used alone or as a mixture of two or more.
〔カーボンブラックナノパーティクル〕
本実施形態の炭素材料分散液には、炭素材料として、平面状グラフェンの代わりに、又は平面状グラフェンとともに、カーボンブラックナノパーティクルを用いることができる。カーボンブラックナノパーティクルは、炭素で形成される微粒子である。カーボンブラックナノパーティクルとして、本実施形態には、平均粒子径が3μm未満の微粒子を用いることが好ましく、2μm未満の微粒子を用いることがより好ましく、1μm未満の微粒子を用いることがさらに好ましい。カーボンブラックナノパーティクルの製造方法には、特に制限はなく、例えば、ファーネス法、チャンネル法、アセチレン法、油煙法、及び松煙法等の公知の製造方法により製造されたカーボンブラックナノパーティクルを用いることができる。カーボンブラックナノパーティクルを炭素材料として用いる場合は、炭素材料分散液に使用する極性溶媒として、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランを挙げることができる。これらの溶媒を1種単独で使用してもよく、2種以上を混合して使用してもよい。 (Carbon black nanoparticles)
In the carbon material dispersion liquid of the present embodiment, as the carbon material, carbon black nanoparticles can be used instead of or together with the planar graphene. Carbon black nanoparticles are fine particles formed of carbon. In the present embodiment, as the carbon black nanoparticles, it is preferable to use fine particles having an average particle diameter of less than 3 μm, more preferably to use fine particles of less than 2 μm, and still more preferably to use fine particles of less than 1 μm. The method for producing carbon black nanoparticles is not particularly limited, and for example, using carbon black nanoparticles produced by a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method. Can be. When carbon black nanoparticles are used as the carbon material, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, and the like are used as polar solvents for the carbon material dispersion. Examples thereof include N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. These solvents may be used alone or as a mixture of two or more.
本実施形態の炭素材料分散液には、炭素材料として、平面状グラフェンの代わりに、又は平面状グラフェンとともに、カーボンブラックナノパーティクルを用いることができる。カーボンブラックナノパーティクルは、炭素で形成される微粒子である。カーボンブラックナノパーティクルとして、本実施形態には、平均粒子径が3μm未満の微粒子を用いることが好ましく、2μm未満の微粒子を用いることがより好ましく、1μm未満の微粒子を用いることがさらに好ましい。カーボンブラックナノパーティクルの製造方法には、特に制限はなく、例えば、ファーネス法、チャンネル法、アセチレン法、油煙法、及び松煙法等の公知の製造方法により製造されたカーボンブラックナノパーティクルを用いることができる。カーボンブラックナノパーティクルを炭素材料として用いる場合は、炭素材料分散液に使用する極性溶媒として、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランを挙げることができる。これらの溶媒を1種単独で使用してもよく、2種以上を混合して使用してもよい。 (Carbon black nanoparticles)
In the carbon material dispersion liquid of the present embodiment, as the carbon material, carbon black nanoparticles can be used instead of or together with the planar graphene. Carbon black nanoparticles are fine particles formed of carbon. In the present embodiment, as the carbon black nanoparticles, it is preferable to use fine particles having an average particle diameter of less than 3 μm, more preferably to use fine particles of less than 2 μm, and still more preferably to use fine particles of less than 1 μm. The method for producing carbon black nanoparticles is not particularly limited, and for example, using carbon black nanoparticles produced by a known production method such as a furnace method, a channel method, an acetylene method, an oil smoke method, and a pine smoke method. Can be. When carbon black nanoparticles are used as the carbon material, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, and the like are used as polar solvents for the carbon material dispersion. Examples thereof include N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. These solvents may be used alone or as a mixture of two or more.
本実施形態の炭素材料分散液は、改変クモ糸フィブロインと炭素材料と極性溶媒を用いて調製する。分散液は、例えば、炭素材料と、極性溶媒とを混合分散機(例えば、ボールミル、ビーズミル、サンドミル、ロールミル、ホモジナイザー、超音波ホモジナイザー、高圧ホモジナイザー、超音波装置、アトライター、デゾルバーテ、ペイントシェーカー等)を用いて混合した後、改変クモ糸フィブロインを添加することで調製することができる。分散液は、改変クモ糸フィブロインと極性溶媒とを混合分散機を用いて混合した溶液に、炭素材料を添加して調製してもよく、炭素材料と極性溶媒とを混合分散機を用いて混合した溶液と、異なる極性溶媒に改変クモ糸フィブロインを溶解させた溶液とを混合して調製してもよい。本実施形態の炭素材料分散液は、液体形状であってもよく、ペースト又はゲルのような半固形状であってもよいが、液体形状であることが好ましい。
炭素 The carbon material dispersion of the present embodiment is prepared by using a modified spider silk fibroin, a carbon material, and a polar solvent. The dispersion is, for example, a mixing and dispersing machine (for example, a ball mill, a bead mill, a sand mill, a roll mill, a homogenizer, an ultrasonic homogenizer, a high-pressure homogenizer, an ultrasonic device, an attritor, a dissolver, a paint shaker, etc.) in which a carbon material and a polar solvent are mixed. And then adding the modified spider silk fibroin. The dispersion may be prepared by adding a carbon material to a solution obtained by mixing a modified spider silk fibroin and a polar solvent using a mixing disperser, and mixing the carbon material and the polar solvent using a mixing disperser. And a solution in which the modified spider silk fibroin is dissolved in a different polar solvent. The carbon material dispersion liquid of the present embodiment may be in a liquid form or a semi-solid form such as a paste or a gel, but is preferably in a liquid form.
本実施形態の炭素材料分散液における炭素材料の含有量の上限値は、改変クモ糸フィブロイン100質量部に対して、25質量部以下であってよく、20質量部以下であってよく、15質量部以下であってよく、10質量部以下であってよく、5質量部以下であってよく、4質量部以下であってよく、3質量部以下であってよく、1質量部以下であってよい。
The upper limit of the content of the carbon material in the carbon material dispersion of the present embodiment may be 25 parts by mass or less, 20 parts by mass or less, and 100 parts by mass of the modified spider silk fibroin. Not more than 10 parts by mass, not more than 5 parts by mass, not more than 4 parts by mass, not more than 3 parts by mass, not more than 1 part by mass, Good.
本実施形態の炭素材料分散液における炭素材料の含有量の下限値は、改変クモ糸フィブロイン100質量部に対して、0.01質量部以上であってよく、0.03質量部以上であってよく、0.05質量部以上であってよく、0.06質量部以上であってよく、0.07質量部以上であってよく、0.08質量部以上であってよく、0.09質量部以上であってよい。
The lower limit of the content of the carbon material in the carbon material dispersion liquid of the present embodiment may be not less than 0.01 part by mass, and not less than 0.03 part by mass, based on 100 parts by mass of the modified spider silk fibroin. Well, it may be 0.05 parts by mass or more, may be 0.06 parts by mass or more, may be 0.07 parts by mass or more, may be 0.08 parts by mass or more, and may be 0.09 parts by mass. Parts or more.
本実施形態の炭素材料分散液における炭素材料の含有量は、例えば、改変クモ糸フィブロイン100質量部に対して、0.01質量部以上25質量部以下であってよく、0.05質量部以上25質量部以下であってよく、0.01質量部以上20質量部以下であってよく、0.05質量部以上20質量部以下であってよく、0.01質量部以上15質量部以下であってよく、0.05質量部以上15質量部以下であってよく、0.01質量部以上10質量部以下であってよく、0.05質量部以上10質量部以下であってよく、0.01質量部以上5質量部以下であってよく、0.05質量部以上5質量部以下であってよく、0.01質量部以上4質量部以下であってよく、0.05質量部以上4質量部以下であってよく、0.01質量部以上3質量部以下であってよく、0.05質量部以上3質量部以下であってよく、0.01質量部以上2質量部以下であってよく、0.05質量部以上2質量部以下であってよく、0.01質量部以上1質量部以下であってよく、0.05質量部以上1質量部以下であってよい。
分散液における炭素材料の含有量が上述の範囲であれば、炭素材料の分散性が良く、また操作性にも優れている。 The content of the carbon material in the carbon material dispersion liquid of the present embodiment, for example, with respect to 100 parts by mass of the modified spider silk fibroin, may be 0.01 to 25 parts by mass, 0.05 parts by mass or more 25 parts by mass or less, may be 0.01 parts by mass or more and 20 parts by mass or less, may be 0.05 parts by mass or more and 20 parts by mass or less, 0.01 parts by mass or more and 15 parts by mass or less May be 0.05 to 15 parts by mass, 0.01 to 10 parts by mass, 0.05 to 10 parts by mass, 0.01 to 5 parts by mass, 0.05 to 5 parts by mass, 0.01 to 4 parts by mass, 0.05 or more parts bymass 4 parts by mass or less, and 0.01 parts by mass or less. 3 parts by mass or less, 0.05 parts by mass or more and 3 parts by mass or less, 0.01 parts by mass or more and 2 parts by mass or less, 0.05 parts by mass or more and 2 parts by mass or less The amount may be 0.01 to 1 part by mass, and may be 0.05 to 1 part by mass.
When the content of the carbon material in the dispersion is in the above range, the dispersibility of the carbon material is good and the operability is also excellent.
分散液における炭素材料の含有量が上述の範囲であれば、炭素材料の分散性が良く、また操作性にも優れている。 The content of the carbon material in the carbon material dispersion liquid of the present embodiment, for example, with respect to 100 parts by mass of the modified spider silk fibroin, may be 0.01 to 25 parts by mass, 0.05 parts by mass or more 25 parts by mass or less, may be 0.01 parts by mass or more and 20 parts by mass or less, may be 0.05 parts by mass or more and 20 parts by mass or less, 0.01 parts by mass or more and 15 parts by mass or less May be 0.05 to 15 parts by mass, 0.01 to 10 parts by mass, 0.05 to 10 parts by mass, 0.01 to 5 parts by mass, 0.05 to 5 parts by mass, 0.01 to 4 parts by mass, 0.05 or more parts by
When the content of the carbon material in the dispersion is in the above range, the dispersibility of the carbon material is good and the operability is also excellent.
本実施形態の炭素材料分散液をそのままドープ液(紡糸原液)として用いる場合、ドープ液における炭素材料の含有量の上限値は、改変クモ糸フィブロイン100質量部に対して、1質量部以下であることが好ましく、0.95質量部以下であってよく、0.9質量部以下であってよく、0.8質量部以下であってよく、0.7質量部以下であってよく、0.6質量部以下であってよく、0.5質量部以下であってよく、0.4質量部以下であってよく、0.3質量部以下であってよく、0.2質量部以下であってよい。また、ドープ液における炭素材料の含有量の下限値は、改変クモ糸フィブロイン100質量部に対して、0.01質量部以上であってよく、0.03質量部以上であってよく、0.05質量部以上であってよく、0.06質量部以上であってよく、0.07質量部以上であってよく、0.08質量部以上であってよく、0.09質量部以上であってよい。また、ドープ液における炭素材料の含有量は、改変クモ糸フィブロイン100質量部に対して、例えば、0.01質量部以上1質量部以下であることが好ましく、0.01質量部以上1質量部未満であることがより好ましく、0.03質量部以上1質量部未満であることがさらに好ましく、0.05質量部以上1質量部未満であることが特に好ましく、0.05質量部以上0.95質量部以下であってよく、0.05質量部以上0.9質量部以下であってよく、0.05質量部以上0.8質量部以下であってよく、0.05質量部以上0.7質量部以下であってよく、0.05質量部以上0.6質量部以下であってよく、0.05質量部以上0.5質量部以下であってよく、0.05質量部以上0.4質量部以下であってよく、又は0.05質量部以上0.3質量部以下であってよく、0.05質量部以上0.2質量部以下であってよい。
When the carbon material dispersion of the present embodiment is used as a dope solution (spinning solution) as it is, the upper limit of the content of the carbon material in the dope solution is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin. Is preferably 0.95 parts by mass or less, 0.9 parts by mass or less, 0.8 parts by mass or less, 0.7 parts by mass or less, and 0.1 part by mass or less. 6 parts by mass or less, 0.5 parts by mass or less, 0.4 parts by mass or less, 0.3 parts by mass or less, and 0.2 parts by mass or less. May be. Further, the lower limit of the content of the carbon material in the dope solution may be 0.01 parts by mass or more, 0.03 parts by mass or more with respect to 100 parts by mass of the modified spider silk fibroin. It may be at least 05 parts by mass, at least 0.06 parts by mass, at least 0.07 parts by mass, at least 0.08 parts by mass, at least 0.09 parts by mass. May be. Further, the content of the carbon material in the dope solution is preferably, for example, 0.01 part by mass or more and 1 part by mass or less, and preferably 0.01 part by mass or more and 1 part by mass with respect to 100 parts by mass of the modified spider silk fibroin. The amount is more preferably less than 0.03 parts by mass and less than 1 part by mass, particularly preferably from 0.05 parts by mass to less than 1 part by mass, and more preferably from 0.05 parts by mass to 0.1 part by mass. 95 parts by mass or less, may be 0.05 parts by mass or more and 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, and may be 0.05 parts by mass or more and 0 parts by mass or less. 0.7 parts by mass or less, 0.05 parts by mass or more and 0.6 parts by mass or less, 0.05 parts by mass or more and 0.5 parts by mass or less, and 0.05 parts by mass or more 0.4 parts by mass or less, or 0.05 May be more than the amount unit or 0.3 part by weight, it may be more than 0.2 parts by mass or more 0.05 part by mass.
本実施形態の炭素材料分散液における改変クモ糸フィブロインの濃度は、分散液全量を100質量%としたとき、1~40質量%であってよく、1~35質量%であってよく、1~30質量%であってよく、1~25質量%であってよく、1~20質量%であってよく、1~15質量%であってよく、1~10質量%であってよく、1~5質量%であってよく、1~3質量%であってよく、1~2質量%であってよい。改変クモ糸フィブロインの濃度が1質量%以上であると、炭素材料の分散性を充分に向上させることができる。改変クモ糸フィブロインの濃度が40質量%以下であると、粘度の著しい増大による炭素材料の分散性の低下を避けることができる。
The concentration of the modified spider silk fibroin in the carbon material dispersion of the present embodiment may be 1 to 40% by mass, 1 to 35% by mass, and 1 to 40% by mass when the total amount of the dispersion is 100% by mass. 30% by mass, 1 to 25% by mass, 1 to 20% by mass, 1 to 15% by mass, 1 to 10% by mass, 1 to 1% by mass It may be 5% by mass, 1-3% by mass, 1-2% by mass. When the concentration of the modified spider silk fibroin is 1% by mass or more, the dispersibility of the carbon material can be sufficiently improved. When the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to avoid a decrease in the dispersibility of the carbon material due to a significant increase in viscosity.
本実施形態の炭素材料分散液をそのままドープ液(紡糸原液)として用いる場合には、改変クモ糸フィブロインの濃度は、ドープ液全量を100質量%としたとき、10~40質量%であることが好ましく、10~35質量%であることがより好ましく、12~35質量%であることがより好ましく、15~35質量%であることがより好ましく、15~30質量%であることがより好ましく、20~35質量%であることがさらに好ましく、20~30質量%であることが特に好ましい。改変クモ糸フィブロインの濃度が10質量%以上であると、紡糸口金からドープ液をより一層安定的に吐出させることができ、生産性が向上する。改変クモ糸フィブロインの濃度が40質量%以下であると、紡糸口金からドープ液を吐出する際に紡糸口金の孔が閉塞するのを避けることができ、生産性が向上する。
When the carbon material dispersion of this embodiment is used as a dope solution (spinning stock solution) as it is, the concentration of the modified spider silk fibroin may be 10 to 40% by mass when the total amount of the dope solution is 100% by mass. More preferably, it is 10 to 35% by mass, more preferably 12 to 35% by mass, more preferably 15 to 35% by mass, more preferably 15 to 30% by mass, The content is more preferably 20 to 35% by mass, and particularly preferably 20 to 30% by mass. When the concentration of the modified spider silk fibroin is 10% by mass or more, the dope solution can be more stably discharged from the spinneret, and the productivity is improved. When the concentration of the modified spider silk fibroin is 40% by mass or less, it is possible to prevent the holes of the spinneret from being closed when the dope is discharged from the spinneret, thereby improving the productivity.
分散液は、溶解を促進するために、ある程度の時間撹拌又は振とうしてもよい。その際、分散液は必要により、使用する改変クモ糸フィブロイン及び極性溶媒に応じて溶解可能な温度に加熱してもよい。分散液は、例えば、30℃以上、40℃以上、50℃以上、60℃以上、70℃以上、80℃以上、又は、90℃以上に加熱してもよい。加熱温度の上限は、例えば、極性溶媒の沸点以下である。
(4) The dispersion may be stirred or shaken for a certain period of time to promote dissolution. At that time, if necessary, the dispersion may be heated to a temperature at which it can be dissolved depending on the modified spider silk fibroin and the polar solvent used. The dispersion may be heated to, for example, 30C or higher, 40C or higher, 50C or higher, 60C or higher, 70C or higher, 80C or higher, or 90C or higher. The upper limit of the heating temperature is, for example, equal to or lower than the boiling point of the polar solvent.
本実施形態の分散液の粘度は、適宜設定してよい。分散液をドープ液(紡糸原液)として用いる場合には、乾式紡糸が可能な粘度であればよく、特に限定されないが、生産性の観点から、25℃において、3000~50000mPa・secであってよく、5000~50000mPa・secであってよく、5000~40000mPa・secであってよく、5000~30000mPa・secであってよく、5000~20000mPa・secであってよく、5000~15000mPa・secであってよく、5000~12000mPa・sec等であってよい。紡糸原液の粘度は、例えば京都電子工業社製の商品名「EMS粘度計」を使用して測定することができる。
粘度 The viscosity of the dispersion of this embodiment may be set as appropriate. When the dispersion is used as a dope solution (spinning stock solution), the viscosity is not particularly limited as long as it allows dry spinning. From the viewpoint of productivity, it may be 3000 to 50,000 mPa · sec at 25 ° C. 5000 to 50000 mPa · sec, 5000 to 40000 mPa · sec, 5000 to 30000 mPa · sec, 5000 to 20000 mPa · sec, and 5000 to 15000 mPa · sec. 5000 to 12000 mPa · sec. The viscosity of the spinning solution can be measured using, for example, a trade name “EMS viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
〔無機塩〕
本実施形態の分散液は、無機塩を更に含有するものであってよい。無機塩は、極性溶媒に対する改変クモ糸フィブロインの溶解促進剤として用いることができる。無機塩は、以下に示すルイス酸とルイス塩基とからなる無機塩であってよい。ルイス塩基としては、例えば、ハロゲン化物イオン等が挙げられる。ルイス酸としては、例えば、アルカリ金属イオン、アルカリ土類金属イオン等の金属イオン等が挙げられる。無機塩としては、例えば、アルカリ金属ハロゲン化物、及びアルカリ土類金属ハロゲン化物等が挙げられる。アルカリ金属ハロゲン化物の具体例としては、塩化リチウム、臭化リチウム等が挙げられる。アルカリ土類金属ハロゲン化物の具体例としては、塩化マグネシウム、塩化カルシウム等が挙げられる。これらの無機塩の中でも、塩化リチウム及び塩化カルシウムが特に好ましい。分散液が無機塩を含有することにより、分散液の調製がより容易になり得る。 (Inorganic salt)
The dispersion of the present embodiment may further contain an inorganic salt. The inorganic salt can be used as a promoter for dissolving the modified spider silk fibroin in a polar solvent. The inorganic salt may be an inorganic salt composed of the following Lewis acid and Lewis base. Examples of the Lewis base include a halide ion and the like. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions. Examples of the inorganic salt include an alkali metal halide and an alkaline earth metal halide. Specific examples of the alkali metal halide include lithium chloride and lithium bromide. Specific examples of the alkaline earth metal halide include magnesium chloride and calcium chloride. Among these inorganic salts, lithium chloride and calcium chloride are particularly preferred. When the dispersion contains an inorganic salt, the preparation of the dispersion may be easier.
本実施形態の分散液は、無機塩を更に含有するものであってよい。無機塩は、極性溶媒に対する改変クモ糸フィブロインの溶解促進剤として用いることができる。無機塩は、以下に示すルイス酸とルイス塩基とからなる無機塩であってよい。ルイス塩基としては、例えば、ハロゲン化物イオン等が挙げられる。ルイス酸としては、例えば、アルカリ金属イオン、アルカリ土類金属イオン等の金属イオン等が挙げられる。無機塩としては、例えば、アルカリ金属ハロゲン化物、及びアルカリ土類金属ハロゲン化物等が挙げられる。アルカリ金属ハロゲン化物の具体例としては、塩化リチウム、臭化リチウム等が挙げられる。アルカリ土類金属ハロゲン化物の具体例としては、塩化マグネシウム、塩化カルシウム等が挙げられる。これらの無機塩の中でも、塩化リチウム及び塩化カルシウムが特に好ましい。分散液が無機塩を含有することにより、分散液の調製がより容易になり得る。 (Inorganic salt)
The dispersion of the present embodiment may further contain an inorganic salt. The inorganic salt can be used as a promoter for dissolving the modified spider silk fibroin in a polar solvent. The inorganic salt may be an inorganic salt composed of the following Lewis acid and Lewis base. Examples of the Lewis base include a halide ion and the like. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions. Examples of the inorganic salt include an alkali metal halide and an alkaline earth metal halide. Specific examples of the alkali metal halide include lithium chloride and lithium bromide. Specific examples of the alkaline earth metal halide include magnesium chloride and calcium chloride. Among these inorganic salts, lithium chloride and calcium chloride are particularly preferred. When the dispersion contains an inorganic salt, the preparation of the dispersion may be easier.
無機塩の含有量は、分散液全量に対して、0.1質量%以上、1質量%以上、2質量%以上、3質量%以上、4質量%以上、7質量%以上、10質量%以上、又は15質量%以上であってよく、20質量%以下、16質量%以下、12質量%以下、又は9質量%以下であってよい。
The content of the inorganic salt is 0.1% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more based on the total amount of the dispersion. Or 15% by mass or more, and may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
〔各種添加剤〕
分散液は、必要に応じて、各種の添加剤を更に含有していてよい。添加剤としては、例えば、可塑剤、レベリング剤、架橋剤、結晶核剤、酸化防止剤、紫外線吸収剤、着色剤、フィラー、合成樹脂が挙げられる。添加剤の含有量は、紡糸原液中の改変フィブロイン全量100質量部に対して、50質量部以下であってよい。 (Various additives)
The dispersion may further contain various additives as necessary. Examples of the additives include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin. The content of the additive may be 50 parts by mass or less based on 100 parts by mass of the total modified fibroin in the spinning solution.
分散液は、必要に応じて、各種の添加剤を更に含有していてよい。添加剤としては、例えば、可塑剤、レベリング剤、架橋剤、結晶核剤、酸化防止剤、紫外線吸収剤、着色剤、フィラー、合成樹脂が挙げられる。添加剤の含有量は、紡糸原液中の改変フィブロイン全量100質量部に対して、50質量部以下であってよい。 (Various additives)
The dispersion may further contain various additives as necessary. Examples of the additives include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a coloring agent, a filler, and a synthetic resin. The content of the additive may be 50 parts by mass or less based on 100 parts by mass of the total modified fibroin in the spinning solution.
〔改変クモ糸フィブロイン〕
原料となる改変クモ糸フィブロインは、特に限定されるものではなく、遺伝子組換え技術により微生物等で製造したフィブロインであってもよく、合成により製造されたフィブロインであってもよい。ただし、改変クモ糸フィブロインから、天然由来のクモ糸フィブロインは除かれる。 (Modified spider silk fibroin)
The modified spider silk fibroin used as a raw material is not particularly limited, and may be fibroin produced by a microorganism or the like by genetic recombination technology, or fibroin produced by synthesis. However, naturally derived spider silk fibroin is excluded from the modified spider silk fibroin.
原料となる改変クモ糸フィブロインは、特に限定されるものではなく、遺伝子組換え技術により微生物等で製造したフィブロインであってもよく、合成により製造されたフィブロインであってもよい。ただし、改変クモ糸フィブロインから、天然由来のクモ糸フィブロインは除かれる。 (Modified spider silk fibroin)
The modified spider silk fibroin used as a raw material is not particularly limited, and may be fibroin produced by a microorganism or the like by genetic recombination technology, or fibroin produced by synthesis. However, naturally derived spider silk fibroin is excluded from the modified spider silk fibroin.
本明細書において「改変クモ糸フィブロイン」とは、天然由来のクモ糸フィブロインとは異なるアミノ酸配列を有するクモ糸フィブロインを意味し、本明細書において「天然由来のクモ糸フィブロイン」とは、天然由来のクモ糸フィブロインと同一のアミノ酸配列を有するクモ糸フィブロインを意味する。
As used herein, the term "modified spider silk fibroin" refers to a spider silk fibroin having an amino acid sequence different from that of a naturally occurring spider silk fibroin. Spider silk fibroin having the same amino acid sequence as the spider silk fibroin.
天然由来のクモ糸フィブロインとしては、例えば、大吐糸管しおり糸タンパク質、横糸タンパク質、及び小瓶状腺タンパク質等のクモ類が産生するクモ糸フィブロインが挙げられる。大吐糸管しおり糸は、結晶領域と非晶領域(無定形領域とも言う。)からなる繰り返し領域を持つため、高い応力と伸縮性を併せ持つ。クモ糸の横糸は、結晶領域を持たず、非晶領域からなる繰り返し領域を持つという特徴を有する。横糸は、大吐糸管しおり糸に比べると応力は劣るが、高い伸縮性を持つ。
Examples of spider silk fibroin of natural origin include spider silk fibroin produced by spiders such as large tubule wicking silk protein, weft silk protein, and small ampullate gland protein. Since the large spinneret thread has a repeating region including a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of spider silk has a feature that it does not have a crystalline region but has a repeating region composed of an amorphous region. The weft has a lower stress than the large spinneret and has a high elasticity.
大吐糸管しおり糸タンパク質は、クモの大瓶状腺で産生され、強靭性に優れるという特徴を有する。大吐糸管しおり糸タンパク質としては、例えば、アメリカジョロウグモ(Nephila clavipes)に由来する大瓶状腺スピドロインMaSp1及びMaSp2、並びに二ワオニグモ(Araneus diadematus)に由来するADF3及びADF4が挙げられる。ADF3は、ニワオニグモの2つの主要なしおり糸タンパク質の一つである。ADF3に由来するクモ糸タンパク質は、比較的合成し易く、また、強伸度及びタフネスの点で優れた特性を有する。
The large spinal cord marker thread protein is produced by the large ampullate gland of spiders, and has the characteristic of excellent toughness. Examples of the large spinal cord marker thread protein include the large ampullate spidroins MaSp1 and MaSp2 derived from the American spider (Nephila laclavipes), and ADF3 and ADF4 derived from Araneus diadematus. ADF3 is one of the two major bookmarker thread proteins of the Japanese spider. The spider silk protein derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and elongation and toughness.
横糸タンパク質は、クモの鞭毛状腺(flagelliform gland)で産生される。横糸タンパク質としては、例えばアメリカジョロウグモ(Nephila clavipes)に由来する鞭毛状絹タンパク質(flagelliform silk protein)が挙げられる。
Weft protein is produced in the flagellar gland of spiders. As the weft protein, for example, a flagellated silk protein (flagelliform @ silk @ protein) derived from the American spider (Nephila @ clavipes) can be mentioned.
クモ類が産生するクモ糸フィブロインの更なる例として、例えば、オニグモ、ニワオニグモ、アカオニグモ、アオオニグモ及びマメオニグモ等のオニグモ属(Araneus属)に属するクモ、ヤマシロオニグモ、イエオニグモ、ドヨウオニグモ及びサツマノミダマシ等のヒメオニグモ属(Neoscona属)に属するクモ、コオニグモモドキ等のコオニグモモドキ属(Pronus属)に属するクモ、トリノフンダマシ及びオオトリノフンダマシ等のトリノフンダマシ属(Cyrtarachne属)に属するクモ、トゲグモ及びチブサトゲグモ等のトゲグモ属(Gasteracantha属)に属するクモ、マメイタイセキグモ及びムツトゲイセキグモ等のイセキグモ属(Ordgarius属)に属するクモ、コガネグモ、コガタコガネグモ及びナガコガネグモ等のコガネグモ属(Argiope属)に属するクモ、キジロオヒキグモ等のオヒキグモ属(Arachnura属)に属するクモ、ハツリグモ等のハツリグモ属(Acusilas属)に属するクモ、スズミグモ、キヌアミグモ及びハラビロスズミグモ等のスズミグモ属(Cytophora属)に属するクモ、ゲホウグモ等のゲホウグモ属(Poltys属)に属するクモ、ゴミグモ、ヨツデゴミグモ、マルゴミグモ及びカラスゴミグモ等のゴミグモ属(Cyclosa属)に属するクモ、及びヤマトカナエグモ等のカナエグモ属(Chorizopes属)に属するクモが産生するスパイダーシルクタンパク質、並びにアシナガグモ、ヤサガタアシナガグモ、ハラビロアシダカグモ及びウロコアシナガグモ等のアシナガグモ属(Tetragnatha属)に属するクモ、オオシロカネグモ、チュウガタシロカネグモ及びコシロカネグモ等のシロカネグモ属(Leucauge属)に属するクモ、ジョロウグモ及びオオジョロウグモ等のジョロウグモ属(Nephila属)に属するクモ、キンヨウグモ等のアズミグモ属(Menosira属)に属するクモ、ヒメアシナガグモ等のヒメアシナガグモ属(Dyschiriognatha属)に属するクモ、クロゴケグモ、セアカゴケグモ、ハイイロゴケグモ及びジュウサンボシゴケグモ等のゴケグモ属(Latrodectus属)に属するクモ、及びユープロステノプス属(Euprosthenops属)に属するクモ等のアシナガグモ科(Tetragnathidae科)に属するクモが産生するスパイダーシルクタンパク質が挙げられる。
Further examples of spider silk fibroins produced by spiders include, for example, spiders belonging to the genus Araneus (Araneus sp.), Such as Orion spiders, Japanese spiders, A. spiders and A. spiders, and the spiders of the Japanese spiders Spiders belonging to the genus (Neoscona), spiders belonging to the genus Argiope (Pronus) such as Argiope serrata, spiders, spiders belonging to the genus Cyrarchachne such as the Torinofundamashi and Otorinofundamashi, and Spiders belonging to the genus Spider spiders (Genus Gasteracantha) such as Tibato spiders, spiders belonging to the genus Ordgarius, such as spiders belonging to the genus Orthodox spiders, such as the spider spider Spider spiders and the spiders spiders spiders, etc. Spiders belonging to the genus Argiope (Argiope) such as Argiope bruennichi and Argiope bruennichi, spiders belonging to the genus Arachnura (genus Arachnura) such as the arachnid spider, spiders such as the spiders belonging to the genus Acusilas and the spiders of the spider spiders belonging to the genus Acusilas such as the spider Spiders belonging to the genus Cytophora, spiders belonging to the spider spider belonging to the genus Cytophora (genus Poltys), spiders belonging to the genus Spiders belonging to the genus Poltys, spiders belonging to the genus Spiders, spiders belonging to the spider, spiders belonging to the genus Cyclos sp. Spider silk proteins produced by spiders belonging to the genus Chorizopes, and spider silk spiders, Asagata spiders, Harabiroashida spiders, and urocore spiders The spiders belonging to the genus Tetragnatha (genus Tetragnatha), the spiders belonging to the genus Tetragnatha, the spiders belonging to the genus Leucauge, the spiders belonging to the genus Leucauge, and the genus E belonging to the spiders sp. The spiders belonging to the genus L, such as spiders belonging to the spiders belonging to the genus Menosira, such as the spider spider, the spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Menosira, the spiders belonging to the spiders belonging to the spiders spiders belonging to the spiders, the black widow spider, the red widow spider, and the black spiders And spiders belonging to the genus Euprostenops (Tetragnathidae), such as spiders belonging to the genus Euprostenops Spider silk proteins produced by spiders.
