JP2022160006A - Synthetic leather and production method thereof - Google Patents

Abstract

To provide a synthetic leather having sufficient hygroscopic properties, allowing the dimensional change caused by contact with water to be suppressed to a minimum extent, and the energy required for production to be reduced.SOLUTION: A synthetic leather comprises a substrate layer including a nonwoven fabric and a polymer material. The nonwoven fabric includes a protein fiber and is shrink-proofed.SELECTED DRAWING: Figure 1

Description

The present invention relates to synthetic leather and its manufacturing method.

BACKGROUND ART Conventionally, synthetic leather such as artificial leather has been used as a substitute for natural leather. Unlike natural leather, which is limited in supply, synthetic leather can be artificially mass-produced. It has come into use.

For example, Patent Document 1 discloses a method for producing artificial leather by impregnating a non-woven fabric pretreated with a sizing macromolecular substance with a polyurethane resin solution, wet-coagulating the non-woven fabric, and then removing the sizing macromolecular substance. into a mixed emulsion of reactive methyl H silicon and a metal catalyst, followed by heating at 100-150°C.

JP-A-10-131059

However, in synthetic leather such as artificial leather as disclosed in Patent Document 1, both the nonwoven fabric and the polymer substance are composed of synthetic resin, so it has poor hygroscopicity and is mainly made of synthetic resin. Since synthetic leather is derived from petroleum, there is a problem that a large amount of energy is required to produce synthetic leather. In order to solve such problems, it is conceivable to configure at least the nonwoven fabric, which is the main material, of the nonwoven fabric and the polymeric material with a protein material. However, some protein materials shrink when they come into contact with water, and there is concern that synthetic leather produced using such protein materials may undergo significant dimensional changes upon contact with water. be.

The present invention has been made in view of the above circumstances, and while having sufficient hygroscopicity, it is possible to suppress dimensional changes due to contact with water as much as possible, and the energy required for manufacturing is reduced. The purpose is to provide synthetic leather. The present invention also provides a method for producing a synthetic leather that has sufficient hygroscopicity, can suppress dimensional changes due to contact with water as much as possible, and can reduce the energy required for production. intended to provide

The present invention relates to, for example, the following inventions.
[1]
Synthetic leather comprising a base layer comprising a non-woven fabric and a polymeric material, wherein the non-woven fabric comprises protein fibers and is shrink-proof.
[2]
The synthetic leather according to [1], wherein the fiber density increase rate defined by the following formula I is 20% or more.
Fiber density increase rate = {(fiber density of non-woven fabric after shrink-proofing/fiber density of non-woven fabric before shrink-proofing) - 1} x 100 (%) (Formula I)
[3]
The synthetic leather according to [1] or [2], wherein the protein fiber has a wet shrinkage of 2% or more as defined by the following formula II.
Wet shrinkage = {1-(length of protein fiber brought into wet state by contact with water/length of protein fiber after spinning but before contact with water)} x 100 (%) (Formula II)
[4]
The synthetic leather according to any one of [1] to [3], wherein the protein fiber has a dry shrinkage of more than 7% as defined by the following formula III.
Dry shrinkage = {1-(length of protein fiber in dry state/length of protein fiber after spinning before contact with water)} x 100 (%) (Formula III)
[5]
The synthetic leather according to any one of [1] to [4], wherein the nonwoven fabric has a fiber density of 0.04 g/cm ² or more.
[6]
The synthetic leather according to any one of [1] to [5], wherein the protein fibers contain modified fibroin.
[7]
The synthetic leather according to [6], wherein the modified fibroin is modified spider silk fibroin.
[8]
A method for producing synthetic leather, comprising the step of impregnating a nonwoven fabric with an impregnating liquid containing a polymeric substance and solidifying the polymeric substance to form a base material layer, wherein the nonwoven fabric contains protein fibers and is shrink-proofed. .
[9]
The method for producing synthetic leather according to [8], wherein the nonwoven fabric is shrink-proofed by water shrinkage due to contact with water.

ADVANTAGE OF THE INVENTION According to the present invention, it is possible to provide a synthetic leather that has sufficient hygroscopicity, can suppress dimensional change due to contact with water as much as possible, and has reduced energy required for manufacturing. can. Further, according to the present invention, a synthetic leather capable of suppressing dimensional change due to contact with water as much as possible while having sufficient hygroscopicity, and capable of reducing the energy required for manufacturing. can provide a manufacturing method of

1 is a schematic cross-sectional view of a synthetic leather according to one embodiment; FIG. 1 is a schematic cross-sectional view of a synthetic leather according to one embodiment; FIG. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 4 is a diagram showing the distribution of z/w (%) values of naturally occurring fibroin. FIG. 4 is a diagram showing the distribution of x/y (%) values of naturally occurring fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of modified fibroin. BRIEF DESCRIPTION OF THE DRAWINGS It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a protein fiber (filament). BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram of the manufacturing method of the synthetic leather which concerns on one Embodiment. BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram of the manufacturing method of the synthetic leather which concerns on one Embodiment. 1 is an explanatory diagram of an electrospinning device according to one embodiment; FIG. It is a graph which shows an example of the result of a moisture absorption heat generation test. 3 is a photograph of synthetic leather 3. FIG.

EMBODIMENT OF THE INVENTION Hereinafter, it demonstrates in detail, referring drawings for the form for implementing this invention. For the sake of convenience, substantially the same elements are given the same reference numerals, and their description may be omitted. In addition, this invention is not limited to the following embodiment.

The synthetic leather according to this embodiment includes a base material layer containing a nonwoven fabric and a polymeric material. The substrate layer can also be said to be a layer in which a nonwoven fabric and an impregnated body made of a polymeric substance impregnated in a nonwoven fabric are integrated, or a nonwoven fabric is embedded in a layer formed of a polymeric substance. It can also be called a layer.

In this embodiment, the nonwoven comprises protein fibers and is shrink proof. In the synthetic leather according to the present embodiment, the nonwoven fabric, which is the main material, contains protein fibers, so that the hygroscopicity is improved and the energy required for manufacturing is reduced. In addition, since the non-woven fabric, which is the main material, of the synthetic leather according to the present embodiment is shrink-proof, dimensional change due to contact with water is suppressed.

Synthetic leather is also called artificial leather. ) By forming a layer, it has a natural leather-like appearance and function. In this specification, synthetic leather refers to so-called artificial leather (a special nonwoven fabric (a base material mainly composed of a fiber layer having a random three-dimensional three-dimensional structure impregnated with polyurethane or a similar flexible polymeric substance) Also included are those that use

FIG. 1 is a schematic cross-sectional view of a synthetic leather according to one embodiment. Synthetic leather 10 shown in FIG. The substrate layer 3 may, for example, consist of a layer of polymeric material 2 in which the nonwoven fabric 1 is embedded. The substrate layer 3 may be a nonporous layer or a porous layer.

(non-woven fabric)
The nonwoven comprises protein fibers and is shrink proof. The nonwoven fabric can be produced by a known production method using, for example, fibers at least partially containing protein fibers. Specifically, for example, a web (including a single-layer web and a laminated web) is formed from fibers at least partially containing protein fibers by a dry method, a wet method, an air-laid method, etc., and then a chemical bond method. (immersion method, spray method, etc.), needle punching method, etc. can be used to bond the fibers of the web to obtain a nonwoven fabric.

Nonwoven fabrics can also be used, for example, by combining proteins with solvents such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), optionally with an inorganic solvent as a solubility enhancer. It can also be obtained by adding it together with a salt and dissolving it to prepare a dope solution, and then spinning the dope solution by an electrospinning method (electrostatic spinning method).

The nonwoven fabric may contain raw material fibers other than protein fibers. Examples of raw fibers other than protein fibers include synthetic fibers such as nylon, polyester, polyamide, polyacrylonitrile, polyolefin, polyvinyl alcohol, polyethylene terephthalate and polytetrafluoroethylene; regenerated fibers such as cupra, rayon and lyocell; Natural fibers such as hemp and silk are included.

The fiber density (basis weight), porosity, bulk density, etc. of the nonwoven fabric can be adjusted, for example, by increasing or decreasing the amount of fibers constituting the web, or by increasing or decreasing the number of laminations in the case of a laminated web.

(shrink-proofing)
Methods for shrink-proofing the non-woven fabric include, for example, a method of irreversibly shrinking protein fibers after spinning and before contact with water by contacting them with water (water shrinkage method); is heated, and the protein fibers in the heated state are relaxed and irreversibly shrunk (dry heat shrink method). Both the water shrinking method and the dry heat shrinking method may be performed on the protein fibers before manufacturing the nonwoven fabric or may be performed on the nonwoven fabric after manufacturing.

Irreversible contraction of protein fibers is considered to occur for the following reasons, for example. That is, one reason is considered to be due to the secondary structure or tertiary structure of protein fibers, and another reason is that, for example, in protein fibers that have residual stress due to drawing in the manufacturing process, residual stress is generated. It is thought that this is caused by relaxation.

The water shrinkage method includes a step of irreversibly shrinking the protein fibers after spinning with water before contacting with water (shrinking step). In the shrinking process, the protein fibers shrink due to contact with water without being affected by an external force. The water to be contacted may be either liquid or gaseous water. The method of bringing the protein fibers into contact with water is also not particularly limited. and exposing it to a high-humidity environment filled with water vapor. Among these methods, the method of immersing the protein fibers in water is preferable because it effectively shortens the shrinkage time and simplifies the processing equipment. As a method for immersing the protein fiber in water, specifically, for example, a method of putting the protein fiber (or non-woven fabric) into a container containing water at a predetermined temperature and bringing it into contact with water. be.

The temperature of the water to be brought into contact with the protein fibers is not particularly limited, but is preferably below the boiling point, for example. At such a temperature, handleability and workability in the shrinking step are improved. Also, the upper limit of the water temperature is preferably 90° C. or lower, more preferably 80° C. or lower. The lower limit of the water temperature is preferably 10°C or higher, more preferably 40°C or higher, and even more preferably 70°C or higher. The temperature of the water that is brought into contact with the protein fibers can be adjusted according to the fibers that make up the protein fibers. Also, the temperature of the water may be constant while the moisture is in contact with the protein fibers, or the temperature of the water may be varied to a predetermined temperature.

The time for which the protein fibers are brought into contact with water is not particularly limited, and may be, for example, 1 minute or longer. The time may be 10 minutes or longer, 20 minutes or longer, or 30 minutes or longer. In addition, the upper limit of the time is not particularly limited, but from the viewpoint of shortening the time of the manufacturing process and eliminating the possibility of hydrolysis of protein fibers, it may be, for example, 120 minutes or less. minutes or less, or 60 minutes or less.

The water shrinkage method may further include a step of contacting the protein fibers with water and then drying them (drying step), following the shrinking step.

The drying method in the drying step is not particularly limited, and for example, natural drying may be used, or forced drying may be performed using drying equipment. The drying temperature is not particularly limited as long as it is lower than the temperature at which the protein is thermally damaged. C., more preferably 60-100.degree. Within this temperature range, the protein fibers can be dried more quickly and efficiently without causing thermal damage or the like to the protein. The drying time is appropriately selected according to the drying temperature and the like, and for example, a time that can eliminate the influence of overdrying of the protein fibers on the quality and physical properties of the nonwoven fabric is adopted.

The dry heat shrinking method includes a step of heating the protein fiber after spinning and before contact with water (heating step), and a step of relaxing the protein fiber in the heated state to irreversibly shrink it (relaxation shrinking step). and

In the heating step, the heating temperature is preferably equal to or higher than the softening temperature of the protein used for the protein fiber. The softening temperature of protein as used herein is the temperature at which contraction due to stress relaxation of the protein fiber starts. In the heat relaxation shrinkage above the softening temperature of the protein, the fiber shrinks to a degree that cannot be obtained simply by removing the moisture in the fiber, and as a result, the obtained protein fiber shrinks due to contact with water, Dimensional change is sufficiently suppressed. The heating temperature is preferably 80°C or higher, more preferably 180°C to 280°C, still more preferably 200°C to 240°C, and even more preferably 220°C to 240°C.

