Esterified protein and preparation method thereof

Abstract

The present invention provides a method for producing a modified protein, which comprises a step for obtaining a modified protein by treating a mixture containing a protein and an acylating agent using a mechanochemical method.

Description

Technical Field

[0001] The invention relates to an esterified protein and a preparation method thereof. Background Art

[0002] In recent years, as concerns about environmental pollution have grown, biodegradable protein materials have attracted attention as an alternative to petroleum-based materials. Protein materials have different physical properties depending on the type of protein, so it is necessary to prepare proteins with the necessary physical properties to suit their uses.

[0003] For example, Patent Document 1 discloses that modified nucleic acids are formulated to ensure expression of a desired amino acid sequence, thereby obtaining modified spider silk proteins as proteins for use in medicine or cosmetics, and also discloses that the modified spider silk proteins are further bound to substances such as polypeptides, polysaccharides, labeling molecules, quantum dots, metals, nucleic acids, lipids, and low-molecular drugs. As described above, by using lysine, serine, and / or cysteine ​​to bind other substances, other properties different from the inherent properties of the protein can be imparted.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2007 / 025719

[0007] Patent Document 2: International Publication No. 2021 / 187502

[0008] Patent Document 3: Japanese Patent No. 5678283

[0009] Patent Document 4: Japanese Patent No. 5782580

[0010] Patent Document 5: Japanese Patent No. 5796147

[0011] Patent Document 6: Japanese Patent No. 5823079

[0012] Patent Document 7: Japanese Patent No. 6830604

[0013] Non-patent literature

[0014] Non-patent literature 1: Ochiai B. et al. ACSOmega4, 2019, 17542-17546.

[0015] Non-patent literature 2: Weissbach U. et al. Beilstein J. Org. Chem. 2017, 13, 1788-1795. Summary of the invention

[0016] Problems to be solved by the invention

[0017] Generally speaking, when modifying a protein, it is necessary to react it with a bound substance in a solvent that can dissolve the protein. However, the solvent that can dissolve the protein is limited, so the degree of freedom of the modification method is small, which leads to the problem that the modified protein that can be manufactured is also limited. In addition, when reacting in a solvent, depending on the type of solvent used, the protein may react with the solvent to generate unnecessary by-products. Specifically, common types of solvents that can dissolve proteins include dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), etc. When the protein is dissolved in these solvents and an acylating agent is added, Swern oxidation-type oxidation reactions, acylation of ethanol solvents, etc. may occur. Therefore, the object of the present invention is to provide a new method for manufacturing a modified protein.

[0018] Methods used to solve problems

[0019] The present inventors have found that a desired modified protein can be obtained by acylation of a protein under mechanochemical conditions. In other words, the present invention provides the following (1) to (66).

[0020] (1) A method for producing a modified protein, comprising the step of treating a mixture containing a protein and an acylating agent using a mechanochemical method to obtain the modified protein.

[0021] (2) According to the method of (1), the mixture further contains a base and / or a reaction accelerator.

[0022] (3) According to the method of (1) or (2), the mixture further comprises a solvent.

[0023] (4) The method according to (3), wherein the amount of the solvent is 1 to 100% by weight relative to the amount of the protein.

[0024] (5) The method according to any one of (1) to (4), further comprising the step of washing the product after the mechanochemical treatment.

[0025] (6) The method according to (5), further comprising a step of drying the washed product after the washing step.

[0026] (7) The method according to any one of (1) to (6), wherein the protein comprises a hydrophobic protein.

[0027] (8) The method according to (7), wherein the hydrophilic index (Hydropathy Index) of the hydrophobic protein exceeds 0.

[0028] (9) The method according to any one of (1) to (8), wherein the protein comprises an artificial protein.

[0029] (10) The method according to (9), wherein the artificial protein comprises an artificial structural protein.

[0030] (11) The method according to any one of (1) to (10), further comprising the step of obtaining the protein by a microbiological method.

[0031] (12) The method for producing a modified protein according to (1), wherein the acylating agent contains a hydrophilic group.

[0032] (13) The method for producing a modified protein according to (1), wherein the acylating agent comprises at least one selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a sulfo group, and a phosphate group.

[0033] (14) The method for producing a modified protein according to (1), wherein the acylating agent contains a hydrophobic group.

[0034] (15) The method for producing a modified protein according to (1), wherein the acylating agent comprises at least one selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, and an aryl group.

[0035] (16) The method for producing a modified protein according to (1), wherein the acylating agent contains a flame retardant element (an atom that can impart flame retardancy).

[0036] (17) The method for producing a modified protein according to (1), wherein the acylating agent contains a sulfur atom or a phosphorus atom.

[0037] (18) The method for producing a modified protein according to (1), wherein the modified protein contains a flame retardant element (an atom that can impart flame retardancy).

[0038] (19) The method for producing a modified protein according to (1), wherein the modified protein contains a sulfur atom or a phosphorus atom.

[0039] (20) The method for producing a modified protein according to (1), wherein the acylating agent is succinic anhydride or maleic anhydride.

[0040] (21) The method for producing a modified protein according to (1), wherein the acylating agent comprises at least one structure selected from the group consisting of a succinyl group and a maleic group.

[0041] (22) The method for producing a modified protein according to (1), wherein the acylating agent contains an alkyl group having 6 or more carbon atoms.

[0042] (23) The method for producing a modified protein according to (1), wherein the acylating agent comprises at least one structure selected from the group consisting of fatty acids and unsaturated fatty acids.

[0043] (24) According to the method described in (1), the treatment using a mechanochemical method is achieved by applying a shear force to the mixture.

[0044] (25) According to the method of (24), a shear force is applied to the mixture using a mixer mill or an extruder.

[0045] (26) A modified protein obtained by the method according to any one of (1) to (25).

[0046] (27) A modified protein composition comprising the modified protein described in (26).

[0047] (28) A modified protein shaped body obtained by shaping the modified protein composition described in (27).

[0048] (29) A molding material comprising the modified protein described in (26).

[0049] (30) The molding material described in (29) is molded to obtain a modified protein molded body. (31) The modified protein molded body described in (28), wherein the modified protein molded body is any one of the group consisting of fibers, films, gels, porous bodies, particles, and resins.

[0050] (32) Fibers comprising a modified protein obtained by the method according to any one of (1) to (25).

[0051] (33) A thin film comprising a modified protein obtained by the method according to any one of (1) to (25).

[0052] (34) A hydrogel comprising a modified protein obtained by the method according to any one of (1) to (25).

[0053] (35) A porous body comprising a modified protein obtained by the method according to any one of (1) to (25).

[0054] (36) Particles comprising a modified protein obtained by the method according to any one of (1) to (25).

[0055] (37) A resin comprising a modified protein obtained by the method according to any one of (1) to (25).

[0056] (38) A method for producing protein fibers, comprising the steps of treating a mixture containing a protein and an acylating agent using a mechanochemical method to obtain a modified protein, and spinning the obtained modified protein.

[0057] (39) A method for producing a solution-state adhesive, comprising the step of dissolving the modified protein obtained by the method described in (1) in a solvent.

[0058] (40) A method for producing a water-dispersible adhesive, comprising the step of dispersing the modified protein obtained by the method described in (1) in an aqueous medium.

[0059] (41) A method for producing a film-like adhesive, comprising the step of forming a film from a solution in which the modified protein obtained by the method described in (1) is dissolved.

[0060] (41-1) A method for producing a film-like adhesive, comprising the steps of obtaining a modified protein by the method described in (1), and dissolving the obtained modified protein in a solvent and forming the resulting film.

[0061] (42) A method for producing a powdered adhesive, comprising the step of obtaining a powder containing a modified protein obtained by the method described in (1).

[0062] (42-1) A method for producing a powdered adhesive, comprising the step of powdering the modified protein obtained by the method described in (1).

[0063] (42-2) A method for producing a powdered adhesive, comprising the steps of obtaining a modified protein by the method described in (1), and powdering the obtained modified protein.

[0064] (42-3) A method for producing a powdered adhesive, comprising the step of obtaining a powder composition containing a modified protein obtained by the method described in (1) and an adhesion enhancer or a bonding enhancer.

[0065] (42-4) A method for producing a powdered adhesive, comprising the steps of adding an adhesion enhancer or a bonding enhancer to the modified protein obtained by the method described in (1) to obtain a composition, and powdering the obtained composition.

[0066] (42-5) A method for producing a powdered adhesive, comprising the steps of obtaining a modified protein by the method described in (1), and obtaining a powder composition containing the obtained modified protein and an adhesion enhancer or bonding enhancer.

[0067] (42-6) A method for producing a powdered adhesive, comprising the steps of obtaining a modified protein by the method described in (1), adding an adhesion enhancer or a bonding enhancer to the obtained modified protein to obtain a composition, and powdering the obtained composition.

[0068] (43) The method for producing a binder according to any one of (39) to (42), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein.

[0069] (44) The method for producing a solution-state adhesive according to (39), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the solvent is an alkaline aqueous solution.

[0070] (45) According to the method for producing a thin film adhesive described in (41) or (41-1), the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the film forming process includes a step of dissolving the modified protein in an alkaline aqueous solution.

[0071] (46) A method for producing an adherend, comprising the steps of placing a solution obtained by dissolving the modified protein obtained by the method described in (1) in a solvent between a plurality of adherends, and then removing the solvent from the solution and solidifying the modified protein to thereby adhere the adherends.

[0072] (46-1) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), dissolving the obtained modified protein in a solvent to obtain a solution, placing the obtained solution between a plurality of adherends, removing the solvent from the solution and solidifying the modified protein to thereby adhere the adherends.

[0073] (47) A method for producing an adherend, comprising the steps of dispersing a modified protein obtained by the method described in (1) in an aqueous medium to obtain an aqueous dispersion, placing the aqueous dispersion between a plurality of adherends, and then removing the aqueous medium from the aqueous dispersion and solidifying the modified protein to thereby adhere the adherends.

[0074] (47-1) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), dispersing the obtained modified protein in an aqueous medium to obtain an aqueous dispersion, placing the obtained aqueous dispersion between a plurality of adherends, removing the aqueous medium from the aqueous dispersion and solidifying the modified protein to thereby adhere the adherends.

[0075] (48) A method for producing an adhesive body, comprising the steps of softening a film containing a modified protein obtained by the method described in (1) by swelling or heating, placing the film between a plurality of adherends, and then allowing the film to harden while being pressed against the adherends, thereby bonding the adherends.

[0076] (48-1) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), dissolving the obtained modified protein in a solvent and forming it into a film, softening the obtained film by swelling or heating and placing it between a plurality of adherends, and then hardening the film while being pressed against the adherends to thereby bond the adherends.

[0077] (49) A method for producing an adherend, characterized in that a powder containing a modified protein obtained by the method described in (1) is placed between a plurality of adherends, the powder is heated, and the powder is pressurized and solidified through the adherends, thereby adhering the adherends.

[0078] (49-1) A method for producing an adhesive body, comprising the steps of powdering a modified protein obtained by the method described in (1), heating the powder while placing it between a plurality of adherends, and pressurizing the powder through the adherends to solidify the powder, thereby bonding the adherends together.

[0079] (49-2) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), powdering the obtained modified protein, and heating the powder while placing it between a plurality of adherends and pressurizing the powder through the adherends to solidify the powder, thereby bonding the adherends together.

[0080] (49-3) A method for producing an adhesive body, comprising the steps of obtaining a powder composition containing a modified protein obtained by the method described in (1) and an adhesion enhancer or a bonding enhancer, and heating the powder composition while placing the obtained powder composition between a plurality of adherends and pressurizing the powder composition through the adherends to solidify the powder composition, thereby bonding the adherends together.

[0081] (49-4) A method for producing an adhesive body, comprising the steps of adding an adhesion enhancer or a bonding enhancer to a modified protein obtained by the method described in (1) to obtain a composition, powdering the obtained composition, and heating the powder composition while placing it between a plurality of adherends and pressurizing the powder composition through the adherends to solidify the powder composition, thereby bonding the adherends together.

[0082] (49-5) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), obtaining a powder composition containing the obtained modified protein and an adhesion enhancer or a bonding enhancer, and heating the powder composition while placing it between a plurality of adherends and pressurizing the powder composition through the adherends to solidify the powder composition, thereby bonding the adherends together.

[0083] (49-6) A method for producing an adhesive body, comprising the steps of obtaining a modified protein by the method described in (1), adding an adhesion enhancer or a bonding enhancer to the obtained modified protein to obtain a composition, powdering the obtained composition, and heating the powder composition while placing it between a plurality of adherends and pressurizing the powder composition through the adherends to solidify the powder composition, thereby bonding the adherends together.

[0084] (50) The method for producing a binder according to any one of (46) to (49), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein.

[0085] (51) The method for producing a binder according to (46), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the solvent is an alkaline aqueous solution.

[0086] (52) According to the method for producing an adhesive body described in (48), the film is obtained by casting a solution obtained by dissolving at least one modified protein selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein in an alkaline aqueous solution.

[0087] (52-1) According to the method for producing an adhesive body described in (48-1), the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the film forming step is a step of dissolving the modified protein in an alkaline aqueous solution and then performing cast molding.

[0088] (53) A method for producing a solution coating, comprising the step of dissolving the modified protein obtained by the method described in (1) in a solvent.

[0089] (53-1) A method for producing a solution coating, comprising the steps of obtaining a modified protein by the method described in (1), and dissolving the obtained modified protein in a solvent.

[0090] (54) A method for producing a water-dispersible coating, comprising the step of dispersing the modified protein obtained by the method described in (1) in an aqueous medium.

[0091] (54-1) A method for producing a water-dispersible coating, comprising the steps of obtaining a modified protein by the method described in (1), and dispersing the obtained modified protein in an aqueous medium.

[0092] (55) A method for producing a thin film coating, comprising the step of forming a thin film from a solution in which the modified protein obtained by the method described in (1) is dissolved.

[0093] (55-1) A method for producing a thin film coating, comprising the steps of obtaining a modified protein by the method described in (1), and dissolving the obtained modified protein in a solvent and forming the resulting film.

[0094] (56) A method for producing a powdered coating, comprising the step of obtaining a powder containing a modified protein obtained by the method described in (1).

[0095] (56-1) A method for producing a powdered coating, comprising the step of powdering the modified protein obtained by the method described in (1).

[0096] (56-2) A method for producing a powdered coating, comprising the steps of obtaining a modified protein by the method described in (1), and powdering the obtained modified protein.

[0097] (56-3) A method for producing a powdered coating, comprising the step of obtaining a powder composition containing a modified protein obtained by the method described in (1) and an adhesion enhancer or a bonding enhancer.

[0098] (56-4) A method for producing a powdered coating, comprising the steps of adding an adhesion enhancer or a bonding enhancer to the modified protein obtained by the method described in (1) to obtain a composition, and powdering the obtained composition.

[0099] (56-5) A method for producing a powdered coating, comprising the steps of obtaining a modified protein by the method described in (1), and obtaining a powder composition containing the obtained modified protein and an adhesion enhancer or bonding enhancer.

[0100] (56-6) A method for producing a powdered coating, comprising the steps of obtaining a modified protein by the method described in (1), adding an adhesion enhancer or a bonding enhancer to the obtained modified protein to obtain a composition, and powdering the obtained composition.

[0101] (57) The method for producing a coating according to any one of (41) to (44), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphoprotein.

[0102] (58) The method for producing a solution coating according to (51), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the solvent is an alkaline aqueous solution.

[0103] (59) According to the method for producing a thin film coating described in (55), the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the method comprises a step of dissolving the modified protein in an alkaline aqueous solution.

[0104] (60) A method for manufacturing a laminate having a substrate and a coating layer laminated on at least a portion of the surface of the substrate, characterized in that a solution formed by dissolving a modified protein obtained by the method described in (1) 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 modified protein is solidified to form the coating layer laminated on at least a portion of the surface of the substrate.

[0105] (60-1) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, comprising the steps of obtaining a modified protein by the method described in (1), dissolving the obtained modified protein in a solvent to obtain a solution, supplying the solution to at least a portion of the surface of the substrate and coating at least a portion of the surface of the substrate with the solution, and removing the solvent from the coating to form the coating layer.

[0106] (61) A method for manufacturing a laminate having a substrate and a coating layer laminated on at least a portion of the surface of the substrate, characterized in that an aqueous dispersion formed by dispersing a modified protein obtained by the method described in (1) in an aqueous medium 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 aqueous dispersion, the aqueous medium is removed from the aqueous dispersion and the modified protein is solidified to thereby laminate the coating layer on at least a portion of the surface of the substrate.

[0107] (61-1) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, comprising the steps of obtaining a modified protein by the method described in (1), dispersing the obtained modified protein in an aqueous medium to obtain an aqueous dispersion, supplying the aqueous dispersion to at least a portion of the surface of the substrate and coating at least a portion of the surface of the substrate with the aqueous dispersion, and removing the aqueous medium from the coating to form the coating layer.

[0108] (62) A method for manufacturing a laminate having a substrate and a coating layer laminated on at least a portion of the surface of the substrate, characterized in that a film containing a modified protein obtained by the method described in (1) is softened by swelling or heating and placed on at least a portion of the surface of the substrate, and then the film is hardened while being pressed onto the substrate to form a coating layer laminated on at least a portion of the surface of the substrate.

[0109] (62-1) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, comprising the steps of obtaining a modified protein by the method described in (1), dissolving the obtained modified protein in a solvent and forming it into a film, softening the film by swelling or heating and placing it on at least a portion of the surface of the substrate, and hardening the film while being pressed against the substrate to form the coating layer.

[0110] (63) A method for manufacturing a laminate having a substrate and a coating layer laminated on at least a portion of the surface of the substrate, characterized in that a powder containing a modified protein obtained by the method described in (1) is placed on at least a portion of the surface of the substrate, the powder is heated, and the powder is pressurized and solidified between a pressurizing body and the substrate, thereby forming a coating layer laminated on at least a portion of the surface of the substrate.

