Fibers for rubber reinforcement, rubber articles, tire cords, and tires

Abstract

The present invention provides fibers for rubber reinforcement which are able to be produced using a bio-derived starting material, and which are not susceptible to breaking even if complexed with a rubber material. First fibers for rubber reinforcement according to the present invention are characterized by using protein fibers that contain a hydrophobic protein. Second fibers for rubber reinforcement according to the present invention are characterized by using protein fibers that have an initial tensile elastic modulus of 2.0 GPa or more in a wet state. These fibers for rubber reinforcement are preferably provided with coating layers in the surfaces, said coating layers being formed from an aqueous adhesive composition.

Description

Technical Field

[0001] The present invention relates to fibers for rubber reinforcement, rubber articles, cords for tires, and tires.

Background Art

[0002] Conventionally, as materials for fibers (fibers for rubber reinforcement) that reinforce rubber articles such as tires, synthetic fibers such as nylon, polyester, and vinylon made from fossil resources such as petroleum have been widely used. However, in consideration of the stable supply of future fossil resources and soaring prices, it is desired to reduce the usage amount of synthetic fibers made from fossil resources as described above.

[0003] Examples of fiber materials made from non-fossil resources include rayon fibers made from wood. However, if most of the currently used synthetic fibers are replaced with wood-derived fibers, it is considered that forest resources will be depleted and the environment will be destroyed. Therefore, there is a strong demand for the supply of sustainable and ethical materials that are naturally derived and do not involve environmental destruction.

[0004] As one way to address such demands, a technology for protein fibers obtained by spinning using a structural protein produced by a fermentation process using microorganisms as a raw material has been developed. More specifically, as disclosed in Patent Document 1 for example, this protein fiber is produced by performing molecular design of a protein amino acid sequence for obtaining various properties as a fiber and a gene sequence for producing the protein molecule, synthesizing the gene, purifying the structural protein produced by fermentation of a microorganism incorporated with the gene, and spinning using this as a raw material. Also, bases for mass-producing structural proteins are being built by the same technology.

[0005] The production of bio-derived raw materials using the fermentation of such microorganisms utilizes biomass resources such as grains or non-edible parts without depleting forest resources, and can also lead to an increase in carbon storage due to the expansion of the soil of crop rotation farmland. Therefore, by conducting the above production, it is possible to ethically provide fibers that contribute to a sustainable society in the future.

[0006] Patent Document 2 discloses that a polypeptide produced using a host transformed with a gene encoding a polypeptide derived from a natural spider silk protein is spun, and the obtained fibroin fiber is used as a reinforcing fiber cord for a pneumatic tire. Patent Document 2 discloses molding a predetermined tire member by topping the above reinforcing fiber cord with a rubber composition for cord topping or the like. Further, in Patent Document 2, it is preferably that the above rubber composition for cord topping contains 3 parts by mass or more of sulfur with respect to 100 parts by mass of the rubber component.

[0007] By the way, among protein fibers, there are some that shrink upon contact with moisture. Also, depending on the type of protein fiber or the method of production, etc., it is conceivable that the fiber may elongate upon contact with moisture. Regarding this point, for example, it has been reported that natural spider silk has the property that its rubbery elasticity increases when swollen with water (Non-Patent Document 1). If unexpected length changes such as shrinkage and elongation due to contact with moisture of such protein fibers occur in the manufacturing process or the product, there is a concern that various problems may occur.

[0008] For example, when manufacturing rubber reinforcing fibers used in tires or the like, before topping (composite with a rubber material) a fiber with a rubber composition containing sulfur, the fiber is coated by dipping or applying an aqueous adhesive composition, and then a step of drying the moisture of the adhesive composition and performing an adhesion treatment by heating is sometimes performed. However, at this time, if there is an unexpected fiber length change due to the type of protein fiber or the method of production (such as moisture content and temperature difference) as described above, there is a risk that workability and cord quality may deteriorate.

[0009] Here, for an adhesive composition used for coating fibers in a tire or the like, in addition to the initial adhesive strength, it is required to maintain adhesion even under distortion caused by various impacts (for example, the rolling of a tire) or under the input of heat. As such an adhesive composition, for example, Patent Documents 3 and 4 disclose an RFL (resorcin - formaldehyde - latex) adhesive composition obtained by aging a mixed liquid containing resorcin, formaldehyde, and rubber latex.

[0010] Also, in order to meet the recent demand for reducing the amount of resorcin used from the perspective of reducing environmental impact, for example, Patent Document 5 discloses a resorcin - free adhesive composition composed of, for example, rubber latex, a blocked isocyanate compound, and an amino - based compound of an epoxy compound and a curing agent.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non - Patent Documents

[0012]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0013] When the fibroin fiber disclosed in Patent Document 2 is compounded with a rubber material, it tends to be easily cut, especially when wet. Further, there is a concern that when the rubber article is used in a harsh environment, its high strength cannot be fully exhibited. Therefore, there was room for improvement in using the above fiber as a reinforcing fiber in rubber articles such as tires.

[0014] In recent years, due to the improvement of tire performance and weight reduction for energy saving, the distortion and heat input to the cord due to tire rotation have become more severe. And, in industrially providing a protein fiber that ensures the cord quality of the rubber reinforcing fiber used in tires, sufficient knowledge has not been obtained so far regarding a fiber material and a method that can be suitably coated by dipping or applying an aqueous adhesive composition.

[0015] Therefore, an object of the present invention is to provide a rubber reinforcing fiber that can be manufactured using a bio-derived raw material and is difficult to cut even when compounded with a rubber material. Another object of the present invention is to provide a high-strength rubber article, a tire cord, and a tire using the above-described rubber reinforcing fiber, respectively.

Means for Solving the Problems

[0016] The gist configuration of the present invention for achieving the above object is as follows.

[0017] That is, the first rubber reinforcing fiber of the present invention is characterized in that a protein fiber containing a hydrophobic protein is used.

[0018] Further, the second rubber reinforcing fiber of the present invention is characterized in that a protein fiber having an initial tensile elastic modulus of 2.0 GPa or more when wet is used.

[0019] Further, the rubber article of the present invention is characterized by including the above rubber reinforcing fiber.

[0020] Further, the tire cord of the present invention is characterized by being made of the above rubber reinforcing fiber.

[0021] Further, the tire of the present invention is characterized by including the above-described tire cord.

Advantages of the Invention

[0022] According to the present invention, it is possible to provide a rubber reinforcing fiber that can be produced using a bio-derived raw material and is difficult to break even when compounded with a rubber material. Further, according to the present invention, it is possible to provide a high-strength rubber article, a tire cord, and a tire using the above-described rubber reinforcing fiber, respectively.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0024] (First Rubber Reinforcing Fiber) The rubber reinforcing fiber according to the first embodiment of the present invention (hereinafter, may be referred to as "the fiber of the first embodiment") is characterized by using a protein fiber containing a hydrophobic protein. In other words, the fiber of the first embodiment is a protein fiber containing a hydrophobic protein.

[0025] The inventor participated in the Innovative Research and Development Promotion Program (ImPACT) and conducted various studies on the technology of conventional protein fiber materials obtained by mimicking the production method of natural spider silk. In particular, the inventor investigated the causes of the problems that conventional protein fibers are prone to breakage when compounded with rubber materials and that they cannot fully exhibit high strength under harsh environments. As a result, it was found that these problems are caused by the contact of the fibers with water. In other words, it was found that the strength of fibers containing polypeptides derived from conventional natural spider silk proteins decreases due to contact with water. More specifically, it was determined that the fibers were prone to breakage during compounding with rubber materials because the adhesive previously coated on the fibers was an aqueous adhesive. It was also determined that the failure to fully exhibit high strength was due to the fibers absorbing moisture under high-temperature and high-humidity conditions, which promoted fiber deterioration.

[0026] Here, the reason for the decrease in fiber strength due to contact with water was considered to be that the molecules (protein molecules) derived from conventional natural spider silk proteins have an amino acid sequence containing many hydrophilic amino acid units. Based on this, the inventor further investigated, particularly focusing on the molecular design of protein molecules, and accumulated and analyzed data on the functions, properties, and productivity of fibers using proteins obtained through a fermentation process using microorganisms. As a result, a protein molecule with an amino acid sequence with reduced hydrophilic amino acid units, that is, a hydrophobic protein, which is industrially feasible and not found in bio-derived proteins, was newly created.

[0027] Natural spider silk is obtained from an aqueous solution of proteins stored in a secretory gland called the silk gland. And this protein can flow as an aqueous solution through the internal organs up to the spinneret while changing its molecular structure in the internal environment due to its hydrophilic function. On the other hand, the above-mentioned hydrophobic protein is insoluble in water like the body fluid of a living organism and aggregates, so it has a molecular structure different from that of natural spider silk.

[0028] Hereinafter, the fiber of the first embodiment will be described in detail based on the embodiment.

[0029] <Hydrophobic protein> The hydrophobic protein contained in the fiber of the first embodiment has been subjected to molecular design, but is not particularly limited, and may be a protein produced by microorganisms or the like by genetic recombination technology, or may be a protein produced by synthesis.

[0030] The hydrophobic protein may be, for example, a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m, or Formula 2: [(A)n motif-REP]m-(A)n motif. The hydrophobic protein of the present embodiment may have an amino acid sequence (N-terminal sequence and C-terminal sequence) further added to either one or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited thereto, but are typically regions without repetition of amino acid motifs and consist of about 100 amino acids.