クモ類が産生するスパイダーシルクタンパク質のより具体的な例としては、例えば、fibroin-3(adf-3)[Araneus diadematus由来](GenBankアクセッション番号AAC47010(アミノ酸配列)、U47855(塩基配列))、fibroin-4(adf-4)[Araneus diadematus由来](GenBankアクセッション番号AAC47011(アミノ酸配列)、U47856(塩基配列))、dragline silk protein spidroin 1[Nephila clavipes由来](GenBankアクセッション番号AAC04504(アミノ酸配列)、U37520(塩基配列))、major ampullate spidroin 1[Latrodectus hesperus由来](GenBankアクセッション番号ABR68856(アミノ酸配列)、EF595246(塩基配列))、dragline silk protein spidroin 2[Nephila clavata由来](GenBankアクセッション番号AAL32472(アミノ酸配列)、AF441245(塩基配列))、major ampullate spidroin 1[Euprosthenops australis由来](GenBankアクセッション番号CAJ00428(アミノ酸配列)、AJ973155(塩基配列))、及びmajor ampullate spidroin 2[Euprosthenops australis](GenBankアクセッション番号CAM32249.1(アミノ酸配列)、AM490169(塩基配列))、minor ampullate silk protein 1[Nephila clavipes](GenBankアクセッション番号AAC14589.1(アミノ酸配列))、minor ampullate silk protein 2[Nephila clavipes](GenBankアクセッション番号AAC14591.1(アミノ酸配列))、minor ampullate spidroin-like protein[Nephilengys cruentata](GenBankアクセッション番号ABR37278.1(アミノ酸配列)等が挙げられる。
More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from amino acid sequence of Nephila claviBAC04A4 and derived from amino acid sequence of ph ), U37520 (base sequence)), major \ ampullate \ spidro n 1 [Derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [Derived from Nephila clavata (GenBank accession number ABA45, amino acid sequence of A23) )), Major \ sample \ spidroin \ 1 [from Euprosthenops \ australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ97155 (base sequence)), and major \ sample \ spidroin \ 2 [Euprosus] ] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor \ silk \ protein \ 1 [Nephila \ clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor \ ampilatte [in] Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampoulate spidroin-like protein [Nephilengys Cruenata] (GenBank Accession No. ABR3727.1.
本実施形態にかかる改変クモ糸フィブロインは、例えば、式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むタンパク質であってもよい。本実施形態にかかる改変クモ糸フィブロインは、ドメイン配列のN末端側及びC末端側のいずれか一方又は両方に更にアミノ酸配列(N末端配列及びC末端配列)が付加されていてもよい。N末端配列及びC末端配列は、これに限定されるものではないが、典型的には、フィブロインに特徴的なアミノ酸モチーフの反復を有さない領域であり、100残基程度のアミノ酸からなる。
なお、本実施形態において、改変フィブロインとして、保温性、吸湿発熱性及び/又は難燃性にも優れることから、好ましくは改変クモ糸フィブロインが用いられる。 The modified spider silk fibroin according to the present embodiment is represented by, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It may be a protein containing a domain sequence. In the modified spider silk fibroin according to the present embodiment, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
In the present embodiment, a modified spider silk fibroin is preferably used as the modified fibroin because it is excellent in heat retention, moisture absorption and heat generation and / or flame retardancy.
なお、本実施形態において、改変フィブロインとして、保温性、吸湿発熱性及び/又は難燃性にも優れることから、好ましくは改変クモ糸フィブロインが用いられる。 The modified spider silk fibroin according to the present embodiment is represented by, for example, Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif. It may be a protein containing a domain sequence. In the modified spider silk fibroin according to the present embodiment, an amino acid sequence (N-terminal sequence and C-terminal sequence) may be further added to one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are, but not limited to, typically a region having no repeat of the amino acid motif characteristic of fibroin, and are composed of about 100 amino acids.
In the present embodiment, a modified spider silk fibroin is preferably used as the modified fibroin because it is excellent in heat retention, moisture absorption and heat generation and / or flame retardancy.
本明細書において「ドメイン配列」とは、フィブロイン特有の結晶領域(典型的には、アミノ酸配列の(A)nモチーフに相当する。)と非晶領域(典型的には、アミノ酸配列のREPに相当する。)を生じるアミノ酸配列であり、式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるアミノ酸配列を意味する。ここで、(A)nモチーフは、アラニン残基を主とするアミノ酸配列を示し、アミノ酸残基数は2~27である。(A)nモチーフのアミノ酸残基数は、2~20、4~27、4~20、8~20、10~20、4~16、8~16、又は10~16であってもよい。また、(A)nモチーフ中の全アミノ酸残基数に対するアラニン残基数の割合は40%以上であればよく、60%以上、70%以上、80%以上、83%以上、85%以上、86%以上、90%以上、95%以上、又は100%(アラニン残基のみで構成されることを意味する。)であってもよい。ドメイン配列中に複数存在する(A)nモチーフは、少なくとも7つがアラニン残基のみで構成されてもよい。REPは2~200アミノ酸残基から構成されるアミノ酸配列を示す。REPは、10~200アミノ酸残基から構成されるアミノ酸配列であってもよく、10~40、10~60、10~80、10~100、10~120、10~140、10~160、又は10~180アミノ酸残基から構成されるアミノ酸配列であってもよい。mは2~300の整数を示し、8~300、10~300、20~300、40~300、60~300、80~300、10~200、20~200、20~180、20~160、20~140又は20~120の整数であってもよい。複数存在する(A)nモチーフは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。複数存在するREPは、互いに同一のアミノ酸配列でもよく、異なるアミノ酸配列でもよい。
As used herein, the term “domain sequence” refers to a crystalline region unique to fibroin (typically, corresponding to the (A) n motif of the amino acid sequence) and an amorphous region (typically, the REP of the amino acid sequence). The amino acid represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Means an array. Here, the (A) n motif indicates an amino acid sequence mainly containing an alanine residue, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in the n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. (A) The ratio of the number of alanine residues to the total number of amino acid residues in the n motif may be 40% or more, and is 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. The REP may be an amino acid sequence composed of 10 to 200 amino acid residues, 10 to 40, 10 to 60, 10 to 80, 10 to 100, 10 to 120, 10 to 140, 10 to 160, or The amino acid sequence may be composed of 10 to 180 amino acid residues. m represents an integer of 2 to 300, and 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 of 20 to 140 or 20 to 120. The plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.
改変クモ糸フィブロインは、例えば、天然由来のクモ糸フィブロインのアミノ酸配列に依拠してそのアミノ酸配列を改変したもの(例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列を改変することによりアミノ酸配列を改変したもの)であってもよく、また天然由来のクモ糸フィブロインに依らず人工的に設計及び合成したもの(例えば、設計したアミノ酸配列をコードする核酸を化学合成することにより所望のアミノ酸配列を有するもの)であってもよい。
The modified spider silk fibroin is, for example, a modified spider silk fibroin whose amino acid sequence is modified based on the amino acid sequence of the spider silk fibroin (for example, by changing the gene sequence of a cloned naturally occurring spider silk fibroin, The amino acid sequence may be modified or artificially designed and synthesized without using spider silk fibroin of natural origin (for example, a desired amino acid sequence can be synthesized by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). May be included).
改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列に対し、例えば、1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変を行うことで得ることができる。アミノ酸残基の置換、欠失、挿入及び/又は付加は、部分特異的突然変異誘発法等の当業者に周知の方法により行うことができる。具体的には、Nucleic Acid Res.10,6487(1982)、Methods in Enzymology,100,448(1983)等の文献に記載されている方法に準じて行うことができる。
The modified spider silk fibroin is, for example, an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues with respect to the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by making modifications. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982) and Methods {in} Enzymology, 100, 448 (1983).
改変クモ糸フィブロインの具体的な例として、クモの大瓶状腺で産生される大吐糸管しおり糸タンパク質に由来する改変クモ糸フィブロイン(第1の改変クモ糸フィブロイン)、グリシン残基の含有量が低減された改変クモ糸フィブロイン(第2の改変クモ糸フィブロイン)、(A)nモチーフの含有量が低減された改変クモ糸フィブロイン(第3の改変クモ糸フィブロイン)、グリシン残基の含有量、及び(A)nモチーフの含有量が低減された改変クモ糸フィブロイン(第4の改変クモ糸フィブロイン)、局所的に疎水性指標の大きい領域を含むドメイン配列を有する改変クモ糸フィブロイン(第5の改変クモ糸フィブロイン)、及びグルタミン残基の含有量が低減されたドメイン配列を有する改変クモ糸フィブロイン(第6の改変クモ糸フィブロイン)が挙げられる。
Specific examples of the modified spider silk fibroin include a modified spider silk fibroin (first modified spider silk fibroin) derived from a large spinal canal thread protein produced in the large ampullate gland of a spider, and the content of glycine residues Spider silk fibroin (second modified spider silk fibroin), (A) modified spider silk fibroin with reduced n- motif content (third modified spider silk fibroin), glycine residue content And (A) a modified spider silk fibroin having a reduced content of the n motif (fourth modified spider silk fibroin), and a modified spider silk fibroin (fifth modified spider silk fibrin having a domain sequence including a region having a locally large hydrophobicity index). Modified spider silk fibroin) and a modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified silk fibroin). Yarn fibroin), and the like.
クモの大瓶状腺で産生される大吐糸管しおり糸タンパク質に由来する改変クモ糸フィブロイン(第1の改変クモ糸フィブロイン)としては、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質が挙げられる。第1の改変クモ糸フィブロインは、式1中、nは3~20の整数が好ましく、4~20の整数がより好ましく、8~20の整数が更に好ましく、10~20の整数が更により好ましく、4~16の整数が更によりまた好ましく、8~16の整数が特に好ましく、10~16の整数が最も好ましい。第1の改変クモ糸フィブロインは、式1中、REPを構成するアミノ酸残基の数は、10~200残基であることが好ましく、10~150残基であることがより好ましく、20~100残基であることが更に好ましく、20~75残基であることが更により好ましい。第1の改変クモ糸フィブロインは、式1:[(A)nモチーフ-REP]mで表されるアミノ酸配列中に含まれるグリシン残基、セリン残基及びアラニン残基の合計残基数がアミノ酸残基数全体に対して、40%以上であることが好ましく、60%以上であることがより好ましく、70%以上であることが更に好ましい。
The modified spider silk fibroin (first modified spider silk fibroin) derived from the large spinal cord marker silk protein produced in the large ampullate of the spider is represented by Formula 1: [(A) n motif-REP] m And proteins containing the domain sequence to be performed. In the first modified spider silk fibroin, in Formula 1, n is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and still more preferably an integer of 10 to 20. , An integer of 4 to 16 is still more preferred, an integer of 8 to 16 is particularly preferred, and an integer of 10 to 16 is most preferred. In the first modified spider silk fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. It is more preferably a residue, and even more preferably 20 to 75 residues. The first modified spider silk fibroin has the total number of glycine, serine and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) n motif-REP] m It is preferably at least 40%, more preferably at least 60%, even more preferably at least 70%, based on the total number of residues.
第1の改変クモ糸フィブロインは、式1:[(A)nモチーフ-REP]mで表されるアミノ酸配列の単位を含み、かつC末端配列が配列番号1~3のいずれかに示されるアミノ酸配列、又は配列番号1~3のいずれかに示されるアミノ酸配列と90%以上の相同性を有するアミノ酸配列である、タンパク質であってもよい。
The first modified spider silk fibroin comprises a unit of the amino acid sequence represented by Formula 1: [(A) n motif-REP] m and has a C-terminal sequence represented by any one of SEQ ID NOs: 1 to 3. The protein may be a sequence or an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOS: 1 to 3.
配列番号1に示されるアミノ酸配列は、ADF3(GI:1263287、NCBI)のアミノ酸配列のC末端の50残基のアミノ酸からなるアミノ酸配列と同一であり、配列番号2に示されるアミノ酸配列は、配列番号1に示されるアミノ酸配列のC末端から20残基取り除いたアミノ酸配列と同一であり、配列番号3に示されるアミノ酸配列は、配列番号1に示されるアミノ酸配列のC末端から29残基取り除いたアミノ酸配列と同一である。
The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is The amino acid sequence shown in SEQ ID NO: 3 is identical to the amino acid sequence shown in SEQ ID NO: 1 by removing 20 residues, and the amino acid sequence shown in SEQ ID NO: 3 is obtained by removing 29 residues from the C-terminal of the amino acid sequence shown in SEQ ID NO: 1. It is identical to the amino acid sequence.
第1の改変クモ糸フィブロインのより具体的な例として、(1-i)配列番号4で示されるアミノ酸配列、又は(1-ii)配列番号4で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。配列同一性は、95%以上であることが好ましい。
As more specific examples of the first modified spider silk fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 having 90% or more sequence identity Modified spider silk fibroin containing an amino acid sequence having sex properties. The sequence identity is preferably 95% or more.
配列番号4で示されるアミノ酸配列は、N末端に開始コドン、His10タグ及びHRV3Cプロテアーゼ(Human rhinovirus 3Cプロテアーゼ)認識サイトからなるアミノ酸配列(配列番号5)を付加したADF3のアミノ酸配列において、第1~13番目の反復領域をおよそ2倍になるように増やすとともに、翻訳が第1154番目アミノ酸残基で終止するように変異させたものである。配列番号4で示されるアミノ酸配列のC末端のアミノ酸配列は、配列番号3で示されるアミノ酸配列と同一である。
The amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence of ADF3 in which an amino acid sequence (SEQ ID NO: 5) comprising an initiation codon, a His10 tag, and an HRV3C protease (Human \ rhinovirus @ 3C protease) recognition site at the N-terminus is added. The 13th repeat region was increased so as to be approximately doubled, and the mutation was mutated so that translation was terminated at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence represented by SEQ ID NO: 4 is the same as the amino acid sequence represented by SEQ ID NO: 3.
(1-i)の改変クモ糸フィブロインは、配列番号4で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (1-i) may have an amino acid sequence represented by SEQ ID NO: 4.
グリシン残基の含有量が低減された改変クモ糸フィブロイン(第2の改変クモ糸フィブロイン)は、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、グリシン残基の含有量が低減されたアミノ酸配列を有する。第2の改変クモ糸フィブロインは、天然由来のクモ糸フィブロインと比較して、少なくともREP中の1又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。
The modified spider silk fibroin having a reduced content of glycine residues (the second modified spider silk fibroin) has a domain sequence in which the content of glycine residues is reduced as compared with a naturally occurring spider silk fibroin. Having an amino acid sequence of The second modified spider silk fibroin has an amino acid sequence corresponding to at least one or more glycine residues in the REP has been replaced with another amino acid residue, as compared to a naturally occurring spider silk fibroin. It can be said.
第2の改変クモ糸フィブロインは、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、REP中のGGX及びGPGXX(但し、Gはグリシン残基、Pはプロリン残基、Xはグリシン以外のアミノ酸残基を示す。)から選ばれる少なくとも一つのモチーフ配列において、少なくとも1又は複数の当該モチーフ配列中の1つのグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものであってもよい。
The second modified spider silk fibroin has GGX and GPGXX in the REP (where G is a glycine residue, P is a proline residue, and X is other than glycine, as compared with the naturally occurring spider silk fibroin. In at least one motif sequence selected from), an amino acid sequence corresponding to the substitution of at least one or a plurality of glycine residues in the motif sequence with another amino acid residue is determined. You may have.
第2の改変クモ糸フィブロインは、上述のグリシン残基が別のアミノ酸残基に置換されたモチーフ配列の割合が、全モチーフ配列に対して、10%以上であってもよい。
は In the second modified spider silk fibroin, the ratio of the motif sequence in which the glycine residue is replaced with another amino acid residue may be 10% or more of the entire motif sequence.
第2の改変クモ糸フィブロインは、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含み、上記ドメイン配列から、最もC末端側に位置する(A)nモチーフから上記ドメイン配列のC末端までの配列を除いた配列中の全REPに含まれるXGX(但し、Xはグリシン以外のアミノ酸残基を示す。)からなるアミノ酸配列の総アミノ酸残基数をzとし、上記ドメイン配列から、最もC末端側に位置する(A)nモチーフから上記ドメイン配列のC末端までの配列を除いた配列中の総アミノ酸残基数をwとしたときに、z/wが30%以上、40%以上、50%以上又は50.9%以上であるアミノ酸配列を有するものであってもよい。(A)nモチーフ中の全アミノ酸残基数に対するアラニン残基数は83%以上であってよいが、86%以上であることが好ましく、90%以上であることがより好ましく、95%以上であることが更に好ましく、100%であること(アラニン残基のみで構成されることを意味する)が更により好ましい。
The second modified spider silk fibroin comprises a domain sequence represented by Formula 1: [(A) n motif-REP] m , and from the above domain sequence, the (A) n motif located at the most C-terminal side to the above The total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminus of the domain sequence is represented by z, When the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located closest to the C-terminus to the C-terminus of the domain sequence is w, z / w is 30% As described above, it may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
第2の改変クモ糸フィブロインは、GGXモチーフの1つのグリシン残基を別のアミノ酸残基に置換することにより、XGXからなるアミノ酸配列の含有割合を高めたものであることが好ましい。第2の改変フィブロインは、ドメイン配列中のGGXからなるアミノ酸配列の含有割合が30%以下であることが好ましく、20%以下であることがより好ましく、10%以下であることが更に好ましく、6%以下であることが更により好ましく、4%以下であることが更によりまた好ましく、2%以下であることが特に好ましい。ドメイン配列中のGGXからなるアミノ酸配列の含有割合は、下記XGXからなるアミノ酸配列の含有割合(z/w)の算出方法と同様の方法で算出することができる。
2 The second modified spider silk fibroin is preferably one in which the content of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, and 6% or less. %, Still more preferably 4% or less, further preferably 2% or less. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGGX below.
z/wの算出方法を更に詳細に説明する。まず、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むクモ糸フィブロイン(改変クモ糸フィブロイン)において、ドメイン配列から、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列を除いた配列に含まれる全てのREPから、XGXからなるアミノ酸配列を抽出する。XGXを構成するアミノ酸残基の総数がzである。例えば、XGXからなるアミノ酸配列が50個抽出された場合(重複はなし)、zは50×3=150である。また、例えば、XGXGXからなるアミノ酸配列の場合のように2つのXGXに含まれるX(中央のX)が存在する場合は、重複分を控除して計算する(XGXGXの場合は5アミノ酸残基である)。wは、ドメイン配列から、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列を除いた配列に含まれる総アミノ酸残基数である。例えば、図1に示したドメイン配列の場合、wは4+50+4+100+4+10+4+20+4+30=230である(最もC末端側に位置する(A)nモチーフは除いている。)。次に、zをwで除すことによって、z/w(%)を算出することができる。
The method of calculating z / w will be described in more detail. First, in a spider silk fibroin containing a domain sequence represented by Formula 1: [(A) n motif-REP] m (modified spider silk fibroin), the (A) n motif located closest to the C-terminal side from the domain sequence The amino acid sequence consisting of XGX is extracted from all the REPs contained in the sequence except for the sequence up to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (there is no duplication), z is 50 × 3 = 150. Further, for example, when there is an X (center X) included in two XGXs as in the case of an amino acid sequence consisting of XGXGX, calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues are used. is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located closest to the C-terminus to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (the (A) n motif located at the most C-terminal side is excluded). Next, z / w (%) can be calculated by dividing z by w.
第2の改変クモ糸フィブロインにおいて、z/wは、50.9%以上であることが好ましく、56.1%以上であることがより好ましく、58.7%以上であることが更に好ましく、70%以上であることが更により好ましく、80%以上であることが更によりまた好ましい。z/wの上限に特に制限はないが、例えば、95%以下であってもよい。
In the second modified spider silk fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70/70. %, Still more preferably 80% or more. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.
第2の改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列から、グリシン残基をコードする塩基配列の少なくとも一部を置換して別のアミノ酸残基をコードするように改変することにより得ることができる。このとき、改変するグリシン残基として、GGXモチーフ及びGPGXXモチーフにおける1つのグリシン残基を選択してもよいし、またz/wが50.9%以上になるように置換してもよい。また、例えば、天然由来のクモ糸フィブロインのアミノ酸配列から上記態様を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のクモ糸フィブロインのアミノ酸配列からREP中のグリシン残基を別のアミノ酸残基に置換したことに相当する改変に加え、更に1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変を行ってもよい。
The second modified spider silk fibroin is obtained, for example, by replacing at least a part of a nucleotide sequence encoding a glycine residue from a cloned naturally occurring spider silk fibroin gene sequence to encode another amino acid residue. It can be obtained by modification. At this time, as the glycine residue to be modified, a GGX motif and one glycine residue in the GPGXX motif may be selected, or the glycine residue may be substituted so that z / w becomes 50.9% or more. In addition, for example, it can be obtained by designing an amino acid sequence satisfying the above aspect from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the replacement of the glycine residue in the REP with another amino acid residue from the amino acid sequence of a naturally occurring spider silk fibroin, one or more amino acid residues are further substituted, The amino acid sequence corresponding to the deletion, insertion and / or addition may be modified.
上記の別のアミノ酸残基としては、グリシン残基以外のアミノ酸残基であれば特に制限はないが、バリン(V)残基、ロイシン(L)残基、イソロイシン(I)残基、メチオニン(M)残基、プロリン(P)残基、フェニルアラニン(F)残基及びトリプトファン(W)残基等の疎水性アミノ酸残基、グルタミン(Q)残基、アスパラギン(N)残基、セリン(S)残基、リシン(K)残基及びグルタミン酸(E)残基等の親水性アミノ酸残基が好ましく、バリン(V)残基、ロイシン(L)残基、イソロイシン(I)残基及びグルタミン(Q)残基がより好ましく、グルタミン(Q)残基が更に好ましい。
The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but includes a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and a methionine ( M) residue, hydrophobic amino acid residue such as proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S ) Residues, lysine (K) residues and hydrophilic amino acid residues such as glutamic acid (E) residues, and valine (V) residues, leucine (L) residues, isoleucine (I) residues and glutamine ( Q) residues are more preferred, and glutamine (Q) residues are even more preferred.
第2の改変クモ糸フィブロインのより具体的な例として、(2-i)配列番号6、配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列、又は(2-ii)配列番号6、配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As more specific examples of the second modified spider silk fibroin, (2-i) the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, or (2-ii) SEQ ID NO: 6 , A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
(2-i)の改変クモ糸フィブロインについて説明する。配列番号6で示されるアミノ酸配列は、天然由来のクモ糸フィブロインに相当する配列番号10で示されるアミノ酸配列のREP中の全てのGGXをGQXに置換したものである。配列番号7で示されるアミノ酸配列は、配列番号6で示されるアミノ酸配列から、N末端側からC末端側に向かって2つおきに(A)nモチーフを欠失させ、更にC末端配列の手前に[(A)nモチーフ-REP]を1つ挿入したものである。配列番号8で示されるアミノ酸配列は、配列番号7で示されるアミノ酸配列の各(A)nモチーフのC末端側に2つのアラニン残基を挿入し、更に一部のグルタミン(Q)残基をセリン(S)残基に置換し、配列番号7の分子量とほぼ同じとなるようにN末端側の一部のアミノ酸を欠失させたものである。配列番号9で示されるアミノ酸配列は、配列番号11で示されるアミノ酸配列中に存在する20個のドメイン配列の領域(但し、当該領域のC末端側の数アミノ酸残基が置換されている。)を4回繰り返した配列のC末端にHisタグが付加されたものである。
The modified spider silk fibroin of (2-i) will be described. The amino acid sequence represented by SEQ ID NO: 6 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin with GQX. The amino acid sequence represented by SEQ ID NO: 7 is obtained by deleting every two (A) n motifs from the N-terminal side to the C-terminal side from the amino acid sequence represented by SEQ ID NO: 6, and further before the C-terminal sequence. In which one [(A) n motif-REP] was inserted. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
配列番号10で示されるアミノ酸配列(天然由来のクモ糸フィブロインに相当)におけるz/wの値は、46.8%である。配列番号6で示されるアミノ酸配列、配列番号7で示されるアミノ酸配列、配列番号8で示されるアミノ酸配列、及び配列番号9で示されるアミノ酸配列におけるz/wの値は、それぞれ58.7%、70.1%、66.1%及び70.0%である。また、配列番号10、配列番号6、配列番号7、配列番号8及び配列番号9で示されるアミノ酸配列のギザ比率(後述する)1:1.8~11.3におけるx/yの値は、それぞれ15.0%、15.0%、93.4%、92.7%及び89.3%である。
Z The value of z / w in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) is 46.8%. The values of z / w in the amino acid sequence represented by SEQ ID NO: 6, the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9 are 58.7%, respectively. 70.1%, 66.1% and 70.0%. In addition, the value of x / y at the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 is as follows: They are 15.0%, 15.0%, 93.4%, 92.7% and 89.3%, respectively.
(2-i)の改変クモ糸フィブロインは、配列番号6、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (2-i) may have an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
(2-ii)の改変クモ糸フィブロインは、配列番号6、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(2-ii)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified spider silk fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(2-ii)の改変クモ糸フィブロインは、配列番号6、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有し、かつREP中に含まれるXGX(但し、Xはグリシン以外のアミノ酸残基を示す。)からなるアミノ酸配列の総アミノ酸残基数をzとし、上記ドメイン配列中のREPの総アミノ酸残基数をwとしたときに、z/wが50.9%以上であることが好ましい。
The modified spider silk 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. When the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
第2の改変クモ糸フィブロインは、N末端及びC末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変クモ糸フィブロインの単離、固定化、検出及び可視化等が可能となる。
2The second modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
タグ配列として、例えば、他の分子との特異的親和性(結合性、アフィニティ)を利用したアフィニティタグを挙げることができる。アフィニティタグの具体例として、ヒスチジンタグ(Hisタグ)を挙げることができる。Hisタグは、ヒスチジン残基が4から10個程度並んだ短いペプチドで、ニッケル等の金属イオンと特異的に結合する性質があるため、金属キレートクロマトグラフィー(chelating metal chromatography)による改変フィブロインの単離に利用することができる。タグ配列の具体例として、例えば、配列番号12で示されるアミノ酸配列(Hisタグ配列及びヒンジ配列を含むアミノ酸配列)が挙げられる。
Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, isolation of a modified fibroin by metal chelation chromatography (chelating @ metal @ chromatography). Can be used for Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 12 (amino acid sequence including a His tag sequence and a hinge sequence).
また、グルタチオンに特異的に結合するグルタチオン-S-トランスフェラーゼ(GST)、マルトースに特異的に結合するマルトース結合タンパク質(MBP)等のタグ配列を利用することもできる。
タ グ Alternatively, tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose binding protein (MBP), which specifically binds to maltose, can be used.
さらに、抗原抗体反応を利用した「エピトープタグ」を利用することもできる。抗原性を示すペプチド(エピトープ)をタグ配列として付加することにより、当該エピトープに対する抗体を結合させることができる。エピトープタグとして、HA(インフルエンザウイルスのヘマグルチニンのペプチド配列)タグ、mycタグ、FLAGタグ等を挙げることができる。エピトープタグを利用することにより、高い特異性で容易に改変クモ糸フィブロインを精製することができる。
Furthermore, an “epitope tag” utilizing an antigen-antibody reaction can be used. By adding a peptide (epitope) showing antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include an HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, and FLAG tag. By using the epitope tag, the modified spider silk fibroin can be easily purified with high specificity.
さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも使用することができる。当該タグ配列を介して吸着したタンパク質をプロテアーゼ処理することにより、タグ配列を切り離した改変クモ糸フィブロインを回収することもできる。
Further, a tag sequence that can be cleaved by a specific protease can be used. By subjecting the protein adsorbed via the tag sequence to protease treatment, the modified spider silk fibroin from which the tag sequence has been separated can also be recovered.
タグ配列を含む第2の改変フィブロインのより具体的な例として、(2-iii)配列番号13、配列番号11、配列番号14若しく配列番号15で示されるアミノ酸配列、又は(2-iv)配列番号13、配列番号11、配列番号14若しく配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As more specific examples of the second modified fibroin containing a tag sequence, (2-iii) the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15, or (2-iv) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15 can be mentioned.
配列番号16、配列番号17、配列番号13、配列番号11、配列番号14及び配列番号15で示されるアミノ酸配列は、それぞれ配列番号10、配列番号18、配列番号6、配列番号7、配列番号8及び配列番号9で示されるアミノ酸配列のN末端に配列番号12で示されるアミノ酸配列(Hisタグ配列及びヒンジ配列を含む)を付加したものである。
The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively. And an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
(2-iii)の改変クモ糸フィブロインは、配列番号13、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (2-iii) may have an amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
(2-iv)の改変クモ糸フィブロインは、配列番号13、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(2-iv)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (2-iv) includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified spider silk fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(2-iv)の改変クモ糸フィブロインは、配列番号13、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有し、かつREP中に含まれるXGX(但し、Xはグリシン以外のアミノ酸残基を示す。)からなるアミノ酸配列の総アミノ酸残基数をzとし、上記ドメイン配列中のREPの総アミノ酸残基数をwとしたときに、z/wが50.9%以上であることが好ましい。
The modified spider silk fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and is contained in REP. When the total number of amino acid residues in the amino acid sequence consisting of XGX (where X represents an amino acid residue other than glycine) is z, and the total number of REP amino acids in the domain sequence is w, z / W is preferably at least 50.9%.
