Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
  • C08G63/685—Polyesters containing atoms other than carbon, hydrogen and oxygen containing nitrogen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/91—Polymers modified by chemical after-treatment
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
  • C08G64/02—Aliphatic polycarbonates
  • C08G64/0208—Aliphatic polycarbonates saturated
  • C08G64/0225—Aliphatic polycarbonates saturated containing atoms other than carbon, hydrogen or oxygen
  • C08G64/0241—Aliphatic polycarbonates saturated containing atoms other than carbon, hydrogen or oxygen containing nitrogen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G64/00—Macromolecular compounds obtained by reactions forming a carbonic ester link in the main chain of the macromolecule
  • C08G64/42—Chemical after-treatment
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
  • C08J5/18—Manufacture of films or sheets
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
  • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
  • D01F6/88—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polycondensation products as major constituent with other polymers or low-molecular-weight compounds
  • D01F6/92—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polycondensation products as major constituent with other polymers or low-molecular-weight compounds of polyesters
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
  • D01F6/88—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polycondensation products as major constituent with other polymers or low-molecular-weight compounds
  • D01F6/94—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polycondensation products as major constituent with other polymers or low-molecular-weight compounds of other polycondensation products

Definitions

• the present disclosure relates to a method for producing a polymer compound, a method for producing a polymer compound solution, a method for producing a film, a method for producing a fiber, and a film and a fiber.
• Structural proteins such as silk fibroin and spider silk fibroin have the potential to exhibit excellent robustness, and are therefore attracting attention as alternatives to structural materials made of synthetic resins and the like.
• advances in recombinant technology have led to the development of mass production techniques for recombinant structural proteins that mimic the structural proteins mentioned above (for example, Patent Document 1).
• Proteins have many functional groups capable of hydrogen bonding, such as amide groups in the main chain and carboxy, amino, hydroxy, and mercapto groups in the side chains, which allows numerous hydrogen bonds to be formed between molecules. For this reason, molded bodies obtained by heating and pressurizing structural proteins are thought to have excellent mechanical strength, but they also have a brittle side, as they crack and break when subjected to impacts. The above-mentioned circumstances are obstacles to using proteins as a substitute for general-purpose plastics.
• Patent Document 2 discloses a method for producing a protein material that includes a step of binding a substance with excellent water resistance to a specific modified fibroin.
• Patent Document 3 discloses a method for producing a polymer in which a thioether bond is generated by a 1,4-addition reaction (Michael addition reaction) between the mercapto group of cysteine constituting the polypeptide backbone and the carbon-carbon double bond of maleimide or a maleic acid derivative, thereby introducing at least one functional group selected from the group consisting of polyether groups, polyester groups, polycarbonate groups, polyamide groups, polyol groups, and modified polysaccharide groups into the polypeptide backbone.
• Patent Document 3 The method described in Patent Document 3 is useful in that it can introduce various functional groups into the polypeptide backbone, but depending on the structure of the Michael addition acceptor that can undergo a Michael addition reaction with the mercapto group of the polypeptide backbone, it may be necessary to more strictly control the reaction and reaction efficiency, etc., and there is room for improvement as a synthesis technology for raw materials for industrial materials.
• the high reactivity of the compound that serves as the Michael addition acceptor can lead to the production of polymer compounds with unexpected long chain growth or branching, in addition to the designed molecule, which can cause an increase in the viscosity of the reaction solution, making it difficult to control the reaction system.
• the proportion of the raw material polypeptide that becomes the desired polymer compound is low, and the raw material conversion rate tends to decrease.
• it is difficult to adequately control the reaction system by adjusting reaction conditions such as the acidity of the reaction solution, the reaction temperature, and the stirring speed.
• the purpose of this disclosure is to provide a manufacturing method that can increase the raw material conversion rate in the production of polymer compounds as described above.
• a polypeptide having at least one mercapto group At least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, having two structures represented by the following general formula (1): [In the general formula (1), M represents any one of H, Na, K, NHEt3 , and NHEtiPr2 ] in dimethyl sulfoxide in the presence of a base and a reducing agent, [2] A polypeptide having at least one mercapto group; At least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, having two structures represented by the following general formula (2): [In the general formula (2), R 1 represents a hydrogen atom or a methyl group] in dimethyl sulfoxide in the presence of a base, a reducing agent and a polymerization inhibitor, [3] A polypeptide having at least one mercapto group; At least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, having two structures represented by the following general formula
• a polypeptide having at least one mercapto group At least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, having two structures represented by the following general formula (2): [In the general formula (2), R 1 represents a hydrogen atom or a methyl group] in dimethyl sulfoxide in the presence of a reducing agent and a polymerization inhibitor, [5] The method for producing a polymer compound according to any one of [1] to [4], wherein the reducing agent is at least one selected from the group consisting of dithiol, sodium sulfate, sodium sulfite, and sodium dithionite.
• [6] The method according to any one of [1] to [5], wherein the amount of the reducing agent is 0.5 equivalents or more relative to the mercapto group of the polypeptide.
• [7] The method according to any one of [1] to [6], wherein the reaction between the polypeptide and the compound is carried out at 50° C. or higher.
• a method for producing a polymer solution comprising isolating a polymer obtained by the production method according to any one of [1] to [7] and then dissolving the isolated polymer in a solvent.
• a method for producing a film comprising forming a film from a polymer compound solution obtained by the production method according to [8].
• a method for producing a fiber comprising spinning a polymer solution obtained by the method for producing a fiber according to [8].
• a method for producing a polymer compound solution by reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (1), in dimethyl sulfoxide in the presence of a base and a reducing agent under heating; and forming a film from the polymer compound solution.
• M represents any one of H, Na, K, NHEt 3 , and NHEtiPr 2.
• a method for producing a polymer compound solution by reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (2), in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor, under heating; and forming a film from the polymer compound solution.
• R 1 represents a hydrogen atom or a methyl group.
• a method for producing a polymer compound solution by reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (1), in dimethyl sulfoxide in the presence of a base and a reducing agent under heating; and spinning the polymer solution.
• M represents any one of H, Na, K, NHEt 3 , and NHEtiPr 2.
• a method for producing a polymer compound solution by reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (2), in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor, under heating; and spinning the polymer solution.
• R 1 represents a hydrogen atom or a methyl group.
• a film comprising a polymer compound, The polymer compound is A polypeptide portion; and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, wherein the polypeptide portion is directly bound to S of the following general formula (3), (4), or (5) via a structure represented by the following general formula (3), (4), or (5):
• M represents any one of H, Na, K, NHEt 3 , and NHEtiPr 2
• R 1 represents H or Me
• a fiber comprising a polymer compound,
• the polymer compound is A polypeptide portion; and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure, wherein the polypeptide portion is directly bound to S of the following general formula (3), (4), or (5) via a structure represented by the following general formula (3), (4), or (5):
• M represents any one of H, Na, K, NHEt 3 , and NHEtiPr 2
• R 1 represents H or Me
• a fiber having an elongation of 400% or more and a breaking strength greater than its yield strength.
• a fiber comprising a synthetic polymer obtained by the production method according to any one of [1] to [8], the fiber having an elongation of 400% or more and a ratio of breaking strength to yield strength of more than 1.
• a film comprising a synthetic polymer obtained by the production method according to any one of [1] to [8], having an elongation of 400% or more and a ratio of breaking strength to yield strength of more than 1.
• a method for producing a solution-type adhesive comprising a step of dissolving a polymer compound obtained by the method according to any one of [1] to [8] in a solvent.
• a method for producing a water-dispersible adhesive comprising a step of dispersing a polymer compound obtained by the method according to any one of [1] to [8] in an aqueous medium.
• a method for producing a film-like adhesive comprising a step of forming a film from a solution in which a polymer compound obtained by the method according to any one of [1] to [8] is dissolved.
• a method for producing a powdery adhesive comprising the step of obtaining a powder composition containing a polymer compound obtained by the method according to any one of [1] to [8].
• a method for producing an adhesive body comprising: placing a solution in which a polymer compound obtained by the method according to any one of [1] to [8] is dissolved in a solvent between a plurality of adherends; and then removing the solvent from the solution to solidify the polymer compound, thereby adhering the adherends to each other.
• a method for producing an adherend comprising: placing an aqueous dispersion in which a polymer compound obtained by the method according to any one of [1] to [8] is dispersed in an aqueous medium between a plurality of adherends; removing the aqueous medium from the aqueous dispersion; and solidifying the polymer compound, thereby adhering the adherends to each other.
• a method for producing an adhesive body comprising the steps of: softening a film containing a polymer compound obtained by the method according to any one of [1] to [8] by swelling or heating; interposing the film between a plurality of adherends; and curing the film while the film is pressed against the adherends, thereby bonding the adherends together.
• a method for producing an adherend comprising: heating a powder composition containing a polymer compound obtained by the method according to any one of [1] to [8], and applying pressure to the powder composition via the adherends to solidify the powder composition in a state in which the powder composition is interposed between the adherends, thereby adhering the adherends to each other.
• a method for producing a solution-type coating agent comprising a step of dissolving the polymer compound obtained by the method according to any one of [1] to [8] in a solvent.
• a method for producing a water-dispersible coating agent comprising a step of dispersing a polymer compound obtained by the method according to any one of [1] to [8] in an aqueous medium.
• a method for producing a film-like coating agent comprising a step of forming a film from a solution in which a polymer compound obtained by the method according to any one of [1] to [8] is dissolved.
• a method for producing a powder coating agent comprising the step of obtaining a powder composition containing the polymer compound obtained by the method according to any one of [1] to [8].
• a method for producing a laminate having a substrate and a coating layer formed on at least a portion of a surface of the substrate comprising the steps of: A method for producing a laminate, comprising: supplying a solution in which a polymer compound obtained by the method according to any one of [1] to [8] is dissolved in a solvent to at least a part of a surface of the substrate; coating at least a part of the surface of the substrate with the solution; and removing the solvent from the solution to solidify the polymer compound, thereby forming a coating layer on at least a part of the surface of the substrate.
• a method for producing a laminate having a substrate and a coating layer formed on at least a portion of a surface of the substrate comprising the steps of:
• a method for producing a laminate comprising: supplying an aqueous dispersion in which a polymer compound obtained by the method according to any one of [1] to [8] is dispersed in an aqueous medium to at least a part of a surface of the substrate; coating at least a part of the surface of the substrate with the aqueous dispersion; and removing the aqueous medium from the aqueous dispersion to solidify the polymer compound, thereby forming a coating layer on at least a part of the surface of the substrate.
• a method for producing a laminate having a substrate and a coating layer formed on at least a portion of a surface of the substrate comprising the steps of: A method for producing a laminate, comprising the steps of: softening a film containing a polymer compound obtained by the method according to any one of [1] to [8] by swelling or heating; placing the film on at least a part of a surface of the substrate; and curing the film in a state of being pressed against the substrate, thereby forming the coating layer on at least a part of the surface of the substrate.
• a method for producing a laminate having a substrate and a coating layer formed on at least a portion of a surface of the substrate comprising the steps of: A method for producing a laminate, comprising: placing a powder composition containing a polymer compound obtained by the method according to any one of [1] to [8] on at least a part of a surface of a substrate; heating the powder composition while pressing the powder composition between a pressurizing body and the substrate to solidify the powder composition, thereby forming the coating layer on at least a part of the surface of the substrate.
• One aspect of the present disclosure provides a method for producing a polymer compound, comprising heating a polypeptide having at least one mercapto group and at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (1), in dimethyl sulfoxide in the presence of a base and a reducing agent: [In the general formula (1), M represents any one of H, Na, K, NHEt3 , and NHEtiPr2 ]
• a method is adopted in which a base and a reducing agent are present in the reaction system when reacting a polypeptide with a specific compound disclosed herein.
• the reducing agent is stable in dimethyl sulfoxide, does not easily undergo a Michael addition reaction with the functional group represented by formula (1), and can inhibit the formation of disulfide bonds between mercapto groups in the polypeptide.
• the base can activate the mercapto group in the polypeptide and promote the Michael addition reaction with the functional group represented by formula (1). Due to this action, a polyether or other skeleton can be introduced into the polypeptide by the method of heating in dimethyl sulfoxide, and a polymer compound can be produced with higher efficiency.
• the desired effect of the present disclosure can be obtained at a higher level by adding a base, but depending on the level of the desired effect, it can be omitted.
• One aspect of the present disclosure provides a method for producing a polymer compound, comprising heating and reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (2), in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor: [In general formula (2), R 1 represents a hydrogen atom or a methyl group.]
• the functional group represented by the general formula (2) is radically polymerized by heating in dimethyl sulfoxide, and self-polymerization of at least one compound selected from the group consisting of polyether, polyester, and polycarbonate having two structures represented by the general formula (2) above can occur.
• the radical polymerization reaction of the functional group represented by the general formula (2) can be suppressed by also having a polymerization inhibitor present in the reaction system.
• a skeleton such as a polyether can be introduced into the polypeptide, and a polymer compound can be produced with higher efficiency.
• the base can be blended to obtain the desired effect of the present disclosure at a higher level, but it can be omitted depending on the level of the desired effect.
• the reducing agent may be at least one selected from the group consisting of dithiol, sodium sulfate, sodium sulfite, and sodium dithionite.
