JP6422291B2 - Method for controlling physical properties of silk - Google Patents

Description

  The present invention relates to a method for producing regenerated silk fibers. In particular, the present invention relates to a method for producing regenerated silk fibers exhibiting changed physical properties. The present invention also relates to a method for evaluating the quality of recycled silk fibers.

Silk II type silk ( B. mori ) silk is composed of silk fibroin and silk sericin. Silk fibroin is the main ingredient, and silk fibrinization has been studied with a focus on changes in the structure and physical properties of silk fibroin. I came. The amino acid composition of the silk fibroin molecule in rabbits is mostly alanine and glycine, and the other amino acids are serine and tyrosine. The portion that precipitates when the silk fibroin molecule is digested with chymotrypsin accounts for 55% of the silk fibroin and mainly consists of a crystal part. Most of them are repeating units of alanine, glycine and serine, that is, Ala-Gly-Ala-Gly-Ser-Gly (SEQ ID NO: 1). On the other hand, the portion dissolved in the supernatant mainly consists of an amorphous part and contains more tyrosine. Even if the repeating unit of Ala-Gly-Ala-Gly-Ser-Gly (SEQ ID NO: 1) is replaced with the repeating unit of Ala-Gly, its spectroscopic characteristics hardly change. In the analysis, an Ala-Gly repeating unit has been used as a crystal part model of silkworm silk.

  Rabbits produce silk fibers in a short time at room temperature from the state of liquid silk (silk before fiberization) in which silk is dissolved in water in the body. Silk fiber is completely different from liquid silk and becomes a solid with high strength and high elasticity. The present inventor has clarified the difference between the liquid silk structure (Silk I type structure) and the silk fiber structure (Silk II type structure). That is, it was revealed that silk fibroin molecules dissolved in water, that is, liquid silk, have not only intermolecular hydrogen bonds but also intramolecular hydrogen bonds. It was also clarified that the β-turn type II structure formed by these two types of hydrogen bonds is repeated throughout the unit consisting of glycine and alanine. Then, the silkworm swings while discharging the liquid silk, and stretches the fibroin (Silk I type structure) in the liquid silk in the direction of the molecular axis, thereby transferring the intramolecular hydrogen bond to the intermolecular hydrogen bond. It was concluded that silk fibers (Silk II type structure) were produced (Patent Document 1).

  Up to now, an antiparallel β sheet structure in which intermolecular hydrogen bonds are formed by Marsh, Pauling, etc. has been proposed as a Silk II type structure, and may be said to be an example of a typical protein structure in a biochemistry textbook. Has been posted. The structure model of Marsh et al. Is based on X-ray analysis data, and one of its major features is that intermolecular hydrogen bonds are formed between identical amino acid residues, that is, between alanine-alanine and between glycine-glycine. It has been proposed (see FIG. 1). However, many subsequent researchers have a large error (R value is 30%) between the X-ray analysis data from which the Marsh model is based and the data predicted from the proposed structure. The Silk II structure of silk fibers has come to be considered to be a more inhomogeneous structure.

Needless to say, silk thread with improved physical properties of silk fibers has extremely excellent characteristics as a material for fabrics. Silk is not only strong and has moderate elasticity and flexibility, but also has high biocompatibility, and is used, for example, as a surgical suture. However, in terms of strength and extensibility, silk yarn still does not reach spider pulling yarn. Accordingly, many attempts have been made to further increase the strength and extensibility of silk.

  As described above, a rabbit performs a swing motion while discharging liquid silk to form a cocoon. However, Shao et al. Limited the swinging motion by fixing the rabbit, forcibly removing the silk thread, and winding the silk fiber at a constant speed under moisture, thereby reducing the stress of the silk thread— It has been reported that strain characteristics can be changed (Non-Patent Document 1).

  Moreover, this inventor is disclosing the method of providing the physical property different from a natural silk fiber with respect to a reproduction silk fiber (patent document 2). Briefly, silk fibroin obtained by refining the cut straw was first dissolved in a salt solution such as lithium bromide, and then dialyzed to remove inorganic salts to obtain a silk fibroin aqueous solution. Silk fibroin film, freeze-dried sponge, etc. are dissolved in hexafluoroisopropanol (HFIP). Next, the solution is discharged through a spinneret toward a coagulation bath in which silk fibroin is insoluble and contains a solvent for dissolving HFIP, such as water, methanol, ethanol, isopropyl alcohol, and acetone. . Regenerated silk fibers can be obtained by spinning silk fibroin solidified in the bath. Furthermore, in Patent Document 2, the regenerated silk fiber is stretched about 2 to 4 times in a wet state immediately after being taken out from the coagulation bath or immediately after being taken out from the coagulation bath, or in a dry state after being taken out from the coagulation bath and air-dried. It is described that it is preferable. It is also described that such treatment can prevent the shrinkage of regenerated silk fibers and can provide stretch-formed silk yarns with very uniform fiber diameters with improved tensile strength.

In addition, in Patent Document 2, AlaCβ (β carbon atom of alanine) has three peaks, that is, a peak in the highest magnetic field region (16.16) in solid ¹³ CCP / NMR measurement using a CMagnetic CMX400 NMR spectrometer. 6 ppm) and two peaks (19.6 ppm and 21.9 ppm) in the low magnetic field region. Patent Document 2 describes that the highest magnetic field peak indicates the presence of a random component in the regenerated silk fibroin molecule, and the remaining two peaks on the low magnetic field side correspond to the β sheet structure. Further, Patent Document 2 makes it possible to orient silk fibroin molecules in the fiber axis direction by stretching the regenerated silk thread, and as a result, more random components can be changed to a β sheet structure. It suggests.

  As described above, so far, the actual Silk II structure has been considered to be more heterogeneous than the model proposed by Marsh et al. However, none of the prior art documents even suggests the possibility that the actual Silk II structure was quite different from the Marsh model.

