JP7281139B2 - Method for producing protein fiber - Google Patents

Description

The present invention relates to a method for producing protein fibers.

BACKGROUND ART Conventionally, wet spinning and dry-wet spinning are known as methods for producing artificial fibers, in which a spinning dope discharged from a nozzle is coagulated in a coagulating bath to form fibers. The wet spinning method and dry-wet spinning method are also used when producing protein fibers containing protein as a main component (see, for example, Patent Document 1).

By the way, it is also known that when artificial fibers are obtained by such wet spinning method, dry-wet spinning method, etc., voids are formed on the surface of the fiber when the fiber is coagulated in the coagulation bath (for example, , see Patent Document 2).

For this reason, for example, when obtaining chemical fibers such as acrylic fibers and acrylonitrile fibers by wet spinning, in order to suppress the generation of voids on the fiber surface, etc., the water content in the spinning dope is reduced (for example, patent Reference 3), and measures have been taken to control the concentration of the solvent in the coagulation bath, which is an aqueous solution of the solvent (see, for example, Patent Reference 4).

Japanese Patent No. 5540154 JP 2016-44382 A JP 2015-132040 A JP 2015-101803 A

However, at present, when protein fibers are obtained by wet spinning, dry-wet spinning, or the like, no measures have been taken to suppress the generation of voids in protein fibers.

Accordingly, an object of the present invention is to provide a method for producing protein fibers in which the generation of voids is sufficiently suppressed.

In order to solve the above problems, the present inventors investigated the cause of void generation in protein fibers. Then, it was presumed that the cause of the generation of voids is that the dissolved solvent is rapidly separated from the spinning dope when the spinning dope in which the protein is dissolved in the dissolving solvent is introduced into the coagulation bath. As a result of intensive studies based on such presumptions, it was found that by containing a dissolving solvent in the coagulation bath, it was possible to slowly remove the solvent from the spinning dope in the coagulation bath over time. The present inventors have found that there is a certain thing, and have completed the present invention based on such findings.

That is, the present invention includes a step of introducing a spinning stock solution containing a protein and a first dissolution solvent into a coagulation bath to coagulate the protein, and the coagulation bath contains the second dissolution solvent. Provided is a method for producing a protein fiber containing.

According to the production method of the present invention, since the coagulation bath contains the dissolving solvent (second dissolving solvent), it is possible to sufficiently suppress the generation of voids in the obtained protein fibers.

In the production method of the present invention, the first dissolution solvent is dimethyl sulfoxide, N,N-dimethylformamide, hexafluoroisopropanol , hexafluoroacetone, formic acid, and these with a dissolution accelerator added, and dissolved in water It may be at least one selected from the group consisting of those with added accelerators, and the second dissolution solvent is dimethylsulfoxide, N,N- dimethylformamide , hexafluoroisopropanol, hexafluoroacetone, formic acid and At least one selected from the group consisting of these with a dissolution enhancer added and water with a dissolution enhancer added may be used.

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 will be exhibited more remarkably.

The temperature of the coagulation bath liquid may be 40° C. or lower, or 0° C. or higher.

A 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 dissolution solvent, and the second dissolution solvent is selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, and these with a dissolution accelerator added. It may be at least one selected.

The coagulation bath liquid may contain an inorganic salt. In this case, it is possible to more easily produce protein fibers in which the generation of voids is sufficiently suppressed.

When the coagulation bath liquid contains an inorganic salt, 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 inorganic salt content in the coagulation bath may be lower than the inorganic salt content in the spinning dope. In this case, the protein is coagulated under milder conditions, which makes it possible to more effectively suppress the generation of voids in protein fibers.

ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the protein fiber which the generation|occurrence|production of a void was fully suppressed can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS It is explanatory drawing which shows roughly an example of the spinning apparatus for manufacturing a protein fiber. FIG. 2 is a diagram showing optical micrographs of spider silk fibroin fibers in Production Examples 1-1 to 1-5. 4 is a box plot showing the results of stress evaluation in Examples. FIG. 4 is a graph showing the evaluation results of the maximum draw ratio in Examples. 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-1. 2 is an optical microscope photograph of spider silk fibroin fibers in Production Example 2-2. 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-3. 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-4. 2 is an optical microscope photograph of spider silk fibroin fibers in Production Example 2-5. 2 is an optical microscope photograph of spider silk fibroin fibers in Production Example 2-6. 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-7. 2 is an optical micrograph of spider silk fibroin fibers in Production Example 2-8.

DETAILED DESCRIPTION OF THE INVENTION Embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

A 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 (dope solvent) into a coagulation bath liquid to coagulate the protein. contains the second dissolution solvent. By containing the dissolving solvent in the coagulation bath, the generation of voids in the obtained protein fibers can be sufficiently suppressed.

