WO2019054503A1

WO2019054503A1 - Molding composition, molded article, and method for producing molded article

Info

Publication number: WO2019054503A1
Application number: PCT/JP2018/034252
Authority: WO
Inventors: 浩一小鷹; 潤一菅原; 純一野場
Original assignee: Ｓｐｉｂｅｒ株式会社; テクノハマ株式会社
Priority date: 2017-09-15
Filing date: 2018-09-14
Publication date: 2019-03-21
Also published as: JPWO2019054503A1

Abstract

A molding composition containing a structural protein and polyhydric alcohols having two or more hydroxy groups and 12 or fewer carbon atoms.

Description

Composition for molding, molding, and method for producing molding

The present invention relates to a composition for molding, a molded body, and a method for producing a molded body.

With the rising awareness of environmental conservation, alternatives to petroleum-derived materials are being studied, and structural proteins that are excellent in terms of strength and the like are listed as candidates. For example, Patent Document 1 discloses a method of compression-molding an aggregate of animal fibers to obtain an animal fiber molding having high mechanical properties such as stress-strain characteristics.

Unexamined-Japanese-Patent No. 2017-110132

However, in the compression molding described in Patent Document 1, it is difficult to obtain a compact having a complicated shape, and there is a problem that productivity is low.

As a molding method for obtaining a molded object having a complicated shape, for example, a molding method such as injection molding is known. However, since fluidity is required for injection molding, it is difficult to apply the aggregate of animal fibers of Patent Document 1 to injection molding.

Then, an object of the present invention is to provide a composition for molding which is applicable to a molding method which contains a structural protein and is required to have fluidity such as injection molding. Another object of the present invention is to provide a molded article molded from the molding composition. Another object of the present invention is to provide a method for producing a molded body using the molding composition.

The present invention provides a molding composition containing a structural protein and a polyhydric alcohol having two or more hydroxy groups and having 12 or less carbon atoms.

The composition for molding is improved in fluidity (particularly at high temperature) by blending a specific polyhydric alcohol, and can be suitably applied to a molding method that requires fluidity, such as injection molding. For this reason, according to the composition for molding, the molding having a complicated shape can be easily obtained by a molding method such as injection molding, and a molding can be obtained with high productivity as compared with compression molding. be able to.

In the composition for molding, the polyhydric alcohol preferably contains at least one selected from the group consisting of lower alcohols, saccharides and sugar alcohols.

In the composition for molding, the structural protein preferably contains a fibroin-like protein.

In the above composition for molding, the fibroin-like protein preferably includes a spider silk fibroin-like protein.

The molding composition is preferably a mixture of the structural protein, the polyhydric alcohol, and water.

The composition for mold molding may be a composition for injection molding.

The present invention also provides a molded article obtained by molding the composition for molding, which comprises a solid material containing the above-mentioned structural protein or a modified product thereof.

The present invention further comprises heating the composition for molding under pressure to obtain a fluid material, injecting the fluid material into the mold, and injecting the material into the mold. Cooling the flowable material to obtain a molded product made of a solid material containing the above-mentioned structural protein or its modified product, and providing a method for producing a molded product.

In the method for producing a molded article, the polyhydric alcohol preferably has a boiling point higher than the heating temperature in the heating step.

According to the present invention, there is provided a molding composition which contains a structural protein and is also applicable to a molding method that requires fluidity such as injection molding. Further, according to the present invention, there is provided a molded body molded from the composition for mold molding. Furthermore, according to the present invention, there is provided a method for producing a molded article using the molding composition.

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following embodiment.

(Composition for molding)
The composition for molding according to this embodiment contains a structural protein and a polyhydric alcohol having 12 or less carbon atoms. The molding composition according to the present embodiment can exhibit fluidity applicable to a molding method such as injection molding by heating and pressing. For this reason, according to the molding composition according to the present embodiment, a molding having a complicated shape can be easily obtained by a molding method such as injection molding, and the productivity is good as compared with compression molding. A molded body can be obtained.

The molding composition according to this embodiment is particularly suitably used for injection molding. That is, the composition for mold formation concerning this embodiment can also be called composition for injection molding.