JP 2001-247664 A International Publication No. WO2008 / 004356 Pamphlet

Nature, Vol. 418, p. 741 (2002)

  The inventor reveals for the first time in detail that the actual Silk II structure is different from the Marsh model. As mentioned above, it has been pointed out so far that the Silk II structure is more heterogeneous than the model proposed by Marsh et al., But the difference revealed by the present inventors goes far beyond that, In fact, it became clear that the Silk II structure does not have the intermolecular structure proposed by Marsh et al.

  Surprisingly, in the actual Silk II structure, an intermolecular hydrogen bond is formed between heterogeneous residues (alanine-glycine), and furthermore, the intermolecular structure has two forms: Form A and Form B Existed. And this inventor discovered that the physical property of regenerated silk fiber could be controlled by changing the content of form A and / or the content of form B.

Therefore, the first aspect of the present invention is
(1) A method for changing the physical properties of regenerated silk fiber, wherein the form A content and / or the form B content in the silk fiber having a Silk II structure is changed.

The content of the form A ^may be measured based on the peak area of about 19.6ppm of AlaCβ in 13 C CP / MAS NMR spectra, also the content of the Form ^B of AlaCβ in 13 C CP / MAS NMR spectrum It can be measured based on a peak area of about 21.7 ppm. Accordingly, a preferred embodiment of the first aspect of the present invention is:
(2) The Form A content is measured based on a peak area of about 19.6 ppm of AlaCβ in the ¹³ C CP / MAS NMR spectrum, and the Form B content is about 21.7 ppm of AlaCβ in the ¹³ C CP / MAS NMR spectrum. It is a method as described in said (1) measured based on the peak area of.

In addition, as described later, in the ¹³ C CP / MAS NMR spectrum of the Silk II structure, AlaCβ, in addition to the two peaks assigned as Form A and Form B described above, In addition to the peak attributed to the “β-turn structure” (Distorted β-turn), the peak attributed to the “distorted β-turn structure”, which was also present in the crystal part, overlapped the highest magnetic field side (about 16 ppm) (see FIG. 2). This corresponds to the random component in Patent Document 2. In addition, the ¹³ C CP / MAS NMR spectrum of AlaCβ shows a “distorted β sheet structure” (Distorted β) in the amorphous part of the silk fibroin molecule between the two peaks assigned as Form A and Form B. -Sheet) was also included.

Furthermore, when manufacturing the regenerated silk fiber, the present inventor can change the stress-strain characteristics of the manufactured regenerated silk fiber by changing either or both of the contents of Form A and Form B. It was confirmed. Accordingly, a further preferred embodiment of the first aspect of the present invention is:
(3) The method according to (1) or (2), wherein the physical property has a stress-strain characteristic of silk fiber.

Moreover, this invention intends the manufacturing method of the regenerated silk fiber which showed the changed physical property paying attention to the content of the said form A and the form B. Therefore, the second aspect of the present invention and preferred embodiments thereof are as follows:
(4) A method for producing a regenerated silk fiber exhibiting changed physical properties, the method comprising the step of changing the form A content and / or the form B content in a silk fiber having a Silk II type structure;
(5) The Form A content is measured based on a peak area of about 19.6 ppm of AlaCβ in the ¹³ C CP / MAS NMR spectrum, and the Form B content is about 21.7 ppm of AlaCβ in the ¹³ C CP / MAS NMR spectrum. The production method according to (4) above, which is measured based on the peak area of the above; and (6) The production method according to (4) or (5), wherein the physical property has stress-strain characteristics of silk fibers. It is.

Furthermore, this invention intends the quality evaluation method of the regenerated silk fiber which used the content of the said form A and the form B as a new parameter | index. As described above, it has been clarified that the contents and properties of Form A and Form B are related. Therefore, the third aspect of the present invention and preferred embodiments thereof are as follows:
(7) A method for evaluating the quality of regenerated silk fibers, comprising quantifying the form A content and / or the form B content in silk fibers having a Silk II structure;
(8) The Form A content is measured based on a peak area of about 19.6 ppm of AlaCβ in a ¹³ C CP / MAS NMR spectrum, and the Form B content is about 21.7 ppm of AlaCβ in a ¹³ C CP / MAS NMR spectrum. (9) the method according to (7) or (8), wherein the quality is related to the stress-strain characteristics of the silk fiber is there.

  According to the present invention, it is possible to change the physical property of the fiber from a viewpoint completely different from the conventional method for modifying the physical property of regenerated silk fiber.

