WO2011105820A2

WO2011105820A2 - Enzyme column and method for manufacturing same

Info

Publication number: WO2011105820A2
Application number: PCT/KR2011/001280
Authority: WO
Inventors: 김중배; 이상원; 전승현; 장문석; 김병찬
Original assignee: 고려대학교 산학협력단
Priority date: 2010-02-26
Filing date: 2011-02-24
Publication date: 2011-09-01
Also published as: WO2011105820A3

Abstract

The present invention relates to an enzyme column and to a method for manufacturing same, and more particularly, to an enzyme column in which polymer nanofibres, which can maximize the hydrolysis efficiency of a sample flowing in the column, are dispersed in the column by dispersing polymer fibers in the column to increase inter-fiber space between fibers, thereby maximizing the amount of an enzyme to be immobilized on the fiber and stabilizing activity, and to a method for manufacturing same.

Description

【Specification】

[Name of invention]

Enzyme column and its manufacturing method

Technology

The present invention relates to an enzyme column and a method for manufacturing the same, and more particularly, to increase the inter-fiber space by dispersing the polymer fibers in the column in detail, thereby immobilizing the fibers. The present invention relates to an enzyme column in which polymer fibers are dispersed well inside a column, which maximizes the amount of enzyme, stabilizes the activity, and reduces the bypass of the sample flowing in the column. ᅳ,

<2>

Background Art

<3> Enzymes are nanocatalysts (catalysts) of 2-20 nm size that promote chemical reactions in living organisms. Enzymes are linear biopolymers of amino acids that self-assemble into one form of structure by the evolution of life. This folding process results in enzyme selectivity, a useful property of enzymes. A binding pocket is formed. The enzyme's selectivity is already being used in a wide variety of fields, and its grandeur is expanding with the development of new disciplines. For example, enzymes are widely used in the pharmaceutical, fine chemical, food and detergent industries, and have a long history in biosensors, biopurification, and biochemical conversion. Recently, new uses of enzymes are spreading in biodiesel production, trypsin digestion in proteomic analysis, polymerase chain reaction, and biofuel cells. '

On the other hand, since the rapid development of nanotechnology since the 1990s, various nanostructured materials having high surface area ratios such as polymer nanofibers, nanoporous materials and magnetic nano Particles (magnet ic nanopar tides) and the like have been developed (see FIG. 1). One of the various advantages of these nanostructured materials is their ability to control nanometer sizes. For example, the size of the pores in the nanoporous material, the thickness control of the nanofibers or nanotubes, the particle size of the nanoparticles can be uniformly adjusted. These uniformly sized nanomaterials and similarly sized enzyme particles combine with other advantages of nanomaterials, such as conductivity and magnetism. It is possible to improve the properties of enzymes, particularly the activity and stability of enzymes in a nano biocatalyst system.

As described above, enzymes can be used in a wide variety of industrial fields. Since such enzymes are relatively expensive catalysts, reuse and recovery of enzymes through immobilized enzymes play an important economic role. In addition, the use of immobilized enzymes makes it very easy to design and control the reaction. Therefore, the immobilization method is an essential element in the use of enzyme as an industrial biocatalyst. As a general enzyme immobilization method using nano-structured materials, a simple adsorption method or a covalent attachment method using a covalent bond is used. The high surface area provided by the nanostructured materials can increase the loading of enzymes and increase the enzyme activity per unit weight. However, there are still many problems with the enzyme stability.

On the other hand, the enzymatic column containing the enzyme immobilized on the surface of the polymer fiber is not only proteolytic process but also L-amino acid production through light splitting of DL-amino acid, isomerization sugar production through fructose conversion of glucose, and cephalosporin. Production of 7-amino sephalosporinic acid by removal of C organic acid, acrylamide through hydrolysis of L-alanine and acrylonitrile, production of palatinose and fructooligosaccharides using fructose, production of cacao butter and fatty acids using vegetable oil, Production of D-aspartic acid by light splitting of DL-aspartic acid, production of maltooligosaccharides using liquefied starch, aspartame production using amino acids, R-ibuprofen production through RS-ibuprofen light splitting, biodiesel production using oil, etc. It is being used as a road.

However, the enzyme column including the enzyme immobilized on the conventional polymer fiber does not fill the column with the polymer fiber packed inside the column, and the bypass phenomenon occurs when the semi-coupling is poured into the column. do. Due to this, there is a problem in that the reaction efficiency of the enzyme reaction is sharply decreased due to a decrease in the chance of encountering the reaction product with the enzyme.

