KR20150073154A - Enzyme-monolithic column and method for manufacturing the same - Google Patents

Abstract

An enzyme-monolithic column of the present invention is synthesized through a method for stably combining a large quantity of enzymes with a monolithic column made of silica material. The same is applied to various industrially utilizable enzymes and an enzyme reaction system requiring a continuous flow reaction, thereby ensuring a high enzyme activity for a long time. For example, an enzyme-monolithic column using trypsin which is a protein hydrolyzing enzyme is prepared to ensure digestion and decomposing functions of small protein molecules. Compared to a protein hydrolyzing enzyme accumulated body which has been industrially limited due to excessively low activities and stability of a protein hydrolyzing enzyme, the same can have an improved protein hydrolyzing enzyme function. Thus, the same is very likely to be industrially utilized, and a support having inexpensive costs is used so that economical problems may be resolved. Moreover, by using a column material used for a liquid chromatography (LC) device for performing a proteome analysis on an actual sample, an experiment for confirming stability by synthesizing an enzyme-monolithic column is performed. When the same is used after being directly connected to a proteome analyzing device, an automated system may be structured, and a large quantity of samples can be analyzed in a short time.

Description

Enzyme-monolithic column and method for manufacturing same

The present invention relates to an enzyme-monolith column, and more particularly, to an enzyme-monolith column that maximizes the amount of an enzyme immobilized in a monolithic column having a network structure capable of providing a large surface area therein, Column. ≪ / RTI >

Enzymes are biocatalysts that catalyze the chemical reaction of organisms, and their selectivity is determined by the binding pouch formed by the folding of amino acid linear biopolymers. The selectivity of enzymes has been applied in various fields and its application range has been gradually widened according to academic progress. Examples include pharmaceuticals, fine chemicals, food and detergent industries, biosensors, biochemical conversion, biochemical conversion, biodiesel production, trypsin hydrolysis in proteomic analysis, polymerase chain reaction, and biofuel cells Research is underway. Despite the wide range of applications due to the variety of chemical reactions determined by the selectivity of enzymes, the decrease in activity and stability due to structural changes due to internal and external factors of the enzyme is a major limiting factor in lowering the applicability.

On the other hand, a column having a diameter of micron is a material having a tube structure serving as a connection passage for flow control of microfluid and continuous flow formation. The interior of the column is categorized as being coated with various materials to perform additional functions and to have no treatment for general flow only. To improve the efficiency of flow reaction, such as a water treatment system, a large number of thin-diameter column bundles are used in order to provide a large contact surface area per unit volume. In order to increase the surface area of the internal space of the column, Studies have also been actively conducted to form a solid monolithic structure using a silica material in a column.

As described above, enzymes can be applied to industrial fields to which various chemical reactions are applied. In order to apply them to actual industries, it is necessary to overcome limitations of reduction in activity and stability due to structural changes due to external factors. However, all of the methods of using the enzyme activity for the actual chemical reaction by fixing the enzyme in the conventional monolithic column developed above are not only low in yield, but also have a considerable deterioration in stability when used over a long period of time.

An enzyme is a biomolecule material whose intrinsic activity is determined by the part of the active bag formed by the folding structure of the protein. Therefore, it is impossible for the enzyme to exhibit continuous activity due to decreased activity due to internal and external influences over time in a general aqueous solution.

DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems. One of the objects of the present invention is to prepare an enzyme-monolith column by stably binding a large amount of enzyme to a monolithic column made of silica, An enzyme-monolithic column using trypsin, which is a digestive enzyme, is prepared to provide an enzyme-monolithic column having digestion and resolution of protein small molecules.

A second object of the present invention is to provide an automated on-line proteomic analysis apparatus and system employing the enzyme-monolithic column of the present invention.

In order to solve the above problems, the present invention proposes an enzyme-monolith column in which an enzyme is stabilized in a column by using an enzyme coating technique.

More specifically, the present invention relates to a method for preparing a porous structure for enzyme immobilization in a monolithic column, maximizing the amount of enzyme immobilized on the porous structure, stabilizing the activity, An enzyme-monolithic column can be provided with the same standard column used.

In an embodiment of the present invention, the enzyme-monolith column includes a monolith column and an enzyme bound to the column, and the monolith column has a network structure inside the column. Thereby forming a high surface area inside.

