KR20040045607A - Polyol/polymer microcapsule and the stabilization method of enzyme using it - Google Patents

Abstract

PURPOSE: Provided are a microcapsule of polyol/polymer and a method for stabilizing an enzyme using the same. Particularly, an enzyme of which stability is dramatically lowered when used in a formulation is stabilized with polyols basically and then into a microcapsule ultimately. Therefore, the enzyme is effectively stabilized. CONSTITUTION: A method for stabilizing an enzyme using a microcapsule of polyol/polymer is characterized by the steps of: dispersing an enzyme in low molecular weight of polyol evenly for stabilization; re-dispersing the stabilized enzyme/polyol in a polymeric solution, followed by emulsification to give an emulsion; and solidifying the enzyme/polyol/polymeric solution to give a hardened polymeric microcapsule.

Description

Polyol / polymer microcapsule and the stabilization method of enzyme using it}

The present invention relates to a technique for preparing polymer microcapsules capable of effectively stabilizing an enzyme having a characteristic of rapidly decreasing stability in use in a formulation.

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 enzyme components It is the biggest factor that lowers the utilization value. Therefore, in order to expand the application of such an enzyme component, research on the construction of an appropriate delivery system has been actively conducted in recent years. Among many enzyme delivery systems, studies using biodegradable polymers as a wall have many advantages in terms of long-term administration (Y.Okawa, M. Yamamoto, H. Okada, T. Yashiki, T. Shimamoto, Chem. Pharmac. Bull. 5 (1988) 1095, H. Okada, Y. Doken, Y. Ogawa, H. Toguchi, Pharmac. Res. 11 (1994) 1143). This delivery system also needs to be assured in terms of manufacturing processes and long term storage. In general, the water-oil-water multiple emulsion method is frequently used for the preparation of enzyme-containing microcapsules, but has a big problem in that the activity of the enzyme is reduced at the water-milk interface (P. Couvreur, MJ Blanco-Proeto). , F. Puisieux, B. Ropues, E. Fattal, Adv. Drug Del. Rev. 28 (1997) 85, 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 activity of the enzyme in the solid state (T. Morita, Y. Sakamura, Y. Horikiri, T. Suzuki, H. Yoshino, J. Control.Rel. 69 (2000) 435). However, these methods of encapsulating solid state enzymes often result in denaturation in many parts of the enzyme due to the nature of the manufacturing process, which exerts heating and physical force.

The present inventors have tried to develop a stabilization system that can prevent the denaturation of such enzymes in the prior art. As a result, the present inventors have found that polyol / polymer microcapsules are suitable for stabilizing enzymes and completed the present invention.

It is an object of the present invention to provide polyol / polymer microcapsules using low molecular weight polyols, high molecular weight polyols and polymer solutions.

It is also an object of the present invention to provide a method for producing polyol / polymer microcapsules using a low molecular weight polyol, a high molecular weight polyol and a polymer solution.

Another object of the present invention is to provide an enzyme stabilized microcapsule system containing enzymes for application in pharmaceutical or cosmetic formulations.

1 is an optical micrograph of polyethylene glycol (400 g / mol) / polycaprolactone microcapsules.

Figure 2 is an optical micrograph of the polyethylene glycol / polycaprolactone microcapsules with a controlled polyethylene glycol mixing ratio (400 g / mol: 8000 g / mol = 5: 5 (w / w)).

3 is an optical micrograph of papain-containing polyethylene glycol / polycaprolactone microcapsules.

4 is a scanning electron micrograph of papain-containing polyethylene glycol / polycaprolactone microcapsules.

The production method of the polyol / polymer microcapsules using the low molecular weight polyol, the high molecular weight polyol and the polymer solution of the present invention is as follows.