<8>

[Detailed Description of the Invention]

[Technical problem]

The present invention has been made to solve the above-mentioned problems, the first problem to be solved of the present invention, by dispersing the polymer fibers in the column by blocking the bypass phenomenon through the semi-efficiency of the sample flowing through the column Enzyme curls to maximize It is to provide a method for producing rum.

The second problem to be solved of the present invention is to provide an enzyme column produced by the above production method.

<11>

Technical Solution

In order to solve the first problem described above, a method for producing an enzyme column according to an embodiment of the present invention, 1) packing the polymer fibers in a column (packing); 2) treating the polymer fibers packed in the column with an alcohol solution to disperse the polymer fibers in the column to increase the inter-fiber space; 3) removing the treated alcohol solution in the column; And 4) adding an enzyme to the polymer fiber packed in the column to fix the enzyme on the surface of the polymer fiber.

According to a preferred embodiment of the present invention, the polymer fiber is a polymer nano fiber or a polymer micron fiber and may have a three-dimensional network structure.

According to another preferred embodiment of the present invention, the polymer fiber is the polymer fiber is polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, Chitosan, polylactic acid, polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polyaniline and poly (styrene-co-maleic anhydride).

According to another preferred embodiment of the present invention, the alcohol solution is

It may include any one or more selected from the group consisting of methanol, ethanol, propanol and butanol, the concentration of the alcohol solution may be 15 ~ 100¾ (v / v). According to another preferred embodiment of the present invention, step 3) may be performed by washing the inside of the column without including a drying step.

According to another preferred embodiment of the present invention, in step 4), a crosslinking agent may be added to the inside of the column in order to fix the enzyme on the surface of the polymer fiber.

According to another preferred embodiment of the present invention, the crosslinking agent is diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethylaminopropylcarbodi Mead and glutaraldehyde may include any one or more selected from the group consisting of.

According to another preferred embodiment of the present invention, the enzyme is trypsin, chemo Proteases such as trypsin, subtilisin, papain and thermolysin, lipases, peroxidases (horse radish peroxidases, soybean peroxidases, chloroperoxidases, manganese peroxides), tyrosinase, laca At least one selected from the group consisting of azeases, celases, xylanases, lactases, organophosphodilases, cholinesterases, glycosylases, alcohol dehydrogenases, glucose dehydrogenases, and glucose isomerases Can be.

In order to solve the above-mentioned second problem, the enzyme column according to an embodiment of the present invention is a column; And polymer fibers packed inside the column, treated with an alcohol solution, dispersed in the column, and having an increased inter-fiber space.

According to another preferred embodiment of the present invention, the polymer fiber is polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene cellulose, chitosan, polylac Tic acid, polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polyaniline, and poly (styrene-co-maleic anhydride).

According to a preferred embodiment of the present invention, an enzyme may be immobilized on the surface of the polymer fiber, in which case the enzyme may be a protease or lipase such as trypsin, chymotrypsin, subtilisin, papain, thermolysin, etc. , Peroxidase (horse radish peroxidase, soybean peroxidase, chloro peroxidase, manganese peroxidase), tyrosinase, lacase, cell 1: lase, xylanase, lactase, organic It may be any one or more selected from the group consisting of phosphohydrolase, cholinesterase, glycosylase, alcohol dehydrogenase, glucose dehydrogenase, glucose isomerase, and the like, but is not limited thereto.

According to a preferred embodiment of the present invention, the enzyme column is L-amino acid production through the hydrolysis process of the protein and DL-amino acid light splitting, isomerized sugar production through fructose conversion of glucose, Separosporin C Production of 7-aminocephalosporinic acid through removal of organic acids, acrylamide through hydrolysis of L-alanine and acrylonitrile, production of palatinose and fructoligosaccharides using fructose, cacao butter and fatty acid production using vegetable oils ， D-aspartic acid production through light splitting of DL-aspartic acid, maltooligosaccharide production using liquefied starch, aspartame production using amino acid, R- ibuprofen production through RS-ibuprofen light splitting, biodiesel production using oil Can be used for production. <25>

Advantageous Effects

The enzyme column of the present invention disperses the polymer fibers in which the enzyme is immobilized in the column, thereby maximizing the space between the high molecular fibers to prevent bypass when a sample such as a protein passes through the column, and at the same time, By maximizing loading capacity and activity, hydrolysis efficiency can be maximized. In addition, the reaction time can be significantly reduced compared to the enzyme column.