[0111] (63-1) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized in that the modified protein obtained by the method described in (1) is powdered, the powder obtained is placed on at least a portion of the surface of the substrate, the powder is heated, and the powder is pressurized between a pressurizing body and the substrate to solidify, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

[0112] (63-2) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized by comprising a step of obtaining a modified protein by the method described in (1), a step of powdering the obtained modified protein, heating the obtained powder while placing it on at least a portion of the surface of the substrate, and pressurizing the powder between a pressurizing body and the substrate to solidify it, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

[0113] (63-3) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized by the steps of obtaining a powder composition containing a modified protein obtained by the method described in (1) and an adhesion enhancer or a bonding enhancer, placing the obtained powder composition on at least a portion of the surface of the substrate, heating the powder composition, and pressurizing the powder composition between a pressurizing body and the substrate to cure it, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

[0114] (63-4) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized by a step of adding an adhesion enhancer or a bonding enhancer to the modified protein obtained by the method described in (1) to obtain a composition, a step of powdering the obtained composition, heating the powder composition while placing the obtained powder composition on at least a portion of the surface of the substrate, and pressurizing the powder composition between a pressurizing body and the substrate to solidify it, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

[0115] (63-5) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized in that the process comprises a step of obtaining a modified protein by the method described in (1), a step of obtaining a powder composition containing the obtained modified protein and an adhesion enhancer or a bonding enhancer, the powder composition obtained 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 the powder composition, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

[0116] (63-6) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, characterized by comprising a step of obtaining a modified protein by the method described in (1), a step of adding an adhesion enhancer or a bonding enhancer to the obtained modified protein to obtain a composition, a step of powdering the obtained composition, a step of heating the obtained powder composition while placing the obtained powder composition on at least a portion of the surface of the substrate, and pressurizing the powder composition between a pressurizing body and the substrate to solidify the powder composition so as to form a coating layer laminated on at least a portion of the surface of the substrate.

[0117] (63-7) A method for manufacturing a laminate having a substrate and a coating layer formed on at least a portion of the surface of the substrate, comprising the steps of powdering a modified protein obtained by the method described in (1), adding an adhesion enhancer or a bonding enhancer to the obtained modified protein to obtain a powder composition, heating the powder composition and placing it on at least a portion of the surface of the substrate, and pressurizing the powder composition between a pressurizing body and the substrate to form the coating layer.

[0118] (64) The method for producing a laminate according to any one of (60) to (63), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein.

[0119] (65) The method for producing a laminate according to (60), wherein the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the solvent is an alkaline aqueous solution.

[0120] (66) According to the method for manufacturing a laminate described in (62), the film is obtained by molding using a solution obtained by dissolving at least one modified protein selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein in an alkaline aqueous solution.

[0121] (66-1) According to the method for manufacturing a laminate described in (62), the modified protein is at least one selected from the group consisting of maleated protein, succinate protein, sulfonate protein and phosphate protein, and the film forming step is a step of dissolving the modified protein in an alkaline aqueous solution and then forming it into a film.

[0122] Effects of the Invention

[0123] The present invention provides a novel method for producing modified proteins, which has a higher degree of freedom in reaction conditions than conventional protein modification in a solvent, and can produce a wider range of modified proteins. In addition, the present invention can produce modified proteins having appropriate physical properties suitable for the desired use.

[0124] Well-known esterification using mechanochemical methods include acetylation of cellulose (see Non-Patent Document 1) and selective esterification of lignin model compounds using enzymes (see Non-Patent Document 2), but no examples of esterification using mechanochemical methods are known for protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] [ Figure 1 ](a) is a photograph showing the appearance of the modified proteins of Comparative Examples 1 and 2 and Examples 1 and 2, and (b) is a graph comparing the GPC retention times of the unmodified protein PRT966 and the modified proteins.

[0126] [ Figure 2 ](a) is the IR spectrum of Comparative Example 1 and unmodified protein PRT966, and (b) is the GPC chromatogram of Comparative Example 1 and unmodified protein PRT966.

[0127] [ Figure 3 ](a) is the IR spectrum of Comparative Example 2 and unmodified protein PRT966, and (b) is the GPC chromatogram of Comparative Example 2 and unmodified protein PRT966.

[0128] [ Figure 4 ](a) is the IR spectrum of Example 1 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 1 and unmodified protein PRT966.

[0129] [ Figure 5 ](a) is the IR spectrum of Example 2 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 2 and unmodified protein PRT966.

[0130] [ Figure 6 ](a) is the IR spectrum of Example 3 and unmodified protein PRT2882, and (b) is the GPC chromatogram of Example 3 and unmodified protein PRT2882.

[0131] [ Figure 7 ](a) is the IR spectrum of Example 4 and unmodified protein PRT2841, and (b) is the GPC chromatogram of Example 4 and unmodified protein PRT2841.

[0132] [ Figure 8 ](a) is the IR spectrum of Example 5 and unmodified protein PRT2662, and (b) is the GPC chromatogram of Example 5 and unmodified protein PRT2662.

[0133] [ Fig. 9 ](a) is the IR spectrum of Example 6 and unmodified protein PRT1000, and (b) is the GPC chromatogram of Example 6 and unmodified protein PRT1000.

[0134] [ Fig.10 ](a) is the IR spectrum of Example 7 and unmodified protein PRT918, and (b) is the GPC chromatogram of Example 7 and unmodified protein PRT918.

[0135] [ Fig.11 ](a) is the IR spectrum of Example 8 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 8 and unmodified protein PRT966.

[0136] [ Fig.12 ](a) is the IR spectrum of Example 9 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 9 and unmodified protein PRT966.

[0137] [ Fig.13 ](a) is the IR spectrum of Example 10 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 10 and unmodified protein PRT966.

[0138] [ Fig.14 ](a) is the IR spectrum of Example 11 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 11 and unmodified protein PRT966.

[0139] [ Fig.15 ](a) is the IR spectrum of Example 12 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 12 and unmodified protein PRT966.

[0140] [ Fig.16 ](a) is the IR spectrum of Example 13 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 13 and unmodified protein PRT966.

[0141] [ Fig.17 ](a) is the IR spectrum of Example 14 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 14 and unmodified protein PRT966.

[0142] [ Fig.18 ](a) is the IR spectrum of Example 15 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 15 and unmodified protein PRT966.

[0143] [ Fig.19 ](a) is the IR spectrum of Example 16 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 16 and unmodified protein PRT966.

[0144] [ Fig. 20 ](a) is the IR spectrum of Example 17 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 17 and unmodified protein PRT966.

[0145] [ Fig.21 ](a) is the IR spectrum of Example 18 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 18 and unmodified protein PRT966.

[0146] [ Fig. 22 ](a) is the IR spectrum of Example 19 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 19 and unmodified protein PRT966.

[0147] [ Fig.23 ](a) is the IR spectrum of Example 20 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 20 and unmodified protein PRT966.

[0148] [ Fig.24 ](a) is the IR spectrum of Example 21 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 21 and unmodified protein PRT966.

[0149] [ Fig.25 ](a) is the IR spectrum of Example 22 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 22 and unmodified protein PRT966.

[0150] [ Fig.26](a) is a photograph showing the state in which water is added to a protein or a modified protein, (b) is a photograph showing the state in which acetone is added to a protein or a modified protein, and (c) is a photograph showing the state in which a 0.5 w / v% sodium hydroxide aqueous solution is added to a protein or a modified protein.

[0151] [ Fig. 27 ](a) is a photograph showing the state of the protein immediately after the addition of DMSO, (b) is a photograph showing the state of the protein dissolved in DMSO, and (c) is a photograph comparing the gelation states of the proteins.

[0152] [ Fig.28 ](a) is the IR spectrum of Example 24 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 24 and unmodified protein PRT966.

[0153] [ Fig.29 ](a) is the IR spectrum of Example 25 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 25 and unmodified protein PRT966.

[0154] [ Fig.30 ] represents a photograph of an extruder screw element. Fig.30 In the figure, FC represents the forward conveying element, R represents the reverse element, K represents the mixing element, and C represents the compression element.

[0155] [ Fig.31 ](a) is the IR spectrum of Example 26 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 26 and unmodified protein PRT966.

[0156] [ Fig.32 ](a) is the IR spectrum of Example 27 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 27 and unmodified protein PRT966.

[0157] [ Fig.33 ](a) is the IR spectrum of Example 28 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 28 and unmodified protein PRT966.

[0158] [ Fig.34 ](a) is the IR spectrum of Example 29 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 29 and unmodified protein PRT966.

[0159] [ Fig.35 ](a) is the IR spectrum of Example 30 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 30 and unmodified protein PRT966.

[0160] [ Fig.36 ](a) is the IR spectrum of Example 31 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 31 and unmodified protein PRT966.

[0161] [ Fig.37 ](a) is the IR spectrum of Example 32 and unmodified protein PRT966, and (b) is the GPC chromatogram of Example 32 and unmodified protein PRT966.

[0162] [ Fig.38 ]a~d are photographs of cast films of PRT966, Ac-PRT, Succ-PRT and St-PRT, respectively.

[0163] [ Fig.39 ](a) is a graph showing the combustion time of each sample, and (b) is a graph showing the mass loss of each sample.

[0164] [ Fig.40 ] is a graph showing the softening point, Tg onset point, and melting temperature Tm of unmodified PRT966 and each modified protein. DETAILED DESCRIPTION

[0165] 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.

[0166] 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 equivalent to each component in the composition, unless otherwise specified, the content of each component in the composition refers to the total amount of the multiple substances present in the composition.

[0167] A first embodiment of the present invention is a method for producing a modified protein, comprising the step of treating a mixture containing a protein and an acylating agent using a mechanochemical method to obtain a modified protein.

[0168] In this specification, "modified protein" refers to a protein in which at least a part or all of the lysine residues, serine residues, threonine residues, tyrosine residues or cysteine ​​residues in the raw material protein are acylated. In other words, the raw material protein has at least one lysine residue, serine residue, threonine residue, tyrosine residue or cysteine ​​residue in its amino acid sequence.

[0169] <Protein>

[0170] Protein can be natural protein or artificial protein. For example, a protein having physical properties close to those required for the desired use can be used. In addition, as a protein, proteins that can be used in industry, proteins that can be used in medicine, etc. can be listed. The so-called industrial use refers to various general materials that can be used indoors or outdoors, etc. As a specific example of protein that can be used in industry, structural proteins can be listed. In addition, as a specific example of structural proteins, spider silk (spider silk), silk, keratin, collagen, elastin, arthropod elastic protein, and proteins derived from them can be listed. As the protein used, it can also be artificial silk fibroin or artificial spider silk fibroin (artificially modified spider silk fibroin). As a specific example of protein that can be used in medicine, enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membranes or transport proteins, antigens for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies or antibody fragments or derivatives can be listed.

[0171] Artificial proteins include recombinant proteins and synthetic proteins. That is, in this specification, the so-called "artificial protein" refers to an artificially manufactured protein. An artificial protein can be a protein whose domain sequence is different from the amino acid sequence of a protein of natural origin, or a protein with the same amino acid sequence as a protein of natural origin. In addition, an "artificial protein" can be a protein that directly utilizes the amino acid sequence of a protein of natural origin, or a protein that modifies the amino acid sequence based on the amino acid sequence of a protein of natural origin (for example, a protein obtained by modifying the amino acid sequence by modifying the gene sequence of a cloned protein of natural origin), or a protein that is artificially designed and synthesized without relying on a protein of natural origin (for example, a protein having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). In addition, unlike natural proteins, artificial proteins can freely design amino acid sequences, so when the artificial protein is included in the molded material or molded body in the present embodiment described later, by appropriately designing the amino acid sequence of the artificial protein, the function or characteristics, physical properties, etc. of the molded material or molded body can be arbitrarily controlled. In addition, since uniform molecular design can be performed frequently, proteins that have high homology with the target protein and meet the target can be stably obtained. Therefore, it is beneficial to stabilize the quality of the molded materials and molded bodies obtained using artificial proteins. From this point of view, it is very advantageous to use artificial structural proteins as artificial proteins.

[0172] The number of amino acid residues of the protein and modified protein involved in the present 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 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 the solvent.

[0173] The molecular weight of the protein involved in this embodiment is not particularly limited, and can be, for example, 2 kDa or more and 500 kDa or less. In addition, 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 less than 500 kDa, less than 400 kDa, less than 360 kDa, less than 300 kDa, or less than 200 kDa.

[0174] The protein involved in the present embodiment contains at least one nucleophilic functional group, such as a serine residue, a threonine residue, a tyrosine residue, a lysine residue and a cysteine ​​residue. The modified protein is a hydroxyl group possessed by a serine residue, a threonine residue or a tyrosine residue in the protein, an amino group possessed by a lysine residue or the like, or a thiol group possessed by a cysteine ​​residue is acylated. When a protein has a serine residue, a threonine residue or a tyrosine residue, its acylation efficiency can be effectively improved by an acylating agent. Considering the improvement of the reactivity of the protein with the acylating agent, the improvement of the productivity of the modified protein, etc., the total content of each serine residue, threonine residue and tyrosine residue of the protein (the ratio of the total residue number 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. In addition, the upper limit of the total content of each of serine residues, threonine residues and tyrosine residues is not particularly limited, but considering the amino acid composition of various proteins that can be preferably used as the proteins and modified proteins involved in this embodiment, it can be, for example, less than 35%, less than 33%, less than 30%, less than 25% or less.

[0175] (Hydrophobic Protein)

[0176] The protein may be, for example, a hydrophobic protein. When the protein is a hydrophobic protein, when the modified protein is used as at least a part of the raw material to manufacture a shaped body, the water resistance of the related shaped body is improved, for example, when the shaped body is used as a general-purpose material for industrial use, it is beneficial to extend its service life. In addition, when the modified protein is combined with a functional substance, for example, the hydrophobicity or hydrophilicity of the entire combination of the functional substance and the modified protein can be arbitrarily adjusted by controlling the hydrophobicity or hydrophilicity of the functional substance. In the case where the protein is hydrophobic (that is, the protein is a hydrophobic protein), the entire combination can be shifted to the hydrophobic side compared to the case where the protein is hydrophilic, so the hydrophobicity or hydrophilicity of the entire combination can be controlled over a larger range.

[0177] The hydrophobicity in a protein can be estimated using the average hydrophilic index (hereinafter also referred to as "HI") as an index. In the present specification, the average hydrophilic index of a hydrophobic protein may be greater than 0, for example, it may be greater than 0.00, greater than 0.10, greater than 0.20, greater than 0.22, greater than 0.25, greater than 0.30, greater than 0.35, greater than 0.40, greater than 0.45, greater than 0.50, greater than 0.55, greater than 0.60, greater than 0.65, or greater than 0.70. In addition, the upper limit is not particularly limited, for example, it may be less than 1.00, or less than 0.7.

[0178] In addition, the average hydropathic index (HI) value of the hydrophobic protein and the hydrophobicity of the repeating sequence unit described later can be obtained by using the known hydrophobicity index of the amino acid residues according to the known method. The known hydrophobicity index of the amino acid residues is 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.

[0179] [Table 1]

[0180] 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

[0181] The hydrophobic protein is preferably a protein with low solubility in a 60°C lithium bromide aqueous solution (concentration: 9M). In other words, the maximum concentration of the hydrophobic protein when dissolved in a 60°C lithium bromide aqueous solution (concentration: 9M) may be, for example, less than 30% by mass, less than 25% by mass, less than 20% by mass, less than 15% by mass, less than 10% by mass, less than 5% by mass, or less than 1% by mass. In addition, as the hydrophobic protein, a protein that is completely insoluble in a 60°C lithium bromide aqueous solution (concentration: 9M) may be used. When the solubility of the hydrophobic protein in a 60°C lithium bromide aqueous solution is low, the obtained modified protein is more likely to obtain good water resistance (especially water resistance suitable for industrial use).

[0182] The hydrophobic protein is preferably a protein having a contact angle with water of 55° or more. The contact angle with water of the hydrophobic protein is more preferably 60° or more, 65° or more, or 70° or more. The contact angle with water can be evaluated by forming a film composed of the hydrophobic protein on a substrate, dropping water on the film, and measuring the contact angle after 5 seconds. When the contact angle with water of the hydrophobic protein is 55° or more, the obtained modified protein is more likely to obtain good water resistance (especially water resistance suitable for industrial use).

[0183] The hydrophobic protein is preferably a protein having excellent hot water resistance. The hot water resistance is preferably a protein that does not decompose for at least 5 hours when a 5 w / v% aqueous dispersion of the hydrophobic protein is heated to 100° C. When the hydrophobic protein is a protein having excellent hot water resistance, the obtained modified protein is more likely to have good water resistance (especially water resistance suitable for industrial use) and hot water resistance.

[0184] <Structural Protein>

[0185] The protein involved in the present embodiment can be, for example, a structural protein. The so-called structural protein is one of the proteins that can be used in industry, 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. For example, a structural protein can be a protein that does not directly participate in biochemical reactions but constitutes nails, hair, skin, tendons, cartilage, and intracellular and extracellular matrices, or a protein such as collagen and keratin molecules that are fibrous but have no hydrophobic core, and a protein polymer that is a globular protein with monomers such as actin but multiple polymers are fibrous. Structural proteins refer to proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. In addition, structural proteins can also refer to proteins that are repeated in motifs composed of characteristic amino acid sequences or amino acid residues to form the skeleton of organisms and materials. Natural structural proteins, for example, can include fibroin, keratin, collagen, elastin, and elastin. Silk fibroin forms fibers with a structure in which the polypeptide chains of β sheets are extended, and multiple polypeptide chains of α helices in keratin are bundled into ropes. The more spherical polypeptide chains in elastin are cross-linked by covalent bonds to form a network as elastic as rubber. In collagen, the slender fibrous polypeptide chains form a triple helix structure (also called "collagen fibrils"), and there is a glycine residue for every 3 residues in each polypeptide chain. Proline residues account for more than 20% of all amino acid residues, of which about half are hydroxyproline, which helps to stabilize the triple helix structure.

