JPWO2018163758A1 - Molded article and method for producing molded article - Google Patents

Abstract

The present invention provides a molded article of a composition containing a polypeptide and an inorganic substance.

Description

  The present invention relates to a molded article and a method for producing the molded article.

  Attempts have been made to replace metal materials with organic materials for the purpose of reducing weight, reducing costs, facilitating molding, and the like. As such an organic material, a phenol resin having high hardness is often used, and a method of adding phenol resin fibers to a matrix resin containing the phenol resin in order to further increase the flexural modulus and flexural strength is known. (For example, see Patent Document 1). Bioplastics, which are non-petroleum-based materials, have been attracting attention due to increasing awareness of environmental conservation. For example, it is known that a molded product having a bending strength of 50.96 MPa can be obtained by converting chicken feather powder into resin. (For example, see Non-Patent Document 1).

JP 2014-80491 A

Shinji Hirai, Monthly Functional Materials Magazine, June 2014, "Environmentally-friendly silk and wool resin using animal protein derived from waste" Science, 2002, Vol. 472-476.

  However, the material disclosed in Patent Literature 1 exhibits a reinforcing effect by including fibers (reinforcing fibers) in the matrix resin, and it is difficult to achieve high strength by the matrix resin itself. Was.

  Therefore, an object of the present invention is to provide a biodegradable molded body exhibiting excellent bending strength and a method for producing the same.

  The present invention relates to a molded article of a composition containing a polypeptide, wherein the polypeptide is at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein. A molded article is provided.

  The molded article has biodegradability, is characterized by being obtained by using a polypeptide and an inorganic substance as raw materials, and being obtained by molding the raw materials. It exhibits extremely high bending strength (for example, more than 100 MPa) without using additional materials such as. Furthermore, since it is possible to impart transparency to the molded product, the applicable applications are higher than those of resins having high strength but lacking transparency, such as phenolic resins and polyetheretherketone (PEEK). There is an advantage that it increases significantly. In addition, since spider silk proteins can be modified in various ways, their performance can be easily optimized according to the end use.

  The molded article may be provided as a heat-press molded article. The molded article refers to a molded article or the like formed by a mold, and can be a molded article having excellent flexural modulus by heating and pressing.

  The molded article can be manufactured by a manufacturing method including a step of heating and pressurizing a composition containing at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.

That is, the present invention provides the following [1] to [14].
[1] A molded article of a composition containing a polypeptide and an inorganic substance.
[2] The molded article according to [1], wherein the polypeptide is a structural protein.
[3] The molded article according to [2], wherein the structural protein includes at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.
[4] The molded article according to any one of [1] to [3], wherein the inorganic substance is a metal oxide or a clay mineral.
[5] The molded article according to [4], wherein the metal oxide contains titanium oxide.
[6] The molded article according to [4], wherein the clay mineral includes smectite.
[7] A method for producing a molded article of a composition containing a polypeptide and an inorganic substance, wherein a step of mixing the polypeptide, the inorganic substance and the alcohol to obtain a mixture, and a step of heating and pressurizing the mixture. , Including.
[8] The method according to [7], wherein the polypeptide is a structural protein.
[9] The method according to [8], wherein the structural protein comprises at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.
[10] The method according to any one of [7] to [9], wherein the inorganic substance is a metal oxide or a clay mineral.
[11] A method for producing a molded article of a composition containing a polypeptide and an inorganic substance, wherein a first amount of the polypeptide, the inorganic substance, and an alcohol are mixed to obtain a first mixture; A method comprising: mixing a second amount of said polypeptide with a first mixture to obtain a second mixture; and heating and pressurizing the second mixture.
[12] The method according to [11], wherein the polypeptide is a structural protein.
[13] The method according to [12], wherein the structural protein comprises at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.
[14] The method according to any one of [11] to [13], wherein the inorganic substance is a metal oxide or a clay mineral.

  According to the present invention, a molded article having excellent bending strength can be obtained.

It is a schematic cross section of a pressure molding machine. (A) is a schematic cross-sectional view of a pressure molding machine before the introduction of the composition, (b) immediately after the introduction of the composition, and (c) in a state where the composition is heated and pressed. It is a graph which shows the measurement result of the bending strength of Example 5-7 and the comparative example 1.

  Hereinafter, a preferred embodiment of the present invention will be described. However, the present invention is not limited to the following embodiments.

  A molded article according to one embodiment of the present invention can be obtained by introducing a composition containing a polypeptide and an inorganic substance into a mold (mold) and performing molding and the like. Alternatively, pressure can be applied. The composition contains a polypeptide and an inorganic substance, and typically has a powdery form (such as a lyophilized powder) or a fibrous form (such as a fiber obtained by spinning). The molded body may be a fusion body of a composition containing the polypeptide and the inorganic substance having such a shape.

  It is preferable that the molded article has transparency. Although the transparency can be visually judged, it is preferable that the transmittance is 50% or more when an optical transmittance measuring device is used and, for example, the integration time is 0.1 second in a wavelength range of 220 to 800 nm. It is.

  Preferably, the polypeptide is a structural protein. A structural protein is a protein having a role of constructing a biological structure, and is different from functional proteins such as enzymes, hormones, and antibodies. Examples of the structural protein include naturally occurring structural proteins such as naturally occurring fibroin, collagen, resilin and elastin. Fibroin produced by insects and spiders is known as a naturally occurring fibroin.

  Examples of the fibroin produced by insects include Bombyx mori, Bombyx mandarina, Tensilae (Antheraea yamamai), tussah (Anterea perinai), and maple (Eriogina mori). ), Silkworms (Samia cynthia), chestnut worms (Caligura japonica), tussah silkworms (Antherea mylitta), silkworms such as Moga silkworms (Antheraea assama), and silkworms produced by silkworms, Vespa serrata, produced by the larvae of the hornet worm, Vespa serrata. Silk proteins.

