JPWO2019117296A1 - Manufacturing method of molded product and molded product - Google Patents

Abstract

A method for producing a molded product is provided, which comprises a step 1 of molding a composition containing a structural protein and a step 2 of compressing and stretching the molding composition obtained in the step 1.

Description

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

Attempts have been made to replace metal materials with organic materials for the purpose of weight reduction, cost reduction, facilitation of molding, and the like. As such an organic material, a phenol resin having a high hardness is often used, and in order to further increase the bending elasticity and bending strength, a method of adding a phenol resin fiber to a matrix resin containing the phenol resin is known. (See, for example, Patent Document 1). In addition, bioplastics, which are non-petroleum materials, are attracting attention due to heightened awareness of environmental conservation. For example, it is known that a molded product having a flexural modulus of 4.5 GPa can be obtained by resinifying silk powder. (See, for example, Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2014-80491

Shinji Hirai, Monthly Functional Materials Magazine, June 2014, "Environmentally Friendly Silk and Wool Resin Using Waste-Derived Animal Proteins" Science, 2002, Volume 295, pp. 472-476

However, the material disclosed in Patent Document 1 exerts a reinforcing effect by containing fibers (reinforcing fibers) in the matrix resin, and it is difficult to achieve high strength with the matrix resin itself. It was. In addition, it was not possible to obtain a molded product having a flexural modulus exceeding 4.5 GPa from a biodegradable material.

Therefore, an object of the present invention is to provide a method for producing a biodegradable molded product having high strength and a biodegradable molded product having high strength.

The present invention provides the following [1] to [10].
[1] A method for producing a molded product, which comprises a step 1 of molding a composition containing a structural protein and a step 2 of compressing and stretching the molding composition obtained in the above step 1.
[2] The method according to [1], wherein the step 1 comprises heating and pressurizing the composition.
[3] The method according to [1] or [2], wherein the step 2 comprises heating the molding composition.
[4] The method according to any one of [1] to [3], wherein the above step 2 is repeated a plurality of times.
[5] The method according to any one of [1] to [4], which comprises a step 3 of drying the molding composition at least before the step 2 and after the step 2.
[6] The method according to any one of [1] to [5], wherein the structural protein is spider silk fibroin.
[7] A molded product of a composition containing a structural protein and having a flexural modulus exceeding 8.47 GPa.
[8] A molded product having a composition containing a structural protein and having a bending strength of more than 141 MPa.
[9] A molded product of a composition containing a structural protein and having an orientation degree of 0.22 or more.
[10] The method according to any one of [7] to [9], wherein the structural protein is spider silk fibroin.

The molded product is biodegradable, is made from a structural protein as a raw material, and is characterized in that the raw material is molded, further compressed and stretched, and is caused by these characteristics. As a result, a molded product having high strength can be obtained without using an additive material such as reinforcing fibers. The molded product having high strength is, for example, a molded product having a flexural modulus of more than 8.47 GPa or a molded product having a bending strength of more than 141 MPa. The molded product having high strength is, for example, a molded product having an orientation degree of 0.22 or more.

By heating and pressurizing in the step of molding the composition, a molded product having higher strength can be obtained. By heating in the step of compressing and stretching the molding composition, a molded product having higher strength can be obtained. By repeating the steps of compressing and stretching the molding composition a plurality of times, a molded product having higher strength can be obtained.

By drying the molding composition before the step of compressing and stretching the molding composition or after the step of compressing and stretching the molding composition, a molded product having higher strength can be obtained. .. The molding composition may be dried before and after the step of compressing and stretching the molding composition.

According to the present invention, there is provided a method for producing a biodegradable molded product having high strength and a biodegradable molded product having high strength without using an additive material such as reinforcing fibers.

