KR20160135104A - Manufacturing method for biocompatible type enzyme complex - Google Patents

Abstract

The present invention relates to a biocompatible enzyme complex and a method for producing the same. More specifically, the biocompatible enzyme complex has excellent biocompatibility and thus is not harmful to a human body. More cross-linking bonds are induced between enzymes compared to a conventional binder, so adhesive force of enzymes is strong. More enzymes can be accumulated in a limited region. At the same time, attached enzymes are not affected by enzyme activity expression, so enzyme properties can be stably expressed for a long time.

Description

Technical Field [0001] The present invention relates to a biocompatible type enzyme complex,

More particularly, the present invention relates to a biocompatible enzyme complex and a method for producing the biocompatible enzyme complex, and more particularly, to a biocompatible enzyme complex which is harmless to the human body due to its excellent biocompatibility and induces more crosslinking between enzymes than conventional binders, The present invention relates to a biocompatible enzyme complex capable of accumulating more enzymes in a limited area and exhibiting an effect of stably exhibiting enzyme characteristics for a long period of time without being influenced by the expression of the enzyme activity. will be.

The selectivity of enzymes as eco-friendly biocatalysts has been successfully used in a wide range of fields such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), glucose sensor, In recent years, applications have been proposed in various fields such as carbon dioxide capture, biofuel cell, biosensor, protein hydrolysis, and antifouling. However, due to the instability of the enzyme itself, various applications of these enzymes are restricted.

In other words, enzymes can be used in various fields such as biosensors, biofuel cells, enzyme columns, enzyme immunoassay (ELISA), biochemical purification equipment and antifouling coatings (antifouling agents), but due to instability due to structural modification of enzymes It is impossible to utilize it continuously for a long time. Therefore, in order to solve this problem, the enzyme is immobilized on the surface and cross-linking between the enzymes is induced, thereby maximizing the enzyme loading and solving the enzyme instability.

Meanwhile, according to the conventional method of attaching and fixing an enzyme through cross-linking, it is possible to attach an enzyme through simple adsorption, or to adsorb the enzyme through glutaraldehyde (GA) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Crosslinking agents are used to induce surface attachment or crosslinking between enzymes. However, in the case of simple adsorption, since the enzyme is not chemically bound, the enzyme easily leaks and may impair applicability.

At this time, using a cross-linking agent such as GA and EDC can chemically attach an enzyme to an arbitrary surface and induce chemical bonding between the enzymes, thereby preventing enzyme leakage and greatly improving stability. However, the cross-linking agents such as GA and EDC and the solvent used to dissolve them have a problem of low biocompatibility. Therefore, bioadhesion or absorption / insertion by the cross-linking agent and solvent after the cross- The various applications required may be limited.

That is, the enzyme is an environmentally friendly biocatalyst. When the enzyme is stably fixed at the time of bioadhesion or absorption and insertion into the body and harmless in vivo, the enzyme can be used as a biocompatible material for biomaterials for diagnosis / It can be applied to various applications such as an electrode material, an antifouling material for medical use, and a material for removing calculus.

Specifically, it is applied to patches of the cosmetics field as an example of application, and it is possible to repeatedly induce an enzyme reaction that decomposes harmful substances, produce biodegradable substances during skin contact, or utilize biodegradable biodegradable crosslinking agents Thereby biodegrading with time to provide the function of continuously transmitting the cosmetic ingredient containing the enzyme or the enzyme to the skin.

Accordingly, the inventors of the present invention have been studying ways to overcome the problems that various applications requiring bioadhesion or absorption / insertion in the body may be limited. In comparison with the conventional enzyme immobilization methods, biocompatibility is improved, The present invention has been completed based on the discovery of a composition for enzyme bonding excellent in enzyme concentration and stability.