  More specific examples of fibroin produced by insects include, for example, silkworm fibroin L chain (GenBank Accession No. M76430 (base sequence), AAA27840.1 (amino acid sequence)).

  Spiders have a maximum of seven types of silk glands, each of which produces fibroin (spider silk protein) with different properties. Spider silk proteins are classified according to their source organs into large ampoule spider proteins (MaSp) having a high toughness, minor ampoule spider proteins (MiSp) having a high degree of elongation, and flagella. It has been named a spider silk protein of flagelliform (Flag), tubular (tubuliform), aggregate (aggregate), grape-like (aciniform) and pear-form (pyriform).

  The large bottle-shaped spider protein is produced by a spider large bottle-shaped line, and has a feature of being excellent in toughness. Examples of large bottle-shaped spider proteins include large bottle-shaped spidroins MaSp1 and MaSp2 derived from the American spider (Nephilaclavipes), and ADF3 and ADF4 derived from Araneus diadematus.

  Weft proteins are those produced in the flagellaform line of spiders. The weft protein includes, for example, a flagellaform silk protein derived from the American spider, Nephila clavipes.

  Examples of the fibroin produced by the spiders include spiders belonging to the genus Araneus (genus Araneus), such as the spiders, the green spider, the red spider, the green spider, and the beetle spider, the genus such as the genus Araneus spp. Spiders belonging to the genus Argiope genus (Genus Pronus), such as spiders belonging to the genus Oribatidae, spiders belonging to the genus Pronus, and spider spiders such as the spiders belonging to the genus Cyrarachne such as the Torinofundamashi and Otrinofundashi, etc. Spiders belonging to the genus Ordgarius, such as spiders belonging to the genus (Gasteracantha), spiders belonging to the genus Oribatida, and spiders belonging to the genus Ordgarius, such as spiders belonging to the genus Ordgarius; Spiders belonging to the genus Argiope such as Argiope spider, spiders belonging to the genus Arachnura such as Argiope spiders, spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as the spiders spider, spiders belonging to the genus Spiders spiders such as the spiders spiders belonging to the genus Acusilas Spiders belonging to the genus Spider belonging to the genus Cytophora, spiders such as the spider spider (Genus Poltys), spiders belonging to the spiders belonging to the genus Poltys, spiders spiders, spiders spiders, spiders spiders belonging to the genus Cyclos sp. Spider silk proteins produced by spiders belonging to the genus Genus), as well as red-eared spiders such as Red-headed Spiders, Red-backed Spiders, Halabiroashida-gugu-suga and Urocore-Red-headed Spiders Spiders belonging to the genus Tetragnatha, spiders belonging to the genus Tetragnatha, spiders belonging to the genus Nephila, such as spiders belonging to the genus Leucauge, such as spiders belonging to the genus Leucauge and spiders belonging to the genus Nephila, such as the spider Spiders belonging to the genus Dyschiriognatha, such as spiders belonging to the genus Menosira and spiders belonging to the genus Dyschiriognatha, such as the spiders belonging to the genus Dyschiriognatha, the spiders belonging to the genus Latus sp. A spider produced by a spider belonging to the family Tetragnathidae, such as a spider belonging to the genus Prostenops. Pider silk protein. Examples of spider silk proteins include dragline proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), and MiSp (MiSp1 and MiSp2).

  More specific examples of fibroin produced by spiders include fibroin-3 (adf-3) [derived from Araneus diadematus] (GenBank Accession No. AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-4 ( adf-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number 75AAC4, amino acid sequence AAC40, amino acid sequence AAC04) Base sequence)), major angu11ate spidroin 1 [Latrodectu Hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spidroin 2 [from Nephila clavata] (GenBank accession number AAL324472 (amino acid sequence), AF4412 (amino acid sequence), AF4412 ampullate spidroin 1 [from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major ampullate spidroin 2 [Euprosthenopus actu M32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplitude silk protein 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amplitude silk protein [Nepalibank protein] Session number AAC14591.1 (amino acid sequence)) and minor ampli- nate spidroin-like protein [Nephilengys crunetata] (GenBank accession number ABR37278.1 (amino acid sequence)).

  More specific examples of naturally occurring fibroin include fibroin in which sequence information is registered in NCBI GenBank. For example, among the sequence information registered in the NCBI GenBank, from sequences including INV as DIVISION, spidroin, ampullate, fibroin, "silk and polypeptide", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a character string of a specific product from a sequence or CDS and extracting a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

  The structural protein may be a polypeptide derived from the above-mentioned natural type structural protein, that is, a recombinant polypeptide. For example, recombinant fibroin is produced in several heterologous protein production systems, and its production method utilizes a transgenic goat, a transgenic silkworm, or a recombinant plant or mammalian cell (Non-patented). Reference 2).

Recombinant fibroin can be obtained, for example, by deleting one or more of the sequences encoding the (A) _n motif from the cloned gene sequence of naturally occurring fibroin. Further, for example, an amino acid sequence corresponding to the deletion of one or more (A) _n motifs from the amino acid sequence of a naturally occurring fibroin is designed, and the nucleic acid encoding the designed amino acid sequence is chemically synthesized. You can also. In each case, in addition to the modification corresponding to the deletion of the (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 modification may be modified. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art, such as partial specific mutagenesis. Specifically, Nucleic Acid Res. 10, 6487 (1982), and Methods in Enzymology, 100, 448 (1983).

Recombinant polypeptides of the major spinal cord marker thread proteins include, for example, proteins containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . Here, in Formula 1, A represents an alanine residue, n is preferably an integer of 2 to 20, and 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16 May be an integer, and (A) the number of alanine residues relative to the total number of amino acid residues in the _n motif is preferably 40% or more, and is 60% or more, 70% or more, 80% or more, or 90% or more; Is also good.