First, a low molecular weight polyol and a high molecular weight polyol are mixed and dissolved in a polymer solution prepared by dissolving a polymer wall in a solvent. At this time, the combined weight of the low molecular weight polyol and the high molecular weight polyol and the amount of the polymer wall of the polymer solution can be controlled considering the thickness of the wall, but it is appropriate to use the same amount. In addition, the combined weight of the low molecular weight polyol and the high molecular weight polyol and the amount of the polymer wall are 20 wt% based on the solvent. The solution in which the polyol is dissolved is placed in an aqueous solution in which a stabilizer is introduced and emulsified using a homogenizer. The emulsion was stirred under reduced pressure in a reduced pressure evaporator for 10 to 120 minutes to filter the dispersion after the solvent was completely removed to recover the capsules, and all other water-soluble substances were removed. The recovered capsules are completely dried for 24 to 48 hours in a room temperature depressurized dryer which is blocked from light.

In addition, the polyol / polymer microcapsules prepared by the above method can be utilized as enzyme or protein stabilizing microcapsules, and it is also possible to introduce this technique to other enzyme application techniques.

To this end, the enzyme forms an internal nucleus, which is coated with a hydrophobic high molecular weight polyol, and finally, a triple microcapsule structure having a structure in which the polymer forms a wall on the outer shell is possible.

The method for preparing the triple microcapsules is as follows.

That is, uniformly dispersing the enzyme in a low molecular weight polyol; Redispersing the stabilized enzyme / polyol in a polymer solution to obtain an emulsion through an emulsification process; And solidifying the enzyme / polyol / polymer solution to recover the hard polymer microcapsules.

Hereinafter, the present manufacturing method will be described in more detail.

First, the enzyme is dispersed in a low molecular weight polyol. In general, enzymes have partial solubility in low molecular weight polyols. In this case, the enzyme forms a spherical dispersion due to the relatively high wettability on the low molecular weight polyol and only the outer layer partially dissolves to form the enzyme / polyol mixed phase. This enzyme / polyol dispersion is dispersed following the polymer solution in which the high molecular weight polyol is dissolved. The polymer solution contains a high molecular weight polyol, a polymer wall (wall polymer) and a solvent. Through this process, the enzymatic phase protected by the low molecular weight polyol can be stably dispersed without being influenced by the solvent of the polymer solution. The high molecular weight polyols act as a buffer to prevent direct contact between enzymes and hydrophobic polymer walls (wall polymers) in the final microcapsules. In the following process, only the solvent is selectively removed from the droplet consisting of enzyme / polyol / polymer / solvent. As the solvent is removed, since the polyol is incompatible with the polymer, phase separation occurs. In this phase separation process, the low molecular weight polyol is discharged to the external water phase through the weakened outer interface due to the high polarity, and the high molecular weight polyol phase remains inside the microcapsules. As a result, the microcapsule is a triple microcapsule structure in which an enzyme forms an inner nucleus, which is coated by a high molecular weight polyol, and finally a polymer forms a wall on its outer shell.

That is, in the present invention, in order to maintain the structural characteristics of the enzyme itself and to fundamentally block interaction with external stimuli by escaping from the simple encapsulation proposed by the existing studies, low molecular weight polyols are formed in the internal pore forming template of the microcapsule ( process and storage in biodegradable polymer microcapsules by using as a template and a dispersion medium of enzymes, and simultaneously introducing high molecular weight polyols as hydrophobic dispersing agents of enzymes and blocking agents that block denaturation by the inner walls of polymers forming walls. Stability of the enzyme can be maintained.

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

Enzymes applicable in the present invention are redox enzymes such as glucose oxidase, xanthine oxidase, D-amino acid oxidase, transfer enzymes such as transaminase, nucleokinase, lipase, amylase, pepsin, trypsin, urease, asparaginana Hydrolyase such as papain, papain and the like, lyases such as aldorase, fumarase, pectinase, etc., lactate ketal isomerase, lactate resease, udapi-di-glucose-4-epimerase Enzymes such as isomerases such as isomers, aspartate ammonia kinase, and synthetic enzymes such as DNA ligase. It is possible to use one or more of these enzymes in combination.

