TECHNICAL FIELD

The present invention relates to an enzyme-porous carbon composite, and more specifically, to an enzyme-porous carbon composite in which an enzyme is effectively immobilized in a porous carbon material by preventing enzyme leakage for a long time as a technique of improving the stability of an enzyme by immobilizing an enzyme in a porous material.

BACKGROUND ART

Enzymes are biocatalysts that promote various chemical reactions and can be used in various industries because the enzymes have high selectivity, precision, and efficiency and promote chemical reactions at very high rates even at room temperature and atmospheric pressure. However, in an industrial reaction environment, the three-dimensional structure of the enzymes is easily denatured due to external environments such as heat, pH, shear stress, and the like, and thus, the enzymes rapidly lose their activity. Therefore, various studies have been conducted to improve the stability of enzymes.

Among techniques of improving the stability of an enzyme, the technique of immobilizing an enzyme on a solid support not only improves the stability of the enzyme but also allows the immobilized enzyme to be easily separated from a reaction solution as a heterogeneous catalyst. In particular, porous materials are being researched as optimal materials for immobilization of an enzyme because it is possible to variously adjust the size and shape of pores of the porous materials in response to the size of enzyme. In addition, when the enzyme is immobilized in the porous material, the stability of the enzyme can be significantly improved due to a crowding effect in which the enzyme is present at a high concentration of several hundred milligrams per unit milliliter in the pores of the porous material. Also, since the porous material can protect the enzyme immobilized therein from physical impact such as external shear stress and the like, an enzymatic reaction can be achieved even in harsh environments.

However, the enzyme loaded in a general porous material may easily leak to the outside, and the leaked enzyme may not be recovered but discarded, or the enzyme structure may be denatured by the external environment, resulting in a decrease in activity of the immobilized enzyme.

To solve these problems, a technique of preventing enzyme leakage by covalently bonding an enzyme to the inside of a porous material was studied. This technique is for preventing the leakage of the enzyme loaded in the porous material by functionalizing the porous material using a physicochemical method and then forming a crosslink between a functional group of the porous material and a functional group of an enzyme side chain using a crosslinking agent.

However, in order to apply the covalent bonding technique, an additional functionalization process needs to be introduced in the process of synthesizing the porous material. In addition, when two or more covalent bonds are induced between a functional group on the inner surface of the pore of the porous material and a functional group on the enzyme surface, the enzyme may be structurally denatured and thus deactivated.

Meanwhile, a nanoscale enzyme reactor technique that applies the covalent bonding technique was studied to prevent the leakage of an enzyme. The nanoscale enzyme reactor technique is a technique of preventing the leakage of the enzyme loaded in a porous material by loading the enzyme in a porous material and forming a crosslink between loaded enzyme molecules before the enzyme leaks to increase the size of crosslinked enzyme molecules compared to the inlet diameter of the pore of the porous material.

However, in the case of the conventional nanoscale enzyme reactor technique, two or more crosslinks may be induced on the enzyme surface by a crosslinking agent, and it is difficult for this technique to stabilize unstable enzymes such as formate dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, and the like, which are easily denatured and thus deactivated.

In addition, when the enzymes are stabilized using a crosslinking agent, there may be a problem of biocompatibility of the crosslinking agent such as glutardialdehyde or the like, and therefore, it is difficult to utilize this technique in the medical field.

DISCLOSURE

Technical Problem

The present invention is directed to providing an enzyme-porous carbon composite in which an enzyme is immobilized in a porous material while minimizing a decrease in enzyme activity, so that the enzyme immobilized in the porous material is prevented from leaking, and the stability of the enzyme immobilized in the porous material is ensured.

[The National R&D Project of Korea supports the present invention]Assignment No: 20182010600430Department name: Ministry of Trade, Industry and EnergyResearch management agency: Korea Institute of Energy Technology Evaluation and PlanningResearch project name: Energy Technology Development ProjectResearch title: Development of CO2conversion process technology based on highly integrated and highly stable enzyme systemSupervising organization: Korea UniversityResearch period: 2019.04.01˜2019.12.31

Technical Solution

One aspect of the present invention provides an enzyme-porous carbon composite which includes a porous carbon carrier and an enzyme loaded in a pore of the porous carbon carrier.

According to an embodiment of the present invention, the enzyme may be loaded by molecules thereof being at least partially adsorbed onto the inside of the pore of the porous carbon carrier.