REP indicates an amino acid sequence, and the number of amino acid residues is preferably 10 to 200 residues, and may be an amino acid sequence consisting of 10 to 150 residues, 20 to 100 residues, or 20 to 75 residues, Represents an integer of 2 to 300. Formula 1: [(A) _n motif-REP] The total number of glycine, serine, and alanine residues contained in the amino acid sequence represented by _m is preferably 40% or more, and more preferably 60%, based on the total number of amino acid residues. Or 70% or more.

The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. A plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Formula 1: [(A) _n motif-REP] includes a unit of the amino acid sequence represented by _m , and has a C-terminal sequence represented by any of SEQ ID NOS: 15 to 17 or any of SEQ ID NOs: 15 to 17 The polypeptide may be an amino acid sequence having 90% or more homology with the amino acid sequence shown in the above.

In the large spinal cord thread, the (A) _n motif corresponds to a crystalline region that forms a crystalline β sheet within the fiber, and the REP is more flexible and largely lacks a regular structure within the fiber. It corresponds to an amorphous region. [(A) _n motif-REP] corresponds to a repetitive region (repeated sequence) composed of a crystal region and an amorphous region, and is a characteristic sequence of a bookmark thread protein.

  The amino acid sequence shown in SEQ ID NO: 15 is the same as the amino acid sequence consisting of 50 amino acids at the C-terminal of the amino acid sequence of ADF3 (NCBI accession number: AAC47010, GI: 1263287), and shown in SEQ ID NO: 16. The amino acid sequence is identical to the amino acid sequence shown in SEQ ID NO: 15 by removing 20 residues from the C-terminal, and the amino acid sequence shown in SEQ ID NO: 17 is the same as the amino acid sequence shown in SEQ ID NO: 15 from the C-terminal. It is identical to the amino acid sequence with 29 residues removed.

  In Formula 1, as the polypeptide having m of 2 or more, for example, a polypeptide having the amino acid sequence shown in SEQ ID NO: 19 can be used. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 19 has an amino acid sequence of ADF3 (NCBI) to which an amino acid sequence (SEQ ID NO: 18) consisting of an initiation codon, a His10 tag, and an HRV3C protease (Humanrhinovirus 3C protease) recognition site is added at the N-terminus. (Accession number: AAC47010, GI: 1263287), which is mutated so that translation terminates at the 543rd amino acid residue.

  Further, a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 19, and having a repeating region consisting of a crystal region and an amorphous region is used. Can be. In the present invention, "1 or more" means, for example, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, Or one or several. In the present invention, "one or several" means 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 ２2 or 1

  In Formula 1, examples of the polypeptide having m of 2 or more include an ADF4-derived recombinant protein having the amino acid sequence shown in SEQ ID NO: 20. The amino acid sequence shown in SEQ ID NO: 20 is a partial amino acid sequence of ADF4 obtained from the NCBI database (NCBI accession number: AAC47011, GI: 1263289), with an initiation codon at the N-terminus, a His10 tag, and an HRV3C protease (Humanrhinovirus). 3C protease) to which an amino acid sequence consisting of a recognition site (SEQ ID NO: 18) has been added. Further, a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 20, and having a repeating region consisting of a crystal region and an amorphous region is used. You may. In Formula 1, examples of the polypeptide having m of 2 or more include a MaSp2-derived recombinant protein having the amino acid sequence shown in SEQ ID NO: 21. The amino acid sequence shown in SEQ ID NO: 21 is a partial sequence of MaSp2 obtained from the NCBI database (NCBI accession number: AAT75313, GI: 50363147), with an initiation codon at the N-terminus, a His10 tag, and an HRV3C protease (Humanrhinovirus 3C). Protease) to which an amino acid sequence consisting of a recognition site has been added. Further, a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 21 and having a repeating region consisting of a crystal region and an amorphous region is used. You may.

  In addition, a polypeptide containing the amino acid sequence represented by Formula 1 and obtained by modification based on the amino acid sequence information of the large spinal canal can be mentioned. Specifically, a polypeptide containing the amino acid sequence represented by SEQ ID NO: 9 can be mentioned. Further, a polypeptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 9 and having a repeating region consisting of a crystal region and an amorphous region is used. You may.

  Examples of the polypeptide derived from the weft thread protein include a polypeptide containing 10 or more, preferably 20 or more, more preferably 30 or more units of the amino acid sequence represented by Formula 3: REP3. When a recombinant protein is produced from a weft thread protein using a microorganism such as Escherichia coli as a host, the molecular weight is preferably 500 kDa or less, more preferably 300 kDa or less, and still more preferably, from the viewpoint of productivity. Is 200 kDa or less.

  In Formula 3, REP3 represents an amino acid sequence composed of Gly-Pro-Gly-Gly-X, and X represents one amino acid selected from the group consisting of Ala, Ser, Tyr, and Val.

  A major feature of spider silk polypeptides is that the weft protein does not have a crystalline region but has a repeating region consisting of an amorphous region. Since a large spinal cord marker thread protein has a repeating region consisting of a crystal region and an amorphous region, it is presumed to have both high stress and elasticity. On the other hand, the weft protein has a lower stress than the large spinal canal thread protein, but has high elasticity. This is thought to be because most of the weft protein is composed of amorphous regions.

  Examples of the polypeptide containing 10 or more units of the amino acid sequence represented by Formula 3: REP3 include a recombinant protein derived from a flagellar silk protein having the amino acid sequence represented by SEQ ID NO: 22. The amino acid sequence shown in SEQ ID NO: 22 is obtained from the N-terminal corresponding to the repeat portion and the motif of the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAF36090, GI: 7106224). The amino acid sequence from residues 1220 to 1659 (referred to as PR1 sequence) and the partial sequence of the flagellar silk protein of the American spider spider obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649) The C-terminal amino acid sequence from the 816th residue to the 907th residue from the C-terminus is linked, and an amino acid sequence (SEQ ID NO: 18) consisting of a start codon, a His10 tag, and an HRV3C protease recognition site is added to the N-terminus of the linked sequence. It is an amino acid sequence. Further, as a polypeptide containing 10 or more units of the amino acid sequence represented by Formula 3: REP3, one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 22. A polypeptide consisting of an amino acid sequence and having a repeating region consisting of an amorphous region may be used.