The heating time in the heating step is preferably 60 seconds or less, more preferably 30 seconds or less, and even more preferably 5 seconds or less, from the viewpoint of the elongation of the fiber after heat treatment. It is believed that this length of heating time does not significantly affect the stress.

In the relaxation contraction step, the relaxation ratio is preferably more than 1-fold, more preferably 1.4-fold or more, still more preferably 1.7-fold or more, and particularly preferably 2-fold or more. The relaxation ratio is understood as, for example, the ratio of the protein fiber delivery speed to the protein fiber winding speed.

(Polymer substance)
A polymeric substance (a layer formed of a polymeric substance) is generally impregnated into a nonwoven fabric in a solution state, solidified (solidified) by removal of the solvent, and adhered to the fibers at least in the interstices between the fibers of the nonwoven fabric. exists in That is, the polymeric substance constitutes the base material layer of the synthetic leather together with the nonwoven fabric as a so-called impregnated body that is integrated with the nonwoven fabric, and connects the fibers of the nonwoven fabric to each other to maintain the shape of the base material layer. , and imparts a predetermined strength to the base material layer. Examples of such polymeric substances include synthetic resins such as polyvinyl chloride, polyolefin, polystyrene, polyurethane, polyester resins, epoxy resins, acrylic polymers, acrylonitrile polymers, and proteins. Examples of polyurethane include polyether-based polyurethane, polyester-based polyurethane, polycarbonate-based polyurethane, and the like, and these may be used alone or in combination of two or more. Polyurethane can be synthesized, for example, by reacting a diisocyanate and a polyol to synthesize a prepolymer, and reacting the prepolymer with a chain extender. Diisocyanates may be aromatic or aliphatic. Polyols may be, for example, polyesters, polyethers, polycarbonates, silicones, fluororesins, and the like. When polyether is used as the polyol, excellent hydrolysis resistance, mold resistance and cold resistance are obtained, and when polycarbonate is used as the polyol, hydrolysis resistance and mold resistance are excellent. Chain extenders can be, for example, glycols, diamines, reaction terminators, and the like. Preferably, the macromolecular substance comprises protein. The protein preferably contains modified fibroin, more preferably modified spider silk fibroin. By containing modified fibroin (preferably modified spider silk fibroin), the substrate layer can be imparted with the functionality of heat retention, moisture absorption heat generation and/or flame retardancy. As a result, the synthetic leather according to the present embodiment can be imparted with the functionality of heat retention, moisture absorption heat generation and/or flame retardancy, and the value as a material is further increased. Preferred embodiments of modified fibroin are described below.

The base material layer may contain a knitted fabric in addition to the nonwoven fabric and the polymeric material. A knitted fabric is a general term for knitted fabrics and woven fabrics. Knitted fabrics include knitted fabrics with weft knitting structures such as weft knitting and circular knitting (also referred to simply as "weft knitted fabrics"), knitted fabrics with warp knitting structures such as tricot and raschel (simply referred to as "warp knitted fabrics"). ) can be either. The fabric may be a fabric having any of a plain weave, a twill weave, or a satin weave. The knitted or woven body may be an unprocessed knitted or woven body itself obtained by knitting or weaving, or may be a knitted or woven body that has undergone a water-repellent finish or the like after knitting or weaving.

A knitted or woven body can be obtained by knitting or weaving raw material yarns. Known methods can be used as the knitting method and the weaving method. As the knitting machine to be used, for example, a circular knitting machine, a warp knitting machine, a flat knitting machine, etc. can be used, and from the viewpoint of productivity, use of a circular knitting machine is preferable. The flat knitting machine includes a forming knitting machine, a seamless knitting machine, and the like, but the use of a seamless knitting machine is more preferable because it can produce a knitted fabric in the form of a final product. The looms used include shuttle looms and shuttleless looms such as gripper looms, rapier looms and air jet looms.

The raw yarn may be a single yarn, a composite yarn (for example, a blended yarn, a mixed yarn, a covering yarn, etc.), or a combination thereof. The single yarn and composite yarn may be a spun yarn obtained by twisting short fibers, or a filament yarn obtained by twisting long fibers. Examples of fibers contained in the raw yarn include protein fibers, synthetic fibers such as nylon, polyester and polytetrafluoroethylene, regenerated fibers such as cupra, rayon and lyocell, and natural fibers such as cotton, hemp and silk.

When the base material layer contains a knitted fabric in addition to the nonwoven fabric and the polymeric substance, the knitted fabric may be included in the base layer as an integrated product obtained by laminating nonwoven fabrics on both sides of the knitted fabric.

The thickness of the substrate layer is not particularly limited, but may be, for example, 100 to 2000 μm, 100 to 1000 μm, 500 to 1000 μm, or 500 to 750 μm.

The substrate layer may have a maximum heat of moisture absorption of more than 0.025° C./g, which is determined according to Formula A below.
Formula A: Maximum heat of moisture absorption = {(maximum sample temperature when the sample is placed in a low-humidity environment until the sample temperature reaches equilibrium and then transferred to a high-humidity environment) - (the sample is After placing in a low humidity environment until the temperature reaches equilibrium, the sample temperature when transferred to a high humidity environment)} (° C.) / sample weight (g)
In formula A, the low-humidity environment means an environment with a temperature of 20°C and a relative humidity of 40%, and the high-humidity environment means an environment with a temperature of 20°C and a relative humidity of 90%.

The substrate layer may have a maximum hygroscopic heat generation of 0.026° C./g or more, 0.027° C./g or more, or 0.028° C./g or more. 0.029° C./g or more, 0.030° C./g or more, 0.035° C./g or more, or 0.040° C./g or more . Although there is no particular upper limit to the maximum heat value of moisture absorption, it is usually 0.060° C./g or less.

The substrate layer has a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, or 26 or more. may be 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, or more than 33 may In the present specification, the LOI value is a value measured in accordance with the test method for particulates or synthetic resins with a low melting point, issued by Fire and Disaster Management Agency Hazardous Materials Regulation Division Director Firefighting Risk No. 50, May 31, 1995.

The base layer may have a heat retention index of greater than 0.18, determined according to Equation B below.
Formula B: heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )
Here, in the present specification, the heat retention rate means the heat retention rate measured by the dry contact method using a thermolab type II tester (under wind of 30 cm/sec), and is measured by the method described in the examples described later. is the value to be

The heat retention index of the base material layer may be 0.20 or more, 0.22 or more, 0.24 or more, 0.26 or more, or 0.28 or more. may be greater than or equal to 0.30, and may be greater than or equal to 0.32. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

In other embodiments, the synthetic leather may have layers other than the base layer. FIG. 2 is a schematic cross-sectional view of synthetic leather according to another embodiment. Synthetic leather 20 shown in FIG. , and a skin layer 5 (first skin layer) laminated on the surface of the adhesive layer 4 (first adhesive layer) opposite to the surface on which the base material layer 3 is laminated. In yet another embodiment, the synthetic leather comprises a base layer 3, an adhesive layer 4 (first adhesive layer) laminated on one side of the base layer 3, and an adhesive layer 4 ( A skin layer 5 (first skin layer) laminated on the surface opposite to the surface on which the substrate layer 3 of the first adhesive layer) is laminated, and an adhesive layer 4 (first adhesive layer) of the substrate layer 3 (first Adhesive layer (second adhesive layer) laminated on the surface opposite to the surface on which the second adhesive layer) is laminated, and the surface opposite to the surface on which the base material layer 3 of the second adhesive layer is laminated and a skin layer (second skin layer) laminated on the side surface. However, if the skin layer 5 can be directly bonded to the base material layer 3 , the adhesive layer 4 does not necessarily need to be interposed between the base material layer 3 and the skin layer 5 . Such synthetic leather is preferably used for clothing or shoes.

In another embodiment, the synthetic leather may comprise a base layer and a polyurethane microporous coating layer (first microporous coating layer) laminated on one side of the base layer. . In addition, the synthetic leather includes a base layer, a polyurethane microporous coating layer (first microporous coating layer) laminated on one side of the base layer, and a first microporous coating layer. and a polyurethane microporous coating layer (second microporous coating layer) laminated on the surface opposite to the coated surface. A polyurethane microporous coating layer refers to a layer having a microporous structure containing polyurethane as a main component. The surface of the polyurethane microporous film layer is subjected to embossing (processing by pressing with hot rolls, etc.), puff suede processing (processing by applying sandpaper, etc.), gravure processing (processing by gravure printing, etc.), etc. A surface treatment may be applied.

In yet another embodiment, the synthetic leather may have a raised/shirred base layer. Since the surface of the base material layer of such synthetic leather is raised, the fineness of the non-woven fabric used for the base material layer affects the texture of the synthetic leather. In addition, such synthetic leather is excellent in breathability.

(protein fiber and protein)
The nonwoven fabric according to the present embodiment is shrink-proofed by performing the shrink-proofing treatment described above, thereby suppressing dimensional change due to contact with water. Thus, protein fibers (and proteins) used in nonwovens may inherently undergo (significant) dimensional changes upon contact with water.

For example, protein fibers may have a wet shrinkage of 2% or more. Wet shrinkage may be 4% or more, 6% or more, 8% or more, 10% or more, or 15% or more. , may be 20% or more, 25% or more, or 30% or more. The upper limit of wet shrinkage is usually 80% or less. The wet shrinkage rate is defined by the following formula II.
Wet shrinkage = {1-(length of protein fiber brought into wet state by contact with water/length of protein fiber after spinning but before contact with water)} x 100 (%) (Formula II)

Also for example, the protein fibers may have a dry shrinkage of greater than 7%. The dry shrinkage may be 15% or more, 25% or more, 32% or more, 40% or more, or 48% or more. , may be 56% or more, 64% or more, or 72% or more. The upper limit of dry shrinkage is usually 80% or less. The drying shrinkage ratio is defined by the following formula III.
Dry shrinkage = {1-(length of protein fiber in dry state/length of protein fiber after spinning before contact with water)} x 100 (%) (Formula III)

Further, for example, the nonwoven fabric may have a fiber density increase rate of 20% or more. The fiber density increase rate may be 30% or more, 40% or more, 50% or more, or 100% or more. The fiber density increase rate is a value defined by Formula I below.
Fiber density increase rate = {(fiber density of non-woven fabric after shrink-proofing/fiber density of non-woven fabric before shrink-proofing) - 1} x 100 (%) (Formula I)

The fiber density (basis weight) of the nonwoven fabric that has undergone shrink-proofing treatment is, for example, 0.04 g/cm ² or more, may be 0.045 g/cm ² or more, may be 0.05 g/cm ² or more, or may be 0.05 g/cm 2 or more. 055 g/cm ² or more. The fiber density (basis weight) is a value defined by the weight per unit area of the nonwoven fabric.

Any protein can be used as the raw material of the protein fiber without any particular limitation. Proteins can include naturally occurring proteins and recombinant proteins (artificial proteins). Recombinant proteins include any proteins that can be produced on an industrial scale, and include, for example, proteins that can be used for industrial purposes, proteins that can be used for medical purposes, structural proteins, and the like. Specific examples of proteins of industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccination, vaccines, antigen binding proteins, immunostimulatory proteins, Allergens, full length antibodies or antibody fragments or derivatives may be mentioned. Specific examples of structural proteins include spider silk (spider silk), silkworm silk, keratin, collagen, elastin, resilin, and proteins derived from these. As the protein to be used, modified fibroin is preferable, and modified spider silk fibroin is more preferable, because it is excellent in heat retention, hygroscopic heat generation and/or flame resistance. By including modified fibroin (preferably, modified spider silk fibroin) in the protein fibers, the nonwoven fabric according to the present embodiment can be further imparted with heat retention, moisture absorption heat generation and/or flame retardancy. As a result, the synthetic leather according to the present embodiment can be imparted with the functionality of heat retention, moisture absorption heat generation and/or flame retardancy, and the value as a material is further increased.

The modified fibroin according to this embodiment has a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. protein containing The modified fibroin may have additional amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and C-terminal sequence are typically, but not limited to, regions that do not have repeated amino acid motifs characteristic of fibroin and consist of about 100 amino acids.

