JP2022024194A - Bicomponent yarn, production method thereof, and fabric - Google Patents

Abstract

To provide a bicomponent yarn and a fabric, which have improved combustion resistance and contain cellulose fibers while maintaining biodegradability.SOLUTION: A bicomponent yarn and a fabric contain artificial fibroin fibers and cellulose fibers, and the artificial fibroin fiber contains modified fibroin and has a history of contraction in which the fiber was irreversibly contracted by contacting water after spinning. A bicomponent yarn producing method has a step to mix the artificial fibroin fibers and the cellulose fibers, and the artificial fibroin fiber contains modified fibroin and has a history of contraction in which the fiber was irreversibly contracted by contacting water after spinning.SELECTED DRAWING: Figure 7

Description

The present invention relates to a composite yarn, a method for producing the same, and a fabric.

Unlike synthetic fibers, natural fibers have merits such as biodegradability, and therefore, demand for various fields is expected to increase in response to the growing awareness of environmental conservation in recent years. Among natural fibers, cellulose fibers such as cotton are used for various purposes including clothing materials because they are soft to the touch, have excellent water absorption, and have excellent dyeability. (For example, Patent Document 1).

Japanese Patent Application Laid-Open No. 2015-212450

However, cellulose fibers have a drawback that they are more flammable than synthetic fibers. In order to make up for the shortcomings related to the combustion resistance of the cellulose fiber, it is conceivable to mix the cellulose fiber and the flame-retardant synthetic fiber. However, when cellulose fibers and synthetic fibers are mixed, the merits obtained by using natural fibers having biodegradability are impaired.

An object of the present invention is to provide a composite yarn and a fabric containing cellulose fibers, which have improved burn resistance while maintaining biodegradability. It is also an object of the present invention to provide a method for producing the composite yarn.

The present inventors have found that modified fibroin has excellent combustion resistance. The present invention is based on this novel finding.

The present invention relates to, for example, the following inventions.
[1]
Contains artificial fibroin fiber and cellulose fiber,
The artificial fibroin fiber is a composite yarn containing modified fibroin and having a shrinkage history of being irreversibly shrunk by contact with water after spinning.
[2]
The composite yarn according to [1], wherein the artificial fibroin fiber has a wet shrinkage rate of 2% or more as defined by the following formula.
Wet shrinkage rate = {1- (length of artificial fibroin fiber that has been brought into a wet state by contact with water / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%)
[3]
The composite yarn according to [1] or [2], wherein the artificial fibroin fiber has a drying shrinkage rate of more than 7% as defined by the following formula.
Dry shrinkage rate = {1- (length of dried artificial fibroin fiber / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%)
[4]
The composite yarn according to any one of [1] to [3], wherein the artificial fibroin fiber is crimped by contact with water.
[5]
The composite yarn according to any one of [1] to [4], wherein the artificial fibroin fiber has a limiting oxygen index (LOI) value of 26.0 or more.
[6]
The composite yarn according to any one of [1] to [5], wherein the artificial fibroin fiber has a maximum heat absorption and heat generation rate of more than 0.025 ° C./g, which is obtained according to the following formula A.
Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)
[In Formula A, a low humidity environment means an environment with a temperature of 20 ° C. and a relative humidity of 40%, and a high humidity environment means an environment with a temperature of 20 ° C. and a relative humidity of 90%. ]
[7]
The composite yarn according to any one of [1] to [6], wherein the modified fibroin is a modified spider silk fibroin.
[8]
The composite yarn according to any one of [1] to [7], which is a blended yarn.
[9]
Contains artificial fibroin fiber and cellulose fiber,
The artificial fibroin fiber is a fabric containing modified fibroin and having a shrinkage history of being irreversibly shrunk by contact with water after spinning.
[10]
Equipped with a process of mixing artificial fibroin fiber and cellulose fiber,
The method for producing a composite yarn, wherein the artificial fibroin fiber contains modified fibroin and has a shrinkage history of being irreversibly shrunk by contact with water after spinning.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a composite yarn and a fabric containing cellulose fibers, which have improved combustion resistance while maintaining biodegradability.

Since the composite yarn and the fabric according to the present invention contain modified fibroin fibers, they are also excellent in hygroscopic heat generation.

It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a figure which shows the distribution of the z / w (%) value of the fibroin of natural origin. It is a figure which shows the distribution of the value of x / y (%) of the fibroin of natural origin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is explanatory drawing which shows typically an example of the spinning apparatus for manufacturing the artificial fibroin raw fiber. It is a figure which shows the example of the length change of the artificial fibroin raw fiber and the artificial fibroin fiber by contact with water. It is explanatory drawing which shows typically an example of the manufacturing apparatus for manufacturing an artificial fibroin fiber. It is explanatory drawing which shows typically an example of the manufacturing apparatus for manufacturing an artificial fibroin fiber. It is a graph which shows an example of the result of the hygroscopic heat generation test.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Composite yarn]
The composite yarn according to the present embodiment contains artificial fibroin fibers and cellulose fibers. The composite yarn includes, for example, a blended yarn, a mixed fiber yarn, a mixed weaving yarn, a mixed weaving yarn, a synthetic twisted yarn, a covering yarn and the like.

(Artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment may contain modified fibroin and have a shrinkage history of being irreversibly shrunk by contact with water after spinning.

(Modified fibroin)
The modified fibroin according to the present embodiment has a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein contained. The modified fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids.

As used herein, the term "modified fibroin" means artificially produced fibroin (artificial fibroin). The modified fibroin may be a fibroin whose domain sequence is different from the amino acid sequence of naturally occurring fibroin, or may be fibroin having the same amino acid sequence as that of naturally occurring fibroin. "Naturally derived fibroin" as used herein is also represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a domain sequence to be used.

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized one (for example, a nucleic acid encoding the designed amino acid sequence) regardless of naturally occurring fibroin. It may have a desired amino acid sequence by chemical synthesis).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically corresponding to (A) _n motif of an amino acid sequence) and an amorphous region (typically to REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to)), and is an amino acid represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, (A) _n motif indicates an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. (A) The number of amino acid residues of the _n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (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). A plurality of (A) _n motifs present in a domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m indicates an integer of 2 to 300, and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

The modified fibroin according to the present embodiment is, for example, an amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues to the cloned naturally occurring fibroin gene sequence. It can be obtained by modifying. Substitutions, deletions, insertions and / or additions of amino acid residues can be carried out by methods well known to those skilled in the art, such as partial mutagenesis. Specifically, Nucleic Acid Res. It can be carried out according to the method described in the literature such as 10, 6487 (1982), Methods in Enzymeology, 100, 448 (1983).

Naturally-derived fibroin is a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Yes, specifically, for example, fibroin produced by insects or spiders.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Anteraea yamamai, Anteraea perni, and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Tussar moth (Antheraea mylitta), Muga silk moth (Antheraea assama), silk moth (Visa) Hornet silk protein can be mentioned.

More specific examples of insect-produced fibroin include, for example, silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

Examples of the fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, etc. Spiders belonging to (Gasteracantha genus), spiders belonging to the spider genus Ordgarius such as Mameitaiseki spider and Mutsutogei sekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, and spiders belonging to the genus Cytophora. ) Spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Tetragnatha, such as the spider Yasagata spider, Harabiroashidaka spider, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha such as spiders, spiders such as spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the family Spiders (Tetragnathidae) such as spiders belonging to the genus Euprostenops. Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2) and the like.

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (nucleotide sequence), U47856 (nucleotide sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence AAC47011) ), U37520 (base sequence)), major amplify protein 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (nucleotide sequence), EF595246 (base sequence)), dragline silk protein-derived from nuclear fibroin 2 Numbers AAL32472 (nucleic acid sequence), AF441245 (nucleic acid sequence)), major aminos protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (nucleotide sequence), AJ973155 (nucleotide sequence)), and major protein (GenBank accession number CAM32249.1 (nucleic acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila clavipes] (GenBank accession number AAC145891 (amino acid sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1. The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally derived fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidoline, amplify, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by silkworm), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferable.

Specific examples of modified fibroin include modified fibroin (first modified fibroin) derived from the large spitting tube bookmark thread protein produced in the large bottle-shaped gland of spider, and a domain sequence with a reduced content of glycine residues. Modified fibroin (second modified fibroin), (A) modified fibroin having a domain sequence with a reduced content of _n motifs (third modified fibroin), content of glycine residues, and (A) _n . Modified fibroin with reduced motif content (fourth modified fibroin), modified fibroin with a domain sequence containing a region with a locally high hydrophobicity index (fifth modified fibroin), and content of glutamine residues. A modified fibroin having a reduced domain sequence (sixth modified fibroin) can be mentioned.