It is sectional drawing which shows typically the pressure molding machine. (A) is a cross section schematically showing a state before the introduction of the composition, (b) a state immediately after the introduction of the composition, and (c) a cross section schematically showing a pressure molding machine in a state where the composition is heated and pressed. It is a figure. (A) is a diagram schematically showing the first stretching step, and (b) is a diagram schematically showing the second stretching step. (A) is a diagram schematically showing another example of the stretching step, and (b) is a diagram schematically showing still another example of the stretching step. It is a flow chart which shows the manufacturing process of the molded article which concerns on Example. (A) is a photograph of a molded product according to a comparative example, and (b) to (d) are photographs of each molded product according to Examples 1 to 3.

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

The molded product according to the present embodiment is obtained by molding a composition containing a structural protein, compressing and stretching the molding composition obtained in this molding step, or the like.

[Structural protein]
Structural proteins are proteins that have a role in constructing biological structures, and are different from functional proteins such as enzymes, hormones, and antibodies. Examples of the structural protein include naturally occurring structural proteins such as fibroin, collagen, resilin, elastin and keratin. As a naturally occurring fibroin, fibroin produced by insects and arachnids is known. In this embodiment, the structural protein preferably contains a spider silk protein.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, silkworm, and silkworm. ), Silkworms (Samia cinthia), Chrysanthemums (Caligra japonica), Chusser silkworms (Antheraea mylitta), Muga silkworms (Antheraea assama) Silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms, silkworms. Silk protein can be mentioned.

More specific examples of fibroin produced by insects include silkworm fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

There are up to seven types of silk glands in spiders, each of which produces fibroin (spider silk protein) with different properties. Spider silk proteins are, according to their source organs, a large bottle of spider protein with high toughness (major amplifier spider protein, MaSp), a small bottle of spider protein with high extensibility (minor amplifier spider protein, MiSp), and a whip. They are named spider silk proteins in the form of fragelliform (Flag), tubular (tubulariform), aggregate (aggregate), grape-like (aciniform) and pear-like (pyriform).

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, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne) such as Torinofundamashi and Otorinofundamashi, spiders belonging to the genus Cyrtarachne, spiders such as spiders 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. belonging to the genus Argiope Spiders belonging to the genus Arachnura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ), 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, 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 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, 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 fibroin produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin- 4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number A) U37520 (base sequence)), major angu11ate spidroin 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline silk protein spiroin 2 [derived from Abrine silk protein spiroin 2 [N] (Amino acid sequence), AF441245 (base sequence)), major operlate spidroin 1 [derived from Euprosthenops australis] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major amplifierSpiroin 2 spiroin Accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplifier silk fibroin 1 [Nephila clavipes] (GenBank accession number AAC14589.1 (amino acid sequence)), minor complete silk fibroin (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplifier spidroin-like productin [Nephilengys cruisentata] (GenBank accession number ABR) 3778.1 (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 structural protein may be a polypeptide derived from the above-mentioned natural structural protein, that is, a recombinant polypeptide. For example, recombinant fibroin is produced in several heterologous protein production systems, and transgenic goats, transgenic silkworms, or recombinant plant or mammalian cells are used as the production method (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 naturally occurring fibroin gene sequence. Further, for example, it is obtained by designing an amino acid sequence corresponding to the deletion of one or more (A) n motifs from the amino acid sequence of naturally occurring fibroin, and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. You can also do it. 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. 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 specific mutagenesis methods. 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).

The recombinant polypeptide of the large spitting tube bookmarking protein is, for example, a protein containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m (here, (A) _{n in} formula 1). The motif shows an amino acid sequence composed of 4 to 20 amino acid residues, and (A) the number of alanine residues is 80% or more of the total number of amino acid residues in the _n motif. REP is 10 to 200 amino acid residues. Indicates an amino acid sequence composed of. M represents an integer of 8 to 300. Multiple existing (A) _n motifs may be the same amino acid sequence or different amino acid sequences. Multiple REPs may be mutually present. It may be expressed as the same amino acid sequence or different amino acid sequences.). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 12 can be mentioned.

