Block copolymer, or method for producing aqueous dispersion, adhesive, coating liquid, coating material or molded body containing block copolymer

Abstract

Provided is a method for producing a block copolymer, the method including a step for mechanochemically treating a mixture containing a protein and a molecule capable of plasticizing the protein.

Description

Technical Field

[0001] The present invention relates to a block copolymer or a method for producing an aqueous dispersion, adhesive, coating liquid, paint or molded body containing the block copolymer. Background Art

[0002] Because structural proteins such as silk fibroin and spider silk fibroin exhibit excellent strength, they have attracted attention as alternatives to structural materials composed of synthetic resins, etc. In recent years, with the development of recombinant technology, mass production technologies for recombinant structural proteins that mimic these structural proteins are being developed (e.g., Patent Document 1).

[0003] Generally speaking, proteins have a structure composed of multiple amino acids, with a continuous structure of amino acids forming the main chain. Proteins have amide groups (peptide bonds) on their main chains, and polar groups such as carboxyl groups, amide groups, amino groups, hydroxyl groups, and thiol groups on their side chains. Proteins maintain their three-dimensional structure through the formation of numerous intramolecular hydrogen bonds, and their physical properties can be altered by the formation of intermolecular hydrogen bonds.

[0004] Molded articles such as resins obtained by heat-pressing structural proteins are considered to have excellent mechanical strength, but they are also brittle and prone to cracking and fissures due to impact, etc. This situation has become an obstacle to the use of proteins as a substitute for general-purpose plastics.

[0005] Similar to general-purpose plastics technology, while adding plasticizers to structural proteins can improve flexibility, plasticizers are generally hydrophobic and therefore have low compatibility with polar group-rich proteins. Consequently, even when plasticizers are mixed with structural proteins, phase separation occurs between the structural protein and the plasticizer, making it difficult to achieve the desired effect.

[0006] In view of this, the inventors discovered that if the following synthetic polymer (block copolymer) is used, which has a first segment containing a polypeptide backbone and one or more second segments bonded to the first segment, wherein the second segment contains a molecule that has a plasticizing function on the polypeptide backbone, then phase separation between the protein and the plasticizer can be avoided, and a molded body with excellent flexibility can be prepared (see Patent Document 2).

[0007] [Prior art literature]

[0008] [Patent Document]

[0009] Patent Document 1: International Publication No. 2015 / 178466

[0010] Patent Document 2: International Publication No. 2021 / 187502

[0011] Patent Document 3: International Publication No. 2019 / 194263 Summary of the Invention

[0012] [Problems to be solved by the invention]

[0013] Further research by the present inventors revealed that even the block copolymers described in Patent Document 2 require dissolution in an organic solvent for preparation, and the available organic solvents are limited by the type of protein, which may reduce the freedom of reaction design. Furthermore, residual organic solvent may still remain in the prepared block copolymers.

[0014] Therefore, the object of the present invention is to provide an optimized preparation method of a novel block copolymer. In addition, another object of the present invention is to provide an optimized production method of an aqueous dispersion, an adhesive, a coating liquid, a coating, or a molded body.

[0015] Methods used to solve problems

[0016] The present inventors have discovered that a powdered block copolymer can be obtained by forming a protein block copolymer under mechanochemical conditions. In other words, the present invention provides the following [1] to

[21] .

[0017] [1]

[0018] A method for producing a block copolymer comprises the step of subjecting a mixture containing a protein and a molecule capable of plasticizing the protein to a mechanochemical treatment.

[0019] [2]

[0020] The method according to [1], wherein the block copolymer has a protein and a molecule capable of plasticizing the protein as unit structures, respectively.

[0021] 〔3〕

[0022] The method according to [1] or [2], further comprising a step of freeze-drying the mechanochemically treated product after the mechanochemical treatment step.

[0023] [4]

[0024] The method according to [1], wherein the number of reactive functional groups contained in the molecule capable of plasticizing the protein is greater than the number of reactive functional groups contained in the protein.

[0025] 〔5〕

[0026] The method according to any one of [1] to [4], wherein the amount of the molecule capable of plasticizing the protein used is greater than the amount of the protein used on a molar basis.

[0027] [6]

[0028] The method according to [5], wherein the reactive functional group in the protein is a nucleophilic functional group, and the reactive functional group in the molecule capable of plasticizing the protein is an electrophilic functional group.

[0029] [7]

[0030] The method according to [6], wherein the nucleophilic functional group is a thiol group and the electrophilic functional group is a thiol-reactive group.

[0031] 〔8〕

[0032] The method according to any one of [1] to [7], wherein the protein includes a hydrophobic protein.

[0033] 〔9〕

[0034] The method according to [8], wherein the hydrophilic index (Hydropathy Index) of the hydrophobic protein is greater than 0.

[0035]

[10]

[0036] The method according to any one of [1] to [9], wherein the protein comprises an artificial protein.

[0037]

[11]

[0038] According to the method described in

[10] , the artificial protein includes an artificial structural protein.

[0039]

[12]

[0040] According to the method described in any one of [1] to

[11] ,

[0041] Among them, proteins contain two or more nucleophilic functional groups, and molecules that can plasticize proteins contain two or more electrophilic functional groups.

[0042]

[13]

[0043] The method according to any one of [1] to

[12] ,

[0044] Among them, proteins contain two or more thiol groups, and molecules that can plasticize proteins contain two or more thiol-reactive groups.

[0045]

[14]

[0046] The method according to any one of [1] to

[13] , wherein the molecular weight of the molecule capable of plasticizing the protein is 10 or greater, when the molecular weight of the protein is 100.

[0047]

[15]

[0048] The method according to any one of [1] to

[14] , wherein the molecular weight of the molecule capable of plasticizing the protein is 100 or less, when the molecular weight of the protein is 100.

[0049]

[16]

[0050] A method for producing a prepolymer comprises the step of subjecting a mixture containing a protein and a molecule capable of plasticizing the protein to a mechanochemical treatment.

[0051]

[17]

[0052] The method according to

[16] , wherein the block copolymer has a protein and a molecule capable of plasticizing the protein as unit structures respectively.

[0053]

[18]

[0054] The method according to

[16] or

[17] , further comprising a step of freeze-drying the mechanochemically treated product after the mechanochemical treatment step.

[0055]

[19]

[0056] The method according to any one of

[16] to

[18] , wherein the number of reactive functional groups contained in the molecule capable of plasticizing the protein is greater than the number of reactive functional groups contained in the protein.

[0057] 〔20〕

[0058] The method according to any one of

[16] to

[19] , wherein the amount of the molecule capable of plasticizing the protein used is greater than the amount of the protein used on a molar basis.

[0059] 〔twenty one〕

[0060] The method according to

[16] to

[20] , wherein the reactive functional group in the protein is a nucleophilic functional group, and the reactive functional group in the molecule capable of plasticizing the protein is an electrophilic functional group.

[0061] 〔twenty two〕

[0062] The method according to

[21] , wherein the nucleophilic functional group is a thiol group and the electrophilic functional group is a thiol-reactive group.

[0063] 〔twenty three〕

[0064] The method according to any one of

[16] to

[22] , wherein the prepolymer is in powder form.

[0065] 〔twenty four〕

[0066] A prepolymer comprising a block copolymer having a protein and a molecule capable of plasticizing the protein as unit structures.

[0067] 〔25〕

[0068] The prepolymer according to

[24] is in powder form.

[0069] 〔26〕

[0070] An adhesive comprising a block copolymer having a protein and a molecule capable of plasticizing the protein as unit structures.

[0071] 〔27〕

[0072] The adhesive according to

[26] is in powder form.

[0073] 〔28〕

[0074] A method for producing an aqueous dispersion of a block copolymer, comprising dispersing the block copolymer obtained by the method described in any one of [1] to

[15] in an aqueous medium.

[0075] 〔29〕

[0076] A method for producing a water-dispersible adhesive, comprising dispersing a block copolymer obtained by the method described in any one of [1] to

[15] in an aqueous medium.

[0077] 〔30〕

[0078] A method for producing a coating liquid, comprising dispersing a block copolymer obtained by the method described in any one of [1] to

[15] in an aqueous medium.

[0079] 〔31〕

[0080] A method for producing a molded article, comprising the step of molding a block copolymer obtained by the method described in any one of [1] to

[15] .

[0081] 〔32〕

[0082] The method according to

[31] , wherein the molding step is a step of heating and pressurizing the block copolymer.

[0083] 〔33〕

[0084] A method for producing an aqueous dispersion of a block copolymer, comprising the following steps:

[0085] A process for obtaining block copolymers by mechanochemical treatment of a mixture containing a protein and a molecule capable of plasticizing the protein;

[0086] A step of dispersing the obtained block copolymer in an aqueous medium.

[0087] 〔34〕

[0088] A method for producing a water-dispersible adhesive comprises the following steps:

[0089] A process for obtaining block copolymers by mechanochemical treatment of a mixture containing a protein and a molecule capable of plasticizing the protein;

[0090] A step of dispersing the obtained block copolymer in an aqueous medium.

[0091] 〔35〕

[0092] A method for producing a coating liquid comprises the following steps:

[0093] A process for obtaining block copolymers by mechanochemical treatment of a mixture containing a protein and a molecule capable of plasticizing the protein;

[0094] A step of dispersing the obtained block copolymer in an aqueous medium.

[0095] 〔36〕

[0096] A method for manufacturing a molded body, comprising the following steps:

[0097] A process for obtaining block copolymers by mechanochemical treatment of a mixture containing a protein and a molecule capable of plasticizing the protein;

[0098] The obtained block copolymer is subjected to a step of heating and pressurizing the block copolymer.

[0099] 〔37〕

[0100] The method according to

[36] , wherein the molding step is a step of heating and pressurizing the block copolymer.

[0101] 〔38〕

[0102] A method for producing a solution-state adhesive, comprising dissolving a block copolymer obtained by the method described in any one of [1] to

[15] in a solvent.

[0103] 〔39〕

[0104] A method for producing a film-like adhesive, comprising the step of forming a film from a solution obtained by dissolving a block copolymer obtained by the method described in any one of [1] to

[15] .

[0105]

[40]

[0106] A method for producing a powdered adhesive, comprising the step of obtaining a powder composition containing a block copolymer obtained by the method described in any one of [1] to

[15] .

[0107]

[41]

[0108] A method for producing an adherend, characterized in that: a block copolymer obtained by any one of the methods described in any one of [1] to

[15] is dissolved in a solvent to form a solution, the solution is placed between a plurality of adherends, the solvent is removed from the solution, and the block copolymer is solidified, thereby adhering the adherends to each other.

[0109]

[42]

[0110] A method for producing an adherend, characterized in that: a block copolymer obtained by any one of the methods described in any one of [1] to

[15] is dispersed in an aqueous medium to form an aqueous dispersion, the aqueous dispersion is placed between a plurality of adherends, the aqueous medium is removed from the aqueous dispersion, and the block copolymer is solidified, thereby adhering the adherends.

[0111]

[43]

[0112] A method for producing an adherend, characterized in that a film containing a block copolymer obtained by the method described in any one of [1] to

[15] is softened by swelling or heating, the film is placed between a plurality of adherends, and the film is hardened while being pressed against the adherends, thereby bonding the adherends.

[0113]

[44]

[0114] A method for producing a bonded body, characterized in that a powder composition containing a block copolymer obtained by the method described in any one of [1] to

[15] is placed between a plurality of adherends, the powder composition is heated, and simultaneously pressurized through the adherends to cure the powder composition, thereby bonding the adherends.

[0115]

[45]

[0116] A method for producing a solution coating, comprising dissolving a block copolymer obtained by the method described in any one of [1] to

[15] in a solvent.

[0117]

[46]

[0118] A method for producing a water-dispersible coating, comprising dispersing a block copolymer obtained by the method described in any one of [1] to

[15] in an aqueous medium.

[0119]

[47]

[0120] A method for producing a film-like coating material, comprising the step of forming a film from a solution in which a block copolymer obtained by the method described in any one of [1] to

[15] is dissolved.

[0121]

[48]

[0122] A method for producing a powdered coating, comprising the step of obtaining a powder composition containing a block copolymer obtained by the method described in any one of [1] to

[15] .

[0123]

[49]

[0124] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on the surface of the substrate, characterized in that a solution formed by dissolving a block copolymer obtained by any one of the methods described in any one of [1] to

[15] in a solvent is supplied to at least a portion of the surface of the substrate, and after coating at least a portion of the surface of the substrate with the solution, the solvent is removed from the solution and the block copolymer is solidified, thereby forming a coating layer laminated on at least a portion of the surface of the substrate.

[0125] 〔50〕

[0126] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on the surface of the substrate, characterized in that an aqueous dispersion formed by dispersing a block copolymer obtained by any one of the methods described in any one of [1] to

[15] in an aqueous medium is supplied to at least a portion of the surface of the substrate; after coating at least a portion of the surface of the substrate with the aqueous dispersion, the aqueous medium is removed from the aqueous dispersion and the block copolymer is cured, thereby forming a coating layer laminated on at least a portion of the surface of the substrate.

[0127] 〔51〕

[0128] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on the surface of the substrate, characterized in that a film containing a block copolymer obtained by any one of the methods described in any one of [1] to

[15] is softened by swelling or heating, and after being placed on at least a portion of the surface of the substrate, the film is hardened while being pressed against the substrate, thereby laminating the coating layer on at least a portion of the surface of the substrate.

[0129] 〔52〕

[0130] A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on the surface of the substrate, characterized in that a powder composition containing a block copolymer obtained by any one of the methods described in any one of [1] to

[15] is placed on at least a portion of the surface of the substrate, the powder composition is heated, and the powder composition is pressurized between a pressurizing body and the substrate to solidify it, thereby laminating the coating layer on at least a portion of the surface of the substrate.

[0131] Effects of the Invention

[0132] According to the manufacture method of the block copolymer involved in the present invention, it is not necessary to dissolve protein and the molecule that can plasticize protein in an organic solvent. Therefore, not only can a wider variety of proteins be utilized as the raw material of the target block copolymer, but also the manufacturing equipment can be significantly simplified. Therefore, according to the method involved in the present invention, the block copolymer of various structures can be prepared in a simpler and low-cost manner. Moreover, (when liquid-assisted grinding is adopted) the residual amount of organic solvent in the obtained block copolymer can be controlled to zero or a very small amount. In addition, according to the present invention, the following materials can be obtained simply and at low cost: the dispersion of the block copolymer is more uniform, or the adhesive with excellent bonding strength, the coating liquid that can form a film with a more uniform thickness, the coating that can form a coating with a more uniform thickness, the molded body with excellent flexibility and strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 These are photographs showing the appearance of each powder and thin film obtained in Comparative Example 1 and Example 1.

[0134] Figure 2 This is a graph showing the GPC analysis results of the thin film before and after the reaction in Comparative Example 1.

[0135] Figure 3 This is a graph showing the GPC analysis results of the powder before and after the reaction in Example 1.

[0136] Figure 4 This is a graph showing the GPC analysis results of the powder before and after the reaction in Example 2.

[0137] Figure 5 Graphs showing the GPC analysis results of the powders before and after the reaction in Examples 3 and 4.

[0138] Figure 6 This is a graph showing the GPC results of the powder prepared in Example 5.

[0139] Figure 7This is a graph showing the GPC results of the powder prepared in Example 6.

[0140] Figure 8 (A) is a photograph showing the products of Example 7 (left) and Comparative Example 2 (right) dispersed in RO water before freeze-drying. (B) is a photograph showing the appearance of the powders of Example 7 (left) and Comparative Example 2 (right) after freeze-drying. (C) is a photograph showing the film obtained in Comparative Example 3 after pulverization. (D) is a photograph showing the gel obtained in Comparative Example 4 after pulverization.

[0141] Figure 9 (A) shows the particle size and its occupancy ratio (cumulative) of Example 7 (solid line) and Comparative Example 4 (dashed line). (B) shows the particle size and its occupancy ratio of Example 7 (solid line) and Comparative Example 4 (dashed line).

[0142] Figure 10 (A) is a micrograph of the powder of Comparative Example 4; (B) is a micrograph of the powder of Example 7.

[0143] Figure 11 It is a graph showing the turbidity test results of Example 7 and Comparative Example 4.

[0144] Figure 12 (A) is a photograph showing the state of each suspension in Example 8 (left) and Comparative Example 5 (right). (B) is a photograph showing the state of the suspension after the Eppendorf tubes in (A) were inverted.

[0145] Figure 13 These are photographs showing the states of the coating films of Example 9 (left) and Comparative Example 6 (right).

[0146] Figure 14 (A) is a photograph showing the state of the resin film of Example 10; (B) is a photograph showing the state of the resin film of Comparative Example 7.

[0147] Figure 15 (A) is a graph showing the results of a tensile test; (B) is a box plot showing the results of the elongation shown in (A).

[0148] Figure 16 It is a graph showing the GPC results of protein PRT2662 (dashed line), Example 11A (dotted line), and Example 11B (solid line).

[0149] Figure 17 (A), (B), and (C) are photographs showing the appearance of the unmodified protein PRT2882, maleated polyethylene glycol, and the product of Example 12, respectively.

[0150] Figure 18Graph showing GPC chromatograms of unmodified protein PRT2882, maleated polyethylene glycol, and the product of Example 12

[0151] Figure 19 This is a graph showing GPC chromatograms of unmodified protein PRT3463, dialdehyde PEG, Example 13, and Comparative Example 8.

[0152] Figure 20 These are photographs showing the state in which Example 13 and Comparative Example 8 were dissolved in DMSO. DETAILED DESCRIPTION

[0153] Hereinafter, specific embodiments of the present disclosure will be described in detail with reference to the accompanying drawings as appropriate. However, the following embodiments are examples for describing the present disclosure, and the present disclosure is not limited to the following contents.

[0154] Unless otherwise specified, the materials exemplified in this specification may be used alone or in combination of two or more. When there are multiple substances corresponding to each component in the composition, the content of each component in the composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified.

[0155] 〔Block copolymer〕

[0156] The block copolymer involved in the present embodiment is formed by the mutual combination of protein and the molecule that can plasticize the protein. The block copolymer may have a repeating structure consisting of unit structure A and unit structure B, or may not have a repeating structure consisting of unit structure A and unit structure B. Wherein, unit structure A is composed of a monomer of any one of protein and the molecule that can plasticize the protein, and unit structure B is composed of another monomer. In addition, the respective quantities of unit structure A and unit structure B contained in the block copolymer, as well as their combination mode, are not particularly limited. These elements, such as in the block copolymer itself, or in the composition or molded body described below obtained using the block copolymer, can be appropriately selected according to the specific required characteristics, etc. That is to say, the block copolymer can have a repeating structure such as AB diblock copolymer, ABA triblock copolymer, ABAB tetrablock copolymer, ABABA pentablock copolymer, etc., or it can have a non-repeating structure such as AABAB, etc. In addition, in the block copolymer involved in the present embodiment, there is no restriction on the binding position of the molecule that can plasticize the protein in the protein backbone. Such binding sites can be appropriately selected based on specific desired properties, such as in the block copolymer itself or in the composition or molded article produced using the block copolymer (described below). Preferred block copolymers have a structure in which protein-plasticizing molecules are attached to both ends of the protein. Such block copolymers are more suitable as prepolymers because proteins and protein-plasticizing molecules react more readily.

