KR102223091B1 - Cosmetic composition for skin whitening comprising lignin peroxidase and decolorizing method of melanin in skin using the same - Google Patents

Abstract

A cosmetic composition for skin whitening containing lignin peroxidase has very few skin side effects, selectively bleaching melanin, and excellent melanin bleaching efficiency. Further, in the case of a composition containing glucose oxidase, since the decolorization efficiency of melanin is further enhanced through continuous supply of low concentration hydrogen peroxide, the composition can be used for skin whitening.
In addition, the method of decolorizing melanin in the skin using the composition also selectively decolorizes melanin, and its efficiency is excellent, so that the skin whitening effect of the applied individual can be achieved.

Description

A cosmetic composition for skin whitening containing lignin peroxidase and a method of bleaching melanin in the skin using the same.

It relates to a cosmetic composition for skin whitening comprising lignin peroxidase and a method for bleaching melanin in the skin using the same.

Melanin plays an important role in protecting the skin from UV rays, which is by filtering out most wavelengths of light, including UV rays. For this reason, most of the melanin is located in the epidermis and determines the color of the skin. There are two types of melanin, eumelanin and pheomelanin, eumelanin represents black or brown, and pheomelanin represents red or yellow.

On the other hand, melanin present in the skin is converted to dopaquinone in the melanin synthesis pathway, L-DOPA (L-3,4-dihydroxyphenylalanine), and is produced by tyrosinase. Accordingly, to date, various types of tyrosinase inhibitors in whitening cosmetics have been most actively studied to reduce melanin production in cells. However, these chemical inhibitors of tyrosinase have many potential side effects, such as vitiligo, and many people suffer from rhododenol, a powerful tyrosinase inhibitor.

For this reason, studies in which enzymes use enzymes instead of chemicals to decolor melanin present on the skin surface are being conducted in order to selectively decolorize melanin present on the skin surface. Other peroxidases such as laccase, dye-decolorizing peroxidase, manganese peroxidase and lignin peroxidase have already been tested, but manganese peroxidase is a manganese heavy metal ion for efficient melanin decolorization. Or require synthetic mediators, which can be toxic to human skin. Lignin peroxidase is a promising catalyst that decolorizes melanin much more effectively than other enzymes because it does not require these substances and even exhibits a high redox potential capable of oxidizing verayl alcohol. In contrast, other enzymes exhibit a less effective redox potential to oxidize melanin. However, despite its high potential as a melanin decolorizing enzyme, lignin peroxidase is only studied as an unpurified enzyme due to difficulty in expression and purification because it biosynthesizes up to 17 other homologous enzymes from Phanerocaete chrysosporium. And have been used. In addition, lignin peroxidase can be easily inactivated by an excessive amount of hydrogen peroxide (H ₂ O ₂ ), which was a major obstacle to inhibiting the increase in melanin bleaching efficiency by inactivating the enzyme.

Accordingly, the purified lignin peroxidase is used to selectively decolorize melanin in the skin within a short time, as well as improve melanin decolorization efficiency to be applied as a major material and technology for whitening cosmetics.

One aspect is to provide a cosmetic composition for skin whitening comprising lignin peroxidase.

Another aspect is to provide a method for depigmenting melanin in the skin comprising applying the composition to the skin of a subject.

Another aspect is to provide a method for depigmenting melanin in skin comprising applying lignin peroxidase, veratrile alcohol, and hydrogen peroxide to the skin of an individual.

One aspect provides a cosmetic composition for skin whitening comprising lignin peroxidase.

The lignin peroxidase catalyzes a reaction in which veratryl alcohol and hydrogen peroxide (H ₂ O ₂ ) react to generate water (H ₂ O) and veratryl alcohol cation radicals.

The generated veratryl alcohol cation reacts with melanin to form veratryl alcohol and decolorizes melanin at the same time, thereby achieving a skin whitening effect.

The composition not only selectively decolorizes melanin, but also has very excellent melanin decolorization efficiency, has excellent skin whitening effect, and has very few skin side effects.

The lignin peroxidase may be derived from nature or may be obtained by various protein synthesis methods well known in the art. As an example, it may be prepared by using a polynucleotide recombination and protein expression system, or by a method of in vitro synthesis through chemical synthesis such as protein synthesis, a cell-free protein synthesis method, and the like. In addition, as an example, the lignin peroxidase may be a product obtained by culturing a peptide, an extract of a tissue or cell derived from a plant, or a microorganism (eg, bacteria or fungi, and particularly yeast).