Furthermore, compared with the conventional in-solution digestion method or commercially available enzyme beads or kits, the reuse is easy and the on-line hydrolysis process or automation in the trypsin hydrolysis process is easy. You can enjoy the process. In addition, it can be applied to various enzymes that can be used industrially as well as tricin, thereby securing the enzyme activity and stability required for industrial production using enzymes.

<28>

[Brief Description of Drawings]

1 illustrates the structure of a conventional nanomaterial.

Figure 2 schematically shows a conventional protein hydrolysis.

3 schematically shows a production process of PS + PSMA nanofibers using an electrospinning method.

4 schematically shows enzyme immobilization using polymer fibers.

FIG. 5 shows an enzyme coating method for (a) an alcohol-coated polymer nanofiber (EC-TR / NF-column) using an enzyme coating method, and (b) an alcohol-treated polymer nanofiber. It is a graph showing the column used (EC-TR / EtOH-NF-column).

6 is a graph showing the results of measuring LOMS / MS through CA-TR / NF for 0 to 59 days as one embodiment of the present invention.

FIG. 7 illustrates an embodiment of the present invention using CA-TR / EtOH-NF for 0 to 59 days.

It is a graph which shows the result of measuring LC-MS / MS.

FIG. 8 shows an embodiment of the present invention using LC-TR / NF for 0-59 days.

It is a graph which shows the result of measuring MS / MS.

9 shows an embodiment of the present invention using EC-TR / EtOH-NF for 0 to 59 days.

It is a graph which shows the result of measuring LC-MS / MS. ᅳ

10 is a graph showing the results of LC-MS / MS after 59 days as one embodiment of the present invention. to be.

11 shows, as one embodiment of the present invention, (a) 0.1 ml / h, (b) 0.5 ml / h, (c) 1 ml / h,

(d) Graph showing LC / MS-MS at 5 ml / h.

12 shows, as one embodiment of the present invention, (a) an EC-TR / NF column, and (b) an EC-TR / EtOH-NF.

Graph showing LC / MS-MS in column.

FIG. 13 is a graph comparing proteolytic ability after stirring EC-TR / EtOH-NF, commercially available trypsin beads, and free enzymes at a high temperature of 50 to confirm thermal stability.

14 is a photograph measuring the degree of dispersion of nanofibers according to the concentration of alcohol.

<43>

[Best form for implementation of the invention]

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

As described above, the enzymatic column including the enzyme immobilized on the conventional polymer fiber does not fill the column with the polymer fiber packed inside the column, and when the semi-coagulum is poured into the column, The fibers are compressed to create voids in the column (the space not layered with polymer fibers), as well as to compress the polymer fibers

^{'' The} inter-fiber space between oil and fiber is reduced and eventually

Bypass will appear. As a result, the reaction product does not come into contact with the enzyme immobilized on the nanofibers while passing through the column.

Thus, the method for producing an enzyme column according to an embodiment of the present invention, 1) packing the high molecular fibers in a column (packing); 2) Treating the polymer fibers packed in the column with an alcohol solution to disperse the polymer fibers in the column to inter-fiber

Increasing inter-fiber space; 3) removing the treated alcohol solution in the column; And 4) adding an enzyme to the polymer fiber packed in the column to fix the enzyme on the surface of the polymer fiber.

As a result, the enzyme column disperses the polymer fibers in which the enzyme is immobilized in the column, thereby maximizing the space between the high molecular fibers to prevent bypass when a sample such as a protein passes through the column and at the same time loading the enzyme such as trypsin. By maximizing the activity, the hydrolysis efficiency can be maximized. In addition, the hydrolysis time can be significantly reduced as compared to the conventional method of hydrolyzing the protein. First, as the step 1), the polymer fiber is packed into the column. According to one embodiment of the present invention, the usable column may have a suitable diameter, shape and material according to the purpose and use, and preferably may have various diameters of cylindrical 50 nm ~ lm, depending on the purpose, The length of the column may also be appropriately adjusted.

According to one embodiment of the invention, polymer fibers are packed to immobilize enzymes in the column. At this time, the polymer fibers used are not limited in kind and diameter as long as they can be used in the enzyme column, but preferably microfibers or nanofibers may be used, and more preferably nanofibers may be used. It is very advantageous to maximize the amount of enzyme immobilized by increasing the specific surface area.