• a specific reducing agent is highly stable in dimethyl sulfoxide and can further inhibit the formation of disulfide bonds between mercapto groups in the polypeptide.
• the amount of the reducing agent may be 0.5 equivalents or more relative to the mercapto groups of the polypeptide.
• reaction between the polypeptide and the compound may be carried out at 50°C or higher.
• One aspect of the present disclosure provides a method for producing a polymer compound solution, which includes dissolving the polymer compound obtained by the above-mentioned production method in a solvent.
• a method for producing a polymer film comprising forming a film from a polymer compound solution obtained by the above-mentioned method for producing a polymer compound solution.
• the forming into the film may be, for example, cast molding.
• One aspect of the present disclosure provides a method for producing polymeric fibers, which includes spinning the polymer compound solution obtained by the above-mentioned production method.
• One aspect of the present disclosure provides a film comprising a polymer compound, the polymer compound having a structure in which a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded via a structure represented by the following formula (3), (4), or (5) such that the polypeptide portion is bonded to S in the following formula (3), (4), or (5), the film having an elongation of 400% or more and a breaking strength greater than a yield strength:
• M represents H, Na, K, NHEt3 , or NHEtiPr2
• R1 represents H or Me.
• One aspect of the present disclosure provides a fiber comprising a polymer compound, or a film comprising the polymer compound, the polymer compound having a structure in which a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded via a structure represented by the following general formula (3), (4), or (5) so that the polypeptide portion is bonded to S in the following general formula (3), (4), or (5), the fiber having an elongation of 400% or more and a breaking strength greater than a yield strength:
• M represents any one of H, Na, K, NHEt3 , or NHEtiPr2
• R1 represents H or Me.
• the present disclosure provides a manufacturing method that can increase the raw material conversion rate in the production of polymer compounds as described above.
• the materials exemplified in this specification may be used alone or in combination of two or more.
• the content of each component in the composition means the total amount of the multiple substances present in the composition, unless otherwise specified.
• the molding material refers to a material used to manufacture a molded body.
• the molding material according to the present disclosure can also be said to be a material in which a polypeptide has been chemically modified.
• the shape of the molded body according to the present specification is not particularly limited, and may be, for example, a film shape, a plate shape, a block shape, a sponge shape, a fiber shape, etc.
• the form of the molding material according to the present specification is also not limited in any way, and may be, for example, a powder, a granule, a liquid, a gel, etc.
• the molding material according to the present disclosure can be made into a molded body of various shapes, for example, by hot pressure molding, cast molding, spinning, etc. A mold may be used during molding, if necessary.
• the method for producing a polymer compound according to the present disclosure utilizes a reaction between a mercapto group (nucleophilic functional group) of a polypeptide and a skeleton such as a polyether having a specific unsaturated bond (electrophilic functional group).
• the first embodiment of the method for producing a polymer compound comprises heating and reacting a polypeptide having at least one mercapto group (hereinafter also referred to as component (A)) with at least one compound selected from the group consisting of polyethers, polyesters and polycarbonates, having two structures represented by the following formula (1) (hereinafter also referred to as component (B1) or, together with component (B2) described below, simply referred to as component (B)) in dimethyl sulfoxide in the presence of a base (hereinafter also referred to as component (C)) and a reducing agent (hereinafter also referred to as component (D)).
• component (A) polypeptide having at least one mercapto group
• component (B1) or, together with component (B2) described below, simply referred to as component (B) in dimethyl sulfoxide in the presence of a base (hereinafter also referred to as component (C)) and a reducing agent (hereinafter also referred to as component (D)).
• the heating reaction is a reaction between component (A) and component (B), and is carried out, for example, by heating to 50°C or higher.
• the timing of heating is not limited to after mixing of components (A) to (D).
• components (A) to (D) may be added to preheated dimethyl sulfoxide, components (C) and (D) may be added to dimethyl sulfoxide, heating may be started, and after a predetermined temperature is reached, components (A) and (B) may be added and reacted, or one of components (A) and (B) may be added to components (C) and (D), and after a predetermined temperature is reached, the remaining one of components (A) and (B) may be added and reacted.
• the inclusion of component (C) can provide a higher level of the desired effect of the present disclosure, but it may be omitted depending on the level of effect required.
• the number of mercapto groups possessed by the above-mentioned polypeptide (component (A)) is 1 or more per molecule, but may be, for example, 2 or more, 4 or more, 8 or more, or 16 or more. When the number of the above-mentioned mercapto groups is within the above range, the contact probability of the reaction points is improved, and the reaction can proceed efficiently.
• the number of mercapto groups possessed by the (A) component may be, for example, 64 or less, or 32 or less per molecule.
• the number of the above-mentioned mercapto groups is within the above range, the production of by-products formed by the reaction between the mercapto groups possessed by the polypeptide can be suppressed, and the desired polymer compound can be obtained more efficiently.
• the number of amino acid residues constituting the (A) component may be, for example, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.
• the number of amino acid residues may be, for example, 5000 or less, 4500 or less, 4000 or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 or less, or 1000 or less. The smaller the number of amino acid residues, the higher the solubility in dimethyl sulfoxide tends to be.
• the production efficiency of the target polymer compound can be further improved when the polypeptide is dissolved in a solvent and reacted with the above compound (component (B1)).
• the molded product composed of the obtained polymer compound can exhibit a higher level of mechanical strength and flexibility.
• the (A) component may be, for example, a hydrophobic polypeptide.
• the (A) component is easier to prepare, and the raw material costs in the above-mentioned manufacturing method can be further reduced.
• the molded article made of the obtained polymer compound can exhibit better flexibility.
• the molded article can also exhibit better water resistance. In this case, the obtained molded article can have a longer service life.
• the hydrophobicity of component (A) can be estimated using the value of the average hydropathy index (hereinafter sometimes referred to as average HI) described below as an index.
• the average HI value of component (A) may be greater than 0, and may be, for example, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more.
• the upper limit of the average HI value of component (A) is not particularly limited, but may be, for example, 1.00 or less, or 0.7 or less.
• the (A) component is preferably one that has low solubility in an aqueous lithium bromide solution (concentration: 9M) at 60°C. This solubility can be evaluated using a polypeptide corresponding to the (A) component.
• the maximum concentration of the polypeptide when dissolved in an aqueous lithium bromide solution (concentration: 9M) at 60°C may be, for example, less than 30% by mass, less than 25% by mass, less than 20% by mass, less than 15% by mass, less than 10% by mass, less than 5% by mass, or less than 1% by mass.
• the (A) component may be one that is completely insoluble in an aqueous lithium bromide solution (concentration: 9M) at 60°C.
• the solubility of the polypeptide portion of the polymer compound obtained by the manufacturing method of the present disclosure can be confirmed by using a polypeptide obtained by decomposing the polymer compound and isolating only the hydrophobic polypeptide.
• the (A) component preferably has a large contact angle with water.
• the contact angle with water can be evaluated by using a polypeptide corresponding to the (A) component on a substrate. More specifically, a film made of the above polypeptide can be formed and evaluated using the film.
• a polypeptide constituting a film that has a contact angle of 55° or more after 5 seconds of dropping water on the film is preferred as the (A) component.
• the contact angle may be, for example, 60° or more, 65° or more, or 70° or more.
• the polymer compound is decomposed, and only the hydrophobic polypeptide is isolated. The above contact angle of the polypeptide portion of the polymer compound can be confirmed by using the polypeptide obtained.
• the (A) component is preferably one that has excellent hot water resistance. Hot water resistance can be evaluated using a polypeptide corresponding to the (A) component.
• a preferred (A) component is a polypeptide that does not decompose even when a dispersion containing the above polypeptide and water is prepared with a content of the above polypeptide of 5% by mass and the dispersion is heat-treated at 100°C for 5 hours.
• the above-mentioned hot water resistance of the polypeptide portion of the polymer compound can be confirmed by using a polypeptide obtained by decomposing the polymer compound and isolating only the hydrophobic polypeptide.
• the weight average molecular weight of the (A) component is, for example, 200 to 1,000,000, more preferably 300 to 900,000, even more preferably 400 to 800,000, even more preferably 500 to 700,000, even more preferably 600 to 600,000, even more preferably 1,000 to 600,000, even more preferably 3,000 to 600,000, even more preferably 5,000 to 600,000, even more preferably 10,000 to 600,000, even more preferably 5,000 to 100,000. If the weight average molecular weight of the (A) component is less than 200, the polypeptide that functions as a hard segment may be too small compared to the polyether structure (soft segment) of the (B1) component. In this case, the rigidity of the molded body molded using the obtained polymer compound (molding material) may be reduced, making it difficult to use it as a structure, for example.
• the weight-average molecular weight in this specification is a value measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
• the electrophoresis is performed according to the following procedure. That is, first, 200 ⁇ L of 2 M lithium chloride DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is added to 2 mg of powder sample, and the sample is dissolved by stirring while heating at 80°C for 60 minutes and then at 95°C for 10 minutes. The sample is then diluted 50 times with 10 M urea solution, further diluted 2 times with sample buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and heated at 95°C for 5 minutes to denature the protein.
• 2 M lithium chloride DMSO manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
• an SDS-PAGE gel (manufactured by Bio-lad) is attached to an electrophoresis device (manufactured by Bio-lad), and the device is filled with SDS buffer, while the electrophoresis device is connected to a power supply device (manufactured by Biocraft).
• 10 ⁇ L of the denatured sample was added to each well of the SDS-PAGE gel, and a current was applied at 30 mA per sheet for 30 minutes.
• the SDS-PAGE gel was removed from the device, immersed in Oriole fluorescent gel stain (Bio-lad), and shaken for 1 hour. The gel was then placed on a UV sample tray (Bio-lad) and a stained image was obtained using a Gel Doc EZ gel imaging device (Bio-lad).
• An XL ladder broad (Aproscience) was used as the standard sample.
• the molecular weight of component (A) may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more.
• the molecular weight of component (A) may be, for example, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less.
• the molecular weight of component (A) may be adjusted within the above range, and may be, for example, 2 to 500 kDa.
• the molecular weight can be measured by a liquid chromatograph mass spectrometer.
• Component (A) may be a natural protein or an artificial protein.
• Component (A) may also be a protein (modified protein) obtained by chemically modifying a natural protein or an artificial protein (e.g., alkylation, etherification, esterification, amidation, thioetherification, thioesterification, etc.).
• the amino acid sequence of the natural protein, artificial protein, or modified protein is not particularly limited as long as it has one or more mercapto groups.
• component (A) for example, a protein having similar physical properties can be used in accordance with the required characteristics according to the application of the polymer compound.
• the polypeptide according to this embodiment include a polypeptide that can be used for medical purposes, a polypeptide that can be used for industrial purposes, etc. "Usable for industrial purposes” means that it can be used, for example, as a variety of general-purpose materials for indoor and outdoor use.
• polypeptides that can be used for medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used in vaccinations, vaccines, antigen-binding proteins, immunostimulating proteins, allergens, and full-length antibodies or antibody fragments or derivatives.
• polypeptides that can be used industrially include structural proteins.
• Structural proteins refer to proteins involved in the structure of living organisms, proteins that constitute structures created by living organisms, or proteins derived from these. Structural proteins also refer to proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles.
• structural proteins can be said to be proteins that form the skeleton of living organisms or materials, with repeated motifs consisting of characteristic amino acid sequences or a certain number of amino acid residues.
• Specific examples of structural proteins include spider silk, silkworm silk, keratin, collagen, elastin, and resilin, as well as proteins derived from these. Structural proteins may be artificial fibroin, or artificial spider silk fibroin (artificially modified spider silk fibroin).
• the structural protein may be an artificial structural protein.
• Artificial structural proteins include synthetic proteins and recombinant structural proteins produced from microorganisms using recombinant gene technology. That is, in this specification, "artificial structural protein” means an artificially produced structural protein.
• An artificial structural protein may have the same amino acid sequence as a natural structural protein, or may be a modified structural protein in which a part of the amino acid sequence has been modified based on the amino acid sequence of a naturally occurring structural protein from the standpoint of productivity, moldability, etc.
• the artificial structural protein may have a glycine residue content of 10-55%.
• the glycine residue content may be, for example, 13%-55%, 15%-55%, 18%-55%, 20%-55%, 22%-55%, or 25%-55%.
• the "glycine residue content" is a value represented by the following formula.
• Glycine residue content (number of glycine residues in artificial structural protein / total number of amino acid residues in polypeptide) x 100 (%)
• the artificial structural protein may have 150 or more amino acid residues.
• the number of amino acid residues may be, for example, 200 or more or 250 or more, and is preferably 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.
• the artificial structural protein may have a total content (total content) of at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any of the serine residue content, the threonine residue content, the tyrosine residue content, the sum of the serine residue content and the threonine residue content, the sum of the serine residue content and the tyrosine residue content, the sum of the threonine residue content and the tyrosine residue content, the sum of the serine residue content, the threonine residue content and the tyrosine residue content), the alanine residue content, and the glycine residue content, which is 40% or more.
• the total content may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. There is no particular upper limit to the total content, but it may be, for example, 90% or less, 85% or less, or 80% or less.
