JP5604294B2 - Protein separation method, protein dissolution method, non-animal fiber fraction collection method, and animal fiber-derived protein - Google Patents

Description

  The present invention relates to a method for collecting keratin and fibroin protein from animal fibers such as wool, feathers, hair, silk, and the like, a method for dissolving proteins, and a method for separately collecting non-animal fibers.

  Keratin proteins are contained in animal hair and feathers such as hair and wool, and fibroin and sericin proteins are contained in silk. For this reason, keratin, fibroin, etc. are taken out from animal fibers (in this specification, animal fibers include animal hair, hair as well as bird feathers) and used for pharmaceuticals, protein reagents, etc. Attempts have been made to try.

  For example, in Patent Document 1, animal hair is dipped in an aqueous solution obtained by adding ammonia to hydrogen peroxide at room temperature to 50 ° C. for 10 to 30 minutes under normal pressure, and further sealed at 130 to 200 ° C. for 30 to 60 minutes. A method is described in which heat treatment is performed to solubilize animal fibers and take out keratin.

  Patent Document 2 discloses a technique in which animal fibers are treated with a solution obtained by adding a reducing agent to a mixed solvent of urea and thiourea to elute keratin protein, and further collect a cuticle site from the residue. .

  Patent Document 3 discloses a technique for dissolving silk with a solution obtained by adding an alkali metal salt such as lithium bromide and a complexing agent such as aminoacetic acid or EDTA to a mixed solvent of a lower aliphatic alcohol and water. Has been. According to this method, a high-concentration silk fibroin solution can be prepared in a short time.

Furthermore, in Patent Document 4, the feather is heated to the glass transition point under pressure to be softened, and after alkali is added and partially hydrolyzed with a protease, it is chemically modified with a hydrophilic substituent, urea or alcohol. A method for obtaining a feather keratin solution by dissolving in an aqueous solution is disclosed. The keratin solution obtained by this method has a feature that the molecular structure of β-keratin is retained.
JP 2000-234268 A JP 2002-114798 A JP 7-173192 A Japanese Patent Laid-Open No. 2005-120286

  However, the conventional methods for collecting proteins from animal fibers use many chemicals such as oxidizing agents, reducing agents and surfactants, and enzymes such as proteases. There is a problem that some components require time and are difficult to remove over a long period of time. In addition, protein collection requires complicated operations, which in turn results in high production costs.

  The present invention has been made in view of the above-described conventional situation, and in a method for separating proteins from animal fibers, it is easy to manufacture without using an oxidizing agent, a reducing agent, a surfactant, or an enzyme. It is an object to provide a method that is low in cost.

  The present inventors paid attention to urea that has been conventionally used for dissolving animal fibers. However, in conventional protein extraction using urea, there is a problem that removal of these agents is difficult because agents such as an oxidizing agent, a reducing agent, and a surfactant are used in the urea aqueous solution. On the other hand, even when animal fibers were treated only with 5-8 M concentrated aqueous urea solution without using these drugs, the yield of protein was poor. Then, when animal fiber was processed by making urea into a molten state instead of an aqueous solution, it was found that animal fiber protein could be completely dissolved, and the present invention was completed.

  That is, the protein fractionation method of the present invention includes a dissolution step of dissolving animal fibers with molten urea to form an animal fiber solution, and a separation step of separating the protein from the animal fiber solution. Features.

  In the protein fractionation method of the present invention, molten urea is used as a solvent for animal fibers instead of urea aqueous solution in the dissolution step. According to the test results of the inventors, molten urea has a much higher ability to dissolve animal fibers than urea aqueous solution. For this reason, animal fibers can be sufficiently dissolved in a short time without using a chemical such as an oxidizing agent, a reducing agent or a surfactant. For this reason, the expense of the chemical | medical agent required for protein collection can be made low, and those removal are also unnecessary. Furthermore, there is little damage regarding the secondary structure of the protein obtained. For example, the inventors have confirmed that when wool is dissolved, the molecular weight of the collected keratin is as high as 75000 Da, and the secondary structure is regenerated. Moreover, since protein can be extracted by simply dissolving animal fibers with molten urea, the operation of the dissolution process is simplified. In the present invention, the method for melting urea is not particularly limited, and includes melting by electromagnetic waves as well as melting by heat. Moreover, the amount of urea with respect to animal fibers is preferably 3 to 20 times by weight and particularly preferably 7 to 12 times. If the proportion of urea is less than 3 times that of animal fibers, the animal fibers cannot be completely immersed in the urea melt, and only partly dissolves and takes time. Moreover, although it can melt | dissolve even if the ratio of urea is 20 times or more with respect to animal fiber, collection | recovery by reprecipitation etc. becomes difficult and cost starts.

