JP2021008678A - Synthetic leather and manufacturing method thereof - Google Patents

Abstract

To provide a synthetic leather excellent in functionality such as water resistance.SOLUTION: A synthetic leather has a substrate layer including a nonwoven fabric and a polymer material. Functionality is imparted to at least one of the nonwoven fabric and the polymer material.SELECTED DRAWING: Figure 1

Description

The present invention relates to synthetic leather and a method for producing the same.

Conventionally, synthetic leather such as artificial leather has been used as a substitute for natural leather. Unlike natural leather, which has a limited supply, synthetic leather can be artificially mass-produced, so in recent years it has become wider for clothing, shoes, accessories such as bags, various covers, furniture, etc. It has come to be used for various purposes.

For example, Patent Document 1 describes a method for producing artificial leather by impregnating a non-woven fabric pretreated with a paste polymer substance with a polyurethane resin solution and wet-coagulating it, and then removing the glue polymer substance to produce artificial leather. A method for producing artificial leather is disclosed, which comprises a step of impregnating a mixed emulsion of reactive methyl H silicon and a metal catalyst and then heating at 100 to 150 ° C.

Japanese Unexamined Patent Publication No. 10-13109

However, many synthetic leathers such as artificial leathers disclosed in Patent Document 1 contain polyurethane and have major drawbacks such as inferior water resistance and weather resistance.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a synthetic leather having excellent functionality such as water resistance. Another object of the present invention is to provide a method for producing synthetic leather, which can easily produce synthetic leather having excellent functionality such as water resistance.

The present invention relates to, for example, the following inventions.
[1]
Synthetic leather comprising a base material layer containing a non-woven fabric and a polymer substance, and at least one of the non-woven fabric and the polymer substance is endowed with functionality.
[2]
The synthetic leather according to [1], wherein the non-woven fabric contains protein fibers.
[3]
The synthetic leather according to [2], wherein the protein fiber contains modified fibroin.
[4]
The synthetic leather according to [3], wherein the modified fibroin is a modified spider silk fibroin.
[5]
The above-mentioned non-woven fabric contains a protein crosslinked product and contains
The protein cross-linked product has a polypeptide skeleton, a first residue which is a residue of a first reactant having two or more first reactive groups capable of reacting with a protein to form a bond, and the above. Each has a plurality of second residues, which are residues of the second reactant having one second reactive group capable of reacting with the first reactive group to form a bond.
At least one of the first residues crosslinks the polypeptide backbone.
The synthetic leather according to any one of [1] to [4], wherein at least one of the first residues is bound to the polypeptide skeleton at one end and to the second residue at the other end. ..
[6]
The synthetic leather according to any one of [1] to [5], wherein the nonwoven fabric contains a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer.
[7]
The synthetic leather according to any one of [1] to [6], wherein the polymer substance contains a protein.
[8]
The synthetic leather according to [7], wherein the protein contains modified fibroin.
[9]
The synthetic leather according to [8], wherein the modified fibroin is a modified spider silk fibroin.
[10]
The polymer substance contains a protein cross-linked product and contains
The protein cross-linked product has a polypeptide skeleton, a first residue which is a residue of a first reactant having two or more first reactive groups capable of reacting with a protein to form a bond, and the above. Each has a plurality of second residues, which are residues of the second reactant having one second reactive group capable of reacting with the first reactive group to form a bond.
At least one of the first residues crosslinks the polypeptide backbone.
The synthetic leather according to any one of [1] to "9", wherein at least one of the first residues is bound to the polypeptide skeleton at one end and to the second residue at the other end. ..
[11]
The synthetic leather according to any one of [1] to [10], wherein the polymer substance contains a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer.
[12]
A step of impregnating a non-woven fabric with an impregnating solution containing a polymer substance and coagulating the polymer substance to form a base material layer is provided, and at least one of the non-woven fabric and the polymer substance is imparted with functionality. There is a method of manufacturing synthetic leather.

According to the present invention, it is possible to provide synthetic leather having excellent functionality such as water resistance. Further, according to the present invention, it is possible to provide a method for producing synthetic leather, which can easily produce synthetic leather having excellent functionality such as water resistance.

It is a schematic cross-sectional view of the synthetic leather which concerns on one Embodiment. It is a schematic cross-sectional view of the synthetic leather which concerns on one Embodiment. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a figure which shows the distribution of the value of z / w (%) of naturally-derived fibroin. It is a figure which shows the distribution of the value of x / y (%) of naturally-derived fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram of the manufacturing method of synthetic leather which concerns on one Embodiment. It is a schematic diagram of the manufacturing method of synthetic leather which concerns on one Embodiment. It is explanatory drawing of the electrospinning apparatus which concerns on one Embodiment. It is a graph which shows the result of having evaluated the water shrinkage rate of the produced protein fiber. It is a graph which shows an example of the result of the hygroscopic heat generation test. It is a photograph of synthetic leather.

Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. For convenience, substantially the same elements may be designated by the same reference numerals and the description thereof may be omitted. The present invention is not limited to the following embodiments.

The synthetic leather according to the present embodiment includes a base material layer containing a non-woven fabric and a polymer substance. The base material layer can be said to be a layer in which the non-woven fabric and the impregnated body made of the polymer substance impregnated in the non-woven fabric are integrated, or the non-woven fabric is contained in the layer formed of the polymer substance. It can also be said that it is a layer in which is buried.

In the present embodiment, at least one of the non-woven fabric and the polymer substance (layer formed of the polymer substance) is provided with functionality. That is, only the non-woven fabric may be endowed with functionality (the polymer substance may not be endowed with functionality), and only the polymer substance may be endowed with functionality (the non-woven fabric may be endowed with functionality). Functionality is not imparted), and both the non-woven fabric and the polymer substance may be those to which functionality is imparted. Further, it is preferable that at least the non-woven fabric as the main material is provided with functionality.

Synthetic leather is also called artificial leather. For example, woven fabric, knitted fabric, non-woven fabric, etc. are used as a base material, and the base material is impregnated or coated with a resin (polymer) such as vinyl chloride or polyurethane, or a resin (polymer). ) By forming a layer, it has a natural leather-like appearance and function. In the present specification, the synthetic leather is referred to as so-called artificial leather (a special non-woven fabric (a base material mainly composed of a fiber layer having a random three-dimensional three-dimensional structure) impregnated with polyurethane or a polymer substance having similar flexibility. Those using) are also included.

FIG. 1 is a schematic cross-sectional view of synthetic leather according to an embodiment. The synthetic leather 10 shown in FIG. 1 includes a base material layer 3 containing a non-woven fabric 1 and a polymer substance 2. The base material layer 3 may be composed of, for example, a layer of a polymer substance 2 in which a non-woven fabric 1 is embedded. The base material layer 3 may be a non-porous layer or a porous layer.

(Non-woven fabric)
The non-woven fabric can be produced from, for example, raw material fibers by a known production method. Specifically, for example, after forming a web (including a single-layer web and a laminated web) from raw material fibers by a dry method, a wet method, an airlaid method, or the like, a chemical bond method (immersion method, spray method, etc.) is used. ) And the fibers of the web are bonded by a needle punching method or the like to obtain a non-woven fabric.

Examples of the raw material fibers include synthetic fibers such as nylon, polyester, polyamide, polyacrylonitrile, polyolefin, polyvinyl alcohol, polyethylene terephthalate and polytetrafluoroethylene, recycled fibers such as cupra, rayon and lyocell, cotton, linen and silk. Examples include natural fibers and protein fibers.

Non-woven fabrics containing protein fibers also promote dissolution of the protein in a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), if necessary. It can also be obtained by adding it together with an inorganic salt as an agent, dissolving it to prepare a dope solution, and then spinning the dope solution by an electrospinning method (electrostatic spinning method).

The fiber density (weight), porosity, bulk density, etc. of the non-woven fabric can be adjusted by, for example, increasing or decreasing the amount of fibers constituting the web, or increasing or decreasing the number of laminated webs.

The non-woven fabric preferably contains protein fibers. The protein fiber preferably contains modified fibroin, and more preferably contains modified spider silk fibroin. By containing the modified fibroin (preferably modified spider silk fibroin), it is possible to impart heat retention, moisture absorption and heat generation and / or flame retardant functionality to the non-woven fabric. As a result, the synthetic leather according to the present embodiment can be imparted with heat retention, hygroscopic heat generation and / or flame retardant functionality, and its value as a material becomes higher. The modified fibroin may be contained in the non-woven fabric as modified fibroin fibers (protein fibers). Preferred embodiments of modified fibroin will be described later.

Examples of the method for imparting functionality to the non-woven fabric include a method in which the non-woven fabric contains modified fibroin (first method) and a method in which the non-woven fabric contains protein fibers containing a predetermined protein crosslinked product (second method). Examples thereof include a method (third method) in which the non-woven fabric contains a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer.

In the first method, for example, a non-woven fabric having added functionality can be obtained by producing a non-woven fabric using a fiber containing a protein fiber containing modified fibroin as a raw material fiber. By containing the modified fibroin, it is possible to impart heat retention, hygroscopic heat generation and / or flame retardant functionality to the non-woven fabric.

The predetermined protein cross-linked product in the second method is a residue of the first reactant having two or more first reactive groups capable of reacting with the protein to form a bond with the polypeptide skeleton. One residue and a second residue, which is a residue of a second reactant having one second reactive group capable of reacting with the first reactive group to form a bond, respectively. Having multiple, at least one of the first residues crosslinks the polypeptide skeleton, at least one of the first residues binds to the polypeptide skeleton at one end and the second residue at the other end. Is combined with.

In the second method, for example, a non-woven fabric to which functionality is imparted can be obtained by producing a non-woven fabric using a fiber containing a predetermined protein crosslinked body as a raw material fiber. In the second method, for example, a fiber containing a protein fiber as a raw material fiber is used to obtain a non-woven fabric (precursor), and a first reactant and a second reaction are made with the non-woven fabric (precursor). By reacting the agent to generate a predetermined protein crosslinked product, a non-woven fabric to which functionality is imparted can be obtained.

In the second method, more specifically, a molded product precursor containing a protein and a first reactant having two or more first reactive groups capable of reacting with the protein to form a bond are used. The first step of reacting to obtain an intermediate, and the intermediate and a second reactant having one second reactive group capable of reacting with the first reactive group to form a bond. The present invention comprises a second step of reacting to obtain a molded product. The molded product in the second method may be, for example, a non-woven fabric containing a protein, a fiber containing a protein (protein fiber), or a predetermined protein crosslinked product itself.

The first step is a step of reacting the protein-containing molded product precursor with the first reactant. The first reactant is a polyfunctional reactant having two or more first reactive groups capable of reacting with the protein to form a bond, and in the first step, the protein is crosslinked by the first reactant. May be done.

