KR100329962B1 - a protease which degrades keratin layers of skin surface, a microorganism which produces the protease and a cosmetic composition containing a culture solution thereof. - Google Patents

Abstract

The present invention relates to a cosmetic composition containing a protease that degrades keratin, a component of a dead cell layer derived from human epidermal cells, a microorganism that produces it, and a protease-containing culture solution thereof.

The present inventors search for strains that produce enzymes that effectively break down keratin, a skin impurity, for use as a composition of cosmetics, and isolate microorganisms that produce new proteolytic enzymes different from those known to date from the soil, Bacillus sp. HB-5, Accession No. KCTC8959P), and the characteristics of the strain and the characteristics of the enzyme were examined, and it was found to have different characteristics from the existing production strains. The strain of the present invention has a single subunit having a molecular weight of 60,000 Daltons (Da) to produce one type of protease, the optimum pH of the reaction is around 11, the optimum temperature is in the range of 50 ℃, pH 8-12, 30 Stable up to -60 ℃ Production media include glucose, sucrose, fructose, starch, nitrogen as yeast extract, protein, phosphate, magnesium salt and human hair. The enzyme of the present invention is a serine proteolytic enzyme which loses enzymatic activity by an enzyme inhibitor such as PMSF, and is a new enzyme having a keratin substrate specificity and stable by a high molecular compound such as glycerin, 1,3-butylene glycol, Casein, gelatin, starch, etc. are decomposed and added to cosmetics, detergents, keratin can be used as a removal component.

Description

A protease which degrades keratin layers of skin surface, a microorganism which produces the protease and a cosmetic composition containing a culture solution .}

The present invention relates to a protease with excellent keratin degrading ability to effectively remove dead skin layer, which is a dead cell layer on the skin surface, and a microorganism producing the same.

Proteolytic enzymes are enzymes that act on proteins or amino acid-binding peptides to hydrolyze peptide bonds. They are those that recognize specific amino acid sequences of proteins with substrate specificity and those that recognize and act on three-dimensional structures of proteins. The degree and mechanism vary. Categorized by the difference of substrate specificity and substrate catalyst mechanism, serine protease is a group having serine residues in the active site and is a protease whose -OH group has a decisive effect on the activity. Proteolytic enzymes belonging to it include trypsin and chymotrypsin, such as animal origin protease, and microbial origin include subtilisin and alkaline protease produced by the genus Bacillus.

Sulfohydryl protease is a group having a -SH group in the active site, and these are mainly plant-proteolytic enzymes. For example, papain, bromelanin, ficin and the like are inhibited by oxidants and heavy metals. Metalloproteinases are a group whose activity is determined by the presence or absence of a metal prosthesis molecule bound to the enzyme. These are inhibited by complex salt forming agents such as EDTA and cyanide. Most of the metal ions found as the supporting molecular group are divalent cations such as Cu ²⁺ and Zn ²⁺ . Depending on the optimal pH, it is divided into acidic, neutral, and alkaline proteolytic enzymes. In addition, they are subdivided into collagenase, elastase, keratinase, and gelatinase according to their specific substrates.

Look out for the metabolism of the skin to remove dead skin cell layers of the stratum corneum of the skin surface and covering the whole body to play a role to protect the bio from the various stimulus or interference from the outside or dry adult is about 1.6m ² in the body It has an area of. The thickness of the skin varies according to age, sex, and area. In general, men's skin is thicker than women. In general, the skin around the eyes is very thin and the palms and soles are very thick. The skin can be divided into three layers of epidermis, dermis and subcutaneous tissue in order from the stomach. The most important physiological function of the epidermis is the formation of an appropriate envelope, the stratum corneum, which is a barrier against various external stimuli, such as drying, ultraviolet, and other physical and chemical stimuli. The epidermis is transformed into basal cells, polar cells, granular cells, and sequential morphological cells, and moves to the surface layer to become keratinocytes. This process of epidermal cell differentiation is called keratinization.

Although keratinocytes continue to be made in turn, they maintain a constant thickness because the outermost keratinocytes sometimes detach, and this new cell layer is always called "Turn Over". In normal skin, this epidermal turnover is known to take about four weeks. If the stratum corneum does not peel off normally and remains on the surface of the skin, the stratum corneum becomes thicker and the face looks dull and dark. Impurities that remain on the skin surface or hair follicles are oxidized or decomposed by oxygen or microorganisms, and these substances cause skin problems such as inflammation. Among these impurities, keratin is a major component of dead cells, which remain on the surface of the skin after the cell dies, and the proteins in the sweat remain on the surface after sweating.