[0157] The block copolymers involved in this embodiment are formed by reacting (binding) reactive functional groups (e.g., nucleophilic functional groups) contained in proteins with reactive functional groups (e.g., electrophilic functional groups) contained in molecules capable of plasticizing proteins. In addition, block copolymers containing modified proteins described below in which reactive functional groups (e.g., electrophilic functional groups) are introduced by chemical modification are formed by reacting (binding) reactive functional groups (e.g., electrophilic functional groups) of the modified proteins with reactive functional groups (e.g., nucleophilic functional groups) of the molecules capable of plasticizing proteins.

[0158] In addition, the number of reactive functional groups (for example, nucleophilic functional groups) contained in the protein and the number of reactive functional groups (for example, electrophilic functional groups) contained in the molecule that can plasticize the protein are not particularly limited and can be appropriately changed according to the structure and form of the target block copolymer. In other words, by appropriately selecting the number of reactive functional groups contained in the protein and the number of reactive functional groups contained in the molecule that can plasticize the protein, a block copolymer with a desired structure and form can be formed. For example, if the number of reactive functional groups contained in the protein and the molecule that can plasticize the protein are the same as each other, the block copolymer obtained is a final form polymer in which the reactive functional groups contained in the protein and the reactive functional groups of the molecule that can plasticize the protein can react completely with each other without any residue. Furthermore, a block copolymer in which the number of reactive functional groups contained in the protein-plasticizing molecule is greater than the number of reactive functional groups contained in the protein can be formed as a prepolymer in which the reactive functional groups of the protein react with the reactive functional groups of the protein-plasticizing molecule, while the protein-plasticizing molecule is formed to contain reactive functional groups that do not react with the reactive functional groups of the protein. Furthermore, the number of reactive functional groups contained in a protein as referred to in this specification refers to the number of reactive functional groups contained in one protein molecule when the block copolymer contains one protein molecule; or, if the block copolymer contains multiple protein molecules, the total number of reactive functional groups contained in each of the multiple protein molecules. In addition, the number of reactive functional groups possessed by a molecule capable of plasticizing proteins as described in this specification refers to: when the molecule capable of plasticizing proteins in the block copolymer is set to be 1 molecule, the number of reactive functional groups possessed by this molecule capable of plasticizing proteins; or, when there are multiple molecules capable of plasticizing proteins in the block copolymer, the total number of reactive functional groups possessed by each of these molecules capable of plasticizing proteins.

[0159] The position of the reactive functional group in the protein or the molecule capable of plasticizing the protein is not subject to any restriction and can be appropriately selected according to the structure and characteristics required by the block copolymer. For example, the reactive functional groups can be respectively located at the two ends of the protein. The reactive functional groups located at the two ends are more likely to appear on the outside of the structure of the protein. Therefore, the protein with reactive functional groups at both ends is more likely to react with the reactive functional groups of the molecule capable of plasticizing the protein, thereby more easily obtaining a block copolymer structure formed by the linear combination of the protein and the molecule capable of plasticizing the protein. Since if the reactive functional groups are both located at the two ends of the protein, the molecule capable of plasticizing the protein can be configured at the two ends of the protein, therefore, when the block copolymer is a prepolymer, the molecule capable of plasticizing the protein is more likely to react with other compounds, making it easier to form a network structure in the reaction utilizing the prepolymer.

[0160] It can be seen from this that the block copolymer involved in the present embodiment can obtain the desired structure and morphology by appropriately selecting the number and arrangement of reactive functional groups contained in proteins or molecules capable of plasticizing proteins. For example, a block copolymer composed of a protein having multiple reactive functional groups and a molecule capable of plasticizing proteins having fewer reactive functional groups can form a structure in which multiple molecules capable of plasticizing proteins are bound to each protein molecule. On the other hand, for example, a block copolymer composed of a molecule capable of plasticizing proteins having multiple reactive functional groups and a protein having fewer reactive functional groups can form a structure in which multiple proteins are bound to each molecule capable of plasticizing proteins. In addition, for example, when a protein with a reactive functional group at each end and a molecule capable of plasticizing proteins are used, the block copolymer formed by the mutual combination of multiple molecules can have the above-mentioned repeating structure or non-repeating structure.

[0161] Furthermore, in the block copolymers of this embodiment, since molecules capable of plasticizing proteins are bound to the proteins, their flexibility allows them to achieve a lower viscosity than proteins in the molten state during heating, pressurization, or shearing processes. Furthermore, the block copolymers, which have undergone viscosity reduction treatment, become flowable, allowing them to be formed into molded articles through extrusion and other methods.

[0162] When the block copolymer according to the present embodiment is extruded, it is not essential to add a plasticizer. However, in order to further improve its fluidity, water or a polyol such as glycol, glycerin, or trimerol may be used.

[0163] The block copolymer according to the present embodiment can be produced by subjecting a mixture containing a protein and a molecule capable of plasticizing the protein to a mechanochemical treatment.

[0164] 〔protein〕

[0165] Proteins can be natural or artificial. Furthermore, they can be modified proteins obtained by chemically modifying natural or artificial proteins. The amino acid sequences of these natural, artificial, and modified proteins are not particularly limited. In this specification, when the term "protein" is used alone, it encompasses natural, artificial, and modified proteins. Examples of proteins used in this embodiment include proteins that can be used for industrial applications and proteins that can be used for medical applications. "Industrially applicable" refers to, for example, various general-purpose materials that can be used indoors or outdoors. Specific examples of proteins that can be used for industrial applications include structural proteins. For example, structural proteins can be proteins with physical properties similar to those required for the intended application. Specific examples of structural proteins include spider silk (spider silk), silkworm silk, keratin, collagen, elastin, elastic protein, and proteins derived from these. The protein used can be artificial silk fibroin or artificial spider silk fibroin (artificially modified spider silk fibroin). Specific examples of proteins that can be used in medicine include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives thereof.

[0166] In this specification, "artificial protein" refers to a protein produced by artificial methods, and its scope includes recombinant proteins and synthetic proteins. Artificial proteins can also be proteins whose domain sequences are different from the amino acid sequences of proteins of natural origin. In addition, "artificial proteins" can be proteins obtained by modifying the amino acid sequence of proteins of natural origin (for example, proteins obtained by modifying the amino acid sequence of cloned natural protein gene sequences), and can also be proteins that are completely artificially designed and synthesized without relying on proteins of natural origin (for example, proteins with the desired amino acid sequence obtained by chemically synthesizing nucleic acids encoding the designed amino acid sequence). In addition, artificial proteins are different from natural proteins in that their amino acid sequences can be freely designed. Therefore, when the artificial protein is used in the molding material or molding body described later, by appropriately designing the amino acid sequence of the artificial protein, the functions, characteristics, physical properties, etc. of the molding material or molding body can be arbitrarily controlled. In addition, since uniform molecular design can be performed frequently, proteins with high homology to the target protein and that meet the target can be stably obtained. Therefore, it is advantageous to achieve stabilization of the quality of the molding material or molded body obtained by using the artificial protein. From this point of view, the use of artificial structural protein as the artificial protein is very advantageous.

[0167] The number of amino acid residues in the protein according to this embodiment is not particularly limited, and may be, for example, 50 or more. Furthermore, the number of amino acid residues may be, for example, 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 also 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. There is a tendency that the fewer the number of amino acid residues, the higher the solubility in a solvent. The preferred number of amino acid residues in the protein is, for example, 100-5000, 150-4500, 200-4000, 250-3500, 300-3000, 350-2500, 400-2000, 450-1500, or 500-1000.

[0168] The molecular weight of the protein involved in this embodiment is not particularly limited, and can be, for example, 2 kDa to 500 kDa. Furthermore, the molecular weight of the protein involved in this embodiment can 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 can be 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less. The preferred molecular weight of the protein can be, for example, 2 kDa to 500 kDa, 3 kDa to 500 kDa, 4 kDa to 500 kDa, 5 kDa to 500 kDa, 6 kDa to 500 kDa, 7 kDa to 500 kDa, 8 kDa to 500 kDa, 9 kDa to 500 kDa, 10 kDa to 500 kDa, 20 kDa to 400 kDa, 30 kDa to 360 kDa, 40 kDa to 360 kDa, 50 kDa to 360 kDa, 60 kDa to 300 kDa, 70 kDa to 300 kDa, 80 kDa to 300 kDa, 90 kDa to 200 kDa, or 100 kDa to 200 kDa.

[0169] The protein involved in the present embodiment comprises at least one nucleophilic functional group, such as serine residues, threonine residues, tyrosine residues, lysine residues and cysteine ​​residues. The modified protein is that the hydroxyl group, lysine residues etc. possessed by the serine residues, threonine residues or tyrosine residues in the protein are acylated. By this acylation reaction, electrophilic functional groups can be introduced into the modified protein. The acylating agent used now, in addition to the position for acylation reaction, can also have electrophilic functional groups such as (methyl) acrylate, (methyl) acrylamide, monomethyl maleate, maleamic acid, maleic diester, maleic diamide, maleimide, alkyl halide in the same molecule, then reactive functional groups can be introduced into the protein. When the protein has serine residues, threonine residues or tyrosine residues, the protein can be acylated more efficiently. From the perspectives of improving the reactivity of the protein with the acylating agent and increasing the productivity of the block copolymer, the total content of serine residues, threonine residues, and tyrosine residues in the protein (the ratio of the total of serine residues, threonine residues, and tyrosine residues to the total number of amino acid residues) can 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 upper limit of the total content of serine residues, threonine residues, and tyrosine residues is not particularly limited, but can be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less, taking into account the amino acid composition of various proteins that can be preferably used in the proteins and block copolymers according to the present embodiment.

[0170] As one aspect of this embodiment, the protein may be a modified protein in which a nucleophilic functional group is introduced into a natural protein or an artificial protein. The introduced nucleophilic functional group may be, for example, a hydroxyl group, an amino group, or a thiol group. The amino group may be a primary amino group (-NH2) or a secondary amino group (-NHR, where R is an alkyl group (e.g., C 1-6 alkyl) or alkenyl (e.g., C 1-6Alkenyl)). In the present embodiment, since the protein has an electrophilic functional group as a reactive functional group, the molecule capable of plasticizing the protein is preferably a protein having a nucleophilic functional group as a reactive functional group. The block copolymer has a structure in which the nucleophilic functional group in the modified protein is combined with the electrophilic functional group in the molecule capable of plasticizing the modified protein. The block copolymer may have a repeating structure consisting of a unit structure A and a unit structure B, or may not have a repeating structure consisting of a unit structure A and a unit structure B. The unit structure A is composed of a monomer of any one of a protein and a molecule capable of plasticizing the protein, and the unit structure B is composed of another monomer. In addition, the respective numbers of the unit structures A and the unit structures B contained in the block copolymer, as well as their combination mode, are not particularly limited. These elements, for example, in the block copolymer itself, or in the composition or molded body described below obtained using the block copolymer, can be appropriately selected according to the specific required properties. That is to say, the block copolymer may have a repeating structure such as AB diblock copolymer, ABA triblock copolymer, ABAB tetrablock copolymer, ABABA pentablock copolymer, etc., or may have a non-repeating structure such as AABAB, etc. In addition, in the block copolymers involved in the present embodiment, there is no restriction on the binding position of the molecule in the protein backbone that can plasticize the protein. Such binding positions, for example, in the block copolymer itself, or in the composition or molded body described below obtained using the block copolymer, can be appropriately selected according to the specific required characteristics. The preferred block copolymer has the following structure: there are molecules that can plasticize the protein at both ends of the protein. This block copolymer is more suitable as a prepolymer because proteins and molecules that can plasticize proteins are more likely to react.

[0171] 〔Hydrophobic protein〕

[0172] Protein can be, for example, a hydrophobic protein. In the case where the protein is a hydrophobic protein, when a block copolymer is used as at least a portion of a raw material to manufacture a molded body, the water resistance of the related molded body is improved, and when the molded body is used as a general-purpose material for industrial use, it is conducive to extending its service life. In addition, when a block copolymer is, for example, combined with a functional substance, by controlling the hydrophobicity or hydrophilicity of the functional substance, the hydrophobicity or hydrophilicity of the entire body of the combination of the functional substance and the block copolymer can be arbitrarily adjusted. When the protein has a hydrophobic situation (that is, when the protein is a hydrophobic protein), compared with the situation where the protein has a hydrophilic situation, the entire body can be converted to the hydrophobic side, and therefore the hydrophobicity or hydrophilicity of the entire body can be controlled in a larger range.

[0173] The hydrophobicity of a protein can be estimated using the average HI value of each amino acid constituting the protein as an indicator. As used herein, the average HI value of a hydrophobic protein is sufficient as long as it is greater than 0. For example, it can be 0.00 or greater, 0.10 or greater, 0.20 or greater, 0.22 or greater, 0.25 or greater, 0.30 or greater, 0.35 or greater, 0.40 or greater, 0.45 or greater, 0.50 or greater, 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. Furthermore, the upper limit is not particularly limited and can be, for example, 1.00 or less or 0.7 or less.

[0174] In addition, the average HI value of a hydrophobic protein and the hydrophobicity of a repeating sequence unit described later can be calculated using known hydrophobicity indices for amino acid residues according to known methods. Known hydrophobicity indices for amino acid residues are shown in Table 1. For example, the hydrophobicity can also be calculated according to the method described in Kyte J, Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132.

[0175] [Table 1]

[0176] amino acids HI amino acids HI Isoleucine (Ile) 4.5 Tryptophan (Trp) -0.9 Valine (Val) 4.2 Tyrosine (Tyr) -1.3 Leucine (Leu) 3.8 Proline (Pro) -1.6 Phenylalanine (Phe) 2.8 Histidine (His) -3.2 Cysteine ​​(Cys) 2.5 Asparagine (Asn) -3.5 Methionine (Met) 1.9 Aspartic acid (Asp) -3.5 Alanine (Ala) 1.8 Glutamine (Gln) -3.5 Glycine (Gly) -0.4 Glutamate (Glu) -3.5 Threonine (Thr) -0.7 Lysine (Lys) -3.9 Serine (Ser) -0.8 Arginine (Arg) -4.5

[0177] The hydrophobic protein preferably has low solubility in a 60°C lithium bromide aqueous solution (concentration: 9 M). In other words, the maximum concentration of the hydrophobic protein when dissolved in a 60°C lithium bromide aqueous solution (concentration: 9 M) can 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. Alternatively, a hydrophobic protein that is completely insoluble in a 60°C lithium bromide aqueous solution (concentration: 9 M) can be used. If the hydrophobic protein has low solubility in a 60°C lithium bromide aqueous solution, the resulting block copolymer is more likely to exhibit good water resistance (especially water resistance suitable for industrial use).

[0178] The hydrophobic protein preferably has a water contact angle of 55° or greater. The water contact angle of the hydrophobic protein is more preferably 60° or greater, 65° or greater, or 70° or greater. The water contact angle can be assessed by forming a film composed of the hydrophobic protein on a substrate, dripping water onto the film, and measuring the contact angle after 5 seconds. When the hydrophobic protein has a water contact angle of 55° or greater, the resulting block copolymer is more likely to have good water resistance (especially water resistance suitable for industrial use).

[0179] The hydrophobic protein is preferably a protein having excellent hot water resistance. For example, the hot water resistance is preferably such that a 5 w / v% aqueous dispersion of the hydrophobic protein is prepared and the dispersion is heated to 100°C for at least 5 hours without decomposing. When the hydrophobic protein is a protein having excellent hot water resistance, the resulting block copolymer is more likely to exhibit excellent water resistance (especially water resistance suitable for industrial use) and hot water resistance.

[0180] 〔Structural proteins〕

[0181] The protein involved in the present embodiment can be, for example, a structural protein. The so-called structural protein is a type of protein that can be used for industrial purposes, and refers to a protein related to the structure of an organism, or a protein that constitutes a structure produced by an organism, or a protein derived from them. Structural protein refers to a protein that self-aggregates under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. In addition, structural protein can also refer to a protein whose motif composed of a characteristic amino acid sequence or amino acid residues is repeated to form the skeleton of an organism and a material. Natural structural proteins, for example, can include fibroin, keratin, collagen, elastin, and elastic protein.

[0182] [Artificial structural protein]

[0183] Structural proteins can also be artificial structural proteins. As used herein, "artificial structural proteins" refer to artificially produced structural proteins, including synthetic proteins and recombinant structural proteins produced by microorganisms using genetic recombination techniques. Artificial structural proteins can also be obtained by partially modifying the amino acid sequence of a naturally occurring structural protein based on factors such as productivity and formability, thereby obtaining modified structural proteins.

[0184] The artificial structural protein according to this embodiment may have a glycine residue content of 10% to 55% based on the number of amino acid residues. In this specification, "glycine residue content" refers to the value represented by the following formula.

[0185] Glycine residue content = (number of glycine residues in the artificial structural protein / total number of amino acid residues in the polypeptide) × 100 (%)

[0186] The number of amino acid residues in the artificial structural protein may be 150 or more. The number of amino acid residues may be, for example, 200 or more or 250 or more, preferably 300 or more, 350 or more, 400 or more, 450 or more, or 500 or more.

[0187] The glycine residue content of the artificial structural protein may be between 10% and 55% based on the number of amino acid residues. For example, the glycine residue content may be 10% to 55%, 13% to 55%, 15% to 55%, 18% to 55%, 20% to 55%, 22% to 55%, or 25% to 55%.

[0188] The artificial structural protein may contain at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine (i.e., any one 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, and 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 (total content) of 40% or more, based on the number of amino acid residues. The total content may be, for example, 45% or more, 50% or more, 55% or more, or 60% or more. The upper limit of the total content is not particularly limited, and for example, it may be 90% or less, 85% or less, or 80% or less.

[0189] In one embodiment, the sum of the serine residue content, the threonine residue content, and the tyrosine residue content of the artificial structural protein, based on the number of amino acid residues, may be 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, or 7% or greater. The sum of the serine residue content, the threonine residue content, and the tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0190] The distribution of serine residues, threonine residues or tyrosine residues in the artificial structural protein involved in this embodiment is even, and the total content of serine residues, threonine residues and tyrosine residues in any consecutive 20 amino acid residues can be greater than 4%, greater than 5%, greater than 10% or greater than 15%, or less than 50%, less than 40%, less than 30% or less than 20%.