• the artificial structural protein may have a total serine residue content, threonine residue content, and tyrosine residue content of 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, or 7% or more.
• the total serine residue content, threonine residue content, and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.
• the artificial structural protein has an average distribution of serine, threonine, or tyrosine residues, and the total content of serine, threonine, and tyrosine residues in any 20 consecutive amino acid residues may be 4% or more, 5% or more, 10% or more, or 15% or more, and may be 50% or less, 40% or less, 30% or less, or 20% or less.
• alanine residue content, serine residue content, threonine residue content, and tyrosine residue content are the same as those obtained by replacing the alanine residue in the above formula with glycine residue, serine residue, threonine residue, and tyrosine residue, respectively.
• the artificial structural protein may have a repetitive sequence.
• the artificial structural protein may have multiple amino acid sequences (repetitive sequence units) with high sequence identity within the artificial structural protein.
• the number of amino acid residues in the repetitive sequence unit is preferably 6 to 200.
• the total number of glycine residues, serine residues, glutamine residues and alanine residues relative to the total number of amino acid residues in the repeat sequence unit may be 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more.
• the sequence identity between the repeat sequence units may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
• the hydrophobicity index of the repeating sequence unit may be, for example, -0.80 or more, -0.70 or more, -0.60 or more, -0.50 or more, -0.40 or more, -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more.
• the upper limit of the hydrophobicity index of the repeating sequence unit is not particularly limited, but may be, for example, 1.0 or less, or 0.7 or less.
• the artificial structural protein may contain an (A)n motif.
• an (A)n motif means an amino acid sequence mainly composed of alanine residues.
• the number of amino acid residues in the (A)n motif may be 2 to 27, and may be an integer of 2 to 20, 2 to 16, or 2 to 12.
• the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).
• the total number of alanine, serine, threonine and valine residues in the (A)n motif may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed of only one or more amino acid residues selected from alanine, serine, threonine and valine residues).
• the (A)n motifs present in the recombinant structural protein according to this embodiment may have the same amino acid sequence or different amino acid sequences. Since the (A)n motif mainly contains alanine residues, it is likely to have an ⁇ -helix structure or a ⁇ -sheet structure.
• the artificial structural protein according to this embodiment has these secondary structures repeatedly. Therefore, when the artificial structural protein is formed into a molded product such as a fiber, film or resin, as described below, it is expected that the secondary structures will provide high strength.
• the alanine residue content may be, for example, 10 to 40%, and may be 12 to 40%, 15 to 40%, 18 to 40%, 20 to 40%, or 22 to 40%.
• the glycine residue content may be, for example, 10 to 55%, and may be 11 to 55%, 13 to 55%, 15 to 55%, 18 to 55%, 20 to 55%, 22 to 55%, or 25 to 55%.
• alanine residue content refers to the number of alanine residues relative to the total number of amino acid residues that make up a protein, and is a value expressed by the following formula.
• Alanine residue content (number of alanine residues in protein / total number of amino acid residues in protein) x 100 (%)
• glycine residue content, serine residue content, threonine residue content, proline residue content and tyrosine residue content are the same as those obtained by replacing the alanine residue in the above formula with glycine residue, serine residue, threonine residue, proline residue and tyrosine residue, respectively.
• the structural protein preferably contains amino acids with relatively large side chains or amino acids with flexibility homogeneously throughout the entire sequence to a certain extent.
• the structural protein may contain a motif containing tyrosine residues, threonine residues, and proline residues in a repeated cycle. Such a structural protein is likely to inhibit the formation of strong intermolecular hydrogen bonds during processing of the molded product obtained by molding, and is likely to improve processability.
• the total content of proline residues, threonine residues, and tyrosine residues in any 20 consecutive amino acid residues may be 5% or more, more than 5.5%, 6.0% or more, more than 6.5%, 7.0% or more, more than 7.5%, 8.0% or more, more than 8.5%, 9.0% or more, 10.0% or more, or 15.0% or more.
• the total content of proline residues, threonine residues, and tyrosine residues in any 20 consecutive amino acid residues may be 50% or less, 40% or less, 30% or less, or 20% or less.
• the total content of serine residues, threonine residues, and tyrosine residues may be, for example, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, or 7% or more.
• the total content of serine residues, threonine residues, and tyrosine residues may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.
• the amount of the polypeptide (component (A)) may be, for example, more than 1 part by mass, 2 parts by mass or more, 5 parts by mass or more, more than 5 parts by mass, 6 parts by mass or more, 7 parts by mass or more, or 8 parts by mass or more, relative to 100 parts by mass of dimethyl sulfoxide.
• the amount of component (A) may be, for example, 50 parts by mass or less, 30 parts by mass or less, 20 parts by mass or less, or 15 parts by mass or less, relative to 100 parts by mass of dimethyl sulfoxide.
• the proportion of the polypeptide (component (A)) in the dimethyl sulfoxide solution may be, for example, 15% by mass or less, less than 15% by mass, 14% by mass or less, 12.5% by mass or less, or 11.5% by mass or less.
• At least one compound (component (B1)) selected from the group consisting of polyethers, polyesters, and polycarbonates, which has two structures represented by formula (1), is capable of reacting with the mercapto group of the above-mentioned polypeptide by having the structure represented by formula (1), and is a compound capable of introducing a polyether structure, polyester structure, or polycarbonate structure into the polypeptide.
• polyethers having two structures represented by formula (1) examples include polyethylene glycol, polytetramethylene glycol, polypropylene glycol, and ethylene glycol-propylene glycol copolymers.
• polyesters having two structures represented by formula (1) include polylactic acid, polyglycolic acid, polybutylene succinate, polycaprolactone, and polyhydroxyalkanoates including 3-hydroxybutanoic acid-3-hydroxyhexanoic acid copolymers.
• polycarbonates having two structures represented by formula (1) include aliphatic polycarbonates such as polyethylene carbonate, polypropylene carbonate, and polytrimethylene carbonate. These compounds have easier molecular motion than the above-mentioned polypeptides, and therefore, when each of them is a compound as described above, the flexibility of the molded body composed of the obtained polymer compound can be further improved.
• the weight average molecular weight of the compound (component (B1)) may be, for example, 100 or more, 1000 or more, or 2000 or more.
• the weight average molecular weight of component (B1) may be, for example, 200,000 or less, 100,000 or less, 50,000 or less, or 10,000 or less.
• the weight average molecular weight of the compound (component (B1)) may be, for example, 0.005 times or more, 0.01 times or more, 0.05 times or more, or 0.1 times or more based on the weight average molecular weight of the polypeptide.
• the weight average molecular weight of component (B1) may be, for example, 20 times or less, 10 times or less, 5 times or less, or 2 times or less based on the weight average molecular weight of the polypeptide.
• the reaction can proceed while further suppressing a decrease in reactivity.
• the weight-average molecular weight of component (B1) may be adjusted within the above-mentioned range, and may be, for example, 0.005 to 20 times, 0.01 to 10 times, 0.05 to 5 times, or 0.1 to 2 times the weight-average molecular weight of the polypeptide.
• the ratio of the number of moles of the structure represented by formula (1) in the above compound (component (B1)) to the number of moles of the mercapto groups in the above polypeptide (component (A)) may be, for example, 0.1 times or more, 0.2 times or more, 0.5 times or more, or 0.7 times or more. By setting the above ratio within the above range, the reaction can proceed while further suppressing the decrease in reactivity.
• the ratio of the number of moles of the structure represented by formula (1) in component (B1) to the number of moles of the mercapto groups in component (A) may be, for example, 5.0 times or less, 3.0 times or less, 1.5 times or less, or 0.8 times or less.
• the reaction can proceed while further suppressing the generation of by-products and gelation.
• the number of polyether structures, etc. in the obtained polymer compound can be adjusted.
• the total number of polyether structures, etc. per polypeptide can be, for example, 1 or more, or 2 or more, and can be 2 to 10, 2 to 8, 2 to 6, or 2 to 4.
• the number of moles of sulfhydryl groups in this specification refers to a value measured by enzymatically cutting out the polypeptide in the measurement sample, converting it into amino acids, and then separating and detecting them using a liquid chromatograph. If you are preparing your own polypeptide, you can determine the number of moles of the above functional groups from the sequence of the designed molecule.
• the number of moles of the structure represented by general formula (1) and the structure represented by general formula (2) in this specification refers to the value measured by MALDI-TOFMS.
• the number of moles of the compound (component (B1)) present in dimethyl sulfoxide may be, for example, 0.1 times or more, 0.2 times or more, 0.5 times or more, or 0.8 times or more relative to the total number of moles of mercapto groups in the polypeptide.
• the number of moles of the component (B1) present in dimethyl sulfoxide may be, for example, 5.0 times or less, 3.0 times or less, 1.5 times or less, or 1.0 times or less relative to the total number of moles of mercapto groups in the polypeptide.
• the base (component (C)) in the above-mentioned production method is a compound that can activate the mercapto group of the polypeptide and promote the Michael addition reaction with the functional group represented by formula (1).
• the base include primary amines, secondary amines, tertiary amines, nitrogen-containing ring compounds, nitrogen-containing aromatic compounds, and inorganic bases.
• primary amines include ethanolamine and hexamethylenediamine.
• Examples of secondary amines include diethylamine, methylethylamine, and N-methylbutylamine.
• tertiary amines include triethylamine, diethylmethylamine, and DIPEA (N,N-diisopropylethylamine).
• nitrogen-containing ring compounds examples include quinuclidine, DABCO (1,4-diazabicyclo[2.2.2]octane), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), and DBN (1,5-diazabicyclo[4.3.0]non-5-ene).
• nitrogen-containing aromatic compounds examples include pyridine and imidazole.
• inorganic bases include sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, tripotassium phosphate, sodium acetate, and potassium acetate.
• the lower limit of the amount of the base may be, for example, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.1 parts by mass or more, or 0.5 parts by mass or more, based on the mass of the polypeptide.
• the upper limit of the amount of the base may be, for example, 50 parts by mass or less, 20 parts by mass or less, 10 parts by mass or less, or 5 parts by mass or less, based on the mass of the polypeptide.
• the amount of the base may be adjusted within the above range, and may be, for example, 0.05 to 50 parts by mass, or 0.1 to 10 parts by mass, based on the mass of the polypeptide.
• the amount of the base may be, for example, 0 to 50 parts by mass, 0 to 10 parts by mass, 0 to 1 part by mass, or 0 to 0.5 parts by mass based on the mass of the polypeptide.
• the reducing agent (component (D)) in the above-mentioned production method is a compound that can stabilize the mercapto group of the polypeptide in dimethyl sulfoxide and inhibit the formation of a disulfide bond between the mercapto groups.
• the reducing agent may contain at least one selected from the group consisting of thiol, dithiol, sodium sulfite, sodium hyposulfite, sodium sulfate, and sodium dithionite, and the reducing agent may contain at least one selected from the group consisting of dithiol and sodium sulfite, and may be dithiol or sodium sulfite.
• the reducing agent is preferably a compound having a mercapto group.
• the reducing agent is preferably a dithiol.
• a compound represented by the following general formula (Y) is preferable.
• Such a compound forms a chemically stable six-membered ring structure in the oxidized form after the reduction reaction, suppressing the reaction between the reducing agent and the Michael addition acceptor in the reaction system, and also suppresses the formation of disulfide bonds between polypeptides by forming a ring structure between the reducing agent and the mercapto group of the polypeptide, making the reaction between the mercapto group of the polypeptide and the Michael addition acceptor significant, and making it possible to more efficiently produce the target polymer compound in accordance with the reaction design.
• dithiols examples include dithiothreitol and 1,4-butanedithiol.
• the compound (dithiol) represented by general formula (Y) is dithiothreitol or 1,4-butanedithiol.
• mercapto groups include, for example, 3-mercaptopropionic acid, 3-mercapto-1,2-propanediol, and pentaerythritol tetra(3-mercaptopropionate).
• the reducing agent may be, for example, tris(2-carboxyethyl)phosphine (TCEP).
• the lower limit of the amount of the reducing agent may be, for example, 0.1 equivalents or more, 0.3 equivalents or more, 0.5 equivalents or more, 0.8 equivalents or more, or 1.0 equivalents or more, relative to the mercapto groups of the polypeptide.
• the upper limit of the amount of the reducing agent may be, for example, 2.4 equivalents or less, 2.0 equivalents or less, 1.6 equivalents or less, 1.2 equivalents or less, or 1.1 equivalents or less, relative to the mercapto groups of the polypeptide.
• the reaction between the polypeptide (component (A)) and the compound (component (B1)) may be carried out, for example, at 55°C or higher. In situations where the reaction temperature exceeds 55°C, disulfide bonds may usually be formed between the mercapto groups of the polypeptide in dimethyl sulfoxide. In contrast, in the production method disclosed herein, the reaction is carried out in the presence of a reducing agent, which can prevent the formation of disulfide bonds. This makes it possible to improve the activity of the reaction between the polypeptide and the compound while suppressing the formation of disulfide bonds, thereby improving the efficiency of synthesis of polymer compounds.
• the reaction temperature of the reaction between component (A)) and component (B1) may be, for example, 20°C or higher, 30°C or higher, 40°C or higher, 50°C or higher, 53°C or higher, 55°C or higher, 57°C or higher, or 60°C or higher.