The first modified fibroin includes a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, (A) the number of amino acid residues of the _n motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, even more preferably an integer of 8 to 20, and an integer of 10 to 20. Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first modified fibroin, the number of amino acid residues constituting REP in Formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first modified fibroin, the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m is an amino acid residue. It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.

The first modified fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C end of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C end of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first modified fibroin, (1-i) the amino acid sequence shown by SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLageNRSH1), or (1-ii) the amino acid sequence shown by SEQ ID NO: 4 and 90. % And above, modified fibroin containing an amino acid sequence having sequence identity can be mentioned. The sequence identity is preferably 95% or more.

The amino acid sequence represented by SEQ ID NO: 4 is the first to the amino acid sequence of ADF3 in which the amino acid sequence (SEQ ID NO: 5) consisting of a starting codon, a His10 tag and an HRV3C protease (Human rhinovirus 3C protease) recognition site is added to the N-terminal. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.

The modified fibroin of (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

The second modified fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. It can be said that the second modified fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP substituted with another amino acid residue as compared with naturally occurring fibroin. ..

The second modified fibroin has a domain sequence of GGX and GPGXX in REP as compared with naturally occurring fibroin (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine. In at least one motif sequence selected from), one having an amino acid sequence corresponding to one or more glycine residues in the motif sequence being replaced with another amino acid residue. May be.

In the second modified fibroin, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more with respect to the total motif sequence.

The second modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and is located closest to the C-terminal side of the domain sequence (A) from the _n motif to the domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. Therefore, when the total number of amino acid residues in the sequence excluding the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The number of alanine residues with respect to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The second modified fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second modified fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX described below.

The method of calculating z / w will be described in more detail. First, in a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , it is located most on the C-terminal side from the domain sequence (A) _n . The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C end of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Further, for example, when X (center X) contained in two XGXs exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). be). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m among all the extracted fibroins, and the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less. From the amino acid sequence of fibroin in No. 1, z / w was calculated by the above-mentioned calculation method. The results are shown in FIG. The horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).

In the second modified fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second modified fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be substituted so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In any case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

The above-mentioned other amino acid residues are not particularly limited as long as they are amino acid residues other than glutamine residues, but are valine (V) residue, leucine (L) residue, isoleucine (I) residue, and methionine ( Hydrophilic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S). ) Residues, hydrophilic amino acid residues such as lysine (K) residues and glutamine (E) residues are preferred, valine (V) residues, leucine (L) residues, isoleucine (I) residues, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

As a more specific example of the second modified fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met). -Contains an amino acid sequence set forth in PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. A modified fibroin can be mentioned.

The modified fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. In the amino acid sequence shown in SEQ ID NO: 7, every two (A) _n motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, and a part of glutamine (Q) residue is further added. It is substituted with a serine (S) residue and a part of the amino acid on the C-terminal side is deleted so as to have almost the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and His tag sequence are added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. Further, the values of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.

The modified fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (2-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of) is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w. Is preferably 50.9% or more.

The second modified fibroin 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 modified fibroin.

As the tag sequence, for example, an affinity tag using specific affinity (binding, affinity) with other molecules can be mentioned. 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, isolation of modified fibroin by metal chelating chromatography (chromatography) is performed. Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).

In addition, 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 modified fibroin 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 protease, the modified fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the modified fibroin containing the tag sequence, the amino acid represented by (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples may be a modified fibroin comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminal of the indicated amino acid sequence.

The modified fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (2-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of) is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w. Is preferably 50.9% or more.

The second modified fibroin 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 third modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motif as compared with naturally occurring fibroin. It can be said that the domain sequence of the third modified fibroin has an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs as compared with the naturally occurring fibroin.

The third modified fibroin may have an amino acid sequence corresponding to a 10-40% deletion of the (A) _n motif from naturally occurring fibroin.

The third modified fibroin has a domain sequence of at least 1 to 3 (A) _n motifs for every 1 to 3 (A) _n motifs from the N-terminal side to the C-terminal side as compared with naturally occurring fibroin. May have an amino acid sequence corresponding to the deletion of.

The third modified fibroin has a domain sequence of at least two consecutive (A) _n -motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the _n -motif being repeated in this order.

The third modified fibroin may have an amino acid sequence corresponding to the deletion of the (A) _n motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

The third modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and two adjacent [(A) _n motifs from the N-terminal side to the C-terminal side. -When the number of amino acid residues in the REP of the unit is sequentially compared and the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 1. When x is the maximum value of the sum of the amino acid residues of two adjacent [(A) _n -motif-REP] units, which is 11.3, and y is the total number of amino acid residues of the domain sequence. In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The number of alanine residues with respect to the total number of amino acid residues in the _n motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residue)-(A) _n motif-second REP (100 amino acid residue)-(A) _n . Motif-Third REP (10 amino acid residues)-(A) _n Motif-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) It has an arrangement called _n motifs.

Two adjacent [(A) _n -motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, there may be a [(A) _n motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP, and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in two selected adjacent [(A) _n motif-REP] units is compared. The comparison is made by finding the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is set to 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparison between the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), the 5th REP is assumed to be 1 when the 4th REP having a smaller number of amino acid residues is 1. The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is 1.8 to _11.3 . Shown by a solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is set to 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than _11.3 . Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of (A) _n motif. be.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximum is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third modified fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least 7 of (A) _n motifs present in a plurality of (A) n motifs are composed only of alanine residues, x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is even more preferable, 70% or more is even more preferable, and 80% or more is used. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

Here, x / y in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroins, x / y from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _m by the above calculation method. Was calculated. The results when the jagged ratio is 1: 1.9 to 4.1 are shown in FIG.

The horizontal axis of FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, x / y in naturally occurring fibroin is less than 64.2% (highest, 64.14%).

The third modified fibroin, for example, deletes one or more of the sequences encoding the (A) _n motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing the nucleic acid encoding the amino acid sequence. In each case, in addition to the modification corresponding to the deletion of (A) _n motif from the amino acid sequence of naturally occurring fibroin, further substitution, deletion, insertion and / or addition of one or more amino acid residues. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the third modified fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met). -Contains an amino acid sequence set forth in PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. A modified fibroin can be mentioned.

The modified fibroin of (3-i) will be described. The amino acid sequence shown by SEQ ID NO: 17 is every two (A) _n from the N-terminal side to the C-terminal side from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. The motif is deleted, and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second modified fibroin.

The value of x / y in the Giza ratio 1: 1.8 to 11.3 of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 15.0%. The value of x / y in the amino acid sequence shown by SEQ ID NO: 17 and the amino acid sequence shown by SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences represented by SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

The modified fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The modified fibroin of (3-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and has N-terminal to C-terminal sides. When the number of amino acid residues of REP of two adjacent [(A) _n -motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The amino acid residue of two adjacent [(A) _n -motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3). When x is the maximum value of the total value obtained by adding the radix and the total number of amino acid residues in the domain sequence is y, it is preferable that x / y is 64.2% or more.

The third modified fibroin may contain the tag sequence described above at either or both of the N-terminus and the C-terminus.

As a more specific example of the modified fibroin containing the tag sequence, the amino acid represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples may be a modified fibroin comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are SEQ ID NO: 11 at the N-terminal of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by (including His tag sequence and hinge sequence) is added.

The modified fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The modified fibroin of (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The modified fibroin of (3-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n -motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is set to 1, the other The maximum value of the total value obtained by adding the number of amino acid residues of two adjacent [(A) _n -motif-REP] units in which the ratio of the number of amino acid residues of REP is 1.8 to 11.3 is x. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third modified fibroin 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 fourth modified fibroin has an amino acid sequence whose domain sequence has a reduced content of (A) _n motifs and a reduced content of glycine residues as compared with naturally occurring fibroin. It has. The domain sequence of the fourth modified fibroin lacks at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, the fourth modified fibroin is a modified fibroin having the characteristics of the above-mentioned second modified fibroin and the third modified fibroin. Specific embodiments and the like are as described in the second modified fibroin and the third modified fibroin.

As more specific examples of the fourth modified fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410). ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. Can be mentioned as a modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in. Specific embodiments of the modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth modified fibroin has a domain sequence in which one or more amino acid residues in REP are replaced with amino acid residues having a high hydrophobicity index as compared with naturally occurring fibroin, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.

It is preferable that the region having a large local hydrophobicity index is composed of consecutive 2 to 4 amino acid residues.

The amino acid residue having a large hydrophobicity index described above is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). It is more preferably a residue.

In the fifth modified fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a large hydrophobicity index as compared with naturally occurring fibroin, and / or one or more amino acid residues in REP. In addition to the modification corresponding to the insertion of an amino acid residue with a high hydrophobicity index, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be a modification of the amino acid sequence corresponding to the above.

