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WO2018221498A1 - Procédé de fabrication de fibre de protéine - Google Patents

Procédé de fabrication de fibre de protéine Download PDF

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Publication number
WO2018221498A1
WO2018221498A1 PCT/JP2018/020502 JP2018020502W WO2018221498A1 WO 2018221498 A1 WO2018221498 A1 WO 2018221498A1 JP 2018020502 W JP2018020502 W JP 2018020502W WO 2018221498 A1 WO2018221498 A1 WO 2018221498A1
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WIPO (PCT)
Prior art keywords
protein
coagulation bath
bath liquid
solution
protein fiber
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2018/020502
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English (en)
Japanese (ja)
Inventor
佑之介 安部
拓弥 齋藤
秀人 石井
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.)
Kojima Industries Corp
Spiber Inc
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Kojima Industries Corp
Spiber Inc
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Priority to JP2019521226A priority Critical patent/JP7281139B2/ja
Publication of WO2018221498A1 publication Critical patent/WO2018221498A1/fr
Anticipated expiration legal-status Critical
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    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • the present invention relates to a method for producing protein fibers.
  • a wet spinning method and a dry-wet spinning method in which a spinning solution discharged from a nozzle is coagulated in a coagulation bath solution to form fibers are known.
  • the wet spinning method and the dry and wet spinning method are also used when producing protein fibers containing protein as a main component (see, for example, Patent Document 1).
  • an object of the present invention is to provide a method for producing a protein fiber in which the generation of voids is sufficiently suppressed.
  • the present inventors examined the cause of voids in protein fibers. Then, it was presumed that the cause of generation of voids was that the dissolved solvent abruptly separated from the spinning dope when the spinning solution in which the protein was dissolved in the dissolving solvent was introduced into the coagulation bath solution. As a result of intensive studies based on such estimation, it is possible to remove the solvent solution of the spinning solution in the coagulation bath liquid slowly over time because the coagulation bath liquid contains the dissolving solvent. The present invention has been found, and the present invention has been completed based on such knowledge.
  • the present invention includes a step of introducing a spinning stock solution containing a protein and a first dissolution solvent into a coagulation bath solution to coagulate the protein, and the coagulation bath solution contains the second dissolution solvent.
  • a method for producing a protein fiber is provided.
  • the coagulation bath liquid contains the dissolving solvent (second dissolving solvent), it is possible to sufficiently suppress the occurrence of voids in the obtained protein fiber.
  • the first dissolution solvent is dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid, a solution in which a dissolution accelerator is added, and dissolution promotion in water.
  • the second dissolving solvent may be dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid, and these. It may be at least one selected from the group consisting of those obtained by adding a solubility promoter and those obtained by adding a solubility promoter to water.
  • the content of the second dissolving solvent in the coagulation bath liquid is preferably 10 to 60% by mass with respect to the total amount of the coagulation bath liquid. In this case, the effect of the present invention is more remarkably exhibited.
  • the temperature of the coagulation bath liquid may be 40 ° C. or lower, or 0 ° C. or higher.
  • the protein may be a structural protein.
  • the structural protein may be fibroin.
  • the fibroin may be spider silk fibroin.
  • the coagulation bath liquid contains methanol and a second dissolving solvent, and the second dissolving solvent is composed of dimethyl sulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution accelerator thereto. It may be at least one selected.
  • the coagulation bath liquid may contain an inorganic salt.
  • protein fibers in which the generation of voids is sufficiently suppressed can be more easily produced.
  • the content of the inorganic salt in the coagulation bath liquid may be more than 0% by mass and 30% by mass or less based on the total amount of the coagulation bath liquid.
  • the spinning dope may contain an inorganic salt, and the content of the inorganic salt in the coagulation bath solution may be lower than the content of the inorganic salt in the spinning dope.
  • the protein coagulates under milder conditions, thereby making it possible to more advantageously suppress the formation of protein fiber voids.
  • FIG. 6 is a view showing optical micrographs of spider silk fibroin fibers in Production Examples 1-1 to 1-5. It is a box-and-whisker diagram which shows the result of evaluation of the stress in an Example. It is a graph which shows the result of evaluation of the maximum draw ratio in an Example.
  • 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-1. It is an optical microscope photograph of the spider silk fibroin fiber in Production Example 2-2. It is an optical microscope photograph of the spider silk fibroin fiber in Production Example 2-3. It is an optical microscope photograph of the spider silk fibroin fiber in Production Example 2-4.