On the other hand, the polymer fibers may have a three-dimensional network structure including an inter-fiber space between the fibers and the fibers, for this purpose, the polymer fibers are produced by electrospinning or melt spinning May be, but is not limited to. The material of the polymer fiber of the present invention is polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid , Polyglycolic acid, polycaprolactone, collagen, polyaniline, poly (styrene-co-maleic anhydride), and copolymers thereof, but may be any one or more selected from the group. Preferably, the use of nanofibers having a three-dimensional network structure made of electrospinning or melt spinning maximizes the space between polymer fibers to prevent bypasses when proteins and samples pass through the column. It is extremely advantageous to maximize the loading and activity of enzymes such as trypsin.

On the other hand, the polymer fibers are packed in a column, and the method of immobilizing the nanofibers on the column is a conventional method, preferably fixing the polymer fibers at both ends of the column, or By physically compressing and packing the polymer fiber in the front of the column can be fixed by pressing the hydraulic pressure so that no empty space. In addition, when the surface of the glass is modified with aminopropyl trimethoxysilane on the glass inside the column and the nanofibers are packed, maleic anhydride of the nanofibers and the surface-modified glass It is apparent to those skilled in the art that the amino group may form a chemical reaction so that the nanofibers may be immobilized inside the column, but the present invention is not limited thereto, and the polymer fibers may be packed into the column through various methods known in the art. Next, in step 2), the polymer fibers packed in the column are treated with an alcohol solution to disperse the polymer fibers in the column to increase the inter-fiber space. Specifically, referring to FIG. 4, when an alcohol solution is added to the inside of the column packed with polymer fibers, the polymer fibers are widely dispersed in the column, thereby significantly increasing the inter-fiber space between the fibers. As a result, when a sample such as a protein passes through the column, the bypass is improved, so that the reaction time is remarkably enjoyed, and the surface area of the polymer fiber that can bind with the enzyme is increased, thereby increasing the amount of enzyme. It can be immobilized on the surface of the fiber.

FIG. 5 (a) is a column without alcohol treatment, and FIG. 5 (b) is a column with alcohol treatment, in which a portion is an outer wall portion of the column and b portion is a hollow portion where polymer fibers are located. This is the part that is performed. In this case, the alcohol-treated enzyme column (FIG. 5b) is dispersed widely enough to fill the inside of the column, whereas the alcohol-treated enzyme column (FIG. 5a) is not dispersed and the polymer fibers are bound together to form a space between fibers. You can see this very small.

On the other hand, according to a preferred embodiment of the present invention, it is very advantageous to prevent the bypass and maximize the efficiency of the polymer fiber is dispersed widely enough to fill the inside of the column through the alcohol treatment. . Specifically, if the dispersed polymer fibers do not fill the inside of the column, a space is formed between the inner wall of the column and the polymer fibers. Without returning to the spaced apart space is reduced efficiency. In addition, if the polymer fibers are not dispersed in a small amount, the space between the fibers may be reduced, which may cause a problem in that the reaction efficiency decreases.

Meanwhile, the alcohol treatment step must be performed after packing the polymer fibers in the column. If the polymer fiber is alcohol-treated and then the polymer fiber is packed in the column, the dispersed polymer fiber is shrunk again during the packing process, resulting in a small space between the fibers. In particular, when the hollow inside the column is used in order to maximize the reaction efficiency as shown in Figure 5b, the polymer fiber is in contact with the inner wall of the column in the packing process may cause a problem that the space between the fibers is significantly smaller.

Meanwhile, the alcohol that can be used in the present invention can be used without limitation as long as it can increase the interfiber space of the polymer fiber, preferably the alcohol solution is methane, ethanol, 1-propanol It may include any one or more selected from the group consisting of 2-propanol and butyl alcohol. On the other hand, according to a preferred embodiment of the present invention, the concentration of the alcohol solution is 15 ~ 100% (v / v) is very advantageous to maximize the dispersion effect of the polymer fiber (see Example 6).

Next, step 3) is performed to remove the alcohol solution treated in the column. If the alcohol treated in step 2) is not removed, the stability of the enzyme is lowered, and the enzyme may adversely affect alcohol-sensitive enzyme reaction, which may denature the enzyme or inhibit the yield of the product. Since the interfiber space of the dispersed polymer fibers may be reduced, the step of removing alcohol treated in step 2) must be performed. To this end, there is a method of evaporating alcohol through a drying method such as washing through a conventional water washing, heating, etc., but when performing the drying step, the polymer fibers dispersed in the column may shrink again, so It is preferable to remove the alcohol.

