JP2013179927A - Modified multi-copper oxidase, and stain for keratin fiber using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide modified multi-copper oxidase, and stain (a hair dye) for keratin fiber, excelling in dyeing property with less damage and eruption of hair and the scalp using the modified multi-copper oxidase.SOLUTION: Multi-copper oxidase raises an isoelectric point by adding basic amino acid or substituting a specific residue of an amino acid sequence for a basic amino acid, and stain (a hair dye) for keratin fiber uses the multi-copper oxidase.

Description

  The present invention relates to a multi-copper oxidase and a keratin fiber dyeing agent using the same, and more specifically, multi-copper oxidase which is difficult to be negatively charged under normal dyeing conditions, hair and scalp damage and rash, and dyeing. The present invention relates to a dyeing agent for keratin fibers having excellent properties.

Conventionally, as this type of dyeing agent, for example, a hair dye, an oxidation type hair dye mainly composed of a diamine-based oxidation dye such as paraphenylenediamine and an oxidizing agent such as hydrogen peroxide has been generally used. Yes. As an oxidizing agent, hydrogen peroxide has been often used in the past, but hydrogen peroxide has a very strong oxidizing action, so when used with alkali, the hair and scalp when used against hair. Will damage it. In addition, the diamine-based oxidation dye acts as an allergen. However, if the scalp is damaged, the diamine-based oxidation dye easily penetrates the scalp, causing a problem of inducing severe rash.
In order to solve such problems, in recent years, keratin fiber oxidation dyeing compositions and hair cosmetics containing laccase, which is a kind of multi-copper oxidase, are used instead of hydrogen peroxide (Patent Documents 1 and 2). ).

In recent years, a composition for dyeing keratin fibers that can oxidize indole or paraphenylenediamine by modifying another type of multi-copper oxidase such as copper eflex oxidase (hereinafter referred to as CueO) or bilirubin oxidase (hereinafter referred to as BOD). A thing is also proposed (patent document 3).
CueO is a protein that functions to protect the periplasmic enzyme of Escherichia coli from copper-induced damage. BOD is a protein obtained from, for example, a microorganism belonging to the genus Myrothecium or the genus Coprinus and used as a therapeutic agent for jaundice, which is a disease in which the blood concentration of bilirubin is increased (Patent Document) 4).

Japanese translation of PCT publication No. 2002-509091 JP 2002-47144 A Republished Patent No. 2007/063614 Japanese Patent No. 2632518

  However, when the above-mentioned conventional oxidase is used as an oxidizing agent for oxidized keratin fiber dyeing agent, the dyeability is inferior to that using hydrogen peroxide, so a large amount of oxidation dye is used to compensate for this. However, when used in a large amount, the diamine-based oxidation dye penetrates into the scalp even when there is no noticeable damage to the scalp, and eventually the problem of rash is not sufficiently solved.

Thus, when intensive research was conducted on the cause of the low dyeability of the stain using oxidase, the isoelectric point of human hair was about 3.0 to 5.5 (usually around 3.7), and the isoelectricity of the oxidase. The hair dyeing process is performed in a higher pH range (about 6.5 to 11.0), so both the hair and the oxidase are negatively charged. It is thought to have an adverse effect on sex.
In detail, if both are strongly negatively charged, the oxidase and the hair repel each other in the dyeing process, so the oxidase moves to a position away from the hair and then oxidizes the oxidative dye. The colored dye molecules are considered to be distributed far away from the hair. As a result, it is presumed that the colored oxidative dye is not easily absorbed by the hair, and many of the colored oxidative dye molecules are washed away when the chemical solution is washed away after dyeing, resulting in low dyeability.

As a result of intensive studies to solve the above problems, the present inventors have found that if the negative charge of the oxidase decreases in the hair dyeing process, preferably if it is positively charged, the oxidase is less likely to repel the hair, As a result, the present inventors have obtained the knowledge that the oxidized dye is easily adsorbed to the hair and the problem can be solved because the colored oxide dye is also distributed in the vicinity of the hair.
That is, the present invention solves the above-mentioned problems of the prior art, provides an oxidase that is difficult to be negatively charged even when used in a high pH environment, and does not damage the hair or scalp during use, and has little rash. An object of the present invention is to provide a keratin fiber dyeing agent excellent in dyeability.

  A feature of the present invention is copper eflex oxidase comprising the amino acid sequence of SEQ ID NO: 1 and further having four or more basic amino acid residues added to the carboxy terminal side.

  Another feature of the present invention is that in the amino acid sequence of SEQ ID NO: 1, at least two of the 69th glutamic acid, 82nd glutamic acid, 328th aspartic acid, and 340th aspartic acid are substituted with basic amino acids. It is a copper eflux oxidase containing the prepared amino acid sequence.

  Still another feature of the present invention is that in the amino acid sequence of SEQ ID NO: 13, the 242st aspartic acid, the 245th aspartic acid, the 261st glutamic acid, the 265th aspartic acid, the 285th aspartic acid, the 310th asparagine Bilirubin oxidase comprising an amino acid sequence in which at least four residues of acid, 322th aspartic acid, 323rd aspartic acid, 328th aspartic acid, and 338th aspartic acid are substituted with basic amino acids.