(protein)
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 microorganisms or the like by genetic recombination technology, may be produced synthetically, or may be a purified protein derived from nature. There may be.

Said protein may be, for example, a structural protein or an artificial structural protein derived from said structural protein. A structural protein means a protein that forms or retains a structure, morphology, etc. in vivo. Examples of structural proteins include fibroin, keratin, collagen, elastin, resilin, and the like. The structural protein is preferably fibroin.

The fibroin may be, for example, one or more selected from the group consisting of silk fibroin, spider silk fibroin, and hornet silk fibroin. In particular, the structural protein may be silk fibroin, spider silk fibroin, or a combination thereof. When silk fibroin and spider silk fibroin are used in combination, the proportion 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. The sericin-removed silk fibroin is purified by removing sericin covering the silk fibroin and other fats. The silk fibroin thus purified is preferably used as a freeze-dried powder. Sericin-unremoved silk fibroin is unpurified silk fibroin from which sericin and the like have not been removed.

Spider silk fibroin may contain a spider silk polypeptide selected from the group consisting of natural spider silk proteins and polypeptides derived from natural spider silk proteins (artificial spider silk proteins).

Natural spider silk proteins include, for example, major duct dragline proteins, weft proteins, and pitular gland proteins. Since the large-discharging guideline yarn has repeated regions composed of crystalline regions and amorphous regions (also referred to as amorphous regions), it has both high stress and elasticity. Spider silk wefts are characterized by having repeating regions consisting of amorphous regions without crystalline regions. The weft thread has a lower stress than the large drawstring guideline thread, but has a high stretchability.

The dragline silk protein is produced in the major pituitary gland of spiders and is characterized by excellent toughness. Major duct dragline proteins include, for example, the major pituitary gland spidroins MaSp1 and MaSp2 from Nephila clavipes, and ADF3 and ADF4 from Araneus diadematus. ADF3 is one of the two major dragline silk proteins of the Arbani spider. Polypeptides derived from naturally occurring spider silk proteins may be polypeptides derived from these dragline silk proteins. Polypeptides derived from ADF3 are relatively easy to synthesize, and have excellent properties in terms of strength and toughness.

Weft proteins are produced in the flagelliform gland of spiders. Weft proteins include, for example, flagelliform silk proteins derived from Nephila clavipes.

A polypeptide derived from a naturally occurring spider silk protein may be a recombinant spider silk protein. Recombinant spider silk proteins include mutants, analogues or derivatives of natural spider silk proteins. A preferred example of such a polypeptide is a recombinant spider silk protein of the major cocoon dragline protein (also referred to as "a polypeptide derived from the cocoon dragline protein").

As fibroin-like proteins, proteins derived from large discharge tube dragline thread and proteins derived from silkworm silk include, for example, formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif- REP] _m -(A) _n motif includes proteins containing a domain sequence. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. (A) The number of amino acid residues in _{the n} motif may be an integer of 2-20, 4-27, 4-20, 8-20, 10-20, 4-16, 8-16, or 10-16 . In addition, (A) the ratio of the number of alanine residues to the total number of amino acid residues in the _n motif may be 40% or more, 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). At least seven of the (A) _n motifs present in the domain sequence may be composed only of alanine residues. REP indicates an amino acid sequence composed of 2-200 amino acid residues. REP may be an amino acid sequence composed of 10-200 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. A plurality of REPs may have the same amino acid sequence or different amino acid sequences. Specific examples of the protein derived from the dragline thread of the large spool tube include proteins containing the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 8.

Examples of proteins derived from weft proteins include proteins containing a domain sequence represented by Formula 3: [REP2] _o (where REP2 is composed of Gly-Pro-Gly-Gly-X in Formula 3). shows an amino acid sequence, X represents one amino acid selected from the group consisting of alanine (Ala), serine (Ser), tyrosine (Tyr) and valine (Val), o represents an integer of 8 to 300). can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:2 can be mentioned. The amino acid sequence shown by SEQ ID NO: 2 is obtained from the N terminal corresponding to the repeat part and the motif of the partial sequence of the flagellated silk protein of Nephila nephila (NCBI accession number: AAF36090, GI: 7106224) obtained from the NCBI database. The amino acid sequence from the 1220th residue to the 1659th residue (referred to as the PR1 sequence), and the partial sequence of the flagellated silk protein of Nephila japonicum obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649). The C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus is bound, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 is added to the N-terminal of the bound sequence. be.