The network can be bound to the enzyme by covalent bonding through a functional group.

The network structure is characterized by being a Si-O bond structure. This is because the monolithic column is produced in the silica system, and thus usually forms the Si-O network structure.

The enzymes bound to the network can form an enzyme aggregate by cross-linking.

The enzyme may be selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, sumolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, lactase, organic phosphohydrolase, cholinesterase, And may be one or more selected from the group consisting of oxidase, alcohol dehydrogenase, glucose dehydrogenase, and glucose isomerase.

The functional group is a functional group capable of covalently bonding with an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenolic group, or an imidazole group of an enzyme.

The enzyme aggregate may be simultaneously covalently bonded to a plurality of functional groups.

The inner diameter of the monolithic column may be between 2 and 700 mu m.

The present invention also provides a method for producing an enzyme-monolithic column in which an enzyme column is formed in a monolithic column.

The method for preparing the enzyme-monolith column comprises the steps of (1) preparing a monolithic column having a network structure; (2) forming a functional group on the network structure of the monolithic column; (3) introducing an enzyme and a cross-linking agent into the monolith column to form an enzyme aggregate by cross-linking between the enzymes.

The enzyme may be added to the monolith column in the step (2).

The enzyme may be selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, sumolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, lactase, organic phosphohydrolase, cholinesterase, And may be one or more protein hydrolytic enzymes selected from the group consisting of oxidase, alcohol dehydrogenase, glucose dehydrogenase, and glucose isomerase.

The network structure is characterized by being a Si-O bond structure. This is because monolithic columns are based on silica-based materials and therefore have a Si-O bond structure in general.

The functional group can be covalently bonded to an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenolic group, or an imidazole group of an enzyme.

When the glutaraldehyde is added in the step of forming the functional group, an aldehyde group can be generated as a functional group, and when (3-aminopropyl) trimethoxysilane is added, an amine group can be formed as a functional group.

The functional group may be an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenolic group, or an imidazole group.

The crosslinking agent may be selected from the group consisting of diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethylaminopropylcarbodiimide, glutaraldehyde, bis (imidoesters) Esters) and diacid chlorides. These compounds may be used alone or in combination of two or more.

According to a preferred embodiment of the present invention, the enzyme-monolithic column is characterized in that it produces L-amino acid through DL-amino acid light-splitting, produces R-ibuprofen through spectroscopic separation of RS-ibuprofen, Production of 7-aminosepa sphosphoric acid through the removal of organic acids of cephalosporin C, production of palatinose and fructooligosaccharides by using L-alanine, acrylamide through hydrolysis of acrylonitrile, fructose, production of cacao butter using vegetable oil And fatty acid production, production of D-aspartic acid by light-splitting of DL-aspartic acid, production of maltooligosaccharide using liquefied starch, production of aspartame using amino acids, and the like.

In order to achieve the second object of the present invention, there is provided a system and system for analyzing proteomics comprising an enzyme monolithic column according to the present invention based on a standardized monolithic column of the present invention. A proteomic analysis apparatus according to the present invention includes an enzyme monolithic column and an analysis apparatus according to the present invention.

The enzyme monolithic column is manufactured using a commercially available column used in a proteomic analysis apparatus and can be directly connected to the analysis apparatus of the proteomic analysis apparatus through a simple connection part.

The analyzer can be used as an automated on-line proteomic analyzer by allowing the sample to be analyzed to pass through an enzyme-monolith column connected to the analyzer and then flowing directly to the analyzer.

Since the enzyme-monolith column of the present invention immobilizes a large amount of enzyme in a monolithic column, it is possible to maximize the efficiency of the enzyme reaction by maximizing the loading amount and the activity of the enzyme, thereby dramatically reducing the reaction time have.

The enzyme-monolithic column of the present invention has high protein resolution as compared with the conventional column, and can maintain long-term stability. Accordingly, the enzyme-monolithic column according to the present invention, which is manufactured to the same size as the column used in the proteomic analysis equipment, can be automated when it is directly connected to the analysis equipment. It is possible to analyze a large amount of samples repeatedly without replacing the column. Thus, the efficiency is very high in terms of economy.

