JP2021080571A - Protein fiber and method for producing the same - Google Patents

Abstract

To provide an improved feeling of a protein fiber containing spider silk protein.SOLUTION: A protein fiber comprises a raw material fiber containing spider silk protein and an amino-modified silicone attached to a surface of the raw material fiber.SELECTED DRAWING: None

Description

The present invention relates to protein fibers and methods for producing the same.

Spider silk protein fibers containing artificial spider silk protein, which have the characteristics of spider silk having excellent strength and high elasticity, have been studied for practical use (see, for example, Patent Document 1). Clothing and the like are being studied as one of the applications of spider silk protein fibers.

Japanese Patent No. 5540154 Japanese Unexamined Patent Publication No. 2006-176916 International Publication No. 2014/61585 Japanese Unexamined Patent Publication No. 2002-609847

However, protein fibers containing spider silk protein are not always sufficient in terms of texture such as flexibility, smoothness, and suppleness, and improvement in this respect is required for application to clothing and the like.

Therefore, an object of one aspect of the present invention is to improve the texture of protein fibers containing spider silk protein.

The present inventors have found that a protein fiber having an improved texture can be obtained by adhering an amino-modified silicone to a raw material fiber containing a spider silk protein. The present invention has been completed based on this novel finding.

That is, one aspect of the present invention relates to a protein fiber containing a raw material fiber containing a spider silk protein and an amino-modified silicone adhering to the surface of the raw material fiber. This protein fiber can have an excellent texture.

Another aspect of the present invention is a step of adhering a treatment liquid containing water and amino-modified silicone to the surface of a raw material fiber containing a spider silk protein, and removing water from the treatment liquid adhering to the surface of the raw material fiber. The present invention relates to a method for producing a protein fiber, which comprises a step of obtaining a protein fiber containing the raw material fiber and the amino-modified silicone adhering to the surface of the raw material fiber. According to this method, a protein fiber containing spider silk protein and having an excellent texture can be produced.

According to the present invention, a protein fiber containing a spider silk protein having a better texture and a method for producing the same are provided.

Hereinafter, some embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

The protein fiber according to one embodiment includes a raw material fiber containing a spider silk protein and an amino-modified silicone adhering to the surface of the raw material fiber. For this protein fiber, for example, a step of spinning a spinning solution containing spider silk protein to form a raw material fiber containing spider silk protein, and a treatment liquid containing water and amino-modified silicone are attached to the surface of the raw material fiber. It is produced by a method including a step and a step of removing water from the treatment liquid adhering to the surface of the raw material fiber to obtain a protein fiber containing the raw material fiber and the amino-modified silicone adhering to the surface of the raw material fiber. be able to.

<Spider silk protein>
The spider silk protein for forming the raw material fiber of the protein fiber is not particularly limited, and is, for example, a protein produced by a microorganism or the like by a gene recombination technique, a protein produced by chemical synthesis, or a naturally derived protein. It may be a spider silk protein. The spider silk protein may be one in which the amino acid sequence of naturally occurring spider fibroin is used as it is, or the amino acid sequence thereof is modified based on the amino acid sequence of naturally occurring spider fibroin (for example, cloned). It may be an amino acid sequence modified by modifying the gene sequence of naturally occurring spider fibroin, or artificially designed and synthesized regardless of naturally occurring spider fibroin (for example, designed amino acid). It may have a desired amino acid sequence by chemically synthesizing the nucleic acid encoding the sequence).

Examples of the natural spider silk protein include a large spider silk thread protein, a weft thread protein, and a vial-shaped gland protein. Since the large spit tube bookmark thread has a repeating region consisting of a crystalline region and an amorphous region (also referred to as an amorphous region), it has both high stress and elasticity. The weft of the spider silk has a characteristic that it does not have a crystalline region but has a repeating region consisting of an amorphous region. The weft thread has a lower stress than the large discharge tube bookmark thread, but has high elasticity.

The large spider canal bookmark thread protein is produced in the large bottle-shaped gland of a spider and has a characteristic of being excellent in toughness. Examples of the large spider thread protein include large bottle-shaped gland spiders MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus. ADF3 is one of the two major bookmark thread proteins of the European garden spider. The polypeptide derived from the natural spider silk protein may be a polypeptide derived from these bookmarked yarn proteins. The polypeptide derived from ADF3 is relatively easy to synthesize and has excellent properties in terms of strength and elongation and toughness.