[0186] The structural protein may also be an artificial structural protein. In this specification, the so-called "artificial structural protein" refers to an artificially produced structural protein. The artificial structural protein may be a structural protein produced by a microorganism using genetic recombination technology, or a protein having the same amino acid sequence as a natural structural protein, or a protein having an amino acid sequence that has been improved from the perspective of productivity or formability.

[0187] When forming a structural protein, the amino acids with relatively smaller side chains are more likely to form hydrogen bonds, and it is easier to obtain a molded body with higher strength. In addition, since the side chains of alanine residues and glycine residues are non-polar amino acids, they are arranged inward during the folding process of the generated polypeptide, and it is easy to form an α-helical structure or a β-sheet structure. Therefore, it is expected to increase the proportion of amino acids such as glycine residues and alanine residues. From the perspective of obtaining a molded body with better strength, the alanine residue content is, for example, 10 to 40%, and can be 12 to 40%, 15 to 40%, 18 to 40%, 20 to 40% or 22 to 40%. From the perspective of obtaining a molded body with better strength, the glycine residue content is, for example, 10 to 55%, and can be 11 to 55%, 13 to 55%, 15 to 55%, 18 to 55%, 20 to 55%, 22 to 55% or 25 to 55%.

[0188] In the present specification, the "alanine residue content" refers to the ratio of the number of alanine residues to the number of all amino acid residues constituting a protein, and is a value represented by the following formula.

[0189] Alanine residue content = (number of alanine residues contained in the protein / total number of amino acid residues in the protein) x 100 (%)

[0190] In addition, the glycine residue content, serine residue content, threonine residue content, proline residue content and tyrosine residue content have the same meanings as those obtained by replacing alanine residue with glycine residue, serine residue, threonine residue, proline residue and tyrosine residue, respectively, in the above formula.

[0191] The structural protein preferably contains a certain degree of amino acids with larger side chains or amino acids with flexibility homogeneously throughout the sequence. Specifically, the structural protein may also be a motif containing tyrosine residues, threonine residues and proline residues that are repeatedly and periodically added. If it is such a structural protein, when the shaped body obtained by forming is processed, it is easy to hinder the formation of stable intermolecular hydrogen bonds, thereby making it easier to process. For example, in any continuous 20 amino acid residues, the total content of proline residues, threonine residues and tyrosine residues may also be 5% or more, greater than 5.5%, 6.0% or more, greater than 6.5%, 7.0% or more, greater than 7.5%, 8.0% or more, greater than 8.5%, 9.0% or more, 10.0% or more or 15.0% or more. In addition, for example, in any continuous 20 amino acid residues, the total content of proline residues, threonine residues and tyrosine residues may be less than 50%, less than 40%, less than 30% or less than 20%.

[0192] The artificial structural protein involved in one embodiment, like the above-mentioned protein, may have a total value of the content of serine residues, threonine residues and tyrosine residues of, 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 from the viewpoint of improving the reactivity with the acylating agent and improving the productivity of the reaction-modified protein obtainable by the reaction with the acylating agent. The total value of the content of serine residues, threonine residues and tyrosine residues may be, for example, 35% or less, 33% or less, 30% or less, 25% or less, or 20% or less.

[0193] 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 consistency in the artificial structural protein. The number of amino acid residues in the repeating sequence unit is preferably 6 to 200. In addition, the sequence consistency between the repeating sequence units can be, for example, 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 degree (hydrophilic index) of the repeating sequence unit can be, for example, more than -0.80, more than -0.70, more than -0.60, more than -0.50, more than -0.40, more than -0.30, more than -0.20, more than -0.10, more than 0.00, more than 0.22, more than 0.25, more than 0.30, more than 0.35, more than 0.40, more than 0.45, more than 0.50, more than 0.55, more than 0.60, more than 0.65 or more than 0.70. In addition, the upper limit of the hydrophobicity degree of the repeating sequence unit is not particularly limited, for example, it can be less than 1.0, or less than 0.7.

[0194] An artificial structural protein according to one embodiment may include (A) n In this specification, (A) n A motif is an amino acid sequence that is primarily composed of alanine residues. (A) n The number of amino acid residues in the motif may be 2 to 27, or may be an integer of 2 to 20, 2 to 16, or 2 to 12. In addition, (A) n The ratio of the number of alanine residues in the motif to the total number of amino acid residues can be 40% or more, or 50% or more, 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).

[0195] Artificial structural protein can also be artificial silk fibroin. As silk fibroin, for example, natural silk fibroin can be listed. As natural silk fibroin, for example, silk fibroin produced by insects or spiders can be listed. Natural silk fibroin is a fibrous protein with a molecular weight of about 370,000. It is composed of 2 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 all amino acid residues. Natural silk fibroin has two different areas, namely a crystalline area rich in amino acid residues with smaller side chains such as glycine, alanine and serine, and an amorphous area with amino acid residues with larger side chains such as tyrosine.

[0196] More specific examples of naturally derived silk fibroin include silk fibroin for which sequence information is registered in NCBI GenBank. For example, it can be confirmed by extracting sequences containing INV as a classification code (DIVISION) in the sequence information registered in NCBI GenBank, and recording "spidroin, ampulla, silk fibroin, "silk and polypeptide" or "silk and protein" as keywords in the definition (DEFINITION), extracting a character string specifying a product from the CDS, and extracting a sequence with a specified character string recorded in the tissue type (TISSUE TYPE) from the source (SOURCE).

[0197] 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 of natural origin, or a silk fibroin having an amino acid sequence identical to that of silk fibroin of natural origin. Artificial silk fibroin may be produced by a known method, for example, by a method described in International Publication No. 2021 / 187502.

[0198] Artificial silk fibroin may be a fibrous protein having a structure suitable for natural silk fibroin, or may be a silk fibroin having a sequence identical to a repeating sequence possessed by natural silk fibroin. The so-called "sequence identical to a repeating sequence possessed by silk fibroin" may actually be a sequence possessed by natural silk fibroin, or may be a sequence similar thereto.

[0199] As long as it has the amino acid sequence specified in the present disclosure, "artificial silk fibroin" may be a silk fibroin obtained by modifying the amino acid sequence of a silk fibroin of natural origin (for example, a silk fibroin obtained by modifying the amino acid sequence of a cloned gene sequence of a silk fibroin of natural origin), or a silk fibroin whose amino acid sequence is artificially designed independently of a silk fibroin of natural origin (for example, a silk fibroin having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence). In addition, a silk fibroin whose amino acid sequence is modified in an artificial silk fibroin may be included in an artificial silk fibroin as long as its amino acid sequence is different from that of a silk fibroin of natural origin. Examples of artificial silk fibroin include artificial silk (silk) fibroin (a silk fibroin whose amino acid sequence of a silk fibroin produced by a silkworm has been modified), and artificial spider silk fibroin (a silk fibroin whose amino acid sequence of a spider silk protein produced by a spider has been modified). Artificial silk fibroin is relatively easy to be fibrillated and has high fiber-forming ability. Therefore, as a molding material, it is preferable to contain artificial spider silk fibroin, and more preferably to consist of artificial spider silk fibroin.

[0200] 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 The protein of the domain sequence represented by the motif. Artificial silk fibroin can further add an amino acid sequence (N-terminal sequence and C-terminal sequence) to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited thereto, and typically do not have a repeated region of the characteristic amino acid motif in silk fibroin, and are composed of about 100 amino acid residues.

[0201] In this specification, "domain sequence" refers to a crystal region that is unique to silk fibroin (typically, corresponding to the amino acid sequence (A) n motif) and an amino acid sequence of an amorphous region (typically, corresponding to the amino acid sequence REP), is the formula 1: [(A) n motif-REP] m Or formula 2: [(A) n motif-REP] m -(A) n The amino acid sequence represented by the motif. Here, (A) n The motif indicates an amino acid sequence mainly composed of alanine residues, with the number of amino acid residues ranging from 2 to 27. (A) nThe 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 can be 50% or more, 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 consist of only alanine residues. REP represents an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 2 to 300, or may be an integer of 3 to 300, 4 to 300, 5 to 300, 6 to 300, or 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.

[0202] Specific examples of artificial silk fibroin include artificial silk fibroin (first artificial silk fibroin) derived from the large silk tube dragline protein produced by the large ampulla gland of a spider described in International Publication No. 2021 / 187502, artificial silk fibroin having a domain sequence with a reduced content of glycine residues (second artificial silk fibroin), and artificial silk fibroin having a domain sequence with a reduced content of (A) n The motif-containing domain sequence of artificial silk fibroin (artificial silk fibroin No. 3), the content of glycine residues is reduced and (A) n The invention relates to an artificial silk fibroin having a motif content (the fourth artificial silk fibroin), an artificial silk fibroin having a domain sequence having a region with a large hydrophobicity index locally (the fifth artificial silk fibroin), and an artificial silk fibroin having a domain sequence with a reduced glutamine residue content (the sixth artificial silk fibroin). The definitions of each of the first to sixth artificial silk fibroins are introduced into the present specification by referring to the contents described in International Publication No. 2021 / 187502.

[0203] Artificial silk fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus, thereby enabling separation, fixation, detection, and visualization of artificial silk fibroin. PRT966 is the sixth artificial silk fibroin containing a tag sequence.

[0204] 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 fibroin by chelating metal chromatography. As a specific embodiment of the tag sequence, for example, the amino acid sequence shown in sequence number 12 (an amino acid sequence comprising a His tag sequence and a hinge sequence) can be listed.

[0205] Alternatively, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose binding protein (MBP) that specifically binds to maltose may be used.

[0206] Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding a peptide (epitope) for displaying antigenicity as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tags, myc tags, FLAG tags, and the like. By utilizing epitope tags, artificial silk fibroin can be easily purified with high specificity.

[0207] Furthermore, a tag obtained by removing a tag sequence using a specific protease may be used. The artificial silk fibroin after the removal of the tag sequence may be recovered by treating the protein adsorbed via the tag sequence with a protease.

[0208] Specific examples of rayon include those shown in Table 2 below.

[0209] [Table 2]

[0210]

[0211] 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.

[0212] The molecular weight of the artificial silk fibroin involved in the present embodiment is not particularly limited, and can be, for example, 2 kDa or more and 700 kDa or less. The molecular weight of the artificial silk fibroin involved in the present 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.

[0213] In this embodiment, the above-mentioned protein is mixed with an acylating agent, and the obtained mixture is treated by a mechanochemical method to chemically modify the protein.

[0214] As the acylating agent, any reagent that can acylate a hydroxyl group, an amino group or a mercapto group may be used, and examples thereof include activated carboxylic acid derivatives such as carboxylic acid halides (e.g., carboxylic acid chlorides and carboxylic acid bromides), carboxylic acid anhydrides (e.g., acid anhydrides and cyclic acid anhydrides), activated sulfonic acid derivatives such as sulfonic acid halides (e.g., sulfonic acid chlorides and sulfonic acid bromides), sulfonic acid anhydrides, phosphorus halides (e.g., phosphorus trichloride and phosphorus oxychloride), and phosphoric acid derivatives such as phosphate triesters (e.g., tris(difluoroethyl) phosphate). These acylating agents can be used to introduce acyl groups corresponding to carboxylic acids, sulfonic acids or phosphoric acids. As carboxylic acids, the following can be cited: saturated monocarboxylic acids such as acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid (n-heptanoic acid), octanoic acid (n-octanoic acid), pelargonic acid (nonanoic acid), capric acid, lauric acid, myristic acid, palmitic acid, heptadecanoic acid, stearic acid, isostearic acid, etc.; unsaturated monocarboxylic acids such as oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, etc.; dicarboxylic acids such as maleic acid. Acid halides corresponding to these carboxylic acids include, for example, acetyl chloride, acetyl bromide, propionyl chloride, propionyl bromide, lauroyl chloride, myristoyl chloride, palmitoyl chloride, stearoyl chloride, etc. Acid anhydrides corresponding to these carboxylic acids include acetic anhydride, propionic anhydride, maleic anhydride, etc. As sulfonic acids, methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, trifluoromethanesulfonic acid, nonafluorobutanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, m-toluenesulfonic acid, o-toluenesulfonic acid, etc. Examples of acid halides corresponding to these sulfonic acids include methanesulfonyl chloride, methanesulfonyl bromide, ethanesulfonyl chloride, ethanesulfonyl bromide, propanesulfonyl chloride, propanesulfonyl bromide, benzenesulfonyl chloride, p-toluenesulfonyl chloride, m-toluenesulfonyl chloride, and o-toluenesulfonyl chloride. Examples of acid anhydrides corresponding to these carboxylic acids include methanesulfonic anhydride, ethanesulfonic anhydride, trifluoromethanesulfonic anhydride, and nonafluorobutanesulfonic anhydride.

[0215] When the acylating agent is maleic anhydride, the obtained modified protein (maleated protein) has a terminal carboxyl group. The maleated protein can be further reacted with sodium bisulfite by a mechanochemical method to be converted into a sulfosuccinylated protein. The maleated protein can be further reacted with a dialkyl phosphite by a mechanochemical method to be converted into a (diethoxyphospho)succinylated protein.

[0216] The amount of the acylating agent used may be 1.5 to 7 equivalents per nucleophilic functional group contained in the protein. The amount of the acylating agent used may 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 acylating agent relative to one nucleophilic functional group contained in the protein. By using the amount of the acylating agent in the above range, the reactivity or reaction efficiency between the protein and the acylating agent in the mechanochemical treatment can be improved, and the target modified protein can be obtained more efficiently.

[0217] Treatment by mechanochemical method refers to treatment of chemical reaction induced by direct absorption of mechanical energy. The mechanical energy can be, for example, energy exerted by impact force or shear force. Such mechanochemical treatment is specifically carried out using a cylindrical ball mill, a medium stirring mill, a planetary mill, a jet mill, a mixing mill, and an extruder (twin screw extruder). Specifically, the protein and the acylating agent are placed in a grinding jar, and medium balls are added according to the type of grinder used. Afterwards, the grinding jar is mounted on the mixing mill and vibrated according to a specified frequency and reaction time. At this time, a solvent, an alkali, a reaction accelerator, etc. can also be further added to the grinding jar. Alternatively, the protein and the acylating agent can also be placed in a mixing mill, the grinding step can be started, and the reactants can be kneaded continuously. At this time, a solvent, an alkali, a reaction accelerator, etc. can also be further added to the mixing mill. In addition, the protein and the acylating agent are placed in an extruder and mixed and kneaded. At this time, a solvent, an alkali, a reaction accelerator, etc. can also be further added to the extruder. Furthermore, if the mechanochemical treatment is performed using an extruder, the target esterified protein (modified protein) can be produced continuously.

[0218] The frequency can be appropriately adjusted by a person skilled in the art according to the reaction, and may 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.

[0219] The reaction time is a time point until the raw material protein disappears or a certain amount of acylated protein (modified protein) is observed in infrared (IR) absorption spectrum, gel filtration chromatography (GPC), etc. The reaction time may be any time, for example, 30 to 150 minutes, 30 to 120 minutes, 30 to 110 minutes, 30 to 100 minutes, 30 to 95 minutes, 60 to 150 minutes, 60 to 120 minutes, 60 to 110 minutes, 60 to 100 minutes, 60 to 95 minutes, 70 to 150 minutes, 70 to 120 minutes, 70 to 110 minutes, 70 to 100 minutes, 70 to 95 minutes, 80 to 150 minutes, 80 to 120 minutes, 80 to 110 minutes, 80 to 100 minutes, or 80 to 95 minutes.

[0220] The solvent can be any solvent that can swell the protein or at least dissolve one of the protein and the acylating agent, but it must be a compound that is liquid at room temperature and pressure and does not chemically react with the protein. The mechanochemical treatment with the addition of such a solvent is called liquid-assisted grinding (LAG), which is a method of more efficiently promoting the desired reaction by adding a small amount of solvent to the grinding tank. The solvent can be listed as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dihydro-levulinone, ionic liquids, etc. The amount of solvent used can be, for example, 0.01g to 1g, preferably 0.01g to 0.8g, more preferably 0.01 to 0.6g, more preferably 0.01 to 0.4g, and optimally 0.01 to 0.2g relative to 1g of protein. In other words, the amount of solvent used can be, for example, 1 to 100 weight percent, preferably 1 to 80 weight percent, more preferably 1 to 60 weight percent, further preferably 1 to 40 weight percent, and most preferably 1 to 20 weight percent relative to the protein. It is believed that a very small amount of solvent forms a micro-reaction field by swelling the protein or partially dissolving it. In addition, it is not necessary to add a solvent. For example, when the acylating agent is a liquid at room temperature and pressure and has the function of serving as the above-mentioned solvent, it may not be necessary to add a solvent.

[0221] The base may be any base that can assist in the acylation of the hydroxyl group of the serine residue, threonine residue or tyrosine residue, the amino group of the lysine residue or the thiol group of the cysteine ​​residue in the protein. Examples of the base include tertiary amines, nitrogen-containing aromatic compounds, inorganic bases, and more specifically, triethylamine, DIPEA (N,N-diisopropylethylamine), DABCO (1,4-diazabicyclo[2.2.2]octane), quinuclidine, DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DBN (1,5-diazabicyclo[4.3.0]non-5-ene), pyridine, imidazole, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, tripotassium phosphate, sodium acetate, potassium acetate, and the like. When the amount of the nucleophilic functional group contained in the protein is 1, the amount of the base used may be, for example, 1 to 10, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. The amount of the base has a preferred range depending on the type of the base used, for example, when an inorganic base such as sodium hydroxide is used as the base, the amount is preferably 1 to 5, more preferably 1 to 2, and when a tertiary amine such as DIPEA is used as the base, the amount is preferably 1 to 5, more preferably 3.5 to 5.

