JP7503287B2 - Flame Retardant Composition - Google Patents

Description

The present invention relates to a flame-retardant composition.

Biodegradable plastics such as polylactic acid have been attracting attention from the perspective of environmental conservation, and are used in a variety of industrial fields in the form of fibers, films, nonwoven fabrics, powders, adhesives, resins, etc.

Biodegradable plastics themselves do not have the same strong flame retardancy as conventional petroleum-based plastic materials, making them difficult to use in components that require flame retardancy.

Patent Document 1 discloses a method for producing flame-retardant biodegradable plastics, which is characterized by adding at least one of aluminum hydroxide powder and magnesium hydroxide powder to pellets made of biodegradable plastic raw materials and molding them.

JP 08-252823 A

When a composition containing a biodegradable plastic such as polylactic acid is combined with a conventional flame retardant obtained by chemical synthesis in order to impart flame retardancy, the biodegradable characteristics may be lost.

Therefore, the present invention aims to provide a flame-retardant composition that is biodegradable and has excellent flame retardancy.

The inventors discovered that modified fibroin has excellent flame retardancy. Applying this knowledge, they developed a novel composition that is both biodegradable and flame retardant.

The present invention relates to, for example, the following inventions.
[1]
A flame retardant composition comprising a modified fibroin and a biodegradable plastic.
[2]
The flame-retardant composition according to [1], wherein the modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more.
[3]
The flame-retardant composition according to [1] or [2], wherein the modified fibroin comprises modified spider silk fibroin.
[4]
The flame-retardant composition according to any one of [1] to [3], wherein the modified fibroin comprises a modified fibroin having an average hydrophobicity index (average HI) of 0 or less.
[5]
The flame-retardant composition according to any one of [1] to [4], wherein the biodegradable plastic comprises at least one selected from the group consisting of biodegradable polyester, biodegradable cellulose, and modified starch.
[6]
The flame-retardant composition according to any one of [1] to [5], wherein the biodegradable plastic comprises at least one selected from the group consisting of biodegradable cellulose and biodegradable polyester.
[7]
The flame-retardant composition according to any one of [1] to [6], wherein the biodegradable plastic is polylactic acid.
[8]
The flame-retardant composition according to any one of [1] to [7], wherein the flame-retardant composition is in the form of a liquid, a powder, a gel, a fiber, a resin, a film, or a porous body.
[9]
The flame-retardant composition according to any one of [1] to [8], wherein the flame-retardant composition is in the form of a solution, a powder, a fiber, or a resin.
[10]
The flame-retardant composition according to any one of [1] to [9], wherein the flame-retardant composition is in the form of a resin.

According to the present invention, it is possible to provide a flame-retardant composition that is biodegradable and has excellent flame retardancy. According to the present invention, it is possible to provide a molded article using the flame-retardant composition.

According to the present invention, for example, by mixing modified fibroin with a biodegradable plastic, a composition that is imparted with flame retardancy can be obtained without impairing biodegradability. In addition, according to the present invention, the degree of flame retardancy of the flame retardant composition or a molded article using the same can be adjusted by adjusting the amount of modified fibroin (active ingredient) contained in the flame retardant composition or a molded article using the same.

The flame-retardant composition of the present invention is imparted with flame retardancy by containing modified fibroin as an active ingredient. That is, the modified fibroin functions as a flame-retardant agent. It has been reported that the addition of many conventional flame-retardant agents reduces mechanical strength and/or heat resistance. On the other hand, modified fibroin is known to have excellent mechanical strength and/or heat resistance. Therefore, it is expected that a flame-retardant composition containing modified fibroin will have improved mechanical strength and/or heat resistance compared to a composition using a conventional flame-retardant agent.

Conventional flame retardants require about 10 to 30 parts by weight, and up to about 50 parts by weight, per 100 parts by weight of material (e.g., resin). In addition, many conventional flame retardants are harmful substances in themselves. For example, some bromine-based flame retardants generate dioxins through thermal decomposition when incinerated, and some phosphorus-based flame retardants pollute the soil environment when decomposed in soil. In contrast, modified fibroin is biodegradable, so when modified fibroin is used as a flame retardant, the environmental impact can be reduced compared to when conventional flame retardants are used.

Conventional flame retardant agents made from natural products such as natural minerals are known, but unlike industrially synthesized products, it is not easy to consistently obtain products of a consistent quality. In contrast, modified fibroin can be mass-produced industrially, making it easier to consistently impart the desired flame retardancy to materials.

FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. FIG. 1 shows the distribution of z/w (%) values of naturally occurring fibroin. FIG. 1 shows the distribution of x/y (%) values of naturally derived fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin. FIG. 2 is a schematic diagram showing an example of a domain sequence of a modified fibroin.

The following describes in detail the embodiments for implementing the present invention. However, the present invention is not limited to the following embodiments.

[Flame-retardant composition]
The flame-retardant composition according to this embodiment contains a modified fibroin and a biodegradable plastic.

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

As used herein, "modified fibroin" refers to 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 a fibroin whose amino acid sequence is the same as that of naturally occurring fibroin. The "naturally occurring fibroin" referred to in this specification is also a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif.

A "modified fibroin" may be one that uses the amino acid sequence of naturally occurring fibroin as is, one that has had its amino acid sequence modified based on the amino acid sequence of naturally occurring fibroin (for example, one that has had its amino acid sequence modified by modifying the gene sequence of a cloned naturally occurring fibroin), or one that has been artificially designed and synthesized without relying on naturally occurring fibroin (for example, one that has a desired amino acid sequence obtained by chemically synthesizing a nucleic acid that codes for a designed amino acid sequence).

As used herein, the term "domain sequence" refers to an amino acid sequence that produces a crystalline region specific to fibroin (typically corresponding to the (A) _n motif in the amino acid sequence) and an amorphous region (typically corresponding to REP in the amino acid sequence), and means an amino acid sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. Here, the (A) _n motif represents an amino acid sequence mainly composed of alanine residues, and has 2 to 27 amino acid residues. The number of amino acid residues in the (A) _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. In addition, the ratio of the number of alanine residues to the total number of amino acid residues in the (A) _n motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). At least seven of the (A) _n motifs present in the domain sequence may be composed of only alanine residues. REP indicates an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m indicates an integer of 2 to 300, and may be an integer of 10 to 300. The (A) _n motifs present in multiple locations may be the same amino acid sequence as each other or different amino acid sequences. The REPs present in multiple locations may be the same amino acid sequence as each other or different amino acid sequences.

The modified fibroin according to this embodiment can be obtained, for example, by modifying the amino acid sequence of the cloned gene sequence of naturally occurring fibroin by, for example, substituting, deleting, inserting, and/or adding one or more amino acid residues. Substitution, deletion, insertion, and/or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, it can be performed according to the methods described in literature, such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

Naturally derived fibroin is a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m -(A) _n motif, and specific examples thereof include fibroins produced by insects or arachnids.

Examples of fibroin produced by insects include silk proteins produced by silkworms such as Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea pernyi, Eriogyna pyretorum, Pilosamia cynthia ricini, Samia cynthia, Caligra japonica, Antheraea mylitta, and Antheraea assama, and silk proteins produced by hornets such as Vespa One example is hornet silk protein excreted by the larvae of the silkworm, Simillima xanthoptera.

A more specific example of fibroin produced by insects is silkworm fibroin L chain (GenBank accession numbers M76430 (base sequence) and AAA27840.1 (amino acid sequence)).

Fibroin produced by spiders includes, for example, spiders belonging to the Araneus genus, such as the Japanese raven spider, the Japanese garden raven spider, the red raven spider, the green raven spider, and the Japanese bean raven spider; spiders belonging to the Neoscona genus, such as the Japanese mountain raven spider, the Japanese house raven spider, the Japanese doyo raven spider, and the Satsuma midamashi spider; spiders belonging to the Pronus genus, such as the Japanese dwarf raven spider; spiders belonging to the Cyrtarachne genus, such as the Japanese spine spider and the Japanese spine spider; spiders belonging to the Gaster genus, such as the Japanese spine spider and the Japanese spine spider; spiders belonging to the genus Acantha, spiders belonging to the genus Ordgarius such as Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Argiope such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Arachnura such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Acusilas such as Orbweaver Spider, spiders belonging to the genus Cytophora such as Orbweaver Spider, Orbweaver Spider and Orbweaver Spider, spiders belonging to the genus Poltys such as Orbweaver Spider spider silk proteins produced by spiders belonging to the genus Cyclosa, such as the bush spider, the four-headed bush spider, the round bush spider, and the black bush spider, and spiders belonging to the genus Chorizopes, such as the Japanese bush spider, as well as spiders belonging to the genus Tetragnatha, such as the long-legged spider, the long-legged spider, the broad-legged spider, and the scale-like long-legged spider; spiders belonging to the genus Leucauge, such as the large white-legged spider, the medium-legged spider, and the small white-legged spider; spiders belonging to the genus Orb Weaver, such as the golden orb spider and the giant orb spider; Examples of spider silk proteins include those produced by spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as the golden spider, spiders belonging to the genus Dyschiriognatha such as the small long-legged spider, spiders belonging to the genus Latrodectus such as the black widow spider, the redback spider, the gray widow spider and the three-spotted latrodectus, and spiders belonging to the family Tetragnathiidae such as spiders belonging to the genus Euprosthenops. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 (adf-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession numbers AAC04504 (amino acid sequence), U37520 (base sequence)), major amplify spidroin 1 [derived from Latrodectus hesperus] (GenBank accession numbers ABR68856 (amino acid sequence), EF595246 (nucleotide sequence)), dragline silk protein spidroin 2 [derived from Nephila clavata] (GenBank accession numbers AAL32472 (amino acid sequence), AF441245 (nucleotide sequence)), major ampullate spidroin 1 [derived from Eurosthenops australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (nucleotide sequence)), and major ampullate spidroin 2 [derived from Eurosthenops australis] (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor ampullate silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor ampullate silk protein 2 [Nephila clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor ampullate spidroin-like protein [Nephilangys cruentata] (GenBank accession number ABR37278.1 (amino acid sequence) and the like.