  The dissolution mechanism of animal fibers in molten urea in the dissolution process is considered as follows. That is, animal fiber is, for example, wool in an absolutely dry state excluding moisture 15% (official moisture content), 82% keratin (hard keratin 45.5%, soft keratin 36.5%), 16% protein other than keratin, 2% consists of lipids. These proteins are intermolecularly bonded mainly by disulfide bonds and hydrogen bonds. Urea is described as cleaving its hydrogen bonds and denaturing proteins. That is, when urea in the molten state cleaves hydrogen bonds as a solvent and reacts with a free amino group or carboxyl group in the protein to chemically bond urea, improving the solubility in molten urea Conceivable. Furthermore, it is estimated that ammonia generated by the decomposition of urea can penetrate into the crystal region of the protein, spread between protein molecules, make it easier for urea molecules to penetrate into it, and promote protein dissolution. The In addition, when the animal fiber is silk, the silk fiber is filament-like compared to keratin, and the fibroin protein has a β structure and forms intermolecular hydrogen bonds in a plane, and has high crystallinity, In particular, the surface layer forms a skin layer with long crystals. For this reason, a high temperature of 180 ° C. and a long time of 50 minutes were required to dissolve silk.

  Thus, the animal fiber solution obtained in the dissolution step is separated into protein and urea in the next separation step. The method is not particularly limited, but a small amount of water is added to the animal fiber solution to prevent coagulation of urea and dispersed in an organic solvent (for example, alcohol such as methanol or ethanol). Proteins that are dissolved in and not dissolved therein can be precipitated and collected by filtration or decantation.

  Alternatively, the separation step can also be performed by removing urea from the animal fiber solution obtained in the dissolution step by dialysis with water and leaving a protein that is a polymer. In the separation process using the dialysis method, since only urea is contained, the dialysis time can be shortened as compared with the method in which other chemicals are added. In the process, some water-insoluble proteins that are hardly soluble in water precipitate, and the water-soluble proteins remain dissolved in water. For this reason, a water-insoluble protein and a water-soluble protein can be fractionated by filtration. In addition, the remaining water-soluble protein can be collected as a fine powder or a solid by using a drying method such as freeze drying, natural drying, heat drying, or spray drying. When animal fiber is animal hair or feathers such as hair or wool, keratin protein is included as the main component, so the water-insoluble protein is mainly composed of hard keratin with a high sulfur content derived from cysteine. It is thought that soft keratin with a low sulfur content is the main component of water-soluble proteins. Further, silk yarn, silk spinning, etc., which are silk fibers, are composed of fibroin and sericin protein, and when used as clothing, sericin is removed by a dilute alkali refining process and used as glossy fibroin. Even when the sericin cocoon containing the sericin is used, it can be completely dissolved by the molten urea, and by adding water, it is possible to separate into water-soluble sericin and water-insoluble fifurin. Even when silk yarn or silk fabric from which sericin has been removed is used, it can be dissolved in molten urea, and silk protein can be finely powdered.

  The temperature in the dissolution step may be appropriately selected according to the type of animal fiber, but it is necessary to set the urea to a temperature at which it is in a molten state. For example, about 150 ° C. is preferable for hair, wool and chicken feathers, and about 180 ° C. is preferable for silk. However, if the temperature is too high, it may cause heat denaturation of the protein, or urea will be decomposed too quickly, so that it is preferably 200 ° C. or lower, and a particularly preferable temperature range is 140 ° C. or higher and 180 ° C. or lower.

  There are no particular limitations on the animal fibers used in the present invention, and animal hair such as hair and wool, feathers, silk, and the like can be used as raw materials.

  Further, by utilizing the fact that animal fibers are dissolved in urea in a molten state, only non-animal fibers can be separately collected from a fiber material composed of animal fibers and non-animal fibers. That is, according to the method for separating and collecting non-animal fibers of the present invention, animal fibers are dissolved by treating a fiber material composed of animal fibers and non-animal fibers with molten urea, and only non-animal fibers are obtained. It is characterized by collecting separately.