In the present invention, a high molecular weight polyol and a low molecular weight polyol are mixed and used. The low molecular weight polyol uniformly disperses the enzyme and flows out of the capsule during capsule formation to serve as a template for forming the capsule more effectively. Low molecular weight polyols that can be used are polymers in the form of polyethers of 1000 g / mol or less, specifically, all low molecular weights including polyethylene glycol, polypropylene glycol and their copolymers and derivatives, butylene glycol, propylene glycol, glycerin, and the like. The compound containing the alcohol group of molecular weight corresponds. The amount of use is appropriately 0.1 to 70% by weight based on the total amount of the capsule. At concentrations of 0.1 wt% or less, it is difficult to effectively disperse the enzyme, and at a concentration of 70 wt% or more, the yield of the final microcapsules is lowered.

The high molecular weight polyol is present in the capsule even without encapsulation into the trauma to impart hydrophobic partitioning effect to the enzyme and to prevent the enzyme from being denatured by the hydrophobic polymer inner wall. As the molecular weight of the polyols increases, the hydrophobicity of the polyol chains also increases proportionally. Therefore, when the high molecular weight polyol is effectively located on the inner wall of the enzyme and the hydrophobic polymer, the enzyme can be effectively protected by the hydrophobic partitioning effect. In order to induce this hydrophobic partitioning effect, the molecular weight of the polyol must be high enough. Suitable polyols are polyether polymers in the form of waxes having a molecular weight of 1000 g / mol or more, specifically polyethylene glycol, polypropylene glycol and their copolymers and derivatives. The amount of use is appropriately 0.1 to 90% by weight relative to the total amount of the capsule. At concentrations below 0.1% by weight it is difficult to expect effective hydrophobic dispensing effects, whereas at concentrations above 90% by weight it is difficult to form hard microcapsules due to their high content.

In the above process, the polymer (polymer wall) used as a wall of the microcapsules is a biodegradable hydrophobic aliphatic polyester, specifically, poly-L-lactic acid, poly-D, L-glycolic acid, poly-L-lactic acid- air prepared from co-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, polycaprolactone, polyvalerolactone, polyhydroxy butyrate, polyhydroxyvalorate, polyetherester and monomers thereof Include coalescing. In addition, possible polymer furnaces (polymer wall) used as the wall of the microcapsules include polystyrene, poly p- or m-methylstyrene, poly p- or m-ethylstyrene, poly p- or m-chlorostyrene, poly p- or m -Chloromethylstyrene, polystyrenesulphonic acid, poly p- or m- or t-butoxystyrene, poly methyl (meth) acrylate, polyethyl (meth) acrylate, polypropyl (meth) acrylate, polyn -Butyl (meth) acrylate, polyisobutyl (meth) acrylate, polyt-butyl (meth) acrylate, poly 2-ethylhexyl (meth) acrylate, polyn-octyl (meth) acrylate, polyla Uryl (meth) acrylate, polystearyl (meth) acrylate, poly2-hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, polyglycidyl (Meth) acrylate, Polydimethylaminoethyl (meth) acrylate, polydiethylaminoethyl (meth) acrylate, polyvinylacetate, polyvinylpropionate, polyvinylbutyrate, polyvinylether, polyallylbutyl ether, polyallylglycidyl ether , Poly (meth) acrylic acid, unsaturated carboxylic acids such as polymaleic acid, polyalkyl (meth) acrylamide, poly (meth) acrylonitrile, and the like, and these may be used in combination. Such polymer walls are suitably in the range of 1-99.99% by weight relative to the total capsule content. It is difficult to form microcapsules at a concentration of 1% by weight or less, and the concentration of enzyme is too low at a concentration of 99.99% by weight or more, so that the effect is difficult to express.