[0191] Furthermore, the alanine residue content, the serine residue content, the threonine residue content, and the tyrosine residue content have the same meanings as in the above formula except that the alanine residue is replaced by a glycine residue, a serine residue, a threonine residue, and a tyrosine residue, respectively.

[0192] The artificial structural protein involved in one embodiment can be a protein with a repeating sequence. That is, the artificial structural protein involved in this embodiment can have multiple amino acid sequences (repeating sequence units) with high sequence identity within the artificial structural protein. The number of amino acid residues in the repeating sequence unit is preferably 6 to 200. The total amount of glycine residues, serine residues, glutamine residues and alanine residues relative to the total amount of amino acid residues in the repeating sequence unit can be more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65% or more than 70%. In addition, the sequence identity between repeating sequence units can be, for example, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99%. In addition, the hydrophobicity of the repeating sequence unit (i.e., the sum of the HI values ​​of the amino acids contained in the repeating sequence) can 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. In addition, the upper limit of the hydrophobicity of the repeating sequence unit is not particularly limited, and for example, it can be 1.0 or less, or 0.7 or less.

[0193] An artificial structural protein according to one embodiment may include (A) n In this specification, (A) n A motif is an amino acid sequence consisting mainly of alanine residues. (A) n The number of amino acid residues in the motif may be 2 to 27, or may be an integer from 2 to 20, 2 to 16, or 2 to 12. n The ratio of the number of alanine residues in the motif to the total number of amino acid residues 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). (A) n The total number of alanine residues, serine residues, threonine residues and valine residues in the motif is relative to (A) nThe ratio of the total number of amino acid residues in the motif may be 80% or more, preferably 85% or more, more preferably 90% or more, further 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 residues, serine residues, threonine residues, and valine residues). The multiple (A) residues present in the recombinant structural protein according to this embodiment n The motifs may be identical amino acid sequences or different amino acid sequences. n The motif mainly contains alanine residues, so it is easy to obtain α-helical structure or β-sheet structure. n The motif is contained in the repeating sequence unit, and the artificial structural protein involved in this embodiment repeatedly has these secondary structures. Therefore, as shown below, when the artificial structural protein is processed into the form of a molded body such as a fiber, film or resin, it is expected to exert high strength through these secondary structures.

[0194] Furthermore, when molding structural proteins, amino acids with relatively small side chains are more likely to form hydrogen bonds, making it easier to obtain stronger molded products. Furthermore, since the side chains of alanine and glycine residues are non-polar amino acids, they tend to be positioned inward during the folding process of the resulting polypeptide, facilitating the formation of α-helical or β-sheet structures. Therefore, it is desirable to increase the proportion of amino acids such as glycine and alanine residues. To achieve a molded product with even greater strength, the alanine residue content can be, for example, 10-40%, and can be 12-40%, 15-40%, 18-40%, 20-40%, or 22-40%. To achieve a molded product with even greater strength, the glycine residue content can be, for example, 10-55%, and can be 11-55%, 13-55%, 15-55%, 18-55%, 20-55%, 22-55%, or 25-55%.

[0195] In this specification, the "alanine residue content ratio" refers to the ratio of the number of alanine residues to the total number of amino acid residues constituting a protein, and is a value represented by the following formula.

[0196] Alanine residue content = (number of alanine residues in the protein / total number of amino acid residues in the protein) x 100 (%)

[0197] In addition, the glycine residue content rate, serine residue content rate, threonine residue content rate, proline residue content rate and tyrosine residue content rate have the same meanings as when alanine residues in the above formula are replaced with glycine residues, serine residues, threonine residues, proline residues and tyrosine residues, respectively.

[0198] Structural proteins preferably contain a certain amount of amino acids with larger side chains or amino acids with flexibility uniformly throughout their sequence. Specifically, structural proteins may also contain a motif containing tyrosine, threonine, and proline residues that is repeatedly and periodically incorporated. Such structural proteins can easily hinder the formation of strong intermolecular hydrogen bonds during processing of the resulting molded product, thereby facilitating processing. For example, the total content of proline, threonine, and tyrosine residues in any consecutive sequence of 20 amino acid residues may be 5% or greater, greater than 5.5%, 6.0% or greater, greater than 6.5%, 7.0% or greater, greater than 7.5%, 8.0% or greater, greater than 8.5%, 9.0% or greater, 10.0% or greater, or 15.0% or greater. Furthermore, for example, the total content of proline, threonine, and tyrosine residues in any consecutive sequence of 20 amino acid residues may be less than 50%, less than 40%, less than 30%, or less than 20%.

[0199] In the artificial structural protein according to one embodiment, similar to the above-mentioned proteins, from the viewpoint of improving reactivity with acylating agents and increasing the productivity of the reactive modified protein obtained by reaction with the acylating agent, the total content of serine residues, threonine residues, and tyrosine residues can 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 can be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0200] Artificial structural protein can also be artificial silk fibroin. As silk fibroin, for example, naturally derived silk fibroin can be cited. As naturally derived silk fibroin, for example, silk fibroin produced by insects or spiders can be cited. Natural silk fibroin is a fibrous protein with a molecular weight of about 370,000. It is composed of two subunits and has a high content of glycine residues, alanine residues, serine residues, and tyrosine residues. These amino acid residues account for nearly 90% of the total number of amino acid residues. Natural silk fibroin has two different areas, namely a crystalline region rich in amino acid residues with smaller side chains such as glycine, alanine, and serine, and an amorphous region having amino acid residues with larger side chains such as tyrosine.

[0201] More specific examples of naturally derived silk fibroin include those for which sequence information is registered in NCBI GenBank. For example, identification can be achieved by extracting sequences containing the classification code "INV" (division) in the sequence information registered in NCBI GenBank, and extracting sequences with keywords "spidroin," "ampullar gland," "fibroin," "silk and polypeptide," or "silk and protein" listed in the definition (DEFINITION), extracting character strings specifying products from the CDS, and extracting sequences with a specified character string listed in the tissue type (TISSUE TYPE) from the source (SOURCE).

[0202] In this specification, "artificial silk fibroin" refers to artificially produced silk fibroin (artificial silk fibroin). Artificial silk fibroin may be a silk fibroin having an amino acid sequence different from that of silk fibroin derived from natural sources, or a silk fibroin having an amino acid sequence identical to that of silk fibroin derived from natural sources. Artificial silk fibroin can be produced by a known method, for example, by the method described in International Publication No. 2019 / 194263.

[0203] Artificial silk fibroin may be a fibrous protein having a structure suitable for naturally derived silk fibroin, or may be a silk fibroin having a sequence identical to a repeating sequence found in naturally derived silk fibroin. The "sequence identical to a repeating sequence found in silk fibroin" may actually be a sequence found in naturally derived silk fibroin or a sequence similar thereto.

[0204] "Artificial silk fibroin" may be obtained by modifying the amino acid sequence of naturally derived silk fibroin as long as it has the amino acid sequence specified in the present disclosure (for example, silk fibroin obtained by modifying the amino acid sequence of cloned naturally derived silk fibroin gene sequence). Alternatively, it may be silk fibroin with an artificially designed amino acid sequence independent of naturally derived silk fibroin (for example, silk fibroin having a desired amino acid sequence obtained by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). In addition, silk fibroin with a modified amino acid sequence of artificial silk fibroin may be included in artificial silk fibroin as long as its amino acid sequence is different from that of naturally derived silk fibroin. Examples of artificial silk fibroin include artificial silk (silk) fibroin (fibroin with a modified amino acid sequence of silk fibroin produced by silkworms) and artificial spider silk fibroin (fibroin with a modified amino acid sequence of spider silk protein produced by spiders). Artificial silk fibroin is relatively easy to fibrillate and has high fiber-forming ability. Therefore, as a molding material, it preferably contains artificial spider silk fibroin, and more preferably consists of artificial spider silk fibroin.

[0205] The artificial silk fibroin involved in this embodiment may be a fibroin comprising formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n A protein having a domain sequence represented by a motif. Artificial silk fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal and C-terminal ends of the domain sequence. The N-terminal and C-terminal sequences are not limited thereto and typically do not have a repeating region of the characteristic amino acid motif of silk fibroin and are composed of approximately 100 amino acid residues.

[0206] In this specification, "domain sequence" is a crystallization region that generates fibroin (usually equivalent to the amino acid sequence (A) n motif) and an amino acid sequence of an amorphous region (usually equivalent to the REP of an amino acid sequence) is represented by the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n The amino acid sequence shown in the motif. Here, (A) n The motif represents an amino acid sequence mainly composed of alanine residues, with the number of amino acid residues ranging from 2 to 27. (A) n The number of amino acid residues in the 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. In addition, as long as (A) n The ratio of the number of alanine residues in the motif to the total number of amino acid residues is sufficient to 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). n At least 7 of the motifs may be composed of only alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300, or an integer of 10 to 300. The presence of multiple (A) n The motifs may be the same amino acid sequence or different amino acid sequences. A plurality of REPs may be the same amino acid sequence or different amino acid sequences.

[0207] Specific examples of artificial silk fibroin include artificial silk fibroin (first artificial silk fibroin) derived from the large spindle silk protein produced by the large ampulla gland of spiders described in International Publication No. 2019 / 194263, artificial silk fibroin having a domain sequence with reduced glycine residue content (second artificial silk fibroin), and artificial silk fibroin having reduced (A) n The motif-containing domain sequence of artificial silk fibroin (the third artificial silk fibroin) has a reduced content of glycine residues and (A) n The invention relates to a rayon having a motif content (the fourth rayon), a rayon having a domain sequence partially containing a region with a large hydrophobicity index (the fifth rayon), and a rayon having a domain sequence with a reduced glutamine residue content (the sixth rayon). The definitions of each of the first to sixth rayon proteins are incorporated herein by reference to the contents of International Publication No. 2019 / 194263.

[0208] Artificial silk fibroin can contain a tag sequence at either or both of its N-terminus and C-terminus. This allows for the separation, immobilization, detection, and visualization of artificial silk fibroin. PRT2662, having the amino acid sequence set forth in SEQ ID NO: 8, is a sixth artificial silk fibroin containing a tag sequence.

[0209] As a tag sequence, for example, an affinity tag that utilizes specific affinity (binding property, affinity) with other molecules can be listed. As a specific example of an affinity tag, a histidine tag (His tag) can be listed. The His tag is a short peptide composed of about 4 to 10 histidine residues arranged in an array, and has the property of specifically binding to metal ions such as nickel, and can therefore be used for the separation of artificial silk core protein by chelating metal chromatography. As a specific example of a tag sequence, for example, the amino acid sequence shown in sequence number 9 (an amino acid sequence comprising a His tag sequence and a hinge sequence) can be listed.

[0210] Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose can be used.

[0211] Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding a peptide (epitope) that displays antigenicity as a tag sequence, antibodies against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, and FLAG tags. By utilizing epitope tags, fibroin can be easily purified with high specificity.

[0212] Furthermore, a tag obtained by removing the tag sequence using a specific protease can also be used. Alternatively, the protein adsorbed via the tag sequence can be treated with a protease to recover the fibroin after the tag sequence has been removed.

[0213] Specific examples of rayon include, for example, those listed in Table 2. Specific examples of rayon include PRT2662 (SEQ ID NO: 8), PRT2882 (SEQ ID NO: 10), and PRT3463 (SEQ ID NO: 11).

[0214] [Table 2]

[0215]

[0216] The rayon may be a rayon having at least two or more characteristics among the characteristics of the first rayon, the second rayon, the third rayon, the fourth rayon, the fifth rayon, and the sixth rayon.

[0217] The molecular weight of artificial silk fibroin is not particularly limited, and for example, it can be 2 kDa or more and 700 kDa or less. The molecular weight of artificial silk fibroin involved in this embodiment can 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 can also be 700 kDa or less, 600 kDa or less, 500 kDa or less, 400 kDa or less, less than 360 kDa, 300 kDa or less, or 200 kDa or less.

[0218] [Producing proteins through microbiological methods]

[0219] The protein involved in this embodiment can be produced by microbiological methods. The protein can be produced by referring to the descriptions of International Publication No. 2017 / 188430, International Publication No. 2017 / 188434, International Publication No. 2017 / 222034, International Publication No. 2018 / 025886, and International Publication No. 2019 / 022163.

[0220] Proteins can be produced, for example, by a method comprising the step of causing a host transformed with an expression vector to express a nucleic acid. In addition to direct expression, other expression methods include secretory production and fusion protein expression, using methods described in the second edition of Molecular Cloning. When expressed in yeast, animal cells, or insect cells, proteins can be obtained as polypeptides with attached sugars or sugar chains.

[0221] The protein involved in this embodiment can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, allowing the protein to be produced and accumulated in the culture medium, and then collecting the protein from the culture medium. The method for culturing the host in the culture medium can be carried out according to methods commonly used for host culture.

[0222] Molecules that can plasticize proteins

[0223] The molecule that can be plasticized to protein involved in the present embodiment can contain a partial structure that can plasticize protein and one or more reactive functional groups (for example, electrophilic functional groups). The molecule that can be plasticized to protein is a molecule in which the intermolecular force between the molecules is less than the intermolecular force between the proteins, and when the two are mixed, the flexibility of the material or the elongation at break during bending and stretching can be made higher than that of a simple protein molecule. In addition, the molecule that can be plasticized to protein is preferably a molecule with biodegradability or derived from biomass. In this way, the block copolymer as a whole has biodegradability, and the manufacturing energy consumption of the block copolymer can be further reduced.

[0224] The molecules capable of plasticizing proteins react with the reactive functional groups contained in the protein through their reactive functional groups to form block copolymers. The molecules containing electrophilic functional groups as reactive functional groups and capable of plasticizing proteins react with the nucleophilic functional groups as reactive functional groups in the protein to form block copolymers. In addition, the molecules containing nucleophilic functional groups as reactive functional groups and capable of plasticizing proteins react with the electrophilic functional groups as reactive functional groups introduced into the modified protein to form block copolymers. As mentioned above, there are no restrictions on the number of reactive functional groups contained in the molecules capable of plasticizing proteins and their positions in the molecules capable of plasticizing proteins. The molecules capable of plasticizing proteins have at least one reactive functional group, preferably two or more reactive functional groups. In addition, the molecules capable of plasticizing proteins preferably have a chain chemical structure and have a reactive functional group at each end of their molecular chain. In this way, the reactivity of reactive functional groups contained in proteins with other compounds can be increased.

[0225] Examples of partial structures capable of plasticizing proteins include polyethers, polyesters, polycarbonates, polyamides, polyols, polyolefins, polyoxymethylenes, polyketals, poly(meth)acrylates, silicones, polyurethanes, polyalkyleneimines, phenolic resins, urea-formaldehyde resins, and melamine resins. Among these, polyethers, polyesters, polycarbonates, polyamides, and polyols are preferably suitable as partial structures capable of plasticizing proteins, and polyethers, polyesters, and polycarbonates are particularly preferably used.

[0226] As polyethers, for example, functional groups derived from polyalkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene oxide / propylene oxide copolymers and polybutylene glycol can be listed. In addition, when these polyether groups are contained in the molecules capable of plasticizing proteins, the polyether can be directly bound to the heteroelements (O, N, S) in the ester or thioester or amide groups of the linkers described later contained in the molecules capable of plasticizing proteins as needed. Alternatively, the polyether can also be directly bound to the heteroelements (O, N, S) in the ester group, thioester group or amide group contained in the protein. In this case, the entire polyether can be easily separated from the linker or protein, thereby increasing the biodegradation rate of the polyether group. The preferred partial structure is polyalkylene glycol. As polyalkylene glycols, for example, polyethylene glycol, polypropylene glycol, polybutylene glycol, etc. can be listed.

[0227] When the partial structure capable of plasticizing protein is polyalkylene glycol, the molecular weight of the polyalkylene glycol part can be 1kDa~100kDa, 2kDa~50kDa, 3kDa~25kDa, and the molecular weight of the molecule capable of plasticizing protein (soft segment) can be 100Da~500kDa, 200Da~250kDa, 300Da~125kDa.

[0228] Examples of polyesters include functional groups derived from polyesters such as polylactic acid, poly(3-hydroxybutyric acid), polyhydroxybutyric acid / hydroxyvaleric acid copolymers, polyhydroxybutyric acid / 4-hydroxybutyric acid copolymers, polyhydroxybutyric acid / hydroxycaproic acid copolymers, polytrimethylene terephthalate, butanediol / long-chain dicarboxylic acid copolymers, polyethylene terephthalate, polybutylene succinate, polybutylene succinate-adipate copolymers, polybutylene adipate-terephthalic acid copolymers, polycaprolactone, and poly(trimethylene furandicarboxylate) (PTF). Among these polyesters, the polyester group is preferably a functional group derived from a biomass plastic or biodegradable plastic such as polycaprolactone.

[0229] Examples of the polycarbonate group include functional groups derived from polycarbonates having an aliphatic hydrocarbon chain as a main skeleton, such as 1,6-hexanediol polycarbonate, 1,5-pentanediol polycarbonate, and 1,10-decanediol carbonate.

[0230] Examples of the polyamide group include functional groups derived from polyamides such as nylon 3, nylon 4, nylon 5, nylon 6, nylon 11, and nylon 610. Among the above polyamides, the polyamide group is preferably a functional group derived from biomass plastics or biodegradable plastics.

[0231] Examples of the polyol group (polyvinyl alcohol group, etc.) include functional groups derived from polyols (polyvinyl alcohols, etc.) such as polyvinyl alcohol and ethylene-vinyl alcohol copolymers. Among the above-mentioned polyols, the polyol group is preferably a functional group derived from biomass plastics or biodegradable plastics.

[0232] Examples of electrophilic functional groups include groups reactive toward hydroxyl groups (hydroxy-reactive groups), groups reactive toward amino groups (amine-reactive groups), and groups reactive toward thiol groups (thiol-reactive groups). Specific examples of electrophilic functional groups include at least one of the structural units represented by any one of formulae (1) to (20), and at least one of the structural units represented by any one of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), and (12). From the perspective of improving biodegradability, it is preferred that the electrophilic functional group include at least one of the structural units represented by any one of formulae (1), (2), (3), (4), (5), and (12).