If, for example, a wool-polyester blend is processed by this method, only the wool is dissolved and removed, leaving only the polyester. For this reason, only polyester can be extracted from the blended product and can be used as a recycled raw material. Further, the thus eluted wool keratin can be used in the fields of medicine / cosmetics, reuse of wool products and the like.
Moreover, what kind of blending form of the fiber containing an animal fiber may be sufficient, and it does not depend on the mixed state of fiber, such as a blend, a twist, and a cross-weaving. The blended product can be used not only for the combination of animal fibers and synthetic fibers, but also for separation of animal fibers and plant fibers.

2 is a solid-state ¹³ C-NMR spectrum of water-soluble protein and water-insoluble protein obtained in Example 1 and raw wool.

  Hereinafter, embodiments embodying the present invention will be described in detail.

-Keratin collection from various animal fibers by dialysis-
Example 1
In Example 1, wool fiber was used as a raw material.
<Dissolution process>
Weigh 10 g of urea in a beaker and heat to 150 ° C. in an oil bath to melt. Then, 1 g of commercially available wool fiber (manufactured by Nippon Kori Co., Ltd.) (including 15% of official moisture) was put therein, heated for 20 minutes, and all were dissolved to obtain an animal fiber solution.

<Separation process>
5 ml of water was added to the animal fiber solution obtained in the dissolution step, filled into a dialysis tube made of cellulose (pore size 2.4 nm, molecular weight 12000 or less cut product), and dialyzed with water. After about 2 days of dialysis, the contents were filtered and separated into a precipitate and a filtrate. And the water-soluble keratin (0.10g) was obtained by washing the precipitate with water and making it heat-dry. The filtrate was freeze-dried to obtain sponge-like water-soluble keratin (0.53 g). In addition, although not only freeze-drying when drying a filtrate but the case where it dried naturally and the case where it heat-dried was implemented, these became a transparent light brown film-like substance.

(Example 2)
In Example 2, hair (Japanese 10-year-old boy) was used as a raw material. Other conditions are the same as those in the first embodiment, and a description thereof will be omitted.

(Example 3)
In Example 3, broiler feathers were used as the raw material, and the heating time of the melting step was 10 minutes. Other conditions are the same as those in the first embodiment, and a description thereof will be omitted.

Example 4
In Example 4, for a silk thread (manufactured by Shinano Kenshi Co., Ltd.) that had been refined with a dilute alkali to remove sericin, the heating temperature in the dissolving step was 180 ° C., and the heating time was 50 minutes. Other conditions are the same as those in the first embodiment, and a description thereof will be omitted.

(Example 5)
In Example 5, silk thread (manufactured by Shinano Kenshi Co., Ltd.) was subjected to the test as it was without refining treatment. Other conditions are the same as those in the fourth embodiment, and a description thereof will be omitted.

-Evaluation-
(1) Dissolution ratio Table 1 shows the dissolution ratio of the water-insoluble protein and water-soluble protein obtained as described above. As can be seen from this table, all of the animal fibers of Examples 1 to 5 could be dissolved.

  Thus, it is thought that it was because urea cut | disconnected the hydrogen bond between protein molecules that it was able to melt | dissolve an animal fiber only with urea. In addition, the heating temperature of 180 ° C. for silk was higher than that of the other examples, and the heating time was increased to 50 minutes. Silk fibroin has a β structure in its secondary structure and forms intermolecular hydrogen bonds in a plane. Because of the formation of a strong structure, it is expected that more temperature and time were required than other animal fibers.

(2) Solid ¹³ C-NMR Measurement With respect to the water-soluble and water-insoluble keratin obtained in Example 1, solid ¹³ C-NMR measurement was performed. For comparison, the raw material wool was also measured. The results are shown in FIG. In the raw wool, carbon peaks of two types of carbonyl groups (C═O) appeared at 174 ppm and 176 ppm. Among them, the peak at 174 ppm is based on the β-sheet structure of the keratin protein secondary structure, and the peak at 176 ppm is based on the α-helix of the keratin protein. On the other hand, since the peak at 174 ppm and the peak at 176 ppm are almost the same in water-soluble keratin, water-soluble keratin has an α-helix structure (considered as soft keratin) and a β-sheet keratin protein ( Equivalent mixture with hard keratin). On the other hand, in water-insoluble keratin, a peak at 174 ppm appears remarkably, and a peak at 176 ppm is unclear. From this, it is considered that the water-insoluble keratin mainly consists of β-sheet keratin (considered as hard keratin). The peaks other than those, the peak of 25ppm is CH _2,3 -C peak of 55ppm is CH _2,3 -N, or CH _2,3 -S or CH _2,3 -O, a peak of 130 ppm C = C Respectively.
From the above solid ¹³ C-NMR measurement results, it was shown in Example 1 that the denatured and dissolved keratin was regenerated by its reversibility and the bonds such as intramolecular hydrogen bonds were maintained.