As used herein, “modified fibroin” means artificially produced fibroin (artificial fibroin). The modified fibroin may be fibroin whose domain sequence differs from the amino acid sequence of naturally occurring fibroin, or may be fibroin whose domain sequence is identical to the amino acid sequence of naturally occurring fibroin. As used herein, "naturally occurring fibroin" 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 that is

"Modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally occurring fibroin (for example, cloned naturally occurring fibroin whose amino acid sequence is modified by modifying the gene sequence of fibroin), or artificially designed and synthesized without relying on naturally occurring fibroin (for example, a nucleic acid encoding a designed amino acid sequence). having a desired amino acid sequence by chemical synthesis).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to the (A) _n motif of the amino acid sequence) and an amorphous region (typically corresponding to the REP of the amino acid sequence). ) and is represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in the _n motif may be an integer of 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16 . In addition, (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, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). At least seven of the (A)n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 amino acid residues. m represents an integer of 2 to 300, and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

The modified fibroin according to the present embodiment has an amino acid sequence corresponding to, for example, substitution, deletion, insertion and/or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. can be obtained by modifying 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 directed mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), Methods in Enzymology, 100, 448 (1983).

Naturally occurring fibroin is a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif. Specific examples include fibroin produced by insects or arachnids.

Examples of fibroin produced by insects include, for example, Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, and Pilosa ), silk proteins produced by silkworms such as Samia cynthia, Caligura japonica, Antheraea mylitta, Antheraea assama, and the larvae of the wasp (Vespa simillima xantoptera) Hornet silk protein.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank Accession No. M76430 (nucleotide sequence) and AAA27840.1 (amino acid sequence)).

Fibroin produced by spiders includes, for example, spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as Araneus spiders, Araneus spiders, red spiders, green spiders, and bean spiders, spiders belonging to the genus Neoscona, such as Araneus spiders, house spiders, loch spiders, and Satsumanomidamashi. Spiders belonging to the genus Pronus, such as the genus Pronus, spiders belonging to the genus Cyrtarachne, such as Torinofundami and the genus Cyrtarachne, and genus Gasteracantha, such as spiny spiders and spiny spiders Spiders belonging to the genus Ordgarius such as spiders belonging to, Ordgarius spiders, and spiders belonging to the genus Ordgarius, spiders belonging to the genus Argiope, such as the argiope spider, the argiope spider, and the argiope spider spiders belonging to the genus), spiders belonging to the genus Acusilas, such as spiders, spiders belonging to the genus Cytophora, such as spiders, spiders, and spiders belonging to the genus Cytophora, spiders belonging to the genus Poltys, such as Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the genus Cyclosa, such as the brown spider, the genus Cyclosa, and the genus Chorizopes, such as the brown spider, and the spider silk protein produced by the spider, Spiders belonging to the genus Tetragnatha, such as the genus Tetragnatha, spiders belonging to the genus Leucauge, such as spiders, spiders, and spiders belonging to the genus Leucauge, spiders belonging to the genus Nephila, such as Nephila Spiders belonging to the genus Menosira, such as spiders belonging to the family, spiders belonging to the genus Menosira, spiders belonging to the genus Dyschiriognatha, such as the lesser paper spider, widow spiders, such as black widow spiders, redback spiders, gray widow spiders, and large widow spiders (Latrodectus genus) Spider silk proteins produced by spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprosthenops and spiders belonging to the genus Euprosthenops be done. Spider silk proteins include, for example, dragline proteins such as MaSp (MaSp1 and MaSp2), ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), AcSp, PySp, Flag, and the like.

More specific examples of spider silk proteins produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession number AAC47010 (amino acid sequence), U47855 (nucleotide sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank Accession No. AAC47011 (amino acid sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank Accession No. AAC04504 (amino acid sequence ), U37520 (nucleotide sequence)), major ampullate spidroin 1 [derived from Latrodectus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (derived from Nephila clavata) No. AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [from Euprosthenops australis] (GenBank Accession No. CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidropin 2 [Euprosthenops australispin] (GenBank Accession No. CAM32249.1 (amino acid sequence), AM490169 (nucleotide sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank Accession No. AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank Accession No. AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilengys crentata] (GenBank Accession and Section No. ABR37278.1 (amino acid sequence).

More specific examples of naturally occurring fibroin include fibroin whose sequence information is registered in NCBI GenBank. For example, among sequence information registered in NCBI GenBank, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION from among sequences containing INV as DIVISION. It can be confirmed by extracting the specific product character string from the sequence, CDS, and the sequence described with the specific character string from SOURCE to TISSUE TYPE.

The modified fibroin according to the present embodiment may be modified silk fibroin (modified silk protein amino acid sequence produced by silkworms), modified spider silk fibroin (spider silk protein produced by spiders) modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferable because it is superior in flame retardancy.

Specific examples of modified fibroin include a modified fibroin (first modified fibroin) derived from the dragline silk protein produced in the major pituitary gland of spiders, and a domain sequence with reduced content of glycine residues. (second modified fibroin), (A) modified fibroin having a domain sequence with reduced content of _n motifs (third modified fibroin), content of glycine residues, and (A) _n Modified fibroin with reduced content of motifs (fourth modified fibroin), modified fibroin having a domain sequence containing a region with a locally large hydrophobicity index (fifth modified fibroin), and content of glutamine residues modified fibroin having a reduced domain sequence (sixth modified fibroin).

A first modified fibroin includes a protein comprising a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, the number of amino acid residues in the (A) _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, still more preferably an integer of 8 to 20, an integer of 10 to 20 is even more preferred, integers from 4 to 16 are even more preferred, integers from 8 to 16 are particularly preferred, and integers from 10 to 16 are most preferred. In the first modified fibroin, the number of amino acid residues constituting REP in formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, 20 to 100 residues and even more preferably 20 to 75 residues. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by Formula 1: [(A) _n motif-REP] _m is amino acid residue It is preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more of the total number.

The first modified fibroin comprises an amino acid sequence unit represented by Formula 1: [(A) _n motif-REP] _m and has an amino acid sequence whose C-terminal sequence is shown in any one of SEQ ID NOs: 1 to 3, or The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in any one of SEQ ID NOs: 1-3.

The amino acid sequence shown in SEQ ID NO: 1 is identical to the amino acid sequence consisting of the C-terminal 50 amino acids of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 has the sequence It is identical to the amino acid sequence shown in No. 1 with 20 residues removed from the C-terminus, and the amino acid sequence shown in SEQ ID No. 3 has 29 residues removed from the C-terminus of the amino acid sequence shown in SEQ ID No. 1. Identical to the amino acid sequence.

As more specific examples of the first modified fibroin, (1-i) the amino acid sequence represented by SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) the amino acid sequence represented by SEQ ID NO: 4 and 90 Modified fibroins comprising amino acid sequences with greater than % sequence identity can be mentioned. Preferably, the sequence identity is 95% or greater.

The amino acid sequence shown by SEQ ID NO: 4 is an amino acid sequence (SEQ ID NO: 5) consisting of an initiation codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus of ADF3. The 13th repeat region was increased to approximately double and mutated so that translation stops at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown by SEQ ID NO:4 is identical to the amino acid sequence shown by SEQ ID NO:3.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO:4.

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to the naturally-occurring fibroin. The second modified fibroin can be said to have an amino acid sequence corresponding to at least one or more glycine residues in REP being replaced with another amino acid residue, as compared with naturally occurring fibroin. .

The second modified fibroin has a domain sequence of GGX and GPGXX (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) in REP compared to naturally occurring fibroin. In at least one motif sequence selected from ), it has an amino acid sequence corresponding to at least one or more glycine residues in the motif sequence being replaced with another amino acid residue may

In the second modified fibroin, the percentage of the motif sequence in which the glycine residue is substituted with another amino acid residue may be 10% or more of the entire motif sequence.

The second modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the domain sequence, the (A) _n motif located most C-terminal to the domain sequence Let z be 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 When w is the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence, z/w is 30% or more, It may have an amino acid sequence that is 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 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

The second modified fibroin preferably has an increased content of the amino acid sequence consisting of XGX by substituting one glycine residue in the GGX motif with another amino acid residue. 6. In the second modified fibroin, the content of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, even more preferably 10% or less. % or less, even more preferably 4% or less, and particularly 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 XGX below.

A method for calculating z/w will be described in more detail. First, in a fibroin (modified fibroin or naturally occurring fibroin) containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , the (A) _n located on the most C-terminal side from the domain sequence An amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminus 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 (no duplication), z is 50×3=150. In addition, for example, when there is an X (central X) contained in two XGX, as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by subtracting the overlap (in the case of XGXGX, 5 amino acid residues be). w is the total number of amino acid residues contained in the domain sequence, excluding the sequence from the (A) _n motif positioned most on the C-terminal side to the C-terminus of the domain sequence. For example, for the domain sequence shown in Figure 3, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the most C-terminal (A) _n motif). Then z/w (%) can be calculated by dividing z by w.

Here, z/w in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Naturally derived fibroin containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m and having an amino acid sequence consisting of GGX in fibroin content of 6% or less among all the extracted fibroin z/w was calculated from the amino acid sequence of fibroin in the above-described calculation method. The results are shown in FIG. The horizontal axis of FIG. 4 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 4, the z/w for naturally-occurring fibroin is all less than 50.9% (the highest is 50.86%).

In the second modified fibroin, z/w is preferably 50.9% or more, more preferably 56.1% or more, even more preferably 58.7% or more, and 70% or more. and even more preferably 80% or more. Although the upper limit of z/w is not particularly limited, it may be, for example, 95% or less.

The second modified fibroin is obtained, for example, by substituting at least part of the nucleotide sequence encoding the glycine residue from the cloned naturally-derived fibroin gene sequence so as to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z/w is 50.9% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above aspect from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the alteration corresponding to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , insertions and/or additions may be made to the amino acid sequence.

The other amino acid residue mentioned above is not particularly limited as long as it is an amino acid residue other than glycine residue, but valine (V) residue, leucine (L) residue, isoleucine (I) residue, methionine ( Hydrophobic amino acid residues such as M) residues, proline (P) residues, phenylalanine (F) residues and tryptophan (W) residues, glutamine (Q) residues, asparagine (N) residues, serine (S ) residues, lysine (K) residues and glutamic acid (E) residues are preferred, and hydrophilic amino acid residues such as valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

More specific examples of the second modified fibroin include (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (2-ii) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (2-i) will be explained. The amino acid sequence shown by SEQ ID NO: 6 is obtained by replacing all GGX in REP of the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin with GQX. The amino acid sequence shown by SEQ ID NO: 7 is obtained by deleting every two (A) _n motifs from the amino acid sequence shown by SEQ ID NO: 6 from the N-terminal side to the C-terminal side, and furthermore, in front of the C-terminal sequence. [(A) _n motif-REP] is inserted into the . The amino acid sequence shown in SEQ ID NO: 8 has two alanine residues inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a partial glutamine (Q) residue. It is obtained by substituting serine (S) residues and deleting some amino acids on the C-terminal side so that the molecular weight is almost the same as that of SEQ ID NO:7. The amino acid sequence shown by SEQ ID NO: 9 is a region of 20 domain sequences present in the amino acid sequence shown by SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and a His tag sequence are added to the C-terminus of a sequence in which is repeated four times.

The z/w value in the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z/w values 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 each 58.7%, 70.1%, 66.1% and 70.0%. In addition, the value of x/y in the jagged ratio (described later) of 1:1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 is 15.0%, 15.0%, 93.4%, 92.7% and 89.8% respectively.

The modified fibroin of (2-i) may consist of the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

The modified fibroin (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and XGX contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

Examples of tag sequences include affinity tags that utilize specific affinity with other molecules. A specific example of the 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 the property of specifically binding to metal ions such as nickel, so isolation of modified fibroin by metal chelating metal chromatography can be used for A specific example of the tag sequence is the amino acid sequence represented by SEQ ID NO: 11 (an amino acid sequence containing a His tag sequence and a hinge sequence).