The fifth modified fibroin retains hydrophobic amino acid residues, for example, one or more hydrophilic amino acid residues in REP (for example, amino acid residues having a negative hydrophobicity index) from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue having a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP. It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth modified fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _m , from the (A) _n motif located most on the C-terminal side to the C-terminal of the above domain sequence. Let p be the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the above domain sequence. When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Dollittle R (1982) “A simple method for dispensing the hydropatic character of a plot. 105-132) is used. Specifically, the hydrophobicity index of each amino acid (hydropathy index, hereinafter also referred to as “HI”) is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m . (Hereinafter referred to as "array A") is used. First, the average value of the hydrophobicity index of consecutive four amino acid residues is calculated for all REPs contained in the sequence A. The average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all four consecutive amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive 4 amino acid residues having an average value of 2.6 or more of hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of 6.6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are present in an overlapping manner by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is present. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif present at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

In the fifth modified fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The fifth modified fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally derived fibroin satisfies the above p / q condition. (Amino acid residue with a negative value) is replaced with a hydrophobic amino acid residue (eg, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acid residues are inserted in the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ), More preferably valine (V), leucine (L) and isoleucine (I).

As a more specific example of the fifth modified fibroin, (5-i) the amino acid sequence represented by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Alternatively, modified fibroin comprising (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.

The modified fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 consists of 3 amino acid residues in every other REP except for the domain sequence of the terminal on the C-terminal side with respect to the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). The amino acid sequence (VLI) is inserted in two places, a part of the glutamine (Q) residue is replaced with a serine (S) residue, and a part of the amino acid on the C-terminal side is deleted. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. be. The amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.

The modified fibroin of (5-i) may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin of (5-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most on the C-terminal side (A) _n . Amino acids contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p, and the total number of amino acid residues contained in the sequence excluding the sequence from the _n -motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

As a more specific example of the modified fibroin comprising a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv). ) Modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The amino acid sequences represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences represented by SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

The modified fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The modified fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The modified fibroin of (5-iv) is also a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin of (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located most on the C-terminal side (A) _n . Amino acids contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p, and the total number of amino acid residues contained in the sequence excluding the sequence from the _n -motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth modified fibroin 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 sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.

The sixth modified fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

When the sixth modified fibroin contains the GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.

In the present specification, the "GPGXX motif content" is a value calculated by the following method.
Formula 1: [(A) _n -motif-REP] _m , or Formula 2: [(A) _n -motif-REP] _m- (A) Fibroin containing a domain sequence represented by _n -motif (modified fibroin or naturally derived). In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from the (A) _n motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) _n motifs.

In the calculation of the GPGXX motif content, the target of "the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is "the most C-terminal side". (A) Sequence from _n motif to C end of domain sequence "(sequence corresponding to REP) may contain a sequence characteristic of fibroin and a sequence having low correlation, and m is small. This is because the case (that is, when the domain sequence is short) affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if the "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing the domain sequence of modified fibroin. The calculation method of the GPGXX motif content will be specifically described with reference to FIG. First, in the domain sequence of the modified fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side”. (A) Since it is included in the "sequence obtained by removing the sequence from the _n motif to the C-terminal of the domain sequence from the domain sequence" (the sequence shown by "region A" in FIG. 5), s is calculated. The number of GPGXX motifs is 7, and s is 7 × 3 = 21. Similarly, all REPs are "sequences obtained by removing the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 5). Since it is contained in.), The total number t of amino acid residues in all REPs excluding the (A) _n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, s / t (%) can be calculated by dividing s by t, which is 21/150 = 14.0% in the case of the modified fibroin of FIG.

The sixth modified fibroin preferably has a glutamine residue content of 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%. ..

In the present specification, the "glutamine residue content" is a value calculated by the following method.
Formula 1: [(A) _n -motif-REP] _m , or Formula 2: [(A) _n -motif-REP] _m- (A) Fibroin containing a domain sequence represented by the _n -motif (modified fibroin or naturally derived). In fibroin), all the sequences contained in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In REP, the total number of glutamine residues contained in the region is u, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n . The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the reason for targeting "the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin corresponds to its domain sequence being deleted from one or more glutamine residues in REP or replaced with other amino acid residues as compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M). ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.

The sixth modified fibroin preferably has a hydrophobicity of REP of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, the "hydrophobicity of REP" is a value calculated by the following method.
Formula 1: [(A) _n -motif-REP] _m , or Formula 2: [(A) _n -motif-REP] _m- (A) Fibroin containing a domain sequence represented by the _n -motif (modified fibroin or naturally derived). In fibroin), all the sequences contained in the sequence excluding the sequence from the (A) _n motif located on the C-terminal side to the C-terminal of the domain sequence from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In REP, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the sequence from the (A) _n motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and further ( A) The hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the _n motif. In the calculation of the hydrophobicity of REP, the reason for targeting "the sequence obtained by excluding the sequence from the (A) _n motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

The sixth modified fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the substitution with another amino acid residue, there may be further modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

The sixth modified fibroin is, for example, deleted one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or other than one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to the above and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth modified fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met). -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or SEQ ID NO: Modified fibroin containing the amino acid sequence represented by No. 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. , A modified fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

The modified fibroin of (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting VL for all the QQs in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with TS, and the remaining Qs are replaced with A. In the amino acid sequence shown by SEQ ID NO: 27, all QQs in the amino acid sequence shown by SEQ ID NO: 7 are replaced with VL, and the remaining Qs are replaced with I. The amino acid sequence shown in SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VI, and the remaining Qs are replaced with L. The amino acid sequence shown in SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VF, and the remaining Qs are replaced with I.

The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all the QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8. In the amino acid sequence shown by SEQ ID NO: 31, all QQs in the amino acid sequence shown by SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

In the amino acid sequence shown in SEQ ID NO: 32, all the QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) are repeated twice are replaced with VF. And the remaining Q is replaced with I.

In the amino acid sequence (Met-PRT917) shown in SEQ ID NO: 41, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with LI, and the remaining Qs are replaced with V. In the amino acid sequence (Met-PRT1028) shown in SEQ ID NO: 42, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with IF, and the remaining Qs are replaced with T.

The amino acid sequences represented by SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all residual glutamine. The group content is 9% or less (Table 2).

The modified fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It may consist of the indicated amino acid sequences.

The modified fibroin of (6-ii) is the same as SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-ii) is also a domain represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

The sixth modified fibroin 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 modified fibroin.

As more specific examples of the modified fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37. (PRT918), modified fibroin comprising the amino acid sequence set forth in SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or (PRT1028). 6-iv) Amino acid sequences and 90s represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. A modified fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned.

The amino acid sequences represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are, respectively, SEQ ID NO: 25. , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence. The amino acid sequence (including His tag sequence and hinge sequence) is added. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39. , The amino acid sequences represented by SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin of (6-iii) is the same as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. It may consist of the indicated amino acid sequences.

The modified fibroin of (6-iv) is the same as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-iv) is also a domain represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif. It is a protein containing a sequence. The sequence identity is preferably 95% or more.

The modified fibroin of (6-iv) preferably has a glutamine residue content of 9% or less. Further, the modified fibroin of (6-iv) preferably has a GPGXX motif content of 10% or more.

The sixth modified fibroin 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 modified fibroin is at least two or more of the characteristics of the first modified fibroin, the second modified fibroin, the third modified fibroin, the fourth modified fibroin, the fifth modified fibroin, and the sixth modified fibroin. It may be a modified fibroin having the above-mentioned characteristics.

The modified fibroin may be a hydrophilic modified fibroin or a hydrophobic modified fibroin. In the present specification, "hydrophobic modified fibroin" is a value obtained by obtaining the total hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. A modified fibroin having (average HI) greater than 0. The hydrophobicity index is as shown in Table 1. Further, the "hydrophilic modified fibroin" is a modified fibroin having an average HI of 0 or less. As the modified fibroin, hydrophilic modified fibroin is preferable from the viewpoint of excellent combustion resistance, and hydrophobic modified fibroin is preferable from the viewpoint of excellent hygroscopic heat generation.

Examples of the hydrophobically modified fibroin include the amino acid sequence represented by SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 43, SEQ ID NO: 35. Examples thereof include modified fibroins comprising the amino acid sequences set forth in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 44.

Examples of the hydrophilic modified fibroin include the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 11, and SEQ ID NO: 14. Or the amino acid sequence represented by SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 7, amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9, amino acid represented by SEQ ID NO: 17, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 15. Examples include modified fibroins comprising the amino acid sequence set forth in the sequence, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The artificial fibroin fiber according to the present embodiment may contain one type of modified fibroin alone, or may contain two or more types of modified fibroin in combination.