The term “protein” refers to a polymer consisting of two or more amino acids linked by amide bonds (or peptide bonds).

The lignin peroxidase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more of the amino acid sequence of lignin peroxidase, respectively. % Or greater, about 98% or greater, or about 99% or greater sequence homology. For example, the lignin peroxidase may be a lignin peroxidase isoenzyme. Specifically, the lignin peroxidase may be a lignin peroxidase derived from Phanerochaete chrysosporium. More specifically , it may be a lignin peroxidase isozyme H8 (LiPH8) derived from Phanerochaete chrysosporium , which may be a protein consisting of the amino acid sequence of SEQ ID NO: 1.

The term "homology" is intended to indicate the degree of similarity with the wild-type amino acid sequence, and comparison of such homology can be performed using a comparison program well known in the art, and homology between two or more sequences It can be calculated as a percentage (%).

In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, titer, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), reduced antigenicity, the N of the lignin peroxidase -Or a protecting group may be bonded to the C-terminus. The protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or a polyethylene glycol (PEG), but the modification of the lignin peroxidase, particularly the stability of lignin peroxidase Any ingredient that can be promoted may be included without limitation.

The term "stability" may mean not only in vivo stability that protects the lignin peroxidase from attack by protein cleavage enzymes in vivo, but also storage stability (eg, room temperature storage stability).

In addition, the lignin peroxidase may additionally include a targeting sequence, a tag, and an amino acid sequence prepared for a specific purpose for a labeled residue.

The concentration in the composition of the lignin peroxidase may be 0.04 U/ml to 0.08 U/ml, 0.05 U/ml to 0.08 U/ml, 0.04 U/ml to 0.07 U/ml, 0.05 U/ml to 0.07 U/ml. have.

In addition, the composition may further include Veratryl Alcohol and hydrogen peroxide (H ₂ O _{2 ).}

The concentration in the composition of the veratril alcohol is 1 mM to 3 mM, 1 mM to 2.5 mM, 1.5 mM to 3 mM, 1.5 mM to 2.5 mM, 1.75 mM to 3 mM, 1.75 mM to 2.5 mM, 1.75 mM to 2.25 mM , 1.5 mM to 2.25 mM, 1 mM to 2.25 mM.

The concentration in the composition of hydrogen peroxide is 200 μM to 300 μM, 200 μM to 275 μM, 225 μM to 300 μM, 225 μM to 275 μM, 240 μM to 300 μM, 240 μM to 275 μM, 240 μM to 260 μM, 200 μM to 260 μM, 225 μM to 260 μM.

When the concentration in the composition of hydrogen peroxide is less than 200 μM, melanin decolorization efficiency may be poor as the specific activity of lignin peroxidase decreases, and when the concentration in the composition of hydrogen peroxide is more than 300 μM, the specific activity of lignin peroxidase is excellent. It can be done, but the melanin bleaching efficiency may not be good.

In addition, the pH of the composition may be 3.5 to 4.5, 3.5 to 4.25, 3.75 to 4.5, 3.75 to 4.25, 3.9 to 4.5, 3.9 to 4.25, 3.9 to 4.1, 3.5 to 4.1, 3.75 to 4.1.

When the pH of the composition is less than 3.5, the specific activity of lignin peroxidase may be excellent, but the veratryl alcohol cation radical does not come into contact with melanin, and the lignin peroxidase and melanin are combined and precipitated, resulting in melanin depigmentation efficiency. This can be bad. In addition, when the pH of the composition exceeds 4.5, melanin decolorization efficiency may not be good as the specific activity of lignin peroxidase is very low.

In addition, the composition may further include glucose oxidase.

The glucose oxidase catalyzes the reaction of glucose and oxygen to produce gluconic acid and hydrogen peroxide, and the generated hydrogen peroxide may be used for a reaction with veratril alcohol catalyzed by the lignin peroxidase. At this time, the generation of hydrogen peroxide using glucose oxidase that catalyzes the reaction of glucose and oxygen can be continuously supplied to lignin peroxidase at low concentration, so that the melanin bleaching efficiency is very excellent, and accordingly, the skin whitening effect of the composition is It can be better.