The protein has at least one reactive functional group from the group consisting of an amide group, a hydroxyl group, a phenolic hydroxyl group, an amino group, a carboxyl group, a thiol group, a selenol group, an imidazolyl group, an indolyl group and a guanidino group. The first reactive group contained in the first reactant may be a group capable of reacting with the reactive functional group to form a bond.

As the first reactive group, an electrophilic group is preferable. When the first reactive group is an electrophilic group, a bond can be easily formed by an addition reaction with the reactive functional group of the protein.

Examples of the first reactive group which is an electrophilic group include the following formulas (A-1), (A-2), (A-3), (A-4), (A-5) or ( The group represented by A-6) is preferable. The wavy line in each equation indicates the bond of each group.

In formula (A-1), X ¹ represents an oxygen atom (O) or a sulfur atom (S). As X ¹ , an oxygen atom is more preferable.

In formula (A-3), X ² represents a leaving group. The leaving group is not particularly limited as long as it is a group capable of a nucleophilic substitution reaction by a reactive functional group of a protein. Examples of the leaving group include a halogen atom (fluorine atom (F), chlorine atom (Cl), bromine atom (Br), iodine atom (I)), sulfonic acid ester group (-OSO ₂ R ¹ ), and carboxylic acid. Examples thereof include an ester group (-OCOR ² ) and a quaternary ammonium group (-NR ³ ₃ ). R ¹ may be, for example, a fluorine atom, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group. R ² may be, for example, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group. R ³ may be, for example, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group. R ¹ , R ² and R ³ may have a substituent. Examples of the substituent include an alkyl group, an alkenyl group, an aryl group, a halogen atom and the like.

As X ² , chlorine atom, bromine atom, iodine atom, ester group and sulfonic acid ester group are more preferable, and bromine atom, iodine atom and sulfonic acid ester group are further preferable. Examples of R ¹ include a fluorine atom, an alkyl group (particularly a methyl group, an ethyl group, a benzyl group and an allyl group), a perfluorooloalkyl group (particularly a trifluoromethyl group and a pentafluoroethyl group), and an aryl group (particularly). Phenyl group, trill group, naphthyl group, fluorophenyl group) and the like are more preferable. Examples of R ² include alkyl groups (particularly methyl group, ethyl group, benzyl group, allyl group), perfluorooloalkyl groups (particularly trifluoromethyl group and pentafluoroethyl group), and aryl groups (particularly phenyl group). Trill group, naphthyl group, fluorophenyl group) and the like are more preferable. The R ^3, an alkyl group (particularly, a methyl group, an ethyl group, a benzyl group, an allyl group), an aryl group (especially phenyl group, a tolyl group, a naphthyl group, a fluorophenyl group) are more preferable.

In formula (A-4), X ³ represents an oxygen atom (O), a sulfur atom (S), a group represented by −NR ⁴ −, or a group represented by −C (R ⁵ ) ₂ −. .. R ⁴ may be, for example, a hydrogen atom, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group, an arylsulfonyl group, an alkylsulfonyl group, an acyl group, or a carbamate group. R ⁵ represents an electron attracting group. Examples of the electron-attracting group include a carbonyl group, a cyano group, an aryl group, an alkenyl group, an alkynyl group and the like. Two R ⁵ may be the same or different from each other. R ⁴ and R ⁵ may have a substituent. Examples of the substituent include an alkyl group, an alkenyl group, an aryl group, a halogen atom and the like.

The X ^3, more preferably an oxygen atom. The R ^4, an arylsulfonyl group, an alkylsulfonyl group, an acyl group, such as a carbamate group is more preferable.

In formula (A-5), X ⁴ represents an oxygen atom (O) or a sulfur atom (S), and Y ¹ is a halogen atom, a hydroxyl group, a group represented by -R ⁶ , and represented by -OR ^6. Indicates a group or a group represented by -OCOR ⁶ . R ⁶ may be, for example, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group. R ⁶ may have a substituent. Examples of the substituent include an alkyl group, an alkenyl group, an aryl group, a halogen atom and the like.

As X ⁴ , an oxygen atom is more preferable. As Y ¹ , a halogen atom, a group represented by −OR ⁶ or a group represented by −OCOR ⁶ and the like are more preferable. As R ⁶ , an alkyl group, an aryl group and the like are more preferable.

In formula (A-6), X ⁵ represents an oxygen atom (O) or a sulfur atom (S), and Y ² represents a group represented by an oxygen atom (O), a sulfur atom (S) or NR ⁷ . R ⁷ may be, for example, an alkylsulfonyl group, an arylsulfonyl group, an acyl group, a carbamate group, an alkyl group, an aryl group, an alkyl halide group or an aryl halide group. R ⁷ may have a substituent. Examples of the substituent include an alkyl group, an alkenyl group, an aryl group, a halogen atom and the like.

As X ⁵ , an oxygen atom is more preferable. As Y ² , an oxygen atom is more preferable. As R ⁷ , an alkylsulfonyl group, an arylsulfonyl group, an acyl group, a carbamate group and the like are more preferable.

The first reactant may be a compound having two or more first reactive groups. The number of the first reactive groups contained in the first reactant is not particularly limited and may be, for example, 2 to 10000, preferably 2 to 1000.

The first step may be carried out, for example, by bringing the first reaction solution containing the first reactant and the molded product precursor into contact with each other and heating them.

The first reaction solution may be solvent-free and may further contain a solvent. The solvent of the first reaction solution is not particularly limited as long as it can dissolve the first reactant and does not inhibit the reaction between the reactive functional group of the protein and the first reactive group. Just do it. When the first reactive group is an electrophilic group, the solvent of the first reaction solution may be, for example, N, N-dimethylacetamide, N, N-dimethylformamide, N-methylpyrrolidone, benzene, toluene, etc. Xylene, mesitylene, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, butyl acetate, propylene glycol monomethyl ether acetate and the like can be preferably used.

The reaction conditions in the first step are not particularly limited as long as they are conditions in which the reactive functional group of the protein reacts with the first reactive group.

In the first step, it is preferred that at least a portion of the first reactant crosslinks the protein and at least a portion of the first reactive group remains in the intermediate. That is, in the first step, it is preferable to obtain an intermediate containing a protein cross-linked by the first reactant and having the first reactive group.

For example, when the first reactant is a compound having two first reactive groups, some of the first reactants crosslink the protein (ie, both of the first reactive groups are with the protein). (Reaction) may be done. Also, the other part of the first reactive group may react with the protein on only one of the first reactive groups, at which time the other first reactive group is unreacted. It may remain in the intermediate.

Also, for example, when the first reactant is a compound having three or more first reactive groups, some of the first reactants crosslink the protein with all the first reactive groups (ie,). , All of the first reactive groups may react with the protein), and in some other parts of the first reactant, some first reactive groups react with the protein and some others The first reactive group of is unreacted and may remain in the intermediate.

The reaction in the first step can also be said to be a reaction in which the reaction agent forms a crosslinked structure or a side chain having the first reactive group starting from the reactive functional group of the protein. The amount of crosslinked structure and side chains can be adjusted by the amount of the first reactant used in the reaction. By reducing the amount of the first reactant used, there is a tendency for fewer side chains and more crosslinked structures to be formed, and by increasing the number of routes for using the first reactant, there are fewer crosslinked structures and sides. Many chains tend to be formed.

The crosslinked structure and the amount of side chains can be adjusted, for example, by having the second reactant as a part of the first reactive group contained in the first reactant prior to carrying out the first step. It can also be realized by performing a pre-step of reacting the second reactive group. By appropriately adjusting the amount of the second reactant used in the previous step with respect to the first reactant, the first reactivity remaining without reacting with the second reactive group in the previous step. You can control the number of groups. Thereby, in the first step, the amount of the first reactive group bound to the protein can be easily adjusted, and as a result, the crosslinked structure of the protein and the amount of side chains can be easily controlled. Will be able to.

That is, in the second method, a part of the first reactant and a part of the second reactant are reacted before the first step, and the first reaction possessed by the first reactant is carried out. A pre-step of reacting a part of the sex group with a part of the second reactive group contained in the second reactant may be further provided.

By forming a large number of crosslinked structures, the water resistance of the molded product (for example, the property of suppressing the amount of shrinkage due to contact with moisture, the property of suppressing the amount of shrinkage during drying after contact with moisture, etc.), the machine Target strength, heat resistance, etc. tend to be improved, and by forming a large number of side chains, the functionality imparted to the molded product (for example, the texture described later) tends to be improved. In the second method, the crosslinked structure and the proportion of side chains may be appropriately adjusted according to the desired properties.

In the first step, an intermediate containing a reactant of the protein and the first reactant is obtained. In the reactant, the protein is crosslinked by the first reactant and the unreacted first reactive group may remain. That is, the above-mentioned reactant contains a polypeptide skeleton derived from a protein, a cross-linked portion that crosslinks the polypeptide skeleton, and a side chain portion that binds to the polypeptide skeleton and has a first reactive group at the end. You can do it.

The second step is a step of reacting the intermediate obtained in the first step with the second reactant. The second reactant has one second reactive group capable of reacting with the first reactive group to form a bond. The second step can also be said to be a step of reacting the first reactive group remaining in the intermediate with the second reactant.

The second reactive group contained in the second reactant is not particularly limited, and may be appropriately changed depending on the type of the first reactive group. For example, when the first reactive group is an electrophilic group, the second reactive group is preferably a nucleophilic group.

Examples of the second reactive group which is a nucleophilic group include a hydroxyl group, a thiol group, an amino group, and a group represented by the following formula (B-1).

In formula (B-1), X ⁶ represents an oxygen atom (O) or a sulfur atom (S).

Examples of the group represented by the formula (B-1) include a group represented by the following formula (B-1-1).

In formula (B-1-1), Y ³ represents a monovalent group. Y ³ may be, for example, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkoxy group, an alkyl sulfide group, an aryl sulfide group, a monosubstituted amino group, a disubstituted amino group and the like, and preferably an alkyl group and an aryl group. A group, an alkoxy group, and a monosubstituted amino group. Y ³ may have a substituent. Examples of the substituent include an alkyl group, an alkenyl group, an aryl group, a halogen atom and the like.

The second reactant may be a compound having one second reactive group and is inactive in the reaction of the second step (reaction between the first reactive group and the second reactive group). It may further have a functional group. According to such a second reactant, the functional group can be easily introduced into the molded product starting from the unreacted first reactive group in the intermediate.

The functional group is not particularly limited, and may be a group that directly imparts functionality to the molded product, or may be a group that imparts reactivity with a further reactant to the molded product.