(Manufacturing method of modified fibroin)
The modified fibroin according to any of the above embodiments is a host transformed with an expression vector having, for example, a nucleic acid sequence encoding the modified fibroin and one or more regulatory sequences operably linked to the nucleic acid sequence. Therefore, it can be produced by expressing the nucleic acid.

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. can do. 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 fibroin obtained from the NCBI web database or the like. Genes can be chemically synthesized by a method of linking automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a starting codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. You may.

The regulatory sequence is a sequence that controls the expression of modified fibroin in the host (for example, promoter, enhancer, ribosome binding sequence, 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 modified fibroin 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.

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. As the expression vector, a vector that can be autonomously replicated in a host cell or can be integrated into the chromosome of the host and contains a promoter at a position where a nucleic acid encoding modified fibroin can be transcribed is preferably used.

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

Preferred examples of prokaryotic hosts include bacteria belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacillus, Corinebacterium, Pseudomonas and the like. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri and the like. Examples of microorganisms belonging to the genus Serratia include Serratia marcescens and the like. Examples of microorganisms belonging to the genus Bacillus include Bacillus satirus and the like. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium, Ammonia Phyllum and the like. Examples of the microorganism belonging to the genus Brevibacterium include Brevibacterium divaricatum and the like. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium and Ammonia Genes. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida and the like.

When a prokaryote is used as a host, the vector into which the nucleic acid encoding the modified fibroin is introduced includes, for example, pBTrp2 (manufactured by Behringer-Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, Examples thereof include pNCO2 (Japanese Unexamined Patent Publication No. 2002-238569).

Examples of eukaryotic hosts include yeast and filamentous fungi (molds and the like). Examples of the yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like. Examples of the filamentous fungus include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

When a eukaryote is used as a host, examples of the vector into which the nucleic acid encoding the modified fibroin is introduced include YEP13 (ATCC37115) and YEp24 (ATCC37051). As a method for introducing an expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. 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 can be mentioned.

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

The modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. 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 culture medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

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

Culturing of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15-40 ° C. The culture time is usually 16 hours to 7 days. The pH of the culture medium during culture is preferably maintained at 3.0 to 9.0. The pH of the culture medium can be adjusted by using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

Further, during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as needed. 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 needed. For example, isopropyl-β-D-thiogalactopyranoside or the like is used when culturing a microorganism transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

Isolation and purification of the expressed modified fibroin can be performed by a commonly used method. For example, when the modified fibroin is expressed in a lysed state in cells, after the culture is completed, the host cells are collected by centrifugation, suspended in an aqueous buffer solution, 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 ammonium sulfate, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -cepharose, 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 resin such as butyl Sepharose, 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.

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble material of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cells, disrupting the cells, and centrifuging the cells. The insoluble material of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified specimen of modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin 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.

(Manufacturing method of artificial fibroin raw fiber)
The artificial fibroin raw fiber according to the present embodiment is obtained by spinning the above-mentioned modified fibroin, and contains the above-mentioned modified fibroin as a main component. The artificial fibroin raw fiber according to the present embodiment is a fiber after spinning and before contact with water.

The artificial fibroin raw fiber according to the present embodiment can be produced by a known spinning method. That is, for example, first, the modified fibroin produced according to the above method is used as a dissolution accelerator in a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), or hexafluoroisopronol (HFIP). It is added together with the inorganic salt of No. 1 and dissolved to prepare a dope solution. Then, using this dope solution, spinning by a known spinning method such as wet spinning, dry spinning, dry wet spinning, or melt spinning, can be used to obtain the desired artificial fibroin raw fiber. Preferred spinning methods include wet spinning or dry-wet spinning.

FIG. 6 is an explanatory diagram schematically showing an example of a spinning device for producing artificial fibroin raw fiber. The spinning device 10 shown in FIG. 6 is an example of a spinning device for dry / wet spinning, and includes an extrusion device 1, an undrawn yarn manufacturing device 2, a moist heat stretching device 3, and a drying device 4.

A spinning method using the spinning device 10 will be described. First, the doping liquid 6 stored in the storage tank 7 is pushed out from the base 9 by the gear pump 8. On a lab scale, a cylinder may be filled with a dope solution and extruded from a nozzle using a syringe pump. Next, the extruded dope liquid 6 is supplied into the coagulation liquid 11 of the coagulation liquid tank 20 through the air gap 19, the solvent is removed, the modified fibroin is coagulated, and a fibrous coagulant is formed. Next, the fibrous solidified body is supplied into the hot water 12 in the stretching bath 21 and stretched. The draw ratio is determined by the speed ratio between the supply nip roller 13 and the take-up nip roller 14. Then, the stretched fibrous coagulant is supplied to the drying device 4 and dried in the yarn path 22, and the artificial fibroin raw fiber is obtained as the wound yarn body 5. 18a to 18g are thread guides.

The coagulation liquid 11 may be any solvent that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. The coagulant 11 may contain water as appropriate. The temperature of the coagulating liquid 11 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 base 9, the extrusion speed is preferably 0.2 to 6.0 ml / hour, and 1.4 to 4.0 ml / hour per hole. It is more preferable that it is time. The distance through which the coagulated protein passes through the coagulation liquid 11 (substantially, the distance from the thread guide 18a to the thread guide 18b) may be long enough to efficiently remove the solvent, for example, 200 to 500 mm. Is. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min. The residence time in the coagulation liquid 11 may be, for example, 0.01 to 3 minutes, preferably 0.05 to 0.15 minutes. Further, stretching (pre-stretching) may be performed in the coagulation liquid 11. The coagulation liquid tank 20 may be provided in multiple stages, and stretching may be performed in each stage or a specific stage, if necessary.

As the stretching performed when obtaining the artificial fibroin raw fiber, for example, in addition to the pre-stretching performed in the coagulation liquid tank 20 and the moist heat stretching performed in the stretching bath 21, dry heat stretching is also adopted.

Moist heat stretching can be performed in warm water, in a solution of warm water containing an organic solvent or the like, or in steam heating. The temperature may be, for example, 50 to 90 ° C, preferably 75 to 85 ° C. In the moist heat drawing, the undrawn yarn (or the pre-drawn yarn) can be drawn, for example, 1 to 10 times, and 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 drawing, the undrawn yarn (or the pre-drawn yarn) can be drawn 0.5 to 8 times, for example, and it is preferable to draw 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 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). It is any of the above, 7 times or more, 8 times or more, and 9 times or more, and the upper limit values are preferably 40 times or less, 30 times or less, 20 times or less, 15 times or less, 14 times or less, and 13 times or less. , 12 times or less, 11 times or less, 10 times or less. When the artificial fibroin raw fiber is a fiber spun at a draw ratio of 2 times or more, the shrinkage rate when the artificial fibroin raw fiber is brought into a wet state by contacting with water becomes higher.

(Manufacturing method of artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment can be obtained by a production method including a shrinking step of shrinking the artificial fibroin raw fiber with water. The shrinkage step may include, for example, a step (contact step) in which the above-mentioned artificial fibroin raw fiber (fiber after spinning and before contact with water) is brought into contact with water and irreversibly shrunk. The shrinkage step may include a step of drying the fibers and further shrinking them (drying step) after the contact step.

FIG. 7 is a diagram showing an example of a change in length of an artificial fibroin raw fiber and an artificial fibroin fiber due to contact with water. The artificial fibroin fiber according to the present embodiment has a property of irreversibly contracting (change in length shown by "primary contraction" in FIG. 7) when it is brought into contact with (wetting) with water. After the primary shrinkage, when it is dried, it further shrinks (the length change shown by "secondary shrinkage" in FIG. 7). The artificial fibroin fiber obtained through the primary shrinkage or the secondary shrinkage expands to the same or similar length as before the secondary shrinkage when it is brought into a wet state by contacting with water, and thereafter, when it is repeatedly dried and wet, it is repeated. The contraction and expansion are repeated with a width similar to that of the secondary contraction (the width indicated by the “stretch ratio” in FIG. 7).

The irreversible shrinkage of the artificial fibroin fiber in the contact step (“primary shrinkage” in FIG. 7) is considered to occur, for example, for the following reasons. That is, one reason is considered to be due to the secondary structure or the tertiary structure of the artificial fibroin raw fiber, and another reason is, for example, in the artificial fibroin raw fiber having residual stress due to stretching in the manufacturing process or the like. It is considered that the residual stress is relaxed by the infiltration of water between the fibers or into the fibers. Therefore, it is considered that the shrinkage rate of the artificial fibroin raw fiber in the shrinking step can be arbitrarily controlled, for example, according to the magnitude of the draw ratio in the manufacturing process of the artificial fibroin raw fiber described above.

