JPH07126296A - Solubilized protein - Google Patents

Abstract

PURPOSE:To produce a soluble protein useful for cosmetics, medicines, etc., as a regenerated protein film or a regenerated fiber by reducing disulfide bonds of a water-insoluble protein and subsequently reacting thioglycolic acid with the generated SH groups in the presence of an oxidizing agent. CONSTITUTION:To a water-insoluble protein (e.g. wool), 1.0M aqueous 2- mercaptoethanol solution adjusted to pH 10 using 3M sodium hydroxide is added and the disulfide bonds (-SS-) of the protein are reduced to sulfhydryl groups (-SH) through a treatment at 30 deg.C for 3hr. After filtration of the insoluble protein expanded through the reduction treatment, pH is adjusted to 10 by adding 3M potassium hydroxide thereto and 0.2M aqueous thioglycolic acid solution is then added thereto so at to apply a further reduction treatment thereto at 30 deg.C for 24hr in the presence of urea. Acetic acid is then added to this treated solution to adjust pH to 7 and 1M aqueous sodium bromate solution is subsequently added thereto. The treated solution is subjected to oxidization treatment at room temperature for 2hr to convert a part or the whole of the SH groups to carboxymethyl disulfide groups, thus producing the solubilized protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a solubilized protein which is insoluble in water and is obtained by solubilizing proteins such as animal hair such as wool having disulfide bonds and hair, and more specifically, regenerated protein. The present invention relates to a novel solubilized protein and a method for producing the same, for the purpose of providing a film and a recycled fiber.

[0002]

2. Description of the Related Art Keratin fibers typified by wool, as is well known, exhibit excellent hygroscopicity or moisture release depending on the amount of water in the air, and are viscoelastic depending on the amount of water in the fibers. By changing the behavior, it brings a rich texture. This is due to the properties of a complex of a keratin fiber (α-helix structure), which is a crystal part in the fiber, and an amorphous matrix protein cross-linked by an intermolecular disulfide bond (-SS-bond). is there.

Heretofore, attempts have been made to solubilize and reuse keratin fibers having such excellent physical properties. For example, in order to increase the reducing power of thioglycolic acid, an aqueous solution of sodium thioglycolate is treated with an oxidizing agent to treat feathers with an aqueous solution of partially oxidized sodium thioglycolate partially converted to sodium dithiodiglycolate. , A method for producing an aqueous solution of high viscosity and high molecular weight keratin (Japanese Patent Laid-Open No. 53-119900), and a method for making an keratin film strong and flexible by containing an aliphatic polyhydric alcohol (Japanese Patent Publication No. 55-33826). No.), a wool is reduced, a protein containing SH groups is dissolved in a neutral buffer solution, and a protein film having a disulfide bond or a cross-linking bond produced by ultraviolet irradiation from this solution is used as a cell culture bed (JP Sho 60-160883
No.) is known.

However, in all of these methods, a common solubilization method in which a disulfide bond in a protein is reduced to an SH group is used. Therefore, firstly, wool or the like has a high crosslink density due to the disulfide bond. Fiber has a low solubilization rate, and there is a drawback that the protein is decomposed when a radical treatment such as heat treatment is performed in order to increase the solubilization rate.

Secondly, there are many disadvantages such that the SH group as it is is difficult to be dissolved in a neutral aqueous solution, and further, a disulfide bond is formed again by air oxidation to make it insoluble.

Furthermore, a method of solubilizing wool by treating it with 100% thioglycolic acid under heating is known (Japanese Patent Laid-Open No. 4-91138), but this method causes a decrease in solution viscosity. , It is considered that the protein has become low molecular weight.

[0007]

Therefore, it is needless to say that even an animal fiber protein having a high cross-linking density by a disulfide bond can be efficiently solubilized, that is, the primary structure of the solubilized keratin protein is maintained, that is, , The peptide bond of the protein is not cleaved, the molecular weight is maintained, and it has an α-helix structure, it is soluble in water, and the solubilized protein takes a cross-linking structure by a disulfide bond again and is used as a regenerated protein product. There has been a demand for the provision of technology that can solve such conditions as possible.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies as a solution to the above problems, and as a result, they have a disulfide bond but are poorly soluble in water, for example, animal hair such as wool and human hair. Keratinized tissue containing keratin proteins such as feathers, hoofs and hoofs is reduced with an aqueous reducing agent solution, and then mixed with thioglycolic acid in the presence of an oxidizing agent to produce mixed disulfides (carboxymethyldisulfide). It was found that the protein obtained by the formation is soluble in water, and the present invention has been completed.