  Yet another feature of the present invention is the above-described multi-copper oxidase wherein the basic amino acid is arginine.

  Still another feature of the present invention is a keratin fiber dyeing agent containing the above-described multi-copper oxidase.

  The multi-copper oxidase of the present invention has a high isoelectric point by adding a basic amino acid residue on the carboxy terminal side or by substituting a specific amino acid residue with a basic amino acid residue. Even when used under conditions of high pH, it is difficult to be negatively charged. Therefore, the repulsive force with negatively charged hair can be reduced or adsorbed even in the hair dyeing process usually performed under alkaline conditions. For this reason, the oxidase is likely to act in the vicinity of the hair, and the colored oxidation dye is also likely to be distributed in the vicinity of the hair, so that the dyeability can be improved.

In the present invention, the keratin fibers include fibers mainly composed of keratin such as hair and wool, and the hair includes wigs composed of hair in addition to hair. In the following description, hair (head hair) will be described as an example of keratin fibers.
The multi-copper oxidase of the present invention is characterized in that a predetermined number of basic amino acids are added to the carboxyl terminus or a specific acidic amino acid residue is substituted with a basic amino acid.
The keratin fiber staining agent of the present invention is characterized in that the above-mentioned multi-copper oxidase is blended.

  In order to obtain an oxidase having a high isoelectric point according to the present invention, a method of adding a basic functional group to the surface of the oxidase can be mentioned. Specifically, a method of adding a basic amino acid at a predetermined location or replacing a specific amino acid residue constituting an oxidase with a basic amino acid can be exemplified. When adding an amino acid, select a location that appears on the surface when folded to form a three-dimensional structure, and that does not change significantly when added (for example, the amino terminus or carboxy terminus). And add. When an amino acid residue is replaced with a basic amino acid, it appears on the surface when it is folded to form a three-dimensional structure, and is not a basic amino acid (preferably an acidic amino acid). Select and replace amino acid residues that do not significantly change the amino acid residues (those that do not affect the ability to oxidize oxidative dyes).

More specifically, the oxidase as described above can be obtained by adding 4 or more, preferably 7 or more basic amino acid residues to the carboxy terminal side of rCueO of SEQ ID NO: 1. The rCueO of SEQ ID NO: 1 is a recombinant protein obtained by removing helix 5, helix 6 and helix 7 from CueO derived from Escherichia coli, and inserting a spacer instead. An example of the base sequence of DNA encoding the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.
The rCueO shown in SEQ ID NO: 1 is that of a mature protein, but the base amino acid sequence may be that of an immature protein, or in addition to the amino terminal side or carboxy terminal side of the amino acid sequence. As long as the enzyme activity and the effect of the present invention are not impaired, an affinity tag such as a histidine tag, a signal peptide, or other structures may be added.
An amino acid sequence of an immature protein with a histidine tag is shown in SEQ ID NO: 3, and an example of the DNA base sequence is shown in SEQ ID NO: 4.

  In detail about the number of amino acid residues to be added, if arginine, which has the highest basicity among basic amino acid residues, is used, the number of residues to be added is small, and if histidine, which has the lowest basicity, is used, it will be added. There is a need to increase the number of residues, and lysine tends to be in the middle. Specifically, when the basic amino acid residue consists only of arginine, it is 4 residues or more, preferably 6 residues or more, and when it consists only of lysine, it is 6 residues or more, preferably 8 residues or more. (The same applies when lysine and arginine are used). When the histidine is used alone, it is 7 residues or more, preferably 10 residues or more (the same applies when histidine, lysine and arginine are mixed).

  Regarding the number of amino acids to be added, there is no particular upper limit in terms of the principle of the present invention, but even if the isoelectric point is made higher than the pH at which the enzyme is deactivated, the effect is small. Or 30 residues or less. Moreover, when the same amino acid residue is continuously added, there is a possibility that the translation efficiency is lowered and the expression level is lowered. In order to prevent this, a large number of the same codons can be consecutively arranged by alternately arranging two or three basic amino acid residues, or by changing the codon used even when the same amino acid residues are adjacent to each other. It is preferable to prevent it from being arranged.

  The residue to be added may be lysine, arginine, or histidine, but arginine having the highest basicity is preferred, followed by lysine. In addition, amino acid residues that are not basic may be mixed in the amino acid residues added to the carboxy terminal side, but it is preferable to add only basic amino acids continuously. When mixing non-basic amino acids, increase the number of basic amino acid residues by that amount (preferably 7 residues or more), or add an arginine residue with particularly high basicity, so that the isoelectric point as a whole Be kept high.

  A preferred amino acid sequence of mCueO obtained by the above method is shown in SEQ ID NO: 5. In this example, six arginine residues are added to the carboxy terminal side of rCueO of SEQ ID NO: 1. An example of a DNA sequence encoding the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 6. The amino acid sequence of the mCueO immature protein of SEQ ID NO: 5 is shown in SEQ ID NO: 7, and an example of the DNA sequence is shown in SEQ ID NO: 8.