As a recombinant polypeptide of collagen, for example, a protein containing a domain sequence represented by the formula 2: [REP2] _o (where o represents an integer of 5 to 300 in the formula 2; REP2 is Gly-X. 1 Indicates an amino acid sequence composed of Y, where X and Y indicate arbitrary amino acid residues other than Gly. Multiple REP2s may have the same amino acid sequence or different amino acid sequences.) Can be done. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 13 can be mentioned. The amino acid sequence shown in SEQ ID NO: 13 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI Genbank accession number: CAA5635.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 5 is added to the N-terminal of the amino acid sequence from the 301st residue to the 540th residue.

As a recombinant polypeptide of resilin, for example, a protein containing a domain sequence represented by the formula 3: [REP3] _p (where, in formula 3, p represents an integer of 4 to 300. REP3 is Ser-J1. The amino acid sequence composed of J-Tyr-Gly-U-Pro is shown. J indicates an arbitrary amino acid residue, and it is particularly preferable that it is an amino acid residue selected from the group consisting of Asp, Ser and Thr. Indicates an arbitrary amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. Multiple REP3s may have the same amino acid sequence or different amino acid sequences. Good.) Can be mentioned. Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 14 can be mentioned. The amino acid sequence shown in SEQ ID NO: 14 replaces Thr at residue 87 with Ser in the amino acid sequence of recillin (Axion No. NP 61157, Gl: 24654243 of NCBI Genbank), and Asn at residue 95. The amino acid sequence (tag sequence) shown by SEQ ID NO: 17 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 with Asp.

Examples of the recombinant polypeptide of elastin include proteins having amino acid sequences such as NCBI Genbank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, a protein containing the amino acid sequence shown in SEQ ID NO: 15 can be mentioned. The amino acid sequence shown by SEQ ID NO: 15 is the amino acid sequence shown by SEQ ID NO: 5 at the N-terminal of the amino acid sequence from the 121st residue to the 390th residue of the amino acid sequence of the accession number AAC98395 of Genbank of NCBI (tag sequence). And the hinge arrangement) are added.

Examples of the recombinant polypeptide of keratin include Type I keratin of Capra hilcus. Specifically, a protein containing the amino acid sequence shown by SEQ ID NO: 16 (amino acid sequence of NCBI Genbank accession number ACY30466) can be mentioned.

The recombinant polypeptide is 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. It may be a recombinant fibroin containing an amino acid sequence having the sequence identity of.

(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 shown in SEQ ID NO: 2 is from the amino acid sequence shown in SEQ ID NO: 1 (corresponding to naturally occurring fibroin) corresponding to naturally occurring fibroin to every other two (corresponding to the N-terminal side to the C-terminal side). A) The n motif is deleted, and one [(A) _n motif-REP] is inserted in front of the C-terminal sequence. The amino acid sequence shown in SEQ ID NO: 4 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 10, 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: 4, and a part of glutamine (Q) residue is further added. It was replaced with a serine (S) residue, and some amino acids on the N-terminal side were deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 4. The amino acid sequence shown in SEQ ID NO: 3 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 1.

(Ii) A recombinant fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10 will be described. (Ii) The recombinant fibroin contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 10. (Ii) 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 recombinant fibroin described above may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization and the like of recombinant fibroin.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. 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 lined up, and since it has the property of specifically binding to a metal ion such as nickel, it is a single recombinant fibroin obtained by metal chelating chromatography. It can be used separately. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 5 (amino acid sequence containing a His tag).

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 an antigenic peptide (epitope) 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, recombinant 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. Recombinant fibroin from which the tag sequence has been separated can also be recovered by treating the protein adsorbed via the tag sequence with a protease.

As a more specific example of the recombinant fibroin comprising a tag sequence, (iii) the amino acid sequence set forth in 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 Examples thereof include recombinant fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by.