In the contact step, the artificial fibroin fibrils after spinning and before contacting with water are brought into contact with water to bring the artificial fibroin fibrils into a wet state. The wet state means that at least a part of the artificial fibroin fiber is wet with water. As a result, the artificial fibroin raw fiber can be contracted without relying on an external force. This contraction is irreversible (corresponding to the "primary contraction" in FIG. 7).

The temperature of the water brought into contact with the artificial fibroin fiber in the contact step may be below the boiling point. This improves handleability, workability in the shrinkage process, and the like. Further, from the viewpoint of sufficiently shortening the shrinkage time, the lower limit of the water temperature is preferably 10 ° C. or higher, more preferably 40 ° C. or higher, still more preferably 70 ° C. or higher. .. The upper limit of the water temperature is preferably 90 ° C. or lower.

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

In the contact step, when the artificial fibroin fiber is brought into contact with water in a relaxed state, the artificial fibroin fiber may not only shrink but also shrink in a wavy manner. In order to prevent the occurrence of such crimps, a contact step is carried out in a state where the artificial fibroin raw fiber is not relaxed, for example, the artificial fibroin raw fiber is brought into contact with water while being tensioned (pulled) in the fiber axial direction. May be good.

The method for producing an artificial fibroin fiber according to the present embodiment may further include a drying step. The drying step is a step of drying the artificial fibroin fiber (or the artificial fibroin fiber obtained through the contact step) that has undergone the contact step and further shrinking it (corresponding to the “secondary shrinkage” in FIG. 7). 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 the temperature at which the protein contained in the artificial fibroin raw fiber is decomposed or the artificial fibroin raw fiber is thermally damaged, for example. Generally, the temperature is in the range of 20 to 150 ° C, and the temperature is preferably in the range of 50 to 100 ° C. By keeping the temperature in this range, the fibers are dried more quickly and efficiently without thermal damage of the fibers or decomposition of the proteins contained in the 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.

FIG. 8 is an explanatory diagram schematically showing an example of a manufacturing apparatus for manufacturing artificial fibroin fiber. The manufacturing apparatus 40 shown in FIG. 8 includes a feed roller 42 for feeding out artificial fibroin raw fiber, a winder 44 for winding artificial fibroin fiber 38, a water bath 46 for performing a contact step, and a dryer 48 for performing a drying step. , Is configured to have.

More specifically, the feed roller 42 is capable of mounting a wound material of the artificial fibroin raw fiber 36, and the wound material of the artificial fibroin raw fiber 36 is turned into an artificial fibroin raw fiber by rotation of an electric motor or the like (not shown). 36 can be sent out continuously and automatically. The winder 44 can continuously and automatically wind the artificial fibroin fiber 38 manufactured through the contact step and the drying step after being fed from the feed roller 42 by the rotation of an electric motor (not shown). .. Here, the delivery speed of the artificial fibroin fiber 36 by the feed roller 42 and the winding speed of the artificial fibroin fiber 38 by the winder 44 can be controlled independently of each other.

The water bath 46 and the dryer 48 are arranged side by side between the feed roller 42 and the winder 44 on the upstream side and the downstream side of the artificial fibroin raw fiber 36 in the feeding direction, respectively. The manufacturing apparatus 40 shown in FIG. 8 has relay rollers 50 and 52 that relay the artificial fibroin raw fiber 36 before and after the contact step traveling from the feed roller 42 toward the winder 44.

The water bath 46 has a heater 54, and the water 47 heated by the heater 54 is housed in the water bath 46. Further, in the water bath 46, the tension roller 56 is installed in a state of being immersed in the water 47. As a result, the artificial fibroin fiber 36 sent out from the feed roller 42 travels toward the winder 44 side while being immersed in the water 47 while being wound around the tension roller 56 in the water bath 46. It has become. The immersion time of the artificial fibroin raw fiber 36 in water 47 is appropriately controlled according to the traveling speed of the artificial fibroin raw fiber 36.

The dryer 48 has a pair of hot rollers 58. The pair of hot rollers 58 are capable of being wound with artificial fibroin fiber 36 that is separated from the water bath 46 and travels toward the winder 44 side. As a result, the artificial fibroin raw fiber 36 immersed in the water 47 in the water bath 46 is heated by a pair of hot rollers 58 in the dryer 48, dried, and then further sent out toward the winder 44. It has become like.

When manufacturing the artificial fibroin fiber 38 using the manufacturing apparatus 40 having such a structure, first, for example, the winding of the artificial fibroin raw fiber 36 spun using the spinning apparatus 10 shown in FIG. The fiber is attached to the feed roller 42. Next, the artificial fibroin raw fiber 36 is continuously sent out from the feed roller 42 and immersed in water 47 in the water bath 46. At this time, for example, the winding speed of the winder 44 is set to be slower than the feeding speed of the feed roller 42. As a result, the artificial fibroin fiber 36 contracts due to contact with water 47 in a state of being tense between the feed roller 42 and the winder 44 so as not to relax, so that the occurrence of crimping can be prevented. Contact with water 47 causes the artificial fibroin fiber 36 to irreversibly contract (corresponding to the "primary contraction" in FIG. 7).

Next, the artificial fibroin fiber 36 (or the artificial fibroin fiber 38 produced through contact with water 47) after contact with water 47 is heated by a pair of hot rollers 58 of the dryer 48. As a result, the artificial fibroin fiber 36 (or the artificial fibroin fiber 38 produced through contact with water 47) after contact with water 47 can be dried and further contracted (“secondary contraction” in FIG. 7). "Equivalent to). At this time, by controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 38 can be further contracted or the length can be kept unchanged. Then, the obtained artificial fibroin fiber 38 is wound by a winder 44 to obtain a wound product of the artificial fibroin fiber 38.

Instead of the pair of hot rollers 58, the artificial fibroin raw fiber 36 after contacting with water 47 using a drying facility consisting only of a heat source such as a drying heat plate 64 as shown in FIG. 9B is used. It may be dried. Also in this case, the artificial fibroin fiber is adjusted by adjusting the relative speed between the feed roller 42 and the winder 44 to take up the winder 44 in the same manner as when the pair of hot rollers 58 are used as the drying equipment. The 38 can be further contracted or the length can be kept unchanged. Here, the drying means is composed of the drying hot plate 64. Moreover, the dryer 48 is not indispensable.

As described above, by using the manufacturing apparatus 40, the target artificial fibroin fiber 38 can be manufactured automatically, continuously, and extremely easily.

FIG. 9 is an explanatory diagram schematically showing another example of a manufacturing apparatus for manufacturing artificial fibroin fiber. FIG. 9A shows a processing apparatus provided in the manufacturing apparatus for performing a contact step, and FIG. 9B shows a drying apparatus provided in the manufacturing apparatus for performing a drying step. The manufacturing apparatus shown in FIG. 9 dries the processing device 60 that carries out the contact step with respect to the artificial fibroin raw fiber 36 and the artificial fibroin raw fiber 36 (or the artificial fibroin fiber 38 manufactured through the contact step) after the contact step. It has a drying device 62 for making them, and they have a structure independent of each other.

More specifically, in the processing apparatus 60 shown in FIG. 9A, the dryer 48 is omitted from the manufacturing apparatus 40 shown in FIG. 8, and the feed roller 42, the water bath 46, and the winder 44 are artificially formed. It has a structure in which the fibroin raw fibers 36 are arranged side by side in order from the upstream side to the downstream side in the traveling direction. Such a processing device 60 is adapted to immerse the artificial fibroin raw fiber 36 sent out from the feed roller 42 in the water 47 in the water bath 46 and shrink it. Then, the obtained artificial fibroin fiber 38 is configured to be wound up by a winder 44.

The drying device 62 shown in FIG. 9B has a feed roller 42, a winder 44, and a drying / heating plate 64. The dry heat plate 64 is arranged between the feed roller 42 and the winder 44 so that the dry heat surface 66 comes into contact with the artificial fibroin fiber 38 and extends along the traveling direction thereof. In this drying device 62, as described above, for example, by controlling the ratio between the feeding speed of the feed roller 42 and the winding speed of the winder 44, the artificial fibroin fiber 38 can be further contracted and lengthened. It is possible not to change the speed.

By using the manufacturing apparatus having such a structure, the artificial fibroin raw fiber 36 can be contracted by the processing apparatus 60 to obtain the artificial fibroin fiber 38, and then the artificial fibroin fiber 38 can be dried by the drying apparatus 62. ..

The processing apparatus 60 may be configured by omitting the feed roller 42 and the winder 44 from the processing apparatus 60 shown in FIG. 9A and using only the water bath 46. When a manufacturing device having such a processing device is used, for example, artificial fibroin fiber is manufactured in a so-called batch system. Further, the drying device 62 shown in FIG. 9B is not essential.