• the reaction between component (A) and component (B1) can be further promoted.
• the upper limit of the reaction temperature of the reaction between component (A)) and component (B1) may be, for example, 110°C or lower, 100°C or lower, 90°C or lower, 85°C or lower, 80°C or lower, 75°C or lower, or 70°C or lower.
• reaction temperature within the above range, the formation of bonds between mercapto groups of the polypeptide as a side reaction can be further suppressed, and the desired polymer compound can be efficiently synthesized.
• the reaction temperature for the reaction between component (A) and component (B1) may be adjusted within the above range, for example, 50 to 90°C, 53 to 80°C, 55 to 75°C, or 55 to 70°C.
• the reaction may proceed at any temperature, but a temperature at which the DMSO solvent does not freeze and the protein does not decompose is desirable.
• the method for producing a polymer compound may further include other steps. Examples of the other steps include separately preparing component (A) and separately preparing component (B1).
• the method for preparing component (A) may include expressing a nucleic acid in a host transformed with an expression vector described below.
• component (A) may be an artificial fibroin.
• the expression method may involve secretory production, fusion protein expression, etc., in accordance with the methods described in Molecular Cloning, 2nd Edition. When expressed in yeast, animal cells, or insect cells, it may be obtained as a polypeptide to which sugar or a sugar chain has been added.
• the artificial fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the artificial fibroin in the culture medium, and harvesting it from the culture medium.
• the method for culturing the host in the culture medium can be performed according to a method normally used for culturing the host.
• the culture medium for the host may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and allows efficient cultivation of the host.
• the carbon source may be anything that can be assimilated by the host.
• Examples of carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
• Nitrogen sources that can be used include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium salts of inorganic or organic acids, as well as other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation bacteria and their digested products.
• inorganic salts examples include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
• Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions, for example, by shaking culture or deep aeration agitation culture.
• the culture temperature is, for example, 15 to 40°C.
• the culture time is usually 16 hours to 7 days.
• the pH of the culture medium during culture is preferably maintained at 3.0 to 9.0.
• the pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
• antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary during cultivation.
• an inducer may be added to the medium as necessary.
• isopropyl- ⁇ -D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.
• Culture media for insect cells include, for example, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell 400, ExCell 405 (both JRH Biosciences), and Grace's Insect Medium (Nature, 195, 788 (1962)).
• Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6 to 7 and a culture temperature of 25 to 30°C.
• antibiotics such as gentamicin can be added to the culture medium during culture as necessary.
• the transformed plant cell When the host is a plant cell, the transformed plant cell may be cultured as is, or may be differentiated into a plant organ and then cultured.
• a medium for culturing the plant cell for example, commonly used Murashige and Skoog (MS) medium, White medium, or these media supplemented with plant hormones such as auxin or cytokinin can be used.
• Animal cells can be cultured for 3 to 60 days under conditions such as a culture medium pH of 5 to 9 and a culture temperature of 20 to 40°C.
• antibiotics such as kanamycin and hygromycin may be added to the medium during culture as necessary.
• Methods for producing artificial fibroin using a host transformed with the above expression vector include a method for producing the artificial fibroin inside the host cell, a method for secreting it outside the host cell, and a method for producing it on the outer membrane of the host cell. Each of these methods can be selected by changing the host cell used and the structure of the artificial fibroin to be produced.
• the artificial fibroin when artificial fibroin is produced inside a host cell or on the outer membrane of the host cell, the artificial fibroin can be modified to be actively secreted outside the host cell by applying the method of Paulson et al. (J. Biol. Chem., 264, 17619 (1989)), the method of Rowe et al. (Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)), or the methods described in JP-A-05-336963 and WO 1994/023021, etc., mutatis mutandis.
• the artificial fibroin can be actively secreted outside the host cell by expressing a polypeptide containing the active site of the artificial fibroin in a form in which a signal peptide has been added using a genetic recombination technique.
• the artificial fibroin produced by the host transformed with the expression vector can be isolated and purified by methods commonly used for isolating and purifying proteins. For example, when the artificial fibroin is expressed in a dissolved state within the cells, after the culture is completed, the host cells are collected by centrifugation and suspended in an aqueous buffer solution, and then disrupted by an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, etc. to obtain a cell-free extract.
• the supernatant obtained by centrifuging the cell-free extract can be used to obtain a purified sample by using methods commonly used for isolating and purifying proteins, such as solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl-Sepharose and phenyl-Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing, either alone or in combination.
• anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-S
• the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the artificial fibroin as a precipitate fraction.
• the recovered insoluble body of the artificial fibroin can be solubilized with a protein denaturant. After this operation, a purified sample of the artificial fibroin can be obtained by the same isolation and purification method as above.
• the artificial fibroin or its derivative can be recovered from the culture supernatant. That is, the culture can be treated by a method such as centrifugation to obtain a culture supernatant, and a purified specimen can be obtained from the culture supernatant by using the same isolation and purification method as described above.
• the (A) component may be artificial fibroin.
• the method may be, for example, a method of artificially synthesizing naturally derived fibroin.
• naturally derived fibroin include fibroin produced by insects or spiders.
• Natural fibroin is a fibrous protein with a molecular weight of about 370,000 and composed of two subunits. It has a high content of glycine residues, alanine residues, serine residues and tyrosine residues, and these amino acid residues account for nearly 90% of the total number of amino acid residues.
• Natural fibroin has a crystalline region rich in amino acid residues with relatively small side chains such as glycine, alanine and serine, and an amorphous region with amino acid residues with relatively large side chains such as tyrosine.
• a more specific example of naturally derived fibroin is a fibroin whose sequence information is registered in NCBI GenBank. For example, it can be confirmed by extracting from among the sequences registered in NCBI GenBank that contain INV as a division, sequences with spidroin, amplify, fibroin, "silk and polypeptide", or "silk and protein" as keywords in DEFINITION, a specific product character string from CDS, and a specific character string in TISSUE TYPE from SOURCE.
• artificial fibroin means artificially produced fibroin (man-made fibroin).
• the artificial fibroin may be a fibroin having an amino acid sequence different from that of naturally occurring fibroin, or may be a fibroin having an amino acid sequence identical to that of naturally occurring fibroin.
• the artificial fibroin may be produced by known methods, for example, by the method described in WO 2019/194263.
• Artificial fibroin may be a fibrous protein having a structure similar to that of naturally occurring fibroin, or may be a fibroin having a sequence similar to the repetitive sequence of naturally occurring fibroin. "A sequence similar to the repetitive sequence of fibroin” may be an actual sequence found in naturally occurring fibroin, or a sequence similar thereto.
• “Artificial fibroin” may be one that has an amino acid sequence specified in this disclosure, but that is based on naturally occurring fibroin and has its amino acid sequence modified (for example, one that has its amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has an amino acid sequence that is artificially designed without relying on naturally occurring fibroin (for example, one that has a desired amino acid sequence by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence). Artificial fibroin that has had its amino acid sequence modified is also included in the category of artificial fibroin, so long as the amino acid sequence is different from that of naturally occurring fibroin.
• artificial fibroin examples include artificial silk fibroin (one that has had the amino acid sequence of silk protein produced by silkworms modified) and artificial spider silk fibroin (one that has had the amino acid sequence of spider silk protein produced by spiders modified). Since artificial fibroin is relatively easy to fibrillate and has a high fiber forming ability, it is preferable for the molding material to include artificial spider silk fibroin, and more preferably consists of artificial spider silk fibroin.
• the artificial fibroin may be a protein containing a domain sequence represented by formula 1: [(A)n motif-REP]m, or formula 2: [(A)n motif-REP]m-(A)n motif.
• the artificial fibroin may further have amino acid sequences (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal sides of the domain sequence.
• the N-terminal sequence and C-terminal sequence are typically, but are not limited to, regions that do not have repetitions of amino acid motifs characteristic of fibroin and consist of approximately 100 amino acid residues.
• domain sequence refers to an amino acid sequence represented by formula 1: [(A)n motif-REP]m or formula 2: [(A)n motif-REP]m-(A)n motif.
• the (A)n motif indicates an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
• the number of amino acid residues in the (A)n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16.
• the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).
• At least seven of the (A)n motifs present in the domain sequence may be composed only of alanine residues.
• REP indicates an amino acid sequence consisting of 2 to 200 amino acid residues.
• REP may be an amino acid sequence consisting of 10 to 200 amino acid residues.
• m indicates an integer from 2 to 300, and may be an integer from 10 to 300.
• Multiple (A)n motifs may be the same amino acid sequence as each other, or may be different amino acid sequences.
• Multiple REPs may be the same amino acid sequence as each other, or may be different amino acid sequences.
• artificial fibroins include artificial fibroins derived from the major paravertebrate silk protein produced in the major ampullate gland of spiders as described in WO 2019/194263 (first artificial fibroin), artificial fibroins having a domain sequence with a reduced content of glycine residues (second artificial fibroin), artificial fibroins having a domain sequence with a reduced content of (A)n motifs (third artificial fibroin), artificial fibroins having a reduced content of glycine residues and a reduced content of (A)n motifs (fourth artificial fibroin), artificial fibroins having a domain sequence including a region with a locally high hydrophobic index (fifth artificial fibroin), and artificial fibroins having a domain sequence with a reduced content of glutamine residues (sixth artificial fibroin).
• first artificial fibroins artificial fibroins derived from the major paravertebrate silk protein produced in the major ampullate gland of spiders as described in WO 2019/194263
• the artificial fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This allows the artificial fibroin to be isolated, immobilized, detected, and visualized.
• An example of a tag sequence is an affinity tag that utilizes specific affinity (binding ability, affinity) with other molecules.
• a specific example of an affinity tag is a histidine tag (His tag).
• His tag is a short peptide with a sequence of about 4 to 10 histidine residues, and has the property of specifically binding to metal ions such as nickel, so it can be used to isolate artificial fibroin by chelating metal chromatography.
• a specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 8 (an amino acid sequence including a His tag sequence and a hinge sequence).
• tag sequences such as glutathione-S-transferase (GST), which specifically binds to glutathione, and maltose-binding protein (MBP), which specifically binds to maltose.
• GST glutathione-S-transferase
• MBP maltose-binding protein
• epitope tags that utilize antigen-antibody reactions.
• an antigenic peptide epitope
• epitope tags include HA tags (peptide sequence of influenza virus hemagglutinin), myc tags, and FLAG tags.
• a tag sequence that can be cleaved with a specific protease can also be used.
• a protease By treating the protein adsorbed via the tag sequence with a protease, it is possible to recover the artificial fibroin from which the tag sequence has been cleaved.
• artificial fibroin examples include the artificial fibroin shown in Table 1.
• the artificial fibroin may be an artificial fibroin having at least two or more characteristics of the first artificial fibroin, the second artificial fibroin, the third artificial fibroin, the fourth artificial fibroin, the fifth artificial fibroin, and the sixth artificial fibroin.
• the molecular weight of the artificial fibroin is not particularly limited, but may be, for example, 2 kDa or more and 700 kDa or less.
• the molecular weight of the artificial fibroin according to this embodiment may be, for example, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, 9 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more, and may be 700 kDa or less, 600 kDa or less, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kD
• the component (B1) is a low molecular weight compound
• the polypeptide has a low molecular weight (for example, 30 kDa or less)
• the yield may decrease during purification in the preparation.
• a purification method for a hydrophobic protein there is a risk of problems such as a decrease in purity.
• a protein (PRT2882) having an amino acid sequence shown in SEQ ID NO: 9 in which a hydrophobic tag (for example, GFILGFIL in SEQ ID NO: 9) is introduced at the N-terminus or C-terminus may be used. Since a low molecular weight recombinant protein having an amino acid sequence including such a hydrophobic tag sequence can be obtained with high yield and purity during purification, the above manufacturing method can obtain a polymer compound with a low content of impurities. As a result, the molded body using such a polymer compound as a molding material can advantageously ensure advantages such as better mechanical properties.
• one or more of the amino acid residues constituting the REP may be hydrophobic amino acid residues.
• the REP contains a hydrophobic amino acid residue.
• a hydrophobic amino acid residue means an amino acid residue with a positive hydrophobic index.
• hydrophobic index hereinafter also referred to as "HI"
• HI hydrophobic index
• hydrophobic amino acid residues examples include isoleucine (HI: 4.5), valine (HI: 4.2), leucine (HI: 3.8), phenylalanine (HI: 2.8), methionine (HI: 1.9), and alanine (HI: 1.8).
• the domain sequence has an amino acid sequence corresponding to the insertion of a cysteine residue in REP, compared to naturally occurring fibroin.
• the domain sequence preferably has an amino acid sequence corresponding to a cysteine residue being inserted at a position adjacent to a glycine residue, serine residue, or alanine residue in REP, and more preferably has an amino acid sequence corresponding to a cysteine residue being inserted at a position adjacent to a glycine residue in REP.
• the cysteine residue in REP may be located between the glycine residue, serine residue, or alanine residue and the glycine residue, serine residue, or alanine residue, or may be located between the serine residue and the glycine residue.
• the domain sequence preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to the hydrophobic amino acid residue in REP.
• the hydrophobic amino acid residues are fixed between the molecules by hydrophobic interaction.