FIG. 1 shows an antiparallel β sheet structure of Silk II type silk fiber proposed by Marsh, Pauling et al. Panels a to c are views of three β sheet layers viewed from three directions, respectively. Large spheres in the figure represent methyl groups. Panel b shows intermolecular hydrogen bonding between silk molecular chains. Below the panel a, the direction of the chain is indicated by an arrow. FIG. 2 shows the peaks separated considering the AlaCβ spectrum and Gaussian distribution in ¹³ C CP / MAS NMR of purified silk fibroin. In the figure, the outermost thick solid line shows the observed spectrum, and the solid solid line shows the spectrum attributed to the crystal part of the chymotrypsin-treated precipitation part. A thick broken line shows a spectrum attributed to an amorphous part. The thin dashed line is the five separated peaks. FIG. 3 shows Form A in silk fibers having a Silk II type structure. Panels a to c are views of three β sheet layers viewed from three directions, respectively. Large spheres in the figure represent methyl groups. Panel b shows the hydrogen bonds between the chains. Below the panel a, the direction of the chain is indicated by an arrow. FIG. 4 shows Form B in silk fibers having a Silk II type structure. Panels df are views in which three β sheet layers are viewed from three directions, respectively. Large spheres in the figure represent methyl groups. Panel e shows the hydrogen bonds between the chains. Below the panel d, the direction of the chain is indicated by an arrow. Figure 5 shows a ^{1 H-DQMAS (Double Quantum Magic} Angle Spinning) spectrum and ¹ H- ¹ H distance correlation taking SilkII structure of silk fibers _{(AG) 15.} ¹ H- ¹ H distance correlation is observed when about a short distance within 4Å, (i) - (ix ) are summarized between such short-range ¹ H- ¹ H. Furthermore, the cross peak part of Hα of alanine and glycine is enlarged. FIG. 6 is a ¹³ C- ¹³ C DARR (Dipolar Assisted Rotational Resonance) spectrum of (AG) ₇ [U- ¹³ C] A [U- ¹³ C] G (AG) ₇ having a Silk II-type structure of silk fibers. . Here, U is a Uniform label, meaning that all the carbons of each residue are ¹³ C-labeled. In addition, the cross peak portions of AlaCβ, Ala, and GlyC═O are enlarged. As a result, it was possible to correlate the two peaks (19.6 ppm and 21.9 ppm) in the low magnetic field region of AlaCβ with Ala and GlyC = O, and to make detailed assignments of Ala and GlyC = O. . Figure 7 is a Double Cross Polarization ¹ H- ¹³ C correlation spectra of taking SilkII structure of silk fibers ^{_{(AG) 7 [3- 13 C}} ] AG (AG) 7. This makes it possible to correlate the two peaks (19.6 ppm and 21.9 ppm) in the low magnetic field region of AlaCβ with Ala Hβ and Hα, and to perform detailed assignment of Ala Hβ and Hα. Figure 8 is a silk fiber take SilkII type structure ^{_{(AG) 7 A [U- 13}} C] Double Cross Polarization 1 H- 13 C correlation spectra of G _{(AG) 7.} This allows correlation between the two peaks of GlyC = O, A, B and Ala Hβ, Gly Hα1, Hα, and NH, and makes detailed assignments of Ala Hβ, Gly Hα1, Hα, and NH. be able to. Figure 9 is a ^{1 H-DQMAS (Double Quantum Magic} Angle Spinning) spectra of silk fibers take SilkII structure of _{^{([2- 2 H] AG)}} 15. It can be seen that no correlation between GlyHα between the molecular chains (boxed) is observed. FIG. 10 shows an (AG) n antiparallel β sheet structure according to the Marsh model. FIG. 11 shows an (AG) n antiparallel β sheet structure that is different from the Marsh model. The upper and lower stages are two types of models packed in such a manner that the (Ala-Gly) n Ala β carbon faces in the opposite direction to the β sheet surface. FIG. 12 shows two types of anti-parallel β-sheet structures in Silk II type that have been structurally optimized while retaining Cell parameters. Form A of the present invention corresponds to Model A (lower stage), and form B corresponds to Model B (upper stage). FIG. 13 shows measured values of solid ¹ H, ¹³ C, ¹⁵ N NMR chemical shifts of antiparallel β sheet structure and (Si) II optimized in the structure of FIG. 12 when (AG) ₁₅ having a Silk II type structure is used as a sample. It is a comparison with the value calculated based on two types of antiparallel β sheet structures in the mold. FIG. 14 shows peaks separated in consideration of the spectrum of AlaCβ and the Gaussian distribution in ¹³ C CP / MAS NMR of Example 2. FIG. 15 shows peaks separated in consideration of the spectrum of AlaCβ and the Gaussian distribution in ¹³ C CP / MAS NMR of Example 3. FIG. 16 shows the peaks separated in consideration of the spectrum of AlaCβ and the Gaussian distribution in ¹³ C CP / MAS NMR of Example 4. FIG. 17 shows the stress-strain curve of purified natural silk fibroin. The vertical axis represents stress and the horizontal axis represents strain. FIG. 18 shows the stress-strain curve of Example 2. The vertical axis represents stress and the horizontal axis represents strain. FIG. 19 shows the stress-strain curve of Example 3. The vertical axis represents stress and the horizontal axis represents strain. FIG. 20 shows the stress-strain curve of Example 4. The vertical axis represents stress and the horizontal axis represents strain.

β sheet structure form A and form B
To date, the Silk II structure has been believed to take the antiparallel β sheet structure proposed by Marsh, Pauling et al. However, as mentioned above, the actual Silk II structure has also been said to be more heterogeneous than the Marsh model.

Actually, in the ¹³ C CP / MAS NMR spectrum of the Silk II type structure of (Ala-Gly) n of the crystal part model compound as well as the crystal part of the silkworm silk fibroin, all AlaCβ peaks were split into three. . And the peak of about 16 ppm on the high magnetic field side was attributed to the distorted β-turn (random coil) (see Patent Document 2).

On the other hand, two of the three peaks of AlaCβ on the low magnetic field side (about 19.6 ppm and about 21.7 ppm) can all be attributed to the antiparallel β sheet structure, but the details are still clear Was not. In other words, in a protein with a known structure, when the Ramachandran Map of chemical shifts in which experimental values of Ala ¹³ Cβ chemical shifts are plotted against internal rotation angles of Ala residues are examined, the internal rotation angle is in the region of antiparallel β sheet structure. It can be seen that even if the internal rotation angle changes, a shift difference as great as 2 ppm does not occur.

Therefore, the large shift difference between the two peaks on the low magnetic field side of AlaCβ was considered to reflect the difference in intermolecular structure. Of course, since the Marsh model has a single intermolecular structure in the first place, according to the Marsh model, the crystal part of the Ala ¹³ Cβ chemical shift gives only a single peak, and there is a big contradiction in this respect. Furthermore, as will be described in detail later, the Marsh model is also problematic in that intermolecular hydrogen bonds form between identical residues (alanine-alanine or glycine-glycine). Thus, the Silk II type structural model had to submit a new model different from the Marsh model.

In Patent Document 2, when the regenerated silk fiber is stretched, the intensity of the peak on the highest magnetic field side of AlaCβ (that is, a distorted β-turn structure or a random component) in the ¹³ C CP / MAS NMR spectrum is reduced. It describes that the intensity of two peaks (19.6 ppm and 21.9 ppm) on the magnetic field side increases. However, Patent Document 2 does not describe at all that these two peaks on the low magnetic field side indicate the presence of Form A and Form B, which are two types of β sheet structures different from the Marsh model. It is noted that the situation or Patent Document 2 does not suggest that the physical properties of the regenerated silk fiber can be further changed by changing the content of either Form A or Form B, or the ratio of both. Should be.