The solvents used during the preparation of the microcapsules are all compounds having a solubility parameter similar to the polymer of choice, specifically, linear alkanes such as hexane, heptane, octane, nonane, decane, butanol, linear or branched pentanol, C4-C10 alcohols such as hexanol, heptanol, octanol, nonanol, tecanol, etc., n-hexyl acetate, 2-ethylhexyl acetate, methyl oleate, dibutyl sebacate, dibutyl adsorbate, ibutyl Alkyl esters having 7 or more carbon atoms such as carbamate, aliphatic ketones such as methyl isobutyl ketone, isobutyl ketone, aromatic hydrocarbons such as benzene, toluene, o- or p-xylene, chlorine compounds such as methylene chloride, chloroform, carbon tetrachloride, etc. Include.

Stabilizers used in the manufacture of microcapsules are all water-soluble polymers that can be introduced into the water phase to improve the dispersion stability of the capsule, such as arabic, tragacanth, karaya, and lach. , Ghatti, locust bean, guar, agar, alginate, carrageenan, furcellaran, pectin, gelatin, Starch and its derivatives; Dextran, xanthan gum and derivatives thereof prepared by microbial fermentation synthesis; And vinyl polymers, acrylic polymers, polyol-containing copolymers and derivatives thereof prepared by radical or ring-opening polymerization, and one or more of them can be selected and used. Preferably polyvinyl alcohol can be used. The dispersion stabilizer may add 0.01% to 30% by weight based on the mass of the microcapsule dispersion aqueous solution. It is difficult to expect excellent dispersion stability in the concentration range below the concentration, it is difficult to proceed the manufacturing process of the microcapsules due to the gelation proceeds in the concentration range above.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited only to these examples.

<Example 1> Preparation of polyethylene glycol-containing polycaprolactone microcapsules

Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polymprolactone (80000 g / mol molecular weight), which is a polymer wall, and in a methylene chloride, which is a solvent, in a weight ratio of 1: 1. Polyethylene glycol and polycaprolactone are introduced in an amount of 15% by weight based on methylene chloride. Each prepared solution was put in an aqueous solution of 1% polyvinyl alcohol (average degree of saponification of 89%) and emulsified at 5000 rpm for 5 minutes using a mechanical homogenizer. At this time, the polyethylene glycol / polycaprolactone / methylene chloride solution has a concentration of 30% by weight in the water phase. After the completion of the emulsification, the emulsion is 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 completion of the vacuum evaporation process, the dispersion is filtered through filter paper to recover only the capsules and remove all water-soluble substances, including water. The recovered capsules are dried for 1 day in a room temperature reduced pressure dryer.

Example 2 Preparation of Polyethylene Glycol Microcapsules

Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polymethylene methacrylate (75000 g / mol molecular weight), which is a polymer wall, and in a weight ratio of 1: 1 to methylene chloride, which is a solvent, to be completely dissolved by stirring at room temperature. Polyethylene glycol and polymethyl methacrylate are introduced in an amount of 20% by weight based on methylene chloride. The procedure is the same as in Example 1 to prepare a polyethylene glycol / polymethyl methacrylate microcapsules.

Example 3 Preparation of Polyethylene Glycol-Containing Polystyrene Microcapsules

Polyethylene glycols having a molecular weight of 400, 4000, 8000, and 12000 g / mol are respectively dissolved in polystyrene (100000 g / mol molecular weight), which is a polymer wall, and in a methylene chloride, which is a solvent, in a weight ratio of 1: 1. Polyethylene glycol and polystyrene are introduced in an amount of 20% by weight based on methylene chloride. The procedure is the same as in Example 1 to prepare a polyethylene glycol / polystyrene microcapsules.