[0222] The reaction accelerator may be any substance that is commonly used in the field of organic chemistry and accelerates the acylation reaction, and an example thereof is 4-dimethylaminopyridine (DMAP). When the acylation is acetylation, it is not necessary to use a reaction accelerator.

[0223] When the acylation is succinylation, the rate of the acylation reaction can be increased by adding a reaction accelerator. In this case, the amount of the reaction accelerator used is greater than 0 equivalents and less than 5 equivalents, or 3.5 equivalents to 5 equivalents, relative to the amount of the nucleophilic functional group contained in the protein (1 amino acid residue having a nucleophilic functional group). Furthermore, the amount of the reaction accelerator used is greater than 0 mol and less than 1 mol, preferably 0.3 mol to 1 mol, relative to 1 mol of the base used.

[0224] When the acylation is stearoylation, the rate of the acylation reaction can be increased by adding a reaction accelerator. In this case, the amount of the reaction accelerator used is 1 to 5 equivalents, preferably 3.5 to 5 equivalents, relative to the amount of the nucleophilic functional group contained in the protein (1 amino acid residue having a nucleophilic functional group). The amount of the reaction accelerator used is 0.2 to 1 mol, or 0.7 to 1 mol, relative to 1 mol of the base used.

[0225] After the reaction is completed, the mixture is taken out from the grinding tank and can be washed with a solvent. By washing, unreacted acylating agent and acid generated by the reaction can be removed. As the solvent used in the washing, water, acetonitrile, acetone, tetrahydrofuran, ethyl acetate, hexane, etc. can be listed. After washing, the solvent used in the washing can be removed by drying the product. Drying can be carried out under reduced pressure.

[0226] When the modified protein has a hydrophilic group (e.g., hydroxyl, carboxyl, amino, sulfonyl, phosphate) at its end, the contact angle becomes smaller. If a coating containing such modified proteins is used, the wettability and hydrophilicity will be enhanced. When the modified protein has a hydrophobic group (e.g., alkyl, alkenyl, alkynyl, cycloalkyl, aryl) at its end, the contact angle becomes larger. If a coating containing such modified proteins is used, the water-repellent and hydrophobic properties will be enhanced. When the contact angle is 90° to 150°, it is suitable for waterproofing and hydrophobic purposes. When it is 110° to 150°, due to the excellent water-repellent property of the material, it is expected to achieve a self-cleaning effect in which water droplets wash away surface dirt. When the contact angle is 70° to 90°, it is suitable for moisturizing purposes. When it is 40° to 90°, due to the improvement of the penetration effect and dispersion effect of the liquid, it is expected to achieve an effect that the material is easier to dye.

[0227] When the modified protein contains a flame retardant element (for example, a sulfonated or phosphorylated succinylated protein, a sulfonated protein, or a phosphorylated protein), the flame retardancy will be enhanced if a coating containing such a modified protein is used. Coatings containing such modified proteins are suitable for use in improving the safety and durability of related products. In the past, it was generally known that flame retardancy can be imparted when polyphosphates or polynucleotide salts are added to proteins. As flame retardants, for example, substances described in the Japan Rubber Association's journal "General Special Edition Organic Additives Flame Retardants" (Nishizawa Jin, 2006, pp. 316-322) are generally known. However, if it is simply added, polyphosphates and polynucleotide salts may flow out after immersion in water, and the effect may be weakened. Using the modified protein involved in this embodiment, it is possible to achieve the effect that the imparted flame retardancy is not easily weakened even if immersed in water. The flame retardant element is an atom commonly found in the structure of a flame retardant (an atom that can impart flame retardancy), and examples of the flame retardant element include a halogen atom, a phosphorus atom, a sulfur atom, a boron atom, and a silicon atom.

[0228] The critical oxygen index (LOI) value of the modified protein may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more. In this specification, the LOI value is a value measured according to the test method for powdered or low-melting synthetic resins in Fire Hazards Regulation Section No. 50 of the Fire Department of Japan dated May 31, 1995.

[0229] When the modified protein has a hydrophilic group at its end (for example, a succinylated protein or a maleated protein), and has a long-chain alkyl group (preferably an alkyl group with more than 4 carbon atoms, more preferably an alkyl group with more than 6 carbon atoms, and further preferably an alkyl group with more than 8 carbon atoms) by taking in water molecules, it is expected to achieve an effect of approaching the action between polymers by reducing the interaction between the chains, so the softening point becomes lower and the glass transition temperature (Tg) becomes lower than that of the unmodified protein. When the unmodified protein is heated to around 150°C, the decomposition behavior becomes obvious, so it is difficult to heat it to more than 150°C to soften it. However, in the case of the modified protein involved in this embodiment, the softening point and glass transition temperature are reduced, so it can be softened at a temperature below 150°C, which is easy to process or shape. The lower the temperature, the better the influence of decomposition can be suppressed, thereby preventing the deterioration of the material, the significant change in color, and the reduction in strength. From this point of view, it is preferred to control the softening point to below 130°C, and more preferably to below 100°C. The modified protein is suitably used for molding using an extruder or the like, or for the purpose of improving the flexibility of a material at a temperature below the softening point.

[0230] Furthermore, when the molded body containing the modified protein further contains a plasticizer, the softening point and glass transition temperature of the molded body will be further reduced. As the plasticizer, components well known in the industry can be used, for example, polyols such as ethylene glycol, propylene glycol, butylene glycol, and glycerol can be listed. However, if the plasticizer is simply added, the plasticizer may flow out during processing or due to changes over time, and the effect is weakened. If it is the modified protein involved in this embodiment, it can be achieved that even if it is assumed that the molded body is heated during processing or stored for a long time, the effect of reducing the softening point and glass transition temperature is not easily weakened.

[0231] The modified protein involved in the present embodiment can be used alone or as a composition composed of other solid or liquid components added or mixed. In other words, the modified protein can constitute a modified protein composition containing it. Such modified protein compositions have a solid or liquid form by modifying components other than the protein. In addition, the modified protein composition can be used as a forming material for forming fibers, films, gels, porous bodies, particles, resins, etc. In other words, by forming the modified protein composition by a specified method, a modified protein composition formed body can be obtained. As other components contained in the solid or liquid modified protein composition, water, organic solvents, ionic liquids, supercritical liquids, etc. can be used. In addition, the modified protein composition, especially when used as an adhesive or coating, can contain an adhesion enhancer or a bonding enhancer. As an adhesion enhancer, for example, there are rosin derivatives, and as a bonding enhancer, for example, there are sugars such as starch and sucrose.

[0232] In addition, the shaped body obtained by shaping the modified protein composition can be manufactured using a known method according to the type of the shaped body. For example, it can be obtained by a known spinning method such as a wet spinning method, a dry-wet spinning method, a dry spinning method, etc. using a spinning solution containing a fiber, a modified protein and a solvent. In addition, a film, a gel, a porous body, a particle, a resin, etc., for example, can be manufactured according to the method described in Japanese Patent No. 5678283, Japanese Patent No. 5782580, Japanese Patent No. 5796147, Japanese Patent No. 5823079, Japanese Patent No. 6830604, etc. In addition, a resin can be obtained by removing a solvent or a dispersion medium from a modified protein composition (e.g., a gel) containing a modified protein and a prescribed solvent or a dispersion medium, and solidifying it.

[0233] The above-mentioned molded body can also be molded using only the modified protein as a material. In this case, the modified protein can also be said to be a molding material for the molded body.

[0234] The modified protein involved in the present embodiment can be used as an adhesive for joining adherends or a coating for laminating a coating layer on a substrate in the state of a solution, a water dispersion, a film or a powder with it as the main component. These solution adhesives or solution coatings, water-dispersible adhesives or water-dispersible coatings, film adhesives or film coatings, powdered adhesives or powdered coatings can be obtained, for example, by the following manufacturing method. In the description of the embodiment of the adhesive shown below, "laminated body" refers to the state of placing more than one film on the substrate and crimping processing, and "adhesive body" refers to the film itself with the property that can be bonded to the substrate by crimping processing. In addition, in the description of the embodiment of the coating, the component that applies pressure to the coating layer in order to make the coating layer closely adhere to the substrate surface is called "extrusion body" or "pressurized body".

[0235] The solution-like adhesive or solution-like coating containing the modified protein can be produced by a method including a step of dissolving the modified protein obtained by the method of this embodiment in a solvent. Using such a method, unlike conventional solution-like adhesives or solution-like coatings containing components derived from petroleum, a solution-like adhesive or solution-like coating having biodegradability can be easily obtained.

[0236] The solvent used for preparing the solution adhesive or solution coating may be any liquid that can dissolve the modified protein, and may include water, alkaline aqueous solution, acidic aqueous solution, aqueous solution containing inorganic salts and other aqueous media, organic solvents, mixed solvents of these liquids, etc. Examples of the organic solvent include formic acid, dimethyl sulfoxide (DMSO), hexafluoroisopropanol (HFIP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dihydro-levulinone, etc.

[0237] The modified protein contained in the solution-like adhesive or solution-like coating is a protein obtained by the process involved in the present embodiment, as long as it is a protein that can be dissolved in the solvent, and there is no particular limitation. The adhesive or coating formed by the modified protein being dissolved in an aqueous medium has excellent advantages such as usability compared to the adhesive or coating formed by the modified protein being dissolved in an organic solvent. Such aqueous solution-like adhesives or aqueous solution-like coatings, for example, preferably use an aqueous solution formed by dissolving modified proteins such as maleated protein, succinate protein, sulfonate protein, and phosphate protein in an alkaline aqueous solution. These modified proteins have high solubility in alkaline aqueous solutions. Therefore, if the aqueous solution-like adhesive or aqueous solution-like coating formed by dissolving the above-mentioned modified protein in an alkaline aqueous solution is used, a higher concentration of adhesive or coating can be applied to the adhered surface or laminate surface of the adherend described later, and it is expected to improve its bonding strength. In particular, if it is an aqueous solution-like adhesive or coating, the molecules of the modified protein are easily released and easily enter the fine depressions present on the surface of the adherend, which can improve the bonding strength.

[0238] The concentration of the modified protein in the solution-like adhesive or solution-like coating can be appropriately determined according to the solubility of the modified protein 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-like adhesive or solution-like coating can contain components other than the modified protein, such as various additives that are known to be contained in the solution-like adhesive or solution-like coating.

[0239] The solution-like adhesive containing modified proteins is used to make an adherend that a plurality of adherends adhere to each other. For example, the solution-like adhesive is placed between a plurality of adherends that need to be adhered, and then the solvent in the solution-like adhesive is removed to solidify the modified protein, so that the adherends can be adhered to each other to obtain an adherend. An advantage of using this type of method is that an adherend can be obtained using an adhesive with biodegradability, so an adherend that can reduce the environmental burden when discarded can be easily manufactured. In addition, the adherend can be an object that can be adhered with the solution-like adhesive containing modified proteins, and its material is not particularly limited, and can be an organic substance (cellulose products such as paper, wood, synthetic resins, protein products), or an inorganic substance (non-metals such as metals and glass).

[0240] The specific method when using solution-like adhesive to make adherends also has no restriction.For example, solution-like adhesive can be applied or dripped on the adhered surface of at least one of the adherends that adhere to each other, or the adhered surface of the adherend is contacted and impregnated with the liquid level of solution-like adhesive, so that there is solution-like adhesive on the adhered surface, then overlap or stick to the adhered surfaces of the adherends, thereby the solution-like adhesive is placed between a plurality of adherends.And, when removing solvent and making the modified protein solidify from the solution-like adhesive placed between a plurality of adherends, for example, in order to ensure close fit between the overlapped or sticky adhered surfaces, it is possible to heat, air-dry or naturally dry under the state of applying pressure to a plurality of adherends from at least any one side of the overlapped or sticky direction of the adhered surface.Certainly, in the case where such pressure is not applied and can be bonded, it is not necessary to apply pressure.

[0241] A solution-like coating containing a modified protein is used to manufacture a laminated body in which a coating layer is formed on all or part of a substrate surface. For example, the solution-like coating is supplied to at least a portion of a substrate surface on which a coating layer is required to be laminated, and after the surface portion of the substrate is coated with the solution-like coating, the solvent in the solution-like adhesive is removed to solidify the modified protein. Thus, a laminated body in which a coating layer is formed on at least a portion of a substrate surface can be obtained. Using such a method has the advantage that a laminated body can also be obtained using a biodegradable coating, so that a laminated body that can reduce the environmental burden when discarded can be easily manufactured. In addition, the substrate can be any object that can be bonded by the solution-like coating containing the modified protein, and its material is not particularly limited, and can be the same material as the adherend bonded with the solution-like adhesive.

[0242] There is no restriction on the specific method of manufacturing the laminate using the solution coating. For example, the solution coating can be applied or dripped onto at least a portion of the substrate surface, or at least a portion of the substrate surface can be contacted or immersed in the liquid surface of the solution coating, and the coating can be supplied and applied to ensure that the solution coating forms a layer of a specified thickness on at least a portion of the substrate. In addition, when the modified protein in the coating layer (solution coating) stacked on the substrate surface is solidified, for example, the coating layer on the substrate surface can be heated, air-dried or naturally dried to remove the solvent in the coating layer. In this case, if necessary, a specified extrusion body (pressurized body) can be placed on the coating layer in a manner that covers the entire coating layer, and the coating layer is extruded (pressurized) to the substrate surface side to ensure that the coating layer is closely attached to the substrate surface. At this time, an extrusion body that does not adhere to the solution coating is preferred. For example, by applying a release agent or pasting a release paper on the contact surface of the extrusion body with the coating layer, or by surface treating the contact surface to ensure that the coating does not adhere, or by using a material that does not adhere to the solution coating as the extrusion body, the solution coating can be prevented from adhering to the extrusion body. In addition, as the extruded body, for example, materials that show release properties to solution-state coatings, such as PTFE (polytetrafluoroethylene), PP (polypropylene), PE (polyethylene), and silicone, can be listed, and as the surface treatment of the extruded body, for example, there is silicone oil coating treatment.

[0243] The water-dispersible adhesive or water-dispersible coating containing the modified protein can be obtained by a method comprising the step of dispersing the modified protein obtained by the method of this embodiment in an aqueous medium (aqueous liquid). Using such a method, unlike conventional water-dispersible adhesives or water-dispersible coatings containing components derived from petroleum, a water-dispersible adhesive or water-dispersible coating having biodegradability can be easily obtained.

[0244] The aqueous medium used for preparing the water-dispersible adhesive or water-dispersible coating includes water, alkaline aqueous solution, acidic aqueous solution, aqueous solution containing inorganic salt, etc., which can disperse the modified protein. When the solvent used dissolves the modified protein, the solvent is suitable for preparing the solution-like adhesive or coating. When the solvent used does not completely dissolve the modified protein, the solvent is suitable for preparing the water-dispersible adhesive or coating.

[0245] The modified protein 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 described aqueous medium. As the modified protein, for example, maleated protein, succinate protein, sulfonated protein, phosphoesterified protein, etc. can be listed. These modified proteins have relatively high water absorption, and therefore can disperse more amounts in aqueous medium. Therefore, it is conducive to improving the containing ratio of the modified protein in the water-dispersible coating containing these modified proteins, and the containing ratio of the aqueous medium can be effectively reduced. And, it is expected to improve the speed of removing the aqueous medium and solidifying the modified protein, so the advantage of improving the bonding speed of the adherend can be obtained. In addition, in the water-dispersible adhesive or the water-dispersible coating, components other than the modified protein such as various additives contained in the known water-dispersible adhesive or the water-dispersible coating can be included when necessary.

[0246] The water-dispersible adhesive containing modified protein is used to make the adherend of multiple adherends mutually adhering. For example, the water-dispersible adhesive is placed between multiple adherends that need to be bonded, and then the aqueous medium in the water-dispersible adhesive is removed to solidify the modified protein, thereby the adherends can be bonded to obtain the adherend. An advantage of using this type of method is that the adherend can be obtained by using the adhesive with biodegradability, so the adherend of the environmental burden when discarding can be easily produced. In addition, as long as the adherend can be bonded with the water-dispersible adhesive containing modified protein, its material is not particularly limited, and can also be the same material as the adherend bonded with the solution-like adhesive containing modified protein.

[0247] The specific method for making an adherend using a water-dispersible adhesive is also not limited. For example, when the water-dispersible adhesive is placed between a plurality of adherends to be bonded, the same method as when the solution-like adhesive is placed between a plurality of adherends can be used. Also, when removing the aqueous medium in the water-dispersible adhesive placed between the adherends and solidifying the modified protein, the same method as when the solvent in the solution-like adhesive placed between the adherends and solidifying the modified protein can be used.

[0248] A water-dispersible coating containing a modified protein is used to produce a laminated body in which a coating layer is formed 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 on which a coating layer is required to be laminated, 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 modified protein. Thus, a laminated body in which a coating layer is formed on at least a portion of the substrate surface can be obtained. Using such a method has the advantage that a laminated body can also be obtained using a biodegradable coating, so that a laminated body that can reduce the environmental burden when discarded can be easily produced. In addition, the substrate can be any object that can be bonded by the water-dispersible coating containing the modified protein, and its material is not particularly limited, and can be the same material as the substrate in which the coating layer is formed using the solution-like coating.

[0249] There is no limitation on the specific method for producing the laminate using the water-dispersible coating. For example, when supplying the 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 in the water-dispersible coating supplied to the substrate surface and solidifying the modified protein, the same method as when removing the solvent in the solution coating supplied to the substrate surface and solidifying the modified protein can be used.