As a collagen-derived protein, for example, a protein comprising a domain sequence represented by Formula 4: [REP3] _p (here, in Formula 4, p represents an integer of 5 to 300. REP3 is Gly-XY X and Y represent any amino acid residue other than Gly.A plurality of REP3s may have the same amino acid sequence or different amino acid sequences.) . Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:3 can be mentioned. The amino acid sequence shown in SEQ ID NO: 3 corresponds to the repeat portion and motif of the partial sequence of human collagen type 4 obtained from the NCBI database (NCBI GenBank accession number: CAA56335.1, GI: 3702452). The amino acid sequence (tag sequence and hinge sequence) of SEQ ID NO: 7 was added to the N-terminus of the amino acid sequence from the 301st residue to the 540th residue.

As a resilin-derived protein, for example, a protein comprising a domain sequence represented by Formula 5: [REP4] _q (here, q is an integer of 4 to 300 in Formula 5. REP4 is Ser-JJ- An amino acid sequence composed of Tyr-Gly-U-Pro, J represents an arbitrary amino acid residue, preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr, U is an arbitrary is preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser.A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. ) can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 4 can be mentioned. The amino acid sequence represented by SEQ ID NO: 4 is obtained by replacing Thr at the 87th residue with Ser and replacing Asn at the 95th residue in the amino acid sequence of resilin (NCBI GenBank accession number NP 611157, Gl: 24654243). The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 is added to the N-terminus of the amino acid sequence from the 19th residue to the 321st residue of the sequence obtained by substituting Asp for Asp.

Examples of elastin-derived proteins include proteins having amino acid sequences of NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine), and the like. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO:5 can be mentioned. The amino acid sequence shown by SEQ ID NO: 5 is the amino acid sequence shown by SEQ ID NO: 7 (tag sequence and hinge sequence) are added.

Examples of keratin-derived proteins include type I keratin of Capra hircus. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 6 (amino acid sequence of accession number ACY30466 in GenBank of NCBI) can be mentioned.

The above structural proteins and proteins derived from the structural proteins may be used singly or in combination of two or more.

A protein that is included as a major component in a protein fiber can be produced, 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.

A method for producing a nucleic acid encoding a protein contained as a main component in the protein fiber is not particularly limited. For example, the nucleic acid can be produced by using a gene encoding a naturally occurring structural protein, amplifying it by polymerase chain reaction (PCR) and cloning, or chemically synthesizing it. The method for chemically synthesizing the nucleic acid is not particularly limited, and for example, AKTA oligopilot plus 10/100 (GE Healthcare Japan Co., Ltd.) based on the amino acid sequence information of the structural protein obtained from the NCBI web database. A gene can be chemically synthesized by a method of linking oligonucleotides automatically synthesized by PCR or the like. At this time, in order to facilitate purification and/or confirmation of the protein, a nucleic acid encoding a protein consisting of an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon and a His10 tag to the N-terminus of the above amino acid sequence is synthesized. good too.

A regulatory sequence is a sequence that controls expression of a recombinant protein in a host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected according to the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the target protein may be used. An inducible promoter is a promoter whose transcription can be controlled by physical factors such as the presence of an inducer (expression inducer), the absence of a repressor molecule, or an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected from plasmid vectors, virus vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc., depending on the type of host. As the expression vector, those capable of autonomous replication in host cells or capable of integration into the chromosome of the host and containing a promoter at a position at which a nucleic acid encoding the desired protein can be transcribed are preferably used. .

As hosts, both prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be suitably used.

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammonium philum. Microorganisms belonging to the genus Brevibacterium include, for example, Brevibacterium divaricatum. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.

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

Eukaryotic hosts include, for example, yeast and filamentous fungi (such as molds). Examples of yeast include yeast belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of filamentous fungi include filamentous fungi belonging to the genus Aspergillus, Penicillium, Trichoderma, and the like.

When a eukaryote is used as a host, examples of vectors for introducing a nucleic acid encoding a target protein include YEP13 (ATCC37115) and YEp24 (ATCC37051). Any method for introducing DNA into the host cell can be used as the method for introducing the expression vector into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation method, spheroplast method, protoplast method, lithium acetate method, competent method and the like.

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

A 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 of culturing the host in the culture medium can be carried out according to a method commonly used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the host so that the host can be efficiently cultured. Either a natural medium or a synthetic medium may be used as long as the medium is used.

Any carbon source may be used as long as it can be assimilated by the above-mentioned transformed microorganism. Examples include carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch and starch hydrolysates, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used. Nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells and their digests can be used. Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

Prokaryotes such as E. coli or eukaryotes such as yeast can be cultured under aerobic conditions such as shaking culture or deep aeration stir culture. The culture temperature is, for example, 15-40°C. Culture time is usually 16 hours to 7 days. The pH of the culture medium during cultivation is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.