As another example of an oxidase having a high isoelectric point, among the amino acid residues of rCueO shown in SEQ ID NO: 1, there are four amino acids: 69th glutamic acid, 82nd glutamic acid, 328th aspartic acid, and 340th aspartic acid. Examples in which at least two of them are replaced with basic amino acids can be exemplified. These amino acid residues are amino acid residues whose isoelectric point increases particularly remarkably when they are replaced in rCueO of SEQ ID NO: 1. When at least two of them are replaced with basic amino acids (arginine) Experiments have shown that the isoelectric point exceeds 7.6. The four amino acid residues correspond to the 97th glutamic acid, the 110th glutamic acid, the 356th aspartic acid, and the 368th aspartic acid in the amino acid sequence of the immature protein shown in SEQ ID NO: 3.
Similar to the above example, the mCueO of this example may also be an amino acid sequence of an immature protein, and on the amino terminal side and carboxy terminal side of the amino acid sequence, unless the enzyme activity and the effect of the present invention are impaired. An affinity tag such as a histidine tag, a signal peptide, and other structures can be added.

  An example in which all four residues (69th, 82nd, 328th, 340th) are substituted with arginine is shown in SEQ ID NO: 9, and an example of a DNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 10. Further, the amino acid sequence of the mCueO immature protein shown in SEQ ID NO: 9 with a histidine tag is shown in SEQ ID NO: 11, and an example of the DNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 12.

As another example of an oxidase having a high isoelectric point, in the amino acid sequence of wtBOD shown in SEQ ID NO: 13, the 242st aspartic acid, the 245th aspartic acid, the 261st glutamic acid, the 265th aspartic acid, 285 Aspartic acid, 310th aspartic acid, 322th aspartic acid, 323rd aspartic acid, 328th aspartic acid, 338th aspartic acid with at least 4 replaced with basic amino acids Can be illustrated. The wtBOD of SEQ ID NO: 13 is a wild-type BOD derived from Myrothecerium verrucaria.
The ten amino acid residues are amino acid residues whose isoelectric points are particularly remarkably increased by substitution with basic amino acid residues in wtBOD of SEQ ID NO: 13, and four of these amino acid residues are basic amino acids (arginine). The experiment revealed that the isoelectric point was 4.9. In addition, the isoelectric point increases as the number of amino acid residues to be substituted increases. An example of the base sequence of DNA encoding the amino acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 14.

The wtBOD shown in SEQ ID NO: 13 is that of a mature protein, but the base amino acid sequence may be that of an immature protein, or in addition, on the amino terminal side or carboxy terminal side of the amino acid sequence, As long as the enzyme activity and the effect of the present invention are not impaired, an affinity tag such as a histidine tag, a signal peptide, or other structures may be added.
The amino acid sequence of the immature protein of wtBOD shown in SEQ ID NO: 13 is shown in SEQ ID NO: 15, and an example of the DNA base sequence is shown in SEQ ID NO: 16. In the amino acid sequence of the immature protein shown in SEQ ID NO: 15, the 10 amino acid residues are 280th aspartic acid, 283rd aspartic acid, 299th glutamic acid, 303th aspartic acid, 323rd asparagine, respectively. Acid, 348th aspartic acid, 360th aspartic acid, 361th aspartic acid, 366th aspartic acid, and 376th aspartic acid.

  The preferred amino acid sequence of mBOD by the above method is shown in SEQ ID NO: 17. In this example, all the 10 residues (242nd, 245th, 261st, 265th, 265th, 285th, 310th, 322nd, 323rd acid, 328th, 338th) of wtBOD of SEQ ID NO: 13 are left as arginine residues. It has been changed based on. An example of a DNA sequence encoding the amino acid sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 18. The amino acid sequence of the mBOD immature protein of SEQ ID NO: 17 is shown in SEQ ID NO: 19, and an example of the DNA sequence is shown in SEQ ID NO: 20.

  A method for obtaining an oxidase in which a basic amino acid residue is added to the carboxy terminus or an oxidase in which a specific amino acid residue is replaced with a basic amino acid is not particularly limited. For example, a base sequence encoding a desired amino acid sequence (gene ) Can be incorporated into an expression vector, introduced into a suitable host, cultured, and then any conventional method for purifying the resulting crude enzyme solution can be suitably employed.

  The base sequence (gene) of an oxidase in which a basic amino acid residue is added to the carboxy terminus can be obtained by a conventional method, but this method is used when an arginine residue is added to the carboxy terminus of rCueO shown in SEQ ID NO: 1. For example, a predetermined number of basic amino acid codons are directly added in front of the termination codon in the nucleotide sequence of SEQ ID NO: 21 (the DNA sequence of SEQ ID NO: 4 plus a termination codon) (SEQ ID NO: 22). Alternatively, as in SEQ ID NO: 23, only a restriction enzyme cleavage site (SplI cleavage site in the case of SEQ ID NO: 23) is added before the stop codon, and a polyarginine linker (sequence Number 24) may be inserted into the restriction enzyme cleavage site.

There is no particular limitation on the method of adding a predetermined number of basic amino acid codons directly as in SEQ ID NO: 22 (6 arginine residues in the example of SEQ ID NO: 22) or adding a restriction enzyme cleavage site as in SEQ ID NO: 23. However, it can be produced by a method applying the PCR method, for example.
Specifically, when a base sequence encoding a base oxidase is amplified by a PCR method, a 3 'primer having a base sequence to be added before a stop codon may be used.
The reaction conditions of the PCR method are not particularly limited, but a unit consisting of denaturation at 96 ° C. for 30 seconds, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 1 minute is defined as one cycle, and this cycle is repeated 25 times. It can be illustrated.