[0250] The film-shaped adhesive or film-shaped coating containing the modified protein can be obtained by a method including the step of forming a film from a modified protein solution obtained by dissolving the modified protein obtained by the method of this embodiment. Using such a method, unlike conventional film-shaped adhesives or film-shaped coatings containing components derived from petroleum, a biodegradable film-shaped adhesive or film-shaped coating can be easily obtained.

[0251] For the manufacture of a solution of a film-like adhesive or a film-like coating, for example, a modified protein solution used as a solution-like adhesive or a solution-like coating is preferably used. Among them, it is preferred to use a solution formed by dissolving modified proteins such as maleated protein, succinate protein, sulfonated protein, and phosphoesterified protein in an alkaline aqueous solution. The film-like adhesive or film-like coating formed by casting such a modified protein solution has a high affinity for water. Therefore, when obtaining an adherend or a laminate as described later, when the film-like adhesive or film-like coating is placed between the adherends or on the substrate surface to make it swollen, the film-like adhesive or film-like coating can absorb more water and fully soften. Thus, it is expected to improve the bonding strength of the film-like adhesive or film-like coating to the adherend or substrate. In addition, as a method for forming a film with a modified protein solution, for example, a method for casting a protein solution can be adopted. When implementing such casting, general known steps and known conditions can be adopted.

[0252] The film-like adhesive containing modified protein is used to make the adhesive of multiple adherends mutually adhering. For example, when the film-like adhesive is placed between the multiple adherends that need to be bonded, the film-like adhesive is then swollen or heated to soften it, and then hardened under the state that the film-like adhesive is pressed onto the adherend, thereby the adhesive can be bonded between the adherends. In addition, the film-like adhesive can also be swollen or heated to soften it in advance, and then placed between the multiple adherends, and then hardened under the state that the film-like adhesive is pressed onto the adherend. Use this type of method, an advantage is that, can use the adhesive with biodegradability to obtain the adhesive, therefore can easily manufacture the adhesive that can reduce the environmental burden when discarded. In addition, the adherend can be the same material as the adherend bonded with the solution-like adhesive containing the modified protein as long as it can be an object bonded with the film-like adhesive containing the modified protein.

[0253] Film-like adhesives can soften when absorbing water or being heated, and then harden by drying or cooling. In addition, the modified protein (also can be the protein before acylation) contained in the film-like adhesive, if it is a protein that shows the characteristic of shrinking behavior after contacting with water or heating when being formed into a film, the film-like adhesive can also shrink by contacting with water or heating. Therefore, the film-like adhesive containing modified protein, when being swollen or heated between adherends, softens and partially sinks into the gap existing on the adherend surface of the adherend, and 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. Then, it can be considered that the film-like adhesive hardens in this state, and due to the anchoring effect (Anchor Effect), the film-like adhesive is bonded to the adherend, and the adherends are bonded. In addition, it can be considered that hydrogen bonds are produced between the modified protein contained in the film-like adhesive and the adherend, so that the film-like adhesive is bonded to the adherend.

[0254] Therefore, when the film-like adhesive is swollen and softened, it is preferred to use an aqueous liquid such as water, a water-containing solution, or a dispersion liquid that can be absorbed by the film-like adhesive and shrink the film-like adhesive. Alternatively, for example, the above-mentioned aqueous liquid may be dripped or immersed in an aqueous liquid to make the film-like adhesive absorb water, thereby making the film-like adhesive swell and soften. Furthermore, when hardening the film-like adhesive softened by swelling, for example, a method of heating and drying, air drying, or natural drying may be adopted in a state where the film-like adhesive is placed between adherends.

[0255] When the film-like adhesive is heated to soften it, the film-like adhesive may be heated before being placed between the adherends, or the adherends and the film-like adhesive may be heated as a whole after the film-like adhesive is placed between the adherends. The heating temperature is not particularly limited as long as it can soften the film-like adhesive and does not negatively affect the modified protein contained in the film-like adhesive (for example, does not decompose it). When hardening the film-like adhesive softened by heating, methods such as cooling by a cooling device or cooling by placing the film-like adhesive may be adopted.

[0256] When the film-like adhesive that softens because of swelling or heating is fully sunk into the gap of the adherend surface of the adherend, preferably the softened film-like adhesive and the interface of the adherend are closely fitted. Then, preferably, for example, any one side in the overlapping direction of the adherend surface or the close-fitting direction applies pressure to a plurality of adherends, and the softened film-like adhesive is pressed onto the adherend to harden it. Here, the pressurization method to the adherend can adopt common arbitrary method.

[0257] The film-like coating containing modified protein is used to manufacture the laminated body that forms coating layer on all or part of substrate surface.For example, under the state that the film-like coating is placed to cover at least a part of substrate surface, the film-like coating is swollen or heated to soften it, and then hardened under the state that the film-like coating is pressed onto the substrate surface, so that the coating layer can be formed on at least a part of substrate surface, and laminated body is obtained.In addition, the film-like coating can also be swollen or heated to soften it in advance, and then placed to cover at least a part of substrate surface, and then hardened under the state that the film-like coating is pressed onto the adherend.Using such method, an advantage is that the coating with biodegradability can be used to obtain laminated body, so the laminated body that can reduce the environmental burden when discarded can be easily manufactured.In addition, as long as the substrate can be an object that can be bonded with the film-like coating containing modified protein, the same material as the adherend bonded with the film-like adhesive containing modified protein can also be used.

[0258] It can be considered that the mechanism by which the film-like coating adheres to the substrate surface is the same as the mechanism by which the film-like adhesive adheres to the adherend surface of the adherend. Therefore, the specific method for manufacturing a laminate using the film-like coating can also be the same as the method for obtaining an adherend using the film-like adhesive. In addition, when the softened film-like coating is pressed onto the substrate surface and hardened, the film-like coating can be squeezed onto the substrate surface side using, for example, an extrusion body that is the same as the extrusion body used when squeezing the solution-like coating applied to the substrate onto the substrate surface side.

[0259] The powdered adhesive or powdered coating containing the modified protein uses a powdered composition containing the modified protein powder obtained by the method of this embodiment. The powdered composition only needs to contain the modified protein as a main component and may contain various additive residues and other secondary components.

[0260] The modified protein contained in the powdered adhesive or powdered coating is not particularly limited as long as it is a protein manufactured by the process involved in the present embodiment. As the modified protein, for example, maleated protein, succinate protein, sulfonate protein, phosphate protein, etc. can be listed. As mentioned above, these modified proteins have high water absorption compared with other modified proteins. Therefore, the powdered adhesive or powdered coating containing such modified proteins has a relatively high water content. Therefore, the heating temperature and pressure required for resinification (curing) can be reduced to an accessible degree when it is placed on the bonded surface of the adherend or the surface of the substrate. Therefore, the use of the powdered adhesive or powdered coating containing the above-mentioned modified protein is expected to bring the advantage of simplifying the bonding operation to the adherend or the substrate. In addition, in the powdered adhesive or powdered coating, components other than the modified protein such as various additives contained in the known powdered adhesive or powdered coating may also be included if necessary.

[0261] The powdered adhesive containing modified protein is also used to make the adherend of multiple adherends mutually adhered.For example, when the powdered adhesive is placed under the state between multiple adherends that need to be adhered, the powdered adhesive is heated and pressed via the adherend simultaneously, so that it is solidified, thereby the adherend can be obtained by bonding between the adherends.An advantage of using this type of method is that the adherend can be obtained by using the adhesive with biodegradability, so the adherend of the environmental burden when discarding can be easily produced.In addition, the adherend can be the same material as the adherend bonded by the solution-like adhesive containing modified protein as long as it is an object bonded by the powdered adhesive containing modified protein.

[0262] The specific method when using powdered adhesive to make adherend does not have any restriction yet.For example, when the powdered adhesive between a plurality of adherends that need to be bonded is heated / pressurized, the powdered adhesive placed between the adherends can be heated together with the adherends by using metal plates etc. to clamp the adherends and heat the metal plates from the two sides opposite to the bonded surface side. Meanwhile, use manual presses etc. to pressurize the metal plates, via the adherends, the powdered adhesive is pressed for a predetermined time.Thus, the powdered adhesive is resinified and solidified, the adherends are bonded.In addition, the heating temperature, pressurization or heating / pressurization time when the powdered adhesive is solidified, according to the kind of the modified protein contained in the powdered adhesive, can be suitably selected from the scope that can make the modified protein resinified.

[0263] The powdered paint containing the modified protein is also used to produce a laminated body in which a paint layer is laminated on all or part of the surface of a substrate. For example, the powdered paint is placed on at least a part of the surface of a substrate, and the powdered paint is heated while being pressurized between a pressurizing body and the substrate to cure the powdered paint, thereby forming a paint layer laminated on at least a part of the surface of the substrate.

[0264] The specific method when using powdered coating to make laminated body is also without any 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 pressurize powdered adhesive for a prescribed time, thereby powdered coating is resinized and solidified.In addition, the heating temperature, pressurization amount or heating / pressurization time of powdered coating are the same as when using powdered adhesive to obtain bonded body.

[0265] [Example]

[0266] The present invention will be described in more detail below.

[0267] Unless otherwise specified, the abbreviations used in the examples are used according to the meanings commonly used in the field of organic chemistry or genetic engineering. Several examples are shown below.

[0268] Ac2O: Acetic anhydride

[0269] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0270] DIPEA: N, N-diisopropylethylamine

[0271] DMAP: 4-dimethylaminopyridine

[0272] DMSO: dimethyl sulfoxide

[0273] FT-IR spectroscopy: Fourier transform infrared spectroscopy

[0274] GPC: Gel Filtration Chromatography

[0275] HFIP: Hexafluoroisopropanol

[0276] RO water: water treated with reverse osmosis membrane (RO membrane)

[0277] TCEP: Tris(2-carboxyethyl)phosphate

[0278] To evaluate the potential of ball mills for chemical modification of recombinant proteins, acylation of recombinant proteins was performed using both mechanochemical and conventional solution methods, and the products were evaluated using various analytical methods.

[0279] 1. Acetylation using solution method

[0280] (Comparative Example 1) Acetylation of 100 kDa recombinant protein (in the absence of alkali)

[0281] The recombinant protein PRT966 (500 mg, 4.80 μmol) was suspended in DMSO (10 mL) while being stirred using a magnetic stirrer / hot plate, and then the mixture was dissolved by continuous heating and stirring (80°C, 1 hour) to obtain a transparent brown solution. Next, Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) was added to the reaction solution, and then stirred (80°C, 6 hours). The reaction mixture was then cooled to room temperature, and RO water (500 mL) treated with ice-cold (0°C) was stirred steadily while slowly separating the liquids to precipitate the product (1 hour). Afterwards, it was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, it was completely dried under reduced pressure in a vacuum oven (80°C, 1 hour) to obtain a sticky brown solid, which was used directly for analysis.

[0282] (Comparative Example 2) Acetylation of 100 kDa recombinant protein (in the presence of alkali)

[0283] The recombinant protein PRT966 (500 mg, 4.80 μmol) was suspended in DMSO (10 mL) while being stirred using a magnetic stirrer / hot plate, and then the mixture was dissolved by continuous heating and stirring (80°C, 1 hour) to obtain a transparent brown solution. Next, Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to the reaction solution, and then stirred (80°C, 6 hours). The reaction mixture was then cooled to room temperature, and RO water (500 mL) treated with ice-cold (0°C) was slowly added while stirring steadily to precipitate the product (1 hour). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a light brown solid, which was directly used for analysis.

[0284] 2. Acetylation using mechanochemical method

[0285] (Example 1) Acetylation of 100 kDa recombinant protein (in the absence of alkali)

[0286] The recombinant protein PRT966 (500 mg, 4.80 μmol) and Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), and 20 stainless steel balls (5 mm in diameter, manufactured by Lechi) were added. The mixture was ground with a mixed grinding ball mill (mixing mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a pale yellowish white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0287] (Example 2) Acetylation of 100 kDa recombinant protein (in the presence of alkali)

[0288] The 100 kDa recombinant protein PRT966 (500 mg, 4.80 μmol), Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group), and DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), and 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added. The mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy brown solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a pale yellowish-white amorphous powder, which was directly used for analysis.

[0289] 3. Comparison of acetylation products between liquid phase method and mechanochemical method

[0290] (1) IR absorption spectrum analysis

[0291] Fourier transform infrared absorption (FT-IR) spectroscopy was performed using a Nicolet iS50 FT-IR spectrometer (trade name, manufactured by Thermo Fisher Scientific). The pure sample was analyzed using attenuated total reflectance (ATR) of diamond crystals at 0.1 cm -1 The wave number of the absorption frequency is recorded in units (cm -1 ). The spectrum is 760-3800cm -1 The range is 4cm -1 The resolution was collected and the average value of 64 scans was calculated.

[0292] (2) GPC analysis

[0293] In the GPC analysis, a liquid chromatography system equipped with a differential refractive index (RI) detector (trade name: Agilent 1260 Infinity II, manufactured by Agilent Technologies Inc.) and a styrene-divinylbenzene copolymer column (inner diameter: 4.6 mm×150 mm) stationary phase equipped with a 0.5 μm guard column filter (trade name: Shodex GPC HK-G, manufactured by Showa Denko K.K.) and HFIP (manufactured by Chuo Glass Co., Ltd.) were used to separate the analytes. The mobile phase was monitored using an Agilent 1260 Infinity II refractive index detector (RID) (manufactured by Agilent Technologies Inc.) at a flow rate of 0.15 mL / min and 40°C. The protein was analyzed in the form of a 0.1 wt% solution (final concentration 1 mM) formed by adding sodium trifluoroacetate to HFIP. For proteins having cysteine ​​residues, TCEP (final concentration 10 mM) was added to prevent the formation of disulfide bonds. The sample was dissolved (45°C, 1500 rpm, 1 hour) using a heating shaker (trade name: Front Lab MyBL-100S, manufactured by AS ONE Co., Ltd.) and passed through a 0.45 μm hydrophilic PTFE membrane filter (trade name: Dismic 25HP, manufactured by Advan Tech) to remove remaining insoluble matter.

[0294] Figure 1 (a) The appearances of Comparative Examples 1 and 2 and Examples 1 and 2 were compared. Figure 1 (b) is a graph of the GPC chromatograms of Comparative Examples 1 and 2, Examples 1 and 2, and the unmodified protein PRT966 superimposed. The modified proteins of Comparative Examples 1 and 2 prepared by the solution method are brown blocks and need to be crushed before use. On the other hand, Examples 1 and 2 prepared by the mechanochemical method obtain white powders and are also excellent in usability.

[0295] The results of IR spectra and GPC analysis of Comparative Examples 1 and 2, and Examples 1 and 2, respectively, are shown in Figures 2 to 5 middle. Figure 2 (a) is the IR spectra of Comparative Example 1 and unmodified protein PRT966, at 1740 cm -1 Since a peak appears, it can be known that acetylation has progressed. Figure 2 (b) is the GPC chromatogram of Comparative Example 1 and the unmodified protein PRT966, and the retention time becomes shorter than that of the unmodified protein. Figure 3 (a) is the IR spectra of Comparative Example 2 and unmodified protein PRT966, at 1730 cm -1Since a peak appears, it can be known that acetylation has progressed. Figure 3 (b) is the GPC chromatogram of Comparative Example 2 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Figure 4 (a) is the IR spectra of Example 1 and the unmodified protein PRT966 at 1740 cm -1 Since a peak appears, it can be known that acetylation has progressed. Figure 4 (b) is the GPC chromatogram of Example 1 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Figure 5 (a) is the IR spectra of Example 2 and the unmodified protein PRT966, at 1743 cm -1 Since a peak appears, it can be known that acetylation has progressed. Figure 5 (b) is the GPC chromatogram of Example 2 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein.

[0296] (3) SDS-PAGE analysis

[0297] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a pre-cast separation gel (trade name: 4-20% Mini-PROTEAN TGX, manufactured by Bio-Rad Laboratories Inc.). The gel was inserted into an electrode assembly (trade name: Mini-PROTEAN Tetra Electrode Assembly, manufactured by Bio-Rad Laboratories Inc.) and installed inside the electrophoresis tank together with SDS electrophoresis buffer (manufactured by Bio-Rad Laboratories Inc.). The protein was dissolved in DMSO, lithium chloride (final concentration 2M) was added and dissolved at a final concentration of 1wt% using a heated oscillator (trade name: TMS-200 Turbo Thermoshaker Incubator, manufactured by Hangzhou Allsheng Instruments Co. Ltd.) (80°C, 2000 rpm, 45 minutes), and then diluted 50 times with a 10M urea aqueous solution. Then, it was mixed with the same amount of sample buffer containing 3-mercapto-1,2-propanediol, heated to 95°C (5 minutes) (trade name: Genius DryBath Incubator MD-01N, manufactured by Major Science Co. Ltd.), supplied to the gel (10 μL), and a molecular weight standard (trade name: XL-Ladder Brood, manufactured by Approscience Co.) was added as a reference. Electrophoresis was performed using a constant voltage (200 V, 30 minutes) (trade name: PowerPac Universal Power Supply, manufactured by Bio-Rad Laboratories Inc.). The protein was stained with Oriole Fluorescent GelStain (Bio-Rad Laboratories Inc.) by gently stirring the gel (trade name: Duomax 1030, Heidolph Instruments) (30 rpm, 1 hour) and visualized using imaging software (GelDoc EZ gel imaging system and ImageLabsoftware (Bio-Rad Laboratories Inc.)).

[0298] According to the results of SDS-PAGE, low molecular weight protein bands generated by decomposition of the protein backbone due to oxidative stress in the system were found in the solution method using DMSO as a solution, but such bands were not found in the mechanochemical method.