In addition, antibiotics such as ampicillin and tetracycline may be added to the culture medium during the culture, if necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using a lac promoter, and indole acrylic when culturing a microorganism transformed with an expression vector using a trp promoter. Acids and the like may be added to the medium.

Isolation and purification of the expressed protein can be performed by commonly used methods. For example, when the protein is expressed in a dissolved state in cells, the host cells are recovered by centrifugation after the completion of culture, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, or a mantongaurin. The host cells are disrupted with a homogenizer, a dyno mill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method commonly used for protein isolation and purification, that is, a solvent extraction method, a salting-out method with ammonium sulfate, a desalting method, an organic solvent Precipitation method, diethylaminoethyl (DEAE)-Sepharose, anion exchange chromatography method using resins such as DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing A purified sample can be obtained by using the methods such as the above alone or in combination.

In addition, when the protein forms an insoluble form in the cells and is expressed, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble form of the protein as a precipitate fraction. The collected insoluble protein can be solubilized with a protein denaturant. After this operation, a purified sample of protein can be obtained by the same isolation and purification method as described above. When the protein is extracellularly secreted, the protein can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

(Method for producing protein fiber)
A 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 to coagulate the protein. , is characterized by containing a second dissolving solvent. The method for producing the protein fiber according to the present embodiment can be carried out according to known spinning methods such as wet spinning and dry-wet spinning.

The spinning dope contains the protein described above and the first dissolution solvent. The spinning stock solution may be a solution obtained by dissolving the above protein in a dissolving solvent. Here, the dissolution solvent means a component (solvent) that dissolves the protein. The dissolution solvent also contains a component that dissolves the protein when used in combination with the dissolution promoter described below. The dissolution solvent may contain a dissolution enhancer. The first dissolving solvent is a component that dissolves the protein in the spinning dope, and is also called a dope solvent.

Dissolving solvent (first dissolving solvent), for example, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), organic solvents such as formic acid can be The first dissolution solvent may be the above organic solvent with a dissolution accelerator added, or water with a dissolution accelerator added. The first dissolution solvent may be used singly or in combination of two or more.

The dissolution solvent (first dissolution solvent) includes dimethyl sulfoxide, N,N-dimethylformamide, hexafluoroisopropanol , hexafluoroacetone, formic acid and these with a dissolution accelerator added, and water with a dissolution accelerator. It may be at least one selected from the group consisting of those added, and at least one selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, and those to which a dissolution accelerator is added. you can When the first dissolution solvent is at least one selected from the group consisting of dimethylsulfoxide, N,N-dimethylformamide, and these with a dissolution accelerator added thereto, since these solvents have a high boiling point, Dissolution of proteins under high temperature conditions becomes possible. As a result, the preparation work for dissolving the protein in the first dissolution solvent to obtain the dope for spinning becomes more efficient. Furthermore, when the first dissolution solvent is at least one selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, and those to which a dissolution accelerator is added, 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 added (containing) a dissolution enhancer. In this case, preparation of the spinning stock solution is facilitated. The dissolution enhancer 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 a Lewis acid and a Lewis base shown below. Lewis bases include, for example, oxoacid ions (nitrate ion, perchlorate ion, etc.), metal oxoacid ions (permanganate ion, etc.), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of Lewis acids include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. When the dissolving solvent is an organic solvent, examples of inorganic salts include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate, calcium chloride, and 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; and aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate, potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and thiocyanate Potassium salts such as potassium acid, sodium salts such as sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate and sodium thiocyanate, zinc chloride, zinc bromide, zinc iodide, zinc nitrate, peroxide Zinc salts such as zinc chlorate and zinc thiocyanate, magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate and magnesium thiocyanate, barium chloride, barium bromide, iodide barium salts such as barium, barium nitrate, barium perchlorate and barium thiocyanate; strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate and strontium thiocyanate; . These inorganic salts are used as dissolution promoters for proteins in dissolution solvents. When the spinning dope contains the dissolution accelerator (the above inorganic salt), the protein can be dissolved in the spinning dope at a high concentration. As a result, it is expected that the production efficiency of protein fibers will be further improved, and that the quality of protein fibers will be improved and physical properties such as stress will be improved. The inorganic salt may be at least one selected from the group consisting of lithium chloride and calcium chloride.

When the dissolution solvent is (or contains) water, the spinning dope may contain, for example, urea, guanidine, or sodium dodecyl sulfate (SDS) as a dissolution enhancer.

The above dissolution accelerators may be used singly 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 stock solution. Well, 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 stock solution (protein solution) may further contain various additives as necessary. Examples of additives include plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, coloring agents, fillers, and synthetic resins. 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 diagram schematically showing an example of a spinning apparatus for producing protein fibers. The spinning device 10 shown in FIG. 1 is an example of a spinning device for dry-wet spinning, and includes an extrusion device 1, a coagulation bath 20, a washing bath (stretching bath) 21, and a drying device 4 in this order from the upstream side. are doing.