  • the protein fiber manufacturing method includes a step of introducing a spinning stock solution containing a protein and a first dissolving solvent (dope solvent) into a coagulation bath solution to coagulate the protein, and the coagulation bath solution Contains a second dissolving solvent.
  • a spinning stock solution containing a protein and a first dissolving solvent dope solvent
  • the coagulation bath solution Contains a second dissolving solvent.
  • the protein fiber manufactured according to the manufacturing method of this embodiment contains protein as a main component.
  • the protein is not particularly limited, and may be produced by a microorganism by genetic recombination technology, may be produced by synthesis, or may be a product obtained by purifying a naturally derived protein. There may be.
  • the protein may be, for example, a structural protein or an artificial structural protein derived from the structural protein.
  • the structural protein means a protein that forms or maintains a structure, a form, or the like in a living body. Examples of the structural protein include fibroin, keratin, collagen, elastin, and resilin.
  • the structural protein is preferably fibroin.
  • the fibroin may be one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin, for example.
  • the structural protein may be silk fibroin, spider silk fibroin or a combination thereof.
  • the ratio of silk fibroin may be, for example, 40 parts by mass or less, 30 parts by mass or less, or 10 parts by mass or less with respect to 100 parts by mass of spider silk fibroin.
  • the silk fibroin may be sericin-removed silk fibroin, sericin-unremoved silk fibroin, or a combination thereof.
  • Sericin-removed silk fibroin is purified by removing sericin covering silk fibroin and other fats.
  • the silk fibroin thus purified is preferably used as a lyophilized powder.
  • the sericin unremoved silk fibroin is an unpurified silk fibroin from which sericin and the like have not been removed.
  • the spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein (artificial spider silk protein).
  • spider silk proteins examples include large sphincter bookmark protein, weft protein, and small bottle-like gland protein. Since the large spout bookmarker has a repeating region composed of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and stretchability.
  • the weft of spider silk has a feature that it does not have a crystalline region but has a repeating region consisting of an amorphous region. The weft thread is less stressed than the large spout bookmarker thread, but has high stretchability.
  • the large sputum bookmark thread protein is produced in spider large bottle-like glands and has the characteristic of excellent toughness.
  • Examples of the large sphincter bookmark thread protein include large bottle-shaped gland spiders MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from two spider spiders (Araneus diadematus).
  • ADF3 is one of the two main dragline proteins of the elder spider.
  • Polypeptides derived from natural spider silk proteins may be polypeptides derived from these bookmark silk proteins.
  • a polypeptide derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and toughness.
  • weft protein is produced in the flagellate gland of spiders.
  • flagellum silk protein derived from the American spider (Nephila clavipes) can be mentioned.
  • the polypeptide derived from a natural spider silk protein may be a recombinant spider silk protein.
  • recombinant spider silk proteins include mutants, analogs or derivatives of natural spider silk proteins.
  • a suitable example of such a polypeptide is a recombinant spider silk protein (also referred to as “polypeptide derived from a large sputum bookmarker protein”).
  • Examples of the protein derived from the large splint bookmark thread and the silkworm silk, which are fibroin-like proteins, include, for example, Formula 1: [(A) n motif-REP] m , or Formula 2: [(A) n motif- REP] m-
  • (A) includes a protein containing a domain sequence represented by the n motif.
  • (A) n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27.
  • the number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 .
  • the ratio of the number of alanine residues to the total number of amino acid residues in the (A) n motif may be 40% or more, such as 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues).
  • a plurality of (A) n motifs present in the domain sequence may be composed of at least seven alanine residues alone.
  • REP indicates an amino acid sequence composed of 2 to 200 amino acid residues.
  • REP may be an amino acid sequence composed of 10 to 200 amino acid residues.
  • m represents an integer of 2 to 300, and may be an integer of 10 to 300.
  • a plurality of (A) n motifs may have the same amino acid sequence or different amino acid sequences.
  • Plural REPs may have the same amino acid sequence or different amino acid sequences.
  • Specific examples of proteins derived from the large sputum bookmarker thread include proteins comprising the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 8.