[0299] 4. Impact of molecular weight difference (from 7.5 kDa to 300 kDa)

[0300] (Example 3) Acetylation of 7.5 kDa recombinant protein

[0301] The recombinant protein PRT2882 (sequence number 8, 500 mg, 50.5 μmol), Ac2O (301 μL, 3.18 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (317 μL, 1.82 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a dark gray block solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a dark gray amorphous powder, which was directly used for analysis without being treated.

[0302] (Example 4) Acetylation of 15 kDa recombinant protein

[0303] The recombinant protein PRT2841 (sequence number 9, 500 mg, 28.1 μmol), Ac2O (316 μL, 3.34 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (333 μL, 1.91 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a light gray block solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a light gray amorphous powder, which was directly used for analysis without treatment.

[0304] (Example 5) Acetylation of 25 kDa recombinant protein

[0305] The recombinant protein PRT2662 (sequence number 10, 500 mg, 18.2 μmol), Ac2O (331 μL, 3.50 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (349 μL, 2.00 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a light gray waxy solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a light gray amorphous powder, which was directly used for analysis without treatment.

[0306] (Example 6) Acetylation of 50 kDa recombinant protein

[0307] The recombinant protein PRT1000 (500 mg, 9.44 μmol), Ac2O (319 μL, 3.37 mmol, 3.5 eq. per hydroxyl group) and DIPEA (336 μL, 1.93 mmol, 2 eq. per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), and 20 stainless steel balls (5 mm in diameter, manufactured by Lechi) were used to grind with a mixed grinding ball mill (trade name: Mixing Mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0308] (Example 7) Acetylation of 50 kDa recombinant protein

[0309] The recombinant protein PRT918 (500 mg, 9.48 μmol), Ac2O (314 μL, 3.32 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (330 μL, 1.90 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (5 mm in diameter, manufactured by Lechi) were added, and a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) was used for grinding (vibration frequency: 30 Hz, 90 minutes) to obtain a white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0310] (Example 8) Acetylation of 100 kDa recombinant protein

[0311] The recombinant protein PRT966 (500 mg, 4.80 μmol), Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (328 μL, 1.88 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a light brown paste. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a light beige amorphous powder, which was directly used for analysis.

[0312] (Example 9) Acetylation of 200 kDa recombinant protein

[0313] The recombinant protein PRT799 (500 mg, 2.37 μmol), Ac2O (305 μL, 3.23 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (321 μL, 1.84 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: Mixing Mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a light gray gummy solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a light gray amorphous powder, which was directly used for analysis without treatment.

[0314] (Example 10) Acetylation of 300 kDa recombinant protein

[0315] The recombinant protein PRT1186 (sequence number 11, 500 mg, 1.59 μmol), Ac2O (307 μL, 3.25 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (324 μL, 1.86 mmol, 2 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a light gray gummy solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous solid, which was directly used for analysis.

[0316] The results of IR spectra and GPC analysis of Examples 3 to 10 are shown in Figures 6 to 13 middle. Figure 6 (a) is the IR spectra of Example 3 and the unmodified protein PRT2882 at 1741 cm -1 Since a peak appears, it can be known that acetylation has progressed. Figure 6 (b) is the GPC chromatogram of Example 3 and the unmodified protein PRT2882, and the retention time becomes slightly shorter than that of the unmodified protein. Figure 7 (a) is the IR spectra of Example 4 and the unmodified protein PRT2841, at 1742 cm -1Since a peak appears, it can be known that acetylation has progressed. Figure 7 (b) is the GPC chromatogram of Example 4 and the unmodified protein PRT2841, and the retention time becomes slightly shorter than that of the unmodified protein. Figure 8 (a) is the IR spectra of Example 5 and the unmodified protein PRT2662, at 1743 cm -1 Since a peak appears, it can be known that acetylation has progressed. Figure 8 (b) is the GPC chromatogram of Example 5 and the unmodified protein PRT2662, in which the retention time becomes slightly shorter than that of the unmodified protein. Fig. 9 (a) is the IR spectra of Example 6 and the unmodified protein PRT1000, at 1744 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig. 9 (b) is the GPC chromatogram of Example 6 and the unmodified protein PRT1000, and the retention time becomes slightly shorter than that of the unmodified protein.

[0317] Fig.10 (a) is the IR spectra of Example 7 and unmodified protein PRT918, at 1744 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.10 (b) is the GPC chromatogram of Example 7 and the unmodified protein PRT918, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.11 (a) is the IR spectra of Example 8 and the unmodified protein PRT966, at 1744 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.11 (b) is the GPC chromatogram of Example 8 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.12 (a) is the IR spectra of Example 9 and the unmodified protein PRT799, at 1730 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.12 (b) is the GPC chromatogram of Example 9 and the unmodified protein PRT799, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.13 (a) is the IR spectra of Example 10 and the unmodified protein PRT1186, at 1731 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.13 (b) is the GPC chromatogram of Example 10 and the unmodified protein PRT1186, and the retention time becomes slightly shorter than that of the unmodified protein.

[0318] 5. Effect of the type of alkali

[0319] (Example 11) Acetylation of 100 kDa recombinant protein

[0320] The recombinant protein PRT966 (500 mg, 4.80 μmol), Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and sodium hydroxide (41.5 mg, 1.04 mmol, 1.1 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) was used for grinding (vibration frequency: 30 Hz, 90 minutes) to obtain a white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous fine powder, which was directly used for analysis.

[0321] (Example 12) Acetylation of 100 kDa recombinant protein

[0322] The recombinant protein PRT966 (500 mg, 4.80 μmol), Ac2O (311 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and sodium carbonate (110 mg, 1.04 mmol, 1.1 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) was used for grinding (vibration frequency: 30 Hz, 90 minutes) to obtain a white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous fine powder, which was directly used for analysis.

[0323] (Example 13) Acetylation of 100 kDa recombinant protein

[0324] The recombinant protein PRT966 (500 mg, 4.80 μmol), Ac2O (445 μL, 4.71 mmol, 5 equivalents per hydroxyl group), DIPEA (820 μL, 4.71 mmol, 5 equivalents per hydroxyl group) and DMAP (575 mg, 4.71 mmol, 5 equivalents per hydroxyl residue) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 8.6 hours) to obtain a dark brown waxy solid. The reaction mixture was removed from the grinding jar and suspended in RO water while stirring (10 minutes). Thereafter, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a brown amorphous powder, which was directly used for analysis.

[0325] The results of IR spectra and GPC analysis of Examples 11 to 13 are shown in Figures 14 to 16 middle. Fig.14 (a) is the IR spectra of Example 11 and the unmodified protein PRT966, at 1743 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.14 (b) is the GPC chromatogram of Example 11 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.15 (a) is the IR spectra of Example 12 and the unmodified protein PRT966, at 1745 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.15 (b) is the GPC chromatogram of Example 12 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.16 (a) is the IR spectra of Example 13 and the unmodified protein PRT966, at 1737 cm -1 Since a peak appears, it can be known that acetylation has progressed. Fig.16 (b) is the GPC chromatogram of Example 13 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein.

[0326] 6. Succinylation using mechanochemical methods

[0327] (Example 14) Succinylation of 100 kDa recombinant protein

[0328] The recombinant protein PRT966 (500 mg, 4.80 μmol), succinic anhydride (330 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group), DIPEA (181 μL, 1.04 mmol, 1.1 equivalents per hydroxyl group) and DMAP (127 mg, 1.04 mmol, 1.1 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a pale yellowish white amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, it was completely dried under reduced pressure using a vacuum oven (80° C., 1 hour) to obtain a white amorphous solid, which was directly used for analysis.

[0329] (Example 15) Succinylation of 100 kDa recombinant protein

[0330] The recombinant protein PRT966 (500 mg, 4.80 μmol), succinic anhydride (330 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group), DIPEA (181 μL, 1.04 mmol, 1.1 equivalents per hydroxyl group) and DBU (155 μL, 1.04 mmol, 1.1 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a viscous dark brown paste. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, it was completely dried under reduced pressure using a vacuum oven (80° C., 1 hour) to obtain a gray-brown amorphous solid, which was directly used for analysis.

[0331] (Example 16) Succinylation of 100 kDa recombinant protein

[0332] The recombinant protein PRT966 (500 mg, 4.80 μmol), succinic anhydride (330 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group) and potassium carbonate (455 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy pale yellowish white solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a pale yellowish white amorphous powder, which was directly used for analysis.

[0333] (Example 17) Succinylation of 100 kDa recombinant protein

[0334] Add recombinant protein PRT966 (500 mg, 4.80 μmol), succinic anhydride (471 mg, 4.71 mmol, 5 equivalents per hydroxyl group), DIPEA (820 μL, 4.71 mmol, 5 equivalents per hydroxyl group), DMAP (575 mg, 4.71 mmol, 5 equivalents per hydroxyl group), add to a 10 mL stainless steel cup (manufactured by Lechi), add 20 stainless steel balls (diameter 5 mm, manufactured by Lechi), grind with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 8.6 hours) to obtain a white amorphous solid. Take out the reaction mixture from the grinding jar and suspend it in RO water (25 mL) while stirring (10 minutes). Afterwards, filter the product under reduced pressure and wash it thoroughly with RO water and acetone. Then, it was completely dried under reduced pressure using a vacuum oven (80° C., 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0335] (Example 18) Succinylation of 100 kDa recombinant protein

[0336] The recombinant protein PRT966 (500 mg, 4.80 μmol), succinic anhydride (471 mg, 4.71 mmol, 5 equivalents per hydroxyl group), DIPEA (820 μL, 4.71 mmol, 5 equivalents per hydroxyl group), and DMAP (575 mg, 4.71 mmol, 5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), and 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added. The mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 20 Hz, 2 hours) to obtain a pale yellow-white paste. The reaction mixture was taken out from the grinding pot and suspended in RO water (25 mL) while stirring (10 minutes). Afterwards, the product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Then, a vacuum oven was used to completely dry it under reduced pressure (80°C, 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0337] The results of IR spectra and GPC analysis of Examples 14 to 18 are shown in Figures 17 to 21 middle. Fig.17 (a) is the IR spectra of Example 14 and the unmodified protein PRT966, at 1713 cm -1 Since a peak appeared, it could be known that succinylation had progressed. Fig.17 (b) is the GPC chromatogram of Example 14 and the unmodified protein PRT966, and the retention time is almost the same as that of the unmodified protein. Fig.18 (a) is the IR spectra of Example 15 and the unmodified protein PRT966 at 1740 cm -1 Since a peak appeared, it could be known that succinylation had progressed. Fig.18 (b) is the GPC chromatogram of Example 15 and the unmodified protein PRT966, and the retention time is almost the same as that of the unmodified protein. Fig.19 (a) is the IR spectra of Example 16 and the unmodified protein PRT966 at 1740 cm -1 Since a peak appeared, it could be known that succinylation had progressed. Fig.19 (b) is the GPC chromatogram of Example 16 and the unmodified protein PRT966, and the retention time is almost the same as that of the unmodified protein. Fig. 20 (a) is the IR spectra of Example 17 and the unmodified protein PRT966, at 1721 cm -1 Since a peak appeared, it could be known that succinylation had progressed. Fig. 20 (b) is the GPC chromatogram of Example 17 and the unmodified protein PRT966, and the retention time is almost the same as that of the unmodified protein. Fig.21(a) is the IR spectra of Example 18 and the unmodified protein PRT966, at 1737 cm -1 Since a peak appeared, it could be known that succinylation had progressed. Fig.21 (b) is the GPC chromatogram of Example 18 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein.

[0338] 7. Stearylation using mechanochemical method

[0339] (Example 19) Stearoylation of 100 kDa recombinant protein

[0340] The recombinant protein PRT966 (500 mg, 4.80 μmol), stearic anhydride (1.82 g, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) was used for grinding (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Then, the crude product was filtered under reduced pressure and thoroughly washed with RO water and acetone. After that, the product was partially dried on filter paper, suspended in ethyl acetate (25 mL), and stirred with a magnetic stirrer (10 minutes). Then, the product was recovered by vacuum filtration, washed sufficiently with ethyl acetate, and passed through a vacuum oven (80° C., 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0341] (Example 20) Stearoylation of 100 kDa recombinant protein

[0342] The recombinant protein PRT966 (500 mg, 4.80 μmol), stearoyl chloride (998 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) was used for grinding (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy yellow paste. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Then, the crude product was filtered under reduced pressure and thoroughly washed with RO water and acetone. After that, the product was partially dried on filter paper, suspended in ethyl acetate (25 mL), and stirred with a magnetic stirrer (10 minutes). Then, the product was recovered by vacuum filtration, washed sufficiently with ethyl acetate, and then dried in a vacuum oven (80° C., 1 hour) to obtain a pale yellowish white amorphous powder, which was used for analysis as it was.

[0343] (Example 21) Stearoylation of 100 kDa recombinant protein

[0344] The recombinant protein PRT966 (500 mg, 4.80 μmol), stearic anhydride (1.82 g, 3.30 mmol, 3.5 equivalents per hydroxyl group), DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DMAP (403 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy amorphous solid. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Then, the crude product was filtered under reduced pressure and thoroughly washed with RO water and acetone. Afterwards, the product was partially dried on filter paper, suspended in ethyl acetate (25 mL), and stirred with a magnetic stirrer (10 minutes). The product was recovered by filtration under reduced pressure, fully washed with ethyl acetate, and dried in a vacuum oven (80° C., 1 hour) to obtain a white amorphous powder, which was directly used for analysis.

[0345] (Example 22) Stearoylation of 100 kDa recombinant protein

[0346] The recombinant protein PRT966 (500 mg, 4.80 μmol), stearoyl chloride (998 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group), DIPEA (574 μL, 3.30 mmol, 3.5 equivalents per hydroxyl group) and DMAP (403 mg, 3.30 mmol, 3.5 equivalents per hydroxyl group) were added to a 10 mL stainless steel cup (manufactured by Lechi), 20 stainless steel balls (diameter 5 mm, manufactured by Lechi) were added, and the mixture was ground with a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) (vibration frequency: 30 Hz, 90 minutes) to obtain a waxy yellow paste. The reaction mixture was taken out from the grinding jar and suspended in RO water (25 mL) while stirring (10 minutes). Then, the crude product was filtered under reduced pressure and thoroughly washed with RO water and acetone. After that, the product was partially dried on filter paper, suspended in ethyl acetate (25 mL), and stirred using a magnetic stirrer (10 minutes). Then, the product was recovered by filtration under reduced pressure, fully washed with ethyl acetate, and dried in a vacuum oven (80° C., 1 hour) to obtain a beige amorphous powder, which was directly used for analysis.

[0347] The results of IR spectra and GPC analysis of Examples 19 to 22 are shown in Figures 22 to 25 middle. Fig. 22 (a) is the IR spectra of Example 19 and the unmodified protein PRT966, at 1733 cm -1 Since a peak appeared, it can be known that stearoylation has progressed. Fig. 22 (b) is the GPC chromatogram of Example 19 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.23 (a) is the IR spectra of Example 20 and the unmodified protein PRT966 at 1740 cm -1 Since a peak appeared, it can be known that stearoylation has progressed. Fig.23 (b) is the GPC chromatogram of Example 20 and the unmodified protein PRT966, and the retention time is almost the same as that of the unmodified protein. Fig.24 (a) is the IR spectra of Example 21 and the unmodified protein PRT966, at 1736 cm -1 Since a peak appeared, it can be known that stearoylation has progressed. Fig.24 (b) is the GPC chromatogram of Example 21 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein. Fig.25 (a) is the IR spectra of Example 22 and the unmodified protein PRT966, at 1742 cm -1 Since a peak appeared, it can be known that stearoylation has progressed. Fig.25(b) is the GPC chromatogram of Example 22 and the unmodified protein PRT966, and the retention time becomes slightly shorter than that of the unmodified protein.

[0348] 8. Degree of esterification

[0349] The degree of esterification of the modified protein is calculated according to the following steps. First, FT-IR spectroscopic analysis is performed on the modified protein and the unmodified protein respectively. Based on the obtained spectra, the peak area of ​​the C=O stretching vibration peak corresponding to the formed ester and the amide bond of the protein is calculated. The peak area of ​​the C=O stretching vibration peak of the ester bond in the modified protein is calculated relative to the peak area of ​​the C=O stretching vibration peak of the amide bond. The calculation formulas are shown below.

[0350] Ester / amide ratio = [(peak area of ​​ester C=O stretching vibration peak of modified protein) - (peak area of ​​ester C=O stretching vibration peak of unmodified protein)] / (peak area of ​​amide C=O stretching vibration peak of modified protein)

[0351] Estimated number of esterified residues = ester / amide ratio × total number of amino acid residues in the protein

[0352] Degree of esterification (%) = (estimated number of esterified residues / total number of hydroxyl groups) × 100

[0353] The esterification degree of each modified protein is shown in Table 3.

[0354] [Table 3]

[0355]

[0356] The degree of esterification is an indicator of the extent to which the hydroxyl groups of the amino acid residues constituting a protein are esterified. Acetylation of nucleophilic functional groups (amino groups, thiol groups) other than hydroxyl groups in proteins forms amide bonds or thioester bonds, and does not affect the C=O stretching vibration peak of esters.

[0357] (4) Modification of protein dispersibility

[0358] The 100 kDa recombinant protein PRT966, the acetylated protein obtained in Example 11, and the succinylated protein obtained in Example 14 were suspended in any one of water, acetone, and 0.5 w / v% sodium hydroxide aqueous solution (final concentration: 1 wt%), and mixed at room temperature using a vortex mixer (Frontlab FLX-S50, manufactured by AS ONE Corporation) (2500 rpm, 30 seconds).

[0359] The results are as follows Fig.26 And as shown in Table 4. Fig.26These are photographs showing the results of adding solvent to the recombinant protein PRT966, the acetylated protein PRT966, and the succinylated protein PRT966. Fig.26 The solvents used in (a), (b), and (c) were water, acetone, and 0.5 w / v% sodium hydroxide aqueous solution, respectively. Proteins were not well dispersed in any solvent, but acetylated proteins were uniformly dispersed in acetone. In addition, succinylated proteins were uniformly dispersed in water and dissolved in alkaline solutions.