Keratin is the most abundant intercellular fibrous linkage protein present in the epidermis, and about 20 kinds of keratin have been found in the human epidermis. Keratin has a high molecular weight and is insoluble because it is connected by cystine disulfide bonds. The keratin has a high molecular weight and is not easily removed by the detergent alone. These protein impurities can be effectively removed by small decomposition. Proteases are thought to be substances with this effect and have been reported to use a stable protease to remove the layer of keratin attached to the skin surface and make it softer (J.S.C.C. 27 (3), 1993).

With the recent emphasis on functional cosmetics, interest in direct cosmetic applications of enzymes produced by microorganisms is also increasing. However, most of the proteolytic enzymes used in cosmetics so far are papains derived from papaya extract, plant-derived proteolytic enzymes, and bromelain derived from pineapple extracts. There was no direct function in the product. Recently, α-hydroxy acid derived from fruit acid used for the purpose of brightening the skin has been restricted due to side effects in the skin due to low pH, unsafe upon exposure to ultraviolet rays, and safety. Currently, protease-producing microorganisms include genus Bacillus and actinomycetes. Proteolytic enzymes produced by these microorganisms are serine proteolytic enzymes, and since there are no cysteine residues readily oxidized in their enzyme activity sites, they can be used as cosmetic raw materials. It seems to be one.

Therefore, the present inventors, unlike the proteolytic enzymes known to date, isolates microorganisms that produce new proteolytic enzymes from the soil in terms of substrate specificity, identify them as Bacillus ( Bacillus sp. KCTC8959P), and examine the properties of the strain and the characteristics of the enzyme. As a result, they found that they had different characteristics from existing production strains, and based on this finding, the present invention was completed. In addition, the application of cosmetics can decompose skin aging tissues to promote the metabolism of the skin, the absorption of nutrients through the promotion of percutaneous absorption of cosmetics, and the removal of melanin by dissolving and removing the stratum corneum of the skin. The invention was completed.

Therefore, the object of the present invention is the Bacillus sp. KCTC8959P produced by Bacillus sp. KCTC8959P molecular weight 60,000 Daltons, pH 6-11 stable, 30-60 ℃ reaction temperature, is stable at 70 ℃ and casein, gelatin, gluten, hemoglobin It is to provide a protease having a keratin-degrading activity, protein and insoluble protein, such as indocyanine.

Another object of the present invention is to provide a microorganism producing the protease.

In addition, another object of the present invention is to provide a cosmetic composition containing the obtained protease-containing culture solution, in particular in the amount of 0.1 to 30% by weight based on the total weight of the composition after culturing the microorganism to remove the cells. have.

In addition, another object of the present invention is to provide a detergent composition containing the protease-containing culture solution obtained after culturing the microorganism to remove the cells.

In addition, another object of the present invention is to provide a cosmetic composition for exfoliation using the culture medium of the microorganism.

Figure 1 is a photo cultured in agar medium suspended casein as a substrate to select strains.

Figure 2 is an electron micrograph showing the morphological characteristics of the protease isolation strain.

Figure 3 shows the enzyme production according to the incubation time of the strain

4 is a diagram showing the results of anion exchange resin (DEAE-cellulose) for purifying the enzyme produced by the strain.

Figure 5 shows the results of gel column chromatography (Sephadex G-100 chromatography) for purification of the enzyme produced by the strain

Figure 6 is a sd es polyacrylamide gel electrophoresis of the purified enzyme protein

Figure 7 shows the keratin degradation pattern of the purified enzyme protein by SD poly acrylamide gel electrophoresis picture.

8 is a graph showing the effect and stability of pH on the reaction of purified enzyme protein

9 is a graph showing the effect on temperature and stability on the reaction of the purified enzyme protein.

Isolation of Strains with Enzyme Activity

The microorganism strain of the present invention is dilute with physiological saline to remove the keratin-degrading activity from the soil of chickens in Gangneung, Gangneung, Korea. After selecting an excellent strain, the casein and keratin degradation activity was measured to finally select a strain having excellent activity. (See Figure 1)

Identification of Strains

The strain of the present invention is a gram-positive rod-shaped bacilli that form spores as a type of bacteria, and exhibited the following characteristics (Table 1).

Analysis of Chemical Components of Strains: The results of analysis of cellular fatty acids of this strain are summarized in Table 2.

As a result of investigating the morphological characteristics of the strain, the strain of the present invention is Bacillus sp. As a microorganism having a morphological characteristic different from that of other protease-producing strains. Phosphate 1g, magnesium salt 0.2g, sodium chloride 0.5g, human hair 2.5g, yeast extract 10g (per 1 liter) and the pH of the medium was 7. The maximum enzyme production was obtained when the culture temperature was 37 ° C., the stirring rate was 150 rpm, and the culture was performed under aerobic conditions of 72 hours shaking culture.