[Structural protein]
The structural protein refers to a protein that forms a biological structure or a protein derived therefrom. That is, the structural protein may be a naturally occurring structural protein, and is a modified protein in which a portion (for example, 10% or less of the amino acid sequence) of the amino acid sequence is altered based on the amino acid sequence of the naturally occurring structural protein. It may be

Specific examples of structural proteins include fibroin (for example, spider silk, silkworm silk and the like), keratin, collagen, elastin and resilin, and proteins derived therefrom.

Examples of fibroin-like proteins (fibroin or proteins derived therefrom) include, for example, proteins containing a domain sequence represented by the formula 1: [(A) _n motif-REP1] _m . Here, in Formula 1, (A) _n motif indicates an amino acid sequence mainly comprising an alanine residue, n is 2 to 20, preferably 4 to 20, more preferably 8 to 20, and still more preferably 10 to It may be an integer of 20, still more preferably 4 to 16, still more preferably 8 to 16, particularly preferably 10 to 16. The ratio of the number of alanine residues to the total number of amino acid residues in (A) _n motif may be 40% or more, preferably 60% or more, and more preferably 70% or more. % Or more is more preferable, 90% or more is still more preferable, and 100% (meaning it is composed of only alanine residues) may be used. REP1 shows an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) _n motifs may be identical to each other or different from each other. The plurality of REP1 may have the same or different amino acid sequences. As a specific example of a fibroin-like protein, for example, a protein comprising the amino acid sequence shown by SEQ ID NO: 1 can be mentioned.

As a collagen-like protein (collagen or a protein derived therefrom), for example, a protein containing a domain sequence represented by the formula 2: [REP2] _p (wherein _p represents an integer of 5 to 300 in the formula 2). REP2 represents an amino acid sequence composed of Gly-X-Y, and X and Y represent any amino acid residues other than Gly. Good) can be mentioned. As a specific example of the collagen like protein, a protein containing the amino acid sequence shown by SEQ ID NO: 2 can be mentioned. In addition, the amino acid sequence shown by SEQ ID NO: 2 is a partial portion of human collagen type 4 sequence obtained from the NCBI database (NCBI GenBank accession numbers: CAA56335.1, GI: 3702452) The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 6 is added to the N-terminus of the corresponding amino acid sequence from the 301st residue to the 540th residue.

As a resilin-like protein (resilin or a protein derived therefrom), for example, a protein containing a domain sequence represented by Formula 3: [REP3] _q (wherein, in Formula 3, q represents an integer of 4 to 300). REP3 represents an amino acid sequence composed of Ser-JJ-Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, and in particular an amino acid residue selected from the group consisting of Asp, Ser and Thr U is preferably any amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. A plurality of REP3s may be identical amino acid sequences to each other. And different amino acid sequences may be mentioned. As a specific example of resilin like protein, the protein containing the amino acid sequence shown by sequence number 3 can be mentioned. The amino acid sequence shown by SEQ ID NO: 3 is the amino acid sequence of resilin (NCBI GenBank accession numbers NP 611 157, Gl: 24654243), wherein Thr at position 87 is substituted with Ser, and Asn at position 95 The amino acid sequence (tag sequence) shown by SEQ ID NO: 7 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence obtained by substituting Asp.

Examples of elastin-like proteins (elastin or proteins derived therefrom) include, for example, proteins having amino acid sequences such as NCBI Accession Nos. AAC98395 (human), I47076 (sheep) and NP786966 (bovine) from GenBank. As a specific example of the elastin-like protein, a protein comprising the amino acid sequence shown in SEQ ID NO: 4 can be mentioned. The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 6 at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank accession number AAC98395 (tag sequence And hinge arrangement) are added.

Examples of the keratin-like protein (keratin or a protein derived therefrom) include, for example, type I keratin of Capra hircus and the like. As a specific example of the keratin-like protein, a protein comprising the amino acid sequence shown in SEQ ID NO: 5 (the amino acid sequence of GenBank Accession No. ACY30466 of NCBI) can be mentioned.

As a structural protein, fibroin-like protein is preferable, and spider silk fibroin-like protein (a spider silk fibroin or a protein derived therefrom) is more preferable. By using such a structural protein, it is possible to obtain a molded body which is further excellent in mechanical strength.