Against this background, the present inventor has clarified the correct Silk II type structure in the present invention. The details will be described in Examples below, but the present inventor analyzed the Silk II type by analyzing a ¹³ C CP / MAS NMR spectrum obtained by combining an ultrafast solid-state NMR microprobe and a 920 MHz ultrahigh magnetic field NMR apparatus. The form A (FIG. 3) and the form B (FIG. 4) in the silk fiber which takes a structure were elucidated.

Content of Form A and Form B As described above, the elucidation of Form A and Form B in the Silk II type structure of silk fibers by the present inventors was mainly based on the analysis of the peak of AlaCβ in the solid ¹³ C CP / MAS NMR spectrum. It was based. That is, the present inventor separated a solid ¹³ C CP / MAS NMR spectrum showing a methyl group of an alanine residue of a silkworm silk fiber assuming a Gaussian distribution. The inventor then examined in more detail the approximately 19.6 ppm peak and the approximately 21.7 ppm peak so separated to establish the presence of Form A and Form B. Therefore, the contents of Form A and Form B in the Silk II type structure can be measured or quantified based on the peak areas of about 19.6 ppm and about 21.7 ppm, respectively. Those skilled in the art can understand that chemical shift measurement values such as 19.6 ppm and 21.7 ppm can cause measurement errors of about ± 0.3 ppm due to the measurement conditions and the characteristics of the equipment used. I will.

The separation of each peak assuming the Gaussian distribution can be performed by optimizing the chemical shift and the half width and height of the peak. FIG. 2 shows an example of peak separation in the solid ¹³ C CP / MAS NMR spectrum of rabbit silk fiber. In the figure, the solid line part is a crystal part occupying 55%, and the broken line part is an amorphous part occupying 45%. A thin solid line portion indicates each peak (a total of five) separated in consideration of the Gaussian distribution.

  From FIG. 2, it can be seen that the silkworm silk fiber is extremely non-uniform, and its crystal part is mainly composed of three components. It can also be seen that the amorphous part is mainly composed of two components. The highest magnetic field side in the spectrum has a distorted β-turn structure (random coil part), and the ratio is 40% in total including the crystal part and the amorphous part. This is basically a portion where the structure before fiber formation remains and is a region that has not sufficiently changed to an antiparallel β structure. Even the crystal part is interesting to contain a 18% distorted β-turn part. On the other hand, the local structure of the molecular chain in the remaining part of the crystal part is an antiparallel β sheet structure, but as described in detail in Examples below, two types of intermolecular structures different from the structures proposed by Marsh and Pauling et al. It is comprised from form A (25%) and form B (13%) which are. In addition, another component other than the β-turn structure of the amorphous part was separated as a wide peak appearing between Form A and Form B, and was attributed to a distorted β-sheet structure (22%).

Regenerated Silk Fiber The regenerated silk fiber whose physical properties have been changed according to the present invention can be typically produced using rabbit silkworm as a raw material.

  Preferably, after cutting the cocoon into small pieces, proteins such as sericin and fat other than silk fibroin are removed. Non-limiting examples of this treatment include about 0.2% (weight / volume) to about 0.5% (weight / volume) soap and optionally about 0.0.degree. C. heated to about 80.degree. C. to about 100.degree. Placing the cocoon pieces in an aqueous solution containing 2% (w / v) to about 0.5% (w / v) sodium carbonate and boiling for about 5 to about 60 minutes with stirring. Thereafter, the silk thread is preferably washed in water heated to about 80 ° C. to about 100 ° C. If desired, this operation may be performed three times and further washed with water for about 5 to about 60 minutes before washing.

  As a raw material for the regenerated silk fiber of the present invention, a silk-like material designed according to the method described in Patent Document 1 and produced by a genetic engineering technique or a chemical technique using E. coli or the like may be used. . That is, for such a silk-like material, a copolymer in which β-turn type II structure is repeated throughout the molecule is selected from alternating copolymers of glycine, which is the first amino acid, and second amino acid. A water-soluble copolymer is selected from those obtained by partially chemically modifying a copolymer in which the β-turn type II structure is repeated throughout the molecule, and the partially chemically modified water-soluble copolymer is selected. Select a copolymer that forms a silk-like material by transferring intramolecular hydrogen bonds in the β-turn type II structure to intermolecular hydrogen bonds when stretched in the presence of water. Can be designed.

  Silk fibroin purified from silkworms by the above treatment and silk-like material produced by genetic engineering or chemical method by the method described in Patent Document 1 are usually dried and dissolved in an aqueous solution of lithium bromide (LiBr) Be made. The preferred concentration of lithium bromide in the aqueous solution is about 5 to about 12M, more preferably about 9M. The concentration of silk fibroin that can be dissolved in the aqueous LiBr solution ranges from about 5% (weight / volume) to about 40% (weight / volume), but is not limited thereto. Silk fibroin is preferably dissolved at a concentration of about 10% (weight / volume). If desired, the LiBr aqueous solution may be heated to about 40 ° C. while shaking to promote the dissolution of silk fibroin. Further, in order to remove the solid content, the LiBr aqueous solution containing dissolved silk fibroin may be filtered with a glass filter or the like.

  The LiBr aqueous solution in which the silk fibroin is dissolved is dialyzed against distilled water for 1 day to several days (for example, 4 days) using a dialysis membrane such as cellulose. By this operation, a silk fibroin aqueous solution from which a salt such as LiBr is removed is obtained.

  After removing water from the silk fibroin aqueous solution by freeze drying or the like, the silk fibroin is dissolved again in a solvent. Examples of suitable solvents include hexafluoroisopropanol (HFIP) and hexafluoroacetone (HFA).