The recombinant polypeptide is 90% or more of the amino acid sequence set forth in (iii) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or the amino acid sequence set forth in (iv) SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. It may be a recombinant fibroin containing an amino acid sequence having the sequence identity of.

The amino acid sequences shown by SEQ ID NOs: 6, 7, 8, 9 and 11 are the amino acid sequences shown by SEQ ID NO: 5 (His tag) at the N-terminal of the amino acid sequences shown by SEQ ID NOs: 1, 2, 3, 4 and 10, respectively. (Including) is added. (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.

The structural protein preferably comprises a recombinant polypeptide. By including the recombinant polypeptide as the structural protein, it is possible to adjust the flexural modulus, bending strength and hardness of the obtained molded product to desired values. Further, the degree of orientation of the obtained molded product can be adjusted to a desired value.

[Recombinant cells expressing structural proteins]
The method for producing the recombinant polypeptide will be described in detail below. The recombinant polypeptide of interest is 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 recombinant polypeptide of interest is not particularly limited. For example, a gene encoding a natural structural protein can be used to amplify and clone it by a polymerase chain reaction (PCR) or the like, or the gene can be produced by chemical synthesis. The chemical synthesis method of the gene is not particularly limited, and for example, based on the amino acid sequence information of the structural protein obtained from the NCBI web database, etc., AKTA oligonucleotide plus 10/100 (GE Healthcare Japan Co., Ltd.), etc. The gene can be chemically synthesized by a method of linking the oligonucleotides automatically synthesized in (1) by PCR or the like. At this time, in order to facilitate purification and confirmation of the protein, a gene encoding a polypeptide having an amino acid sequence consisting of a start 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, etc.), and can be appropriately selected depending on the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the protein of interest may be used. An inducible promoter is a promoter that can control transcription by the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

The type of expression vector can be appropriately selected depending on the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a phosmid vector, and an artificial chromosome vector. The expression vector preferably contains a promoter at a position capable of autonomous replication in the host cell, integration into the host chromosome, and transcription of the gene encoding the recombinant polypeptide of interest. Used for.

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

Examples of the vector into which the gene encoding the recombinant polypeptide of interest is introduced include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, and pNCO2 (specially manufactured). Kai 2002-238569) and the like can be mentioned.

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.

Examples of the vector 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 a gene 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 target recombinant polypeptide is 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 for culturing the host according to the present invention in a culture medium can be carried out according to the method usually used for culturing the host.

When the host according to the present invention is a prokaryotic organism such as Escherichia coli or a eukaryotic organism such as yeast, the culture medium of the host according to the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated 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 assimilated by the transforming microorganisms, for example, glucose, fructose, sucrose, carbohydrates such as molasses, starch and starch hydrolyzate containing them, acetic acid and propionic acid. Organic acids and alcohols such as ethanol and propanol can be used.

Nitrogen sources include, for example, 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, corn steep liquor, etc. Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and their digests 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 as needed 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 needed. For example, isopropyl-β-D-thiogalactopyranoside and the like are used when culturing microorganisms transformed with an expression vector using the lac promoter, and indol acrylic is used when culturing microorganisms transformed with an expression vector using the trp promoter. Acids and the like may be added to the medium.

The recombinant polypeptide according to the present invention can be isolated and purified by a method usually used for isolating and purifying a protein. For example, when the recombinant polypeptide 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 or a French press. , Mantongaulin homogenizer, dynomil and the like to disrupt host cells to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, a method usually used for isolating and purifying a protein, that is, a solvent extraction method, a salting out method using ammonium sulfate or the like, a desalting method, or an organic solvent is used. Precipitation method, anion exchange chromatography method using a resin such as diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), positive using a resin such as S-Sepharose FF (manufactured by Pharmacia) Ion exchange chromatography method, hydrophobic chromatography method using resin such as butyl Sepharose, phenyl Sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric point electrophoresis Purified preparations can be obtained by using the above methods alone or in combination.