Next, as step 4), the enzyme is added to the polymer fiber packed in the column to fix the enzyme on the surface of the polymer fiber. Enzymes that can be applied to the present invention can be used without limitation as long as it can be immobilized on a polymer fiber, preferably the enzyme is trypsin, chymotrypsin, subtilisin, papain, thermolysine) such as protease, lipase , Peroxidase (horse radish peroxidase, soybean peroxidase, chloro peroxidase, manganese peroxidase), tyrosinase, laccase, celerase, xylanase, lactase, organophosphohydrate Relase choline, at least one selected from the group consisting of steases, glycosylases, alcohol dehydrogenases, glucose dehydrogenases, and glucose isomerases, typically trypsin is used as an indicator of the enzyme column. That is, the enzyme column in which trypsin can be used can be used in different types of enzymes. Preferably, the enzyme that can be used at this time may have a size of more than lnm.

On the other hand, the method of immobilizing the enzyme on the surface of the polymer fiber is not limited, and preferably, the enzyme is added to the inside of the column to immobilize the enzyme on the surface of the polymer fiber through the adsorption and / or covalent bond between the polymer fiber and the enzyme You can. More preferably, by adding a crosslinking agent to form crosslinks between the enzymes to immobilize the enzymes on the surface of the polymer fibers, the enzymes may be entrained on the surface of the polymer fibers and immobilized through the enzyme coating. This is called. The crosslinking agent that can be used at this time can be used without any limitation as long as it can form crosslinking between enzymes without inhibiting the activity of the enzyme. A specific example of this is diisocyane. Yit, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, glutaraldehyde, a mixture thereof, and the like, and glutaraldehyde is more preferable. However, the present invention is not limited thereto.

According to a preferred embodiment of the present invention, after preparing a high molecular nanofiber (polymer nanofiber) that can be attached to the amino group of the enzyme by electrospinning, if a simple treatment using alcohol polymer nanofiber (nanofiber ) Disperse increases surface area and inter-fiber space. The dispersed polymer nanofibers increase the surface area to which enzymes can attach, thereby increasing the amount of enzyme per unit weight of nanofibers.

Then, after removing the alcohol added by washing with water, the enzyme trypsin solution is flowed, and the glutaraldehyde and trypsin mixture are flowed into the column to crosslink _. The enzyme is coated on the polymer nanofibers within. The advantage of this method is that if the polymer nanofibers are not dispersed through alcohol treatment, when the protein solution flows into the column, the polymer nanofibers are compressed by hydraulic pressure to generate empty space in the column. The protein solution is bypassed to decrease the hydrolysis efficiency (see FIG. 5A). On the other hand, if the polymer nanofibers are dispersed through alcohol treatment, even if hydraulic pressure is generated when they are thrown in the column of the protein solution, the polymer nanofibers are dispersed and filled in the column to prevent bypass. Hydrolysis efficiency can be maximized (see FIG. 5B).

The enzyme column produced by the above-described manufacturing method of the present invention is packed into a column and the inside of the column, and treated with an alcohol solution to disperse the column and increase the inter-fiber space (inter-fiber space). It includes. In addition, the target enzyme can be immobilized on the surface of the polymer fiber of the enzyme column thus prepared.

According to a preferred embodiment of the present invention, it is very advantageous to prevent the bypass and maximize the efficiency of the polymer fiber is dispersed widely enough to fill the inside of the column through the alcohol treatment. Specifically, when the polymer fiber is unable to fill the inside of the column, a space is formed between the inner wall of the column and the polymer fiber. Therefore, when the reaction product is flowed into the column, some reaction products do not react with the enzyme immobilized on the polymer fiber. Without being taken out into the spaced apart, the efficiency is reduced. In addition, if the polymer fibers are not dispersed in a small amount, the space between the fibers may be small, resulting in a decrease in reaction efficiency. ^' This improves the bypass when samples such as proteins pass through the column, significantly reducing reaction time and increasing the surface area of the polymer fibers that can bind the enzyme, resulting in much higher amounts of enzyme. It can be immobilized on the surface of the polymer fiber. This leads to a marked increase in activity. Furthermore, the sustainability of the activity is remarkably improved compared to conventional alcohol-free enzyme columns, in-solution digestion methods or enzyme beads. Since the reaction between the reaction material and the enzyme is made intimately, the reaction time can be reduced.

