KR20160092652A - chitosan coated enzyme-porous material complex and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a chitosan coated enzyme-porous material composite and a method of manufacturing the same. The method of manufacturing the same comprises the steps of: (A) making enzymes to be adsorbed inside the pores of a porous carrier; (B) making chitosan to be adsorbed on a surface of the porous carrier with the enzyme adsorbed; and (C) adding glutaric dialdehyde to the chitosan and performing a crosslinking between the amine group of the chitosan, thereby minimizing denaturalization of the enzymes and effectively preventing the leakage of the enzymes adsorbed inside the porous carrier. Furthermore, the composition can be applied to various items such as biologically-converted materials, biologically-purifying materials, anti-fouling pigments, biocatalytic materials for detergents, enzyme-based columns, packing materials for the conversion of carbon dioxide, biosensors, and bio-fuel cells.

Description

TECHNICAL FIELD The present invention relates to a chitosan-coated enzyme-porous material composite and a method for producing the same,

The present invention relates to a crosslinked chitosan-coated enzyme-porous material composite capable of effectively preventing leakage of an enzyme adsorbed inside a porous carrier, and a method for producing the same.

In order to improve the economical efficiency of the enzyme, it is necessary to reuse and recover the enzyme through the immobilized enzyme. In addition, the use of the immobilized enzyme can be very simple in designing and controlling the reactor, which is an essential element for industrially utilizing the enzyme.

Porous materials utilize the high surface area of the pores formed in the interior to realize a much higher enzyme loading than conventional methods. As a general enzyme immobilization method using the porous material, there is a method of adsorption or covalent attachment using covalent bonding. However, the simple adsorption method can not prevent the leakage of the enzyme in the pores, and there is a problem that the pores of the porous material can not be efficiently utilized by attaching the enzyme only to the inner surface of the porous material through the covalent bond.

To solve this problem, the Nanoscale Enzyme Reactor (NER) approach has been devised in a very simple and effective way to increase the amount of enzyme loaded in the porous material and prevent the leakage of the enzyme. The NER technique, also referred to as a ship-in-a-bottle approach, can stabilize the enzyme activity by effectively preventing the enzyme from leaking by cross-linking the enzyme in the pores of the porous material. This technique has various advantages such as not only the stability of the enzyme activity is improved but also the high concentration of the enzyme and the reduction of the mass transfer resistance of the reaction substrate. However, in the cross-linking between the enzymes, degradation of the enzyme by structural modification may occur. Particularly, in the case of some enzymes in which an amino group capable of reacting with a cross-linking agent exists in the active site, May occur.

Therefore, there is a need for an innovative enzyme immobilization technology capable of preventing leakage of an enzyme accumulated in a porous material and preventing structural modification of the enzyme by cross-linking.

Korean Patent Publication No. 2014-0055566 Korea Patent Publication No. 2013-0130640

It is an object of the present invention to provide a method for producing an enzyme-porous material composite coated with crosslinked chitosan so as to effectively prevent leakage of an enzyme adsorbed inside the porous carrier.

Another object of the present invention is to provide a chitosan-coated enzyme-porous material composite produced by the above-described method.

In order to accomplish the above object, the present invention provides a method of producing a chitosan-coated enzyme-porous material composite, comprising the steps of: (A) adsorbing an enzyme in pores of a porous carrier; (B) adsorbing chitosan on the surface of the porous support on which the enzyme is adsorbed; And (C) reacting the chitosan with a cross-linking agent to cross-link the amine groups of the chitosan.

The crosslinking agent is selected from the group consisting of glutaric dialdehyde, diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, bis (imidoesters), bis (succinimidyl ester) It can be more than a species.

The chitosan solution may have a concentration of 0.01 to 10.0 mg / mL, preferably 0.1 to 1.0 mg / mL.

The crosslinking agent may be a solution of glutaric dialdehyde in an amount of 0.0001 to 10.0% (w / w), preferably 0.001 to 1.0% (w / w).

The enzyme may be selected from the group consisting of carbonic anhydrase, trypsin, glucose oxidase, alcohol dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase And may be at least one selected from the group consisting of

A cofactor may be further adsorbed inside the pores of the porous carrier.

The cofectors may be at least one selected from the group consisting of hydrogen transfer coenzyme and enzyme coenzyme.

The porous carrier may be one selected from the group consisting of a porous silica carrier, a porous carbon carrier, and a polymer carrier.

According to another aspect of the present invention, there is provided a chitosan-coated enzyme-porous material composite according to the present invention.