When the recombinant polypeptide is expressed by forming an insoluble matter in the cell, the host cell is similarly recovered, crushed, and centrifuged to obtain an insoluble matter of the recombinant polypeptide as a precipitate fraction. To collect. The insoluble form of the recovered recombinant polypeptide can be solubilized with a protein denaturing agent. After the 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 purified sample can be obtained by treating the culture by a method such as centrifugation to obtain a culture supernatant and using the same isolation and purification method as described above from the culture supernatant.

[Manufacturing method of molded product]
The method for producing a molded product according to one embodiment is a step 1 in which a composition containing a structural protein is introduced into, for example, a mold, and a molding composition obtained in the step 1 is compressed and stretched. The step 2 of obtaining a molded product is included.

Step 1 is a step of introducing a composition containing a structural protein into a mold (mold) and molding it to obtain a molding composition (molded product). In this molding process, the composition may be heated and / or pressurized.

The composition may include a structural protein. The composition may be a structural protein only, may contain a structural protein and any additive component (eg, a plasticizer, a colorant, a filler, a synthetic resin, etc.), and may contain a plurality of types of structural proteins. You may. The content of the additive component is preferably 50% by mass or less of the total amount of structural proteins. The composition typically has a powdery (lyophilized powder, etc.) or fibrous (spinning, etc.) shape. The molding composition can be a fusion of compositions containing structural proteins of such shape.

The composition may contain an inorganic additive component such as TiO ₂ or montmorillonite.

The molding composition can be molded using, for example, a pressure molding machine. FIG. 1 is a schematic cross-sectional view of a pressure molding machine that can be used to produce a molding composition. The pressure molding machine 10 shown in FIG. 1 includes a mold 2 having a through hole formed and capable of heating, and an upper pin 4 and a lower pin 6 capable of moving up and down in the through hole of the mold 2. Is. In the pressure molding machine 10, the composition containing the structural protein is introduced into the gap formed by inserting the upper pin 4 or the lower pin 6 into the through hole of the mold 2, and the mold 2 is heated while being heated. A molded composition can be obtained by compressing the composition with the upper pin 4 and the lower pin 6.

FIG. 2 shows a process diagram for obtaining a molding composition, in which (a) is before the introduction of the composition, (b) is immediately after the introduction of the composition, and (c) is heating and pressurizing the composition. It is a schematic sectional view of the pressure molding machine in a state. As shown in FIG. 2A, the composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the composition 8a before heating and pressurizing is formed through the through hole. Heat and pressurize inside. The upper pin 4 is lowered until a predetermined pressing force is reached, and heating and pressurization are continued until the composition reaches a predetermined temperature in the state shown in FIG. 2C, and the composition after heating and pressurizing. Obtain 8b. After that, the temperature of the mold 2 is lowered by using a cooler (for example, a spot cooler), and when the molding composition 8b reaches a predetermined temperature, the upper pin 4 or the lower pin 6 is removed from the mold 2 and the contents are removed. Take out things. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lower pin 4 may be lowered and the lower pin 6 may be raised. The removed contents are dried to obtain a molding composition. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, but both the lower pin 4 may be lowered and the lower pin 6 may be raised.

The heating in step 1 is preferably performed at a mold temperature of 80 to 300 ° C., more preferably 100 to 180 ° C., and even more preferably 100 to 130 ° C. Pressurization is preferably performed at 5 kN or higher, more preferably 10 kN or higher, and even more preferably 20 kN or higher. Further, after reaching a predetermined heating and pressurizing condition, the time (heat retention condition) for continuing the treatment under that condition is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, still more preferably 5 to 30 minutes.

Before step 1, a step of adding water to the composition containing the structural protein may be performed. The amount of water added in the step is preferably 10 to 40 wt% and more preferably 20 to 30 wt% with respect to the mass of the structural protein.