The enzyme column of the present invention can be used not only for the use of protein hydrolysis columns, but also for the production of L-amino acids through DL-amino acid light splitting, the production of isomerized sugars through the conversion of fructose to glucose, and the removal of organic acids from cephalosporin C. Production of aminocephalosporinic acid, hydrolysis of L-alanine and acrylonitrile, production of palatinose and fructose using fructose, sugar production, cacao butter and fatty acid production using vegetable oil, and optical splitting of DL-aspartic acid It can be widely used for production of D-aspartic acid production, maltooligosaccharide production using liquefied starch, aspartame production using amino acid, R-ibuprofen production through RS-ibuprofen light splitting, and biodiesel production using oil.

As described above, the enzyme column according to the present invention can not only shorten the total proteomic analysis time by hydrolyzing the target protein within a short time, but also stabilize the enzyme activity using the enzyme coating method. The lifespan of these products is greatly increased, making them useful for the proteomic analysis industry. In addition, the enzyme column of the present invention not only proteolysis, but also bioconversion, bioremediation, biosensors, high fructose corn syrup production, pharmaceutical industry , Food industry, chemical industry, etc.) can be used for the treatment of various enzymes.

<68>

[Form for implementation of invention]

Hereinafter, preferred embodiments will be presented to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example 1 Preparation of Enzyme Column

<71> 1-1. Preparation of the ingredients

(1) enzymes

Enzymes for enzyme immobilization are high purity derived from porcine pancreas. Trypsin was used. Trypsin was purchased from Sigma-Aldr ich (St. Louis, Mo., USA).

(2) Polymer nano fiber

<75> The polymer for making the polymer nanofiber is PS molecular weight (丽) =

950,400) and poly (styrene-co-maleic anhydride) (PSMA, Mw = 224,000) were used, and the organic solvent for dissolving the polymer was tetrahydrofuran (THF). And acetone were used. These materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

(3) Buffers and Chemicals

Phosphate buffer (PB) 10, 100 mM, pH 7.9 was used as the buffer, and glutaraldehyde was used to use the enzyme coating. Tris buffer (OOmM, pH 7.9) was used to prevent maleic anhydride groups in polymer nanofibers after enzyme coating. Nci-benzoyl-L-arginine 4-nitroanilide hydro, chloride (N a_Benzoyl_L_arginine 4-nitroani 1 ide hydrochloride, L—BAPNA) was used as a semi-ung substrate for enzyme activity and physical properties. In the proteomic analysis, the model protein used in the proteolytic process was enolase, which was all purchased from Sigma-Aldr ich (St. Louis, MO, USA).

<78> 1-2. Device used

In order to produce polymer nanofibers, a polymer solvent is used for the electrospinning method. For this, a high-voltage supply and a syringe pump (syringe pump, PHD-2000 Infusion, Harvard Apparatus, Holliston, MA) are used. , USA).

In order to confirm the characteristics of the enzyme catalyst, the product obtained by reacting trypsin immobilized with the enzyme coating method and L-BAPNA, which was an anti-ungwoo substrate, was measured using a spectrophotometer (Sspectrophotometer, Shimadzu, UV-2450). Hydrolyzed peptides after hydrolysis of the model proteins using immobilized enzymes for protein hydrolysis were used nanoAQUITY UPLC (Waters) and 7-tesla LTQ-FT (Thermo).

<8i> 2-1. Fabrication of Trypsin Hydrolysis Column

(1) Preparation of PS + PSMA Nanofibers by Electrospinning Method and Alcohol Treatment of Polymer Nanofibers

<83> Nanofibers for enzyme immobilization include polystyrene (PS) and poly (styrene).

-ex) -maleic anhydride) (Poly (styrene-co-maleic anhydride), PSMA) The maleic anhydride of PSMA is covalently immobilized with the amine group of the enzyme.

Figure 3 shows the production of PS + PSMA nanofibers using the electrospinning method. The production method of PS + PSMA nanofiber is as follows. PS and PSMA were mixed at a weight ratio of 2: 1 at room temperature, dissolved in tetrahydrofuran (THF), and mixed for about 3 hours using a magnetic stirrer. Thereafter, the acetone solution was mixed to lower the viscosity of the polymer solution, and then the polymer solution was filled with a 5 ml syringe with 30 gauge stainless steel needles. The operating condition of the voltage was 7 kV, and the flow rate was 0.1 ml / hr using a syringe pump. The nanofibers from the electrospinning were collected in clean aluminum foil.

