WO2019054503A1 - Composition de moulage, article moulé et procédé de production d'article moulé - Google Patents

Composition de moulage, article moulé et procédé de production d'article moulé Download PDF

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Publication number
WO2019054503A1
WO2019054503A1 PCT/JP2018/034252 JP2018034252W WO2019054503A1 WO 2019054503 A1 WO2019054503 A1 WO 2019054503A1 JP 2018034252 W JP2018034252 W JP 2018034252W WO 2019054503 A1 WO2019054503 A1 WO 2019054503A1
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WIPO (PCT)
Prior art keywords
molding
protein
composition
structural protein
amino acid
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Ceased
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PCT/JP2018/034252
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English (en)
Japanese (ja)
Inventor
浩一 小鷹
潤一 菅原
純一 野場
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tekunohama Co Ltd
Spiber Inc
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Tekunohama Co Ltd
Spiber Inc
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Priority to JP2019542322A priority Critical patent/JPWO2019054503A1/ja
Publication of WO2019054503A1 publication Critical patent/WO2019054503A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
    • B29C39/22—Component parts, details or accessories; Auxiliary operations
    • B29C39/24—Feeding the material into the mould
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
    • B29C45/17—Component parts, details or accessories; Auxiliary operations
    • B29C45/18—Feeding the material into the injection moulding apparatus, i.e. feeding the non-plastified material into the injection unit
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/04—Oxygen-containing compounds
    • C08K5/05—Alcohols; Metal alcoholates
    • C08K5/053—Polyhydroxylic alcohols
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof

Definitions

  • the present invention relates to a composition for molding, a molded body, and a method for producing a molded body.
  • 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.
  • a molding method for obtaining a molded object having a complicated shape for example, a molding method such as injection molding is known.
  • a molding method such as injection molding is known.
  • fluidity is required for injection molding, it is difficult to apply the aggregate of animal fibers of Patent Document 1 to injection molding.
  • 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.
  • the polyhydric alcohol preferably contains at least one selected from the group consisting of lower alcohols, saccharides and sugar alcohols.
  • the structural protein preferably contains a fibroin-like protein.
  • 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.
  • the polyhydric alcohol preferably has a boiling point higher than the heating temperature in the heating step.
  • 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.
  • composition for molding 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.
  • 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
  • structural proteins include fibroin (for example, spider silk, silkworm silk and the like), keratin, collagen, elastin and resilin, and proteins derived therefrom.
  • fibroin-like proteins examples include, for example, proteins containing a domain sequence represented by the formula 1: [(A) n motif-REP1] m .
  • (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.
  • 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.
  • a fibroin-like protein for example, a protein comprising the amino acid sequence shown by SEQ ID NO: 1 can be mentioned.
  • 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.
  • a protein containing the amino acid sequence shown by SEQ ID NO: 2 can be mentioned.
  • 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.
  • 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.
  • 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.
  • elastin-like proteins examples include, for example, proteins having amino acid sequences such as NCBI Accession Nos. AAC98395 (human), I47076 (sheep) and NP786966 (bovine) from GenBank.
  • 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.
  • keratin-like protein examples include, for example, type I keratin of Capra hircus and the like.
  • 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.
  • fibroin-like protein is preferable, and spider silk fibroin-like protein (a spider silk fibroin or a protein derived therefrom) is more preferable.
  • spider silk fibroin-like protein a spider silk fibroin or a protein derived therefrom
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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. .
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.
  • prokaryote examples include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Microbacterium, Brevibacterium, Corynebacterium and Pseudomonas.
  • 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).
  • yeast the yeast which belongs to Saccharomyces genus, Pichia genus, Schizosaccharomyces genus etc. can be mentioned, for example.
  • filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.
  • examples of vectors into which a nucleic acid encoding a target protein is introduced include YEP13 (ATCC 37115), YEp24 (ATCC 37051), and the like.
  • any method of introducing DNA into the host cell can be used.
  • a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method competent method and the like.
  • 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.
  • the culture medium 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.
  • 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.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed.
  • an inducer may be added to the medium as needed.
  • 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.
  • anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -sepharose, DIAION HP
  • the host cell 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.
  • a purified preparation of protein can be obtained by the same isolation and purification method as described above.
  • the protein 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.
  • 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.
  • monosaccharide glucose, fructose etc. can be mentioned, for example.
  • disaccharides include sucrose and the like.
  • sugar alcohols examples include glycerin, erythritol, xylitol and the like.
  • the structural protein 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.
  • 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.
  • the structural protein is highly dispersed in the composition.
  • 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
  • pulverization mixing shows that a structural protein is grind
  • 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.
  • 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
  • the shaped body may be composed of a solid material containing a structural protein and polyhydric alcohols.
  • the structural protein preferably does not exist as a powder but is integrated.
  • the mechanical strength is poor and appearance problems such as white turbidity also occur.
  • 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.
  • the method of molding is not particularly limited, and may be, for example, the following method.
  • 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.
  • the flowable material is injected into the mold.
  • the mold be heated in order to prevent the fluid material from solidifying before the inside of the mold is sufficiently filled.
  • 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.
  • 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.
  • 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
  • amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 6 is added to the N-terminus.
  • 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.
  • 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.
  • 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- ⁇ -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.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • 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.
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl
  • 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.
  • ethylene glycol made by Tokyo Chemical Industry Co., Ltd., carbon atom number: 2
  • IFM-800DG made by Iwatani Sangyo Co., Ltd.
  • 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.
  • 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. .
  • A is obtained when a uniform solid material without white turbidity is obtained
  • B when white turbidity is observed in a part
  • white turbidity is observed in a state where the powder is pressed or solidified.
  • the formability was evaluated as C, in the case of
  • 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.
  • 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.
  • polyvinyl alcohol manufactured by Wako Pure Chemical Industries, polyvinyl alcohol (polymerization degree: about 2000), complete saponification
  • 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.
  • 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.
  • trimellitic acid ester ADKA, Adekaizer C-9N, carbon atom number: 36
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Manufacturing & Machinery (AREA)
  • Mechanical Engineering (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Injection Moulding Of Plastics Or The Like (AREA)