(Crispy)
The artificial fibroin fiber according to the present embodiment may be crimped by contact with water. The degree of crimping is not limited, and the number of crimps may be, for example, 10 to 100 pieces / 40 mm, or 20 to 40 pieces / 40 mm. The artificial fibroin fiber crimped by contact with water can be produced, for example, by contacting the artificial fibroin raw fiber with an aqueous medium. The aqueous medium is a liquid or gas (steam) medium containing water (including water vapor). The aqueous medium may be water or a mixed solution of water and a hydrophilic solvent. Further, as the hydrophilic solvent, for example, a volatile solvent such as ethanol and methanol or a vapor thereof can be used. The aqueous medium is preferably a mixed solution of water and ethanol. By using an aqueous medium containing a volatile solvent or its vapor, the artificial fibroin fiber dries quickly, resulting in a softer finish. The ratio of water to the volatile solvent or its vapor is not particularly limited, and for example, the water: volatile solvent or its vapor may have a mass ratio of 10:90 to 90:10.

When the aqueous medium is a liquid, a known oil such as an oil for process passage (for example, for antistatic) or for finishing may be dispersed in the aqueous medium. That is, instead of the aqueous medium, an oil agent dispersion liquid containing an aqueous medium and an oil agent dispersed in the aqueous medium can also be used. By using such an oil agent dispersion, the artificial fibroin raw fiber can be crimped and the oil agent can be attached to the artificial fibroin fiber obtained by crimping the artificial fibroin raw fiber. By adhering the oil agent to the artificial fibroin fiber, various properties can be imparted to the artificial fibroin fiber. The amount of the oil agent is not particularly limited, and may be, for example, 1 to 10% by mass or 2 to 5% by mass with respect to the total amount of the aqueous medium and the oil agent.

The temperature of the aqueous medium may be 10 ° C. or higher, 25 ° C. or higher, 40 ° C. or higher, 60 ° C. or higher, or 100 ° C. or higher, and may be 230 ° C. or lower, 120 ° C. or lower, or 100 ° C. or lower. More specifically, when the aqueous medium is a gas (steam), the temperature of the aqueous medium is preferably 100 to 230 ° C, more preferably 100 to 120 ° C. When the steam of the aqueous medium is 230 ° C. or lower, heat denaturation of the artificial fibroin fiber can be prevented. When the aqueous medium is a liquid, the temperature of the aqueous medium is preferably 10 ° C. or higher, 25 ° C. or higher, or 40 ° C. or higher from the viewpoint of efficiently imparting crimping, and from the viewpoint of maintaining high fiber strength of the artificial fibroin fiber. , 60 ° C or lower is preferable. The time for contacting the artificial fibroin raw fiber with the aqueous medium is not particularly limited, but may be 30 seconds or longer, 1 minute or longer, or 2 minutes or longer, and preferably 10 minutes or shorter from the viewpoint of productivity. The contact of the aqueous medium with the artificial fibroin fiber may be carried out under normal pressure or under reduced pressure (for example, vacuum).

As a method of bringing the artificial fibroin raw fiber into contact with the aqueous medium, a method of immersing the artificial fibroin raw fiber in water, a method of spraying the steam of the aqueous medium on the artificial fibroin raw fiber, or an environment filled with steam of the aqueous medium. Examples include a method of exposing artificial fibroin raw fiber. When the aqueous medium is steam, contact of the aqueous medium with the artificial fibroin fiber can be performed using a common steam set device. Specific examples of the steam set device include devices such as a product name: FMSA type steam setter (manufactured by Fukushin Kogyo Co., Ltd.) and a product name: EPS-400 (manufactured by Tsujii Dyeing Machinery Co., Ltd.). As a specific example of the method of crimping the artificial fibroin fiber by the steam of the aqueous medium, the artificial fibroin fiber is accommodated in a predetermined accommodation chamber, and the steam of the aqueous medium is introduced into the accommodation chamber to introduce the steam of the aqueous medium to the temperature in the accommodation chamber. The temperature is adjusted to the above-mentioned predetermined temperature (for example, 100 ° C. to 230 ° C.), and steam is brought into contact with the artificial fibroin raw fiber.

The crimping step of the artificial fibroin fiber by contact with the aqueous medium is preferably in a state where no tensile force is applied to the artificial fibroin fiber (no tension is applied in the fiber axial direction), or a predetermined size. It is carried out with only added (tensioned by a predetermined amount in the fiber axis direction). At that time, by adjusting the tensile force applied to the artificial fibroin fiber, it is possible to control the degree of crimping. As a method of adjusting the tensile force applied to the artificial fibroin fiber, for example, a method of adjusting the load applied to the artificial fibroin fiber by suspending a weight of various weights on the artificial fibroin fiber, etc. A method of fixing both ends of the artificial fibroin fiber in a slackened state and changing the amount of slack in various ways. A method of appropriately changing (tightening force to the paper tube or bobbin) and the like can be mentioned.

After contacting the artificial fibroin raw fiber with an aqueous medium, the artificial fibroin raw fiber may be dried. The drying method is not particularly limited, and natural drying may be used, or artificial fibroin raw fibers may be forcibly dried using a drying facility. Crisping with an aqueous medium and subsequent drying can be performed continuously. Specifically, for example, the artificial fibroin fiber can be sent out from the bobbin, immersed in an aqueous medium, and then blown with hot air or sent out onto a hot roller to be dried. The drying temperature is not particularly limited, and may be, for example, 20 to 150 ° C, preferably 40 to 120 ° C, and more preferably 60 to 100 ° C.

As is clear from the above, the water-based shrinkage step of the artificial fibroin fiber, which is carried out to obtain the artificial fibroin fiber, is other than the step of shortening (not shrinking) the length of the artificial fibroin fiber. Also included is the step of crimping (crimping to shorten the length) the artificial fibroin fiber. Depending on the setting of conditions, a shrinking step (contact step, drying step) of shrinking (shortening the length) the artificial fibroin raw fiber with water and a step of shrinking the artificial fibroin fiber can be carried out simultaneously or stepwise.

(Embodiment of artificial fibroin fiber)
The artificial fibroin fiber according to the present embodiment may have a wet shrinkage rate of 2% or more as defined by the following formula.
Wet shrinkage rate = {1- (length of artificial fibroin fiber that has been brought into a wet state by contact with water / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%)

The artificial fibroin fiber according to the present embodiment has a wet shrinkage rate of 2.5% or more, 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more. , Or 6% or more. The upper limit of the wet shrinkage rate is not particularly limited, but is 80% or less, 60% or less, 40% or less, 20% or less, 10% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3%. It may be:

The artificial fibroin fiber according to the present embodiment may have a drying shrinkage rate of more than 7% as defined by the following formula.
Dry shrinkage rate = {1- (length of dried artificial fibroin fiber / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%)

The artificial fibroin fiber according to the present embodiment has a drying shrinkage rate of 8% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 37% or more, 38% or more. , Or 39% or more. The upper limit of the drying shrinkage rate is not particularly limited, but may be 80% or less, 70% or less, 60% or less, 50% or less, or 40% or less.

The artificial fibroin fiber according to the present embodiment may have a limiting oxygen index (LOI) value of 18 or more, 20 or more, 22 or more, or 24 or more. It may be 26 or more, 28 or more, 29 or more, or 30 or more. In the present specification, the LOI value is a value measured in accordance with the test method of powder granules or synthetic resin having a low melting point on May 31, 1995, Fire and Disaster Management Agency Dangerous Goods Regulation Section Manager Fire Danger No. 50.

The artificial fibroin fiber according to the present embodiment may have a maximum moisture absorption calorific value obtained according to the following formula A of more than 0.025 ° C./g.
Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)
In the formula A, the low humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 40%, and the high humidity environment means an environment having a temperature of 20 ° C. and a relative humidity of 90%.

The artificial fibroin fiber according to the present embodiment has a maximum moisture absorption and heat generation degree of 0.026 ° C./g or more, may be 0.027 ° C./g or more, and may be 0.028 ° C./g or more. It may be 0.029 ° C./g or higher, 0.030 ° C./g or higher, 0.035 ° C./g or higher, 0.040 ° C./g or higher. May be. The upper limit of the maximum heat absorption and heat generation is not particularly limited, but is usually 0.060 ° C./g or less.

(Cellulose fiber)
Examples of the cellulose fiber in the present specification include natural cellulose fibers such as cotton, hemp, flax, Manila hemp, palm, and rush. That is, the cellulose fibers in the present invention are distinguished from regenerated cellulose fibers such as rayon, cupra, lyocell, and acetate. Cellulose fibers may be, for example, short fibers, medium fibers, and long fibers.