The alcohol treatment process for making dispersed nanofibers is as follows. Nanofibers were placed in a vial containing 50% v / v alcohol solution (ethanol), followed by shaking for 10 minutes at 200 rpm. Once the nanofibers were completely dispersed, they were washed without drying until alcohol was completely removed from the solution. The dispersed nanofibers were stored in buffer solution until trypsin immobilization.

(2) Enzyme Immobilization Using Polymer Nanofibers

Enzyme (trypsin) immobilization using nanofibers (NF) was carried out using the following four types.

[88] [Coupling Enzyme Immobilization Using Alkali-treated Nanofibers (CA-

TR / NF), covalent enzyme fixation method using alcohol-treated nanofibers (CA-TR / EtOH-NF), enzyme coating method (EC-TR / NF) using alcohol-free nanofibers, alcohol-treated nanofibers Enzyme coating method using fiber (EC-TR / EtOH-NF)].

4 is a conceptual diagram for trypsin immobilization using polymer nanofibers. After preparing the same weight (5 mg) alcohol-free nanofibers (NF) and alcohol-treated nanofibers ( _Et0H - _NF ) for trypsin immobilization, trypsin solution (10 mg / ml, 10 _mM sodium phosphate buffer (sodium phosphate) buffer, pH 7.9)). After shaking the vial containing trypsin solution and nanofibers at 200 rpm for 30 minutes, the trypsin was incubated at 4 ^° C for 2 hours to immobilize the trypsin to the nanofibers. Thereafter, for the synthesis of enzyme coating (EC-TR), a glutaraldehyde solution (0.5% w / v) as a crosslinker was added thereto, and then incubated overnight at 4 ^° C. 90> For covalent attached trypsin (CA—TR) A buffer solution was added instead of a glutaraldehyde solution. The biocatalyst nanofibers (CA-TR, EC-TR) incubated overnight were washed with lOOmM sodium phosphate buffer (pH 7.9), and the unreacted aldehyde groups remained on the nanofibers. Shaking was performed for 30 minutes using lOOmM sodium phosphate buffer (pH 7.9) to prevent this. The finished biocatalyst nanofibers were completely washed with 10 mM sodium phosphate buffer (pH 7.9) and stored at 4 ^° C.

(3) Determination of activity of immobilized trypsin

Trypsin activity of the biocatalyst nanofibers was measured by hydrolysis of L-BAPNA, the semi-manipulator of trypsin in aqueous buffer. The L-BAPNA solution was used by diluting 1/100 of a solution in which L-BAPNA was dissolved with DMF (10 mg / ml in DMF) using 10 mM sodium phosphate buffer (pH 7.9). Enzyme immobilized on nano-structured material is impossible to measure in real time, so 10ml L-BAPNA solution is added with biocatalytic nanofibers and reacted at 200rpm. Used. Thereafter, after diluting to 1/10 with 10 mM sodium phosphate buffer (pH 7.9), the absorbance was measured at 410 nm using a spectrophotometer.

(4) Protein Hydrolysis Using Biocatalytic Nanofibers

Experiments were carried out to apply proteolytic processes in proteomic analysis using biocatalyst nanofibers. The protein to be hydrolyzed was model protein Enolaa low Kenolase), and the hydrolysis method was in-solut ion digestion method. The experimental method is as follows.

In a vial containing biocatalyst nanofibers, 2 ml of enolase solution (0.1 mg / ml in lOOmM ammonium bicarbonate (pH 8.0)) was added. The reaction was overnight at 200 rpm and phase. After reaction, 1.5 ml of supernatant was added to a new vial and formic acid was added to prevent further reaction. The resulting samples were stored at -70 ^° C until analysis using LC—MS / MS. The biocatalyst nanofibers used were washed clean and stored at 4 ^° C until the next use.

(5) Protein hydrolysis using enzyme column

The conventional in-solut ion digestion method has a long time problem. Chromatography of nanofibers to solve this problem Enzyme column was prepared by enzymatic coating method after put into a chromatography column. The enzyme column is connected to a syringe pump that can flow protein solution into the column at a constant rate. In the chromatography column, frits are formed at the inlet and outlet of the column to prevent nanofibers from coming out of the column, so that only the product solution decomposed by the enzymatic column can come out. Enzymatic Columns Columns (EC-TR / NF-column) for enzyme-coated nanofibers on small alcohol-treated nanofibers (EC—TR / EtOH-NF-columns) It was produced in two ways (FIG. 5).