[0031] In the present specification, the "domain sequence" refers to an amino acid sequence that gives rise to a crystalline region (typically corresponding to the (A)n motif of an amino acid sequence) and an amorphous region (typically corresponding to REP of an amino acid sequence), and means an amino acid sequence represented by Formula 1: [(A)n motif - REP]m, or Formula 2: [(A)n motif - REP]m - (A)n motif. Here, the (A)n motif represents an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2 to 27. The number of amino acid residues of the (A)n motif may be 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. Also, the ratio of the number of alanine residues to the total number of amino acid residues in the (A)n motif may be 40% or more, and may be 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, 86% or more, 90% or more, 95% or more, or 100% (meaning composed only of alanine residues). At least seven of the (A)n motifs present in multiple in the domain sequence may be composed only of alanine residues. REP represents an amino acid sequence composed of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer from 2 to 300, and may be an integer from 10 to 300. The multiple (A)n motifs may have the same amino acid sequence as each other, or may have different amino acid sequences. The multiple REPs may have the same amino acid sequence as each other, or may have different amino acid sequences.

[0032] The hydrophobic protein may be, for example, one obtained by modifying its amino acid sequence based on the amino acid sequence of naturally occurring spider fibroin (for example, one obtained by modifying the amino acid sequence by modifying the cloned gene sequence of naturally occurring spider fibroin), or may be one artificially designed and synthesized without relying on naturally occurring spider fibroin (for example, one having a desired amino acid sequence by chemically synthesizing a nucleic acid encoding the designed amino acid sequence).

[0033] Hydrophobic proteins can be obtained, for example, by modifying the amino acid sequence corresponding to the cloned gene sequence of natural spider fibroin, such as substituting, deleting, inserting, and / or adding one or more amino acid residues. 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 the site-directed mutagenesis method. Specifically, it can be carried out according to the methods described in documents such as Nucleic Acid Res. 10, 6487 (1982) and Methods in Enzymology, 100, 448 (1983).

[0034] Specific examples of hydrophobic proteins include modified proteins having a domain sequence with a reduced content of glutamine residues with respect to the major ampullate spidroin protein produced in the major ampullate gland of spiders.

[0035] It is preferable that the modified protein contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

[0036] When the modified protein contains the GPGXX motif in REP, the GPGXX motif content rate is usually 1% or more, may be 5% or more, and is preferably 10% or more. There is no particular limitation on the upper limit of the GPGXX motif content rate, and it may be 50% or less, or may be 30% or less.

[0037] In this specification, the "GPGXX motif content rate" is a value calculated by the following method. In the spider fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, for all REPs contained in the sequence obtained by removing the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence, the number obtained by multiplying the total number of GPGXX motifs contained in that region by 3 (i.e., corresponding to the total number of G and P in the GPGXX motif) is defined as s. When the total number of amino acid residues of all REPs after removing the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence and further removing the (A)n motif is defined as t, the GPGXX motif content rate is calculated as s / t.

[0038] In the calculation of the GPGXX motif content rate, the target of "the sequence obtained by removing the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is because the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence (the sequence corresponding to REP) may contain a sequence with low correlation with the characteristic sequence of spider fibroin, and when m is small (i.e., when the domain sequence is short), it affects the calculation result of the GPGXX motif content rate. Therefore, this influence is excluded. When the "GPGXX motif" is located at the C-terminal of REP, even if "XX" is, for example, "AA", it is treated as the "GPGXX motif".

[0039] FIG. 1 is a schematic diagram showing the domain arrangement of spider fibroin. The method for calculating the GPGXX motif content rate will be specifically described with reference to FIG. 1. First, in the domain arrangement of spider fibroin shown in FIG. 1 (of the type "[(A)n motif-REP]m-(A)n motif"), since all REPs are included in the sequence obtained by removing the sequence from the C-terminal of the domain arrangement to the (A)n motif located on the most C-terminal side from the domain arrangement (the sequence indicated by "Region A" in FIG. 1), the number of GPGXX motifs for calculating s is 7, and s is 7×3 = 21. Similarly, since all REPs are included in the sequence obtained by removing the sequence from the C-terminal of the domain arrangement to the (A)n motif located on the most C-terminal side from the domain arrangement (the sequence indicated by "Region A" in FIG. 1), the total number t of amino acid residues of all REPs after further removing the (A)n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150. Next, by dividing s by t, s / t (%) can be calculated, and in the case of the fibroin in FIG. 1, it is 21 / 150 = 14.0%.

[0040] The modified protein preferably has a glutamine residue content rate of 9% or less, more preferably 7% or less, still more preferably 4% or less, and particularly preferably 0%.

[0041] In the present specification, the "glutamine residue content rate" is a value calculated by the following method. In the spider fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, in all REPs contained in the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence excluding the sequence from the domain sequence (the sequence corresponding to "Region A" in Figure 1), when the total number of glutamine residues contained in that region is u, and the total number of amino acid residues of all REPs excluding the sequence from the domain sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence and further excluding the (A)n motif is t, the glutamine residue content rate is calculated as u / t. The reason for targeting the "sequence excluding the sequence from the domain sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence" in the calculation of the glutamine residue content rate is the same as the reason described above.

[0042] The modified protein may have an amino acid sequence corresponding to the deletion of one or more glutamine residues in the REP or substitution with other amino acid residues in its domain sequence as compared with the naturally-derived spider fibroin.

[0043] "Other amino acid residues" may be any amino acid residues other than glutamine residues, but are preferably amino acid residues having a higher hydrophobicity index (HI) than glutamine residues. The hydrophobicity index (HI) of amino acid residues is as shown in Table 1.

[0044]

Table 1

[0045] As shown in Table 1, examples of amino acid residues having a higher hydrophobicity index than the glutamine residue include amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P), and histidine (H). Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M), and alanine (A) are more preferable, and amino acid residues selected from isoleucine (I), valine (V), leucine (L), and phenylalanine (F) are even more preferable.

[0046] The modified protein preferably has a hydrophobicity degree of REP of -0.8 or more, more preferably -0.7 or more, even more preferably 0 or more, even more preferably 0.3 or more, and particularly preferably 0.4 or more. There is no particular limitation on the upper limit of the hydrophobicity degree of REP, and it may be 1.0 or less, or may be 0.7 or less.

[0047] In this specification, the "hydrophobicity degree of REP" is a value calculated by the following method. In a protein such as spider fibroin containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif, for all REPs contained in the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence excluding the sequence from the domain sequence (the sequence corresponding to "Region A" in Figure 1), when the sum of the hydrophobicity indices of each amino acid residue in that region is v, and when the total number of amino acid residues of all REPs excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence and further excluding the (A)n motif is t, the hydrophobicity degree of REP is calculated as v / t. In the calculation of the hydrophobicity degree of REP, the reason for targeting "the sequence excluding the sequence from the (A)n motif located at the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the same as the reason described above.

[0048] Note that the above "hydrophobicity degree of REP" is synonymous with "average HI" in this specification.

[0049] The modified protein may have an amino acid sequence modification corresponding to substitution, deletion, insertion, and / or addition of one or more amino acid residues in addition to the modification corresponding to deletion of one or more glutamine residues in REP and / or substitution of one or more glutamine residues in REP with other amino acid residues, as compared with the domain sequence of naturally occurring spider fibroin.

[0050] The modified protein can be obtained, for example, by deleting one or more glutamine residues in REP from the gene sequence of cloned naturally occurring spider fibroin and / or substituting one or more glutamine residues in REP with other amino acid residues. Also, for example, it can be obtained by designing an amino acid sequence corresponding to deletion of one or more glutamine residues in REP and / or substitution of one or more glutamine residues in REP with other amino acid residues from the amino acid sequence of naturally occurring spider fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

[0051] More specific examples of the modified protein include (6-i) modified fibroin containing the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 21, or (6-ii) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 21.

[0052] The modified fibroin of (6-i) will be described.

[0053] The amino acid sequence represented by SEQ ID NO: 1 (Met-PRT410) is based on the nucleotide sequence and amino acid sequence of Nephila clavipes (GenBank accession number: P46804.1, GI: 1174415), which is a naturally occurring fibroin. Amino acid modifications were made to improve productivity, such as making the number of consecutive alanine residues in the (A)n motif five by making the alanine residues consecutive. On the other hand, since no modification was made to the glutamine residue (Q) in Met-PRT410, the glutamine residue content is about the same as that of the naturally occurring fibroin.

[0054] The amino acid sequence represented by SEQ ID NO: 5 (M_PRT888) is obtained by substituting all QQ in Met-PRT410 (SEQ ID NO: 1) with VL.

[0055] The amino acid sequence represented by SEQ ID NO: 6 (M_PRT965) is obtained by substituting all QQ in Met-PRT410 (SEQ ID NO: 1) with TS and substituting the remaining Q with A.

[0056] The amino acid sequence represented by SEQ ID NO: 7 (M_PRT889) is obtained by substituting all QQ in Met-PRT410 (SEQ ID NO: 1) with VL and substituting the remaining Q with I.

[0057] The amino acid sequence (M_PRT916) represented by SEQ ID NO: 8 is obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 1) with VI and replacing the remaining Q with L.

[0058] The amino acid sequence (M_PRT918) represented by SEQ ID NO: 9 is obtained by replacing all QQ in Met-PRT410 (SEQ ID NO: 1) with VF and replacing the remaining Q with I.

[0059] The amino acid sequence (M_PRT699) represented by SEQ ID NO: 10 is obtained by replacing all QQ in M_PRT525 (SEQ ID NO: 12) with VL. The amino acid sequence (M_PRT525) represented by SEQ ID NO: 12 is obtained by inserting two alanine residues into the region (A5) where alanine residues are continuous with respect to Met-PRT410 (SEQ ID NO: 1), deleting two C-terminal domain sequences so as to have approximately the same molecular weight as Met-PRT410, and replacing 13 glutamine residues (Q) with serine residues (S) or proline residues (P).

[0060] The amino acid sequence (M_PRT698) represented by SEQ ID NO: 11 is obtained by replacing all QQ in M_PRT525 (SEQ ID NO: 12) with VL and replacing the remaining Q with I.