第2の改変クモ糸フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。
The second modified spider silk 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 the host.
(A)nモチーフの含有量が低減された改変クモ糸フィブロイン(第3の改変クモ糸フィブロイン)は、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、(A)nモチーフの含有量が低減されたアミノ酸配列を有する。第3の改変フィブロインのドメイン配列は、天然由来のクモ糸フィブロインと比較して、少なくとも1又は複数の(A)nモチーフが欠失したことに相当するアミノ酸配列を有するものということができる。
(A) n motifs modified spider silk fibroin content is reduced (Third modified spider silk fibroin), the domain sequence is compared to the naturally occurring silk fibroin, containing (A) n motif It has a reduced amino acid sequence. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or a plurality of (A) n motifs as compared to a naturally occurring spider silk fibroin.
第3の改変クモ糸フィブロインは、天然由来のクモ糸フィブロインから(A)nモチーフを10~40%欠失させたことに相当するアミノ酸配列を有するものであってもよい。
The third modified spider silk fibroin may have an amino acid sequence corresponding to 10 to 40% deletion of the (A) n motif from a naturally occurring spider silk fibroin.
第3の改変クモ糸フィブロインは、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、少なくともN末端側からC末端側に向かって1~3つの(A)nモチーフ毎に1つの(A)nモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。
The third modified spider silk fibroin has a domain sequence of at least one for every 1-3 (A) n motifs from the N-terminus to the C-terminus compared to naturally occurring spider silk fibroin. A) It may have an amino acid sequence corresponding to the deletion of the n motif.
第3の改変クモ糸フィブロインは、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、少なくともN末端側からC末端側に向かって2つ連続した(A)nモチーフの欠失、及び1つの(A)nモチーフの欠失がこの順に繰り返されたことに相当するアミノ酸配列を有するものであってもよい。
The third modified spider silk fibroin has a deletion of the (A) n motif whose domain sequence is at least two contiguous from the N-terminal side to the C-terminal side, as compared with the naturally-derived spider silk fibroin, and It may have an amino acid sequence corresponding to the deletion of one (A) n motif repeated in this order.
第3の改変クモ糸フィブロインは、そのドメイン配列が、少なくともN末端側からC末端側に向かって2つおきに(A)nモチーフが欠失したことに相当するアミノ酸配列を有するものであってもよい。
The third modified spider silk fibroin has an amino acid sequence whose domain sequence corresponds to the deletion of the (A) n motif at least every other region from the N-terminal to the C-terminal. Is also good.
第3の改変クモ糸フィブロインは、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含み、N末端側からC末端側に向かって、隣合う2つの[(A)nモチーフ-REP]ユニットのREPのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないREPのアミノ酸残基数を1としたとき、他方のREPのアミノ酸残基数の比が1.8~11.3となる隣合う2つの[(A)nモチーフ-REP]ユニットのアミノ酸残基数を足し合わせた合計値の最大値をxとし、ドメイン配列の総アミノ酸残基数をyとしたときに、x/yが20%以上、30%以上、40%以上又は50%以上であるアミノ酸配列を有するものであってもよい。(A)nモチーフ中の全アミノ酸残基数に対するアラニン残基数は83%以上であってよいが、86%以上であることが好ましく、90%以上であることがより好ましく、95%以上であることが更に好ましく、100%であること(アラニン残基のみで構成されることを意味する)が更により好ましい。
The third modified spider silk fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m , and two adjacent [(A) The number of amino acid residues of the REP of the [ n motif-REP] unit is sequentially compared, and when the number of amino acid residues of the REP having a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1. The maximum value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units of 8 to 11.3 is x, and the total number of amino acid residues in the domain sequence is y. Then, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues relative to the total number of amino acid residues in the n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and more preferably 95% or more. More preferably, it is even more preferably 100% (meaning that it is composed of only alanine residues).
x/yの算出方法を図1を参照しながら更に詳細に説明する。図1には、クモ糸フィブロインからN末端配列及びC末端配列を除いたドメイン配列を示す。当該ドメイン配列は、N末端側(左側)から(A)nモチーフ-第1のREP(50アミノ酸残基)-(A)nモチーフ-第2のREP(100アミノ酸残基)-(A)nモチーフ-第3のREP(10アミノ酸残基)-(A)nモチーフ-第4のREP(20アミノ酸残基)-(A)nモチーフ-第5のREP(30アミノ酸残基)-(A)nモチーフという配列を有する。
The method of calculating x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from spider silk fibroin. From the N-terminal side (left side), the domain sequence is (A) n motif-first REP (50 amino acid residues)-(A) n motif-second REP (100 amino acid residues)-(A) n Motif-third REP (10 amino acid residues)-(A) n motif-fourth REP (20 amino acid residues)-(A) n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called an n motif.
隣合う2つの[(A)nモチーフ-REP]ユニットは、重複がないように、N末端側からC末端側に向かって、順次選択する。このとき、選択されない[(A)nモチーフ-REP]ユニットが存在してもよい。図1には、パターン1(第1のREPと第2のREPの比較、及び第3のREPと第4のREPの比較)、パターン2(第1のREPと第2のREPの比較、及び第4のREPと第5のREPの比較)、パターン3(第2のREPと第3のREPの比較、及び第4のREPと第5のREPの比較)、パターン4(第1のREPと第2のREPの比較)を示した。なお、これ以外にも選択方法は存在する。
Two adjacent [(A) n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so that there is no overlap. At this time, there may be an unselected [(A) n motif-REP] unit. FIG. 1 shows pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP with the fourth REP), pattern 2 (comparison of the first REP and the second REP, and Pattern 4 (comparison of the second REP with the third REP, and comparison of the fourth REP with the fifth REP), pattern 4 (the comparison of the fourth REP with the fifth REP), and pattern 4 (the first REP with the fifth REP). Second REP comparison). Note that there are other selection methods.
次に各パターンについて、選択した隣合う2つの[(A)nモチーフ-REP]ユニット中の各REPのアミノ酸残基数を比較する。比較は、よりアミノ酸残基数の少ない方を1としたときの、他方のアミノ酸残基数の比を求めることによって行う。例えば、第1のREP(50アミノ酸残基)と第2のREP(100アミノ酸残基)の比較の場合、よりアミノ酸残基数の少ない第1のREPを1としたとき、第2のREPのアミノ酸残基数の比は、100/50=2である。同様に、第4のREP(20アミノ酸残基)と第5のREP(30アミノ酸残基)の比較の場合、よりアミノ酸残基数の少ない第4のREPを1としたとき、第5のREPのアミノ酸残基数の比は、30/20=1.5である。
Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of the other amino acid residues when the smaller number of the amino acid residues is set to 1. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP is The ratio of the number of amino acid residues is 100/50 = 2. Similarly, when comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP having a smaller number of amino acid residues is set to 1, the fifth REP Is 30/20 = 1.5.
図1中、よりアミノ酸残基数の少ない方を1としたときに、他方のアミノ酸残基数の比が1.8~11.3となる[(A)nモチーフ-REP]ユニットの組を実線で示した。以下このような比をギザ比率と呼ぶ。よりアミノ酸残基数の少ない方を1としたときに、他方のアミノ酸残基数の比が1.8未満又は11.3超となる[(A)nモチーフ-REP]ユニットの組は破線で示した。
In FIG. 1, when the smaller number of amino acid residues is set to 1, the [(A) n motif-REP] unit pair in which the ratio of the other amino acid residues is 1.8 to 11.3 is set. Shown by solid line. Hereinafter, such a ratio is referred to as a jagged ratio. Assuming that the smaller number of amino acid residues is 1, the pair of [(A) n motif-REP] units in which the ratio of the other amino acid residues is less than 1.8 or more than 11.3 is indicated by a broken line. Indicated.
各パターンにおいて、実線で示した隣合う2つの[(A)nモチーフ-REP]ユニットの全てのアミノ酸残基数を足し合わせる(REPのみではなく、(A)nモチーフのアミノ酸残基数もである。)。そして、足し合わせた合計値を比較して、当該合計値が最大となるパターンの合計値(合計値の最大値)をxとする。図1に示した例では、パターン1の合計値が最大である。
In each pattern, the total number of amino acid residues of two adjacent [(A) n motif-REP] units shown by a solid line is added (not only the REP but also the number of amino acid residues of the (A) n motif. is there.). Then, the sum total is compared, and the total value (maximum value of the total values) of the patterns having the maximum total value is x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.
次に、xをドメイン配列の総アミノ酸残基数yで除すことによって、x/y(%)を算出することができる。
Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.
第3の改変クモ糸フィブロインにおいて、x/yは、50%以上であることが好ましく、60%以上であることがより好ましく、65%以上であることが更に好ましく、70%以上であることが更により好ましく、75%以上であることが更によりまた好ましく、80%以上であることが特に好ましい。x/yの上限に特に制限はなく、例えば、100%以下であってよい。ギザ比率が1:1.9~11.3の場合には、x/yは89.6%以上であることが好ましく、ギザ比率が1:1.8~3.4の場合には、x/yは77.1%以上であることが好ましく、ギザ比率が1:1.9~8.4の場合には、x/yは75.9%以上であることが好ましく、ギザ比率が1:1.9~4.1の場合には、x/yは64.2%以上であることが好ましい。
In the third modified spider silk fibroin, x / y is preferably at least 50%, more preferably at least 60%, further preferably at least 65%, and more preferably at least 70%. Even more preferably, it is even more preferably 75% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited, and may be, for example, 100% or less. When the indentation ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the indentation ratio is 1: 1.8 to 3.4, x / y is x / y. / Y is preferably at least 77.1%, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably at least 75.9%, and the jagged ratio is 1 In the case of 1.9 to 4.1, x / y is preferably at least 64.2%.
第3の改変クモ糸フィブロインが、ドメイン配列中に複数存在する(A)nモチーフの少なくとも7つがアラニン残基のみで構成される改変クモ糸フィブロインである場合、x/yは、46.4%以上であることが好ましく、50%以上であることがより好ましく、55%以上であることが更に好ましく、60%以上であることが更により好ましく、70%以上であることが更によりまた好ましく、80%以上であることが特に好ましい。x/yの上限に特に制限はなく、100%以下であればよい。
When the third modified spider silk fibroin is a modified spider silk fibroin in which at least seven of the (A) n motifs present in a plurality in the domain sequence are composed of only alanine residues, x / y is 46.4% Or more, more preferably 50% or more, still more preferably 55% or more, still more preferably 60% or more, even more preferably 70% or more, It is particularly preferred that it is 80% or more. The upper limit of x / y is not particularly limited, and may be 100% or less.
第3の改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列から、x/yが64.2%以上になるように(A)nモチーフをコードする配列の1又は複数を欠失させることにより得ることができる。また、例えば、天然由来のクモ糸フィブロインのアミノ酸配列から、x/yが64.2%以上になるように1又は複数の(A)nモチーフが欠失したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のクモ糸フィブロインのアミノ酸配列から(A)nモチーフが欠失したことに相当する改変に加え、更に1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変を行ってもよい。
The third modified spider silk fibroin is, for example, one or more of the sequences encoding the (A) n motif such that x / y is 64.2% or more from the cloned spider silk fibroin gene sequence. Can be obtained by deleting Further, for example, 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 spider silk fibroin derived from nature. Alternatively, it can be obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the deletion of the (A) n motif from the amino acid sequence of spider silk fibroin of natural origin, one or more amino acid residues are further substituted, deleted, inserted and / or substituted. Alternatively, the amino acid sequence corresponding to the addition may be modified.
第3の改変クモ糸フィブロインのより具体的な例として、(3-i)配列番号18、配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列、又は(3-ii)配列番号18、配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変フィブロインを挙げることができる。
As more specific examples of the third modified spider silk fibroin, (3-i) the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or (3-ii) SEQ ID NO: 18 , A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.
(3-i)の改変クモ糸フィブロインについて説明する。配列番号18で示されるアミノ酸配列は、天然由来のクモ糸フィブロインに相当する配列番号10で示されるアミノ酸配列から、N末端側からC末端側に向かって2つおきに(A)nモチーフを欠失させ、更にC末端配列の手前に[(A)nモチーフ-REP]を1つ挿入したものである。配列番号7で示されるアミノ酸配列は、配列番号18で示されるアミノ酸配列のREP中の全てのGGXをGQXに置換したものである。配列番号8で示されるアミノ酸配列は、配列番号7で示されるアミノ酸配列の各(A)nモチーフのC末端側に2つのアラニン残基を挿入し、更に一部のグルタミン(Q)残基をセリン(S)残基に置換し、配列番号7の分子量とほぼ同じとなるようにN末端側の一部のアミノ酸を欠失させたものである。配列番号9で示されるアミノ酸配列は、配列番号11で示されるアミノ酸配列中に存在する20個のドメイン配列の領域(但し、当該領域のC末端側の数アミノ酸残基が置換されている。)を4回繰り返した配列のC末端にHisタグが付加されたものである。
The modified spider silk fibroin (3-i) will be described. The amino acid sequence represented by SEQ ID NO: 18 is different from the amino acid sequence represented by SEQ ID NO: 10 corresponding to naturally occurring spider silk fibroin in that every two (A) n motifs are deleted from the N-terminal side to the C-terminal side. And one [(A) n motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 7 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 18 with GQX. The amino acid sequence represented by SEQ ID NO: 8 has two alanine residues inserted at the C-terminal side of each (A) n motif of the amino acid sequence represented by SEQ ID NO: 7, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some of the N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 7. The amino acid sequence represented by SEQ ID NO: 9 has a region of 20 domain sequences existing in the amino acid sequence represented by SEQ ID NO: 11 (however, several amino acid residues on the C-terminal side of the region are substituted). Is a sequence obtained by adding a His tag to the C-terminal of a sequence obtained by repeating the above four times.
配列番号10で示されるアミノ酸配列(天然由来のクモ糸フィブロインに相当)のギザ比率1:1.8~11.3におけるx/yの値は15.0%である。配列番号18で示されるアミノ酸配列、及び配列番号7で示されるアミノ酸配列におけるx/yの値は、いずれも93.4%である。配列番号8で示されるアミノ酸配列におけるx/yの値は、92.7%である。配列番号9で示されるアミノ酸配列におけるx/yの値は、89.3%である。配列番号10、配列番号18、配列番号7、配列番号8及び配列番号9で示されるアミノ酸配列におけるz/wの値は、それぞれ46.8%、56.2%、70.1%、66.1%及び70.0%である。
X The amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring spider silk fibroin) has an x / y value of 15.0% at a giza ratio of 1: 1.8 to 11.3. The value of x / y in the amino acid sequence represented by SEQ ID NO: 18 and the amino acid sequence represented by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence represented by SEQ ID NO: 9 is 89.3%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66. 1% and 70.0%.
(3-i)の改変クモ糸フィブロインは、配列番号18、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (3-i) may have an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.
(3-ii)の改変クモ糸フィブロインは、配列番号18、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(3-ii)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. The modified spider silk fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(3-ii)の改変クモ糸フィブロインは、配列番号18、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有し、かつN末端側からC末端側に向かって、隣合う2つの[(A)nモチーフ-REP]ユニットのREPのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないREPのアミノ酸残基数を1としたとき、他方のREPのアミノ酸残基数の比が1.8~11.3(ギザ比率が1:1.8~11.3)となる隣合う2つの[(A)nモチーフ-REP]ユニットのアミノ酸残基数を足し合わせた合計値の最大値をxとし、ドメイン配列の総アミノ酸残基数をyとしたときに、x/yが64.2%以上であることが好ましい。
The modified spider silk fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has C-terminal sequence from the N-terminal side. When the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared toward the terminal side, when the number of amino acid residues of REP having a small number of amino acid residues is 1, Of two adjacent [(A) n motif-REP] units having a ratio of the number of amino acid residues of the other REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3). It is preferable that x / y be 64.2% or more, where x is the maximum value of the sum of the number of amino acid residues and y is the total number of amino acid residues in the domain sequence.
第3の改変クモ糸フィブロインは、N末端及びC末端のいずれか一方又は両方に上述したタグ配列を含んでいてもよい。
The third modified spider silk fibroin may include the above-described tag sequence at one or both of the N-terminus and the C-terminus.
タグ配列を含む第3の改変クモ糸フィブロインのより具体的な例として、(3-iii)配列番号17、配列番号11、配列番号14若しくは配列番号15で示されるアミノ酸配列、又は(3-iv)配列番号17、配列番号11、配列番号14若しくは配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As more specific examples of the third modified spider silk fibroin including the tag sequence, (3-iii) the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, or (3-iv) ) Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
配列番号16、配列番号17、配列番号13、配列番号11、配列番号14及び配列番号15で示されるアミノ酸配列は、それぞれ配列番号10、配列番号18、配列番号6、配列番号7、配列番号8及び配列番号9で示されるアミノ酸配列のN末端に配列番号12で示されるアミノ酸配列(Hisタグ配列及びヒンジ配列を含む)を付加したものである。
The amino acid sequences represented by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively. And an amino acid sequence represented by SEQ ID NO: 12 (including a His tag sequence and a hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 9.
(3-iii)の改変クモ糸フィブロインは、配列番号17、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (3-iii) may have an amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15.
(3-iv)の改変クモ糸フィブロインは、配列番号17、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(3-iv)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (3-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15. The modified spider silk fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(3-iv)の改変クモ糸フィブロインは、配列番号17、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列と90%以上の配列同一性を有し、かつN末端側からC末端側に向かって、隣合う2つの[(A)nモチーフ-REP]ユニットのREPのアミノ酸残基数を順次比較して、アミノ酸残基数が少ないREPのアミノ酸残基数を1としたとき、他方のREPのアミノ酸残基数の比が1.8~11.3となる隣合う2つの[(A)nモチーフ-REP]ユニットのアミノ酸残基数を足し合わせた合計値の最大値をxとし、ドメイン配列の総アミノ酸残基数をyとしたときに、x/yが64.2%以上であることが好ましい。
The modified spider silk fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 15, and has C-terminal sequence from the N-terminal side. When the number of amino acid residues of REP of two adjacent [(A) n motif-REP] units is sequentially compared toward the terminal side, when the number of amino acid residues of REP having a small number of amino acid residues is 1, The maximum value of the sum total of the number of amino acid residues of two adjacent [(A) n motif-REP] units whose ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 is defined as Assuming that x is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably at least 64.2%.
第3の改変クモ糸フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。
The third modified spider silk 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 the host.
グリシン残基の含有量、及び(A)nモチーフの含有量が低減された改変クモ糸フィブロイン(第4の改変クモ糸フィブロイン)は、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、(A)nモチーフの含有量が低減されたことに加え、グリシン残基の含有量が低減されたアミノ酸配列を有するものである。第4の改変クモ糸フィブロインのドメイン配列は、天然由来のクモ糸フィブロインと比較して、少なくとも1又は複数の(A)nモチーフが欠失したことに加え、更に少なくともREP中の1又は複数のグリシン残基が別のアミノ酸残基に置換されたことに相当するアミノ酸配列を有するものということができる。すなわち、第4の改変クモ糸フィブロインは、上述したグリシン残基の含有量が低減された改変クモ糸フィブロイン(第2の改変クモ糸フィブロイン)と、(A)nモチーフの含有量が低減された改変クモ糸フィブロイン(第3の改変クモ糸フィブロイン)の特徴を併せ持つ改変クモ糸フィブロインである。具体的な態様等は、第2の改変クモ糸フィブロイン、及び第3の改変クモ糸フィブロインで説明したとおりである。
The modified spider silk fibroin (the fourth modified spider silk fibroin) in which the content of the glycine residue and the content of the (A) n motif are reduced has a domain sequence that is lower than that of a naturally derived spider silk fibroin. , (A) having an amino acid sequence in which the content of glycine residues is reduced in addition to the reduced content of the n motif. The domain sequence of the fourth modified spider silk fibroin differs from the naturally occurring spider silk fibroin in that at least one or more (A) n motifs have been deleted, and at least one or more of the It can be said that the glycine residue has an amino acid sequence corresponding to the substitution of another amino acid residue. That is, in the fourth modified spider silk fibroin, the modified spider silk fibroin (the second modified spider silk fibroin) in which the content of the glycine residue was reduced and the content of the (A) n motif were reduced. A modified spider silk fibroin having the characteristics of the modified spider silk fibroin (third modified spider silk fibroin). Specific embodiments and the like are as described for the second modified spider silk fibroin and the third modified spider silk fibroin.
第4の改変クモ糸フィブロインのより具体的な例として、(4-i)配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列、(4-ii)配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。配列番号7、配列番号8若しくは配列番号9で示されるアミノ酸配列を含む改変クモ糸フィブロインの具体的な態様は上述のとおりである。
As more specific examples of the fourth modified spider silk fibroin, (4-i) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, (4-ii) SEQ ID NO: 7, SEQ ID NO: 8 or Modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 9 can be mentioned. Specific embodiments of the modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 are as described above.
局所的に疎水性指標の大きい領域を含むドメイン配列を有する改変クモ糸フィブロイン(第5の改変クモ糸フィブロイン)は、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、REP中の1又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び/又はREP中に1又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する、局所的に疎水性指標の大きい領域を含むアミノ酸配列を有するものであってよい。
The modified spider silk fibroin having a domain sequence including a region having a locally large hydrophobicity index (fifth modified spider silk fibroin) has a domain sequence of 1 in REP compared to naturally occurring spider silk fibroin. Or local substitution corresponding to substitution of a plurality of amino acid residues with amino acid residues having a large hydrophobicity index and / or insertion of one or more amino acid residues having a large hydrophobicity index into REP. May have an amino acid sequence containing a region having a large hydrophobicity index.
局所的に疎水性指標の大きい領域は、連続する2~4アミノ酸残基で構成されていることが好ましい。
領域 A region having a locally large hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.
上述の疎水性指標の大きいアミノ酸残基は、イソロイシン(I)、バリン(V)、ロイシン(L)、フェニルアラニン(F)、システイン(C)、メチオニン(M)及びアラニン(A)から選ばれるアミノ酸残基であることがより好ましい。
The amino acid residue having a large hydrophobicity index is selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A). More preferably, it is a residue.
第5の改変フィブロインは、天然由来のクモ糸フィブロインと比較して、REP中の1又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び/又はREP中に1又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に、天然由来のクモ糸フィブロインと比較して、1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変があってもよい。
The fifth modified fibroin is that one or more amino acid residues in the REP have been replaced with amino acid residues having a large hydrophobicity index, and / or Or, in addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, further, compared with naturally occurring spider silk fibroin, substitution or deletion of one or more amino acid residues, insertion, And / or there may be an amino acid sequence modification corresponding to the addition.
第5の改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列からREP中の1又は複数の親水性アミノ酸残基(例えば、疎水性指標がマイナスであるアミノ酸残基)を疎水性アミノ酸残基(例えば、疎水性指標がプラスであるアミノ酸残基)に置換すること、及び/又はREP中に1又は複数の疎水性アミノ酸残基を挿入することにより得ることができる。また、例えば、天然由来のクモ糸フィブロインのアミノ酸配列からREP中の1又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び/又はREP中に1又は複数の疎水性アミノ酸残基を挿入したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のクモ糸フィブロインのアミノ酸配列からREP中の1又は複数の親水性アミノ酸残基を疎水性アミノ酸残基に置換したこと、及び/又はREP中に1又は複数の疎水性アミノ酸残基を挿入したことに相当する改変に加え、更に1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変を行ってもよい。
The fifth modified spider silk fibroin is obtained by, for example, removing one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in REP from the cloned gene sequence of naturally occurring spider silk fibroin. It can be obtained by substituting a hydrophobic amino acid residue (for example, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues into REP. Further, for example, one or more hydrophilic amino acid residues in REP are replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acids are contained in REP. It can also be obtained by designing an amino acid sequence corresponding to insertion of a residue and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of the naturally occurring spider silk fibroin, and / or one or more hydrophobic amino acid residues in the REP. In addition to the modification corresponding to the insertion of a sex amino acid residue, the amino acid sequence may further be modified corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues.
第5の改変フィブロインは、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含み、最もC末端側に位置する(A)nモチーフから上記ドメイン配列のC末端までの配列を上記ドメイン配列から除いた配列に含まれる全てのREPにおいて、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域に含まれるアミノ酸残基の総数をpとし、最もC末端側に位置する(A)nモチーフから上記ドメイン配列のC末端までの配列を上記ドメイン配列から除いた配列に含まれるアミノ酸残基の総数をqとしたときに、p/qが6.2%以上であるアミノ酸配列を有してもよい。
The fifth modified fibroin contains a domain sequence represented by Formula 1: [(A) n motif-REP] m and extends from the (A) n motif located at the most C-terminal side to the C-terminus of the domain sequence. In all REPs included in the sequence excluding the sequence from the domain sequence, the total number of amino acid residues included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is p, When the total number of amino acid residues contained in the sequence excluding the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence is defined as q, p / q is 6 .2% or more.
アミノ酸残基の疎水性指標については、公知の指標(Hydropathy index:Kyte J,&Doolittle R(1982)“A simple method for displaying the hydropathic character of a protein”,J.Mol.Biol.,157,pp.105-132)を使用する。具体的には、各アミノ酸の疎水性指標(ハイドロパシー・インデックス、以下「HI」とも記す。)は、下記表1に示すとおりである。
Regarding the hydrophobicity index of amino acid residues, a publicly known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for display, the hydropathic charactor of aa protein, J.Pol.Mol. 105-132). Specifically, the hydropathic index (hydropathic index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.
p/qの算出方法を更に詳細に説明する。算出には、式1:[(A)nモチーフ-REP]mで表されるドメイン配列から、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列を除いた配列(以下、「配列A」とする)を用いる。まず、配列Aに含まれる全てのREPにおいて、連続する4アミノ酸残基の疎水性指標の平均値を算出する。疎水性指標の平均値は、連続する4アミノ酸残基に含まれる各アミノ酸残基のHIの総和を4(アミノ酸残基数)で除して求める。疎水性指標の平均値は、全ての連続する4アミノ酸残基について求める(各アミノ酸残基は、1~4回平均値の算出に用いられる。)。次いで、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域を特定する。あるアミノ酸残基が、複数の「疎水性指標の平均値が2.6以上となる連続する4アミノ酸残基」に該当する場合であっても、領域中には1アミノ酸残基として含まれることになる。そして、当該領域に含まれるアミノ酸残基の総数がpである。また、配列Aに含まれるアミノ酸残基の総数がqである。
The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Formula 1: [(A) n motif-REP] m (Hereinafter, referred to as “sequence A”). First, in all REPs included in sequence A, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated. The average value of the hydrophobicity index is determined by dividing the total sum of HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is determined for all four consecutive amino acid residues (each amino acid residue is used for calculating the average value one to four times). Next, a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more is specified. Even when a certain amino acid residue corresponds to a plurality of “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more”, it is included as one amino acid residue in the region. become. Then, the total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.
例えば、「疎水性指標の平均値が2.6以上となる連続する4アミノ酸残基」が20カ所抽出された場合(重複はなし)、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域には、連続する4アミノ酸残基(重複はなし)が20含まれることになり、pは20×4=80である。また、例えば、2つの「疎水性指標の平均値が2.6以上となる連続する4アミノ酸残基」が1アミノ酸残基だけ重複して存在する場合、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域には、7アミノ酸残基含まれることになる(p=2×4-1=7。「-1」は重複分の控除である。)。例えば、図2に示したドメイン配列の場合、「疎水性指標の平均値が2.6以上となる連続する4アミノ酸残基」が重複せずに7つ存在するため、pは7×4=28となる。また、例えば、図2に示したドメイン配列の場合、qは4+50+4+40+4+10+4+20+4+30=170である(C末端側の最後に存在する(A)nモチーフは含めない)。次に、pをqで除すことによって、p/q(%)を算出することができる。図2の場合28/170=16.47%となる。
For example, when “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” are extracted at 20 locations (without duplication), the average value of the hydrophobicity index of the 4 consecutive amino acid residues is 2 The region of .6 or more would contain 20 consecutive 4 amino acid residues (no duplication), and p would be 20 × 4 = 80. For example, when two “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” overlap by one amino acid residue, the hydrophobicity index of four consecutive amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “−1” is a deduction for the overlap). For example, in the case of the domain sequence shown in FIG. 2, there are seven consecutive “4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without overlapping, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 2, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) n motif present at the C-terminal end). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 2, 28/170 = 16.47%.
第5の改変クモ糸フィブロインにおいて、p/qは、6.2%以上であることが好ましく、7%以上であることがより好ましく、10%以上であることが更に好ましく、20%以上であることが更により好ましく、30%以上であることが更によりまた好ましい。p/qの上限は、特に制限されないが、例えば、45%以下であってもよい。
In the fifth modified spider silk fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Is still more preferred, and even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.
第5の改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインのアミノ酸配列を、上記のp/qの条件を満たすように、REP中の1又は複数の親水性アミノ酸残基(例えば、疎水性指標がマイナスであるアミノ酸残基)を疎水性アミノ酸残基(例えば、疎水性指標がプラスであるアミノ酸残基)に置換すること、及び/又はREP中に1又は複数の疎水性アミノ酸残基を挿入することにより、局所的に疎水性指標の大きい領域を含むアミノ酸配列に改変することにより得ることができる。また、例えば、天然由来のクモ糸フィブロインのアミノ酸配列から上記のp/qの条件を満たすアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。いずれの場合においても、天然由来のクモ糸フィブロインと比較して、REP中の1又は複数のアミノ酸残基が疎水性指標の大きいアミノ酸残基に置換されたこと、及び/又はREP中に1又は複数の疎水性指標の大きいアミノ酸残基が挿入されたことに相当する改変に加え、更に1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当する改変を行ってもよい。
The fifth modified spider silk fibroin is, for example, one or more hydrophilic amino acid residues in REP (for example, the amino acid sequence of the cloned naturally-derived spider silk fibroin is changed so as to satisfy the above-mentioned p / q conditions). Replacing an amino acid residue with a negative hydrophobicity index with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acids in the REP It can be obtained by inserting a residue to locally modify the amino acid sequence to include a region having a large hydrophobicity index. Further, for example, it can be obtained by designing an amino acid sequence satisfying the above-mentioned p / q condition from the amino acid sequence of spider silk fibroin derived from nature, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP were replaced with amino acid residues having a higher hydrophobicity index as compared to naturally occurring spider silk fibroin, and / or one or more amino acid residues in the REP. In addition to the modification corresponding to the insertion of a plurality of amino acid residues having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues was performed. Is also good.
疎水性指標の大きいアミノ酸残基としては、特に制限はないが、イソロイシン(I)、バリン(V)、ロイシン(L)、フェニルアラニン(F)、システイン(C)、メチオニン(M)及びアラニン(A)が好ましく、バリン(V)、ロイシン(L)及びイソロイシン(I)がより好ましい。
The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A Is preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.