• the cysteine residue in REP may be located next to the hydrophobic amino acid residue, may be located between the hydrophobic amino acid residue and an amino acid residue other than the hydrophobic amino acid residue, may be located between the hydrophobic amino acid residue and a glycine residue, a serine residue, or an alanine residue, or may be located between the hydrophobic amino acid residue and a glycine residue.
• the hydrophobic amino acid residue may be one selected from the group consisting of isoleucine residues, valine residues, leucine residues, phenylalanine residues, methionine residues, and alanine residues.
• the domain sequence may have an amino acid sequence corresponding to the insertion of a cysteine residue in the REP located near the N-terminus and/or C-terminus of the domain sequence, as compared with naturally occurring fibroin.
• the molecular chain can be lengthened.
• the REP located near the N-terminus of the domain sequence means the REP located 1st to 3rd from the N-terminus of the domain sequence.
• the cysteine residue may be located in the REP located 1st to 2nd from the N-terminus of the domain sequence.
• the REP located near the C-terminus of the domain sequence means the REP located 1st to 3rd from the C-terminus of the domain sequence.
• the cysteine residue may be located in the REP located 1st to 2nd from the C-terminus of the domain sequence. It is preferable that the cysteine residue is located in the REP located at the most N-terminus and/or the most C-terminus of the domain sequence.
• the domain sequence may have an amino acid sequence corresponding to a cysteine residue inserted in the center or near the center of the REP compared to naturally-occurring fibroin.
• near the center of the amino acid sequence in the REP refers to the first to fifth positions from the amino acid residue located in the center of the REP (the amino acid residue on the N-terminal side when there are two amino acid residues located in the center) toward the N-terminal side, or the first to fifth positions from the amino acid residue located in the center of the REP (the amino acid residue on the C-terminal side when there are two amino acid residues located in the center).
• the cysteine residue may be located in the center of the REP, or may be located in the first to third positions or the first to second positions toward the N-terminal side or C-terminal side from the amino acid residue located in the center of the REP.
• a first segment that includes a polypeptide backbone in which cysteine residues are inserted so that, for example, one cysteine residue is located on each of the N-terminal and C-terminal sides of the domain sequence
• a synthetic polymer can be obtained in which such first segments and second segments are linked so that they are alternately located one by one. Molded articles (e.g., fibers, films, gels, etc.) obtained using this synthetic polymer are expected to have improved elongation.
• a synthetic molecule in which a second segment is linked to such a first segment is expected to have improved solubility in a solvent.
• the hydrophobicity index (hydropathy index) of the REP of the artificial fibroin may be, for example, -0.80, -0.70, -0.06 or more, -0.50 or more, -0.40 or more, -0.30 or more, -0.20 or more, -0.10 or more, 0.00 or more, 0.10 or more, 0.20 or more, 0.22 or more, 0.25 or more, 0.30 or more, 0.35 or more, 0.40 or more, 0.45 or more, 0.50 or more, 0.55 or more, 0.60 or more, 0.65 or more, or 0.70 or more.
• There is no particular upper limit to the hydrophobicity of the REP and it may be 1.0 or less, or 0.7 or less.
• hydrophobiccity of REP is a value calculated by the following method.
• Formula 1 [(A) n motif-REP] m
• Formula 2 [(A) n motif-REP] m- (A) n motif.
• the hydrophobicity of REP is calculated as e/f.
• the reason for targeting the "sequence obtained by removing from the domain sequence the sequence from the (A) n motif located at the most C-terminus side to the C-terminus of the domain sequence" in calculating the hydrophobicity of REP is the same as that described above.
• the domain sequence may have an amino acid sequence corresponding to 1 to less than 16 cysteine residues inserted into the REP compared to naturally occurring fibroin. That is, the total number of cysteine residues corresponding to insertion into the REP may be 1 to less than 16.
• the total number of cysteine residues corresponding to insertion into the REP may be 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 2 to 4.
• the number of cysteine residues per REP in the domain sequence may be, for example, 1 to 3, 1 to 2, or 1.
• the total number of cysteine residues in the artificial fibroin of this embodiment may be 1 or more and less than 16, 1 or more and less than 12, 1 or more and less than 10, 1 or more and less than 8, 1 or more and less than 6, or 2 or more and less than 4.
• the artificial fibroin according to this embodiment may further have modifications in the amino acid sequence equivalent to the substitution, deletion, insertion and/or addition of one or more amino acid residues compared to naturally occurring fibroin.
• the artificial fibroin according to this embodiment preferably has an amino acid sequence corresponding to the insertion of a cysteine residue.
• the method for preparing component (B1) may be a method that includes esterifying maleic anhydride with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates.
• an acid or a base may be used, and the reaction may be carried out without a solvent.
• Purification may be performed, for example, by washing with a solvent such as cyclopentyl methyl ether, tetrahydrofuran, ethyl acetate, or ethanol to remove impurities.
• a second embodiment of the method for producing a polymer compound includes reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, having two structures represented by the following general formula (2) (hereinafter also referred to as component (B2)), by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor (hereinafter also referred to as component (E)).
• component (B2) a polymerization inhibitor
• R 1 represents a hydrogen atom or a methyl group.
• the heating reaction is a reaction between component (A) and component (B2), and is carried out, for example, by heating to 50° C. or higher.
• the timing of heating is not limited to after components (A) to (E) are mixed.
• components (A) to (E) may be added to preheated dimethyl sulfoxide; components (C), (D), and (E) may be added to dimethyl sulfoxide, heating may be initiated, and after a predetermined temperature is reached, components (A) and (B) may be added and reacted; or one of components (A) and (B) and components (C), (D), and (E) may be added, heating may be initiated, and after a predetermined temperature is reached, the remaining one of components (A) and (B) may be added and reacted.
• polyethers having two structures represented by formula (2) include polyethylene glycol, polytetramethylene glycol, polypropylene glycol, and ethylene glycol-propylene glycol copolymers.
• polyesters having two structures represented by formula (2) include polylactic acid, polyglycolic acid, polybutylene succinate, polycaprolactone, and polyhydroxyalkanoates including 3-hydroxybutanoic acid-3-hydroxyhexanoic acid copolymers.
• polycarbonates having two structures represented by formula (2) include aliphatic polycarbonates such as polyethylene carbonate, polypropylene carbonate, and polytrimethylene carbonate. These compounds have easier molecular motion than the above-mentioned polypeptides, and therefore, when they are the above-mentioned compounds, the flexibility of the molded article made of the obtained polymer compound can be further improved.
• component (B2) With regard to the amount of component (B2) to be blended, the explanation given for component (B1) in the first embodiment above can be interpreted as being for component (B2) and applied.
• the polymerization inhibitor (component (E)) in the above-mentioned manufacturing method is a compound that inhibits the self-polymerization of component (B2).
• component (E) include hydroquinone, methoquinone, 4-tert-butylpyrocatechol, tert-butylhydroquinone, 1,4-benzoquinone, dibutylhydroxytoluene, methoquinol, phenothiazine, and 1,1-diphenyl-2-picrylhydrazyl.
• the content of the (E) component may be, for example, 0.01 parts by mass or more, 0.05 parts by mass or more, 0.1 parts by mass or more, or 0.2 parts by mass or more, relative to 100 parts by mass of the above compound (component (B2)).
• component (B2) By setting the content of the polymerization inhibitor within the above range, the initiation of radical polymerization of the structure represented by general formula (2) is suppressed, and it may be possible to make the reaction between the structure represented by general formula (2) and the mercapto group of the polypeptide more dominant.
• the content of the (E) component may be, for example, 25 parts by mass or less, 10 parts by mass or less, 5 parts by mass or less, or 1 part by mass or less, relative to 100 parts by mass of component (B2). By setting the content of the (E) component within the above range, it is possible to further suppress the (E) component from inhibiting the reaction in the system.
• the method for preparing component (B2) may be a method that includes esterifying (meth)acrylic acid, (meth)acrylic anhydride, (meth)acryloyl chloride, and at least one compound selected from the group consisting of polyether, polyester, and polycarbonate.
• the preparation of component (B2) does not require the introduction of a protective group into the polyether, and a simple method can be adopted. Purification was performed by removing organic salts through hot extraction with tetrahydrofuran, and then adding n-hexane to cause reprecipitation.
• the polymer compound synthesized by the above-mentioned production method has a structure in which a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded by a structure represented by the following general formula (3), (4), or (5) so that the polypeptide portion is bonded to S in the following general formula (3), (4), or (5).
• M represents any one of H (hydrogen atom), Na (sodium atom), K (potassium atom), NHEt3 , or NHEtiPr2
• R1 represents H or Me.
• the Me represents a methyl group
• the Et represents an ethyl group
• the iPr represents an isopropyl group.
• the above-mentioned polymer compound can also be called a block copolymer since it has a polypeptide and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure. If the structural unit consisting of the polypeptide is A and the structural unit containing at least one of the polyether structure, the polyester structure, and the polycarbonate structure is B, the above-mentioned polymer compound only needs to have at least one each of A and B.
• the above-mentioned polymer compound may be, for example, an A-B diblock copolymer, an A-B-A triblock copolymer, an A-B-A-B tetrablock copolymer, an A-B-A-B-A pentablock copolymer, etc., or may be a copolymer such as A-A-B-B-B-A-B-B-A-A-A.
• the polymer compound may be a linear polymer or a comb polymer, and may have a three-dimensional network structure. From the viewpoint of further improving the flexibility of the molded article made of the polymer compound, it is preferable that the polymer be a linear polymer or a comb polymer, and from the viewpoint of improving the elastic modulus of the molded article, it is preferable that the polymer have a three-dimensional network structure.
• the position of at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure that is introduced into the polypeptide portion is not particularly limited, and may be the end of the polypeptide portion or a position other than the end of the polypeptide portion, but is preferably the end of the polypeptide portion in terms of ease of production of the polymer compound.
• the polymer compound synthesized by the above-mentioned manufacturing method can be used as a molding material.
• the shape of the molded product using the above-mentioned polymer compound as a molding material is not particularly limited, and may be, for example, a film, fiber, etc.
• One aspect of the present disclosure provides a method for producing a polymer solution, which includes dissolving the polymer obtained by the above-mentioned production method in a solvent.
• the polymer solution obtained by such a method can be used as a so-called dope liquid when preparing a molded body.
• solvents used for preparing the dope liquid include formic acid, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, hexafluoro-2-propanol, and N-methyl-2-pyrrolidone.
• the target polymer is obtained in a state dissolved in dimethyl sulfoxide (as a dimethyl sulfoxide solution) by the above-mentioned heating reaction. Therefore, the above-mentioned production method for a polymer solution includes a step of dissolving the polymer extracted from dimethyl sulfoxide by, for example, a known extraction method or separation method, in a predetermined solvent.
• a polymer film may be produced by a method including a step of forming a film from the polymer compound solution obtained by the above-mentioned method for producing a polymer compound solution.
• the forming into the film may be, for example, cast molding.
• Polymer fibers may be produced by a method including a step of spinning the polymer solution obtained by the above-mentioned method for producing a polymer solution.
• the dimethyl sulfoxide solution of the polymer compound obtained by the heating reaction in the above-mentioned method for producing a polymer compound can be used as it is as a dope for producing a film or fiber. This allows the polymer film to be produced more easily and at low cost.
• the film can also be formed by, for example, cast molding.
• One aspect of the present disclosure provides a method for producing a film, comprising: reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (1), by heating in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution (a dimethyl sulfoxide solution of a polymer compound), and forming a film from the polymer compound solution.
• M represents any one of H, Na, K, NHEt3 , and NHEtiPr2 .
• One aspect of the present disclosure provides a method for producing a film, comprising: reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (2), by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor to obtain a polymer compound solution (a dimethyl sulfoxide solution of a polymer compound), and forming a film from the polymer compound solution.
• R 1 represents a hydrogen atom or a methyl group.
• One aspect of the present disclosure provides a method for producing a fiber, comprising: reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (1), by heating in dimethyl sulfoxide in the presence of a base and a reducing agent to obtain a polymer compound solution (a dimethyl sulfoxide solution of a polymer compound), and spinning the polymer compound solution.
• M represents any one of H, Na, K, NHEt3 , and NHEtiPr2 .
• One aspect of the present disclosure provides a method for producing a fiber, comprising: reacting a polypeptide having at least one mercapto group with at least one compound selected from the group consisting of polyethers, polyesters, and polycarbonates, the compound having two structures represented by the following general formula (2), by heating in dimethyl sulfoxide in the presence of a base, a reducing agent, and a polymerization inhibitor to obtain a polymer solution (a dimethyl sulfoxide solution of a polymer), and spinning the polymer solution.
• R 1 represents a hydrogen atom or a methyl group.
• One aspect of the present disclosure provides a film comprising a polymer compound, the polymer compound having a structure in which a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded via a structure represented by the following formula (3), (4), or (5) such that the polypeptide portion is bonded to S in the following formula (3), (4), or general formula (5), the film having an elongation of 400% or more and a ratio of breaking strength to yield strength exceeding 1.
• M represents any one of H, Na, K, NHEt3 , or NHEtiPr2
• R1 represents H or Me.