Examples of the functional group include a hydrocarbon group such as an alkyl group, an alkenyl group and an alkynyl group; a group having a ring structure such as an aryl group and a heterocyclic group; and a reactive group protected by a protective group (hydroxy group and amino). Group, thiol group, etc.); carbonyl group (-C (= O)-), ether bond (-O-), amide bond (> NC (= O)-), urethane bond (> NC (= O) O-) ), Urea bond (> N (C = O) N <) group having a structure such as carbonate bond (-OC (= O) O-); alkoxysilyl group, sulfonyl group (-S (= O)-), Examples thereof include a carboxyl group (-C (= O) OH), a sulfonic acid group (-S (= O) ₂ OH), and a quaternary ammonium group.

For example, when the functional group is an alkyl group, the texture of the molded product is improved. Therefore, for example, when the molded product is fibrous or cloth-like, a material having an excellent texture and a good texture can be obtained by using a second reactant having an alkyl group as a functional group.

When the water resistance and strength of the conventional protein material are improved by cross-linking, the texture of the material deteriorates and it may be difficult to use it for applications that come into contact with human skin. However, according to the second method, an excellent texture and texture can be obtained by the functional group while maintaining the water resistance and mechanical strength due to cross-linking, and a material suitable for use in contact with human skin can be obtained. be able to.

The second step may be carried out, for example, by bringing the second reaction solution containing the second reactant and the intermediate into contact with each other and heating them.

The second reaction solution may be solvent-free and may further contain a solvent. The solvent of the second reaction solution is not particularly limited as long as it can dissolve the second reactant and does not inhibit the reaction between the first reactive group and the second reactive group. Just do it. When the first reactive group is an electrophilic group and the second reactive group is a nucleophilic group, the solvent of the second reaction solution is, for example, N, N-dimethylacetamide, N, N-. Dimethylformamide, N-methylpyrrolidone, benzene, toluene, xylene, mesitylene, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, butyl acetate, propylene glycol monomethyl ether acetate and the like can be preferably used.

In the second step, the amount of the second reactant used is not particularly limited. The amount of the second reactant used may be, for example, greater than the amount of the first reactive group in the intermediate.

In the second step, some or all of the first reactive groups in the intermediate are consumed by reacting with the second reactive groups. It is desirable that the first reactive group does not remain in the molded product as much as possible. For this reason, in the second method, it is preferable that all of the first reactive groups are consumed by the reaction with the second reactive group or other side reactions.

By the second step, a molded product containing a protein crosslinked product is obtained.

In the description of the second method, the first residue shows the remaining structure of the first reactant minus the first reactive group. In addition, the second residue shows the remaining structure obtained by removing the second reactive group from the second reactant.

The polypeptide skeleton and the first residue are bonds formed by the reaction of the reactive functional group and the first reactive group of the protein (for example, the reactive functional group is an amino group and the first reactive group is. In the case of an isocyanate group, it is bonded by a urea bond). Further, the first residue and the second residue are bonds formed by the reaction of the first reactive group and the second reactive group (for example, the first reactive group is an isocyanate group and the second residue is the first. When the second reactive group is a hydroxyl group, it is bonded by a urethane bond).

In the second method, the protein is crosslinked by the first reactant to obtain a molded product having excellent water resistance, mechanical strength, heat resistance and the like. Further, in the second method, a functional group can be imparted by a second reactant starting from the first reactive group remaining in the intermediate, so that a molded product having various functions can be easily obtained. Can be done.

The modified hydroxyl group-containing polymer in the third method is a polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer. The modified hydroxyl group-containing polymer can be obtained, for example, by reacting the hydroxyl group-containing polymer with a reactant having a functional functional group.

The hydroxyl group-containing polymer can be used without particular limitation as long as it is a polymer compound having a hydroxyl group. Specific examples of the hydroxyl group-containing polymer include polysaccharides such as starch, glycogen, cellulose, chitin, agarose, hyaluronic acid, chondroitin sulfate, pectin and carrageenan, and synthetic polymers such as polyvinyl alcohol (PVA) and phenol resin. Can be mentioned.

As the hydroxyl group-containing polymer, a polysaccharide is preferable from the viewpoint of having biodegradability. Further, as the hydroxyl group-containing polymer, starch is preferable from the viewpoint of having high biodegradability and high solubility.

The functional functional group is a functional group having characteristics (for example, hydrophobicity, hydrophilicity) corresponding to the functionality to be imparted (for example, water resistance, hydrophilicity, lipophilicity, oil resistance), and the functionality to be imparted. It can be appropriately selected according to the above.

For example, when it is desired to improve water resistance, the functional functional groups include, for example, an alkyl group such as a methyl group, an ethyl group, an n-propyl group and an isopropyl group, an aromatic group such as a phenyl group and a naphthyl group, and an acetyl group. , Hydrophobic functional groups such as acyl groups such as propanoyl groups and benzoyl groups can be used.

A reactant having a functional functional group is a compound having a functional functional group and further having a binding functional group capable of binding to a hydroxyl group-containing polymer. The binding functional group may be a functional group that can be bonded to the hydroxyl group-containing polymer by a hydrogen bond or a covalent bond, but is preferably a functional group that can be covalently bonded to the hydroxyl group-containing polymer, and is contained in the hydroxyl group-containing polymer. It is more preferable that the functional group can be covalently bonded to the hydroxyl group of the above.

Examples of the reactant having a functional functional group include isocyanate having a functional functional group (RN = C = O: R is a functional functional group) and an acid anhydride (RC (= O) -O). -C (= O) -R: R is a functional functional group), epoxides, aziridines, alkyl halides and the like. As the reactant having a functional functional group, isocyanate and anhydrous acetic acid having a functional functional group are preferable because they can be covalently bonded to the hydroxyl group in the hydroxyl group-containing polymer, and any functional functional group is further preferable. Is more preferable because it can be introduced into an isocyanate having a functional functional group.

In the third method, for example, a non-woven fabric having added functionality can be obtained by producing a non-woven fabric using a mixture of a raw material fiber and a modified hydroxyl group-containing polymer. In the third method, for example, a spinning raw material mixed with a modified hydroxyl group-containing polymer is spun to obtain a raw material fiber containing the modified hydroxyl group-containing polymer, and then the nonwoven fabric is produced using the raw material fiber. As a result, a non-woven fabric to which functionality is imparted can be obtained.

The content of the modified hydroxyl group-containing polymer contained in the non-woven fabric is not particularly limited, and may be appropriately set according to the functionality to be imparted, the type of fiber contained in the non-woven fabric, and the like. The content of the modified hydroxyl group-containing polymer may be, for example, 0.001 to 70% by mass, 0.01 to 65% by mass, or 0.1 to 60% by mass based on the total amount of the non-woven fabric. It may be there.

When the non-woven fabric contains protein fibers, it is preferable that the modified hydroxyl group-containing polymer and the protein fibers are hydrogen-bonded. This further improves functionality. The hydrogen bond is, for example, a functional group in a modified hydroxyl group-containing polymer (for example, a hydroxyl group, a functional group, a functional group in a binding functional group, etc.) and a functional group in a protein (for example,). , Amino group, carboxyl group, etc.) can be formed.

In the third method, the non-woven fabric may further contain a hydroxyl group-containing polymer. The hydroxyl group-containing polymer is preferably a polymer of the same type as the hydroxyl group-containing polymer that is the raw material of the modified hydroxyl group-containing polymer. When the non-woven fabric contains a hydroxyl group-containing polymer, the content of the hydroxyl group-containing polymer may be 50% by mass or more, and 60% by mass, based on 100% by mass of the total amount of the modified hydroxyl group-containing polymer and the hydroxyl group-containing polymer. % Or more, 70% by mass or more, or 80% by mass or more. The upper limit may be 90% by mass or less.

(Polymer substance)
The polymer substance is generally impregnated in a non-woven fabric in a solution state, solidified (solidified) by desolvation, and exists in a state of being fixed to the fibers in the gaps between the fibers of the non-woven fabric. That is, the polymer substance forms a base layer of synthetic leather together with the non-woven fabric as a so-called impregnated body integrated with the non-woven fabric, and the fibers of the non-woven fabric are connected to each other to maintain the shape of the base layer. Moreover, a predetermined strength is imparted to the base material layer. Examples of such polymer substances include synthetic resins such as polyvinyl chloride, polyolefin, polystyrene, polyurethane, polyester resin, epoxy resin, acrylic polymer, and acrylonitrile polymer, and proteins. Examples of the polyurethane include polyether polyurethane, polyester polyurethane, polycarbonate 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 with a polyol to synthesize a prepolymer, and then reacting the prepolymer with a chain extender. The diisocyanate may be an aromatic compound or an aliphatic compound. The polyol may be, for example, polyester, polyether, polycarbonate, silicone, fluororesin or the like. When a polyether is used as the polyol, it is excellent in hydrolysis resistance, mold resistance and cold resistance, and when a polycarbonate is used as the polyol, it is excellent in hydrolysis resistance and mold resistance. The chain extender may be, for example, glycol, diamine, reaction terminator or the like. The polymer substance preferably contains a protein. As the protein, it is preferable to contain modified fibroin, and it is more preferable to contain modified spider silk fibroin. By containing the modified fibroin (preferably modified spider silk fibroin), the base material layer can be imparted with heat retention, hygroscopic heat generation and / or flame retardant functionality. As a result, the synthetic leather according to the present embodiment can be imparted with heat retention, hygroscopic heat generation and / or flame retardant functionality, and its value as a material becomes higher. Preferred embodiments of modified fibroin will be described later.

As a method of imparting functionality to the polymer substance (layer formed of the polymer substance), for example, a method of incorporating the modified fibroin into the polymer substance (layer formed of the polymer substance) (first method). ), A method in which a polymer substance (layer formed of a polymer substance) contains a protein containing a predetermined protein crosslinked body (second method), and a polymer substance (layer formed of a polymer substance). Examples thereof include a method (third method) of incorporating a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer. Specific embodiments of the first method, the second method and the third method are as described above.

The base material layer may contain a knitted body in addition to the non-woven fabric and the polymer substance. The knitted body is a general term for knitted fabrics and woven fabrics. The knitted fabric is a knitted fabric having a weft knitting structure such as a horizontal knitting or a circular knitting (also simply referred to as a "weft knitting fabric"), or a knitted fabric having a warp knitting structure such as a tricot or Russell (also simply called a "warp knitted fabric"). It may be any of.). The woven fabric may be a woven fabric having any of a plain weave, a twill weave, and a satin weave. The knitted body may be an unprocessed knitted body itself obtained by knitting or weaving, or may be a knitted body that has been knitted or woven and then subjected to a process such as water repellent treatment.

The braided body can be obtained by knitting or weaving a raw material yarn. A known method 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 and the like can be used, and from the viewpoint of productivity, the use of a circular knitting machine is preferable. Examples of the flat knitting machine include a molding knitting machine and a non-sewn knitting machine, but it is more preferable to use a non-sewn knitting machine because the knitted fabric can be manufactured in the form of a final product. Examples of the loom used include a shuttle loom and a non-shuttle loom such as a gripper loom, a rapier loom, and an air jet loom.