The weft protein is produced in the flagelliform gland of the spider. Examples of the weft protein include flagellar silk protein derived from Nephila clavipes in the United States.

The polypeptide derived from the natural spider silk protein may be a recombinant spider silk protein. Examples of the recombinant spider silk protein include variants, analogs and derivatives of the natural spider silk protein. A preferred example of such a polypeptide is a recombinant spider silk protein of a large spit tube bookmark thread protein (also referred to as a "polypeptide derived from a large spit tube bookmark thread protein").

As a protein derived from spider silk, which is an artificial spider silk protein, for example, _{a protein containing a domain sequence represented by the formula 1: [(A) n} motif-REP] _m (here, in the formula 1, (A) _n motif). Indicates an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues is 80% or more of the total number of amino acid residues in the _{n motif. REP is from 10 to 200 amino acid residues.} The constituent amino acid sequence is shown. M represents an integer of 8 to 300. Multiple existing (A) _n motifs may be the same amino acid sequence or different amino acid sequences. The multiple REPs are the same as each other. The amino acid sequence of the above may be used, or a different amino acid sequence may be used.) Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 1 (M_PRT918) can be mentioned.

The spider silk protein described above may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, and the like of the target protein.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, it is a simple spider silk protein obtained by metal chelating chromatography. It can be used separately. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 3. An example of a spider silk protein having an amino acid sequence containing this is a protein having the amino acid sequence shown in SEQ ID NO: 2 (hereinafter, may be referred to as "PRT918").

The amino acid sequence shown by SEQ ID NO: 2 is shown by SEQ ID NO: 1 designed based on the nucleotide sequence and amino acid sequence of the naturally occurring fibroin Nephila clavipes (GenBank accession number: P4684.1, GI: 11744415). The tag sequence shown in SEQ ID NO: 3 is added to the N-terminal of the amino acid sequence.

Tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

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

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the spider silk protein from which the tag sequence has been separated can also be recovered.

The spider silk protein described above may contain a secretory signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The above-mentioned natural spider silk protein and the protein derived from the spider silk protein can be used alone or in combination of two or more.

A protein contained as a main component in a protein fiber (raw material fiber) is transformed with, for example, an expression vector having a nucleic acid sequence encoding the protein and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by expressing the nucleic acid in the resulting host.

The method for producing a nucleic acid encoding a protein contained as a main component in a protein fiber (raw material fiber) is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding a natural structural protein, or a method of chemically synthesizing the nucleic acid. The chemical synthesis method of nucleic acid is not particularly limited, and for example, AKTA oligonucleotide 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 or the like. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized in (1) 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 in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. May be good.

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

<Production of spider silk protein>
The spider silk protein can be produced, for example, by expressing a predetermined nucleic acid in a host transformed with an expression vector. As an expression method, in addition to direct expression, secretory production, fusion protein expression, etc. can be performed according to the method described in Molecular Cloning 2nd Edition. When expressed by yeast, animal cells, or insect cells, spider silk protein can be obtained as a polypeptide to which sugar or sugar chain is added.

The spider silk protein is produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the spider silk protein according to the present invention in the culture medium, and collecting the spider silk protein from the culture medium. Can be done. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the host culture medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and the culture of the host is made efficient. Either a natural medium or a synthetic medium may be used as long as the medium can be used.

The carbon source may be any assimilated by the host, for example, glucose, fructose, sucrose, carbohydrates containing them such as molasses, starch and starch hydrolyzate, and organic acids such as acetic acid and propionic acid. , And alcohols such as ethanol and propanol can be used.

The expression vector has a nucleic acid sequence for producing a spider silk protein and one or more regulatory sequences operably linked to the nucleic acid sequence. The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, etc.), and can be appropriately selected depending on the type of host. The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector.

The host is one transformed with the above expression vector. As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.

As the expression vector, a vector capable of autonomous replication in a host cell or integrated into the chromosome of the host and containing a promoter at a position where the nucleic acid according to the present invention can be transcribed is preferably used.