The structural protein can be expressed, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the structural protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by

There are no particular restrictions on the method for producing the nucleic acid encoding the target protein. For example, the nucleic acid can be produced by a method of amplification and cloning by polymerase chain reaction (PCR) or the like, or a method of chemical synthesis, using a gene encoding a natural structural protein. The method for chemically synthesizing nucleic acid is not particularly limited, and, for example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. based on the amino acid sequence information of structural proteins obtained from the NCBI web database etc. A nucleic acid can be chemically synthesized by a method of linking oligonucleotides that are automatically synthesized in the above by PCR or the like. At this time, in order to facilitate purification and confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence having an amino acid sequence consisting of an initiation codon and a His10 tag added to the N terminus of the above amino acid sequence may be synthesized. .

The regulatory sequence is a sequence that controls the expression of a recombinant protein in a host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. As a promoter, an inducible promoter which functions in a host cell and is capable of inducing expression of a target protein may be used. An inducible promoter is a promoter that can control transcription due to the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as temperature, osmotic pressure or an increase or decrease in pH value.

The type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector and the like. As the expression vector, a vector capable of autonomous replication in a host cell or capable of integration into the host chromosome and containing a promoter at a position capable of transcribing a nucleic acid encoding a target protein is suitably used. .

As a host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of the prokaryote include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.

When a prokaryote is used as a host, examples of vectors for introducing a nucleic acid encoding a target protein include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Application Laid-Open No. 2002-238569) and the like can be mentioned.

Eukaryotic hosts can include, for example, yeast and filamentous fungi (molds and the like). As a yeast, the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

When a eukaryote is used as a host, examples of vectors into which a nucleic acid encoding a target protein is introduced include YEP13 (ATCC 37115), YEp24 (ATCC 37051), and the like.

As a method of introducing the expression vector into the host cell, any method of introducing DNA into the host cell can be used. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.

As a method for expressing a nucleic acid by a host transformed with an expression vector, in addition to direct expression, secretion production, fusion protein expression and the like can be performed according to the method described in Molecular Cloning 2nd Edition, etc. .

The target protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, causing the protein to be produced and accumulated in the culture medium, and collecting the protein from the culture medium. The method of culturing the host in a culture medium can be carried out according to a method usually used for culturing the host.

When the host is a prokaryote such as E. coli or a eukaryote such as yeast, the culture medium contains a carbon source which can be used by the host, a nitrogen source, inorganic salts and the like, and the medium can efficiently culture the host. If it is, either a natural culture medium or a synthetic culture medium may be used.

The carbon source may be any as long as the above-mentioned transformed microorganism can assimilate, for example, glucose, fructose, sucrose and molasses containing them, carbohydrates such as starch and starch hydrolysate, acetic acid and propionic acid etc. Organic acids and alcohols such as ethanol and propanol can be used. Nitrogen sources include, for example, ammonium, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used. As inorganic salts, for example, potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

The culture of a prokaryote such as E. coli or a eukaryote such as yeast can be performed under aerobic conditions such as shake culture or submerged aeration culture, for example. The culture temperature is, for example, 15 to 40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. Adjustment of the pH of the culture medium can be carried out using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia and the like.

Also, during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. When a microorganism transformed with an expression vector using an inducible promoter as a promoter is cultured, an inducer may be added to the medium as needed. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside etc., and culturing a microorganism transformed with an expression vector using a trp promoter, indole acrylic An acid or the like may be added to the medium.

Protein isolation and purification can be carried out by a commonly used method. For example, when the protein is expressed in a dissolved state in cells, after completion of culture, host cells are recovered by centrifugation and suspended in an aqueous buffer, and then sonicator, French press, Manton Gaulin homogenizer Then, the host cells are disrupted by Dinomill et al. To obtain a cell-free extract. Methods commonly used for isolation and purification of proteins from supernatants obtained by centrifuging cell-free extracts, ie solvent extraction methods, salting out methods such as ammonium sulfate, desalting methods, precipitation with organic solvents Method, anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -sepharose, DIAION HPA-75 (made by Mitsubishi Kasei Corp.), cation using resin such as S-Sepharose FF (made by Pharmacia) Exchange chromatography method, hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc. Using one of the methods of It is possible to obtain.

When the protein is expressed in the form of an insoluble form in cells, the host cell is similarly recovered and then disrupted and centrifuged to recover the insoluble form of the protein as a precipitate fraction. The recovered insoluble form of protein can be solubilized with a protein denaturant. After the operation, a purified preparation of protein can be obtained by the same isolation and purification method as described above.