The raw material yarn may be a single yarn, a composite yarn (for example, a blended yarn, a mixed fiber yarn, a covering yarn, etc.), or a combination thereof may be used. The single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or filament yarns in which long fibers are twisted. Examples of the fibers contained in the raw material 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 braided body other than the non-woven fabric and the polymer substance, the knitted body may be included in the base material layer as, for example, a non-woven fabric bonded to both sides thereof and integrated.

The thickness of the base material 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 base material layer may have a maximum heat absorption and heat generation degree of 0.025 ° C./g, which is determined according to the following formula A.
Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)
In the formula A, the low humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 40%, and the high humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 90%.

The base material layer may have a maximum heat absorption and heat generation rate of 0.026 ° C./g or higher, 0.027 ° C./g or higher, 0.028 ° C./g or higher, and 0. It may be .029 ° C./g or higher, 0.030 ° C./g or higher, 0.035 ° C./g or higher, or 0.040 ° C./g or higher. .. The upper limit of the maximum heat absorption and heat generation is not particularly limited, but is usually 0.060 ° C./g or less.

The base material layer has a critical oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, or 26 or more. It may be 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more or 33 or more. You may. In this specification, the LOI value is a value measured in accordance with the test method of powdery granules or synthetic resin having a low melting point on May 31, 1995, Fire and Disaster Management Agency Dangerous Goods Regulation Division Chief Fire Danger No. 50.

The base material layer may have a heat retention index of more than 0.18 determined according to the following formula B.
Formula B: Heat retention index = heat retention rate (%) / sample basis weight (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 testing machine (under windward of 30 cm / sec), and is measured by the method described in 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, 0.28 or more. It may be 0.30 or more, and it may be 0.32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

In other embodiments, the synthetic leather may include a layer other than the substrate layer. FIG. 2 is a schematic cross-sectional view of synthetic leather according to another embodiment. The synthetic leather 20 shown in FIG. 2 includes a base material layer 3 containing a non-woven fabric 1 and a polymer substance 2, and an adhesive layer 4 (first adhesive layer) laminated on one surface of the base material layer 3. A skin layer 5 (first skin layer) laminated on the surface opposite to the surface on which the base material layer 3 of the adhesive layer 4 (first adhesive layer) is laminated is provided. In still another embodiment, the synthetic leather comprises a base material layer 3, an adhesive layer 4 (first adhesive layer) laminated on one surface of the base material layer 3, and an adhesive layer 4 ( The skin layer 5 (first skin layer) laminated on the surface opposite to the surface on which the base material layer 3 of the first adhesive layer) is laminated, and the adhesive layer 4 (first) of the base material layer 3. The opposite of the surface on which the adhesive layer (second adhesive layer) laminated on the surface opposite to the surface on which the adhesive layer) is laminated and the surface on which the base material layer 3 of the second adhesive layer is laminated. It may include an epidermis layer (second epidermis 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 have to be interposed between the two layers 3 and 5. Such synthetic leather is preferably used for clothing or shoes.

In another embodiment, the synthetic leather may include a base material layer and a polyurethane microporous coating layer (first microporous coating layer) laminated on one surface of the base material layer. .. Further, in synthetic leather, a base material layer, a polyurethane microporous coating layer (first microporous coating layer) laminated on one surface of the base material layer, and a first microporous coating layer are laminated. A polyurethane microporous coating layer (second microporous coating layer) laminated on the surface opposite to the surface to which the surface is formed may be provided. The polyurethane microporous coating layer refers to a layer having a microporous structure containing polyurethane as a main component. The surface of the polyurethane microporous coating layer is embossed (processed by pressing with a hot roll, etc.), puff suede processed (processed by applying sandpaper, etc.), gravure processed (processed by gravure printing, etc.), etc. The surface may be processed.

In still other embodiments, the synthetic leather may have a brushed / 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. Moreover, such synthetic leather is excellent in breathability.

(protein)
The protein contained in the non-woven fabric and / or the polymer substance is not particularly limited, and any protein can be used. 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 retardancy in addition to waterproof and moisture permeable. When the protein fiber contains modified fibroin (preferably modified spider silk fibroin), the synthetic leather according to the present embodiment can be further imparted with heat retention, heat absorption and heat generation and / or flame retardancy. The value as synthetic leather becomes higher.

The modified fibroin according to the present embodiment has a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein contained. The modified fibroin may further have an amino acid sequence (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 the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be fibroin having the same amino acid sequence as naturally occurring fibroin. “Naturally derived fibroin” as used herein is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m − (A) _n motif. It is a protein containing the domain sequence to be used.

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized product that does not depend on naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence). It may have 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 (A) _n motif of an amino acid sequence) and an amorphous region (typically, REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to.), And is represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the _n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (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). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. A plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying. Substitution, deletion, insertion and / or addition of amino acid residues can be carried out by methods well known to those skilled in the art such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Energy, 100, 448 (1983).

Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Yes, specifically, for example, fibroin produced by insects or arachnids.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, tussah, and tussah. ), Silk moth (Samia cinthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama), silk moth (Antheraea assama), silk moth Hornet silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, the silk moth fibroin L chain (GenBank accession number M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

Examples of fibroin produced by arachnids include spider silk proteins produced by spiders belonging to the order Araneae. More specifically, spiders belonging to the genus Araneus, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Pronus, such as spiders, spiders belonging to the genus Trinofundamashi, such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders, spiders, spiders, spiders, etc. Spiders belonging to the genus Ordgarius, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus), spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora such as spiders, spiders, spiders and spiders, and spiders belonging to the genus Poltys such as spiders. Spider silk proteins produced by spiders belonging to the genus Cyclosa, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Tetragnatha, such as Ashidaka spider and Urokoa shinagagumo, spiders belonging to the genus White spider (genus Leucage), spiders belonging to the genus Leucage, spiders belonging to the genus Leucage, spiders belonging to the genus Leucage Spiders belonging to the genus Azumi (Menosira genus) such as spiders and spiders, spiders belonging to the genus Dyschiriognatha such as Himea shinagamo, spiders belonging to the genus Dyschiriognatha, spiders, spiders, sea urchins, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spider silk proteins produced by spiders belonging to the genus Euprostenops and spiders belonging to the family Tetragnathidae, such as spiders belonging to the genus Euprostenops. Be done. Examples of spider silk proteins include traction thread proteins such as MaSp (MaSp1 and MaSp2) and 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, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (nucleic acid sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (nucleic acid sequence)), dragline silk protein spidroin 1 [from Nephila clavipes] (GenBank sequence No. AAC4011) (Nucleotide sequence) ), U37520 (nucleic acid sequence)), major amplifier spidroin 1 [derived from Latrodictus protein] (GenBank accession number ABR68856 (nucleic acid sequence), EF595246 (nucleic acid sequence)), dragline silk protein (derived from radinic protein) Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major protein spidroin 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleus acid sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (nucleic acid sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC14589.1 (nucleotide sequence)), minor amplifiernepilate clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank accession) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally occurring fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by spiders), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferable because it is more excellent in flame retardancy.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmarker thread protein produced in the large bottle-shaped gland of spiders, and a domain sequence with a reduced content of glycine residues. Modified fibroin with (second modified fibroin), (A) modified fibroin with a domain sequence with reduced _n motif content (third modified fibroin), glycine residue content, and (A) _n Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin with a domain sequence that locally contains a region with a high hydrophobicity index (fifth modified fibroin), and content of glutamine residues. Examples include modified fibroin (sixth modified fibroin) having a reduced domain sequence.

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

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

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C end of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C end of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first modified fibroin, the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLargeNRSH1), or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 and 90 A modified fibroin containing an amino acid sequence having% or more sequence identity can be mentioned. The sequence identity is preferably 95% or more.

The amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of the start codon, the His10 tag and the HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The C-terminal amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in 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 naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP replaced with another amino acid residue as compared with naturally occurring fibroin. ..

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

In the second modified fibroin, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more of the total motif sequence.

The second modified fibroin contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the domain sequence from the (A) _n motif located closest to the C-terminal side from the above domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. The content ratio of the amino acid sequence consisting of GGX in the domain sequence of the second modified fibroin is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.

The method of calculating z / w will be described in more detail. First, in the fibroin (modified fibroin or naturally-derived fibroin) containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m, it is located most on the C-terminal side from the domain sequence (A) _n. The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Further, for example, when X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). is there). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 3, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w in naturally derived fibroin will be described. First, as described above, when the fibroin in which the amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types of arachnid-derived fibroin were extracted) were extracted. Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m among all the extracted fibroins, and the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less. Z / w was calculated from the amino acid sequence of fibroin according to the above calculation method. The result is 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 in naturally-derived fibroin is 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, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue 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. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment 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 modification corresponding to the substitution of 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. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

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

As a more specific example of the second modified fibroin, (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) -Contains an amino acid sequence represented by PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Modified fibroin can be mentioned.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. The amino acid sequence shown by SEQ ID NO: 7 is such that every two (A) _n motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added. It is substituted with a serine (S) residue and a part of the amino acid on the C-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and His tag sequence are added to the C-terminal of the sequence obtained by repeating the above four times.

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

The modified fibroin of (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

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

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).

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

Furthermore, an "epitope tag" utilizing 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 the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. By utilizing the epitope tag, the modified fibroin can be easily purified with high specificity.

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the modified fibroin containing the tag sequence, the amino acids represented by (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples may be modified fibroins comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

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

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

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

The second modified fibroin may contain a secretory 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 third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motif as compared with naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs as compared with naturally occurring fibroin.

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

The third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal C-terminal one to three toward the side of the (A) _n motif every one (A) _n motif It may have an amino acid sequence corresponding to the deletion of.

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

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

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -REP] When the number of amino acid residues in the REP of the unit is sequentially compared and the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 1. When x is the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units, which is 11.3, and y is the total number of amino acid residues in the domain sequence. In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues with respect 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. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 3 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (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 an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side to the C-terminal side so as not to overlap. At this time, there may be an unselected [(A) _n motif-REP] unit. In FIG. 3, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. 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. The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the fourth REP (20 amino acid residues) and the fifth REP (30 amino acid residues), when the fourth REP with a smaller number of amino acid residues is set to 1, the fifth REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 3, a set of [(A) _n motif-REP] units in which the ratio of the number of amino acid residues to the other is 1.8 to 11.3 when the one having the smaller number of amino acid residues is 1. Shown by solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3. The set of [(A) _n motif-REP] units is indicated by a broken line. Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of (A) _n motif. is there.). Then, the total values added are compared, and the total value of the pattern in which the total value is maximized (maximum value of the total value) is defined as x. In the example shown in FIG. 3, the total value of pattern 1 is the maximum.

