KR20030055442A - Stabilized enzyme producing method using a polymer and a composition contained these enzyme - Google Patents

Abstract

PURPOSE: A process for preparing a stabilized enzyme using polysaccharides and a composition containing the same stabilized enzyme are provided, thereby stabilizing enzymes to be used in cosmetics or medicines. CONSTITUTION: A process for preparing a stabilized enzyme using polysaccharides comprises the steps of: purifying natural polysaccharides having linear chain backbone with simple side chains; oxidizing the purified polysaccharides with NaIO4; reacting the modified polysaccharides with an enzyme; and separating and purifying the reaction product, wherein the saccharides used is xanthan gum, gum guar or alginate; the enzyme is papain, bromelain, lysozyme and cellulase; and the weight ratio of the enzyme to polysaccharides is 1: 2 to 1:7. A composition containing the stabilized enzyme is prepared by the addition of the stabilized enzyme into cosmetics, functional drinks or medicament for prevention or treatment of diseases.

Description

Stabilized enzyme producing method using a polymer and a composition contained these enzyme}

The present invention relates to a method for preparing a stabilizing enzyme using a polysaccharide and to a composition to which these stabilizing enzymes are added, and more particularly, to entrap the enzyme using xanthan gum, guar gum, and alginate to enzymatically protect the environmental and Prevents changes due to chemical factors, and also relates to the use of the enzyme protein produced therein applied to cosmetics and the like.

Conventionally, cosmetics have been added with various enzymes according to their purpose of use, for example, with the common skin exfoliation ability of papain, bromelain, lysozyme and cellulase. Bromelain has the function of anti-inflammatory agent, so it can be expected to improve the skin. Lysozyme and cellulase destroy microbial contamination by destroying the cell wall of bacteria, and harmful microorganisms ( Staphylococcus aureus) on the skin , Propionibacterium acnes ) can control the growth.

By the way, the enzyme added to cosmetics for the above purpose is composed of a protein, so that its stability is extremely low, such as being easily decomposed and denatured by temperature, chemicals, etc., and thus is difficult to use. Therefore, various peripheral factors such as cosmetic composition, storage and distribution temperature of cosmetics may inhibit the stability of the enzyme and thereby decompose or deactivate the added enzyme in a short time, thereby not exhibiting any function of the enzyme. .

Therefore, it is very important to apply the product to the product by removing the environmental and chemical instability of the enzyme, and furthermore, the technology to increase the added value of the product by providing various effects on the formulation and function of the product is not only in the cosmetics industry, but also in the pharmaceutical field. It is an important technique that can be used effectively.

In response to these demands, various attempts have been made to remove enzyme instability depending on storage temperature and ambient conditions. The method of stabilizing the enzyme using a polysucrose called Ficoll 400 is P.V. Reported by Sundaram et al. (Protein Engineering, vol. 8, no. 10, 1039-1047, 1995). This method is also described by SC-glucan (Pacific, Korea) reported by Sim et al. (Biotechnology letter, 22: 137-140, 2000). The enzyme was stabilized by a method similar to that of Sundaram, and the present inventors also attempted to denature nonionic polysaccharides produced by lactic acid bacteria having a specific structure of an alternant to increase the stability of the enzyme (Korean Patent Application No. 2001-45929). I've done it. However, there are not many polysaccharides that can be used for stabilizing the enzymes according to the conventional methods, and there have been very expensive problems to date.

As described above, a technique for stabilizing enzymes to maintain their long-term enzyme activity by increasing their stability in cosmetics such as creams and lotions is very necessary and important, and is applicable to enzymes having various properties using polysaccharides. Adding to a product with a variety of functionalities is an important technology, but the production method of the stabilizing enzyme having a satisfactory result in terms of technical or economic has not yet been provided.

Meanwhile, xanthan gum, gum guar gum, and alginate, which have been conventionally added to skin cosmetics, increase the stability of cosmetic formulations due to their viscosity and properties, and have a function of coating the skin when applied to the skin. Improves skin moisturizing power. These polysaccharides have a regular linear backbone and side chains of monosaccharides, so that if these enzymes can be stabilized using these polysaccharides, the stability of the formulation of the product to which these stabilized enzymes are added Of course, the moisturizing function and the skin's epidermis, which is the main function of the enzyme, can be obtained at the same time, such as the prevention of wrinkles and the removal of harmful microorganisms on the skin.