Spider silk proteins produced by a host transformed with an expression vector can be isolated and purified by methods commonly used for protein isolation and purification. For example, when spider silk protein is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, turbid in an aqueous buffer, and then an ultrasonic crusher, a French press, or a manton. Crush the host cells with a gaulin homogenizer, dynomil, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting-out method using sulfuric acid, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia). Ion exchange chromatography method, hydrophobic chromatography method using resins such as butyl Sepharose and phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

As the above chromatography, column chromatography using phenyl-toyopearl (Tosoh), DEAE-toyopearl (Tosoh), or Sephadex G-150 (Pharmacia Biotech) is preferably used.

When the spider silk protein is expressed by forming an insoluble matter in the cell, the host cell is similarly recovered, crushed, and centrifuged to recover the insoluble matter of the spider silk protein as a precipitation fraction. The insoluble form of the recovered spider silk protein can be solubilized with a protein denaturing agent. After the operation, a purified sample of spider silk protein can be obtained by the same isolation and purification method as described above.

When the spider silk protein or a derivative having a sugar chain added to the spider silk protein is secreted extracellularly, the spider silk protein or a derivative thereof can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

<Spinning>
Spider silk proteins can be spun by the methods commonly used for spinning fibroin. For example, by spinning a spinning solution in which a spider silk protein is dissolved in a solvent, a raw material fiber formed of the spider yarn protein can be obtained.

The spinning solution is prepared by adding a solvent to the spider silk protein to adjust the viscosity so that it can be spun. The solvent may be any solvent capable of dissolving the spider silk protein. Solvents include, for example, hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, urea, guanidine, sodium dodecylsulfate (SDS), bromide. Examples thereof include an aqueous solution containing lithium, calcium chloride, lithium thiocyanate and the like.

Inorganic salts may be added to the spinning solution as needed. Examples of the inorganic salt include the following inorganic salts composed of Lewis acid and Lewis base. Examples of the Lewis base include oxoacid ion (nitrate ion, perchlorate ion, etc.), metal oxoacid ion (permanganate ion, etc.), halide ion, thiocyanate ion, cyanate ion, and the like. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, and complex ions. Specific examples of the inorganic salt composed of Lewis acid and Lewis base 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 chlorides, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron salts such as iron thiocyanate, 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 potassium thiocyanate. Potassium salts, sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate, and sodium salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, perchloric acid, etc. Zinc and zinc salts such as zinc thiocyanate, magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium salts such as magnesium thiocyanate, barium chloride, barium bromide, barium iodide, Examples thereof include barium salts such as barium nitrate, barium perchlorate, and barium thiocyanate, and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate.

The viscosity of the spinning liquid may be appropriately set according to the spinning method, and can be, for example, 100 to 15,000 cP (centipoise) at 35 ° C. The viscosity of the spinning liquid can be measured using, for example, the trade name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

The spinning method is not particularly limited as long as it can spin a spider silk protein, and examples thereof include dry spinning, melt spinning, and wet spinning. Wet spinning can be mentioned as a preferable spinning method.

In wet spinning, a solvent in which a spider yarn protein is dissolved is extruded from a spinneret (nozzle) into a coagulation liquid (coagulation liquid tank), and the spider yarn protein is solidified in the coagulation liquid to solidify the undrawn yarn in the shape of a yarn. Can be obtained. The coagulant may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol. Water may be added to the coagulating liquid as appropriate. The temperature of the coagulating liquid is preferably 0 to 30 ° C. When a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm is used as the spinneret, the extrusion speed is preferably 0.2 to 6.0 ml / hour, and 1.4 to 4.0 ml / hour per hole. More preferably, it is time. The length of the coagulation liquid tank may be as long as it can efficiently remove the solvent, and is, for example, 200 to 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, preferably 1 to 3 m / min. The residence time may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, the raw material fiber may be stretched in the coagulating liquid. In order to suppress the evaporation of the lower alcohol, the coagulation liquid may be kept at a low temperature and taken in the state of undrawn yarn. The coagulation liquid tank may be provided in multiple stages, and stretching may be performed in each stage or in a specific stage, if necessary.

The undrawn raw material fiber obtained by the above method can be made into a drawn yarn through a drawing step. Examples of the stretching method include wet heat stretching and dry heat stretching.