In that case, as shown in FIG. 2A, the water-containing composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2, but water is added before or after the introduction. Stir on top. Subsequently, as shown in FIG. 2B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the water-containing composition 8a before heating and pressurizing is penetrated. Heat and pressurize in the hole. The procedure of the subsequent step 1 may be the same as that of the above embodiment.

After the step 1 following the step of adding water, a step of drying the molding composition obtained in the step 1 may be performed. For drying, it is preferable to leave it in a vacuum and / or in an environment of 100 ° C. for 1 minute to 10 days.

Step 2 is a step of compressing and stretching the molding composition obtained in the above step 1. In step 2, for example, a stretching machine having two cylindrical rolls is used. In step 2, for example, the molding composition (molded article) is sandwiched between two rolls, and while applying pressure, the two rolls are rotated and sent out. As a result, the entire molding composition is compressed and stretched. The compression and stretching may be performed only once, but the compression and stretching may be performed a plurality of times. That is, step 2 may be repeated a plurality of times. Step 2 may be performed without heating the molding composition. On the other hand, in step 2, the molding composition may be heated. In that case, a heating and stretching machine can be used. The stretching machine may be of a type in which the rollers are manually rotated, or may be of a type in which the rollers are automatically rotated by electric power or the like. The delivery speed in the stretching machine can be adjusted arbitrarily. When a heating and stretching machine is used, for example, a heater is built in a roller. The temperature of the rollers can be adjusted arbitrarily.

3A and 3B show a step of obtaining a molded product, in which FIG. 3A is a diagram schematically showing a first stretching step, and FIG. 3B is a diagram schematically showing a second stretching step. The stretching machine 20 shown in FIG. 3A includes an upper roll 10A and a lower roll 10B. The positions (intervals) of the upper roll 10A and the lower roll 10B can be set to stretch the molding composition 8b at a predetermined plate thickness ratio, that is, a reduction ratio. In the stretching machine 20, the molding composition 8b can be compression-stretched by feeding and sandwiching the molding composition 8b between the upper roll 10A and the lower roll 10B. When the step 2 is performed only once, it can be stretched to obtain a molded product 8c having a plate thickness thinner than the plate thickness of the molding composition 8b. When the step 2 is performed a plurality of times, as shown in FIG. 3 (b), the molded body 8c is fed again between the upper roll 10A and the lower roll 10B. The positions (intervals) of the upper roll 10A and the lower roll 10B are set so as to stretch the molded body 8c at a predetermined plate thickness ratio, that is, a reduction ratio. The plate thickness ratio here may be the same as or different from the plate thickness ratio in the previous stretching. After that, it is stretched to obtain a molded body 8d having a plate thickness thinner than that of the molded body 8c.

In step 2, the higher the reduction rate and the number of stretches, the higher the strength, but it can be determined according to the application.

When heating is performed in step 2, the heating is preferably performed at 80 to 200 ° C., more preferably 100 to 180 ° C., and even more preferably 120 to 160 ° C.

The step 2 is not limited to the form using two rolls, and may be performed in another form capable of performing compression stretching. For example, as shown in FIG. 4A, a stretching machine 20A provided with a flat plate-shaped substrate and a roll 11 is used, a molding composition is placed on the substrate, and the roll 11 is pressed while rolling from above. The molding composition may be compression-stretched. Further, as shown in FIG. 4B, a stretching machine 20B provided with a flat upper mold 12A and a lower mold 12B is used, and the molding composition is sandwiched between the upper mold 12A and the lower mold 12B to apply pressure. The molding composition may be compression-stretched by adding.

A step 3 of drying the molding composition may be performed between the steps 1 and 2 (before the step 2). In that case, the drying is preferably allowed to stand in a vacuum and / or in an environment of 100 ° C. for 30 minutes to 3 days. After the step 2, the step 3 of drying the molding composition may be performed. In that case, the drying is preferably allowed to stand in a vacuum and / or in an environment of 100 ° C. for 30 minutes to 3 days. Both of these, i.e., before and after step 2, may be performed step 3 to dry the molding composition.