Accordingly, the object of the present invention is to solve the problems of the prior art as described above, so that the enzyme maintains its structural stability for a long time even under conditions that are unfavorable for maintaining the activity of enzymes such as cosmetics and functional beverages, and the characteristics of each enzyme are used in the formulation of the product. It is to provide a method for preparing a stabilizing enzyme that can be represented well in.

In addition, the present invention is to find a polysaccharide having a lower cost and superior characteristics than a conventional enzyme entrapping agent to provide a method for easily preparing a large amount of enzyme stabilizing agent, so that the activity of the enzyme is long-term storage. An object of the present invention is to provide a manufacturing method which can supply a stabilized enzyme to be maintained at a low cost.

In particular, it is an object of the present invention to provide a method for producing a stabilizing enzyme-containing cosmetic that can maintain the stability of the enzyme appropriately even under environmental conditions or circulation conditions that are disadvantageous for maintaining the enzyme added to the cosmetics.

As described above, the present invention is to provide a method that can more easily increase the stability of the enzyme added to cosmetics, functional drinks, etc. The present inventors, such as the polysaccharide added to a specific polysaccharide added to cosmetics, functional drinks, etc. The present invention provides a method for preparing a stabilizing enzyme that can maintain long-term activity at room temperature by selecting and purifying appropriately to be used for enzyme stabilization, treating with a specific method, and then reacting with the enzyme to further enhance stability of the enzyme. Was completed.

1 is a photograph confirming the increase in molecular weight of the stabilizing enzyme prepared according to the present invention,

2 is a graph showing the molecular weight fractionation enzymaticity of the stabilizing enzyme prepared according to the present invention,

3 is a graph showing the skin exfoliation activity of the stabilizing enzyme prepared according to the present invention,

4 is a graph showing the growth inhibitory activity of harmful microorganisms of the stabilizing enzyme prepared according to the present invention.

In order to achieve the above object, the present invention provides a relatively uncomplicated side chain in a structurally straight backbone among polysaccharides used in the cosmetic industry in enzyme entrapping for stabilizing enzymes. The natural polysaccharide having the structure is purified to be suitable for use in enzyme stabilization, chemical modification (oxidation) with sodium periodate (NaIO ₄ ), and the product obtained by reacting with the enzyme is separated and purified according to the molecular weight. Is characterized by obtaining a stabilized enzyme encapsulation formulation.

In the above constitution, a polysaccharide which can be used as an enzyme encapsulation agent is a xanthan as a polysaccharide capable of stabilizing the enzyme in a range that does not inhibit the activity of the enzyme except for a polysaccharide which prevents the enzyme activity from appearing in the enzyme-polysaccharide bond. Xanthan gum, gum guar, arginate and the like are preferred.

In addition, in the above configuration, the enzyme that can be preferably encompassed in the polysaccharide used as the enzyme encapsulation agent is characterized in that papain (broin), bromelain (lyezyme), cellulase (cellulase).

Furthermore, the present invention is characterized in that the enzyme stabilizing agent prepared as described above is added to cosmetics, functional beverages, and therapeutic or prophylactic agents under conditions to which enzymes are normally added to sustain the activity of the enzyme for a longer time.

As described above, the enzyme bound to the polysaccharide is entrapped in the form of the enzyme bound to the polysaccharide so that the enzyme is not decomposed or inactivated even under conditions such as a temperature of room temperature and other chemical compositions.

Furthermore, in the above configuration, since xanthan gum and guar gum are insoluble, it is preferable to remove them by a series of precipitation processes after reaction with primary sodium periodate (NaIO ₄ ) to obtain a water-soluble polysaccharide, When the polysaccharide is reacted with the enzyme according to the structure of the present invention, not only the enzyme can be stabilized, but also the cosmetics can be added to cosmetics by maintaining the viscosity of the cosmetics and improving the colloidal stability and moisturizing function.