  Further, the method for obtaining the base sequence of oxidase in which a specific amino acid residue is replaced with a basic amino acid is not particularly limited, and a site-specific mutation may be introduced into a gene encoding a normal oxidase according to a known method. . Such site-specific mutation can be easily performed using a commercially available kit. Examples of commercially available kits that can be used include QuikChange (trade name) Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) and Transformer (trade name) Site-Directed Mutagenesis Kit (manufactured by CONTECH).

The base sequence prepared as described above is incorporated into an appropriate expression vector. Although an expression vector that can be used is not particularly limited, examples of a plasmid that can be used as an expression vector for E. coli include pBR322, pBluescript, pUC19, pUC18, and pUC119. Among these, pUC18 is preferable.
Examples of plasmids that can be used as an expression vector for yeast include pPIC9K, pPIC3.5K, pPIC6, pAO815, and pGAPZ. Of these, pPIC9K is preferable.

  A method for incorporating a base sequence encoding a desired oxidase into the expression vector is not particularly limited, and a well-known method can be used. For example, Takara Ligation Kit Ver. 2.1 (trade name: manufactured by Takara Bio Inc.) can be used by reacting at 16 ° C. overnight.

An expression vector (recombinant vector) in which a desired base sequence is incorporated is introduced into an appropriate host. The host to be used is not particularly limited, and any of microorganisms, animal cells, and plant cells may be used, but microorganisms are preferably used in terms of handleability, and Escherichia coli and methanol-utilizing yeast (Pichia pastoris) are particularly preferable. Is preferred.
The strain is not particularly limited, but when E. coli is used, XL1-Blue, BL21, JM109, NM522, DH5α, HB101, and DH10B can be exemplified, and among these, BL21 is preferable. When using methanol assimilation yeast, GS115, KM71, SMD1168 can be illustrated, and GS115 is preferable among these.

  The method for introducing the recombinant vector into the host is not particularly limited, and all known methods such as lipofection, electroporation, microinjection, and particle gun can be preferably used.

The host (transformant) into which the recombinant vector has been introduced as described above is cultured according to a conventional method. The medium to be used is not particularly limited. Specifically, when the host is Escherichia coli, well-known media such as LB medium and M9-glucose medium can be exemplified. Moreover, you may add chemical | medical agents, such as copper chloride, an isopropyl thio- (beta) -D-galacside (IPTG), and ampicillin, to a culture medium as needed. The culture conditions are not particularly limited, but may be cultured at 28 to 37 ° C. for 1 to 24 hours. Moreover, you may perform ventilation | gas_flowing and stirring as needed.
When the host is methanol-utilizing yeast, examples of the medium that can be used include well-known media such as YPD media, MD media, and MBB media. Moreover, you may add chemical | medical agents, such as copper chloride, methanol, and G418, to a culture medium. The culture conditions are not particularly limited, but may be cultured at 20 to 32 ° C., preferably 30 ° C. for 2 to 7 days. Moreover, you may perform ventilation | gas_flowing and stirring as needed.

The multi-copper oxidase of the present invention can be obtained from a medium in which the above host is cultured. The method for obtaining a desired oxidase from the medium is not particularly limited, and examples thereof include a method for purifying a protein obtained from the supernatant of the culture solution and a protein obtained by destroying the host by the osmotic shock method.
The purification method is not particularly limited, but salting out, solvent precipitation, dialysis, ultrafiltration, polyacrylamide electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing All known methods can be suitably used.

The multi-copper oxidase prepared as described above is useful as an oxidizing agent in a keratin fiber staining agent.
As the oxidative dye used in the keratin fiber dyeing agent of the present invention, any oxidative dye can be used as long as it can be oxidized by the multi-copper oxidase. Specifically, paraphenylenediamine, 5-aminoorthocresol, orthoaminophenol, metaaminophenol, paraaminophenol, 2,4-diaminophenol, 2,6-diaminopyridine, 5- (2-hydroxyethylamino)- 2-methylphenol, N, N-bis (β-hydroxy) -paraphenylenediamine sulfate, paranitro-orthophenylenediamine, paranitro-2 ′, 4′-diaminoazobenzene sodium sulfate, toluene-2,5-diamine, 5 -Aminoorcresol sulfate, paraaminophenol sulfate, orthochloro-paraphenylenediamine sulfate, 4,4'-diaminodiphenylamine sulfate, paramethylaminophenol sulfate, paraphenylenediamine sulfate, metaphenylenediamine Acid salt, toluene-2,5-diamine sulfate, 2,4-diaminophenoxyethanol hydrochloride, toluene-2,5-diamine hydrochloride, metaphenylenediamine hydrochloride, 2,4-diaminophenol hydrochloride, 3,3 '-Iminodiphenol, paraphenylenediamine hydrochloride, N-phenyl-paraphenylenediamine hydrochloride, N-phenyl-paraphenylenediamine acetate, 1,5-dihydroxynaphthalene, tolylene-3,4-diamine, paramethylamino Examples include phenol, N, N'-bis (4-aminophenyl) -2,5-diamino-1,4-quinonediimine, orthoaminophenol sulfate, 2,4-diaminophenol sulfate, and metaaminophenol sulfate. However, due to substrate specificity, paraphenylenediamine, para Minophenol, orthoaminophenol, 2,4-diaminophenol, toluene-2,5-diamine and salts thereof are preferred. These may be used alone or in combination of two or more as required.