-Collecting proteins from various animal fibers by alcohol injection method-
(Example 6)
<Dissolution process>
Weigh 10 g of urea in a beaker and heat to 150 ° C. in an oil bath to melt. Then, 1 g of commercially available wool fiber (manufactured by Nippon Koryo Co., Ltd.) was put therein, heated for 20 minutes, and all were dissolved to obtain an animal fiber solution.

<Separation process>
After adding 10 ml of water to the animal fiber solution obtained in the dissolution step to prevent solidification of urea, the solution was poured into 50 ml of methanol to precipitate proteins. Thereafter, the mixture was filtered off, and the precipitate was washed with methanol and water, and then dried by heating. The filtrate was vacuum dried to obtain a protein powder.

-Wool-Separated collection of wool from polyester blended fabric-
(Example 7)
In Example 7, a dissolution test using urea was performed on the wool-polyester blended fabric. That is, 20 g of urea is measured in a beaker and melted by heating to 150 ° C. on an oil bath. Then, 2 g of 50% wool-50% polyester blended fabric was put therein, and heated for 20 minutes to dissolve the wool.

  The textile residue and the filtrate were separated by filtration, and the filtrate was put into methanol to precipitate the protein. Thereafter, the mixture was filtered, washed with alcohol, and vacuum dried to obtain a protein powder. Further, the textile residue separated by filtration was washed with water, dried and then observed with a stereomicroscope. As a result, the wool fibers disappeared and only the polyester fibers remained.

  As described above, only the polyester can be collected from the polyester blend fiber by immersing the wool-polyester blend (50% / 50%) fabric in molten urea. The extracted protein can also be collected separately. Therefore, if this method is used, protein and non-animal fibers can be separated and recovered from the textile product waste, and the textile product can be recycled.

(Example 8)
A dissolution test using urea was performed on a fabric using a twisted fiber of cotton-silk (80%, 20%). Other conditions are the same as in Example 7. As a result, as in Example 7, only animal fibers were dissolved, and cotton as plant fibers could be separated and recovered.

  The present invention is not limited to the description of the embodiments of the invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

  According to the protein fractionation method of the present invention, protein can be collected from animal fibers with molten urea, and pure protein can be obtained. For this reason, it can be applied to fields such as medical fields such as artificial skin, pharmaceuticals and cosmetics, and reuse of animal fiber products.

Claims (7)

A dissolution process in which animal fibers that have not been treated with an oxidizing agent or a reducing agent are dissolved in molten urea to form animal fiber solutions;
A separation step of separating the protein from the animal fiber solution.   2. The protein fractionation method according to claim 1, wherein the dissolving step is performed at a temperature not lower than a temperature at which urea is in a molten state and not higher than 200 ° C. The protein separation method according to claim 1 or 2, wherein in the separation step, water is added to the animal fiber solution to obtain an animal fiber aqueous solution, and then the protein is precipitated by adding an organic solvent.   The protein separation method according to claim 1 or 2, wherein the separation step comprises collecting water by adding water to the animal fiber solution to precipitate the protein.   The protein separation method according to claim 1 or 2, wherein the separation step is performed by dialysis of an animal fiber solution with water to remove urea, thereby separating and collecting the water-insoluble protein and the water-soluble protein. . A method for dissolving a protein, comprising dissolving animal fibers that have not been treated with an oxidizing agent or a reducing agent with urea in a molten state. A fiber material composed of animal fibers that have not been treated with an oxidizing agent or a reducing agent and non-animal fibers is treated with molten urea to dissolve the animal fibers and separate only the non-animal fibers. A method for separating non-animal fibers, which is characterized by being collected.

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