Next, 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, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

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 of only alanine residues, the x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

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

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

The third modified fibroin, for example, deletes one or more of the sequences encoding the (A) _n motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs so that x / y is 64.2% or more is designed and designed 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. In each case, in addition to the modification corresponding to the deletion of (A) _n motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -Contains an amino acid sequence represented by PRT799) or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Modified fibroin can be mentioned.

The modified fibroin of (3-i) will be described. The amino acid sequence shown by SEQ ID NO: 17 is from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin, every other (A) _n from the N-terminal side to the C-terminal side. The motif is deleted, and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.

The value of x / y in the Giza ratio of 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. It is 1% and 70.0%.

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

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

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other The amino acid residue of two adjacent [(A) _n- motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When the maximum value of the total value obtained by adding the radix is x and the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

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

As a more specific example of the modified fibroin containing the tag sequence, the amino acids represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples may be modified fibroins comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

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

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

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other The maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) _n motif-REP] units in which the ratio of the number of amino acid residues of REP in REP is 1.8 to 11.3 is x. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third modified fibroin may contain a secretory 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 as compared with naturally occurring fibroin. To have. The domain sequence of the fourth modified fibroin lacked at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the above-mentioned 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.

As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Examples thereof include modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth 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.

The fifth modified fibroin had its domain sequence replaced with one or more amino acid residues in the REP compared to naturally occurring fibroin, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.

The region having a large local hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.

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

In the fifth modified fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index as compared with naturally occurring fibroin, and / or one or more amino acid residues in REP. In addition to the modification corresponding to the insertion of an amino acid residue with a high hydrophobicity index, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be a modification of the amino acid sequence corresponding to the above.

The fifth modified fibroin, for example, leaves one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino 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 each case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , from the (A) _n motif located closest to the C-terminal side to the C-terminal of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the above domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more is defined as p. When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic karacter of protein7. B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by Equation 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, the average value of the hydrophobicity index of four consecutive amino acid residues is calculated for all REPs contained in the sequence A. The average value of the hydrophobicity index is obtained by dividing the total 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 consecutive 4 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 hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. The total number of amino acid residues contained in sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are present in an overlapping manner by only one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 6, p is 7 × 4 = because there are seven “consecutive four amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, 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 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, 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, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally occurring fibroin satisfies the above p / q condition. (Amino acid residue with a negative value) is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acid residues are inserted in the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

As a more specific example of the fifth modified fibroin, (5-i) the amino acid sequence set forth in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666). Alternatively, a modified fibroin containing (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.

The modified fibroin of (5-i) will be described. The amino acid sequence shown by SEQ ID NO: 19 consists of 3 amino acid residues every other REP, except for the domain sequence of the terminal 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) is inserted at two places, a part of the glutamine (Q) residue is replaced with a serine (S) residue, and a part of the amino acid on the C-terminal side is deleted. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. is there. The amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.

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

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

As a more specific example of the modified fibroin containing a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv). ) A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences shown in SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

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

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth 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 the domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

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

The fifth modified fibroin may contain a secretory 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 sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.

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

When the sixth modified fibroin contains the GPGXXX motif in the REP, the content of the GPGXXX motif is usually 1% or more, may be 5% or more, and is 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.

In the present specification, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) Fibroin containing a domain sequence represented by _n- motif (modified fibroin or naturally derived) In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The GPGXX motif content 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, "the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is targeted at "the most C-terminal side". The sequence from (A) _n motif to the C end of the domain sequence located in (A) (sequence corresponding to REP) may include a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 7 is a schematic diagram showing the domain sequence of modified fibroin. The calculation method of the GPGXX motif content will be specifically described with reference to FIG. 7. First, in the domain sequence of the modified fibroin shown in FIG. 7 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side”. (A) Since the sequence from the _n motif to the C-terminal of the domain sequence is excluded from the domain sequence ”(the sequence shown by“ region A ”in FIG. 7), s is to be calculated. The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are "sequences obtained by removing the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 7). Since it is contained in (.), The total number t of amino acid residues in all REPs excluding the (A) _n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, which is 21/150 = 14.0% in the case of the modified fibroin of FIG.

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

In the present specification, the "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) fibroin containing a domain sequence represented by _n motif (modified fibroin or naturally derived fibroin) In fibroin), all the sequences from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 7). In the REP, the total number of glutamine residues contained in the region is u, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal 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 the motif. In the calculation of the glutamine residue content, the reason why "the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin corresponds to its domain sequence lacking one or more glutamine residues in the REP or substituting for other amino acid residues as compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M). ) Amino acid residues selected from alanine (A), glutamine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). it 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 preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.

In the sixth modified fibroin, the hydrophobicity of REP is preferably −0.8 or more, more preferably −0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, 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) fibroin containing a domain sequence represented by _n motif (modified fibroin or naturally derived fibroin) In fibroin), all the sequences from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 7). In the REP of, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal 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 of all REPs excluding the _n motif. In the calculation of the hydrophobicity of REP, the reason why "the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the replacement of one or more amino acid residues with another amino acid residue, there may be a modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

The sixth modified fibroin, for example, deletes one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or removes one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to this and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth modified fibroin, (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 SEQ ID NO: A modified fibroin containing the amino acid sequence represented by No. 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. , A modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

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

The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

In the amino acid sequence shown by SEQ ID NO: 32, all the QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) are repeated twice are replaced with VF. And the remaining Q is replaced with I.

In the amino acid sequence (Met-PRT917) shown in SEQ ID NO: 41, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with LI, and the remaining Qs are replaced with V. In the amino acid sequence (Met-PRT1028) shown in SEQ ID NO: 42, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with IF, and the remaining Qs are replaced with T.

The amino acid sequences shown in 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 residual glutamine. The group content is 9% or less (Table 2).

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

The modified fibroins of (6-ii) are in 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 contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-ii) is also a domain represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), Modified fibroin containing the amino acid sequence set forth in 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), or (PRT1028). 6-iv) Amino acid sequences and 90s shown by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. A modified fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned.

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

The modified fibroins of (6-iii) are 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 or SEQ ID NO: 44. It may consist of the indicated amino acid sequence.

The modified fibroins of (6-iv) are 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 or SEQ ID NO: 44. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-iv) is also a domain represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a secretory 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 is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the above-mentioned characteristics.

The modified fibroin may be hydrophilic modified fibroin or hydrophobic modified fibroin. In the present specification, "hydrophilic modified fibroin" is a value obtained by obtaining the total hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. It is a modified fibroin having (average HI) of 0 or less. The hydrophobicity index is as shown in Table 1. Further, the "hydrophobic modified fibroin" is a modified fibroin having an average HI of more than 0. Hydrophilic modified fibroin is particularly excellent in flame retardancy. Hydrophobic modified fibroin is particularly excellent in hygroscopic heat generation and heat retention.

Examples of the hydrophilic modified fibroin include the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, amino acid sequence shown in 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, SEQ ID NO: 18, SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9, amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. Examples include modified fibroins comprising the amino acid sequence set forth in the sequence, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

Examples of the hydrophobic modified fibroin include the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, SEQ ID NO: 35, Examples thereof include modified fibroins containing the amino acid sequences set forth 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.

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

The protein fiber can be obtained, for example, by dissolving the protein in a soluble solvent to prepare a dope solution, and spinning by a known spinning method such as wet spinning, dry spinning, dry wet spinning, or melt spinning. Examples of the solvent capable of dissolving the protein include dimethyl sulfoxide (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.

(Manufacturing method of synthetic leather)
In the method for producing synthetic leather according to the present embodiment, for example, a step of impregnating a non-woven fabric with an impregnating solution containing a polymer substance and then coagulating the polymer substance to form a base material layer (base material layer forming step) is performed. Be prepared. The non-woven fabric, the polymer substance and the base material layer can be applied in the same manner as those described above.

In the manufacturing method according to the present embodiment, only the non-woven fabric may be endowed with functionality (the polymer substance is not endowed with functionality), and only the polymer substance is endowed with functionality. It may be (the non-woven fabric is not imparted with functionality), and both the non-woven fabric and the polymer substance may be imparted with functionality. Among them, it is preferable that at least the non-woven fabric as the main material is provided with functionality.

In the base material layer forming step, for example, the non-woven fabric is immersed in the impregnating liquid or the non-woven fabric is sprayed with the impregnating liquid to bring the non-woven fabric into contact with the impregnating liquid to impregnate the non-woven fabric with the impregnating liquid. It can be carried out by a method (wet method) or the like in which the components in the impregnating liquid are solidified by contacting (for example, dipping) with a liquid for solidification to form a base material layer. Further, depending on the type of solvent (for example, one having a low boiling point), the non-woven fabric impregnated with the impregnating solution as described above is desolvated by volatilizing the solvent of the impregnating solution without contacting the coagulating solution. It is also possible to carry out the base material layer forming step by a method of solidifying the components in the impregnating liquid to form the base material layer or the like.

The solvent used for the impregnating solution can be appropriately selected depending on the type of polymer substance, for example, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, alcohol, or hexafluoroisopropanol (HFIP). Organic solvents such as.

FIG. 8 is a schematic view 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. 8, first, the non-woven fabric 81 is pulled out from the roll on which the non-woven fabric 81 is wound, and the non-woven fabric 81 is immersed in the impregnation tank 82 containing the impregnating liquid. Next, the polymer substance impregnated in the non-woven fabric 81 is coagulated in the coagulation tank 83 containing the coagulation liquid. Then, after washing through a hot water washing tank 84 containing a washing liquid, the synthetic leather is dried by a dryer 85, and the synthetic leather 86 is wound on a roll to produce the synthetic leather.

The type of coagulating liquid can be appropriately selected depending on the types of the non-woven fabric and the polymer substance, and the type of the solvent of the impregnating liquid. For example, when the polymer substance contains polyurethane, for example, N, N-dimethylformamide can be used as the solvent for the impregnating solution, and water (hot water) can be used as the coagulating solution. In addition, for example, when the polymer substance contains a protein, for example, dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP) as a solvent for the impregnating solution, and dissolution promotion thereof. A product to which an inorganic salt is added as an agent, lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, acetone, and water (hot water) can be used as the coagulation liquid. Various additives may be added to the coagulation liquid as needed.