The extrusion device 1 has a storage tank 7 in which a spinning stock solution (dope solution) 6 is stored. The spinning dope 6 is obtained by dissolving the above protein in a dissolving solvent (dope solvent). A coagulation bath liquid 11 is stored in the coagulation bath 20 . The spinning stock solution 6 is pushed out from a nozzle 9 provided with an air gap 19 between it and the coagulation bath solution 11 by a gear pump 8 attached to the lower end of the storage tank 7 . The extruded spinning dope 6 is supplied (introduced) into the coagulation bath liquid 11 of the coagulation bath 20 through the air gap 19 . Solvent is removed from the spinning dope in the coagulation bath 11 to coagulate the protein. The coagulated protein is guided to the washing bath 21, washed with the washing liquid 12 in the washing bath 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 bath 21. be done. At this time, for example, if the rotation speed of the second nip roller 14 is set faster than the rotation speed of the first nip roller 13, protein fibers 36 drawn at a ratio corresponding to the rotation speed ratio can be obtained. The protein fibers stretched in the washing liquid 12 are removed from the washing bath 21, dried when passing through the drying device 4, and then wound up by a winder. In this way protein fibers are obtained by the spinning device 10 as a winding 5 which is finally wound on a winder. Note that 18a to 18g are thread guides.

The coagulation bath liquid 11 contains a second dissolution solvent. Here, the second dissolution solvent is a dissolution solvent contained in the coagulation bath liquid 11 and can also be said to be a solvent capable of dissolving the protein in the spinning dope 6 . As the second dissolution solvent, the same solvents as those exemplified for the first dissolution solvent can be exemplified. The second dissolving solvent may be the same solvent as the first dissolving solvent (dope solvent), or may be a different solvent. When the dope solution 6 is introduced into the coagulation bath solution 11, the first dissolving solvent in the dope solution 6 is introduced into the coagulation bath solution 11 together with the dope solution 6. Before the stock solution 6 is introduced, it contains a second dissolution solvent in advance. That is, in the present embodiment, the coagulation bath liquid 11 is added with the second dissolving solvent before the spinning stock solution 6 is introduced into the coagulation bath liquid 11 . By containing the second dissolving solvent in the coagulation bath liquid 11, the generation of voids in the obtained protein fibers is sufficiently suppressed.

The second dissolution solvent is dimethyl sulfoxide, N,N-dimethylformamide, hexafluoroisopropanol , hexafluoroacetone, formic acid and these with a dissolution enhancer added, and water with a dissolution enhancer added. It may be at least one selected from the group consisting of dimethylsulfoxide, N,N-dimethylformamide and at least one selected from the group consisting of these with a dissolution accelerator added.

The coagulation bath liquid 11 may be any solution as long as it contains the second dissolving solvent and the solvent can be removed. It's okay. The coagulation bath liquid 11 may contain water as appropriate. The coagulation bath liquid 11 may contain methanol and a second dissolution solvent. In this case, the second dissolution solvent is preferably at least one selected from the group consisting of dimethylsulfoxide, N,N-dimethylformamide, and these with a dissolution accelerator added thereto.

The content of the second dissolving solvent in the coagulation bath liquid 11 may be 10 to 60 mass % with respect to the total amount of the coagulation bath liquid 11 . 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, or 50% by mass. Below, it may be 40 mass % or less, or 30 mass % or less. When the content of the second dissolving solvent is within the range described above, the generation of voids in the protein fibers is more significantly suppressed. When the content of the second dissolving solvent in the coagulation bath liquid 11 is 15% by mass or more with respect to the total amount of the coagulation bath liquid 11, the maximum draw ratio becomes even higher. becomes even larger. The stress as used herein means a value (unit: g/D) obtained by dividing the maximum load until the protein fiber breaks due to tensile external force in the fiber axis direction divided by the weight per 9000 m of the protein fiber.

The coagulation bath liquid 11 may contain a dissolution accelerator. As a dissolution accelerator in the coagulation bath liquid 11, for example, the inorganic salts described above may be used. Further, 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 singly or in combination of two or more.

When the spinning dope contains an inorganic salt as a dissolution accelerator, the coagulation bath solution 11 may contain the same inorganic salt as the spinning dope, or may contain a different inorganic salt. The coagulation bath solution 11 may contain an inorganic salt even if the spinning dope does not contain an inorganic salt as a dissolution accelerator.

When 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 of voids in the obtained protein fiber is further suppressed. In addition, the stress and maximum draw ratio of the obtained protein fibers are further increased. By adjusting the content of the inorganic salt in the coagulation bath liquid 11, it becomes easier to adjust the coagulation rate of the protein in the coagulation bath liquid 11, and the production of protein fibers in which the occurrence of voids is suppressed is further enhanced. become easier.