Furthermore, since it can be applied not only to trypsin but also to various enzymes that can be applied industrially, it can be applied to an enzyme reaction system requiring a continuous flow reaction, and high enzyme activity can be secured for a long time.

1 is a schematic diagram showing a process for modifying a conventional commercially available silica column into a monolithic column.
2 is a schematic view showing a conventional method of producing an enzyme-monolithic column.
3 is a schematic view showing a method of producing an enzyme-monolith column according to a preferred embodiment of the present invention.
FIG. 4 shows an enzyme-monolith column connected with a syringe and a syringe pump to confirm the activity and digestion performance of the protein hydrolase immobilized on the enzyme-monolith column.
FIG. 5 is a graph showing the activity stability of EC-TR / MonoC (enzyme coatings of trypsin on monolithic column) samples according to the flow rate.
FIG. 6 is a graph showing the activity stability of CA-TR / MonoC (covalent attachments of trypsin on monolithic column) and EC-TR / MonoC (enzyme coatings of trypsin on monolithic column)
FIG. 7 is a result of hydrolyzing a model protein, enolase, using CA-TR / MonoC (covalent attachments of trypsin on monolithic column) samples.
FIG. 8 is a result of hydrolyzing a model protein, enolase, using EC-TR / MonoC (enzyme coatings of trypsin on monolithic column) samples.
Figure 9 is a schematic diagram showing an automated on-line proteomic analysis system in which an enzyme-monolith column is directly connected to a proteomic analysis instrument.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

An enzyme coating (EC) is a technique for attaching an enzyme through a covalent bond within a nanostructured material, and then crosslinking the enzyme with an attached enzyme by using a crosslinking agent to immobilize the enzyme. The nanostructure has a wide contact surface area and can fix the enzyme intensively inside and outside the substance. It can reduce the external influence that induces the decrease of the enzyme activity by the multi-layered structure, The stability of the activity can be maximized.

An enzyme-monolithic column, which is immobilized on a monolithic column with a structure for enzyme immobilization inside the column material, is immobilized with enzyme protease, trypsin, by an enzyme coating method, Which is essential for the trypsin digestion process, which is considered to be a protein-hydrolyzing enzyme. In addition, automated on-line extinguishing will be possible by producing an enzyme-monolithic column using the same standard column used in real-world proteomic analysis equipment (LC-MS).

[Preparation example]

Preparation of Monolithic Columns

A monolithic column was prepared in the same manner as in Fig. In order to immobilize the protein hydrolyzing enzyme, it is a process of making voids for attaching enzymes inside the monolithic column. The 1 M NaOH solution was flowed into the prepared column at 40 DEG C for 30 minutes and aged for 2 hours and 30 minutes. Then, the column was washed with distilled water for 1 hour at room temperature, and then 1 M HCl solution was flowed for 20 minutes and aged for 1 hour.

A solution was prepared so as to form a void in the column subjected to the pretreatment described above. To prepare the solution, 10 ml of 0.01 M acetic acid is prepared, 1.06 g of polyethylene glycol is dissolved, 4 ml of tetramethoxysilane (TMOS) is added, and the mixture is stirred at 4 ° C for 45 minutes. The prepared solution was put in a prepared column and aged overnight at 40 ° C.

Thereafter, distilled water was flown at room temperature for 30 minutes to form a gap inside the column, and a solution of 0.01 M ammonium hydroxide solution at 120 ° C for 3 hours, ethanol and HCl 40: 1 was flowed at room temperature for 30 minutes , Air dried, dried at 330 ° C overnight.

A 10% (3-aminopropyl) trimethoxysilane solution was prepared in toluene to form a functional group for covalent bonding to the formed pores, followed by drying at 70 ° C. overnight and thorough washing to prepare a monolithic column Respectively.

[Comparative Example]

Preparation of enzyme-monolithic column (CA-TR / MonoC (covalent attachments of trypsin on monolithic column)) using covalent attachment (CA)

As shown in FIG. 2, an enzyme-monolithic column using a covalent bond fixing method was prepared by introducing a flow of a synthesis solution fluid into a column using a device and a connection as shown in FIG.