More specific examples of naturally derived fibroin include fibroins whose sequence information is registered in NCBI GenBank. For example, from among the sequences registered in NCBI GenBank that contain INV as a division, it can be confirmed by extracting sequences in which spidroin, amplify, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION, a specific product character string from CDS, and a specific character string in TISSUE TYPE from SOURCE.

The modified fibroin according to this embodiment may be modified silk fibroin (a silk protein produced by silkworms with a modified amino acid sequence) or modified spider silk fibroin (a spider silk protein produced by spiders with a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferred because of its superior flame retardancy.

Specific examples of modified fibroins include a modified fibroin derived from a major spinal duct dragline silk protein produced in the major ampullate gland of spiders (first modified fibroin), a modified fibroin having a domain sequence with a reduced content of glycine residues (second modified fibroin), a modified fibroin having a domain sequence with a reduced content of (A) _n motifs (third modified fibroin), a modified fibroin having a reduced content of glycine residues and a reduced content of (A) _n motifs (fourth modified fibroin), a modified fibroin having a domain sequence including a region with a locally high hydrophobicity index (fifth modified fibroin), and a modified fibroin having a domain sequence with a reduced content of glutamine residues (sixth modified fibroin).

The first modified fibroin includes a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m . In the first modified fibroin, the number of amino acid residues in the (A) _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, even more preferably an integer of 10 to 20, even more preferably an integer of 4 to 16, particularly preferably an integer of 8 to 16, and most preferably an integer of 10 to 16. 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, even more preferably 20 to 100 residues, and even more preferably 20 to 75 residues. The first modified fibroin is preferably such that the total number of glycine residues, serine residues and alanine residues contained in the amino acid sequence represented by formula 1: [(A) _n motif-REP] _m is 40% or more, more preferably 60% or more, and even more preferably 70% or more, of the total number of amino acid residues.

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

The amino acid sequence shown in SEQ ID NO:1 is identical to the amino acid sequence consisting of 50 amino acid residues at the C-terminus of the amino acid sequence of ADF3 (GI:1263287, NCBI), the amino acid sequence shown in SEQ ID NO:2 is identical to the amino acid sequence obtained by removing 20 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:3 is identical to the amino acid sequence obtained by removing 29 residues from the C-terminus of the amino acid sequence shown in SEQ ID NO:1.

A more specific example of the first modified fibroin is (1-i) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 4 (recombinant spider silk protein ADF3KaiLargeNRSH1), or (1-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The sequence identity is preferably 95% or more.

The amino acid sequence shown in SEQ ID NO:4 is an amino acid sequence of ADF3 with an amino acid sequence (SEQ ID NO:5) consisting of an initiation codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site added to the N-terminus, in which the repeat region 1 to 13 has been increased by approximately 2 times and mutated so that translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminus of the amino acid sequence shown in SEQ ID NO:4 is identical to the amino acid sequence shown in SEQ ID NO:3.

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

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

The second modified fibroin may have an amino acid sequence in which, compared to naturally occurring fibroin, at least one glycine residue in at least one motif sequence selected from GGX and GPGXX (wherein G represents a glycine residue, P represents a proline residue, and X represents an amino acid residue other than glycine) in REP is replaced with another amino acid residue in at least one or more of the motif sequences.

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

The second modified fibroin may have an amino acid sequence having a z _{/ w} ratio of 30% or more, 40% or _more , 50% or more, or 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in all REPs in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence, and w is the total number of amino acid residues in the sequence excluding the sequence from the (A) n motif located at the most C-terminus side to the C-terminus of the domain sequence from the domain sequence. The number of alanine residues relative to the total number of amino acid residues in the (A) _n motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

The second modified fibroin is preferably one in which one glycine residue in the GGX motif is replaced with another amino acid residue to increase the content of the amino acid sequence consisting of XGX. The second modified fibroin preferably has a content of the amino acid sequence consisting of GGX in the domain sequence of 30% or less, more preferably 20% or less, even more preferably 10% or less, even more preferably 6% or less, even more preferably 4% or less, and particularly preferably 2% or less. The content of the amino acid sequence consisting of GGX in the domain sequence can be calculated in the same manner as the calculation method for the content (z/w) of the amino acid sequence consisting of XGX described below.

The calculation method of z/w will be explained in more detail. First, in a fibroin (modified fibroin or naturally derived fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , an amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from 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 overlap), z is 50 x 3 = 150. In addition, for example, in the case of an amino acid sequence consisting of XGXGX, when there is an X (central X) contained in two XGX, the overlap is deducted from the calculation (in the case of XGXGX, it is 5 amino acid residues). w is the total number of amino acid residues contained in the sequence excluding the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in Figure 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 (excluding the (A) _n motif located at the most C-terminus). 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, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified, and 663 types of fibroin (of which, 415 types of fibroin derived from spiders) were extracted. Among all the extracted fibroins, z/w was calculated from the amino acid sequence of naturally derived fibroin containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , in which the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less, by the above-mentioned calculation method. The results are shown in FIG. 2. The horizontal axis of FIG. 2 indicates z/w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, z/w in all naturally derived fibroins 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, even more preferably 58.7% or more, even more preferably 70% or more, and even more preferably 80% or more. There is no particular upper limit to z/w, but it may be, for example, 95% or less.

The second modified fibroin can be obtained, for example, by modifying the gene sequence of a cloned naturally occurring fibroin by replacing at least a part of the base sequence encoding a glycine residue to encode another amino acid residue. In this case, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or the glycine residue may be substituted so that z/w is 50.9% or more. Alternatively, for example, an amino acid sequence satisfying the above-mentioned aspects may be designed from the amino acid sequence of a naturally occurring fibroin, and a nucleic acid encoding the designed amino acid sequence may be chemically synthesized to obtain the second modified fibroin. In either case, in addition to the modification equivalent to replacing the glycine residue in REP with another amino acid residue from the amino acid sequence of a naturally occurring fibroin, the amino acid sequence may be modified by further substituting, deleting, inserting, and/or adding one or more amino acid residues.

The other amino acid residue is not particularly limited as long as it is an amino acid residue other than a glycine residue, but is preferably a hydrophobic amino acid residue such as a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a methionine (M) residue, a proline (P) residue, a phenylalanine (F) residue, or a tryptophan (W) residue, or a hydrophilic amino acid residue such as a glutamine (Q) residue, an asparagine (N) residue, a serine (S) residue, a lysine (K) residue, or a glutamic acid (E) residue, more preferably a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, a phenylalanine (F) residue, or a glutamine (Q) residue, and even more preferably a glutamine (Q) residue.

More specific examples of the second modified fibroin include (2-i) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by replacing all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally derived fibroin with GQX. The amino acid sequence shown in SEQ ID NO: 7 is obtained by deleting every third (A) _n motif from the N-terminus side to the C-terminus side of the amino acid sequence shown in SEQ ID NO: 6, and further inserting one [(A) _n motif-REP] before the C-terminus sequence. The amino acid sequence shown in SEQ ID NO: 8 is obtained by inserting two alanine residues on the C-terminus side of each (A) _n motif of the amino acid sequence shown in SEQ ID NO: 7, further substituting some glutamine (Q) residues with serine (S) residues, and deleting some amino acids on the C-terminus side so as to have a molecular weight approximately the same as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO:9 is a sequence in which a region of 20 domain sequences present in the amino acid sequence shown in SEQ ID NO:7 is repeated four times (with some amino acid residues on the C-terminal side of the region being replaced), to which a specific hinge sequence and a His tag sequence are added at the C-terminus.

The z/w value 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, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 are 58.7%, 70.1%, 66.1%, and 70.0%, respectively. Furthermore, the x/y values in the jagged ratio (described below) of 1:1.8 to 11.3 in 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 15.0%, 15.0%, 93.4%, 92.7%, and 89.8%, respectively.

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

The modified fibroin of (2-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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 comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (2-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and has a z/w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

The second modified fibroin may contain a tag sequence at either or both of the N-terminus and C-terminus. This allows the modified fibroin to be isolated, immobilized, detected, visualized, etc.

Examples of tag sequences include affinity tags that utilize specific affinity (binding ability, affinity) with other molecules. A specific example of an affinity tag is a histidine tag (His tag). A His tag is a short peptide with about 4 to 10 histidine residues lined up, and has the property of specifically binding to metal ions such as nickel, and can be used to isolate modified fibroin by chelating metal chromatography. A specific example of a tag sequence is the amino acid sequence shown in SEQ ID NO: 11 (an amino acid sequence including a His tag sequence and a hinge sequence).

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

Furthermore, an "epitope tag" that utilizes an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of epitope tags include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. By using an epitope tag, modified fibroin can be easily purified with high specificity.

Furthermore, a tag sequence that can be cleaved with a specific protease can also be used. By treating the protein adsorbed via the tag sequence with a protease, the modified fibroin from which the tag sequence has been cleaved can be recovered.

More specific examples of modified fibroins containing a tag sequence include (2-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (2-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The amino acid sequences shown in SEQ ID NO:16 (PRT313), SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including the His tag sequence and hinge sequence) to the N-terminus 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, respectively.