In order to achieve the above-mentioned other objects, the chitosan-coated enzyme-porous material composite according to the present invention may be used for bio-conversion material, biocompatible material, antifouling coating material, biocatalyst material for detergent, Packing material, biosensor or biofuel cell.

The chitosan-coated enzyme-porous material composite of the present invention can be prepared by coating the surface of a porous carrier on which an enzyme is adsorbed in pores with a chitosan coating layer by using a porous carrier and glutaric dialdehyde to effectively prevent leakage of the adsorbed enzyme And can enhance enzyme activity and stability.

In addition, the chitosan-coated enzyme-porous material composite of the present invention can be used as a bio-conversion material, an antifouling coating material, a biocatalyst material for detergent, an enzyme-based column, A carbon dioxide conversion packing material, a biosensor or a biofuel cell.

1 is a view illustrating a process for preparing a chitosan-coated enzyme-porous material composite according to an embodiment of the present invention.
2A is a graph showing the initial activity of the immobilized carbonic anhydrase of the complex prepared according to Examples and Comparative Examples of the present invention.
FIG. 2B is a graph showing the stability of the immobilized carbonic anhydrase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
FIG. 3 is a graph showing the content of carbonate produced according to a cycle using a carbonic anhydrase loaded on and immobilized on a complex prepared according to another embodiment of the present invention.
4A is a graph showing the initial activity of immobilized trypsin of the complex prepared according to the examples and comparative examples of the present invention.
FIG. 4B is a graph showing the stability of immobilized trypsin of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
FIG. 5A is a graph showing the initial activity of the immobilized glucose oxidase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
FIG. 5B is a graph showing the stability of immobilized glucose oxidase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
6A is a graph showing the initial activity of the immobilized alcohol dehydrogenase of the complex prepared according to Examples and Comparative Examples of the present invention.
FIG. 6B is a graph showing the stability of the immobilized alcohol dehydrogenase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
FIG. 7A is a graph showing the initial activity of the immobilized formate dehydrogenase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.
7B is a graph showing the stability of the immobilized formate dehydrogenase of the complex prepared according to Examples and Comparative Examples of the present invention.
8A is a graph showing the initial activity of the immobilized formaldehyde dehydrogenase of the complex prepared according to the examples and comparative examples of the present invention.
FIG. 8B is a graph showing the stability of the immobilized formaldehyde dehydrogenase of the complex prepared according to Examples and Comparative Examples of the present invention. FIG.

The present invention relates to a chitosan-coated enzyme-porous material composite capable of minimizing denaturation of an enzyme upon cross-linking and effectively preventing leakage of an enzyme adsorbed inside the porous carrier, and a method for producing the same.

Hereinafter, the present invention will be described in detail.

The method for preparing a chitosan-coated enzyme-porous material composite according to the present invention comprises the steps of: (A) adsorbing an enzyme in pores of a porous carrier; (B) adsorbing chitosan on the surface of the porous support on which the enzyme is adsorbed; And (C) reacting the chitosan with a cross-linking agent to cross-link the amine groups of the chitosan.

First, in step (A), the enzyme is adsorbed in the pores of the porous carrier.

The enzyme may be at least one selected from the group consisting of carbonic anhydrase, trypsin, glucose oxidase, alcohol dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase.

In addition to the enzymes used in the present invention, the enzyme may be selected from the group consisting of subtilisin, papain, lipase, horseradish peroxidase, soybean peroxidase, chloroperoxidase, manganese peroxidase, thyroxinase, lactase, cellulase, xylanase, A cholesterol oxidase, a cholesterol oxidase, a polyphenol oxidase, a monoamine oxidase, a xanthine oxidase, a cytochrome oxidase, an ascorbic acid oxidase, a cholesterol oxidase, , And D-arabino-1,4-lactone oxidase can also be used.

In addition, by using one kind selected from the group consisting of a porous silica carrier, a porous carbon carrier, and a polymer carrier as the porous carrier, chitosan is coated on the surface of the porous carrier when the cross-linking agent is used as much as possible to prevent leakage of the enzyme to the outside of the porous carrier do. At this time, a porous alumina carrier, a niobium carrier, a tantalum carrier, a zirconium carrier, a titanium carrier, and a vinyl polymer carrier may be used instead of the silica carrier, the carbon carrier and the polymer carrier as the porous carrier.

A cofactor may be adsorbed together with the enzyme in the pores of the porous carrier.

The cofectors may be at least one selected from the group consisting of hydrogen transfer coenzyme and enzyme coenzyme.

Next, in step (B), chitosan is adsorbed on the surface of the porous carrier on which the enzyme is adsorbed.