The composite yarn (blended yarn, mixed fiber yarn, mixed weave yarn, mixed weave yarn, combined twisted yarn, covering yarn, etc.) according to the present embodiment is manufactured according to a conventional method except that the above-mentioned artificial fibroin fiber and cellulose fiber are used. can do. The composite yarn according to the present embodiment may further contain fibers other than artificial fibroin fibers and cellulose fibers. Examples of other fibers include synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, linen, silk, wool and cashmere. It is preferable to use these other fibers as long as the effects of the present invention are not impaired.

The content ratio of the artificial fibroin fiber and the cellulose fiber in the composite yarn according to the present embodiment may be 20% by mass or more, based on the total amount of the artificial fibroin fiber and the cellulose fiber. % Or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more. It may be 80% by mass or more. The upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the cellulose fiber. It may be 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.

Regarding the content of the artificial fibroin fiber and the cellulose fiber in the composite yarn according to the present embodiment, the total content of the artificial fibroin fiber and the cellulose fiber may be 50% by mass or more based on the total amount of the composite yarn, and is 60% by mass. % Or more, 70% by mass or more, 80% by mass or more, and 90% by mass or more. The upper limit of the total content of the artificial fibroin fiber and the cellulose fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the composite yarn.

[Fabric]
The fabric according to this embodiment contains the above-mentioned artificial fibroin fiber and cellulose fiber. Fabrics include woven fabrics, knitted fabrics (knitted fabrics), laces, felts, non-woven fabrics and the like.

The fabric according to the present embodiment may further contain fibers other than the above-mentioned artificial fibroin fiber and cellulose fiber. Examples of other fibers include synthetic fibers such as polyester and polyamide fibers, and natural fibers such as cotton, linen, silk, wool and cashmere. It is preferable to use these other fibers as long as the effects of the present invention are not impaired.

The fabric according to the present embodiment may contain artificial fibroin fibers and cellulose fibers by, for example, being produced by mixing the above-mentioned artificial fibroin fibers and cellulose fibers. The fabric according to the present embodiment also contains artificial fibroin fibers and cellulose fibers, for example, by producing a mixture of the above-mentioned yarns formed from artificial fibroin fibers and yarns formed from cellulose fibers. May be good. The fabric according to the present embodiment may also contain artificial fibroin fibers and cellulose fibers by producing, for example, using the composite yarn according to the present invention. Further, the above-mentioned yarn formed from the artificial fibroin fiber and the yarn formed from the cellulose fiber may be a single yarn or a composite yarn with other fibers. Further, in any of the production methods, other fibers and / or threads formed from other fibers may be used in combination, if necessary.

The fabric (woven fabric, knitted fabric, lace, felt, non-woven fabric, etc.) according to the present embodiment can be manufactured according to a conventional method except that the above-mentioned fibers and / or yarns are used.

The content ratio of the artificial fibroin fiber and the cellulose fiber in the fabric according to the present embodiment may be 20% by mass or more, based on the total amount of the artificial fibroin fiber and the cellulose fiber, and the content of the artificial fibroin fiber may be 10% by mass or more. It may be more than or equal to 30% by mass, may be 40% by mass or more, may be 50% by mass or more, may be 60% by mass or more, and may be 70% by mass or more. , 80% by mass or more. The upper limit of the content of the artificial fibroin fiber may be 90% by mass or less, 80% by mass or less, or 70% by mass or less based on the total amount of the artificial fibroin fiber and the cellulose fiber. It may be 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, and 20% by mass or less.

Regarding the content of the artificial fibroin fiber and the cellulose fiber in the fabric according to the present embodiment, the total content of the artificial fibroin fiber and the cellulose fiber may be 50% by mass or more, and 60% by mass or more, based on the total amount of the fabric. It may be 70% by mass or more, 80% by mass or more, and 90% by mass or more. The upper limit of the total content of the artificial fibroin fiber and the cellulose fiber is not particularly limited, and may be 100% by mass or 95% by mass or less based on the total amount of the fabric.

The fabric according to this embodiment may be used as a material for, for example, clothing, medical products, sanitary products, interior products, bedding, ornaments, bags, accessories, miscellaneous goods, vehicle parts, composite products with resins, and the like. can.

Hereinafter, the present invention will be described in more detail based on Examples and the like. However, the present invention is not limited to the following examples.

[Test Example 1: Production of artificial fibroin fiber and evaluation of shrinkage]
(1) Production of modified fibroin (synthesis of nucleic acid encoding modified fibroin and construction of expression vector)
Modified fibroin having the amino acid sequence shown by SEQ ID NO: 18 (PRT399), modified fibroin having the amino acid sequence shown by SEQ ID NO: 12 (PRT380), modified fibroin having the amino acid sequence shown by SEQ ID NO: 13 (PRT410), SEQ ID NO: A modified fibroin (PRT799) having the amino acid sequence shown by 15 and a modified fibroin (PRT918) having the amino acid sequence shown by SEQ ID NO: 37 were designed.

Nucleic acids encoding the five designed modified fibroins were synthesized. NdeI sites were added to the nucleic acid at the 5'end, and EcoRI sites were added downstream of the stop codon. Each of these five types of nucleic acids 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 the protein expression vector pET-22b (+) to obtain each expression vector.

(Expression of modified fibroin)
Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture broth was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was carried out until the OD ₆₀₀ reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 ml of the production medium (Table 5 below) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. In addition, 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 kept at 37 ° C., and the culture was controlled at a constant pH of 6.9. The culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. 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 desired modified fibroin. Twenty hours after the addition of IPTG, the culture broth 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 appearance of a band of a size corresponding to the desired modified fibroin dependent on the addition of IPTG resulted in the appearance of the target modified fibroin. Expression was confirmed.

(Purification of modified fibroin)
The cells recovered 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 at 60 ° C. Stir with a stirrer for 30 minutes 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. Moisture was removed from the recovered aggregated protein with a lyophilizer to obtain a lyophilized powder of modified fibroin.

(2) Production of artificial fibroin raw fiber (preparation of doping solution)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and lyophilized powder of modified fibroin was added thereto to a concentration of 18% by mass or 24% by mass (Table). 6), dissolved for 3 hours using a shaker. Then, insoluble matter and bubbles were removed to obtain a modified fibroin solution.

(spinning)
The obtained modified fibroin solution was used as a dope solution (spinning stock solution), and spun and stretched artificial fibroin raw fibers were produced by dry-wet spinning using a spinning device according to the spinning device 10 shown in FIG. In the spinning device 10 shown in FIG. 6, the spinning device used was a second undrawn yarn manufacturing device (a second undrawn yarn manufacturing device) between the undrawn yarn manufacturing device 2 (first bath) and the moist heat stretching device 3 (third bath). It is equipped with a second bath). The conditions for dry-wet spinning are as follows.
Extrude nozzle diameter: 0.2 mm
Liquids and temperatures in the first to third baths: see Table 6 Total draw ratio: see Table 6 Drying temperature: 60 ° C.

(3) Production and Evaluation of Artificial Fibroin Fibers Each artificial fibroin raw fiber obtained in Production Examples 1 to 19 is subjected to a contact step in which it is brought into contact with water (hereinafter, may be referred to as "primary shrinkage"). Alternatively, the artificial fibroin fiber was produced by subjecting the contact step to a drying step of drying at room temperature (hereinafter, may be referred to as "secondary shrinkage").

<Primary contraction>
A plurality of artificial fibroin raw fibers having a length of 30 cm were cut out from the wound products of the artificial fibroin raw fibers obtained in Production Examples 1 to 19. A plurality of these artificial fibroin raw fibers were bundled to obtain an artificial fibroin raw fiber bundle having a fineness of 150 denier. A 0.8 g lead weight was attached to each artificial fibroin raw fiber bundle, and in that state, each artificial fibroin raw fiber bundle was immersed in water at the temperatures shown in Tables 7 to 10 for 10 minutes (contact step). Then, the length of each artificial fibroin fiber bundle was measured in water. The length measurement of the artificial fibroin fiber bundle in water was carried out with a 0.8 g lead weight attached to the artificial fibroin fiber bundle in order to eliminate the crimping of the artificial fibroin fiber bundle. Next, the primary shrinkage rate (%) of each artificial fibroin fiber was calculated according to the following formula I. In Formula I, L0 indicates the length of the artificial fibroin fiber bundle (here, 30 cm) after spinning and before contact with water, and Lw indicates the length of the artificial fibroin fiber bundle that has undergone primary contraction. The primary shrinkage rate is synonymous with the wet shrinkage rate.
Formula I: Primary shrinkage rate = {1- (Lw / L0)} x 100 (%)

<Secondary contraction>
After immersion in water (contact step) in the primary shrinkage, the artificial fibroin fiber bundle was removed from the water. The removed artificial fibroin fiber bundle was dried at room temperature for 2 hours with a 0.8 g lead weight attached (drying step). After drying, the length of each artificial fibroin fiber bundle was measured. Next, the secondary shrinkage rate (%) of each artificial fibroin fiber was calculated according to the following formula II. In Formula II, L0 indicates the length of the artificial fibroin fiber bundle after spinning and before contact with water (here, 30 cm), and Lwd indicates the length of the artificial fibroin fiber bundle that has undergone secondary contraction. The secondary shrinkage rate is synonymous with the dry shrinkage rate.
Formula II: Secondary shrinkage rate = {1- (Lwd / L0)} × 100 (%)

The results are shown in Tables 7-10.