In the present invention, an indoline compound or an indole compound can also be used as a dye.
The indoline compound is not particularly limited. For example, indoline, 5,6-dihydroxyindoline, N-methyl-5,6-dihydroxyindoline, N-ethyl-5,6-dihydroxyindoline, N-butyl-5,6- Dihydroxyindoline, 4-hydroxy-5-methoxyindoline, 6-hydroxy-7-methoxyindoline, 6,7-dihydroxyindoline, 4,5-dihydroxyindoline, 4-methoxy-6-hydroxyindoline, N-hexyl-5 6-dihydroxyindoline, 2-methyl-5,6-dihydroxyindoline, 3-methyl-5,6-dihydroxyindoline, 4-hydroxyindoline, 2,3-dimethyl-5,6-dihydroxyindoline, 2-methyl-5 -Ethyl-6-hydroxyindo 2-methyl-5-hydroxy-6-β-hydroxyethylindoline, 4-hydroxypropylindoline, 2-hydroxy-3-methoxyindoline, 6-hydroxy-5-methoxyindoline, 6-hydroxyindoline, 5-hydroxy Indoline, 7-hydroxyindoline, 7-aminoindoline, 5-aminoindoline, 4-aminoindoline, 5,6-dihydroxyindoline carboxylic acid, 1-methyl-5,6-dihydroxyindoline, salts thereof and the like it can. These may be used alone or in combination of two or more as required.

  The indole compound is not particularly limited, and examples thereof include 4,5-dihydroxyindole, 5,6-dihydroxyindole, 6,7-dihydroxyindole, N-methyl-5,6-dihydroxyindole, N-ethyl-5,6. -Dihydroxyindole, N-hexyl-5,6-dihydroxyindole, 2-methyl-5,6-dihydroxyindole, 3-methyl-5,6-dihydroxyindole, 4-hydroxyindole, 2,3-dimethyl-5, 6-dihydroxyindole, 2-methyl-5-ethyl-6-hydroxyindole, 2-methyl-5-hydroxy-6-β-hydroxyethylindole, 4-hydroxypropylindole, 2-hydroxy-3-methoxyindole, 4 -Hydroxy-5-methoxyin , 6-hydroxy-7-methoxyindole, 6-hydroxy-5-methoxyindole, 6-hydroxyindole, 5-hydroxyindole, 7-hydroxyindole, 7-aminoindole, 5-aminoindole, 4-aminoindole 5,6-dihydroxyindolecarboxylic acid, 1-methyl-5,6-dihydroxyindole, and salts thereof. These may be used alone or in combination of two or more as required.

The dye for keratin fibers of the present invention may contain a dye or a pigment other than the oxidation dye, if necessary. Examples of these pigments and dyes include tar pigments, HC dyes, basic dyes, direct dyes, etc., and these may be used alone or in combination of two or more as required. By containing these pigments and dyes, hair can be dyed in various colors.
Examples of such a tar dye include those specified by the Ministry of Health and Welfare Ordinance No. 30 “Ministerial Ordinance for Determining Tar Dyes that can be Used for Pharmaceuticals” promulgated on August 31, 1966. Examples of the HC dye include HC blue 2, HC orange 1, HC red 1, HC red 3, HC yellow 2, HC yellow 4, and the like. As the basic dye, basic blue 99, basic brown 16 , Basic brown 17, basic red 51, basic red 76, basic yellow 57, etc., and direct dyes include 2-amino-6-chloro-4-nitrophenol, 3-methylamino-4- Nitrophenoxyethanol, 2-amino-3-nitrophenol, 4-hydroxypropylamino-3-nitrophenol and the like can be mentioned. These may be used alone or in combination of two or more as required.

The keratin fiber dyeing agent of the present invention can contain various components usually used in cosmetics and keratin fiber dyeing agents as long as the effects of the present invention are not impaired.
For example, surfactants include cationic surfactants such as lauryltrimethylammonium chloride and stearyltrimethylammonium chloride; polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene behenyl ether, polyoxyethylene castor oil, polyoxyethylene castor oil, Nonionic surfactants such as oxyethylene hydrogenated castor oil and sorbitan stearate; Oils such as cetyl alcohol, cetostearyl alcohol, stearyl alcohol, petrolatum, paraffin, liquid paraffin, cetyl palmitate, octyl palmitate; xanthan gum, succinoglucan , Guar gums such as hydroxypropyl guar gum, cationized guar gum, carboxymethyl cellulose, hydroxyethyl cellulose, cationized cell Thickeners such as celluloses such as cellulose; humectants such as 1,3-BG, PG, DPG, glycerin; chelating agents such as EDTA, EDTA-2Na, EDTA-4Na, hydroxyethanediphosphonic acid: parabens, A preservative such as methyl isothiazolinone; a solvent such as ethanol and isopropyl alcohol; a fragrance and the like, which can be arbitrarily combined as needed.