[0233] [Chemical Formula 1]

[0234]

[0235] In formulas (1) to (20), R 2 In formulas (2) to (8) and (13) to (15), Y independently represents an oxygen atom, a sulfur atom or NR 1 , R 1 represents a hydrogen atom, a hydrocarbon group (e.g., C 1-6 alkyl), aryl (e.g., phenyl, naphthyl, pyridyl), carbonyl or sulfonyl. In formulas (2) to (6), (10) to (15), R each independently represents a hydrogen atom, a hydrocarbon group (e.g., a linear or branched C 1-6 In formulas (8) and (16), Z represents a hydrogen atom, a hydrocarbon group (e.g., C 1-6alkyl), aryl (e.g., phenyl, naphthyl, pyridyl), halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom), sulfonate group and fluorine-containing carboxylate group. In formula (19), X represents a halogen atom. The aryl group may be further replaced by a halogen atom, cyano group, nitro group, alkyl (e.g., C 1-6 alkyl), haloalkyl (e.g., trifluoromethyl), etc.

[0236] The following are non-limiting examples of the chemical structure of molecules that can plasticize proteins. For example, when a molecule that can plasticize proteins has one electrophilic functional group, it reacts with a nucleophilic functional group in the protein. For example, when a molecule that can plasticize proteins has two or more electrophilic functional groups, each electrophilic functional group can be reacted with a nucleophilic functional group contained in multiple molecules of the protein, thereby allowing each molecule that can plasticize proteins to bind to multiple proteins. In addition, when a molecule that can plasticize proteins has two or more electrophilic functional groups, at least one of the multiple electrophilic functional groups in the molecule that can plasticize proteins can be made not to react with the nucleophilic functional group in the protein and remain, and the molecule that can plasticize proteins can be combined with the protein. This polymer is suitable for use as a prepolymer.

[0237] [Chemical Formula 2]

[0238]

[0239] Molecules capable of plasticizing proteins preferably have an electrophilic functional group at each end of their protein-plasticizing structure. Particularly preferred molecules capable of plasticizing proteins are the compounds shown below. In the formula, n can be 22 to 2273, 45 to 1136, or 68 to 568.

[0240] [Chemical Formula 3]

[0241]

[0242] The molecular weight of the protein-plasticizing molecule can be, for example, 200-700,000, 250-500,000, 300-400,000, 350-350,000, 400-300,000, 500-200,000, 600-1,000,000, 700-50,000, 800-10,000, 900-7,500, or 200-5,000. When the molecular weight of the protein-plasticizing molecule is 200 or greater, the protein-plasticizing portion of the structure becomes more easily localized within the three-dimensional structure of the block copolymer, thereby ensuring that the material achieves the desired functional level without excessively increasing the weight proportion of the protein-plasticizing molecule. Therefore, there is no need to extend the reaction time or increase the required reaction temperature during the block copolymer manufacturing process. On the other hand, when the molecular weight of the protein-plasticizing molecule is 700,000 or less, the binding reactivity between the protein-plasticizing molecule and the protein is less likely to decrease. In addition, the molecular weight of the protein-plasticizing molecule mentioned above is a weight-average molecular weight, which is usually obtained by a known method using GPC.

[0243] The ratio of the molecular weight of the molecule capable of plasticizing the protein to the molecular weight of the protein can be appropriately adjusted according to the purpose of the block copolymer, etc. The molecular weight of the molecule (the total molecular weight if two or more molecules capable of plasticizing the protein are bound to each protein) is based on the molecular weight of the protein as 100, for example, 1 to 10,000, 1.5 to 9,000, 2 to 8,000, 3 to 7,000, 5 to 5,000, 7 to 3,000, or 10 to 2,000. If the molecular weight of the molecule is less than 1 relative to the molecular weight of the protein, the molded body (for example, fiber, film, resin, etc.) obtained by the block copolymer may lack sufficient plasticity (flexibility). On the other hand, if it is greater than 10,000, the rigidity of the molded body may decrease due to excessive plasticity. Within the above range, a molded body with more excellent flexibility can be prepared. Furthermore, when the molecular weight of the protein is 100, the molecular weight of the molecule may be, for example, 1.0 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more, 5.0 or more, 10 or more, 20 or more, 30 or more, or 40 or more. Furthermore, although the upper limit is not particularly limited, it may be, for example, 200 or less, 150 or less, 100 or less, 80 or less, 70 or less, 60 or less, or 50 or less. Furthermore, when the molecular weight of the protein is 100, the molecular weight of the molecule may be in the range of 1 to 200, or may be 1.5 to 150, 5 to 130, 10 to 100, or 10 to 70. When the ratio of the molecular weight of the protein to the molecular weight of the molecule is within the above range, for example, in a molded article produced using the block copolymer, the overall flexibility or extensibility of the block copolymer can be improved while fully maintaining the properties (e.g., high mechanical strength) attributed to the inclusion of the protein. Furthermore, the above molecular weight ratio of the molecule is calculated based on the weight-average molecular weight, with the molecular weight of the protein being 100.

[0244] In block copolymer, the content ratio of protein and the molecule can be appropriately adjusted according to the purposes of block copolymer. About content ratio, in mass ratio, when the molecule is set to 100, protein can be, for example, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 110, more than 150, more than 200, more than 250, more than 300, more than 300, more than 400, more than 450, more than 500, more than 550, more than 600. The upper limit of the correlation value is not particularly limited and can be less than 1000, less than 900, less than 800, less than 700. By controlling the content ratio of protein and the molecule in block copolymer within the above range, the waste of the molecule can be suppressed, so as to reduce the manufacturing cost of block copolymer. In addition, reducing the content ratio of the molecule relative to protein in block copolymer can be achieved by reducing the molecular weight of protein.

[0245] The molecular weight of the protein is, for example, preferably 200 to 1,000,000, more preferably 300 to 900,000, further preferably 400 to 800,000, further preferably 500 to 700,000, further preferably 600 to 600,000, more preferably 1,000 to 600,000, further preferably 3,000 to 600,000, further preferably 5,000 to 600,000, further preferably 10,000 to 600,000, more preferably 5,000 to 100,000. In addition, if the molecular weight of the protein is less than 200, the protein (hard segment) may be too small relative to the molecule (soft segment) that can plasticize the protein. In this case, the rigidity of the molded body molded using the block copolymer (molding material) becomes small, and it may be difficult to use it as a structural body, for example. On the other hand, when the molecular weight of the protein exceeds 1,000,000, the reactivity of the ligation reaction is reduced, and therefore the reaction cannot be completed within a commercially viable production time as a chemical engineering material, which may result in the localization of unreacted molecules capable of plasticizing the protein remaining in the molded body. In addition, the molecular weight of the protein can be, for example, 1,000 or more, 2,000 or more, 3,000 or more, 4,000 or more, 5,000 or more, 6,000 or more, 7,000 or more, 8,000 or more, 9,000 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, or 100,000 or more. Further, the molecular weight of the protein can be 400,000 or less, less than 360,000, 300,000 or less, or 200,000 or less. If the molecular weight of the protein is below 200,000, or below 100,000, it is expected that the amount of molecules that can plasticize the protein can be minimized in order to improve the flexibility of the block copolymer. In addition, the molecular weight of the protein is the weight average molecular weight.

[0246] The molecular weights used in this specification are values ​​measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This electrophoresis was performed according to the following procedure. First, 200 μL of a 2 M lithium chloride DMSO solution was added to 2 mg of a powdered sample. The sample was heated at 80°C for 60 minutes, and then at 95°C for 10 minutes while stirring to dissolve the sample. The sample was then diluted 50-fold with a 10 M urea solution, diluted 2-fold with sample buffer (Fujifilm Wako Pure Chemical Industries, Ltd.), and heated at 95°C for 5 minutes to denature the protein. Next, an SDS-PAGE gel (Bio-lad) was mounted on an electrophoresis apparatus (Bio-lad), filled with SDS buffer, and connected to a power supply (Biocraft). 10 μL of the denatured sample was added to each well of the SDS-PAGE gel, and a current was applied at 30 mA per well for 30 minutes. After electrophoresis, the SDS-PAGE gel was removed from the apparatus and immersed in Oriole fluorescent gel stain (Bio-lad), with shaking for 1 hour. The gel was then placed on a UV sample tray (Bio-lad), and the stained image was acquired using a GelDocEZ gel imaging device (Bio-lad).

[0247] [Method for producing block copolymer]

[0248] A first embodiment of the present invention is a method for producing a block copolymer, which includes a step of mechanochemically treating a mixture containing a protein and a molecule capable of plasticizing the protein.

[0249] The definition of protein used in the method involved in this embodiment can refer to the definition of protein mentioned above. The protein preferably comprises a hydrophobic protein. In addition, the hydrophilic index of the hydrophobic protein is preferably greater than 0. The protein can be an artificial protein, preferably comprising an artificial structural protein.

[0250] In a specific embodiment, for example, the protein has two or more nucleophilic functional groups, and the molecule capable of plasticizing the protein has two or more electrophilic functional groups. In this embodiment, the nucleophilic functional groups and the electrophilic functional groups are combined to form a block copolymer containing the protein and the molecule capable of plasticizing the protein as monomer units. In a preferred embodiment, the protein has two or more hydroxyl, amino, or thiol groups, and the molecule capable of plasticizing the protein has two or more hydroxyl-reactive groups, amine-reactive groups, or thiol-reactive groups.

[0251] The electrophilic functional group may be, for example, a group represented by formula (1) to (20), preferably a group represented by formula (1), (2), (6) to (8), (16), or (19), and more preferably a group represented by formula (1), (2), or (6). Preferred combinations of nucleophilic functional groups and electrophilic functional groups are as follows: a hydroxyl group and a hydroxyl-reactive group represented by any one of formulas (2) to (9), (13) to (17), and (19); an amino group and an amine-reactive group represented by any one of formulas (2) to (9), (13) to (17), and (19); and a thiol group and a thiol-reactive group represented by any one of formulas (1) to (6), (8), (10) to (15), and (18).

[0252] [Chemical Formula 4]

[0253]

[0254] In formulas (1) to (20), R 2 In formulas (2) to (8) and (13) to (15), Y independently represents an oxygen atom, a sulfur atom or NR 1 , R 1 represents a hydrogen atom, a hydrocarbon group (e.g., C 1-6 alkyl), aryl (e.g., phenyl, naphthyl, pyridyl), carbonyl or sulfonyl. In formulas (2) to (6), (10) to (15), R each independently represents a hydrogen atom, a hydrocarbon group (e.g., a linear or branched C 1-6 In formulas (8) and (16), Z represents a hydrogen atom, a hydrocarbon group (e.g., C 1-6 alkyl), aryl (e.g., phenyl, naphthyl, pyridyl), halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom), sulfonate group and fluorine-containing carboxylate group. In formula (19), X represents a halogen atom. The aryl group may be further replaced by a halogen atom, cyano group, nitro group, alkyl (e.g., C 1-6 alkyl), haloalkyl (e.g., trifluoromethyl), etc.

[0255] The amount of the molecule capable of plasticizing the protein can be used in an amount of 1.5 to 7 equivalents per nucleophilic functional group contained in the protein. The amount of the molecule capable of plasticizing the protein can preferably be 1.5 to 6.5 equivalents, 1.5 to 6 equivalents, 1.5 to 5.5 equivalents, 1.5 to 5 equivalents, 1.5 to 4 equivalents, 2 to 7 equivalents, 2 to 6.5 equivalents, 2 to 6 equivalents, 2 to 5.5 equivalents, 2 to 5 equivalents, 2 to 4.5 equivalents, 2.5 to 7 equivalents, 2.5 to 6.5 equivalents, 2.5 to 6 equivalents, 2.5 to 5.5 equivalents, 2.5 to 5 equivalents or 2.5 to 4.5 equivalents per nucleophilic functional group. "Equivalent per nucleophilic functional group" refers to the molar equivalent of the molecule capable of plasticizing the protein relative to one nucleophilic functional group contained in the protein. By controlling the amount of the protein-plasticizing molecule within the above range, the reactivity and even the reaction efficiency between the protein and the protein-plasticizing molecule during the mechanochemical treatment can be improved, thereby more efficiently obtaining the target block copolymer.

[0256] As mentioned above, when manufacturing a prepolymer, by making the number of reactive functional groups (e.g., electrophilic functional groups) possessed by the molecules capable of plasticizing proteins in the mixture excessive relative to the number of reactive functional groups (e.g., nucleophilic functional groups) contained in the protein, it is possible to retain unreacted reactive functional groups in the molecules capable of plasticizing proteins after the binding reaction between the molecules capable of plasticizing proteins and the protein. For example, this can be achieved by adjusting the respective usage amounts (moles) of the protein and the molecules capable of plasticizing proteins in the mixture. That is, for example, the usage amount (moles) of the molecules capable of plasticizing proteins in the mixture is made greater than the usage amount (moles) of the protein. When the usage amount (moles) of the molecules capable of plasticizing proteins is large, the electrophilic functional groups are excessive relative to the nucleophilic functional groups of the protein, resulting in unreacted reactive functional groups remaining in the molecules capable of plasticizing proteins that are bound to the protein. Furthermore, as long as the number of reactive functional groups (e.g., electrophilic functional groups) in the protein-plasticizing molecules in the mixture is greater than the number of reactive functional groups (e.g., nucleophilic functional groups) in the protein in the mixture, even if the amount (in moles) of the protein-plasticizing molecules used is the same as or less than the amount (in moles) of the protein used, unreacted reactive functional groups can remain in the protein-plasticizing molecules already bound to the protein. In this way, prepolymers can also be produced.

[0257] Mechanochemical treatment refers to a treatment that involves a chemical reaction initiated by the direct absorption of mechanical energy. This mechanical energy can be, for example, energy exerted by impact or shear forces. Specifically, this mechanochemical treatment can be performed using a tubular ball mill, a media agitator mill, a planetary mill, a jet mill, a mixer grinder, an extruder (twin-screw extruder), or the like. Specifically, a protein and a molecule capable of plasticizing the protein are placed in a grinding jar, and depending on the type of grinder used, media balls, etc., are added. The grinding jar is then mounted on the mixer grinder and vibrated at a prescribed frequency and reaction time. At this point, a solvent, base, reaction accelerator, etc. may be further added to the grinding jar. Alternatively, the protein and the molecule capable of plasticizing the protein may be placed in the mixer grinder, the grinding step may be initiated, and the reactants may be continuously kneaded. At this point, a solvent, base, reaction accelerator, etc. may be further added to the mixer grinder. Alternatively, the protein and the molecule capable of plasticizing the protein may be placed in an extruder and mixed. At this point, a solvent, base, reaction accelerator, etc. may be further added to the extruder. Furthermore, if an extruder is used for mechanochemical processing, continuous production of the target block copolymer can be achieved.

[0258] The frequency can be appropriately adjusted by those skilled in the art according to the reaction, and can be, for example, 10-50 Hz, 10-45 Hz, 10-40 Hz, 10-35 Hz, 15-50 Hz, 15-45 Hz, 15-40 Hz, 15-35 Hz, 20-50 Hz, 20-45 Hz, 20-40 Hz or 20-35 Hz.

[0259] The reaction time is set to a time period until the starting protein disappears or a certain amount of block copolymer is observed in infrared (IR) absorption spectroscopy, gel filtration chromatography (GPC), etc. The reaction time may be any time period, for example, 30 to 240 minutes, 30 to 210 minutes, 30 to 180 minutes, 30 to 150 minutes, 30 to 120 minutes, 30 to 110 minutes, 30 to 100 minutes, 30 to 95 minutes, 60 to 240 minutes, 60 to 210 minutes, 60 to 180 minutes, 60 to 150 minutes, 60 to 120 minutes, 60 to 110 minutes, or 60 to 100 minutes. , 60-95 minutes, 70-240 minutes, 70-210 minutes, 70-180 minutes, 70-150 minutes, 70-120 minutes, 70-110 minutes, 70-100 minutes, 70-95 minutes, 80-240 minutes, 80-210 minutes, 80-180 minutes, 80-150 minutes, 80-120 minutes, 80-110 minutes, 80-100 minutes, or 80-95.

[0260] The solvent can be any solvent that can swell the protein or at least dissolve the protein and a molecule that can plasticize the protein, but it must be a compound that is liquid at room temperature and pressure and does not chemically react with the protein. Mechanochemical processing with the addition of such a solvent is called liquid-assisted grinding (LAG), a method that more efficiently promotes the desired reaction by adding a small amount of solvent to the grinding tank. Examples of such solvents include alcohols such as methanol and ethanol, and aprotic polar solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and dihydrolevulinone. The amount of solvent used can be, for example, 0.01g to 1g per 1g of protein, or 0.01g to 0.8g, 0.01g to 0.6g, 0.01g to 0.4g, or 0.01g to 0.2g. In other words, the amount of solvent used can be, for example, 1-100% by weight, 1-80% by weight, 1-60% by weight, 1-40% by weight, or 1-20% by weight relative to the protein. It is believed that a very small amount of solvent forms a micro-reaction field by causing the protein to swell or partially dissolve. Furthermore, the addition of a solvent is not always necessary. For example, when a molecule capable of plasticizing the protein is liquid at room temperature and pressure and has the function of serving as the solvent, the addition of a solvent may not be necessary.

[0261] After the reaction is completed, the mixture is removed from the grinding tank and can be washed with a solvent. By washing, unreacted substances and unnecessary by-products produced during the reaction can be removed. Examples of solvents used in washing include water, methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, ethyl acetate, and hexane. After washing, the solvent used in washing can be evaporated and removed by drying the product. Drying can be carried out under reduced pressure.

[0262] The average particle size of the block copolymer involved in the present embodiment is preferably 1 to 80 μm, 1 to 50 μm, 2 to 50 μm, 2 to 40 μm, 4 to 25 μm, 5 to 25 μm, 8 to 25 μm or 8 to 16 μm. When the average particle size is within the above range, it has excellent handling performance. The average particle size can be measured by, for example, the following method. After the particles and even the powder of the block copolymer are uniformly dispersed on a glass plate by the attraction of a vacuum chamber, a dry and wet image analysis particle size distribution instrument (product name: DW-200nano, manufactured by Jasco International Co., Ltd.) is used to measure 5 times each, and a 10 million pixel camera is used to capture the projected image, and the resulting projected image is analyzed using image analysis software.