The molded product obtained by the present embodiment is a resin-like molded product. A resin-like molded product is a molded product having an appearance similar to that of a synthetic resin.

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

FIG. 5 is a flow chart showing a manufacturing process of the molded product according to the embodiment. First, artificial spider silk powder was prepared by the following procedure (step S01).

(1) Preparation of Structural Protein Expressing Strain The nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P4684.1, GI: 11744415) derived from Nephila clavipes are obtained from the GenBank web database and then produced. Amino acids represented by SEQ ID NO: 12 are substituted, inserted and deleted for the purpose of improving sex, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 5 is added to the N-terminal. A recombinant fibroin having a sequence (hereinafter, also referred to as “PRT410”) was designed.

Next, the gene encoding PRT410 was outsourced for synthesis. As a result, a gene having an NdeI site immediately upstream of the 5'end and an EcoRI site immediately downstream of the 3'end was obtained. After cloning the gene into a cloning vector (pUC118), it was treated with restriction enzymes NdeI and EcoRI and recombined into a protein expression vector pET-22b (+).

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the pET22b (+) expression vector containing the gene encoding PRT410 obtained above. The transformed Escherichia coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours, and then the same culture solution was added to 100 mL of the seed culture medium shown in Table 1 so that the OD ₆₀₀ was 0.005. The temperature of the culture solution was kept at 30 ° C., and the cells were further cultured in a flask for about 15 hours until the OD ₆₀₀ reached 5, to obtain a seed culture solution.

The obtained seed culture solution was added to a jar fermenter to which 500 mL of the production medium shown in Table 2 was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., controlled to be constant at pH 6.9, and the culture was carried out so that the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration. As the antifoaming agent, ADEKANOLG-295S (manufactured by ADEKA Corporation) was used.

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

<Making a molded product>
Next, the artificial spider silk powder was filled in a stainless steel jig, and heated and pressurized at a temperature of 170 ° C. and a pressure of 30 MPa using a hot press (step S02). As a result, a molding composition (intermediate molded product) was obtained (step S03). The size of the molding composition was a disk shape having a diameter of 20 mm and a thickness of 3 mm, and the mass was 1.2 g.

Next, the molding composition was dried under vacuum at a temperature of 100 ° C. under reduced pressure for 6 hours using a vacuum dryer (AVO-250N manufactured by AS ONE Corporation) (step S04).

Next, using a manual heating and stretching machine (manufactured by Imoto Seisakusho Co., Ltd., IMC-1989 type), the molding composition was directly sandwiched between rolls and stretched (step S05). The stretching conditions were a temperature of 150 ° C., a number of stretching times of 1 to 9, and a final reduction rate of 30 to 72%. As a result, a stretched molded product was obtained. Details of the stretching conditions in each example will be described later.

After stretching, the molded product was dried again in the above vacuum dryer at a temperature of 100 ° C. under reduced vacuum for 12 hours (step S06). After drying, the molded product was stored in a desiccator for 12 hours, and then a molded product used for the test was obtained (step S07). Then, using a diamond cutter, the central portion of the molded product was cut out in a strip shape to prepare a test piece for a bending test.

<Bending test>
A tabletop precision universal testing machine (manufactured by Shimadzu Corporation, Autograph AGS-X 1kN) was used to perform a three-point bending test on the cut out test piece, and the bending strength and flexural modulus were measured. The measurement conditions were a distance between fulcrums of 14 mm and a test speed of 1 mm / min. The size of the test piece was 18 mm in length, 4 mm in width, and 0.6 to 2 mm in thickness.

<Measurement of hardness>
The Vickers hardness of the cut out test piece was measured with a Micro Vickers hardness tester (manufactured by Shimadzu Corporation, trade name: HMV-G).