KR 10-2013-0136991 A (Disclosure date 2013.12.13)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a biocompatibility, which is harmless to the human body and induces more cross- Which is capable of accumulating more enzymes in a limited region, and at the same time, the attached enzymes are not affected by the expression of enzyme activity, and exhibit an effect of expressing the enzyme characteristics stably for a long period of time, and a method for producing the same .

In order to solve the above-described problems, the present invention provides a method for producing a biocompatible enzyme complex comprising preparing a biocompatible enzyme adhesive and an enzyme, and immobilizing the enzyme through a biocompatible enzyme adhesive.

According to a preferred embodiment of the present invention, the biocompatible enzyme adhesive is a compound containing dopamine, a compound containing a dopamine-derived catechol, genipin, and ethylene glycol diglycidyl ether (EGDGE) And may include at least one selected.

According to another preferred embodiment of the present invention, the enzyme is selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, suomolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, , Organic phosphohydrolase, choline esterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, glucose isomerase and horseradish peroxidase.

According to another preferred embodiment of the present invention, the enzyme may be a non-aggregating enzyme or an enzyme aggregate.

According to another preferred embodiment of the present invention, the enzyme aggregate is selected from the group consisting of ammonium sulfate, methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, sodium chloride, And aggregating the enzyme with a precipitating agent comprising at least one selected from the group consisting of sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate.

According to another preferred embodiment of the present invention, the step of preparing the biocompatible enzyme adhesive and the enzyme, respectively, further comprises the steps of preparing a support and modifying the support to prepare a surface-modified support .

According to another preferred embodiment of the present invention, the support may include at least one selected from nanofibers and chitosan nanoparticles.

The biocompatible enzyme complex of the present invention and its preparation method are harmless to the human body due to their excellent biocompatibility and induce more cross-linking between enzymes than conventional binders, The enzymes capable of accumulating the enzyme and being attached thereto are not affected by the expression of the enzyme activity, so that they can exhibit stable enzyme characteristics for a long period of time.

1 is a polymer nanofiber according to a preferred embodiment of the present invention.
2 is a flow chart for modifying chitosan nanoparticles according to a preferred embodiment of the present invention.
FIG. 3A is a photograph of the nanofiber before enzyme adhesion, and FIG. 3B is a photograph of the enzyme complex according to Example 1. FIG.
4 is a graph showing the adhesive ability of the enzyme complex according to Example 1. Fig.
5 to 13 are graphs showing the activity measurement of the enzyme complex according to Examples 2 to 28 and Comparative Examples 1 to 5, respectively.
14 is a graph showing the stability of the enzyme complex according to Examples 13 to 16 and Comparative Example 5.
15 is a flow chart for fixing an enzyme to a covalent conjugate according to a preferred embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

As described above, according to the conventional method, by attaching an enzyme through simple adsorption or by using a crosslinking agent such as glutaraldehyde (GA) or 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) Adhesion or cross-linking between enzymes. However, in the case of simple adsorption, since the enzyme is not chemically bound, the enzyme easily leaks and may impair applicability. On the other hand, by using a cross-linking agent such as GA and EDC, it is possible to chemically attach an enzyme to an arbitrary surface and induce chemical bonding between enzymes, thereby preventing enzyme leakage and greatly improving stability. However, the biocompatibility of the crosslinking agent and the solvent to be dissolved therewith is poor, and the crosslinking agent used for the coupling and the crosslinking agent and the solvent remaining after the synthesis may limit various applications requiring bioadhesion or absorption / insertion in the body have.

Accordingly, the present invention provides a method for preparing a biocompatible enzyme complex comprising preparing a biocompatible enzyme adhesive and an enzyme, and immobilizing the enzyme through a biocompatible enzyme adhesive, thereby solving the above-mentioned problems. As a result, unlike the conventional invention, it is harmless to the human body due to its excellent biocompatibility. It induces more crosslinking between enzymes than conventional binders, and thus has strong adhesive force of enzymes and can accumulate more enzymes in a limited area At the same time, the attached enzymes are not influenced by the expression of the enzyme activity, so that the effect of stably expressing the enzyme characteristics for a long period of time can be achieved.