Also, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

Furthermore, an "epitope tag" using an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, FLAG tag, and the like. Modified fibroin can be easily purified with high specificity by using an epitope tag.

Furthermore, a tag sequence that can be cleaved by a specific protease can also be used. The modified fibroin from which the tag sequence has been cut off can also be recovered by treating the protein adsorbed via the tag sequence with protease.

More specific examples of modified fibroin containing a tag sequence include: (2-iii) amino acids represented by SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (2-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences shown in SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in SEQ ID NO: 11 (including His tag sequence and hinge sequence) was added to the N-terminus of the amino acid sequence shown.

The modified fibroin of (2-iii) may consist of the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (2-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(2-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is contained in REP ( where X represents an amino acid residue other than glycine). is preferably 50.9% or more.

The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A third modified fibroin has an amino acid sequence in which the domain sequence has a reduced content of (A) _n motifs compared to the naturally occurring fibroin. The domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to deletion of at least one or more (A) _n motifs compared to the naturally occurring fibroin.

A third modified fibroin may have an amino acid sequence corresponding to 10-40% deletion of the (A) _n motif from the naturally-occurring fibroin.

A third modified fibroin has a domain sequence at least one (A) _n motif for every 1-3 (A) _n motifs from the N-terminal side to the C-terminal side as compared to the naturally-occurring fibroin. may have an amino acid sequence corresponding to the deletion of

The third modified fibroin has at least two consecutive (A) _n motif deletions from the N-terminal side to the C-terminal side and one (A ) may have an amino acid sequence corresponding to deletion of _n motifs repeated in this order.

The third modified fibroin may have an amino acid sequence corresponding to deletion of at least every two (A) _n motifs from the N-terminal side to the C-terminal side of the domain sequence. .

A third modified fibroin comprises a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , with two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] unit sequentially compares the number of amino acid residues of REP, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other REP is 1.8 to 11. When the maximum value of the total sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units equal to 3 is x, and the total number of amino acid residues of the domain sequence is y Furthermore, 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 95% or more. 100% (meaning composed only of alanine residues) is even more preferred.

A method for calculating x/y will be described in more detail with reference to FIG. FIG. 3 shows the domain sequence of the modified fibroin with the N-terminal and C-terminal sequences removed. The domain sequence is, from the N-terminal side (left side), (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n Motif-third REP (10 amino acid residues)-(A) _n motif-fourth REP (20 amino acid residues)-(A) _n motif-fifth REP (30 amino acid residues)-(A) It has a sequence called _n motif.

Two adjacent [(A) _n -motif-REP] units are selected sequentially from the N-terminal side to the C-terminal side so that there is no overlap. At this time, unselected [(A) _n motif-REP] units may be present. In FIG. 3, pattern 1 (comparison of the first REP and the second REP, and comparison of the third REP and the fourth REP), pattern 2 (comparison of the first REP and the second REP, and comparison of the fourth REP and the fifth REP), pattern 3 (comparison of the second REP and the third REP, and comparison of the fourth REP and the fifth REP), pattern 4 (comparison of the first REP and A second REP comparison) was shown. Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two adjacent [(A) _n motif-REP] units selected is compared. Comparison is carried out by determining the ratio of the number of amino acid residues to the number of amino acid residues of the other. For example, when comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP with fewer amino acid residues is set to 1, the second REP is The ratio of amino acid residue numbers 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 with fewer amino acid residues is set to 1, the fifth REP is 30/20=1.5.

In FIG. 3, a set of [(A) _n motif-REP] units having a ratio of 1.8 to 11.3 for the number of amino acid residues of the other is set to 1 for the one with the smaller number of amino acid residues. It is indicated by a solid line. This ratio is referred to herein as the serration ratio. A set of [(A) _n motif-REP] units in which the other amino acid residue number ratio is less than 1.8 or greater than 11.3 when the number of amino acid residues is less than 1 is indicated by a dashed line. Indicated.

In each pattern, the numbers of all amino acid residues of two adjacent [(A) _n motif-REP] units indicated by solid lines are summed up (not only REP but also the number of amino acid residues of (A) _n motifs). be.). Then, the added total values are compared, and the total value (maximum total value) of the pattern with the maximum total value is defined as x. In the example shown in FIG. 3, the total value of pattern 1 is the largest.

Then x/y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, and even more preferably 70% or more. Preferably, 75% or more is even more preferable, and 80% or more is particularly preferable. The upper limit of x/y is not particularly limited, and may be, for example, 100% or less. When the serration ratio is 1:1.9 to 11.3, x/y is preferably 89.6% or more, and when the serration ratio is 1:1.8 to 3.4, x /y is preferably 77.1% or more, and when the serration ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and the serration ratio is 1 : 1.9 to 4.1, x/y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least 7 of the (A) _n motifs present in the domain sequence are composed only of alanine residues, x/y is 46.4% or more. is preferably 50% or more, more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and 80% or more It is particularly preferred to have The upper limit of x/y is not particularly limited as long as it is 100% or less.

Here, x/y in naturally occurring fibroin will be explained. First, as described above, fibroin whose amino acid sequence information is registered in NCBI GenBank was confirmed by the method exemplified, and 663 types of fibroin (among them, 415 types of arachnid-derived fibroin) were extracted. Of all the extracted fibroin, from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , x/y was calculated. FIG. 5 shows the results when the serration ratio is 1:1.9 to 4.1.

The horizontal axis of FIG. 5 indicates x/y (%), and the vertical axis indicates frequency. As is clear from FIG. 5, the x/y ratios for naturally occurring fibroin are all less than 64.2% (the highest is 64.14%).

The third modified fibroin, for example, deletes one or more of the (A) _n motif-encoding sequences from the cloned naturally occurring fibroin gene sequence such that x/y is 64.2% or more. can be obtained by Alternatively, for example, an amino acid sequence corresponding to deletion of one or more (A) _n motifs is designed such that x/y is 64.2% or more from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence described above. In any case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are substituted, deleted, inserted and/or added. Alterations in the amino acid sequence corresponding to what has been done may be made.

More specific examples of the third modified fibroin include (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met -PRT799), or (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. Mention may be made of modified fibroin.

The modified fibroin of (3-i) will be explained. The amino acid sequence represented by SEQ ID NO: 17 is every two (A) _n The motif was deleted and one [(A) _n motif-REP] was inserted in front of the C-terminal sequence. The amino acid sequence shown by SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.

The x/y value of the amino acid sequence represented by SEQ ID NO: 10 (corresponding to naturally-occurring fibroin) is 15.0% at the jagged ratio of 1:1.8 to 11.3. The x/y values of the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 are both 93.4%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 8 is 92.7%. The value of x/y in the amino acid sequence shown by SEQ ID NO: 9 is 89.8%. The z/w values in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1%, 66.0%, respectively. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and from the N-terminal side to the C-terminal side , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Amino acid residues of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (Giza ratio is 1:1.8 to 11.3) It is preferable that x/y is 64.2% or more, where x is the maximum sum of the cardinal numbers and y is the total number of amino acid residues in the domain sequence.

The third modified fibroin may contain the tag sequence described above at either or both of the N-terminus and C-terminus.

As more specific examples of modified fibroin containing a tag sequence, (3-iii) the amino acid shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799) sequence, or (3-iv) a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. .

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 have SEQ ID NO: 11 at the N-terminus of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown in (including His tag sequence and hinge sequence) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

(3-iv) modified fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

(3-iv) modified fibroin has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and , the numbers of amino acid residues of REP of two adjacent [(A) _n motif-REP] units are sequentially compared, and when the number of amino acid residues of REP with a small number of amino acid residues is set to 1, the number of amino acid residues of the other Let x be the maximum value of the total sum of the amino acid residue numbers of two adjacent [(A) _n motif-REP] units with a ratio of the number of amino acid residues of REP of 1.8 to 11.3. , x/y is preferably 64.2% or more, where y is the total number of amino acid residues in the domain sequence.

The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motifs and a reduced content of glycine residues compared to naturally occurring fibroin. have. The domain sequence of the fourth modified fibroin is that at least one or more (A) _n motifs are deleted, and at least one or more glycine residues in REP are deleted compared to the naturally occurring fibroin. It can be said to have an amino acid sequence corresponding to substitution with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having both the features of the above-described second modified fibroin and the third modified fibroin. Specific aspects and the like are as described in the second modified fibroin and the third modified fibroin.

More specific examples of the fourth modified fibroin include (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410 ), the amino acid sequence shown in SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in . Specific embodiments of the modified fibroin comprising the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

A fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a larger hydrophobicity index than in naturally occurring fibroin, and/or REP It may have an amino acid sequence that includes regions of high local hydrophobicity index corresponding to the insertion of one or more amino acid residues with high hydrophobicity index therein.

A region with a locally high hydrophobicity index is preferably composed of 2 to 4 consecutive amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). A residue is more preferred.

A fifth modified fibroin is obtained by replacing one or more amino acid residues in REP with amino acid residues having a higher hydrophobicity index than in naturally occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, one or more amino acid residues are substituted, deleted, inserted and / or added as compared to naturally occurring fibroin There may be alterations in the amino acid sequence corresponding to what has been done.

For the fifth modified fibroin, for example, one or more hydrophilic amino acid residues (eg, amino acid residues with a negative hydrophobicity index) in REP from the cloned naturally occurring fibroin gene sequence are replaced with hydrophobic amino acid residues. by substituting a group (for example, an amino acid residue with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into REP. Further, for example, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acid residues in REP It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and/or one or more hydrophobic amino acids in REP In addition to modifications corresponding to residue insertions, amino acid sequence modifications corresponding to substitutions, deletions, insertions and/or additions of one or more amino acid residues may also be made.

The fifth modified fibroin comprises a domain sequence represented by Formula 1: [(A) _n motif-REP] _m , and from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence Let p be the total number of amino acid residues contained in a region where the average value of the hydrophobic index of four consecutive amino acid residues is 2.6 or more in all REPs contained in the sequences excluding the sequences from the domain sequence, p/q is 6, where q is the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. It may have an amino acid sequence that is greater than or equal to .2%.

Hydropathy indexes of amino acid residues are known (Hydropathy index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 157). 105-132). Specifically, the hydropathic index (hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

A method for calculating p/q will be described in more detail. For the calculation, the sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence (hereinafter referred to as “sequence A”) is used. First, in all REPs contained in sequence A, the average value of the hydrophobic index of 4 consecutive amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the 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 obtained for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobic index of 4 consecutive amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to multiple "4 consecutive amino acid residues with an average hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Also, the total number of amino acid residues contained in sequence A is q.

For example, if 20 “continuous 4 amino acid residues with an average hydrophobic index of 2.6 or more” are extracted (no overlap), the average hydrophobic index of 4 consecutive amino acid residues is 2 A region that is 0.6 or greater will contain 20 consecutive 4-amino acid residues (no overlap), and p is 20×4=80. Further, for example, when two "consecutive 4 amino acid residues having an average hydrophobicity index of 2.6 or more" exist overlapping by one amino acid residue, the hydrophobic index of the consecutive 4 amino acid residues A region with an average of 2.6 or more contains 7 amino acid residues (p=2×4−1=7, where “−1” is duplicate subtraction). For example, in the case of the domain sequence shown in FIG. 6, there are seven non-overlapping “continuous 4 amino acid residues with an average hydrophobicity index of 2.6 or more”, so p is 7 × 4 = 28. For example, in the case of the domain sequence shown in FIG. 6, q is 4+50+4+40+4+10+4+20+4+30=170 (not including the (A) _n motif present at the end of the C-terminal side). Then p/q (%) can be calculated by dividing p by q. In the case of FIG. 6, 28/170=16.47%.

In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, and 20% or more. Even more preferably, it is still more preferably 30% or more. Although the upper limit of p/q is not particularly limited, it may be, for example, 45% or less.