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

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

The modified fibroin (2-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and preferably has a z/w ratio of 50.9% or more, where z is the total number of amino acid residues in the amino acid sequence consisting of XGX (wherein X represents an amino acid residue other than glycine) contained in REP, and w is the total number of amino acid residues in REP in the domain sequence.

The second modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

The third modified fibroin has an amino acid sequence in which the content of (A) _n motifs is reduced compared to naturally-occurring fibroin, and the domain sequence of the third modified fibroin can be said to have an amino acid sequence corresponding to the deletion of at least one or more (A) _n motifs compared to naturally-occurring fibroin.

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

The third modified fibroin may have an amino acid sequence in which the domain sequence is such that, compared to a naturally occurring fibroin, at least one (A) _n motif is deleted from the N-terminus to the C-terminus for every one to three (A) _n motifs.

The third modified fibroin may have an amino acid sequence in which the domain sequence corresponds to at least two consecutive (A) _n motifs deleted from the N-terminus to the C-terminus, and one (A) _n motif deleted in this order, compared to naturally occurring fibroin.

The third modified fibroin may have an amino acid sequence in which the domain sequence is such that at least every third (A) _n motif is deleted from the N-terminus to the C-terminus.

The third modified fibroin may have an amino acid sequence comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , and when the number of amino acid residues of the REP of two adjacent [(A) _n motif-REP] units is compared from the N-terminus side to the C-terminus side, and the number of amino acid residues of the REP having the fewer number of amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues of the two adjacent [(A) _n motif-REP] units having a ratio of the number of amino acid residues of the other REP to the number of amino acid residues of 1.8 to 11.3 is set to x, and the total number of amino acid residues of the domain sequence is set to y, the ratio of x/y may be 20% or more, 30% or more, 40% or more, or 50% or more. The number of alanine residues relative to the total number of amino acid residues in the (A) _n motif may be 83% or more, but is preferably 86% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 100% (meaning that it is composed only of alanine residues).

The method of calculating x/y will be explained in more detail with reference to Figure 1. Figure 1 shows the domain sequence of modified fibroin excluding the N-terminal sequence and the C-terminal sequence. The domain sequence has, from the N-terminus (left side), the following sequence: (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(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) _n motif.

Two adjacent [(A) _n motif-REP] units are selected in sequence from the N-terminus to the C-terminus so as not to overlap. At this time, there may be an [(A) _n motif-REP] unit that is not selected. FIG. 1 shows pattern 1 (comparison of the first REP with the second REP, and the third REP with the fourth REP), pattern 2 (comparison of the first REP with the second REP, and the fourth REP with the fifth REP), pattern 3 (comparison of the second REP with the third REP, and the fourth REP with the fifth REP), and pattern 4 (comparison of the first REP with the second REP). Note that there are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in the two adjacent selected [(A) _n motif-REP] units is compared. The comparison is performed by determining the ratio of the number of amino acid residues of the other REP to the number of amino acid residues of the one having fewer amino acid residues, which is set to 1. For example, in the case of a comparison between a first REP (50 amino acid residues) and a second REP (100 amino acid residues), when the first REP having fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the second REP is 100/50=2. Similarly, in the case of a comparison between a fourth REP (20 amino acid residues) and a fifth REP (30 amino acid residues), when the fourth REP having fewer amino acid residues is set to 1, the ratio of the number of amino acid residues of the fifth REP is 30/20=1.5.

In Figure 1, a pair of [(A) _n- motif-REP] units in which the ratio of the number of amino acid residues in the other is 1.8 to 11.3 when the number of amino acid residues in the other is set to 1 is shown by a solid line. In this specification, this ratio is referred to as the "jaggy ratio." A pair of [(A) _n- motif-REP] units in which the ratio of the number of amino acid residues in the other is less than 1.8 or more than 11.3 when the number of amino acid residues in the other is set to 1 is shown by a dashed line.

In each pattern, the numbers of all amino acid residues in the two adjacent [(A) _n motif-REP] units indicated by solid lines are added together (not only the numbers of amino acid residues in REP but also the numbers of amino acid residues in the (A) _n motif). The sums are then compared, and the sum of the pattern with the largest sum (the maximum sum) is designated as x. In the example shown in FIG. 1, the sum of pattern 1 is the largest.

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

In the third modified fibroin, x/y is preferably 50% or more, more preferably 60% or more, even more preferably 65% or more, even more preferably 70% or more, even more preferably 75% or more, and particularly preferably 80% or more. There is no particular upper limit to x/y, and it 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, when the jagged ratio is 1:1.8 to 3.4, x/y is preferably 77.1% or more, when the jagged ratio is 1:1.9 to 8.4, x/y is preferably 75.9% or more, and when the jagged ratio is 1:1.9 to 4.1, x/y is preferably 64.2% or more.

When the third modified fibroin is a modified fibroin in which at least seven of the (A) _n motifs present in the domain sequence are composed of only alanine residues, x/y is preferably 46.4% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 70% or more, and particularly preferably 80% or more. There is no particular upper limit to x/y, and it is sufficient that it is 100% or less.

Here, x/y in naturally occurring fibroin will be described. First, as described above, fibroins whose amino acid sequence information is registered in NCBI GenBank were confirmed by the method exemplified above, and 663 types of fibroin (of which, 415 types were spider-derived fibroin) were extracted. From all the extracted fibroins, x/y was calculated from the amino acid sequence of naturally occurring fibroin composed of a domain sequence represented by formula 1: [(A) _n motif-REP] _m by the calculation method described above. The results when the jagged ratio was 1:1.9 to 4.1 are shown in FIG. 3.

The horizontal axis of Figure 3 indicates x/y (%), and the vertical axis indicates frequency. As is clear from Figure 3, the x/y ratio in all naturally derived fibroins is less than 64.2% (the highest is 64.14%).

The third modified fibroin can be obtained, for example, by deleting one or more of the sequences encoding the (A) _n motif from the gene sequence of the cloned naturally-occurring fibroin so that x/y is 64.2% or more. Alternatively, for example, it can be obtained by designing an amino acid sequence corresponding to the deletion of one or more (A) _n motifs from the amino acid sequence of the naturally-occurring fibroin so that x/y is 64.2% or more, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the deletion of the (A) _n motif from the amino acid sequence of the naturally-occurring fibroin, the amino acid sequence may be modified by further substituting, deleting, inserting and/or adding one or more amino acid residues.

More specific examples of the third modified fibroin include (3-i) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met-PRT799), or (3-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The modified fibroin (3-i) will now be described. The amino acid sequence shown in SEQ ID NO:17 is obtained by deleting every third (A) _n motif from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO:10 (Met-PRT313) corresponding to naturally-occurring fibroin, and further inserting one [(A) _n motif-REP] just before the C-terminus sequence. The amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 is as described in the second modified fibroin.

The x/y value in the jagged ratio of 1:1.8-11.3 for the amino acid sequence shown in SEQ ID NO:10 (corresponding to naturally occurring fibroin) is 15.0%. The x/y value in the amino acid sequence shown in SEQ ID NO:17 and the amino acid sequence shown in SEQ ID NO:7 is 93.4%. The x/y value in the amino acid sequence shown in SEQ ID NO:8 is 92.7%. The x/y value in the amino acid sequence shown in SEQ ID NO:9 is 89.8%. The z/w values in the amino acid sequences shown in 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%, 66.1% and 70.0%, respectively.

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

The modified fibroin of (3-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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 comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (3-ii) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and when the numbers of amino acid residues of REP in two adjacent [(A) _n- motif-REP] units are sequentially compared from the N-terminus to the C-terminus, and the number of amino acid residues of the REP having the fewer number of amino acid residues is taken as 1, the maximum value of the sum of the numbers of amino acid residues of two adjacent [(A) _n- motif-REP] units in which the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 (Jagged ratio of 1:1.8 to 11.3) is taken as x, and y is the total number of amino acid residues in the domain sequence, x/y is 64.2% or more.

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

More specific examples of modified fibroins containing a tag sequence include (3-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (3-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The amino acid sequences shown in SEQ ID NO:18, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:17, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

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

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

The modified fibroin (3-iv) preferably has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and when the numbers of amino acid residues of REP in two adjacent [(A) _n motif-REP] units are sequentially compared from the N-terminus to the C-terminus, and the number of amino acid residues of the REP having the fewer number of amino acid residues is set to 1, the maximum value of the sum of the numbers of amino acid residues of two adjacent [(A) _n motif-REP] units such that the ratio of the number of amino acid residues of the other REP is 1.8 to 11.3 is defined as x, and the total number of amino acid residues in the domain sequence is y, the ratio x/y is 64.2% or more.

The third modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

The fourth modified fibroin has an amino acid sequence in which the content of (A) _n motifs is reduced and the content of glycine residues is reduced, compared with naturally-derived fibroin. The domain sequence of the fourth modified fibroin can be said to have an amino acid sequence in which at least one or more (A) _n motifs are deleted and at least one or more glycine residues in REP are replaced with other amino acid residues, compared with naturally-derived fibroin. That is, the fourth modified fibroin is a modified fibroin having both the characteristics of the second modified fibroin and the third modified fibroin described above. Specific aspects are as described for the second modified fibroin and the third modified fibroin.

Specific examples of the fourth modified fibroin include (4-i) modified fibroins containing an amino acid sequence represented by SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by 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. Specific embodiments of modified fibroins containing the amino acid sequence represented by 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 may have an amino acid sequence whose domain sequence includes a region with a high local hydrophobicity index, which corresponds to one or more amino acid residues in REP being replaced with amino acid residues with a high hydrophobicity index and/or one or more amino acid residues with a high hydrophobicity index being inserted into REP, compared to naturally occurring fibroin.