That is, the first object of the present invention is to reduce the disulfide bond of a water-insoluble protein containing a disulfide bond to an SH group, and a part or all of it is represented by the following formula -SSCH ₂ COOH. It is to provide a solubilized protein obtained by using a carboxymethyl disulfide group.

A second object of the present invention is to treat a water-insoluble protein containing a disulfide bond with an appropriate aqueous solution of a reducing agent to reduce it to an SH group, and then to treat it in the presence of an oxidizing agent. Glycolic acid is reacted and S of protein
It is intended to provide a method for producing a solubilized protein, which comprises binding thioglycolic acid to part or all of H groups.

The water-insoluble protein used in the present invention is a natural keratin protein such as birds and animals' hair, feathers,
Examples include hoofs, horns, and nails. The animal hair is not particularly limited, but examples thereof include wool such as Merino and Lincoln and human hair. These water-insoluble proteins are shredded or crushed if necessary, and then immersed in an appropriate reducing agent aqueous solution for reduction.

The reducing agent used for the reduction may be of any type as long as it is soluble in water, but thioglycolic acid or its salt, thiolactic acid or its salt, thioglycerol, dithiothreitol, 2 -Mercaptoethanol, cysteamine, glutathione, thiourea, tri-n-butylphosphine, sodium borohydride and the like are preferable, and thioglycolic acid or a salt thereof is most preferable. Examples of the salts of thioglycolic acid and thiolactic acid include ammonium salt, sodium salt, potassium salt and the like.

The amount of the reducing agent to be used can be determined by appropriately calculating the disulfide bond in the water-insoluble protein from the amount of SH groups, but it is 0.00 per 1 g of the water-insoluble protein.
05-0.5 mole, especially 0.005-0.05 mole
e is preferred. The concentration of the solution is usually 0.01M to 1
It is preferably 0M, particularly 0.1M to 1M.

When thioglycolic acid is used as a reducing agent, the amount of thioglycolic acid is necessary to form carboxymethyl disulfide by oxidation even after reducing the disulfide bond in the water-insoluble protein to SH group. It is also possible to leave the amount remaining.

The pH of the alkaline aqueous solution of the reducing agent is 7-1.
It is preferable that the pH is in the range of 3, particularly pH 9-12. When an acid is used as the reducing agent, the alkaline agent used to adjust the pH of the aqueous solution is not particularly limited, but potassium hydroxide, sodium hydroxide, ammonia, monoethanolamine, diethanolamine, and arginine can be used. It is preferred to use one kind or a combination of two or more kinds of basic amino acids such as lysine and lysine and salts such as sodium bicarbonate and ammonium bicarbonate. The reduction temperature is about 10 to 60 ° C, and the reduction time is 10 minutes to
It may be about 48 hours.

In order to improve the solubilization efficiency of water-insoluble proteins, a protein denaturing agent may be used after the reduction operation, if necessary. As such a protein denaturing agent, a 2-8 M urea aqueous solution or a 2-5 M guanidine hydrochloride aqueous solution is used. The treatment with the protein denaturing agent is carried out by treating the reduced water-insoluble protein at 0 to 40 ° C. for 1 to 48 times.
It is carried out by immersing while stirring for about a time.

After the above reduction reaction is completed, the protein is reacted with thioglycolic acid in the presence of an oxidizing agent. At this time, when thioglycolic acid is used as the reducing agent in the above-mentioned reduction treatment and a sufficient amount of thioglycolic acid remains, this thioglycolic acid can be used for the reaction. On the other hand, when a reducing agent other than thioglycolic acid was used in the above reduction treatment, the reduced protein was collected by filtration,
After sufficiently removing the reducing agent aqueous solution, it is immersed in an aqueous thioglycolic acid solution.

The amount of thioglycolic acid used should be such that SH groups in the water-insoluble protein can be oxidized to form carboxymethyl disulfide, and the amount is 0.1 per 1 g of the water-insoluble protein. 0005 ~
The amount is preferably 0.5 moles, and particularly preferably 0.005-0.05 moles. The solution concentration is preferably 0.01M to 10M, particularly preferably 0.1M to 1M.

Next, an acid is added to the aqueous thioglycolic acid solution to adjust the pH of the solution to 5-9, preferably 6-8. The acid is not particularly limited, but organic acids such as acetic acid, lactic acid, citric acid, succinic acid and phosphoric acid are preferable.

Although the oxidizing agent is not particularly limited,
Examples thereof include air, oxygen, sodium bromate, potassium bromate, hydrogen peroxide and the like.