The manufacturing method according to the present embodiment may further include a finishing step (surface grinding, coating, dyeing, etc.) after the base material layer forming step. For example, as a finishing step, a step of applying a skin layer resin on a paper pattern, a step of applying an adhesive resin on the surface of the skin layer resin opposite to the paper pattern, and a skin layer resin of the adhesive resin are A synthetic leather can be produced by a method including a step of stacking a base material layer on the opposite surface and heat-pressing. The synthetic leather produced by such a production method may be the synthetic leather shown in FIG. FIG. 9 is a schematic view 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. 9, first, the paper pattern 91 is pulled out from the roll from which the paper pattern is wound, the skin layer resin (polymer) 92 is applied on the paper pattern 91, and the dryer 93. Dry with. Next, the 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 in the dryer 93. Then, the base material layer 96 is pulled out from the roll around which the base material layer 96 is wound, and is laminated 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 the base material layer 96, the adhesive layer resin 94, the skin layer resin (polymer) 92, and the release paper 91 are laminated in this order is rolled into a roll by heat-bonding with a hot press roll 95. Synthetic leather can be manufactured by winding it up.

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

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, 5 to 30 μm. It may be 20 μm.

It should be noted that the synthetic leather according to the present embodiment can be manufactured discontinuously without using the manufacturing apparatus or the like shown in FIGS. 8 or 9 or by using an apparatus other than those. For example, after immersing the entire non-woven fabric of a predetermined size that has not been wound up in an impregnating liquid in an impregnating tank having an independent structure, the polymer substance impregnated in the non-woven fabric is used or used with a coagulating liquid. Coagulate without. Then, if necessary, the desired synthetic leather can be obtained by washing and drying.

When the non-woven fabric contains protein fibers, the non-woven fabric can be formed by, for example, 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 a dope liquid extruded from the spinneret. Is charged and blown off to the collecting side electrode. At this time, the doping solution is stretched to form fibers. The applied voltage is usually 5 to 100 kV, preferably 10 to 50 kV. The distance between the electrodes is usually 1 to 25 cm, preferably 2 to 20 cm.

FIG. 10 is an explanatory diagram of the electrospinning device 100 according to the embodiment. A voltage is applied by the power supply 35 between the metal base nozzle 33 (supply side electrode) and the metal net 38 (collection side electrode). The doping liquid 32 in the microsyringe 31 is moved in the direction of arrow P using a syringe pump, the doping liquid 32 is pushed out from the metal mouthpiece nozzle 33, and the doping liquid is expanded by electric charge to form a fibrous material 36 into a metal net 38. By accumulating on the surface of the above, a non-woven fabric 39 containing protein fibers can be obtained. The obtained non-woven fabric may then remove the solvent. Examples of the method for desorbing the solvent include drying under reduced pressure or immersion in a desolvation tank.

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

The synthetic leather according to the present embodiment has a maximum moisture absorption and heat generation degree of 0.026 ° C / g or more, 0.027 ° C / g or more, or 0.028 ° C / g or more. It may be 0.029 ° C / g or higher, 0.030 ° C / g or higher, 0.035 ° C / g or higher, or 0.040 ° C / g or higher. There may be. The upper limit of the maximum heat absorption and heat generation is not particularly limited, but is usually 0.060 ° C./g or less.

The synthetic leather according to the present embodiment has a critical oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, 24 or more, 26. It may be more than, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, or It may be more than 33. In this specification, the LOI value is a value measured in accordance with the test method of powdery granules or synthetic resin having a low melting point on May 31, 1995, Fire and Disaster Management Agency Dangerous Goods Regulation Division Chief Fire Danger No. 50.

The synthetic leather according to the present embodiment may have a heat retention index of more than 0.18 obtained according to the following formula B.
Formula B: Heat retention index = heat retention rate (%) / sample basis weight (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 testing machine (under windward of 30 cm / sec), and is measured by the method described in 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. It may be .28 or more, 0.30 or more, and 0.32 or more. The upper limit of the heat retention index is not particularly limited, but may be, for example, 0.60 or less, or 0.40 or less.

(Use of synthetic leather)
The synthetic leather according to the present embodiment can be used for the same purposes as the conventional synthetic leather (for example, synthetic leather composed of synthetic resin). The synthetic leather according to the present embodiment can be used, for example, for clothing, decorative items such as shoes and bags, various covers and furniture, and automobile interior materials.

Hereinafter, the present invention will be described in more detail 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]
Designed a modified fibroin (PRT918) having the amino acid sequence shown by SEQ ID NO: 37, a modified fibroin (PRT966) having the amino acid sequence shown by SEQ ID NO: 40, and a modified fibroin (PRT799) having the amino acid sequence shown by SEQ ID NO: 15. did. Nucleic acids encoding the designed modified fibroin were synthesized. An NdeI site was added to the nucleic acid at the 5'end and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into the protein expression vector pET-22b (+) to obtain an expression vector.

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

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. In addition, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL at 60 ° C. Was stirred with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white agglutinating protein obtained after dialysis was recovered by centrifugation, water was removed by a lyophilizer, and the lyophilized powder was recovered to obtain modified fibroin (PRT918, PRT966 and PRT799).

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

[Test Example 2: Manufacture and evaluation of a base material to which functionality has been imparted]
(Manufacturing Example 1)
The above-mentioned spider fibroin (PRT799) was added to dimethyl sulfoxide (DMSO) to a concentration of 24% by mass, and then LiCl was added as a dissolution accelerator to a concentration of 4.0% by mass. Then, using a shaker, the spider silk fibroin was dissolved over 3 hours to obtain a DMSO solution. Dust and bubbles in the obtained DMSO solution were removed to prepare a doping solution. The solution viscosity of the dope solution was 5000 cP (centipores) at 90 ° C.

Dry-wet spinning was performed using the doping solution obtained as described above and a known dry-wet spinning apparatus to obtain a monofilament made of spider silk fibroin. Here, dry-wet spinning was performed under the following conditions.
Coagulant (methanol) temperature: 5-10 ° C
Stretching ratio: 6 times Drying temperature: 80 ° C

A spun yarn is produced by a known method using the modified spider silk fibroin fiber obtained as described above, and a knitted fabric is knitted by weft knitting using a spun yarn made of this modified spider yarn fibroin fiber and a known knitting machine. (5 cm square) was knitted. The count of the spun yarn made of the modified spider yarn fibroin fiber was 58.1 Nm, and the gauge number of the knitting machine was 18.

(Test Example 2-1)
The knitted fabric obtained in Production Example 1 was immersed in 20 mL of hexanediisoanate (HDI). Next, the knitted fabric impregnated with HDI was sandwiched between aluminum foils and heated at 130 ° C. for 30 minutes. After heating, the knitted fabric was taken out, immersed in 20 mL of butanol (BuOH), and reacted at 100 ° C. for 240 minutes. The test sample after the reaction was washed with THF to obtain a knitted fabric (Test Example 2-1) imparted with functionality.

(Test Example 2-2)
For comparison, the knitted fabric obtained in Production Example 1 was evaluated as the knitted fabric of Test Example 2-2.

(Test Example 2-3)
For further comparison, the knitted fabric obtained in Production Example 1 was immersed in 20 mL of hexanediisoanate (HDI). Next, the knitted fabric impregnated with HDI was sandwiched between aluminum foils and heated at 130 ° C. for 30 minutes. Then, the knitted fabric was washed with THF to obtain a knitted fabric of Test Example 2-3.

The water resistance and texture of the knitted fabrics of Test Examples 2-1 to 3 were evaluated by the following test methods.

<Water resistance evaluation>
A 3 cm square was drawn on the knitted fabric with a pencil and used as an evaluation sample. The evaluation sample was washed in the washing mode "house cleaning" of a Panasonic washing machine (Na-VG1100L). Then, it was dehydrated in the same washing machine for 15 minutes and naturally dried for 120 minutes. The vertical and horizontal lengths of the squares before and after washing were measured, and the shrinkage rates in the vertical and horizontal directions were determined. The same test was performed three times, and the average value of the three times was used as the evaluation result. The results are shown in Table 6.

<Texture evaluation>
The texture of the knitted fabric was evaluated on a three-point scale. The texture of the knitted fabric of Comparative Example 1 was evaluated as the reference (B), the knitted fabric having a better texture was evaluated as A, and the knitted fabric having a rough texture and inferior texture was evaluated as C. The results are shown in Table 6.

The evaluation of Test Example 2-3 was carried out on the knitted fabric using the modified fibroin fiber, but it is understood that the same effect can be obtained on the woven fabric, the non-woven fabric and the like using the modified fibroin fiber.

[Test Example 3: Production and evaluation of protein fibers with added functionality]
(Test Example 3-1)
<Preparation of undiluted spinning solution (doping solution)>
After dissolving 200 mg of starch (manufactured by Wako Pure Chemical Industry Co., Ltd.) in 11400 mg of solvent (dimethyl sulfoxide (DMSO) containing 4 wt% LiCl), 400 mg of phenylisocyanate (manufactured by Tokyo Chemical Industry Co., Ltd.) is added thereto. Then, the reaction was carried out by stirring at 90 ° C. for 4 hours. As a result, the hydroxyl group of the starch and the isocyanate group of the phenyl isocyanate were reacted to obtain a modified starch (modified hydroxyl group-containing polymer) in which the phenyl group (functional functional group) was bonded via a urethane bond. The modified starch had a modification rate (ratio of hydroxyl groups converted to functional functional groups) determined from the charging ratio of 100%.

After cooling the reaction solution to room temperature, 300 mg of the modified fibroin (PRT799) powder obtained above was added to the reaction solution, and the mixture was stirred and dissolved at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. The content of the modified starch in the spinning stock solution is 17% by mass based on the total content of the modified starch and the starch.

<Manufacturing of protein fibers>
The prepared spinning stock solution is filtered at 60 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100 using nitrogen gas from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into a mass% methanol coagulation bath. The discharge temperature was 60 ° C. and the discharge pressure was 0.3 MPa. After coagulation, the obtained raw yarn was wound at a winding speed of 3.00 m / min and air-dried to obtain protein fibers (fibroin fibers).

<Water shrinkage test>
The obtained protein fiber was cut to a length of about 10 cm, and the length (cm) of the thread before immersion in water was measured. The yarn was then immersed in a water bath at 40 ° C. for 1 minute. Then, the yarn was taken out from the water bath, vacuum dried at room temperature for 15 minutes, and then the length of the dried yarn was measured. The water shrinkage rate of the protein fiber was calculated according to the following formula.
Water shrinkage rate (%) = {(length before immersion / length after immersion / drying) -1} x 100

(Test Example 3-2)
<Preparation of undiluted spinning solution (doping solution)>
After dissolving 253 mg of starch (manufactured by Wako Pure Chemical Industries, Ltd.) in 7600 mg of solvent (dimethyl sulfoxide (DMSO) containing 4 wt% LiCl), 147 mg of acetic anhydride (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto. It was added and reacted at 90 ° C. with stirring for 4 hours. As a result, the hydroxyl group of starch and acetic anhydride reacted to obtain modified starch (modified hydroxyl group-containing polymer) to which an acetyl group (functional functional group) was bonded. The modified starch had a modification rate (ratio of hydroxyl groups converted to functional functional groups) determined from the charging ratio of 100%.