Moist heat stretching can be carried out in warm water, in a solution obtained by adding an organic solvent or the like to warm water, or in steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In the moist heat stretching, the undrawn raw material fiber can be stretched, for example, 1 to 10 times, preferably 2 to 8 times.

Dry heat stretching can be performed using an electric tube furnace, a dry heat plate, or the like. The temperature may be, for example, 140 ° C. to 270 ° C., preferably 160 ° C. to 230 ° C. In the dry heat stretching, the unstretched raw material fiber can be stretched 0.5 to 8 times, preferably 1 to 4 times.

Wet heat stretching and dry heat stretching may be performed individually, or they may be performed in multiple stages or in combination. That is, the first-stage stretching is performed by moist heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by moist heat stretching, the second stage stretching is performed by moist heat stretching, and the third stage stretching is performed by dry heat stretching. Wet heat stretching and dry heat stretching can be appropriately combined.

The final draw ratio in the drawing step is, for example, 5 to 20 times, preferably 6 to 11 times, that of the undrawn raw material fiber.

<Raw material fiber>
The raw material fiber of protein fiber contains spider silk protein as a main component. The proportion of spider silk protein in the raw material fiber may be 60 to 100% by mass based on the mass of the raw material fiber. The raw material fiber of the protein fiber may be a long fiber or a short fiber obtained by cutting the long fiber into a predetermined length. The raw material fiber of the protein fiber may be a non-crimped fiber or a crimped fiber. For example, a plurality of long fibers may be bundled or twisted to form a filament yarn or the like. The short fiber may be, for example, a crimped fiber that has been crimped, and may be a spun yarn such as a spun yarn.

Any known method can be adopted for the crimping process on the raw material fiber. For example, a conventional mechanical crimping method or the like can be adopted. The crimping process can also be carried out, for example, by bringing the raw material fibers into contact with moisture below the boiling point (hereinafter, also referred to as “contact step”). Further, the crimping process including the contact step with moisture further includes drying the raw material fibers after contact with moisture (after the contact step) (hereinafter, also referred to as “drying step”). May be good.

The temperature of the water that comes into contact with the raw material fibers in the contact step may be lower than the boiling point. As a result, handleability, workability of crimping, and the like are improved. Further, from the viewpoint of sufficiently shortening the crimping time, the lower limit of the water temperature is preferably 10 ° C. or higher, more preferably 40 ° C. or higher, and further preferably 70 ° C. or higher. preferable. The upper limit of the temperature of water is preferably 90 ° C. or lower.

In the contact step, the method of bringing the moisture into contact with the raw material fiber is not particularly limited. Examples of the method include a method of immersing the raw material fiber in water, a method of spraying the raw material fiber with water at room temperature or in a heated steam state, and a high humidity environment in which the raw material fiber is filled with water vapor. Examples include a method of exposing to the bottom. Among these methods, in the contact step, the method of immersing the raw material fiber in water is preferable because the crimping time can be effectively shortened and the processing equipment can be simplified.

In the contact step, when the raw material fibers are brought into contact with moisture in a relaxed state, the raw material fibers may be crimped in a wavy manner, and a crimped yarn can be obtained.

The drying step is a step of drying the raw material fibers that have undergone the contact step. The drying may be, for example, natural drying, or forced drying using a drying facility. As the drying equipment, any known contact type or non-contact type drying equipment can be used. Further, the drying temperature is not limited as long as it is lower than, for example, a temperature at which the protein contained in the raw material fiber is decomposed or the raw material fiber is thermally damaged, but in general, 20 The temperature is in the range of ~ 150 ° C, preferably in the range of 50 to 100 ° C. When the temperature is in this range, the raw material fibers are dried more quickly and efficiently without causing thermal damage to the raw material fibers or decomposition of the proteins contained in the raw material fibers. The drying time is appropriately set according to the drying temperature and the like, and for example, a time during which the influence of overdrying on the quality and physical properties of the artificial fibroin fiber can be eliminated as much as possible is adopted.

<Treatment solution containing amino-modified silicone>
By adhering a treatment liquid containing water and amino-modified silicone to the surface of the raw material fiber and then removing water from the treatment liquid adhering to the surface of the raw material fiber, amino adhering to the surface of the raw material fiber and the raw material fiber. Protein fibers containing modified silicones can be obtained.