This oxidation reaction is carried out at 0 to 40 ° C. while passing air through the reaction system or while adding the above-mentioned oxidizing agent aqueous solution. The oxidation reaction may be completed until the mercapto odor is no longer felt, but a portion of the solution is sampled and added to a neutral 0.1 M sodium dinitrobisbenzoate aqueous solution in an appropriate amount to obtain 412
The disappearance of the SH group may be confirmed by measuring the absorbance at nm, and the process may be terminated.

The solubilized protein thus obtained is
Purification may be carried out using an appropriate purification means, if necessary. For example, the solubilized protein aqueous solution may be subjected to dialysis means such as electrodialysis to remove the reducing agent oxide, protein denaturing agent such as urea, low molecular weight polypeptide, and electrolyte. If necessary, insoluble matter can be removed by filtration or centrifugation.

The solubilized protein of the present invention obtained as described above has the following properties. (1) Amino Acid Analysis of Solubilized Protein In the solubilized protein, a disulfide bond compound between cysteine and thioglycolic acid, which is originally not contained in wool protein, is formed. (Fig. 1) (2) Molecular weight: 40,000 to 60,000 (SDS-polyacrylamide gel electrophoresis method) It should be noted that color development in a low molecular weight region of tens of thousands of molecular weight caused by hydrolysis of protein in the treatment process is detected. Not done. (3) Circular dichroism (CD): It has negative extreme values at 209 nm and 222 nm and has an α-helix structure in water. (Fig. 2)

As is clear from the above results, in the solubilized protein of the present invention, a part or all of the disulfide bond (-SS-bond) of the water-insoluble protein is cleaved, and the SH derived from this disulfide bond is cleaved. The group is -SSCH ₂ COO
It has a structure that replaces the H group. And this-
Since the SSCH ₂ COOH group does not return to the disulfide bond of cystine under normal conditions, it can be stably stored in a solution state for a long period of time.

Further, although the solubilized protein of the present invention is water-soluble, it has the same properties as the original water-insoluble protein, because the disulfide bond of cystine is formed again under a certain oxidizing condition to replace the insoluble polymer. It can be used as a raw material for a regenerated protein product such as a regenerated protein film or a regenerated fiber having any shape.

In the case of producing such a recycled film or recycled fiber, the solubilized protein of the present invention is used as it is or after it is reduced with a new reducing agent and dialyzed with water, and then oxidized within or within the polypeptide. It is preferable to regenerate the disulfide bond between them or to regenerate the film or fiber and then similarly regenerate the disulfide bond.

To prepare a film from the solubilized protein aqueous solution of the present invention, first, an aqueous solution containing 1 to 5% by weight of the solubilized protein is added to the aqueous solution of the solubilized protein as a plasticizer such as methyl cellosolve, ethyl cellosolve, propyl cellosolve, carbitol, and glycerin. 1 to 3% by weight, cast this solution on a Teflon, polypropylene or polycarbonate plate and dry on silica gel in a desiccator for 1 to 3 days,
A solubilized protein film is formed on the plate.

Next, the obtained film is sprayed with a reducing agent solution prepared by adding 5 to 20% by weight of a plasticizer to a mixed solution of methanol and an aqueous thioglycolic acid solution (pH 8 to 11) or is reduced together with a cast plate. The carboxymethyl disulfide group is reduced by immersing in an agent solution,
The SH group is generated.

The concentration of the thioglycolic acid aqueous solution prepared as a mixed solution is preferably about 0.1 to 2 M, and the volume mixing ratio of the methanol / thioglycolic acid aqueous solution in the mixed solution is 90/10 to 70. About / 30 is preferable. Further, the treatment by spraying or dipping is performed at room temperature for 1/6 to 1/2 hour.

Subsequent to the above treatments, the film on the plate is washed with a solution of the methanol / water mixture containing 5-20% by weight of a plasticizer. The volume mixing ratio of the methanol / water mixed solution used for washing is 90/10 to 70.
About / 30 is preferable.

After washing, the film is dried in air at a temperature of 80 to 100 ° C. for 1/10 to 1/4 hours to oxidize the --SH group and convert it to a --SS-- bond. After drying, a colorless and transparent keratin film is obtained by immersing it in water together with a cast plate.

In the above treatment method, thioglycol may be used as a reducing agent instead of thioglycolic acid, and ethanol, isopropanol, n-propanol or acetone may be used instead of methanol.

At the time of regeneration, other existing proteins such as collagen, eslatin, silk protein, etc., natural polymer compounds such as polysaccharides and their derivatives, or existing water-soluble synthetic polymer compounds are used in combination to form a complex. It is also possible to achieve this.