  The regenerated silk fiber of the present invention is preferably spun from a solution of silk fibroin dissolved in HFIP or HFA. As shown in the examples below, it is clear that the ratio of Form A and Form B in the Silk II type structure is different between when HFIP is used as a solvent for dissolving silk fibroin and when HFA is used. became. These regenerated silk fibers exhibited different stress-strain characteristics. Therefore, changing the solvent in which silk fibroin is dissolved is a preferred embodiment for adjusting the content of Form A and Form B of the present invention.

  More specifically, when spinning from the silk fibroin solution described above, any of a wet spinning method, a dry jet spinning method, a dry spinning method, and an electrospinning method can be used. In particular, when producing long fibers, it is preferable to use a wet spinning method. That is, the regenerated silk of the present invention can be spun by discharging the silk fibroin solution toward a coagulation bath through a spinneret. Moreover, when manufacturing a nonwoven fabric, it is preferable to utilize an electrospinning method.

  The bath liquid used for the coagulation bath of the wet spinning method may be any liquid as long as the silk fibroin is insoluble and the solvent of the silk fibroin solution is soluble. For example, when HFIP is selected as the solvent for the silk fibroin solution, water, methanol, ethanol, isopropyl alcohol, acetone, or the like can be used as the bath liquid. Among these, it is preferable to use environmentally safe and inexpensive methanol in addition to the high coagulation ability of silk fibroin.

  The regenerated silk fiber of the present invention can be obtained by allowing it to stand in a coagulation bath for several hours and then drying in order to sufficiently remove the solvent (HFIP or the like). However, it is also a preferred embodiment to stretch the regenerated silk fiber during drying. It has been found that the ratio of Form A and Form B in the Silk II type structure can be changed by applying different draw ratios, as shown in the examples below. These regenerated silk fibers exhibited different stress-strain characteristics. Therefore, changing the stretch ratio of the regenerated silk fiber is also a preferred embodiment for adjusting the contents of Form A and Form B of the present invention. Typically, the regenerated silk fiber of the present invention can be stretched to about 1 to about 4 times, preferably about 1.5 to about 3 times. It is also possible to perform cold drawing while being taken out of the coagulation bath and still wet, or to be drawn in the coagulation bath.

  Thus, it is possible to produce a regenerated silk fiber that changes the contents of Form A and Form B of the present invention and thereby exhibits changed physical properties.

Uses of the Regenerated Silk Fiber of the Present Invention The regenerated silk fiber of the present invention can be used not only for materials such as fabrics but also as surgical sutures. Alternatively, the regenerated silk fiber of the present invention can be useful as an artificial blood vessel or regenerative medical equipment. That is, the regenerated silk fiber of the present invention is thin and strong, and can exhibit various elasticity and flexibility.

  Those skilled in the art given the above description can fully implement the present invention. The following examples are given for illustrative purposes only.

Example 1: Form A and Form B
Sample preparation The crystal part sample of silkworm fibroin (Cp fraction) was prepared by dissolving scoured silk thread in 9M LiBr aqueous solution, and then regenerating silk fibroin aqueous solution through dialysis, and treating it with α-chymotrypsin. %). Rabbit silk crystal part model compound (Ala-Gly) ₁₅ was synthesized by a solid phase method. Selective ¹³ C labeling and ² H labeling of Ala and Gly residues were obtained by appropriately introducing the corresponding labeled amino acids during synthesis.

Solid NMR measurement Ultra high-speed solid-state NMR probe (super high-speed rotation at 70 KHz, manufactured by JEOL RESONANCE Co., Ltd.) and JEOL JNM-installed by Molecular Science Laboratories (38 Meijidai-cho, Okazaki, Aichi, Japan) By combining an ECA 920 MHz ultra-high magnetic field solid-state NMR apparatus, ¹ HDQ (Double Quantum) / MAS (Magic Angle Spinning) NMR spectra were obtained for silk silk part of the silkworm and the model compound (Ala-Gly) ₁₅ Silk II type. Thereby, all solid ¹ H chemical shifts are obtained, and assignment of ¹ H chemical shifts is optionally performed using double cross polarization ¹ H- ¹³ C, optionally with ¹³ C-labeled (Ala-Gly) _15. The correlation method was used. The assignment of ¹³ C peak was performed by ¹³ C DARR (Dipolar Assisted Rotational Resonance) method using (Ala-Gly) ₁₅ selectively labeled with ¹³ C in addition to the conventional assignment result. ¹³ C CP / MAS NMR and DARR measurements were carried out using a JEOL ECX 400 NMR apparatus under MAS 8 kHz.

NMR chemical shift calculation Using the GIPAW (Gauge-inclusion projector augmented wave) method (using a commercially available program: Accelrys Inc., San Diego, CA, USA), which is an NMR chemical shift calculation method for crystal structures. ¹ H, ¹³ C, ¹⁵ N solid chemical shift calculation was performed on Silk II type crystal part (Ala-Gly) n of silkworm fibroin. Finally, the Silk II heterogeneous structure of the silkworm silk fibroin crystal part satisfying all the solid-state NMR data was determined.