[0061] The amino acid sequence (Met-PRT966) represented by SEQ ID NO: 21 is obtained by replacing all QQ in the amino acid sequence represented by SEQ ID NO: 2 (the amino acid sequence before the amino acid sequence represented by SEQ ID NO: 20 is added to the C-terminus) with VF and replacing the remaining Q with I.

[0062] Note that the amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 21 all have a glutamine residue content of 9% or less, as shown in Table 2.

[0063]

Table 2

[0064] The modified fibroin of (6-i) may consist of an amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 21.

[0065] The modified fibroin of (6-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 21. The modified fibroin of (6-ii) is also a protein containing a domain sequence represented by Formula 1: [(A)n motif-REP]m or Formula 2: [(A)n motif-REP]m-(A)n motif. The above sequence identity is preferably 95% or more.

[0066] The modified fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Also, the modified fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

[0067] The modified protein may contain a tag sequence at either or both of the N-terminus and the C-terminus. Thereby, isolation, immobilization, detection, visualization, etc. of the modified protein become possible.

[0068] More specific examples of the modified protein containing a tag sequence include (6-iii) modified fibroin containing an amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 22, or (6-iv) modified fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 22.

[0069] The amino acid sequences represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22 are each the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 21 with the amino acid sequence represented by SEQ ID NO: 3 (including His tag sequence and hinge sequence) added to the N-terminus. Since only a tag sequence is added to the N-terminus, there is no change in the glutamine residue content rate, and the amino acid sequences represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22 all have a glutamine residue content rate of 9% or less (Table 3).

[0070]

Table 3

[0071] (6-iii) The modified fibroin may consist of the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 22.

[0072] (6-iv) The modified fibroin includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 22. The modified fibroin of (6-iv) is also a protein including a domain sequence represented by Formula 1: [(A)n motif-REP]m, or Formula 2: [(A)n motif-REP]m-(A)n motif. The above sequence identity is preferably 95% or more.

[0073] (6-iv) The modified fibroin preferably has a glutamine residue content rate of 9% or less. Also, the modified fibroin of (6-iv) preferably has a GPGXX motif content rate of 10% or more.

[0074] The modified protein may contain a secretion signal for releasing the protein produced in the recombinant protein production system to the outside of the host. The sequence of the secretion signal can be appropriately set according to the type of the host.

[0075] The hydrophobic protein is preferably a protein such as a spider silk protein in which the sum of the hydrophobicity indices (HI) of all constituent amino acid residues is determined and the value (average HI) obtained by dividing the sum by the total number of amino acid residues is 0 or more. In this case, the resulting fiber becomes more difficult to break. The hydrophobicity index (HI) is as shown in Table 1.

[0076] The hydrophobic protein can be produced, for example, by expressing the nucleic acid in a host transformed with an expression vector having a nucleic acid sequence encoding the hydrophobic protein and one or more regulatory sequences operably linked to the nucleic acid sequence.

[0077] The method for producing the nucleic acid encoding the hydrophobic protein is not particularly limited. For example, the nucleic acid can be produced by a method of amplifying and cloning it by polymerase chain reaction (PCR) or the like using a gene encoding a natural spider silk protein, or by a method of chemically synthesizing it. The chemical synthesis method of the nucleic acid is also not particularly limited. For example, based on the amino acid sequence information of the spider silk protein obtained from the NCBI web database or the like, the gene can be chemically synthesized by a method of ligating oligonucleotides automatically synthesized by AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Co., Ltd.) or the like by PCR or the like. At this time, in order to facilitate the purification and / or confirmation of the hydrophobic protein, a nucleic acid encoding a hydrophobic protein having an amino acid sequence with an amino acid sequence consisting of a start codon and a His10 tag added to the N-terminus may be synthesized.

[0078] The regulatory sequence is a sequence that controls the expression of the recombinant protein in the host (for example, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected according to the type of host. As the promoter, an inducible promoter that functions in the host cell and can induce the expression of the target hydrophobic protein may be used. The inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure, or pH value.

[0079] The type of expression vector can be appropriately selected according to the type of host, such as plasmid vector, viral vector, cosmid vector, fosmid vector, artificial chromosome vector, etc. As the expression vector, those that can replicate autonomously in the host cell or can be integrated into the host chromosome and contain a promoter at a position where the nucleic acid encoding the spider silk protein can be transcribed are preferably used.

[0080] As the host, any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells can be preferably used.

[0081] When using a prokaryote such as bacteria as the host, the expression vector is preferably a vector that can replicate autonomously in the prokaryote and at the same time contains a promoter, a ribosome binding sequence, a nucleic acid encoding a hydrophobic protein, and a transcription termination sequence. It may also contain a gene that controls the promoter.

[0082] Examples of prokaryotes include microorganisms belonging to the genera Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas, etc. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli, etc. Examples of microorganisms belonging to the genus Brevibacillus include Brevibacillus agri, etc. Examples of microorganisms belonging to the genus Serratia include Serratia liquefaciens, etc. Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis, etc. Examples of microorganisms belonging to the genus Microbacterium include Microbacterium ammoniaphilum, etc. Examples of microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatum, etc. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes, etc. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas putida, etc.

[0083] When using prokaryotes as hosts, examples of vectors for introducing nucleic acids encoding hydrophobic proteins include pBTrp2 (manufactured by Boehringer Mannheim), pGEX (manufactured by Pharmacia), pUC18, pBluescriptII, pSupex, pET22b, pCold, pUB110, pNCO2 (Japanese Patent Laid-Open No. 2002-238569), etc.

[0084] Examples of eukaryotic hosts include yeasts and filamentous fungi (such as molds). Examples of yeasts include yeasts belonging to the genera Saccharomyces, Pichia, Schizosaccharomyces, etc. Examples of filamentous fungi include filamentous fungi belonging to the genera Aspergillus, Penicillium, Trichoderma, etc.

[0085] When using eukaryotes as the host, examples of vectors for introducing nucleic acids encoding hydrophobic proteins include YEp13 (ATCC37115), YEp24 (ATCC37051), etc. As methods for introducing the expression vector into the host cell, any method for introducing DNA into the host cell can be used. For example, methods using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation, spheroplast method, protoplast method, lithium acetate method, competent method, etc. can be mentioned.

[0086] As methods for expressing nucleic acids by a host transformed with an expression vector, in addition to direct expression, secretion production, fusion protein expression, etc. can be carried out according to the methods described in Molecular Cloning, 2nd Edition, etc.

[0087] Hydrophobic proteins can be produced, for example, by culturing the transformed host in a culture medium, generating and accumulating the hydrophobic protein in the culture medium, and collecting it from the culture medium. The method of culturing the transformed host in a culture medium can be carried out according to the methods usually used for culturing the host.

[0088] When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, as the culture medium, a natural medium or a synthetic medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that the host can assimilate and can efficiently culture the host.

[0089] As the carbon source, any that the host can assimilate can be used. For example, carbohydrates such as glucose, fructose, sucrose, and molasses containing these, starch and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.

[0090] As the nitrogen source, for example, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented microbial cells and their digests can be used.

[0091] As the inorganic salts, for example, potassium dihydrogen phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

[0092] The culture of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out, for example, 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 culture at 3.0 to 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0093] In addition, if necessary during the culture, antibiotics such as ampicillin and tetracycline may be added to the culture medium. When culturing a microorganism transformed with an expression vector using an inducible promoter as the promoter, an inducer may be added to the medium if necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like may be added to the medium, and when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacrylic acid or the like may be added to the medium.

[0094] The hydrophobic protein produced by the transformed host can be isolated and purified by the methods commonly used for protein isolation and purification. For example, when the hydrophobic protein is expressed in a soluble state within the cell, after completion of the culture, the host cells are recovered by centrifugation, suspended in an aqueous buffer, and then the host cells are disrupted using an ultrasonic disruptor, French press, Manton Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, methods commonly used for protein isolation and purification, namely, solvent extraction method, salting-out method using ammonium sulfate, etc., desalting method, precipitation method using an organic solvent, anion exchange chromatography method using resins such as diethylaminoethyl (DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography method using resins such as S-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatography method using resins such as butyl Sepharose, phenyl Sepharose, gel filtration method using a molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis methods such as isoelectric focusing electrophoresis, etc. are used alone or in combination to obtain a purified preparation.

[0095] As the above chromatography, column chromatography using phenyl-Toyopearl (Tosoh), DEAE-Toyopearl (Tosoh), Sephadex G-150 (Pharmacia Biotech) is preferably used.

[0096] Also, when the hydrophobic protein is expressed by forming an insoluble body within the cell, similarly, after recovering the host cells, disrupting them, and performing centrifugation, the insoluble body of the hydrophobic protein is recovered as a precipitate fraction. The recovered insoluble body of the hydrophobic protein can be solubilized with a protein denaturant. After this operation, a purified preparation of the hydrophobic protein can be obtained by the same isolation and purification method as described above.

[0097] When a hydrophobic protein is secreted extracellularly, the hydrophobic protein can be recovered from the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a method such as centrifugation, and a purified standard can be obtained from the culture supernatant by using the same isolation and purification method as described above.

[0098] <Protein fiber> The fiber of the first embodiment is a protein fiber containing the above-described hydrophobic protein, and can be produced by spinning the hydrophobic protein. Since the fiber of the first embodiment contains a hydrophobic protein having the above-described specific molecular design, a decrease in fiber strength when water is included by immersing in water or the like is significantly suppressed. Therefore, the fiber of the first embodiment is not easily broken even when it is compounded with a rubber material, particularly when an operation requiring contact with an aqueous component is performed.

[0099] The spinning method is not particularly limited as long as it can spin a hydrophobic protein. For example, dry spinning, melt spinning, wet spinning, etc. can be mentioned. A preferable spinning method can be wet spinning. Also, in any method, a spinning dope (dope solution) in which a hydrophobic protein is dissolved in a solvent can be used.