第5の改変クモ糸フィブロインの具体的な例として、(5-i)配列番号19、配列番号20若しくは配列番号21で示されるアミノ酸配列、又は(5-ii)配列番号19、配列番号20若しくは配列番号21で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
Specific examples of the fifth modified spider silk fibroin include (5-i) the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or (5-ii) SEQ ID NO: 19, SEQ ID NO: 20 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 21 can be mentioned.
(5-i)の改変クモ糸フィブロインについて説明する。配列番号22で示されるアミノ酸配列は、天然由来のクモ糸フィブロインの(A)nモチーフ中のアラニン残基が連続するアミノ酸配列をアラニン残基が連続する数を5つになるよう欠失したものである。配列番号19で示されるアミノ酸配列は、配列番号22で示されるアミノ酸配列に対し、REP一つ置きにそれぞれ3アミノ酸残基からなるアミノ酸配列(VLI)を2カ所挿入し、かつ配列番号22で示されるアミノ酸配列の分子量とほぼ同じとなるようにC末端側の一部のアミノ酸を欠失させたものである。配列番号23で示されるアミノ酸配列は、配列番号22で示されるアミノ酸配列に対し、各(A)nモチーフのC末端側に2つのアラニン残基を挿入し、更に一部のグルタミン(Q)残基をセリン(S)残基に置換し、かつ配列番号22で示されるアミノ酸配列の分子量とほぼ同じとなるようにC末端側の一部のアミノ酸を欠失させたものである。配列番号20で示されるアミノ酸配列は、配列番号23で示されるアミノ酸配列に対し、REP一つ置きにそれぞれ3アミノ酸残基からなるアミノ酸配列(VLI)を1カ所挿入したものである。配列番号21で示されるアミノ酸配列は、配列番号23で示されるアミノ酸配列に対し、REP一つ置きにそれぞれ3アミノ酸残基からなるアミノ酸配列(VLI)を2カ所挿入したものである。
The modified spider silk fibroin of (5-i) will be described. The amino acid sequence represented by SEQ ID NO: 22 is obtained by deleting the amino acid sequence having consecutive alanine residues in the (A) n motif of a spider silk fibroin of natural origin so that the number of consecutive alanine residues becomes five. It is. The amino acid sequence represented by SEQ ID NO: 19 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues at every other REP into the amino acid sequence represented by SEQ ID NO: 22; A part of the amino acids at the C-terminal side are deleted so that the molecular weight of the amino acid sequence to be obtained is almost the same. The amino acid sequence represented by SEQ ID NO: 23 is different from the amino acid sequence represented by SEQ ID NO: 22 by inserting two alanine residues at the C-terminal side of each (A) n motif, and further including a part of glutamine (Q) residue. In this group, a group is substituted with a serine (S) residue, and some amino acids on the C-terminal side are deleted so that the molecular weight of the amino acid sequence shown in SEQ ID NO: 22 is almost the same. The amino acid sequence represented by SEQ ID NO: 20 is obtained by inserting one amino acid sequence (VLI) consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23. The amino acid sequence represented by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of three amino acid residues every other REP into the amino acid sequence represented by SEQ ID NO: 23.
(5-i)の改変クモ糸フィブロインは、配列番号19、配列番号20又は配列番号21で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (5-i) may be composed of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
(5-ii)の改変クモ糸フィブロインは、配列番号19、配列番号20又は配列番号21で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(5-ii)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified spider silk fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(5-ii)の改変クモ糸フィブロインは、配列番号19、配列番号20又は配列番号21で示されるアミノ酸配列と90%以上の配列同一性を有し、かつ最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれる全てのREPにおいて、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域に含まれるアミノ酸残基の総数をpとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をqとしたときに、p/qが6.2%以上であることが好ましい。
The modified spider silk fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most C-terminal (A ) 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 REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more. And the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
第5の改変クモ糸フィブロインは、N末端及びC末端のいずれか一方又は両方にタグ配列を含んでいてもよい。
5The fifth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus.
タグ配列を含む第5の改変クモ糸フィブロインのより具体的な例として、(5-iii)配列番号24、配列番号25若しくは配列番号26で示されるアミノ酸配列、又は(5-iv)配列番号24、配列番号25若しくは配列番号26で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As more specific examples of the fifth modified spider silk fibroin containing a tag sequence, (5-iii) the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, or (5-iv) SEQ ID NO: 24 , A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 26.
配列番号24、配列番号25及び配列番号26で示されるアミノ酸配列は、それぞれ配列番号19、配列番号20及び配列番号21で示されるアミノ酸配列のN末端に配列番号12で示されるアミノ酸配列(Hisタグ配列及びヒンジ配列を含む)を付加したものである。
The amino acid sequences represented by SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 correspond to the amino acid sequence represented by SEQ ID NO: 12 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Sequences and hinge sequences).
(5-iii)の改変クモ糸フィブロインは、配列番号24、配列番号25若しくは配列番号26で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (5-iii) may have an amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
(5-iv)の改変クモ糸フィブロインは、配列番号24、配列番号25若しくは配列番号26で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(5-iv)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]mで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. The modified spider silk fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) n motif-REP] m . The sequence identity is preferably 95% or more.
(5-iv)の改変クモ糸フィブロインは、配列番号24、配列番号25若しくは配列番号26で示されるアミノ酸配列と90%以上の配列同一性を有し、かつ最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれる全てのREPにおいて、連続する4アミノ酸残基の疎水性指標の平均値が2.6以上となる領域に含まれるアミノ酸残基の総数をpとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれるアミノ酸残基の総数をqとしたときに、p/qが6.2%以上であることが好ましい。
The modified spider silk fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and is located most C-terminal (A ) 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 REP is included in a region where the average value of the hydrophobicity index of four consecutive amino acid residues is 2.6 or more. And the total number of amino acid residues contained in the sequence obtained by removing the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is denoted by q. Sometimes, it is preferable that p / q is 6.2% or more.
第5の改変クモ糸フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。
(5) The fifth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
グルタミン残基の含有量が低減されたドメイン配列を有する改変クモ糸フィブロイン(第6の改変クモ糸フィブロイン)は、天然由来のクモ糸フィブロインと比較して、グルタミン残基の含有量が低減されたアミノ酸配列を有する。
The modified spider silk fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified spider silk fibroin) has a reduced content of glutamine residues as compared to naturally occurring spider silk fibroin Has an amino acid sequence.
第6の改変クモ糸フィブロインは、REPのアミノ酸配列中に、GGXモチーフ及びGPGXXモチーフから選ばれる少なくとも一つのモチーフが含まれていることが好ましい。
6 The sixth modified spider silk fibroin preferably contains at least one motif selected from GGX motif and GPGXX motif in the amino acid sequence of REP.
第6の改変クモ糸フィブロインが、REP中にGPGXXモチーフを含む場合、GPGXXモチーフ含有率は、通常1%以上であり、5%以上であってもよく、10%以上であるのが好ましい。GPGXXモチーフ含有率の上限に特に制限はなく、50%以下であってよく、30%以下であってもよい。
場合 When the sixth modified spider silk fibroin contains a GPGXX motif in the REP, the content of the GPGXX motif is usually 1% or more, and may be 5% or more, and preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited, and may be 50% or less, or 30% or less.
本明細書において、「GPGXXモチーフ含有率」は、以下の方法により算出される値である。
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれる全てのREPにおいて、その領域に含まれるGPGXXモチーフの個数の総数を3倍した数(即ち、GPGXXモチーフ中のG及びPの総数に相当)をsとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、GPGXXモチーフ含有率はs/tとして算出される。 In the present specification, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal (A) In all REPs included in the sequence except for the sequence from the n motif to the C-terminus of the domain sequence from the n motif to the domain sequence, the total number of GPGXX motifs contained in the region was tripled ( That is, s is defined as s (corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n Assuming that the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is calculated as s / t.
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列に含まれる全てのREPにおいて、その領域に含まれるGPGXXモチーフの個数の総数を3倍した数(即ち、GPGXXモチーフ中のG及びPの総数に相当)をsとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、GPGXXモチーフ含有率はs/tとして算出される。 In the present specification, the “GPGXX motif content” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal (A) In all REPs included in the sequence except for the sequence from the n motif to the C-terminus of the domain sequence from the n motif to the domain sequence, the total number of GPGXX motifs contained in the region was tripled ( That is, s is defined as s (corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence, and further (A) n Assuming that the total number of amino acid residues of all REPs excluding the motif is t, the GPGXX motif content is calculated as s / t.
GPGXXモチーフ含有率の算出において、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」を対象としているのは、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列」(REPに相当する配列)には、クモ糸フィブロインに特徴的な配列と相関性の低い配列が含まれることがあり、mが小さい場合(つまり、ドメイン配列が短い場合)、GPGXXモチーフ含有率の算出結果に影響するので、この影響を排除するためである。なお、REPのC末端に「GPGXXモチーフ」が位置する場合、「XX」が例えば「AA」の場合であっても、「GPGXXモチーフ」として扱う。
In the calculation of the content ratio of the GPGXX motif, “the sequence obtained by removing the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence” to “the most C-terminal side” (A) sequence from the n motif to the C-terminus of the domain sequence ”(sequence corresponding to REP) may include a sequence having low correlation with a sequence characteristic of spider silk fibroin. Is small (that is, when the domain sequence is short), which affects the calculation result of the GPGXX motif content, so that this effect is eliminated. When the “GPGXX motif” is located at the C-terminus of the REP, even when “XX” is, for example, “AA”, it is treated as a “GPGXX motif”.
図3は、クモ糸フィブロインのドメイン配列を示す模式図である。図3を参照しながらGPGXXモチーフ含有率の算出方法を具体的に説明する。まず、図3に示したクモ糸フィブロインのドメイン配列(「[(A)nモチーフ-REP]m-(A)nモチーフ」タイプである。)では、全てのREPが「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」(図3中、「領域A」で示した配列。)に含まれているため、sを算出するためのGPGXXモチーフの個数は7であり、sは7×3=21となる。同様に、全てのREPが「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」(図3中、「領域A」で示した配列。)に含まれているため、当該配列から更に(A)nモチーフを除いた全REPのアミノ酸残基の総数tは50+40+10+20+30=150である。次に、sをtで除すことによって、s/t(%)を算出することができ、図3のフィブロインの場合21/150=14.0%となる。
FIG. 3 is a schematic diagram showing the domain sequence of spider silk fibroin. The method of calculating the content rate of the GPGXX motif will be specifically described with reference to FIG. First, in the domain sequence of spider silk fibroin ("[(A) n motif-REP] m- (A) n motif" type) shown in FIG. 3, all REPs are located at the "most C-terminal position". (A) A sequence obtained by removing the sequence from the n motif to the C-terminus of the domain sequence from the domain sequence ”(the sequence shown as“ region A ”in FIG. 3). The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are “sequences in which the sequence from the (A) n motif located at the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” (the sequence shown as “region A” in FIG. 3). ), The total number t of amino acid residues of all REPs excluding the (A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, and in the case of the fibroin of FIG. 3, 21/150 = 14.0%.
第6の改変クモ糸フィブロインは、グルタミン残基含有率が9%以下であることが好ましく、7%以下であることがより好ましく、4%以下であることが更に好ましく、0%であることが特に好ましい。
The sixth modified spider silk fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, still more preferably 4% or less, and more preferably 0%. Particularly preferred.
本明細書において、「グルタミン残基含有率」は、以下の方法により算出される値である。
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列(図3の「領域A」に相当する配列。)に含まれる全てのREPにおいて、その領域に含まれるグルタミン残基の総数をuとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、グルタミン残基含有率はu/tとして算出される。グルタミン残基含有率の算出において、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, the “glutamine residue content” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs included in the sequence (sequence corresponding to “region A” in FIG. 3) in which the sequence from the (A) n motif to the C-terminus of the domain sequence located on the side of the (A) is excluded from the domain sequence, the total number of glutamine residues in the u, most C located at the end side, except the sequence of (a) from n motif to the C-terminal domain sequence from domain sequence, further the total REP excluding (a) n motif Glutamine residue content is calculated as u / t, where t is the total number of amino acid residues. In the calculation of the glutamine residue content, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列(図3の「領域A」に相当する配列。)に含まれる全てのREPにおいて、その領域に含まれるグルタミン残基の総数をuとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、グルタミン残基含有率はu/tとして算出される。グルタミン残基含有率の算出において、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, the “glutamine residue content” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs included in the sequence (sequence corresponding to “region A” in FIG. 3) in which the sequence from the (A) n motif to the C-terminus of the domain sequence located on the side of the (A) is excluded from the domain sequence, the total number of glutamine residues in the u, most C located at the end side, except the sequence of (a) from n motif to the C-terminal domain sequence from domain sequence, further the total REP excluding (a) n motif Glutamine residue content is calculated as u / t, where t is the total number of amino acid residues. In the calculation of the glutamine residue content, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as described above. The same is true.
第6の改変クモ糸フィブロインは、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、REP中の1又は複数のグルタミン残基を欠失したこと、又は他のアミノ酸残基に置換したことに相当するアミノ酸配列を有するものであってよい。
The sixth modified spider silk fibroin had one or more glutamine residues in the REP deleted or substituted with other amino acid residues in the domain sequence compared to the naturally occurring spider silk fibroin. It may have an amino acid sequence corresponding to this.
「他のアミノ酸残基」は、グルタミン残基以外のアミノ酸残基であればよいが、グルタミン残基よりも疎水性指標の大きいアミノ酸残基であることが好ましい。アミノ酸残基の疎水性指標は表1に示すとおりである。
The “other amino acid residue” may be an amino acid residue other than a glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than a glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.
表1に示すとおり、グルタミン残基よりも疎水性指標の大きいアミノ酸残基としては、イソロイシン(I)、バリン(V)、ロイシン(L)、フェニルアラニン(F)、システイン(C)、メチオニン(M)アラニン(A)、グリシン(G)、スレオニン(T)、セリン(S)、トリプトファン(W)、チロシン(Y)、プロリン(P)及びヒスチジン(H)から選ばれるアミノ酸残基を挙げることができる。これらの中でも、イソロイシン(I)、バリン(V)、ロイシン(L)、フェニルアラニン(F)、システイン(C)、メチオニン(M)及びアラニン(A)から選ばれるアミノ酸残基であることがより好ましく、イソロイシン(I)、バリン(V)、ロイシン(L)及びフェニルアラニン(F)から選ばれるアミノ酸残基であることが更に好ましい。
As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it can. Among them, an amino acid residue selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) is more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F).
第6の改変クモ糸フィブロインは、REPの疎水性度が、-0.8以上であることが好ましく、-0.7以上であることがより好ましく、0以上であることが更に好ましく、0.3以上であることが更により好ましく、0.4以上であることが特に好ましい。REPの疎水性度の上限に特に制限はなく、1.0以下であってよく、0.7以下であってもよい。
In the sixth modified spider silk fibroin, the hydrophobicity of REP is preferably -0.8 or more, more preferably -0.7 or more, still more preferably 0 or more. It is still more preferably 3 or more, and particularly preferably 0.4 or more. The upper limit of the hydrophobicity of REP is not particularly limited, and may be 1.0 or less, or may be 0.7 or less.
本明細書において、「REPの疎水性度」は、以下の方法により算出される値である。
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列(図3の「領域A」に相当する配列。)に含まれる全てのREPにおいて、その領域の各アミノ酸残基の疎水性指標の総和をvとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、REPの疎水性度はv/tとして算出される。REPの疎水性度の算出において、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, “REP hydrophobicity” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs contained in the sequence (sequence corresponding to “region A” in FIG. 3) in which the sequence from the (A) n motif to the C-terminus of the domain sequence located at the side of the (A) is excluded from the domain sequence, The sum of the hydrophobicity indices of each amino acid residue is defined as 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 the (A) n motif is further removed. Assuming that the total number of amino acid residues in all REPs is t, the hydrophobicity of the REP is calculated as v / t. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むクモ糸フィブロインにおいて、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列(図3の「領域A」に相当する配列。)に含まれる全てのREPにおいて、その領域の各アミノ酸残基の疎水性指標の総和をvとし、最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除き、更に(A)nモチーフを除いた全REPのアミノ酸残基の総数をtとしたときに、REPの疎水性度はv/tとして算出される。REPの疎水性度の算出において、「最もC末端側に位置する(A)nモチーフからドメイン配列のC末端までの配列をドメイン配列から除いた配列」を対象としている理由は、上述した理由と同様である。 In the present specification, “REP hydrophobicity” is a value calculated by the following method.
Formula 1: [(A) n motif -rep] m, or Formula 2: [(A) n motif -REP] m - (A) in the spider silk fibroin comprising a domain sequence represented by n motifs, the most C-terminal In all REPs contained in the sequence (sequence corresponding to “region A” in FIG. 3) in which the sequence from the (A) n motif to the C-terminus of the domain sequence located at the side of the (A) is excluded from the domain sequence, The sum of the hydrophobicity indices of each amino acid residue is defined as 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 the (A) n motif is further removed. Assuming that the total number of amino acid residues in all REPs is t, the hydrophobicity of the REP is calculated as v / t. In the calculation of the degree of hydrophobicity of the REP, the reason why the “sequence in which the sequence from the (A) n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence” is targeted is as follows. The same is true.
第6の改変クモ糸フィブロインは、そのドメイン配列が、天然由来のクモ糸フィブロインと比較して、REP中の1又は複数のグルタミン残基を欠失したこと、及び/又はREP中の1又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当する改変に加え、更に1又は複数のアミノ酸残基を置換、欠失、挿入及び/又は付加したことに相当するアミノ酸配列の改変があってもよい。
The sixth modified spider silk fibroin may have a domain sequence that lacks one or more glutamine residues in the REP, and / or one or more glutamine residues in the REP, as compared to the naturally occurring spider silk fibroin. In addition to the modification corresponding to the substitution of a glutamine residue with another amino acid residue, the modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues is also included. There may be.
第6の改変クモ糸フィブロインは、例えば、クローニングした天然由来のクモ糸フィブロインの遺伝子配列からREP中の1又は複数のグルタミン残基を欠失させること、及び/又はREP中の1又は複数のグルタミン残基を他のアミノ酸残基に置換することにより得ることができる。また、例えば、天然由来のクモ糸フィブロインのアミノ酸配列からREP中の1又は複数のグルタミン残基を欠失したこと、及び/又はREP中の1又は複数のグルタミン残基を他のアミノ酸残基に置換したことに相当するアミノ酸配列を設計し、設計したアミノ酸配列をコードする核酸を化学合成することにより得ることもできる。
The sixth modified spider silk fibroin may be, for example, by deleting one or more glutamine residues in the REP from the cloned naturally occurring spider silk fibroin gene sequence, and / or one or more glutamine in the REP. It can be obtained by replacing a residue with another amino acid residue. In addition, for example, one or more glutamine residues in REP have been deleted from the amino acid sequence of naturally occurring spider silk fibroin, and / or one or more glutamine residues in REP have been replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the substitution and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.
第6の改変クモ糸フィブロインのより具体的な例として、(6-i)配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33若しくは配列番号43で示されるアミノ酸配列を含む、改変クモ糸フィブロイン、又は(6-ii)配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33若しくは配列番号43で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As a more specific example of the sixth modified spider silk fibroin, (6-i) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: A modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 43, or (6-ii) SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: Modified spider silk fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 43 can be mentioned.
(6-i)の改変クモ糸フィブロインについて説明する。
The modified spider silk fibroin of (6-i) will be described.
配列番号7で示されるアミノ酸配列(Met-PRT410)は、天然由来のフィブロインであるNephila clavipes(GenBankアクセッション番号:P46804.1、GI:1174415)の塩基配列及びアミノ酸配列に基づき、(A)nモチーフ中のアラニン残基が連続するアミノ酸配列をアラニン残基が連続する数を5つにする等の生産性を向上させるためのアミノ酸の改変を行ったものである。一方、Met-PRT410は、グルタミン残基(Q)の改変は行っていないため、グルタミン残基含有率は、天然由来のフィブロインのグルタミン残基含有率と同程度である。
The amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) is based on the nucleotide sequence and amino acid sequence of a naturally occurring fibroin, Nephila clavipes (GenBank Accession Number: P468804.1, GI: 1174415), and (A) n The amino acid sequence in which the alanine residues in the motif are consecutive has been modified such that the number of consecutive alanine residues is changed to 5, for example, to improve productivity. On the other hand, in Met-PRT410, the glutamine residue (Q) is not modified, so that the glutamine residue content is almost the same as that of naturally occurring fibroin.
配列番号27で示されるアミノ酸配列(M_PRT888)は、Met-PRT410(配列番号7)中のQQを全てVLに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 27 (M_PRT888) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VL.
配列番号28で示されるアミノ酸配列(M_PRT965)は、Met-PRT410(配列番号7)中のQQを全てTSに置換し、かつ残りのQをAに置換したものである。
ア ミ ノ 酸 The amino acid sequence (M_PRT965) represented by SEQ ID NO: 28 is obtained by substituting all QQs in Met-PRT410 (SEQ ID NO: 7) with TS and replacing the remaining Qs with A.
配列番号29で示されるアミノ酸配列(M_PRT889)は、Met-PRT410(配列番号7)中のQQを全てVLに置換し、かつ残りのQをIに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 29 (M_PRT889) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VL and replacing the remaining Qs with I.
配列番号30で示されるアミノ酸配列(M_PRT916)は、Met-PRT410(配列番号7)中のQQを全てVIに置換し、かつ残りのQをLに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 30 (M_PRT916) is obtained by substituting all QQ in Met-PRT410 (SEQ ID NO: 7) with VI and substituting the remaining Q with L.
配列番号31で示されるアミノ酸配列(M_PRT918)は、Met-PRT410(配列番号7)中のQQを全てVFに置換し、かつ残りのQをIに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 31 (M_PRT918) is obtained by replacing all QQs in Met-PRT410 (SEQ ID NO: 7) with VF and replacing the remaining Qs with I.
配列番号34で示されるアミノ酸配列(M_PRT525)は、Met-PRT410(配列番号7)に対し、アラニン残基が連続する領域(A5)に2つのアラニン残基を挿入し、Met-PRT410の分子量とほぼ同じになるよう、C末端側のドメイン配列2つを欠失させ、かつグルタミン残基(Q)13箇所をセリン残基(S)又はプロリン残基(P)に置換したものである。
The amino acid sequence (M_PRT525) represented by SEQ ID NO: 34 is obtained by inserting two alanine residues into a region (A 5 ) in which alanine residues are continuous with Met-PRT410 (SEQ ID NO: 7), In this example, two C-terminal domain sequences were deleted, and 13 glutamine residues (Q) were replaced with a serine residue (S) or a proline residue (P).
配列番号32で示されるアミノ酸配列(M_PRT699)は、M_PRT525(配列番号34)中のQQを全てVLに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 32 (M_PRT699) is obtained by replacing all QQs in M_PRT525 (SEQ ID NO: 34) with VL.
配列番号33で示されるアミノ酸配列(M_PRT698)は、M_PRT525(配列番号34)中のQQを全てVLに置換し、かつ残りのQをIに置換したものである。
ア ミ ノ 酸 The amino acid sequence represented by SEQ ID NO: 33 (M_PRT698) is obtained by substituting all QQs in M_PRT525 (SEQ ID NO: 34) with VL and replacing the remaining Qs with I.
配列番号43で示されるアミノ酸配列(Met-PRT966)は、配列番号9で示されるアミノ酸配列(C末端に配列番号42で示されるアミノ酸配列が付加される前のアミノ酸配列)中のQQを全てVFに置換し、かつ残りのQをIに置換したものである。
The amino acid sequence represented by SEQ ID NO: 43 (Met-PRT966) is obtained by converting all the QQs in the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence before the amino acid sequence represented by SEQ ID NO: 42 is added to the C-terminus) to VF , And the remaining Q is replaced by I.
配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33及び配列番号43で示されるアミノ酸配列は、いずれもグルタミン残基含有率は9%以下である(表2)。
The amino acid sequences represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 all have a glutamine residue content of 9% or less. (Table 2).
(6-i)の改変クモ糸フィブロインは、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33又は配列番号43で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (6-i) consists of the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 It may be something.
(6-ii)の改変クモ糸フィブロインは、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33又は配列番号43で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(6-ii)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (6-ii) has an amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 and 90 % Or more. The modified spider silk fibroin of (6-ii) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence. The sequence identity is preferably 95% or more.
(6-ii)の改変クモ糸フィブロインは、グルタミン残基含有率が9%以下であることが好ましい。また、(6-ii)の改変クモ糸フィブロインは、GPGXXモチーフ含有率が10%以上であることが好ましい。
The modified spider silk fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified spider silk fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.
第6の改変クモ糸フィブロインは、N末端及びC末端のいずれか一方又は両方にタグ配列を含んでいてもよい。これにより、改変クモ糸フィブロインの単離、固定化、検出及び可視化等が可能となる。
6The sixth modified spider silk fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. As a result, the modified spider silk fibroin can be isolated, immobilized, detected, visualized, and the like.
タグ配列を含む第6の改変クモ糸フィブロインのより具体的な例として、(6-iii)配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41若しくは配列番号44で示されるアミノ酸配列を含む、改変フィブロイン、又は(6-iv)配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41若しくは配列番号44で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含む、改変クモ糸フィブロインを挙げることができる。
As more specific examples of the sixth modified spider silk fibroin including the tag sequence, (6-iii) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: A modified fibroin comprising the amino acid sequence represented by SEQ ID NO: 41 or SEQ ID NO: 44, or (6-iv) SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or A modified spider silk fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 44 can be mentioned.
配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41及び配列番号44で示されるアミノ酸配列は、それぞれ配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33及び配列番号43で示されるアミノ酸配列のN末端に配列番号12で示されるアミノ酸配列(Hisタグ配列及びヒンジ配列を含む)を付加したものである。N末端にタグ配列を付加しただけであるため、グルタミン残基含有率に変化はなく、配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41及び配列番号44で示されるアミノ酸配列は、いずれもグルタミン残基含有率が9%以下である(表3)。
The amino acid sequences represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44 are SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, respectively. The amino acid sequence represented by SEQ ID NO: 12 (including the His tag sequence and the hinge sequence) was added to the N-terminal of the amino acid sequence represented by SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 43 Things. Since only a tag sequence was added to the N-terminus, there was no change in the glutamine residue content, and SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 And the amino acid sequence represented by SEQ ID NO: 44 has a glutamine residue content of 9% or less (Table 3).
(6-iii)の改変クモ糸フィブロインは、配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41又は配列番号44で示されるアミノ酸配列からなるものであってもよい。
The modified spider silk fibroin of (6-iii) consists of the amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44 It may be something.
(6-iv)の改変クモ糸フィブロインは、配列番号35、配列番号36、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41又は配列番号44で示されるアミノ酸配列と90%以上の配列同一性を有するアミノ酸配列を含むものである。(6-iv)の改変クモ糸フィブロインもまた、式1:[(A)nモチーフ-REP]m、又は式2:[(A)nモチーフ-REP]m-(A)nモチーフで表されるドメイン配列を含むタンパク質である。上記配列同一性は、95%以上であることが好ましい。
The modified spider silk fibroin of (6-iv) has an amino acid sequence represented by SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44, and 90 % Or more. The modified spider silk fibroin of (6-iv) is also represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m- (A) n motif Is a protein containing a domain sequence. The sequence identity is preferably 95% or more.
(6-iv)の改変クモ糸フィブロインは、グルタミン残基含有率が9%以下であることが好ましい。また、(6-iv)の改変クモ糸フィブロインは、GPGXXモチーフ含有率が10%以上であることが好ましい。
The modified spider silk fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. The modified spider silk fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.
第6の改変クモ糸フィブロインは、組換えタンパク質生産系において生産されたタンパク質を宿主の外部に放出するための分泌シグナルを含んでいてもよい。分泌シグナルの配列は、宿主の種類に応じて適宜設定することができる。
(6) The sixth modified spider silk fibroin may contain a secretion signal for releasing a protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.
改変クモ糸フィブロインは、第1の改変クモ糸フィブロイン、第2の改変クモ糸フィブロイン、第3の改変クモ糸フィブロイン、第4の改変クモ糸フィブロイン、第5の改変クモ糸フィブロイン、及び第6の改変クモ糸フィブロインが有する特徴のうち、少なくとも2つ以上の特徴を併せ持つ改変クモ糸フィブロインであってもよい。
The modified spider silk fibroin comprises a first modified spider silk fibroin, a second modified spider silk fibroin, a third modified spider silk fibroin, a fourth modified spider silk fibroin, a fifth modified spider silk fibroin, and a sixth modified spider silk fibroin. Among the characteristics of the modified spider silk fibroin, the modified spider silk fibroin having at least two or more characteristics may be used.
改変クモ糸フィブロインは、親水性改変クモ糸フィブロインであってもよく、疎水性改変クモ糸フィブロインであってもよい。疎水性改変クモ糸フィブロインとは、改変クモ糸フィブロインを構成する全てのアミノ酸残基の疎水性指標(HI)の総和を求め、次にその総和を全アミノ酸残基数で除した値(平均HI)が0以上である改変クモ糸フィブロインである。疎水性指標は表1に示したとおりである。また、親水性改変クモ糸フィブロインとは、上記の平均HIが0未満である改変クモ糸フィブロインである。
The modified spider silk fibroin may be a hydrophilic modified spider silk fibroin or a hydrophobic modified spider silk fibroin. Hydrophobic modified spider silk fibroin refers to a value obtained by calculating the sum of the hydrophobicity indexes (HI) of all amino acid residues constituting the modified spider silk fibroin, and then dividing the sum by the total number of amino acid residues (average HI). ) Is 0 or more. The hydrophobicity index is as shown in Table 1. The hydrophilic modified spider silk fibroin is a modified spider silk fibroin having the above average HI of less than 0.
疎水性改変クモ糸フィブロインとしては、例えば、上述した第6の改変フィブロインを挙げることができる。疎水性改変クモ糸フィブロインのより具体的な例としては、配列番号27、配列番号28、配列番号29、配列番号30、配列番号31、配列番号32、配列番号33又は配列番号43で示されるアミノ酸配列、配列番号35、配列番号37、配列番号38、配列番号39、配列番号40、配列番号41又は配列番号44で示されるアミノ酸配列を含む改変クモ糸フィブロインが挙げられる。
Examples of the hydrophobic modified spider silk fibroin include the sixth modified fibroin described above. More specific examples of the hydrophobically modified spider silk fibroin include amino acids represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43 Modified spider silk fibroin comprising the amino acid sequence represented by the sequence, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.