The enzyme complex according to the production method of the present invention may or may not include a support.

According to a preferred embodiment of the present invention, in the case of including a support, before preparing the biocompatible enzyme adhesive and enzyme, preparing a support, and modifying the support to prepare a surface-modified support As shown in FIG.

On the other hand, the support may be any kind of support that can be biocontacted or absorbed and inserted into a living body, and may be one kind of support selected from the group consisting of planar, spherical, bead, polygonal, And more preferably at least one selected from the group consisting of nanofibers and chitosan nanoparticles.

When the nanofiber is used as the support, the nanofiber may be any nanofiber prepared by a method capable of producing nanofibers. Preferably, the nanofiber may be nanofibers prepared by electrospinning. have.

When the chitosan nanoparticles are used as the support, the chitosan nanoparticles are mixed with a surfactant containing chitosan, tripolyphosphate (TPP) and polysorbate in a weight ratio of 1: 0.01 to 10: 1 to 20 , Preferably from 1: 0.01 to 0.3: 5 to 15, by weight. If the weight ratio of the chitosan nanofibers and the tripolyphosphate is less than 1: 0.01, nanoparticles may not be formed. If the weight ratio is more than 1:10, coagulation may occur and chitosan nanoparticles may be easily produced Problems may arise. In addition, if the weight ratio of the chitosan nanofibers and the surfactant exceeds 1:20, bubbles may be generated excessively.

Next, the step of modifying the support to prepare a support having a modified surface will be described.

FIG. 2 is a flow chart for modifying chitosan nanoparticles according to a preferred embodiment of the present invention. When the support is chitosan nanoparticles, the modification may include reacting the chitosan nanoparticles with ethylene glycol diglycidyl ether, And reacting the reacted chitosan nanoparticles with an enzyme to prepare a covalent attachment (CA).

Meanwhile, the ethylene glycol diglycidyl ether may be used in an amount of 1 to 1000 parts by weight, preferably 110 to 550 parts by weight, based on 100 parts by weight of the chitosan nanoparticles. If the amount of the ethylene glycol diglycidyl ether is less than 1 part by weight, the surface of the chitosan nanoparticles may not be well modified. If the amount of the ethylene glycol diglycidyl ether is more than 1,000 parts by weight, aggregation may occur, Problems can arise. The enzyme may be used in an amount of 1 part by weight or more, preferably 100 to 1000 parts by weight, based on 100 parts by weight of the chitosan nanoparticles. If the amount of the enzyme is less than 1 part by weight, it may be difficult to produce a covalent conjugate.

Next, steps of preparing the biocompatible enzyme adhesive and enzyme, respectively, will be described.

The biocompatible enzyme adhesive is preferably a biocompatible material in which an enzyme is required to be in contact with a living body or can be absorbed and inserted into a living body. Preferably, the biocompatible enzyme adhesive is a compound containing dopamine, a dopamine-derived catechol group, a genipin ), And ethylene glycol diglycidyl ether (EGDGE). According to the present invention, an enzyme complex capable of being safely introduced into a living body and capable of supporting a remarkably large amount of enzyme can be produced without a supporter as well as an extremely large amount of enzyme can be supported and fixed on a support, Since the immobilized enzyme can not easily flow out, its stability is maintained even after a long period of time has elapsed.

The enzyme is selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, sumolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, lactase, organic phosphohydrolase, choline esterase , At least one selected from the group consisting of glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, glucose isomerase and horseradish peroxidase, preferably at least one selected from trypsin and glucose oxidase .

Alternatively, the enzyme may be a non-aggregating enzyme or an enzyme aggregate, the non-aggregating enzyme may include trypsin, and the enzyme aggregate may include a glucose oxidase.