1-a) Assignment of ^{1 A} NMR spectrum of (Ala-Gly) ₁₅ Silk II
Assignment of ¹ H chemical shift was performed by ¹ H-DQMAS NMR and double cross polarization ¹ H- ¹³ C correlation method using (Ala-Gly) ₁₅ and selectively ¹³ C-labeled (Ala-Gly) _15. (FIGS. 5, 7, 8, 9). For that purpose, the ¹³ C spectrum was assigned by ¹³ C DARR method using (Ala-Gly) ₁₅ selectively labeled with ¹³ C in addition to the conventional assignment result (FIG. 6). As a sample, (AG) ₇ [1,2,3- ¹³ C] A [1,2- ¹³ C] G (AG) ₇ was used. ^{The 1} H spectrum has three regions, HN, Hα, and Hβ, from the low magnetic field side. Hα can be easily assigned to AlaHα (around 5 ppm) and GlyHα (divided into two at around 4 ppm) from the correlation with the ¹³ C axis. Since HN is not directly bonded to ¹³ C, the strength is weak. However, Ala-HN is correlated with Gly-CO (169 ppmm) through two bonds, and Gly-HN is correlated with Ala-CO (172 ppm) through two bonds. , AlaHN and GlyHN can be assigned. As a result, it can be seen that the HN chemical shifts roughly match between Ala and Gly residues. Ala-Hβ can be assigned a ¹ H spectrum from the correlation between a peak derived from an amorphous part or a turn part and two kinds of crystal peaks, corresponding to a ¹³ Cβ peak split into three. That is, 1.2 ppm ( ¹ H) -16.1 ppm ( ¹³ C), 1.0 ppm ( ¹ H) -19.2 ppm ( ¹³ C), and 1.3 ppm ( ¹ H) -22.3 ppm ( ¹³ C) It is matched. On the other hand, the correlation between the two carbonyl (C = O) peaks of Gly (corresponding to Form A and Form B) indicates that Gly Hα1 is separated into A and B and simultaneously Gly Hα2 is not separated. . Thereby, Gly Hα can be assigned.

1-b) Comparison with conventional Silk II model based on ¹ H NMR spectrum
Next, in order to obtain knowledge about the structure of detailed SilkII type ^was examined ¹ H- ¹ H correlation by 1 H-DQMAS spectrum (Fig. 5). First, the chemical shift of HN showed no difference between Ala and Gly residues. The chemical shift of HN reflects the interatomic distance between the intermolecular hydrogen-bonded carbonyl groups, indicating that there is no difference in intermolecular hydrogen bond distance between Ala and Gly residues in the β sheet. . More interestingly, of two non-equivalent Gly Hα hydrogens, a correlation peak was observed between In plane Gly-Hα (4.6 ppm) and Ala-Hα (5.0 ppm). is there. As described later, from the ¹ H chemical shift calculation, among the two non-equivalent Gly Hα hydrogens, the low magnetic field side was assigned as In plane, and the high magnetic field side was assigned as Out plane. As shown in the panel a of FIG. 1, since the intermolecular hydrogen bond is formed between the same residues (Ala-Ala or Gly-Gly) in the Marsh model, both protons are 4.5 and 5 in the β sheet. .9 cm, far enough from the DQMAS observable range (~ 4.0 cm). This proposed model can explain this measurement result. Furthermore, look between the molecules. In the Marsh model, Out plane Gly-Hα (3.9 ppm) and Ala-Hα are closer, 2.2 、, and if the correlation between In plane Gly-Hα and Ala-Hα is found, this correlation Should of course be observed. Actually, no correlation was found between Gly-Hα of Out plane and Ala-Hα, and only a correlation with Gly-Hα of In plane was observed. Therefore, between the same residues (Ala-Ala or Gly- It was concluded that the structure was not a Marsh-type structure in which an intermolecular hydrogen bond was taken in (Gly) but a structure in which an intermolecular hydrogen bond was formed between different residues ((Ala-Gly)). From the ¹ H-DQMAS spectrum of all delaminated Ala-Hα ([2-d] Ala-Gly) ₁₅ , it can be confirmed more clearly (FIG. 9). If a hydrogen bond is formed, the interplane autocorrelation of In planeGly-Hα should appear as a diagonal peak, which is apparent from the fact that it was not observed in FIG.

1-c) Construction of Silk II structure model In the ¹³ C CP / MAS NMR spectrum of silk silk fibroin, its crystal part and (Ala-Gly) n Silk II type structure of the crystal part model compound, there are three AlaCβ peaks. Split into two. The peak at about 16 ppm on the high magnetic field side has been attributed to a distorted β-turn or random coil. On the other hand, the two peaks (19.6 ppm and 21.7 ppm) on the low magnetic field side are all attributed to the antiparallel β sheet structure, but the details have not yet been attributed. Examining the Ramachandran Map in which the experimental values of Ala ¹³ Cβ chemical shifts are plotted against the internal rotation angle of the Ala residue, a large shift of 2 ppm is observed even if the internal rotation angle changes in the region of the antiparallel β sheet structure. There is no difference. Therefore, this large shift difference was considered to reflect the difference in intermolecular structure. Of course, since the Marsh model has a single intermolecular structure, according to the Marsh model, the crystal part of the Ala ¹³ Cβ chemical shift gives only a single peak, which is contradictory. As already mentioned, the Marsh model also has the problem that intermolecular hydrogen bonds are formed between identical residues (Ala-Ala or Gly-Gly). As described above, the Silk II structural model has to be submitted with a new model different from the Marsh model.

Therefore, in order to elucidate the correct structure of Silk II, for (Ala-Gly) n, the internal rotation angles of the Ala and Gly residues of the molecular chain are represented by the angles of typical antiparallel β sheet structures (φ, ψ = 140 ° , 140 °), and two crystal structures having different intermolecular structures were prepared. As reported values for the unit cell, the values of Marsh et al. (Fiber axis b: 6.97Å, c: 9.20Å, a: 9.40Å, symmetry: P2 ₁ (x, y, z −x, y + 1/2, -Z) and the values of Takahashi et al. (Fiber axis c: 6.98 ：, b: 9.49Å, a: 9.38 対 称, symmetry: P2 ₁ (x, y, z -x, y + 1/2, -z) However, there is not much difference, and here we have adopted the newer unit cell of the latter.