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

The amino acid residues with a high hydrophobicity index are more preferably selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A).

The fifth modified fibroin may have, in addition to modifications corresponding to the replacement of one or more amino acid residues in REP with amino acid residues having a high hydrophobicity index and/or the insertion of one or more amino acid residues having a high hydrophobicity index into REP, modifications in the amino acid sequence corresponding to the replacement, deletion, insertion and/or addition of one or more amino acid residues compared to naturally occurring fibroin.

The fifth modified fibroin can be obtained, for example, by substituting one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP from the gene sequence of cloned naturally-occurring fibroin with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and/or by inserting one or more hydrophobic amino acid residues into REP. It can also be obtained, for example, by designing an amino acid sequence corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-occurring fibroin and/or the insertion of one or more hydrophobic amino acid residues into REP, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the substitution of one or more hydrophilic amino acid residues in REP with hydrophobic amino acid residues from the amino acid sequence of naturally-occurring fibroin and/or the insertion of one or more hydrophobic amino acid residues into REP, the amino acid sequence may be further modified by substituting, deleting, inserting and/or adding one or more amino acid residues.

The fifth modified fibroin may have an amino acid sequence comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , in which, in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence, p is the total number of amino acid residues contained in a region having an average hydrophobicity index of 2.6 or more of four consecutive amino acid residues, and q is the total number of amino acid residues contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence, such that p/q is 6.2% or more.

The hydrophobicity index of amino acid residues is determined using a known index (Hydrophilicity index: Kyte J, & Doolittle R (1982) "A simple method for displaying the hydropathic character of a protein", J. Mol. Biol., 157, pp. 105-132). Specifically, the hydrophobicity index (hereinafter also referred to as "HI") of each amino acid is as shown in Table 1 below.

The calculation method of p/q will be explained in more detail. For the calculation, a sequence (hereinafter referred to as "sequence _A ") is used, which is obtained by removing the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence represented by formula ₁ : [(A) n motif-REP] m. First, the average value of the hydrophobicity index of the consecutive 4 amino acid residues is calculated for all REPs included in sequence A. The average value of the hydrophobicity index is calculated by dividing the sum of the HI of each amino acid residue included in the consecutive 4 amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is calculated for all the consecutive 4 amino acid residues (each amino acid residue is used for calculating the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of the consecutive 4 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 the hydrophobicity index of 2.6 or more", it is included as one amino acid residue in the region. The total number of amino acid residues included in the region is p. The total number of amino acid residues included in sequence A is q.

For example, if 20 "contiguous four amino acid residues with an average hydrophobicity index of 2.6 or more" are extracted (without overlap), the region in which the average hydrophobicity index of the contiguous four amino acid residues is 2.6 or more contains 20 contiguous four amino acid residues (without overlap), and p is 20×4=80. Also, for example, if two "contiguous four amino acid residues with an average hydrophobicity index of 2.6 or more" overlap by one amino acid residue, the region in which the average hydrophobicity index of the contiguous four amino acid residues is 2.6 or more contains seven amino acid residues (p=2×4−1=7, where "−1" is the deduction of the overlap). For example, in the case of the domain sequence shown in FIG. 4, there are seven "contiguous four amino acid residues with an average hydrophobicity index of 2.6 or more" without overlap, and p is 7×4=28. 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 (not including the (A) _n motif at the end of the C-terminus). Next, p/q (%) can be calculated by dividing p by q. In the case of FIG. 4, it is 28/170=16.47%.

In the fifth modified fibroin, p/q is preferably 6.2% or more, more preferably 7% or more, even more preferably 10% or more, even more preferably 20% or more, and 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 can be obtained, for example, by modifying the amino acid sequence of a cloned naturally-occurring fibroin to an amino acid sequence containing a region with a high hydrophobicity index locally by replacing one or more hydrophilic amino acid residues (e.g., amino acid residues with a negative hydrophobicity index) in REP with hydrophobic amino acid residues (e.g., amino acid residues with a positive hydrophobicity index) and/or inserting one or more hydrophobic amino acid residues into REP so as to satisfy the above-mentioned p/q condition. It can also be obtained, for example, by designing an amino acid sequence that satisfies the above-mentioned p/q condition from the amino acid sequence of a naturally-occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In either case, in addition to the modification corresponding to the replacement of one or more amino acid residues in REP with amino acid residues with a high hydrophobicity index and/or the insertion of one or more amino acid residues with a high hydrophobicity index into REP, a modification corresponding to the replacement, deletion, insertion and/or addition of one or more amino acid residues may be further performed.

There are no particular limitations on the amino acid residues with a high hydrophobicity index, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are preferred, with valine (V), leucine (L) and isoleucine (I) being more preferred.

A more specific example of the fifth modified fibroin is (5-i) a modified fibroin containing an amino acid sequence shown in SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), or (5-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The modified fibroin (5-i) will be described. The amino acid sequence shown in SEQ ID NO:19 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP in two places in the amino acid sequence shown in SEQ ID NO:7 (Met-PRT410) except for the domain sequence at the C-terminus, and further replacing some glutamine (Q) residues with serine (S) residues and deleting some amino acids at the C-terminus. The amino acid sequence shown in SEQ ID NO:20 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP in one place in the amino acid sequence shown in SEQ ID NO:8 (Met-PRT525). The amino acid sequence shown in SEQ ID NO:21 is obtained by inserting an amino acid sequence (VLI) consisting of three amino acid residues at every other REP in two places in the amino acid sequence shown in SEQ ID NO:8.

The modified fibroin (5-i) may consist of the amino acid sequence shown in 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 shown in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. The modified fibroin of (5-ii) is also a protein comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

The modified fibroin (5-ii) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, and preferably has a p/q ratio of 6.2% or more, where p is the total number of amino acid residues contained in a region having an average hydrophobicity index of 2.6 or more for four consecutive amino acid residues in all REPs contained in the sequence obtained by excluding from the domain sequence _the sequence from the (A) _{n motif located at the most C-terminal side to the C-terminus of the domain sequence, and q is the total number of amino acid residues contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) n} motif located at the most C-terminus side to the C-terminus of the domain sequence.

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

More specific examples of modified fibroins containing a tag sequence include (5-iii) modified fibroins containing an amino acid sequence shown in SEQ ID NO:22 (PRT720), SEQ ID NO:23 (PRT665) or SEQ ID NO:24 (PRT666), or (5-iv) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

The amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24 are obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including the His tag sequence and hinge sequence) to the N-terminus of the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

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

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

The modified fibroin (5-iv) has a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, and preferably has a p/q ratio of 6.2% or more, where p is the total number of amino acid residues contained in a region having an average hydrophobicity index of 2.6 or more for four consecutive amino acid residues in all REPs contained in the sequence obtained by excluding from the domain sequence _the sequence from the (A) _{n motif located at the most C-terminal side to the C-terminus of the domain sequence, and q is the total number of amino acid residues contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) n} motif located at the most C-terminus side to the C-terminus of the domain sequence.

The fifth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

The sixth modified fibroin has an amino acid sequence with a reduced content of glutamine residues 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 a 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. There is no particular upper limit to the GPGXX motif content, and it may be 50% or less, or may be 30% or less.

In this specification, the "GPGXX motif content" is a value calculated by the following method.
In a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, the GPGXX motif content is calculated as s/t, where s is three times the total number of GPGXX motifs contained in all REPs contained in the sequence obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence (i.e., equivalent to the total number of G and P in the GPGXX motif), and t is the total number of amino acid residues in all REPs obtained by excluding from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif.

In calculating the GPGXX motif content, the target is "the sequence excluding the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence from the domain sequence" because the "sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (sequence corresponding to REP) may contain a sequence that has low correlation with the sequence characteristic of fibroin, and when m is small (i.e., when the domain sequence is short), this affects the calculation result of the GPGXX motif content, and this influence is to be eliminated. Note that when the "GPGXX motif" is located at the C-terminus of REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing the domain sequence of the modified fibroin. A method for calculating the GPGXX motif content will be specifically described with reference to FIG. 5. First, in the domain sequence of the modified fibroin shown in FIG. 5 (which is of the "[(A) _n motif-REP] _m -(A) _n motif" type), all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 5), so the number of GPGXX motifs for calculating s is 7, and s is 7×3=21. Similarly, all REPs are included in the "sequence obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" (the sequence shown in "Region A" in FIG. 5), so the total number t of amino acid residues of all REPs obtained by further removing 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, even more preferably 4% or less, and particularly preferably 0%.

In this specification, the "glutamine residue content" is a value calculated by the following method.
In a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, in all REPs contained in a sequence (sequence corresponding to "region A" in Figure 5) obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence, the total number of glutamine residues contained in the region is u, and the total number of amino acid residues in all REPs removed from the domain sequence from the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif is t, the glutamine residue content is calculated as u/t. The reason for targeting "the sequence removed from the domain sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" in calculating the glutamine residue content is the same as that described above.

The sixth modified fibroin may have an amino acid sequence whose domain sequence corresponds to the deletion of one or more glutamine residues in REP or the substitution of other amino acid residues, compared to naturally occurring fibroin.

The "other amino acid residue" may be any amino acid residue other than glutamine residue, but is preferably an amino acid residue with a higher hydrophobicity index than 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 higher hydrophobicity index than glutamine residues include those selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, it is more preferable to use amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A), and it is even more preferable to use amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F).

In the sixth modified fibroin, the hydrophobicity of the REP is preferably -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular upper limit to the hydrophobicity of the REP, and it may be 1.0 or less, or 0.7 or less.