The glucose oxidase may be derived from nature or may be obtained by various protein synthesis methods well known in the art. As an example, it may be prepared by using a polynucleotide recombination and protein expression system, or by a method of in vitro synthesis through chemical synthesis such as protein synthesis, a cell-free protein synthesis method, and the like. In addition, as an example, the glucose oxidase may be a product obtained by culturing a peptide, an extract of a plant-derived tissue or cell, or a microorganism (eg, bacteria or fungi, and particularly yeast).

The glucose oxidase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more of the amino acid sequence of glucose oxidase, respectively. % Or greater, about 98% or greater, or about 99% or greater sequence homology. Specifically, the glucose oxidase may be a glucose oxidase (GOx) derived from black mold (Aspergillus niger ), and more specifically, may be a protein consisting of the amino acid sequence of SEQ ID NO: 2.

In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, titer, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), reduced antigenicity, the N of the glucose oxidase -Or a protecting group may be bonded to the C-terminus. The protecting group may be an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or a polyethylene glycol (PEG), but the modification of the glucose oxidase, especially the stability of glucose oxidase Any ingredient that can be promoted may be included without limitation.

The term "stability" may mean not only in vivo stability that protects the glucose oxidase from attack by protein cleavage enzymes in vivo, but also storage stability (eg, room temperature storage stability).

In addition, the glucose oxidase may additionally include a targeting sequence, a tag, and an amino acid sequence prepared for a specific purpose for a labeled residue.

The concentration in the composition of the glucose oxidase is 0.2 U/ml to 0.3 U/ml, 0.2 U/ml to 0.275 U/ml, 0.225 U/ml to 0.3 U/ml, 0.225 U/ml to 0.275 U/ml, 0.24 U/ml to 0.3 U/ml, 0.24 U/ml to 0.275 U/ml, 0.24 U/ml to 0.26 U/ml, 0.2 U/ml to 0.26 U/ml, 0.225 U/ml to 0.26 U/ml have. The melanin bleaching efficiency may be excellent within the range of the glucose oxidase.

In addition, the glucose may be glucose present in or generated in the subject to which the composition is to be applied, but may be included in the composition.

The term "individual" refers to all organisms such as mice, mice, livestock, cells, including humans, which are capable of possessing melanin. As a specific example, it may be a mammal, including humans, and includes not only in vivo but also in vitro conditions.

When the glucose is included in the composition, the concentration in the composition is 100 mM to 500 mM, 100 mM to 400 mM, 200 mM to 500 mM, 200 mM to 400 mM, 100 mM to 350 mM, 200 mM to 350 mM, 250 mM to 350 mM, 250 mM to 500 mM, 250 mM to 400 mM. The melanin bleaching efficiency may be excellent within the concentration range of glucose.

In addition, the composition may further include _{oxygen (O 2) in addition to the glucose peroxidase.} The glucose and glucose oxidase of the composition may react with oxygen in the air or outside to produce hydrogen peroxide, and may react with oxygen in the composition to produce hydrogen peroxide.

In addition, the cosmetic composition may additionally include components conventionally added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and conventional adjuvants and carriers such as fragrances.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils , Powder foundation, emulsion foundation, wax foundation, and spray may be formulated, but are not limited thereto. More specifically, lotions such as flexible lotion or nutrient lotion, emulsions such as facial lotion and body lotion, creams such as nutritional cream, moisture cream, eye cream, etc., essence, cosmetic ointment, spray, gel, pack, sunscreen, make-up It can be prepared in a formulation such as a foundation, a powder, such as a base, a liquid type, a solid type, or a spray type. In this case, the cosmetic composition is an emulsifier, a soap acid, a solvent, a colorant, a preservative, an antioxidant, an antifoaming agent, an antibacterial agent, an anti-redeposition agent, an enzyme, a plant or mineral oil, a fat, a fluorescent substance, a fungicide, a hydrophobic substance, a moisturizer, It may contain excipients including fragrances, fragrance carriers, preservatives, proteins, silicones, solubilizers, sugar derivatives, sunscreen agents, vitamins, plant extracts, waxes, and the like.

When the formulation of the cosmetic composition is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide, etc. are used as carrier components. Can be used.

When the formulation of the cosmetic composition is a powder or spray, toss, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, Propellants such as propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like may be used.

Another aspect provides a method of depigmenting melanin in skin comprising applying the composition to the skin of a subject.

The term “application” includes methods of administration or application to the skin of a subject, such as application by finger, brush, cotton ball, pad, spray, sponge, cotton swab, roll-on, and the like. A person skilled in the art can readily determine the appropriate method to apply.