親水性改変クモ糸フィブロインとしては、例えば、上述した第1の改変フィブロイン、第2の改変フィブロイン、第3の改変フィブロイン、第4の改変フィブロイン、及び第5の改変フィブロインを挙げることができる。親水性クモ糸タンパク質のより具体的な例としては、配列番号4で示されるアミノ酸配列、配列番号6、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列、配列番号13、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列、配列番号18、配列番号7、配列番号8又は配列番号9で示されるアミノ酸配列、配列番号17、配列番号11、配列番号14又は配列番号15で示されるアミノ酸配列、配列番号19、配列番号20又は配列番号21で示されるアミノ酸配列を含む改変クモ糸フィブロインが挙げられる。
Examples of the hydrophilic modified spider silk fibroin include the above-mentioned first modified fibroin, second modified fibroin, third modified fibroin, fourth modified fibroin, and fifth modified fibroin. More specific examples of the hydrophilic spider silk protein include an amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, an amino acid sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: Modified spider silk fibroin comprising the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
上述した改変クモ糸フィブロインは、1種を単独で、又は2種以上を組み合わせて用いることができる。
は The above-mentioned modified spider silk fibroin can be used alone or in combination of two or more.
改変クモ糸フィブロインは、例えば、当該改変クモ糸フィブロインをコードする核酸配列と、当該核酸配列に作動可能に連結された1又は複数の調節配列とを有する発現ベクターで形質転換された宿主により、当該核酸を発現させることにより生産することができる。
The modified spider silk fibroin is, for example, a host transformed with an expression vector having a nucleic acid sequence encoding the modified spider silk fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing a nucleic acid.
改変クモ糸フィブロインをコードする核酸の製造方法は、特に制限されない。例えば、改変クモ糸フィブロインをコードする遺伝子を利用して、ポリメラーゼ連鎖反応(PCR)等で増幅しクローニングする方法、又は、化学的に合成する方法によって、当該核酸を製造することができる。核酸の化学的な合成方法も特に制限されず、例えば、NCBIのウェブデータベースなどより入手したクモ糸タンパク質のアミノ酸配列情報をもとに、AKTA oligopilot plus 10/100(GEヘルスケア・ジャパン株式会社)等で自動合成したオリゴヌクレオチドをPCR等で連結する方法によって遺伝子を化学的に合成することができる。この際に、改変クモ糸フィブロインの精製及び/又は確認を容易にするため、N末端に開始コドン及びHis10タグからなるアミノ酸配列を付加したアミノ酸配列からなる改変クモ糸フィブロインをコードする核酸を合成してもよい。
方法 The method for producing the nucleic acid encoding the modified spider silk fibroin is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by polymerase chain reaction (PCR) using a gene encoding a modified spider silk fibroin, or a method of chemically synthesizing. The method for chemically synthesizing nucleic acids is not particularly limited. For example, based on amino acid sequence information of spider silk proteins obtained from the NCBI web database or the like, AKTA oligopilot plus 10/100 (GE Healthcare Japan KK) Genes can be chemically synthesized by a method of linking oligonucleotides synthesized automatically by PCR or the like by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified spider silk fibroin, a nucleic acid encoding the modified spider silk fibroin consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus was synthesized. You may.
調節配列は、宿主における組換えタンパク質の発現を制御する配列(例えば、プロモーター、エンハンサー、リボソーム結合配列、転写終結配列等)であり、宿主の種類に応じて適宜選択することができる。プロモーターとして、宿主細胞中で機能し、目的とする改変クモ糸フィブロインを発現誘導可能な誘導性プロモーターを用いてもよい。誘導性プロモーターは、誘導物質(発現誘導剤)の存在、リプレッサー分子の非存在、又は温度、浸透圧若しくはpH値の上昇若しくは低下等の物理的要因により、転写を制御できるプロモーターである。
The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and is capable of inducing the expression of the desired modified spider silk fibroin may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor such as an increase or decrease in temperature, osmotic pressure, or pH value.
発現ベクターの種類は、プラスミドベクター、ウイルスベクター、コスミドベクター、フォスミドベクター、人工染色体ベクター等、宿主の種類に応じて適宜選択することができる。発現ベクターとしては、宿主細胞において自立複製が可能、又は宿主の染色体中への組込みが可能で、改変クモ糸フィブロインをコードする核酸を転写できる位置にプロモーターを含有しているものが好適に用いられる。
種類 The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. As the expression vector, those capable of autonomous replication in a host cell or integration into a host chromosome and containing a promoter at a position where a nucleic acid encoding a modified spider silk fibroin can be transcribed are suitably used. .
宿主として、原核生物、並びに酵母、糸状真菌、昆虫細胞、動物細胞及び植物細胞等の真核生物のいずれも好適に用いることができる。
As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be suitably used.
細菌等の原核生物を宿主として用いる場合は、発現ベクターは、原核生物中で自立複製が可能であると同時に、プロモーター、リボソーム結合配列、改変クモ糸フィブロインをコードする核酸、及び転写終結配列を含むベクターであることが好ましい。プロモーターを制御する遺伝子が含まれていてもよい。
When a prokaryote such as a bacterium is used as a host, the expression vector is capable of autonomous replication in the prokaryote and contains a promoter, a ribosome binding sequence, a nucleic acid encoding a modified spider silk fibroin, and a transcription termination sequence. It is preferably a vector. A gene that controls the promoter may be included.
原核生物としては、エシェリヒア属、ブレビバチルス属、セラチア属、バチルス属、ミクロバクテリウム属、ブレビバクテリウム属、コリネバクテリウム属及びシュードモナス属等に属する微生物を挙げることができる。エシェリヒア属に属する微生物として、例えば、エシェリヒア・コリ等を挙げることができる。ブレビバチルス属に属する微生物として、例えば、ブレビバチルス・アグリ等を挙げることができる。セラチア属に属する微生物として、例えば、セラチア・リクエファシエンス等を挙げることができる。バチルス属に属する微生物として、例えば、バチルス・サチラス等を挙げることができる。ミクロバクテリウム属に属する微生物として、例えば、ミクロバクテリウム・アンモニアフィラム等を挙げることができる。ブレビバクテリウム属に属する微生物として、例えば、ブレビバクテリウム・ディバリカタム等を挙げることができる。コリネバクテリウム属に属する微生物として、例えば、コリネバクテリウム・アンモニアゲネス等を挙げることができる。シュードモナス(Pseudomonas)属に属する微生物として、例えば、シュードモナス・プチダ等を挙げることができる。
Prokaryotes include microorganisms belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Microorganisms belonging to the genus Serratia include, for example, Serratia requestifaciens and the like. Examples of microorganisms belonging to the genus Bacillus include, for example, Bacillus subtilis. Microorganisms belonging to the genus Microbacterium include, for example, Microbacterium ammonia phyllum. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum. Examples of the microorganism belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
原核生物を宿主とする場合、改変クモ糸フィブロインをコードする核酸を導入するベクターとしては、例えば、pBTrp2(ベーリンガーマンハイム社製)、pGEX(Pharmacia社製)、pUC18、pBluescriptII、pSupex、pET22b、pCold、pUB110、pNCO2(特開2002-238569号公報)等を挙げることができる。
When a prokaryote is used as a host, as a vector for introducing a nucleic acid encoding a modified spider silk fibroin, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 (JP-A-2002-238569) and the like.
真核生物の宿主としては、例えば、酵母及び糸状真菌(カビ等)を挙げることができる。酵母としては、例えば、サッカロマイセス属、ピキア属、シゾサッカロマイセス属等に属する酵母を挙げることができる。糸状真菌としては、例えば、アスペルギルス属、ペニシリウム属、トリコデルマ(Trichoderma)属等に属する糸状真菌を挙げることができる。
Examples of eukaryotic hosts include yeast and filamentous fungi (such as mold). Examples of the yeast include yeast belonging to the genus Saccharomyces, the genus Pichia, the genus Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
真核生物を宿主とする場合、改変クモ糸フィブロインをコードする核酸を導入するベクターとしては、例えば、YEp13(ATCC37115)、YEp24(ATCC37051)等を挙げることができる。上記宿主細胞への発現ベクターの導入方法としては、上記宿主細胞へDNAを導入する方法であればいずれも用いることができる。例えば、カルシウムイオンを用いる方法〔Proc. Natl. Acad. Sci. USA,69,2110(1972)〕、エレクトロポレーション法、スフェロプラスト法、プロトプラスト法、酢酸リチウム法、コンピテント法等を挙げることができる。
場合 When a eukaryote is used as a host, examples of a vector into which a nucleic acid encoding a modified spider silk fibroin is introduced include YEp13 (ATCC 37115) and YEp24 (ATCC 37051). As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. {Natl. {Acad. {Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, competent, and the like.
発現ベクターで形質転換された宿主による核酸の発現方法としては、直接発現のほか、モレキュラー・クローニング第2版に記載されている方法等に準じて、分泌生産、融合タンパク質発現等を行うことができる。
As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .
改変クモ糸フィブロインは、例えば、形質転換された宿主を培養培地中で培養し、培養培地中に改変クモ糸フィブロインを生成蓄積させ、該培養培地から採取することにより製造することができる。形質転換された宿主を培養培地中で培養する方法は、宿主の培養に通常用いられる方法に従って行うことができる。
The modified spider silk fibroin can be produced, for example, by culturing the transformed host in a culture medium, producing and accumulating the modified spider silk fibroin in the culture medium, and collecting the modified spider silk fibroin from the culture medium. The method of culturing the transformed host in a culture medium can be performed according to a method generally used for culturing a host.
宿主が、大腸菌等の原核生物又は酵母等の真核生物である場合、培養培地として、該宿主が資化し得る炭素源、窒素源及び無機塩類等を含有し、該宿主の培養を効率的に行える培地であれば天然培地、合成培地のいずれを用いてもよい。
When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the host, thereby efficiently culturing the host. Either a natural medium or a synthetic medium may be used as long as the medium can be used.
炭素源としては、該宿主が資化し得るものであればよく、例えば、グルコース、フラクトース、スクロース、及びこれらを含有する糖蜜、デンプン及びデンプン加水分解物等の炭水化物、酢酸及びプロピオン酸等の有機酸、並びにエタノール及びプロパノール等のアルコール類を用いることができる。
The carbon source may be any as long as the host can assimilate it.Examples include glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolysates, and organic acids such as acetic acid and propionic acid. And alcohols such as ethanol and propanol.
窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物を用いることができる。
As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
無機塩類としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムを用いることができる。
As the inorganic salts, for example, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.
大腸菌等の原核生物又は酵母等の真核生物の培養は、例えば、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことができる。培養温度は、例えば、15~40℃である。培養時間は、通常16時間~7日間である。培養中の培養培地のpHは3.0~9.0に保持することが好ましい。培養培地のpHの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。
培養 Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culturing time is usually 16 hours to 7 days. The pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
また、培養中必要に応じて、アンピシリン及びテトラサイクリン等の抗生物質を培養培地に添加してもよい。プロモーターとして誘導性のプロモーターを用いた発現ベクターで形質転換した微生物を培養するときには、必要に応じてインデューサーを培地に添加してもよい。例えば、lacプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはイソプロピル-β-D-チオガラクトピラノシド等を、trpプロモーターを用いた発現ベクターで形質転換した微生物を培養するときにはインドールアクリル酸等を培地に添加してもよい。
中 Further, if necessary, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. An acid or the like may be added to the medium.
形質転換された宿主により生産された改変クモ糸フィブロインは、タンパク質の単離精製に通常用いられている方法で単離及び精製することができる。例えば、改変クモ糸フィブロインが、細胞内に溶解状態で発現した場合には、培養終了後、宿主細胞を遠心分離により回収し、水系緩衝液にけん濁した後、超音波破砕機、フレンチプレス、マントンガウリンホモゲナイザー及びダイノミル等により宿主細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られる上清から、タンパク質の単離精製に通常用いられている方法、すなわち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル(DEAE)-セファロース、DIAION HPA-75(三菱化成社製)等のレジンを用いた陰イオン交換クロマトグラフィー法、S-Sepharose FF(Pharmacia社製)等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィー法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の方法を単独又は組み合わせて使用し、精製標品を得ることができる。
改 変 The modified spider silk fibroin produced by the transformed host can be isolated and purified by a method usually used for protein isolation and purification. For example, when the modified spider silk fibroin is expressed in a dissolved state in the cells, after completion of the culture, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, The host cells are crushed with a Manton-Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for isolating and purifying proteins, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent Precipitation method, anion-exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION @ HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose @ FF (manufactured by Pharmacia). Electrophoretic methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. Purification using methods such as alone or in combination It is possible to obtain the goods.
上記クロマトグラフィーとしては、フェニル-トヨパール(東ソー)、DEAE-トヨパール(東ソー)、セファデックスG-150(ファルマシアバイオテク)を用いたカラムクロマトグラフィーが好ましく用いられる。
カ ラ ム As the above chromatography, column chromatography using phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), and Sephadex G-150 (Pharmacia Biotech) is preferably used.
また、改変クモ糸フィブロインが細胞内に不溶体を形成して発現した場合は、同様に宿主細胞を回収後、破砕し、遠心分離を行うことにより、沈殿画分として改変クモ糸フィブロインの不溶体を回収する。回収した改変クモ糸フィブロインの不溶体は蛋白質変性剤で可溶化することができる。該操作の後、上記と同様の単離精製法により改変クモ糸フィブロインの精製標品を得ることができる。
Further, when the modified spider silk fibroin is expressed by forming an insoluble body in the cells, the host cells are similarly recovered, crushed, and centrifuged to obtain an insoluble body of the modified spider silk fibroin as a precipitate fraction. Collect. The recovered insoluble form of the modified spider silk fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the modified spider silk fibroin can be obtained by the same isolation and purification method as described above.
改変クモ糸フィブロインが細胞外に分泌された場合には、培養上清から改変クモ糸フィブロインを回収することができる。すなわち、培養物を遠心分離等の手法により処理することにより培養上清を取得し、該培養上清から、上記と同様の単離精製法を用いることにより、精製標品を得ることができる。
When the modified spider silk fibroin is secreted extracellularly, the modified spider silk fibroin can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.
〔改変クモ糸フィブロイン繊維の製造方法〕
本実施形態に係る改変クモ糸フィブロイン繊維の製造方法は、上述した改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液を準備する工程と、当該分散液から原繊維を形成させる工程と、を少なくとも備える。 (Production method of modified spider silk fibroin fiber)
The method for producing a modified spider silk fibroin fiber according to the present embodiment includes a step of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming a raw fiber from the dispersion. And at least a step.
本実施形態に係る改変クモ糸フィブロイン繊維の製造方法は、上述した改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液を準備する工程と、当該分散液から原繊維を形成させる工程と、を少なくとも備える。 (Production method of modified spider silk fibroin fiber)
The method for producing a modified spider silk fibroin fiber according to the present embodiment includes a step of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming a raw fiber from the dispersion. And at least a step.
本明細書において、「原繊維」とは、紡糸原液(ドープ液)として用いる分散液から、分散液の冷却、溶媒の蒸発(気化)、化学反応等により形成された繊維状の固体のことをいう。分散液から原繊維を形成させる工程は、上述した分散液を吐出させることを含んでいてもよく、上述した分散液から原繊維を引き出すことを含んでいてもよく、分散液を吐出させること及び分散液から原繊維を引き出すことの両方を含んでいてもよい。分散液から形成させた原繊維を、そのまま改変クモ糸フィブロイン繊維として任意の用途に用いてもよいし、後述する延伸等の加工を原繊維に対して施したものを改変クモ糸フィブロイン繊維としてもよい。
In the present specification, “fibrils” refers to fibrous solids formed from a dispersion used as a spinning dope (dope) by cooling the dispersion, evaporating a solvent (vaporizing), a chemical reaction, or the like. Say. The step of forming fibrils from the dispersion may include discharging the above-described dispersion, or may include drawing the fibrils from the above-described dispersion, discharging the dispersion, and It may include both drawing fibrils from the dispersion. The fibrils formed from the dispersion may be used as modified spider silk fibroin fibers as they are, or modified spider silk fibroin fibers obtained by subjecting the fibrils to processing such as stretching described below. Good.
本実施形態に係る改変クモ糸フィブロイン繊維の製造方法は、紡糸原液(ドープ液)として用いる分散液を脱泡する工程(脱泡工程)をさらに備えていてもよい。
方法 The method for producing a modified spider silk fibroin fiber according to the present embodiment may further include a step of defoaming a dispersion used as a spinning stock solution (dope solution) (defoaming step).
〔分散液からの原繊維の引き出し〕
本明細書において「原繊維を分散液から引き出す」とは、分散液の液体表面に対して、針等の器具の先の尖った先端を接触させ、該器具を分散液との接触点から離れる方向に引き抜くことにより、分散液と器具の先端との接触点から液体が、器具の先端に付着したまま、細く繊維状に伸びて引き出されることを意味する。繊維状に引き出された液体が固体に変化し、この繊維状の固体が原繊維となる。原繊維を引き出すことを、空気等の気体中でおこなってもよく、液体中でおこなってもよい。液体中でおこなう場合は、該液体は、分散液とは親和性の低い液体、例えば、非極性溶媒であることが好ましい。原繊維を引き出すことを気体中でおこなう場合には、分散液と器具との接触点は、気体/分散液の界面であってもよく、原繊維を引き出すことを液体中でおこなう場合には、分散液と器具との接触点は、該液体/分散液の界面であってもよい。原繊維を引き出すことによって、原繊維の繊維径をより細くすることができる。 [Drawing of fibrils from dispersion]
As used herein, "pulling the fibrils out of the dispersion" refers to bringing the pointed tip of an instrument such as a needle into contact with the liquid surface of the dispersion and moving the instrument away from the point of contact with the dispersion. Withdrawing in the direction means that the liquid is drawn out from the contact point between the dispersion liquid and the tip of the device, while being attached to the tip of the device, extending in a thin fibrous form. The liquid drawn into a fibrous state changes to a solid, and the fibrous solid becomes fibrils. The extraction of the fibrils may be performed in a gas such as air, or may be performed in a liquid. When performed in a liquid, the liquid is preferably a liquid having a low affinity for the dispersion, for example, a non-polar solvent. When drawing fibrils is performed in a gas, the point of contact between the dispersion and the device may be at the gas / dispersion interface, and when drawing fibrils in a liquid, The point of contact between the dispersion and the device may be at the liquid / dispersion interface. By drawing the fibrils, the fiber diameter of the fibrils can be made smaller.
本明細書において「原繊維を分散液から引き出す」とは、分散液の液体表面に対して、針等の器具の先の尖った先端を接触させ、該器具を分散液との接触点から離れる方向に引き抜くことにより、分散液と器具の先端との接触点から液体が、器具の先端に付着したまま、細く繊維状に伸びて引き出されることを意味する。繊維状に引き出された液体が固体に変化し、この繊維状の固体が原繊維となる。原繊維を引き出すことを、空気等の気体中でおこなってもよく、液体中でおこなってもよい。液体中でおこなう場合は、該液体は、分散液とは親和性の低い液体、例えば、非極性溶媒であることが好ましい。原繊維を引き出すことを気体中でおこなう場合には、分散液と器具との接触点は、気体/分散液の界面であってもよく、原繊維を引き出すことを液体中でおこなう場合には、分散液と器具との接触点は、該液体/分散液の界面であってもよい。原繊維を引き出すことによって、原繊維の繊維径をより細くすることができる。 [Drawing of fibrils from dispersion]
As used herein, "pulling the fibrils out of the dispersion" refers to bringing the pointed tip of an instrument such as a needle into contact with the liquid surface of the dispersion and moving the instrument away from the point of contact with the dispersion. Withdrawing in the direction means that the liquid is drawn out from the contact point between the dispersion liquid and the tip of the device, while being attached to the tip of the device, extending in a thin fibrous form. The liquid drawn into a fibrous state changes to a solid, and the fibrous solid becomes fibrils. The extraction of the fibrils may be performed in a gas such as air, or may be performed in a liquid. When performed in a liquid, the liquid is preferably a liquid having a low affinity for the dispersion, for example, a non-polar solvent. When drawing fibrils is performed in a gas, the point of contact between the dispersion and the device may be at the gas / dispersion interface, and when drawing fibrils in a liquid, The point of contact between the dispersion and the device may be at the liquid / dispersion interface. By drawing the fibrils, the fiber diameter of the fibrils can be made smaller.
分散液と接触させる器具としては、分散液との接触点を小さく形成させるために先端が尖っているものが好ましく、例えば、針、ピペットチップ、ピンセット、串、楊枝、細い棒等を挙げられるが、これらに限定されない。また、器具の材質として、プラスチック、金属、ガラス、木等が挙げられるが、これらに限定されない。
The device to be brought into contact with the dispersion is preferably one having a sharp tip in order to form a small point of contact with the dispersion, and examples thereof include a needle, a pipette tip, tweezers, a skewer, a toothpick, and a thin stick. However, it is not limited to these. In addition, examples of the material of the device include, but are not limited to, plastic, metal, glass, and wood.
器具を分散液との接触点から離れる方向に引き抜く速度を、分散液の粘度によって調整することが好ましい。また、引き抜く速度を一定とすることが好ましい。引き抜く速度を一定とすることで、繊維のよれ、たるみ又は切れ等を防止し、また、繊維径のばらつきを抑えることができる。引き抜く速度として、例えば、0.1cm/秒以上、0.1cm/秒~15m/秒、1cm/秒~5m/秒、3cm/秒~1m/秒、5cm/秒~50cm/秒、5cm/秒~35cm/秒が挙げられる。
速度 It is preferable that the speed at which the device is withdrawn in the direction away from the point of contact with the dispersion is adjusted by the viscosity of the dispersion. Further, it is preferable that the drawing speed is constant. By keeping the pulling speed constant, it is possible to prevent the fiber from being twisted, sagged or cut, and to suppress the variation in the fiber diameter. As the drawing speed, for example, 0.1 cm / sec or more, 0.1 cm / sec to 15 m / sec, 1 cm / sec to 5 m / sec, 3 cm / sec to 1 m / sec, 5 cm / sec to 50 cm / sec, 5 cm / sec 3535 cm / sec.
分散液に器具の先端を接着させて器具を引き抜いた直後には、原繊維の先端と器具の先端とは接着しているが、原繊維がいったん引き出された後には、器具の先端と原繊維の先端とが接着していなくともよい。例えば、原繊維の長さが5cm以上形成された後には、該原繊維を引っ張り続けることにより、器具の先端と原繊維の先端とが接着していなくとも、分散液から原繊維が引き出され続ける。繊維径を一定させるためには、好ましくは器具を引き抜く速度と同じ速度で、原繊維を引っ張り続けることが好ましい。
Immediately after the tip of the instrument is attached to the dispersion liquid and the instrument is pulled out, the tip of the fibril and the tip of the instrument are adhered, but once the fibril is pulled out, the tip of the instrument and the Does not have to be bonded to the tip. For example, after the length of the fibrils is formed to be 5 cm or more, by continuing to pull the fibrils, the fibrils continue to be drawn out of the dispersion liquid even if the tip of the device and the tips of the fibrils are not bonded. . In order to keep the fiber diameter constant, it is preferable to continue pulling the raw fiber at a speed preferably equal to the speed at which the device is pulled out.
〔分散液の吐出〕
本明細書において「分散液を吐出させる」とは、分散液に対して圧力を加えて又は加えないで、ノズルから分散液を吐出させることを意味する。ノズルを通って細く吐出された分散液が溶媒の蒸発(気化)、化学反応等によって固体となることにより、原繊維が形成される。分散液を吐出させる先は、空気中に吐出させてもよく、液体(凝固液)中に吐出させてもよい。 (Discharge of dispersion liquid)
In the present specification, “discharging the dispersion liquid” means discharging the dispersion liquid from the nozzle with or without applying pressure to the dispersion liquid. The fine fibers are formed by the dispersion liquid finely discharged through the nozzle becoming solid by evaporation (vaporization) of the solvent, chemical reaction, or the like. The destination where the dispersion liquid is discharged may be discharged into the air or may be discharged into a liquid (coagulating liquid).
本明細書において「分散液を吐出させる」とは、分散液に対して圧力を加えて又は加えないで、ノズルから分散液を吐出させることを意味する。ノズルを通って細く吐出された分散液が溶媒の蒸発(気化)、化学反応等によって固体となることにより、原繊維が形成される。分散液を吐出させる先は、空気中に吐出させてもよく、液体(凝固液)中に吐出させてもよい。 (Discharge of dispersion liquid)
In the present specification, “discharging the dispersion liquid” means discharging the dispersion liquid from the nozzle with or without applying pressure to the dispersion liquid. The fine fibers are formed by the dispersion liquid finely discharged through the nozzle becoming solid by evaporation (vaporization) of the solvent, chemical reaction, or the like. The destination where the dispersion liquid is discharged may be discharged into the air or may be discharged into a liquid (coagulating liquid).
ノズルから分散液を吐出する方法に特に制限はないが、例えば、紡糸原液の送液手段として定量ポンプを用いる方法を使用することができる。吐出量は生産速度に応じて適宜調整することができる。
方法 The method of discharging the dispersion liquid from the nozzle is not particularly limited. For example, a method using a metering pump as a means for sending the spinning solution can be used. The discharge amount can be appropriately adjusted according to the production speed.
分散液を吐出させるノズルとして、紡糸口金を用いてもよい。紡糸口金の口金形状、ホール形状、ホール数などは特に限定されるものではなく、所望の繊維径及び単糸本数等に応じて適宜選択できる。
紡 A spinneret may be used as a nozzle for discharging the dispersion. The spinneret shape, hole shape, number of holes, and the like of the spinneret are not particularly limited, and can be appropriately selected according to the desired fiber diameter, the number of single yarns, and the like.
紡糸口金のホール形状が円形である場合は、紡糸口金の孔径として0.01mm以上0.6mm以下を例示できる。孔径が0.01mm以上であると、圧力損失を低減することができ設備費用を抑えることができる。孔径が0.6mm以下であると、繊維径を細くするための延伸操作の必要性を低減することができ、吐出から巻き取りまでの間で延伸切れを起こす可能性を低減することができる。
When the hole shape of the spinneret is circular, the hole diameter of the spinneret can be, for example, 0.01 mm or more and 0.6 mm or less. When the hole diameter is 0.01 mm or more, the pressure loss can be reduced and the equipment cost can be reduced. When the pore diameter is 0.6 mm or less, it is possible to reduce the necessity of a stretching operation for reducing the fiber diameter, and it is possible to reduce the possibility of stretching breakage from discharge to winding.
紡糸口金を通過する際の分散液の温度、及び紡糸口金の温度は、特に限定されるものではなく、用いる分散液の濃度及び粘度、分散液に含まれる極性溶媒の種類等により適宜調整すればよい。当該温度は、改変クモ糸フィブロインの劣化等を防止するという観点から、30℃~100℃が好ましい。また、当該温度は、溶媒の揮発による圧力上昇、分散液の固形化による配管内の閉塞が発生する可能性を低減するという観点から、用いる溶媒の沸点に満たない温度を上限とすることが好ましい。これにより工程安定性が向上する。
The temperature of the dispersion when passing through the spinneret, and the temperature of the spinneret are not particularly limited, and may be appropriately adjusted depending on the concentration and viscosity of the dispersion used, the type of the polar solvent contained in the dispersion, and the like. Good. The temperature is preferably from 30 ° C. to 100 ° C. from the viewpoint of preventing the modified spider silk fibroin from deteriorating. In addition, the temperature is preferably set to an upper limit of a temperature lower than the boiling point of the solvent to be used, from the viewpoint of reducing the pressure increase due to the volatilization of the solvent and the possibility of clogging in the pipe due to solidification of the dispersion. . This improves the process stability.
なお、本実施形態においては、分散液を吐出させることと原繊維を引き出すことのどちらか一方をおこなって原繊維を形成してもよく、両方をおこなってもよい。分散液を吐出させること及び原繊維を引き出すことの両方をおこなう場合には、例えば、分散液をノズルから吐出させ、吐出された分散液に対して器具の先端を接触させ、該器具を分散液から離れる方向に引き抜き、接触点から原繊維を引き出すことによって、原繊維を形成させることができる。
In the present embodiment, either the discharging of the dispersion or the extraction of the fibrils may be performed to form the fibrils, or both may be performed. When performing both the discharging of the dispersion and the drawing out of the fibrils, for example, the dispersion is discharged from a nozzle, the tip of the device is brought into contact with the discharged dispersion, and the device is dispersed. By pulling out the fiber in a direction away from the fiber and pulling out the fiber from the contact point, the fiber can be formed.
〔乾式紡糸法〕
乾式紡糸法は、紡糸原液(ドープ液)中の溶媒を蒸発(気化)させることによって紡糸(原繊維を形成)する方法である。上述のように、本実施形態においては、原繊維を形成させる際に、原繊維を分散液から引き出すことを空気等の気体中でおこなってもよく、また、分散液の吐出において、分散液を吐出させる先として、空気等の気体中に吐出させてもよい。このように気体中で原繊維を形成させる際には、分散液中の溶媒を蒸発させることにより、原繊維が形成される。すなわち、本実施形態においては、原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させてもよい。溶媒を蒸発させる方法として、熱風乾燥、加熱乾燥、送風乾燥、自然乾燥等の公知の乾式紡糸法に用いられる乾燥方法を挙げることができる。ただし、本実施形態においては、形成される原繊維が極めて細径であるため、熱風乾燥、加熱乾燥、送風乾燥等の積極的な乾燥を行わなくとも、自然に溶媒が蒸発し、原繊維を形成することができ、効率がよい。
特に、分散液に含まれる炭素材料としてカーボンブラックナノパーティクルを用いる場合は、乾式紡糸法によって原繊維を形成させることが好ましい。 (Dry spinning method)
The dry spinning method is a method of spinning (forming fibrils) by evaporating (vaporizing) a solvent in a spinning solution (dope solution). As described above, in the present embodiment, when forming the fibrils, the drawing of the fibrils from the dispersion may be performed in a gas such as air. The discharge may be performed in a gas such as air. When fibrils are formed in a gas as described above, the fibrils are formed by evaporating the solvent in the dispersion. That is, in the present embodiment, in the step of forming the fibrils, the fibrils may be formed by a dry spinning method. Examples of the method for evaporating the solvent include drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying. However, in the present embodiment, since the formed fibrils have an extremely small diameter, the solvent evaporates spontaneously without performing aggressive drying such as hot-air drying, heat-drying, and air-drying, and the fibrils are removed. It can be formed and is efficient.
In particular, when carbon black nanoparticles are used as the carbon material contained in the dispersion, it is preferable to form the fibrils by a dry spinning method.