In general, epoxy-modified silicone is often used for the treatment of protein-containing fibers, but for protein fibers containing spider silk protein, an excellent texture can be obtained by using amino-modified silicone. ..

The amino-modified silicone may be a compound having a dimethyl silicone chain and an amino group introduced into the terminal and / or side chain of the dimethyl silicone chain. Preferred amino-modified silicones have the following general formula (I):

で表される、側鎖Ｘにアミノ基を導入してなる化合物である。式（Ｉ）中、Ｒは、それぞれ独立して、―Ｈ、―ＯＨ、−ＣＨ₃及び−Ｓｉ（ＣＨ₃）₃からなる群より選ばれる一価の基であり、Ｘは、―（ＣＨ₂）_a―ＮＨ₂（ａは０〜３の整数である）、又は、―（ＣＨ₂）_a―ＮＨ（ＣＨ₂）_bＮＨ₂（ｂは１〜３の整数である）を示し、ｎは１〜１５００であり、ｍは１〜２０である。アミノ変性シリコーンが、ジメチルシリコーン鎖の両末端に配置されたアミノ基を有していてもよい。

It is a compound in which an amino group is introduced into the side chain X, which is represented by. In formula (I), R is a monovalent group independently selected from the group consisting of _{—H, —OH, −CH 3} and −Si (CH ₃ ) _{3, and X is − (CH). 2} ) _a ―NH ₂ (a is an integer of 0 to 3) or − (CH ₂ ) _a ―NH (CH ₂ ) _b NH ₂ (b is an integer of 1 to 3), n Is 1 to 1500 and m is 1 to 20. The amino-modified silicone may have amino groups located at both ends of the dimethyl silicone chain.

The amino group equivalent (molecular weight of the amino-modified silicone per mol of amino group) of the amino-modified silicone is preferably 8000 g / mol or less, more preferably 5000 g / mol or less, and further preferably 3000 g / mol or less. .. The amino group equivalent may be 100 g / mol or more. The viscosity of the amino-modified silicone at 25 ° C. is preferably 100 to 1000 Pa.

The concentration of the amino-modified silicone in the treatment liquid is preferably 0.5 to 3.0% by mass based on the mass of the treatment liquid. When the concentration of the amino-modified silicone is 0.5% by mass or more, the texture of the protein fiber is particularly effectively improved. When the concentration of the amino-modified silicone is 3.0% by mass or less, fiber entanglement tends to be less likely to occur.

The treatment liquid containing the amino-modified silicone may be an emulsion in which the amino-modified silicone is dispersed in water. Therefore, when the amino-modified silicone does not have self-emulsifying property, various surfactants and the like may be added to the treatment liquid as an emulsifier component for forming an emulsion. The surfactant contained in the treatment liquid is preferably at least one of a nonionic surfactant and a cationic surfactant.

The nonionic surfactant is not particularly limited as long as it can emulsify the main component, and examples thereof include polyoxyalkylene aliphatic ether and polyoxyalkylene aromatic ether. Preferred specific examples include polyoxyethylene tridecyl alcohol.

Examples of the cationic surfactant include a quaternary ammonium sulfate salt, a quaternary ammonium phosphate salt, and a tertiary amine type surfactant. Of these, a quaternary ammonium salt-type cationic surfactant is preferable. Examples of the quaternary ammonium salt type cationic surfactant include alkyl chlorides such as Alucard 12-37W (trade name, manufactured by Lion Axo Co., Ltd.) and Alucard T-800 (trade name, manufactured by Lion Axo Co., Ltd.). Trimethylammonium, didecyldimethylammonium chloride such as Alucard 210-80E (trade name, manufactured by Lion Axo Co., Ltd.), palm alkyldimethylbenzylammonium chloride such as Alucard CB-50 (trade name, manufactured by Lion Axo Co., Ltd.), etc. Can be mentioned. One type of cationic surfactant may be used alone, or two or more types may be used in combination.

The treatment liquid may contain, for example, an antistatic agent, other additives, a pH adjuster and the like as other components. These types are not particularly limited, and examples thereof include the following.