  • Examples of the protein derived from the weft protein include a protein containing a domain sequence represented by Formula 3: [REP2] o (where REP2 is composed of Gly-Pro-Gly-Gly-X in Formula 3) X represents an amino acid sequence, X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr), and valine (Val), and o represents an integer of 8 to 300. Can be mentioned. Specific examples include a protein comprising the amino acid sequence represented by SEQ ID NO: 2.
  • amino acid sequence shown in SEQ ID NO: 2 is from the N-terminal corresponding to the repeat part and the motif of the partial sequence (NCBI accession number: AAF36090, GI: 7106224) of a partial sequence of the American flagella silk protein obtained from the NCBI database.
  • PR1 sequence An amino acid sequence from the 1220th residue to the 1659th residue (referred to as PR1 sequence) and a partial sequence of American flagella silk protein obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649)
  • a C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminal is linked, and the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence) is added to the N-terminus of the combined sequence. is there.
  • a protein containing a domain sequence represented by Formula 4: [REP3] p (wherein, in Formula 4, p represents an integer of 5 to 300.
  • REP3 represents Gly-XY
  • X and Y represent any amino acid residue other than Gly, and a plurality of REP3 may be the same amino acid sequence or different amino acid sequences.
  • a protein containing the amino acid sequence represented by SEQ ID NO: 3 can be exemplified.
  • the amino acid sequence represented by SEQ ID NO: 3 corresponds to the repeat part and motif of the partial sequence of human collagen type 4 (NCBI GenBank accession number: CAA56335.1, GI: 3702452) obtained from the NCBI database.
  • An amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence from the 301st residue to the 540th residue.
  • a protein comprising a domain sequence represented by Formula 5: [REP4] q (wherein q represents an integer of 4 to 300.
  • REP4 represents Ser-JJ- An amino acid sequence composed of Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr. In particular, it is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.
  • Plural REP4s may have the same or different amino acid sequences. ). Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 4.
  • the amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of resilin (NCBI GenBank accession number NP 611157, Gl: 24654243), in which Thr at the 87th residue is replaced with Ser, and the Asn at the 95th residue.
  • the amino acid sequence represented by SEQ ID NO: 7 (tag sequence and hinge sequence) is added to the N-terminus of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced with Asp.
  • elastin-derived proteins include proteins having amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specific examples include a protein containing the amino acid sequence represented by SEQ ID NO: 5.
  • the amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence represented by SEQ ID NO: 7 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 a hinge arrangement).
  • keratin-derived proteins examples include Capra hircus type I keratin. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 6 (amino acid sequence of NCBI GenBank accession number ACY30466) can be mentioned.
  • structural proteins and proteins derived from the structural proteins can be used singly or in combination of two or more.
  • the protein contained as the main component in the protein fiber is, for example, by a host transformed with an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. Can be produced by expressing the nucleic acid.
  • the method for producing a nucleic acid encoding a protein contained as a main component in a protein fiber is not particularly limited.
  • the nucleic acid can be produced by a method of amplification and cloning by polymerase chain reaction (PCR) using a gene encoding a natural structural protein, or a method of chemical synthesis.
  • the method for chemically synthesizing nucleic acids is not particularly limited.
  • AKTA oligopilot plus 10/100 GE Healthcare Japan Co., Ltd.
  • a gene can be chemically synthesized by a method of linking oligonucleotides automatically synthesized in step 1 by PCR or the like.
  • nucleic acid encoding a protein consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N terminus of the above amino acid sequence is synthesized. Also good.
  • Regulatory sequences are sequences that control the expression of recombinant proteins in the host (for example, promoters, enhancers, ribosome binding sequences, transcription termination sequences, etc.), and can be appropriately selected depending on the type of host.
  • an inducible promoter that functions in a host cell and can induce expression of a target protein may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value.
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, virus vector, cosmid vector, fosmid vector, artificial chromosome vector, and the like.
  • An expression vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a target protein can be transcribed is preferably used. .
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • vectors for introducing a nucleic acid encoding a target protein include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescript II, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238696) and the like can be mentioned.
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of a vector into which a nucleic acid encoding a target protein is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051).
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [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, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd edition, etc. .
  • the protein can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium.
  • the method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the host, and can efficiently culture the host. If so, either a natural medium or a synthetic medium may be used.
  • Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism.
  • Examples thereof include glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolyzate, acetic acid and propionic acid, etc.
  • Organic acids and alcohols such as ethanol and propanol can be used.