The enzyme aggregate is selected from the group consisting of ammonium sulfate, methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate As the precipitating agent containing at least one kind of precipitating agent, preferably at least one selected from the group consisting of ammonium sulfate, methanol, ethanol, 1-propanol, 2-propanol and butyl alcohol, And then coagulating the enzyme with a precipitating agent containing ammonium sulfate.

Next, the step of immobilizing the enzyme through a biocompatible enzyme adhesive will be described.

The immobilization step is not usually limited as long as it is a method capable of producing an enzyme complex.

First, a case where a support is included will be described.

Wherein the immobilization step comprises immobilizing at least 1 part by weight of non-aggregating enzyme and at least 1 part by weight of a biocompatible enzyme adhesive to 100 parts by weight of the modified support, Preferably, 100 to 1000 parts by weight of the non-aggregating enzyme and 100 to 2000 parts by weight of the biocompatible enzyme adhesive are reacted, but the present invention is not limited thereto.

If the amount of the non-aggregated enzyme is less than 1 part by weight with respect to 100 parts by weight of the modified support, a problem may occur that the enzyme activity at the desired level is not exhibited. If the biocompatible enzyme adhesive is less than 1 part by weight, A non-fixed problem may occur.

When the enzyme is an enzyme aggregate, the immobilization step comprises reacting the enzyme and the precipitating agent at a weight ratio of 1: 1 to 150, preferably 1: 75 to 125, to prepare an enzyme aggregate; and 1 part by weight or more of an enzyme aggregate and 1 part or more by weight of a biocompatible enzyme adhesive, preferably 100 to 1000 parts by weight of an enzyme aggregate and 100 to 2000 parts by weight of a biocompatible enzyme adhesive are reacted with 100 parts by weight of the modified support But is not limited thereto.

If the weight ratio of the enzyme and the precipitating agent is less than 1: 1 in the step of preparing the enzyme aggregate, there may arise a problem that the enzyme is not precipitated well. If the amount of the enzyme aggregate is less than 1 part by weight based on 100 parts by weight of the modified support, there may arise a problem that the desired level of enzyme activity is not exhibited. If the amount of the biocompatible enzyme adhesive is less than 1 part by weight, There is no problem.

Next, a case where a support is not included will be described.

If the support is not included, the immobilization may be performed by reacting 100 to 15000 parts by weight, preferably 7500 to 12500 parts by weight, of the precipitating agent with respect to 100 parts by weight of the enzyme to prepare an enzyme aggregate, Type enzyme adhesive at a weight ratio of 1: 0.1 ~ 5, preferably 1: 1 ~ 2. If the amount of the precipitating agent is less than 100 parts by weight based on 100 parts by weight of the enzyme in the step of preparing the enzyme aggregate, the problem that the enzyme does not precipitate well may occur. In addition, if the weight ratio of the enzyme aggregate and the biocompatible enzyme adhesive is less than 1: 0.1, the enzyme may not be fixed well.

Meanwhile, the immobilization step can be used without limitation as long as it is a conventional enzyme immobilization condition, preferably at 25 to 75 rpm at 1 to 10 ° C for 13 to 21 hours, more preferably at 15 to 75 rpm at 2 to 7 ° C. To 19 hours, preferably 35 to 65 rpm, to prepare an enzyme complex, but the present invention is not limited thereto.

Hereinafter, the present invention will be described with reference to the following examples. The following examples are provided to illustrate the invention, but the scope of the present invention is not limited by the following examples.

[ Example ]

Preparation Example 1: Nanofiber

Polystyrene and styrene maleic anhydride copolymer at a weight ratio of 1: 0.5 was electrospun at a voltage of 7 kV and a flow rate of 0.1 ml / hour to prepare nanofibers.

Preparation Example  2: chitosan nanoparticles

Chitosan, tripolyphosphate (TPP) and polysorbate were reacted at a weight ratio of 1: 0.12: 11 at 4 캜 for 17 hours at 50 rpm and reacted to prepare chitosan nanoparticles.