Antistatic agent: Alcohols having 8 to 32 carbon atoms and these alkylene oxide adducts having 2 to 4 carbon atoms (for example, 1 to 20 molar additions) phosphates (for example, lauryl alcohol phosphate potassium salt, stearyl alcohol) Sodium phosphate salt of EO2 mol adduct, potassium phosphate salt of EO7 mol adduct of isostearyl alcohol, etc.), (thio) phosphite having 9 to 90 carbon atoms (for example, triphenylphosphite, trilauryl) Trithiophosphite, etc.), fatty acid soap with 8 to 32 carbon atoms (counterions are, for example, ammonium, sodium, potassium, ammonia, etc.) (for example, ammonium laurate soap, potassium oleate soap, sodium castor oil soap, etc.), carbon Imidazoline compounds of number 8 to 32 (eg, lauryl imidazoline, oleyl imidazoline, etc.), sulfate esters having 8 to 32 carbon atoms and salts thereof (eg, sodium lauryl alcohol sulfate, ammonium oleyl alcohol sulfate, etc.), carbon Examples of the sulfonic acid number 8 to 32 and salts thereof (for example, sodium lauryl sulfonate salt, dodecylbenzene sulfonic acid and sodium salt thereof, sodium sulfosuccinate di-2-ethylhexyl ester salt, etc.) can be mentioned. The amount of the antistatic agent is preferably 10% by mass or less based on the total mass of the treatment liquid.

Other Additives: Antioxidants (eg, hindered phenolic 2,6-di-t-butylphenol, triethylene glycol-bis [3- (3-t-butyl-5-methyl-4-hydroxyphenyl) propionate) , 1,6-Hexanediol-bis [3- (3,5-di-t-butyl-4-hydroxyphenyl) propionate], etc., amine-based 2,4-bis- (n-octylthio) -6- ( 4-Hydroxy-3,5-di-t-butylanilino) -1,3,5-triazine, etc.], UV absorber (eg, benzotriazole-based 2- (3,5-di-t-amyl) hydroxyphenyl) Etc., hindered amine-based bis (2,2,6,6-tetramethyl-4-piperidyl) sebacate, etc.), fluorine compounds (eg, perfluoroethane, perfluorooctane, etc.), polyether-modified polydimethylsiloxane, etc. The amount of other additives is preferably 10% by mass or less, more preferably 0.01 to 5% by mass, based on the total mass of the treatment liquid.

Acidity regulators: lower fatty acids (2-8 carbon atoms) and derivatives thereof (eg, acetic acid, lactic acid, hydrochloric acid, malic acid, sodium acetate, etc.), ammonia and alkali metal hydroxides (eg, sodium hydroxide, hydroxide) Potassium, lithium hydroxide, magnesium hydroxide, calcium hydroxide, etc.). The amount of the pH adjuster is preferably 10% by mass or less, more preferably 5% by mass or less, based on the total mass of the treatment liquid.

The total amount of the antistatic agent, other additives, pH adjuster and the like is preferably 30% by mass or less based on the total mass of the treatment liquid.

The method for preparing the treatment liquid is not particularly limited, and a usual method can be applied. The treatment liquid can be an aqueous solution or an aqueous emulsion. The aqueous emulsion is obtained, for example, by a method of stirring and mixing or a method of emulsion polymerization.

Amino-modified silicones and emulsions containing them are readily available on the market and can also be synthesized. Commercially available products of amino-modified silicone include, for example, SF-8417, BY16-203, BY16-849, BY16-892, FZ-3785 or BY16-890 (all manufactured by Toray Dow Corning Silicone), KF-864, Examples thereof include KF-860, KF-880, KF-8004, KF-8002, KF-8005, KF-867, KF-869, KF-861, and KF-8610 (all manufactured by Shin-Etsu Chemical Co., Ltd.). Commercially available emulsions containing amino-modified silicone include, for example, POLO-MF-18T, X-51-1264, POLO-MF-33, POLO-MF-23, POLO-MF-56, KM-2002-T, KM-9772, KM-2002-L-1, POLO N-MF-14, POLO N-MF-14EC, KM-9771, POLO N-MF-63 (all manufactured by Shin-Etsu Chemical Co., Ltd.), FZ-4658, IE-4174 EMULSION, IE-7045, SM 7036EX, SM 7060EX, IE-7046T, HV496, SM8701EX, MEM-1997 (above, manufactured by Toray Dow Corning Silicone), WACKER FC201, WACKER FC218 (manufactured by Asahi Kasei Wacker Silicone). ..