As shown in FIG. 1, the chitosan is located on the surface of the porous carrier but is not in intimate contact with the porous carrier or tightly bonded to the chitosan.

Next, in step (C), chitosan located on the surface of the porous carrier is crosslinked using a crosslinking agent.

When the chitosan and the cross-linking agent are reacted, cross-linking between the amine groups of the chitosan is performed, so that the chitosan is tightly bonded to each other to form a chitosan coating layer on the surface of the nanoporous support.

The crosslinking agent may be selected from the group consisting of glutaric dialdehyde, diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, bis (imidoester), bis (succinimidyl ester) and diacid chloride , And preferably at least one selected from glutaric dialdehyde.

In the present invention, since the crosslinking agent is used, the chitosan coating layer and the surface of the porous carrier are adhered without being bonded.

The chitosan solution may have a concentration of 0.01 to 10.0 mg / mL, preferably 0.1 to 1.0 mg / mL. When the concentration of the chitosan solution is less than the lower limit, the chitosan coating layer may not be formed. If the concentration exceeds the upper limit, the chitosan coating layer and the surface of the porous support may not be adhered to each other.

The crosslinking agent may be a solution of glutaric dialdehyde in an amount of 0.0001 to 10.0% (w / w), preferably 0.001 to 1.0% (w / w). When the concentration of the crosslinking agent solution is less than the lower limit, the activity and stability of the enzyme-porous material composite coated with chitosan may be lowered. If the concentration exceeds the upper limit, the chitosan coating layer is closely adhered to the surface of the porous carrier, And the flow-in of the products may be limited.

In particular, the lower the concentration of the cross-linking agent solution is, the less easily the cross-linking of chitosan can be prevented and the leakage of the enzyme can not be effectively prevented. When the amino group capable of bonding with the cross-linking agent exists in the reaction site such as an alcohol dehydrogenase, The activity is greatly reduced because it also affects the internal enzymes. Therefore, it is preferable to set the concentration of the crosslinking agent suitable for each enzyme.

The chitosan-coated enzyme-porous material composite prepared according to the above-described method can enhance enzyme activity and stability, and can be used as a bio-conversion material, a biocompatible material, an antifouling coating material, a detergent Can be applied to the development of various materials such as biocatalyst material, enzyme-based column, packing material for carbon dioxide conversion, biosensor, and biofuel cell.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

<Carbonic anhydrase >

Example 1. Carbonic anhydrase

To immobilize the carbonic anhydrase, 2 mL of a carbonic anhydrase solution (4 mg _enzyme / ml _PB , 100 mM sodium phosphate buffer, PB) was added to 4 mg of spherical nanoporous silica capable of magnetic separation. Gt; 200 &lt; / RTI &gt; After that, the enzyme which was not adsorbed was removed by magnetic separation and 2 mL of 0.5 mg / mL chitosan solution was added. The chitosan was removed by magnetic separation after stirring at 200 rpm for 15 hours at room temperature to sufficiently adsorb the chitosan molecules on the surface of the silica. Thereafter, 0.001% glutaraldehyde (GA) was added as a crosslinking agent and stirring was carried out at 200 rpm for 2 hours to induce crosslinking between the chitosan, and the remaining glutaric dialdehyde To inactivate, 2 mL of PB was added and stirred at 200 rpm for 2 hours and further stirred at 200 rpm for 1 hour using Tris buffer (100 mM Tris buffer) to prepare an enzyme-porous material composite coated with chitosan . Subsequently, the enzyme-immobilized substance was washed with PB and stored at 4 ° C.

Example 2. Carbonic anhydrase

The procedure of Example 1 was repeated except that 0.01% glutaric dialdehyde was used instead of 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example 3. Carbonic anhydrase

The procedure of Example 1 was repeated except that 0.1% glutaric dialdehyde was used instead of the 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Comparative Example 1. Chitosan and glutaric dialdehyde not used

2 mL of a carbonic anhydrase solution (4 mg _enzyme / ml _PB , 100 mM sodium phosphate buffer, PB) was added to 4 mg of spherical nanoporous silica particles capable of magnetic separation, and the mixture was stirred at 200 rpm for 1 hour Complex. The complex was then washed with PB and stored at 4 &lt; 0 &gt; C.