As a collagen recombinant polypeptide, for example, a protein containing a domain sequence represented by Formula 2: [REP2] _o (where, in Formula 2, o represents an integer of 5 to 300. REP2 is Gly-X X and Y each represent an amino acid residue other than Gly. A plurality of REP2s may have the same amino acid sequence or different amino acid sequences.) Can be. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 12 can be mentioned. The amino acid sequence represented by SEQ ID NO: 12 corresponds to the repeat portion and the motif of the partial sequence of human collagen type 4 obtained from the NCBI database (Genbank accession number of NCBI: CAA56335.1, GI: 3702452). The amino acid sequence represented by SEQ ID NO: 5 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from residues 301 to 540.

As a recombinant polypeptide of resilin, for example, a protein containing a domain sequence represented by Formula 4: [REP4] _p (where p represents an integer of 4 to 300 in Formula 4, and REP4 is Ser-J- 1 shows an amino acid sequence composed of J-Tyr-Gly-U-Pro, wherein J represents an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. Represents any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser.A plurality of REP4s may have the same amino acid sequence or different amino acid sequences. Good). Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 13 can be mentioned. In the amino acid sequence represented by SEQ ID NO: 13, in the amino acid sequence of resilin (NCBI Genbank, Accession No. NP 611157, Gl: 246654243), Thr at the 87th residue is replaced with Ser, and Asn at the 95th residue is substituted. In which the amino acid sequence represented by SEQ ID NO: 5 (tag sequence and hinge sequence) is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which is replaced by Asp.

  Examples of the elastin recombinant polypeptide include NCBI Genbank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine) and other proteins having an amino acid sequence. Specifically, a protein containing the amino acid sequence represented by SEQ ID NO: 14 can be mentioned. The amino acid sequence represented by SEQ ID NO: 14 corresponds to the amino acid sequence represented by SEQ ID NO: 5 (tag sequence) at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of Genbank accession number AAC98395 of NCBI. And a hinge sequence).

  The recombinant polypeptide has 90% or more of (i) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10, or (ii) the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 May be a recombinant fibroin comprising an amino acid sequence having the following sequence identity.

(I) Recombinant fibroin containing the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 10 will be described. The amino acid sequence represented by SEQ ID NO: 2 is obtained from the amino acid sequence represented by SEQ ID NO: 1 corresponding to naturally-occurring fibroin (corresponding to naturally-occurring fibroin), from the N-terminal side to the C-terminal side every two ( A) The _n- motif is deleted, and one [(A) _n- motif-REP] is inserted before the C-terminal sequence. The amino acid sequence represented by SEQ ID NO: 4 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 2 with GQX. The amino acid sequence represented by SEQ ID NO: 10 has two alanine residues inserted at the C-terminal side of each (A) _n motif of the amino acid sequence represented by SEQ ID NO: 4, and further has a partial glutamine (Q) residue. It has been replaced with a serine (S) residue, and some N-terminal amino acids have been deleted so that the molecular weight becomes almost the same as that of SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 3 is obtained by substituting all GGXs in the REP of the amino acid sequence represented by SEQ ID NO: 1 with GQX.

(Ii) A description will be given of a recombinant fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 10. (Ii) The recombinant fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 10. (Ii) The recombinant fibroin is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

  The above-mentioned recombinant fibroin may include a tag sequence at one or both of the N-terminus and the C-terminus. Thereby, isolation, immobilization, detection, visualization, and the like of the recombinant fibroin can be performed.

  Examples of the tag sequence include an affinity tag utilizing specific affinity (binding property, affinity) with another molecule. A specific example of the affinity tag is a histidine tag (His tag). The His tag is a short peptide in which about 4 to 10 histidine residues are arranged, and has a property of specifically binding to a metal ion such as nickel. Therefore, a single tag of a recombinant fibroin by metal chelating chromatography (chelating metal chromatography) is used. Can be used for separation. Specific examples of the tag sequence include, for example, the amino acid sequence represented by SEQ ID NO: 5 (amino acid sequence including a His tag).

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

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

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

  As more specific examples of the recombinant fibroin containing the tag sequence, (iii) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or (iv) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 And a recombinant fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.

  The recombinant polypeptide has 90% or more of the amino acid sequence represented by (iii) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or (iv) the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 May be a recombinant fibroin comprising an amino acid sequence having the following sequence identity.

The amino acid sequences represented by SEQ ID NOs: 6, 7, 8, 9 and 11 correspond to the amino acid sequence represented by SEQ ID NO: 5 (His tag) Is included). (Iv) Recombinant fibroin is also a protein containing a domain sequence represented by Formula 1: [(A) _n motif-REP] _m . The sequence identity is preferably 95% or more.

  Preferably, the structural protein comprises a recombinant polypeptide. By including the recombinant polypeptide as a structural protein, it is possible to adjust the flexural modulus, flexural strength and hardness of the obtained molded article to desired values.

[Recombinant cells expressing structural proteins]
The method for producing the recombinant polypeptide is described in detail below. The recombinant polypeptide of interest can be prepared, for example, by a host transformed with an expression vector having a gene sequence encoding a structural protein and one or more regulatory sequences operably linked to the gene sequence. It can be produced by expressing a gene.

  The method for producing the gene encoding the desired recombinant polypeptide is not particularly limited. For example, the gene can be produced by a method of amplifying and cloning by the polymerase chain reaction (PCR) using a gene encoding a natural structural protein, or by chemical synthesis. The method for chemically synthesizing genes is not particularly limited. For example, AKTA oligopilot plus 10/100 (GE Healthcare Japan) based on amino acid sequence information of structural proteins obtained from the NCBI web database or the like. The gene can be chemically synthesized by a method of linking oligonucleotides automatically synthesized by the method such as PCR. At this time, in order to facilitate purification and confirmation of the protein, a gene encoding a polypeptide having an amino acid sequence consisting of an initiation codon and a His10 tag added to the N-terminal of the above amino acid sequence may be synthesized.