The composition may be a composition within the above-described range, and through application of the composition, the skin whitening effect of the applied individual may be achieved through decolorization of melanin in the skin as described above.

Another aspect provides a method for depigmenting melanin in skin comprising applying lignin peroxidase, veratrile alcohol, and hydrogen peroxide to the skin of an individual.

The "lignin peroxidase", "veratryl alcohol", "hydrogen peroxide" and the like may be within the above-described range.

In the method of bleaching melanin in the skin, the application of the hydrogen peroxide may be applying a hydrogen peroxide solution of 80 μM to 300 μM.

The hydrogen peroxide solution refers to a solution containing hydrogen peroxide as a solute, and the solvent of the solution is not limited, but may be water, specifically distilled water, and more specifically tertiary distilled water.

The concentration of the hydrogen peroxide solution is 80 μM to 300 μM, 100 μM to 300 μM, 80 μM to 250 μM, 100 μM to 250 μM, 80 μM to 200 μM, 100 μM to 200 μM, 150 μM to 200 μM, 150 μM To 300 μM, 150 μM to 250 μM.

In addition, in the method of bleaching melanin in the skin, the application of the hydrogen peroxide may be to apply a hydrogen peroxide solution at intervals of 1 minute to 90 minutes, specifically 1 minute to 90 minutes, 5 minutes to 90 minutes, 1 minute It may be made at intervals of to 60 minutes, 5 minutes to 60 minutes, 1 minute to 30 minutes, 5 minutes to 30 minutes, 10 minutes to 30 minutes, 10 minutes to 90 minutes, and 10 minutes to 60 minutes.

In addition, in the method of bleaching melanin in the skin, the application of the hydrogen peroxide may be the application of the hydrogen peroxide solution 1 to 30 times, specifically 1 to 30 times, 1 to 25 times, It may be applied 5 to 30 times, 5 to 25 times, 1 to 20 times, 5 to 20 times, 10 to 20 times, 10 to 30 times, 10 to 25 times.

As described above, lignin peroxidase catalyzes the reaction of veratryl alcohol and hydrogen peroxide to produce veratryl alcohol cation radicals and water. The veratryl alcohol cation radical may decolorize melanin by acting with melanin in the skin. Through this, it is possible to achieve the skin whitening effect of the applied object.

A cosmetic composition for skin whitening containing lignin peroxidase has very few skin side effects, selectively bleaching melanin, and excellent melanin bleaching efficiency. Further, in the case of a composition containing glucose oxidase, since the decolorization efficiency of melanin is further enhanced through continuous supply of low concentration hydrogen peroxide, the composition can be used for skin whitening.

In addition, the method of decolorizing melanin in the skin using the composition also selectively decolorizes melanin, and its efficiency is excellent, so that the skin whitening effect of the applied individual can be achieved.

1 is a diagram illustrating the mechanism of melanin decolorization by lignin peroxidase, (a) is a diagram showing the melanin decolorization mechanism in which lignin peroxidase catalyzes a catalytic action using VA cationic radicals, which are redox mediators, (b) Is a diagram showing the mechanism of melanin decolorization catalyzed by lignin peroxidase _{with H 2} O ₂ generated by glucose oxidase.
2 is a diagram showing the pH-dependent melanin bleaching efficiency and specific activity of LiPH8 using veratryl alcohol.
3 is a diagram showing the effect of _{H 2} O ₂ concentration on the bleaching efficiency of melanin and the activity of LiPH8 for veratryl alcohol.
4 is a diagram showing a melanin decolorization reaction occurring _{by intermittently adding H 2} O _2.
5 is a diagram showing the melanin decolorization efficiency over time by LiPH catalyzed with _{GO X.}

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1. Production and separation and purification of recombinant lignin peroxidase

The synthetic gene encoding the lignin peroxidase isozyme H8 (Lignin Peroxidase isozyme H8: LiPH8, SEQ ID NO: 1) derived from Phanerochaete chrysosporium was prepared by Bioneer (Korea) (gene and protein base sequences are UniPortKB. entry: shown in P06181). The refolding and purification method applied mutatis mutandis to the previously published paper (WA Doyle, AT Smith, Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca ²⁺ and haem, Biochem J 315(1). (1996) 15-19.).