[0263] The block copolymer involved in the present embodiment is a powder with a smaller and more uniform average particle size obtained by freeze-drying a mixture containing a protein and a molecule capable of plasticizing the protein after mechanochemical treatment. The average particle size of the block copolymer powder obtained by freeze-drying can be, for example, 1 to 30 μm, 1 to 20 μm, 1 to 10 μm or about 2 to 8 μm. This powdery block copolymer has better operability and can adjust the usage more accurately. Moreover, the block copolymer after freeze-drying not only has a smaller average particle size, but also can loosen the tertiary structure of the protein, so that its dispersibility in aqueous media (aqueous liquids) such as water, alkaline aqueous solutions, acidic aqueous solutions, neutral aqueous solutions containing inorganic salts, and solubility in solvents can also be improved. In particular, when the block copolymer powder obtained by mechanochemical treatment, or the block copolymer powder obtained by further freeze-drying is dispersed in an aqueous medium to form an aqueous dispersion, it can be applied on the surface of the substrate more evenly without uneven conditions. In this way, a coating film with almost no unevenness and more uniformity can be formed. That is to say, the aqueous dispersion of block copolymer powder can also be used as a coating liquid for forming a coating film. In addition, after the aqueous dispersion of block copolymer powder is applied on the surface of the substrate, another substrate is attached, and by hardening the block copolymer, two substrates can be bonded together. In other words, the aqueous dispersion of the block copolymer involved in this embodiment can also be used as a water-dispersible adhesive. In addition, when the aqueous dispersion of block copolymer is used as a coating liquid, for example, a specific colorant or an additive commonly used for coating liquids can also be added, coordinated, or mixed therein. In addition, when the aqueous dispersion of block copolymer is used as a water-dispersible adhesive, an additive commonly used for adhesives can also be added, coordinated, or mixed therein. The coating liquid or water-dispersible adhesive composed of the aqueous dispersion of block copolymer involved in this embodiment, owing to the use of aqueous media such as water as solvent, is suitable for coating on materials with lower organic solvent resistance and bonding materials with lower organic solvent resistance, and can also reduce the peculiar smell of organic solvents.

[0264] Block copolymers prepared by mechanochemical methods do not use organic solvents during the manufacturing process, or use only a very small amount of organic solvent (sufficient to swell the protein or molecules that can plasticize the protein). Therefore, organic solvent residue is less likely to remain and it is easier to purify it in powder form. In addition, as described above, the block copolymers involved in this embodiment can also be purified into particles with a uniform particle size distribution and smaller particle size.

[0265] When the block copolymer of this embodiment is applied to a wood surface and bonded to another piece of wood, the average elongation in a tensile test is preferably 3.5% or more, 3.6% or more, 3.7% or more, 3.8% or more, 3.9% or more, 4.0% or more, 4.1% or more, 4.2% or more, 4.3% or more, 4.4% or more, 4.5% or more, 4.6% or more, or 4.7% or more. When the elongation in the tensile test reaches 3.5% or more, it can be considered to have sufficient adhesion. Because the block copolymer involved in this embodiment is less likely to have residual organic solvent, it is more suitable for bonding materials with low organic solvent resistance compared to conventional aqueous dispersions, water-dispersible adhesives, and coating solutions, and can also reduce the unique odor of organic solvents.

[0266] In addition, if traditional solution method is adopted to prepare block copolymer, because organic solvent is easily absorbed by block copolymer, therefore, even if being dried under reduced pressure, it is also difficult to remove organic solvent, and easily forms larger block.This type of block copolymer, for example, due to being subjected to the influence of partial structure with plasticity introduced to the molecule that can plasticize protein, its hardness is reduced than the state of protein monomer, therefore it is difficult to pulverize, and powder with uniform particle size distribution cannot be made, also be difficult to be coated on substrate surface with uniform thickness.In addition, owing to being unable to fully dry by methods such as freeze drying, therefore be difficult to obtain particle diameter less and powder with uniform particle size distribution, also be difficult to maintain equal quality and stably carry out good production of repeatability.

[0267] [Prepolymers and finished polymers]

[0268] As described above, according to the production method of this embodiment, the block copolymer can be produced as a finished polymer or as a prepolymer.

[0269] Prepolymer is a kind of polymer obtained by stopping polyreaction at an appropriate stage midway, and it contains unreacted reactive functional groups, so that it is easy to be molded, usually stops polyreaction under the plastic state with suitable purposes, thus manufactures.This prepolymer can be adjusted to desired shape by carrying out residual polyreaction in the molding process.The prepolymer of block copolymer contains unreacted reactive functional groups (for example, electrophilic functional groups), such as by making these unreacted reactive functional groups react with other compounds containing nucleophilic reactive functional groups, it is easy to form any structure between block copolymer. In addition, by being attached to the unreacted reactive functional groups of block copolymer prepolymer with the functional functional groups of desired functions, it is possible to realize the desired function corresponding to the functional functional groups in the block copolymer finally formed. In addition, the prepolymer of block copolymer can also improve the solubility to specific solvents.

[0270] A prepolymer of a block copolymer having two or more unreacted electrophilic functional groups can also be further mixed with a compound having two or more nucleophilic functional groups to produce a finished polymer. The prepolymer having two or more unreacted electrophilic functional groups can react with the compound having two or more nucleophilic functional groups to form a block copolymer (finished polymer) with a higher degree of polymerization by linking multiple prepolymers.

[0271] The compound having two or more nucleophilic functional groups can be any substance containing two or more nucleophilic functional groups in its chemical structure, and can be selected from the molecules exemplified above for the protein plasticizing molecules. Examples of the nucleophilic functional groups include hydroxyl groups, amino groups, and thiol groups.

[0272] Examples of the compound having two or more nucleophilic functional groups include diols such as polyethylene glycol, polypropylene glycol, and polybutylene glycol; dithiols such as dithiothreitol and 2,2′-(ethylenediyldioxy)bisethanethiol; and diamines such as polyethylenediamine.

[0273] The compound having two or more nucleophilic functional groups may be used in an amount of 1.5 to 7 equivalents based on the amount of each nucleophilic functional group contained in the prepolymer. Preferably, the compound having two or more nucleophilic functional groups may be used in an amount of 1.5 to 6.5 equivalents, 1.5 to 6 equivalents, 1.5 to 5.5 equivalents, 1.5 to 5 equivalents, 1.5 to 4 equivalents, 2 to 7 equivalents, 2 to 6.5 equivalents, 2 to 6 equivalents, 2 to 5.5 equivalents, 2 to 5 equivalents, 2 to 4.5 equivalents, 2.5 to 7 equivalents, 2.5 to 6.5 equivalents, 2.5 to 6 equivalents, 2.5 to 5.5 equivalents, 2.5 to 5 equivalents, or 2.5 to 4.5 equivalents per nucleophilic functional group.

[0274] Block copolymers (finished polymers) can be produced, for example, by dissolving a prepolymer in a solvent (e.g., DMSO), adding a compound having two or more nucleophilic functional groups, and then stirring. If the polymerization reaction proceeds slowly, the reaction solution can also be heated. In addition, as a method for producing a finished polymer, a prepolymer can be dissolved in a solvent, after adding a compound having two or more nucleophilic functional groups, cast on a substrate surface, and heat-dried to produce a resin film.

[0275] One embodiment of the present invention is a method for producing a molded article, comprising the steps of: mechanochemically treating a mixture containing a protein and a molecule capable of plasticizing the protein to obtain a block copolymer; and molding the block copolymer. Furthermore, in the method for producing a molded article according to this embodiment, a composition containing the block copolymer may be obtained before molding the block copolymer, and then molding the composition.

[0276] The block copolymer involved in the present embodiment, for example, can constitute a solid composition alone. In addition, the block copolymer can also be coordinated, added or mixed with other solid or liquid components to constitute a solid or liquid composition. The other components contained in the block copolymer composition are not subject to any restriction, and any known components can be used. In addition, the solid composition of the block copolymer can be, for example, in the form of powder, particles, blocks, gels, etc. In addition, the liquid composition of the block copolymer can be, for example, in the form of solution, dispersion, etc. As other components contained in the block copolymer liquid composition, water, organic solvents, ionic liquids, supercritical fluids, etc., which are commonly used when solid is constituted into a liquid composition, can be used.

[0277] The composition of block copolymer can be used as the molding material for preparing specific molded bodies. The molded body molded using the block copolymer composition as a molding material, for example, can be a fiber, a film, a resin, a gel, a porous body, a microparticle, etc. These molded bodies can be molded by, for example, a known molding method using a composition containing a protein material as a molding material. Fiber can be obtained by using a spinning solution containing a block copolymer composition (preferably a powdered composition consisting only of block copolymers) and a solvent, using a known spinning method such as wet spinning, dry-wet spinning, dry spinning, etc. Resin, for example, can be obtained by heating and pressing to mold (manufacture) a composition containing a block copolymer (preferably a powdered composition consisting only of block copolymers). In addition, a film, for example, can also be molded (manufactured) by referring to the method described in Japanese Patent No. 5678283. In addition, gels, porous bodies, microparticles, etc. can be molded (produced) by, for example, referring to the methods described in Japanese Patent No. 5782580, Japanese Patent No. 5796147, Japanese Patent No. 5823079, etc. In addition, resins can be obtained, for example, by removing the solvent or dispersion medium from a composition (e.g., in a gel form) containing a block copolymer and a specific solvent or dispersion medium, and solidifying the resulting composition.

[0278] The block copolymers of this embodiment can be used as adhesives for bonding adherends or coatings for forming a coating layer on a substrate in the form of a solution, aqueous dispersion, film, or powder containing the block copolymer as a main component. These solution-like adhesives or coatings, water-dispersible adhesives or coatings, film-like adhesives or coatings, and powder-like adhesives or coatings can be obtained, for example, by the following production methods.

[0279] A solution-like adhesive or solution-like coating containing a block copolymer can be produced by a method comprising dissolving the block copolymer obtained by the method of this embodiment in a solvent. This method allows for convenient biodegradable solution-like adhesives or solution-like coatings, unlike conventional solution-like adhesives or solution-like coatings containing components derived from petroleum.

[0280] Examples of solvents used to prepare solution-form adhesives or solution-form coatings include aqueous media such as water, alkaline aqueous solutions, acidic aqueous solutions, and neutral aqueous solutions containing inorganic salts, which can dissolve the block copolymer, and organic solvents. Examples of organic solvents include formic acid, dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and dihydrolevulinone.

[0281] The block copolymer contained in solution-like adhesive or solution-like coating can be obtained by the process involved in the present embodiment, as long as it can be dissolved in the aqueous medium or organic solvent, it is not particularly limited.Block copolymer is dissolved in the adhesive or coating formed in the aqueous medium, compared to the adhesive or coating formed by block copolymer dissolving in the organic solvent, there is the advantage of excellent operability etc. In addition, the block copolymer involved in the present embodiment is owing to being combined with the molecule that can make protein plasticization on protein, so its water solubility is higher than protein.Therefore, the aqueous solution-like adhesive or aqueous solution-like coating formed by block copolymer dissolving in the aqueous medium, compared with the aqueous solution-like adhesive or aqueous solution-like coating formed by only dissolving protein in the aqueous medium, it is possible to improve the content of protein therein.Therefore, compared with using the aqueous solution-like adhesive or aqueous solution-like coating formed by dissolving protein in the aqueous medium, using the aqueous solution-like adhesive or aqueous solution-like coating formed by dissolving block copolymer in the aqueous medium, it is possible to improve the bonding strength of adhesive or coating to the adherend surface or laminate surface of adherend described later.

[0282] The concentration of the block copolymer in the solution adhesive or solution coating can be appropriately adjusted according to the solubility of the block copolymer in the solvent, etc. The concentration can be, for example, 5 to 40% by mass, 10 to 35% by mass, 15 to 20% by mass, or 20 to 25% by mass. Furthermore, if necessary, the solution adhesive or solution coating may contain components other than the block copolymer, such as various additives known to be contained in the solution adhesive or solution coating.

[0283] The solution-like adhesive containing block copolymer is used to make the adherend of a plurality of adherends that adhere to each other.For example, the solution-like adhesive is placed between the a plurality of adherends that need to bond, the solvent in the solution-like adhesive is removed afterwards, and the block copolymer is solidified, thereby adherends can be bonded to each other to obtain adherends. An advantage of using this type of method is that, an adherend can be obtained using an adhesive with biodegradability, so the adherend of the environmental burden when the discarded can be easily manufactured. In addition, as long as adherend can be bonded with the solution-like adhesive containing block copolymer, its material is not particularly limited, and can be an organic substance (cellulose products such as paper, timber or synthetic resins, protein products), or an inorganic substance (non-metals such as metals or glass).

[0284] The specific method when using solution-like adhesive to manufacture adherend does not have any restriction.For example, can be coated with or drip solution-like adhesive on the adhered surface of at least one of them in the adherend that adheres to each other, or make the adhered surface of adherend contact, impregnate the liquid level of solution-like adhesive, make there is solution-like adhesive on the adhered surface, then overlap or be close to between the adhered surface of adherend, thereby solution-like adhesive is placed between multiple adherends.And, from the solution-like adhesive placed between multiple adherends, remove solvent and make block copolymer solidify, for example, in order to ensure close fit between the adhered surface of overlapping or docking, can heat, air-dry or naturally dry under the state that multiple adherends are applied pressure from at least any one side of the overlapping of adhered surface or docking direction.Certainly, when not applying such pressure and also can bond, then do not need to apply pressure.

[0285] The solution-like coating containing block copolymer is used to manufacture the laminated body that forms coating on all or part of substrate surface.For example, solution-like coating is supplied to at least a portion of substrate surface that needs lamination coating, after coating the surface portion of this substrate with solution-like coating, remove the solvent in solution-like adhesive, and solidify block copolymer.Thus, the laminated body that forms coating on at least a portion of substrate surface can be obtained.Using this type of method, an advantage is that it is also possible to use biodegradable coating to obtain laminated body, therefore it is possible to easily manufacture the laminated body that can reduce the environmental burden when discarded.In addition, as long as substrate can be bonded with solution-like coating containing block copolymer, its material is not particularly limited and can be identical with the adherend bonded with described solution-like adhesive.

[0286] The specific method when using solution-like coating to manufacture laminated body is also without any restriction.For example, solution-like coating can be applied or dripped onto at least a portion of substrate surface, or at least a portion of substrate surface is contacted or impregnated with the liquid level of solution-like coating, coating is supplied and applied, to ensure that solution-like coating forms a layer of prescribed thickness on at least a portion of substrate.And, when the block copolymer in the coating layer (solution-like coating) stacked on substrate surface is solidified, for example, the coating layer on substrate surface can be heated, air-dried or naturally dried to remove the solvent in the coating layer. In this case, if necessary, a prescribed extrusion body (pressurized body) can be placed on the coating layer in a manner covering the entire coating layer, and the coating layer is squeezed (pressurized) to the substrate surface side to ensure that the coating layer closely adheres to the substrate surface. At this time, the extrusion body of the solution-like coating is preferably not adhered. For example, by applying a release agent or pasting release paper on the contact surface of the extrusion body with the coating layer, or surface treatment is carried out on the contact surface to ensure that the coating does not adhere, or the material of the solution-like coating is used as the extrusion body, thereby preventing the solution-like coating from adhering to the extrusion body.

[0287] A water-dispersible adhesive or water-dispersible coating containing a block copolymer can be produced by a method comprising dispersing the block copolymer obtained by the method of this embodiment in an aqueous medium. This method allows for convenient production of a biodegradable water-dispersible adhesive or water-dispersible coating, unlike conventional water-dispersible adhesives or water-dispersible coatings containing petroleum-derived components.

[0288] Examples of the aqueous medium used for producing the water-dispersible adhesive or water-dispersible coating include water in which the block copolymer can be dispersed, or alkaline aqueous solutions, acidic aqueous solutions, and aqueous solutions containing inorganic salts.

[0289] The block copolymer contained in the water-dispersible adhesive or the water-dispersible coating is not particularly limited as long as it can be manufactured by the process involved in the present embodiment and can be dispersed in the aqueous medium. The content of the block copolymer in the water-dispersible adhesive or the water-dispersible coating is appropriately adjusted according to factors such as the adhesion of the water-dispersible adhesive to the adherend, the adhesion of the water-dispersible coating to the substrate, and even the fixation. In addition, the water-dispersible adhesive or the water-dispersible coating may contain components other than the block copolymer such as various additives contained in the known water-dispersible adhesive or the water-dispersible coating, if necessary.

[0290] In addition, the block copolymer involved in the present embodiment is owing to being combined with the molecule that can make protein plasticization on protein, therefore, compared to protein, it has higher affinity to the aqueous medium containing water.Therefore, the water-dispersible adhesive or the water-dispersible coating containing this block copolymer, compared to the water-dispersible adhesive or the water-dispersible coating that only protein is dispersed in aqueous medium to form, not only can improve the content (dispersion amount) of protein, block copolymer can also be made to be uniformly dispersed in aqueous medium.Therefore, compared with the water-dispersible adhesive or the water-dispersible coating that protein is dispersed in aqueous medium to form, the water-dispersible adhesive or the water-dispersible coating that block copolymer is dispersed in aqueous medium to form, can improve the bonding strength of adhesive or coating to the adherend surface or laminate surface of adherend described later.

[0291] The water-dispersible adhesive that contains block copolymer is used to make the adherend that a plurality of adherends adhere to each other.For example, water-dispersible adhesive is placed between the a plurality of adherends that need bonding, remove the aqueous medium in the water-dispersible adhesive afterwards, make the block copolymer solidify, thereby can be bonded between the adherends and obtain adherend.An advantage of using this type of method is, can use the adhesive with biodegradability to obtain adherend, therefore can easily produce the adherend that can reduce the environmental burden when discarding.In addition, as long as adherend can be bonded with the water-dispersible adhesive that contains block copolymer, its material is not particularly limited, also can be identical with the adherend that is bonded with the solution-like adhesive that contains block copolymer.

[0292] The concrete method when using the water-dispersible adhesive to make adherend does not have any restriction yet.For example, when the water-dispersible adhesive is placed between a plurality of adherends of need bonding, the same method as when the described solution-shaped adhesive is placed between a plurality of adherends can be adopted.And, when removing the aqueous medium in the water-dispersible adhesive that places between the adherends and making the block copolymer solidify, the same method as when the solvent in the solution-shaped adhesive that places between the adherends can be adopted and the block copolymer solidify can be adopted.

[0293] The water-dispersible coating containing block copolymers is used to manufacture a laminate formed by stacking a coating on all or part of a substrate surface. For example, the water-dispersible coating is supplied to at least a portion of the substrate surface where a coating needs to be stacked, and after the surface portion of the substrate is coated with a solution-like coating, the aqueous medium in the water-dispersible adhesive is removed to solidify the block copolymer. Thus, a laminate formed by stacking a coating on at least a portion of the substrate surface can be obtained. Using such a method, one advantage is that a laminate can also be obtained using a biodegradable coating, so that a laminate that can reduce the environmental burden when discarded can be easily manufactured. In addition, as long as the substrate can be bonded with the water-dispersible coating containing block copolymers, its material is not particularly limited and can be the same as the substrate formed by the solution-like coating.

[0294] There are no restrictions on the specific method for producing the laminate using a water-dispersible coating. For example, when supplying a water-dispersible coating to at least a portion of the substrate surface, the same method as when supplying the solution coating to the substrate surface can be used. Furthermore, when removing the aqueous medium from the water-dispersible coating supplied to the substrate surface and solidifying the block copolymer, the same method as when removing the solvent from the solution coating supplied to the substrate surface and solidifying the block copolymer can be used.