乾式紡糸法は、紡糸原液(ドープ液)中の溶媒を蒸発(気化)させることによって紡糸(原繊維を形成)する方法である。上述のように、本実施形態においては、原繊維を形成させる際に、原繊維を分散液から引き出すことを空気等の気体中でおこなってもよく、また、分散液の吐出において、分散液を吐出させる先として、空気等の気体中に吐出させてもよい。このように気体中で原繊維を形成させる際には、分散液中の溶媒を蒸発させることにより、原繊維が形成される。すなわち、本実施形態においては、原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させてもよい。溶媒を蒸発させる方法として、熱風乾燥、加熱乾燥、送風乾燥、自然乾燥等の公知の乾式紡糸法に用いられる乾燥方法を挙げることができる。ただし、本実施形態においては、形成される原繊維が極めて細径であるため、熱風乾燥、加熱乾燥、送風乾燥等の積極的な乾燥を行わなくとも、自然に溶媒が蒸発し、原繊維を形成することができ、効率がよい。
特に、分散液に含まれる炭素材料としてカーボンブラックナノパーティクルを用いる場合は、乾式紡糸法によって原繊維を形成させることが好ましい。 (Dry spinning method)
The dry spinning method is a method of spinning (forming fibrils) by evaporating (vaporizing) a solvent in a spinning solution (dope solution). As described above, in the present embodiment, when forming the fibrils, the drawing of the fibrils from the dispersion may be performed in a gas such as air. The discharge may be performed in a gas such as air. When fibrils are formed in a gas as described above, the fibrils are formed by evaporating the solvent in the dispersion. That is, in the present embodiment, in the step of forming the fibrils, the fibrils may be formed by a dry spinning method. Examples of the method for evaporating the solvent include drying methods used in known dry spinning methods such as hot air drying, heat drying, air drying, and natural drying. However, in the present embodiment, since the formed fibrils have an extremely small diameter, the solvent evaporates spontaneously without performing aggressive drying such as hot-air drying, heat-drying, and air-drying, and the fibrils are removed. It can be formed and is efficient.
In particular, when carbon black nanoparticles are used as the carbon material contained in the dispersion, it is preferable to form the fibrils by a dry spinning method.
〔巻き取り〕
形成された原繊維は、ワインダー等の巻き取り装置で巻き取ってもよい。巻き取り装置を用いることで、繊維を連続的に製造できる。また、原繊維を引き出した後、巻き取り装置で原繊維を巻き取ることで、原繊維を引き出し続けることができる。巻き取り装置として、ワインダーを用いてもよい。ワインダーでは、適宜張力及び接圧等の巻き取り条件を調整して巻き取ることができる。ワインダーとしては、公知のワインダーを用いてもよい。ワインダーを用いることで、原繊維の繊維径をコントロールできる。具体的には、巻き取り速度を速くすることで繊維径をより細くすることができ、巻き取り速度を遅くすることで繊維径の太い繊維も製造することができる。 〔Take-up〕
The formed fibrils may be wound by a winding device such as a winder. By using a winding device, fibers can be continuously produced. Further, after the raw fiber is drawn out, the raw fiber can be continuously drawn out by winding the raw fiber with a winding device. A winder may be used as the winding device. In the winder, winding can be performed by appropriately adjusting winding conditions such as tension and contact pressure. As the winder, a known winder may be used. By using a winder, the fiber diameter of the fibrils can be controlled. Specifically, the fiber diameter can be made thinner by increasing the winding speed, and a fiber having a large fiber diameter can be manufactured by reducing the winding speed.
形成された原繊維は、ワインダー等の巻き取り装置で巻き取ってもよい。巻き取り装置を用いることで、繊維を連続的に製造できる。また、原繊維を引き出した後、巻き取り装置で原繊維を巻き取ることで、原繊維を引き出し続けることができる。巻き取り装置として、ワインダーを用いてもよい。ワインダーでは、適宜張力及び接圧等の巻き取り条件を調整して巻き取ることができる。ワインダーとしては、公知のワインダーを用いてもよい。ワインダーを用いることで、原繊維の繊維径をコントロールできる。具体的には、巻き取り速度を速くすることで繊維径をより細くすることができ、巻き取り速度を遅くすることで繊維径の太い繊維も製造することができる。 〔Take-up〕
The formed fibrils may be wound by a winding device such as a winder. By using a winding device, fibers can be continuously produced. Further, after the raw fiber is drawn out, the raw fiber can be continuously drawn out by winding the raw fiber with a winding device. A winder may be used as the winding device. In the winder, winding can be performed by appropriately adjusting winding conditions such as tension and contact pressure. As the winder, a known winder may be used. By using a winder, the fiber diameter of the fibrils can be controlled. Specifically, the fiber diameter can be made thinner by increasing the winding speed, and a fiber having a large fiber diameter can be manufactured by reducing the winding speed.
〔延伸工程〕
改変クモ糸フィブロイン繊維の製造方法は、形成させた原繊維を延伸する工程(延伸工程)を更に含むものであってよい。任意の延伸倍率に原繊維を延伸し、延伸して完成された繊維を本実施形態の改変クモ糸フィブロイン繊繊として任意の用途に用いることができる。延伸方法としては、湿熱延伸、乾熱延伸等をあげることができる。 (Stretching step)
The method for producing the modified spider silk fibroin fiber may further include a step of drawing the formed raw fiber (drawing step). The original fiber is drawn at an arbitrary draw ratio, and the drawn fiber can be used as a modified spider yarn fibroin fiber of the present embodiment for any use. Examples of the stretching method include wet heat stretching and dry heat stretching.
改変クモ糸フィブロイン繊維の製造方法は、形成させた原繊維を延伸する工程(延伸工程)を更に含むものであってよい。任意の延伸倍率に原繊維を延伸し、延伸して完成された繊維を本実施形態の改変クモ糸フィブロイン繊繊として任意の用途に用いることができる。延伸方法としては、湿熱延伸、乾熱延伸等をあげることができる。 (Stretching step)
The method for producing the modified spider silk fibroin fiber may further include a step of drawing the formed raw fiber (drawing step). The original fiber is drawn at an arbitrary draw ratio, and the drawn fiber can be used as a modified spider yarn fibroin fiber of the present embodiment for any use. Examples of the stretching method include wet heat stretching and dry heat stretching.
湿熱延伸は、温水中、温水に有機溶剤等を加えた溶液中、又はスチーム加熱中で行うことができる。湿熱延伸の温度は50~90℃であることが好ましく、75~85℃がより好ましい。該温度が50℃以上であると、繊維中の細孔径を小さく安定させることができる。また、温度が90℃以下であると、温度設定が容易であり紡糸安定性が向上する。
Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or steam heating. The wet heat stretching temperature is preferably from 50 to 90 ° C, more preferably from 75 to 85 ° C. When the temperature is 50 ° C. or more, the pore diameter in the fiber can be made small and stable. When the temperature is 90 ° C. or lower, the temperature can be easily set and spinning stability is improved.
湿熱延伸における延伸倍率は、未延伸糸(又は前延伸糸)に対して、例えば、1~30倍であってよく、1~25倍であってよく、1~20倍であってよく、1~15倍であってよく、1~10倍であってよく、2~10倍であってよく、2~8倍であってよく、2~6倍であってよく、2~4倍であってよく、2~3倍であってよい。
The draw ratio in the wet heat drawing is, for example, 1 to 30 times, 1 to 25 times, 1 to 20 times, or 1 to 30 times with respect to the undrawn yarn (or pre-drawn yarn). 1515 times, 1 倍 10 times, 2 ~ 10 times, 2 ~ 8 times, 2 ~ 6 times, 2、24 times And may be 2-3 times.
乾熱延伸は、接触型の熱板、及び非接触型の炉等の熱源を備えた装置を用いて、空気中で延伸することにより行うことができるが、装置は特に限定されるものではなく、繊維を所定の温度まで昇温させ、かつ所定の倍率で延伸が可能な装置であればよい。乾熱延伸を行う温度としては、例えば、100℃~270℃であってよく、140℃~230℃であってよく、140℃~200℃であってよく、160℃~200℃であってよく、160℃~180℃であってよい。
Dry heat drawing can be performed by drawing in the air using a device equipped with a heat source such as a contact hot plate and a non-contact furnace, but the device is not particularly limited. Any device can be used as long as it can raise the temperature of the fiber to a predetermined temperature and can draw the fiber at a predetermined magnification. The temperature at which the dry heat stretching is performed may be, for example, 100 ° C to 270 ° C, 140 ° C to 230 ° C, 140 ° C to 200 ° C, or 160 ° C to 200 ° C. , 160 ° C to 180 ° C.
乾熱延伸工程における延伸倍率は、未延伸糸(又は前延伸糸)に対して、例えば、1~30倍であってよく、1~25倍であってよく、1~20倍であってよく、1~15倍であってよく、1~10倍であってよく、2~10倍であってよく、2~8倍であってよく、2~6倍であってよく、2~4倍であってよく、2~3倍であってよい。
The draw ratio in the dry heat drawing step may be, for example, 1 to 30 times, 1 to 25 times, or 1 to 20 times with respect to the undrawn yarn (or pre-drawn yarn). 1 to 15 times, 1 to 10 times, 2 to 10 times, 2 to 8 times, 2 to 6 times, 2 to 4 times And may be 2-3 times.
延伸工程は、湿熱延伸及び乾熱延伸を、それぞれ単独で行うものであってもよく、またこれらを多段で、又は組み合わせて行うものであってもよい。すなわち、延伸工程として、一段目延伸を湿熱延伸で行い、二段目延伸を乾熱延伸で行う、又は一段目延伸を湿熱延伸で行い、二段目延伸を湿熱延伸で行い、更に三段目延伸を乾熱延伸で行う等、湿熱延伸及び乾熱延伸を適宜組み合わせて行うことができる。
In the stretching step, the wet heat stretching and the dry heat stretching may be performed individually, or may be performed in multiple stages or in combination. That is, as the stretching step, 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, and the second-stage stretching is performed by wet-heat stretching. Stretching can be performed by appropriately combining wet heat stretching and dry heat stretching, such as by performing dry heat stretching.
延伸工程を経た繊維の最終的な延伸倍率の下限値は、未延伸糸(又は前延伸糸)に対して、例えば、1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、又は9倍のうちの何れかであってよい。延伸工程を経た繊維の最終的な延伸倍率の上限値は、例えば、40倍、30倍、20倍、15倍、14倍、13倍、12倍、11倍、又は10倍のうちの何れかであってよい。また、例えば、最終的な延伸倍率は3~40倍であってよく、3~30倍であってよく、5~30倍であってよく、5~20倍であってよく、5~15倍であってよく、5~13倍であってよい。ただし、延伸倍率は、所望する繊維の太さ、機械物性などの特性が得られる範囲であれば限定されるものではない。
The lower limit of the final draw ratio of the fiber after the drawing step is, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or more with respect to the undrawn yarn (or pre-drawn yarn). It may be any of double, eight, or nine times. The upper limit of the final draw ratio of the fiber after the drawing step is, for example, any one of 40 times, 30 times, 20 times, 15 times, 14 times, 13 times, 12 times, 11 times, or 10 times. It may be. In addition, for example, the final stretching magnification may be 3 to 40 times, 3 to 30 times, 5 to 30 times, 5 to 20 times, or 5 to 15 times. And may be 5 to 13 times. However, the draw ratio is not limited as long as the desired properties such as fiber thickness and mechanical properties can be obtained.
延伸工程の前又は後に、必要に応じて、繊維に対して、帯電抑制性、収束性及び潤滑性等を付与する目的で油剤を付与してもよい。付与する油剤の種類及び付与する量等は、特に限定されるものではなく、繊維を使用する用途、繊維の取扱い性等を考慮し適宜調整することができる。
前 Before or after the drawing step, an oil agent may be added to the fiber, if necessary, for the purpose of imparting antistatic properties, convergence properties, lubricity and the like. The type of oil agent to be applied, the amount to be applied, and the like are not particularly limited, and can be appropriately adjusted in consideration of the use of the fiber, the handleability of the fiber, and the like.
以上のようにして、本実施形態に係る改変クモ糸フィブロイン繊維を製造することができる。上述の方法は一例であり、本実施形態に係る改変クモ糸フィブロイン繊維は、上述の方法以外によって製造されたものであってもよい。
改 変 As described above, the modified spider silk fibroin fiber according to the present embodiment can be manufactured. The above-described method is an example, and the modified spider silk fibroin fiber according to the present embodiment may be manufactured by a method other than the above-described method.
〔改変クモ糸フィブロイン繊維〕
本実施形態に係る改変クモ糸フィブロイン繊維は、改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される炭素材料とを含む繊維である。炭素材料の含有量は、改変クモ糸フィブロインの含有量を基準として、0.01質量部以上1質量部以下であってよく、0.01質量部以上1質量部未満であってよく、0.03質量部以上1質量部未満であってよく、0.05質量部以上1質量部未満であってよく、0.05質量部以上0.95質量部以下であってよく、0.05質量部以上0.9質量部以下であってよく、0.05質量部以上0.8質量部以下であってよく、0.05質量部以上0.7質量部以下であってよく、0.05質量部以上0.6質量部以下であってよく、0.05質量部以上0.5質量部以下であってよく、又は0.05質量部以上0.3質量部以下であってよい。 (Modified spider silk fibroin fiber)
The modified spider silk fibroin fiber according to the present embodiment is a fiber containing the modified spider silk fibroin and one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles. The content of the carbon material may be from 0.01 part by mass to 1 part by mass, from 0.01 part by mass to less than 1 part by mass, based on the content of the modified spider silk fibroin. 03 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and 0.95 parts by mass or less, and may be 0.05 parts by mass. It may be 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, may be 0.05 parts by mass or more and 0.7 parts by mass or less, and may be 0.05 parts by mass or less. It may be not less than 0.6 part by mass and not less than 0.05 part by mass, and may be not less than 0.05 part by mass and not more than 0.3 part by mass.
本実施形態に係る改変クモ糸フィブロイン繊維は、改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される炭素材料とを含む繊維である。炭素材料の含有量は、改変クモ糸フィブロインの含有量を基準として、0.01質量部以上1質量部以下であってよく、0.01質量部以上1質量部未満であってよく、0.03質量部以上1質量部未満であってよく、0.05質量部以上1質量部未満であってよく、0.05質量部以上0.95質量部以下であってよく、0.05質量部以上0.9質量部以下であってよく、0.05質量部以上0.8質量部以下であってよく、0.05質量部以上0.7質量部以下であってよく、0.05質量部以上0.6質量部以下であってよく、0.05質量部以上0.5質量部以下であってよく、又は0.05質量部以上0.3質量部以下であってよい。 (Modified spider silk fibroin fiber)
The modified spider silk fibroin fiber according to the present embodiment is a fiber containing the modified spider silk fibroin and one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles. The content of the carbon material may be from 0.01 part by mass to 1 part by mass, from 0.01 part by mass to less than 1 part by mass, based on the content of the modified spider silk fibroin. 03 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and less than 1 part by mass, may be 0.05 parts by mass or more and 0.95 parts by mass or less, and may be 0.05 parts by mass. It may be 0.9 parts by mass or less, may be 0.05 parts by mass or more and 0.8 parts by mass or less, may be 0.05 parts by mass or more and 0.7 parts by mass or less, and may be 0.05 parts by mass or less. It may be not less than 0.6 part by mass and not less than 0.05 part by mass, and may be not less than 0.05 part by mass and not more than 0.3 part by mass.
改変クモ糸フィブロイン繊維の平均繊維径は、3μm以下であってよく、2μm以下であってよく、1~3μmであってよく、2~3μmであってよく、1~2μmであってよい。上述の本実施形態にかかる炭素材料分散液を用いることによって、極めて細い繊維径を有する改変クモ糸フィブロイン繊維を簡便に製造することができる。改変クモ糸フィブロイン繊維の繊維径は、例えば、走査電子顕微鏡(SEM)を用いて測定することができる。
(4) The average fiber diameter of the modified spider silk fibroin fiber may be 3 μm or less, 2 μm or less, 1 to 3 μm, 2 to 3 μm, or 1 to 2 μm. By using the above-described carbon material dispersion according to the present embodiment, a modified spider silk fibroin fiber having an extremely small fiber diameter can be easily produced. The fiber diameter of the modified spider silk fibroin fiber can be measured using, for example, a scanning electron microscope (SEM).
〔改変クモ糸フィブロイン不織布の製造方法〕
本実施形態にかかる改変クモ糸フィブロイン不織布の製造方法は、上述した、改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液を準備する工程と、前記分散液から原繊維を形成させる工程と、前記原繊維又は前記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、を少なくとも備える。製造された改変クモ糸フィブロイン不織布は、そのまま任意の用途に用いてもよい。原繊維を形成させる工程は、上述した分散液を吐出させることを含んでいてもよく、上述した分散液から原繊維を引き出すことを含んでいてもよく、分散液を吐出させること及び分散液から原繊維を引き出すことの両方を含んでいてもよい。また、本実施形態に係る改変クモ糸フィブロイン不織布は、原繊維又は原繊維から製造した改変クモ糸フィブロイン繊維を用いて、公知の不織布の製造方法により製造してもよい。具体的には、例えば、原繊維又は改変クモ糸フィブロイン繊維を用いて、乾式法、湿式法及びエアレイド法等でウェブ(単層ウェブ、積層ウェブを含む)を形成させた後、ケミカルボンド法(浸漬法、スプレー法等)及びニードルパンチ法等によりウェブの繊維間を結合させて、不織布を得ることができる。 (Production method of modified spider silk fibroin nonwoven fabric)
The method for producing a modified spider silk fibroin nonwoven fabric according to the present embodiment includes the steps of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming fibrils from the dispersion. And at least a step of intertwining the fibrils or modified spider silk fibroin fibers produced from the fibrils. The manufactured modified spider silk fibroin nonwoven fabric may be used for any purpose as it is. The step of forming the fibrils may include discharging the dispersion described above, and may include extracting the fibrils from the dispersion described above, and discharging the dispersion and from the dispersion. It may include both drawing fibrils. Further, the modified spider silk fibroin nonwoven fabric according to the present embodiment may be manufactured by a known nonwoven fabric manufacturing method using raw fibers or modified spider silk fibroin fibers manufactured from raw fibers. Specifically, for example, a web (including a single-layer web and a laminated web) is formed using a raw fiber or a modified spider silk fibroin fiber by a dry method, a wet method, an air laid method, and the like, and then a chemical bond method ( The fibers of the web can be bonded together by a dipping method, a spraying method, etc.) and a needle punching method to obtain a nonwoven fabric.
本実施形態にかかる改変クモ糸フィブロイン不織布の製造方法は、上述した、改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液を準備する工程と、前記分散液から原繊維を形成させる工程と、前記原繊維又は前記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、を少なくとも備える。製造された改変クモ糸フィブロイン不織布は、そのまま任意の用途に用いてもよい。原繊維を形成させる工程は、上述した分散液を吐出させることを含んでいてもよく、上述した分散液から原繊維を引き出すことを含んでいてもよく、分散液を吐出させること及び分散液から原繊維を引き出すことの両方を含んでいてもよい。また、本実施形態に係る改変クモ糸フィブロイン不織布は、原繊維又は原繊維から製造した改変クモ糸フィブロイン繊維を用いて、公知の不織布の製造方法により製造してもよい。具体的には、例えば、原繊維又は改変クモ糸フィブロイン繊維を用いて、乾式法、湿式法及びエアレイド法等でウェブ(単層ウェブ、積層ウェブを含む)を形成させた後、ケミカルボンド法(浸漬法、スプレー法等)及びニードルパンチ法等によりウェブの繊維間を結合させて、不織布を得ることができる。 (Production method of modified spider silk fibroin nonwoven fabric)
The method for producing a modified spider silk fibroin nonwoven fabric according to the present embodiment includes the steps of preparing a dispersion containing the above-described modified spider silk fibroin, a carbon material, and a polar solvent, and forming fibrils from the dispersion. And at least a step of intertwining the fibrils or modified spider silk fibroin fibers produced from the fibrils. The manufactured modified spider silk fibroin nonwoven fabric may be used for any purpose as it is. The step of forming the fibrils may include discharging the dispersion described above, and may include extracting the fibrils from the dispersion described above, and discharging the dispersion and from the dispersion. It may include both drawing fibrils. Further, the modified spider silk fibroin nonwoven fabric according to the present embodiment may be manufactured by a known nonwoven fabric manufacturing method using raw fibers or modified spider silk fibroin fibers manufactured from raw fibers. Specifically, for example, a web (including a single-layer web and a laminated web) is formed using a raw fiber or a modified spider silk fibroin fiber by a dry method, a wet method, an air laid method, and the like, and then a chemical bond method ( The fibers of the web can be bonded together by a dipping method, a spraying method, etc.) and a needle punching method to obtain a nonwoven fabric.
不織布の製造工程は、不織布の繊維密度を調整する工程(調整工程)をさらに備えていてもよい。繊維密度(目付)は、不織布の単位面積当たりの重量で定義される値である。不織布の繊維密度の調整は、例えば、ウェブを構成する繊維量を増減することで調整することができ、積層ウェブの場合は、積層数を増減することにより調整することができる。
The manufacturing process of the nonwoven fabric may further include a process of adjusting the fiber density of the nonwoven fabric (adjustment process). The fiber density (basis weight) is a value defined by the weight per unit area of the nonwoven fabric. The fiber density of the nonwoven fabric can be adjusted by, for example, increasing or decreasing the amount of fibers constituting the web, and in the case of a laminated web, can be adjusted by increasing or decreasing the number of layers.
〔製品〕
本実施形態に係る改変クモ糸フィブロイン繊維を含む、各種の製品を製造することができる。このような製品としては、例えば、繊維、糸、織物等の布帛、編み物、組み物、不織布、紙、及び綿を挙げることができる。繊維としては、例えば、長繊維、短繊維、モノフィラメント、又はマルチフィラメント等を挙げることができ、糸としては、紡績糸、撚糸、仮撚糸、加工糸、混繊糸、又は混紡糸等を挙げることができる。さらに、これらの繊維や糸から、織物等の布帛、編物、組み物、若しくは不織布等、紙及び綿等を製造することができる。これらの製品は、公知の方法により製造することができる。 [Product]
Various products including the modified spider silk fibroin fiber according to the present embodiment can be manufactured. Such products include, for example, fabrics such as fibers, yarns, and fabrics, knits, braids, non-woven fabrics, paper, and cotton. Examples of the fiber include a long fiber, a short fiber, a monofilament, and a multifilament, and examples of the yarn include a spun yarn, a twisted yarn, a false twisted yarn, a processed yarn, a mixed fiber, and a mixed spun yarn. Can be. Furthermore, from these fibers and yarns, fabrics such as woven fabrics, knits, braids, non-woven fabrics, and the like, paper, cotton, and the like can be manufactured. These products can be manufactured by a known method.
本実施形態に係る改変クモ糸フィブロイン繊維を含む、各種の製品を製造することができる。このような製品としては、例えば、繊維、糸、織物等の布帛、編み物、組み物、不織布、紙、及び綿を挙げることができる。繊維としては、例えば、長繊維、短繊維、モノフィラメント、又はマルチフィラメント等を挙げることができ、糸としては、紡績糸、撚糸、仮撚糸、加工糸、混繊糸、又は混紡糸等を挙げることができる。さらに、これらの繊維や糸から、織物等の布帛、編物、組み物、若しくは不織布等、紙及び綿等を製造することができる。これらの製品は、公知の方法により製造することができる。 [Product]
Various products including the modified spider silk fibroin fiber according to the present embodiment can be manufactured. Such products include, for example, fabrics such as fibers, yarns, and fabrics, knits, braids, non-woven fabrics, paper, and cotton. Examples of the fiber include a long fiber, a short fiber, a monofilament, and a multifilament, and examples of the yarn include a spun yarn, a twisted yarn, a false twisted yarn, a processed yarn, a mixed fiber, and a mixed spun yarn. Can be. Furthermore, from these fibers and yarns, fabrics such as woven fabrics, knits, braids, non-woven fabrics, and the like, paper, cotton, and the like can be manufactured. These products can be manufactured by a known method.
〔炭素材料の分散方法〕
本発明はまた、改変クモ糸フィブロインと、改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含む、炭素材料の分散方法も提供する。上記炭素材料は、上述したように、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される。上記特定の炭素材料及び改変クモ糸フィブロインを特定の割合で、極性溶媒と混合することにより、上記炭素材料を長い時間均一に極性溶媒中に分散させることができる。また、該方法により分散された分散液は、操作性が容易であり、上記炭素材料を原料とする各種用途に好適に使用することができる。 (Method of dispersing carbon material)
The present invention also provides a method for dispersing a carbon material, comprising a step of mixing a modified spider silk fibroin, a carbon material of 25 parts by mass or less based on 100 parts by weight of the modified spider silk fibroin, and a polar solvent. As described above, the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles. By mixing the specific carbon material and the modified spider silk fibroin in a specific ratio with a polar solvent, the carbon material can be uniformly dispersed in the polar solvent for a long time. Further, the dispersion liquid dispersed by the method has easy operability, and can be suitably used for various applications using the above-mentioned carbon material as a raw material.
本発明はまた、改変クモ糸フィブロインと、改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含む、炭素材料の分散方法も提供する。上記炭素材料は、上述したように、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される。上記特定の炭素材料及び改変クモ糸フィブロインを特定の割合で、極性溶媒と混合することにより、上記炭素材料を長い時間均一に極性溶媒中に分散させることができる。また、該方法により分散された分散液は、操作性が容易であり、上記炭素材料を原料とする各種用途に好適に使用することができる。 (Method of dispersing carbon material)
The present invention also provides a method for dispersing a carbon material, comprising a step of mixing a modified spider silk fibroin, a carbon material of 25 parts by mass or less based on 100 parts by weight of the modified spider silk fibroin, and a polar solvent. As described above, the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles. By mixing the specific carbon material and the modified spider silk fibroin in a specific ratio with a polar solvent, the carbon material can be uniformly dispersed in the polar solvent for a long time. Further, the dispersion liquid dispersed by the method has easy operability, and can be suitably used for various applications using the above-mentioned carbon material as a raw material.
〔分散補助剤〕
本発明はまた、改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤も提供する。上記炭素材料は、上述したように、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される。改変クモ糸フィブロインにより、炭素材料の極性溶媒への分散性が向上する。すなわち、改変クモ糸フィブロインは、上記炭素材料の分散補助剤として使用することができる。 (Dispersion aid)
The present invention also provides a dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin. As described above, the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles. The modified spider silk fibroin improves the dispersibility of the carbon material in the polar solvent. That is, the modified spider silk fibroin can be used as a dispersion aid for the carbon material.
本発明はまた、改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤も提供する。上記炭素材料は、上述したように、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される。改変クモ糸フィブロインにより、炭素材料の極性溶媒への分散性が向上する。すなわち、改変クモ糸フィブロインは、上記炭素材料の分散補助剤として使用することができる。 (Dispersion aid)
The present invention also provides a dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin. As described above, the carbon material is selected from one or more layers of planar graphene and carbon black nanoparticles. The modified spider silk fibroin improves the dispersibility of the carbon material in the polar solvent. That is, the modified spider silk fibroin can be used as a dispersion aid for the carbon material.
〔細径化剤〕
本発明はまた、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される炭素材料を含む、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤も提供する。上記特定の炭素材料は、改変クモ糸フィブロイン繊維を製造するにあたり、繊維径を極めて細くすることができる。すなわち、上記特定の炭素材料は、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤として使用することができる。 (Thinning agent)
The present invention also provides a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles. . In producing the modified spider silk fibroin fiber, the specific carbon material can have an extremely small fiber diameter. That is, the specific carbon material can be used as a diameter reducing agent for producing a modified spider silk fibroin fiber having a reduced diameter.
本発明はまた、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルから選択される炭素材料を含む、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤も提供する。上記特定の炭素材料は、改変クモ糸フィブロイン繊維を製造するにあたり、繊維径を極めて細くすることができる。すなわち、上記特定の炭素材料は、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤として使用することができる。 (Thinning agent)
The present invention also provides a thinning agent for producing a reduced modified spider silk fibroin fiber, comprising one or more layers of planar graphene and a carbon material selected from carbon black nanoparticles. . In producing the modified spider silk fibroin fiber, the specific carbon material can have an extremely small fiber diameter. That is, the specific carbon material can be used as a diameter reducing agent for producing a modified spider silk fibroin fiber having a reduced diameter.
以下、実施例に基づいて本発明をより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
〔改変クモ糸フィブロインの製造〕
(1)発現ベクターの作製
ネフィラ・クラビペス(Nephila clavipes)由来のフィブロイン(GenBankアクセッション番号:P46804.1、GI:1174415)の塩基配列及びアミノ酸配列に基づき、配列番号15で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT799」ともいう。)、配列番号39で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT918」ともいう。)及び配列番号44で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT966」ともいう。)を設計した。なお、配列番号15で示されるアミノ酸配列は、ネフィラ・クラビペス由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したアミノ酸配列を有し、さらにN末端に配列番号12で示されるアミノ酸配列(タグ配列及びヒンジ配列)が付加されている。 (Production of modified spider silk fibroin)
(1) Preparation of Expression Vector Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 11744415) derived from Nephila clavipes, it has an amino acid sequence represented by SEQ ID NO: 15. A modified spider silk fibroin (hereinafter, also referred to as “PRT799”), a modified spider silk fibroin having an amino acid sequence represented by SEQ ID NO: 39 (hereinafter, also referred to as “PRT918”), and an amino acid sequence represented by SEQ ID NO: 44 A modified spider silk fibroin (hereinafter, also referred to as “PRT966”) was designed. The amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 12 (tag sequence and hinge sequence) is added to the N-terminus.
(1)発現ベクターの作製
ネフィラ・クラビペス(Nephila clavipes)由来のフィブロイン(GenBankアクセッション番号:P46804.1、GI:1174415)の塩基配列及びアミノ酸配列に基づき、配列番号15で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT799」ともいう。)、配列番号39で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT918」ともいう。)及び配列番号44で示されるアミノ酸配列を有する改変クモ糸フィブロイン(以下、「PRT966」ともいう。)を設計した。なお、配列番号15で示されるアミノ酸配列は、ネフィラ・クラビペス由来のフィブロインのアミノ酸配列に対して、生産性の向上を目的としてアミノ酸残基の置換、挿入及び欠失を施したアミノ酸配列を有し、さらにN末端に配列番号12で示されるアミノ酸配列(タグ配列及びヒンジ配列)が付加されている。 (Production of modified spider silk fibroin)
(1) Preparation of Expression Vector Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 11744415) derived from Nephila clavipes, it has an amino acid sequence represented by SEQ ID NO: 15. A modified spider silk fibroin (hereinafter, also referred to as “PRT799”), a modified spider silk fibroin having an amino acid sequence represented by SEQ ID NO: 39 (hereinafter, also referred to as “PRT918”), and an amino acid sequence represented by SEQ ID NO: 44 A modified spider silk fibroin (hereinafter, also referred to as “PRT966”) was designed. The amino acid sequence represented by SEQ ID NO: 15 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues with respect to the amino acid sequence of fibroin derived from Nephila clavipes for the purpose of improving productivity. Further, an amino acid sequence represented by SEQ ID NO: 12 (tag sequence and hinge sequence) is added to the N-terminus.