  The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, a promoter, an enhancer, a ribosome binding sequence, a transcription termination sequence, and the like), and can be appropriately selected depending on the type of the host. An inducible promoter that functions in a host cell and can induce the expression of a target protein may be used as the promoter. An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or a physical factor 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 virus vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like. Expression vectors that can replicate autonomously in a host cell or can be integrated into the host chromosome, and that contain a promoter at a position where the gene encoding the recombinant polypeptide of interest can be transcribed are preferred. Used for

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

  Preferred examples of prokaryotes include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, and the like.

  Examples of a vector into which a gene encoding a recombinant polypeptide of interest is introduced include, for example, pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSuex, pET22b, pCold, pUB110, pNCO2 See JP-A-2002-238569) and the like.

  Eukaryotic hosts include, for example, yeasts and filamentous fungi (such as mold).

  Examples of the yeast include yeast belonging to the genera Saccharomyces, Pichia, Schizosaccharomyces, and the like. Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and the like.

  Examples of the vector include YEP13 (ATCC37115), YEp24 (ATCC37051) and the like.

  As a method for introducing the expression vector into the host cell, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast, protoplast, lithium acetate, and competent methods.

  As a method for expressing a gene using a host transformed with an expression vector, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition, and the like. .

  The recombinant polypeptide of interest can be obtained, for example, by culturing a host transformed with the expression vector according to the present invention in a culture medium, producing and accumulating the protein in the culture medium, and collecting the protein from the culture medium. Can be manufactured. The method of culturing the host according to the present invention in a culture medium can be performed according to a method generally used for culturing a host.

  When the host according to the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium of the host according to the present invention contains a carbon source, nitrogen source, inorganic salts, and the like that can be utilized by the host. However, either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the host.

  The carbon source may be any as long as the transformed microorganism can assimilate, for example, glucose, fructose, sucrose, and molasses containing these, carbohydrates such as starch and starch hydrolyzate, acetic acid and propionic acid, and the like. Organic acids and alcohols such as ethanol and propanol can be used.

  As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermentation cells and digests thereof can be used.

  As the inorganic salts, for example, potassium potassium phosphate, potassium potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

  Cultivation of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is, for example, 15 to 40 ° C. The culture time is usually 16 hours to 7 days. It is preferable to maintain the pH of the culture medium during the culturing 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.

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

  The recombinant polypeptide according to the present invention can be isolated and purified by a method generally used for isolating and purifying proteins. For example, when the recombinant polypeptide is expressed in a dissolved state in the cells, after culturing, the host cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated by a sonicator, French press. The host cells are crushed with a Manton Gaulin homogenizer and Dynomill to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a method usually used for isolating and purifying proteins, that is, a solvent extraction method, a salting out method using ammonium sulfate or the like, a desalting method, an organic solvent Precipitation method, anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia). Electrophoresis methods such as ion exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, isoelectric focusing, etc. And other methods alone or in combination. To obtain a purified sample.

  When the recombinant polypeptide is expressed as an insoluble form in the cells, the host cells are similarly recovered, crushed, and centrifuged to obtain an insoluble form of the recombinant polypeptide as a precipitate fraction. Collect. The recovered insoluble form of the recombinant polypeptide can be solubilized with a protein denaturant. After this operation, a purified preparation of the recombinant polypeptide can be obtained by the same isolation and purification method as described above.

  When the recombinant polypeptide is secreted extracellularly, the recombinant polypeptide can be recovered from the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation, and a purified sample can be obtained from the culture supernatant by using the same isolation and purification method as described above.

  The inorganic substance used in the present invention is preferably a metal oxide or a clay mineral.

  The metal oxide is not limited as long as it is a metal oxide that can exist stably. Preferred metal oxides are transition metal oxides. Examples of transition metal oxides include scandium oxide, yttrium oxide, titanium oxide, zirconium oxide, hafnium oxide, vanadium oxide, niobium oxide, tantalum oxide, chromium oxide, molybdenum oxide, tungsten oxide, and manganese (II) oxide. Manganese (III), technetium oxide, rhenium oxide, iron (II) oxide, iron (III) oxide, triiron tetroxide, ruthenium oxide, cobalt (II) oxide, cobalt (III) oxide, rhodium oxide, iridium oxide, oxide Nickel (II), nickel (III) oxide, palladium oxide, platinum oxide, copper (I) oxide, copper (II) oxide, silver (I) oxide, silver (II) oxide, silicon dioxide, magnesium oxide, aluminum oxide ( Alumina), calcium oxide, zinc oxide, bismite, cerium oxide, Of calcium, tungsten carbide, and boron oxide. A preferred metal oxide is titanium oxide.

  The crystal form of the metal oxide is not particularly limited. Preferred metal oxides are rutile crystals. When the metal oxide is a rutile-type crystal, it is more excellent in physical and chemical stability. The metal oxide is preferably subjected to a surface treatment. For example, by surface treatment, hydrophilicity or water repellency is added by aluminum hydroxide, stearic acid or the like. Specific examples of such metal oxides include TTO-51A, TTO-51C, TTO-55A, TTO-55B, TTO-55C, and TTO-55D (all trade names, manufactured by Ishihara Sangyo Co., Ltd.). Can be

  The clay mineral is a mineral mainly composed of a phyllosilicate mineral. In addition, calcite (calcite), dolomite (dolomite), feldspar (feldspar), quartz (quartz), and zeolite (zeolite) are also included in the clay minerals. A preferred clay mineral is smectite, and a more preferred clay mineral is montmorillonite or bentonite. Smectite is a swellable clay mineral, and specific examples include montmorillonite, beidellite, nontronite, saponite, hectorite, sauconite, and stevensite. In addition, bentonite is a clay containing accompanying minerals such as quartz, cristobalite, zeolite, feldspar and calcite in addition to montmorillonite which is a main mineral.