[0295] The film-like adhesive or film-like coating containing block copolymer can be manufactured by comprising the method for the operation of film-shaped block copolymer solution that dissolves the block copolymer obtained by the method for the present embodiment.Use this type of method, different from the film-like adhesive or film-like coating containing the composition derived from petroleum, it is possible to easily obtain the film-like adhesive or film-like coating with biodegradability.In addition, the forming method of the above-mentioned film can be cast molding.

[0296] The film-forming solution used to produce the film-forming adhesive or film-forming coating is preferably a block copolymer solution, such as a solution-forming adhesive or coating. The block copolymer solution can be cast using the same known methods and conditions as for protein solution casting.

[0297] The film-like adhesive that contains block copolymer is used to make the adherend that a plurality of adherends adhere to each other.For example, when film-like adhesive is placed under the state between a plurality of adherends that need to bond, film-like adhesive is swollen or heated to soften it, then hardened under the state that makes film-like adhesive be pressed onto on the adherend, thereby can be bonded between the adherend to obtain adherend.In addition, film-like adhesive can also be swollen or heated to soften it in advance, then placed between a plurality of adherends, then hardened under the state that makes film-like adhesive be pressed onto on the adherend.Use this type of method, an advantage having is, can use the adhesive with biodegradability to obtain adherend, therefore can easily manufacture the adherend that can reduce the environmental burden when discarding.In addition, as long as adherend can be bonded with the film-like adhesive that contains block copolymer, also can be identical with the adherend that is bonded with the solution-like adhesive that contains block copolymer.

[0298] Film-like adhesives absorb moisture or soften when heated, and then harden by drying or cooling. In addition, if the block copolymers contained in the film-like adhesive, particularly have the characteristic of showing shrinkage behavior when the molded body is in contact with water or heated, the film-like adhesive will also shrink when in contact with water or heated. Therefore, the film-like adhesive containing block copolymers, under the state of swelling or heating and softening between adherends, locally sinks into the gap existing on the adherend surface of the adherend, especially when the modified protein has the above-mentioned shrinkage characteristics, shrinks under the state of being trapped in the gap of the adherend surface. If the film-like adhesive hardens in this state, due to the anchoring effect (Anchor Effect), the film-like adhesive is bonded to the adherend, thereby bonding between the adherends. In addition, the film-like adhesive can play a full flexibility because it contains a block copolymer that is combined with proteins to plasticize molecules as a main component. Therefore, this type of film-like adhesive can show higher flexibility in a softened state, can more fully embed in the gap of the adherend surface, thereby is expected to obtain higher bonding strength. Furthermore, the film-like adhesive has the following excellent function: due to its excellent flexibility, it can be peeled from the adherend after being bonded to the adherend and can be reused as a film-like adhesive. In addition, when the film-like adhesive is reused, it can be washed after being peeled from the adherend. In addition, the adhesion of the film-like adhesive to the adherend may be due to the formation of hydrogen bonds between the adherend and the block copolymer.

[0299] When swelling and softening the film adhesive, it is preferable to use an aqueous liquid such as water, a water-containing solution, or a dispersion that can be absorbed by the film adhesive or that can cause the film adhesive to shrink. Alternatively, the film adhesive can be swollen and softened by, for example, dripping the aqueous liquid onto the film adhesive or immersing multiple adherends in the aqueous liquid to cause the film adhesive to absorb water. Furthermore, when hardening the film adhesive softened by swelling, for example, the film adhesive can be dried by heating, air-drying, or naturally drying while placed between adherends.

[0300] When heating the film adhesive to soften it, the film adhesive may be heated before being placed between the adherends, or the entire adherend and film adhesive may be heated after the film adhesive is placed between the adherends. The heating temperature is not particularly limited, as long as it softens the film adhesive and does not negatively affect the protein contained in the film adhesive (e.g., does not decompose it). To harden the film adhesive softened by heating, cooling using a cooling device or allowing it to cool can be used.

[0301] When making the film-like adhesive that softens because of swelling or heating fully sink into the gap of the adherend's bonded surface, preferably make the interface of softening film-like adhesive and adherend closely fit.Then, preferably for example from the overlapping direction of the adherend's surface or any one side in the docking direction, a plurality of adherends are exerted with pressure, the film-like adhesive that softens is pressed onto the adherend, and it is hardened.Here said pressurization method to adherend, can adopt common arbitrary method.

[0302] The film-like coating containing block copolymer is used to manufacture the laminated body that forms coating on all or part of substrate surface.For example, when placing film-like coating so that it covers at least a portion of substrate surface, film-like coating is swollen or heated, softened, then hardened under the state that film-like coating is pressed onto substrate surface, thereby can form coating on at least a portion of substrate surface, obtain laminated body.In addition, film-like coating can also be swollen or heated in advance so that it softens, then placed so that it covers at least a portion of substrate surface, then hardened under the state that film-like coating is pressed onto adherend.Use this type of method, an advantage having is that, can use coating with biodegradability to form laminated body, therefore can easily manufacture the laminated body that can reduce environmental burden when discarding.In addition, as long as substrate can be the material that can be bonded with the film-like coating containing block copolymer, also can be identical with the adherend bonded with the film-like adhesive containing block copolymer.

[0303] It can be thought that the mechanism that film-like coating adheres to substrate surface is identical with the mechanism that film-like adhesive adheres to the adhered surface of adherend.Therefore, the concrete method when using film-like coating to make laminate can also adopt the same method when obtaining adherend with film-like adhesive.In addition, the film-like coating after softening is pressed onto the substrate surface and when hardening, for example, the extrusion body identical with the extrusion body used when the solution-like coating applied to the substrate is extruded onto the substrate surface side can be used to extrude the film-like coating onto the substrate surface side.In addition, the film-like coating is also identical with the film-like adhesive, not only has higher bond strength to substrate, and can be peeled off after bonding, even can be reused.

[0304] A powdered adhesive or powdered coating containing a block copolymer uses a powder composition containing the block copolymer powder obtained by the method of this embodiment. The powder composition only needs to contain the block copolymer as a main component and may also contain various auxiliary components such as residual additives.

[0305] The block copolymer contained in powdered adhesive or powdered coating, as long as it is the block copolymer obtained by the process manufacturing involved in the present embodiment, is not particularly limited.For example, the block copolymer contained in solution adhesive or solution coating, water-dispersible adhesive or water-dispersible coating, film adhesive or film coating can be used.In addition, as mentioned above, the block copolymer involved in the present embodiment shows an affinity higher than protein for aqueous aqueous medium (aqueous liquid).Therefore, the powdered adhesive or powdered coating containing this block copolymer, compared to the water-dispersible adhesive or water-dispersible coating formed by only protein dispersion in aqueous medium, not only can improve the content (dispersion amount) of protein, block copolymer can also be made to be uniformly dispersed in aqueous medium.Therefore, compared with the water-dispersible adhesive or water-dispersible coating formed by protein dispersion in aqueous medium, the water-dispersible adhesive or water-dispersible coating formed by block copolymer dispersion in aqueous medium can improve the bonding strength of adhesive or coating to the adhered surface of adherend described later or laminate surface.

[0306] The powdered adhesive that contains modified protein is also used to make the adherend that a plurality of adherends adhere to each other.For example, when powdered adhesive is placed under the state between a plurality of adherends that need bonding, powdered adhesive is heated simultaneously via adherend pressurization, is solidified, thereby can be bonded between adherend and obtain adherend.An advantage of using this type of method is, can use the adhesive with biodegradability to obtain adherend, therefore can easily produce the adherend that can reduce the environmental burden when discarding.In addition, adherend can be identical with the adherend bonded by the solution-like adhesive that contains block copolymer as long as can be bonded with the powdered adhesive that contains block copolymer.

[0307] The concrete method when using powdered adhesive to make adherend does not have any restriction yet.For example, when the powdered adhesive between the multiple adherends that need to be bonded is heated / pressurized, the powdered adhesive that places between the adherends can be heated together with the adherend by using metal plates etc. from the two sides opposite to the bonded surface side to clamp the adherend and heat the metal plates. Meanwhile, use manual presses etc. to pressurize the metal plates, via adherend, the powdered adhesive is carried out the pressurization of the prescribed time. Thus, the powdered adhesive is resinized and solidified, bonding adherend. In addition, the heating temperature, pressurization amount or heating / pressurization time when the powdered adhesive is solidified can be suitably selected from the scope that can make the modified protein resinization according to the kind of the modified protein that contains in the powdered adhesive.

[0308] A powdered coating containing a block copolymer is used to produce a laminated body in which a coating is formed on all or part of a substrate surface. For example, the powdered coating is placed on at least a portion of a substrate surface and then heated while simultaneously applying pressure between a pressurizing body and the substrate to cure the powdered coating, thereby forming a coating on at least a portion of the substrate surface.

[0309] The specific method when using powdered coating to make laminated body also has no restriction.For example, configuration metal plate etc. makes it cover all powdered adhesives placed on substrate surface, and metal plate is heated, thereby heats powdered adhesive.At the same time, use manual press etc. between metal plate and substrate to carry out pressurization of powdered adhesive for prescribed time, thereby powdered coating resinization and solidification.In addition, the heating temperature, pressurization amount or heating / pressurization time of powdered coating are the same as when using powdered adhesive to obtain adherend.

[0310] [Example]

[0311] Hereinafter, the present disclosure will be described in more detail based on Examples, etc. However, the present disclosure is not limited to the following Examples.

[0312] Unless otherwise noted, the abbreviations used in the examples are used according to their commonly used meanings in the field of organic chemistry or genetic engineering. Several examples are shown below.

[0313] DCM: dichloromethane

[0314] DMSO: dimethyl sulfoxide

[0315] GPC: Gel Filtration Chromatography

[0316] HFIP: Hexafluoroisopropanol

[0317] PEG: polyethylene glycol

[0318] RO water: water treated with reverse osmosis membrane (RO membrane)

[0319] TCEP: Tris(2-carbonylethyl)phosphine hydrochloride

[0320] THF: Tetrahydrofuran

[0321] In the Examples and Comparative Examples, a compound represented by the following formula (hereinafter also referred to as "bismaleimide PEG") was used as a molecule capable of plasticizing proteins.

[0322] [Chemical Formula 5]

[0323]

[0324] 1. Protein Block Copolymer Reaction

[0325] (1) Preparation of block copolymers

[0326] As described below, a protein having two thiol groups was reacted with polyethylene glycol (bismaleimide PEG) having two maleimide groups via a solution method or a mechanochemical method to prepare a block copolymer. The resulting block copolymer was evaluated using GPC. The following formula is a schematic diagram of the reaction between a protein having two thiol groups and PEG having two maleimide groups.

[0327] [Chemical Formula 6]

[0328]

[0329] Comparative Example 1: Preparation of block copolymers by solution method

[0330] The protein PRT2662 (SEQ ID NO: 8, 255 mg, 9.3 μmol) with a molecular weight of 27461 Da and bismaleimide PEG (186 mg, 9.3 μmol) of approximately 20 kDa were suspended in DMSO (4.6 mL). The mixture was then heated to 100°C in an oil bath and stirred for 15 minutes using a HERAXLES stirrer (Model HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.) to allow the protein and bismaleimide PEG to react, resulting in a brown, transparent solution in which the reactants were dissolved. The resulting solution was then thinly coated onto a metal plate with a release film (Teijin Film Solutions Co., Ltd., 38 μm thick) using a doctor blade (manufactured by Imoto Seisakusho Co., Ltd., coating width 80 mm, gap 400 μm) to form a coating film. Next, the coating film was dried at 60°C for more than 2 hours in a constant temperature air drying oven, and then the solvent was further removed at 80°C for 15 hours in a vacuum oven to obtain a brown transparent film-like block copolymer (Comparative Example 1).

[0331] Example 1: Preparation of block copolymers by mechanochemical method (addition of water)

[0332] A pulverizer (product name: Mixer Mill MM400, manufactured by Verder Scientific Co., Ltd.) was used to prepare a powder of protein PRT2662 (100 mg, 3.6 μmol) with a molecular weight of 27461 Da, and a powder of bismaleimide PEG (73 mg, 3.6 μmol) with a molecular weight of approximately 20 kDa. The bismaleimide PEG powder was placed in a 1.5 mL ball mill container of the pulverizer. RO water (100 μL) was then added dropwise to the powder in the container as a swelling solvent to wet the entire powder. Next, ten 2 mmφ grinding balls (manufactured by As One Co., Ltd.) were placed in the container. After the container was sealed, the pulverizer was vibrated at a frequency of 30 Hz to allow the protein and bismaleimide PEG to react. After 3 hours, the reactants were vacuum dried at room temperature to obtain a white powdery block copolymer (Example 1).

[0333] Example 2: Preparation of block copolymers by mechanochemical method (with addition of DMSO)

[0334] The protein PRT2662 (100 mg, 3.6 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (73 mg, 3.6 μmol) of approximately 20 kDa were placed in a 1.5 mL ball mill container in the same mill used to prepare the block copolymer in Example 1. DMSO (100 μL) was then added dropwise as a swelling solvent to wet the entire powder. Ten 2 mm diameter grinding balls (manufactured by As One Co., Ltd.) were placed in the container, sealed, and the mill was vibrated at a frequency of 30 Hz to react the protein and bismaleimide PEG. After 3 hours, the reaction mixture was vacuum-dried at room temperature to obtain a white powdery block copolymer (Example 2).

[0335] Example 3: Preparation of block copolymers by mechanochemical method (with addition of ethanol)

[0336] The protein PRT2662 (100 mg, 3.6 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (73 mg, 3.6 μmol) of approximately 20 kDa were placed in a 1.5 mL ball mill container in the same mill used to prepare the block copolymer in Example 1. Ethanol (100 μL) was then added dropwise as a swelling solvent to wet the entire powder. Ten 2 mm diameter grinding balls (manufactured by As One Co., Ltd.) were placed in the container, sealed, and the mill was vibrated at a frequency of 30 Hz to react the protein and bismaleimide PEG. After 3 hours, the reaction mixture was vacuum-dried at room temperature to obtain a white powdery block copolymer (Example 3).

[0337] Example 4: Preparation of block copolymers by mechanochemical method (with addition of methanol)

[0338] The protein PRT2662 (100 mg, 3.6 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (73 mg, 3.6 μmol) of approximately 20 kDa were placed in a 1.5 mL ball mill container in the same mill used to prepare the block copolymer in Example 1. Methanol (100 μL) was then added dropwise as a swelling solvent to wet the entire powder. Ten 2 mm diameter grinding balls (manufactured by As One Co., Ltd.) were placed in the container, sealed, and the mill was vibrated at a frequency of 30 Hz to react the protein and bismaleimide PEG. After 3 hours, the reaction mixture was vacuum-dried at room temperature to obtain a white powdery block copolymer (Example 4).

[0339] (2) Evaluation

[0340] (2-1) Appearance of Block Copolymer Film and Block Copolymer Powder

[0341] Figure 1 The following are photos showing the appearance of the block copolymer film of Comparative Example 1 and the appearance of the block copolymer powder of Example 1. Figure 1 In the figure, A is a photograph of protein PRT2662 powder before reaction, B is a photograph of bismaleimide PEG powder before reaction, C is a photograph of the block copolymer film of Comparative Example 1, and D is a photograph of the appearance of the block copolymer powder of Example 1.

[0342] (2-2) GPC measurement

[0343] For GPC analysis, analytes were separated using an Agilent 1260 Infinity II liquid chromatography system (Agilent Technologies, Inc.) equipped with a refractive index (RI) detector and a styrene-divinylbenzene copolymer column (inner diameter: 4.6 mm × 150 mm) equipped with a 0.5 μm guard column filter (product name: Shodex GPCHK-G, manufactured by Showa Denko K.K.) as the stationary phase and HFIP. The mobile phase was monitored using an Agilent 1260 Infinity II refractive index detector (RID) (Agilent Technologies, Inc.) at a flow rate of 0.15 mL / min and 40°C. Proteins were analyzed as a 0.1 wt% solution (final concentration: 1 mM) of sodium trifluoroacetate in HFIP. For proteins containing cysteine ​​residues, TCEP (final concentration: 10 mM) was added to prevent disulfide bonds. The sample was dissolved using a heating shaker (Front Lab MyBL-100S, manufactured by AS ONE Co., Ltd.) (45°C, 1500 rpm, 1 hour) and passed through a hydrophilic PTFE membrane filter with a pore size of 0.45 μm (product name: Dismic25HP45AN, manufactured by AdvanTech) to remove residual insoluble matter.

[0344] (2-3) Analysis

[0345] GPC analysis was performed on the block copolymer film of Comparative Example 1, the block copolymer powders of Examples 1 to 4, and the protein (PRT2662) used in the production of the block copolymer film of Comparative Example 1 and the block copolymer powders of Examples 1 to 4. The results are shown in Table 1. Figures 2 to 5 As shown. Figure 2 As shown in FIG, the block copolymer film peak (solid line) of Comparative Example 1 can be confirmed within a shorter retention time than the protein peak (dashed line). Figure 3As shown in FIG, the block copolymer powder peak (solid line) of Example 1 can be confirmed within a shorter retention time than the protein peak (dashed line). Figure 4 As shown in FIG, the block copolymer powder peak (solid line) of Example 2 can be confirmed within a shorter retention time than the protein peak (dashed line). Figure 5 As shown, the peaks of the block copolymer powders of Examples 3 and 4 (dashed line: Example 3, dotted line: Example 4) can be confirmed at a shorter retention time than the protein peak (solid line). This shows that both the block copolymer film and the block copolymer powder are higher molecular weight substances formed by the binding of bismaleimide PEG to protein.

[0346] 2. Discussion on the conditions of mechanochemical reaction

[0347] As in Example 1, the block copolymer was prepared by a mechanochemical method by changing the vibration frequency and reaction time conditions, and the degree of reaction was evaluated using GPC.

[0348] 2. The effect of vibration frequency on the degree of reaction

[0349] (1) Preparation of block copolymer (Example 5)

[0350] After the protein PRT2662 (100 mg, 3.6 μmol) with a molecular weight of 27461 Da and the bismaleimide PEG (73 mg, 3.6 μmol) of about 20 kDa were placed in a 1.5 mL ball mill container of the same grinder used to prepare the block copolymer in Example 1, RO water (100 μL) was added dropwise as a swelling solvent to wet all the powders. After 10 2 mmφ grinding balls (manufactured by As One Co., Ltd.) were placed in the container and sealed, the grinder was vibrated at a vibration frequency of 10 Hz, 20 Hz or 30 Hz. After 3 hours, the reactants were vacuum dried at room temperature to obtain three white powdery block copolymers. The block copolymer powders obtained under the conditions of 10 Hz, 20 Hz and 30 Hz vibration frequencies are referred to as Examples 5A, 5B and 5C, respectively.