次に、設計した配列番号15、配列番号39及び配列番号44で示されるアミノ酸配列を有する改変クモ糸フィブロインPRT799、PRT918、PRT966をコードする核酸を合成した。当該核酸には、5’末端にNdeIサイト及び終止コドン下流にEcoRIサイトを付加した。当該核酸をクローニングベクター(pUC118)にクローニングした。その後、同核酸をNdeI及びEcoRIで制限酵素処理して切り出した後、それぞれタンパク質発現ベクターpET-22b(+)に組換えて発現ベクターを得た。
Next, nucleic acids encoding the designed spider silk fibroins PRT799, PRT918, and PRT966 having the designed amino acid sequences represented by SEQ ID NO: 15, SEQ ID NO: 39, and SEQ ID NO: 44 were synthesized. An NdeI site at the 5 'end and an EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was treated with NdeI and EcoRI with restriction enzymes, cut out, and recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
(2)改変クモ糸フィブロインの発現
(1)で得られた発現ベクターで、大腸菌BLR(DE3)を形質転換した。当該形質転換大腸菌を、アンピシリンを含む2mLのLB培地で15時間培養した。当該培養液を、アンピシリンを含む100mLのシード培養用培地(表4)にOD600が0.005となるように添加した。培養液温度を30℃に保ち、OD600が5になるまでフラスコ培養を行い(約15時間)、シード培養液を得た。 (2) Expression of modified spider silk fibroin Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
(1)で得られた発現ベクターで、大腸菌BLR(DE3)を形質転換した。当該形質転換大腸菌を、アンピシリンを含む2mLのLB培地で15時間培養した。当該培養液を、アンピシリンを含む100mLのシード培養用培地(表4)にOD600が0.005となるように添加した。培養液温度を30℃に保ち、OD600が5になるまでフラスコ培養を行い(約15時間)、シード培養液を得た。 (2) Expression of modified spider silk fibroin Escherichia coli BLR (DE3) was transformed with the expression vector obtained in (1). The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of a seed culture medium containing ampicillin (Table 4) so that the OD 600 became 0.005. The temperature of the culture was maintained at 30 ° C., and the flask was cultured until the OD 600 reached 5 (about 15 hours) to obtain a seed culture.
当該シード培養液を500mLの生産培地(表5)を添加したジャーファーメンターにOD600が0.05となるように添加した。培養液温度を37℃に保ち、pH6.9で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の20%に維持するようにした。
The seed culture solution was added to a jar fermenter to which 500 mL of a production medium (Table 5) had been added so that the OD 600 was 0.05. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, the concentration of dissolved oxygen in the culture solution was maintained at 20% of the saturated concentration of dissolved oxygen.
生産培地中のグルコースが完全に消費された直後に、フィード液(グルコース455g/1L、Yeast Extract 120g/1L)を1mL/分の速度で添加した。培養液温度を37℃に保ち、pH6.9で一定に制御して培養した。また培養液中の溶存酸素濃度を、溶存酸素飽和濃度の20%に維持するようにし、20時間培養を行った。その後、1Mのイソプロピル-β-チオガラクトピラノシド(IPTG)を培養液に対して終濃度1mMになるよう添加し、改変クモ糸フィブロインを発現誘導させた。IPTG添加後20時間経過した時点で、培養液を遠心分離し、菌体を回収した。IPTG添加前とIPTG添加後の培養液から調製した菌体を用いてSDS-PAGEを行い、IPTG添加に依存した目的とする改変クモ糸フィブロインサイズのバンドの出現により、目的とする改変クモ糸フィブロインの発現を確認した。
フ ィ ー ド Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / 1 L of glucose, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The temperature of the culture was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9. Further, 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. Thereafter, 1M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified spider silk fibroin. Twenty hours after the addition of IPTG, the culture was centrifuged to collect the cells. SDS-PAGE was carried out using the cells prepared from the culture solution before and after the addition of IPTG, and the appearance of the band of the desired modified spider silk fibroin size dependent on the addition of IPTG revealed that the desired modified spider silk fibroin Was confirmed.
(3)改変クモ糸フィブロインの精製
IPTGを添加してから2時間後に回収した菌体を20mM Tris-HCl buffer(pH7.4)で洗浄した。洗浄後の菌体を約1mMのPMSFを含む20mM Tris-HCl緩衝液(pH7.4)に懸濁させ、高圧ホモジナイザー(GEA Niro Soavi社製)で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで20mM Tris-HCl緩衝液(pH7.4)で洗浄した。洗浄後の沈殿物を100mg/mLの濃度になるように8M グアニジン緩衝液(8M グアニジン塩酸塩、10mM リン酸二水素ナトリウム、20mM NaCl、1mM Tris-HCl、pH7.0)で懸濁し、60℃で30分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ(三光純薬株式会社製のセルロースチューブ36/32)を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収することにより、改変クモ糸フィブロイン(PRT799、PRT918、PRT966)を得た。 (3) Purification of modified spider silk fibroin Two hours after the addition of IPTG, the recovered cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain modified spider silk fibroin (PRT799, PRT918, PRT966).
IPTGを添加してから2時間後に回収した菌体を20mM Tris-HCl buffer(pH7.4)で洗浄した。洗浄後の菌体を約1mMのPMSFを含む20mM Tris-HCl緩衝液(pH7.4)に懸濁させ、高圧ホモジナイザー(GEA Niro Soavi社製)で細胞を破砕した。破砕した細胞を遠心分離し、沈殿物を得た。得られた沈殿物を、高純度になるまで20mM Tris-HCl緩衝液(pH7.4)で洗浄した。洗浄後の沈殿物を100mg/mLの濃度になるように8M グアニジン緩衝液(8M グアニジン塩酸塩、10mM リン酸二水素ナトリウム、20mM NaCl、1mM Tris-HCl、pH7.0)で懸濁し、60℃で30分間、スターラーで撹拌し、溶解させた。溶解後、透析チューブ(三光純薬株式会社製のセルロースチューブ36/32)を用いて水で透析を行った。透析後に得られた白色の凝集タンパク質を遠心分離により回収し、凍結乾燥機で水分を除き、凍結乾燥粉末を回収することにより、改変クモ糸フィブロイン(PRT799、PRT918、PRT966)を得た。 (3) Purification of modified spider silk fibroin Two hours after the addition of IPTG, the recovered cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL, and then suspended at 60 ° C. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected to obtain modified spider silk fibroin (PRT799, PRT918, PRT966).
〔分散液の調製〕
(実施例1)
ギ酸(富士フィルム和光純薬株式会社製)3.16gと酸化グラフェン(Sigma-Aldrich社製、製品番号:796034)1mgを混合し、超音波洗浄装置(BRANSON社製)を用いて、温度60℃で100分間超音波振動によりそれぞれ撹拌した。ただちに、上述の製造工程で得られた改変クモ糸フィブロイン(PRT799)1gを添加し、攪拌しながら40℃のアルミブロックヒーターで1時間加温して溶解させ、酸化グラフェンの分散液を調製した。改変クモ糸フィブロインの添加量は、分散液全量に対して24質量%とした。改変フィブロインに対する酸化グラフェンの割合は0.1質量%とした。 (Preparation of dispersion liquid)
(Example 1)
3.16 g of formic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide (manufactured by Sigma-Aldrich, product number: 796034) were mixed, and the temperature was 60 ° C. using an ultrasonic cleaning device (manufactured by BRANSON). For 100 minutes by ultrasonic vibration. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-described production step was added, and the mixture was heated and dissolved with an aluminum block heater at 40 ° C. for 1 hour while stirring to prepare a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
(実施例1)
ギ酸(富士フィルム和光純薬株式会社製)3.16gと酸化グラフェン(Sigma-Aldrich社製、製品番号:796034)1mgを混合し、超音波洗浄装置(BRANSON社製)を用いて、温度60℃で100分間超音波振動によりそれぞれ撹拌した。ただちに、上述の製造工程で得られた改変クモ糸フィブロイン(PRT799)1gを添加し、攪拌しながら40℃のアルミブロックヒーターで1時間加温して溶解させ、酸化グラフェンの分散液を調製した。改変クモ糸フィブロインの添加量は、分散液全量に対して24質量%とした。改変フィブロインに対する酸化グラフェンの割合は0.1質量%とした。 (Preparation of dispersion liquid)
(Example 1)
3.16 g of formic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide (manufactured by Sigma-Aldrich, product number: 796034) were mixed, and the temperature was 60 ° C. using an ultrasonic cleaning device (manufactured by BRANSON). For 100 minutes by ultrasonic vibration. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-described production step was added, and the mixture was heated and dissolved with an aluminum block heater at 40 ° C. for 1 hour while stirring to prepare a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
(実施例2)
改変クモ糸フィブロインの添加量を分散液全量に対して5質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を0.5質量%とした他は、実施例1と同様にして分散液を調製した。具体的には、ギ酸の添加量を1.9gとし、改変クモ糸フィブロインの添加量を0.1gとし、酸化グラフェンの添加量を0.5mgとした。 (Example 2)
A dispersion was prepared in the same manner as in Example 1, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 0.5% by mass. did. Specifically, the amount of formic acid added was 1.9 g, the amount of modified spider silk fibroin was 0.1 g, and the amount of graphene oxide was 0.5 mg.
改変クモ糸フィブロインの添加量を分散液全量に対して5質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を0.5質量%とした他は、実施例1と同様にして分散液を調製した。具体的には、ギ酸の添加量を1.9gとし、改変クモ糸フィブロインの添加量を0.1gとし、酸化グラフェンの添加量を0.5mgとした。 (Example 2)
A dispersion was prepared in the same manner as in Example 1, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 0.5% by mass. did. Specifically, the amount of formic acid added was 1.9 g, the amount of modified spider silk fibroin was 0.1 g, and the amount of graphene oxide was 0.5 mg.
(実施例3)
改変クモ糸フィブロインの添加量を分散液全量に対して1質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を1質量%とした他は、実施例1と同様にして分散液を調製した。 (Example 3)
A dispersion was prepared in the same manner as in Example 1, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 1% by mass.
改変クモ糸フィブロインの添加量を分散液全量に対して1質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を1質量%とした他は、実施例1と同様にして分散液を調製した。 (Example 3)
A dispersion was prepared in the same manner as in Example 1, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 1% by mass.
(実施例4)
ジメチルスルホキシド(富士フィルム和光純薬株式会社製)3.16gと酸化グラフェン1mgを混合し、超音波洗浄装置を用いて、温度60℃で100分間超音波振動によりそれぞれ撹拌した。ただちに、上記改変フィブロインの製造工程で得られた改変フィブロイン(PRT799)1gを添加し、攪拌しながら90℃のアルミブロックヒーターで3時間加温して溶解させ、酸化グラフェンの分散液を調製した。改変クモ糸フィブロインの添加量は、分散液全量に対して24質量%とした。改変フィブロインに対する酸化グラフェンの割合は0.1質量%とした。 (Example 4)
3.16 g of dimethyl sulfoxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide were mixed, and stirred by ultrasonic vibration at a temperature of 60 ° C. for 100 minutes using an ultrasonic cleaning device. Immediately, 1 g of the modified fibroin (PRT799) obtained in the process of producing the modified fibroin was added, and the mixture was heated with a 90 ° C. aluminum block heater for 3 hours to dissolve with stirring, thereby preparing a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
ジメチルスルホキシド(富士フィルム和光純薬株式会社製)3.16gと酸化グラフェン1mgを混合し、超音波洗浄装置を用いて、温度60℃で100分間超音波振動によりそれぞれ撹拌した。ただちに、上記改変フィブロインの製造工程で得られた改変フィブロイン(PRT799)1gを添加し、攪拌しながら90℃のアルミブロックヒーターで3時間加温して溶解させ、酸化グラフェンの分散液を調製した。改変クモ糸フィブロインの添加量は、分散液全量に対して24質量%とした。改変フィブロインに対する酸化グラフェンの割合は0.1質量%とした。 (Example 4)
3.16 g of dimethyl sulfoxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) and 1 mg of graphene oxide were mixed, and stirred by ultrasonic vibration at a temperature of 60 ° C. for 100 minutes using an ultrasonic cleaning device. Immediately, 1 g of the modified fibroin (PRT799) obtained in the process of producing the modified fibroin was added, and the mixture was heated with a 90 ° C. aluminum block heater for 3 hours to dissolve with stirring, thereby preparing a dispersion of graphene oxide. The amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dispersion. The ratio of graphene oxide to the modified fibroin was 0.1% by mass.
(実施例5)
改変クモ糸フィブロインの添加量を分散液全量に対して5質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を4質量%とした他は、実施例4と同様にして分散液を調製した。 (Example 5)
A dispersion was prepared in the same manner as in Example 4, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 4% by mass.
改変クモ糸フィブロインの添加量を分散液全量に対して5質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を4質量%とした他は、実施例4と同様にして分散液を調製した。 (Example 5)
A dispersion was prepared in the same manner as in Example 4, except that the added amount of the modified spider silk fibroin was 5% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 4% by mass.
(実施例6)
改変クモ糸フィブロインの添加量を分散液全量に対して1質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を20質量%とした他は、実施例4と同様にして分散液を調製した。 (Example 6)
A dispersion was prepared in the same manner as in Example 4, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 20% by mass.
改変クモ糸フィブロインの添加量を分散液全量に対して1質量%とし、改変クモ糸フィブロインに対する酸化グラフェンの割合を20質量%とした他は、実施例4と同様にして分散液を調製した。 (Example 6)
A dispersion was prepared in the same manner as in Example 4, except that the amount of the modified spider silk fibroin was 1% by mass relative to the total amount of the dispersion, and the ratio of graphene oxide to the modified spider silk fibroin was 20% by mass.
(比較例1)
比較例として、ギ酸単独溶媒に対する酸化グラフェンの分散性を評価した。改変クモ糸フィブロインを添加しなかった他は、実施例1と同様にして酸化グラフェン分散液を調製した。 (Comparative Example 1)
As a comparative example, the dispersibility of graphene oxide in a formic acid single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 1, except that the modified spider silk fibroin was not added.
比較例として、ギ酸単独溶媒に対する酸化グラフェンの分散性を評価した。改変クモ糸フィブロインを添加しなかった他は、実施例1と同様にして酸化グラフェン分散液を調製した。 (Comparative Example 1)
As a comparative example, the dispersibility of graphene oxide in a formic acid single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 1, except that the modified spider silk fibroin was not added.
(比較例2)
比較例として、DMSO単独溶媒に対する酸化グラフェンの分散性を評価した。改変クモ糸フィブロインを添加しなかった他は、実施例4と同様にして酸化グラフェン分散液を調製した。 (Comparative Example 2)
As a comparative example, the dispersibility of graphene oxide in a DMSO single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 4, except that the modified spider silk fibroin was not added.
比較例として、DMSO単独溶媒に対する酸化グラフェンの分散性を評価した。改変クモ糸フィブロインを添加しなかった他は、実施例4と同様にして酸化グラフェン分散液を調製した。 (Comparative Example 2)
As a comparative example, the dispersibility of graphene oxide in a DMSO single solvent was evaluated. A graphene oxide dispersion was prepared in the same manner as in Example 4, except that the modified spider silk fibroin was not added.
〔分散性の評価〕
分散液における酸化グラフェンの分散性を評価した。分散性の評価は、撹拌後の分散液における酸化グラフェンの沈降物を目視で確認して行なった。表6に分散性の評価結果を示した。
分散性の評価基準は、以下のとおりである。
◎:攪拌後、1ヶ月以上静置しても分散状態を維持。
○:攪拌後、14日間以上静置しても分散状態を維持。
△:撹拌後、48時間以内に沈降。
×:撹拌後、24時間以内に沈降。 (Evaluation of dispersibility)
The dispersibility of graphene oxide in the dispersion was evaluated. The dispersibility was evaluated by visually confirming the precipitate of graphene oxide in the dispersion liquid after stirring. Table 6 shows the evaluation results of the dispersibility.
The evaluation criteria for dispersibility are as follows.
:: After the stirring, the dispersion state is maintained even after being left for one month or more.
:: After stirring, the dispersion state is maintained even if the mixture is allowed to stand for 14 days or more.
Δ: sedimented within 48 hours after stirring.
×: sedimentation within 24 hours after stirring.
分散液における酸化グラフェンの分散性を評価した。分散性の評価は、撹拌後の分散液における酸化グラフェンの沈降物を目視で確認して行なった。表6に分散性の評価結果を示した。
分散性の評価基準は、以下のとおりである。
◎:攪拌後、1ヶ月以上静置しても分散状態を維持。
○:攪拌後、14日間以上静置しても分散状態を維持。
△:撹拌後、48時間以内に沈降。
×:撹拌後、24時間以内に沈降。 (Evaluation of dispersibility)
The dispersibility of graphene oxide in the dispersion was evaluated. The dispersibility was evaluated by visually confirming the precipitate of graphene oxide in the dispersion liquid after stirring. Table 6 shows the evaluation results of the dispersibility.
The evaluation criteria for dispersibility are as follows.
:: After the stirring, the dispersion state is maintained even after being left for one month or more.
:: After stirring, the dispersion state is maintained even if the mixture is allowed to stand for 14 days or more.
Δ: sedimented within 48 hours after stirring.
×: sedimentation within 24 hours after stirring.
ギ酸単独溶媒、及びDMSO単独溶媒に酸化グラフェンを分散させた分散液(比較例1及び2)では、酸化グラフェンが48時間以内に沈降した。一方で、改変クモ糸フィブロインを添加した酸化グラフェンの分散液(実施例1~6)では、酸化グラフェンの分散状態を14日間以上維持し、非常に良好な分散性を示した。特に実施例1及び2においては酸化グラフェンの分散状態を1ヶ月以上維持し、極めて良好な分散性を示した。
In the formic acid single solvent and the dispersion liquid in which graphene oxide was dispersed in DMSO single solvent (Comparative Examples 1 and 2), graphene oxide precipitated within 48 hours. On the other hand, in the dispersion of graphene oxide to which the modified spider silk fibroin was added (Examples 1 to 6), the dispersion state of graphene oxide was maintained for 14 days or more, and very good dispersibility was exhibited. In particular, in Examples 1 and 2, the dispersion state of graphene oxide was maintained for one month or more, and extremely good dispersibility was exhibited.
〔繊維の製造及び評価〕
(実施例7)
(1)ドープ液(紡糸原液)の調製
ギ酸3.16gと酸化グラフェン1mgを混合し、超音波洗浄装置を用いて、温度60℃で99分間超音波振動により撹拌し、酸化グラフェン/ギ酸分散液を調製した。ただちに、上記改変フィブロインの製造工程で得られた改変クモ糸フィブロイン(PRT799)1gを、酸化グラフェン/ギ酸分散液に添加し、攪拌しながら40℃のアルミブロックヒーターで2時間加温して溶解させ、ドープ液(分散液)を調製した。得られたドープ液は、粘性のある黒色の液体を呈し、25℃での粘度は6,230[mPa・sec]であった。改変クモ糸フィブロインの添加量は、ドープ液全量に対して24質量%とした。酸化グラフェンの添加量は、改変フィブロインに対して0.1質量%とした。
(2)乾式紡糸
乾式紡糸を、紡糸装置を使用して室温で行なった。調製した酸化グラフェン含有ドープ液(分散液)をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させた。ノズル先端に液滴が形成されたところで、液滴の端に針の先端を接触させて、ドープ液を繊維状に引き出した。引き出した繊維をワインダーで巻き取ることで、ドラフトをかけながら連続的に繊維を形成させ、改変クモ糸フィブロイン繊維を得た。ワインダーの巻き取り速度は、最大速度の20.82m/分(34.7cm/秒)とした。 (Fiber production and evaluation)
(Example 7)
(1) Preparation of dope solution (spinning stock solution) 3.16 g of formic acid and 1 mg of graphene oxide were mixed, and the mixture was stirred by ultrasonic vibration at a temperature of 60 ° C. for 99 minutes using an ultrasonic cleaning device, and a graphene oxide / formic acid dispersion was obtained. Was prepared. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-mentioned modified fibroin production step was added to the graphene oxide / formic acid dispersion, and heated with a 40 ° C aluminum block heater for 2 hours with stirring to dissolve. And a dope liquid (dispersion liquid) was prepared. The obtained dope liquid was a viscous black liquid and had a viscosity at 25 ° C. of 6,230 [mPa · sec]. The added amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dope solution. The addition amount of graphene oxide was 0.1% by mass based on the modified fibroin.
(2) Dry spinning Dry spinning was performed at room temperature using a spinning apparatus. The prepared graphene oxide-containing dope liquid (dispersion liquid) was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle. When the droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet to draw out the dope solution into a fibrous form. The drawn fiber was wound up by a winder to form a fiber continuously while drafting, thereby obtaining a modified spider silk fibroin fiber. The winding speed of the winder was set to the maximum speed of 20.82 m / min (34.7 cm / sec).
(実施例7)
(1)ドープ液(紡糸原液)の調製
ギ酸3.16gと酸化グラフェン1mgを混合し、超音波洗浄装置を用いて、温度60℃で99分間超音波振動により撹拌し、酸化グラフェン/ギ酸分散液を調製した。ただちに、上記改変フィブロインの製造工程で得られた改変クモ糸フィブロイン(PRT799)1gを、酸化グラフェン/ギ酸分散液に添加し、攪拌しながら40℃のアルミブロックヒーターで2時間加温して溶解させ、ドープ液(分散液)を調製した。得られたドープ液は、粘性のある黒色の液体を呈し、25℃での粘度は6,230[mPa・sec]であった。改変クモ糸フィブロインの添加量は、ドープ液全量に対して24質量%とした。酸化グラフェンの添加量は、改変フィブロインに対して0.1質量%とした。
(2)乾式紡糸
乾式紡糸を、紡糸装置を使用して室温で行なった。調製した酸化グラフェン含有ドープ液(分散液)をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させた。ノズル先端に液滴が形成されたところで、液滴の端に針の先端を接触させて、ドープ液を繊維状に引き出した。引き出した繊維をワインダーで巻き取ることで、ドラフトをかけながら連続的に繊維を形成させ、改変クモ糸フィブロイン繊維を得た。ワインダーの巻き取り速度は、最大速度の20.82m/分(34.7cm/秒)とした。 (Fiber production and evaluation)
(Example 7)
(1) Preparation of dope solution (spinning stock solution) 3.16 g of formic acid and 1 mg of graphene oxide were mixed, and the mixture was stirred by ultrasonic vibration at a temperature of 60 ° C. for 99 minutes using an ultrasonic cleaning device, and a graphene oxide / formic acid dispersion was obtained. Was prepared. Immediately, 1 g of the modified spider silk fibroin (PRT799) obtained in the above-mentioned modified fibroin production step was added to the graphene oxide / formic acid dispersion, and heated with a 40 ° C aluminum block heater for 2 hours with stirring to dissolve. And a dope liquid (dispersion liquid) was prepared. The obtained dope liquid was a viscous black liquid and had a viscosity at 25 ° C. of 6,230 [mPa · sec]. The added amount of the modified spider silk fibroin was 24% by mass based on the total amount of the dope solution. The addition amount of graphene oxide was 0.1% by mass based on the modified fibroin.
(2) Dry spinning Dry spinning was performed at room temperature using a spinning apparatus. The prepared graphene oxide-containing dope liquid (dispersion liquid) was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle. When the droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet to draw out the dope solution into a fibrous form. The drawn fiber was wound up by a winder to form a fiber continuously while drafting, thereby obtaining a modified spider silk fibroin fiber. The winding speed of the winder was set to the maximum speed of 20.82 m / min (34.7 cm / sec).
(3)繊維径評価
上記(2)で得られた繊維を、SEM(Phenome ProX Desktop SEM、Thermo Fisher Scientific社製)を用い、加速電圧15kVの条件で、1700倍の倍率で観察した。得られたSEM画像をImageJソフトウェアのDiameterJプラグインを使用して解析することで、平均繊維径を算出した。図4に実施例7で得られた改変クモ糸フィブロイン繊維のSEM画像を示した。表7に図4のSEM画像の解析結果(繊維径)を示した。
なお、ImageJソフトウェアについては下記の文献に記載がある。
Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012), “Fiji: an open-source platform for biological-image analysis”, Nature methods 9(7):p.676-682, PMID 22743772, doi:10.1038/nmeth.2019、
Schneider, C. A.; Rasband, W. S. & Eliceiri, K. W. (2012), “NIH Image to ImageJ: 25 years of image analysis”, Nature methods 9(7): 671-675, PMID 22930834。
また、DiameterJプラグインについては、下記の文献に記載がある。
Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: A validated open source nanofiber diameter measurement tool. Biomaterials 2015;61:p.327-38. doi:10.1016/j.biomaterials.2015.05.015。 (3) Evaluation of Fiber Diameter The fiber obtained in the above (2) was observed at a magnification of 1700 times using an SEM (Phenome ProX Desktop SEM, manufactured by Thermo Fisher Scientific) at an acceleration voltage of 15 kV. The average fiber diameter was calculated by analyzing the obtained SEM image using DiameterJ plug-in of ImageJ software. FIG. 4 shows an SEM image of the modified spider silk fibroin fiber obtained in Example 7. Table 7 shows the analysis result (fiber diameter) of the SEM image of FIG.
The ImageJ software is described in the following document.
Schindelin, J .; Arganda-Carreras, I .; & France, E.C. et al. (2012), "Fiji: an open-source platform for biological-image analysis", Nature methods 9 (7): p. 676-682, PMID 22743772, doi: 10.1038 / nmeth. 2019,
Schneider, C .; A. Rasband, W .; S. & Eliceiri, K .; W. (2012), "NIH Image to Image J: 25 years of image analysis", Nature methods 9 (7): 671-675, PMID 2293834.
The DiameterJ plug-in is described in the following document.
Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: Validated open source nanofiber diameter measurement tool. Biomaterials 2015; 61: p. 327-38. doi: 10.1016 / j. biomaterials. 2015.05.015.
上記(2)で得られた繊維を、SEM(Phenome ProX Desktop SEM、Thermo Fisher Scientific社製)を用い、加速電圧15kVの条件で、1700倍の倍率で観察した。得られたSEM画像をImageJソフトウェアのDiameterJプラグインを使用して解析することで、平均繊維径を算出した。図4に実施例7で得られた改変クモ糸フィブロイン繊維のSEM画像を示した。表7に図4のSEM画像の解析結果(繊維径)を示した。
なお、ImageJソフトウェアについては下記の文献に記載がある。
Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012), “Fiji: an open-source platform for biological-image analysis”, Nature methods 9(7):p.676-682, PMID 22743772, doi:10.1038/nmeth.2019、
Schneider, C. A.; Rasband, W. S. & Eliceiri, K. W. (2012), “NIH Image to ImageJ: 25 years of image analysis”, Nature methods 9(7): 671-675, PMID 22930834。
また、DiameterJプラグインについては、下記の文献に記載がある。
Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: A validated open source nanofiber diameter measurement tool. Biomaterials 2015;61:p.327-38. doi:10.1016/j.biomaterials.2015.05.015。 (3) Evaluation of Fiber Diameter The fiber obtained in the above (2) was observed at a magnification of 1700 times using an SEM (Phenome ProX Desktop SEM, manufactured by Thermo Fisher Scientific) at an acceleration voltage of 15 kV. The average fiber diameter was calculated by analyzing the obtained SEM image using DiameterJ plug-in of ImageJ software. FIG. 4 shows an SEM image of the modified spider silk fibroin fiber obtained in Example 7. Table 7 shows the analysis result (fiber diameter) of the SEM image of FIG.
The ImageJ software is described in the following document.
Schindelin, J .; Arganda-Carreras, I .; & France, E.C. et al. (2012), "Fiji: an open-source platform for biological-image analysis", Nature methods 9 (7): p. 676-682, PMID 22743772, doi: 10.1038 / nmeth. 2019,
Schneider, C .; A. Rasband, W .; S. & Eliceiri, K .; W. (2012), "NIH Image to Image J: 25 years of image analysis", Nature methods 9 (7): 671-675, PMID 2293834.
The DiameterJ plug-in is described in the following document.
Hotaling NA, Bharti K, Kriel H, Simon Jr. CG. DiameterJ: Validated open source nanofiber diameter measurement tool. Biomaterials 2015; 61: p. 327-38. doi: 10.1016 / j. biomaterials. 2015.05.015.
(比較例3)
(1)ドープ液の調製
比較例として、酸化グラフェンを含有しないドープ液を調製した。酸化グラフェンを添加しなかった他は、実施例7と同様の手順でドープ液を調製した。
(2)乾式紡糸
乾式紡糸を、実施例7と同様に卓上の紡糸装置を使用して室温で行なった。調製したドープ液をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させた。ノズル先端に液滴が形成されたところで、液滴の端に針の先端を接触させ、ドープ液を繊維状に引き出そうと試みたが、ドープ液は液滴状のまま重力方向に落下するのみで、ドープ液を繊維状に引き出すことはできず、繊維を形成させることはできなかった。 (Comparative Example 3)
(1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 7 except that graphene oxide was not added.
(2) Dry spinning Dry spinning was performed at room temperature using a tabletop spinning apparatus in the same manner as in Example 7. The prepared dope solution was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle. When a droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet, and an attempt was made to draw out the dope solution in a fibrous form. In addition, the dope solution could not be drawn out into a fibrous form, and fibers could not be formed.
(1)ドープ液の調製
比較例として、酸化グラフェンを含有しないドープ液を調製した。酸化グラフェンを添加しなかった他は、実施例7と同様の手順でドープ液を調製した。
(2)乾式紡糸
乾式紡糸を、実施例7と同様に卓上の紡糸装置を使用して室温で行なった。調製したドープ液をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させた。ノズル先端に液滴が形成されたところで、液滴の端に針の先端を接触させ、ドープ液を繊維状に引き出そうと試みたが、ドープ液は液滴状のまま重力方向に落下するのみで、ドープ液を繊維状に引き出すことはできず、繊維を形成させることはできなかった。 (Comparative Example 3)
(1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 7 except that graphene oxide was not added.
(2) Dry spinning Dry spinning was performed at room temperature using a tabletop spinning apparatus in the same manner as in Example 7. The prepared dope solution was filled in a syringe and defoamed by centrifugation. Using a syringe pump, the dope solution was discharged into the air from a 0.2 mm diameter monohole nozzle. When a droplet was formed at the tip of the nozzle, the tip of the needle was brought into contact with the end of the droplet, and an attempt was made to draw out the dope solution in a fibrous form. In addition, the dope solution could not be drawn out into a fibrous form, and fibers could not be formed.