  • the nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • Isolation and purification of the expressed protein can be performed by a commonly used method.
  • the host cell is recovered by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Manton Gaurin.
  • the host cells are disrupted with a homogenizer, dynomill, or the like to obtain a cell-free extract.
  • a method usually used for protein isolation and purification that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, an organic solvent, etc.
  • Precipitation method anion exchange chromatography method using resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using resin such as S-Sepharose FF (manufactured by Pharmacia)
  • Electrophoresis methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing Using methods such as these alone or in combination, purification It is possible to obtain the goods.
  • the host cell when the protein is expressed by forming an insoluble substance in the cell, the host cell is similarly collected and then crushed and centrifuged to collect the protein insoluble substance as a precipitate fraction.
  • the recovered protein insoluble matter can be solubilized with a protein denaturant.
  • a purified protein preparation 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 with a technique 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.
  • the protein fiber is obtained by spinning the above-described protein and contains the above-described protein as a main component.
  • the method for producing a protein fiber according to the present embodiment includes a step of introducing a spinning stock solution containing a protein and a first dissolving solvent into a coagulation bath solution to coagulate the protein. And a second dissolving solvent.
  • the method for producing a protein fiber according to the present embodiment can be carried out according to a known spinning method such as wet spinning or dry wet spinning.
  • the spinning dope contains the aforementioned protein and the first dissolving solvent.
  • the spinning dope may be obtained by dissolving the above-described protein in a dissolving solvent.
  • the dissolution solvent means a component (solvent) that dissolves the protein.
  • the dissolution solvent includes a component that dissolves the protein when used in combination with a dissolution accelerator described later.
  • the dissolution solvent may contain a dissolution accelerator.
  • the first dissolving solvent is a component that dissolves the protein in the spinning dope and is also referred to as a dope solvent.
  • the dissolving solvent is, for example, an organic solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), hexafluoroisopronol (HFIP), hexafluoroacetone (HFA), formic acid or the like. It may be.
  • the first dissolution solvent may be a solution obtained by adding a dissolution accelerator to the above organic solvent, or a solution obtained by adding a dissolution accelerator to water.
  • the first dissolving solvent may be used alone or in combination of two or more.
  • Dissolving solvent is dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and those added with a dissolution accelerator, and a dissolution accelerator is added to water And at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a solubility enhancer thereto. Good.
  • the first dissolution solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide, and those obtained by adding a dissolution accelerator thereto, these solvents have a high boiling point, Proteins can be dissolved under high temperature conditions.
  • the first dissolving solvent is at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a dissolution accelerator to these, the safety of the solvent is high. The production workability is improved and the cost of the solvent itself is low, so that the production cost of the protein fiber is reduced.
  • the first dissolution solvent may be one containing a dissolution accelerator (containing one). In this case, preparation of the spinning dope becomes easy.
  • the dissolution promoter can be appropriately selected according to the type of protein and dissolution solvent.
  • the dissolution accelerator may be, for example, an inorganic salt composed of the following Lewis acid and Lewis base.
  • the Lewis base include oxo acid ions (nitrate ions, perchlorate ions, etc.), metal oxo acid ions (permanganate ions, etc.), halide ions, thiocyanate ions, cyanate ions, and the like.
  • the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, and the like.
  • examples of inorganic salts include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, calcium bromide.
  • Calcium salts such as calcium iodide, calcium nitrate, calcium perchlorate and calcium thiocyanate
  • iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate and iron thiocyanate
  • aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate
  • Potassium salts such as potassium acid, sodium chloride, sodium bromide
  • Sodium salts such as zinc, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate
  • zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate and zinc thiocyanate
  • Magnesium salts such as
  • the inorganic salts are used as protein promoters for dissolution solvents.
  • the spinning dope contains a dissolution accelerator (the above inorganic salt)
  • the protein can be dissolved in the spinning dope at a high concentration. Thereby, the production efficiency of protein fibers is further improved, and the quality of protein fibers and the improvement of physical properties such as stress are expected.
  • the inorganic salt may be at least one selected from the group consisting of lithium chloride and calcium chloride.
  • the spinning dope may contain, for example, urea, guanidine, or sodium dodecyl sulfate (SDS) as a dissolution accelerator.
  • SDS sodium dodecyl sulfate
  • the above-mentioned dissolution promoter may be used alone or in combination of two or more.