  The inorganic material preferably has a number average particle size of 30 nm or less, more preferably 20 nm or less, and even more preferably 18 nm or less.

  The composition serving as a raw material of the molded article may be a composition consisting of only a polypeptide and an inorganic substance, but further contains additional components (for example, a plasticizer, a colorant, a filler, moisture, a synthetic resin, and the like). It may be a composition. When an additional component such as a plasticizer is used, the content of the additional component is preferably set to 50% by mass or less of the total amount of the polypeptide. When an additional component such as a plasticizer is used, the content of the additional component is, for example, 0.1% by mass or more of the total amount of the polypeptide from the viewpoint of practicality. Further, contaminants generated in the process of obtaining the polypeptide may be included.

[Method for producing composition]
The composition according to the present embodiment can be produced by mixing a polypeptide and an inorganic substance. When mixing, it is preferable to use a crusher (mill). Examples of the mill include a roller mill, a jet mill, a hammer mill, a pin mill, a rotary mill, a vibration mill, a planetary mill, an attritor mill (also referred to as an attritor), and a bead mill. The preferred mill is an attritor mill. The attritor mill may be a dry attritor mill or a wet attritor mill.

  The mixing is usually carried out while putting a predetermined amount of the polypeptide and the inorganic substance in a mill and then pulverizing. It is preferable to stir the polypeptide and the inorganic substance in a solvent before removing the predetermined amount of the polypeptide and the inorganic substance into the mill, and then remove the solvent. By performing this operation before the pulverization and mixing in the mill, the inorganic substance is more uniformly dispersed in the polypeptide, so that the flexural strength and flexural modulus of the molded article are further improved.

  The solvent may be any one that does not react with the polypeptide and the inorganic substance, and is preferably one that can be easily removed by drying or the like. Examples of the solvent include alcohols such as methanol, ethanol, and propanol; ethers such as diisopropyl ether; ketones such as acetone; and water. These solvents may be used alone or in a combination of two or more. The solvent may be used in any amount as long as the polypeptide and the inorganic substance can be easily mixed. The used amount of the solvent is, for example, 20 to 100 mL, 30 to 80 mL, or 40 to 65 mL based on the total mass of the polypeptide and the inorganic substance of 10 g.

  In addition, after mixing a part of the used amount of the polypeptide (the first amount of the polypeptide) and the inorganic substance in advance, the remaining amount of the polypeptide (the second amount of the polypeptide) is added and mixed. Is more preferable. More specifically, after a part of the used amount of the polypeptide (the first amount of the polypeptide) and the inorganic substance are mixed in a solvent, the solvent is removed to prepare a premix (also referred to as a master batch). I do. The residue of the polypeptide (the second amount of the polypeptide) is added to the obtained premix, and the mixture is further mixed using a mill. By performing this operation, the inorganic substance is more uniformly dispersed in the polypeptide, so that the flexural strength and flexural modulus of the molded article are further improved.

  The amount of polypeptide used to prepare the premix (the first amount) may be 1-50% of the total amount of polypeptide used, 1-40%, 2-30%, 3-10%. It may be 20%, 4-15%, 5-15%, 6-13%, or 8-11%.

[Method of manufacturing molded article]
The molded article can be manufactured using a pressure molding machine. FIG. 1 is a schematic sectional view of a pressure molding machine that can be used for producing a molded article. The press forming machine 10 shown in FIG. 1 includes a mold 2 having a through hole formed therein and capable of heating, and an upper pin 4 and a lower pin 6 which can move up and down in the through hole of the mold 2. A composition containing a polypeptide is introduced into a space formed by inserting the upper pin 4 or the lower pin 6 into the mold 2, and the upper pin 4 and the lower side are heated while the mold 2 is heated. By compressing the composition with the pin 6, a molded article can be obtained.

  FIG. 2 shows a process chart for obtaining a molded article, wherein (a) is before introduction of the composition, (b) is immediately after introduction of the composition, and (c) is heating and pressing of the composition. It is a schematic cross section of a pressure molding machine in a state. As shown in FIG. 2A, the composition is introduced into the through hole of the mold 2 with only the lower pin 6 inserted into the through hole. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through-hole of the mold 2 and lowered to start heating of the mold 2, and the composition 8a before heating and pressurizing is passed through the through-hole. Heat and pressurize inside. The upper pin 4 is lowered until a predetermined pressure is applied, and heating and pressing are continued until the composition reaches a predetermined temperature in the state shown in FIG. 8b is obtained. Thereafter, the temperature of the mold 2 is lowered using a cooler (for example, a spot cooler), and when the composition 8b has reached a predetermined temperature, the upper pin 4 or the lower pin 6 is extracted from the mold 2, and the contents are removed. Take out. Pressing may be performed by lowering the upper pin 4 with the lower pin 6 fixed, but both lowering of the upper pin 4 and raising of the lower pin 6 may be performed.

  The heating is preferably performed at a mold temperature of 80 to 300C, more preferably 100 to 180C, and still more preferably 100 to 130C. The pressurization is preferably performed at 5 kN or more, more preferably 10 kN or more, even more preferably 20 kN or more. In addition, after reaching a predetermined heating and pressurizing condition, the time for continuing the treatment under the condition (warming condition) is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, even more preferably 5 to 30 minutes.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples unless departing from the technical idea of the present invention.