Example 2. Determination of optimal pH conditions for lignin peroxidase activity

Lignin peroxidase oxidizes Veratryl Alcohol (VA) to VA cationic radicals (VA · ⁺ ), which acts as a redox mediator to decolorize melanin (Fig. 1(a)). Since VA is a natural compound produced from white rot fungi and has fewer potential side effects than synthetic mediators, lignin peroxidase using VA was used, and in this example, the pH effect on LiPH8 catalyzed decolorization of melanin was investigated.

Specifically, melanin bleaching was performed for 1 hour in 50 mg/L synthetic melanin, 2 mM veratryl alcohol, 250 μM H ₂ O ₂ and 0.06 U/ml LiPH8 BR buffer, and the enzyme activity was 2 mM veratryl alcohol, BR Measurements were made using 250 μM H ₂ O _{2 in buffer.}

As a result, LiPH8 showed the highest specific activity for VA under acidic conditions (pH 3.0) as shown in FIG. 2. However, melanin decolorization did not occur in the pH 3 buffer solution, and melanin precipitation appeared. In addition, the formation of these melanin precipitates was accelerated by the addition of enzymes. This means that the formation of a complex between the solid melanin and the enzyme took place. Therefore, it was found that direct contact between melanin and a redox mediator is important for proper decolorization of melanin. Since VA cation radicals, which are redox mediators, have a very short lifespan, it was found that melanin decolorization does not occur if there is no direct contact between melanin and VA cation radicals.

In addition, LiPH8 at pH 4.0 showed the highest melanin bleaching efficiency of 30% or more within 1 hour, and LiPH8 decreased melanin bleaching efficiency at pH 5.0 and 6.0, whereas the specific activity of LiPH8 was significantly reduced compared to pH 4.0. Could. In conclusion, it was found that the melanin decolorization efficiency of LiPH8 was highest at pH 4.0, and all subsequent melanin decolorization reactions were performed in pH 4.0 buffer.

Example 3. Determination of Optimal Hydrogen Peroxide Concentration and Injection Method

As shown in Fig. 1(a), lignin peroxidase catalyzes the reaction of VA and hydrogen peroxide to form water and VA cation radicals. Therefore, the melanin decolorization reaction of lignin peroxidase requires hydrogen peroxide, but lignin peroxidase is easily inactivated by _{excessive concentration of H 2} O _{2 through rupture of heme structure or formation of inactive compound III.} Accordingly, this example was carried out to determine the optimal concentration of hydrogen peroxide, which is a substrate of lignin peroxidase for obtaining high efficiency of melanin decolorization reaction.

Specifically, melanin bleaching was performed for 1 hour in 50 mg/L synthetic melanin, 2 mM veratryl alcohol, and 0.06 U/ml LiPH8 BR buffer (pH 4.0), and the activity of the enzyme was 2 mM in BR buffer (pH 3.0). It was measured with veratryl alcohol.

As a result, although H ₂ O ₂ is an essential substrate as the final electron acceptor during melanin decolorization, the melanin decolorization efficiency gradually decreases with increasing _{H 2} O ₂ concentration, and more than 30% in _{250 μM H 2} O ₂ It was close to the highest level. However, the initial specific activity for VA _{steadily increased as the H 2} O ₂ concentration increased (FIG. 3).

This means that H ₂ O ₂ is an important substrate for short-term enzyme activity (within 1 minute), but inhibited enzyme activity during long-term reaction (within 1 hour). For high melanin bleaching efficiency, although it can act as an enzyme inhibitor at the same time paradoxically, a high concentration of H ₂ O ₂ is required, so it was possible to conceive of a continuous supply of _{low concentration H 2} O ₂ that improves the overall melamine bleaching efficiency. .

Intermittent addition of _{H 2} O ₂ was performed to confirm whether maintenance of low level H ₂ O ₂ by continuous H ₂ O ₂ supply can improve melanin decolorization efficiency. Based on the results shown in FIG. 3, _{the critical concentration of H 2} O ₂ capable of inactivating LiPH8 was assumed to be 300 to 350 μM. Specifically, melanin decolorization was performed with 50 mg/L of synthetic melanin, 2 mM veratryl alcohol, and 0.06 U/ml of LiPH8 in BR buffer at pH 4.0.