  • the content of the dissolution accelerator is 0.1% by mass or more, 1% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more with respect to the total amount of the spinning dope. It may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.
  • the spinning dope (protein solution) may further contain various additives as necessary.
  • the additive include a plasticizer, a leveling agent, a crosslinking agent, a crystal nucleating agent, an antioxidant, an ultraviolet absorber, a colorant, a filler, and a synthetic resin.
  • the content of the additive may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of protein in the spinning dope.
  • FIG. 1 is an explanatory view schematically showing an example of a spinning device for producing protein fibers.
  • a spinning device 10 shown in FIG. 1 is an example of a spinning device for dry and wet spinning, and has an extrusion device 1, a coagulating bath 20, a washing bath (stretching bath) 21, and a drying device 4 in order from the upstream side. is doing.
  • the extrusion apparatus 1 has a storage tank 7 in which a spinning solution (dope solution) 6 is stored.
  • the spinning dope 6 is obtained by dissolving the above-described protein in a dissolving solvent (dope solvent).
  • the coagulation bath liquid 11 is stored in the coagulation bath 20.
  • the spinning solution 6 is pushed out by a gear pump 8 attached to the lower end of the storage tank 7 from a nozzle 9 provided with an air gap 19 between the coagulating bath solution 11.
  • the extruded spinning solution 6 is supplied (introduced) into the coagulation bath liquid 11 of the coagulation bath 20 through the air gap 19.
  • the solvent is removed from the spinning dope to coagulate the protein.
  • the coagulated protein is guided to the washing tub 21, washed with the washing liquid 12 in the washing tub 21, and then sent to the drying device 4 by the first nip roller 13 and the second nip roller 14 installed in the washing tub 21. It is done. At this time, for example, if the rotation speed of the second nip roller 14 is set to be higher than the rotation speed of the first nip roller 13, the protein fiber 36 drawn at a magnification corresponding to the rotation speed ratio is obtained.
  • the protein fibers drawn in the washing liquid 12 are dried when passing through the drying device 4 after leaving the washing bath 21 and then wound by a winder. In this way, protein fibers are obtained as a wound product 5 that is finally wound around a winder by the spinning device 10.
  • Reference numerals 18a to 18g are thread guides.
  • the coagulation bath liquid 11 contains a second dissolving solvent.
  • the second dissolving solvent is a dissolving solvent contained in the coagulation bath liquid 11, and can also be referred to as a solvent capable of dissolving the protein in the spinning dope 6.
  • the second dissolving solvent include the same ones as exemplified for the first dissolving solvent.
  • the second dissolving solvent may be the same type of solvent as the first dissolving solvent (dope solvent) or a different type of solvent.
  • the coagulation bath solution 11 is obtained by adding the second dissolving solvent before introducing the spinning dope 6 into the coagulation bath solution 11.
  • dissolution solvent is fully suppressed.
  • the second dissolving solvent is composed of dimethyl sulfoxide, N, N-dimethylformamide, hexafluoroisopronol, hexafluoroacetone, formic acid and those having a solubility accelerator added thereto, and those having a solubility accelerator added to water. It may be at least one selected from the group, and is preferably at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a solubility enhancer thereto.
  • the coagulation bath solution 11 only needs to contain a second dissolving solvent and can be desolvated, and includes, for example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, acetone and the like. It may be.
  • the coagulation bath liquid 11 may appropriately contain water.
  • the coagulation bath liquid 11 may contain methanol and a second dissolving solvent.
  • the second dissolution solvent is preferably at least one selected from the group consisting of dimethyl sulfoxide, N, N-dimethylformamide and those obtained by adding a dissolution accelerator to these.
  • the content of the second dissolving solvent in the coagulation bath liquid 11 may be 10 to 60% by mass with respect to the total amount of the coagulation bath liquid 11. Further, the content of the second dissolving solvent may be 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more with respect to the total amount of the coagulation bath liquid 11, and 50% by mass. Hereinafter, it may be 40% by mass or less, or 30% by mass or less. When the content of the second dissolving solvent is within the above range, the generation of voids in the protein fiber is further remarkably suppressed.
  • the stress in this specification means the value (unit: g / D) which remove
  • the coagulation bath liquid 11 may contain a dissolution accelerator.
  • the dissolution accelerator in the coagulation bath solution 11 for example, the above-described inorganic salt may be used.