(1) Preparation of Strain Expressing Structural Protein The nucleotide sequence and amino acid sequence of fibroin (GenBank Accession No .: P468804.1, GI: 11744415) derived from Nephila clavipes were obtained from the GenBank web database and then produced. The amino acid sequence represented by SEQ ID NO: 9 was subjected to substitution, insertion and deletion of amino acid residues for the purpose of improving the sex, and the amino acid sequence represented by SEQ ID NO: 5 (tag sequence and hinge sequence) was added to the N-terminus. A recombinant fibroin having a sequence (hereinafter, also referred to as “PRT410”) was designed.

  Next, a gene encoding PRT410 was synthesized. That is, a gene was obtained in which an NdeI site was added immediately upstream of the 5 'end of the gene and an EcoRI site was added immediately downstream of the 3' end of the gene. After cloning the gene into a cloning vector (pUC118), it was treated with NdeI and EcoRI with restriction enzymes, and recombined into a protein expression vector pET-22b (+).

(2) Protein Expression Escherichia coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT410 obtained above. Transformed E. coli, after 15 hours of culture in LB medium 2mL containing ampicillin, and the culture medium, the medium 100mL seed culture shown in Table _1, was added to _{an OD 600} of 0.005. The temperature of the culture was maintained at 30 ° C., and culturing was continued for about 15 hours in a flask until the OD ₆₀₀ reached 5, to obtain a seed culture.

The obtained seed culture solution was added to a jar fermenter to which 500 mL of the production medium shown in Table 2 had been added so that the OD ₆₀₀ was 0.05. The temperature of the culture was maintained at 37 ° C., the pH was controlled to be constant at 6.9, and the culture was performed so that the dissolved oxygen concentration in the culture was maintained at 20% of the dissolved oxygen saturation concentration. In addition, ADECANOL LG-295S (made by ADEKA Corporation) was used as an antifoaming agent.

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

(3) Purification of structural protein Two hours after the addition of IPTG, the recovered cells were washed with a 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in a 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM phenylmethylsulfonyl fluoride (PMSF), and the cells were disrupted with a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with a 20 mM Tris-HCl buffer (pH 7.4) until it became highly pure. The precipitate after washing is suspended in an 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) so as to have a concentration of 100 mg / mL. For 30 minutes with a stirrer to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (Cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after the dialysis was collected by centrifugation, the water was removed with a freeze dryer, and the freeze-dried powder was collected.

(4) Production of a molded article (Comparative Example 1)
1.35 g of the freeze-dried powder (hereinafter, referred to as “sample”) obtained above was weighed, and the sample was placed in a mold 2 of a press-forming machine 10 shown in FIG. It has a rectangular through hole with a cross section of 35 mm × 15 mm.) At this time, the sample was added so that the thickness became uniform. After introducing all the samples into the through holes, the heating of the mold 2 is started, and the upper pin 4 and the lower pin 4 are moved using a hand press (trade name: NT-100H-V09, manufactured by NPa System Co., Ltd.). The sample was pressurized by inserting the side pin 6 into the through hole. At this time, the pressurizing condition of the sample was controlled to be 40 kN (27.55 MPa). When the temperature of the sample reached 130 ° C., the heating was stopped, the sample was cooled with a spot cooler (trade name: TS-25EP-1, manufactured by Trusco Nakayama Co., Ltd.), and removed when the temperature of the sample reached 50 ° C. After deburring, a rectangular parallelepiped molded article of 35 mm × 15 mm × 2 mm was obtained. That is, the heating temperature was 130 ° C., and the annealing time was 5 minutes.

(Example 1)
After mixing 9.9 g of freeze-dried powder and 0.1 g of titanium dioxide instead of freeze-dried powder, 1.35 g of the obtained composition was weighed and used as a sample. A molded article was obtained in the same manner as in Comparative Example 1 except that the heating was stopped when the temperature of the sample reached 200 ° C. The content of titanium dioxide was 1% by mass based on the total mass of the mixture, the heating temperature was 200 ° C, and the annealing time was 0 minutes.

(Example 2)
Instead of lyophilized powder, 9.9 g of lyophilized powder and 0.1 g of silica-coated titanium dioxide (trade name: TTO-51A, average particle size: 20 nm, hydrophilic, manufactured by Ishihara Sangyo Co., Ltd.) were mixed, and then 1.35 g of the composition obtained by grinding at 100 rpm for 10 minutes with a lighter mill was weighed out and used as a sample. Other than that was carried out similarly to Example 1, and obtained the molded body. The content of the silica-coated titanium dioxide was 1% by mass based on the total mass of the mixture, the heating temperature was 200 ° C, and the annealing time was 0 minutes.

(Example 3)
Instead of lyophilized powder, 9.9 g of lyophilized powder, 0.1 g of silica-coated titanium dioxide and 50 mL of ethanol were mixed, heated to 40 ° C. using a vacuum drier, and polished at 100 rpm for 10 minutes with an attritor mill. 1.35 g of the composition obtained by crushing was weighed and used as a sample. Other than that was carried out similarly to Example 1, and obtained the molded body. The content of the silica-coated titanium dioxide was 1% by mass based on the total mass of the mixture, the heating temperature was 200 ° C, and the annealing time was 0 minutes.

<Measurement method of flexural modulus and flexural strength>
The molded article obtained as described above was used as a test piece, and allowed to stand in a thermo-hygrostat (EHL, LHL-113) at 20 ° C./65% for one day, followed by autographing (Shimadzu Corporation) A three-point bending test was performed using a cage jig at AG-X manufactured by Co., Ltd.). The load cell used was 50 kN. At this time, the distance between the fulcrums of the three-point bending was fixed at 27 mm, and the measurement speed was 1 mm / min. In addition, the size of the molded product was measured with a micro-caliper, and the measurement was performed by setting the size on a jig. The flexural modulus was determined from the displacement (strain) from 0.05 to 0.25%.

<Measurement of water content>
The moisture content of the test piece at the time of measuring the bending strength was measured using a Karl Fischer moisture meter (trade name: 860 KF Thermoprep, 851 Titrando, 801 Stirrer, manufactured by Metrohm). At this time, the test piece was heated to 180 ° C.