The keratin fiber dyeing agent of the present invention may be of a single agent type or a multi-agent type. However, in the case of the present invention, even if a dye and an enzyme are mixed, color does not develop if there is no oxygen. Particularly suitable as a dye-type dyeing agent.
The dosage form can also be any form such as liquid, emulsion, cream, gel, foam, aerosol, etc. The container can also be in a bag, bottle, pump container, tube, spray can Anything can be adopted.

  EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated in detail, it cannot be overemphasized that this invention is not restrict | limited at all by these. In the following description, “%” means “% by weight”.

Example 1
The modified multi-copper oxidase of the present invention was prepared by the following procedure.
(1) Preparation of 6XArg-added mCueO gene rCueO expression plasmid pUCCueO Δα5-7 (same as pUC18-CueO Δα5-7 described in Patent Document 3. For the production method, see [0076] to [0096]) Using a CueO F (+) primer of SEQ ID NO: 25 and a 6XArg tag-introduced primer of SEQ ID NO: 26 as a template, a gene fragment of about 0.7 kbp in the latter half of 6XArg-added mCueO described in SEQ ID NO: 8 by PCR Was amplified.

The rCueO expression plasmid pUCCueO Δα5-7 refers to the gene (SEQ ID NO: 4) encoding the protein (SEQ ID NO: 3) in which the histidine tag (6XHis tag) is added after the carboxy terminal amino acid Val of rCueO. A restriction enzyme EcoRI cleavage sequence was introduced before, a restriction enzyme BamHI cleavage sequence was introduced after the stop codon, and inserted into the EcoRI-BamHI cleavage site of the cloning vector pUC18.
The CueOF (+) primer of SEQ ID NO: 25 corresponds to the 748-768th nucleotide sequence in the ORF of rCueO (SEQ ID NO: 4).
In the 6XArg tag introduction primer base sequence of SEQ ID NO: 26, the 3rd to 8th bases from the 5 'end (gatcc) are BamHI cleavage sites, the 9th to 11th bases (tta) are anticodons to the stop codon, 12 The ˜29th base (acgacgacgacgagacg) is a repeat of Arg's anticodon 6 times, and the 30th and subsequents are complementary sequences to the carboxy terminal region coding sequence of CueO.

The PCR reaction was performed in the following reaction cycle using the PCR reaction solution shown below.
(2) PCR reaction solution Pwo Super Yield PCR Buffer 5.0 μl
pUCCueO Δα5-7 (10 ng / μl) 1.0 μl
CueO F (+) primer (10 pmol / μl) 1.0 μl
6 × Arg tag introduction primer (10 pmol / μl) 1.0 μl
dNTP mix. (2 mM, manufactured by Toyobo Co., Ltd.) 5.0 μl
Sterile distilled water 36.5 μl
Pwo Super Yield DNA Polymerase 0.5 μl
Total 50.0μl

(3) PCR reaction cycle 1) 95 ° C, 5 min preheating 2) 95 ° C, 1 min denaturation 3) 52 ° C, 1 min annealing 4) 72 ° C, 1 min extension → Return to 2) (25 cycles)
5) 72 ° C, 5 min extension

(4) Purification of PCR amplified fragment After completion of the above reaction, the reaction solution was subjected to 1% agarose gel electrophoresis to separate the amplified fragment, and the amplified fragment was extracted and purified from the gel using QIAEXII Gel Extraction Kit manufactured by Qiagen. . The operation was performed according to the instructions of Qiagen.

(5) TA Cloning of PCR Amplified Fragment The purified amplified fragment was ligated with Promega pGEM-T Easy Vector. The ligation operation was performed according to the instructions of Promega. After completion of the ligation reaction, 5 μl of the reaction solution was used to transform E. coli XL10-Gold competent cells. Transformants were incubated overnight at 37 ° C. using LB agar medium containing ampicillin.

(6) Restriction enzyme digestion Plasmids were isolated from the grown colonies by alkaline SDS method, digested with restriction enzymes NcoI and BamHI, and then subjected to 1% agarose gel electrophoresis to separate digested fragments. Subsequently, an about 0.7 kbp digested fragment was extracted from the gel and purified using a QIAEXII Gel Extraction Kit.
Separately, pUCCueO Δα5-7 was digested with restriction enzymes NcoI and BamHI, and a fragment of about 3.4 kbp was purified by the same operation.

(7) Production of 6XArg-added mCueO Expression Plasmid Using DNA Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.), approximately 0.7 kbp digested fragment obtained from the grown colonies according to the Takara Bio Inc. instruction manual And a fragment of about 3.4 kbp obtained from pUCCueO Δα5-7 was ligated. After completion of the ligation reaction, 5 μl of the reaction solution was used to transform E. coli XL10-Gold competent cells. The transformant was spread on an LB agar medium containing ampicillin and incubated overnight at 37 ° C.

(8) Confirmation of 6XArg addition type mCueO expression plasmid A plasmid was isolated from the grown colonies by the alkaline SDS method, and the sequence of the mutant gene was confirmed in the obtained plasmid. The confirmed plasmid is designated as pUCCueO Δα5-7 6R. The amino acid sequence of 6XArg addition type mCueO encoded by pUCCueO Δα5-7 6R is shown in SEQ ID NO: 7, and the amino acid sequence of mature 6XArg addition type mCueO is shown in SEQ ID NO: 5.