Example  1: Preparation of enzyme complex

The nanofibers prepared according to Preparation Example 1 were treated with ethanol to prepare polymer nanofibers having expanded fiber spaces as shown in FIG. 3A. Subsequently, 200 parts by weight of dopamine as a biocompatible enzyme adhesive and 100 parts by weight of glucose oxidase were reacted with 100 parts by weight of nanofibers to prepare an enzyme complex as shown in FIG. 3B.

Example  2

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 110 parts by weight of ethylene glycol diglycidyl ether and 500 parts by weight of glucose oxidase were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the glucose oxidase to prepare a covalent conjugate.

Then, ammonium sulfate was reacted with a glucose oxidase and a precipitating agent at a weight ratio of 1: 125 to prepare a glucose oxidase aggregate. To 100 parts by weight of the covalent conjugate, 500 parts by weight of a glucose oxidase aggregate, Were reacted at 50 rpm at 4 캜 for 17 hours and reacted to prepare an enzyme complex.

Example  3

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 110 parts by weight of ethylene glycol diglycidyl ether and 500 parts by weight of trypsin were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the trypsin to prepare a covalent conjugate.

Thereafter, 500 parts by weight of trypsin and 1400 parts by weight of Jenny pin were racked at 50 rpm for 17 hours at 4 ° C and reacted with 100 parts by weight of the covalent conjugate to prepare an enzyme complex.

Example  4

The nanofibers prepared according to Preparation Example 1 were treated with ethanol to prepare polymer nanofibers having expanded fiber spaces as shown in FIG. 3A. Thereafter, ammonium oxidase and ammonium sulfate were reacted at a weight ratio of 1:20 to prepare a glucose oxidase aggregate. To 100 parts by weight of the nanofiber, 6000 parts by weight of glucose oxidase aggregate and 3450 by weight of EGDGE Was shaken at 25 rpm for 6 days at 100 rpm and reacted to prepare an enzyme complex.

Example  5

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 460 parts by weight of ethylene glycol diglycidyl ether and 5000 parts by weight of glucose oxidase were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the glucose oxidase to prepare a covalent conjugate.

Thereafter, ammonium oxidase and ammonium sulfate were reacted at a weight ratio of 1:75 to prepare a glucose oxidase aggregate. To 100 parts by weight of the covalent conjugate, 5,000 parts by weight of glucose oxidase aggregate and 4,000 to 5,000 parts by weight of EGDGE, 4370 parts by weight were racked at 35 rpm for 4 hours at 50 rpm and reacted to prepare an enzyme complex.

Example  6

The reaction was carried out in the same manner as in Example 5 except that ammonium sulfate was used as a glucose oxidase and a precipitating agent at a weight ratio of 1: 100.

Example  7

Was prepared in the same manner as in Example 5 except that ammonium sulfate was used as a glucose oxidase and a precipitating agent at a weight ratio of 1: 125.

Example  8

The procedure of Example 5 was repeated except that no precipitating agent was used.

Example  9

The procedure of Example 5 was repeated except that 460 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  10

The procedure of Example 6 was repeated except that 460 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  11

Was carried out in the same manner as in Example 7, except that 460 parts by weight of the covalently bonded conjugate was reacted with 100 parts by weight of the covalent conjugate.

Example  12

The procedure of Example 9 was repeated except that no precipitating agent was used.

Example  13

The reaction was carried out in the same manner as in Example 5 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  14

The reaction was carried out in the same manner as in Example 6 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  15

The reaction was carried out in the same manner as in Example 7 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  16

The procedure of Example 13 was repeated except that no precipitating agent was used.

Example  17

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 460 parts by weight of ethylene glycol diglycidyl ether and 5000 parts by weight of glucose oxidase were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the glucose oxidase to prepare a covalent conjugate.