Abstract

L'invention concerne une composition de moulage contenant une protéine structurale et des polyols ayant deux groupes hydroxy ou plus et 12 atomes de carbone ou moins.
PCT/JP2018/034252 2017-09-15 2018-09-14 Composition de moulage, article moulé et procédé de production d'article moulé Ceased WO2019054503A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023513579A (ja) * 2020-02-12 2023-03-31 ボルト スレッズ インコーポレイテッド 組換えシルク固体及びフィルム
JP2025084724A (ja) * 2023-11-22 2025-06-03 キヤノンバージニア， インコーポレイテッド フィブロイン成形体の製造方法
JP2025084725A (ja) * 2023-11-22 2025-06-03 キヤノンバージニア， インコーポレイテッド 熱分解性排水物質及びフィブロインを含む成形用の組成物を用いた成形体の製造

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JPH0592057A (ja) * 1991-10-02 1993-04-16 U S:Kk ゴルフ用テイおよびその製造方法
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JP2002512929A (ja) * 1998-05-05 2002-05-08 ナチュラル ポリマー インターナショナル コーポレイション タンパク質およびデンプンを主成分とした生物分解性熱可塑性組成物
JP2003286407A (ja) * 2002-03-28 2003-10-10 Dainippon Ink & Chem Inc 大豆蛋白樹脂組成物及びそれを用いた大豆蛋白成形品
JP2004083734A (ja) * 2002-08-27 2004-03-18 National Food Research Institute 耐水性に優れた生分解性成形品とその製造方法
JP2009529592A (ja) * 2006-03-13 2009-08-20 ナトゥリン ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー 生分解性タンパク質系熱硬化性組成物、その製造方法および用途
WO2017047504A1 (fr) * 2015-09-18 2017-03-23 Ｓｐｉｂｅｒ株式会社 Article moulé et procédé de fabrication d'article moulé
WO2017047503A1 (fr) * 2015-09-18 2017-03-23 Ｓｐｉｂｅｒ株式会社 Article moulé et son procédé de fabrication

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS51121064A (en) * 1975-04-17 1976-10-22 Sumitomo Bakelite Co Ltd Protein and starch dual molding compositions
JPH0592057A (ja) * 1991-10-02 1993-04-16 U S:Kk ゴルフ用テイおよびその製造方法
JPH07188422A (ja) * 1993-12-27 1995-07-25 Shin Nippon Shokuhin Kk 蛋白質から得られる熱可塑性材料、その成形品、及びそれらの製造方法
JP2001517253A (ja) * 1995-11-29 2001-10-02 ミッドウエスト グレイン プロダクツ，アイエヌシー． 穀物タンパク質を主成分とする生分解性固形製品及びその成形方法
JP2002512929A (ja) * 1998-05-05 2002-05-08 ナチュラル ポリマー インターナショナル コーポレイション タンパク質およびデンプンを主成分とした生物分解性熱可塑性組成物
JP2003286407A (ja) * 2002-03-28 2003-10-10 Dainippon Ink & Chem Inc 大豆蛋白樹脂組成物及びそれを用いた大豆蛋白成形品
JP2004083734A (ja) * 2002-08-27 2004-03-18 National Food Research Institute 耐水性に優れた生分解性成形品とその製造方法
JP2009529592A (ja) * 2006-03-13 2009-08-20 ナトゥリン ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー 生分解性タンパク質系熱硬化性組成物、その製造方法および用途
WO2017047504A1 (fr) * 2015-09-18 2017-03-23 Ｓｐｉｂｅｒ株式会社 Article moulé et procédé de fabrication d'article moulé
WO2017047503A1 (fr) * 2015-09-18 2017-03-23 Ｓｐｉｂｅｒ株式会社 Article moulé et son procédé de fabrication

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023513579A (ja) * 2020-02-12 2023-03-31 ボルト スレッズ インコーポレイテッド 組換えシルク固体及びフィルム
JP2025084724A (ja) * 2023-11-22 2025-06-03 キヤノンバージニア， インコーポレイテッド フィブロイン成形体の製造方法
JP2025084725A (ja) * 2023-11-22 2025-06-03 キヤノンバージニア， インコーポレイテッド 熱分解性排水物質及びフィブロインを含む成形用の組成物を用いた成形体の製造

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