[Test Example 2: Evaluation of flame retardancy of artificial fibroin fiber]
(Manufacturing of artificial fibroin fiber)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and the modified fibroin (PRT799) produced in "(1) Production of modified fibroin" of Test Example 1 was frozen therein. Dry powder was added to a concentration of 24% by weight and dissolved using a shaker for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 60 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100% by mass methanol solidified from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 60 ° C. After coagulation, the obtained raw yarn was wound up and air-dried to obtain artificial fibroin raw fiber.

An artificial fibroin fiber (raw material fiber) was produced by subjecting the obtained artificial fibroin raw fiber to a contact step of contacting it with water and then subjecting it to a drying step of drying it at room temperature.

(Manufacturing of knitted fabric)
Using the obtained raw material fibers, a knitted fabric was manufactured by circular knitting using a circular knitting machine. The knitted fabric had a thickness of 180 denier and a gauge of 18. 20 g was cut out from the obtained knitted fabric and used as a test piece.

(Combustibility test)
The flammability test was based on the Fire and Disaster Management Agency Dangerous Goods Regulation Division Manager Fire Danger No. 50, the test method for powdered or granular synthetic resin with a low melting point on May 31, 1995. The test was carried out under the conditions of a temperature of 22 ° C., a relative humidity of 45% and an atmospheric pressure of 1021 hPa. Table 11 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).

As a result of the flame retardancy test, the limit oxygen index (LOI) value of the knitted fabric knitted with artificial fibroin fiber containing modified fibroin (PRT799) was 27.2. Generally, if the LOI value is 26 or more, it is considered to be flame-retardant. From this result, it can be seen that the artificial fibroin fiber has excellent flame retardancy. Therefore, by using a composite yarn or fabric in which artificial fibroin fiber and cellulose fiber are mixed, a composite yarn or fabric having excellent flame retardancy can be obtained.

[Test Example 3: Evaluation of hygroscopic heat generation of artificial fibroin fiber]
(Manufacturing of artificial fibroin fiber)
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and the modified fibroin (PRT918 or PRT799) produced in "(1) Production of modified fibroin" of Test Example 1 was prepared therein. The lyophilized powder of No. 1 was added to a concentration of 24% by mass, and dissolved using a shaker for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 60 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then nitrogen gas is used from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into a mass% methanol coagulation bath. The discharge temperature was 60 ° C. and the discharge pressure was 0.3 MPa. After solidification, the obtained raw yarn was wound at a winding speed of 3.00 m / min and air-dried to obtain artificial fibroin raw fiber.

An artificial fibroin fiber (raw material fiber) was produced by subjecting the obtained artificial fibroin raw fiber to a contact step of contacting it with water and then subjecting it to a drying step of drying it at room temperature.

For comparison, commercially available wool fiber, cotton fiber, tencel fiber, rayon fiber and polyester fiber were prepared as raw material fibers.

(Manufacturing of knitted fabric)
Using each raw material fiber, a knitted fabric was manufactured by weft knitting using a flat knitting machine. The knitted fabric using PRT918 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 18. The knitted fabric using PRT799 fiber as the raw material fiber had a thickness of 1 / 30N (hair count single yarn) and a gauge number of 16. The thickness and the number of gauges of the knitted fabric using the other raw material fibers were adjusted so as to have substantially the same cover factor as the knitted fabric using the PRT918 fiber and the PRT799 fiber. Specifically, it is as follows.
Wool thickness: 2 / 30N (double yarn), gauge number: 14
Cotton thickness: 2 / 34N (double thread), gauge number: 14
Tencel thickness: 2 / 30N (double thread), gauge number: 15
Rayon thickness: 1 / 38N (single thread), gauge number: 14
Polyester Thickness: 1 / 60N (single yarn), Gauge number: 14

(Hygroscopic heat generation test)
Two knitted fabrics cut into 10 cm × 10 cm were combined, and the four sides were sewn together to form a test piece (sample). After leaving the test piece in a low humidity environment (temperature 20 ± 2 ° C, relative humidity 40 ± 5%) for 4 hours or more, move it to a high humidity environment (temperature 20 ± 2 ° C, relative humidity 90 ± 5%) and test piece. The temperature was measured at 1-minute intervals for 30 minutes using a temperature sensor attached to the center of the inside.

From the measurement results, the maximum degree of heat absorption and heat generation was determined according to the following formula A.
Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)

FIG. 10 is a graph showing an example of the result of the hygroscopic heat generation test. The horizontal axis of the graph shows the time (minutes) left in the high-humidity environment, where 0 is the time when the sample is moved from the low-humidity environment to the high-humidity environment. The vertical axis of the graph shows the temperature (sample temperature) measured by the temperature sensor. In the graph shown in FIG. 10, the point indicated by M corresponds to the maximum value of the sample temperature.

Table 12 shows the calculation results of the maximum heat absorption and heat generation.

It can be seen that the artificial fibroin fiber containing the modified fibroin (PRT918 or PRT799) has a higher maximum hygroscopic heat generation rate and is excellent in hygroscopic heat generation property as compared with the existing materials. Therefore, by mixing artificial fibroin fiber and cellulose fiber to form a composite yarn or fabric, it is possible to obtain a composite yarn or fabric having excellent hygroscopic heat generation.

1 ... Extruder, 2 ... Unstretched yarn manufacturing equipment, 3 ... Wet and heat stretching equipment, 4 ... Drying equipment, 6 ... Dope liquid, 10 ... Spinning equipment, 20 ... Coagulation liquid tank, 21 ... Stretching bath, 36 ... Artificial fibroin raw material Fiber, 38 ... Artificial fibroin fiber, 40 ... Manufacturing equipment, 42 ... Feed roller, 44 ... Winder, 46 ... Water bath, 48 ... Dryer, 54 ... Heater, 56 ... Tension roller, 58 ... Hot roller, 60 ... Processing equipment , 62 ... Drying device, 64 ... Drying plate.

Claims (10)

Contains artificial fibroin fiber and cellulose fiber,
The artificial fibroin fiber is a composite yarn containing modified fibroin and having a shrinkage history of being irreversibly shrunk by contact with water after spinning. The composite yarn according to claim 1, wherein the artificial fibroin fiber has a wet shrinkage rate of 2% or more as defined by the following formula.
Wet shrinkage rate = {1- (length of artificial fibroin fiber that has been brought into a wet state by contact with water / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%) The composite yarn according to claim 1 or 2, wherein the artificial fibroin fiber has a drying shrinkage rate of more than 7% as defined by the following formula.
Dry shrinkage rate = {1- (length of dried artificial fibroin fiber / length of artificial fibroin fiber after spinning and before contact with water)} x 100 (%) The composite yarn according to any one of claims 1 to 3, wherein the artificial fibroin fiber is crimped by contact with water. The composite yarn according to any one of claims 1 to 4, wherein the artificial fibroin fiber has a limiting oxygen index (LOI) value of 26.0 or more. The composite yarn according to any one of claims 1 to 5, wherein the artificial fibroin fiber has a maximum heat absorption and heat generation rate of more than 0.025 ° C./g, which is obtained according to the following formula A.
Formula A: Maximum heat absorption and heat generation = {(Maximum value of sample temperature when the sample is placed in a low humidity environment until the sample temperature reaches equilibrium and then moved to a high humidity environment)-(Sample, sample Sample temperature when moving to a high humidity environment after being placed in a low humidity environment until the temperature reaches equilibrium)} (° C) / sample weight (g)
[In Formula A, a low humidity environment means an environment with a temperature of 20 ° C. and a relative humidity of 40%, and a high humidity environment means an environment with a temperature of 20 ° C. and a relative humidity of 90%. ] The composite yarn according to any one of claims 1 to 6, wherein the modified fibroin is a modified spider silk fibroin. The composite yarn according to any one of claims 1 to 7, which is a blended yarn. Contains artificial fibroin fiber and cellulose fiber,
The artificial fibroin fiber is a fabric containing modified fibroin and having a shrinkage history of being irreversibly shrunk by contact with water after spinning. Equipped with a process of mixing artificial fibroin fiber and cellulose fiber,
A method for producing a composite yarn, wherein the artificial fibroin fiber contains modified fibroin and has a shrinkage history of being irreversibly shrunk by contact with water after spinning.

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