In this specification, the "hydrophobicity of REP" is a value calculated by the following method.
In a fibroin (modified fibroin or naturally-derived fibroin) containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif, in all REPs contained in a sequence (sequence corresponding to "region A" in Figure 5) obtained by removing from the domain sequence the sequence from the (A) _n motif located at the most C-terminal side to the C-terminus of the domain sequence, the sum of the hydrophobicity indices of each amino acid residue in the region is v, and the total number of amino acid residues in all REPs removed from the domain sequence from the sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence and further excluding the (A) _n motif is t, the hydrophobicity of the REP is calculated as v/t. The reason for targeting "the sequence removed from the domain sequence from the (A) _n motif located at the most C-terminus side to the C-terminus of the domain sequence" in calculating the hydrophobicity of the REP is the same as that described above.

The sixth modified fibroin may have a domain sequence that, compared to a naturally occurring fibroin, is modified by deleting one or more glutamine residues in REP and/or substituting one or more glutamine residues in REP with other amino acid residues, and may also have a modification in the amino acid sequence that is equivalent to substituting, deleting, inserting and/or adding one or more amino acid residues.

The sixth modified fibroin can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of a cloned naturally-occurring fibroin and/or substituting one or more glutamine residues in REP with other amino acid residues. It can also be obtained, for example, by designing an amino acid sequence corresponding to the deletion of one or more glutamine residues in REP from the amino acid sequence of a naturally-occurring fibroin and/or the substitution of one or more glutamine residues in REP with other amino acid residues, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

More specific examples of the sixth modified fibroin include (6-i) modified fibroin containing the amino acid sequence shown in 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:42 (Met-PRT1028), or (6-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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.

The modified fibroin (6-i) will be described. The amino acid sequence shown in SEQ ID NO:25 is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 (Met-PRT410) with VL. The amino acid sequence shown in SEQ ID NO:26 is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with TS and the remaining Q with A. The amino acid sequence shown in SEQ ID NO:27 is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with VL and the remaining Q with I. The amino acid sequence shown in SEQ ID NO:28 is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with VI and the remaining Q with L. The amino acid sequence shown in SEQ ID NO:29 is obtained by replacing all QQ in the amino acid sequence shown in SEQ ID NO:7 with VF and the remaining Q with I.

The amino acid sequence shown in SEQ ID NO:30 is the amino acid sequence shown in SEQ ID NO:8 (Met-PRT525) in which all QQs have been replaced with VL. The amino acid sequence shown in SEQ ID NO:31 is the amino acid sequence shown in SEQ ID NO:8 in which all QQs have been replaced with VL and the remaining Qs have been replaced with I.

The amino acid sequence shown in SEQ ID NO:32 is a sequence in which the 20 domain sequence regions present in the amino acid sequence shown in SEQ ID NO:7 (Met-PRT410) are repeated twice, with all QQs replaced with VF and the remaining Qs replaced with I.

The amino acid sequence shown in SEQ ID NO:41 (Met-PRT917) is the amino acid sequence shown in SEQ ID NO:7 in which all QQs have been replaced with LI and the remaining Qs have been replaced with Vs. The amino acid sequence shown in SEQ ID NO:42 (Met-PRT1028) is the amino acid sequence shown in SEQ ID NO:7 in which all QQs have been replaced with IF and the remaining Qs have been replaced with Ts.

The amino acid sequences shown in 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 all have a glutamine residue content of 9% or less (Table 2).

The modified fibroin (6-i) may consist of the amino acid sequence shown in 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.

The modified fibroin of (6-ii) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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. The modified fibroin of (6-ii) is also a protein comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The sequence identity is preferably 95% or more.

The modified fibroin (6-ii) preferably has a glutamine residue content of 9% or less. The modified fibroin (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 C-terminus. This allows the modified fibroin to be isolated, immobilized, detected, visualized, etc.

More specific examples of modified fibroins containing a tag sequence include (6-iii) modified fibroins containing the amino acid sequence shown in 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), 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 (6-iv) modified fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in 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.

The amino acid sequences shown in 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 obtained by adding the amino acid sequence shown in SEQ ID NO:11 (including a His tag sequence and a hinge sequence) to the N-terminus of the amino acid sequences shown in 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, respectively. Since only a tag sequence has been added to the N-terminus, there is no change in the glutamine residue content, and the amino acid sequences shown in 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 all have a glutamine residue content of 9% or less (Table 3).

The modified fibroin (6-iii) may consist of the amino acid sequence shown in 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.

The modified fibroin (6-iv) comprises an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in 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. The modified fibroin (6-iv) is also a protein comprising a domain sequence represented by formula 1: [(A) _n motif-REP] _m , or formula 2: [(A) _n motif-REP] _m- (A) _n motif. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set depending on the type of host.

The modified fibroin may be a modified fibroin having 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.

The modified fibroin is preferably a hydrophilic modified fibroin because it has better flame retardancy. In this specification, "hydrophilic modified fibroin" refers to modified fibroin in which the sum of the hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin is calculated and then the sum is divided by the total number of amino acid residues, resulting in a value (average HI) of 0 or less. The hydrophobicity index is as shown in Table 1. Modified fibroin with an average HI of more than 0 is also sometimes called hydrophobic modified fibroin.

Examples of hydrophilic modified fibroins include modified fibroins containing the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:15, the amino acid sequence shown in SEQ ID NO:18, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:11, SEQ ID NO:14, or SEQ ID NO:15, the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

Examples of hydrophobic modified fibroins include modified fibroins containing the amino acid sequence shown in 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, and the amino acid sequence shown in SEQ ID NO:35, 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.

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

The method for producing a nucleic acid encoding a modified fibroin is not particularly limited. For example, the nucleic acid can be produced by a method in which a gene encoding a natural fibroin is amplified and cloned by polymerase chain reaction (PCR) or the like, and modified by genetic engineering techniques, or by a chemical synthesis method. The method for chemically synthesizing a nucleic acid is also not particularly limited. For example, a gene can be chemically synthesized by a method in which oligonucleotides automatically synthesized using AKTA oligopilot plus 10/100 (GE Healthcare Japan, Ltd.) or the like are linked by PCR or the like based on amino acid sequence information of fibroin obtained from the NCBI web database or the like. At this time, in order to facilitate purification and/or confirmation of the modified fibroin, a nucleic acid encoding a modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of an initiation codon and a His10 tag is added to the N-terminus of the above amino acid sequence may be synthesized.

The regulatory sequence is a sequence (e.g., promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.) that controls the expression of the modified fibroin in the host, and can be selected appropriately depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the modified fibroin may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducer (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 according to the type of host, such as a plasmid vector, a virus vector, a cosmid vector, a fosmid vector, or an artificial chromosome vector. As an expression vector, one that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a modified fibroin can be transcribed is preferably used.

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

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

When a prokaryote is used as a host, examples of vectors into which a nucleic acid encoding a modified fibroin can be introduced include pBTrp2 (Boehringer Mannheim), pGEX (Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (JP Patent Publication No. 2002-238569).

Examples of eukaryotic hosts include yeasts and filamentous fungi (molds, etc.). Examples of yeasts include yeasts belonging to the genera Saccharomyces, Pichia, and Schizosaccharomyces. Examples of filamentous fungi include filamentous fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma.

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

In addition to direct expression, nucleic acids can be expressed by a host transformed with an expression vector through secretion production, fusion protein expression, etc., in accordance with the methods 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 it from the culture medium. The method for culturing the host in the culture medium can be performed according to a method normally used for culturing the host.

When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and allows efficient cultivation of the host.

The carbon source may be any that can be assimilated by the transformed microorganism, and examples of such carbon sources include carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch, and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of such nitrogen sources include inorganic acids or ammonium salts of organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal, soybean meal hydrolysate, various fermentation bacteria, and digests thereof. Examples of such inorganic salts include potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

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

During the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a lac promoter, and indoleacrylic acid or the like may be added to the medium when culturing a microorganism transformed with an expression vector using a trp promoter.

The expressed modified fibroin can be isolated and purified by a commonly used method. For example, when the modified fibroin is expressed in a dissolved state within the cells, after the culture is completed, the host cells are collected by centrifugation and suspended in an aqueous buffer solution, and then disrupted using an ultrasonic homogenizer, French press, Manton Gaulin homogenizer, Dyno Mill, or the like to obtain a cell-free extract. The supernatant obtained by centrifuging the cell-free extract can be used to obtain a purified sample by using methods commonly used for isolating and purifying proteins, such as solvent extraction, salting out with ammonium sulfate or the like, desalting, precipitation with an organic solvent, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei Corporation), cation exchange chromatography using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography using resins such as butyl Sepharose and phenyl Sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing, either alone or in combination.

In addition, when the modified fibroin is expressed by forming an insoluble body within the cells, the host cells are similarly recovered, disrupted, and centrifuged to recover the insoluble body of the modified fibroin as a precipitate fraction. The recovered insoluble body of the modified fibroin can be solubilized with a protein denaturant. After this operation, a purified preparation of the modified fibroin can be obtained by the same isolation and purification method as above. When the modified fibroin is secreted outside the cells, the modified fibroin can be recovered from the culture supernatant. That is, the culture is treated by a method such as centrifugation to obtain a culture supernatant, and a purified preparation can be obtained from the culture supernatant by the same isolation and purification method as above.

The flame-retardant composition according to this embodiment may contain one type of modified fibroin alone, or may contain a combination of two or more types of modified fibroin.