[0100] Hereinafter, it will be mainly described by taking wet spinning as an example.

[0101] In wet spinning, first, the spinning dope is extruded from a spinneret (nozzle) into a coagulation liquid (coagulation liquid tank), and an undrawn yarn in the shape of a thread can be obtained by solidifying the hydrophobic protein in the coagulation liquid.

[0102] The content of the hydrophobic protein in the spinning dope may be 1% by mass or more, 2% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more, and may be 40% by mass or less, 35% by mass or less, 30% by mass or less, or 25% by mass or less.

[0103] The solvent used for the spinning dope is not particularly limited as long as it can dissolve or disperse hydrophobic proteins. Specifically, examples of the solvent include organic solvents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMAc), hexafluoro-2-propanol (HFIP), hexafluoroacetone (HFA), and formic acid. Further, the solvent may be water to which a dissolution accelerator described later is added. The solvent can be used alone or in combination of two or more kinds.

[0104] In particular, it is preferable that the solvent is at least one selected from the group consisting of dimethyl sulfoxide, formic acid, and those obtained by adding a dissolution accelerator thereto.

[0105] The spinning dope may further contain a dissolution accelerator. The dissolution accelerator makes it easier to prepare the spinning dope.

[0106] The dissolution promoter may be, for example, an inorganic salt composed of the following Lewis acid and Lewis base. Examples of the Lewis base include oxo acid ions (such as nitrate ions and perchlorate ions), metal oxo acid ions (such as permanganate ions), halide ions, thiocyanate ions, cyanate ions, and the like. Examples of the Lewis acid include metal ions such as alkali metal ions and alkaline earth metal ions, polyatomic ions such as ammonium ions, complex ions, and the like. Examples of the inorganic salts include lithium salts such as lithium chloride, lithium bromide, lithium iodide, lithium nitrate, lithium perchlorate, and lithium thiocyanate; calcium salts such as calcium chloride, calcium bromide, calcium iodide, calcium nitrate, calcium perchlorate, and calcium thiocyanate; iron salts such as iron chloride, iron bromide, iron iodide, iron nitrate, iron perchlorate, and iron thiocyanate; aluminum salts such as aluminum chloride, aluminum bromide, aluminum iodide, aluminum nitrate, aluminum perchlorate, and aluminum thiocyanate; potassium salts such as potassium chloride, potassium bromide, potassium iodide, potassium nitrate, potassium perchlorate, and potassium thiocyanate; sodium salts such as sodium chloride, sodium bromide, sodium iodide, sodium nitrate, sodium perchlorate, and sodium thiocyanate; zinc salts such as zinc chloride, zinc bromide, zinc iodide, zinc nitrate, zinc perchlorate, and zinc thiocyanate; magnesium salts such as magnesium chloride, magnesium bromide, magnesium iodide, magnesium nitrate, magnesium perchlorate, and magnesium thiocyanate; barium salts such as barium chloride, barium bromide, barium iodide, barium nitrate, barium perchlorate, and barium thiocyanate; and strontium salts such as strontium chloride, strontium bromide, strontium iodide, strontium nitrate, strontium perchlorate, and strontium thiocyanate. These inorganic salts are used as dissolution promoters for hydrophobic proteins in formic acid. By containing a dissolution promoter (the above inorganic salt) in the spinning dope, the hydrophobic protein can be dissolved in the spinning dope at a high concentration. As a result, the production efficiency of protein fibers is further improved, and an improvement in the quality of protein fibers and physical properties such as stress is expected.The inorganic salt may be at least one selected from the group consisting of lithium chloride and calcium chloride. The dissolution promoter can be used alone or in combination of two or more.

[0107] The content of the dissolution promoter in the spinning dope may be 0.1% by mass or more, 1% by mass or more, 4% by mass or more, 7% by mass or more, 10% by mass or more, or 15% by mass or more, and may be 20% by mass or less, 16% by mass or less, 12% by mass or less, or 9% by mass or less.

[0108] The spinning dope may further contain various additives as needed. Examples of the additives include plasticizers, leveling agents, crosslinking agents, crystal nucleating agents, antioxidants, ultraviolet absorbers, colorants, fillers, synthetic resins, and the like. The total content of the additives may be 50 parts by mass or less with respect to 100 parts by mass of the total amount of proteins in the spinning dope.

[0109] Also, the spinning dope may further contain alcohol as long as the effects of the present invention are not impaired.

[0110] The viscosity of the spinning dope may be appropriately set according to the spinning method. For example, it can be 100 to 15,000 cP (centipoise) at 35°C. The viscosity of the spinning dope can be measured, for example, using a product named "EMS Viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

[0111] As the coagulating liquid, any solution that can be desolvated can be used. For example, lower alcohols with 1 to 5 carbon atoms such as methanol, ethanol, and 2-propanol, and acetone can be mentioned. Appropriate water may be added to the coagulating liquid. The temperature of the coagulating liquid is preferably 0 to 30°C. When using a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm as the spinneret, the extrusion rate per hole is preferably 0.2 to 6.0 ml / hour, and more preferably 1.4 to 4.0 ml / hour. The length of the coagulating liquid tank may be any length that allows efficient desolvation, for example, 200 to 500 mm. The take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min. The residence time may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes. Also, stretching (pre-stretching) may be performed in the coagulating liquid to obtain a pre-stretched yarn. To suppress the evaporation of the lower alcohol, the coagulating liquid may be maintained at a low temperature and taken up in the state of the undrawn yarn. The coagulating liquid tank may be provided in multiple stages, and stretching may be performed at each stage or a specific stage as necessary.

[0112] Next, the obtained undrawn yarn (or pre-stretched yarn) can be stretched to obtain a stretched yarn (protein fiber). Examples of the stretching method include wet heat stretching and dry heat stretching.

[0113] Wet heat stretching can be performed in warm water, a solution obtained by adding an organic solvent or the like to warm water, or during steam heating. The temperature may be, for example, 50 to 90°C, and preferably 75 to 85°C. In wet heat stretching, the undrawn yarn (or pre-stretched yarn) can be stretched, for example, 1 to 10 times, and preferably 2 to 8 times.

[0114] Dry heat stretching can be performed using an electric tubular furnace, a hot plate, or the like. The temperature may be, for example, 140°C to 270°C, and preferably 160°C to 230°C. In dry heat stretching, the undrawn yarn (or pre-stretched yarn) can be stretched, for example, 0.5 to 8 times, and preferably 1 to 4 times.

[0115] Wet heat drawing and dry heat drawing may be carried out independently, or may be carried out in multiple stages or in combination. For example, the first stage drawing may be wet heat drawing, and the second stage drawing may be dry heat drawing, or the first stage drawing may be wet heat drawing, the second stage drawing may be wet heat drawing, and the third stage drawing may be dry heat drawing. That is, drawing can be carried out by appropriately combining wet heat drawing and dry heat drawing.

[0116] The final draw ratio after drawing is, for example, 5 to 20 times, preferably 6 to 11 times, based on the undrawn yarn (or pre-drawn yarn).

[0117] In addition, the fiber after drawing can be used as the fiber of the first embodiment, but after drawing, it can also be chemically cross-linked between polypeptide molecules in the obtained protein fiber to be used as the fiber of the first embodiment. Examples of functional groups that can be cross-linked include amino groups, carboxyl groups, thiol groups, and hydroxy groups. For example, the amino group of the lysine side chain contained in the polypeptide can be cross-linked by an amide bond through dehydration condensation with the carboxyl group of the glutamic acid or aspartic acid side chain. Cross-linking may be carried out by performing a dehydration condensation reaction under vacuum heating, or may be carried out using a dehydration condensing agent such as carbodiimide.

[0118] Cross-linking between polypeptide molecules may be carried out using a cross-linking agent such as carbodiimide or glutaraldehyde, or may be carried out using an enzyme such as transglutaminase. Carbodiimide is a compound represented by the general formula R1N=C=NR2 (wherein R1 and R2 each independently represent an organic group containing an alkyl group or cycloalkyl group having 1 to 6 carbon atoms). Specific examples of carbodiimide include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropylcarbodiimide (DIC), and the like. Among these, as carbodiimide, EDC and DIC are preferred because they have a high ability to form amide bonds between polypeptide molecules and are easy to undergo cross-linking reactions.

[0119] The crosslinking treatment is preferably carried out by applying a crosslinking agent to the protein fiber and then performing vacuum heating and drying. The crosslinking agent may be applied to the protein fiber in its pure form, or it may be diluted to a concentration of 0.005 to 10% by mass with a lower alcohol having 1 to 5 carbon atoms, a buffer solution, etc. and then applied to the protein fiber. The crosslinking treatment is preferably carried out at a temperature of 20 to 45 °C for 3 to 42 hours. By the crosslinking treatment, higher stress (strength) can be imparted to the protein fiber.

[0120] As described above, by spinning using a dope solution in which protein is dissolved, β-sheet crystals are oriented, so the obtained fiber has an initial tensile elastic modulus close to that of a high-strength fiber obtained by liquid crystal spinning with fiber drawing. Furthermore, the obtained fiber has a strong tendency against compressive strain input in the fiber axis direction, and is characterized in that a fracture surface where cracks occur along the fiber axis direction is difficult to generate. This is presumably because the proteins act on each other between the β-sheet crystals oriented in the fiber axis direction, or because the proteins have a higher-order structure.

[0121] (Second rubber-reinforcing fiber) In addition, the rubber-reinforcing fiber according to the second embodiment of the present invention (hereinafter sometimes referred to as "the fiber of the second embodiment") is characterized by using a protein fiber having an initial tensile elastic modulus of 2.0 GPa or more when wet. In other words, the fiber of the second embodiment is a protein fiber having an initial tensile elastic modulus of 2.0 GPa or more when wet. Since the fiber of the second embodiment has a high initial tensile elastic modulus when wet as described above, a decrease in fiber strength when water is contained by immersing it in water or the like is significantly suppressed. Therefore, the fiber of the second embodiment is difficult to break even when it is compounded with a rubber material, particularly when an operation requiring contact with an aqueous component is performed.