Example 4 Preparation of Two Polyethylene Glycol-Containing Polycaprolactone Microcapsules

Polycaprolactone / consisting of a polymer wall and a solvent by mixing polyethylene glycol having a molecular weight of 400 g / mol, which is a low molecular weight polyol, and polyethylene glycol having a high molecular weight, of 8000 g / mol, with a weight ratio of 7: 3, 5: 5, 3: 7. Introduced to methylene chloride polymer solution. At this time, the weight of the low molecular weight and high molecular weight polyethylene glycol plus the weight of the polycaprolactone which is a polymer wall is used in the same amount. In addition, the polyethylene glycol and polycaprolactone are introduced by 20% by weight based on the methylene chloride as a solvent. The following process was carried out in the same manner as in Example 1 to prepare a polyethylene glycol / polycaprolactone microcapsules.

Example 5 Preparation of Papain-Containing Polyethylene Glycol / Polycaprolactone Microcapsules

Papain is used as a model enzyme. Papain is first dispersed in low molecular weight polyethylene glycols having a molecular weight of 400 g / mol. At this time, the content of papain is introduced in 1, 3, 5% by weight relative to the total capsule weight. Subsequently, the papain / polyethylene glycol dispersion is redispersed in the methylene chloride aqueous solution in which a high molecular weight polyethylene glycol having a molecular weight of 8000 g / mol and a polycaprolactone as a polymer wall are dissolved. The content of the components used in this process is according to the composition of Example 4. The following procedure was carried out in the same manner as in Example 1 to prepare a papain enzyme-containing polyethylene glycol / polycaprolactone microcapsules.

Test Example 1 Characterization of Polyethylene Glycol / Polymer Microcapsules

The capsule form of the polyethyleneglycol / polymer microcapsules prepared in Examples 1-3 was observed under an optical microscope. As shown in FIG. 1, the prepared polyethylene glycol / polymer microcapsules are spherical bodies having an average particle size of 5 to 20 mm. Each sphere forms a polyethyleneglycol domain therein. However, the content of polyethylene glycol introduced showed a result depending on the molecular weight. The effect of the amount introduced into the polymer microcapsules according to the molecular weight of polyethylene glycol was summarized in Table 1 by quantitative analysis using thin layer chromatography.

Polyethylene glycol introduction amount (%) 400 g / mol 4000g / mol 8000g / mol 12000g / mol Example 1 Composition 0 0.5 14 21 Example 2 Composition 0 0.7 18 20 Example 3 Composition 0 0.3 9 16

As can be seen from Table 1, the amount of polyethylene glycol introduced was highly dependent on the molecular weight. As the molecular weight increased, the polarization of the polyethyleneglycol molecules decreased, and the amount of introduction increased. However, in the optical micrograph, even when low molecular weight polyethylene glycol was used, the result of forming a domain inside was obtained. This microcapsule formation behavior is because polyethylene glycol phase separates inside the capsule during the removal of the solvent to form a specific domain and then slowly flows out again. The low molecular weight polyethylene glycol is believed to have a template function to induce internal domain formation during capsule formation. High molecular weight polyethylene glycol showed excellent supporting results inside regardless of the process.

Test Example 2 Content Analysis According to Polyethylene Glycol Mixing Ratio

In order to confirm the behavior of the capsule contained in the polyethylene glycol mixing ratio, the polyethylene glycol / polycaprolactone microcapsules prepared in Example 4 were quantitatively analyzed using thin layer chromatography, and the results are summarized in Table 2. The amount introduced into the polyethylene glycol capsule was detected according to the mixing ratio. However, in the optical micrograph analysis of FIG. 2, the capsule form could be observed regardless of the mixing ratio. These results indicate that low molecular weight polyethylene glycol serves as a template for domain formation in the capsule and high molecular weight polyethylene glycol remains in the template.