For the fifth modified fibroin, for example, the amino acid sequence of the cloned naturally occurring fibroin is modified so as to satisfy the p/q conditions described above, so that one or more hydrophilic amino acid residues in REP (eg, hydrophobicity index). negative amino acid residue) with a hydrophobic amino acid residue (e.g., an amino acid residue with a positive hydrophobicity index), and/or inserting one or more hydrophobic amino acid residues in REP can be obtained by locally modifying an amino acid sequence containing a region with a large hydrophobicity index. Alternatively, for example, it can be obtained by designing an amino acid sequence that satisfies the above p/q conditions from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, one or more amino acid residues in REP have been replaced with amino acid residues having a higher hydrophobicity index compared to naturally-occurring fibroin, and/or one or more In addition to the modification corresponding to the insertion of an amino acid residue with a large hydrophobicity index, further modification corresponding to the substitution, deletion, insertion and/or addition of one or more amino acid residues may be performed. .

Amino acid residues with a large hydrophobicity index are not particularly limited, but areoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) are preferred, and valine (V), leucine (L) and isoleucine (I) are more preferred.

As more specific examples of the fifth modified fibroin, (5-i) an amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665), or SEQ ID NO: 21 (Met-PRT666); or (5-ii) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-i) will be explained. The amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP except for the terminal domain sequence on the C-terminal side with respect to the amino acid sequence shown by SEQ ID NO: 7 (Met-PRT410). The amino acid sequence (VLI) was inserted at two sites, some glutamine (Q) residues were substituted with serine (S) residues, and some amino acids on the C-terminal side were deleted. The amino acid sequence represented by SEQ ID NO: 20 is the amino acid sequence represented by SEQ ID NO: 8 (Met-PRT525) in which an amino acid sequence (VLI) consisting of three amino acid residues is inserted at every other REP. be. The amino acid sequence shown by SEQ ID NO: 21 is obtained by inserting two amino acid sequences (VLI) each consisting of 3 amino acid residues every other REP into the amino acid sequence shown by SEQ ID NO: 8.

The modified fibroin of (5-i) may consist of the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus.

More specific examples of modified fibroin containing a tag sequence include (5-iii) the amino acid sequence represented by SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665), or SEQ ID NO: 24 (PRT666), or (5-iv ) modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are added to the N-terminals of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, where the amino acid sequence shown in SEQ ID NO: 11 (His tag sequence and hinge sequence) are added.

The modified fibroin of (5-iii) may consist of the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The modified fibroin (5-iv) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located on the most C-terminal side (A) _n Amino acids contained in a region where the average hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the domain sequence excluding the sequence from the motif to the C-terminus of the domain sequence Let p be the total number of residues, and q be the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence. , p/q is preferably 6.2% or more.

The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

A sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues compared to naturally occurring fibroin.

The sixth modified fibroin preferably contains at least one motif selected from a GGX motif and a GPGXX motif in the REP amino acid sequence.

When the sixth modified fibroin contains a GPGXX motif in REP, the GPGXX motif content is usually 1% or more, 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.

As used herein, 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) _n motif fibroin (modified fibroin or naturally occurring fibroin), the number of GPGXX motifs contained in the region in all REPs contained in the sequence excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence The number obtained by multiplying the total by three (that is, the total number of G and P in the GPGXX motif) is defined as s, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is taken from the domain sequence. The GPGXX motif content rate is calculated as s/t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content rate, the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is "the most C-terminal side (A) The sequence from the _n motif to the C-terminus of the domain sequence” (the sequence corresponding to REP) may include sequences that are poorly correlated with sequences characteristic of fibroin, and m is small If the domain sequence is short (that is, if the domain sequence is short), it affects the calculation result of the GPGXX motif content rate, so this effect is to be eliminated. When a "GPGXX motif" is located at the C-terminus of REP, it is treated as a "GPGXX motif" even if "XX" is, for example, "AA".

FIG. 7 is a schematic diagram showing the domain sequence of modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. First, in the modified fibroin domain sequence (“[(A) _n motif-REP] _m -(A) _n motif” type) shown in FIG. (A) The sequence obtained by removing the sequence from the _n motif to the C-terminus of the domain sequence from the domain sequence” (sequence shown as “region A” in FIG. 7). The number of GPGXX motifs is 7, and s is 7×3=21. Similarly, all REPs are "sequences obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" (in FIG. 7, the sequence indicated by "region A" .), the total number of amino acid residues t of all REPs further 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, which is 21/150=14.0% for the modified fibroin in FIG.

The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, even more preferably 4% or less, and particularly preferably 0%. .

As used herein, "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) _n motif fibroin (modified fibroin or naturally occurring In fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 7). REP, the total number of glutamine residues contained in the region is u, the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and (A) _n The glutamine residue content is calculated as u/t, where t is the total number of amino acid residues in all REPs excluding motifs. In the calculation of the glutamine residue content, the target is the "sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because of the reasons described above. It is the same.

A sixth modified fibroin corresponds to a domain sequence that lacks one or more glutamine residues in REP or replaces them with other amino acid residues as compared to the naturally-occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than a glutamine residue, but preferably an amino acid residue with a higher hydrophobicity index than that of a glutamine residue. Hydrophobicity indexes of amino acid residues are shown in Table 1.

As shown in Table 1, amino acid residues having a higher hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M ) amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H); can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferred. , isoleucine (I), valine (V), leucine (L) and phenylalanine (F).

The sixth modified fibroin preferably has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, and 0.3 or more. is even more preferable, and 0.4 or more is particularly preferable. There is no particular upper limit to the hydrophobicity of REP, and it may be 1.0 or less, or 0.7 or less.

As used herein, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif fibroin (modified fibroin or naturally occurring In fibroin), the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence (the sequence corresponding to "region A" in FIG. 7). In the REP, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v/t, where t is the total number of amino acid residues in all REPs excluding _n motifs. In calculating the hydrophobicity of REP, the reason for targeting "the sequence obtained by removing the sequence from the (A) _n motif located on the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" is the reason described above. It is the same.

A sixth modified fibroin has a domain sequence lacking one or more glutamine residues in REP and/or one or more glutamine residues in REP compared to naturally-occurring fibroin In addition to the modification corresponding to the substitution of another amino acid residue, there may be modifications of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. .

A sixth modified fibroin is obtained, for example, by deleting one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and/or by deleting one or more glutamine residues in REP. can be obtained by substituting the amino acid residue of Also, for example, deletion of one or more glutamine residues in REP from the amino acid sequence of naturally occurring fibroin, and/or substitution of one or more glutamine residues in REP with other amino acid residues It can also be obtained by designing an amino acid sequence corresponding to , and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

More specific examples of the sixth modified fibroin include (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or sequence Modified fibroin comprising the amino acid sequence represented by number 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42, and a modified fibroin comprising an amino acid sequence having 90% or more sequence identity.

The modified fibroin of (6-i) will be explained. The amino acid sequence shown by SEQ ID NO:25 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 (Met-PRT410) with VL. The amino acid sequence shown by SEQ ID NO: 26 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with TS, and replacing the remaining Q with A. The amino acid sequence shown by SEQ ID NO: 27 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VL, and replacing the remaining Q with I. The amino acid sequence shown by SEQ ID NO:28 is obtained by substituting VI for all QQs in the amino acid sequence shown by SEQ ID NO:7 and L for the remaining Qs. The amino acid sequence shown by SEQ ID NO: 29 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with VF, and replacing the remaining Q with I.

The amino acid sequence shown by SEQ ID NO:30 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:8 (Met-PRT525) with VL. The amino acid sequence shown by SEQ ID NO: 31 is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 8 with VL, and replacing the remaining Q with I.

The amino acid sequence represented by SEQ ID NO: 32 consists of 20 domain sequence regions present in the amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410) that are repeated twice, and all QQ in the sequence are replaced with VF, And the remaining Q is replaced with I.

The amino acid sequence shown by SEQ ID NO: 41 (Met-PRT917) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO: 7 with LI and the remaining Q with V. The amino acid sequence shown by SEQ ID NO:42 (Met-PRT1028) is obtained by replacing all QQs in the amino acid sequence shown by SEQ ID NO:7 with IF, and replacing the remaining Q with T.

SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all glutamine residues The group content is below 9% (Table 2).

The modified fibroin of (6-i) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 It may consist of the amino acid sequence shown.

The modified fibroin of (6-ii) is represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-ii) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-ii) preferably has a GPGXX motif content of 10% or more.

The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This enables isolation, immobilization, detection, visualization, and the like of modified fibroin.

More specific examples of modified fibroin containing a tag sequence include (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028) modified fibroin comprising the amino acid sequence, or ( 6-iv) SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 and 90 Modified fibroins comprising amino acid sequences with greater than % sequence identity can be mentioned.

The amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 at the N-terminus of SEQ ID NO: 11 The amino acid sequence (including the His-tag sequence and hinge sequence) is added. Since the tag sequence was only added to the N-terminus, there was no change in the glutamine residue content, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 It may consist of the amino acid sequence shown.

The modified fibroin of (6-iv) is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44 Amino acid sequences having 90% or greater sequence identity with the indicated amino acid sequences are included. The modified fibroin of (6-iv) also has a domain represented by Formula 1: [(A) _n motif-REP] _m or Formula 2: [(A) _n motif-REP] _m -(A) _n motif A protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin (6-iv) preferably has a glutamine residue content of 9% or less. In addition, the modified fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The modified fibroin has at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the characteristics of

Modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin. As used herein, "modified hydrophilic fibroin" refers to a value obtained by obtaining the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the sum by the total number of amino acid residues. (average HI) of 0 or less. The hydrophobicity index is as shown in Table 1. A “hydrophobic modified fibroin” is a modified fibroin with an average HI of greater than zero. Hydrophilic modified fibroin is particularly excellent in flame retardancy. Hydrophobic-modified fibroin is particularly excellent in hygroscopic heat generation and heat retention.

Examples of modified hydrophilic fibroin include the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence shown in 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, and SEQ ID NO: 14. or the amino acid sequence represented by SEQ ID NO: 15, the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, the amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15 A modified fibroin comprising the amino acid sequence shown in the sequence SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 is included.

Examples of hydrophobically modified fibroin include 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, or SEQ ID NO: 43, SEQ ID NO: 35, A modified fibroin comprising the amino acid sequence shown in 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 is included.

A protein according to this embodiment can be produced by a conventional method using a nucleic acid encoding the protein. A nucleic acid encoding the protein may be chemically synthesized based on base sequence information, or may be synthesized using a PCR method or the like.

A protein fiber can be obtained by, for example, dissolving a protein in a solvent capable of dissolving the protein to form a dope solution, and spinning by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. Solvents capable of dissolving proteins include, for example, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, hexafluoroisopropanol (HFIP), and the like. An inorganic salt may be added to the solvent as a dissolution accelerator.

FIG. 8 is an explanatory diagram schematically showing an example of a spinning apparatus for producing protein fibers. A spinning device 1000 shown in FIG. 8 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 101 , an undrawn yarn manufacturing device 102 , a wet heat drawing device 103 and a drying device 104 .

A spinning method using the spinning apparatus 1000 will be described. First, the dope liquid 106 stored in the storage tank 107 is pushed out from the mouthpiece 109 by the gear pump 108 . On a lab scale, the dope solution may be filled into a cylinder and pushed out through a nozzle using a syringe pump. Then, the extruded dope liquid 106 is fed through the air gap 119 into the coagulation liquid 111 of the coagulation liquid bath 120, the solvent is removed, the protein is coagulated, and a fibrous coagulum is formed. The fibrous coagulum is then fed into the warm water 112 in the stretching bath 121 and stretched. The draw ratio is determined by the speed ratio between the supply nip roller 113 and the take-up nip roller 114 . Thereafter, the stretched fibrous coagulum is supplied to the drying device 104 and dried in the yarn path 122 to obtain the protein fibers 136 as the wound body 105 . 118a-118g are thread guides.

(Manufacturing method of synthetic leather)
The method for producing a synthetic leather according to the present embodiment includes, for example, a step of impregnating a nonwoven fabric with an impregnating liquid containing a polymeric substance, and then solidifying the polymeric substance to form a base layer (base layer forming step). Prepare. The nonwoven fabric, polymeric substance and base material layer can be applied in the same manner as described above.