[0351] (2) Evaluation

[0352] The GPC results of the block copolymer powders of Examples 5A, 5B and 5C are as follows: Figure 6 As shown. Figure 6 In the figure, the solid line represents the protein peak, the dotted line represents the peak of the block copolymer powder of Example 5A obtained by reacting at a vibration frequency of 10 Hz, the single-point dashed line represents the peak of the block copolymer powder of Example 5B obtained by reacting at a vibration frequency of 20 Hz, and the dotted line represents the peak of the block copolymer powder of Example 5C obtained by reacting at a vibration frequency of 30 Hz. Figure 6As shown in Figure 3, no matter how the vibration frequency during reaction, each block copolymer powder peak can be confirmed within a retention time shorter than the protein peak. In addition, the higher the vibration frequency during reaction, the block copolymer powder peak can be detected within a shorter retention time. This shows that, within a certain range, no matter how the vibration frequency during reaction, the reaction between protein and bismaleimide PEG can be effectively carried out, and within a certain range, the higher the vibration frequency, the easier it is for the reaction between protein and bismaleimide PEG to be carried out.

[0353] 3. The effect of reaction time on the degree of reaction

[0354] (1) Preparation of block copolymer (Example 6)

[0355] The protein PRT2662 (100 mg, 3.6 μmol) with a molecular weight of 27461 Da and the bismaleimide PEG (73 mg, 3.6 μmol) of about 20 kDa were placed in a 1.5 mL ball mill container of the same grinder used to prepare the block copolymer in Example 1, and then RO water (100 μL) was added dropwise as a swelling solvent to wet all the powders. After 10 2 mm φ grinding balls (manufactured by As One Co., Ltd.) were placed in the container and sealed, the grinder was vibrated at a vibration frequency of 30 Hz. After 1 hour or 3 hours, the reactants were vacuum dried at room temperature to obtain two white powdery block copolymer powders. The block copolymer powders obtained under reaction times of 1 hour and 3 hours are referred to as Examples 6A and 6B, respectively.

[0356] (2) Evaluation

[0357] The GPC results of the block copolymer powders of Examples 6A and 6B are as follows: Figure 7 As shown. Figure 7 In the figure, the solid line represents the protein peak, the dotted line represents the block copolymer powder peak of Example 6A obtained by reaction at a reaction time of 1 hour, and the dashed line represents the block copolymer powder peak of Example 6B obtained by reaction at a reaction time of 3 hours. Figure 7 As shown in the figure, regardless of the reaction time, each block copolymer powder peak can be confirmed within a retention time shorter than the protein peak. In addition, the longer the reaction time, the block copolymer powder peak can be detected within a shorter retention time. This shows that, within a certain range, regardless of the length of the reaction time, the reaction between the protein and the bismaleimide PEG can be effectively carried out, and within a certain range, the longer the reaction time, the easier it is for the reaction between the protein and the bismaleimide PEG to proceed.

[0358] 4. Comparison of block copolymer powders

[0359] (1) Preparation of block copolymers

[0360] Comparative Example 2: Preparation of block copolymer powder by solution method and freeze drying method

[0361] The protein PRT2662 (255 mg, 9.3 μmol) with a molecular weight of 27461 Da and bismaleimide PEG (186 mg, 9.3 μmol) of about 20 kDa were suspended in DMSO (4.6 mL). Subsequently, the mixture was placed in an oil bath and heated to 100°C, and stirred for 15 minutes using a HERAXLES stirrer (product name: MODEL HERAXLES / 16G, manufactured by Koike Precision Machinery Manufacturing Co., Ltd.) to react with the protein and bismaleimide PEG to obtain a brown transparent solution after the reactants were dissolved. Subsequently, about 50 mL of acetone was added dropwise to the obtained solution to reduce the solubility of the solute, and the solution was precipitated by centrifugation (110 rpm, 5 minutes). Then, the supernatant was discarded, and an equal amount of RO water was added after sufficient shaking, and centrifugation was performed (150 rpm, 10 minutes). The same steps were repeated 3 times. An equal amount of RO water was added to the obtained gel-like reaction product, and the mixture was shaken thoroughly to disperse the mixture. The mixture was then dried in a freeze-drying apparatus using liquid nitrogen for more than 48 hours to obtain a white powdery block copolymer (Comparative Example 2).

[0362] Comparative Example 3: Powderization of Block Copolymer Film Prepared by Solution Method

[0363] The protein PRT2662 (255 mg, 9.3 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (186 mg, 9.3 μmol) of approximately 20 kDa were suspended in DMSO (4.6 mL). The mixture was then heated to 100°C in an oil bath and stirred for 15 minutes using a HERAXLES stirrer (Model HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.) to allow the protein and bismaleimide PEG to react, resulting in a brown, transparent solution containing the dissolved reactants. Using a doctor blade (manufactured by Imoto Seisakusho Co., Ltd., coating width 80 mm, gap 400 μm), the resulting solution was thinly applied to a metal plate fitted with a release film (manufactured by Teijin Film Solutions Co., Ltd., thickness 38 μm) to form a coating film. Next, the coating film was dried at 60°C for more than 2 hours using a constant temperature air drying oven, and then the solvent was further removed in a vacuum oven at 80°C for 15 hours to obtain a brown transparent film. The film was placed in a 5 mL ball mill container (manufactured by Verder Scientific Co., Ltd.) together with two 10 mmφ grinding balls (manufactured by Verder Scientific Co., Ltd.) and pulverized using a ball mill (product name: MM400, manufactured by Verder Scientific Co., Ltd.) at a vibration frequency of 30 Hz. After 30 minutes, the pulverized product was vacuum dried at room temperature to obtain a brown transparent powdered block copolymer (Comparative Example 3).

[0364] Comparative Example 4: Powderization of Block Copolymer Gel Prepared by Solution Method

[0365] The protein PRT2662 (255 mg, 9.3 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (186 mg, 9.3 μmol) of approximately 20 kDa were suspended in DMSO (4.6 mL). The mixture was then heated to 100°C in an oil bath and stirred for 15 minutes using a HERAXLES stirrer (Model HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.) to allow the protein and bismaleimide PEG to react, yielding a brown, transparent solution containing the dissolved reactants. This solution was poured into a mold composed of a metal plate, a release film (Teijin Film Solutions Co., Ltd., 38 μm thick), and a cut-out silicone sheet (Togawa Rubber Co., Ltd.), and allowed to stand at room temperature for at least 15 hours. The mold containing the solution was then placed in room-temperature ethanol and shaken for 3 hours. The mixture was then rinsed with 60°C RO water for 10 minutes, and this process was repeated twice to yield a colorless, transparent block copolymer gel. The resulting gel was dried in a forced air constant temperature drying oven at 60°C for more than 2 hours, and then the solvent was further removed in a vacuum oven at 80°C for 15 hours to obtain a brown, transparent solid block copolymer. This solid was placed in a 5 mL ball mill container (manufactured by Verder Scientific Co., Ltd.) along with two 10 mmφ grinding balls (manufactured by Verder Scientific Co., Ltd.) and pulverized using a ball mill (product name: MM400, manufactured by Verder Scientific Co., Ltd.) at a vibration frequency of 30 Hz. After 30 minutes, the pulverized product was vacuum dried at room temperature to obtain a light brown powdered block copolymer (Comparative Example 4).

[0366] Example 7: Preparation of block copolymer powder by mechanochemical and freeze-drying methods

[0367] The protein PRT2662 (300 mg, 10.8 μmol), with a molecular weight of 27461 Da, and bismaleimide PEG (216 mg, 10.8 μmol) with a molecular weight of approximately 20 kDa were placed in a 1.5 mL ball mill container of the same mill used to prepare the block copolymer in Example 1. RO water (300 μL) was then added dropwise as a swelling solvent to wet the entire powder. Ten 2 mm diameter grinding balls (manufactured by As One Co., Ltd.) were placed in the container, sealed, and the mill was vibrated at a frequency of 30 Hz to react the protein and bismaleimide PEG. After 3 hours, the reaction mixture was vacuum-dried at room temperature to obtain a white powdery block copolymer (Example 7).

[0368] (2) Evaluation

[0369] (2-1) State of fine powder

[0370] The block copolymers of Comparative Examples 2 to 4 and Example 7 were observed and compared through images. Figure 8 (A) is a photograph showing the state of the product dispersed in RO water before freeze-drying in Example 7 (left) and Comparative Example 2 (right). Figure 8 (B) is a photograph of the appearance of the freeze-dried powder of Example 7 (left) and Comparative Example 2 (right). Figure 8 (A), the block copolymer powder of Example 7 was well dispersed to form a uniform white suspension, while the block copolymer of Comparative Example 2 was observed to have gel attached to the wall. Figure 8 (B) The block copolymer of Example 7 formed fine powder, while the block copolymer of Comparative Example 2 formed larger lumps. Figure 8 (C) is a photograph showing the morphology of the pulverized block copolymer film of Comparative Example 3. Since the block copolymer film of Comparative Example 3 was soft, the force of the milling balls was not reflected, and thus no fine powder could be obtained. Figure 8 (D) is a photograph showing the morphology of the pulverized block copolymer gel of Comparative Example 4. Compared with the block copolymer powder of Example 7, the obtained block copolymer powder is slightly colored.

[0371] (2-2) Particle size distribution measurement

[0372] After the block copolymer powder of Example 7 and the block copolymer powder of Comparative Example 4 were uniformly dispersed on a glass plate by suction in a vacuum chamber, they were measured 5 times each using a dry and wet dual-purpose image analysis particle size distribution analyzer (product name: DW-200nano, manufactured by Jasco International Co., Ltd.), and the projected image was captured using a 10-megapixel camera. Finally, the obtained projected image was analyzed using image analysis software.

[0373] Figure 9 : is the particle size distribution diagram of each block copolymer powder of Example 7 and Comparative Example 4. Figure 9 In (A), the solid line represents the particle size and its occupancy ratio (cumulative) of the block copolymer powder of Example 7, and the dotted line represents the particle size and its occupancy ratio (cumulative) of the block copolymer powder of Comparative Example 4. Figure 9 In (B), the solid line represents the particle size and the occupancy ratio of the block copolymer powder of Example 7, and the dotted line represents the particle size and the occupancy ratio of the block copolymer powder of Comparative Example 4. Figure 9In (B), particles with a particle size of less than 1 μm, which are difficult to accurately measure, are excluded. As shown in the chart of (A), the block copolymer powder of Comparative Example 4 has particles with a particle size exceeding 10 μm, accounting for approximately 15% of the total, while the block copolymer powder of Example 7 has particles with a particle size exceeding 10 μm, accounting for only approximately 3%. As shown in the chart of (B), the block copolymer powder of Comparative Example 4 has a relatively gentle peak (standard deviation 6.1) at the position of 10-20 μm, while the block copolymer powder of Example 7 has a narrower peak (standard deviation 3.6) at the position of 4 μm, indicating a smaller particle size and more uniform distribution.

[0374] Figure 10 (A) is a micrograph of the block copolymer powder of Comparative Example 4. Figure 10 (B) is a microscopic photograph of the block copolymer powder of Example 7. It can be seen intuitively that the block copolymer powder of Example 7 has more small particles than the block copolymer powder of Comparative Example 4, and the particle size distribution is more uniform.

[0375] 5. Comparison of physical properties

[0376] The block copolymers obtained above were processed according to methods known to those skilled in the art, and the physical properties of the processed products were compared.

[0377] (1) Comparison of residual organic solvents

[0378] The amount of residual organic solvent in the block copolymer film of Comparative Example 1 was evaluated using a gas chromatograph (product name: GC-2010Plus, manufactured by Shimadzu Corporation). Specifically, 43 mg of the block copolymer film of Comparative Example 1 was cut to prepare a sample. After soaking the sample in methanol (10 mL) for more than 24 hours, the methanol solution was heated to 240°C at a rate of 10°C / min and maintained at this temperature for 2 minutes to volatilize the solution, and the residual amount of DMSO in the sample was measured. The measurement conditions are shown in Table 3. Two measurements were performed, and the average value of the DMSO residual amount was recorded.

[0379] [Table 3]

[0380]

[0381] The results of gas chromatography are shown in Table 4. As shown in Table 4, the residual amount of DMSO in the block copolymer film of Comparative Example 1 is an average of 0.106wt%. The block copolymer prepared by the solution method is prone to residual organic solvent inside because it is necessary to use an organic solvent such as DMSO to dissolve the protein during the preparation process. On the other hand, the block copolymer prepared by the mechanochemical method does not require an organic solvent to dissolve the protein during the preparation process, so it can be considered that there is no residual organic solvent inside.

[0382] [Table 4]

[0383]

[0384] (2) Water dispersibility test

[0385] 0.1g of the block copolymer powder of Example 7 is put into Eppendorf Tubes, and RO water is then added to make the solid content concentration reach 8%. Subsequently, the centrifuge tube is gently shaken by hand to prepare a block copolymer aqueous dispersion (Example 8). In addition, a block copolymer aqueous dispersion (Comparative Example 5) is prepared according to the same method as above, only replacing the block copolymer powder of Example 7 with the block copolymer powder of Comparative Example 4. Then, for confirming the dispersed state of block copolymer in the block copolymer aqueous dispersion of Example 8 obtained and the block copolymer aqueous dispersion of Comparative Example 5, a turbidity test is carried out.

[0386] <Turbidity test>

[0387] The turbidity of the dispersion was measured using the turbidity measurement method specified in JIS K0101, "Test Methods for Industrial Water." Appropriate amounts of RO water (manufactured by UnimatLife Co., Ltd., ROPure Rainbow) were added to the block copolymer powders of Comparative Example 4 and Example 7, respectively, to achieve solid concentrations of 0.5%, 1.0%, 1.5%, and 2.0%, respectively. The mixtures were then placed in a sample cell for absorbance measurement (Kartell Co., Ltd., disposable sample cell 1.5 mL, model 2-478-11), shaken thoroughly, and allowed to stand for 1 minute. The absorbance was then measured using a UV-visible spectrophotometer (Shimadzu Corporation, model UV-2600). Because the solution in this experimental system produces precipitation, the absorbance at a position 15 mm below the sample cell was analyzed.

[0388] Figure 11 The results of dispersing the block copolymer powders of Comparative Example 4 and Example 7 in RO water at concentrations of 0.5 to 2.0%, respectively, and comparing their turbidity by absorbance analysis are shown. The solid line represents the result of Example 7, and the dotted line represents the result of Comparative Example 4. At any concentration, Comparative Example 4 will precipitate quickly after oscillation, so the part through which the laser passes is almost colorless and has a low absorbance. On the other hand, Example 7 can still maintain a turbid state well even after a certain period of time after oscillation, and its absorbance is also higher than that of the solution system reactant. However, when Example 7 reaches a certain concentration or above, the absorbance will no longer change. According to the above results, when the concentration of the block copolymer powder obtained by mechanochemical treatment in RO water is 1%, the absorbance (Abs.) of the aqueous dispersion is preferably greater than 22.0.

[0389] Figure 12 (A) is a photograph of the dispersed state of the block copolymer in the block copolymer aqueous dispersion of Example 8 (left) and the block copolymer aqueous dispersion of Comparative Example 5 (right). Figure 12 (B) is to Figure 12 (A) is a photograph of the aqueous dispersion state when each Eppendorf tube (Eppendorf Tubes) is placed upside down. Figure 12 As shown in (A) and (B), the aqueous dispersion of Example 8 exhibits a uniform, turbid dispersion, while the aqueous dispersion of Comparative Example 5 contains gel-like insoluble matter adhering to the centrifuge tube wall. This demonstrates that the block copolymer prepared by the mechanochemical method has higher water dispersibility than the block copolymer prepared by the solution method.

[0390] (3) Preparation of coating film

[0391] The block copolymer aqueous dispersion of Example 8 was thinly applied to a metal plate with a release film, placed in a blast constant temperature drying oven, and dried at 60° C. for 2 hours to obtain a block copolymer coating (Example 9). In addition, a block copolymer coating (Comparative Example 6) was obtained according to the same method as above, except that the block copolymer aqueous dispersion of Comparative Example 5 was used instead of the block copolymer aqueous dispersion of Example 8. Next, the properties of the obtained block copolymer coatings of Example 9 and Comparative Example 6 were confirmed by visual inspection.

[0392] Figure 13 (Left) is a photograph showing the properties of the block copolymer coating film of Example 9. Figure 13 (Right side) is a photograph showing the properties of the block copolymer coating film of Comparative Example 6. Figure 13 (left) and Figure 13 As shown in the figure (right), the coating film thickness of Example 9 is uniform, while the coating film thickness of Comparative Example 6 is non-uniform and has localized areas where no film has formed. This indicates that the block copolymer aqueous dispersion prepared by the mechanochemical method can be used as a coating liquid capable of forming a coating film of uniform thickness, unlike the block copolymer aqueous dispersion prepared by the solution method.

[0393] (4) Preparation of resin

[0394] On the inner side of a 1.5 mm × 3.5 mm mold (manufactured by Global Machine Co., Ltd.), a thin layer of mold release agent DAIFREE (product name: MS-600, manufactured by Daikin Industries, Ltd.) is applied, and baked to fix, and the anti-sticking treatment is completed. 2 g of the block copolymer powder of Example 7 is placed in a mold that has completed the anti-sticking treatment at room temperature, and the metal cover connected to the pressure jack is closed. Subsequently, a pressure molding machine (product name: NT-100H, manufactured by NPa Systems Co., Ltd.) is used to apply a pressure of 15 MPa and heat to 130 ° C. After reaching 130 ° C, the temperature and pressure are kept constant, and the molded body is heated and molded. After 3 minutes, it is cooled to room temperature and the pressure is released, and the molded body is taken out to obtain a brown transparent block copolymer molded body (resin, Example 10). In addition, a block copolymer molded body (Comparative Example 7) is prepared according to the same method as above, using only the block copolymer powder of Comparative Example 4 instead of the block copolymer powder of Example 7.

[0395] Figure 14 (A) is a photograph showing the properties of the block copolymer molded product of Example 10, Figure 14 (B) is a photograph showing the properties of the block copolymer molded product of Comparative Example 7. Figure 14 (A) and Figure 14 As shown in (B), the block copolymer molded article of Example 10 showed no turbidity or cracks, while the block copolymer molded article of Comparative Example 7 was highly turbid, had extremely low strength, and easily cracked. This indicates that the block copolymer molded article prepared by the mechanochemical method has lower turbidity and higher strength than the block copolymer molded article prepared by the solution method.