Total H ₂ O ₂ was evenly divided and supplied at intervals of 60, 30, 20, 15 and 12 minutes within 1 hour, specifically, based on a total reaction volume of 2 ml, a total of 10 μl of a hydrogen peroxide solution was supplied. The hydrogen peroxide solution was used by diluting 30% hydrogen peroxide (9.8 M) in tertiary distilled water. The hydrogen peroxide solution was prepared in tertiary distilled water at 200 times the reaction concentration (240 mM in the case of 1200 μM). The volume of hydrogen peroxide supplied per time is different for each supply time interval.In the case of 60 minutes, it is supplied once, so 10 μl is supplied per time, and in the case of 30 minutes, it is supplied twice, so it is supplied 5 μl per time, and in the case of 20 minute intervals, it is supplied 3 times. Therefore, 3.33 μl was supplied per once, and 2.5 μl was supplied four times in the case of 15-minute intervals, and 2 μl was supplied per time because it was supplied five times in the case of 12-minute intervals.

As a result, when the H ₂ O ₂ concentration was 1400 μM and the time interval was 12 minutes _{, the inhibitory effect was observed even though the instant H 2} O ₂ concentration was only 280 μM (FIG. 4). It was thought that the LiPH8 added in this example was not sufficient to consume _{280 μM of H 2} O _{2 within 12 minutes.} Nevertheless, melanin decolorization efficiency reached 70% saturated at about 1000 μM and 1200 μM H ₂ O _{2 , so even though 280 μM of H 2} O ₂ was consumed per 12 minutes, it was not efficient to add more enzymes. Except for this, _{the instantaneous concentration of H 2} O ₂ less than 300 μM did not show any inhibitory effect on melanin decolorization.

This indicated that intermittent supply _{of H 2} O ₂ at regular time intervals was very effective in improving melanin pigment bleaching by inhibiting the inactivation of LiPH8 due to _{excessive H 2} O _2. However, in the case of whitening cosmetics, it is almost impossible to supply _{H 2} O ₂ intermittently according to time intervals, which means that it is necessary to continuously and simultaneously generate _{low concentration H 2} O _{2 for LiPH8.}

Example 4. Determination of optimal glucose oxidase activity for hydrogen peroxide production by glucose oxidase (GOx)

In this example, a black mold producing _{D-gluconic acid and H 2} O ₂ by catalyzing the oxidation of β-D-glucose by using the _{molecule O 2} for continuous in-situ generation _{of low concentration H 2} O _{2 (} Aspergillus niger ) derived glucose oxidase (Glucose Oxidase: GOx, Sigma Aldrich, #G6766, SEQ ID NO: 2) (gene and protein nucleotide sequences are shown in UniPortKB entry: P13006) was used. The generated H ₂ O ₂ was used by lignin peroxidase to decolor melanin as an electron acceptor (Fig. 1(b)).

Specifically, melanin decolorization was performed with 50 mg/L of synthetic melanin, 2 mM veratryl alcohol, and 0.06 U/ml of LiPH8 in BR buffer at pH 4.0.

As a result, GOx also retained its activity at pH 4.0-7.0, which means that it is applicable to melanin decolorization at pH 4.0. The optimal amount of GOx required was determined by the melanin bleaching efficiency in various GOx units as shown in Table 1. As the unit of GOx increased, the bleaching efficiency increased proportionally. However, without the addition of GOx, LiPH8 did not show any activity against melanin decolorization (Table 1). These results clearly indicate that _{GO X supplies H 2} O _2, which is essential for LiPH8 catalyzed melanin decolorization.

Glucose oxidase Unit (U/ml) Melanin decolorization efficiency (%) 0 0 0.03 31.94 ± 0.42 0.06 49.70 ± 0.63 0.12 57.01 ± 2.96 0.24 63.43 ± 1.06

Example 5. Determination of Optimal Glucose Concentration for Hydrogen Peroxide Production by Glucose Oxidase

Since the amount of H ₂ O ₂ produced depends on the glucose concentration used by the fixed GOx concentration, in this example, melanin decolorization was performed while changing the initial glucose concentration. Specifically, melanin decolorization was performed in the BR buffer solution at pH 4.0. Synthetic melanin 50 mg/L, veratryl alcohol 2 mM, LiPH8 0.06 U/ml and 0.24 U/ml glucose oxidase. In addition, since a high concentration of glucose can produce hydrogen peroxide at a high concentration, the glucose concentration was tested from 0 to 300 mM. Among the properties of the purchased glucose oxidase, the Km value was 30 to 100 mM, and it is known that the maximum activity is maintained when a substrate is added three times the Km value, so the test was performed up to the maximum value of 300 mM.