In the manufacturing method according to this embodiment, the nonwoven fabric contains protein fibers and is shrink-proof. Protein fibers and shrink-proofing methods can be applied in the same manner as described above.

In the base material layer forming step, for example, the nonwoven fabric is immersed in the impregnating liquid or the nonwoven fabric is sprayed with the impregnating liquid to bring the nonwoven fabric and the impregnating liquid into contact with each other, impregnate the nonwoven fabric with the impregnating liquid, and then solidify. It can be carried out by a method (wet method) in which the components in the impregnating liquid are solidified by contacting (for example, immersing) the impregnating liquid to form the substrate layer. Depending on the type of solvent (for example, one with a low boiling point), the nonwoven fabric impregnated with the impregnating liquid as described above may be desolvated by volatilizing the solvent of the impregnating liquid without contacting the coagulating liquid. The base material layer forming step can also be carried out by a method of solidifying the components in the impregnating liquid to form the base material layer.

The solvent used for the impregnating liquid can be appropriately selected depending on the type of polymer substance, and examples thereof include dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, alcohol, or hexafluoroisopropanol (HFIP). organic solvents such as

FIG. 9 is a schematic diagram showing an example of a manufacturing apparatus for manufacturing synthetic leather by a wet method. In the manufacturing method using the manufacturing apparatus 800 shown in FIG. 9, first, the nonwoven fabric 81 is pulled out from the roll on which the nonwoven fabric 81 is wound, and the nonwoven fabric 81 is immersed in the impregnation bath 82 containing the impregnation liquid. Next, the polymer substance with which the nonwoven fabric 81 is impregnated is coagulated in a coagulation bath 83 containing a coagulation liquid. Then, after washing through a hot water washing bath 84 containing a washing liquid, the synthetic leather 86 is dried in a dryer 85 and wound around a roll, whereby the synthetic leather can be manufactured.

The type of coagulating liquid can be appropriately selected according to the types of the nonwoven fabric and polymer substance, and the type of solvent of the impregnating liquid. For example, when the polymeric substance contains polyurethane, for example, N,N-dimethylformamide can be used as the impregnating liquid solvent, and water (hot water) can be used as the coagulating liquid. Further, for example, when the macromolecular substance contains protein, for example, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP) as solvents for the impregnation solution, and dissolution promotion in these An inorganic salt added as the agent, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, acetone, and water (hot water) can be used as the coagulating liquid. Various additives may be added to the coagulation liquid as required.

The manufacturing method according to the present embodiment may further include a finishing step (surface grinding, coating, dyeing, etc.) after the substrate layer forming step. For example, as a finishing process, a step of applying a skin layer resin on a release paper, a step of applying an adhesive resin to the surface of the skin layer resin opposite to the release paper, and a step of applying an adhesive resin to the surface of the skin layer resin A synthetic leather can be produced by a method comprising a step of superimposing a base material layer on the opposite side and thermocompression bonding. The synthetic leather manufactured by such a manufacturing method may be the synthetic leather shown in FIG. FIG. 10 is a schematic diagram showing an example of a manufacturing apparatus for manufacturing the synthetic leather shown in FIG. In the manufacturing method using the manufacturing apparatus 900 shown in FIG. Dry with Next, an adhesive layer resin 94 is applied to the surface of the skin layer resin (polymer) 92 (the surface opposite to the surface in contact with the release paper 91 ) and dried by the dryer 93 . Then, the base material layer 96 is pulled out from the roll on which the base material layer 96 is wound, and is placed on the surface of the adhesive layer resin 94 (the surface opposite to the surface in contact with the skin layer resin (polymer) 92), A synthetic leather 97 in which a substrate layer 96, an adhesive layer resin 94, a skin layer resin (polymer) 92, and a release paper 91 are laminated in this order by bonding by thermal compression using a hot press roll 95 is formed on a roll. Synthetic leather can be manufactured by winding.

The adhesive layer resin 94 is not particularly limited, and adhesives used for conventional synthetic leather can be used. Examples of adhesives include synthetic resins such as polyolefin, polystyrene, polyurethane, acrylic, and EVA (ethylene-vinyl acetate copolymer). As the skin layer resin (polymer) 92, for example, the above-described polymer substance (polymer substance contained in the base material layer) can be used.

The thickness of the adhesive layer formed by the adhesive layer resin 94 is not particularly limited, but may be, for example, 1 to 50 μm, 1 to 40 μm, 5 to 30 μm, or 5 to 50 μm. It may be 20 μm.

The synthetic leather according to the present embodiment can also be manufactured discontinuously without using the manufacturing apparatus or the like shown in FIGS. 8 and 9, or using a different apparatus. For example, after immersing the entire unwound non-woven fabric of a predetermined size in an impregnating liquid in an impregnating tank having an independent structure, the polymer material impregnated in the non-woven fabric is coagulated using or using a coagulating liquid. solidify without After that, the target synthetic leather can be obtained by washing and drying as necessary.

A nonwoven fabric containing protein fibers can be formed, for example, by an electrospinning method. In the electrospinning method (electrostatic spinning method), a voltage is applied between a supply side electrode (which can also be used as a spinneret) and a collection side electrode (for example, a metal roll or a metal net), and the dope liquid is extruded from the spinneret. is given an electric charge and blown away to the collection side electrode. At this time, the dope is stretched to form fibers. The applied voltage is usually 5-100 kV, preferably 10-50 kV. The distance between the electrodes is usually 1-25 cm, preferably 2-20 cm.

FIG. 11 is an illustration of an electrospinning device 100 according to one embodiment. A voltage is applied by a power supply 35 between the metal nozzle 33 (supply side electrode) and the metal net 38 (collection side electrode). The dope 32 in the microsyringe 31 is moved in the direction of arrow P by using a syringe pump, the dope 32 is extruded from the metal mouthpiece nozzle 33 , and the dope is stretched by electric charge to form a fibrous material 36 into a metal net 38 . By accumulating on the surface of , a nonwoven fabric 39 containing protein fibers can be obtained. The resulting nonwoven may then be stripped of solvent. Methods for desorbing the solvent include, for example, drying under reduced pressure or immersion in a desolvation bath.

The synthetic leather according to the present embodiment may have a maximum hygroscopic heat value of more than 0.025° C./g, which is obtained according to the following formula A.
Formula A: Maximum heat of moisture absorption = {(maximum sample temperature when the sample is placed in a low-humidity environment until the sample temperature reaches equilibrium and then transferred to a high-humidity environment) - (the sample is After placing in a low humidity environment until the temperature reaches equilibrium, the sample temperature when transferred to a high humidity environment)} (° C.) / sample weight (g)
In formula A, the low-humidity environment means an environment with a temperature of 20°C and a relative humidity of 40%, and the high-humidity environment means an environment with a temperature of 20°C and a relative humidity of 90%.

The synthetic leather according to the present embodiment may have a maximum hygroscopic heat generation of 0.026° C./g or more, may be 0.027° C./g or more, or may be 0.028° C./g or more. may be 0.029° C./g or more, 0.030° C./g or more, 0.035° C./g or more, or 0.040° C./g or more There may be. Although there is no particular upper limit to the maximum heat value of moisture absorption, it is usually 0.060° C./g or less.

The synthetic leather according to the present embodiment has a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, may be 28 or more, may be 29 or more, may be 30 or more, may be 31 or more, may be 32 or more, may be 33 or more, or It may be greater than 33.

The synthetic leather according to the present embodiment may have a heat retention index greater than 0.18, which is calculated according to the following formula B.
Formula B: heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )
Here, in the present specification, the heat retention rate means the heat retention rate measured by the dry contact method using a thermolab type II tester (under wind of 30 cm/sec), and is measured by the method described in the examples described later. is the value to be

The heat retention index of the synthetic leather according to the present embodiment may be 0.20 or more, 0.22 or more, 0.24 or more, 0.26 or more, and 0 0.28 or greater, 0.30 or greater, or 0.32 or greater. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

(Uses of synthetic leather)
The synthetic leather according to the present embodiment can be used for the same applications as conventional synthetic leather (for example, synthetic leather made of synthetic resin). The synthetic leather according to the present embodiment can be used for applications such as clothing, accessories such as shoes and bags, various covers and furniture, and automotive interior materials.

Hereinafter, the present invention will be described more specifically based on test examples and the like. However, the present invention is not limited to the following test examples.

[Test Example 1: Production of modified fibroin]
Modified spider silk fibroin (PRT399) having the amino acid sequence shown by SEQ ID NO: 18, modified spider silk fibroin (PRT380) having the amino acid sequence shown by SEQ ID NO: 12, modified spider silk fibroin having the amino acid sequence shown by SEQ ID NO: 13 (PRT410), modified fibroin having the amino acid sequence shown in SEQ ID NO: 37 (PRT918), modified fibroin having the amino acid sequence shown in SEQ ID NO: 40 (PRT966), and modified fibroin having the amino acid sequence shown in SEQ ID NO: 15 ( PRT799) was designed. A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

E. coli BLR (DE3) was transformed with the resulting expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin to an OD ₆₀₀ of 0.005. The temperature of the culture solution was kept at 30° C., and flask culture was performed until OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) to an OD ₆₀₀ of 0.05. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture solution was kept at 37° C. and the pH was constantly controlled to 6.9 for culturing. Also, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation concentration, and culture was carried out for 20 hours. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the modified fibroin. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of the desired modified fibroin size band depending on the addition of IPTG. did.

Two hours after the addition of IPTG, the collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and heated at 60°C. for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed with a freeze dryer, and the freeze-dried powder was recovered to obtain modified fibroin (PRT399, PRT380, PRT410, PRT918, PRT966 and PRT799). rice field.

PRT918 and PRT966 are hydrophobically modified fibroins with average HI greater than zero. PRT799 is a hydrophilic modified fibroin with an average HI of 0 or less.

[Test Example 2: Production of Modified Fibroin Fiber and Evaluation of Contractility]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and lyophilized powder of modified fibroin (PRT399, PRT380, PRT410 or PRT799) was added thereto at a concentration of 18% by mass or 24% by mass. % by mass and dissolved for 3 hours using a shaker. After that, insoluble matter and bubbles were removed to obtain a modified fibroin solution.

The obtained modified fibroin solution was used as a dope solution (spinning stock solution), and a modified spider fibroin fiber spun and drawn was produced by dry-wet spinning using a spinning apparatus according to the spinning apparatus 1000 shown in FIG. The spinning apparatus used was the spinning apparatus 1000 shown in FIG. 8, in which a second undrawn yarn manufacturing apparatus ( second bath). The conditions for dry-wet spinning are as follows.
Extrusion nozzle diameter: 0.2mm
Coagulation bath temperature: 2-15°C
Total draw ratio: 1 to 4 times Drying temperature: 60°C

(Shrinkage rate evaluation)
The resulting modified fibroin fibers (manufacturing examples 1 to 19) were evaluated for shrinkage. That is, each modified fibroin fiber is subjected to a shrinking process in which it is brought into contact with water (contact step) and then dried (drying step), and the shrinkage rate of the wet modified fibroin fiber and the wet state After that, the shrinkage of the dried modified fibroin fibers was determined.