[0360] [Table 4]

[0361]

[0362] (5) Modification of protein dispersibility

[0363] The 100 kDa recombinant protein PRT966, acetylated protein, and succinylated protein were suspended in DMSO (final concentration: 5 wt%), and then heated using a heating shaker (90° C., 1500 rpm, 1 hour).

[0364] The state of the solution after heating and shaking is as follows Fig. 27 shown. Fig. 27 (a) is a photograph showing the state of the protein just after adding DMSO. Fig. 27 (b) is a photograph showing the state of being dissolved in DMSO, Fig. 27 (c) is a photograph comparing the gelation states of each protein. All are transparent viscous solutions (gel-like), and no precipitation is observed. The degree of gelation is from high to low: Example 11 (acetylated protein), Example 12 (acetylated protein), unmodified protein PRT966, Comparative Example 2 (acetylated protein), Example 14 (succinylated protein).

[0365] 9. Spinning

[0366] The recombinant protein PRT966 and Example 8 (acetylated PRT966) were mixed at a predetermined ratio (by weight). DMSO (final concentration: 20 wt%) and lithium chloride (final concentration: 4 wt%) were added to the resulting mixture, and the mixture was heated and stirred (100°C, 30 minutes) using a magnetic stirrer / hot plate to completely dissolve the protein, thereby obtaining a spinning solution (final concentration: 20 wt%). The obtained spinning solution was loaded into a plastic syringe (trade name: ss-05Lz, manufactured by Terumo Corporation) equipped with a disposable 22G Luer lock needle (trade name: Neolus NN-2225R, manufactured by Terumo Corporation, inner diameter 0.413 mm). Then, the tip of the needle was immersed in a coagulation bath (RO water at 75°C), and the spinning solution was slowly and evenly injected into the coagulation bath while the needle was slowly moved around the coagulation bath.

[0367] The results are shown in Table 5. The greater the proportion of acetylated protein, the more adhesion after spinning can be suppressed. In addition, it can be found that the unmodified protein presents a flat shape in the coagulation bath, but when the spinning solution containing acetylated protein is used, the cross-sectional shape after coagulation is closer to a circle.

[0368] [Table 5]

[0369] example protein Fiber grade 1 PRT966 only 3 2 PRT966 and acetylated PRT966 (2:1) 4 3 PRT966 and acetylated PRT966 (1:1) 4 4 PRT966 and acetylated PRT966 (1:2) 5 5 Only acetylated PRT966 4

[0370] 10. Adhesives containing modified proteins

[0371] <Manufacturing of Solution Adhesive>

[0372] In a 50 mL stainless steel cup (manufactured by Latsch), 100 kDa recombinant protein PRT966 (7.5 g, 72.1 μmol) and sodium hydroxide (621 mg, 15.5 mmol, 1.1 equivalents per hydroxyl group) were added, and a stainless steel ball (25 mm in diameter, manufactured by Latsch) was placed, and the mixture was ground with a mixed grinding ball mill (mixing mill MM400, manufactured by Latsch) (vibration frequency: 30 Hz, 30 minutes) to obtain a uniform off-white amorphous powder. Then, maleic anhydride (4.85 g, 49.4 mmol, 3.5 equivalents per hydroxyl group) was added to the stainless steel cup, and then further ground with a mixed grinding ball mill (mixing mill MM400, manufactured by Latsch) (vibration frequency: 30 Hz, 60 minutes) to obtain a uniform off-white amorphous powder. The reaction mixture was taken out of the stainless steel cup, put into RO water (200 mL), and suspended while stirring (5 minutes). Next, 1M HCl aqueous solution (50 mL) was added to the obtained suspension to acidify the suspension, and stirring was continued for further 5 minutes. Subsequently, acetonitrile (100 mL) was added to the suspension, and then vacuum filtered (KIRIYAMA 5A, Filter paper, Kiriyama Manufacturing: PG201 diaphragm vacuum pump, manufactured by Yamato Scientific Co., Ltd.) to recover the product. Then, the recovered product is thoroughly washed with RO water and acetone, and then completely dried under reduced pressure (80°C, 1 hour) in a vacuum oven (VOS-310C vacuum oven, EYELA Tokyo Rika Instruments Co., Ltd.: GCD-051X oil rotary vacuum pump, manufactured by Aifac Co., Ltd.) to obtain a maleated (maleic acid esterified) protein powder consisting of a light yellow amorphous solid. Next, 1 g of the maleated protein powder thus obtained is put into a 1M sodium hydroxide aqueous solution (5 mL) heated to 80°C and rapidly stirred for 1 hour to dissolve it. A solution-like adhesive in which the maleated protein is dissolved in an alkaline aqueous solution at a concentration of 20wt% is thus obtained. This is referred to as Example 23.

[0373] <Manufacturing of Adhesive Body>

[0374] Two longitudinally rectangular cardboard sheets (20.4 cm × 5.9 cm × 0.3 cm) and two longitudinally rectangular glass sheets (7.6 cm × 2.6 cm × 0.1 cm) were prepared as adherends. Then, the solution adhesive of Example 23 was applied to a portion of one side of one cardboard sheet and then left for 3 minutes. Next, another cardboard sheet was stacked on the surface of the cardboard sheet coated with the adhesive, and then the two cardboard sheets were clamped with a clip and left overnight so that all the overlapping parts of the two cardboard sheets could be pressed against each other. In this way, the solution adhesive was dried, thereby removing the solvent in the solution adhesive, allowing the solution adhesive to solidify, and the two cardboard sheets were bonded together, thereby obtaining a bonded body A formed by bonding two cardboard sheets. In addition, except that two glass sheets were used instead of two cardboard sheets, the same operation as when obtaining the bonded body A was carried out to obtain a bonded body B formed by bonding two glass sheets.

[0375] <Strength of Adhesive Body A>

[0376] Next, the upper end of one cardboard sheet in the bonded body A was fixed and hung in the air, and a 770g weight was hung on the lower end of the other cardboard sheet to observe whether the bonded portion located in the middle of the vertical direction would peel off. The results showed that the bonded portion did not peel off and had sufficient bonding strength.

[0377] <Strength of Adhesive Body B>

[0378] Next, the bonded body B was placed horizontally, and in this state, the part of the lower cardboard sheet opposite to the bonded surface was fixed, and a 190g weight was placed on the end of the upper cardboard sheet opposite to the bonded surface to observe whether the bonded part would peel off. The results showed that the bonded part did not peel off and had sufficient bonding strength.

[0379] 11. Mechanochemical esterification using a roller mixer mill

[0380] In this reaction, a test 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 type) was used as a counter-rotating roll mixer.

[0381] (Example 24) Acetylation of PRT966

[0382] Before the reaction, the temperature in the feed chamber (60 mL) of the mixer mill was adjusted to 65°C. While the blades of the mixer mill were slowly rotated (10 rpm), a crushed mixture of 100 kDa recombinant protein PRT966 (20.0 g, 0.192 mmol) and sodium hydroxide (1.66 g, 41.4 mmol, 1.1 equivalents per nucleophilic residue) was added into the feed chamber. Then, acetic anhydride (17.8 mL, 188 mmol, 5 equivalents per nucleophilic residue) was carefully added in batches. The reaction mixture was slowly rotated (10 rpm) until it became uniform. After confirming that the blades of the mixer could rotate freely and that both the torque and the temperature were stable, the grinding step was started and the reaction mixture was continuously kneaded (50 rpm, 65°C, 1.5 hours) to obtain a beige amorphous powder. The reaction mixture was taken out of the feed chamber and suspended in RO water (200 mL) while stirring for 10 minutes. The product was recovered by vacuum filtration, washed thoroughly with RO water and acetone, and then completely dried in a vacuum oven under reduced pressure (1 hour, 80° C.) to obtain a fine off-white amorphous powder. The obtained powder was not further processed and was used for FT-IR and GPC analysis.

[0383] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.28 In the IR spectrum of Example 24, at 1750 cm -1 A new peak was observed nearby, and it can be considered that acetylation has advanced.

[0384] (Example 25) Succinylation of PRT966

[0385] Before the reaction, the temperature in the feed chamber (60 mL) of the mixer mill was adjusted to 65°C. While the blades of the mixer mill were slowly rotated (10 rpm), a crushed mixture of 100 kDa recombinant protein PRT966 (20.0 g, 0.192 mmol) and sodium hydroxide (1.66 g, 41.4 mmol, 1.1 equivalents per nucleophilic residue) was added into the feed chamber. Then, succinic anhydride (18.8 g, 188 mmol, 5 equivalents per nucleophilic residue) was carefully added in batches. The reaction mixture was slowly rotated (10 rpm) until it became uniform. After confirming that the blades of the mixer could rotate freely and that both the torque and the temperature were stable, the grinding step was started and the reaction mixture was continuously kneaded (50 rpm, 65°C, 1.5 hours) to obtain a beige amorphous powder. The reaction mixture was taken out of the feed chamber and suspended in RO water (200 mL) while stirring for 5 minutes. The suspension was then acidified by adding 1M hydrochloric acid (50 mL) and stirred for another 5 minutes. Acetonitrile (200 mL) was added to the suspension and stirred for another 5 minutes. After the product was recovered by vacuum filtration, it was fully washed with RO water and acetone, and then completely dried under reduced pressure in a vacuum oven (1 hour, 80° C.) to obtain a fine off-white amorphous powder. The obtained powder was not further processed and was used for FT-IR and GPC analysis.

[0386] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.29 In the IR spectrum of Example 25, at 1750 cm -1 A new peak was observed nearby, suggesting that succinylation has advanced.

[0387] 12. Mechanochemical esterification using twin-screw extruder

[0388] In this reaction, a test kneading machine (laboratory kneading extruder 3S150, manufactured by Toyo Seiki Seisaku-sho, Ltd.) equipped with a co-rotating twin-screw extruder (2D15W model, manufactured by Toyo Seiki Seisaku-sho, Ltd.) with the die removed was used. The screw element of the extruder is constructed as follows: Fig.30 As shown, the staggered angle between adjacent discs in the mixing section is adjusted to 45°. Fig.30 In the figure, "FC" represents the forward conveying element, "R" represents the reverse element, "K" represents the mixing element, and "C" represents the compression element.

[0389] (Example 26) Succinylation of PRT966

[0390] Before the reaction, the temperature of the extruder barrel was adjusted to 160 ° C. During the entire reaction, the speed of the screw (100 rpm) remained unchanged. In order to lubricate the screw, succinic anhydride (5.0 g, 50.0 mmol) was gradually added to the extruder. After confirming that the molten succinic anhydride was extruded, ensuring that the torque did not exceed 40 N m, a mixture of 100 kDa recombinant protein PRT966 (50.0 g, 0.480 mmol), sodium hydroxide (4.14 g, 103 mmol, 1.1 equivalents per nucleophilic residue), and succinic anhydride (33.0 g, 330 mmol, 3.5 equivalents per nucleophilic residue) was gradually added to the extruder within 15 minutes. The reaction mixture was extruded and recovered in the form of a light brown amorphous powder, and suspended in RO water (400 mL) under stirring for 10 minutes. Afterwards, the suspension was acidified by adding 1M hydrochloric acid (100 mL) and stirred for another 5 minutes. Acetonitrile (400 mL) was added to the suspension and stirred for another 10 minutes. The product was recovered by vacuum filtration, washed thoroughly with RO water and acetone, and then completely dried under reduced pressure in a vacuum oven (1 hour, 80° C.) to obtain a fine off-white amorphous powder. The obtained powder was not further processed and was used for FT-IR and GPC analysis.

[0391] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.31 In the IR spectrum of Example 26, at 1750 cm -1 A new peak was observed nearby, suggesting that succinylation has advanced.

[0392] 13. Acylation using mechanochemical methods

[0393] (Example 27) Sulfonation of 100 kDa recombinant protein

[0394] The recombinant protein PRT966 (7.5 g, 72.1 μmol), sulfur trioxide pyridine complex (7.87 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) were mixed with a stainless steel grinding medium ( The mixture was mixed with 2-ethylhexanoic acid (8.2 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) and put into a 125 mL hard steel grinding jar (manufactured by Lechi Co., Ltd.), and crushed using a mixed grinding ball mill (trade name: mixed grinding mill MM500 controlled type, manufactured by Lechi Co., Ltd.) (vibration frequency: 30 Hz, 90 minutes), then allowed to stand for 30 minutes, and crushed again (vibration frequency: 30 Hz, 90 minutes) to obtain an off-white amorphous powder. Next, sodium 2-ethylhexanoate (8.2 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) was added to the grinding jar, and the reactant was crushed (vibration frequency: 30 Hz, 10 minutes) to obtain an off-white soft paste. The reaction mixture was taken out of the grinding jar, and the crude product was suspended, separated and washed with CLYNSOLVE (registered trademark) P-7 (manufactured by Japan Alcohol Sales Co., Ltd., 2×100 mL), RO water (2×100 mL) and acetone (2×100 mL) in turn. Then, the mixture was filtered by vacuum filtration (KIRIYAMA 5A, The product was recovered by using filter paper, manufactured by Kiriyama Seisakusho; PG201 diaphragm vacuum pump, manufactured by Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain an off-white fine amorphous powder (NaSO 3- PRT) and used it directly for analysis.

[0395] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.32 (a) and (b).

[0396] (Example 28) Sulfonation of 100 kDa recombinant protein

[0397] The recombinant protein PRT966 (7.5 g, 72.1 μmol), sulfur trioxide pyridine complex (7.87 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) were mixed with a stainless steel grinding medium ( The mixture was mixed with 2-ethylhexanoic acid (8.2 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) and put into a 125 mL hard steel grinding jar (manufactured by Lechi Co., Ltd.), and crushed using a mixed grinding ball mill (trade name: mixed grinding mill MM500 controlled type, manufactured by Lechi Co., Ltd.) (vibration frequency: 30 Hz, 90 minutes), then allowed to stand for 30 minutes, and crushed again (vibration frequency: 30 Hz, 90 minutes) to obtain an off-white amorphous powder. Next, sodium 2-ethylhexanoate (8.2 g, 49.4 mmol, 3.5 equivalents per nucleophilic residue) was added to the grinding jar, and the reactant was crushed (vibration frequency: 30 Hz, 10 minutes) to obtain an off-white soft paste. The reaction mixture was taken out of the grinding jar, and the crude product was suspended, separated and washed with CLYNSOLVE (registered trademark) P-7 (manufactured by Japan Alcohol Sales Co., Ltd., 2×100 mL), RO water (2×100 mL) and acetone (2×100 mL) in turn. The crude product was suspended in 0.1 M hydrochloric acid (100 mL) to acidify it and stirred for 5 minutes. The product was recovered by filtration (filter paper, Kiriyama Manufacturing Co., Ltd.; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain an off-white fine amorphous powder (HSO3-PRT), which was directly used for analysis.

[0398] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.33 (a) and (b).

[0399] (Example 29) Stearoylation of 100 kDa recombinant protein

[0400] Stearic acid (7.50 g, 26.4 mmol, 3.5 equivalents per nucleophilic residue) and 1,1-carbonyldiimidazole (4.27 g, 26.4 mmol, 3.5 equivalents per nucleophilic residue) were placed in a 50 mL stainless steel grinding jar (manufactured by Lach Co., Ltd.) and a stainless steel grinding medium ( Steel ball, manufactured by Lechi Co., Ltd.), use a mixed grinding ball mill (trade name: mixing mill MM400, manufactured by Lechi Co., Ltd.) to crush (vibration frequency: 30 Hz, 30 minutes) to obtain a colorless amorphous powder. Next, 100 kDa recombinant protein PRT966 (4.0 g, 38.4 μmol) was added to the grinding jar and crushed (vibration frequency: 30 Hz, 90 minutes) to obtain a colorless amorphous powder. The reaction mixture was taken out of the grinding jar and suspended in tetrahydrofuran (200 mL) for 10 minutes while stirring for 10 minutes. Thereafter, the mixture was filtered by vacuum filtration (KIRIYAMA No. 5A, The product was recovered by filtration (filter paper, Kiriyama Seisakusho; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain an off-white fine amorphous powder (St-PRT), which was used directly for analysis.

[0401] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.34 (a) and (b).

[0402] (Example 30) Oleoylation of 100 kDa recombinant protein

[0403] Under an inert nitrogen atmosphere, oleic acid (8.32 mL, 26.4 mmol, 3.5 equivalents per nucleophilic residue) and 1,1-carbonyldiimidazole (4.27 g, 26.4 mmol, 3.5 equivalents per nucleophilic residue) were placed in a sealed flask and stirred using a magnetic stirrer / hot plate. The mixture was heated and stirred at 60°C until carbon dioxide stopped being produced in the reaction mixture. The reaction mixture and recombinant protein PRT966 (4.0 g, 38.4 μmol) were placed in a 50 mL stainless steel grinding cup (manufactured by Lach), and stainless steel grinding media ( The mixture was pulverized (vibration frequency: 30 Hz, 90 minutes) using a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi) to obtain a granular brown powder. The reaction mixture was taken out from the grinding pot and suspended in tetrahydrofuran (200 mL) for 10 minutes while stirring. Then, the mixture was filtered by vacuum filtration (Kiriyama 5A, The product was recovered by filtration (filter paper, Kiriyama Manufacturing Co., Ltd.; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain an off-white fine amorphous powder (Ol-PRT), which was used directly for analysis.

[0404] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.35 (a) and (b).

