JP2021054986A - Flame-retardant composition - Google Patents

Abstract

To provide a flame-retardant composition having biodegradability and excellent flame retardancy.SOLUTION: There is provided a flame-retardant composition comprising a modified fibroin and a biodegradable plastic.SELECTED DRAWING: None

Description

The present invention relates to flame-retardant compositions.

From the viewpoint of environmental protection, biodegradable plastics such as polylactic acid are attracting attention, and are used in various industrial fields in the form of fibers, films, non-woven fabrics, powders, adhesives, resins and the like.

Biodegradable plastic itself does not have strong flame retardancy like conventional plastic materials made from petroleum, and it is difficult to use it for members that require flame retardancy.

Patent Document 1 discloses a method for producing a flame-retardant biodegradable plastic, which comprises adding at least one of aluminum hydroxide powder or magnesium hydroxide powder to pellets made of a biodegradable plastic raw material and molding the pellet. ..

Japanese Patent Application Laid-Open No. 08-252823

When a composition containing a biodegradable plastic such as polylactic acid is combined with a conventional flame-retardant imparting agent obtained by chemical synthesis for the purpose of imparting flame retardancy, the biodegradability characteristics may be impaired. there were.

Therefore, an object of the present invention is to provide a flame-retardant composition having biodegradability and excellent flame retardancy.

The present inventors have found that modified fibroin has excellent flame retardancy. Applying this knowledge, we have developed a new composition that has both biodegradability and flame retardancy.

The present invention relates to, for example, the following inventions.
[1]
A flame-retardant composition comprising modified fibroin and a biodegradable plastic.
[2]
The flame-retardant composition according to [1], wherein the modified fibroin has a critical oxygen index (LOI) value of 26.0 or more.
[3]
The flame-retardant composition according to [1] or [2], wherein the modified fibroin contains modified spider silk fibroin.
[4]
The flame-retardant composition according to any one of [1] to [3], wherein the modified fibroin contains a modified fibroin having an average value (mean HI) of 0 or less as a hydrophobicity index.
[5]
The flame-retardant composition according to any one of [1] to [4], wherein the biodegradable plastic contains 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 contains 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 form of the flame-retardant composition is liquid, powder, gel, fiber, resin, film or porous body.
[9]
The flame-retardant composition according to any one of [1] to [8], wherein the form of the flame-retardant composition is a solution, powder, fiber, or resin.
[10]
The flame-retardant composition according to any one of [1] to [9], wherein the form of the flame-retardant composition is a resin.

According to the present invention, it is possible to provide a flame-retardant composition having biodegradability and excellent flame retardancy. According to the present invention, it is possible to provide a molded product using the flame-retardant composition.

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

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

A conventional flame retardant imparting agent may require about 10 to 30 parts by weight with respect to 100 parts by weight of a material (for example, resin), and in many cases, about 50 parts by weight. In addition, conventional flame retardant-imparting agents are often harmful substances in their own right. For example, some bromine-based flame retardant-imparting agents generate dioxins by thermal decomposition during incineration, and phosphorus-based flame-retardant imparting agents pollute the soil environment when soil is decomposed. On the other hand, since the modified fibroin has biodegradability, when the modified fibroin is used as a flame retardant imparting agent, the environmental load should be reduced as compared with the case where the conventional flame retardant imparting agent is used. Can be done.

As a conventional flame-retardant-imparting agent, a flame-retardant-imparting agent made of natural products such as natural minerals is also known, but unlike industrial synthetic products, it is necessary to stably obtain a certain quality. Was not easy. On the other hand, since the modified fibroin can be mass-produced industrially, it is easier to stably impart the desired flame retardancy to the material.

It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a figure which shows the distribution of the value of z / w (%) of naturally-derived fibroin. It is a figure which shows the distribution of the value of x / y (%) of naturally-derived fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin. It is a schematic diagram which shows an example of the domain sequence of modified fibroin.