• One aspect of the present disclosure provides a fiber comprising a polymer compound, the polymer compound having a structure in which a polypeptide portion and at least one structural unit selected from the group consisting of a polyether structure, a polyester structure, and a polycarbonate structure are directly bonded to each other by a structure represented by the following formula (3), (4), or (5) such that the polypeptide portion is bonded to S in the following formula (3), (4), or general formula (5), the fiber having an elongation of 400% or more and a ratio of breaking strength to yield strength of more than 1.
• R1 represents a hydrogen atom or a methyl group.
• the ratio of breaking strength to yield strength is greater than 1, but may be, for example, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, or 1.5 or more. In the above film and fiber, the ratio of breaking strength to yield strength (breaking strength [MPa]/yield strength [MPa]) may be, for example, 3 or less, or 2 or less.
• the polymer compound according to this embodiment can be used as an adhesive for adhering an adherend or as a coating agent for laminating and forming a coating layer on a substrate, in the form of a solution, aqueous dispersion, film, or powder, containing the polymer compound as a main component.
• solution-type adhesives and solution-type coating agents, water-dispersible adhesives and water-dispersible coating agents, film-type adhesives and film-type coating agents, and powder-type adhesives and powder-type coating agents can be obtained, for example, by the following manufacturing methods.
• a solution-type adhesive or solution-type coating agent containing a polymer compound can be manufactured by a method including a step of dissolving the polymer compound obtained by the method of this embodiment in a solvent. According to such a method, unlike conventional solution-type adhesives or solution-type coating agents containing petroleum-derived components, a solution-type adhesive or solution-type coating agent having biodegradability can be easily obtained.
• the target polymer compound is obtained in a state dissolved in dimethyl sulfoxide (as a dimethyl sulfoxide solution) by the above-mentioned heating reaction.
• the manufacturing method of the solution-type adhesive or coating agent according to this embodiment includes a step of dissolving the polymer compound extracted from dimethyl sulfoxide by, for example, a known extraction method or separation method in a predetermined solvent.
• the solution-type adhesive or solution-type coating agent according to this embodiment may be composed of a dimethyl sulfoxide solution of the polymer compound obtained by the heating reaction in the manufacturing method of the polymer compound.
• Solvents used in the manufacture of solution adhesives and solution coating agents include aqueous media such as water in which polymer compounds can be dissolved, basic aqueous solutions, acidic aqueous solutions, and neutral aqueous solutions containing inorganic salts, as well as organic solvents.
• organic solvents include formic acid, dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpiperidone (NMP), and dihydrolevoglucosenone.
• the polymer compound contained in the solution adhesive or solution coating agent is obtained by the manufacturing method according to this embodiment, and is not particularly limited as long as it is soluble in the aqueous medium or organic solvent described above.
• An adhesive or coating agent in which a polymer compound is dissolved in an aqueous medium has the advantage of being easier to handle than an adhesive or coating agent in which a polymer compound is dissolved in an organic solvent.
• the polymer compound according to this embodiment has a molecule that can plasticize a protein bound to the protein, and therefore has higher water solubility than a protein.
• an aqueous solution adhesive or aqueous solution coating agent in which a polymer compound is dissolved in an aqueous medium can have a higher protein content than an aqueous solution adhesive or aqueous solution coating agent in which a protein is simply dissolved in an aqueous medium. Therefore, by using an aqueous solution adhesive or aqueous solution coating agent in which a polymer compound is dissolved in an aqueous medium, it is expected that the adhesive strength of the adhesive or coating agent to the adherend surface of the adherend or the laminate surface described below will be improved compared to when an aqueous solution adhesive or aqueous solution coating agent in which a protein is dissolved in an aqueous medium is used.
• the concentration of the polymer compound in the solution adhesive or solution coating agent is appropriately determined based on the solubility of the polymer compound in the solvent, etc. Such a concentration may be, for example, 5 to 40 mass%, 10 to 35 mass%, 15 to 20 mass%, or 20 to 25 mass%.
• the solution adhesive or solution coating agent may contain components other than the polymer compound, such as various additives contained in known solution adhesives or solution coating agents, as necessary.
• the upper limit of the above concentration may also be, for example, less than 30 mass%, 28 mass% or less, 25 mass% or less, or 23 mass% or less.
• a solution-type adhesive containing a polymer compound is used to manufacture an adhesive in which multiple adherends are bonded together.
• the solution-type adhesive is placed between multiple adherends to be bonded, and then the solvent in the solution-type adhesive is removed and the polymer compound is solidified to bond the adherends together to obtain an adhesive.
• This method has the advantage that an adhesive can be easily manufactured, since an adhesive that is biodegradable can be used to obtain an adhesive that reduces the environmental impact when disposed of.
• the material of the adherend is not particularly limited as long as it can be bonded with a solution-type adhesive containing a polymer compound, and may be an organic substance (e.g., cellulose products such as paper and wood, synthetic resins, protein products, etc.) or an inorganic substance (non-metallic substances such as metal and glass).
• organic substance e.g., cellulose products such as paper and wood, synthetic resins, protein products, etc.
• inorganic substance non-metallic substances such as metal and glass
• the specific method for producing an adhesive using a solution-type adhesive is not limited in any way.
• the solution-type adhesive may be applied or dripped onto at least one of the adherends to be bonded to each other, or the adherend surface of the adherend may be brought into contact with or immersed in the liquid surface of the solution-type adhesive to make the solution-type adhesive present on the adherend surface, and then the adherends may be overlapped or butted together with their adherend surfaces to interpose the solution-type adhesive between the adherends.
• the adherends when removing the solvent in the solution-type adhesive interposed between the adherends to solidify the polymer compound, the adherends may be heated, air-dried, or naturally dried while applying pressure to at least one of the overlapping or butting directions of the adherend surfaces so that the overlapped or butted adherend surfaces are in close contact with each other.
• pressure may be applied to at least one of the overlapping or butting directions of the adherend surfaces so that the overlapped or butted adherend surfaces are in close contact with each other.
• the solution-type coating agent containing a polymer compound is used to manufacture a laminate in which a coating layer is formed on the entire or part of the surface of a substrate.
• the solution-type coating agent is supplied to at least a part of the surface of the substrate on which the coating layer is to be formed, and the surface part of the substrate is coated with the solution-type coating agent, and then the solvent in the solution-type adhesive is removed to solidify the polymer compound.
• a laminate can be obtained by forming a coating layer on at least a part of the surface of the substrate.
• This method also has the advantage that a laminate can be easily manufactured, which can reduce the environmental load at the time of disposal, because a laminate can be obtained using a biodegradable coating agent.
• the material of the substrate is not particularly limited as long as it is capable of being adhered to the solution-type coating agent containing a polymer compound, and may be the same as the adherend to which the solution-type adhesive is adhered.
• the specific method for producing a laminate using a solution-type coating agent is not limited in any way.
• the solution-type coating agent may be applied or dropped onto at least a part of the substrate surface, or at least a part of the substrate surface may be brought into contact with or immersed in the liquid surface of the solution-type coating agent, thereby supplying the solution-type coating agent to at least a part of the substrate to form a layer of a predetermined thickness.
• the solvent in the coating agent layer may be removed, for example, by heating, air-drying, or naturally drying the coating agent layer on the substrate surface.
• a predetermined pressing body (pressurizing body) may be placed on the coating agent layer so as to cover the entire surface of the coating agent layer, and the coating agent layer may be pressed (pressurized) toward the substrate surface so that the coating agent layer adheres to the substrate surface.
• the pressing body does not adhere to the solution-type coating agent.
• the adhesion of the solution-type coating agent to the pressing body can be prevented by applying a release agent to the contact surface of the pressing body with the coating agent layer, attaching release paper to the contact surface, or by subjecting the contact surface to a surface treatment that prevents the coating agent from adhering, or by using a pressing body made of a material that does not adhere to the solution-type coating agent.
• a water-dispersible adhesive or water-dispersible coating agent containing a polymer compound can be manufactured by a method including a step of dispersing the polymer compound obtained by the method of this embodiment in an aqueous medium. According to such a method, unlike conventional water-dispersible adhesives or water-dispersible coating agents containing petroleum-derived components, a water-dispersible adhesive or water-dispersible coating agent having biodegradability can be easily obtained. As described above, in the method for manufacturing a polymer compound according to this embodiment, the target polymer compound is obtained in a state dissolved in dimethyl sulfoxide (as a dimethyl sulfoxide solution).
• the method for manufacturing a water-dispersible adhesive or water-dispersible coating agent includes a step of dispersing the polymer compound extracted from dimethyl sulfoxide by, for example, a known extraction method or separation method, in a predetermined aqueous medium.
• aqueous media used in the manufacture of water-dispersible adhesives and water-dispersible coating agents include water in which polymer compounds can be dispersed, basic aqueous solutions, acidic aqueous solutions, and aqueous solutions containing inorganic salts.
• the polymeric compound contained in the water-dispersible adhesive or water-dispersible coating agent is obtained by the manufacturing method according to this embodiment, and is not particularly limited as long as it is dispersible in the aqueous medium described above.
• the content of the polymeric compound in the water-dispersible adhesive or water-dispersible coating agent is appropriately determined taking into consideration the adhesiveness of the water-dispersible adhesive to the adherend, and the adhesiveness or fixation of the water-dispersible coating agent to the substrate.
• the water-dispersible adhesive or water-dispersible coating agent may contain components other than the polymeric compound, such as various additives contained in known water-dispersible adhesives or water-dispersible coating agents, as necessary.
• the polymer compound according to this embodiment has a higher affinity for aqueous media containing water than proteins, since the molecules capable of plasticizing proteins are bound to the proteins. Therefore, water-dispersible adhesives and water-dispersible coating agents containing such polymer compounds can have a higher protein content (dispersion amount) and can disperse the polymer compound uniformly in the aqueous medium, compared to water-dispersible adhesives and water-dispersible coating agents that are simply formed by dispersing proteins in an aqueous medium.
• Water-dispersible adhesives containing polymer compounds are used to manufacture an adhesive in which multiple adherends are bonded together. For example, a water-dispersible adhesive is placed between multiple adherends to be bonded, and then the aqueous medium in the water-dispersible adhesive is removed and the polymer compound is solidified, thereby bonding the adherends together to obtain an adhesive.
• This method has the advantage that an adhesive can be easily manufactured, since an adhesive that is biodegradable can be used to obtain an adhesive that reduces the environmental impact when disposed of.
• the material of the adherend is not particularly limited as long as it can be bonded with a water-dispersible adhesive containing a polymer compound, and it may be the same as an adherend bonded with a solution-based adhesive containing a polymer compound.
• the specific method for producing an adhesive using a water-dispersible adhesive is not limited in any way.
• a method similar to that used when interposing the above-mentioned solution-type adhesive between multiple adherends can be used.
• a method similar to that used when removing the solvent from the solution-type adhesive interposed between the adherends and solidifying the polymer compound can be used.
• the water-dispersible coating agent containing a polymer compound is used to manufacture a laminate in which a coating layer is formed on the entire or part of the surface of a substrate.
• the water-dispersible coating agent is supplied to at least a part of the surface of the substrate on which the coating layer is to be formed, and the surface part of the substrate is coated with the solution-like coating agent, and then the aqueous medium in the water-dispersible adhesive is removed to solidify the polymer compound.
• a laminate can be obtained by forming a coating layer on at least a part of the surface of the substrate.
• This method also has the advantage that a laminate can be easily manufactured, since a laminate can be obtained using a biodegradable coating agent, which can reduce the environmental load when discarded.
• the material of the substrate is not particularly limited as long as it is capable of adhering to the water-dispersible coating agent containing a polymer compound, and may be the same as the substrate to which the coating layer is formed with the solution-like coating agent described above.
• the specific method for producing a laminate using a water-dispersible coating agent is not limited in any way.
• a method similar to that used when supplying the solution-like coating agent to the substrate surface described above can be used.
• a method similar to that used when removing the solvent from the solution-like coating agent supplied to the substrate surface to solidify the polymer compound can be used.
• a film-like adhesive or film-like coating agent containing a polymer compound can be manufactured by a method including a step of forming a film from a polymer compound solution in which the polymer compound obtained by the method of this embodiment is dissolved. According to such a method, unlike conventional film-like adhesives and film-like coating agents containing petroleum-derived components, a film-like adhesive or film-like coating agent having biodegradability can be easily obtained.
• the method for forming the above-mentioned film may be known cast molding.
• a film forming solution used in the production of a film adhesive or a film coating agent for example, a polymer compound solution used as a solution adhesive or a solution coating agent is preferably used. Therefore, such a film forming solution may be a dimethyl sulfoxide solution of a polymer compound obtained by the method for producing a polymer compound according to this embodiment, or may be a solution obtained by dissolving a polymer compound extracted from such dimethyl sulfoxide by, for example, a known extraction method or separation method in a specified solvent.
• known methods and known conditions similar to those used when casting a protein solution may be used.
• the film-like adhesive containing a polymer compound is used to manufacture an adhesive in which multiple adherends are bonded together.
• the film-like adhesive is placed between multiple adherends to be bonded, and then the film-like adhesive is swelled or heated to soften it, and then the film-like adhesive is pressed against the adherends and cured, thereby bonding the adherends together to obtain an adhesive.