表7に示すとおり、酸化グラフェンを添加していないドープ液(比較例3)においては、乾式紡糸により繊維を形成させることができなかったのに対し、酸化グラフェンを添加したドープ液(実施例7)においては、乾式紡糸により繊維を形成させることができた。さらに、得られた改変クモ糸フィブロイン繊維の平均繊維径は、2~3μmという細径であり、予想されない秀逸な結果を得ることができた。加えて、繊維径が極めて細径であったため、ドープ液から溶媒を脱離(気化)させるための加熱工程は不要であった。また、繊維の巻き取りをワインダーの最大速度で行ったが、糸切れが発生することはなかった。巻き取り速度をさらに高速にすることで、繊維の更なる細径化が可能であることが示唆された。
As shown in Table 7, in the dope solution to which graphene oxide was not added (Comparative Example 3), fibers could not be formed by dry spinning, whereas the dope solution to which graphene oxide was added (Example 7) In the case of ()), fibers could be formed by dry spinning. Furthermore, the average fiber diameter of the obtained modified spider silk fibroin fiber was as small as 2-3 μm, and an unexpectedly excellent result could be obtained. In addition, since the fiber diameter was extremely small, a heating step for desorbing (vaporizing) the solvent from the dope solution was unnecessary. The winding of the fiber was performed at the maximum speed of the winder, but no yarn breakage occurred. It was suggested that the fiber could be further reduced in diameter by increasing the winding speed.
〔不織布の製造及び評価〕
(実施例8)
(1)ドープ液(紡糸原液)の調製
上記実施例3と同様に、酸化グラフェン/ギ酸/改変クモ糸フィブロイン(PRT799)ドープ液を調製した。
(2)乾式紡糸
乾式紡糸は、市販のわたあめ菓子製造装置(株式会社アズマ製)を使用して行なった。装置の電源を入れ、わたあめ菓子製造装置の鍋の内部のディスク状回転部を回転させた。該ディスク状回転部とは、わたあめ菓子製造装置を用いて通常のようにわたあめを製造する際には、ザラメ等を投入する投入口の周囲の回転部のことである。調製した酸化グラフェン含有ドープ液をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させ、ノズル先端に液滴が形成されたところで、該液滴をわたあめ菓子製造装置のディスク状回転部に接触させ、ディスク状回転部の回転を利用して繊維を連続的に形成させ、改変クモ糸フィブロイン不織布を得た。この製造方法により、実施例7に記載した繊維形成方法よりもより細径の繊維を得ることができた。
(3)繊維径評価
上記(2)で得られた不織布を、SEMを用い、加速電圧15kVの条件で1700倍の倍率で観察した。得られたSEM画像をImageJソフトウェアのDiameterJプラグインを使用して解析することで、平均繊維径を算出した。図5に実施例8で得られた不織布のSEM画像を示した。表8に図5のSEM画像の解析結果を示した。
(比較例4)
(1)ドープ液の調製
比較例として、酸化グラフェンを含有しないドープ液を調製した。酸化グラフェンを添加しなかった他は、実施例8と同様の手順でドープ液を調製した。
(2)乾式紡糸
調製したドープ液をシリンジに充填し、遠心分離により脱泡した。乾式紡糸は、実施例2と同様に、市販のわたあめ菓子製造装置を使用して行なった。装置の電源を入れ、回転鍋を回転させた。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させ、ノズル先端に液滴が形成されたところで、液滴をわたあめ菓子製造装置のディスク状回転部に接触させ、繊維の形成を試みたが、繊維を形成させることはできなかった。 (Production and evaluation of nonwoven fabric)
(Example 8)
(1) Preparation of Dope Solution (Spinning Stock Solution) In the same manner as in Example 3, a dope solution of graphene oxide / formic acid / modified spider silk fibroin (PRT799) was prepared.
(2) Dry spinning Dry spinning was performed using a commercially available cotton candy manufacturing apparatus (manufactured by Azuma Co., Ltd.). The apparatus was turned on, and the disk-shaped rotating section inside the pan of the cotton candy manufacturing apparatus was rotated. The disk-shaped rotating part is a rotating part around an inlet for feeding a rough or the like when a cotton candy is manufactured using a cotton candy confectionery apparatus as usual. The prepared graphene oxide-containing dope solution was filled in a syringe, and defoamed by centrifugation. Using a syringe pump, a dope solution is discharged into the air from a 0.2-mm-diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet contacts the disk-shaped rotating part of the cotton candy manufacturing device. Then, the fibers were continuously formed by utilizing the rotation of the disk-shaped rotating portion to obtain a modified spider silk fibroin nonwoven fabric. By this manufacturing method, a fiber having a smaller diameter than that of the fiber forming method described in Example 7 could be obtained.
(3) Evaluation of Fiber Diameter The nonwoven fabric obtained in the above (2) was observed at a magnification of 1700 times using an SEM at an acceleration voltage of 15 kV. The average fiber diameter was calculated by analyzing the obtained SEM image using DiameterJ plug-in of ImageJ software. FIG. 5 shows an SEM image of the nonwoven fabric obtained in Example 8. Table 8 shows the analysis result of the SEM image of FIG.
(Comparative Example 4)
(1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 8, except that graphene oxide was not added.
(2) Dry spinning The prepared dope solution was filled in a syringe and defoamed by centrifugation. The dry spinning was performed using a commercially available cotton candy manufacturing apparatus in the same manner as in Example 2. The apparatus was turned on and the rotating pan was rotated. Using a syringe pump, the dope liquid is discharged into the air from a 0.2 mm diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet is brought into contact with a disk-shaped rotating portion of a cotton candy manufacturing apparatus. An attempt was made to form fibers, but no fibers could be formed.
(実施例8)
(1)ドープ液(紡糸原液)の調製
上記実施例3と同様に、酸化グラフェン/ギ酸/改変クモ糸フィブロイン(PRT799)ドープ液を調製した。
(2)乾式紡糸
乾式紡糸は、市販のわたあめ菓子製造装置(株式会社アズマ製)を使用して行なった。装置の電源を入れ、わたあめ菓子製造装置の鍋の内部のディスク状回転部を回転させた。該ディスク状回転部とは、わたあめ菓子製造装置を用いて通常のようにわたあめを製造する際には、ザラメ等を投入する投入口の周囲の回転部のことである。調製した酸化グラフェン含有ドープ液をシリンジに充填し、遠心分離により脱泡した。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させ、ノズル先端に液滴が形成されたところで、該液滴をわたあめ菓子製造装置のディスク状回転部に接触させ、ディスク状回転部の回転を利用して繊維を連続的に形成させ、改変クモ糸フィブロイン不織布を得た。この製造方法により、実施例7に記載した繊維形成方法よりもより細径の繊維を得ることができた。
(3)繊維径評価
上記(2)で得られた不織布を、SEMを用い、加速電圧15kVの条件で1700倍の倍率で観察した。得られたSEM画像をImageJソフトウェアのDiameterJプラグインを使用して解析することで、平均繊維径を算出した。図5に実施例8で得られた不織布のSEM画像を示した。表8に図5のSEM画像の解析結果を示した。
(比較例4)
(1)ドープ液の調製
比較例として、酸化グラフェンを含有しないドープ液を調製した。酸化グラフェンを添加しなかった他は、実施例8と同様の手順でドープ液を調製した。
(2)乾式紡糸
調製したドープ液をシリンジに充填し、遠心分離により脱泡した。乾式紡糸は、実施例2と同様に、市販のわたあめ菓子製造装置を使用して行なった。装置の電源を入れ、回転鍋を回転させた。シリンジポンプを用いて、0.2mm径のモノホールノズルからドープ液を空気中に吐出させ、ノズル先端に液滴が形成されたところで、液滴をわたあめ菓子製造装置のディスク状回転部に接触させ、繊維の形成を試みたが、繊維を形成させることはできなかった。 (Production and evaluation of nonwoven fabric)
(Example 8)
(1) Preparation of Dope Solution (Spinning Stock Solution) In the same manner as in Example 3, a dope solution of graphene oxide / formic acid / modified spider silk fibroin (PRT799) was prepared.
(2) Dry spinning Dry spinning was performed using a commercially available cotton candy manufacturing apparatus (manufactured by Azuma Co., Ltd.). The apparatus was turned on, and the disk-shaped rotating section inside the pan of the cotton candy manufacturing apparatus was rotated. The disk-shaped rotating part is a rotating part around an inlet for feeding a rough or the like when a cotton candy is manufactured using a cotton candy confectionery apparatus as usual. The prepared graphene oxide-containing dope solution was filled in a syringe, and defoamed by centrifugation. Using a syringe pump, a dope solution is discharged into the air from a 0.2-mm-diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet contacts the disk-shaped rotating part of the cotton candy manufacturing device. Then, the fibers were continuously formed by utilizing the rotation of the disk-shaped rotating portion to obtain a modified spider silk fibroin nonwoven fabric. By this manufacturing method, a fiber having a smaller diameter than that of the fiber forming method described in Example 7 could be obtained.
(3) Evaluation of Fiber Diameter The nonwoven fabric obtained in the above (2) was observed at a magnification of 1700 times using an SEM at an acceleration voltage of 15 kV. The average fiber diameter was calculated by analyzing the obtained SEM image using DiameterJ plug-in of ImageJ software. FIG. 5 shows an SEM image of the nonwoven fabric obtained in Example 8. Table 8 shows the analysis result of the SEM image of FIG.
(Comparative Example 4)
(1) Preparation of Dope Solution As a comparative example, a dope solution containing no graphene oxide was prepared. A dope solution was prepared in the same procedure as in Example 8, except that graphene oxide was not added.
(2) Dry spinning The prepared dope solution was filled in a syringe and defoamed by centrifugation. The dry spinning was performed using a commercially available cotton candy manufacturing apparatus in the same manner as in Example 2. The apparatus was turned on and the rotating pan was rotated. Using a syringe pump, the dope liquid is discharged into the air from a 0.2 mm diameter monohole nozzle, and when a droplet is formed at the tip of the nozzle, the droplet is brought into contact with a disk-shaped rotating portion of a cotton candy manufacturing apparatus. An attempt was made to form fibers, but no fibers could be formed.
表8に示すとおり、酸化グラフェンを添加していないドープ液(比較例4)においては、乾式紡糸により不織布を形成させることができなかったのに対し、酸化グラフェンを添加したドープ液(実施例8)においては、乾式紡糸により不織布を形成させることができた。さらに、得られた改変クモ糸フィブロイン不織布の平均繊維径は、1~2μmという細径であり、予想されない秀逸な結果を得ることができた。加えて、不織布の繊維径は十分に細径であったため、ドープ液から溶媒を脱離(気化)させるための加熱工程は不要であった。
As shown in Table 8, the dope solution to which no graphene oxide was added (Comparative Example 4) failed to form a nonwoven fabric by dry spinning, whereas the dope solution to which graphene oxide was added (Example 8). In ()), a nonwoven fabric could be formed by dry spinning. Furthermore, the average fiber diameter of the obtained modified spider silk fibroin nonwoven fabric was as small as 1 to 2 μm, and unexpectedly excellent results could be obtained. In addition, since the nonwoven fabric had a sufficiently small fiber diameter, a heating step for removing (vaporizing) the solvent from the dope solution was unnecessary.
参考例1:改変フィブロインの燃焼性試験
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロイン(PRT799)の凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 1: Flammability test of modified fibroin A freeze-dried powder of modified fibroin (PRT799) was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was added. Used and mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロイン(PRT799)の凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 1: Flammability test of modified fibroin A freeze-dried powder of modified fibroin (PRT799) was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was added. Used and mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
得られた紡糸原液を90℃に加熱し、目開き5μmの金属フィルターで濾過し、次いで30mLのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径0.2mmのソリッドノズルから100質量%メタノール凝固浴槽中へ吐出させた。吐出温度は90℃であった。凝固後、得られた原糸を巻き取り、自然乾燥させて改変フィブロイン繊維(原料繊維)を得た。
The obtained spinning stock solution was heated to 90 ° C., filtered through a metal filter having a mesh size of 5 μm, and allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 90 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
原料繊維を撚り合せた撚糸を使用して、丸編機を使用した丸編みで編地(太さ:180デニール、ゲージ数:18)を製造した。得られた編地を20g切り出して、試験片として使用した。
(4) A knitted fabric (thickness: 180 denier, gauge number: 18) was manufactured by circular knitting using a circular knitting machine by using a twisted yarn obtained by twisting raw fibers. 20 g of the obtained knitted fabric was cut out and used as a test piece.
燃焼性試験は、「消防危50号(平成7年5月31日付け)」に記載の「粉粒状又は融点の低い合成樹脂の試験方法」に準拠した。試験は、温度22℃、相対湿度45%、気圧1021hPaの条件下で実施した。測定結果(酸素濃度(%)、燃焼率(%)、換算燃焼率(%))を表9に示す。
The flammability test was based on the “Test method for synthetic resin with powdery or low melting point” described in “Fire Danger No. 50 (May 31, 1995)”. The test was performed under the conditions of a temperature of 22 ° C., a relative humidity of 45%, and an air pressure of 1021 hPa. Table 9 shows the measurement results (oxygen concentration (%), burning rate (%), reduced burning rate (%)).
燃焼性試験の結果、改変フィブロイン(PRT799)繊維で編んだ編地の限界酸素指数(LOI)値は27.2であった。一般にLOI値が26以上であると、難燃性であると知られている。改変フィブロインは、難燃性に優れていることが分かる。
As a result of the flammability test, the knitted fabric made of the modified fibroin (PRT799) fiber had a limiting oxygen index (LOI) value of 27.2. Generally, when the LOI value is 26 or more, it is known to be flame retardant. It can be seen that the modified fibroin has excellent flame retardancy.
参考例2:改変フィブロインの吸湿発熱性評価
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロインの凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 2: Evaluation of heat generation by moisture absorption of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethyl sulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was used. And mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロインの凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 2: Evaluation of heat generation by moisture absorption of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethyl sulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and a shaker was used. And mixed for 3 hours to dissolve. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
得られた紡糸原液を60℃に加熱し、目開き5μmの金属フィルターで濾過し、次いで30mLのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径0.2mmのソリッドノズルから100質量%メタノール凝固浴槽中へ吐出させた。吐出温度は60℃であった。凝固後、得られた原糸を巻き取り、自然乾燥させて改変フィブロイン繊維(原料繊維)を得た。
The obtained spinning dope was heated to 60 ° C., filtered with a metal filter having a mesh size of 5 μm, and then allowed to stand in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
比較のため、原料繊維として、市販されているウール繊維、コットン繊維、テンセル繊維、レーヨン繊維及びポリエステル繊維を用意した。
市 販 For comparison, commercially available wool fibers, cotton fibers, Tencel fibers, rayon fibers, and polyester fibers were prepared as raw fibers.
各原料繊維を使用して、横編機を使用した横編みで編地をそれぞれ製造した。PRT918繊維又はPRT799繊維を使用した編地の太さ及びゲージ数を表10に示すとおりである。その他の原料繊維を使用した編地は、改変フィブロイン繊維の編地とほぼ同一のカバーファクターとなるように太さ及びゲージ数を調整した。具体的には、以下のとおりである。
Using each raw material fiber, a knitted fabric was produced by flat knitting using a flat knitting machine. Table 10 shows the thickness and gauge number of the knitted fabric using PRT918 fiber or PRT799 fiber. The thickness and the number of gauges of the knitted fabric using other raw material fibers were adjusted so that the same cover factor as the knitted fabric of the modified fibroin fiber was obtained. Specifically, it is as follows.
10cm×10cmに裁断した編地を2枚合わせにし、四辺を縫い合わせて試験片(試料)とした。試験片を低湿度環境(温度20±2℃、相対湿度40±5%)で4時間以上放置した後、高湿度環境(温度20±2℃、相対湿度90±5%)に移し、試験片内部中央に取り付けた温度センサーにより30分間、1分間隔で温度の測定を行った。
Two knitted fabrics cut to 10 cm × 10 cm were aligned, and four sides were sewn to obtain a test piece (sample). After leaving the test specimen in a low humidity environment (temperature 20 ± 2 ° C, relative humidity 40 ± 5%) for 4 hours or more, it was moved to a high humidity environment (temperature 20 ± 2 ° C, relative humidity 90 ± 5%) The temperature was measured at 1 minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.
測定結果から、下記式Aに従って、最高吸湿発熱度を求めた。
式A: 最高吸湿発熱度={(試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値)-(試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度)}(℃)/試料重量(g) From the measurement results, the maximum heat of moisture absorption was calculated according to the following formula A.
Formula A: Maximum heat of moisture absorption = {(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (℃) / sample weight (g)
式A: 最高吸湿発熱度={(試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移したときの試料温度の最高値)-(試料を、試料温度が平衡に達するまで低湿度環境下に置いた後、高湿度環境下に移すときの試料温度)}(℃)/試料重量(g) From the measurement results, the maximum heat of moisture absorption was calculated according to the following formula A.
Formula A: Maximum heat of moisture absorption = {(Maximum value of sample temperature when sample is placed in low humidity environment until sample temperature reaches equilibrium and then moved to high humidity environment) − (Sample is sample Sample temperature when placed in a low humidity environment until the temperature reaches equilibrium and then transferred to a high humidity environment)} (℃) / sample weight (g)
図6は、吸湿発熱性試験の結果の一例を示すグラフである。グラフの横軸は、試料を低湿度環境から高湿度環境に移した時点を0とし、高湿度環境での放置時間(分)を示す。グラフの縦軸は、温度センサーで測定した温度(試料温度)を示す。図6に示したグラフ中、Mで示した点が、試料温度の最高値に対応している。
FIG. 6 is a graph showing an example of the result of the moisture absorption / heating test. The horizontal axis of the graph represents the time (minute) of leaving the sample in the high humidity environment as 0 when the time when the sample was transferred from the low humidity environment to the high humidity environment. The vertical axis of the graph indicates the temperature (sample temperature) measured by the temperature sensor. In the graph shown in FIG. 6, the point indicated by M corresponds to the maximum value of the sample temperature.
各編地の最高吸湿発熱度の算出結果を表11に示す。
Table 11 shows the calculation results of the maximum heat of moisture absorption of each knitted fabric.
表11に示すとおり、改変フィブロイン(PRT918及びPRT799)は、既存の材料と比べて、最高吸湿発熱度が高く、吸湿発熱性に優れていることが分かる。
と お り As shown in Table 11, it can be seen that the modified fibroin (PRT918 and PRT799) has a higher maximum heat of moisture absorption and excellent heat generation by moisture absorption than the existing materials.
参考例3:改変フィブロインの保温性評価
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロインの凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 3: Evaluation of heat retention of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was shaken using a shaker. The mixture was dissolved by mixing for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
塩化リチウムのジメチルスルホキシド溶液(濃度:4.0質量%)に、改変フィブロインの凍結乾燥粉末を、濃度24質量%となるよう添加し、シェーカーを使用して3時間混合することにより、溶解させた。その後、不溶物と泡を取り除き、改変フィブロイン溶液(紡糸原液)を得た。 Reference Example 3: Evaluation of heat retention of modified fibroin A freeze-dried powder of modified fibroin was added to a solution of lithium chloride in dimethylsulfoxide (concentration: 4.0% by mass) to a concentration of 24% by mass, and the mixture was shaken using a shaker. The mixture was dissolved by mixing for 3 hours. Thereafter, insolubles and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).
得られた紡糸原液を60℃に加熱し、目開き5μmの金属フィルターで濾過し、次いで30mLのステンレスシリンジ内で静置し、脱泡させた後に、ニードル径0.2mmのソリッドノズルから100質量%メタノール凝固浴槽中へ吐出させた。吐出温度は60℃であった。凝固後、得られた原糸を巻き取り、自然乾燥させて改変フィブロイン繊維(原料繊維)を得た。
The obtained spinning dope was heated to 60 ° C., filtered through a metal filter having a mesh size of 5 μm, and then left standing in a 30 mL stainless syringe to remove bubbles. % Methanol was discharged into a coagulation bath. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound and air-dried to obtain a modified fibroin fiber (raw fiber).
比較のため、原料繊維として、市販されているウール繊維、シルク繊維、綿繊維、レーヨン繊維及びポリエステル繊維を用意した。
市 販 For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers and polyester fibers were prepared as raw material fibers.
各原料繊維を使用して、横編機を使用した横編みで編地をそれぞれ製造した。PRT966繊維又はPRT799繊維を使用した編地の番手、撚り本数、ゲージ数、目付けは、表12に示すとおりである。その他の原料繊維を使用した編地は、改変フィブロイン繊維の編地とほぼ同一のカバーファクターとなるように調整した。具体的には、以下のとおりである。
Using each raw material fiber, a knitted fabric was produced by flat knitting using a flat knitting machine. Table 12 shows the count, the number of twists, the number of gauges, and the basis weight of the knitted fabric using the PRT966 fiber or the PRT799 fiber. The knitted fabric using other raw material fibers was adjusted to have almost the same cover factor as the knitted fabric of the modified fibroin fiber. Specifically, it is as follows.
保温性は、カトーテック株式会社製のKES-F7サーモラボII試験機を使用し、ドライコンタクト法(皮膚と衣服が乾燥状態で直接触れた時を想定した方法)を用いて評価した。20cm×20cmの矩形に裁断した編地1枚を試験片(試料)として使用した。試験片を、一定温度(30℃)に設定した熱板にセットし、風洞内風速30cm/秒の条件で、試験片を介して放散された熱量(a)を求めた。試験片をセットしない状態で、上記同様の条件で放散された熱量(b)を求め、下記式Bに従い保温率(%)を算出した。
式B: 保温率(%)=(1-a/b)×100 The heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothing directly touched in a dry state). One knitted fabric cut into a rectangle of 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) radiated through the test piece was determined under the condition of a wind speed in the wind tunnel of 30 cm / sec. Without setting the test piece, the amount of heat (b) radiated under the same conditions as above was determined, and the heat retention (%) was calculated according to the following formula B.
Formula B: Heat retention rate (%) = (1−a / b) × 100
式B: 保温率(%)=(1-a/b)×100 The heat retention was evaluated using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd., using a dry contact method (a method assuming that the skin and clothing directly touched in a dry state). One knitted fabric cut into a rectangle of 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) radiated through the test piece was determined under the condition of a wind speed in the wind tunnel of 30 cm / sec. Without setting the test piece, the amount of heat (b) radiated under the same conditions as above was determined, and the heat retention (%) was calculated according to the following formula B.
Formula B: Heat retention rate (%) = (1−a / b) × 100
測定結果から、下記式Cに従って、保温性指数を求めた。
式C: 保温性指数=保温率(%)/試料の目付け(g/m2) From the measurement results, the heat retention index was determined according to the following formula C.
Formula C: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
式C: 保温性指数=保温率(%)/試料の目付け(g/m2) From the measurement results, the heat retention index was determined according to the following formula C.
Formula C: Insulation index = Insulation rate (%) / Sample weight (g / m 2 )
保温性指数の算出結果を表11に示す。保温性指数が高いほど、保温性に優れる材料と評価することができる。
Table 11 shows the calculation results of the heat retention index. The higher the heat retention index, the more the material can be evaluated as having excellent heat retention.
表13に示すとおり、改変フィブロイン(PRT966及びPRT799)は、既存の材料と比べて、保温性指数が高く、保温性に優れていることが分かる。
と お り As shown in Table 13, it can be seen that the modified fibroin (PRT966 and PRT799) has a high heat retention index and is excellent in heat retention as compared with existing materials.
参考例1~3に示したとおり、改変フィブロインが改変クモ糸フィブロインであると、保温性、吸湿発熱性及び/又は難燃性がより優れるものとすることができる。改変クモ糸フィブロインを用いて本発明の繊維とすることで、保温性、吸湿発熱性及び/又は難燃性により優れ、かつ細径の繊維を得ることができる。
示 し As shown in Reference Examples 1 to 3, when the modified fibroin is modified spider silk fibroin, the heat retention, the moisture absorption and heat generation and / or the flame retardancy can be more excellent. By using the modified spider silk fibroin to produce the fiber of the present invention, a fiber having excellent heat retention, moisture absorption and heat generation and / or flame retardancy, and a small diameter can be obtained.
Claims (17)
- 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
前記炭素材料が一層又は二層以上の平面状グラフェンである、炭素材料分散液。 A modified spider silk fibroin, a carbon material, and a polar solvent,
A carbon material dispersion, wherein the carbon material is one or more planar graphene. - 前記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、請求項1に記載の分散液。 The dispersion according to claim 1, wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide.
- 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含み、
前記炭素材料がカーボンブラックナノパーティクルであり、
前記極性溶媒が、N,N-ジメチルアセトアミド、1,3-ジメチル-2-イミダゾリドン、N-メチル-2-ピロリドン、アセトニトリル、N-メチルモルホリンN-オキシド、ギ酸、エチレングリコール、テトラヒドロフランからなる群から選ばれる少なくとも1種である、炭素材料分散液。 A modified spider silk fibroin, a carbon material, and a polar solvent,
The carbon material is carbon black nanoparticles,
The polar solvent is selected from the group consisting of N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-pyrrolidone, acetonitrile, N-methylmorpholine N-oxide, formic acid, ethylene glycol, and tetrahydrofuran. A carbon material dispersion which is at least one selected from the group consisting of: - ドープ液である、請求項1~3のいずれか一項に記載の分散液。 4. The dispersion according to any one of claims 1 to 3, which is a dope solution.
- 改変クモ糸フィブロインと、一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料と、を含み、平均繊維径が3μm以下である、改変クモ糸フィブロイン繊維。 A modified spider yarn comprising a modified spider yarn fibroin and at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles, and having an average fiber diameter of 3 μm or less. Fibroin fiber.
- 前記炭素材料の含有量が、前記改変クモ糸フィブロイン100質量部に対して1質量部以下である、請求項5に記載の改変クモ糸フィブロイン繊維。 The modified spider silk fibroin fiber according to claim 5, wherein the content of the carbon material is 1 part by mass or less based on 100 parts by mass of the modified spider silk fibroin.
- 前記平面状グラフェンが、グラフェン、酸化グラフェン、還元型酸化グラフェン、機能化酸化グラフェン及び還元型機能化酸化グラフェンからなる群から選ばれる少なくとも1種である、請求項5又は6に記載の改変クモ糸フィブロイン繊維。 The modified spider silk according to claim 5, wherein the planar graphene is at least one selected from the group consisting of graphene, graphene oxide, reduced graphene oxide, functionalized graphene oxide, and reduced functionalized graphene oxide. Fibroin fiber.
- 請求項5~7のいずれか一項に記載の改変クモ糸フィブロイン繊維を含む、製品。 (8) A product comprising the modified spider silk fibroin fiber according to any one of (5) to (7).
- 前記製品が、繊維、糸、布帛、編み物、組み物、不織布、紙、及び綿からなる群から選択される少なくとも1種である、請求項8に記載の製品。 The product according to claim 8, wherein the product is at least one selected from the group consisting of fibers, yarns, fabrics, knits, braids, nonwoven fabrics, paper, and cotton.
- 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程を備え、前記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン繊維の製造方法。 Modified spider silk fibroin, comprising a step of forming fibrils from a dispersion containing a carbon material and a polar solvent, wherein the carbon material is one or more layers of planar graphene, modified spider silk fibroin fibers Production method.
- 前記原繊維を形成させる工程において、乾式紡糸法によって原繊維を形成させる、請求項10に記載の改変クモ糸フィブロイン繊維の製造方法。 The method for producing a modified spider silk fibroin fiber according to claim 10, wherein in the step of forming the raw fiber, the raw fiber is formed by a dry spinning method.
- 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程を備え、前記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン繊維の製造方法。 Modified spider silk fibroin, a carbon material, comprising a step of forming a fibril by a dry spinning method from a dispersion containing a polar solvent, wherein the carbon material is carbon black nanoparticles, production of modified spider silk fibroin fiber Method.
- 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から原繊維を形成させる工程と、
前記原繊維又は前記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、前記炭素材料が一層又は二層以上の平面状グラフェンである、改変クモ糸フィブロイン不織布の製造方法。 Modified spider silk fibroin, a carbon material, and a polar solvent, a step of forming fibrils from a dispersion liquid,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
Wherein the carbon material is one or two or more layers of planar graphene. - 改変クモ糸フィブロインと、炭素材料と、極性溶媒と、を含む分散液から乾式紡糸法によって原繊維を形成させる工程と、
前記原繊維又は前記原繊維から製造された改変クモ糸フィブロイン繊維を絡合させる工程と、
を備え、前記炭素材料がカーボンブラックナノパーティクルである、改変クモ糸フィブロイン不織布の製造方法。 Modified spider silk fibroin, a carbon material, a polar solvent, a step of forming fibrils by a dry spinning method from a dispersion containing,
Entangling the fibrils or modified spider silk fibroin fibers produced from the fibrils,
And a method for producing a modified spider silk fibroin nonwoven fabric, wherein the carbon material is carbon black nanoparticles. - 改変クモ糸フィブロインと、前記改変クモ糸フィブロイン100質量部に対して25質量部以下の炭素材料と、極性溶媒と、を混合する工程を含み、
前記炭素材料が、一層又は二層以上の平面状グラフェンである、炭素材料の分散方法。 Modified spider silk fibroin, comprising the step of mixing a carbon material of not more than 25 parts by mass with respect to 100 parts by mass of the modified spider silk fibroin, and a polar solvent,
A method for dispersing a carbon material, wherein the carbon material is one or two or more layers of planar graphene. - 改変クモ糸フィブロインを含む、炭素材料を極性溶媒に分散させるための分散補助剤であって、前記炭素材料が一層又は二層以上の平面状グラフェンである、分散補助剤。 (4) A dispersion aid for dispersing a carbon material in a polar solvent, comprising a modified spider silk fibroin, wherein the carbon material is a single layer or two or more layers of planar graphene.
- 一層又は二層以上の平面状グラフェン及びカーボンブラックナノパーティクルからなる群から選択される少なくとも1種の炭素材料を含む、細径化された改変クモ糸フィブロイン繊維を製造するための細径化剤。
A thinning agent for producing a modified spider silk fibroin fiber having a reduced diameter, comprising at least one carbon material selected from the group consisting of one or more layers of planar graphene and carbon black nanoparticles.
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CN108452374A (en) * | 2018-05-09 | 2018-08-28 | 海南蛛王药业有限公司 | A kind of spider silk composition and preparation method thereof |
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WO2018164234A1 (en) * | 2017-03-10 | 2018-09-13 | カジナイロン株式会社 | Method for producing protein fiber, and method for shrinking protein fiber |
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