When the coagulation bath liquid 11 contains an inorganic salt, the content of the inorganic salt in the coagulation bath liquid 11 is not particularly limited. It may be 25% by mass or less, or 10% by mass or more and less than 20% by mass. When the spinning dope 6 contains an inorganic salt, the content of the inorganic salt in the coagulation bath liquid 11 is preferably lower than the inorganic salt content in the spinning dope 6 . In this case, when the dope 6 for spinning is introduced into the coagulation bath 11, protein coagulation is not hindered in the coagulation bath 11, and protein coagulation becomes possible under milder conditions. Thereby, generation of voids in protein fibers is more advantageously suppressed.

The temperature of the coagulation bath liquid 11 is not particularly limited, but may be 40° C. or less, 30° C. or less, 25° C. or less, 20° C. or less, 10° C. or less, or 5° C. or less. 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. preferable. If the temperature of the coagulation bath liquid 11 is within the above range, it becomes easier to produce protein fibers in which the occurrence of voids is sufficiently suppressed. By adjusting the temperature of the coagulation bath liquid 11 within the above range, the generation of voids in the protein fibers is sufficiently suppressed, and the stress of the obtained protein fibers is further increased. The temperature of the coagulation bath liquid can be adjusted, for example, by using the spinning apparatus 10 having a coagulation bath 20 internally provided with a heat exchanger and a cooling circulation device. For example, the temperature can be adjusted within 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 be done. In this case, more efficient cooling becomes possible by circulating the solvent (for example, methanol) used for the coagulation bath liquid 11 as a medium.

A plurality of coagulation baths 20 in which the coagulation bath liquid 11 is stored may be provided. In this case, the coagulation bath liquid (first coagulation bath liquid) in the coagulation bath to which the spinning dope 6 extruded from the nozzle 9 is directly supplied (introduced) may contain the second dissolving solvent. That is, when a plurality of coagulation baths 20 in which the coagulation bath liquid 11 is stored are provided, the coagulation bath liquids other than the first coagulation bath liquid (other coagulation bath liquids) do not contain the second dissolution solvent. may The temperature of other coagulation bath liquids may be 40°C or lower, 20°C or lower, 10°C or lower, or 5°C or lower, and may be 0°C or higher, or higher than 40°C.

The distance that the coagulated protein passes through the coagulation bath liquid 11 (substantially, the distance from the yarn guide 18a to the yarn guide 18b) allows efficient desolvation, and the extrusion speed of the spinning dope from the nozzle 9 is sufficient. It may be determined according to (ejection speed) or the like. The retention time of the coagulated protein (or spinning dope) in the coagulation bath solution 11 is determined according to the distance the coagulated protein passes through the coagulation bath solution 11, the extrusion speed of the spinning dope 6 from the nozzle 9, and the like. for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation bath liquid 11 .

The drawing performed in the washing bath 21 when obtaining the protein fibers may be so-called moist heat drawing, which is performed in hot water or in a solution of hot water to which an organic solvent or the like is added. The temperature for this wet heat stretching may be, for example, 50 to 90°C, preferably 75 to 85°C. In wet heat drawing, the undrawn yarn (or pre-drawn yarn) can be drawn, for example, 1 to 10 times, preferably 2 to 8 times.

The lower limit of the final draw ratio is preferably more than 1 times, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times with respect to the undrawn yarn (or pre-drawn yarn). 7 times or more, 8 times or more, 9 times or more, and the 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.

EXAMPLES The present invention will be described below based on Examples, but the present invention is not limited to these Examples.

1. Production of spider silk protein (spider silk fibroin: PRT410) (synthesis of gene encoding spider silk protein and construction of expression vector)
Based on the nucleotide sequence and amino acid sequence of Nephila clavipes-derived fibroin (GenBank Accession Number: P46804.1, GI: 1174415), a modified fibroin having the amino acid sequence shown in SEQ ID NO: 1 (hereinafter referred to as "PRT410 ”) was designed.

The amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence obtained by subjecting the amino acid sequence of Nephila clavipes-derived fibroin to amino acid residue substitutions, insertions and deletions for the purpose of improving productivity, and its N-terminus. and an amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 added to.

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 same nucleic acid was digested with restriction enzymes NdeI and EcoRI, excised, and then recombined with the protein expression vector pET-22b(+) to obtain an expression vector.

E. coli BLR(DE3) was transformed with the resulting pET22b(+) expression vector. 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 temperature of the culture solution was kept at 30° C., and flask culture was performed for about 15 hours until OD ₆₀₀ reached 5 to obtain a seed culture solution.

