KR20110060257A - Method for stabilizing enzyme forming cross-linked hydrogel with enzyme - Google Patents

Abstract

PURPOSE: A method for stabilizing an enzyme is provided to enhance enzyme stabilitiy and to block enzyme denaturation. CONSTITUTION: A method for stabilizing an enzyme comprises: a step of dispersing the enzyme into polyol to prepare solution 1; a step of dissolving hydrogel in a solvent to prepare solution 2; a step of redispersing solution 1 to solution 2; and a step of adding cross-linking solution and stirring at 40-70°C for 30 minutes to one hour to prepare cross-linked hydrogel. The enzyme is oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. The polyol is polyethylene glycol, polypropylene glycol, or copolymer thereof. The hydrogel is gelatin, acacia gum, collagen, or carrageenan gum.

Description

Method for stabilizing enzyme forming cross-linked hydrogel with enzyme}

The present invention relates to a method for stabilizing enzymes, and more specifically, to stabilize the enzyme by uniformly dispersing the enzyme in a polyol and redispersing it again in a hydrogel solution, and then adding a crosslinking agent to undergo hydrogel crosslinking through a curing process by crosslinking. The present invention relates to a method for stabilizing enzymes by producing a sieve.

With the development of the bio industry, a variety of enzymes are being used for treatment / healing. However, most enzymes have a very short half-life, which tends to denature before the ultimate expression of efficacy, and this low stability of the enzyme component is the biggest factor degrading its utility value. Therefore, researches on the construction of an appropriate delivery system have been actively conducted in recent years in order to expand the application of enzymes. Among many enzyme delivery systems, studies using biodegradable polymers as a wall have many advantages in terms of long-term administration (Y. Okawa, et. al ., Chem . Pharmac . Bull . 5 (1988) 1095; And H. Okada, et al ., Pharmac . Res . 11 (1994) 1143). This delivery system also needs to be assured in terms of manufacturing processes and long term storage.

In general, a water-oil-water multiple emulsion method is frequently used for the preparation of enzyme-containing microcapsules, but the activity of the enzyme is reduced at the water-milk interface (P. Couvreur, et al ., Adv . Drug Del . Rev. 28 (1997) 85; And H. Sah, J. Pharmac . Sci . 88 (1999) 1320). Therefore, recently, the solid-oil-water emulsion method is more realistically accepted due to the advantage of maintaining the enzyme activity in the solid state (T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, H. Yoshino, J. Control.Rel. 69 (2000) 435). However, the methods for encapsulating the enzyme in the solid state mostly occur due to the characteristics of the manufacturing process produced in the chemical environment. In addition, when the polyol is introduced with the enzyme in an aqueous solution, it is chemically found that the enzyme titer is rapidly increased (Jai K. Kaushik, et. al ., J. Phys . Chem . B 1998, 102, 7058-7066), various enzyme stabilization methods have been proposed (Korean Patent Publication No. 2004-0084364). However, the enzyme stabilized by such an aqueous polyol solution is very unstable because the interface of the enzyme is modified by the interfacial tension in the cosmetic formulation containing the surfactant and oil, the amount of enzyme that can be introduced when using a polymer microcapsules This limited, organic solvent environment used results in denaturation.

Therefore, the present inventors have tried to develop a stabilization system to prevent the denaturation of the enzyme, as a result of uniform dispersion of the enzyme in the polyol to stabilize it and redispersed again in a hydrogel solution and then adding a crosslinking agent to the curing process by crosslinking The present invention was completed by discovering that the enzyme can be stabilized by generating a hydrogel crosslinked product.

It is therefore an object of the present invention to provide a method of stabilizing enzymes for application in pharmaceutical or cosmetic formulations.

In order to achieve the above object, the present invention comprises the steps of 1) dispersing an enzyme in a polyol; 2) dissolving the hydrogel in a solvent; 3) redispersing the solution of 1) in 2); And 4) adding a crosslinking agent solution to 3), followed by stirring at 40 to 70 ° C. for 30 minutes to 1 hour to produce a hydrogel crosslinked product.