Mixing ratio 400 g / mol: 8000 g / mol Polyethylene glycol introduction amount (%) 400 g / mol 8000 g / mol 10: 0 0 0 7: 3 0 4 5: 5 0 6.5 3: 7 0 9.5 0: 10 0 14

<Test Example 3> Papain introduction amount analysis in the polyethylene glycol / polycaprolactone capsule

An optical micrograph of the papain-containing polyethylene glycol / polycaprolactone microcapsules prepared in Example 5 is shown in FIG. 3. Unlike in FIG. 1, the papain region can be easily observed inside the manufactured microcapsules. 4 shows a scanning electron micrograph of papain-containing microcapsules. The capsule outer wall is formed of smooth polycaprolactone and no leakage of papain or polyethylene glycol to the outside of the capsule is observed, indicating that papain and polyethylene glycol are effectively collected by the polycaprolactone wall. The amount of papain introduced into the microcapsules was analyzed as follows. First, 50 ml of papain-containing microcapsules were added to 1 ml of dimethyl sulfoxide, incubated for 1 hour, and then 2 ml of 0.05% sodium dodecyl sulfate / 0.01 N sodium hydroxide solution was added and left at room temperature for 1 hour to completely polymer and enzyme. Dissolve. Protein is then quantified using micro-BCA. Papain content quantified by this method is shown in Table 3 below. As can be seen from Table 3, the method proposed in the present invention shows that the enzyme is effectively captured.

Papain content (% by weight, calculated) Papain introduction amount (% by weight, measured value) Introduction rate (%) One 0.99 99 3 2.79 93 5 4.55 91

Test Example 4 Analysis of Papain Stability in Polyethyleneglycol / Polycaprolactone Capsules

Papain activity in the polyethyleneglycol / polycaprolactone microcapsules prepared in Example 5 was confirmed according to storage temperature. In this case, papain 3% containing microcapsules were selected to measure the activity. Papain activity in the capsule was analyzed as follows. 20 ml of papain-containing microcapsules were placed in 1 ml of acetone, and ultrasonic waves were injected for 10 seconds. It was then centrifuged for 5 minutes at 12000 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 as a casein substrate. The papain activity obtained according to the storage conditions is shown in Table 4 below. Papain-containing polyethyleneglycol / polycaprolactone microcapsules showed good activity even under long-term storage conditions. This good activity means that polyols such as polyethylene glycol effectively stabilize the enzyme in solid form in the microcapsules. It is interpreted that the polyol is properly positioned between the enzyme and the polymer wall such as polycaprolactone to impart hydrophobic partitioning effect, thereby blocking the direct interaction between the enzyme and the polymer and consequently improving the stability of the enzyme.

Save time aridity Wet Room temperature 40 ℃ Room temperature 40 ℃ After manufacturing 100 100 100 100 after 2 weeks 97 ± 3 92 ± 4 93 ± 5 89 ± 5 4 weeks later 95 ± 4 91 ± 2 90 ± 3 82 ± 4

As described above, the polyol / polymer microcapsules are a system capable of effectively introducing an enzyme component therein. In particular, the molecular weight of the polyol can be appropriately controlled to provide a hydrophobic partition effect between the enzyme and the hydrophobic polymer wall after introduction of the enzyme component and the enzyme component during capsule formation. In addition, the prepared enzyme-containing polyol / polymer microcapsules are expected to give excellent stability to the supported enzyme to further increase the utilization of the enzyme in practical applications. In particular, it is expected to be used as a reaction control system in various bioengineering fields using enzymes as catalysts, and in dermatology fields using enzymes as decomposition catalysts.

Claims (20)