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

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

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

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

The "modified fibroin" may be one in which the amino acid sequence of naturally-derived fibroin is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of naturally-derived fibroin (for example, cloned naturally-derived). It may be an amino acid sequence modified by modifying the gene sequence of fibroin, or an artificially designed and synthesized product that does not depend on naturally occurring fibroin (for example, a nucleic acid encoding the designed amino acid sequence). It may have a desired amino acid sequence by chemical synthesis).

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to the (A) n} motif of an amino acid sequence) and an amorphous region (typically to the REP of an amino acid sequence). It is an amino acid sequence that produces (corresponding to.), And is represented by the formula 1: [(A) _n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed only of alanine residues). A plurality of (A) _n motifs present in the domain sequence may be composed of at least seven alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

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

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

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

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

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

More specific examples of spider silk proteins produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)). fibroin-4 (aff-4) [derived from Araneus diadematus] (GenBank accession numbers AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank sequence No. AAC47011 (amino acid sequence), U47856 (base sequence)) ), U37520 (base sequence)), major amplifier spidroin 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein (derived from radinic protein) Numbers AAL32472 (amino acid sequence), AF441245 (base sequence)), major amplifier protein 1 [derived from Europe protein australis] (GenBank accession numbers CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major protein (GenBank accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila proteins] (GenBank accession number AAC1458.91 (amino acid sequence)), minor amplifier clavipes] (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nefilengys cruisetata] (GenBank access) The number ABR3778.1 (amino acid sequence) and the like can be mentioned.

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

The modified fibroin according to the present embodiment may be modified silk fibroin (modified amino acid sequence of silk protein produced by spiders), or modified spider silk fibroin (spider silk protein produced by spiders). It may be a modified amino acid sequence). As the modified fibroin, modified spider silk fibroin is preferable because it is more excellent in flame retardancy.

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

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

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

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

As a more specific example of the first modified fibroin, the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk product ADF3 KaiLargeNRSH1), or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 and 90 A modified fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned. The sequence identity is preferably 95% or more.

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

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

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

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

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

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

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

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

Here, z / w in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types of arachnid-derived fibroin were extracted) were extracted. Naturally derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m among all the extracted fibroins, and the content ratio of the amino acid sequence consisting of GGX in the fibroin is 6% or less. From the amino acid sequence of fibroin, z / w was calculated by the above-mentioned calculation method. The result is shown in FIG. The horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).

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

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

The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residue and glutamate (E) residue are preferred, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

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

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

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

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

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

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

The second modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of modified fibroin.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of modified fibroin by metal chelating chromatography (chelating metal chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).

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

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

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

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

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

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

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

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

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

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

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

The third modification fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal C-terminal one to three toward the side of the (A) _n motif every one (A) _n motif It may have an amino acid sequence corresponding to the deletion of.

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

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

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

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from the modified fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid 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) _It has an arrangement called n motifs.

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

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

In Figure 1, when the 1 more lesser of amino acid residues, the ratio of the other amino acid residues is from 1.8 to 11.3 a set of _{[(A) n} motif -rep] Unit Shown by solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are the N-terminals 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 amino acid sequence shown by (including His tag sequence and hinge sequence) is added.

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

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

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

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

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

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

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

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.

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

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

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

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

For the hydrophobicity index of amino acid residues, a known index (Hydropathic index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic protein, B. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

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

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The amino acid sequences 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 are all residual glutamine. The group content is 9% or less (Table 2).

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

The modified fibroins of (6-ii) are 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. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-ii) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

The sixth modified fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of modified fibroin.

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

The amino acid sequences shown by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are, respectively, SEQ ID NO: 25. , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence. The amino acid sequence (including His tag sequence and hinge sequence) is added. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39. , 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 fibroins of (6-iii) are 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. It may consist of the indicated amino acid sequence.

The modified fibroins of (6-iv) are 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. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The modified fibroin of (6-iv) is also a domain represented by _{formula 1: [(A) n} motif-REP] _m or formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

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

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

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

The modified fibroin is preferably a hydrophilic modified fibroin because it is more excellent in flame retardancy. In the present specification, "hydrophilic modified fibroin" is a value obtained by obtaining the total hydrophobicity index (HI) of all amino acid residues constituting the modified fibroin, and then dividing the total by the total number of amino acid residues. It is a modified fibroin having (average HI) of 0 or less. The hydrophobicity index is as shown in Table 1. In addition, modified fibroin having an average HI of more than 0 may be referred to as hydrophobic modified fibroin.