[0405] (Example 31) Sulfosuccinylation of 100 kDa Recombinant Protein

[0406] 100 kDa maleated recombinant protein (5.0 g, 40.5 μmol) and sodium bisulfite (4.13 g, 39.7 mmol, 5.0 equivalents per nucleophilic residue) were added to a 50 mL stainless steel grinding jar (manufactured by Lach). The mixture was pulverized by mixing with a mixing mill (product name: mixing mill MM400, manufactured by Lechi) using RO water (1.0 mL) for liquid-assisted pulverization (vibration frequency: 30 Hz, 10 minutes) to obtain a light beige amorphous powder. The reaction mixture was removed from the grinding jar and suspended in RO water (200 mL) for 10 minutes while stirring. Acetonitrile (100 mL) was added to the suspension and the mixture was filtered by vacuum filtration (KIRIYAMA 5A, The product was recovered by filtration (filter paper, Kiriyama Seisakusho; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain an off-white fine amorphous powder (SulfoSucc-PRT), which was used directly for analysis.

[0407] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.36 (a) and (b).

[0408] (Example 32) (Diethoxyphosphoryl)succinylation of 100 kDa recombinant protein

[0409] 100 kDa maleated PRT966 (5.0 g, 40.5 μmol), diethyl phosphite (3.59 mL, 27.9 mmol, 3.5 equivalents per nucleophilic residue), and sodium ethoxide (570 mg, 8.76 mmol, 1.1 equivalents per nucleophilic residue) were added to a 50 mL stainless steel grinding jar (manufactured by Lach). The mixture was pulverized with liquid aid (vibration frequency: 30 Hz, 10 minutes) using a mixed grinding ball mill (trade name: mixed grinding mill MM400, manufactured by Lechi Co., Ltd.) with the aid of RO water (1.0 mL) to obtain a light beige amorphous powder. The reaction mixture was taken out of the grinding tank and suspended in RO water (200 mL) for 10 minutes while stirring. Acetonitrile (100 mL) was added to the suspension and vacuum filtered (KIRIYAMA 5A, The product was recovered by filtration (filter paper, Kiriyama Seisakusho; PG201 diaphragm vacuum pump, Yamato Scientific Co., Ltd.) and washed thoroughly with acetone. The product was dried at 80°C for 1 hour in a vacuum oven under reduced pressure to obtain a grayish white fine amorphous powder (DEPS-PRT). The obtained amorphous powder was used directly for analysis.

[0410] The results of FT-IR analysis and GPC analysis of the obtained amorphous powder and the results of the recombinant protein PRT966 used as a raw material are shown in Fig.37 (a) and (b).

[0411] 14. Surface wettability test

[0412] (1) Preparation of samples for analysis

[0413] The recombinant protein derivative to be analyzed was ground with stainless steel grinding media ( The mixture was added with 200 mL of 1% tantalum and 1% tantalum (made by Lechi Co., Ltd.) and added to a 50 mL stainless steel grinding jar (made by Lechi Co., Ltd.), and crushed using a mixed grinding ball mill (trade name: mixed grinding mill MM400, made by Lechi Co., Ltd.) (vibration frequency: 30 Hz, 5 minutes) to make it into a uniform powder. The crushed sample was stored in a constant humidity and thermostatic chamber (temperature range: 20-25°C ± 3°C, humidity range: 50-65% ± 5% RH) and allowed to reach equilibrium with the atmosphere after 1 hour.

[0414] (2) Cast film

[0415] The obtained uniform recombinant protein powder was dissolved in DMSO for 1 hour under stirring at 80°C to obtain a 15 wt% doping solution. The doping solution was cast onto a substrate sheet made of polyethylene terephthalate (trade name: SundayPET PG1, transparent color, thickness 0.5 mm, manufactured by Acrysunday Co., Ltd.). The thickness of the film was controlled by a doctor blade coater (80 mm×400 μm, manufactured by Imoto Seisakusho Co., Ltd.) to form a film with a uniform film thickness of 400 μm. The obtained film was first dried at 60°C for 4 hours using a constant temperature forced convection oven (DN411H, manufactured by Yamato Scientific Co., Ltd.), and then dried at 80°C for 8 hours under reduced pressure using a vacuum oven. The obtained film was cut into 35 mm×15 mm sizes and fixed to a slide glass using tape to keep it flat during contact angle analysis. Fig.38 a to d show the photographs of cast films of PRT966, Ac-PRT, Succ-PRT and St-PRT, respectively.

[0416] (3) Compressed powder

[0417] The obtained uniform recombinant protein powder (1.2 g) was added to a stainless steel mold (manufactured by Globalmachine Co., Ltd.), compressed at a pressure of 50 MPa using a desktop Newton press (trade name: NT-100H, manufactured by Sanzhuang Industry Co., Ltd.), and kept under pressure for 1 minute. The mold used was cylindrical and had a rectangular through hole with a cross-section of 35 mm × 15 mm. After that, the pressure was released, and the sample was taken out of the mold to obtain a compressed sample in the shape of a rectangular parallelepiped of 35 mm × 15 mm × 2 mm.

[0418] (4) Contact angle measurement experiment

[0419] Prior to analysis, the analysis samples were stored in a constant humidity and thermostatic chamber (temperature range: 20-25°C ± 3°C, humidity range: 50-65% ± 5% RH) and allowed to reach equilibrium with the atmosphere for 1 hour. Surface wettability was evaluated by measuring the static contact angle between 1 drop of RO water (about 2 μL) and the sample surface. When measuring the contact angle, a commercially available contact angle meter (trade name: DMs-601, manufactured by Kyowa Interface Science Co., Ltd.) and image capture and data fitting software (FAMAS, manufactured by Kyowa Interface Science Co., Ltd.) were used to implement the static drop method. A total of 6 measurements were performed for each recombinant protein derivative, and the average contact angle was calculated. The experimental results are expressed in the form of mean ± standard deviation, and a contact angle of <90° is evaluated as "hydrophilic", while a contact angle of ≥90° is evaluated as "hydrophobic".

[0420] (5) Results

[0421] The water contact angles of the compressed powder samples and cast films prepared from the recombinant protein PRT966, Ac-PRT (Example 8), Succ-PRT (Example 14) and St-PRT (Example 29) were measured. Fig.38 a to d show the photographs of cast films of PRT966, Ac-PRT, Succ-PRT and St-PRT, respectively.

[0422] The water contact angles measured for each recombinant protein derivative (n=6) are summarized in Table 6. Errors represent standard deviations. The "contact ellipse" in Table 6 is the average of the left and right contact angles. According to the steepness of the measured water contact angles, contact angle analysis was performed using compressed powder samples, and the samples were arranged in order from the most hydrophobic to the most hydrophilic, with the results being Ac-PRT>St-PRT>PRT966>Succ-PRT. Regardless of the experiment, it was observed that Ac-PRT and St-PRT were more hydrophobic than PRT966, while Succ-PRT was more hydrophilic.

[0423] [Table 6]

[0424]

[0425] 15. Flame retardancy test

[0426] (1) Method for preparing analytical samples

[0427] The recombinant protein derivative to be analyzed was ground with stainless steel grinding media ( The obtained uniform protein powder (1.2 g) was added to a stainless steel mold (Globalmachine Co., Ltd.) and pressurized at a pressure of 50 MPa for 1 minute using a desktop Newton press (trade name: NT-100H, manufactured by Sanzhuang INDUSTRY Co., Ltd.). The mold used was cylindrical and had a dumbbell-shaped through-hole with a cross-section of 45 mm × 15 mm. After that, the pressure was released, and the sample was taken out of the mold to obtain a dumbbell-shaped compressed sample of 45 mm × 15 mm × 2 mm.

[0428] (2) Flame retardancy analysis

[0429] Prior to analysis, the analytical sample was placed in a constant humidity and temperature chamber (20-25°C ± 3°C, 50-65% ± 5% RH) and allowed to reach equilibrium with the atmosphere for 1 hour. Each sample was placed in a support fixture with the length of the sample horizontal, and the strip sample was allowed to extend at least 40 mm from the support fixture. A laboratory burner with a 25 mm orange flame was used to ignite the extended end of the sample. The flame was allowed to contact the sample for 30 seconds, then the flame was removed, and the time until the flame went out and the change in sample mass were recorded. Each recombinant protein derivative was measured (n = 6), and the average value of the burning time and mass loss was calculated. The experimental results are expressed as mean ± standard error.

[0430] (3) First flame retardancy test

[0431] In the first flame retardancy test, the flame retardancy of unmodified PRT 966, NaSO3-PRT (Example 27), HSO3-PRT (Example 28), and SulfoSucc-PRT (Example 31) was evaluated in an environment of 5 to 10°C and 30 to 35% RH.

[0432] The moisture content, combustion time, and mass loss of each recombinant protein (n=6) are summarized in Table 7. Fig.39 (a) is a graph showing the burning time of each sample, Fig.39 (b) is a graph showing the mass loss of each sample. Errors represent standard deviations. Based on the measured burning time and mass loss, all protein derivative samples showed improved fire resistance compared to PRT966, with Example 31 showing the most outstanding fire resistance. The order of fire resistance was Example 31 > Example 28 > Example 27 > PRT966.

[0433] [Table 7]

[0434]

[0435] (4) Second flame retardancy test

[0436] In the second flame retardancy test, the flame retardancy of unmodified PRT966, SulfoSucc-PRT (Example 31) and DEPS-PRT (Example 32) was evaluated. The ambient conditions during the experiment were 20-25°C and 70-75% RH.

[0437] The moisture content, burning time, and mass loss of each recombinant protein derivative (n=6) are summarized in Table 8. Errors represent standard deviations. Based on the measured burning time and mass loss, the modified protein derivatives all showed improved fire resistance compared to the unmodified PRT966, and Example 32 showed the most outstanding fire resistance. The order of fire resistance was Example 32> Example 31> PRT966.

[0438] [Table 8]

[0439]

[0440] 15. Thermoplasticity test

[0441] (1) Preparation of resin samples for analysis

[0442] The recombinant protein derivatives to be analyzed (pure products or containing 20 wt% glycerol as a plasticizer) were ground with stainless steel grinding media ( The mixture was added with a 50 mL stainless steel grinding jar (made by Lechi) and crushed using a mixed grinding ball mill (trade name: mixed grinding mill MM400, made by Lechi) (vibration frequency: 30 Hz, 5 minutes) to obtain a uniform mixture. The obtained uniform mixture (3.0 g) was added to a stainless steel mold (Global Machine Co., Ltd.) that had been thinly coated with a fluorine-based release agent (trade name: DAIFREE MS-600, made by Daikin Industries, Ltd.), and pressurized to 30 MPa using a desktop Newton press (trade name: NT-100H, made by Sanzhuang Industry Co., Ltd.). The mold used was cylindrical and had a rectangular through hole with a cross-section of 35 mm × 15 mm. The compressed sample was heated to 170°C using a temperature control controller (trade name: MTCD15-EN, made by Misumi Group Headquarters Co., Ltd.) and maintained at this temperature for 5 minutes. While maintaining the pressure at 30 MPa, the sample was cooled to 60°C. After that, the pressure was released, and the sample was taken out of the mold to obtain a resin sheet with a size of 35 mm × 15 mm × 4.5 mm. The formed resin sheet was cut into analysis samples of 5 mm × 5 mm × 4.5 mm using a table circular saw (trade name: K-210, manufactured by HOZAN CO., LTD.) equipped with a diamond disc cutter (particle size: #150, trade name: K-210-3, manufactured by HOZAN CO., LTD.).

[0443] (2) Thermomechanical analysis

[0444] The glass transition temperature (Tg), onset temperature and softening point of each recombinant protein resin sample were determined by using a thermomechanical analyzer (trade name: TMA4000, manufactured by NETZSCH Japan Co., Ltd.) equipped with a quartz compression load probe ( The top of the compression test rod (P / N T1442-04; compression support tube (P / N T1442-03, manufactured by NETZSCH Japan Co., Ltd.) is flat and can press the sample with a load of 4.9N. Then, the sample is pressed at a constant speed (10K min -1 ) Heat the sample to 220°C and record the deformation of the sample. A total of 6 measurements were performed on each recombinant protein derivative analyzed, and the average values ​​of the onset temperature of Tg and the softening point were calculated. The experimental results are expressed as mean ± standard deviation. Thermomechanical analysis was performed on each resin sample made using unmodified PRT966, Ac-PRT (Example 8), Succ-PRT (Example 14), Mal-PRT (Example 23), St-PRT (Example 29) and Ol-PRT (Example 30). Pure protein resin samples and resin samples plasticized with 20wt% glycerol were analyzed.

[0445] (3) Results

[0446] The Tg onset temperatures and softening points of each recombinant protein resin sample (n=6) are summarized in Table 9. Errors represent standard deviations. Fig.40 It is a graph showing the softening point, Tg starting point, and melting temperature Tm of unmodified PRT966 and each modified protein. According to the thermomechanical analysis, for both the unmodified and plasticized recombinant protein resin samples, all modified derivatives have lower glass transition temperatures (Tg) and softening points than unmodified PRT966, and Example 30 has the lowest Tg starting point and softening point. In addition, the analysis results show that the thermal processing properties of the recombinant protein resin can be improved by adding 20wt% glycerol as a plasticizer, and it has been confirmed that the softening points of all plasticized samples are below the highest temperature (220°C).

[0447] [Table 9]

[0448]

Claims

1. A method for producing a modified protein, comprising treating a mixture containing a protein and an acylating agent using a mechanochemical method to obtain the modified protein.

2. The method according to claim 1, wherein the mixture further comprises a base and / or a reaction accelerator. The method of claim 1 , wherein the protein comprises a hydrophobic protein. The method according to claim 3 , wherein the hydrophilicity index of the hydrophobic protein is greater than 0.

5. The method of claim 1, wherein the protein comprises an artificial protein. The method of claim 5 , wherein the artificial protein comprises an artificial structural protein.

7. The method according to any one of claims 1 to 6, further comprising the step of obtaining the protein by a microbiological method.

8. The method according to claim 1, wherein the treatment using a mechanochemical method is achieved by applying a shear force to the mixture.

9. The method of claim 8, wherein the shear force is applied to the mixture using a mixer grinder or an extruder.

10. A modified protein obtained by the method according to any one of claims 1 to 6.

11. The modified protein obtained by the method according to claim 7.

12. A modified protein composition comprising the modified protein according to claim 10.

13. A modified protein composition comprising the modified protein according to claim 11.

14. A modified protein shaped body obtained by shaping the modified protein composition according to claim 12.

15. A modified protein shaped body obtained by shaping the modified protein composition according to claim 13.

16. A molding material comprising the modified protein according to claim 10.

17. A molding material comprising the modified protein according to claim 11.

18. A modified protein shaped body obtained by shaping the shaping material according to claim 16.

19. A modified protein shaped body obtained by shaping the shaping material according to claim 17.

20. A method for producing a solution-state adhesive, comprising the step of dissolving the modified protein obtained by the method according to claim 1 in a solvent.

21. A method for producing a water-dispersible adhesive, comprising the step of dispersing the modified protein obtained by the method according to claim 1 in an aqueous medium.

22. A method for producing a film-like adhesive, comprising the step of forming a film from a solution in which the modified protein obtained by the method according to claim 1 is dissolved.

23. A method for producing a powdered adhesive, comprising the step of obtaining a powder comprising the modified protein obtained by the method according to claim 1.

24. A method for producing a bonded body, characterized in that: The modified protein obtained by the method of claim 1 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 modified protein, thereby adhering the adherends.

25. A method for producing a bonded body, characterized in that: The modified protein obtained by the method of claim 1 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 modified protein, thereby adhering the adherends.

26. A method for producing a bonded body, characterized in that: The thin film containing the modified protein obtained by the method of claim 1 is softened by swelling or heating, and the thin film is placed between a plurality of adherends, and then the thin film is cured while being pressed against the adherends, thereby bonding the adherends.

27. A method for producing an adhesive body, characterized in that: A powder containing the modified protein obtained by the method of claim 1 is placed between a plurality of adherends, and the powder is heated and pressed through the adherends to solidify the powder, thereby adhering the adherends.

28. A method for producing a solution-like coating, comprising the step of dissolving the modified protein obtained by the method according to claim 1 in a solvent.

29. A method for producing a water-dispersible coating, comprising the step of dispersing the modified protein obtained by the method according to claim 1 in an aqueous medium.

30. A method for producing a film-like coating, comprising the step of forming a film from a solution in which the modified protein obtained by the method according to claim 1 is dissolved.

31. A method for producing a powdered coating, comprising the step of obtaining a powder containing the modified protein obtained by the method according to claim 1.

32. A method for producing a laminate having a substrate and a coating layer laminated on at least a portion of a surface of the substrate, characterized in that: A solution in which the modified protein obtained by the method according to claim 1 is dissolved 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 to solidify the modified protein, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

33. A method for producing a laminate having a substrate and a coating layer laminated on at least a portion of a surface of the substrate, characterized in that: An aqueous dispersion in which the modified protein obtained by the method according to claim 1 is dispersed in an aqueous medium 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 aqueous dispersion, the aqueous medium is removed from the aqueous dispersion to solidify the modified protein, thereby forming a coating layer stacked on at least a portion of the surface of the substrate.

34. A method for producing a laminate having a substrate and a coating layer laminated on at least a portion of a surface of the substrate, characterized in that: The coating layer is formed by laminating on at least a portion of the surface of the substrate by softening the film containing the modified protein obtained by the method according to claim 1 by swelling or heating and placing it on at least a portion of the surface of the substrate, and then hardening the film while being pressed onto the substrate.

35. A method for producing a laminate having a substrate and a coating layer laminated on at least a portion of a surface of the substrate, characterized in that: While a powder containing the modified protein obtained by the method according to claim 1 is placed on at least a portion of the surface of the substrate, the powder is heated and simultaneously pressurized between a pressurizing body and the substrate to solidify the powder, thereby forming the coating layer by stacking on at least a portion of the surface of the substrate.

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