(Test Example 1)
In Test Example 1, each of Examples 1 to 3 was heated and stretched only once to change the reduction rate. In Example 1, the reduction rate was 30%, in Example 2, the reduction rate was 50%, and in Example 3, the reduction rate was 68%. In Comparative Example, a molded product was obtained in the same manner as in Examples 1 to 3 except that heat stretching was not performed after pressure molding. The test results are shown in Table 3. As shown in Table 3, in all the examples in which the reduction ratio was changed, higher values were obtained in both the bending strength and the flexural modulus than in the comparative example.

6 (a) is a photograph of a molded product according to a comparative example, and FIGS. 6 (b) to 6 (d) are photographs of each molded product according to Examples 1 to 3. In Examples 1 to 3, the transparency was improved as compared with Comparative Examples. The larger the reduction rate, the higher the transparency.

(Test Example 2)
In Test Example 2, in Examples 3, 4, 5, and 6, the final reduction ratio was set to be close to 70%, and heat stretching was performed 1, 3, 5, and 9, respectively. The test results are shown in Table 4. As shown in Table 4, at the same reduction rate, the bending strength increased as the number of heat-stretching steps increased.

Further, in Examples 7 and 8, the number of heat stretching was set to 5 times, respectively, and the final reduction rate was set to be close to 70%, and the results were 65% and 72%, respectively. The test results are shown in Table 5. As shown in Table 5, when the number of heat-stretching steps was the same, the larger the reduction ratio, the larger both the bending strength and the bending elastic modulus.

The test results are summarized in Table 6.

(Test Example 3)
In Test Example 3, the molded product was dried in the same manner as described above, except that the first drying using a vacuum dryer (step S04) and the drying using a vacuum dryer after stretching (step S06) were not performed. Was produced. That is, in Test Example 3, two drying steps were omitted. In Examples 9 and 10, each of the heating and stretching was performed only once to change the reduction rate. In Example 9, the reduction rate was 41%, and in Example 10, the reduction rate was 60%. In Comparative Example, a molded product was obtained in the same manner as in Examples 9 and 10 except that heat stretching was not performed after pressure molding. The above bending test was performed on these test pieces. Further, in Test Example 3, the degree of orientation of the test piece was measured using a wide-angle X-ray scattering device (XtaLAB manufactured by Rigaku Co., Ltd.). The test results are shown in Table 7. As shown in Table 7, when the number of heat-stretching steps was the same, the larger the reduction rate, the larger the degree of orientation. Further, in Examples 9 and 10, the bending strength is lower than that in Examples 1 to 8. It is considered that this is because the test piece contained a large amount of water due to the omission of the drying step.

According to the present invention, there is provided a method for producing a biodegradable molded product having high strength and a biodegradable molded product having high strength without using an additive material such as reinforcing fibers.

8a ... composition before molding, 8b ... molding composition before compression stretching, 8c, 8d ... molded body after compression stretching, 10 ... pressure molding machine, 10A ... upper roll, 10B ... lower roll, 11 ... roll, 12A ... upper mold, 12B ... lower mold, 20 ... stretching machine, 20A, 20B ... stretching machine.

Claims (10)

Step 1 of molding a composition containing a structural protein,
A method for producing a molded product, comprising the step 2 of compressing and stretching the molding composition obtained in the step 1. The method of claim 1, wherein step 1 comprises heating and pressurizing the composition. The method according to claim 1 or 2, wherein the step 2 comprises heating the molding composition. The method according to any one of claims 1 to 3, wherein the step 2 is repeated a plurality of times. The method according to any one of claims 1 to 4, which comprises a step 3 of drying the molding composition at least before the step 2 and after the step 2. The method according to any one of claims 1 to 5, wherein the structural protein is spider silk fibroin. A molded product of a composition containing a structural protein and having a flexural modulus exceeding 8.47 GPa. A molded product of a composition containing a structural protein and having a bending strength of more than 141 MPa. A molded product of a composition containing a structural protein and having a degree of orientation of 0.22 or more. The molded product according to any one of claims 7 to 9, wherein the structural protein is spider silk fibroin.