ENZYME-CARRIER COMPLEX

Abstract

The present invention relates to an enzyme-carrier complex, and more particularly to the adsorption and stabilization of an enzyme on the surface of a carrier, and an enzyme-carrier complex with secured enzyme stability so that an enzyme immobilized by a hydrophobic interaction exhibits long-term enzymatic activity.

Claims

1. An enzyme-carrier complex comprising: a hydrophobic carrier; and an enzyme adsorbed on a surface of the hydrophobic carrier.

2. The enzyme-carrier complex of claim 1, wherein the enzyme comprises one or more enzymes selected from the group consisting of acylase, trypsin, chymotrypsin, pepsin, lipases, glucose oxidase, pyranose oxidase, horseradish peroxidase, thyroxinase, carbonic anhydrase, formaldehyde dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, cholesterol dehydrogenase, lactonase, proteases, peroxidases, aminopeptidases, phosphatases, transaminases, serine-endopeptidase, cysteine-endopeptidase, and metalloendopeptidases.

3. The enzyme-carrier complex of claim 1, wherein the hydrophobic carrier comprises one or more material selected from the group consisting of carbon nanotubes, fullerenes, graphene, porous carbon, polycarbonate, polyimide, polystyrene, polydimethylsiloxane, and polyethylene terephthalate.

4. The enzyme-carrier complex of claim 1, wherein the hydrophobic carrier further comprises a first functional group on a surface thereof to induce a hydrophobic interaction with the enzyme.

5. The enzyme-carrier complex of claim 4, wherein the enzyme further comprises a second functional group for inducing a hydrophobic interaction with the first functional group.

6. The enzyme-carrier complex of claim 5, wherein the second functional group for inducing a hydrophobic interaction comprises one or more functional groups selected from the group consisting of a halogenated alkyl group, an organosilicon group, an alkyl group, a vinyl group, an allyl group, and an aryl group.

7. The enzyme-carrier complex of claim 1, wherein the enzyme-carrier complex is any one selected from the group consisting of an acylase-carbon nanotube complex, a trypsin-carbon nanotube complex, a lipase-carbon nanotube complex, a glucose oxidase-carbon nanotube complex, a pyranose oxidase-carbon nanotube complex, a horseradish peroxidase-carbon nanotube complex, a tyrosinase-carbon nanotube complex, a carbonic anhydrase-carbon nanotube complex, and a formaldehyde dehydrogenase-carbon nanotube complex.

8. An electrode for a biofuel cell, the electrode comprising the enzyme-carrier complex according to claim 1.

9. An electrode for a biosensor, the electrode comprising the enzyme-carrier complex according to claim 1.

10. A carbon dioxide conversion system comprising the enzyme-carrier complex according to claim 1.

11. An antifouling system comprising the enzyme-carrier complex according to claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a view of an enzyme-carrier complex according to an embodiment of the present invention.

[0023] FIGS. 2 and 3 are graphs for evaluating enzyme stability by comparing an enzyme-carrier complex according to an embodiment of the present invention with an enzyme in a free state and each of enzyme-carrier complexes according to comparative examples.

[0024] FIG. 4 illustrates an image and view showing a state in which an enzyme-carrier complex according to an embodiment of the present invention is dispersed in water.

[0025] FIG. 5 illustrates an image and a view showing a state in which carbon nanotubes used as a carrier in the complex of FIG. 4 are dispersed alone in water.

BEST MODE

[0026] Hereinafter, embodiments of the present invention will be described in detail in such a manner that the invention can be carried out by one of ordinary skill in the art to which the present invention pertains, without undue difficulty. The present invention may be embodied in many different forms and is not limited by embodiments set forth herein.

[0027] Referring to FIG. 1, an enzyme-carrier complex according to the present invention includes a hydrophobic carrier and an enzyme adsorbed on a surface of the hydrophobic carrier. In one embodiment, the absorption may occur by a hydrophobic interaction between a hydrophobic moiety of the enzyme and the surface of the hydrophobic carrier.