<Contact step>
A plurality of test modified fibroin fibers each having a length of 30 cm were cut from each modified fibroin fiber roll. A plurality of modified fibroin fibers were bundled to obtain a modified fibroin fiber bundle having a fineness of 150 denier. A lead weight of 0.8 g was attached to each modified fibroin fiber bundle, and each modified fibroin fiber bundle was immersed in water at temperatures shown in Tables 6 to 9 for 10 minutes. The length of each modified fibroin fiber bundle was then measured in water. The measurement was performed with a 0.8 g lead weight attached to the modified fibroin fiber bundle in order to eliminate crimping of the modified fibroin fiber bundle. Next, the shrinkage rate of the modified fibroin fiber in wet state (wet shrinkage rate) was calculated according to the following formula V. In formula V, L0 indicates the length (30 cm) of the modified fibroin fiber bundle before immersion in water, and Lw indicates the length of the modified fibroin fiber bundle that has been immersed in water to make it wet.
Wet shrinkage rate (%) = {1-(Lw/L0)} x 100 (Formula V)

<Drying step>
After the contacting step, the modified fibroin fiber bundle was removed from the water. The removed modified fibroin fiber bundle was dried at room temperature for 2 hours with a 0.8 g lead weight attached. After drying, the length of each modified fibroin fiber bundle was measured. Next, the shrinkage rate (dry shrinkage rate) of the modified fibroin fiber that was dried after being moistened was calculated according to the following formula VI. In formula VI, L0 indicates the length (30 cm) of the modified fibroin fiber bundle before being immersed in water, and Lwd is the length of the modified fibroin fiber bundle after being immersed in water to make it wet and then dried. .
Dry shrinkage (%) = {1-(Lwd/L0)} x 100 (%) (formula VI)

The results are shown in Tables 6-9. In Tables 6 to 9, "total draw ratio" indicates the total draw ratio in the spinning process.

The modified fibroin fibers had high wet and dry shrinkage. On the other hand, the modified fibroin fibers that underwent the aforementioned shrinkability evaluation (shrinkage step) had a sufficiently reduced shrinkage rate when brought into contact with water again. It is believed that the shrinking process described above alleviates the residual stress caused by drawing during spinning.

[Test Example 3: Shrink-proofing and Evaluation of Molded Article Using Modified Fibroin Fiber]
(1) Production of Molded Body (Nonwoven Fabric) Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent. % by mass and dissolved for 3 hours using a shaker. After that, insoluble matter and bubbles were removed to obtain a modified fibroin solution.

The obtained modified fibroin solution was used as a dope solution (spinning stock solution), and dry-wet spinning was performed using a spinning apparatus according to the spinning apparatus 1000 shown in FIG. 8 to obtain a modified fibroin fiber (24 multifilaments). The conditions for dry-wet spinning are as follows.
Temperature of coagulation liquid (methanol): 5-10°C
Stretch ratio: 5 times Drying temperature: 80°C

The obtained modified fibroin fibers (24 multifilaments) were cut into a predetermined length to make short fibers. Thereafter, carding was performed using a known carding machine to obtain a plurality of webs in which short fibers were entangled.

Then, one sheet of the obtained web was punched with a known needle punch machine to obtain a nonwoven fabric 1. The fiber density (basis weight) of this nonwoven fabric 1 was 350 g/m ² .

In addition, after laminating three sheets of the obtained web while tilting at angles of 0, 45 and 90 degrees to obtain a laminated web, this laminated web is punched twice by a known needle punch machine to form a nonwoven fabric. got 2. The fiber density (basis weight) of the nonwoven fabric 2 was 530 g/m ² .

(2) Shrinkproof Treatment and Evaluation Test pieces each measuring 35 mm long and 15 mm wide were cut out from nonwoven fabrics 1 and 2, and their weights were measured. After each test piece was immersed in water at 40°C for 10 minutes, it was dried at room temperature for 12 hours to obtain two types of highly shrinkable nonwoven fabrics 1 and 2 (shrink-proof nonwoven fabrics 1 and 2) with different fiber densities. rice field.

The vertical and horizontal lengths and weights of the obtained high shrinkage nonwoven fabrics 1 and 2 were measured. Next, the shrinkage rate, fiber density, and fiber density increase rate of the high shrinkage nonwoven fabrics 1 and 2 were calculated (average value of n=3). Table 10 shows the results. The shrinkage rate and fiber density increase rate were calculated according to Formula VII and Formula I below, respectively.
Shrinkage ratio = {1-(longitudinal (horizontal) length of highly shrinkable nonwoven fabric/longitudinal (horizontal) length of raw material nonwoven fabric)} x 100 (%) (Formula VII)
Fiber density increase rate = {(fiber density of high-density nonwoven fabric / fiber density of raw nonwoven fabric) - 1} x 100 (%) (Formula I)

By subjecting a molded article (nonwoven fabric) using modified fibroin fibers to a shrink-proof treatment (water shrinkage method), the molded article (nonwoven fabric) was highly shrunk and the fiber density was increased. It should be noted that the molded article (non-woven fabric) that has undergone the above-described shrink-proofing treatment had a sufficiently reduced shrinkage rate when brought into contact with water again. It is believed that the above-mentioned shrink-proofing treatment relieved the residual stress caused by drawing during spinning.

[Test Example 4: Flame Retardancy Evaluation of Modified Fibroin Knitted Fabric]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and freeze-dried powder of modified fibroin (PRT799) was added thereto to a concentration of 24% by mass. Used and dissolved for 3 hours. Thereafter, insoluble matter and foam were removed to obtain a modified fibroin solution (spinning dope).

The prepared spinning dope was filtered at 90° C. through a metal filter with an opening of 5 μm, then allowed to stand still in a 30 mL stainless steel syringe to degas, and then 100% by mass of methanol solidified from a solid nozzle with a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 90°C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain a modified fibroin fiber (raw material fiber).

Using the obtained raw material fibers (twisted filament yarns), a knitted fabric was produced by circular knitting using a circular knitting machine. The knitted fabric had a thickness of 180 denier and a gauge number of 18. A 20 g piece was cut out from the obtained knitted fabric to obtain a test piece.

The flammability test complied with the test method for particulates or synthetic resins with a low melting point issued on May 31, 1995, Hazardous Materials Regulation Division Director, Fire and Disaster Management Agency, Shoken No. 50. The test was performed under the conditions of a temperature of 22° C., a relative humidity of 45% and an atmospheric pressure of 1021 hPa. Table 11 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).

As a result of the flame retardancy test, the limiting oxygen index (LOI) value of the knitted fabric made of the modified fibroin (PRT799) fiber was 27.2. In general, if the LOI value is 26 or more, it is considered to be flame retardant. It can be seen that the modified fibroin is excellent in flame retardancy.

[Test Example 5: Evaluation of moisture absorption heat generation of modified fibroin knitted fabric]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and freeze-dried powder of modified fibroin was added thereto so as to have a concentration of 24% by mass, followed by shaking using a shaker. Allow to dissolve for 3 hours. Thereafter, insoluble matter and foam were removed to obtain a modified fibroin solution (spinning dope).

The prepared spinning dope was filtered at 60° C. through a metal filter with an opening of 5 μm, then allowed to stand still in a 30 mL stainless steel syringe to degas, and then 100% by mass of methanol was coagulated from a solid nozzle with a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60°C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain a modified fibroin fiber (raw material fiber).

For comparison, commercially available wool fibers, cotton fibers, Tencel fibers, rayon fibers and polyester fibers were prepared as raw material fibers.

Using each raw material fiber, a knitted fabric was produced by flat knitting using a flat knitting machine. The knitted fabric using PRT918 fiber as the raw material fiber had a thickness of 1/30 N (hair count single yarn) and a gauge number of 18. The knitted fabric using the PRT799 fiber as the raw material fiber had a thickness of 1/30 N (hair count single yarn) and a gauge number of 16. Knitted fabrics using other raw material fibers were adjusted in thickness and gauge number so as to have almost the same cover factor as knitted fabrics using PRT918 fibers and PRT799 fibers. Specifically, it is as follows.
Wool thickness: 2/30N (two-ply yarn), gauge: 14
Cotton thickness: 2/34N (two-ply yarn), gauge number: 14
Tencel thickness: 2/30N (two-ply yarn), gauge number: 15
Rayon thickness: 1/38N (single yarn), gauge number: 14
Polyester thickness: 1/60N (single yarn), gauge number: 14

Two knitted fabrics cut to 10 cm×10 cm were put together, and the four sides were sewn together to obtain a test piece (sample). After leaving the test piece in a low humidity environment (temperature 20 ± 2 ° C, relative humidity 40 ± 5%) for 4 hours or more, transfer 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 interior.

From the measurement results, the maximum hygroscopic heat generation was determined according to the following formula A.
Formula A: Maximum heat of moisture absorption = {(maximum sample temperature when the sample is placed in a low-humidity environment until the sample temperature reaches equilibrium and then transferred to a high-humidity environment) - (the sample is After placing in a low humidity environment until the temperature reaches equilibrium, the sample temperature when transferred to a high humidity environment)} (° C.) / sample weight (g)

FIG. 12 is a graph showing an example of the results of the moisture absorption heat generation test. The horizontal axis of the graph indicates the time (minutes) left in the high-humidity environment, with 0 being the point at which 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. 12, the point indicated by M corresponds to the maximum sample temperature.

Table 12 shows the calculation results of the maximum heat value of moisture absorption.

As shown in Table 12, the modified fibroins (PRT918 and PRT799) have a higher maximum moisture heat generation rate and are superior in moisture heat generation compared to existing materials.

[Test Example 6: Evaluation of heat retention of modified fibroin fabric]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and freeze-dried powder of modified fibroin was added thereto so as to have a concentration of 24% by mass, followed by shaking using a shaker. Allow to dissolve for 3 hours. Thereafter, insoluble matter and foam were removed to obtain a modified fibroin solution (spinning dope).

The prepared spinning dope was filtered at 60° C. through a metal filter with an opening of 5 μm, then allowed to stand still in a 30 mL stainless steel syringe to degas, and then 100% by mass of methanol was coagulated from a solid nozzle with a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60°C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain a modified fibroin fiber (raw material fiber).

For comparison, commercially available wool fibers, silk fibers, cotton fibers, rayon fibers and polyester fibers were prepared as raw material fibers.

Using each raw material fiber, a knitted fabric was produced by flat knitting using a flat knitting machine. The knitted fabric using PRT966 fibers as raw fibers had a yarn count of 30 Nm, a number of twists of 1, a gauge number of 18 GG, and a basis weight of 90.1 g/m ² . The knitted fabric using PRT799 fibers as raw fibers had a yarn count of 30 Nm, a number of twists of 1, a gauge number of GG of 16, and a basis weight of 111.0 g/m ² . Knitted fabrics using other raw fibers were adjusted in thickness and gauge number so as to have almost the same cover factor as knitted fabrics using PRT966 fibers and PRT799 fibers. Specifically, it is as follows.
Wool count: 30 Nm, number of twists: 2, gauge number: 14 GG, basis weight: 242.6 g/m ²
Silk count: 60 Nm, number of twists: 2, gauge number: 14 GG, basis weight: 225.2 g/m ²
Cotton count: 34 Nm, number of twists: 2, gauge number: 14 GG, basis weight: 194.1 g/m ²
Rayon count: 38 Nm, twist number: 1, gauge number: 14 GG, basis weight: 181.8 g/m ²
Polyester count: 60 Nm, number of twists: 1, number of gauges: 14 GG, basis weight: 184.7 g/m ²

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 direct contact between skin and clothes in a dry state). A piece of knitted fabric cut to 20 cm×20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30° C.), and the amount of heat (a) dissipated through the test piece was determined under the conditions of a wind speed of 30 cm/sec in the wind tunnel. The amount of heat (b) dissipated under the same conditions as above was determined without setting the test piece, and the heat retention rate (%) was calculated according to the following formula.
Heat retention rate (%) = (1-a/b) x 100

From the measurement results, the heat retention index was obtained according to the following formula B.
Formula B: heat retention index = heat retention rate (%) / basis weight of sample (g/m ² )

Table 13 shows the calculation results of the heat retention index. The higher the heat retention index, the more excellent the heat retention of the material can be evaluated.

As shown in Table 13, the modified fibroins (PRT966 and PRT799) have a higher heat retention index and are superior in heat retention compared to existing materials.