According to the production method of the present invention, a water-soluble protein can be produced with high efficiency. Further, since the reaction product is not strongly alkaline or strongly acidic, the subsequent treatment can be carried out safely and simply.

[0035]

INDUSTRIAL APPLICABILITY The solubilized protein of the present invention is soluble in water, and can be converted into the original water-insoluble protein by reducing it to the desired shape and then oxidizing it. It is extremely useful as a raw material for regenerated protein products such as films and regenerated fibers.

Since it is similar to human hair, nails and skin tissue, it can also be used in the fields of cosmetics and medicine. Furthermore, in its structure -SS
Since it has a CH ₂ COOH group and can be insolubilized in water if necessary, it can also be used as a scavenger for heavy metals.

[0037]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 To 1.0 g of wool was added 50 ml of a 0.2 M thioglycolic acid aqueous solution whose pH was adjusted to 9.6 with 3 M potassium hydroxide in advance, and the mixture was treated at 30 ° C. for 3 hours.
Next, 24 g of urea was added to this treatment liquid so as to have a concentration of 8 M, and the mixture was further stirred at 30 ° C. for 24 hours to carry out a reduction treatment. Thereafter, pH was adjusted to 7 while adding acetic acid dropwise to this solution, and further, oxidation treatment was performed for 3 days while passing air. At this point the mercapto odor disappeared.

The obtained solution was treated with a dialysis membrane capable of removing a molecular weight of 10,000 or less to remove urea and the like in the solution. Next, the water-insoluble portion precipitated in the dialysis process and the water-soluble portion were separated by centrifugation (2500 rpm, 10
And the target water-solubilized wool keratin protein was obtained.

Further, the powder of the solubilized protein was obtained by freeze-drying, and the yield was calculated by weighing. As a result, the solubilization rate was 51.4%. In addition, SDS-
As a result of measuring the molecular weight by polyacrylamide gel electrophoresis, bands specific to keratin protein were detected at molecular weights of 40,000 and 60,000, and color development in lower molecular regions caused by protein hydrolysis was not detected. .

Example 2 To 1.0 g of wool, 50 ml of a 1.0 M 2-mercaptoethanol aqueous solution whose pH had been adjusted to 10 with 3 M potassium hydroxide in advance was added and treated at 30 ° C. for 3 hours. Next, the wool expanded by the reduction treatment was filtered off, and 50 ml of a 0.2 M thioglycolic acid aqueous solution whose pH was adjusted to 10 with 3 M potassium hydroxide was added to the treated wool. Of urea,
Further, reduction treatment was performed by stirring at 30 ° C. for 24 hours. After that, acetic acid was added dropwise to the treatment solution to adjust the pH to 7, and 50 ml of a 1 M sodium bromate aqueous solution was added dropwise to carry out an oxidation treatment at room temperature for 2 hours. At this point the mercapto odor disappeared.

Next, urea and the like in the treatment liquid were removed by using a dialysis membrane capable of removing a molecular weight of 10,000 or less. Next, the water-insoluble portion precipitated in the dialysis process and the water-soluble portion are separated by centrifugation (2500 rpm, 10 minutes),
A solubilized product of the target wool keratin protein in water was obtained. Further, the powder of solubilized protein was obtained by freeze-drying, and the yield was calculated by weighing.
%Met.

Further, the molecular weight was measured by SDS-polyacrylamide gel electrophoresis in the same manner as in Example 1. As a result, bands specific to keratin protein were detected at molecular weights of 40,000 and 60,000, which were caused by hydrolysis of the protein. No color development was detected in the low molecular weight region.

Experimental Example 1 Amino Acid Composition of Solubilized Wool Keratin Protein Solubilized Wool Keratin Protein Obtained in Example 1 1.4 m
g was hydrolyzed with 1 ml of 6N hydrochloric acid, dried under reduced pressure, and then dried.
It was dissolved in 2 ml of 0.02N hydrochloric acid. Of which 10 μl
Was injected into an amino acid analyzer (L-8500 type Hitachi, Ltd.). The analysis results are shown in FIG. In the solubilized protein,
A disulfide bond compound between cysteine and thioglycolic acid, which is originally not contained in wool proteins, is produced. (Fig. 1)

Experimental Example 2 Immune activity against hard keratin antibody in human hair matrix cells:
After separating the solubilized protein according to the SDS-polyacrylamide gel electrophoresis method of Example 1, the protein was transferred to a polyvinyl difluorite membrane, and the reactivity to an antibody obtained from hard keratin in human hair mother cells was detected. The common law (J. Cel
Biol. 103, 2593 (1987)). As a result,
The reactivity between the keratin protein and the antibody could be confirmed only in the sample subjected to the reduction treatment.