[0028] Any hydrophobic carrier may be used without limitation as long as it is an insoluble material that is commonly referred to as exhibiting hydrophobicity in the art. In the present invention, the hydrophobic carrier refers to a carrier capable of having a van der Waals interaction or a pi-pi interaction, among non-covalent bonds, with the surface of the enzyme on the surface thereof that can contact the surface of the enzyme. In addition, any hydrophobic carrier may be used without limitation as long as it is a material as defined above, and may include, for example, one or more materials selected from the group consisting of carbon nanotubes, fullerenes, graphene, porous carbon such as nanoporous carbon or activated carbon, polycarbonate, polyimide, polystyrene, polydimethylsiloxane, and polyethylene terephthalate.

[0029] The shape of the hydrophobic carrier is not limited, and may be, for example, one or more shapes selected from the group consisting of spherical, plate, rod, tubular, and amorphous shapes. In addition, the hydrophobic carrier may further include nano-sized pores in the surface thereof, but the present invention is not limited thereto.

[0030] In addition, the size of the hydrophobic carrier may vary from nanoscale to microscale, but the present invention is not particularly limited thereto.

[0031] In addition, the hydrophobic carrier may further include, on the surface thereof, a first functional group to induce a hydrophobic interaction with the enzyme or to further enhance the hydrophobic interaction. The hydrophobic interaction minimizes or prevents an effect on the steric structure of an enzyme compared to covalent bonding, and thus is advantageous in that it can prevent or minimize problems such as denaturation of the steric structure of an enzyme and deterioration or loss of enzymatic activity, which occur according to strong binding affinity caused when an enzyme is immobilized on a support via conventional covalent bonding or when enzymes are crosslinked via covalent bonding. In this regard, the first functional group included in the hydrophobic carrier is not limited as long as it is a functional group capable of inducing a hydrophobic interaction, and may include, for example, one or more functional groups selected from the group consisting of a halogenated alkyl group, an organosilicon group, an alkyl group, a vinyl group, an allyl group, and an aryl group.

[0032] In addition, the first functional group may be introduced onto the surface of the hydrophobic carrier through a known technique, but the present invention is not particularly limited thereto.

[0033] As the enzyme, any known enzyme may be employed without limitation, and may include, for example, one or more enzymes selected from the group consisting of acylase, trypsin, chymotrypsin, pepsin, lipases, glucose oxidase, pyranose oxidase, horseradish peroxidase, thyroxinase, carbonic anhydrase, formaldehyde dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, cholesterol dehydrogenase, lactonase, proteases, peroxidases, aminopeptidases, phosphatases, transaminases, serine-endopeptidase, cysteine-endopeptidase, and metalloendopeptidases. More preferably, the enzyme may include one or more selected from the group consisting of acylase, trypsin, lipases, glucose oxidase, pyranose oxidase, horseradish peroxidase, thyroxinase, carbonic anhydrase, and formaldehyde dehydrogenase, and even more preferably, the enzyme may be acylase.

[0034] In addition, according to one embodiment of the present invention, the enzyme may further include a second functional group for enhancing a hydrophobic interaction with the hydrophobic carrier. The second functional group may function to minimize the effect on the steric structure of the enzyme while minimizing dissociation of the enzyme from the hydrophobic carrier, and stably exhibit enzymatic activity. The second functional group may include one or more functional groups selected from the group consisting of a halogenated alkyl group, an organosilicon group, an alkyl group, a vinyl group, an allyl group, and an aryl group. In addition, the second functional group may be selected from those that are the same as or different from the first functional group that may be included in the hydrophobic carrier, but the present invention is not particularly limited thereto.

[0035] According to one embodiment of the present invention, the enzyme and the hydrophobic carrier in the above-described enzyme-carrier complex may be any one combination selected from an acylase-carbon nanotube complex, a trypsin-carbon nanotube complex, a lipase-carbon nanotube complex, a glucose oxidase-carbon nanotube complex, a pyranose oxidase-carbon nanotube complex, a horseradish peroxidase-carbon nanotube complex, a tyrosinase-carbon nanotube complex, a carbonic anhydrase-carbon nanotube complex, and a formaldehyde dehydrogenase-carbon nanotube complex, and due to an enhanced interaction between the enzyme and the surface of the carrier in such a combination, the enzyme may be stably immobilized, and there is an advantage that long-term enzymatic activity may be exhibited.