(9) Production and purification of 6XArg addition type mCueO
(i) Culture of transformants E. coli BL21 (DE3) competent cells were transformed with pUCCueO Δα5-7 6R, and the transformed bacteria were spread on LB agar medium containing ampicillin and incubated at 37 ° C. overnight. The resulting colony was cultured in the following two stages to express 6XArg-added mCueO.
Pre-culture (test tube): shaking culture was performed overnight at 37 ° C. in 4 ml of LB medium containing 0.1 mg / ml ampicillin.
Main culture (conical flask with ₂ L baffle): Shaking culture was performed at 32 ° C. for 12 hours in 400 ml of the above medium supplemented with 1 mM CuCl ₂ and 0.5 mM IPTG.
After completion of the culture, the cells were collected by centrifugation, washed with a 0.85% aqueous sodium chloride solution, and immediately subjected to the following osmotic shock.

(ii) Osmotic shock Suspension of cells in ice-cooled 20% sucrose, 100 mM Tris-H ₂ SO ₄ buffer (pH 7.5) containing 10 mM EDTA, left in ice water for 10 minutes, and then centrifuged. The cells were collected. Next, the suspension was suspended in ice-cold distilled water containing a protease inhibitor, allowed to stand in ice water for 10 minutes, and the supernatant was collected by centrifugation. The obtained supernatant was further subjected to ultracentrifugation at 90,000 Xg for 30 minutes, and the supernatant was used as a crude enzyme solution.

(iii) Cation exchange chromatography Toyopearl SP-650M (manufactured by Tosoh Corporation) was added potassium phosphate buffer (pH 6.0) to the above crude enzyme solution so as to have a final concentration of 20 mM and equilibrated with the same buffer solution. ) Overlaid on a column (100 ml) and washed with the same buffer. Thereafter, the adsorbed protein was extracted with a linear gradient of NaCl from 0 to 500 mM (400 ml). The active fractions were collected and concentrated, dialyzed against 100 mM potassium phosphate buffer (pH 6.0), and used as a purified sample of mCueO.

Example 2
Another example of the modified multi-copper oxidase of the present invention was prepared by the following procedure.
First, a DNA fragment encoding mature BOD is obtained by the method described in [0022] and [0023] of Japanese Patent No. 4437652, and the 265th, 285th, and 323rd positions in SEQ ID NO: 13 are obtained using the QuikChange method. The codon corresponding to the 338th amino acid residue was replaced with the codon aga of arginine.
Thereafter, a purified preparation of 4-residue-substituted mBOD was obtained according to the method of the same publication [0024] to [0031].

Comparative Example 1
A purified sample of rCueO was obtained by the same method as mCueO (1) described in [0076] to [0101] of Patent Document 3 (Republished Patent Publication No. 2007/063614). The amino acid sequence of the obtained rCueO is shown in SEQ ID NO: 27.

Comparative Example 2
BOD obtained by the method described in [0022] to [0031] of Japanese Patent No. 4437652 was used as wt BOD of Comparative Example 2. The amino acid sequence of the obtained wtBOD is shown in SEQ ID NO: 13.

Example 3
A two-agent type keratin fiber dyeing agent comprising a first agent containing paraphenylenediamine as a dye intermediate and a second agent containing oxidase was prepared.
As shown in Table 1, the first agent is 2 g of polyoxyethylene hydrogenated castor oil, 1 g of lactic acid, 1 g of hydroxyethyl cellulose, 0.9 g of monoethanolamine, 1 g of paraphenylenediamine, and mixed with purified water to a total of 25 g. Prepared. The pH of the first agent is 9.8.
The second agent is the mCueO dilute solution prepared in Example 1 (concentration in use is 0.5 units / g). The isoelectric point (pI) of mCueO used in this example is 8.1.

Example 4
As shown in Table 1, a keratin fiber staining agent was prepared in the same manner as in Example 3 except that 0.5 g of resorcin was included as a coupler in the first agent.

Example 5
As shown in Table 1, a keratin fiber staining agent was prepared in the same manner as in Example 3 except that 0.3 g of paraaminophenol was contained in the first agent as a dye intermediate.

Example 6
As shown in Table 1, a keratin fiber staining agent was prepared in the same manner as in Example 3 except that 0.3 g of metaaminophenol was included as a coupler in the first agent.

Example 7
As shown in Table 1, a keratin fiber dye was prepared in the same manner as in Example 3 except that the first agent contained 0.2 g of 2,4 diaminophenoxyethanol hydrochloride as a coupler.

Example 8
As shown in Table 1, a keratin fiber staining agent was prepared in the same manner as in Example 3 except that 0.3 g of pAOC (paraaminoorthocresol) was included as a coupler in the first agent.

Example 9
As shown in Table 1, a keratin fiber staining agent was prepared in the same manner as in Example 8 except that 3 g of 28% aqueous ammonia was used instead of monoethanolamine.