Comparative Example 2. Chitosan not used

2 mL of a carbonic anhydrase solution (4 mg _enzyme / ml _PB , 100 mM sodium phosphate buffer, PB) was added to 4 mg of spherical nanoporous silica particles capable of magnetic separation, and the mixture was stirred at 200 rpm for 1 hour . The enzyme, which was not adsorbed, was then removed by magnetic separation and 2 mL of 0.1% glutaric dialdehyde solution was added. Thereafter, the mixture of the enzyme adsorbing material and glutaric dialdehyde solution was allowed to stand for 30 minutes, stirred at 200 rpm for 30 minutes, and then stirred at 200 rpm for 1 hour using Tris buffer solution (100 mM Tris buffer) . The complex was then washed with PB and stored at 4 &lt; 0 &gt; C.

Comparative Example  3. Glutaric Dialdehyde  use Do not

2 mL of a carbonic anhydrase solution (4 mg _enzyme / ml _PB , 100 mM sodium phosphate buffer, PB) was added to 4 mg of spherical porous silica capable of magnetic separation, and the mixture was stirred at 200 rpm for 1 hour. Then, the non-adsorbed enzyme was removed by magnetic separation, and 2 mL of a 0.5 mg / mL chitosan solution was added. The chitosan was removed by magnetic separation after stirring at 200 rpm for 15 hours at room temperature to sufficiently adsorb the chitosan molecules on the surface of the silica. Then, 2 mL of PB was added, and the mixture was stirred at 200 rpm for 2 hours, and further stirred at 200 rpm for 1 hour using Tris buffer (100 mM Tris buffer) to prepare a complex. The complex was then washed with PB and stored at 4 &lt; 0 &gt; C.

Test Example  1. Immobilized Of carbonic anhydrase  Activity and stability measurement

FIG. 2A is a graph showing the initial activity of the immobilized carbonic anhydrase of the complex prepared according to Examples 1 to 3 and Comparative Examples 1 to 3, and FIG. 2B is a graph showing the initial activity of the immobilized carbonic anhydrase according to Examples 1 to 3 and Comparative Examples 1 to 3 FIG. 3 is a graph showing the stability of the immobilized carbonic anhydrase of the complex prepared. FIG. In the immobilized carbonic anhydrase, 'immobilization' means adsorbed inside the pores of the nanoporous silica carrier and immobilized in the pores to prevent leakage to the outside by the chitosan coating layer.

The activity of the hydrolytic enzyme was measured by hydrolysis of 4-nitrophenyl acetate (4-NA) as a reaction substrate of carbonic anhydrase in aqueous buffer. 4-NA solution was prepared by dissolving 4-NA in acetonitrile (10.9 mg / mL in acetonitrile) with 1/20 dilution with 100 mM sodium phosphate buffer (pH 7.6). In the case of an enzyme immobilized on spherical nanoporous silica, since it is impossible to measure in real time, it is possible to measure by taking a sample in 0.1 mL per hour after reacting the complex with 2 mL of 4-NA solution at 200 rpm Were used. Next, the diluted solution was diluted 1/10 with 100 mM sodium phosphate buffer (pH 7.6), and the absorbance of the product was measured at 348 nm using a spectrophotometer. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 2, the composites prepared according to Examples 1 to 3 of the present invention were found to be superior in activity to those of the composites of Comparative Examples 1 and 3, and in particular, Examples using 0.1% glutaric dialdehyde 3 showed the best activity compared to the other examples.

In addition, the complexes of Examples 1 to 3 showed almost no leakage of the enzyme up to 80 days compared with the complexes of Comparative Examples 1 and 3, and it was confirmed that the complexes of Examples 1 to 3 exhibit excellent stability. This property was most excellent in Example 3.

In FIG. 2B, free CA is an enzyme liberated, for example, an enzyme dissolved in a solvent without immobilization. When stability is checked under stirring conditions, the activity rapidly decreases.

Test Example 2. Carbonate conversion using immobilized carbonic anhydrase

FIG. 3 is a graph showing the content of carbonate produced according to the cycle using the carbonic anhydrase loaded and immobilized on the complex prepared according to Example 3 of the present invention. FIG.

2 mg of the complex of Example 3 was placed in 20 mL of 100 mM Tris buffer (pH 7.6), and 100 v / v% CO ₂ gas was aerated in the bottle at a rate of 0.15 L / min for 5 minutes to remove carbon dioxide The bicarbonate ion is separated from the complex of Example 3 through magnetic separation after conversion to bicarbonate ion. Then, the aqueous bicarbonate ion solution is added to 10 mL of a 670 mM aqueous calcium chloride solution and stirred at 200 rpm overnight at room temperature so that bicarbonate ions can be sufficiently converted into calcium carbonate crystals. The stirred solution was filtered through a paper filter using a vacuum pump, and the paper filter was dried at 100 ° C overnight to measure its mass.