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

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g/1 L, Yeast Extract 120 g/1 L) was added at a rate of 1 mL/min. The temperature of the culture medium was maintained at 37° C. and the pH was constantly controlled to 6.9 for culturing. Cultivation was performed for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. After that, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of PRT410. After 20 hours from the addition of IPTG, the culture solution was centrifuged to collect the cells. SDS-PAGE was performed using the cells prepared from the culture solutions before and after the addition of IPTG, and the expression of PRT410 was confirmed by the appearance of a band of a size corresponding to PRT410 depending on the addition of IPTG.

(Purification of spider silk fibroin)
Two hours after the addition of IPTG, the collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The precipitate after washing was 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 heated at 60°C. 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.). The white aggregated protein (PRT410) obtained after dialysis was recovered by centrifugation. Water was removed from the collected aggregated protein using a freeze dryer to obtain a freeze-dried powder of PRT410.

2. Production of spider silk fibroin fiber (preparation of spinning dope)
After adding the spider silk fibroin (PRT410) described above to dimethyl sulfoxide (DMSO) as a dissolution solvent (first dissolution solvent) to a concentration of 24% by mass, lithium chloride (LiCl) as a dissolution accelerator was added to a concentration of 4. 0% by mass, and then shaken for 3 hours using a shaker for dissolution. After that, dust and bubbles were removed to obtain a spinning solution (dope solution). The solution viscosity of the spinning dope was 5000 cP (centipoise) at 90°C.

(spinning)
Using the obtained spinning dope, spun and drawn protein fibers (spider silk fibroin fibers) were produced by dry-wet spinning using a spinning apparatus according to the spinning apparatus 10 shown in FIG. The spinning apparatus used was the spinning apparatus 10 shown in FIG. 1, in which a second undrawn yarn manufacturing apparatus (second 2 coagulation bath) and a third undrawn yarn manufacturing device (third coagulation bath). A solution containing methanol (MeOH) and DMSO (second dissolving solvent) in the composition (mass ratio) shown in Table 3 was used as the first coagulation bath. Table 3 shows the content of the second dissolving solvent with respect to the total amount of the coagulation bath. MeOH was used as the coagulation bath liquid in the second coagulation bath and the third coagulation bath. Water was used as the cleaning liquid in the cleaning bath. Other dry-wet spinning conditions are as follows.
Extrusion nozzle diameter: 0.2mm
Residence time, temperature and stretch ratio of the first coagulation bath: see Table 3 Stretch ratio: see Table 3 Drying temperature: 95°C

3. Evaluation of Voids Voids of the spider silk fibroin fibers in Production Examples 1-1 to 1-5 were evaluated by surface observation using an optical microscope. Specifically, voids were evaluated by observing the fibers after coagulation and washing using an optical microscope and evaluating the ratio of voids of 1 μm or more per about 1000 μm ² . Optical micrographs of spider silk fibroin fibers in Production Examples 1-1 to 1-5 are shown in FIG. The fibers after coagulation are fibers obtained by coagulation in the first coagulation bath. Note that the scale bar in the optical micrograph after washing of Production Example 1-1 in FIG. 2 is 50 μm.

As shown in FIG. 2, when the coagulation bath contains DMSO as a dissolution solvent (second dissolution solvent) (Production Examples 1-2 to 1-5), when the coagulation bath does not contain DMSO (Production Example Compared to 1-1), the generation of voids in spider silk fibroin fibers was suppressed. In addition, when the coagulation bath contained 10% by mass or more of DMSO (Production Examples 1-4 and 1-5), the generation of voids in the spider silk fibroin fibers was significantly suppressed.

4. Evaluation of Stress The protein fibers obtained in Production Examples 1-1 to 1-8 were evaluated for stress. The stress was measured 10 times with an INSTRON 3342 tensile tester at a test piece length of 100 mm and a tensile speed of 100 mm/min, and the average value was calculated. Regarding significant differences between mean values, comparisons between two levels were performed by the 2-sapmlet test, and comparisons of three or more levels were performed by the analysis of variance test. A significance level (p value) of less than 0.05 was judged to indicate a significant difference. FIG. 3 is a box plot showing the evaluation results of stress. The stress as used herein means a value (unit: g/D) obtained by dividing the maximum load until the protein fiber breaks due to tensile external force in the fiber axis direction divided by the weight per 9000 m of the protein fiber. In FIG. 3, * indicates an outlier.

Spider silk fibroin fibers (manufacturing examples 1-4 to 1-8) obtained using the first coagulation bath having a DMSO content of 20% by mass or more were obtained from the first coagulation bath having a DMSO content of less than 20% by mass. The stress was higher than that of the spider silk fibroin fibers (manufacturing examples 1-1 to 1-3) obtained using the coagulation bath solution No. 1. In addition, it was shown that the stress becomes even higher when a coagulation bath having a DMSO content of 30% by mass or more is used in the first coagulation bath.