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

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

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

The method for producing the nucleic acid encoding the modified fibroin is not particularly limited. For example, the nucleic acid is produced by a method of amplifying and cloning by a polymerase chain reaction (PCR) or the like using a gene encoding natural fibroin and modifying it by a genetic engineering method, or a method of chemically synthesizing it. can do. The chemical synthesis method of nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of fibroin obtained from NCBI's web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. Genes can be chemically synthesized by ligating automatically synthesized oligonucleotides by PCR or the like. At this time, in order to facilitate purification and / or confirmation of the modified fibroin, a nucleic acid encoding the modified fibroin consisting of an amino acid sequence in which an amino acid sequence consisting of a start codon and a His10 tag is added to the N-terminal of the above amino acid sequence is synthesized. You may.

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

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. As the expression vector, a vector containing a promoter at a position capable of autonomous replication in a host cell, integration into a host chromosome, and transcription of a nucleic acid encoding modified fibroin is preferably used.

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

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

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

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

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

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

The modified fibroin can be produced, for example, by culturing a host transformed with an expression vector in a culture medium, producing and accumulating the modified fibroin in the culture medium, and collecting the modified fibroin from the culture medium. The method of culturing the host in the culture medium can be carried out according to the method usually used for culturing the host.

When the host is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and the host can be efficiently cultured. If so, either a natural medium or a synthetic medium may be used.

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

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

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

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

When the modified fibroin is expressed by forming an insoluble matter in the cells, the insoluble matter of the modified fibroin is recovered as a precipitate fraction by similarly collecting the host cell, crushing it, and centrifuging it. The insoluble form of the recovered modified fibroin can be solubilized with a protein denaturing agent. After the operation, a purified preparation of modified fibroin can be obtained by the same isolation and purification method as described above. When the modified fibroin is secreted extracellularly, the modified fibroin can be recovered from the culture supernatant. That is, a purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant, and using the same isolation and purification method as described above from the culture supernatant.

The flame-retardant composition according to the present embodiment may contain one modified fibroin alone, or may contain two or more modified fibroins in combination.

The critical oxygen index (LOI) value of the modified fibroin may be 18 or higher, 20 or higher, 22 or higher, 24 or higher, or 26 or higher. , 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 of powdery granules or synthetic resin having a low melting point on May 31, 1995, Fire and Disaster Management Agency Hazardous Material Regulation Division Chief Fire Danger No. 50.

The content of the modified fibroin in the flame-retardant composition can be appropriately set according to the use of the flame-retardant composition and the like.

The content of the modified fibroin in the flame-retardant composition is, 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 based on the total amount of the flame-retardant composition. , 90% by mass or more, 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. ..

(Biodegradable plastic)
The flame-retardant composition according to this embodiment contains a biodegradable plastic. Biodegradable plastics are plastics that are completely consumed by microorganisms and produce only natural by-products (carbon dioxide, methane, water, biomass, etc.). Biodegradable plastics do not contain proteins.

The biodegradable plastic is not particularly limited, and examples thereof include biodegradable polyester, biodegradable cellulose, starch material (for example, modified starch such as starch, starch acetate, distarch phosphate, and dextrin), and polyvinyl alcohol. .. As the biodegradable plastic, natural materials such as wood pulp, non-wood fibers, microalgae by-products, and seaweed by-products, or cellulosic materials such as rayon can also be used.

Examples of the biodegradable polyester include hydroxyalkylcarboxylic acids such as lactic acid, glycolic acid and hydroxybutylcarboxylic acid lactate, aliphatic lactones such as glycolide, lactide, butyrolactone and caprolactone, ethylene glycol, propylene glycol, butanediol and hexanediol. Examples thereof include materials obtained by polymerizing a polymerization raw material such as an aliphatic dicarboxylic acid such as an aliphatic diol, succinic acid, adipic acid, and sebacic acid.