Thereafter, ammonium oxidase and ammonium sulfate were reacted at a weight ratio of 1:75 to prepare a glucose oxidase aggregate. To 100 parts by weight of the covalent conjugate, 5,000 parts by weight of glucose oxidase aggregate and 4,000 to 5,000 parts by weight of EGDGE, 4370 parts by weight was racked at 35 rpm for 8 hours at 50 rpm and reacted to prepare an enzyme complex.

Example  18

The reaction was carried out in the same manner as in Example 17 except that ammonium sulfate was used as a glucose oxidase and a precipitating agent at a weight ratio of 1: 100.

Example  19

The reaction was carried out in the same manner as in Example 17 except that ammonium sulfate was used as a glucose oxidase and a precipitating agent at a weight ratio of 1: 125.

Example  20

The procedure of Example 17 was repeated except that no precipitating agent was used.

Example  21

The procedure of Example 17 was repeated except that 460 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  22

The procedure of Example 18 was repeated except that 460 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  23

The reaction was carried out in the same manner as in Example 19 except that 460 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  24

The procedure of Example 21 was repeated except that no precipitating agent was used.

Example  25

The reaction was carried out in the same manner as in Example 17 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  26

The reaction was carried out in the same manner as in Example 18 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  27

The reaction was carried out in the same manner as in Example 19 except that 862.5 parts by weight of EGDGE was reacted with 100 parts by weight of the covalent bond.

Example  28

The procedure of Example 25 was repeated except that no precipitating agent was used.

Comparative Example  One

The nanofibers prepared according to Preparation Example 1 were treated with ethanol to prepare polymer nanofibers having expanded fiber spaces as shown in FIG. 3A.

Comparative Example  2

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 110 parts by weight of ethylene glycol diglycidyl ether and 500 parts by weight of glucose oxidase were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the glucose oxidase to prepare a covalent conjugate.

Comparative Example  3

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 110 parts by weight of ethylene glycol diglycidyl ether and 500 parts by weight of trypsin were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the trypsin to prepare a covalent conjugate.

Comparative Example  4

The nanofibers according to Preparation Example 1 were treated with ethanol to prepare polymer nanofibers having expanded fiber spaces as shown in FIG. 3A, and 6000 parts by weight of glucose oxidase was reacted with 100 parts by weight of the nanofibers to obtain an enzyme complex .

Comparative Example  5

To 100 parts by weight of the chitosan nanoparticles according to Preparation Example 2, 460 parts by weight of ethylene glycol diglycidyl ether and 5000 parts by weight of glucose oxidase were prepared. The chitosan nanoparticles and ethylene glycol diglycidyl ether were reacted, and the reacted chitosan nanoparticles were reacted with the glucose oxidase to prepare a covalent conjugate.

Experimental Example  1: Measurement of activity

TMB, glucose, horseradish peroxid and the enzymatic complexes according to Examples 2 to 28 and Comparative Examples 2 to 5 were reacted at a weight ratio of 9: 1 and then irradiated at a wavelength of 655 nm at 23 ° C to give a mixture Was measured and the activity was measured.

FIG. 5 is a graph showing the activity of the enzyme complex according to Example 2 and Comparative Example 2. As shown in FIG. 5, the activity of the enzyme complex according to Example 2 was higher than that of the enzyme complex according to Comparative Example 2 .

FIG. 6 is a graph showing the activity measurement of the enzyme complex according to Example 3 and Comparative Example 3. As shown in FIG. 6, the activity of the enzyme complex according to Example 3 was significantly higher than that of the enzyme complex according to Comparative Example 3 It can be seen that it is high.

7 is a graph showing the activity measurement of the enzyme complex according to Example 4 and Comparative Example 4. As shown in FIG. 7, the activity of the enzyme complex according to Example 4 is significantly higher than that of the enzyme complex according to Comparative Example 4 It can be seen that it is high.