"Dyeing test"
Using the keratin fiber dyeing agents of Examples 3 to 9 described above, a hair dyeing test was performed on a 1 g bundle of burax white hair.
First, as a pretreatment, the hair bundle is washed with running water at 38 ° C. for 30 seconds, rinsed with 1% SDS solution for 1 minute, washed again with running water at 38 ° C. for 30 seconds, washed away with SDS solution, and then air-dried. did.
Next, 0.5 g of the first agent and 1.5 g of the second agent were weighed, put into a cup, mixed with a brush, and the mixed solution was applied to the hair bundle with the brush. The hair bundle was spread in a fan shape so that it could be applied as uniformly as possible.
After coating, the mixture was allowed to stand at room temperature (26.5 ° C.) for 15 minutes, and then the hair bundle was turned over to spread the mixed solution uniformly with a brush, and further allowed to stand at room temperature for 15 minutes.
Thereafter, the hair bundle is washed with running water at 38 ° C. for 30 seconds to wash away the keratin fiber dyeing agent, 1% SDS solution is applied, combed with a comb 20 times and foamed, and then washed with running water at 38 ° C. The comb was washed 20 times with a comb to wash away the 1% SDS solution. After washing, moisture was wiped off with a towel and dried.
After drying, the Lab value was measured with a spectrocolorimeter (manufactured by Konica Minolta, CM-2600d), and the color difference ΔE based on untreated gray hair was calculated. The results are shown in Table 2.
As shown in Table 2, the hair bundle is dyed in various colors, and it can be seen that the hair can be dyed to a desired color desired by the user.

Example 10 and Comparative Example 3
The amount of monoethanolamine was changed so that the pH of the first agent was 9.0, and the diluted solution of the enzyme obtained in Example 1 and Comparative Example 1 was used as the second agent (the concentration during use was 0.00. The keratin fiber dyes of Example 10 and Comparative Example 3 were prepared in the same manner as in Example 3 except that 25 units / g) was used.

Example 11 Comparative Example 4
As the first agent, instead of paraphenylenediamine (PPD), a compound containing 0.3 g of 5,6-dihydroxyindole (5,6-DHI) was used, and the second agent was obtained in Example 2 and Comparative Example 2. The keratin fiber staining agents of Example 11 and Comparative Example 4 were prepared in the same manner as in Example 10 except that the diluted enzyme solution (concentration when used was 0.25 units / g) was used.

Using the dyeing agents for keratin fibers of Examples 10 and 11 and Comparative Examples 3 and 4 described above, a dyeing test was performed in the same manner as the above dyeing test. Table 3 shows the isoelectric point (pI) of the enzyme and the test results.
As shown in Table 3, it can be seen that if multi-copper oxidase having a high isoelectric point is used, the dyeability is superior compared to the case where the isoelectric point is low.

In the above Examples, Comparative Examples, and Reference Examples, the concentration of each enzyme was measured and prepared under the following conditions.
0.875 mL of 200 mM acetate buffer (pH 5.5), aqueous solution of each enzyme (rCueO, mCueO, wtBOD, rBOD) (0.5 mg / mL) in a cuvette (1.5 mL UV disposable cell, manufactured by Top) 025 mL and each substrate solution 0.1 mL were mixed, and the amount of change in absorbance at each measurement wavelength was measured using a spectrophotometer (UV-2459, manufactured by SHIMADZU) (measurement wavelength is paraphenylenediamine). (470 nm, 300 nm for 5,6-dihydroxyindole).
Then, compared with the amount of change in absorbance of wtCueO and wtBOD at the already known target concentrations (0.5 and 0.25 units / g), each aqueous enzyme solution was diluted so as to be the same as the amount of change in absorbance. The enzyme solution was considered as the target concentration.

Since the modified multi-copper oxidase of the present invention has a high isoelectric point, it is difficult to be negatively charged even under alkaline conditions, or is positively charged and hardly repels or adsorbs to keratin fibers under such conditions. It can be suitably used as an oxidizing agent for the second agent in the fiber dyeing agent.
The staining agent for keratin fibers of the present invention oxidizes an oxidative dye with an oxidase having a high isoelectric point, so that the hair and scalp are less damaged and rashed, and the repulsive force between the hair and the oxidase is small or conversely adsorbed. As a result, colored oxidase is distributed in the vicinity of the hair, so that it is excellent in dyeability.

Claims (5)

  A copper eflex oxidase comprising the amino acid sequence of SEQ ID NO: 1 and further having four or more basic amino acid residues added to the carboxy terminal side.   The amino acid sequence of SEQ ID NO: 1 includes an amino acid sequence in which at least two residues of the 69th glutamic acid, the 82nd glutamic acid, the 328th aspartic acid, and the 340th aspartic acid are substituted with basic amino acids. Features copper eflux oxidase.   In the amino acid sequence of SEQ ID NO: 13, the 242nd aspartic acid, the 245th aspartic acid, the 261st glutamic acid, the 265th aspartic acid, the 285th aspartic acid, the 310th aspartic acid, the 322nd aspartic acid, 323 A bilirubin oxidase comprising an amino acid sequence in which at least four residues of the first aspartic acid, the 328th aspartic acid, and the 338th aspartic acid are substituted with basic amino acids.   The multi-copper oxidase according to any one of claims 1 to 3, wherein the basic amino acid is arginine.   A staining agent for keratin fibers, comprising the multi-copper oxidase according to any one of claims 1 to 4.