As shown in FIG. 3, the complex of Example 3 was continuously subjected to a process of converting carbon dioxide to calcium carbonate using carbonic anhydrase for 30 times, and constantly produced a certain amount of calcium carbonate (120-160 mg) .

<Trypsin >

Example 4. Preparation of trypsin, 0.001% glutaric dialdehyde

The enzyme-porous material composite coated with chitosan was prepared using trypsin instead of the carbonic anhydrase in the same manner as in Example 1.

Example 5. Preparation of trypsin, 0.01% glutaric dialdehyde

The procedure of Example 4 was repeated except that 0.001% glutaric dialdehyde was replaced with 0.01% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example 6. Preparation of trypsin, 0.1% glutaric dialdehyde

The procedure of Example 4 was repeated except that 0.1% glutaric dialdehyde was used instead of 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Comparative Example 4. Chitosan and glutaric dialdehyde not used

A complex was prepared in the same manner as in Comparative Example 1 except that the trypsin solution was used instead of the carbonic anhydrase solution.

Comparative Example 5: Not using chitosan

The complex was prepared in the same manner as in Comparative Example 2 except that the trypsin solution was used instead of the carbonic anhydrase solution.

Comparative Example 6. Glutaric dialdehyde not used

A complex was prepared in the same manner as in Comparative Example 3 except that the trypsin solution was used instead of the carbonic anhydrase solution.

Test Example 3. Measurement of activity and stability of immobilized trypsin

4A is a graph showing the initial activity of immobilized trypsin of the complexes prepared according to Examples 4 to 6 and Comparative Examples 4 to 6, FIG. 4B is a graph showing the initial activity of the complex prepared according to Examples 4 to 6 and Comparative Examples 4 to 6. FIG. Lt; RTI ID = 0.0 &gt; immobilized trypsin. &Lt; / RTI &gt;

Measurements of trypsin activity were determined by the reaction of N-Benzoyl-L-arginine 4-nitroanilide ahydrochloride (BAPNA), the reaction substrate of trypsin in aqueous buffer. BAPNA solution was prepared by dissolving BAPNA in N, N-dimethylformamide (DMF) (10 mg / mL in DMF) diluted 1/100 with 100 mM sodium phosphate buffer (pH 7.9). In the case of enzymes immobilized on spherical nanoporous silica, it is not possible to measure in real time. Therefore, it is necessary to react the complex with 2 mL of BAPNA solution at 200 rpm and take a sample of 0.1 mL per time Respectively. Next, the diluted solution was diluted 1/10 with 100 mM sodium phosphate buffer (pH 7.9), and the absorbance of the product was measured at 410 nm using a spectrophotometer. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 4, it was confirmed that the complexes prepared according to Examples 5 and 6 of the present invention were superior in activity to the complexes of Comparative Examples 4 to 6, and in particular, the compositions using 0.1% glutaric dialdehyde 6 showed the best activity compared to the other examples.

Further, it was confirmed that the composite of Examples 5 and 6 exhibited excellent stability up to 120 days compared with the composite of Comparative Examples 4 and 6. This property was most excellent in Example 6.

In Fig. 4B, free TR is an enzyme liberated in the solvent, for example, without an immobilization process. When stability is checked under stirring conditions, the activity rapidly decreases.

& Lt; Sugar oxidase &gt;

Example 7. Preparation of glucose oxidase, 0.001% glutaric dialdehyde

The enzyme-porous material composite coated with chitosan was prepared in the same manner as in Example 1, except that the enzyme was replaced by a saccharide-oxidizing enzyme instead of the carbonic anhydrase.

Example 8. Preparation of glucose oxidase, 0.01% glutaric dialdehyde

The procedure of Example 7 was repeated except that 0.01% glutaric dialdehyde was used instead of 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example 9. Preparation of glucose oxidase, 0.1% glutaric dialdehyde

The procedure of Example 7 was repeated except that 0.1% glutaric dialdehyde was used instead of 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Comparative Example 7: Not using chitosan and glutaric dialdehyde

A complex was prepared in the same manner as in Comparative Example 1 except that the saccharide-oxidizing enzyme solution was used instead of the carbonic anhydrase solution.

Comparative Example 8. Chitosan not used

A complex was prepared in the same manner as in Comparative Example 2 except that the saccharide-oxidizing enzyme solution was used instead of the carbonic anhydrase solution.

Comparative Example 9. Glutaric dialdehyde not used

The complex was prepared in the same manner as in Comparative Example 3 except that the saccharide-oxidizing enzyme solution was used instead of the carbonic anhydrase solution.