Examples of biodegradable plastics include biodegradable polyester, biodegradable cellulose, modified starch, and polyvinyl alcohol (PVA). Examples of biodegradable polyesters include polyhydroxyalkanoic acid (PHA) 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 has biodegradability and contains biomass as at least a part of the raw material. Bioplastics are produced with almost no reliance on petroleum resources, the plants that can be used as raw materials absorb carbon dioxide and grow, and even when they are discarded by incineration, they generally have low calories burned and generate carbon dioxide. It is attracting attention as a plastic that is friendly to the global environment because of its small amount, and it is possible to further reduce the environmental load. Examples of the biodegradable biomass plastic include biodegradable cellulose, biodegradable polyester, polyethylene terephthalate succinate, modified starch and the like. Examples of the biodegradable polyester as a biodegradable biomass plastic include polyhydroxyalkanoic acid (PHA) such as polylactic acid (PLA), polybutylene adipate terephthalate, polybutylene succinate, polyhydroxybutyrate copolymer, and poly. Examples thereof include butylene succinate adipate.

From the viewpoint of material strength, availability, and the like, the biodegradable plastic may contain 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 if necessary. For example, the biodegradable plastic may be processed into a resin or a fibrous form.

The content of the biodegradable plastic in the flame-retardant composition can be appropriately set according to the use of the flame-retardant composition and the like.

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

(Flame-retardant composition)
The flame-retardant composition according to the present embodiment may further contain other additives (other than modified fibroin and biodegradable plastic) depending on its form, use and the like. Additives include, for example, plasticizers, leveling agents, cross-linking agents, crystal nucleating agents, antioxidants, UV absorbers, colorants, fillers, and synthetic resins. The content of the additive may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of the flame-retardant composition, and may be 20 parts by mass or less from the viewpoint of environmental protection.

In the flame-retardant composition according to the present embodiment, the modified fibroin may be in any form of, for example, powder, paste, or liquid (for example, suspension, solution). The modified fibroin may also be in the form of, for example, fibers, films, gels, porous bodies, particles and the like. The form of the modified fibroin may be appropriately set according to the use of the flame-retardant composition and the like. The modified fibroin may be, for example, at least one form selected from the group consisting of liquids (eg, suspensions, solutions), powders, gels, fibers, resins, films, and porous bodies.

When the modified fibroin according to the present embodiment is prepared in powder form, for example, the protein obtained by the above-mentioned method for producing modified fibroin may be dried and made into powder. The protein powder may contain other additives, if desired.

When the modified fibroin according to the present embodiment is prepared in the form of a liquid (for example, a solution), for example, the protein obtained by the above-mentioned method for producing modified fibroin is dissolved in a solvent in which the modified fibroin can be dissolved to form a liquid (modified fibroin). It may be a solution). The modified fibroin solution may contain other additives, if desired. Examples of the solvent capable of dissolving the modified fibroin include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, hexafluoroisopropanol (HFIP) and the like. An inorganic salt may be added to the solvent as a dissolution accelerator.

When the modified fibroin according to the present embodiment is prepared in the form of fibers, for example, the above-mentioned modified fibroin solution is used as a dope solution and spun by a known spinning method such as wet spinning, dry spinning, dry wet spinning or melt spinning. It may be a fiber (modified fibroin fiber). The form of the fiber may be a single yarn, a composite yarn such as a blended yarn, a mixed fiber yarn, a mixed weaving yarn, a mixed weaving yarn, a twisted yarn, a covering yarn, or a non-woven fabric. Good.