[0122] The fibers of the second embodiment include the fibers of the first embodiment described above, and may further include fibers other than the fibers of the first embodiment. For example, the fibers of the second embodiment may include protein fibers containing proteins other than the hydrophobic proteins described above (such as hydrophilic proteins), and fibers formed by coating the surface thereof with components that are difficult to dissolve in water, etc.

[0123] In this specification, the "initial tensile modulus" refers to the Young's modulus measured by performing a tensile test on a fiber with a fiber length of 300 mm under the condition of a tensile speed of 300 mm / min. In this specification, "(fibers) when wet" refers to fibers that have been immersed in water for 15 minutes to be sufficiently hydrated. On the other hand, "(fibers) when dry" in this specification refers to fibers that have not been subjected to special treatments such as immersion. The above tensile test can be performed, for example, using Instron3345 manufactured by Instron Corporation. Furthermore, the above initial tensile modulus can be calculated, for example, by analyzing the data obtained by Instron3345 using analysis software such as "Instron Bluehill Le".

[0124] The fibers of the second embodiment preferably have an initial tensile modulus when wet of 3.0 GPa or more, more preferably 4.0 GPa or more. Also, the initial tensile modulus when wet of the fibers of the second embodiment can be, for example, 12.0 GPa or less.

[0125] Also, the fibers of the second embodiment preferably have a breaking strength when wet of 50 MPa or more. In this case, the strength of the rubber article can be more effectively reinforced. Also, the breaking strength when wet of the fibers of the second embodiment can be, for example, 350 MPa or less. Furthermore, the fibers of the second embodiment preferably have a breaking strength when dry of 100 MPa or more. In this case, the strength of the rubber article can be more effectively reinforced. Also, the breaking strength when dry of the fibers of the second embodiment can be, for example, 700 MPa or less. In addition, in this specification, the "breaking strength" refers to the strength when the tensile strength decreases by 15% in a tensile test on a fiber with a fiber length of 300 mm under the condition of a tensile speed of 300 mm / min. The above tensile test can be carried out, for example, using Instron 3345 manufactured by Instron Corporation.

[0126] The initial tensile modulus and breaking strength of the above-mentioned fiber when dry / wet can be adjusted, for example, by appropriately selecting the type of solvent used in the dope solution during spinning, adjusting the draw ratio during spinning, and the like.

[0127] The method for manufacturing the fiber of the second embodiment is the same as that described above for the fiber of the first embodiment.

[0128] Hereinafter, matters common to the fiber of the first embodiment and the fiber of the second embodiment (hereinafter, may be collectively referred to simply as "fiber") will be described.

[0129] The above fiber is not particularly limited, and can be, for example, staple fiber; non-woven fabric; cord fabric; film; cord such as canvas monofilament cord, twisted cord formed by twisting one or more multifilaments; and the like. In particular, when reinforcing rubber articles such as tires and conveyor belts, the above fiber is preferably a cord, and more preferably a twisted cord formed by twisting a plurality of filaments. Further, the total fineness of the above fiber is not particularly limited, but is preferably 500 to 5,000 dtex.

[0130] When used for compounding with a rubber material, the above fiber preferably has a fiber length of more than 6.0 mm, more preferably more than 10 mm, and even more preferably 35 mm or more. As described above, the fiber of the present embodiment has the effect of particularly improving the tendency to be easily cut when wet, and this effect is more significantly exhibited in longer fibers than in short fibers that have already been cut. Regarding the fiber length, in the 2nd Edition Fiber Handbook (edited by the Fiber Society; published by Maruzen Co., Ltd.; in 1994; page 66), fibers with a length exceeding 10 mm are classified as "long fibers", and fibers with a length less than 10 mm are classified as "short fibers". And in this embodiment, classification can also be performed based on the same criteria as in the above-mentioned document. Note that fibers with a length exactly 10 mm are not mentioned in the above-mentioned document, but in this specification, they are considered to be included in "short fibers". Also, "long fibers" shall include continuous fibers.

[0131] In order to obtain a higher-strength rubber article by embedding the above fibers in a rubber material, the fibers may be provided with a coating layer made of an adhesive composition on the surface. Also, when forming the coating layer, the coating surface of the above fibers may be pretreated in advance by electron beam, microwave, corona discharge, plasma, etc. Further, the thickness of the coating layer is preferably 0.5 to 50 μm, more preferably 1 to 10 μm.

[0132] From the viewpoint of facilitating coating, the adhesive composition is preferably dissolved in a solvent and used after reducing the viscosity. Also, from the viewpoint of reducing the environmental load, the above solvent preferably consists of water. That is, from the viewpoint of reducing the environmental load, the adhesive composition is preferably an aqueous adhesive composition. Since the decrease in fiber strength is significantly suppressed when the above fibers contain water as described above, the fibers are not easily broken even when an aqueous adhesive composition is used. In this specification, the "aqueous adhesive composition" refers to an adhesive composition in which the proportion of moisture in the whole is 30% by mass or more.

[0133] The above aqueous adhesive composition is not particularly limited as long as it can improve the adhesiveness to the rubber material. For example, an aqueous adhesive composition containing components usually used when adhering rubber and fibers can be mentioned. In particular, the above aqueous adhesive composition preferably contains a rubber latex ((A) component) like rubber paste. In other words, the above aqueous adhesive composition preferably contains a rubber component. In this case, the adhesiveness can be enhanced more effectively.

[0134] Examples of the above-mentioned (A) rubber latex (rubber component) include rubber latex that can be vulcanized with sulfur. More specifically, natural rubber latex, synthetic rubber latex having an unsaturated diene, etc. may be mentioned. These rubber latexes may be used alone or in combination of two or more.

[0135] The above-mentioned natural rubber latex is not particularly limited. For example, field latex, ammonia-treated latex, centrifugally concentrated latex, deproteinized latex treated with a surfactant and / or an enzyme, and those obtained by modifying these may be mentioned.

[0136] Examples of the above-mentioned synthetic rubber latex having an unsaturated diene are not particularly limited. For example, styrene-butadiene copolymer rubber latex, vinylpyridine-styrene-butadiene copolymer rubber latex, carboxyl group-modified styrene-butadiene copolymer rubber latex, nitrile rubber latex, chloroprene rubber latex, etc. may be mentioned. These synthetic rubber latexes may be used alone or in combination of two or more.

[0137] The content (solid content concentration) of the above-mentioned (A) rubber latex in the total solid content of the aqueous adhesive composition is preferably 25% by mass or more. In this case, the rubber component of the rubber latex contained in the adhesive composition that interacts with the rubber composition for topping (the rubber material used for compounding) and vulcanizes becomes sufficiently large, and adhesion due to co-vulcanization of the rubber components can be obtained, and the adhesion state of the coating rubber in the composite of the rubber-reinforcing fiber and the rubber material can be made better. From the same viewpoint, the content (solid content concentration) of the above-mentioned (A) rubber latex in the total solid content of the aqueous adhesive composition is more preferably 30% by mass or more.

[0138] In addition, it is preferable that the above aqueous adhesive composition further contains (B) resorcinol and (C) formaldehyde in addition to (A) rubber latex. In this case, the adhesiveness can be enhanced more effectively. Such an aqueous adhesive composition is not particularly limited, and a conventionally used known resorcinol-formaldehyde-latex mixture (RFL composition) can be used.

[0139] When the aqueous adhesive composition contains (A) rubber latex, (B) resorcinol, and (C) formaldehyde, the content (solid content concentration) of the above (A) rubber latex in the total solid content of the aqueous adhesive composition is preferably 25% by mass or more and preferably 80% by mass or less. If the above content is 25% by mass or more, the rubber component of the rubber latex contained in the adhesive composition that vulcanizes by interacting with the rubber composition for topping (the rubber material used for compounding) becomes sufficiently large, and adhesion due to co-vulcanization of the rubber components can be obtained, and the adhesion state of the coating rubber in the composite of the rubber-reinforcing fiber and the rubber can be made better. Further, if the above content is 80% by mass or less, it becomes possible to secure a certain amount or more of (B) resorcinol and (C) formaldehyde relative to each other. As a result, the cohesive failure resistance of the coating layer composed of the adhesive composition is sufficiently ensured, and it becomes difficult for breakage to occur within the coating layer, and sufficient adhesiveness can be obtained. From the same viewpoint, the content (solid content concentration) of the above (A) rubber latex in the total solid content of the aqueous adhesive composition is more preferably 30% by mass or more, still more preferably 40% by mass or more, more preferably 70% by mass or less, and still more preferably 65% by mass or less.

[0140] When the aqueous adhesive composition contains (A) a rubber latex, (B) resorcinol, and (C) formaldehyde, the molar ratio of (C) formaldehyde to (B) resorcinol ((C) formaldehyde / (B) resorcinol) is preferably in the range of 0.8 to 1.5. In this case, the polycondensation of (B) resorcinol with (C) formaldehyde becomes appropriate, and the cohesive failure resistance of the coating layer composed of the adhesive composition can be further improved.

[0141] Alternatively, from the viewpoint of reducing the environmental load, it is also preferable that the above aqueous adhesive composition does not contain (B) resorcinol.

[0142] Furthermore, in addition to (A) the rubber latex, the aqueous adhesive composition (D) an aqueous compound having a (blocked) isocyanate group, (E) an epoxy compound, and (F) an amine compound can further contain one or more components selected from the group consisting of. Such an aqueous adhesive composition is not particularly limited, and for example, the adhesive composition described in International Publication No. 2010 / 125992 can be used.