When the protein is secreted extracellularly, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified preparation can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[Polyhydric alcohols]
In the present embodiment, polyhydric alcohols have 2 or more hydroxy groups, and the number of carbon atoms of polyhydric alcohols is 12 or less.

The number of carbon atoms of the polyhydric alcohol is preferably 10 or less, more preferably 7 or less, and still more preferably 6 or less. Moreover, it is preferable that the carbon atom number of polyhydric alcohols is two or more.

The polyhydric alcohols preferably contain at least one selected from the group consisting of lower alcohols, sugars and sugar alcohols. By using such polyhydric alcohols, a composition excellent in injection moldability can be easily obtained.

Lower alcohols are alcohols having 5 or less carbon atoms. As the lower alcohol, for example, ethylene glycol, glycerin, erythritol, xylitol and the like can be mentioned.

The saccharides include monosaccharides and disaccharides, and among these, monosaccharides are more preferably used. As a monosaccharide, glucose, fructose etc. can be mentioned, for example. Examples of disaccharides include sucrose and the like.

Examples of sugar alcohols include glycerin, erythritol, xylitol and the like.

In this embodiment, by adding polyhydric alcohols to the structural protein, the structural protein can be applied to a molding method that requires fluidity such as injection molding. The amount of polyhydric alcohol added is not particularly limited as long as the composition for molding can exhibit fluidity that can be applied to injection molding and the like. The amount of polyhydric alcohol added is, for example, preferably 20 parts by mass or more, and more preferably 25 parts by mass or more with respect to 100 parts by mass of the structural protein. The amount of polyhydric alcohol added is, for example, preferably 40 parts by mass or less, and more preferably 35 parts by mass or less, with respect to 100 parts by mass of the structural protein.

The molding composition according to the present embodiment may further contain water. Fluidity tends to be further improved by adding water to structural proteins in combination with polyhydric alcohols.

The addition amount of water is not particularly limited, and for example, the amount is preferably 7 parts by mass or more, and more preferably 10 parts by mass or more with respect to 100 parts by mass of the structural protein. The amount of water added is, for example, preferably 35 parts by mass or less, more preferably 20 parts by mass or less, and still more preferably 15 parts by mass or less with respect to 100 parts by mass of the structural protein . If the amount of water added is too large, air bubbles may be mixed into the molded product, and the molded product may be easily shrunk, but if it is in the above range, sufficient fluidity improvement effect can be obtained while avoiding these problems. Can.

In the present embodiment, the method for producing the molding composition is not particularly limited, and the structural protein and the polyhydric alcohol may be mixed by a known method. That is, the composition for molding may be a mixture of a structural protein and a polyhydric alcohol.

In the composition for molding according to the present embodiment, it is preferable that the structural protein is highly dispersed in the composition. From this viewpoint, the composition for molding is preferably a mixture of powder of structural protein and polyhydric alcohol, or a mixture of pulverized and structural protein and polyhydric alcohol, and structural protein It is more preferable that it is the mixture which grind | pulverized and mixed the powder and polyhydric alcohols. In addition, a grinding | pulverization mixing shows that a structural protein is grind | pulverized during mixing, and micronization or diameter reduction is carried out here.

In the present embodiment, water may be simultaneously mixed at the time of mixing of the structural protein and the polyhydric alcohol.

The molding composition according to the present embodiment is a composition that can exhibit fluidity applicable to injection molding and the like by heating and pressing. The composition for molding is, for example, a composition that develops the above-mentioned fluidity by heating at 120 to 150 ° C. (more preferably 130 to 140 ° C.) and pressing at 20 to 45 MPa (more preferably 25 to 30 MPa). It is preferable that it is a thing. By performing molding in such a temperature range, it is possible to manufacture a molded body having more excellent mechanical properties.

(Molded body)
The molded object which concerns on this embodiment is a molded object which mold-formed the above-mentioned composition for mold molding (preferably injection molding). The formed body is composed of a solid material containing the above-mentioned structural protein or its modified body. The denatured body refers to a structural protein in the composition for molding which has been denatured by heat and pressure during molding, and is, for example, a thermally denatured structural protein, a reaction product of a structural protein with a polyhydric alcohol, etc. You may

In a preferred embodiment, the shaped body may be composed of a solid material containing a structural protein and polyhydric alcohols.