In the present invention, the enzyme is uniformly dispersed in a polyol, stabilized, and redispersed again in a hydrogel solution, and then a crosslinking agent is added to form a hydrogel crosslinked through crosslinking curing to effectively introduce the enzyme into the enzyme. The polyol gave a hydrophobic orientation effect between the enzyme and the hydrogel, thereby preventing the denaturation of the enzyme and increasing its activity.

The present invention relates to a method of stabilizing an enzyme by uniformly dispersing an enzyme in a polyol and stabilizing it again in a hydrogel solution and then adding a crosslinking agent to undergo a curing process by crosslinking to generate a hydrogel crosslinked product.

The method of stabilizing an enzyme provided in the present invention includes the following steps:

1) dispersing the enzyme in a polyol;

2) dissolving the hydrogel in a solvent;

3) redispersing the solution of 1) in 2); And

4) adding a crosslinking agent solution to 3) and stirring the mixture at 40 to 70 ° C. for 30 minutes to 1 hour to form a hydrogel crosslinked product.

Looking at the above process in more detail as follows.

First, the enzyme is evenly dispersed in the polyol in step 1). Enzymes to be used include enzymes such as oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. For example, papain, cerato pectidases and lipases may be used. But it is not limited thereto. The enzymes may be used alone or in combination of two or more.

The polyol is effectively oriented between the enzyme and water by the hydrophobicity imparted by the high molecular weight in the hydrogelation process, thereby effectively protecting the enzyme by the "solvophobic effect". In order to induce this hydrophobic orientation effect, the molecular weight of the polyol must be high enough, and a suitable polyol is a polyester polymer having a molecular weight of 5,000 g / mol, specifically polyethylene glycol, polypropylene glycol and copolymers and derivatives thereof. do.

In step 2), the hydrogel is dissolved in a solvent such as water, aqueous alkali solution or ethanol.

In this case, the water-soluble polymer used as a hydrogel may include gelatin, acacia gum, collagen or carrageenan gum, and these may be used alone or in combination of two or more.

The polyol solution in which the enzyme is dispersed is then redispersed in the hydrogel solution.

At this time, the amount of the water-soluble polymer, that is, the hydrogel is appropriate to use the amount of 20 to 30% by weight of the combined weight of the enzyme and the polyol. In addition, the combined weight of the polyol / enzyme and the amount of the water-soluble polymer are introduced at 30 to 40% by weight based on water.

The crosslinking agent solution which melt | dissolved the crosslinking agent in solvent, such as water or alkali aqueous solution, is added to the said dispersion liquid, and it stirred at 40-70 degreeC for 30 minutes-one hour, and produces a hydrogel crosslinked body.

The crosslinking agent may form a condensation reaction with an amine group, and may include glyoxal, genipin, glutaraldehyde, or glycerol, and is 0.7 to 1.5 wt.% Based on water-soluble polymer. Introduced in%. When the cross-linking agent is introduced in excess of 1.5% by weight, the strength of the cross-linking body becomes very strong, so that the activity of the enzyme can not be effectively expressed in the formulation. It can not be effective. That is, the crosslinking agent may form a network structure by condensation reaction with the water-soluble polymer and some enzymes.

In the present invention, a relatively high content of enzyme / polyol / hydrogel crosslinked product was obtained through the above process. In the crosslinked product of the present invention, the enzyme and the hydrogel form a crosslinked product through crosslinking, and the polyol is oriented between the enzyme and the water therein to effectively protect the enzyme. Initially introduced polyols have a chemically-physically combined crosslinker that effectively orients the polyol at the enzyme / water interface so that the conformation of enzymes in the crosslinker is maintained.

The hydrogel crosslinked product prepared by the above method can be utilized as an enzyme (protein) stabilizing hydrogel, and it is also possible to introduce this technique into other enzyme application techniques.

In addition, it is expected that the enzyme stabilized hydrogel crosslinked body prepared in the present invention may be usefully used in pharmaceutical and cosmetic compositions.

Hereinafter, the content of the present invention will be described in more detail through Examples and Test Examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 Preparation of Papain-containing Polypropylene Glycol / Gelatin Hydrogel Crosslinked Body

Papain was dispersed in high molecular weight polypropylene glycol having a molecular weight of 5,000 g / mol. At this time, the content of papain was 50% of the total polypropylene. Subsequently, 6% gelatin was dissolved in distilled water, and the papain / polypropylene glycol mixture was mixed with an aqueous gelatin solution at 30% of the total weight. Thereafter, an additional 1.5% of 40% glyoxal aqueous solution was introduced and stirred at room temperature for about 1 hour to finally complete the reaction.