[0143] The "(blocked) isocyanate group" of the above aqueous compound having a (blocked) isocyanate group means a blocked isocyanate group or an isocyanate group, (i) a blocked isocyanate group formed by the reaction of an isocyanate group with a blocking agent for the isocyanate group, (ii) an isocyanate group in which the isocyanate group is unreacted with a blocking agent for the isocyanate group, (iii) an isocyanate group formed by the dissociation of a blocking agent from a blocked isocyanate group, (iv) an isocyanate group, is included.

[0144] The term "aqueous" of the aqueous compound having the above (D) (blocked) isocyanate group indicates that it is water-soluble or water-dispersible. The above water-solubility does not necessarily mean complete water-solubility, but also means being partially water-soluble or not phase-separating in the aqueous solution of the adhesive composition.

[0145] The above-mentioned blocking agent is not particularly limited as long as it is a blocking agent compound that can protect isocyanate groups from any chemical reaction and dissociate the blocking agent by heat treatment as necessary to restore the isocyanate groups. Examples of such blocking agents include, but are not limited to, alcohols, phenols, active methylene compounds, oximes, lactams, amines, etc. Specifically, lactams such as ε-caprolactam, δ-valerolactam, γ-butyrolactam; phenols such as phenol, cresol, ethylphenol, butylphenol, octylphenol, nonylphenol, dinonylphenol, thiophenol, chlorophenol, amylphenol; oximes such as methyl ethyl ketoxime, acetoxime, acetophenone oxime, benzophenone oxime, cyclohexanone oxime; alcohols such as methanol, ethanol, butanol, isopropyl alcohol, butyl alcohol, cyclohexanol; dialkyl malonates such as dimethyl malonate, diethyl malonate; active methylene compounds such as methyl acetoacetate, ethyl acetoacetate, acetylacetone; mercaptans such as butyl mercaptan, dodecyl mercaptan; amides such as acetanilide, acetic acid amide; imides such as succinimide, phthalimide, maleimide; sulfites such as sodium bisulfite; cellosolves such as methyl cellosolve, ethyl cellosolve and butyl cellosolve; pyrazoles such as pyrazole, 3,5-dimethylpyrazole, 3-methylpyrazole, 4-benzyl-3,5-dimethylpyrazole, 4-nitro-3,5-dimethylpyrazole, 4-bromo-3,5-dimethylpyrazole and 3-methyl-5-phenylpyrazole; amines such as dimethylamine, diethylamine, di-n-propylamine, diisopropylamine, dicyclohexylamine, diphenylamine, xylylidene, N,N-diethylhydroxyamine, N,N'-diphenylformamidine, 2-hydroxypyridine, 3-hydroxypyridine and 2-mercaptopyridine; and triazoles such as 1,2,4-triazole, etc. The blocking agent may be used alone or in combination of two or more. In particular, as the blocking agent, phenol, ε-caprolactam, and ketoxime, which can easily and stably obtain the thermosetting of the adhesive composition by thermal dissociation upon heating, can be preferably used.

[0146] The aqueous compound having the above (D) (blocked) isocyanate group is preferably a water-dispersible (blocked) isocyanate compound that is an addition product of (D-1) a polyisocyanate having an aromatic ring and a blocking agent having one or more active hydrogen groups. Here, regarding the above "water-dispersible (blocked) isocyanate compound that is an addition product of (D-1) a polyisocyanate having an aromatic ring and a blocking agent having one or more active hydrogen groups", the "active hydrogen group" refers to a group containing hydrogen that becomes active hydrogen (atomic hydrogen (hydrogen radical) and hydride ion (hydride)) when placed under suitable conditions. Examples of the above active hydrogen group include an amino group and a hydroxyl group.

[0147] In addition, the above-mentioned "aqueous dispersible (blocked) isocyanate compound, which is an addition product of a polyisocyanate having an aromatic ring and a blocking agent having one or more active hydrogen groups" specifically includes aromatic polyisocyanates or araliphatic polyisocyanates. Examples of aromatic polyisocyanates include phenylenediisocyanates such as m-phenylenediisocyanate and p-phenylenediisocyanate; tolylenediisocyanates such as 2,4-tolylenediisocyanate and 2,6-tolylenediisocyanate (TDI); diphenylmethane diisocyanates such as 2,4'-diphenylmethane diisocyanate, 4,4'-diphenylmethane diisocyanate (MDI), dialkyldiphenylmethane diisocyanate, and tetraalkyldiphenylmethane diisocyanate; polymethylene polyphenyl polyisocyanate (polymeric MDI); m- or p-isocyanatophenylsulfonyl isocyanates; diisocyanatobiphenyls such as 4,4'-diisocyanatobiphenyl and 3,3'-dimethyl-4,4'-diisocyanatobiphenyl; naphthalene diisocyanates such as 1,5-naphthalene diisocyanate; and the like. Examples of araliphatic polyisocyanates include xylylene diisocyanates such as m-xylylene diisocyanate, p-xylylene diisocyanate (XDI), and tetramethylxylylene diisocyanate; diethylbenzene diisocyanate; and α,α,α,α-tetramethylxylylene diisocyanate (TMXDI); and the like. Further, modified products such as carbodiimide, polyol, and allophanate of the above polyisocyanate are also included. Among these polyisocyanates containing an aromatic ring in the molecule, from the viewpoint of the code focusing property of the adhesive composition, aromatic polyisocyanates are preferred, more preferably tolylenediisocyanate (TDI), diphenylmethane diisocyanate (MDI), or polymethylene polyphenyl polyisocyanate (polymeric MDI), and particularly preferably diphenylmethane diisocyanate (MDI).

[0148] Furthermore, the aqueous compound having the above (D) (blocked) isocyanate group is more preferably an aqueous urethane compound having a (D-2) (blocked) isocyanate group.

[0149] Examples of commercially available products of the aqueous urethane compound having the above (D-2) (blocked) isocyanate group include DM-6400 manufactured by Meisei Chemical Industry Co., Ltd., Elastron BN69 manufactured by Daiichi Kogyo Seiyaku Co., Ltd., GRILBOND IL-6 manufactured by EMS, etc. Furthermore, as the aqueous urethane compound having the above (D-2) (blocked) isocyanate group, Elastron BN27, BN77, BN11, etc. manufactured by Daiichi Kogyo Seiyaku Co., Ltd. can also be used.

[0150] The aqueous urethane compound having the above (D-2) (blocked) isocyanate group can be obtained, for example, by reacting an organic polyisocyanate compound (α) having a molecular structure in which aromatics are bonded by an alkylene chain, a compound (β) having a plurality of active hydrogens, and a thermal dissociable blocking agent (γ) for an isocyanate group. In the organic polyisocyanate compound (α), the bond of the alkylene chain is preferably a methylene bond.

[0151] The aqueous urethane compound having the above (D-2) (blocked) isocyanate group can be produced by a known method, such as the method described in Japanese Patent Publication No. 63-51474.

[0152] When compounded, the content (solid content concentration) of the aqueous compound having the above (D) (blocked) isocyanate group in the total solid content in the aqueous adhesive composition is preferably 5% by mass or more, and preferably 75% by mass or less. If the above content is 5% by mass or more, the adhesiveness during the compounding of the fiber and the rubber material can be further improved. Also, if the above content is 75% by mass or less, it becomes possible to secure a certain amount or more of (A) rubber latex relative to it, so that the adhesiveness during the compounding of the fiber and the rubber material can be maintained well. From the same viewpoint, the content (solid content concentration) of the aqueous compound having the above (D) (blocked) isocyanate group in the total solid content in the aqueous adhesive composition is more preferably 10% by mass or more, still more preferably 20% by mass or more, preferably 75% by mass or less, more preferably 60% by mass or less, and still more preferably 50% by mass or less.

[0153] The above (E) epoxy compound refers to a compound having an oxacyclopropane (oxirane) (epoxy group), which is a 3-membered ring ether, in its structural formula.

[0154] The above (E) epoxy compound preferably is a compound containing two or more epoxy groups in one molecule. In this case, due to polyfunctionalization, the fracture resistance of the coating layer composed of the adhesive composition is further increased, and the adhesive strength at high temperature also becomes higher. From the same viewpoint, the above (E) epoxy compound more preferably is a compound containing four or more epoxy groups in one molecule.

[0155] Specific examples of the above-mentioned (E) epoxy compound include, for example, reaction products of polyhydric alcohols such as diethylene glycol diglycidyl ether, polyethylene diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, polyglycerol polyglycidyl ether, pentaerythritol polyglycidyl ether, diglycerol polyglycidyl ether, sorbitol polyglycidyl ether, etc. and epichlorohydrin; novolak-type epoxy resins such as phenol novolak-type epoxy resin, cresol novolak-type epoxy resin, etc.; bisphenol A-type epoxy resin, etc. The above-mentioned (E) epoxy compound is preferably a reaction product of polyhydric alcohols and epichlorohydrin, or a novolak-type epoxy resin. Commercially available products can be used for the above-mentioned sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, novolak-type epoxy resin, etc.

[0156] When compounded, the content (solid content concentration) of the (E) epoxy compound in the total solid content of the aqueous adhesive composition is preferably 0.3% by mass or more and preferably 45% by mass or less. If the above content is 0.3% by mass or more, the adhesiveness can be further improved. Also, if the above content is 45% by mass or less, it becomes possible to secure a certain amount or more of other components such as (A) rubber latex relative to each other, so that the adhesiveness during the composite of the fiber and the rubber material can be maintained well. From the same viewpoint, the content (solid content concentration) of the (E) epoxy compound in the total solid content of the aqueous adhesive composition is more preferably 0.5% by mass or more and more preferably 40% by mass or less.