8 is a graph showing the activity measurement of the enzyme complex according to Examples 5 to 8 and Comparative Example 5. As shown in FIG. 8, the activity of the enzyme complex according to Examples 5 to 8 is higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

9 is a graph showing the activity of the enzyme complex according to Examples 9 to 12 and Comparative Example 5. As shown in FIG. 9, the activity of the enzyme complex according to Examples 9 to 12 is higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

10 shows the activity measurement of the enzyme complex according to Examples 13 to 16 and Comparative Example 5. As shown in FIG. 10, the activity of the enzyme complex according to Examples 13 to 16 was higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

11 is a graph showing the activity measurement of the enzyme complex according to Examples 17 to 20 and Comparative Example 5. As can be seen from FIG. 11, the activity of the enzyme complex according to Examples 17 to 20 is higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

12 shows the activity measurement of the enzyme complex according to Examples 21 to 24 and Comparative Example 5. As shown in FIG. 12, the activity of the enzyme complex according to Examples 21 to 24 is higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

FIG. 13 is a graph showing the activity of the enzyme complex according to Examples 25 to 28 and Comparative Example 5. As shown in FIG. 10, the activity of the enzyme complex according to Examples 25 to 28 is higher than that of the enzyme complex according to Comparative Example 5 Which is higher than the activity.

Experimental Example  2: Measurement of adhesion ability

The adhesive capacity of the enzyme complex according to Example 1 and Comparative Example 1 was measured according to the following relational expression (1).

[Relation 1]

Adhesion capacity = (amount of enzyme reacted - amount of enzyme reduced after washing) / (amount of enzyme reacted)

4 is a graph showing the adhesive ability of the enzyme conjugate according to Example 1 and Comparative Example 1. As shown in FIG. 4, the enzyme conjugate according to Example 1 has an adhesive ability Is improved by three times or more.

Experimental Example 3: Measurement of enzyme stability

When the enzyme complexes according to Examples 13 to 16 and Comparative Example 5 were stored at a high temperature of 60 ° C, the enzyme stability was measured according to changes in enzyme activity.

As can be seen from FIG. 14, the enzymatic complexes according to Examples 13 to 16 have improved stability by about 20% or more as compared with the enzyme complex according to Comparative Example 5.

Claims (7)

Preparing a biocompatible enzyme adhesive and an enzyme, respectively; And
And immobilizing the enzyme through a biocompatible enzyme adhesive. The biocompatible enzyme adhesive according to claim 1, wherein the biocompatible enzyme adhesive comprises at least one compound selected from the group consisting of dopamine, a compound including a dopamine-derived catechol group, genipin, and ethylene glycol diglycidyl ether (EGDGE) Wherein the method comprises the steps of: The method of claim 1, wherein the enzyme is selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, sumolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, lactase, Wherein the enzyme is at least one selected from the group consisting of glucose, glucose, glucose, glucose, glucose, glucose, glucose, glucose, glucose, glucose, dextrose, glucose, dextrose, glucose, dextrose, glucose dehydrogenase and horseradish peroxidase. The method according to claim 1, wherein the enzyme is a non-aggregating enzyme or an enzyme aggregate. 5. The method of claim 4 wherein the enzyme aggregate is selected from the group consisting of ammonium sulfate, methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, sodium chloride, sodium sulfate, sodium phosphate, Wherein the enzyme is produced by agglutinating an enzyme with an oxidizing agent comprising at least one selected from the group consisting of potassium sulfate and potassium phosphate. 2. The method according to claim 1, wherein before preparing the biocompatible enzyme adhesive and the enzyme,
Preparing a support; And
Preparing a support having a surface modified by modifying the support; and preparing a support having the modified surface by modifying the support. The biocompatible material according to claim 6, wherein the support comprises at least one selected from polymer fibers, graphene, monolithic columns, carbon paper, carbon nanotubes, fumed silica, polymer beads and chitosan nanoparticles Type enzyme complex.

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