Thus, unlike the Marsh model, two types of models packed in such a manner that the Ala β carbon of (Ala-Gly) n is alternately oriented in the opposite direction to the β sheet surface were examined and used as the initial structure. These structures are obtained by rotating the lower right molecular chain in the left panel of FIG. 10 by 180 ° around the molecular chain axis. The model preparation procedure is as follows.
1. Of the coordinates of the two molecules specified in the Marsh model shown in FIG. 10, only the molecule on the right side of the lower part of the left panel is rotated 180 ° around the molecular axis. The upper two molecules are automatically generated by a symmetrical operation, and the upper model 1 of FIG. 11 is created (the molecular chain axis of the Marsh model is the same b axis as the left panel of FIG. 11). The molecular chain rotated 180 ° is shifted by one residue in the molecular chain direction in order to align the hydrogen bonding position.
2. By rotating the coordinates of the two molecules created in 1 by 90 ° around the x-axis (a-axis), a structure in which the molecular chain axis faces the z-axis (c-axis) is generated. The remaining two molecules are automatically generated by symmetrical operation, and model 2 in the lower part of FIG. 11 is created. Note that the created coordinates are shifted by ½ residue in the molecular chain direction (c-axis) so that the created coordinates do not collide with the coordinates generated by the symmetry operation by the axis rotation.
3. For symmetry, a axis of the crystal cell molecular chain axis, b-axis, but three different models to suit the c-axis is considered in the case of P2 ₁ x, z (a.c) are equivalent and molecular Two models in which the chain axis is aligned with the b-axis and the c-axis may be examined.

  With respect to the above two models, the structure shown in FIG. 12 was obtained as a result of the structure minimization while retaining the cell parameters with the product name “Discover” (pcff force field) manufactured by Accelrys. In addition, Model A (the lower part of FIG. 12) was 2.04 kcal / mol more stable than Model B (the upper part of FIG. 12). From the results of this energy calculation, of the two peaks (19.6 ppm and 21.7 ppm) on the low magnetic field side of the AlaCβ peak, the peak at 19.6 ppm with the stronger peak intensity is model A, and the peak intensity is weaker. It was assumed that it was reasonable to assign the 21.7 ppm peak to Model B. And this attribution was established by the result of the chemical shift calculation described below.

1-d) ¹ H, ¹³ C, ¹⁵ N solid-state NMR chemical shift calculation Further, regarding the intermolecular structure of Model A and Model B of (Ala-Gly) n, the GIPAW (Gauge-inclusion projector augmented wave) method (commercially available) Using the program: Accelrys Inc., San Diego, CA, USA) was used to optimize energy and then perform chemical shift calculations. The measurement result of ¹ H solid-state NMR chemical shift carried out in this study is added to the already reported measurement result of ¹³ C and ¹⁵ N solid-state NMR chemical shift of (Ala-Gly) ₁₅ to compare with the calculation result. This was done using the bar spectrum (FIG. 13).

First, a comparison result between actual measurement and calculation of ¹ H solid-state NMR chemical shift was examined in FIG. As a result, in the ¹³ C CP / MAS NMR spectrum of (Ala-Gly) n, the Ala ¹ HCβ peak (FIG. 5) corresponding to the 19.6 ppm peak, which is the main peak of Ala ¹³ Cβ, is more stable in terms of energy. It is considered appropriate to attribute the Ala ¹ HCβ peak (FIG. 5) to Model B, which corresponds to the 21.7 ppm peak of Ala ¹³ Cβ having a weaker peak intensity. This was reproduced. That is, the latter obtained a calculation result that the magnetic field shift is lower by about 0.3 ppm, and it was found that the actual measurement can be explained well. Compared to the peak intensity of Model A, the peak intensity of Model B was displayed as a bar spectrum by reducing the peak intensity to about half reflecting the actual measurement result. In particular, two non-equivalent Gly Hα hydrogens could be clearly assigned against the background of the good agreement with the actual measurement of the ¹ H chemical shift calculation. That is, the low magnetic field side could be attributed to In plane, and the high magnetic field side could be attributed to Out plane (FIG. 13).

On the other hand, as for Model B, it was shown that the calculation result and the measurement result clearly coincided with each other at the Ala ¹ HCβ peak and the Gly ¹ Hα peak. Furthermore, the tendency of the shift difference between Model A and Model B was well reproduced for the two actually measured non-equivalent Gly Hα hydrogens. As for NH amide hydrogen, Model B appears slightly in the high magnetic field as compared with Model A, but this tendency is also well reproduced in the calculation results.

The agreement between the observed and calculated results is further emphasized in ¹³ C and ¹⁵ N solid state NMR chemical shifts. In FIG. 13, the comparison result between the actual measurement and calculation of ¹³ C solid-state NMR chemical shift was next examined. It was found that the actual measurement result of ^13C CP / MAS NMR spectrum of (Ala-Gly) n can be well reproduced by the calculation results of Model A and Model B. That is, the calculation result of the chemical shift is that the peak of 19.6 ppm, which is the main peak of Ala ¹³ Cβ, is attributed to Model A, which is more energetically stable, and the peak of 21.7 ppm of Ala ¹³ Cβ whose peak intensity is about half. Can be attributed to Model B. In the region other than Ala ¹³ Cβ, the shift difference between Model A and Model B was well reproduced in the calculation results for Gly and Ala ¹³ C═O. For all other ¹³ C peaks, the calculation results of Model A and Model B showed that the actual measurements could be reproduced well.

Finally, the results of comparison between actual measurement and calculation of ¹⁵ N solid-state NMR chemical shift were examined in FIG. From the calculation results of Model A and Model B, the latter is expected to appear on the low magnetic field side in the Ala peak and on the high magnetic field side in the Gly peak. The actual measurement results correspond to it.

As described above, it was concluded that the calculation results of ¹ H, ¹³ C and ¹⁵ N solid-state NMR chemical shifts sufficiently reproduced the actual measurement results. This means that the quantum chemical calculation starting from this model structure can reproduce the result of the actually measured NMR chemical shift with extremely high accuracy. Thus, since the above model A and Model B coexistence model was established as a model of the silk II structure antiparallel β sheet structure of silkworm silk, the former was named Form A and the latter was named Form B.