Test Example 4. Measurement of activity and stability of immobilized glucose oxidase

FIG. 5A is a graph showing the initial activity of the immobilized glucose oxidase of the complexes prepared according to Examples 7 to 9 and Comparative Examples 7 to 9, FIG. 5B is a graph showing the initial activity of the immobilized glucose oxidase prepared according to Examples 7 to 9 and Comparative Examples 7 to 9 Lt; RTI ID = 0.0 &gt; of &lt; / RTI &gt; immobilized glucose oxidase.

Measurement of glucose oxidase activity was measured through the reaction of glucose, which is a reaction substrate of glucose oxidase, in an aqueous buffer. First, 0.96 mg of 3,3,5,5-tetramethylbenzidine (TMB) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and diluted with 19 mL of 100 mM sodium phosphate buffer (PB, pH 7.0) . 2.5 mL of glucose solution (110.3 w / v% in PB) was mixed with 12 mL of the prepared TMB solution to prepare a reaction substrate solution. Peroxidase (POD) was dissolved in PB to make 3.79 mg / mL. The enzyme activity was measured with the two solutions prepared as described above. In order to measure the activity of the enzyme, 890 uL reaction substrate solution, 10 uL POD solution, and 100 uL of glucose oxidase immobilized solution were mixed and the absorbance was measured at 655 nm by a spectrophotometer. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 5, the composites prepared according to Examples 8 and 9 of the present invention were found to be superior in activity to the composites of Comparative Examples 7 to 9, and in particular, the compositions using 0.1% glutaric dialdehyde 9 showed the best activity compared to the other examples.

Further, it was confirmed that the composite of Examples 8 and 9 exhibited excellent stability up to 120 days compared with the composite of Comparative Examples 1 and 3. This property was most excellent in Example 9.

< Alcohol dehydrogenase >

Example 10. Alcohol dehydrogenase, 0.01% glutaric dialdehyde

The enzyme-porous material composite coated with chitosan was prepared in the same manner as in Example 2 except that the alcohol dehydrogenase was used instead of the carbonic anhydrase.

Example 11. Alcohol dehydrogenase, 0.05% glutaric dialdehyde

The procedure of Example 10 was repeated except that 0.05% glutaric dialdehyde was used instead of the 0.01% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example 12. Alcohol dehydrogenase, 0.1% glutaric dialdehyde

The procedure of Example 10 was repeated except that 0.1% glutaric dialdehyde was used instead of the 0.01% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Comparative Example 10. Chitosan and glutaric dialdehyde not used

A complex was prepared in the same manner as in Comparative Example 1 except that the alcohol dehydrogenase solution was used instead of the carbonic anhydrase solution.

Comparative Example 11. Chitosan not used

A complex was prepared in the same manner as in Comparative Example 2 except that the alcohol dehydrogenase solution was used instead of the carbonic anhydrase solution.

Comparative Example 12. Glutaric dialdehyde not used

The complex was prepared in the same manner as in Comparative Example 3 except that an alcohol dehydrogenase solution was used instead of the carbonic anhydrase solution.

Test Example 5 Measurement of activity and stability of immobilized alcohol dehydrogenase

FIG. 6A is a graph showing the initial activities of the immobilized alcohol dehydrogenase of the complex prepared according to Examples 10 to 12 and Comparative Examples 10 to 12, FIG. 6B is a graph showing the initial activity of the immobilized alcohol dehydrogenase prepared according to Examples 10 to 12 and Comparative Examples 10 to 12 Lt; / RTI &gt; of the immobilized alcohol dehydrogenase.

Measurement of alcohol dehydrogenase activity was measured by the reaction of ethanol as a reaction substrate of alcohol dehydrogenase in aqueous buffer. First, dissolve 10 mg of β-nicotinamide adenine dinucleotide hydrate (NAD ⁺ ) in 1 mL of distilled water. Subsequently, 0.2 mL of 99.8% ethanol, 0.8 mL of NAD ⁺ aqueous solution, and 1.6 mL of sodium phosphate buffer (PB, pH 7.6) are mixed to prepare a reaction substrate solution. In the case of an enzyme immobilized on spherical nanoporous silica, it is impossible to measure in real time. Therefore, the above complex is put into 2 mL of a reaction substrate solution, reacted at 200 rpm, and a sample is taken at 0.1 mL per time Respectively. Next, the solution was diluted 1/10 with 100 mM sodium phosphate buffer (pH 7.6), and the concentration of the product was measured at 340 nm using a spectrophotometer. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 6, the composites prepared according to Examples 10 to 12 of the present invention were found to be superior in activity to those of the composites of Comparative Examples 10 to 12, and in particular, Examples using 0.01% glutaric dialdehyde 10 showed the best activity compared to the other examples.