Bacillus sp of the present invention. Proteolytic enzymes produced by HB-5 are enzymes with a single subunit of molecular weight of about 60,000 Daltons. The optimum reaction pH and temperature are pH 11 and 50 ° C.

The strain of the present invention and the protease produced by the strain will be described in more detail with reference to the following examples.

Example 1

When the strain of the present invention was incubated in the medium, the maximum enzyme was obtained at the incubation temperature of 37 ° C. for 72 hours. In order to measure enzyme activity, 5 ml of casein solution was preheated to 37 ° C. for 30 minutes, and then 1 ml of enzyme solution was added and reacted at 37 ° C. for 10 minutes, and then 5 ml of prepared TCA solution was added to stop the reaction. After standing at room temperature for 30 minutes, centrifugation was performed at 12,000 g, 1 ml of supernatant, 5 ml of sodium carbonate solution, and 1 ml of polyline reagent were added to stabilize color development at 38 ° C. for about 30 minutes, and then absorbance was measured at 660 nm using a spectrophotometer. The activity is calculated by subtracting the blank from the measured absorbance of each sample and converting it to the amount of free tyrosine using the standard curve. Enzyme titer was defined as the amount of enzyme required to release 1 ug of tyrosine in 1.0 ml of reaction solution per minute from casein under the above conditions.

In order to measure the keratin degradation activity, 0.8 ml of substrate solution was preheated to 37 ° C. for 30 minutes, and then 0.2 ml of enzyme solution was added thereto and reacted at 37 ° C. for 1 hour, followed by addition of 0.2 ml of prepared TCAsolution. After standing at room temperature for 30 minutes, centrifugation was performed at 12,000 g, and then 1 ml of the supernatant was taken and the absorbance at 595 nm was measured using a spectrophotometer.

Example 2

Bacillus sp. Of the present invention as the optimum enzyme production culture conditions. HB-5 was incubated. 3 shows the results of observing the growth of the strain, enzyme production and pH change over time. As can be seen in Figure 3, the growth of the fungi increased up to 60 hours after inoculation and entered the stationary phase, and the production of enzymes started to increase after about 48 hours of incubation and maximum activity (72 U / ml) when incubated for 72 hours. After that, the activity was decreased. The pH change of the medium also increased from the initial 7 to 8.2.

Example 3

In order to separate the enzyme from 2 L of culture medium of the optimum enzyme production medium, the filter was filtered with 0.45 um filter paper (Wattman), and the culture solution was concentrated using an ultrafiltration system (membrane ultrafiltraion system) having a molecular weight of 10,000 Daltons or more. Anion exchange resin (DEAE-cellulose, Sigma, see Figure 4), gel column chromatography (Sephadex-G100 Chromatography, Sigma, see Figure 5). Purification As a result, the enzyme was separated into one enzyme on gel column chromatography. As a result of confirming the characteristics of the enzyme, the enzyme was identified as SDS-polyacylamide gel electrophoresis (SDS-polyacylamide gel electrophoresis) having a single subunit (molecular weight) of about 60,000 Daltons. The experimental results are shown in FIG.

Example 4

In order to investigate the effect of enzyme activity by the inhibitor of this protease, perform the same procedure as in enzyme activity assay, but dissolve each inhibitor to the corresponding concentration in the reaction solution to which the substrate is added, and then react with the enzyme for 15 minutes at 37 ° C. The remaining enzyme activity was measured. This enzyme was confirmed to be a serine protease because the enzyme activity was strongly inhibited by PMSF, an inhibitor specifically acting on serine protease (see Table 3).

PMSF: Phenylmethylsulfonyl fluoride NEM: N-ethylmaleimide

EDTA: Ethylenedimethyltetraacetic acid

In addition, the pH stability of the protease was measured after 2 hours of 25 ℃ after mixing the same amount of the reaction solution and enzyme of several pH, the pH which shows the optimum enzyme activity is 11. This result is shown in FIG. Stability for temperature was measured by mixing the same amount of the reaction solution and the enzyme at the optimum pH, and then treated for 30 minutes at each reaction temperature of 30-70 ℃, the temperature indicating the optimum enzyme activity was 50 ℃. This result is shown in FIG.