[0157] As the above (F) amine compound, for example, amines known as amine-based curing agents can be used. Examples include aliphatic amines, alicyclic amines, aromatic amines, heterocyclic amines, Mannich bases, and the like. Among these, as the (F) amine compound, it is preferable to use a heterocyclic amine.

[0158] Examples of the heterocyclic amine include piperazine, 1-aminoethylpiperazine, 1,4-diaminoethylpiperazine, 3-aminopyrrolidine, 2-(2-aminoethyl)pyrrolidine, 4,4'-bipiperazine, 4,4'-ethylenedipiperidine, 4,4'-trimethylenedipiperidine, 4-(aminomethyl)piperidine, 3-(4-aminobutyl)piperidine, and the like.

[0159] When blended, the content (solid content concentration) of the (F) amine compound in the total solid content of the aqueous adhesive composition is preferably 0.2% by mass or more and 10% by mass or less. If the above content is 0.2% by mass or more, the (F) amine compound can further enhance the curing as a curing agent for the (D) (blocked) isocyanate group-containing aqueous compound and / or the (E) epoxy compound. Also, if the above content is 10% by mass or less, it is possible to effectively suppress the embrittlement of the aqueous adhesive composition and thus the decrease in adhesive strength.

[0160] Further, when the above aqueous adhesive composition contains (A) rubber latex, it is preferably free of sulfur (elemental sulfur). When sulfur is used, there is a risk that powdered sulfur will precipitate in the (A) rubber latex, or when the rubber particles of the (A) rubber latex are vulcanized with sulfur by heating or drying, it may become difficult for the rubber component in the topping rubber material to interact with the rubber component in the adhesive composition.

[0161] The coating layer composed of the above adhesive composition can be formed by dipping, brushing, casting, spraying, roll coating, knife coating, etc. Hereinafter, dipping will be taken as an example for explanation.

[0162] Figure 2 shows a schematic view of an example of an apparatus for forming a coating layer made of an adhesive composition on the surface of a fiber (cord) using the dipping method. In the apparatus 10 shown in Figure 2, the cord is unwound, the tension per cord is adjusted to 0.20 to 1.2 Kg, and the cord is dipped into the adhesive composition (liquid) in the liquid tank 5. Next, after adjusting the amount of the adhesive composition adhering to the cord by the squeezing pressure between the rolls by the squeezing roll 4, the cord is dried in the drying zone 1 at, for example, 110°C to 140°C, preferably 110 to 120°C, and then heat-treated in the hot zone 2 and the normalization zone 3 at, for example, 130°C to 170°C, preferably 130 to 150°C. Thereafter, it may be cooled appropriately. Note that if the tension of the cord exceeds 1.2 Kg, the cord strength may be reduced, and if it is less than 0.20 Kg, slack may occur in the running cord, and there is a risk of frequent derailment from the cord path. Note that when the tension of the cord during dipping is lower, the deformation of the protein fiber in the direction of the tension in contact with water is smaller. Therefore, the tension of the cord is preferably 0.20 to 0.30 Kg.

[0163] Then, in order to reinforce the obtained rubber article, the above fiber can be combined (topping) with the rubber material by being embedded in the rubber material or the like, and vulcanized and integrated by a conventional method. The rubber component contained in the rubber material is not particularly limited, and examples thereof include conjugated diene rubbers such as natural rubber (NR), polyisoprene rubber (IR), polybutadiene rubber (BR), styrene-butadiene rubber (SBR), acrylonitrile-butadiene rubber (NBR), chloroprene rubber (CR), and butyl rubber (IIR), and further ethylene-propylene copolymer rubber (EPM), ethylene-propylene-diene copolymer rubber (EPDM), polysiloxane rubber, and the like. Among these, conjugated diene synthetic rubbers are preferred. These rubber components may be used alone or in combination of two or more.

[0164] In addition, various compounding agents such as fillers such as carbon black, silica, and aluminum hydroxide, vulcanizing agents, vulcanization accelerators, anti-aging agents, and softening agents, which are usually used in the rubber industry, can be appropriately compounded with the above rubber component.

[0165] Examples of the vulcanizing agent include inorganic vulcanizing agents such as sulfur, tellurium polysulfide compounds such as tetramethylthiuram disulfide and dipentamethylene thiuram tetrasulfide, and organic vulcanizing agents such as 4,4-dithiomorpholine, p-quinonedioxime, p,p'-dibenzoquinonedioxime, and cyclic sulfur imide. Among these, it is preferable to use sulfur as the vulcanizing agent.

[0166] When the above fiber has a coating layer made of an adhesive composition on its surface, when vulcanizing together with the rubber material, the vulcanizing agent contained in the rubber material migrates to the adhesive composition, and the adhesive composition is crosslinked by the vulcanizing agent, so that higher adhesiveness can be exhibited.

[0167] (Rubber article) A rubber article according to an embodiment of the present invention is characterized by including the above-described rubber reinforcing fiber. Since the above rubber article includes the above-described rubber reinforcing fiber, it has high strength. The above rubber article is not particularly limited except for including the above-described rubber reinforcing fiber. The rubber article is not particularly limited, and examples thereof include tires, conveyor belts, belts, hoses, air springs, rubber crawlers, seismic isolation rubbers, and vibration isolation rubbers.

[0168] (Tire cord and tire) Further, a tire cord according to an embodiment of the present invention is characterized by being made of the above-described rubber reinforcing fiber. Since the above tire cord is made of the above-described rubber reinforcing fiber, it has high strength. Note that the above tire cord may be the above-described rubber reinforcing fiber. Furthermore, a tire according to an embodiment of the present invention is characterized by including the above-described tire cord. Since the above tire includes the above-described tire cord, it has high strength. The above tire is not particularly limited except for including the above-described tire cord in an arbitrary member. Also, the manufacturing method of the above tire is not particularly limited, and a tire can be manufactured using a known tire manufacturing method.

Examples

[0169] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples and can be appropriately modified without changing the gist thereof.

[0170] (1-1) Synthesis of Nucleic Acid Encoding Hydrophobic Protein and Construction of Expression Vector A nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 17 designed was synthesized. An NdeI site was added to the 5' end of the nucleic acid, and an EcoRI site was added downstream of the stop codon. These five types of nucleic acids were cloned into a cloning vector (pUC118). Then, the nucleic acid was excised by restriction enzyme treatment with NdeI and EcoRI, and then recombined into the protein expression vector pET-22b(+) to obtain an expression vector.

[0171] (1-2) Expression of Hydrophobic Protein E. coli BLR(DE3) was transformed with the pET22b(+) expression vector containing the nucleic acid encoding the protein having the amino acid sequence shown in SEQ ID NO: 17. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (shown in Table 4) containing ampicillin so that OD600 became 0.005. The culture solution temperature was maintained at 30 °C, and flask culture was performed until OD600 reached 5 (about 15 hours) to obtain a seed culture solution.

[0172]

Table 4

[0173] The seed culture solution was added to a jar fermenter supplemented with 500 mL of production medium (shown in Table 5) so that OD600 became 0.05, and the transformed E. coli was inoculated. The culture solution temperature was maintained at 37 °C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

[0174]

Table 5

[0175] Immediately after the glucose in the production medium was completely consumed, a feed solution (455 g / L glucose, 120 g / L Yeast Extract) was added at a rate of 1 mL / min. The temperature of the culture solution was maintained at 37°C and cultured under constant control at pH 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M 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. At the time point 20 hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. SDS-PAGE was performed using the cells prepared from the culture solution before and after the addition of IPTG, and the expression of the target protein (PRT918) was confirmed by the appearance of a band of the target protein size depending on the addition of IPTG. In addition, for the expressed protein (modified protein), the value (average HI) obtained by dividing the sum of the hydrophobicity indices (HI) of all constituent amino acid residues by the total number of amino acid residues was 0.45, confirming that it is a hydrophobic protein.

[0176] (1-3) Purification of Hydrophobic Protein The cells harvested 2 hours after adding 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 approximately 1 mM PMSF, and the cells were disrupted using a high-pressure homogenizer (GEA Niro Soavi). The disrupted cells were centrifuged to obtain a precipitate. The obtained precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity was achieved. The washed precipitate was suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) at a concentration of 100 mg / mL, stirred with a stirrer at 60 °C for 30 minutes, and dissolved. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36 / 32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, and the moisture was removed using a freeze dryer to recover the freeze-dried powder.

[0177] Regarding the target protein in the obtained freeze-dried powder, the purification degree was confirmed by image analysis of the results of polyacrylamide gel electrophoresis of the powder using Totallab (nonlinear dynamics ltd.). As a result, the purification degree was approximately 85%.

[0178] (1-4) Preparation of spinning dope DMSO in which lithium chloride was dissolved to a concentration of 4% by mass was used as the solvent. The freeze-dried powder of the hydrophobic protein of PRT918 (SEQ ID NO: 17) prepared above was added to the solvent so that the content became 24% by mass. Then, after dissolving with a 90 °C aluminum block heater for 1 hour, insolubles and bubbles were removed to obtain a spinning dope.

[0179] (1-5) Spinning (preparation of protein fiber) The spinning dope was filled into a reserve tank and discharged from a monofilament nozzle with a diameter of 0.1 or 0.2 mm into a 100% by mass methanol coagulation bath using a gear pump. The discharge rate was adjusted to 0.01 - 0.08 mL / min. After coagulation, washing and stretching were performed in a 100% by mass methanol washing bath. After washing and stretching, it was dried using a hot plate, and the obtained raw yarn (protein fiber) was wound up.

[0180] (2) Preparation of protein fibers containing hydrophilic proteins A nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 4 was synthesized, and a protein (PRT799) was expressed in substantially the same manner as described above, except that this nucleic acid was used. The expressed protein (modified protein) was confirmed to be a hydrophilic protein rather than a hydrophobic protein, with an average hydrophobicity index (HI) value of -0.8, which is obtained by dividing the sum of the hydrophobicity indices (HI) of all constituent amino acid residues by the total number of amino acid residues. For this modified protein, purification and spinning were performed in the same manner as above to obtain raw yarn (protein fiber).