The treatment agent may be diluted with a diluent. As the diluent, an organic solvent (for example, methanol, ether, hexane, toluene, chloroform, etc.) and low-viscosity mineral oil (for example, liquid paraffin having 10 cst / 25 ° C., for example, liquid paraffin having 10 to 15 carbon atoms) can be used. The ratio of the diluent is preferably 20 to 90% by mass, more preferably 40 to 70% by mass, based on the total mass of the treatment liquid containing the diluent.

The raw material fiber to which the treatment liquid containing the amino-modified silicone is attached may be a short fiber or a long fiber, or may be a non-crimped fiber or a crimped fiber. The treatment liquid may be attached to the raw material fiber at the same time as the crimping process of the raw material fiber or after the crimping process. In particular, by contacting the raw material fiber containing spider silk protein before crimping with a treatment liquid which is an emulsion in which amino-modified silicone is dispersed in water, the treatment liquid adheres to the raw material fiber and the raw material fiber is rolled. Shrinking can be performed at the same time.

The method of adhering the treatment liquid to the raw material fiber is not particularly limited, and examples thereof include a dip nip method and a shower-like injection method. In the case of crimped yarn to which amino-modified silicone is attached, problems such as yarn breakage and increase in fluff can be significantly improved even in continuous production through a card or roller in the processing process. By removing water from the treatment liquid attached to the raw material fiber by heating or the like, a protein fiber to which the amino-modified silicone is attached can be obtained.

The application of amino-modified silicone to the raw material fiber containing the spider silk protein is performed at least once in each manufacturing process of the protein fiber. That is, the method according to the present embodiment can be regarded as a method for processing protein fibers, which comprises applying amino-modified silicone to the raw material fibers to improve the texture of the protein fibers.

<Spun yarn>
When the protein fiber is a crimped fiber, various general spinning methods are used to obtain a spun yarn using the crimped fiber. The spun yarn may be composed of only protein fibers containing spider silk protein, or may be a blend of other fibers. Examples of other fibers to be blended include acrylic fibers, cellulosic fibers, and animal hair fibers. The other fibers to be blended may be a single fiber, or may be a plurality of fibers in a combination of the same type of fibers or a combination of different types of fibers.

<Raw material knitted fabric>
The protein fiber according to this embodiment is used as a filament yarn or a spun yarn, for example, in a woven fabric, a knitted fabric, a braid, a non-woven fabric, or the like.

Further, the filament yarn and the spun yarn used in the production of these products can be a single yarn composed of only protein fibers, a composite yarn composed of a combination of protein fibers and other fibers, or a combination thereof. Filament yarns are preferably used as the single yarns and the composite yarns. Here, the other fiber means a fiber or the like that does not contain protein. Examples thereof include synthetic fibers such as nylon and polyester, recycled fibers such as cupra and rayon, and natural fibers such as cotton and hemp. When used in combination with other fibers, the content of protein fibers is preferably 5% by mass or more, more preferably 20% by mass or more, based on the total amount of fibers containing protein fibers. It is more preferably 50% by mass or more. By setting the content of the protein fiber in the above range, the shrinkage rate in the shrinkage step of the raw material knitted fabric can be adjusted. Further, the composite yarn includes, for example, a blended yarn, a mixed fiber yarn, and a covering yarn.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

1. 1. Preparation of protein fiber (1) Synthesis of nucleic acid encoding spider silk protein and construction of expression vector Nucleic acid encoding protein (PRT918) having the amino acid sequence shown in SEQ ID NO: 2 was synthesized. In this nucleic acid, an NdeI site was added at the 5'end and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the obtained 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 OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., and flask culture was carried out for about 15 hours until _{the OD 600 reached 5, to obtain a seed culture solution.}

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 2) was added so that the OD ₆₀₀ was 0.05, and transformed Escherichia coli was inoculated. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The crushed 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 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 30 at 60 ° C. Stir for 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 obtained after dialysis was recovered by centrifugation, water was removed by a lyophilizer, and the lyophilized powder was recovered.

(4) Preparation of Spinning Solution A lyophilized powder of protein (PRT918) was added to formic acid to a concentration of 26% by mass, and this was dissolved by heating at 40 ° C. for 1 hour to prepare a protein solution having a concentration of 26% by mass. Obtained. Unnecessary substances and bubbles were removed from the protein solution to prepare a spinning solution.