After cooling the reaction solution to room temperature, 2000 mg of the modified fibroin (PRT799) powder obtained above was added to the reaction solution, and the mixture was stirred and dissolved at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. The content of the modified starch in the spinning stock solution is 17% by mass based on the total content of the modified starch and the starch.

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Test Example 3-1.

(Test Example 3-3)
<Preparation of undiluted spinning solution (doping solution)>
215 mg of starch (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 7600 mg of a solvent (dimethyl sulfoxide (DMSO) containing 4% by weight of LiCl), and then 185 mg of acetic anhydride (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto. It was added and reacted at 90 ° C. with stirring for 4 hours. As a result, the hydroxyl group of starch and acetic anhydride reacted to obtain modified starch (modified hydroxyl group-containing polymer) to which an acetyl group (functional functional group) was bonded. The modified starch had a modification rate (ratio of hydroxyl groups converted to functional functional groups) determined from the charging ratio of 50%.

After cooling the reaction solution to room temperature, 2000 mg of the modified fibroin (PRT799) powder obtained above was added to the reaction solution, and the mixture was stirred and dissolved at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. The content of the modified starch in the spinning stock solution is 17% by mass based on the total content of the modified starch and the starch.

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Example 3-1.

(Test Example 3-4)
<Preparation of undiluted spinning solution (doping solution)>
After dissolving 128 mg of polyvinyl alcohol (PVA) (manufactured by Wako Pure Chemical Industry Co., Ltd.) in 7600 mg of solvent (dimethyl sulfoxide (DMSO) containing 4% by weight of LiCl), phenyl isocyanate (manufactured by Tokyo Chemical Industry Co., Ltd.) is dissolved therein. ) 272 mg was added, and the mixture was reacted by stirring at 90 ° C. for 4 hours. As a result, the hydroxyl group of PVA and phenylisocyanate reacted to obtain a modified PVA (modified hydroxyl group-containing polymer) in which the phenyl group (functional functional group) was bonded via a urethane bond. The modified PVA had a modification rate (ratio of hydroxyl groups converted into functional functional groups) determined from the charging ratio of 100%.

After cooling the reaction solution to room temperature, 2000 mg of the modified fibroin (PRT799) powder obtained above was added to the reaction solution, and the mixture was stirred and dissolved at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. The content of modified PVA in the spinning stock solution is 17% by mass based on the total content of modified PVA and PVA.

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Example 3-1.

(Test Example 3-5)
<Preparation of undiluted spinning solution (doping solution)>
After dissolving 193 mg of polyvinyl alcohol (PVA) (manufactured by Wako Pure Chemical Industry Co., Ltd.) in 7600 mg of solvent (dimethyl sulfoxide (DMSO) containing 4% by weight of LiCl), phenylisocyanate (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved therein. ) 207 mg was added, and the mixture was reacted by stirring at 90 ° C. for 4 hours. As a result, the hydroxyl group of PVA and phenylisocyanate reacted to obtain a modified PVA (modified hydroxyl group-containing polymer) in which the phenyl group (functional functional group) was bonded via a urethane bond. The modified PVA had a modification rate (ratio of hydroxyl groups converted to functional functional groups) determined from the charging ratio of 50%.

After cooling the reaction solution to room temperature, 2000 mg of the modified fibroin (PRT799) powder obtained above was added to the reaction solution, and the mixture was stirred and dissolved at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. The content of modified PVA in the spinning stock solution is 17% by mass based on the total content of modified PVA and PVA.

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Example 3-1.

(Test Example 3-6)
<Preparation of undiluted spinning solution (doping solution)>
1200 mg of the modified fibroin (PRT799) powder obtained above was added to a solvent (dimethyl sulfoxide (DMSO) containing 4% by weight of LiCl) and dissolved by stirring at 90 ° C. for 12 hours to obtain a transparent spinning stock solution. ..

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Test Example 3-1 for comparison.

(Test Example 3-7)
<Preparation of undiluted spinning solution (doping solution)>
3000 mg of the modified fibroin (PRT799) powder obtained above and 600 mg of starch (manufactured by Wako Pure Chemical Industries, Ltd.) were added to a solvent (dimethyl sulfoxide (DMSO) containing 4% by weight of LiCl) at 90 ° C. for 12 hours. The mixture was stirred and dissolved to obtain a transparent undiluted spinning solution.

<Protein fiber production and water shrinkage test>
Using the prepared spinning stock solution, protein fiber production and a water shrinkage test were carried out in the same procedure as in Test Example 3-1 for comparison.

The results are shown in FIGS. 11 and 7.

Protein fibers are prepared using a spinning stock solution containing a modified hydroxyl group-containing polymer (modified starch or modified PVA) to which a hydrophobic functional group (phenyl group or acetyl group) is bonded as a functional functional group and a protein (modified fibroin). By production (Test Examples 3-1 to 3-5), when a spinning stock solution containing only protein is used (Test Example 3-6), and a spinning stock solution containing a protein and an unmodified hydroxyl group-containing polymer. (Test Example 3-7), the water shrinkage rate was reduced, and a protein fiber having water resistance was obtained. By producing a non-woven fabric using these protein fibers, a non-woven fabric having functionality (water resistance) can be obtained. Further, these things can be obtained by producing a synthetic leather having a base material layer containing the non-woven fabric and a polymer substance using such a non-woven fabric, and thereby having functional (water resistance). It clearly shows that. Further, as the impregnating liquid impregnated in the non-woven fabric, a modified hydroxyl group-containing polymer (modified starch or modified PVA) to which a hydrophobic functional group (phenyl group or acetyl group) is bonded as a functional functional group, and a protein (modified fibroin) It is clear from the above that a synthetic leather having functionality (water resistance) can also be obtained by using an impregnating solution containing a predetermined solvent.

[Test Example 4: Production of modified fibroin fiber and evaluation of flame retardancy]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and a freeze-dried powder of modified fibroin (PRT799) was added thereto to a concentration of 24% by mass, and a shaker was added. It was used and dissolved for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 90 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100% by mass methanol solidified from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 90 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fibers.

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

The flammability test was based on the Fire and Disaster Management Agency Dangerous Goods Regulation Division Chief Fire Danger No. 50, the test method for powdered or low melting point synthetic resins on May 31, 1995. The test was carried out under the conditions of a temperature of 22 ° C., a relative humidity of 45% and an atmospheric pressure of 1021 hPa. Table 8 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).

As a result of the flame retardancy test, the critical oxygen index (LOI) value of the modified fibroin (PRT799) fiber was 27.2. Generally, if the LOI value is 26 or more, it is considered to be flame-retardant. It can be seen that the modified fibroin fiber is excellent in flame retardancy. By producing a non-woven fabric using fibers containing modified fibroin, a non-woven fabric having functionality (flame retardancy) can be obtained.

[Test Example 5: Production of modified fibroin fiber and evaluation of heat absorption and heat generation]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and lyophilized powder of modified fibroin was added thereto to a concentration of 24% by mass, and a shaker was used. It was dissolved for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 60 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100% by mass methanol solidified from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fibers.

For comparison, commercially available wool fibers, cotton fibers, tencel fibers, rayon fibers and polyester fibers were prepared as fibers.

Each fiber was used to produce an evaluation knitted fabric by weft knitting using a flat knitting machine. The evaluation knitted fabric using PRT918 fiber as the fiber had a thickness of 1/30 N (hair count single yarn) and a gauge number of 18. The evaluation knitted fabric using PRT799 fiber as the fiber had a thickness of 1/30 N (hair count single yarn) and a gauge number of 16. The thickness and the number of gauges of the evaluation knitted fabric using the other fibers were adjusted so as to have substantially the same coverage factor as the evaluation knitted fabric using the PRT918 fiber and the PRT799 fiber. Specifically, it is as follows.
Wool thickness: 2 / 30N (double yarn), gauge number: 14
Cotton thickness: 2 / 34N (double thread), gauge number: 14
Tencel thickness: 2 / 30N (double thread), number of gauges: 15
Rayon thickness: 1 / 38N (single thread), number of gauges: 14
Polyester Thickness: 1 / 60N (single thread), Gauge number: 14

Two evaluation knitted fabrics cut into 10 cm × 10 cm were combined, and the four sides were sewn together to form a test piece (sample). The test piece is left in a low humidity environment (temperature 20 ± 2 ° C., relative humidity 40 ± 5%) for 4 hours or more, and then transferred to a high humidity environment (temperature 20 ± 2 ° C., relative humidity 90 ± 5%). The temperature was measured at 1-minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.

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

FIG. 12 is a graph showing an example of the results of the hygroscopic heat generation test. The horizontal axis of the graph is 0 when the sample is moved from the low humidity environment to the high humidity environment, and indicates the leaving time (minutes) in the high humidity environment. The vertical axis of the graph shows 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 value of the sample temperature.

Table 9 shows the calculation results of the maximum heat absorption and heat generation.

As shown in Table 9, the modified fibroin (PRT918 and PRT799) fibers had a higher maximum hygroscopic heat generation rate and excellent hygroscopic heat generation properties as compared with the existing fiber materials. It can be seen that the modified fibroin fiber is excellent in hygroscopic heat generation. By producing a non-woven fabric using fibers containing modified fibroin, a non-woven fabric having functionality (moisture absorption and heat generation) can be obtained.

[Test Example 6: Production of modified fibroin fiber and evaluation of heat retention]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and lyophilized powder of modified fibroin was added thereto to a concentration of 24% by mass, and a shaker was used. It was dissolved for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 60 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100% by mass methanol solidified from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fibers.

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

Each raw material fiber was used to produce an evaluation knitted fabric by weft knitting using a flat knitting machine. The evaluation knitted fabric using PRT966 fiber as the fiber had a count of 30 Nm, a number of twists of 1, a gauge of 18 GG, and a basis weight of 90.1 g / m ² . The evaluation knitted fabric using PRT799 fiber as the fiber had a count of 30 Nm, a number of twists of 1, a gauge number of GG: 16, and a basis weight of 111.0 g / m ² . The thickness and the number of gauges of the evaluation knitted fabric using the other fibers were adjusted so as to have substantially the same coverage factor as the evaluation knitted fabric using the PRT966 fiber and the PRT799 fiber. Specifically, it is as follows.
Wool count: 30 Nm, number of twists: 2, number of gauges: 14 GG, basis weight: 242.6 g / m ²
Silk count: 60 Nm, number of twists: 2, number of gauges: 14 GG, basis weight: 225.2 g / m ²
Cotton count: 34 Nm, number of twists: 2, number of gauges: 14 GG, basis weight: 194.1 g / m ²
Rayon count: 38 Nm, number of twists: 1, number of gauges: 14 GG, basis weight: 181.8 g / m ²
Polyester count: 60 Nm, number of twists: 1, number of gauges: 14 GG, basis weight: 184.7 g / m ²

The heat retention was evaluated by using a KES-F7 Thermolab II tester manufactured by Kato Tech Co., Ltd. and using a dry contact method (a method assuming direct contact between the skin and clothes in a dry state). One knitted fabric cut into 20 cm × 20 cm was used as a test piece (sample). The test piece was set on a hot plate set at a constant temperature (30 ° C.), and the amount of heat (a) dissipated through the test piece was determined under the condition of a wind speed of 30 cm / sec in the wind tunnel. The amount of heat (b) dissipated under the same conditions as described 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 result, the heat retention index was obtained according to the following formula B.
Formula B: Heat retention index = heat retention rate (%) / sample basis weight (g / m ² )

The calculation results of the heat retention index are shown in Table 10. The higher the heat retention index, the more excellent the heat retention material can be evaluated.