(A) mixing a low molecular weight polyol and a high molecular weight polyol and dissolving it in a polymer solution; (B) emulsifying the solution in which the polyol is dissolved in an aqueous solution containing a stabilizer; (C) stirring the emulsion in a reduced pressure evaporator to remove the solvent; (D) filtering the dispersion from which the solvent is removed to remove all the water-soluble substances to recover the capsules; And (E) drying the recovered capsules at room temperature under reduced pressure to obtain a polyol / polymer microcapsules; wherein the polymer solution of (a) is prepared by dissolving a polymer wall (wall polymer) in a solvent. Method for producing a polyol / polymer microcapsules, characterized in that. The method for producing polyol / polymer microcapsules according to claim 1, wherein the weight of the low molecular weight polyol and the high molecular weight polyol is equal to the weight of the polymer wall. The method according to claim 1, wherein the weight of the low molecular weight polyol and the high molecular weight polyol and the weight of the polymer wall are 20% by weight with respect to the solvent, respectively. According to claim 1, wherein the (b) stabilizer is arabic (arabic), tragacanth (karaga), karaya (larch), gatti, locust bean (locust bean) , Guar, agar, alginate, carrageenan, furcellaran, pectin, gelatin, starch and derivatives thereof; Dextran, xanthan gum and derivatives thereof prepared by microbial fermentation synthesis; And vinyl polymers, acrylic polymers, polyol-containing copolymers and derivatives thereof prepared by the radical or ring-opening polymerization method. The method of claim 1, wherein the stabilizer of step (b) is a method for producing a polyol / polymer microcapsule, characterized in that 0.01 to 30% by weight based on the mass of the microcapsule dispersion aqueous solution. A polyol / polymer microcapsule, which is prepared by the process according to any one of claims 1 to 5. A method for stabilizing an enzyme or a protein, wherein the enzyme or protein is collected using the polyol / polymer microcapsules of claim 6. (A) uniformly dispersing the enzyme in a low molecular weight polyol; (B) redispersing the stabilized enzyme / polyol in a polymer solution and then emulsifying to obtain an emulsion; And (C) solidifying the enzyme / polyol / polymer solution and recovering it as a hard polymer microcapsules; wherein the polymer solution of step (b) is prepared by dissolving a high molecular weight polyol and a polymer wall (wall polymer) in a solvent Method for producing an enzyme stabilized microcapsule, characterized in that. The enzyme stabilized microcapsules according to claim 8, prepared by the above-mentioned preparation method. [Claim 10] The enzyme stabilizing microcapsules of claim 8, wherein the enzyme is at least one selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Manufacturing method. The method of claim 8, wherein the low molecular weight polyol is a polyether form of 1000 g / mol or less, and serves as a template during microcapsule formation and serves as a dispersion medium of the enzyme. [Claim 9] The enzyme stabilized microcapsule of claim 8, wherein the low molecular weight polyol is at least one selected from the group consisting of polyethylene glycol, polypropylene glycol and copolymers and derivatives thereof, butylene glycol, propylene glycol and glycerin. Manufacturing method. The method of claim 8, wherein the low molecular weight polyol is 0.1 to 70% by weight based on the total capsule capsule manufacturing method of the enzyme stabilized microcapsules. The method of claim 8, wherein the high molecular weight polyol is present in the form of a polyether of 1000g / mol or more in the microcapsule to impart hydrophobic partitioning effect to the enzyme and to block denaturation by the inner wall of the polymer forming a wall Method for producing an enzyme stabilized microcapsule, characterized in that. 9. The method of claim 8, wherein the high molecular weight polyol is at least one selected from the group consisting of polyethylene glycol and polypropylene glycol, copolymers and derivatives thereof. The method of claim 8, wherein the high molecular weight polyol is 0.1 to 90% by weight based on the total capsule capsule manufacturing method of the enzyme stabilized microcapsules. 9. The method of claim 8, wherein the polymer wall (wall polymer) forms a wall of microcapsules. 9. The enzyme stabilizing microcapsule according to claim 8, wherein the polymer wall (wall polymer) is at least one selected from the group consisting of polyester, polyacrylate, polyvinyl ether, unsaturated carboxylic acid, copolymers and derivatives thereof. Method of preparation. 9. The method according to claim 8, wherein the polymer wall (wall polymer) is 1 to 99.99% by weight based on the total amount of the capsule. Enzymes into the inner nucleus; Hydrophobic high molecular weight polyol as the coating material of the enzyme; Triple capsule microcapsules, characterized in that the polymer having a wall structure;