The modified fibroin fiber may be a short fiber or a long fiber. Moreover, the modified fibroin fiber may be the modified fibroin fiber alone or may be combined with other fibers. That is, a single yarn composed of only modified fibroin fibers and a composite yarn composed of a combination of modified fibroin fibers and other fibers may be used alone or in combination thereof. The single yarn and the composite yarn may be spun yarns in which short fibers are twisted, or filament yarns in which long fibers are twisted or not twisted. Further, the modified fibroin fiber, whether it is a short fiber or a long fiber, can be used as a fiber alone or in combination with other fibers without being processed into a yarn. Examples of other fibers include synthetic fibers such as nylon and polyester, recycled fibers such as cupra and rayon, and natural fibers such as cotton and linen. 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, and more preferably 40% by mass, based on the total amount of fibers. It is more preferably more than that, and even more preferably 50% by mass or more.

When the modified fibroin is prepared in the form of a film, gel, porous body, particles, etc., for example, Japanese Patent Application Laid-Open No. 2009-505668, Japanese Patent Application Laid-Open No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 46338735, etc. It can be manufactured according to the method described in 1.

The flame-retardant composition according to the present embodiment can be produced, for example, by a method including mixing modified fibroin and a biodegradable plastic. The flame-retardant composition according to the present embodiment may be in any form of, for example, powder, paste, liquid (for example, suspension, solution), or sheet. The flame-retardant composition according to the present 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), particles or the like.

The powdery flame-retardant composition is not particularly limited, and for example, a method of mixing powdery modified fibroin and powdery biodegradable plastic, a dry or wet ball mill of a material having a pellet shape, or the like. It can be produced by a method of mixing while reducing the particle size.

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

The content of the modified fibroin in the mixed solution in the dissolution step 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 with respect to the total amount of the mixed solution.

In the lysis step, purified modified fibroin may be used as the modified fibroin to be dissolved, or modified fibroin in a host cell expressing the modified fibroin may be used. The purified modified fibroin may be modified fibroin purified from a host cell expressing the modified fibroin. When the modified fibroin in the host cell is lysed as the target protein, the host cell is brought into contact with a solvent to lyse the modified fibroin in the host cell in the solvent. The host cell may be any cell that expresses the target protein, and may be, for example, an intact cell or a cell that has undergone a treatment such as a destruction treatment. Further, the cells may have already undergone a simple purification treatment.

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

In the dissolution step, DMSO, HFIP, carboxylic acid and the like can be used as a solvent that dissolves both modified fibroin and biodegradable plastic as a solvent that can be used. As the carboxylic acid, formic acid, acetic acid, dichloroacetic acid, trifluoroacetic acid, propionic acid, butanoic acid, isobutyric acid, pentanoic acid and other dicarboxylic acids can be used. Carboxylic acids may be in the form of acid anhydrides or acid chlorides.

In the dissolution step, by dissolving the modified fibroin and the biodegradable plastic in a solvent, a mixed solution containing the modified fibroin and the biodegradable plastic is obtained. The mixed solution containing the modified fibroin and the biodegradable plastic dissolves the biodegradable plastic by a method such as heating without using a solvent, and the modified fibroin and the modified fibroin are added to the dissolved biodegradable plastic. It may be prepared by adding a protein solution containing a dissolved solvent.

The melting step may be carried out at room temperature or kept at various heating temperatures to dissolve the modified fibroin and the biodegradable plastic in a 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 further preferable. The holding time of the heating temperature may be appropriately set, for example, under the condition that the modified fibroin is sufficiently dissolved and the contaminants (other than the target protein) are less dissolved.

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

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

When the modified fibroin in the host cell expressing the modified fibroin is lysed, the amount of the solvent added is the ratio of the solvent (mL) to the weight (g) of the host cell (volume (mL) / weight (g)). It may be 1 to 100 times, 1 to 50 times, 1 to 25 times, 1 to 10 times, and 1 to 5 times.

The insoluble matter may be removed from the mixed solution containing the modified fibroin and the biodegradable plastic obtained in the dissolution step, if necessary. That is, the method for producing a liquid flame-retardant composition may include, if necessary, a step of removing the insoluble matter from the mixed solution after the dissolution step. Examples of the method for removing the insoluble matter from the mixed solution include general methods such as centrifugation, filter filtration such as a drum filter and a press filter. In the case of filter filtration, insoluble matter can be removed more efficiently from the mixed solution by using a filtration aid such as Celite and diatomaceous earth and a precoating agent in combination.