In the solid material constituting the molded body, the structural protein preferably does not exist as a powder but is integrated. When the compact is in a state in which the powder of the structural protein is compacted, the mechanical strength is poor and appearance problems such as white turbidity also occur. In this embodiment, by adding a polyhydric alcohol to the above-mentioned composition for molding, the structural protein becomes easy to flow at the time of molding, and the molding having an excellent appearance (for example, an appearance like a plaster) You can get the body.

In the present embodiment, the method of molding is not particularly limited, and may be, for example, the following method.

In a preferred embodiment, the molded body is heated in a mold by heating the above-mentioned molding composition under pressure to obtain a fluid material, injecting the fluid material into the mold, and injecting the material into the mold. Cooling the injected flowable material to obtain a compact comprising the above-mentioned solid material.

The heating temperature in the heating step is not particularly limited, and it may be a temperature at which the molding composition can exhibit sufficient fluidity (ie, a temperature at which a fluid material having sufficient fluidity can be obtained). The heating temperature may be, for example, 120 ° C. or more, and preferably 130 ° C. or more. The heating temperature may be, for example, 150 ° C. or less, and preferably 140 ° C. or less.

The pressurizing condition in the heating step is not particularly limited as long as vaporization of polyhydric alcohol by heating is sufficiently suppressed and the composition for molding can exhibit sufficient fluidity. The pressurizing condition may be, for example, 20 MPa or more, and preferably 25 MPa or more. The pressure condition may be, for example, 45 MPa or less, and more preferably 30 MPa or less.

In the injection step, the flowable material is injected into the mold. At this time, it is preferable to apply pressure to the fluid material so that the fluid material is injected to the details in the mold. In addition, in the injection step, it is preferable that the mold be heated in order to prevent the fluid material from solidifying before the inside of the mold is sufficiently filled. These pressure and heating conditions can be appropriately adjusted in accordance with the flowability of the flowable material, the shape in the mold, and the like.

In the cooling step, the flowable material injected into the mold is cooled and solidified to obtain a molded body having a shape corresponding to the shape in the mold. The cooling method is not particularly limited, and can be appropriately selected from known methods.

In the above-mentioned manufacturing method, it is preferable that polyhydric alcohols contained in the composition for mold formation have a boiling point higher than the heating temperature in a heating process. Thereby, vaporization of polyhydric alcohols in the heating step and the pouring step is suppressed, and deterioration of the external shape of the molded product due to foaming and the like can be avoided.

As mentioned above, although the suitable embodiment of the present invention was described, the present invention is not limited to the above-mentioned embodiment.

Hereinafter, the present invention will be more specifically described by way of examples, but the present invention is not limited to the examples.

<Production of spider silk fibroin-like protein>
(1) Preparation of expression strain Based on the base sequence and amino acid sequence of fibroin (GenBank accession number: P46804.1, GI: 1174415) derived from Nephila clavipes, it has the amino acid sequence shown in SEQ ID NO: 1 A modified fibroin (hereinafter also referred to as "PRT410") was designed. In addition, the amino acid sequence shown by SEQ ID NO: 1 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving the productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes Furthermore, the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 6 is added to the N-terminus.

Next, the nucleic acid encoding PRT410 was synthesized. The NdeI site at the 5 'end and the EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with NdeI and EcoRI, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Protein expression E. coli BLR (DE3) was transformed with pET22b (+) expression vector containing a nucleic acid encoding a protein having the amino acid sequence shown by SEQ ID NO: 1. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 1) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 ml of production medium (Table 2) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 ml / min. The temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein. Twenty hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. SDS-PAGE was performed using cells prepared from the culture solution before IPTG addition and after IPTG addition, and expression of a target protein was confirmed by appearance of a band of the target protein size depending on IPTG addition.

(3) Purification of Protein Two hours after addition of IPTG, the cells collected were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) to high purity. The washed precipitate is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL, and 30 at 60 ° C. Stir with a stirrer for a minute to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Pure Chemical Industries, Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, the water was removed by a lyophilizer, and the lyophilized powder was recovered. The freeze-dried powder was used as a powder of spider silk fibroin-like protein "PRT410" in Examples and Comparative Examples.

Example 1
70% by weight of the above protein powder, 20% by weight of ethylene glycol (made by Tokyo Chemical Industry Co., Ltd., carbon atom number: 2) and 10% by weight of water are charged into a household miller (IFM-800DG made by Iwatani Sangyo Co., Ltd.) and mixed, A composition for molding was obtained.