Table 3 below summarizes the results of Examples 1 to 3 and Comparative Example 1. The numerical value is an average value of the results obtained by performing three tests, and is described together with the standard deviation.

(Example 4)
Instead of the freeze-dried powder, after mixing 9.9 g of the freeze-dried powder and 0.1 g of montmorillonite (trade name: Montmorillonite (Aluminum Pillared Clay), manufactured by Sigma-Aldrich Japan), weigh 1.35 g of the obtained composition. This was used as a sample. Other than that was carried out similarly to Example 1, and obtained the molded body. The content of montmorillonite was 1% by mass based on the total mass of the mixture, the mixture heating temperature was 200 ° C, and the annealing time was 0 minutes.

Table 4 below summarizes the results of Example 4 and Comparative Example 1. The numerical value is an average value of the results obtained by performing three tests, and is described together with the standard deviation.

(Example 5)
Instead of the freeze-dried powder, 9.9 g of the freeze-dried powder and 0.1 g of titanium oxide (trade name: TTO-51A, average particle diameter: 20 nm, hydrophilic, manufactured by Ishihara Sangyo Co., Ltd.) were mixed, and then the mixture was mixed with an attritor mill. 1.35 g of the composition obtained by grinding at 1000 rpm for 10 minutes was weighed and used as a sample. A molded article was obtained in the same manner as in Example 1 except that the heating was stopped when the temperature of the sample reached 190 ° C. The content of titanium oxide was 1% by mass based on the total mass of the mixture, the mixture heating temperature was 190 ° C, and the annealing time was 0 minutes.

(Example 6)
Instead of the freeze-dried powder, 0.9 g of the freeze-dried powder, 0.1 g of titanium oxide (trade name: TTO-51A, average particle size: 20 nm, hydrophilic, manufactured by Ishihara Sangyo Co., Ltd.) and 50 mL of ethanol were mixed, and the pressure was reduced. After heating to 40 ° C. using a drier, 9.0 g of freeze-dried powder was added, and 1.35 g of a composition obtained by grinding at 1000 rpm for 10 minutes with an attritor mill was weighed out, and this was taken as a sample. used. Otherwise in the same manner as in Example 5, a molded article was obtained. The content of titanium oxide was 1% by mass based on the total mass of the mixture, the mixture heating temperature was 190 ° C, and the annealing time was 0 minutes.

(Example 7)
Instead of the freeze-dried powder, 9.9 g of the freeze-dried powder, 0.1 g of titanium oxide (trade name: TTO-51A, average particle size: 20 nm, hydrophilic, manufactured by Ishihara Sangyo Co., Ltd.) and 50 mL of ethanol were mixed, and the pressure was reduced. After heating to 40 ° C. using a dryer, 1.35 g of a composition obtained by grinding with an attritor mill at 1000 rpm for 10 minutes was weighed and used as a sample. Otherwise in the same manner as in Example 5, a molded article was obtained. The content of titanium oxide was 1% by mass based on the total mass of the mixture, the mixture heating temperature was 190 ° C, and the annealing time was 0 minutes.

<Measurement method of flexural modulus and flexural strength>
The molded article obtained as described above was used as a test piece, and allowed to stand in a thermo-hygrostat (EHL, LHL-113) at 20 ° C./65% for one day, followed by autographing (Shimadzu Corporation) A three-point bending test was performed using a cage jig at AG-X manufactured by Co., Ltd.). The load cell used was 50 kN. At this time, the distance between the fulcrums of the three-point bending was fixed at 27 mm, and the measurement speed was 1 mm / min. In addition, the size of the molded product was measured with a micro-caliper, and the measurement was performed by setting the size on a jig. The flexural modulus was determined from the displacement (strain) from 0.05 to 0.25%.

The results are shown in FIG. In FIG. 3, a bar graph shows bending strength (unit: MPa), and a line graph shows bending elastic modulus (unit: GPa). All of the molded products of Examples 5 to 7 had higher flexural strength and flexural modulus than the molded product of Comparative Example 1. Above all, Example 6 showed higher values in bending strength and flexural modulus. In Table 5, the numerical values are the average values of the results obtained by performing three tests, and are described together with the standard deviation.

  2 ... mold, 4 ... upper pin, 6 ... lower pin, 8a ... composition before heating and pressing, 8b ... composition after heating and pressing, 10 ... press molding machine.

Claims (14)

  A molded article of a composition containing a polypeptide and an inorganic substance.   The molded article according to claim 1, wherein the polypeptide is a structural protein.   The molded article according to claim 2, wherein the structural protein includes at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.   The molded article according to any one of claims 1 to 3, wherein the inorganic substance is a metal oxide or a clay mineral.   The molded article according to claim 4, wherein the metal oxide includes titanium oxide.   The molded article according to claim 4, wherein the clay mineral includes smectite. A method for producing a molded article of a composition containing a polypeptide and an inorganic substance,
Mixing the polypeptide, the inorganic substance and an alcohol to obtain a mixture,
Heating and pressurizing the mixture.   The method according to claim 7, wherein the polypeptide is a structural protein.   9. The method according to claim 8, wherein the structural protein comprises at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.   The method according to any one of claims 7 to 9, wherein the inorganic substance is a metal oxide or a clay mineral. A method for producing a molded article of a composition containing a polypeptide and an inorganic substance,
Mixing a first amount of the polypeptide, the mineral and an alcohol to obtain a first mixture;
Mixing a second amount of said polypeptide with a first mixture to obtain a second mixture;
Heating and pressurizing the second mixture.   The method according to claim 11, wherein the polypeptide is a structural protein.   The method according to claim 12, wherein the structural protein includes at least one selected from the group consisting of a natural spider silk protein and a polypeptide derived from the natural spider silk protein.   The method according to any one of claims 11 to 13, wherein the inorganic substance is a metal oxide or a clay mineral.

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