Test Example 1 Papain Introduction

The amount of papain introduced into the polypropylene glycol / gelatin hydrogel prepared in Example 1 was analyzed as follows. First, 50 ml of papain-containing polypropylene glycol / gelatin hydrogel prepared in Example 1 was placed in 1 ml of dimethyl sulfoxide and incubated for 1 hour, followed by 0.05% sodium dodecyl sulfate / 0.01 N sodium hydroxide solution 2 ml was added and left at room temperature for 1 hour to completely dissolve the enzyme. Subsequently, the content of papain was quantified by protein quantification using micro-BCA, and the results of calculating the papain introduction rate based on Equation 1 are shown in Table 1 below.

Introduction rate (%) = Papain actual measurement (weight) / (polypropylene glycol / gelatin hydrogel) weight × 100

Papain Introduction
(% By weight, theoretical) Papain Introduction
(% By weight, calculated value) % Adoption 15 13 86

In the results of Table 1, it was confirmed that the enzyme was effectively captured through the stabilization system according to the present invention.

[Example 2]

In Example 1, a papain-containing polypropylene glycol / gelatin hydrogel crosslinked product was prepared in the same manner as in Example 1 except that the amount of papain introduced was adjusted to 15% by weight relative to the total crosslinked product.

Comparative Example 1

Comparative Example 1 was prepared by dispersing 1% by weight of papain in 1% by weight of high molecular weight polypropylene glycol having a molecular weight of 5,000 g / mol.

Comparative Example 2

Papain was dispersed in polyethyleneglycol having a molecular weight of 400 g / mol. The dispersion was completely dissolved by stirring at room temperature in methylene chloride at a weight ratio of polymethylmethacrylate (75,000 g / mol molecular weight) and 3: 7. Polyethylene glycol and polymethyl methacrylate were introduced in an amount of 20% by weight based on methylene chloride.

The solution was placed in an aqueous solution of 1% polyvinyl alcohol (average degree of saponification of 89%) and emulsified at 5,000 rpm for 5 minutes using a mechanical homogenizer. At this time, the polyethyleneglycol / polymethylmethacrylate / methylenechloride solution has a concentration of 30% by weight in the water phase. After the completion of emulsification, the emulsion was transferred to a reduced pressure evaporator and stirred under reduced pressure at room temperature for 30 minutes to completely remove methylene chloride as a solvent. After the vacuum evaporation process was completed, the dispersion was filtered through filter paper to recover only the capsules and to remove all water-soluble substances including water. The recovered capsules were dried for 1 day in a room temperature vacuum dryer.

[Formulation Example 1 and Comparative Formulation Examples 1 to 3]

According to the composition shown in Table 2 below, the cosmetic preparations of Formulation Example 1 and Comparative Formulation Examples 1 to 3 were prepared (unit: wt%).

division Compounding ingredient Formulation Example 1 Comparative Formulation Example 1 Comparative Formulation Example 2 Comparative Formulation Example 3 Awards Example 2 2.0 - - - Awards Comparative Example 1 - 2.0 - - Awards Comparative Example 2 - - - 2.0 Awards Papain - - 1.0 - Awards Purified water Balance Balance Balance Balance Paid Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 Paid Stearic acid 0.60 0.60 0.60 0.60 Paid Glycerol Stearate 1.00 1.00 1.00 1.00 Paid Cetearyl Alcohol 2.00 2.00 2.00 2.00 Paid Arachidyl behenyl alcohol and
Arachidil Glucoside 1.00 1.00 1.00 1.00 Paid Cetearyl alcohol and
Cetearylglucoside 2.00 2.00 2.00 2.00 Paid PEG-100 stearate,
Glycerol Olate and Propylene Glycol 1.50 1.50 1.50 1.50 Paid Caprylic / Capric Triglycerides 11.00 11.00 11.00 11.00 Paid Cyclomethicone 6.00 6.00 6.00 6.00 Awards Preservative, incense Quantity Quantity Quantity Quantity Awards Triethanolamine 0.10 0.10 0.10 0.10

<Manufacturing Method>

1) The aqueous phase components were uniformly mixed at room temperature and heated to 60 ° C.