[0396] (5) Preparation of water-dispersible adhesive

[0397] The block copolymer aqueous dispersion of Example 8 was thinly applied to a 15 mm x 15 mm area at one end of a piece of wood (Hinoki cypress, 1.5 mm x 40 mm x 2 mm), and then another piece of wood of the same size was superimposed. The two pieces of wood were then secured with a clamp and placed in a forced-air constant-temperature drying oven, where they were dried at 60°C for 48 hours to bond the two pieces together. Separately, two pieces of wood were bonded together using the same method as above, except that the block copolymer aqueous dispersion of Comparative Example 5 was used instead of the block copolymer aqueous dispersion of Example 8.

[0398] (6) Tensile test of bonded wood

[0399] Wood pieces bonded with the block copolymer aqueous dispersion of Example 8 and wood pieces bonded with the block copolymer aqueous dispersion of Comparative Example 5 were stored at room temperature (20°C) and humidity (65%) for at least 24 hours. Tensile tests were then repeated 10 times in a tensile testing machine (AG-X plus 50kN, manufactured by Shimadzu Corporation). The tensile tests were conducted under the following conditions: a load cell of 50kN, a grip spacing of 57.5mm, and a tensile speed of 10mm / min.

[0400] Figure 15 (A) is a graph showing the results of a tensile test. Figure 15 In (A), the vertical axis represents stress, the horizontal axis represents elongation, the solid line represents the tensile test results of wood bonded with the block copolymer aqueous dispersion of Example 8, and the dotted line represents the tensile test results of wood bonded with the block copolymer aqueous dispersion of Comparative Example 5. Figure 15 As shown in (A), the elongation at break of the wood bonded with the block copolymer aqueous dispersion of Example 8 is about 11.8%, while the elongation at break of the wood bonded with the block copolymer aqueous dispersion of Comparative Example 5 is only about 5.5%. Figure 15 (B) Yes Figure 15 (A) Box plot of the elongation results shown, e.g. Figure 15 As shown in Figure (B), the average elongation of wood bonded using the block copolymer aqueous dispersion of Example 8 (left) is 4.7%, while the average elongation of wood bonded using the block copolymer aqueous dispersion of Comparative Example 5 (right) is 3.3%. These results indicate that the block copolymer aqueous dispersion of Example 8 has higher strength than the block copolymer aqueous dispersion of Comparative Example 5 and is therefore suitable for use as a wood adhesive.

[0401] 6. Preparation of prepolymer

[0402] An excess of polyethylene glycol (PEG) with two maleimide groups was added to a protein with two thiol groups and reacted to prepare a prepolymer with reactive maleimide groups at both ends. An equimolar amount of a curing agent with two thiol groups was then added to the resulting prepolymer to produce a finished polymer. The progress of these reactions was assessed using GPC. The reactions are shown below.

[0403] [Chemical Formula 7]

[0404]

[0405] (2) Synthesis of prepolymer by mechanochemical method (Example 11)

[0406] The 27461Da protein PRT2662 (211 mg, 7.7 μmol) and the approximately 20kDa bismaleimide PEG (307 mg, 15.4 μmol) were placed in a 5mL ball mill container in the same mill used to prepare the block copolymer in Example 1. RO water (300 μL) was added dropwise as a swelling solvent to wet the entire powder. Two 10mmφ grinding balls (Verder Scientific Co., Ltd.) were added, the container sealed, and the mill was vibrated at 30 Hz to allow the protein and bismaleimide PEG to react. After 3 hours, the reaction mixture was vacuum-dried at room temperature to obtain a white powdery block copolymer prepolymer (Example 11A).

[0407] Separately, 80 mg of the block copolymer prepolymer from Example 11A was suspended in DMSO (909 mL). The resulting suspension was heated to 70°C in an oil bath and stirred for 15 minutes using a Heraxles stirrer (Model Heraxles / 16G, manufactured by Koike Precision Machinery Co., Ltd.). The suspension was then cooled to room temperature. After cooling, 2,2'-(ethylenedioxy)bis(ethylenedithiol) (1.4 mg, 7.7 μmol) was added to the suspension and stirred for 30 minutes to allow the block copolymer prepolymer and ethylenedithiol to react, yielding a brown, transparent solution of the melted block copolymer. The resulting block copolymer solution was then coated onto a release film (Teijin Film Solutions, Ltd., 38 μm thick) attached to a metal plate using a doctor blade (manufactured by Imoto Seisakusho Co., Ltd.) with a coating width of 80 mm and a gap of 400 μm. The coated metal plate was placed in a constant temperature drying oven with forced draft, dried at 60°C for more than 2 hours, and then further removed from the solvent in a vacuum oven at 80°C for 15 hours to obtain a brown transparent block copolymer finished polymer film (Example 11B).

[0408] (3) GPC analysis

[0409] Figure 16 Figure 1 shows the GPC results for protein PRT2662 (dashed line), Example 11A (dotted line), and Example 11B (solid line). The results for Example 11A and Example 11 show that the retention time gradually decreases as the reaction proceeds, indicating that the molecular weight increases as the reaction proceeds, consistent with design expectations and indicating effective reaction control.

[0410] 7. Mechanochemical Polymerization Using a Mixer Grinder

[0411] Block copolymers were prepared by reacting a protein having one or more thiol groups with polyethylene glycol (PEG) having one or more maleate groups using a mixer mill under mechanochemical conditions and evaluated by gel permeation chromatography (GPC). For this reaction, a laboratory kneader (laboratory kneading extruder 3S150, manufactured by Toyo Seiki Seisaku-sho, Ltd.) equipped with a roll mixer model R60 (manufactured by Toyo Seiki Seisaku-sho, Ltd., blade shape: roll) was used as a counter-rotating roll mixer.

[0412] The reaction is summarized as follows: The resulting block copolymer has a repeating unit structure represented by the following formula, which is linked at the asterisk (*).

[0413] [Chemical Formula 8]

[0414]

[0415] (Example 12)

[0416] 10kDa protein PRT2882 (15g, 16mmol), 11kDa maleated polyethylene glycol (15g, 1.4mmol) and reducing agent dithiothreitol (430mg, 2.8mmol) were weighed separately and mixed thoroughly. Before carrying out the reaction, the temperature in the feed chamber (60mL) of the mixing mill was adjusted to 85°C. While slowly rotating the blades of the mixing mill (10rpm), a mixture containing 100kDa recombinant protein PRT2882 was added to the feed chamber in batches, and triethylamine (1.63mL, 11.7mmol) was added dropwise. Slow rotation (10rpm) was performed until the reactants became uniform. After confirming that both the torque and the temperature were stable, the grinding step was started, and the reactants were continuously kneaded (50rpm, 85°C, 1 hour) to obtain a brown solid product. After the reaction was completed, a portion of the product was peeled off and GPC determination was performed.

[0417] Figure 17 The appearance of the obtained product, the unmodified protein PRT2882 and the maleated polyethylene glycol are shown. Figure 17 In the figure, (A) is a photograph of the appearance of the unmodified protein PRT2882, (B) is a photograph of the appearance of maleated polyethylene glycol, and (C) is a photograph of the appearance of the product.

[0418] The obtained product was analyzed by GPC using the above method. Figure 18 As shown. Figure 18In the figure, the dashed line is the chromatogram of the unmodified protein PRT2882, the dotted line is the chromatogram of maleated polyethylene glycol, and the solid line is the chromatogram of the product. The chromatogram of the product shows peaks at shorter retention times than those of the starting materials, indicating that the reaction has progressed and a block copolymer has been formed.

[0419] 8. Reaction of amines with aldehydes

[0420] (1) Synthesis of dialdehyde PEG

[0421] Step 1: Nucleophilic substitution reaction

[0422] Polyethylene glycol 10000 (20 g, 2.00 mmol, also known as "PEG10K") was added to a mixture of sodium hydroxide (480 mg, 12.00 mmol) and water (48 μL) under ambient conditions, stirred and heated to 80°C. After the PEG10K was completely melted and formed a uniform suspension, chloroacetaldehyde dimethyl acetal (2.05 mL, 9.00 mmol) was stirred at 80°C and added dropwise to the molten suspension, and stirring was continued for 2 hours. Subsequently, 2-chloroacetaldehyde dimethyl acetal (2.05 mL, 9.00 mmol) was added to the molten suspension for a second time and stirring was continued for 2 hours. After cooling, the reaction mixture was dissolved in DCM (150 mL) and salts were removed by gravity filtration. After the filtrate was concentrated in vacuo, the resultant was poured into hexane, stirred rapidly and crushed. The resultant was isolated by vacuum filtration and washed with hexane. The collected solid was partially dried on the filter, then transferred to a round-bottom flask and dried under reduced pressure. It was first dried using a rotary evaporator (45°C) until visibly dry, and then thoroughly dried using an oil pump vacuum line to remove residual solvent and moisture, ultimately yielding PEG10K bis(dimethyl acetal) as a colorless, amorphous solid.

[0423] Project 2: Acetal hydrolysis under acid catalyst

[0424] Under ambient conditions, PEG10K bis(dimethyl acetal) (18 g, 1.77 mmol) was suspended in a 1 M hydrochloric acid solution (20 mL, 20.0 mmol) at room temperature with rapid stirring, and the reaction temperature was raised to 80°C and stirring was continued for 3 hours until the reaction was complete. After the reaction was cooled, the product was extracted with DCM (4×50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product was then quickly added to ice-cooled hexane (200 mL) and rapidly stirred to crush. The product was filtered and separated under reduced pressure and washed with hexane. The collected solid was partially dried on the filter and transferred to a round-bottom flask. Under reduced pressure, the solid was first dried to a visible dry state using a rotary evaporator (45°C), and then thoroughly dried using an oil pump vacuum line to remove residual solvent and moisture, ultimately obtaining PEG10K dialdehyde (PEG-I) as a white amorphous solid.

[0425] (3-1) Synthesis of block copolymers in liquid phase

[0426] The protein PRT3463 (518 mg, 48 μmol), with a molecular weight of 10,763 kDa, and the dialdehyde PEG (482 mg, 48 μmol), with a molecular weight of approximately 10 kDa, were suspended in DMSO (8.18 mL). The mixture was then heated to 100°C in an oil bath and stirred for 5 hours using a HERAXLES stirrer (Model HERAXLES / 16G, manufactured by Koike Precision Machinery Co., Ltd.) to allow the protein and dialdehyde PEG to react, resulting in a brown, transparent solution containing the dissolved reactants. The resulting solution was then thinly coated onto a metal plate with a release film (Teijin Film Solutions Co., Ltd., 38 μm thick) using a doctor blade (manufactured by Imoto Seisakusho Co., Ltd., coating width 80 mm, gap 400 μm) to form a film. Next, the coating film was dried at 60°C for more than 2 hours in a constant temperature air drying oven, and then the solvent was further removed at 80°C for 15 hours in a vacuum oven to obtain a brown transparent film-like block copolymer (Comparative Example 8).

[0427] (3-2) Mechanochemical Synthesis of Block Copolymers

[0428] The 10763 kDa protein PRT3463 (518 mg, 48 μmol, SEQ ID NO: 11), the approximately 10 kDa dialdehyde PEG (482 mg, 48 μmol), and 0-5 wt% THF were weighed and thoroughly mixed. The mixture was placed in a ball mill container (Varder Scientific Co., Ltd.), and grinding balls (As One, Φ = 2 mm, 10 balls in total) were added. The container was sealed and the reaction was carried out using a ball mill (Varder Scientific Co., Ltd., product name: MM400) at a vibration frequency of 30 Hz. After 1 hour, the reactants were dried under reduced pressure at room temperature to obtain a white powdery block copolymer (Example 13).

[0429] (4) GPC analysis

[0430] The reaction product obtained in the above (3-1) or (3-2) was subjected to gel permeation chromatography (GPC) analysis. The results of GPC are as follows: Figure 19 As shown in Figure 3 , regardless of the reaction conditions, a peak indicating a higher molecular weight was observed compared to the peaks of the protein PRT3463 and dialdehyde PEG before the reaction. This result demonstrates that block copolymers can be prepared by reacting amines with aldehydes through a mechanochemical reaction.

[0431] (5) Comparison of DMSO solubility

[0432] The film obtained in (3-1) was pre-ground into a fine powder using a pestle and mortar. The powder obtained in (3-2) was used directly. A 5% DMSO solution of the protein powder (50 mg of protein PRT3463, 864 μL of DMSO) was prepared and heated to 85°C in an oil bath for 15 minutes. The state of the solution was visually observed.

[0433] The results are as follows Figure 20 As shown, the powder from Example 13 completely dissolved in DMSO, while the powder from the film of Comparative Example 8 exhibited a gel-like state. The powder from Example 13 had smaller particle size and a loose multidimensional structure between proteins, making it easily soluble. In contrast, the powder from the film of Comparative Example 8 had larger particles and, due to the formation of numerous multidimensional structures between proteins during film formation, reduced solubility. These results confirm that mechanochemical methods are more advantageous for synthesizing high-molecular-weight compounds than liquid-phase reactions in the reaction of amines with aldehydes.

Claims

1. A method for producing a block copolymer, comprising the step of subjecting a mixture containing a protein and a molecule capable of plasticizing the protein to a mechanochemical treatment.

2. The method according to claim 1, wherein The block copolymer includes protein and a molecule capable of plasticizing protein as unit structures.

3. The method according to claim 1, wherein After the mechanochemical treatment step, the method further includes a step of freeze-drying the mechanochemically treated product.

4. The method according to claim 1, wherein The number of reactive functional groups contained in the molecule capable of plasticizing the protein is greater than the number of reactive functional groups contained in the protein.

5. The method according to claim 4, wherein The amount of the molecule capable of plasticizing the protein used is greater than the amount of the protein used on a molar basis.

6. The method according to claim 4, wherein: The reactive functional groups in the protein are nucleophilic functional groups, and the reactive functional groups in the molecules capable of plasticizing the protein are electrophilic functional groups.

7. The method according to claim 6, wherein: The nucleophilic functional group is a thiol group, and the electrophilic functional group is a thiol reactive group.

8. The method according to claim 6, wherein: The nucleophilic functional group is an amino group, and the electrophilic functional group is an amine reactive group.

9. The method of claim 1, wherein the protein comprises a hydrophobic protein.

10. The method according to claim 9, wherein the hydropathic index of the hydrophobic protein is greater than 0.

11. The method of claim 1, wherein the protein comprises an artificial protein.

12. The method of claim 11, wherein the artificial protein comprises an artificial structural protein.

13. The method of claim 1, wherein the mechanochemical treatment is achieved by applying shear force to the mixture.

14. The method of claim 13, wherein the shear force is applied to the mixture using a mixer mill or an extruder. 15 . A method for producing an aqueous dispersion of a block copolymer, comprising dispersing the block copolymer obtained by the method according to claim 1 in an aqueous medium. 16 . A method for producing a water-dispersible adhesive, comprising dispersing the block copolymer obtained by the method according to claim 1 in an aqueous medium. 17 . A method for producing a coating liquid, comprising dispersing the block copolymer obtained by the method according to claim 1 in an aqueous medium.

18. A method for producing a molded article, comprising the step of molding a block copolymer obtained by the method according to any one of claims 1 to 12.

19. The method according to claim 18, wherein The molding step is a step of heating and pressurizing the block copolymer.

20. A method for producing a solution-state adhesive, comprising dissolving the block copolymer obtained by the method according to any one of claims 1 to 12 in a solvent.

21. A method for producing a film-like adhesive, comprising the step of forming a film from a solution obtained by dissolving the block copolymer obtained by the method according to any one of claims 1 to 12.

22. A method for producing a powdered adhesive, comprising the step of obtaining a powder composition containing a block copolymer obtained by the method according to any one of claims 1 to 12.

23. A method for producing an adhered body, characterized in that: The block copolymer obtained by the method according to any one of claims 1 to 12 is dissolved in a solvent to form a solution, and the solution is placed between a plurality of adherends. The solvent is then removed from the solution to solidify the block copolymer, thereby adhering the adherends.

24. A method for producing an adhered body, characterized in that: The block copolymer obtained by the method according to any one of claims 1 to 12 is dispersed in an aqueous medium to form an aqueous dispersion, and the aqueous dispersion is placed between a plurality of adherends. The aqueous medium is then removed from the aqueous dispersion to solidify the block copolymer, thereby adhering the adherends.

25. A method for manufacturing an adhesive body, characterized in that: A film containing a block copolymer obtained by the method according to any one of claims 1 to 12 is softened by swelling or heating, placed between a plurality of adherends, and cured while being pressed against the adherends, thereby bonding the adherends.

26. A method for producing an adhered body, characterized in that: A powder composition containing the block copolymer obtained by the method according to any one of claims 1 to 12 is placed between a plurality of adherends, and the powder composition is heated and simultaneously pressurized via the adherends to cure the powder composition, thereby adhering the adherends.

27. A method for producing a solution coating, comprising the step of dissolving the block copolymer obtained by the method according to any one of claims 1 to 12 in a solvent.

28. A method for producing a water-dispersible coating, comprising dispersing the block copolymer obtained by the method according to any one of claims 1 to 12 in an aqueous medium.

29. A method for producing a film-like coating, comprising the step of forming a film from a solution in which the block copolymer obtained by the method according to any one of claims 1 to 12 is dissolved.

30. A method for producing a powdered coating, comprising the step of obtaining a powder composition containing a block copolymer obtained by the method according to any one of claims 1 to 12.

31. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: A solution in which the block copolymer obtained by the method according to any one of claims 1 to 12 is dissolved in a solvent is supplied to at least a portion of the surface of the substrate; after coating at least a portion of the surface of the substrate with the solution, the solvent is removed from the solution and the block copolymer is cured, thereby forming a coating layer on at least a portion of the surface of the substrate.

32. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: An aqueous dispersion in which the block copolymer obtained by the method according to any one of claims 1 to 12 is dispersed in an aqueous medium is supplied to at least a portion of the surface of the substrate; after coating at least a portion of the surface of the substrate with the aqueous dispersion, the aqueous medium is removed from the aqueous dispersion, and the block copolymer is cured, thereby forming a coating layer on at least a portion of the surface of the substrate.

33. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, wherein: A film containing the block copolymer obtained by the method according to any one of claims 1 to 12 is softened by swelling or heating, placed on at least a portion of the surface of the substrate, and then hardened while being pressed against the substrate, thereby forming the coating layer on at least a portion of the surface of the substrate.

34. A method for manufacturing a laminate comprising a substrate and a coating layer at least partially laminated on a surface of the substrate, characterized in that: A powder composition containing the block copolymer obtained by the method according to any one of claims 1 to 12 is placed on at least a portion of the surface of the substrate, the powder composition is heated, and simultaneously pressurized between a pressurizing body and the substrate to cure the powder composition, thereby forming the coating layer on at least a portion of the surface of the substrate.

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