[Test Example 7: Production of synthetic leather]
Using the nonwoven fabrics and impregnating liquids shown in Tables 14 and 15, synthetic leathers 1-1 to 1-4 and synthetic leathers 2-1 to 2-4 consisting of only nonwoven fabrics and polymeric substrate layers were prepared as follows. Manufactured by Further, a synthetic leather 3 was produced by laminating a skin layer on the surface of a base material layer as follows. Details of their manufacturing methods are given below. In Table 14, nonwoven fabric 3 (modified fibroin nonwoven fabric) is a nonwoven fabric produced from fibers obtained by spinning using PRT799 as follows. In Table 15, nonwoven fabric 4 (modified fibroin nonwoven fabric) is a nonwoven fabric manufactured from shrink-proof fibers spun using PRT966 as follows. The compositions of impregnating liquid 1 (modified fibroin impregnating liquid) and impregnating liquid 2 (polyurethane (PU) impregnating liquid) are as follows.
- Impregnation liquid 1 (modified fibroin impregnation liquid): containing 5% by mass of PRT799, the solvent being a mixed solvent of ethanol/water (3/7 (w/w)).
- Impregnation liquid 2 (polyurethane (PU) impregnation liquid): contains PU resin (final concentration: 5% by mass), solvent is a mixed solvent of MP-865PS (manufactured by DIC) / DMF (1/5 (w / w) .

<Production of synthetic leather 1-1>
(Manufacture of nonwoven fabric 3)
Lyophilized powder of modified fibroin (PRT799) was added to formic acid to a concentration of 28% by mass and dissolved using a shaker for 3 hours. Thereafter, insoluble matter and foam were removed to obtain a modified fibroin solution (spinning dope). The resulting modified spider fibroin solution was used as a dope solution (spinning stock solution), and modified spider fibroin fibers were produced by dry-wet spinning using a known dry-wet spinning apparatus. Next, the resulting modified fibroin fibers were cut into short fibers of 50 mm, and a sheet (web) was obtained by a known carding process. Thereafter, the obtained sheet was punched with a known needle punch machine to produce a nonwoven fabric 3 made of modified fibroin fibers. The basis weight (fiber density) of the nonwoven fabric 3 was 100 to 200 g/m ² . The conditions for dry-wet spinning are as follows.
Coagulation bath temperature: 2-15°C
Total draw ratio: 1 to 4 times Drying temperature: 100°C

(Manufacture of synthetic leather 1-1)
The nonwoven fabric 3 obtained as described above was impregnated with the impregnating liquid 1 so that the pick-up rate was 400%. After that, the nonwoven fabric 3 impregnated with the impregnating liquid 1 was immersed in water for 15 minutes to remove the solvent from the impregnating liquid 1 and solidify the modified fibroin in the impregnating liquid 1 . Next, the nonwoven fabric 3 was taken out of water and then dried at room temperature. As a result, a synthetic leather 1-1 (consisting only of a base material layer) composed of the modified fibroin fiber non-woven fabric and the polymer substance composed of the modified fibroin impregnated and integrated therewith was manufactured. In this synthetic leather 1-1, the non-woven fabric 3 and the synthetic leather as a whole are not subjected to any shrink-proofing treatment (not shrink-proofed).

<Production of synthetic leather 1-2>
The synthetic leather 1-1 obtained as described above was immersed in water at 40° C. for 10 minutes for water shrinkage treatment, and then dried at room temperature. As a result, a synthetic leather 1-2 (consisting only of a base material layer) composed of the modified fibroin fiber nonwoven fabric and the modified fibroin polymer was produced. In the synthetic leather 1-2, the non-woven fabric 3 and the synthetic leather as a whole are shrink-proofed (shrink-proofed) by water shrinkage treatment.

<Production of synthetic leather 1-3>
The nonwoven fabric 3 obtained as described above was impregnated with the impregnating liquid 2 so that the pick-up rate was 400%. After that, the nonwoven fabric 3 impregnated with the impregnating liquid 2 is immersed in a 15% by mass N,N-dimethylformamide (DMF) aqueous solution for 5 minutes to remove the solvent from the impregnating liquid 2 and remove the polyurethane in the impregnating liquid 2. The resin was allowed to solidify (harden). Next, the nonwoven fabric 3 was taken out from the DMF aqueous solution and then dried at room temperature. As a result, a synthetic leather 1-3 (consisting only of the base material layer) composed of the modified fibroin fiber non-woven fabric and the polymeric material made of polyurethane resin impregnated and integrated therewith was produced. In the synthetic leather 1-3, the non-woven fabric 3 and the synthetic leather as a whole are not subjected to any shrink-proof treatment (not shrink-proof).

<Production of synthetic leather 1-4>
The synthetic leather 1-3 obtained as described above was immersed in water at 40° C. for 10 minutes for water shrinkage treatment, and then dried at room temperature. As a result, a synthetic leather 1-4 (consisting only of the base material layer) composed of the modified fibroin fiber nonwoven fabric and the polymeric substance composed of the polyurethane resin was produced. In the synthetic leather 1-4, the non-woven fabric 3 and the synthetic leather as a whole are subjected to shrink-proofing (shrink-proofing) by water shrinkage treatment.

<Production of synthetic leather 1-1>
(Manufacture of nonwoven fabric 4)
Lyophilized powder of modified fibroin (PRT966) was added to formic acid to a concentration of 28% by mass and dissolved using a shaker for 3 hours. Thereafter, insoluble matter and foam were removed to obtain a modified fibroin solution (spinning dope). The resulting modified spider fibroin solution was used as a dope solution (spinning stock solution), and modified spider fibroin fibers were produced by dry-wet spinning using a known dry-wet spinning apparatus. Thereafter, the obtained modified spider fibroin fiber was subjected to water shrinkage treatment by immersing it in water at 90° C. for 10 minutes, and then dried at room temperature. Next, the resulting modified fibroin fibers were cut into short fibers of 50 mm, and a sheet (web) was obtained by a known carding process. After that, the obtained sheet was punched with a known needle punch machine to produce a nonwoven fabric 4 made of modified fibroin fibers. The nonwoven fabric 4 is shrink-proofed by being constructed using modified fibroin fibers subjected to water shrinkage treatment. The basis weight (fiber density) of the nonwoven fabric 4 was 100 to 200 g/m ² . The conditions for dry-wet spinning are as follows.
Coagulation bath temperature: 2-15°C
Total draw ratio: 1 to 4 times Drying temperature: 100°C

(Manufacture of synthetic leather 2-1)
A synthetic leather 2-1 was produced in the same manner as the synthetic leather 1-1 except that the nonwoven fabric 4 obtained as described above was used. This synthetic leather 2-1 is composed of a shrink-proof modified fibroin fiber non-woven fabric and a polymeric material composed of modified fibroin (consisting of only a base material layer).

<Manufacture of synthetic leather 2-2>
The synthetic leather 2-1 obtained as described above was subjected to water shrinkage treatment by immersing it in water at 40° C. for 10 minutes, and then dried at room temperature. As a result, a synthetic leather 2-2 (consisting only of a base material layer) composed of the shrink-proof modified fibroin fiber nonwoven fabric and the modified fibroin polymeric material was produced. This synthetic leather 2-2 is a synthetic leather that has undergone a shrink-proofing treatment (shrink-proofing) by a water-shrinking treatment.

<Production of synthetic leather 2-3>
A synthetic leather 2-3 was produced in the same manner as the synthetic leather 1-3 except that the nonwoven fabric 4 obtained as described above was used. This synthetic leather 2-3 is composed of a shrink-proof modified fibroin fiber non-woven fabric and a polymeric substance composed of a polyurethane resin (consisting of only a base material layer).

<Production of synthetic leather 2-4>
The synthetic leather 2-3 obtained as described above was subjected to water shrinkage treatment by immersing it in water at 40° C. for 10 minutes, and then dried at room temperature. As a result, a synthetic leather 2-4 (consisting only of a base material layer) composed of the shrink-proof modified fibroin fiber non-woven fabric and the polymeric substance composed of polyurethane resin was produced. This synthetic leather 2-2 is a synthetic leather that has undergone a shrink-proofing treatment (shrink-proofing) by a water-shrinking treatment.

<Production of synthetic leather 3>
(Production of base material layer)
First, synthetic leather 2-1 was produced in the same manner as described above, and this was used as the base material layer.

(Preparation of resin for skin layer)
A lyophilized powder of modified fibroin (PRT799) was added to formic acid to a concentration of 20% by mass and dissolved using a shaker at 40° C. for 1 hour or more to obtain a modified fibroin solution. After that, the pigment was added to the modified fibroin solution so as to be 5% by mass, and then insoluble matter and foam were removed. Thus, a skin layer resin was prepared from a modified fibroin solution containing a pigment.

(Manufacture of synthetic leather 3)
The resin for the skin material layer obtained as described above is coated on the release paper pasted on the iron plate so as to have a thickness of 0.5 mm, and then dried at 90 ° C. for 10 minutes to form the skin material layer. The resin was incompletely solidified. After that, the skin layer resin was laminated on the substrate layer obtained as described above together with the release paper attached to the iron plate, and heated at 90° C. for 1 minute. As a result, the skin layer resin was completely solidified and adhered to the substrate layer. After that, the iron plate and the release paper were peeled off from the skin layer resin. Thus, the intended synthetic leather 3 was produced, in which the skin layer was adhered to the surface of the base material layer. The photograph is shown in FIG. The iron plate was used to improve the surface smoothness of the skin layer.

[Test Example 8: Water shrinkage test of synthetic leather]
Each synthetic leather was immersed in a 40° C. water bath for 10 minutes. After that, the synthetic leather was taken out from the water bath and vacuum-dried at room temperature for 15 minutes, and then the length of the dried synthetic leather was measured. The water shrinkage rate of synthetic leather was calculated according to the following formula. Results are shown in Tables 14 and 15.
Water shrinkage rate (%) = {(length before immersion / length after immersion and drying) - 1} x 100

DESCRIPTION OF SYMBOLS 1...Nonwoven fabric 2...Polymer substance 3...Base layer 4...Adhesive layer 5...Skin layer 10, 20...Synthetic leather 31...Microsyringe 32, 106...Dope liquid 33...Made of metal Base nozzle 35 Power supply 36 Fibrous material 38 Metal net 39 Nonwoven fabric 81 Nonwoven fabric 82 Impregnation tank 83 Coagulation tank 84 Hot water washing tank 85 Dryer 86 Synthetic leather 91 Release paper 92 Skin layer resin (polymer) 93 Dryer 94 Adhesive layer resin 95 Hot press roll 96 Base material layer 97 Synthetic leather 100 Electro Spinning device 101 Extruding device 102 Undrawn yarn manufacturing device 103 Wet heat drawing device 104 Drying device 120 Coagulation liquid tank 121 Drawing bath 136 Modified fibroin fiber 800, 900 Synthetic leather manufacturing equipment, 1000 ... Spinning equipment.

Claims (9)

comprising a substrate layer comprising a nonwoven fabric and a polymeric material;
Synthetic leather, wherein the non-woven fabric contains protein fibers and is shrink-proof. The synthetic leather according to claim 1, wherein the fiber density increase rate defined by the following formula I is 20% or more.
Fiber density increase rate = {(fiber density of non-woven fabric after shrink-proofing/fiber density of non-woven fabric before shrink-proofing) - 1} x 100 (%) (Formula I) The synthetic leather according to claim 1 or 2, wherein the protein fiber has a wet shrinkage of 2% or more as defined by the following formula II.
Wet shrinkage = {1-(length of protein fiber brought into wet state by contact with water/length of protein fiber after spinning but before contact with water)} x 100 (%) (Formula II) The synthetic leather according to any one of claims 1 to 3, wherein said protein fibers have a dry shrinkage of greater than 7% as defined by Formula III below.
Dry shrinkage = {1-(length of protein fiber in dry state/length of protein fiber after spinning before contact with water)} x 100 (%) (Formula III) The synthetic leather according to any one of claims 1 to 4, wherein the nonwoven fabric has a fiber density of 0.04 g/cm ² or more. The synthetic leather according to any one of claims 1 to 5, wherein said protein fibers comprise modified fibroin. 7. The synthetic leather according to claim 6, wherein said modified fibroin is modified spider silk fibroin. A step of impregnating the nonwoven fabric with an impregnating liquid containing a polymeric substance and solidifying the polymeric substance to form a base layer;
A method for producing synthetic leather, wherein the nonwoven fabric comprises protein fibers and is shrink-proof. The method for producing synthetic leather according to claim 8, wherein the nonwoven fabric is shrink-proofed by water shrinkage due to contact with water.

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