As shown in Table 10, the modified fibroin (PRT966 and PRT799) fibers had a high heat retention index and excellent heat retention as compared with the existing fiber materials. It can be seen that the modified fibroin fiber has excellent heat retention. By producing a non-woven fabric using fibers containing modified fibroin, a non-woven fabric having functionality (heat retention) can be obtained.

[Test Example 7: Production of synthetic leather]
Using the non-woven fabric and the impregnating liquid shown in Table 11, synthetic leather 1-1 to 1-4 composed of only the non-woven fabric and the base material layer of the polymer substance was produced as follows. In addition, synthetic leather 1-5 formed by laminating a skin layer on the surface of a base material layer via an adhesive layer was produced as follows. Details of these manufacturing methods are shown below. In Table 11, the non-woven fabric 1 (modified fibroin non-woven fabric) is a non-woven fabric produced from fibers obtained by spinning with PRT966 as described below. The non-woven fabric 2 (PET non-woven fabric) is a non-woven fabric produced by the same method as the non-woven fabric 1 using commercially available polyethylene terephthalate fibers. The composition of the impregnating solution 1 (modified fibroin impregnating solution) and the impregnating solution 2 (polyurethane (PU) impregnating solution) is as follows.
Impregnated solution 1 (modified fibroin impregnated solution): Contains 5% by mass PRT799, and the solvent is 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), and the solvent is a mixed solvent of MP-865PS (manufactured by DIC Corporation) / DMF (1/5 (w / w)). ..

<Manufacturing of synthetic leather 1-1>
(Manufacturing of non-woven fabric 1)
A lyophilized powder of modified fibroin (PRT966) was added to formic acid to a concentration of 28% by weight and dissolved using a shaker for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution). The obtained modified spider silk fibroin solution was used as a dope solution (spinning stock solution), and modified spider silk fibroin fibers were produced by dry-wet spinning using a known dry-wet spinning apparatus. Next, the obtained modified fibroin fiber was cut into short fibers of 50 mm, and then a sheet (web) was obtained by a known card treatment. Then, the obtained sheet was punched with a known needle punching machine to produce a nonwoven fabric 1 made of modified fibroin fibers. The basis weight (fiber density) of the non-woven fabric 1 was 100 to 200 g / m ² . The conditions for dry-wet spinning are as follows.
Coagulation bath temperature: 2 to 15 ° C
Total draw ratio: 1 to 4 times Drying temperature: 100 ° C

(Manufacturing of synthetic leather 1-1)
The non-woven fabric 1 obtained as described above was impregnated with the above-mentioned impregnating liquid 1 so that the pickup rate was 400%. Then, the non-woven fabric 1 impregnated with the impregnating solution 1 was immersed in water for 15 minutes to remove the solvent from the impregnating solution 1 to solidify the modified fibroin in the impregnating solution 1. Next, the non-woven fabric 1 was taken out of water and then dried at room temperature. As a result, synthetic leather 1-1 (consisting only of the base material layer) composed of the modified fibroin fiber non-woven fabric and the polymer substance composed of the modified fibroin impregnated and integrated therein was produced.

<Manufacturing of synthetic leather 1-2>
(Manufacturing of non-woven fabric 2)
A commercially available polyethylene terephthalate fiber was cut into short fibers of 50 mm, and then a sheet was obtained by card treatment. Then, using needle punching, a non-woven fabric 2 having a basis weight of 100 to 200 g / m ^{2 made} of polyethylene terephthalate fiber was produced by a known needle punching method.

(Manufacturing of synthetic leather 1-2)
The non-woven fabric 2 obtained as described above was impregnated with the above-mentioned impregnating liquid 1 so that the pickup rate was 400%. Then, the modified fibroin in the impregnating liquid 1 impregnated in the nonwoven fabric 2 was coagulated and then dried in the same manner as in the production of the synthetic leather 1-1. As a result, a synthetic leather 1-2 composed of a polyethylene terephthalate fiber non-woven fabric and a polymer substance made of modified fibroin impregnated therein and integrated (consisting only of a base material layer) was produced.

<Manufacturing of synthetic leather 1-3>
(Manufacturing of non-woven fabric 1)
Nonwoven fabric 1 was produced in the same manner as in the production of synthetic leather 1-1.

(Manufacturing of synthetic leather 1-3)
The non-woven fabric 1 obtained as described above was impregnated with the above-mentioned impregnating liquid 2 so that the pickup rate was 400%. Then, the non-woven fabric 1 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 the polyurethane in the impregnating liquid 2 is removed. The resin was solidified (solidified). Next, the non-woven fabric 2 was taken out from the aqueous DMF solution and then dried at room temperature. As a result, synthetic leather 1-3 (consisting only of the base material layer) composed of the modified fibroin fiber non-woven fabric and the polymer substance made of the polyurethane resin impregnated and integrated therein was produced.

<Manufacturing of synthetic leather 1-4>
(Manufacturing of non-woven fabric 2)
Nonwoven fabric 2 was produced in the same manner as in the production of synthetic leather 1-2.

(Manufacturing of synthetic leather 1-4)
Using the non-woven fabric 2 obtained as described above, the non-woven fabric 2 is impregnated with the impregnating liquid 2 in the same manner as in the production of synthetic leather 1-3, and then the impregnating liquid 2 is solidified (solidified) and further solidified. It was dried. As a result, synthetic leather 1-4 (consisting only of the base material layer) composed of a polyethylene terephthalate fiber non-woven fabric and a polymer substance such as a polyurethane resin impregnated and integrated therein was produced.

Synthetic leather 1-1, 1-2, 1-3 in which at least one of the non-woven fabric and the polymer substance thus obtained is modified fibroin, and synthetic leather in which both the non-woven fabric and the polymer substance are made of polyurethane resin. When the appearances of 1-4 were observed, it was confirmed that the former synthetic leather had the same appearance as the latter synthetic leather.

<Manufacturing synthetic leather 1-5>
(Manufacturing of base material layer)
First, synthetic leather 1-1 was produced in the same manner as described above, and this was used as a base material layer.

(Preparation of resin for epidermis layer)
A lyophilized powder of modified fibroin (PRT799) was added to formic acid to a concentration of 20% by mass and dissolved at 40 ° C. for 1 hour or longer using a shaker to obtain a modified fibroin solution. Then, the pigment was added to the modified fibroin solution in an amount of 5% by mass, and then insoluble matter and bubbles were removed. As a result, a resin for the epidermis layer composed of a modified fibroin solution containing a pigment was prepared.

(Manufacturing of synthetic leather 1-5)
The resin for the skin material layer obtained as described above is coated on a release paper pasted on an iron plate to a thickness of 0.5 mm, and then dried at 90 ° C. for 10 minutes to obtain a skin material layer. The resin for use was incompletely solidified. Then, the resin for the epidermis layer was laminated on the base material layer obtained as described above together with the paper pattern attached to the iron plate, and heated at 90 ° C. for 1 minute. As a result, the resin for the epidermis layer was completely solidified and adhered to the base material layer. Then, the iron plate and the paper pattern were peeled off from the resin for the skin layer. Thus, the desired synthetic leather 1-5 was produced in which the epidermis 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 epidermis layer.

1 ... Nonwoven fabric, 2 ... Polymer material, 3 ... Base material layer, 4 ... Adhesive layer, 5 ... Skin layer, 10, 20 ... Synthetic leather, 31 ... Microsyring, 32 ... Dope liquid, 33 ... Metal base nozzle , 35 ... power supply, 36 ... fibrous material, 38 ... metal net, 39 ... non-woven fabric, 81 ... non-woven 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 ... Heat press roll, 96 ... Base layer, 97 ... Synthetic leather, 100 ... Electro spinning device , 800,900 ... Synthetic leather manufacturing equipment.

Claims (12)

It has a base material layer containing a non-woven fabric and a polymer substance,
Synthetic leather to which at least one of the non-woven fabric and the polymer substance is endowed with functionality. The synthetic leather according to claim 1, wherein the non-woven fabric contains protein fibers. The synthetic leather according to claim 2, wherein the protein fiber contains modified fibroin. The synthetic leather according to claim 3, wherein the modified fibroin is modified spider silk fibroin. The non-woven fabric contains a protein crosslinked product and contains
The protein cross-linked product has a polypeptide skeleton, a first residue which is a residue of a first reactant having two or more first reactive groups capable of reacting with a protein to form a bond, and the above-mentioned Each has a plurality of second residues, which are residues of the second reactant having one second reactive group capable of reacting with the first reactive group to form a bond.
At least one of the first residues crosslinks the polypeptide backbone.
The synthetic leather according to any one of claims 1 to 4, wherein at least one of the first residues is bound to the polypeptide skeleton at one end and to the second residue at the other end. .. The synthetic leather according to any one of claims 1 to 5, wherein the nonwoven fabric contains a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer. The synthetic leather according to any one of claims 1 to 6, wherein the polymer substance contains a protein. The synthetic leather according to claim 7, wherein the protein contains modified fibroin. The synthetic leather according to claim 8, wherein the modified fibroin is modified spider silk fibroin. The polymer substance contains a protein crosslinked product and contains
The protein cross-linked product has a polypeptide skeleton, a first residue which is a residue of a first reactant having two or more first reactive groups capable of reacting with a protein to form a bond, and the above-mentioned Each has a plurality of second residues, which are residues of the second reactant having one second reactive group capable of reacting with the first reactive group to form a bond.
At least one of the first residues crosslinks the polypeptide backbone.
The synthetic leather according to any one of claims 1 to 9, wherein at least one of the first residues is bound to the polypeptide skeleton at one end and to the second residue at the other end. .. The synthetic leather according to any one of claims 1 to 10, wherein the polymer substance contains a modified hydroxyl group-containing polymer in which a functional functional group is bonded to the hydroxyl group-containing polymer. A step of impregnating a non-woven fabric with an impregnating solution containing a polymer substance and coagulating the polymer substance to form a base material layer is provided.
A method for producing synthetic leather, wherein at least one of the non-woven fabric and the polymer substance is endowed with functionality.