In addition, the porous carbon carrier may be mesoporous carbon and/or microporous carbon and may, for example, have a pore size of 1 to 5,000 nm. In addition, the enzyme may include one or more enzymes selected from the group consisting of trypsin, chymotrypsin, pepsin, a lipase, glucose oxidase, horseradish peroxidase, tyrosinase, carbonic anhydrase, formaldehyde dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, cholesterol dehydrogenase, acylase, lactonase, a protease, a peroxidase, an aminopeptidase, a phosphatase, a transaminase, serine endopeptidase, cysteine endopeptidase, and a metalloendopeptidase.

In addition, the porous carbon carrier may further include a first functional group on a surface of the pore thereof to induce one or more of a hydrophobic interaction and a hydrophilic interaction with the enzyme.

In addition, the first functional group may include at least one of one or more hydrophobic interaction-inducing functional groups selected from the group consisting of an alkyl group, a phenyl group, a benzyl group, a vinyl group, and a halo group and one or more hydrophilic interaction-inducing functional groups selected from the group consisting of a hydroxy group, a carbonyl group, a halide group, a carboxyl group, a methoxy group, a hydroperoxy group, an amine group, an imine group, an imide group, a nitrile group, a nitro group, a thiol group, a sulfino group, and a phosphate group.

In addition, the enzyme may further include a second functional group to induce one or more of a hydrophobic interaction and a hydrophilic interaction with a pore surface of the porous carbon carrier.

In addition, the second functional group may include at least one of one or more hydrophobic interaction-inducing functional groups selected from the group consisting of an alkyl group, a phenyl group, a benzyl group, a vinyl group, and a halo group and one or more hydrophilic interaction-inducing functional groups selected from the group consisting of a hydroxy group, a carbonyl group, a halide group, a carboxyl group, a methoxy group, a hydroperoxy group, an amine group, an imine group, an imide group, a nitrile group, a nitro group, a thiol group, a sulfino group, and a phosphate group.

In addition, the enzyme-porous carbon composite may exhibit an enzyme release amount of less than 33% compared to before stirring, as measured after being stirred in a mixed state with a buffer solution for 12 days.

In addition, the enzyme-porous carbon composite may exhibit an enzyme activity of 16% or more compared to before stirring, as measured after being stirred in a mixed state with a buffer solution for 12 days.

In addition, the enzyme-porous carbon composite may exhibit an enzyme activity of 72% or more compared to before stirring, as measured after being stirred in a mixed state with a buffer solution for 45 days.

Another aspect of the present invention provides an electrode for a biofuel cell or a biosensor which includes the enzyme-porous carbon composite according to the present invention.

Still another aspect of the present invention provides a carbon dioxide conversion system which includes the enzyme-porous carbon composite according to the present invention.

Yet another aspect of the present invention provides an anti-fouling system which includes the enzyme-porous carbon composite according to the present invention.

Advantageous Effects

An enzyme-porous carbon composite according to the present invention can be suitably used to immobilize an enzyme in porous carbon without denaturation of an active site of the enzyme and stabilize the enzyme. In addition, the composite can be used in the medical field where the use thereof is limited by immobilizing and stabilizing the enzyme without using a component having a problem of biocompatibility, such as a crosslinking agent applied to a conventional enzyme stabilization technique. Additionally, when the enzyme-porous carbon composite is utilized to immobilize enzymes such as glucose oxidase and the like for the purpose of producing electrodes for glucose-based biofuel cells and biosensors for measuring blood glucose, a very high stabilization effect can be achieved. In addition, when the enzyme-porous carbon composite is utilized to immobilize enzymes such as carbonic anhydrase capable of converting carbon dioxide into bicarbonate, formate dehydrogenase capable of converting bicarbonate into formic acid, and the like, it is possible to stably maintain the activity of the enzymes for a long time compared to a conventional immobilization method using a porous material, and thus it can be used as a catalyst material for a carbon dioxide conversion and utilization system. Additionally, when the enzyme-porous carbon composite is utilized to immobilize an enzyme such as acylase which is capable of suppressing biofilm formation by decomposing a signaling molecule that plays a role in quorum sensing, it is possible to stably maintain the activity of the enzyme compared to the conventional immobilization method using a porous material, and thus it can be used as a catalyst material in an anti-fouling system for the purpose of suppressing biofilm formation on a membrane surface.