Further, it was confirmed that the composite of Examples 11 and 12 exhibited excellent stability up to 50 days compared with the composite of Comparative Examples 10 and 12. This property was most excellent in Example 12.

In Comparative Example 11, unlike Comparative Examples 2, 5 and 8, structural modification of the enzyme occurred due to cross-linking between the enzymes and the activity could not be confirmed. In the case of the alcohol dehydrogenase, since the amino group participating in the reaction exists in the active site, the activity can be easily lost by the cross-linking agent.

< Formate dehydrogenase >

Example 13. Formate dehydrogenase, 0.001% glutaric dialdehyde

The enzyme-porous material composite coated with chitosan was prepared in the same manner as in Example 1 except that formic acid dehydrogenase was used instead of the carbonic anhydrase.

Example 14. Formate dehydrogenase, 0.005% glutaric dialdehyde

Except that 0.005% glutaric dialdehyde was used in place of the 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example  15. Formic acid dehydrogenase , 0.01% Glutaric Dialdehyde

The procedure of Example 10 was repeated except that 0.001% glutaric dialdehyde was replaced with 0.01% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Example  16. Formic acid dehydrogenase , 0.02% Glutaric Dialdehyde

The procedure of Example 10 was repeated except that 0.002% glutaric dialdehyde was used instead of 0.001% glutaric dialdehyde to prepare an enzyme-porous material composite coated with chitosan.

Comparative Example 13. Chitosan and glutaric dialdehyde not used

A complex was prepared in the same manner as in Comparative Example 1 except that a solution of formic acid dehydrogenase was used instead of the solution of carbonic anhydrase.

Comparative Example 14. Glutaric dialdehyde not used

A complex was prepared in the same manner as in Comparative Example 3 except that a solution of formic acid dehydrogenase was used in place of the solution of carbonic anhydrase.

Test Example 6. Measurement of activity and stability of immobilized formate dehydrogenase

FIG. 7A is a graph showing the initial activity of the immobilized formate dehydrogenase of the complex prepared according to Examples 13 to 16 and Comparative Examples 13 and 14, and FIG. 7B is a graph showing the initial activity of the immobilized formate dehydrogenase prepared according to Examples 13 to 16 and Comparative Examples 13 and 14 Lt; RTI ID = 0.0 &gt; formate &lt; / RTI &gt; dehydrogenase.

Measurement of formate dehydrogenase activity was measured by the reaction of formic acid, the reaction substrate of formic acid dehydrogenase in aqueous buffer. First, dissolve 51.0 mg sodium formate (SF) and 1.5 mg β-nicotinamide adenine dinucleotide hydrate (NAD ⁺ ) in 1 mL of distilled water, respectively. Subsequently, 0.2 mL of each of SF solution and NAD + aqueous solution and 1.6 mL of 100 mM sodium phosphate buffer (PB, pH 7.6) are mixed to prepare a reaction substrate solution. In the case of an enzyme immobilized on spherical nanoporous silica, it is impossible to measure in real time. Therefore, the above complex is put into 2 mL of a reaction substrate solution, reacted at 200 rpm, and a sample is taken at 0.1 mL per time Respectively. After that, it was diluted 1/10 with 100 mM sodium phosphate buffer (pH 7.6), and the concentration of the product was measured by a spectrophotometer at 340 nm. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 7, the composites prepared according to Examples 13 to 16 of the present invention were found to be superior in activity to those of the composites of Comparative Examples 13 and 14, and in particular, the compositions using 0.01% glutaric dialdehyde 15 showed the best activity compared to the other examples.

In addition, it was confirmed that the composite of Examples 13 to 16 exhibited excellent stability up to 25 days compared with the composite of Comparative Examples 13 and 14.

< Formaldehyde dehydrogenase >

Example 17. Formaldehyde dehydrogenase, 0.01% glutaric dialdehyde

The enzyme-porous material composite coated with chitosan was prepared in the same manner as in Example 2 except that formaldehyde dehydrogenase was used instead of the carbonic anhydrase.

Comparative Example 15. Chitosan and glutaric dialdehyde not used

The complex was prepared in the same manner as in Comparative Example 1 except that a formaldehyde dehydrogenase solution was used instead of the carbonic anhydrase solution.