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

The flame-retardant composition which is a molded product 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, powdery or liquid. The powdery or liquid composition can be produced, for example, by the same method as the method for producing the powdery or liquid flame-retardant composition described above. The molded body is not particularly limited, and examples thereof include fibers, films, porous bodies, and resins. The concentrations of the modified fibroin and the biodegradable plastic in the raw material composition, the viscosity of the raw material composition, and the like may be appropriately adjusted depending on the intended use of the molded product and the like.

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 for increasing the concentration of the modified fibroin by volatilizing the solvent by distillation. , A method of using a modified fibroin having a high concentration in the dissolution step can be used. It is also possible to use a method in which the amount of the solvent added is smaller than the amount of the modified fibroin.

The fibrous flame-retardant composition is 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). be able to. For example, when the doping liquid is applied to the coagulating liquid, the modified fibroin and the biodegradable plastic in the doping liquid coagulate. At this time, by applying the dope liquid as a filamentous liquid to the coagulation liquid, the mixture of the modified fibroin and the biodegradable plastic is solidified into a filament, and a yarn (undrawn yarn) can be formed. The solvent may contain a suitable inorganic salt exemplified above.

The undrawn yarn can be formed, for example, according to the method described in Japanese Patent No. 5584932.

The viscosity of the doping solution may be appropriately adjusted to a viscosity suitable for spinning. The viscosity of the doping solution may be, for example, 10 to 50,000 cP (centipoise). The viscosity can be measured using, for example, the trade name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd. When the viscosity of the doping liquid is not within the range of 10 to 10,000 cP (centipoise), the viscosity of the doping liquid may be adjusted to a viscosity that allows spinning. The method for adjusting the viscosity of the doping solution is not particularly limited. The concentration of the modified fibroin and the concentration of the biodegradable plastic in the doping solution may be appropriately adjusted to a concentration capable of spinning. The method for adjusting the concentration in the doping solution is not particularly limited.

Hereinafter, 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, but the spinning method is not limited to wet spinning and is dry-wet. Spinning or the like can also be used.

The fibrous flame-retardant composition produced by wet spinning is obtained, for example, by applying a dope liquid as a filamentous liquid to a coagulating liquid to obtain an undrawn yarn, and to draw an undrawn yarn to obtain a drawn yarn. It can be manufactured by a method including.

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

The method of applying the doping liquid as a thread-like liquid is not particularly limited, and examples thereof include a method of extruding the doping liquid from a spinneret into a coagulating liquid in a solvent removal tank. Undrawn yarn is obtained by solidifying the modified fibroin and biodegradable plastic in the doping solution. The extrusion speed when the doping liquid is extruded into the coagulating liquid can be appropriately set according to the diameter of the base, the viscosity of the doping liquid, and the like. For example, in the case of a syringe pump having a nozzle having a diameter of 0.1 to 0.6 mm, the extrusion speed is preferably 0.2 to 6.0 mL / h per hole from the viewpoint of spinning stability. More preferably, 1.4 to 4.0 mL / h. The length of the desolvation tank (coagulant tank) in which the coagulant is placed is not particularly limited, but may be, for example, 200 to 500 mm. The take-up speed of the undrawn yarn formed by solidification of the modified fibroin and the 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. The take-up speed of the undrawn yarn is preferably 1 to 3 m / min from the viewpoint of solvent removal efficiency. The undrawn yarn formed by coagulation of the modified fibroin and the biodegradable plastic may be further drawn (pre-stretched) in a solid liquid, but from the viewpoint of suppressing evaporation of the lower alcohol used in the coagulation liquid, the coagulation liquid is used. It is preferable to keep the temperature at a low temperature and remove the undrawn yarn from the coagulating liquid.

(B) Stretching The stretching may be one-step stretching or multi-step stretching of two or more steps. When drawn in multiple stages, the molecules can be oriented in multiple stages and the total draw ratio can be increased, which is suitable for producing fibers with high toughness.