As a result, as the concentration of glucose increased, the melanin bleaching efficiency increased, and it was found to be saturated at a glucose concentration of about 100 mM (Table 2). However, at a very low glucose concentration of less than 1 mM, the melanin pigment removal efficiency was negative, which means that the color of the melanin pigment became dark due to insufficient oxidation of melanin. In addition, the optimal GOx concentration was 0.24 U/ml (Table 1), and it was found that the optimal glucose concentration was 100 mM to 300 mM (Table 2). This is because sufficient amounts of GOx and glucose did not cause other effects in melanin depigmentation.

Glucose concentration (mM) Melanin decolorization efficiency (%) 0 0 0.1 -2.35 ± 0.44 0.5 -0.78 ± 0.44 One -0.78 ± 0.44 5 21.35 ± 1.11 10 47.10 ± 0.67 20 57.30 ± 0.44 100 62.48 ± 0.22 300 62.17 ± 4.22

Example 6. Melanin bleaching according to reaction time using glucose oxidase and recombinant lignin peroxidase

In this example, melanin bleaching was performed at an optimal glucose oxidase and glucose concentration for melanin bleaching.

Specifically, melanin decolorization was performed with synthetic melanin 50 mg/L, veratryl alcohol 2 mM, LiPH8 0.06 U/ml, 300 mM β-D-glucose, and 0.24 U/ml glucose oxidase in BR buffer at pH 4.0.

As a result, the melanin decolorization efficiency was clearly brightened and gradually increased as the color of the melanin solution passed over time as shown in FIG. 5 (the photos in FIG. 5 are 0, 10, 20, 30, 40, 50 from the left. , Is a photo of the reaction solution after 60 minutes). A total of 40% melanin pigment removal efficiency was achieved within 20 minutes, and the overall efficiency reached about 60% in 1 hour. The enzyme showed sufficient activity to achieve 80% after 23 hours even though the rate of bleaching was slow because the melanin concentration was low after 20 minutes. This indicates that the production of H ₂ O _{2 by GO X} is very effective in maintaining the activity of LiPH8.

<110> ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY <120> COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING LIGNIN PEROXIDASE AND DECOLORIZING METHOD OF MELANIN IN SKIN USING THE SAME <130> UTP19140KR <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 372 <212> PRT <213> Phanerochaete chrysosporium <220> <221> MISC_FEATURE <222> (1)..(372) <223> Lignin Peroxidase isozyme H8 <400> 1 Met Ala Phe Lys Gln Leu Phe Ala Ala Ile Ser Leu Ala Leu Leu Leu 1 5 10 15 Ser Ala Ala Asn Ala Ala Ala Val Ile Glu Lys Arg Ala Thr Cys Ser 20 25 30 Asn Gly Lys Thr Val Gly Asp Ala Ser Cys Cys Ala Trp Phe Asp Val 35 40 45 Leu Asp Asp Ile Gln Gln Asn Leu Phe His Gly Gly Gln Cys Gly Ala 50 55 60 Glu Ala His Glu Ser Ile Arg Leu Val Phe His Asp Ser Ile Ala Ile 65 70 75 80 Ser Pro Ala Met Glu Ala Gln Gly Lys Phe Gly Gly Gly Gly Ala Asp 85 90 95 Gly Ser Ile Met Ile Phe Asp Asp Ile Glu Thr Ala Phe His Pro Asn 100 105 110 Ile Gly Leu Asp Glu Ile Val Lys Leu Gln Lys Pro Phe Val Gln Lys 115 120 125 His Gly Val Thr Pro Gly Asp Phe Ile Ala Phe Ala Gly Arg Val Ala 130 135 140 Leu Ser Asn Cys Pro Gly Ala Pro Gln Met Asn Phe Phe Thr Gly Arg 145 150 155 160 Ala Pro Ala Thr Gln Pro Ala Pro Asp Gly Leu Val Pro Glu Pro Phe 165 170 175 His Thr Val Asp Gln Ile Ile Asn Arg Val Asn Asp Ala Gly Glu Phe 180 185 190 Asp Glu Leu Glu Leu Val Trp Met Leu Ser Ala His Ser Val Ala Ala 195 200 205 Val Asn Asp Val Asp Pro Thr Val Gln Gly Leu Pro Phe Asp Ser Thr 210 215 220 Pro Gly Ile Phe Asp Ser Gln Phe Phe Val Glu Thr Gln Leu Arg Gly 225 230 235 240 Thr Ala Phe Pro Gly Ser Gly Gly Asn Gln Gly Glu Val Glu Ser Pro 245 250 255 Leu Pro Gly Glu Ile Arg Ile Gln Ser Asp His Thr Ile Ala Arg Asp 260 265 270 Ser Arg Thr Ala Cys Glu Trp Gln Ser Phe Val Asn Asn Gln Ser Lys 275 280 285 Leu Val Asp Asp Phe Gln Phe Ile Phe Leu Ala Leu Thr Gln Leu Gly 290 295 300 Gln Asp Pro Asn Ala Met Thr Asp Cys Ser Asp Val Ile Pro Gln Ser 305 310 315 320 Lys Pro Ile Pro Gly Asn Leu Pro Phe Ser Phe Phe Pro Ala Gly Lys 325 330 335 Thr Ile Lys Asp Val Glu Gln Ala Cys Ala Glu Thr Pro Phe Pro Thr 340 345 350 Leu Thr Thr Leu Pro Gly Pro Glu Thr Ser Val Gln Arg Ile Pro Pro 355 360 365 Pro Pro Gly Ala 370 <210> 2 <211> 605 <212> PRT <213> Aspergillus niger <220> <221> MISC_FEATURE <222> (1)..(605) <223> Glucose Oxidase <400> 2 Met Gln Thr Leu Leu Val Ser Ser Leu Val Val Ser Leu Ala Ala Ala 1 5 10 15 Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr 20 25 30 Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly 35 40 45 Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro 50 55 60 Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg 65 70 75 80 Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser 85 90 95 Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln 100 105 110 Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val 115 120 125 Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp 130 135 140 Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala 145 150 155 160 Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile 165 170 175 Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr 180 185 190 Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val 195 200 205 Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys 210 215 220 Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr 225 230 235 240 Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu 245 250 255 Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val 260 265 270 Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly 275 280 285 Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys 290 295 300 His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu 305 310 315 320 Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile 325 330 335 Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr 340 345 350 Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly 355 360 365 Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser 370 375 380 Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu 385 390 395 400 Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile 405 410 415 Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr 420 425 430 Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp 435 440 445 Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp 450 455 460 Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu 465 470 475 480 Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile 485 490 495 Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro 500 505 510 Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr 515 520 525 Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser 530 535 540 Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val 545 550 555 560 Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr 565 570 575 Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys 580 585 590 Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln 595 600 605