Meanwhile, in the present invention, the activity of papain and bromelain, which are proteolytic enzymes, is measured by N, N-dimethylated casein (Sigma) and Nα-benzoyl-DL-arginine-ρ-nitroanilide (BAPNA). After the reaction for a certain period of time using a Sigma company), absorbance was measured at 280 nm for N, N-dimethylated casein and 405 nm for BAPNA using a spectrophotometer (CE 1020, manufactured by Cecil). Lysozyme, a lytic enzyme, is dispersed as a substrate in 0.1% Micrococcus lysodeikticus ( Sigma Co. , Ltd. ) in 0.05M, pH 6.2 buffer, and the substrate and enzyme solution are added to the enzyme solution in a ratio of 20: 1 at 25 ° C. The reaction was carried out for 30 minutes, and the absorbance was measured at 450 nm using a spectrophotometer. The activity of the enzyme was measured according to the decrease in absorbance. Cellulase activity measurement was performed by dissolving carboxymethyl cellulose (Sigma) in 0.05M, pH 6.2 buffer solution to 0.5%, adding enzyme solution and reacting at 37 ° C for 1 hour, followed by dinitrosalicylic acid ( The activity was confirmed by quantification by DNS) method.

The selected polysaccharide is purified, oxidized with sodium periodate (NaIO ₄ ), separated and purified, and a polysaccharide having a molecular weight of 100 K or more is reacted with an enzyme. Since xanthan gum and guar gum contain many insoluble substances, it is necessary to adjust conditions such as concentration of sodium iodide (NaIO ₄ ) and reaction time to remove these insoluble substances, obtain a water-soluble part, and use it for reaction with enzyme. More effective. The oxidized polysaccharide easily forms a covalent bond with the enzyme, and the enzyme is encapsulated in the polysaccharide to increase stability. The enzyme solution obtained in this manner was ultrafiltered with MWCO (molecular weight cut-off) 100,000 (Satorius AG, Germany) and washed with citrate buffer (0.1M, pH 6.0) of 10 times the enzyme solution volume. Remove less than 100,000 filtrates and recover only MW 100,000 or more. The increase in molecular weight due to the binding of the enzyme and the polysaccharide of the recovered enzyme complex can be confirmed by SDS-PAGE electrophoresis. The molecular weight of the enzymes used in the present invention are all MW 50,000 or less, papain is MW about 20,700, bromelain is about MW 33,000, lysozyme is about MW 14,307, cellulase was about MW 31,000, the enzyme solution treated according to the present invention, That is, it can be seen that the molecular weight of the enzyme stabilized by being encapsulated in the polysaccharide was greatly increased. In addition, when comparing the enzyme activity of the recovered enzyme solution with the activity of the enzyme added to the reaction, the production yield was calculated in consideration of the yield of Xanthan gum about 65%, gum guar about 52%, alginate about 76%. there was.

Hereinafter, the present invention will be described in more detail with examples and test examples, but the present invention is not limited thereto.

Example

Xanthan gum, guar gum, and alginate selected for use in this example were reacted with high sodium sodium iodide (NaIO ₄ ) at 25 ° C. for 5-16 hours, and not by ultrafiltration of MWCO 50,000. Remove the remaining NaIO ₄ and low molecular weight polysaccharide below MW 50,000, and separate the high molecular weight polysaccharide of MW 50,000 or more. The polysaccharide obtained in this manner is reacted with the enzyme at 25 ° C., and the ratio of the polysaccharide to the enzyme is added in a ratio of 1: 2 to 1: 7 depending on the enzyme. After reacting for 1 to 8 hours depending on the enzyme, sodium borohydride (NaBH ₄ ) is treated at 25 ° C. for about 30 minutes, and after a certain time, the enzyme is stabilized by leaving it at 4 ° C. for a long time.

The enzyme polysaccharide reaction solution obtained in this manner is separated by an ultrafiltration method to recover a fraction of molecular weight of 100,000 (MW 100,000), thereby separating the enzyme that is not bound to the stabilizing enzyme bound to the polysaccharide. Since the various enzymes used have a molecular weight of about MW 20,000 to 33,000, it is considered that the enzyme having a molecular weight of 100,000 (MW 100,000) or more is combined with a polysaccharide and the enzyme having a molecular weight of 100,000 (MW 100,000) or less is an enzyme that cannot be bound. More than 100,000 fractions were used in cosmetic formulations and the change in molecular weight was confirmed by electrophoresis (SDS-PAGE).