The method for forming the flame-retardant composition in the form of a film is not particularly limited, but a predetermined fibroin solution containing a modified fibroin, a biodegradable plastic and a solvent in which these are dissolved is placed on a solvent-resistant flat plate. Examples thereof include a method of obtaining a film having a predetermined thickness by applying the film to a thickness to form a coating film and removing the solvent from the coating film. If necessary, the modified fibroin solution may be adjusted to a concentration and viscosity capable of filming.

Examples of the method for forming a film having a predetermined thickness include a casting method. When forming a film by the casting method, a modified fibroin solution is cast on a flat plate to a thickness of several microns or more using a jig such as a doctor coat or a knife coater to form a cast film, and then dried under reduced pressure or removed. A film-like flame-retardant composition can be obtained by desorbing the solvent by immersing it in a solvent tank. The film can be formed according to the method described in Japanese Patent No. 5678283.

The method for forming the flame-retardant composition which is a porous body is not particularly limited. For example, a method for obtaining a porous body by adding an appropriate amount of a foaming agent to a modified fibroin solution containing modified fibroin, a biodegradable plastic and a solvent in which they are dissolved, and removing the solvent, or patent No. 5796147. Examples include performing according to the method used. The modified fibroin solution may be adjusted to a concentration and viscosity suitable for porosification.

The method for forming the flame-retardant composition which is a resin molded product is not particularly limited. For example, by introducing a powder containing modified fibroin and biodegradable plastic into a pressure molding machine and then pressurizing and heating using a hand press machine or the like, the dried protein powder reaches the required temperature, and the resin molded product A flame-retardant composition is obtained. The flame-retardant composition, which is a resin molded product, can be formed according to the method described in Patent Document (WO2017 / 047503).

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

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

Hereinafter, as an example of a method for producing a flame-retardant composition as a fiber-reinforced resin molded product, a modified fibroin fiber which is a modified fibroin in the form of a fiber and a biodegradable plastic impregnated in the modified fibroin fiber are included. A method for manufacturing a fiber-reinforced resin molded product will be described.

The fiber-reinforced resin molded product can be produced, for example, by a method including a step of impregnating modified fibroin fibers with biodegradable plastic.

As the impregnation method, a known impregnation method such as a solvent method or a hot melt method can be used. For example, a solution for a base resin containing, for example, a biodegradable plastic and a solvent may be uniformly dropped onto the modified fibroin fibers arranged on a plate-like base material such as a stainless steel plate. Then, when the solvent is volatilized and removed, a fiber-reinforced resin molded product can be formed as a prepreg. The obtained fiber-reinforced resin molded product may be removed from the plate-like base material by cutting an excess portion.

As a method of arranging the modified fibroin fibers on the plate-like substrate, for example, filament winding can be used. For example, the modified fibroin fiber may be wrapped around a plate-like base material such as a stainless steel plate so as to form a gap while applying tension. The modified fibroin fiber used here is preferably a modified spider silk fibroin fiber from the viewpoint of imparting flame retardancy.

The above-mentioned fiber-reinforced resin molded product may be laminated to form a laminated body. The number of layers in the laminate may be any number and may be set as appropriate. The fiber-reinforced resin molded product does not have to be laminated. When the base resin is thermosetting, the base resin and the modified fibroin fiber can be brought into close contact with each other by heating the laminate.

The method for producing the fiber reinforced resin is not limited to the above method, and an existing processing method suitable for the material may be used. For example, when thermoplastic polylactic acid is used as the base resin, it is not always necessary to impregnate the fibers in advance. For example, a film-like polylactic acid is placed on both sides of a fiber layer formed of modified fibroin fibers one by one, and then pressed by a small press to make the modified fibroin fibers impregnated with polylactic acid. A reinforced resin molded body can be obtained.

The flame-retardant composition according to the present embodiment can be used as it is as, for example, a flame-retardant molded product, or can be used as a raw material for various flame-retardant molded products. Since the flame-retardant composition according to the present embodiment is excellent in flame retardancy, flame retardancy can be imparted to the material or article by incorporating it into the material (raw material) or article.