2) The oily ingredients were heated to 60 ° C. to uniformly dissolve and mix.

3) 1) was added to 2) under stirring to emulsify uniformly.

Test Example 2 Papain Stability Analysis

Papain activity in the cosmetic preparations prepared in Formulation Example 1 and Comparative Formulation Examples 1 to 3 was confirmed at a storage temperature of 45 ° C. according to the storage period.

Papain activity in the hydrogel was analyzed as follows. 20 ml of Formulation Example 1 and Comparative Formulation Examples 1 to 3 were added to 1 ml of acetone, and ultrasound was injected for 10 seconds. Then, it was centrifuged for 5 minutes at 12,000 rpm. This process was repeated three times to obtain only pure enzyme. The obtained pure enzyme was measured for activity using a UV absorber at 280 nm with casein as a substrate. The activity of papain obtained according to the storage conditions is shown in Table 3 below.

division Early 1 week 2 weeks 4 Weeks 8 Weeks 16 Weeks Formulation Example 1 100% 95% 85% 81% 81% 80% Comparative Formulation Example 1 100% 67% 59% 51% 27% 15% Comparative Formulation Example 2 100% 49% 21% 5% - - Comparative Formulation Example 3 100% 90% 86% 75% 69% 71%

In the results of Table 3, Formulation Example 1 containing the papain-containing polypropylene glycol / gelatin hydrogel prepared in Formulation Example 1 showed excellent activity even in long-term storage conditions compared to Comparative Formulation Examples 1-3.

As such, the good activity of the enzyme means that the polyol, such as polyethylene glycol, effectively stabilizes the enzyme in solid form in the hydrogel. That is, it was confirmed that the polyol is properly positioned between the enzyme and the polymer wall such as the hydrogel to impart a hydrophobic partitioning effect, thereby blocking the direct interaction between the enzyme and the polymer, thereby improving the stability of the enzyme.

Hydrogel crosslinked product according to the present invention is a system that can effectively introduce the enzyme component therein. In particular, in order to effectively exhibit the solubility effect of the polyol in the cosmetic formulations containing a lot of surfactants and oils, it can be found in the aspect that can prevent the modification of the enzyme by giving a hydrophobic orientation effect between the enzyme and the hydrogel. .

In addition, the hydrogel crosslinked body according to the present invention is expected to be able to further enhance the utilization of the enzyme in practical applications by providing excellent stability to the enzyme loaded in a high content therein. In particular, it can be used as a reaction control system in various bioengineering fields using enzymes as catalysts, and in dermatology fields using enzymes as decomposition catalysts, it is expected to be used as keratin care and other active ingredient skin absorption accelerators. .

Claims (6)

1) dispersing the enzyme in a polyol; 2) dissolving the hydrogel in a solvent; 3) redispersing the solution of 1) in 2); And 4) adding a crosslinker solution to 3) and then stirring at 40 to 70 ° C. for 30 minutes to 1 hour to produce a hydrogel crosslinked product; Method to stabilize the enzyme, including. According to claim 1, wherein the enzyme used in step 1) stabilizes the enzyme, characterized in that selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase Way. According to claim 1, wherein the polyol used in step 1) is a method for stabilizing the enzyme, characterized in that selected from the group consisting of polyethylene glycol, polypropylene glycol and copolymers thereof. The method of claim 1, wherein the hydrogel used in step 2) is gelatin, acacia gum, collagen and carrageenan gum stabilizing enzyme, characterized in that at least one member selected from the group consisting of. According to claim 1, wherein the crosslinking agent used in step 4) stabilizes the enzyme, characterized in that selected from the group consisting of glyoxal (glyoxal), genipin (genipin), glutaraldehyde (glutaraldehyde) and glycerol (glycerol) How to let. An enzyme / polyol / hydrogel crosslinked product prepared by the method according to any one of claims 1 to 5 and having a structure in which enzymes, polyols and hydrogel crosslinked bodies are sequentially arranged from the inside.