Comparative Example 16. Chitosan not used

A complex was prepared in the same manner as in Comparative Example 2 except that a formaldehyde dehydrogenase solution was used instead of the carbonic anhydrase solution.

Comparative Example 17. Glutaric dialdehyde not used

The complex was prepared in the same manner as in Comparative Example 3 except that a solution of formaldehyde dehydrogenase was used instead of the solution of carbonic anhydrase.

Test Example 7. Measurement of activity and stability of immobilized formaldehyde dehydrogenase

FIG. 8A is a graph showing the initial activity of the immobilized formaldehyde dehydrogenase of the complex prepared according to Example 17 and Comparative Examples 15 to 17, and FIG. 8B is a graph showing the initial activity of the complex prepared according to Example 17 and Comparative Examples 15 to 17 FIG. 5 is a graph showing the stability of immobilized formaldehyde dehydrogenase. FIG.

Measurement of formaldehyde dehydrogenase activity was measured by the reaction of formaldehyde, the reaction substrate of formaldehyde dehydrogenase, in aqueous buffer. First, 0.057 mL of formaldehyde and 1.5 mg of β-nicotinamide adenine dinucleotide hydrate (NAD ⁺ ) are dissolved in 1 mL of distilled water, respectively. Then, 0.2 mL of formaldehyde aqueous solution and 0.2 mL of NAD + aqueous solution, and 1.6 mL of 100 mM sodium phosphate buffer (PB, pH 7.6) are mixed to prepare a reaction substrate solution. In the case of an enzyme immobilized on spherical nanoporous silica, it is impossible to measure in real time. Therefore, the above complex is put into 2 mL of a reaction substrate solution, reacted at 200 rpm, and a sample is taken at 0.1 mL per time Respectively. After that, it was diluted 1/10 with 100 mM sodium phosphate buffer (pH 7.6), and the concentration of the product was measured by a spectrophotometer at 340 nm. The stability of the enzyme was measured by two methods: a method of measuring the activity by time as described above, a method of reusing the same sample to measure its activity and comparing with the initial activity.

As shown in FIG. 8, the composite prepared according to Example 17 of the present invention was superior to the composite of Comparative Examples 15 to 17 in activity.

In Comparative Example 16, structural modification of the enzyme occurred due to crosslinking between the enzymes.

In addition, it was confirmed that the composite of Example 17 exhibited excellent stability up to 50 days as compared with the composite of Comparative Examples 15 to 17.

Claims (12)

(A) adsorbing an enzyme in pores of a porous carrier;
(B) adsorbing chitosan on the surface of the porous support on which the enzyme is adsorbed; And
(C) reacting the chitosan with a cross-linking agent to perform cross-linking between the amine groups of the chitosan, thereby producing a chitosan-coated enzyme-porous material composite. The method of claim 1, wherein the crosslinking agent is selected from the group consisting of glutaric dialdehyde, diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, bis (imidoester), bis (succinimidyl ester) Wherein the chitosan-coated enzyme-porous material composite is coated with a chitosan-coated enzyme-porous material composite. The method of claim 1, wherein the cross-linking agent is a 0.0001 to 10.0% (w / w) glutaric dialdehyde solution. The method of claim 1, wherein the cross-linking agent is a 0.001 to 1.0% (w / w) glutaric dialdehyde solution. The method of claim 1, wherein the concentration of the chitosan is 0.01 to 10.0 mg / mL. The method of claim 1, wherein the concentration of the chitosan is 0.1 to 1.0 mg / mL. 2. The chitosan-coated enzyme-coated product according to claim 1, wherein the enzyme is at least one selected from the group consisting of carbonic anhydrase, trypsin, saccharide oxidase, alcohol dehydrogenase, formate dehydrogenase and formaldehyde dehydrogenase. A method for manufacturing a porous material composite. The method of claim 1, wherein a cofactor is additionally adsorbed in the pores of the porous carrier. [Claim 9] The method of claim 8, wherein the cofectors are at least one selected from the group consisting of hydrogen transfer coenzyme and enzyme coenzyme. The method according to claim 1, wherein the porous carrier is one selected from the group consisting of a porous silica carrier, a porous carbon carrier, and a polymer carrier. A chitosan-coated enzyme-porous material composite prepared according to any one of claims 1 to 10. 12. The method of claim 11, wherein the chitosan-coated enzyme-porous material composite comprises at least one of a bio-converting material, a biocompatible material, an antifouling coating material, a biocatalyst material for detergents, an enzyme- Or a biofuel cell, the chitosan-coated enzyme-porous material composite.

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