The limiting oxygen index (LOI) value of the modified fibroin may be 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 29 or more, or 30 or more. In this specification, the LOI value is a value measured in accordance with the test method for powdered or low-melting-point synthetic resins, Fire and Disaster Management Agency ...

The content of modified fibroin in the flame-retardant composition can be set appropriately depending on the application of the flame-retardant composition.

The content of modified fibroin in the flame retardant composition may be, for example, 10% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, and 99% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 30% by mass or less, 10% by mass or less, or 5% by mass or less, based on the total amount of the flame retardant composition.

(Biodegradable plastics)
The flame-retardant composition according to the present embodiment includes a biodegradable plastic. A biodegradable plastic is a plastic that can be completely consumed by microorganisms and produces only natural by-products (carbon dioxide, methane, water, biomass, etc.). Biodegradable plastic does not contain proteins.

Biodegradable plastics are not particularly limited, but examples include biodegradable polyesters, biodegradable cellulose, starch materials (e.g., starch, starch acetate, distarch phosphate, modified starches such as dextrin), and polyvinyl alcohol. Natural materials such as wood pulp, non-wood fibers, microalgae by-products, and seaweed by-products, as well as cellulosic materials such as rayon, can also be used as biodegradable plastics.

Examples of biodegradable polyesters include materials obtained by polymerizing polymerization raw materials such as hydroxyalkyl carboxylic acids such as lactic acid, glycolic acid, and lactic acid hydroxybutyl carboxylic acid; aliphatic lactones such as glycolide, lactide, butyrolactone, and caprolactone; aliphatic diols such as ethylene glycol, propylene glycol, butanediol, and hexanediol; and aliphatic dicarboxylic acids such as succinic acid, adipic acid, and sebacic acid.

Examples of biodegradable plastics include biodegradable polyesters, biodegradable cellulose, modified starch, and polyvinyl alcohol (PVA). Biodegradable polyesters include polyhydroxyalkanoates (PHAs) such as polylactic acid (PLA) and polyglycolic acid, polycaprolactone, polybutylene adipate/terephthalate (PBAT), polyethylene terephthalate succinate, and biopolybutylene succinate (PBS).

The biodegradable plastic is preferably a biodegradable biomass plastic that is biodegradable and contains biomass as at least a part of the raw material. Biomass plastics are attracting attention as environmentally friendly plastics because they are produced with little reliance on petroleum resources, the plants that can be used as raw materials grow by absorbing carbon dioxide, and even when disposed of by incineration, they generally have a low combustion calorie and generate a small amount of carbon dioxide, making it possible to further reduce the environmental load. Examples of biodegradable biomass plastics include biodegradable cellulose, biodegradable polyester, polyethylene terephthalate succinate, modified starch, etc. Examples of biodegradable polyesters as biodegradable biomass plastics include polyhydroxyalkanoic acids (PHAs) such as polylactic acid (PLA), polybutylene adipate terephthalate, polybutylene succinate, polyhydroxybutyrate copolymers, polybutylene succinate adipate, etc.

From the standpoint of material strength, availability, etc., the biodegradable plastic may include at least one selected from the group consisting of polylactic acid and biodegradable cellulose.

The biodegradable plastic contained in the flame retardant composition may be processed as necessary. For example, the biodegradable plastic may be processed into a resin or fiber.

The content of biodegradable plastic in the flame-retardant composition can be set appropriately depending on the application of the flame-retardant composition.

The content of biodegradable plastic in the flame retardant composition may be, for example, 5% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, and 99% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 30% by mass or less, 10% by mass or less, or 5% by mass or less, based on the total amount of the flame retardant composition.

(Flame-retardant composition)
The flame-retardant composition according to the present embodiment may further contain other additives (other than modified fibroin and biodegradable plastics) depending on the form, application, etc. Examples of additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, and synthetic resins. The content of the additives may be 50 parts by mass or less relative to 100 parts by mass of the total amount of the flame-retardant composition, and may be 20 parts by mass or less from the viewpoint of environmental conservation.

In the flame-retardant composition according to this embodiment, the modified fibroin may be in any of the following forms: powder, paste, or liquid (e.g., suspension, solution). The modified fibroin may also be in the form of, for example, fibers, films, gels, porous bodies, particles, etc. The form of the modified fibroin may be appropriately set depending on the application of the flame-retardant composition, etc. The modified fibroin may be in at least one form selected from the group consisting of, for example, liquid (e.g., suspension, solution), powder, gel, fibers, resin, film, and porous body.

When preparing the modified fibroin according to this embodiment in powder form, for example, the protein obtained by the above-mentioned method for producing modified fibroin may be dried to form a powder. The protein powder may contain other additives as necessary.

When the modified fibroin according to this embodiment is prepared in the form of a liquid (e.g., a solution), for example, the protein obtained by the above-mentioned method for producing modified fibroin may be dissolved in a solvent capable of dissolving the modified fibroin to form a liquid (modified fibroin solution). The modified fibroin solution may contain other additives as necessary. Examples of solvents capable of dissolving modified fibroin include dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP). The solvent may contain an inorganic salt as a dissolution promoter.

When the modified fibroin according to this embodiment is prepared in the form of a fiber, for example, the modified fibroin solution described above may be used as a dope liquid and spun into a fiber (modified fibroin fiber) by a known spinning method such as wet spinning, dry spinning, dry-wet spinning, or melt spinning. The form of the fiber may be a single yarn, a composite yarn such as a blended yarn, a blended fiber yarn, a blended woven yarn, an interwoven yarn, a ply-twisted yarn, or a covering yarn, or a nonwoven fabric, etc.

The modified fibroin fiber may be a short fiber or a long fiber. The modified fibroin fiber may be a modified fibroin fiber alone or may be combined with other fibers. That is, a single thread consisting of only modified fibroin fiber and a composite thread consisting of a combination of modified fibroin fiber and other fibers may be used alone or in combination. The single thread and the composite thread may be a spun thread in which short fibers are twisted together, or a filament thread in which long fibers are twisted together or not twisted together. The modified fibroin fiber, whether short or long, may be used as it is without being processed into a thread, alone or in combination with other fibers. Examples of other fibers include synthetic fibers such as nylon and polyester, regenerated fibers such as cupra and rayon, and natural fibers such as cotton and hemp. When used in combination with other fibers, the content of the modified fibroin fiber is preferably 20% by mass or more, more preferably 30% by mass or more, even more preferably 40% by mass or more, and even more preferably 50% by mass or more, based on the total amount of fibers.

When preparing the modified fibroin in the form of a film, gel, porous body, particle, etc., it can be produced in accordance with the methods described in, for example, JP 2009-505668 A, JP 2009-505668 A, Japanese Patent No. 5678283 A, Japanese Patent No. 4638735 A, etc.

The flame-retardant composition according to this embodiment can be produced, for example, by a method including mixing modified fibroin with a biodegradable plastic. The flame-retardant composition according to this embodiment may be in any form, for example, a powder, a paste, a liquid (for example, a suspension, a solution), or a sheet. The flame-retardant composition according to this embodiment may also be in the form of, for example, a fiber, a film, a gel, a porous body, a resin molded body (for example, a fiber-reinforced resin molded body), a particle, etc.

The powdered flame-retardant composition is not particularly limited, but can be produced, for example, by a method of mixing powdered modified fibroin with powdered biodegradable plastic, or by mixing pellet-shaped materials while reducing the particle size using a dry or wet ball mill, etc.

A liquid flame-retardant composition can be produced, for example, by a method including a dissolution step in which modified fibroin and a biodegradable plastic (e.g., polylactic acid) are mixed in a solvent to obtain a mixed liquid. The mixed liquid contains modified fibroin, biodegradable plastic, and a solvent (dissolution solvent) in which they are dissolved or dispersed. The mixed liquid may contain impurities that were contained together with the modified fibroin in the dissolution step. The mixed liquid may be a solution for molding a protein molded body.

The content of modified fibroin in the mixed solution in the dissolution process may be 5% by mass or more and 35% by mass or less, or 5% by mass or more and 50% by mass or less, based on the total amount of the mixed solution.

In the dissolution step, the modified fibroin to be dissolved may be purified modified fibroin, or modified fibroin in a host cell that has expressed the modified fibroin. The purified modified fibroin may be modified fibroin purified from a host cell that has expressed the modified fibroin. When the modified fibroin in the host cell is dissolved as the target protein, the host cell is contacted with a solvent, and the modified fibroin in the host cell is dissolved in the solvent. The host cell may be one that expresses the target protein, and may be, for example, an intact cell, or a cell that has been subjected to a treatment such as a disruption treatment. Alternatively, the host cell may be a cell that has already been subjected to a simple purification treatment.

The method for purifying a target protein from a host cell expressing the target protein is not particularly limited, but for example, the methods described in Japanese Patent No. 6077570 and Japanese Patent No. 6077569 can be used.

In the dissolution process, solvents that can be used to dissolve both the modified fibroin and the biodegradable plastic include DMSO, HFIP, and carboxylic acids. Examples of carboxylic acids that can be used include dicarboxylic acids such as formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, and pentanoic acid. The carboxylic acid may be in the form of an acid anhydride or an acid chloride.

In the dissolving step, the modified fibroin and the biodegradable plastic are dissolved in a solvent to obtain a mixed solution containing the modified fibroin and the biodegradable plastic. The mixed solution containing the modified fibroin and the biodegradable plastic may also be prepared by dissolving the biodegradable plastic by a method such as heating without using a solvent, and adding a protein solution containing the modified fibroin and the solvent in which the modified fibroin has been dissolved to the dissolved biodegradable plastic.