Experimental Example 3 Measurement of Circular Dichroism (CD) 2 mg of the freeze-dried powder of the solubilized product of the wool keratin protein obtained in Example 1 in water was dissolved again in 2 ml of ion-exchange distilled water, Using a 1 mm cell of this solution, using a JASCO A-20 type CD / ORD spectrometer, 200-250
It was measured in the wavelength range of nm. The result is shown in FIG.

As is clear from the figure, the spectrum of the solution has negative extreme values at 209 nm and 222 nm,
It was found that the solubilized protein has an α-helix structure in water.

Example 3 To 1.0 g of hair, 50 ml of 3M aqueous potassium hydroxide solution was added in advance to adjust the pH to 11. To this solution, 50 ml of 0.2 M aqueous thioglycolic acid solution was added, treated at 50 ° C. for 20 minutes, 24 g of urea was added to this solution to 8 M, and further stirred at 30 ° C. for 24 hours. , Reduction treatment was performed. After that, acetic acid was added dropwise to this solution to adjust the pH to 7, and oxidation treatment was performed for 3 days while passing air. At this point the mercapto odor disappeared.

The obtained solution was treated with a dialysis membrane capable of removing a molecular weight of 10,000 or less to remove urea and the like in the solution.
Then, the water-insoluble portion precipitated in the dialysis process and the water-soluble portion were separated by centrifugation (2500 rpm, 10 minutes) to obtain the desired solubilized hair keratin protein. As a result of calculating the yield by measuring the weight of the solubilized protein powder obtained by freeze-drying, the solubilization rate was 3
It was 1.7%.

Example 4 2% by weight of glycerin was added to 3% by weight of a solubilized protein aqueous solution, and cast on a Teflon plate. After drying, ethanol and 1M thioglycolic acid aqueous solution (pH 9.0)
Glycerin 10 was added to the mixed solution of (mixing volume ratio 90:10).
The solution added with wt% was sprayed onto the film and allowed to stand for 10 minutes.

Then, the film was washed by immersing it in a mixed solution of ethanol and water (mixing volume ratio 80:20) containing 10% by weight of glycerin, and dried in air at 100 ° C. for 10 minutes. Finally, washing with water gave a keratin film which was colorless and transparent, swollen with water, but insoluble in water.

The number of cystine residues in the water-solubilized protein used was 29 residues per 1000 residues as a result of automatic amino acid analysis. The one in the made film is
According to the analysis results by the polarographic method using methylmercury iodide, the number of residues per 1000 residues was 20 and the number of cysteine residues was 12 residues.

The Young's modulus of the obtained keratin film having a thickness of 25 μ was 1 at a relative humidity of 65% and a temperature of 20 ° C.
0.0 × 10 ⁷ N / m ² , breaking strength 3.5 × 10 ⁶ N / m ²
m ² and the breaking elongation were 15%.

[Brief description of drawings]

FIG. 1 is a drawing showing the amino acid composition of solubilized wool keratin protein.

FIG. 2 Circular dichroism (CD) of the solubilized protein of the present invention
It is drawing which shows a spectrum. that's all

Claims (6)

[Claims] 1. A disulfide bond (-SS-) of a water-insoluble protein is reduced to a sulfhydryl group (-SH),
Solubilized protein obtained by the part or whole and carboxymethyl disulfide group represented by the following formula -SSCH ₂ COOH. 2. Water-insoluble proteins are animal hair, feathers, horns,
The solubilized protein according to claim 1, which is selected from hoofs and nails. 3. A disulfide bond (-SS-) of a water-insoluble protein is reduced to a sulfhydryl group (-SH), and then thioglycolic acid is present in the presence of an oxidizing agent on a part or all of the produced SH group. A method for producing a solubilized protein, which comprises reacting 4. The method for producing a solubilized protein according to claim 3, wherein the reducing agent for converting a disulfide bond (-SS-) into a sulfhydryl group (-SH) is thioglycolic acid. 5. The method for producing a solubilized protein according to claim 3, wherein the oxidizing agent is air or oxygen. 6. A carboxymethyl disulfide group (-S
SCH ₂ COOH) containing solubilized protein is reduced,
Next, a method for producing a regenerated protein product, which comprises regenerating a disulfide bond (-SS-) within a polypeptide or between the polypeptides by oxidation.