[0181] (3) Preparation of twisted cords In Example 1, protein fibers containing the above-described hydrophobic protein were used, and in Comparative Examples 1 and 2, protein fibers containing hydrophilic proteins were used to obtain cords (twisted cords) twisted at 2000 dtex / 2, with 42 turns / cm of S-twist and 42 turns / cm of Z-twist, respectively.

[0182] (4) Preparation of aqueous adhesive composition 500 parts by mass of water and 500 parts by mass of natural rubber latex (Field latex) with a solid content concentration of 20% by mass were mixed to obtain a rubber latex containing natural rubber (NR latex). In Example 1 and Comparative Example 2, the NR latex was used as the aqueous adhesive composition.

[0183] To 756 parts by mass of water, 244 parts by mass of a vinyl pyridine-styrene-butadiene copolymer rubber latex (manufactured by Sumitomo A&L, Inc., "PYLATEX") having a solid content concentration of 41% by mass was mixed to obtain a rubber latex (Vp latex) containing a vinyl pyridine-styrene-butadiene copolymer rubber. In Example 2 and Comparative Example 3, the Vp latex was used as an aqueous adhesive composition.

[0184] 588.29 parts by mass of water, 16.61 parts by mass of resorcinol (manufactured by Tokyo Chemical Industry Co., Ltd.), 20.80 parts by mass of formalin (manufactured by Tokyo Chemical Industry Co., Ltd., "Formaldehyde Solution (37%)"), and 3.79 parts by mass of caustic soda (10%) were mixed and then aged at room temperature for 7 hours to obtain an aged liquid containing resorcinol and formaldehyde. Next, 370.51 parts by mass of a vinyl pyridine-styrene-butadiene copolymer rubber latex (manufactured by Sumitomo A&L, Inc., "PYLATEX") was added to 629.49 parts by mass of the above-aged liquid, and then aged at room temperature for 16 hours to obtain an RFL composition (resorcinol-formaldehyde-latex mixture). In Example 3 and Comparative Example 4, the RFL composition was used as an aqueous adhesive composition.

[0185] 462.39 parts by mass of water, 242.17 parts by mass of a compound having a (blocked) isocyanate group (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., "Elastron BN27", solid content concentration 30%, a heat-reactive aqueous urethane resin having a methylene diphenyl molecular structure), 8.55 parts by mass of an epoxy compound (manufactured by Nagase ChemteX Corporation, "Denacol EX614B", sorbitol polyglycidyl ether), 1.99 parts by mass of an amine compound (manufactured by Tokyo Chemical Industry Co., Ltd., piperazine (anhydrous)), and 284.90 parts by mass of a vinyl pyridine-styrene-butadiene copolymer rubber latex (manufactured by Sumitomo A&L, Inc., "PYLATEX") were sequentially mixed to obtain a latex-containing composition. The latex-containing composition has a composition according to Example 5 of International Publication No. 2010 / 125992 and does not contain resorcinol. In Example 4 and Comparative Example 5, the latex-containing composition was used as an aqueous adhesive composition.

[0186] (5) Formation of a coating layer on the protein fiber The apparatus 10 shown in Fig. 2 was used. Using the twisted yarn cord prepared in the above (3), a tension of 0.25 kg / strand (cord tension) was applied and it was immersed in the adhesive liquid stored in the liquid tank 5. As the adhesive liquid, it was appropriately selected and used from the aqueous adhesive composition obtained in the above (4). Next, the twisted yarn cord with the adhesive liquid attached was dried in the drying zone 1 (130 °C, 120 seconds) while applying a tension of 0.6 kg / strand (cord tension), and then heat-treated in the hot zone 2 (150 °C, 40 seconds) and the normalization zone 3 (140 °C, 40 seconds). In this way, a coating layer made of the aqueous adhesive composition was formed on the surface of the twisted yarn cord.

[0187] (6) Manufacture of fiber-reinforced rubber The obtained twisted yarn cord was embedded in an unvulcanized rubber composition (unvulcanized) containing a rubber component composed of natural rubber and styrene-butadiene rubber, carbon black, and a crosslinking agent, and vulcanized at 160 °C for 20 minutes to obtain fiber-reinforced rubber.

[0188] And in carrying out the above operations, the following measurements etc. were performed.

[0189] (7) Measurement of the initial tensile elastic modulus and breaking strength of the protein fiber (before formation of the coating layer) when dry / wet Regarding the obtained protein fiber, a tensile test was performed using Instron 3345 manufactured by Instron under the conditions of a fiber length of 300 mm and a tensile speed of 300 mm / min in an environment of a temperature of 20 °C and a humidity of 65%. The data obtained from this tensile test was analyzed with the analysis software "Instron Bluehill Le" to calculate the Young's modulus and obtain the "initial tensile elastic modulus when dry". Also, after immersing the obtained protein fibers in water for 15 minutes to sufficiently hydrate them, the Young's modulus of the fibers was calculated in the same manner as above, and the "initial tensile elastic modulus when wet" was determined. Furthermore, when the same tensile test as above was performed on the hydrated fibers, the strength at which the tensile strength decreased by 15% was measured as the "breaking strength when wet". The results are shown in Table 7.

[0190] (8) Evaluation of the frayability of protein fibers Regarding the immersion of the twisted cord in (5) above, the presence or absence of cord breakage when a 100 m twisted cord was subjected to the immersion treatment was visually confirmed, and the frayability was evaluated according to the following criteria. The results are shown in Table 7. ○: No cord breakage occurred ×: Cord breakage occurred

[0191] (9) Evaluation of adhesive strength and rubber adhesion Finally, a cord was dug out from the obtained fiber-reinforced rubber (vulcanizate), and the cord was peeled off from the vulcanizate by pulling it at a speed of 300 mm / min, and the peeling resistance per cord was determined as the adhesive strength (N / cord). Furthermore, the rubber adhesion state and the presence or absence of cutting of the cord after peeling were observed, and ranking was performed according to the criteria shown in Table 6. The results are shown in Table 7.

[0192]

Table 6

[0193]

Table 7

[0194] From Table 7, it can be seen that the fibers of Examples 1 to 4 are composed of hydrophobic proteins and have an initial tensile elastic modulus of 2.0 GPa or more when wet. Therefore, even when they are compounded with a rubber material, especially when an operation involving contact with an aqueous component is performed, it is found that they are difficult to break. Furthermore, from Table 7, it can be seen that the fibers of Examples 1 to 4 retain high fiber strength and can exhibit high adhesiveness to a rubber material even when a coating layer composed of an aqueous adhesive composition is formed.

Industrial Applicability

[0195] According to the present invention, it is possible to provide rubber reinforcing fibers that can be produced using bio-derived raw materials and are difficult to break even when compounded with a rubber material. Further, according to the present invention, it is possible to provide high-strength rubber articles, tire cords, and tires using the above-described rubber reinforcing fibers, respectively.

Explanation of Symbols

[0196] 10 apparatus; 1 drying zone; 2 hot zone; 3 normalization zone; 4 drawing roll; 5 liquid bath

Claims

1. A hydrophobic protein that has its amino acid sequence modified based on the amino acid sequence of naturally occurring spider fibroin or is artificially designed and synthesized without relying on naturally occurring spider fibroin, wherein the value (average HI) obtained by dividing the sum of the hydrophobicity indices (HI) of all constituent amino acid residues by the total number of amino acid residues is 0 or more, and a protein fiber containing the hydrophobic protein is used. A fiber for rubber reinforcement, characterized by this.

2. A protein fiber containing a protein that has its amino acid sequence modified based on the amino acid sequence of naturally occurring spider fibroin or is artificially designed and synthesized without relying on naturally occurring spider fibroin, and having an initial tensile modulus of elasticity when wet of 2.0 GPa or more. A fiber for rubber reinforcement, characterized by using this.

3. The fiber for rubber reinforcement according to claim 1 or 2, comprising a coating layer made of an aqueous adhesive composition on the surface.

4. The fiber for rubber reinforcement according to claim 3, wherein the aqueous adhesive composition contains (A) a rubber latex.

5. The fiber for rubber reinforcement according to claim 4, wherein the aqueous adhesive composition further contains (B) resorcinol and (C) formaldehyde.

6. The aqueous adhesive composition is (D) an aqueous compound having a (blocked) isocyanate group, (E) an epoxy compound, and (F) an amine compound The fiber for rubber reinforcement according to claim 4 or 5, further containing one or more components selected from the group consisting of.

7. The fiber for rubber reinforcement according to claim 6, wherein the aqueous compound having a (blocked) isocyanate group is a water-dispersible (blocked) isocyanate compound that is an addition product of (D-1) a polyisocyanate having an aromatic ring and a blocking agent having one or more active hydrogen groups.

8. The fiber for rubber reinforcement according to claim 6, wherein the aqueous compound having a (blocked) isocyanate group is a (blocked) isocyanate group-containing aqueous urethane compound (D-2).

9. The fiber for rubber reinforcement according to claim 6, wherein the (E) epoxy compound is a compound having two or more epoxy groups in one molecule.

10. The fiber for rubber reinforcement according to claim 6, wherein the (F) amine compound is a heterocyclic amine.

11. The rubber reinforcing fiber according to any one of claims 4 to 10, wherein the aqueous adhesive composition does not contain sulfur.

12. The rubber reinforcing fiber according to any one of claims 1 to 11, wherein the fiber length is more than 10 mm.

13. A rubber article comprising the rubber reinforcing fiber according to any one of claims 1 to 12.

14. A tire cord comprising the rubber reinforcing fiber according to any one of claims 1 to 12.

15. A tire comprising the tire cord according to claim 14.

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