Mode of Invention

[0036] Hereinafter, the present invention will be described in detail with reference to the following examples. However, these examples are not intended to limit the scope of the present invention.

EXAMPLE 1

Preparation of Enzyme-Carrier Complex Using Acylase and Carbon Nanotubes (ADS-AC/CNTs)

[0037] A carbon nanotube (CNT) solution (8 mg/mL) and an acylase (AC) solution (40 mg/mL), prepared using phosphate-buffered saline (100 mM, pH 7.0) as a solvent, were mixed in the same volume ratio, followed by stirring at 200 rpm for 1 hour to thereby adsorb acylase onto the surfaces of carbon nanotubes. Subsequently, acylase unattached to the carbon nanotubes was removed using phosphate-buffered saline, followed by stirring with Tris buffer (100 mM, pH 7.4) at 200 rpm for 30 minutes, causing unreacted functional groups to be capped. Thereafter, the prepared enzyme-carrier complex was centrifuged to remove the supernatant, washed with phosphate-buffered saline, and then stored at 4° C.

COMPARATIVE EXAMPLE 1

Free Acylase Solution (Free AC)

[0038] An acylase solution dissolved in phosphate-buffered saline (100 mM, pH 7.0) was prepared.

EXPERIMENTAL EXAMPLE 1

Measurement of Enzymatic Activity and Stability of Enzyme-Carrier Complex Using Acylase and Carbon Nanotubes

[0039] As an enzyme, acylase (AC), which decomposes bacterial quorum-sensing signaling material, was used. AC activity was measured using fluorescence emitted as a result of reaction of L-methionine produced by hydrolysis of N-acetyl-L-methionine with o-phthalaldehyde (OPA).

[0040] Enzyme stability was evaluated by measuring a decrease in enzymatic activity while continuing to stir at 200 rpm after a sample was dispersed in phosphate-buffered saline. Specifically, relative activity with respect to initial activity of the enzyme-carrier complex of Example 1 using acylase and carbon nanotubes and the free acylase solution of Comparative Example 1 was evaluated for 200 days, and the results thereof are shown in FIG. 2.

[0041] As can be confirmed from FIG. 2, the enzyme in the case of Comparative

[0042] Example 1 was non-activated within 19 days, whereas the case of Example 1 maintained a relative activity of 20% for 200 days. Specifically, the case of Example 1 exhibited a relative activity reduced to 23% for the first 40 days, and then stably maintained a relative activity of 23% until 200 days had elapsed.

EXPERIMENTAL EXAMPLE 2

[0043] The enzymatic activity and stability of each of the enzyme-carrier complexes according to Example 1 and Comparative Example 2 were measured in the same manner as in Experimental Example 1, and the results thereof are shown in FIG. 3.

[0044] As can be confirmed from FIG. 3, the case of Example 1 maintained a relative activity of 20% for 200 days, whereas the case of Comparative Example 2 did not exhibit enzymatic activity after being immobilized.

COMPARATIVE EXAMPLE 3

Carbon Nanotube (CNT) Solution

[0045] A solution in which carbon nanotubes (8 mg/mL) were added to phosphate-buffered saline (100 mM, pH 7.0) was prepared.

EXPERIMENTAL EXAMPLE 3

[0046] Each of the enzyme-carrier complex according to Example 1 and the carbon nanotubes according to Comparative Example 3 was added to a water-containing container and stirred at 200 rpm for 1 hour, and then each container was left on a table for 1 minute, followed by photographing, and acquired images are shown in FIGS. 4 and 5.

[0047] As illustrated in FIG. 4, it can be confirmed that the enzyme-carrier complex of Example 1, in which acylase is adsorbed on surfaces of the carbon nanotubes, is uniformly dispersed in water, from which it can be seen that the enzyme-carrier complexes are not aggregated.

[0048] In contrast, as illustrated in FIG. 5, it can be confirmed that, when carbon nanotubes alone are dispersed in water, most settle on the bottom, and even when carbon nanotubes do not settle, the carbon nanotubes are present as grains aggregated therebetween.