Electrophoresis (SDS-PAGE) to confirm the molecular weight change of the stabilizing enzyme used a separating gel containing 8% of acrylamide solution, which separated the protein of 20,000-90,000 KD at this concentration. This is because an enzyme with a changed molecular weight is expected to have very little movement since it is a suitable region for this (Ref: BioMedical Research, Yu-Joon Yoo). 4 × separating buffer and 4 × stacking buffer were used with 1.5M tris-Cl (pH 8.8) and 0.5M tris-Cl (pH 6.8), respectively, and the tank buffer composition was 0.025M tris-Cl, 0.192M glycine, 0.1% SDS, calibrated to pH 8.8. The composition of the 2 × SDS sample buffer is also 0.125M tris-Cl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 2% bromophenol blue. Two 1.5 mm gels were loaded and electrophoresed with a current of 60 mA and a voltage of 70 to 80 volts. As shown in FIG. 1, the stabilizing enzyme solution produced was analyzed by electrophoresis (SDS-PAGE) to confirm the increase in molecular weight. ①, ② and ③ in this figure is a result that can confirm that the molecular weight of the enzyme is increased by combining the enzyme and the polysaccharide, it can be confirmed that each enzyme is stabilized through this result. ④ is the molecular weight marker (maker), ⑤ is papain with a molecular weight of about 20,000 enzymes. As a result, the binding between the enzyme and the polysaccharide can be confirmed.

<Test Example 1>

Stabilization Enzyme Activity Test

Changes in the molecular weight of the material produced by the bond between the enzyme and the polysaccharide affect the reaction rate and all mechanical relationships of the enzyme. In order to confirm the approximate molecular weight of the stabilizing enzyme produced according to the above example, the following experiment was performed. First, a fraction of 100,000 or more molecular weight and a fraction of 100,000 or less molecular weight were separated by MWCO 100,000 ultrafiltration. At this time, two or more times 0.1M pH 6.0 citrate buffer (citrate buffer), and washed with a fraction of less than 100,000 molecular weight, this fraction was again fractional fraction of 50,000 or more and less than 10 and less than 50,000 by MWCO 50,000 ultrafiltration. To decompose. In addition, the molecular weight of the enzyme-polysaccharide obtained by investigating the enzymatic activity of each fraction was as shown in FIG.

Except for lysozyme, papain, bromelain, and cellulase were found to have the most activity in the fractions of 100,000 or more. This means that after the NaIO ₄ treatment of the polysaccharide, the polysaccharide having a molecular weight of 50,000 or less was removed, so that the binding between the enzyme and the polysaccharide was efficiently performed. Therefore, it can be judged that the stabilization of the enzyme was performed efficiently. However, lysozyme showed high enzymatic activity even in fractions of 50,000 to 100,000. This is due to the very low molecular weight of the lysozyme (about MW 14,407), but as can be seen by the electrophoresis analysis, it seems that most of the enzyme is combined with the polysaccharide.

<Test Example 2>

Skin exfoliation test by stabilizing enzyme

The dehydroxyacetone method (Dihydroxyacetine, GEPierard, Dermatology, 186, 133-137, 1993) was used to perform skin exfoliation test by stabilizing enzyme. The dihydroxyacetone method was used to remove dihydroxyacetone at a concentration of 10%. When added to the cream formulation and applied to the skin, keratin protein and dihydroxyacetone cause a maillard reaction, turning the skin to a dark brown color. This blackened skin becomes thinner and fades away as keratin is removed. This cycle is about 7-10 days, the same as the original skin. However, application of enzymes can shorten the cycle. Therefore, it can be confirmed by the naked eye to show the activity of the stabilized enzyme in the cosmetic formulation. Corneal was removed by applying twice a day from 1 day after the application of dihydroxy acetone, and the enzyme action time was 10 minutes.

In order to test whether the produced enzyme can maintain its activity on the cosmetic formulation, the comparison of the exfoliation efficacy by adding each stabilizing enzyme to the lotion formulation is shown in gray level. The enzymes used in the experiments were papain, lysozyme and cellulase. Skin Visiometer SV600 (CK electronics GmbH, Germany) and Sif＼gmascan pro 3.0 (Sigma) were used to quantify the exfoliation capacity. Co. Ltd), and the gray level was measured as shown in FIG. 3.

As shown in the figure, the color of the stabilizing enzyme-added group was thinner than the control group, and when analyzed with gray level, the curve of the stabilizing enzyme-added group was shifted to the right, that is, the white direction. Therefore, it was confirmed that the stabilizing enzyme-added group removes keratin at a faster rate, and it can be confirmed that the cosmetic formulation can stably exhibit exfoliation ability.