  • the coagulation bath liquid 11 may contain a dissolution accelerator by using a second dissolution solvent containing a dissolution accelerator.
  • the coagulation bath liquid 11 may contain an inorganic salt, and may contain at least one selected from the group consisting of lithium chloride and calcium chloride.
  • the dissolution accelerator in the coagulation bath liquid 11 may be used alone or in combination of two or more.
  • the coagulation bath solution 11 may contain the same inorganic salt as that contained in the spinning stock solution, or may contain a different inorganic salt. Even when the spinning dope does not contain an inorganic salt as a dissolution accelerator, the coagulation bath solution 11 may contain an inorganic salt.
  • the coagulation bath liquid 11 contains an inorganic salt
  • protein coagulation in the coagulation bath liquid 11 can be performed under milder conditions. Thereby, generation
  • the content of the inorganic salt in the coagulation bath liquid 11 is not particularly limited, but exceeds 0% by mass and 30% by mass or less, and 5% by mass or more based on the total amount of the coagulation bath liquid 11. It may be 25% by mass or less, or 10% by mass or more and less than 20% by mass.
  • the content of the inorganic salt in the coagulation bath solution 11 is preferably lower than the content of the inorganic salt in the spinning dope 6. In this case, when the spinning solution 6 is introduced into the coagulation bath solution 11, protein coagulation can be performed under milder conditions without inhibiting protein coagulation in the coagulation bath solution 11. Thereby, generation
  • the temperature of the coagulation bath liquid 11 is not particularly limited, but may be 40 ° C. or lower, 30 ° C. or lower, 25 ° C. or lower, 20 ° C. or lower, 10 ° C. or lower, or 5 ° C. or lower.
  • the temperature of the coagulation bath liquid 11 is not particularly limited, but may be ⁇ 30 ° C. or higher, ⁇ 20 ° C. or higher, or ⁇ 10 ° C. or higher, and is 0 ° C. or higher from the viewpoint of workability, cooling cost, and the like. preferable.
  • the temperature of the coagulation bath liquid 11 is within the above range, it becomes even easier to produce protein fibers in which the generation of voids is sufficiently suppressed.
  • the temperature of coagulation bath liquid can be adjusted by using the spinning apparatus 10 which has the coagulation bath 20 which equips an inside with a heat exchanger, and a cooling circulation apparatus, for example.
  • the temperature is adjusted to the above range by heat exchange between the coagulation bath liquid and the heat exchanger by flowing a medium cooled to a predetermined temperature by a cooling circulation device through a heat exchanger installed in the coagulation bath. Can do. In this case, more efficient cooling is possible by circulating a solvent (for example, methanol) used for the coagulation bath liquid 11 as a medium.
  • a solvent for example, methanol
  • a plurality of coagulation baths 20 in which the coagulation bath liquid 11 is stored may be provided.
  • the coagulation bath liquid (first coagulation bath liquid) in the coagulation bath to which the spinning stock solution 6 extruded from the nozzle 9 is directly supplied (introduced) may contain the second dissolving solvent. That is, when there are a plurality of coagulation baths 20 in which the coagulation bath liquid 11 is stored, coagulation bath liquids (other coagulation bath liquids) other than the first coagulation bath liquid do not contain the second dissolving solvent. May be.
  • the temperature of the other coagulation bath liquid may be 40 ° C. or lower, 20 ° C. or lower, 10 ° C. or lower, or 5 ° C. or lower, or 0 ° C. or higher, or higher than 40 ° C.
  • the distance through which the coagulated protein passes through the coagulation bath solution 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) can be efficiently removed from the solvent, and the extrusion speed of the spinning dope from the nozzle 9 is good. It may be determined according to (discharge speed) or the like.
  • the residence time of the coagulated protein (or spinning solution) in the coagulation bath solution 11 is determined according to the distance that the coagulated protein passes through the coagulation bath solution 11, the extrusion speed of the spinning solution 6 from the nozzle 9, and the like. For example, it may be 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation bath solution 11.
  • stretching performed in the solution which added the organic solvent etc. to warm water may be sufficient as extending
  • the wet heat stretching temperature may be, for example, 50 to 90 ° C., and preferably 75 to 85 ° C.
  • an undrawn yarn (or predrawn yarn) can be drawn, for example, 1 to 10 times, and preferably 2 to 8 times.