An enzyme-monolith column with covalent binding of protein hydrolytic enzymes was synthesized by mixing the monolithic column prepared in Preparation Example with proteinase (trypsin). Specifically, to wash the monolithic column prepared in the above Preparation Example, 100 mM phosphoric acid buffer was flowed at room temperature for 30 minutes. Then, 0.1% glutaraldehyde solution was added to form a functional group for covalent bonding with the enzyme 100 mM phosphate buffer, and the mixture was allowed to flow at room temperature for 30 minutes. To remove unreacted glutaraldehyde, 100 mM phosphate buffer solution is again flowed at room temperature for 30 minutes. To induce the covalent bond between the monolith column and the protein hydrolyzing enzyme, 2.5 mg / ml trypsin solution was flowed for 10 minutes and aged for 1 hour and 50 minutes. 100 mM phosphate buffer is allowed to flow at room temperature for 20 minutes to remove unreacted trypsin. 100 mM Tris buffer was flowed for 60 minutes and capped. After completion of the reaction, the remaining Tris buffer solution was washed with 100 mM phosphate buffer for 20 minutes. The covalent attachments of trypsin on monolithic column (CA-TR / MonoC) were covalently bound to the prepared protein hydrolytic enzymes.

Preparation of enzyme-monolithic columns (EC-TR / MonoC) using enzymatic coating (EC) after covalent binding of protein hydrolytic enzymes

The cross-linked enzyme-monolith column as shown in FIG. 3 was prepared by introducing the flow of the synthesis solution fluid into the column using the apparatus and connection as shown in FIG.

4 shows a syringe pump, a syringe, a monolithic column, and a connecting part, which connects the end of the monolithic column to the syringe through a connecting part, and uses a syringe pump to flow the fluid into the monolithic column It is a device used for induction.

Preparative monolithic column and protein hydrolytic enzyme (trypsin, trypsin, TR) were mixed and protein hydrolytic enzyme was added by covalent binding enzyme coating method, covalently bonded protein hydrolytic enzyme The enzyme coatings of trypsin on monolithic column (EC-TR / MonoC) were synthesized by adding cross-linking agent to form cross-linking between quasi-proteolytic enzymes. Specifically, an enzyme-monolithic column in which a protein hydrolase was covalently bonded was synthesized by mixing the monolithic column prepared in the above Production Example with a protein hydrolyzing enzyme (trypsin). Specifically, to wash the monolithic column prepared in the above preparation example, 100 mM phosphoric acid buffer was flown at room temperature for 30 minutes. Then, 0.1% glutaraldehyde solution was added to 100 mM phosphoric acid to form a functional group for covalent bonding with the enzyme Was prepared as a buffer solution and allowed to flow at room temperature for 30 minutes. To remove unreacted glutaraldehyde, 100 mM phosphate buffer solution is again flowed at room temperature for 30 minutes. To induce the covalent bond between monolithic column and protein hydrolytic enzyme and cross-linking between covalently bound enzymes, 2 μl of 25% glutaraldehyde solution was added to trypsin at a concentration of 2.5 mg / ml, And matured for 1 hour and 50 minutes. 100 mM phosphate buffer is allowed to flow at room temperature for 20 minutes to remove unreacted trypsin. After completion of the capping reaction, 100 mM Tris buffer solution was flowed for 60 minutes, and washed with 100 mM phosphate buffer solution for 20 minutes to remove remaining Tris buffer solution. The monolithic column (EC-TR / MonoC) coated with the protein hydrolytic enzyme prepared by this process was stored at 4 ° C and stored until used without any decrease in activity.

Active measurement

* Protein hydrolase is covalently attached to enzyme-monolith column (covalent attachments of trypsin on monolithic column) using covalent attachment (CA) (enzyme coatings of trypsin on monolithic column) using an enzyme coating (EC) method using an apparatus and a connection as shown in FIG. 4, and the activity of the enzyme-monolith column (EC-TR / And the reacted solution was measured using an ultraviolet / visible spectrophotometer (Shimadzu UV-2450).