The dissolution process may be carried out at room temperature, or may be held at various heating temperatures to dissolve the modified fibroin and biodegradable plastic in the solvent. The holding time of the heating temperature is not particularly limited, but may be 10 minutes or more, and in consideration of industrial production, 10 to 120 minutes is preferable, 10 to 60 minutes is more preferable, and 10 to 30 minutes is even more preferable. The holding time of the heating temperature may be set appropriately, for example, under conditions in which the modified fibroin is sufficiently dissolved and little dissolution of impurities (substances other than the target protein) is observed.

The amount of solvent added to dissolve the modified fibroin is not particularly limited, as long as it is an amount that can dissolve the modified fibroin.

When dissolving purified modified fibroin, the amount of solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the ratio of the volume (mL) of the solvent to the weight (g) of the modified fibroin (dry powder containing modified fibroin) (volume (mL)/weight (g)).

When dissolving the modified fibroin in host cells expressing the modified fibroin, the amount of solvent added may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, or 1 to 5 times the ratio of solvent (mL) to the weight (g) of the host cells (volume (mL)/weight (g)).

If necessary, insoluble matter may be removed from the mixture containing the modified fibroin and the biodegradable plastic obtained in the dissolving step. In other words, the method for producing a liquid flame-retardant composition may include a step of removing insoluble matter from the mixture after the dissolving step, if necessary. Methods for removing insoluble matter from the mixture include common methods such as centrifugation and filter filtration using a drum filter, press filter, etc. When using filter filtration, insoluble matter can be removed more efficiently from the mixture by using a filter aid such as celite or diatomaceous earth in combination with a pre-coating agent, etc.

The liquid flame-retardant composition may contain a biodegradable plastic, a modified fibroin, and a dispersion medium in which either or both of them are dispersed. In this case, the liquid flame-retardant composition may be produced, for example, by a method including a dispersion step in which the biodegradable plastic, the modified fibroin, and either or both of them are dispersed in a dispersion medium. The biodegradable plastic, the modified fibroin, and either or both of them may be dispersed by adding them in the form of a powder to the dispersion medium.

The flame-retardant composition, which is a molded body, can be produced, for example, by a method including a molding step of molding a raw material composition containing modified fibroin and a biodegradable plastic. The raw material composition may be, for example, in a powder or liquid form. The powder or liquid composition can be produced, for example, by a method similar to the method for producing the powder or liquid flame-retardant composition described above. The molded body is not particularly limited, but examples thereof include fibers, films, porous bodies, resins, etc. The concentrations of the modified fibroin and biodegradable plastic in the raw material composition, the viscosity of the raw material composition, etc. may be appropriately adjusted depending on the application of the molded body, etc.

The method for adjusting the concentration of the modified fibroin in the raw material composition is not particularly limited, but when the raw material composition is liquid, for example, a method of increasing the concentration of the modified fibroin by volatilizing the solvent by distillation, or a method of using a modified fibroin with a high concentration in the dissolution process can be used. In addition, a method of reducing the amount of solvent added relative to the amount of modified fibroin can also be used.

The fibrous flame-retardant composition can be produced, for example, by a method including a step of spinning a dope solution containing modified fibroin, a biodegradable plastic, and a solvent in which they are dissolved (spinning step). For example, when the dope solution is applied to a coagulation liquid, the modified fibroin and biodegradable plastic in the dope solution are coagulated. In this case, by applying the dope solution as a thread-like liquid to the coagulation liquid, the mixture of modified fibroin and biodegradable plastic is coagulated into a thread-like shape, and a thread (undrawn thread) can be formed. The solvent may contain the suitable inorganic salts exemplified above.

The undrawn yarn can be formed, for example, according to the method described in Japanese Patent No. 5,584,932.

The viscosity of the dope liquid may be appropriately adjusted to a viscosity suitable for spinning. The viscosity of the dope liquid may be, for example, 10 to 50,000 cP (centipoise). The viscosity can be measured, for example, using an "EMS Viscometer" manufactured by Kyoto Electronics Manufacturing Co., Ltd. If the viscosity of the dope liquid is not within the range of 10 to 10,000 cP (centipoise), the viscosity of the dope liquid may be adjusted to a viscosity that allows spinning. There is no particular limitation on the method of adjusting the viscosity of the dope liquid. The concentration of the modified fibroin and the concentration of the biodegradable plastic in the dope liquid may be appropriately adjusted to a concentration that allows spinning. There is no particular limitation on the method of adjusting the concentration in the dope liquid.

As an example of a method for producing a fibrous flame-retardant composition, a method for producing a fibrous flame-retardant composition by wet spinning will be described below. However, the spinning method is not limited to wet spinning, and dry-wet spinning, etc. can also be used.

A fibrous flame-retardant composition produced by wet spinning can be produced, for example, by a method that includes applying a dope liquid as a filamentous liquid to a coagulation liquid to obtain an undrawn yarn, and drawing the undrawn yarn to obtain a drawn yarn.

The coagulation liquid may be any solution that can remove the solvent. The coagulation liquid preferably contains a lower alcohol having 1 to 5 carbon atoms, such as methanol, ethanol, or 2-propanol, or acetone. The coagulation liquid may contain water. From the viewpoint of spinning stability, the temperature of the coagulation liquid is preferably 5 to 30°C.

The method of applying the dope as a thread-like liquid is not particularly limited, but for example, it can be extruded from a spinning nozzle into the coagulation liquid of a desolvation tank. The undrawn thread is obtained by coagulating the modified fibroin and biodegradable plastic in the dope. The extrusion speed when extruding the dope into the coagulation liquid can be appropriately set according to the diameter of the nozzle and the viscosity of the dope. For example, in the case of a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm, the extrusion speed is preferably 0.2 to 6.0 mL/h per hole, and more preferably 1.4 to 4.0 mL/h per hole, from the viewpoint of spinning stability. The length of the desolvation tank (coagulation liquid tank) in which the coagulation liquid is placed is not particularly limited, but may be, for example, 200 to 500 mm. The take-up speed of the undrawn thread formed by coagulation of the modified fibroin and biodegradable plastic may be, for example, 1 to 14 m/min, and the residence time may be, for example, 0.01 to 0.15 min. From the viewpoint of the efficiency of desolvation, the take-up speed of the undrawn yarn is preferably 1 to 3 m/min. The undrawn yarn formed by the coagulation of the modified fibroin and the biodegradable plastic may be further drawn in the solid liquid (pre-drawing), but from the viewpoint of suppressing the evaporation of the lower alcohol used in the coagulation liquid, it is preferable to maintain the coagulation liquid at a low temperature and take up the yarn in the undrawn state from the coagulation liquid.

(b) Drawing The drawing may be a single-stage drawing or a multi-stage drawing of two or more stages. Multi-stage drawing is suitable for producing a fiber with high toughness because the molecules are oriented in multiple stages and the total draw ratio can be increased.

Methods for forming a film-like flame-retardant composition include, but are not limited to, a method in which a modified fibroin solution containing modified fibroin, a biodegradable plastic, and a solvent in which they are dissolved is applied to a solvent-resistant flat plate to a predetermined thickness to form a coating film, and the solvent is removed from the coating film to obtain a film of a predetermined thickness. If necessary, the modified fibroin solution may be adjusted to a concentration and viscosity that allows it to be formed into a film.

A method for forming a film of a predetermined thickness is, for example, a casting method. When forming a film by the casting method, a modified fibroin solution is cast onto a flat plate using a tool such as a doctor coater or knife coater to a thickness of several microns or more to form a cast film, and then the solvent is removed by drying under reduced pressure or immersion in a solvent removal tank to obtain a film-like flame-retardant composition. The film can be formed in accordance with the method described in Japanese Patent No. 5678283.

The method for forming the flame-retardant composition as a porous body is not particularly limited. For example, a method of adding an appropriate amount of a foaming agent to a modified fibroin solution containing modified fibroin, a biodegradable plastic, and a solvent that dissolves them, and removing the solvent to obtain a porous body, or a method similar to the method described in Japanese Patent No. 5796147, etc. may be used. The modified fibroin solution may be adjusted to a concentration and viscosity suitable for making the fibroin porous.

The method for forming the flame-retardant composition as a resin molded body is not particularly limited. For example, a powder containing modified fibroin and a biodegradable plastic is introduced into a pressure molding machine, and then pressure and heating are performed using a hand press or the like, so that the dry protein powder reaches the required temperature, and a flame-retardant composition as a resin molded body can be obtained. The flame-retardant composition as a resin molded body can be formed in accordance with the method described in patent document (WO2017/047503).

The method for forming the gel-like flame-retardant composition is not particularly limited. For example, the gel-like flame-retardant composition can be produced by a method including a solution generation step of dissolving modified fibroin and a biodegradable plastic in a dissolving solvent to obtain a modified fibroin solution, and a replacement step of replacing the dissolving solvent in the modified fibroin solution with a water-soluble solvent (e.g., water). At this time, between the solution generation step and the replacement step, a step of pouring the modified fibroin solution into a mold and molding it into a predetermined shape may be included. After the replacement step, a step of cutting the obtained gel-like material and molding it into a predetermined shape may be included. The formation of the gel-like flame-retardant composition can be performed, for example, in accordance with the method described in Japanese Patent No. 5782580.

The flame-retardant composition can also be used, for example, as a fiber-reinforced resin molded body. The flame-retardant composition as a fiber-reinforced resin molded body may contain modified fibroin fiber, which is modified fibroin in the form of fiber, and a biodegradable plastic. The biodegradable plastic may be impregnated into the modified fibroin fiber.

As an example of a method for producing a flame-retardant composition as a fiber-reinforced resin molded body, a method for producing a fiber-reinforced resin molded body containing modified fibroin fiber, which is modified fibroin in the form of fiber, and a biodegradable plastic impregnated into the modified fibroin fiber, is described below.