(5) Spinning The obtained spinning liquid is filled in a reserve tank, and a 0.1 or 0.2 mm diameter monohole nozzle connected to the reserve tank is used in a coagulation bath containing 100% by mass of methanol using a gear pump. The spinning liquid was discharged toward the surface to form a raw material fiber composed of the solidified spinning liquid. The discharge rate was adjusted to 0.01 to 0.08 mL / min. The raw material fibers were washed and stretched in a washing bath containing 100% by mass methanol. The washed and stretched raw yarn was dried on a drying plate, and the dried raw yarn of protein fiber (raw material fiber) was wound up.

2. Adhesion of Treatment Liquid to Raw Material Fiber The following silicone emulsion (manufactured by Shin-Etsu Chemical Co., Ltd.) containing amino-modified silicone was prepared as the treatment liquid of Examples 1 to 7 or Comparative Examples 2 and 3 shown in Table 3. The treatment liquids of Examples 5 to 7 were prepared by diluting the silicone emulsion B with water so as to have the concentration shown in Table 3. The concentration shown in Table 3 is the concentration of the silicone oil agent based on the mass of the treatment liquid.
Silicone emulsion A: POLO N-MF14
Silicone emulsion B: POLO N-MF14EC
Silicone emulsion C: KM-9771
Silicone emulsion D: POLO-MF-63
Silicone emulsion E: POLO N-MF-18T
Silicone emulsion F: X-51-1264

The functional group equivalent shown in Table 3 is the molecular weight (amino group equivalent or epoxy group equivalent) of the modified silicone per 1 mol of amino group or epoxy group, and is shown according to the following classification.
High: 10000 g / mol or more Medium: More than 3000 g / mol and less than 10000 g / mol Low: 3000 g / mol or less

The ionicity shown in Table 3 indicates the type of surfactant contained in the treatment liquid.

Raw fiber containing spider silk protein was cut to obtain short fiber. The obtained short fibers were put into each treatment liquid, and the treatment liquid was gently stirred so that the silicone spreads over the entire fibers. As a result, silicone adhered to the surface of the short fibers and the short fibers were crimped. The short fibers recovered from the treatment liquid were lightly squeezed and then dehydrated by a dehydrator. Then, the short fibers were dried under constant temperature and humidity conditions of 40 ° C. and 60% RH for 18 hours to obtain short fibers of protein fibers to which silicone was attached. Comparative Example 1 is a raw material fiber to which silicone is not attached.

3. 3. Evaluation Texture Based on the following criteria, the degree of improvement in texture of each protein fiber compared with the untreated raw material fiber of Comparative Example 1 was sensory evaluated.
◎: The improvement of texture is felt very strongly 〇: The improvement of texture is strongly felt △: The improvement of texture is slightly felt

Card Passability The protein fibers of Examples 5-7 and Comparative Examples 1 and 3 were passed through a comb for carding. The short fibers of Examples 5 and 6 passed through the comb smoothly. In the case of the protein fiber (untreated raw material fiber) of Comparative Example 1, fiber breakage occurred. In the case of the protein fiber of Comparative Example 3, entanglement of the fiber occurred.

Claims (6)

A protein fiber containing a raw material fiber containing a spider silk protein and an amino-modified silicone adhering to the surface of the raw material fiber. The protein fiber according to claim 1, wherein the amino-modified silicone has an amino group equivalent of 8000 g / mol or less. The protein fiber according to claim 1 or 2, which is a crimped fiber. A step of adhering a treatment liquid containing water and amino-modified silicone to the surface of a raw material fiber containing spider silk protein, and
A step of removing water from the treatment liquid adhering to the surface of the raw material fiber to obtain a protein fiber containing the raw material fiber and the amino-modified silicone adhering to the surface of the raw material fiber.
A method for producing a protein fiber. The method for producing a protein fiber according to claim 4, wherein the concentration of the amino-modified silicone in the treatment liquid is 0.5 to 3.0% by mass based on the mass of the treatment liquid. The method for producing a protein fiber according to claim 4 or 5, wherein the treatment liquid further contains at least one of a nonionic surfactant and a cationic surfactant.