5. Evaluation of Maximum Draw Ratio The maximum draw ratio was evaluated at two points in the spinning apparatus 10 shown in FIG. The points evaluated were the stretch between 18b and 18c (hardening bath stretch) at the first point and the stretch between 14 and 18e (wash bath stretch) at the second point. The evaluation was not performed at two points simultaneously, and when one was evaluated, the other was fixed at a draw ratio close to the same. As an evaluation method, the draw ratio was increased by 1 time, and the draw ratio at the time of fiber breakage was defined as the maximum draw ratio. The results are shown in FIG.

When the coagulation bath contained 15% by mass or more of DMSO, the maximum draw ratio was higher than when the coagulation bath contained less than 15% by mass of DMSO.

6. Evaluation of void generation amount when adding inorganic salt to coagulation bath liquid Spider silk fibroin fibers (protein fibers) were produced in the same manner as in Production Example 1-1 except that the conditions shown in Table 4 were used (Production Examples 2-1 to 2-8). Evaluation of voids was performed in the same manner as in Production Examples 1-1 to 1-5. The evaluation results of voids in the protein fibers obtained in Production Examples 2-1 to 2-8 are shown in FIGS. 5 to 12, respectively.

As shown in FIGS. 5 to 12, even when the coagulation bath liquid (first coagulation bath liquid) contains DMSO as a dissolution solvent (second dissolution solvent) and contains an inorganic salt, voids are sufficiently generated. was shown to be suppressed by When 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, or the content of calcium chloride (CaCl ₂ ) is 10%. By setting it to % by mass, the generation of voids was suppressed more remarkably (comparison between Production Examples 2-2 to 2-4 and Production Example 2-1). In addition, when the composition MeOH/DMSO (mass ratio) of the first coagulation bath liquid was 80/20, the generation of voids was suppressed more significantly by setting the LiCl content to 5% by mass or more (manufacturing Comparison between Examples 2-6 to 2-8 and Production Example 2-5).

DESCRIPTION OF SYMBOLS 1... Extrusion apparatus, 2... Undrawn yarn manufacturing apparatus, 3... Wet heat drawing apparatus, 4... Drying apparatus, 6... Spinning dope, 10... Spinning apparatus, 20... Coagulation bath, 21... Washing bath, 36... Protein fiber.

Claims (10)

introducing a spinning stock solution containing a protein and a first dissolution solvent into a coagulation bath to coagulate the protein;
the coagulation bath contains a second dissolution solvent;
The first dissolution solvent is dimethylsulfoxide, N,N-dimethylformamide, hexafluoroisopropanol , hexafluoroacetone, formic acid, and inorganic salts added thereto, and inorganic salts in water, urea, guanidine, and At least one selected from the group consisting of the addition of at least one selected from the group consisting of sodium dodecyl sulfate,
The second dissolving solvent is at least one selected from the group consisting of dimethylsulfoxide, N,N-dimethylformamide, hexafluoroisopropanol , hexafluoroacetone, formic acid, and inorganic salts added thereto. the law of nature,
The coagulation bath liquid contains methanol and the second dissolving solvent, and the second dissolving solvent is composed of dimethylsulfoxide, N,N-dimethylformamide, and inorganic salts added thereto. A method for producing a protein fiber, which is at least one selected from the group . 2. The method for producing protein fibers according to claim 1 , wherein the content of said second dissolving solvent is 10 to 60% by mass with respect to the total amount of said coagulation bath liquid. The method for producing protein fibers according to claim 1 or 2 , wherein the temperature of the coagulation bath liquid is 40°C or less. The method for producing protein fibers according to any one of claims 1 to 3 , wherein the temperature of the coagulation bath liquid is 0°C or higher. The method for producing a protein fiber according to any one of claims 1 to 4 , wherein the protein is a structural protein. 6. The method for producing a protein fiber according to claim 5 , wherein said structural protein is fibroin. The method for producing a protein fiber according to claim 6 , wherein the fibroin is spider silk fibroin. The method for producing protein fibers according to any one of claims 1 to 7 , wherein the coagulation bath contains an inorganic salt. 9. The method for producing protein fibers according to claim 8 , wherein the content of said inorganic salt in said coagulation bath liquid is more than 0% by mass and 30% by mass or less based on the total amount of said coagulation bath liquid. The spinning stock solution contains an inorganic salt,
10. The method for producing a protein fiber according to claim 8 , wherein the content of said inorganic salt in said coagulation bath is lower than the content of said inorganic salt in said spinning dope.

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