Fiber-reinforced resin moldings can be produced, for example, by a method that includes a step of impregnating modified fibroin fibers with a biodegradable plastic.

As the impregnation method, known impregnation methods such as the solvent method and hot melt method can be used. For example, a solution for the substrate resin containing a biodegradable plastic and a solvent may be dripped evenly onto modified fibroin fibers arranged on a plate-shaped substrate such as a stainless steel plate. The solvent is then volatilized and removed to form a fiber-reinforced resin molded body as a prepreg. The excess portion of the obtained fiber-reinforced resin molded body may be cut off and removed from the plate-shaped substrate.

As a method for arranging the modified fibroin fibers on a plate-shaped substrate, for example, filament winding can be used. For example, the modified fibroin fibers may be wound around a plate-shaped substrate such as a stainless steel plate while applying tension to the fibers so that no gaps are formed. From the viewpoint of imparting flame retardancy, the modified fibroin fibers used here are preferably modified spider silk fibroin fibers.

The above-mentioned fiber-reinforced resin molded bodies may be stacked to form a laminate. The number of layers in the laminate may be any number and may be set appropriately. The fiber-reinforced resin molded bodies do not have to be stacked. When the base resin is thermosetting, the base resin and the modified fibroin fiber can be bonded to each other by heating the laminate.

The manufacturing method of the fiber-reinforced resin is not limited to the above method, and existing processing methods suited to the material may be used. For example, when using thermoplastic polylactic acid as the base resin, it is not necessarily necessary to impregnate the fibers in advance. For example, a film-like polylactic acid can be placed on each side of a fiber layer formed from modified fibroin fibers, and then pressed with a small press to obtain a fiber-reinforced resin molded body in which the modified fibroin fibers are impregnated with polylactic acid.

The flame-retardant composition according to this embodiment can be used as it is as a flame-retardant molded body, for example, or can be used as a raw material for various flame-retardant molded bodies. Since the flame-retardant composition according to this embodiment has excellent flame retardancy, it can impart flame retardancy to a material (raw material) or an article by being contained in the material or article.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

[Production of modified fibroin]
(1) Preparation of an expression vector A modified fibroin (PRT799) having the amino acid sequence shown in SEQ ID NO: 15 was designed. A nucleic acid encoding the designed modified fibroin was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the termination codon. This nucleic acid was cloned into a cloning vector (pUC118). The nucleic acid was then excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

(2) Protein Expression E. 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 solution was added to 100 mL of seed culture medium containing ampicillin (Table 4) so that the OD ₆₀₀ was 0.005. The culture solution temperature was kept at 30° C., and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture was added to a jar fermenter containing 500 mL of production medium (Table 5) so that the OD ₆₀₀ was 0.05. The culture temperature was kept at 37° C., and the pH was controlled to be constant at 6.9. The dissolved oxygen concentration in the culture 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 culture temperature was kept at 37°C, and the culture was controlled to a constant pH of 6.9. The dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was continued for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of the modified fibroin. After 20 hours had passed after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the desired modified fibroin was confirmed by the appearance of a band of the desired modified fibroin size depending on the addition of IPTG.

(3) Protein purification The cells harvested 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 disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg/mL, and stirred with a stirrer at 60 ° C. for 30 minutes to dissolve. After dissolution, the mixture was dialyzed against water using a dialysis tube (Cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.) The white aggregated protein obtained after dialysis was collected by centrifugation, and the water was removed using a freeze-dryer. The freeze-dried powder was collected to obtain the modified fibroin (PRT799).

[Production of protein fibers]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved to a concentration of 4.0% by mass was prepared as a solvent, and the freeze-dried powder of modified fibroin was added thereto to a concentration of 24% by mass, and dissolved using a shaker for 3 hours. Insoluble matter and bubbles were then removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning solution was filtered at 90°C through a metal filter with 5 μm mesh, then allowed to stand in a 30 mL stainless steel syringe to degas it, and then discharged from a solid nozzle with a needle diameter of 0.2 mm into a 100% by mass methanol coagulation bath. The discharge temperature was 90°C. After coagulation, the obtained raw yarn was wound and naturally dried to obtain modified fibroin fiber (raw fiber).

[Production of knitted fabric]
The obtained raw fiber (twisted filament yarn) was used to produce a knitted fabric by circular knitting using a circular knitting machine. The knitted fabric had a thickness of 180 denier and a gauge number of 18. 20 g of the obtained knitted fabric was cut out as a test piece.

[Flammability test]
The flammability test was conducted in accordance with the test method for powdered or low melting point synthetic resins, Fire and Disaster Management Agency, Hazardous Materials Regulation Division Chief, Fire and Disaster Management Agency, Fire and Disaster Management Agency, No. 50, May 31, 1995. The test was conducted under conditions of temperature 22°C, relative humidity 45%, and atmospheric pressure 1021 hPa. The measurement results (oxygen concentration (%), flammability rate (%), converted flammability rate (%)) are shown in Table 6.

As a result of the flame retardancy test, the limiting oxygen index (LOI) value of the fabric knitted with the modified fibroin (PRT799) fiber was 27.2. Generally, an LOI value of 26 or more is considered to be flame retardant. It is clear that the modified fibroin has excellent flame retardancy. Therefore, a composition with excellent flame retardancy can be obtained by manufacturing a composition by mixing the modified fibroin with other materials, etc.

[Method for producing fiber reinforced resin]
A fiber-reinforced resin molding comprising the above-mentioned modified fibroin fiber and polylactic acid impregnated into the modified fibroin fiber was produced by the following method. Although polylactic acid was used below, the biodegradable plastic to be combined with the modified fibroin fiber is not limited to polylactic acid.

(1) Filament winding Filament winding was performed to arrange the modified fibroin fibers in a plate shape. A 150 mm x 150 mm x 4 mm stainless steel plate was placed in a filament winder (FWM-1500LF, Asahi Kasei Engineering Co., Ltd.), and the modified fibroin fibers were wound around the stainless steel plate while applying tension to the fibers so that no gaps were formed. The basis weight was approximately 210 g/ ^m2 .

(2) Preparation of polylactic acid film Pellet-shaped REVODE110 (Zhejiang Haizheng Biomaterials Co., Ltd.; melting point 163°C) was used as polylactic acid. 2.2 g of polylactic acid pellets were sandwiched between Teflon (registered trademark) sheets and preheated at 200°C for 10 minutes using a small press (H300, AS ONE Corporation), and then pressed at 10 MPa for 5 minutes to prepare a polylactic acid film. At this time, the thickness of the film was adjusted by sandwiching a 130 μm thick Teflon (registered trademark) sheet as a spacer. The obtained polylactic acid film had a size of about 140 mm x 110 mm and a thickness of about 120 μm.

(3) Preparation of prepreg One polylactic acid film prepared as described above was placed on each of the upper and lower surfaces of the fibers arranged on a stainless steel plate by filament winding. After that, the fibers were preheated at 160°C for 20 minutes using a small press (H300, AS ONE Corporation), and then pressed at 2 MPa for 5 minutes to impregnate the modified fibroin fibers with polylactic acid to obtain a prepreg. The excess part of the prepreg was cut off and removed from the stainless steel plate, and it was stored in a desiccator until use.

(4) Preparation of test pieces A release agent (Frecoat 770NC, Henkel Japan Co., Ltd.) was applied sufficiently to the inside of a 100 mm x 20 mm x 20 mm mold. The prepreg was cut to the same size as the mold, and eight sheets were laid out in the mold. After preheating at 160°C for 20 minutes using a small press (H300, AS ONE Co., Ltd.), it was pressurized at 2 MPa and maintained for 5 minutes, and then cooled to room temperature.

By using the above method, a prepreg consisting of modified fibroin fiber and polylactic acid impregnated into the modified fibroin fiber was obtained.

Claims (8)

The present invention relates to a biodegradable plastic, the biodegradable plastic comprising: a modified fibroin; and a biodegradable plastic (excluding starch and modified starch) ,
The flame-retardant composition, wherein the modified fibroin is a protein containing a domain sequence represented by formula 1: [(A) _n motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _n motif, and the domain sequence is different from the amino acid sequence of naturally occurring fibroin .
[In formula 1 and formula 2, (A) _n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. The ratio of the number of alanine residues to the total number of amino acid residues in (A) _n motif is 40% or more. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. m represents an integer of 2 to 300. A plurality of (A) _n motifs may be the same amino acid sequence as each other or different amino acid sequences. A plurality of REPs may be the same amino acid sequence as each other or different amino acid sequences.] The flame retardant composition according to claim 1, wherein the modified fibroin has a limiting oxygen index (LOI) value of 26.0 or more. The flame retardant composition according to claim 1 or 2, wherein the modified fibroin comprises modified spider silk fibroin. The flame-retardant composition according to any one of claims 1 to 3, wherein the modified fibroin contains modified fibroin having an average hydrophobicity index (average HI) of 0 or less. The flame-retardant composition according to any one of claims 1 to 4 , wherein the biodegradable plastic comprises at least one selected from the group consisting of biodegradable cellulose and biodegradable polyester. The flame-retardant composition according to any one of claims 1 to 5 , wherein the biodegradable plastic is polylactic acid. The flame retardant composition according to any one of claims 1 to 6 , wherein the flame retardant composition is in the form of a liquid, a powder, a gel, a fiber , a film, or a porous body. The flame retardant composition according to any one of claims 1 to 7 , wherein the flame retardant composition is in the form of a solution, a powder, or a fiber.

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