• the film-like adhesive may be softened by swelling or heating beforehand, and then placed between multiple adherends, and then the film-like adhesive may be pressed against the adherends and cured.
• the film-like adhesive softens when it absorbs moisture or is heated, and hardens when it is subsequently dried or cooled.
• the polymer compound contained in the film-like adhesive has the property of shrinking when contacted with water or heated, especially when it is formed into a molded body, the film-like adhesive also shrinks when contacted with water or heated. For this reason, when a film-like adhesive containing a polymer compound is placed between adherends in a swollen or heated and softened state, it partially bites into the gaps present on the adherend's surface, and in particular, if the decorative protein has the above-mentioned shrinkage property, it shrinks while biting into the gaps on the adherend's surface.
• the film-like adhesive hardens in this state, the film-like adhesive and the adherend are bonded together by the anchor effect.
• the film-like adhesive exhibits sufficient flexibility by containing as its main component a polymer compound in which a molecule that plasticizes the protein is bonded to the protein. Therefore, such a film-like adhesive exhibits higher flexibility when softened, and is therefore expected to be able to penetrate more fully into the gaps on the adherend's surface, resulting in even higher adhesive strength.
• the film-like adhesive has excellent flexibility, it can be peeled off from the adherend after adhering to the adherend, and furthermore, it exhibits the excellent function of being able to be reused as a film-like adhesive.
• the film-like adhesive When reusing the film-like adhesive, it may be washed with water after being peeled off from the adherend. It is also thought that the adhesion of the film-like adhesive to the adherend is due to the formation of hydrogen bonds between the adherend and the polymer compound.
• an aqueous liquid such as water or a solution or dispersion containing water, which is absorbed by the film-like adhesive and can cause the film-like adhesive to shrink
• the above-mentioned aqueous liquid may be dropped onto the film-like adhesive or multiple adherends may be immersed in the aqueous liquid, causing the film-like adhesive to absorb water and thus swelling and softening the film-like adhesive.
• a method may be used in which the film-like adhesive is placed between adherends and then heated and dried, air-dried, or naturally dried.
• the film-like adhesive When the film-like adhesive is heated to soften it, it may be heated before being interposed between the adherends, or the adherends and the film-like adhesive may be heated as a whole after the film-like adhesive is interposed between the adherends.
• the heating temperature There are no particular limitations on the heating temperature, so long as it is a temperature that can soften the film-like adhesive and does not adversely affect (e.g., does not decompose) the proteins contained in the film-like adhesive.
• a method of cooling it with a cooling device or allowing it to cool can be used.
• the film-like adhesive softened by swelling or heating In order for the film-like adhesive softened by swelling or heating to penetrate sufficiently into the gaps in the adherend's surface, it is desirable to bring the interface between the softened film-like adhesive and the adherend into intimate contact. For example, it is preferable to apply pressure to multiple adherends from at least one side in the overlapping or butting direction of the adherend's surfaces, and to press the softened film-like adhesive against the adherend while hardening it. Any common method can be used as the method for applying pressure to the adherends.
• the film-like coating agent containing a polymer compound is used to manufacture a laminate in which a coating layer is formed on the entire or part of the surface of a substrate.
• the film-like coating agent is placed so as to cover at least a part of the surface of the substrate, and the film-like coating agent is swelled or heated to soften it, and then the film-like coating agent is pressed against the substrate surface and hardened to obtain a laminate in which a coating layer is formed on at least a part of the substrate surface.
• the film-like coating agent may be softened by swelling or heating in advance, and then placed so as to cover at least a part of the substrate surface, and then hardened in a state in which the film-like coating agent is pressed against the adherend.
• a laminate can be obtained using a coating agent having biodegradability, and therefore there is an advantage that a laminate that can reduce the environmental load at the time of disposal can be easily manufactured.
• the substrate may be one to which the film-like coating agent containing a polymer compound can be adhered, and may be the same as the adherend to which the film-like adhesive containing a polymer compound is adhered.
• the mechanism by which a film-like coating agent adheres to the surface of a substrate is believed to be similar to the mechanism by which a film-like adhesive adheres to the surface of an adherend. Therefore, the specific method for producing a laminate using a film-like coating agent can be the same as that for obtaining an adhesive using a film-like adhesive.
• the film-like coating agent may be pressed against the surface of the substrate using a pressing body similar to the pressing body used when pressing the solution-like coating agent coated on the substrate against the surface of the substrate.
• the film-like coating agent is not only expected to have high adhesive strength to the substrate, but can also be peeled off after adhesion and can be reused.
• a powder composition containing a powder of a polymer compound obtained by the method of this embodiment is used for a powder adhesive or powder coating agent containing a polymer compound.
• This powder composition may contain sub-components such as various residual additives, so long as it contains a polymer compound as the main component.
• the target polymer compound is obtained in a state dissolved in dimethyl sulfoxide (as a dimethyl sulfoxide solution). Therefore, the method for producing a powder adhesive or powder coating agent according to this embodiment includes a step of powdering the polymer compound extracted from dimethyl sulfoxide by, for example, a known extraction method or separation method, by a known method.
• the polymer compound contained in the powder adhesive or powder coating agent is not particularly limited as long as it is obtained by the manufacturing method according to this embodiment.
• any of the polymer compounds contained in the solution adhesive or solution coating agent, the water-dispersible adhesive or water-dispersible coating agent, and the film adhesive or film coating agent may be used.
• the polymer compound according to this embodiment has a higher affinity for an aqueous medium (aqueous liquid) containing water than proteins.
• the powder adhesive or powder coating agent containing such a polymer compound can have a higher protein content (dispersion amount) and can disperse the polymer compound uniformly in the aqueous medium compared to the water-dispersible adhesive or water-dispersible coating agent in which the protein is simply dispersed in the aqueous medium. Therefore, by using the water-dispersible adhesive or water-dispersible coating agent in which the polymer compound is dispersed in the aqueous medium, the adhesive strength of the adhesive or coating agent to the adherend surface of the adherend or the laminate surface described later can be expected to be improved compared to the case in which the water-dispersible adhesive or water-dispersible coating agent in which the protein is dispersed in the aqueous medium is used.
• Powdered adhesives containing modified proteins are also used to produce an adhesive in which multiple adherends are bonded together.
• the powdered adhesive can be heated and pressed through the adherends to solidify, thereby bonding the adherends together to produce an adhesive.
• This method has the advantage that an adhesive can be easily produced that reduces the environmental impact when disposed of, since an adhesive can be obtained using a biodegradable adhesive.
• the adherends may be any that can be bonded with a powdered adhesive containing a polymer compound, and may be similar to adherends that are bonded with a solution-based adhesive containing a polymer compound, for example.
• the specific method for producing an adhesive using a powdered adhesive is not limited in any way.
• the adherends are sandwiched between metal plates or the like from both sides opposite the adherend surface, and the metal plates are heated to heat the powdered adhesive between the adherends together with the adherends.
• the metal plate is pressed with a hand press or the like, and the powdered adhesive is pressed through the adherends for a predetermined period of time. This causes the powdered adhesive to resinify and solidify, adhering the adherends.
• the heating temperature, amount of pressure, and heating and pressurizing time for solidifying the powdered adhesive are appropriately selected from within the ranges that can resinify the modified protein, depending on the type of modified protein contained in the powdered adhesive.
• Powdered coating agents containing polymeric compounds are also used to manufacture laminates in which a coating layer is laminated over the entire or part of the surface of a substrate.
• the powdered coating agent is placed on at least a part of the surface of a substrate, and the powdered coating agent is heated and pressed between a pressurizing body and the substrate to solidify, thereby forming a coating layer on at least a part of the surface of the substrate.
• the specific method for producing a laminate using a powder coating agent is not limited in any way.
• a metal plate or the like is placed so as to cover the entire powder adhesive placed on the surface of the substrate, and the metal plate is heated to heat the powder adhesive.
• the powder coating agent is resinified and solidified by pressing the powder adhesive between the metal plate and the substrate for a predetermined period of time using a hand press or the like.
• the heating temperature and amount of pressure, or the heating and pressing time, of the powder coating agent are the same as those used when obtaining an adhesive body using a powder adhesive.
• the average hydropathic index value of the artificial fibroin having the amino acid sequence shown in SEQ ID NO:9 is 0.47.
• a nucleic acid encoding an artificial fibroin having the amino acid sequence shown in SEQ ID NO:9 was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the polypeptide expression vector pET-22b(+) to obtain an expression vector.
• the resulting expression vector was used to transform E. coli BLR (DE3).
• 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 2) containing ampicillin so that the OD 600 was 0.005.
• the culture solution temperature was kept at 30° C., and flask culture was continued until the OD 600 reached 5 (about 15 hours) to obtain a seed culture solution.
• the seed culture was added to a jar fermenter containing 500 mL of production medium (Table 3) so that the OD 600 was 0.05.
• the culture temperature was kept at 37° C., and the pH was controlled to be constant at 6.9.
• the dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration.
• the feed solution (glucose 455 g/1 L, yeast extract 120 g/1 L) was added at a rate of 1 mL/min.
• the culture temperature was kept at 37°C, and the culture was controlled to a constant pH of 6.9.
• the dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours.
• 1 M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of artificial fibroin.
• IPTG isopropyl- ⁇ -thiogalactopyranoside
• the cells harvested 20 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4).
• the washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi).
• the disrupted cells were centrifuged to obtain a precipitate.
• the resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure.
• the washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and stirred with a stirrer at 60 ° C. for 30 minutes to dissolve. After dissolution, the solution was dialyzed against water using a dialysis tube (Cellulose tube 36/32, manufactured by Sanko Junyaku Co., Ltd.).
• a dialysis tube Cellulose tube 36/32, manufactured by Sanko Junyaku Co., Ltd.
• the white aggregated polypeptide obtained after dialysis was collected by centrifugation, the water was removed using a freeze-dryer, and the freeze-dried powder was collected to obtain powdered artificial fibroin (PRT2882). It was confirmed that disulfide bonds were formed between molecules of the artificial fibroin into which cysteine residues had been inserted. It was confirmed that the artificial fibroin contained 13.8 parts by mass of dimers, 2.1 parts by mass of trimers, and 0.8 parts by mass of tetramers per 100 parts by mass of monomers. The formation of disulfide bonds was measured by SDS-PAGE.
• Example I-1 In a two-necked flask, 1 g (0.1 mmol) of artificial fibroin (polypeptide, 10 kDa) having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 prepared as described above and 12.5 mg (0.08 mmol) of dithiothreitol (DTT) as a reducing agent were added, and 12.5 g of dimethyl sulfoxide (DMSO) was added, and the mixture was heated and stirred at 70°C for 30 minutes to prepare solution A containing the polypeptide. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the mercapto group of the polypeptide.
• DTT dithiothreitol
• Solution A and solution B were mixed, and 110 ⁇ L (0.08 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 7 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the amount of base was adjusted to 8 parts by mass based on the total mass of the polypeptide.
• the reaction solution was heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to remove the precipitate, and a solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bonded was obtained.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Industrial Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• Example I-1 The reaction solution prepared in Example I-1 was degassed by centrifugation, and 3 g of the degassed reaction solution was dropped and spread on a disposable tray (manufactured by AS ONE Corporation, product name: Balance Dish SCC, external dimensions 80 mm x 80 mm x 24 mm). Thereafter, the solution was pre-dried at 60°C for 8 hours, and further dried under reduced pressure at 80°C for 3 hours to evaporate the solvent, thereby producing a film having a thickness of 0.09 mm.
• the measurement conditions for the GPC measurement were as follows: Equipment: Agilent 1260 Infinity II liquid chromatography system (Agilent Technologies, Inc.) Detector: Agilent 1260 Infinity II reflective index detector (RID) (Agilent Technologies, Inc.) Column: 0.5 ⁇ m guard column filter (Shodex GPC HK-G, Showa Denko K.K.), styrene-divinylbenzene copolymer column (Shodex GPC HK404L x 2, inner diameter: 4.6 mm x length: 150 mm, Showa Denko K.K.) Eluent: TFANa-HFIP solution (85.3 mg of sodium trifluoroacetate (TFANA) dissolved in 1000 g of hexafluoroisopropanol (HFIP, Central Glass Co., Ltd.)) ⁇ Flow rate: 0.15mL/min ⁇ Measurement temperature: 40°C
• Example I-2 A polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bound was obtained in the same manner as in Example I-1, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 4.
• Example I-3 In a two-necked flask, 1 g (0.1 mmol) of the artificial fibroin (polypeptide, 10 kDa) having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 prepared as described above and 7.2 mg (0.08 mmol) of butanediol as a reducing agent were added, and 9.0 g of dimethyl sulfoxide (DMSO) was added, and the mixture was heated and stirred at 70°C for 30 minutes to prepare solution C containing the polypeptide. The amount of the reducing agent was adjusted to 0.8 equivalents relative to the mercapto group of the polypeptide.
• DMSO dimethyl sulfoxide
• Solution C and solution D were mixed, and 110 ⁇ L (0.08 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 7 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the amount of base was adjusted to 8 parts by mass based on the total mass of the polypeptide.
• the reaction solution was heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to remove the precipitate, and a solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bonded was obtained.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Kogyo Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• Example I-4 A solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety are bound was obtained in the same manner as in Example I-2, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 4.