The flowability and moldability of the obtained molding composition were evaluated by the following method. As a result of evaluation, the fluidity and the formability were both A.

<Evaluation method>
The molding composition was charged into a pressure-resistant container having an extrusion port with a diameter of 5 mm, heated and pressurized while the extrusion port was closed, and held for 1 minute under conditions of a heating temperature of 140 ° C. and a pressure of 5.6 MPa. The composition was then extruded from the extrusion port at a maximum pressure of 40 MPa, and the composition extruded out of the container was cooled to room temperature.

The flowability was evaluated as A when the composition could be sufficiently extruded from the extrusion port, B when the composition remained in the pressure-resistant container although partially extruded, and C when the composition could not be extruded as C. . In addition, the composition after cooling is observed, A is obtained when a uniform solid material without white turbidity is obtained, B when white turbidity is observed in a part, and white turbidity is observed in a state where the powder is pressed or solidified. The formability was evaluated as C, in the case of

In addition, about the composition whose flowability evaluation is A, it confirmed that a molded object was producible by injection molding. Moreover, in the composition whose evaluation of fluidity | liquidity is B or C, manufacture of the molded object by injection molding was difficult.

(Example 2)
A molding composition was obtained in the same manner as in Example 1, except that ethylene glycol was replaced with 20% by weight of glycerol (manufactured by Wako Pure Chemical Industries, Ltd., carbon atom number: 3). The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, both the flowability and the moldability were A.

(Example 3)
A molding composition was obtained in the same manner as in Example 1, except that 20% by weight of glucose (manufactured by Wako Pure Chemical Industries, D-glucose, carbon number: 6) was used instead of ethylene glycol. The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, both the flowability and the moldability were A.

(Comparative example 1)
A mold-forming composition was obtained in the same manner as in Example 1, except that 20% by weight of polyvinyl alcohol (polyvinyl alcohol 1000, partial saponification type, manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of ethylene glycol. The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, the fluidity was B, and the moldability was C.

(Comparative example 2)
Composition for mold molding in the same manner as in Example 1 except that polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, polyvinyl alcohol (polymerization degree: about 2000), complete saponification) 20% by weight was used instead of ethylene glycol. I got The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, both the flowability and the moldability were C.

(Comparative example 3)
A molding composition was obtained in the same manner as in Example 1, except that 20% by weight of cellulose nanofibers (Cerish KY100G manufactured by Daicel Finechem Co., Ltd.) was used instead of ethylene glycol. The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, the flowability was C and the moldability was B.

(Comparative example 4)
A composition for mold molding was prepared in the same manner as in Example 1 except that 20% by weight of trimellitic acid ester (ADEKA, Adekaizer C-9N, carbon atom number: 36) was used instead of ethylene glycol. Obtained. The obtained mold forming composition was evaluated by the same evaluation method as in Example 1. As a result, the fluidity was B, and the moldability was C.

The results of Examples and Comparative Examples are shown in Table 3.

The composition for molding according to the present invention can be applied to a molding method which contains a structural protein and which requires fluidity such as injection molding. For this reason, the molding composition according to the present invention can be suitably used as a substitute for petroleum-derived materials.

Claims

Structural protein,
Polyhydric alcohols having 2 or more hydroxy groups and having 12 or less carbon atoms,
The composition for moldings containing.
The molding composition according to claim 1, wherein the polyhydric alcohol comprises at least one selected from the group consisting of lower alcohols, saccharides and sugar alcohols.
The molding composition according to claim 1, wherein the structural protein comprises a fibroin-like protein.
The molding composition according to claim 3, wherein the fibroin-like protein comprises spider silk fibroin-like protein.
The molding composition according to any one of claims 1 to 4, which is a mixture of the structural protein, the polyhydric alcohol, and water.
The molding composition according to any one of claims 1 to 5, which is for injection molding.
It is a molded object which mold-molded the composition for mold formation as described in any one of Claims 1-6,
A molded body comprising a solid material containing the structural protein or a modified product thereof.
A heating step of heating the molding composition according to any one of claims 1 to 6 under pressure to obtain a fluid material;
Injecting the fluid material into a mold;
Cooling the flowable material injected into the mold to obtain a compact made of a solid material containing the structural protein or its modified product;
A method of producing a molded body, comprising
The manufacturing method according to claim 8, wherein the polyhydric alcohol has a boiling point higher than the heating temperature in the heating step.