Example 5

Enzyme stability experiments were carried out on 50% glycerin, 50% 1,3-butylene glycol, 50% propylene glycol, 1,3-butylene glycol: propylene glycol 30: 20%) was added to the added reaction solution, and the remaining enzyme activity was measured over time. At the time of addition of 50% glycerin, more than 80% of the total activity remained over 20 days. The results are shown in Table 4.

In addition, in the stability test of enzymes for surfactants, the enzyme solution was added to the reaction solution containing 1% of surfactant and 18% of 1,3-butylene glycol, and the remaining enzyme activity over time. Measure After 1 day, the enzyme activity remained over 90%, but after 2 days, the enzyme activity was lost. The results are shown in Table 5.

Example 6

In order to examine the substrate specificity of the protease, human epithelial cell-derived keratin-based substrates were identified by SDS-polyacylamide gel electrophoresis. Human epithelial cell derived keratin has four polypeptides with a molecular weight of 73, 56, 51, 45 kD. After 10 minutes of reaction with the enzyme solution, four major peptides of 35, 33, 30, and 28 kD are newly formed. After 20 minutes of reaction, the original keratin disappears and a peptide smaller than 15 kD is produced. After 30 minutes of reaction, almost all keratin and their fragments disappear. These results indicate that the enzyme effectively hydrolyzes keratin. This result is shown in FIG.

Example 7

The composition (A), (B) of Formulation Example 1 and the same composition as Formulation Example 1 (A), 2% papain in Comparative Example 1 instead of proteolytic enzyme, 2% or less added to the comparative formulation 2% and A milk lotion containing Comparative Example 3 was prepared and tested for improving skin color of the cosmetic. After artificial tanning using a cosmetic gel containing DHA, the color difference of the skin color before and after application of the milk lotion containing the protease culture against a panel of 18-30 year old female test agents was applied. Skin-Turn Over (%) was obtained. When the cosmetic composition according to the present invention is applied to the skin, it can be seen that it exhibits an excellent skin color improvement effect. The results are shown in Table 6.

Preparation Method: The raw material 1-4 is heated to 80 ℃ and stirred to form an aqueous solution. The raw material 5-9 is stirred at 80 ° C. to form an oily solution, and then mixed with an aqueous solution to emulsify to form a particle-like solution of oily components. The mixture was cooled to 45 ° C., and raw material 10 and raw material 11-13 were mixed to prepare a milk lotion containing a protease culture solution and a comparative example.

Hereinafter, it can be seen from the results of Table 6 that the cosmetic composition according to the present invention exhibits an excellent skin color improving effect even when applied to the skin.

Example 8

In order to measure the skin cosmetic effect of the cosmetic composition prepared in Formulation Example 1, a panel of 18-30 year-old female inspection agents was used twice a day, that is, evenly applied to the face after morning and evening face wash. The skin care effect was evaluated by the 5-point evaluation method. The results are shown in Table 7. The larger the number, the better and satisfactory effect. The lower the number, the poorer the effect. It can be seen from the results of Table 7 that the cosmetic composition according to the present invention exhibits excellent skin cosmetic effects even when applied to the skin.

As described above, the microorganism according to the present invention produces new proteolytic enzymes in physical and chemical terms from the proteolytic enzymes known to date, and this degrading enzyme decomposes casein, gelatin, starch, etc. Since it specifically decomposes keratin, it can be used as an ingredient that effectively removes keratin by adding it to cosmetics and detergents.

Claims (7)

Bacillus sp. HB-5 (Bcillus sp. HB-5) KCTC 8959P microorganism having the form of FIG. 1 and producing protease, particularly keratinase The microorganism of claim 1 produces 60,000 Daltons in molecular weight, optimum activity around pH 11, pH 8-11 stability, optimum reaction temperature of 50 ° C, reaction temperature of 30-60 ° C, and casein, gelatin, gluten, hemoglobin, India Proteolytic enzymes having keratin-degrading activity which are proteins such as cyanine and insoluble proteins. From the culture of the microorganism of claim 1, it is stable even at a molecular weight of 60,000 Daltons, near optimum pH of 11, pH 8-11, optimal reaction temperature of 50 ° C, reaction temperature of 30-60 ° C, and casein, gelatin, gluten, hemoglobin and delivery time. A method for separating proteases with keratin degrading activity, which are non- and insoluble proteins. A cosmetic composition comprising the protease of claim 2 as an active ingredient. The cosmetic composition according to claim 4, comprising 0.1-30% by weight of the protease-containing culture solution obtained after culturing the microorganism of claim 1 to remove the cells. The cosmetic composition according to claim 4 or 5, which is particularly intended for exfoliation. A detergent composition comprising the enzyme of claim 2 as an active ingredient.