  • the final draw ratio of the lower limit of the undrawn yarn (or predrawn yarn) is preferably more than 1 time, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times. Above, 7 times or more, 8 times or more, 9 times or more, and upper limit is preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, 13 times or less 12 times or less, 11 times or less, and 10 times or less.
  • spider silk protein spike silk fibroin: PRT410
  • PRT410 spider silk protein-encoding gene and construction of expression vector
  • a modified fibroin having the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as “PRT410”) based on the base sequence and amino acid sequence of fibroin (GenBank accession numbers: P46804.1, GI: 1174415) derived from Nephila clavipes. Was also designed.)
  • the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence obtained by performing substitution, insertion and deletion of amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, and its N-terminal. And the amino acid sequence shown in SEQ ID NO: 7 (tag sequence and hinge sequence).
  • a nucleic acid encoding the designed PRT410 was synthesized.
  • the nucleic acid was added with an NdeI site at the 5 'end and an EcoRI site downstream of the stop codon.
  • the nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the nucleic acid was cleaved by restriction enzyme treatment with NdeI and EcoRI, and then recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • Escherichia coli BLR (DE3) was transformed with the obtained pET22b (+) expression vector.
  • the transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours.
  • the culture solution was added to 100 mL of a seed culture medium (Table 1) containing ampicillin so that the OD 600 was 0.005.
  • the culture temperature was kept at 30 ° C., and the flask culture was performed for about 15 hours until the OD 600 reached 5 to obtain a seed culture.
  • the seed culture 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 culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min.
  • the culture solution temperature was maintained at 37 ° C., and the culture was performed at a constant pH of 6.9.
  • the culture was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration.
  • 1M isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of PRT410.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the washed precipitate was suspended in 8M guanidine buffer (8M 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 60 ° C. And stirred for 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). White aggregated protein (PRT410) obtained after dialysis was recovered by centrifugation. Water was removed from the recovered aggregated protein with a freeze dryer to obtain a freeze-dried powder of PRT410.
  • 8M guanidine buffer 8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0
  • spun and stretched protein fibers were produced by dry and wet spinning using a spinning device similar to the spinning device 10 shown in FIG.
  • the spinning device used is a second undrawn yarn production device (first unwinding device) between the undrawn yarn production device 2 (first coagulation bath) and the wet heat drawing device 3 (washing bath) in the spinning device 10 shown in FIG. 2 coagulation bath) and a third undrawn yarn production apparatus (third coagulation bath).
  • first coagulation bath liquid a solution containing methanol (MeOH) and DMSO (second dissolving solvent) in the composition (mass ratio) shown in Table 3 was used.
  • Table 3 shows the content of the second dissolving solvent with respect to the total amount of the coagulation bath liquid.
  • MeOH was used as the coagulation bath liquid for the second coagulation bath and the third coagulation bath.
  • water was used as the cleaning liquid for the cleaning bath.
  • Other dry and wet spinning conditions are as follows. Extrusion nozzle diameter: 0.2 mm Residence time of first coagulation bath liquid, temperature and draw ratio: see Table 3 draw ratio: see Table 3 Drying temperature: 95 ° C.
  • the coagulation bath liquid contains DMSO as the dissolving solvent (second dissolving solvent) (Production Examples 1-2 to 1-5)
  • the coagulation bath liquid does not contain DMSO (Production Example).
  • the coagulation bath solution contained 10% by mass or more of DMSO (Production Examples 1-4 to 1-5), the generation of voids in the spider silk fibroin fiber was more significantly suppressed.
  • FIG. 3 is a box-and-whisker diagram showing the evaluation results of stress.
  • the stress in this specification means the value (unit: g / D) which remove
  • * indicates an outlier.
  • the spider silk fibroin fibers (Production Examples 1-4 to 1-8) obtained using the first coagulation bath liquid having a DMSO content of 20% by mass or more have a DMSO content of less than 20% by mass.
  • the stress was higher than that of spider silk fibroin fibers (Production Examples 1-1 to 1-3) obtained using one coagulation bath solution. Further, it was shown that when the coagulation bath liquid having a DMSO content of 30% by mass or more in the first coagulation bath liquid was used, the stress was further increased.
  • the maximum draw ratio was evaluated at two locations of the spinning device 10 shown in FIG.
  • the evaluated locations are stretching (solidification bath stretching) between 18b and 18c at the first location, and stretching (cleaning bath stretching) between 14 and 18e at the second location.