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

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

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

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the cells were cultured under constant pH 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

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

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL at 60 ° C. Was stirred with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white agglutinated protein obtained after dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain modified fibroin (PRT799).

[Manufacturing of protein fibers]
Dimethyl sulfoxide (DMSO) in which LiCl was dissolved so as to be 4.0% by mass was prepared as a solvent, and lyophilized powder of modified fibroin was added thereto to a concentration of 24% by mass, and a shaker was used. It was dissolved for 3 hours. Then, the insoluble matter and bubbles were removed to obtain a modified fibroin solution (spinning stock solution).

The prepared spinning stock solution is filtered at 90 ° C. with a metal filter having an opening of 5 μm, then allowed to stand in a 30 mL stainless syringe to defoam, and then 100% by mass methanol solidified from a solid nozzle having a needle diameter of 0.2 mm. It was discharged into the bathtub. The discharge temperature was 90 ° C. After solidification, the obtained raw yarn was wound up and air-dried to obtain modified fibroin fiber (raw material fiber).

[Manufacturing of knitted fabric]
Using the obtained raw material fibers (twisted filament yarns), a knitted fabric was produced by circular knitting using a circular knitting machine. The knitted fabric had a thickness of 180 denier and a gauge of 18. 20 g was cut out from the obtained knitted fabric to prepare a test piece.

[Combustibility test]
The flammability test was based on the Fire and Disaster Management Agency Dangerous Goods Regulation Division Chief Fire Danger No. 50, the test method for powdered or low melting point synthetic resins on May 31, 1995. The test was carried out under the conditions of a temperature of 22 ° C., a relative humidity of 45% and an atmospheric pressure of 1021 hPa. Table 6 shows the measurement results (oxygen concentration (%), combustion rate (%), converted combustion rate (%)).

As a result of the flame retardancy test, the critical oxygen index (LOI) value of the knitted fabric knitted with the modified fibroin (PRT799) fiber was 27.2. Generally, if the LOI value is 26 or more, it is considered to be flame-retardant. It can be seen that the modified fibroin is excellent in flame retardancy. Therefore, a composition having excellent flame retardancy can be obtained by producing a composition by mixing the modified fibroin with another material or the like.

[Manufacturing method of fiber reinforced plastic]
A fiber-reinforced resin molded product composed of the above-mentioned modified fibroin fiber and polylactic acid impregnated in the modified fibroin fiber was produced by the following method. In the following, polylactic acid is used, but 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 × 150 mm × 4 mm stainless steel plate was installed in a filament winder (FWM-1500LF, Asahi Kasei Engineering Co., Ltd.), and the modified fibroin fiber was wound around the stainless steel plate so as not to have a gap while applying tension. The basis weight was about 210 g / m ² .

(2) Preparation of Polylactic Acid Film As polylactic acid, pelletized REVODE110 (Zhejiang Haichang Biomaterials Co., Ltd .; melting point 163 ° C.) was used. 2.2 g of polylactic acid pellets are sandwiched between Teflon (registered trademark) sheets, preheated at 200 ° C. for 10 minutes by a small press (H300, AS ONE Corporation), and then pressurized at 10 MPa for 5 minutes to form a polylactic acid film. Made. At this time, the thickness of the film was adjusted by sandwiching a Teflon (registered trademark) sheet having a thickness of 130 μm as a spacer. The obtained polylactic acid film had a size of about 140 mm × 110 mm and a thickness of about 120 μm.

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

(4) Preparation of test piece A mold release agent (Frecoat 770NC, Henkel Japan Ltd.) was sufficiently applied to the inside of a 100 mm × 20 mm × 20 mm mold. After cutting the prepreg to the same size as the mold, eight prepregs were spread on the mold. After preheating at 160 ° C. for 20 minutes with a small press (H300, AS ONE Corporation), the mixture was pressurized at 2 MPa, maintained for 5 minutes, and then allowed to cool to room temperature.

By the above method, a prepreg composed of modified fibroin fiber and polylactic acid impregnated in the modified fibroin fiber was obtained.

Claims (10)

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

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