Specifically, CA-TR / MonoC and EC-TR / MonoC, which were stored at 4 ° C, were washed with 10 mM phosphate buffer. N α -benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) is prepared as a reaction solution by dissolving 10 mg of BAPNA in dimethylformamide (DMF). The resulting solution is poured into an enzyme-monolith column using a syringe pump to confirm the activity of the protein hydrolyzing enzyme. N-benzoyl-L-arginine 4-nitroanilide hydrochloride is decomposed into 4-nitroanilide by a protein hydrolyzing enzyme, and the decomposed 4-nitroanilide is a substance which is detected in the light absorption region at 410 nm. As the reaction progresses The absorbance increases.

Fig. 5 is a graph showing the results of measuring the activity of the EC-TR / MonoC sample.

Measurement of thermal stability

In order to confirm the thermal stability of CA-TR / MonoC and EC-TR / MonoC, after high-temperature treatment, fluid flow was induced inside the column using the apparatus and connection as shown in FIG. 4, and the reaction solution was measured with a UV / Vis spectrophotometer ultraviolet / visible spectrophotometer, Shimadzu UV-2450).

The enzyme-monolith column prepared in Examples 1 and 2 was heat-treated at 40 ° C and 50 ° C, and its activity was measured and then heat-treated again to confirm the thermal stability. Specifically, two kinds of enzyme-monolithic columns were placed in an oven at the same temperature for the same period of time, and heat treatment was performed by water bath method. After washing, the activity was confirmed. The column which had been confirmed to be active was washed once, The method was repeated.

FIG. 6 is a graph showing the thermal stability of two samples, wherein the CA-TR / MonoC sample exhibited an active half-life of less than 5 days at both 40 ° C and 50 ° C conditions, while the EC- Stability.

Measurement of protein hydrolysis capacity

Protein hydrolysis of CA-TR / MonoC and EC-TR / MonoC was confirmed by model protein digestion (enolase).

As shown in FIGS. 7 and 8, when the model protein hydrolysis experiment was performed using the enzyme monolith column, the CA-TR / MonoC sample showed a protein coverage value of 9% and the EC-TR / MonoC sample 16% protein coverage.

Claims (13)

A monolithic column having a network structure in which the network structure has a functional group; And
An enzyme bound to the functional group of the network structure; and an enzyme-monolith column. The enzyme-monolith column according to claim 1, wherein the network structure is a Si-O bond structure. The enzyme-monolith column according to claim 1, wherein the enzyme bound to the functional group of the network forms an enzyme aggregate by crosslinking. 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-monolith column is one or more selected from the group consisting of lysolecithin, cholinesterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, and glucose isomerase. The enzyme-monolith column according to claim 1, wherein the functional group can be covalently bonded to an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenol group or an imidazole group of the enzyme. 6. The enzyme-monolith column according to claim 5, wherein the functional group is at least one selected from the group consisting of an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenol group and an imidazole group. (1) preparing a monolithic column having a network structure;
(2) forming a functional group on the network structure of the monolithic column;
(3) introducing the enzyme into the monolith column in which the functional group is formed; And
(4) introducing an enzyme and a crosslinking agent into the monolith column to form an enzyme aggregate by cross-linking between enzymes;
≪ / RTI > wherein the enzyme-monolith column is an enzyme-monolith column. The method of claim 7, wherein the step (2)
2-1) forming a functional group on the network structure of the monolithic column; And
2-2) introducing the enzyme into the monolithic column.  8. The method of claim 7, wherein the enzyme is selected from the group consisting of trypsin, chymotrypsin, subtilisin, papain, suramolysin, lipase, peroxidase, tyrosinase, lacase, cellulase, xylanase, Wherein the enzyme is one or more selected from the group consisting of cholesterol, cholinesterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase and glucose isomerase. The method according to claim 7, wherein the network structure is a Si-O bond structure. The method of claim 7, wherein the crosslinking agent is selected from the group consisting of diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3- dimethylaminopropylcarbodiimide, glutaraldehyde, bis (imidoesters) , Bis (succinimidyl ester), and diacid chloride. The method of producing an enzyme-monolith column according to claim 1, The method for producing an enzyme-monolith column according to claim 7, wherein the functional group is covalently bonded to an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenolic group or an imidazole group of an enzyme How to. The method for producing an enzyme-monolith column according to claim 7, wherein the functional group is at least one selected from the group consisting of an amino group, a carboxyl group, an aldehyde group, a thiol group, a threonine group, an indole group, a phenol group and an imidazole group How to.

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