• Example I-5 1 g (0.1 mmol) of the polyethylene glycol bisacrylate prepared as described above and 25 mg (0.23 mmol) of hydroquinone (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) as a polymerization inhibitor were weighed into a flask, and 9.0 g of dimethyl sulfoxide (DMSO, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added and dissolved by heating and stirring at 70° C. for 10 minutes. In this way, solution E containing polyethylene glycol bisacrylate was prepared.
• DMSO dimethyl sulfoxide
• the solution A prepared in the same manner as in Example I-1 was mixed with the above solution E, and 110 ⁇ L (0.08 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the polypeptide was adjusted to 7 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the reaction solution was then heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to remove the precipitate, and a solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bonded was obtained.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Kogyo Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• Examples I-6 to I-7) A solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety are bound was obtained in the same manner as in Example I-5, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 4.
• the amount of dimethyl sulfoxide refers to the total amount in the solution after mixing (e.g., the sum of the amount of DMSO in solution A and the amount of DMSO in solution B).
• the solution A prepared in the same manner as in Example I-1 was mixed with the above solution F, and 110 ⁇ L (0.08 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 7 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the reaction solution was then heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to obtain a polymer compound in which the polypeptide portion and the polyethylene glycol portion were bonded as a precipitate.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Industrial Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• the above solution G and solution H were mixed, and 110 ⁇ L (0.08 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 7 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the reaction solution was then heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to obtain a polymer compound in which a polypeptide portion and a polyethylene glycol portion were bonded as a precipitate.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Industrial Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• Comparative Examples I-1 and I-2 the post-reaction solutions obtained were evaluated in the same manner as in Example I-1. The results are shown in Table 5.
• Table 5 for Comparative Example 1, the peak intensity ratio in GPC is marked "-", which indicates that the measurement itself could not be performed due to precipitation and gel-like matter in the reaction system.
• Example I-8 The reaction solution prepared in Example I-3 was degassed by centrifugation, and 3 g of the degassed reaction solution was dropped and spread on a disposable tray (As One Corporation, product name: Balance Dish SCC, external dimensions 80 mm x 80 mm x 24 mm). Thereafter, the solution was dried at 60°C for 8 hours, and further dried under reduced pressure at 80°C for 3 hours to evaporate the solvent, thereby producing a film having a thickness of 0.09 mm.
• a disposable tray As One Corporation, product name: Balance Dish SCC, external dimensions 80 mm x 80 mm x 24 mm.
• ⁇ Film evaluation> The film prepared as described above was measured for breaking elongation, elastic modulus, and toughness using a tensile tester (manufactured by Shimadzu Corporation, product name: AG-X plus 50kN). The film was stored for 24 hours or more under an environment of 20°C and relative humidity: 65% before use. After storage, a dumbbell-shaped test piece was prepared by punching using a punching machine (manufactured by Tester Sangyo Co., Ltd., product name: SA-1008) so that the film distance at the measurement site was 25 mm and the width was 10 mm.
• the test piece was set in a tensile tester, and a tensile test was performed under the conditions of a load cell: 50 kN, a gripper distance: 25 mm, and a tensile speed: 10 mm/min. The results are shown in Table 6.
• Example I-9 A film was prepared in the same manner as in Example I-8, except that the polymer material was changed to that prepared in Example I-7. The obtained film was evaluated in the same manner as in Example I-8. The results are shown in Table 6.
• Example I-10 100 g of a solution prepared in the same manner as in Example I-1 was gradually dropped into 100 g of ethyl acetate prepared in a 300 mL beaker while stirring.
• the precipitate formed in the beaker was finely pulverized using a homogenizer (manufactured by IKA, product name: T-18 Digital).
• the precipitate was then collected by suction filtration.
• the collected precipitate was poured into 100 g of ethyl acetate and stirred and washed. After washing with ethyl acetate twice, the precipitate was collected by filtration and vacuum dried at 40° C. for 2 hours to obtain a powder of a purified polymer compound.
• the viscosity of the dope solution was measured by filling the dope solution into a glass tube containing a spherical probe ( ⁇ 4.7 mm) using an EMS viscometer (Kyoto Electronics Manufacturing Co., Ltd., product name: EMS-1000S) at a measurement temperature of 45°C.
• the syringe pump kept at 45°C was operated to extrude the dope at a linear velocity of 1.00 m/min.
• the filament was passed through a drying line at 275°C via two rollers and wound up by a winder with a rotation speed of 15 m/min to obtain a fiber (filament).
• the mass per meter of the wound fiber was measured, and the mass per 10,000 m (fineness, unit: dtex) was calculated.
• a sample was prepared by bundling five of the above filaments, and cross-sectional analysis was performed using a polarizing microscope (Nikon Corporation, product name: ECLIPSE LV100ND) to determine the circular equivalent diameter of the five filaments, and the arithmetic average was used as the circular equivalent diameter of the filament.
• ⁇ Fiber evaluation: stress relaxation measurement> The stress relaxation of the sample obtained by bundling five filaments was evaluated using a tensile tester (manufactured by Shimadzu Corporation, product name: AG-X plus 50kN). The sample was stored for 24 hours or more in an environment of 20°C and early withdrawal humidity: 65% before use. The sample was set in the tester, and a tensile test was carried out under the conditions of load cell: 50kN, gripper distance: 50mm, and tensile speed: 50cm/min. At this time, the stress X when the 50mm sample was stretched 300% was measured. Then, the sample was held in that state for 30 seconds, and the stress Y immediately after the holding was measured. This measurement was carried out three times, and the stress relaxation was calculated using the formula (X-Y)/Xx100, and the arithmetic average value was taken as the stress relaxation. The results are shown in Table 7.
• Example II-1 To 3.13 g of a solution containing a polymer compound in which a polypeptide portion and a polyethylene glycol portion are bonded, prepared in the same manner as in Example I-1, 50 mg of a water-soluble thermoplastic resin (manufactured by Meisei Chemical Industry Co., Ltd., product name: Alkox E-75) was added, and the mixture was stirred at 70° C. for 1 hour to prepare a DMSO solution (dope solution). The viscosity of the obtained dope solution at a temperature of 25° C. was 4130 mPa ⁇ s. The viscosity of the dope solution here is a value measured at a measurement temperature of 25° C. using a digital viscometer (manufactured by Ametech Co., Ltd., product name: Brookfield DV2TLVTJ0).
• the above dope solution was transferred to a 100 mL glass beaker.
• the dope was then stirred slowly using a stainless steel spatula to avoid introducing air bubbles, and the dope was stretched into fibers by slowly lifting it up, taking advantage of its viscosity.
• the stretched fibrous dope was placed on a release film and vacuum dried for 15 hours in a vacuum oven at 80°C to obtain a filament (a polymer compound formed into a fiber shape).
• a sample was prepared by bundling five of the above filaments, and cross-sectional analysis was performed using a polarizing microscope (Nikon Corporation, product name: ECLIPSE LV100ND) to determine the circular equivalent diameter of the five filaments, and the arithmetic mean was used as the circular equivalent diameter of the filament.
• Example I-10 The five resulting filaments were bundled together into a sample, and the fibers were evaluated in the same manner as in Example I-10. The results are shown in Table 8. In Table 8, "-" indicates that no measurement was performed.
• Examples III-1 to III-4 A solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety are bound was obtained in the same manner as in Example I-1, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 9.
• Example III-1 to III-4 the resulting post-reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 9.
• Examples III-5 to III-6 A solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety are bound was obtained in the same manner as in Example I-1, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 10.
• Example III-5 to III-6 the resulting post-reaction solutions were evaluated in the same manner as in Example I-1. The results are shown in Table 10.
• Examples III-9 to III-11 A solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety are bound was obtained in the same manner as in Example I-1, except that the components, their amounts, and the reaction temperature were adjusted as shown in Table 11.
• Example III-9 to III-11 the post-reaction solutions obtained were evaluated in the same manner as in Example I-1. The results are shown in Table 11. For reference, Table 11 also shows the results of Examples I-1 and III-2.
• Example I-11 ⁇ Production of Film Adhesive>
• 2.5 g (0.25 mmol) of artificial fibroin (polypeptide, 10 kDa) having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 prepared as described above and 31.2 mg (0.2 mmol) of dithiothreitol (DTT) as a reducing agent were added, and 25 g of dimethyl sulfoxide (DMSO) was added, and the mixture was heated and stirred at 70°C for 30 minutes to prepare solution I containing the polypeptide.
• the amount of the reducing agent was adjusted to 0.8 equivalents relative to the mercapto group of the polypeptide.
• Solution I and solution J were mixed, and 275 ⁇ L (0.2 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was further added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 6 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the amount of base was adjusted to 8 parts by mass based on the total mass of the polypeptide.
• the reaction solution was heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to degas it, and a DMSO solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bonded was obtained.
• the centrifugation was performed using a micro high-speed refrigerated centrifuge (Tomy Kogyo Co., Ltd., product name: MX-307) at 15,000 rpm for 10 minutes.
• the DMSO solution of the obtained polymer compound was dropped onto a stainless steel plate covered with a PET sheet (Teijin Film Solutions Limited, Purex, A54 type, thickness 38 ⁇ m) and spread using a frame applicator with an analog micrometer (Imoto Manufacturing, 250 mm coating width, 1 mm gap) and a coating machine (Imoto Manufacturing, IMC-7370, coating speed 10 mm/1 sec). It was then dried at 60°C for 8 hours and further dried under reduced pressure at 80°C for 3 hours to evaporate the DMSO, yielding a film-like adhesive with a thickness of 0.08 mm.
• a PET sheet Teijin Film Solutions Limited, Purex, A54 type, thickness 38 ⁇ m
• a frame applicator with an analog micrometer (Imoto Manufacturing, 250 mm coating width, 1 mm gap) and a coating machine (Imoto Manufacturing, IMC-7370, coating speed 10 mm/1 sec). It was then dried at 60°C for 8 hours and further dried under reduced pressure at 80°C for 3 hours to evaporate the
• the film-like adhesive obtained as described above was cut to 15 mm x 15 mm, and the cut film-like adhesive was immersed in water for 5 minutes to swell and soften.
• the softened film-like adhesive was placed on one end of a wooden board (made of cypress, 1.5 mm x 40 mm x 2 mm) in an area of 15 mm x 15 mm, and another wooden board of the same size was placed on top of it so that the film-like adhesive was interposed between the two wooden boards.
• the two wooden boards were fixed with clips so that the film-like adhesive was pressed against each wooden board, and placed in a constant temperature thermostat and dried at 60 ° C for 1 hour to harden the film-like adhesive.
• ⁇ Tensile test of adhesive bond> The thus obtained bonded body A and bonded body B were each set in a tensile tester (AG-X plus 50kN, manufactured by Shimadzu Corporation) at room temperature of 20°C and humidity of 65%, and a tensile test was carried out. The tensile test was carried out under the conditions of a load cell of 50kN, a gripping distance of 57.5mm, and a tensile speed of 10mm/min. As a result, the tensile strength of bonded body A was 10 times or more greater than the tensile strength of bonded body B. This demonstrated that the film-like adhesive has a sufficiently large adhesive force (adhesive strength) compared to commercially available double-sided tapes and the like.
• Example I-12 ⁇ Production of Powder Adhesive> 319.3 g of dimethyl sulfoxide (DMSO) was weighed out into a three-necked separable flask, and 419 mg (2.72 mmol) of dithiothreitol (DTT) was added as a reducing agent and stirred to dissolve. Then, 27.5 g (2.75 mmol) of artificial fibroin (polypeptide, 10 kDa) having the amino acid sequence (PRT2882) shown in SEQ ID NO: 9 prepared as described above was added, and the mixture was heated and stirred at 70°C for 30 minutes to prepare solution K containing the polypeptide. The amount of the reducing agent was adjusted so that it was approximately 1 equivalent to the mercapto group of the polypeptide.
• DMSO dimethyl sulfoxide
• DTT dithiothreitol
• Solution K and solution L were mixed, and 4,400 mg (43.5 mmol) of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a base to prepare a reaction solution.
• the amount of polypeptide in the mixed solution was adjusted to 5.5 parts by mass per 100 parts by mass of dimethyl sulfoxide.
• the amount of base was adjusted to 16 parts by mass based on the total mass of the polypeptide.
• the reaction solution was heated and stirred at 70°C for 90 minutes to allow the reaction to proceed.
• the solution after the reaction was centrifuged to remove the precipitate, and a DMSO solution containing a polymer compound in which a polypeptide moiety and a polyethylene glycol moiety were bonded was obtained. The centrifugation was performed at 15,000 rpm for 10 minutes.
• the obtained DMSO solution of the polymer compound was washed in acetone and centrifuged five times, and then the supernatant was removed by decantation to separate the precipitate.
• the obtained precipitate was spread on a tray and dried in a draft for several hours, and then dried overnight in a vacuum oven at 40°C to obtain a powder of the polymer compound.
• the residual DMSO in this powder was 3% by mass or less.
• the obtained powder of the polymer compound was used as a powdered adhesive.
• the present disclosure provides a manufacturing method that can increase the raw material conversion rate in the production of polymer compounds as described above.

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