Reference Example 1: Content of Form A and Form B in Rabbit Silk Fiber The AlaCβ peak in the ¹³ C CP / MAS NMR spectrum of purified rabbit silk fibroin was separated assuming a Gaussian distribution. The calculation was performed by optimizing the chemical shift and the half width and height of the peak. The separated peak is shown in FIG. In the figure, the solid line is the crystal part of the chymotrypsin-treated precipitation part occupying about 55 to 56% (percentage of peak area; hereinafter the same), and the broken line is the amorphous part occupying about 44 to 45%. The thin broken line indicates each peak (total of 5) separated in consideration of the Gaussian distribution. FIG. 2 shows that rabbit silk fibroin is very uneven and its crystal part is mainly composed of three components. Moreover, it turns out that an amorphous part is mainly comprised from two components. The highest magnetic field side in the spectrum is a distorted β-turn structure (random coil part), and the ratio is 40% in total including the crystal part (18%) and the amorphous part (22%). there were. The molecular chain structure of the remaining part of the crystal part was an antiparallel β sheet structure, and was composed of Form A (25%) and Form B (13%). Components other than the distorted β-turn structure of the amorphous part were separated as a peak between Form A and Form B, and were attributed to the distorted β sheet structure (22%).

Examples 2-4: Relationship between Form A and Form B Content and Physical Properties The effect of the content of Form A and Form B on the physical properties of regenerated silk fibers was examined.
Manufacture of regenerated silk fiber B. mori cocoons are cut into small pieces and placed in a solution containing 0.25% ( w / v ) sodium carbonate and 0.25% ( w / v ) Marcel soap. And treated at 85 ° C. for 15 minutes. By this treatment, components other than silk fibroin such as silk sericin were removed. The obtained silk fibroin is sufficiently washed with pure water, and then the silk fibroin is dissolved in 9M lithium bromide aqueous solution at 40 ° C. over 1 hour to obtain a 10% (weight / volume) silk fibroin solution. It was. The silk fibroin solution was dialyzed against deionized water at 4 ° C. for 4 days using a cellulose membrane. The silk fibroin concentration in the silk fibroin aqueous solution after dialysis was adjusted to 2.0% (weight / volume) and freeze-dried.

  Example 2: The freeze-dried silk fibroin was dissolved over 2 days in analytical grade hexafluoroisopropanol (HFIP) purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and 15% (weight / volume) ) Silk fibroin solution. After degassing the solution, the silk fibroin solution was extruded from a stainless steel spinneret having an inner diameter of 0.45 mm toward a coagulation bath containing methanol at room temperature using a syringe pump. After winding the silk fiber regenerated in the coagulation bath at a speed of 20 m / min, the obtained regenerated silk fiber is kept immersed in the coagulation bath for 3 hours, thereby diffusing HFIP molecules in methanol. Removed from silk fibers. Next, the regenerated silk fiber was soaked in distilled water, and stretched to 3.0 times the length of the original regenerated silk fiber at 40 ° C. using a stretching machine (Imoto Seisakusho Co., Ltd., Kyoto, Japan). The stretched regenerated silk fiber was wound around a bobbin and dried overnight at room temperature.

  Example 3 The lyophilized silk fibroin was dissolved in analytical grade hexafluoroacetone (HFA) purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) over 2 days. The subsequent processing was the same as that in Example 2.

  Example 4: The freeze-dried silk fibroin was dissolved over 2 days in analytical grade hexafluoroisopropanol (HFIP) purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and 15% (weight / volume) ) Silk fibroin solution. After degassing the solution, the silk fibroin solution was extruded from a stainless steel spinneret having an inner diameter of 0.45 mm toward a coagulation bath containing methanol at room temperature using a syringe pump. After winding the silk fiber regenerated in the coagulation bath at a speed of 20 m / min, the obtained regenerated silk fiber is kept immersed in the coagulation bath for 3 hours, thereby diffusing HFIP molecules in methanol. Removed from silk fibers. Next, the regenerated silk fiber is immersed in distilled water, and using a stretching machine (manufactured by Imoto Seisakusho Co., Ltd., Kyoto, Japan) at 40 ° C., about 1 to 1.5 times the length of the original regenerated silk fiber. Lightly stretched at a rate. The stretched regenerated silk fiber was wound around a bobbin and dried overnight at room temperature.

Content of β sheet structure form A and form B in regenerated silk fiber In the same manner as in Reference Example 1, the peak of AlaCβ in the ¹³ C CP / MAS NMR spectrum of the regenerated silk fiber of Examples 2 to 4 was measured, and a Gaussian distribution was obtained. Assuming separation into 5 peaks (FIGS. 14-16). The results shown in Table 1 indicate that the content and ratio of β sheet structure form A and form B in the regenerated silk fiber are changed by changing the type of solvent for dissolving silk fibroin and / or the stretch ratio of regenerated silk fiber. It can be changed.

Measurement of physical properties of regenerated silk fibers Using an EZ-Graph tension tester (using a 5N load cell) manufactured by Shimadzu Corporation (Kyoto, Japan), at room temperature, stress (strain) -strain (Strain) ) The response was measured. The measurement was performed under the condition of a test speed of 10 mm / sec using each sample having a length of 25 mm. For comparison, the stress-strain curve of natural silk fiber measured under the same conditions is also shown (FIG. 17). The stress-strain curve of Examples 2-4 was shown in FIGS. These results are expressed as the average of 10 measurements.

  Comparing Reference Example 1, Example 2 (FIG. 18) and Example 3 (FIG. 19), the greater the content of β sheet structure form B, the smoother the stress-strain curve, and the shoulders of those curves ( The stress was around 200 MPa, and the strain was around a few to 10%. In addition, Example 4 (FIG. 20) shows that when the content of the β sheet structure form B decreases, the strain characteristics improve, but the stress tends to decrease.

  According to the present invention, it is possible to produce silk fibers having changed physical properties. Thus, the present invention can be useful in the textile and medical equipment industries.

Claims (2)

A method for evaluating the quality of a regenerated silk fiber, comprising measuring a peak area of about 19.6 ppm of AlaCβ and a peak area of about 21.7 ppm of AlaCβ in a ¹³ C CP / MAS NMR spectrum of a silk fiber having a Silk II type structure , Said method comprising quantifying the ratio of the two . The method of claim 1 , wherein the quality is related to the stress-strain characteristics of silk fibers.

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