Protease, such as papain, has been reported to remove keratin by breaking down proteins on the surface of the skin, and lysozyme was treated with enzymes with high titer removal ability and relatively high titer given by the characteristics of lytic enzymes derived from egg whites. High exfoliation ability in the In addition, the cellulase has relatively little exfoliation ability, but the cellulase may be usefully used to have a function of controlling the growth of harmful bacteria present in the skin.

<Test Example 3>

Hazardous bacteria inhibition

In theory, lysozyme is a lytic enzyme that has the ability to inhibit the growth of harmful bacteria by decomposing peptidoglycans on the cell walls of harmful bacteria present in the skin with N-acetyl hexosamidase activity. Papain, a protease, can also inhibit the growth of bacteria by degrading the peptide bonds of peptidoglycans on the bacterial cell wall (enzymatics, Chung Dong-hyo, 1993, Advanced Culture History). In this regard, staphylococcus aureus and propionibacterium acnes , which are harmful bacteria present in the skin after stabilizing lysozyme and papain, and sacca to measure fungal inhibitory activity, although not harmful bacteria Sacchaomyces serevisiae was tested for growth inhibition of three microorganisms.

Bacteria were cultured with Brain Heart Infusion medium (Difco Co. Ltd.), yeasts with fungi were cultured with Malt extrat medium (Difco Co. Ltd.), and the concentration of microorganisms was tested at 3%. . For use in the microbial growth inhibition test of the stabilizing enzyme was sterilized with a sterilized 0.2 ㎛ filter (Minisart plus, manufactured by Sartorius, Germany) and added to the medium at 5% concentration and tested. As a control, a polymer was added in the same ratio in place of the enzyme solution to inoculate microorganisms and then cultured under the same conditions. Microorganisms were cultured anaerobicly at 37 ° C for Staphylococcus aureus and Propionibacterium acnes, and at 30 ° C for Sacchaomyces serevisiae. After anaerobic culture, three microorganisms were cultured for 24 hours, and then absorbance was measured at 620 nm using a spectrophotometer (CE 1020, Cecil Co. Ltd.). The result is shown in FIG. 4.

Results in Figure 4 is stabilized enzyme lysozyme and papain are microorganisms Stein present in the skin Philo aureus (Staphylococcus aureus) and propionyl sludge tumefaciens acre Ness (Propionibacterium acnes) and in my process serenity busy with saccharide (Sacchaomyces serevisiae ) can be suppressed. Although slightly different for each enzyme and microorganism, the growth of microorganisms is suppressed from at least about 12% to up to about 45%. Therefore, when used in cosmetics by stabilizing the enzymes can be used to suppress harmful bacteria of the skin, it can be expected to reduce the skin trouble and prevent acne.

The present invention according to the above constitution is a technology that can easily stabilize a very unstable enzyme protein according to conditions such as temperature and chemical conditions, polysaccharide itself by easily encapsulating the enzyme using xanthan gum, guar gum and alginate It is a useful method to stabilize the enzyme protein while maintaining the excellent properties of the so that it can be used in the product. In particular, the present invention can not only add additional functionality by using polysaccharides currently used in the cosmetic and pharmaceutical industries, but also be useful in the cosmetic and pharmaceutical industries because the activity of the enzyme can be continuously maintained in the formulation for a long time. It is a useful invention that can be.

Claims (5)

In the method of entrapment of enzyme, sodium polyiodate is purified by appropriately purifying a natural polysaccharide having a side chain structure that is relatively uncomplicated in the structural backbone of the polysaccharide to be used for enzyme stabilization. A method for producing a stabilizing enzyme using a polysaccharide, characterized in that the product obtained by reacting with an enzyme after oxidizing with (NaIO ₄ ) is separated and purified according to molecular weight to obtain an enzyme-stabilized enzyme-encapsulating agent. . The method of claim 1, wherein the polysaccharide that can be used as an enzyme encapsulating agent is xanthan gum, gum guar, or arginate. The method of claim 1, wherein the enzyme is papain (broin), bromelain (bromelain), lysozyme (lysozyme), cellulase (cellulase) characterized in that the production method of the stabilizing enzyme using a polysaccharide. The method according to any one of claims 1 to 3, wherein the ratio of the enzyme and the polysaccharide is 1: 2 to 1: 7. A stabilizing enzyme-added composition, which is obtained by adding the stabilizing enzyme prepared according to claim 1 to cosmetics, functional beverages, and therapeutic or prophylactic preparations under the condition that the enzyme is normally added.