  • the evaluation was not performed at two locations simultaneously, and when one was evaluated, the other was fixed at a draw ratio close to the same magnification.
  • the draw ratio was increased by 1 time, and the draw ratio when the yarn was cut was defined as the maximum draw ratio. The results are shown in FIG.
  • the maximum draw ratio was higher than when DMSO was contained in a proportion of less than 15% by mass.
  • first coagulation bath liquid contains DMSO as a dissolving solvent (second dissolving solvent) and contains an inorganic salt
  • second dissolving solvent a dissolving solvent
  • voids are sufficiently generated. It was shown to be suppressed.
  • the composition MeOH / DMSO (mass ratio) of the first coagulation bath liquid is 90/10
  • the content of LiCl in the coagulation bath liquid is 10% by mass or more
  • the content of calcium chloride (CaCl 2 ) is 10

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Textile Engineering (AREA)
  • Artificial Filaments (AREA)
  • Peptides Or Proteins (AREA)
  • Spinning Methods And Devices For Manufacturing Artificial Fibers (AREA)

Abstract

La présente invention concerne un procédé de fabrication d'une fibre de protéine qui comprend une étape consistant à introduire une solution de filage qui contient une protéine et un premier solvant de dissolution dans un liquide de bain de solidification et à solidifier la protéine, le liquide de bain de solidification contenant un second solvant de dissolution.
PCT/JP2018/020502 2017-05-30 2018-05-29 Procédé de fabrication de fibre de protéine Ceased WO2018221498A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2019151440A1 (fr) * 2018-01-31 2019-08-08 Ｓｐｉｂｅｒ株式会社 Méthode de production d'un article moulé de protéine, méthode de production de solution de protéine et méthode de production de protéine
CN113260745A (zh) * 2019-01-09 2021-08-13 丝芭博株式会社 改造丝心蛋白
CN114341412A (zh) * 2019-06-28 2022-04-12 丝芭博株式会社 布料、立体造型布料及其制造方法

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JPH09268426A (ja) * 1996-03-28 1997-10-14 Kuraray Co Ltd 繊維の製造方法
WO2003060099A2 (fr) * 2002-01-11 2003-07-24 Nexia Biotechnologies, Inc. Procedes et appareils de filage d'une proteine de soie d'araignee
WO2013065650A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution polypeptidique, procédé de production d'une fibre polypeptidique synthétique, et procédé d'affinage de polypeptide
JP2016531845A (ja) * 2013-09-17 2016-10-13 ボルト スレッズ インコーポレイテッド 改良シルク繊維を合成するための方法および組成物
WO2017030197A1 (fr) * 2015-08-20 2017-02-23 国立研究開発法人理化学研究所 Procédé de fabrication de composition polypeptidique présentant une structure de type fibroïne

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JPH09268426A (ja) * 1996-03-28 1997-10-14 Kuraray Co Ltd 繊維の製造方法
WO2003060099A2 (fr) * 2002-01-11 2003-07-24 Nexia Biotechnologies, Inc. Procedes et appareils de filage d'une proteine de soie d'araignee
WO2013065650A1 (fr) * 2011-11-02 2013-05-10 スパイバー株式会社 Solution polypeptidique, procédé de production d'une fibre polypeptidique synthétique, et procédé d'affinage de polypeptide
JP2016531845A (ja) * 2013-09-17 2016-10-13 ボルト スレッズ インコーポレイテッド 改良シルク繊維を合成するための方法および組成物
WO2017030197A1 (fr) * 2015-08-20 2017-02-23 国立研究開発法人理化学研究所 Procédé de fabrication de composition polypeptidique présentant une structure de type fibroïne

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Publication number Priority date Publication date Assignee Title
WO2019151440A1 (fr) * 2018-01-31 2019-08-08 Ｓｐｉｂｅｒ株式会社 Méthode de production d'un article moulé de protéine, méthode de production de solution de protéine et méthode de production de protéine
CN113260745A (zh) * 2019-01-09 2021-08-13 丝芭博株式会社 改造丝心蛋白
US12319718B2 (en) 2019-01-09 2025-06-03 Spiber Inc. Modified fibroin
CN114341412A (zh) * 2019-06-28 2022-04-12 丝芭博株式会社 布料、立体造型布料及其制造方法

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