Claims (16)

As a cosmetic composition for skin whitening comprising lignin peroxidase consisting of the amino acid sequence of SEQ ID NO: 1,
The pH of the composition is 3.9 to 4.1, a cosmetic composition for skin whitening. delete The cosmetic composition of claim 1, further comprising Veratryl Alcohol and hydrogen peroxide (H ₂ O _{2 ).} The method according to claim 3, wherein the concentration in the composition of the hydrogen peroxide is 200 μM to 300 μM, a cosmetic composition for skin whitening. delete The cosmetic composition for skin whitening of claim 1, further comprising glucose oxidase. The method according to claim 6, wherein the glucose oxidase is a protein consisting of the amino acid sequence of SEQ ID NO: 2, skin whitening cosmetic composition. The method according to claim 6, wherein the concentration in the composition of the glucose oxidase is 0.2 U / ml to 0.3 U / ml, the cosmetic composition for skin whitening. The cosmetic composition according to claim 6, further comprising glucose. The method of claim 9, wherein the concentration of the glucose in the composition is 100 mM to 500 mM, a cosmetic composition for skin whitening. The cosmetic composition of claim 6, _{further comprising oxygen (O 2 ).} A cosmetic method through depigmentation of melanin in the skin comprising the step of applying the composition of any one of claims 1, 3, 4, 6 to 11 to the skin of an individual. A cosmetic method through depigmentation of melanin in the skin comprising the step of applying lignin peroxidase consisting of the amino acid sequence of SEQ ID NO: 1 to the skin of an individual, veratrile alcohol, and hydrogen peroxide,
The application is made under the conditions of pH 3.9 to 4.1, a cosmetic method through decolorization of melanin in the skin. The method of claim 13, wherein the hydrogen peroxide is applied by applying a hydrogen peroxide solution of 80 μM to 300 μM. The method of claim 13, wherein the application of the hydrogen peroxide is to apply the hydrogen peroxide solution once to 30 times. The method of claim 13, wherein the application of the hydrogen peroxide is to apply the hydrogen peroxide solution at intervals of 1 minute to 90 minutes.

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