First, the polymer nanofibers are filled into a chromatography column, and in the case of the EC-TR / EtOH-NF-column, 50% v / v alcohol solution is poured overnight at 1 ml / h flow rate to disperse the nanofibers in the column. gave. After washing with 10 mM phosphate buffer (pH 7.9) to ensure that no alcohol solution remains in the column, trypsin solution (10 mg / ml in 10 mM phosphate buffer (pH 7.9)) is filled with nanofibers. It was held for 2 hours at a flow rate of 2.5 ml / h into a column. For the synthesis of enzyme coating, the solution containing trypsin and glutaraldehyde was flowed for 10 minutes and then incubated for 50 minutes. Then, as in the enzyme coating method, lOOmM Tris-HCKpH 7.9) was used to prevent aldehyde groups of unfinished nanofibers for 1 hour. The enzyme column was washed with 5 ml / h flow rate using PB buffer and stored at room temperature until use.

Example 2 Activity Measurement of Trypsin Hydrolysis Column

In the case of free enzyme, the enzyme is dissolved in water, so it can be measured in real time using a spectrophotometer, but in the case of an enzyme immobilized on a nanostructured material, it cannot be measured in real time. Therefore, after reacting at 200 rpm, a method of taking and measuring samples by lOOul was used every hour. Then, after diluting to 1/10 with 10mM sodium phosphate (sodium phosphate, pH 7.9), the absorbance was measured with a spectrophotometer at 410nm.

Table 1

<i03> Table 1 shows EC-TR / EtOH-NF. Initial Activity EC— The initial activity of the TR / NF and the kinetic values are shown. First, the initial activity of EC-TR / EtOH-NF is about 8.2 times higher than the initial activity of EOTR / NF. This proves that the maleic anhydride group contained in the nanofiber is exposed to the aqueous phase because the surface area is increased by spreading the nanofiber through alcohol treatment. Since the amine groups of the enzyme are immobilized to maleic anhydride, this means that the ability of the dispersed nanofibers to immobilize trypsin is improved. Compared to the enzyme coating on the enzyme loading amount is greatly improved.

<| 04> Reaction rate constants of immobilized enzymes (V _max and K can be obtained by michaelis-menten analysis. Because the enzyme coating was crosslinked by glutaraldehyde treatment, hop intensity It is not possible to measure the exact amount of loading by the BCA method or the BCA method, and the method of measuring the initial activity is also affected by the mass transfer limitation. Therefore, measuring the loading of the immobilized enzyme by obtaining V _max may be a method of obtaining a relatively accurate enzyme loading.

As a result, the V _max of EC-TR / EtOH-NF was about 21.7 times higher than that of Vmax of EC-TR / NF. Assuming that the turnover number (kcat) at V _max is constant in the immobilization system, the maximum velocity value (Vmax) is proportional to the enzyme loading (V _max = k _cat > [E ] o]). Therefore, since the surface area was increased by the nanofiber spread through the alcohol treatment, it can be confirmed that the amount of trypsin adhered to the dispersed nanofibers is 19 times higher than the amount of trypsin adhered to the conventional nanofibers.

^ Is information that can be used to determine mass transfer resistance when an enzyme reacts with a substrate. The lower the Km value, the lower the mass transfer resistance, and the higher the reaction efficiency of enzyme and substrate. As a result, EC-TR / EtOH-NF was found to be about 5.8 times higher than ^ of EC-TR / NF. It is confirmed that the mass transfer resistance is high because the dispersed nanofibers are attached with a large amount of cross-linked trypsin.

Example 3 Measurement of Stability, Efficiency and Reusability of Enzyme Columns Trypsin can be used in the process of analyzing proteins in the proteomics, and in the process of making proteins into fabtide through trypsin hydrolysis process. In this experiment, we examined the utility of the trypsin hydrolysis process in proteomic analysis using immobilized trypsin. For this purpose, the model protein enolase

hydrolysis efficiency of four different immobilized trypsin (EC-TR / EtOH-NF, EC-TR / NF, CA-TR / EtOH-NF, CA-TR / EtOH-NF) with enolase, Experiments were conducted on stability and reusability.

After Enolase hydrolysis, the solution containing the hydrolyzed product was stored at -70 ^° C for IX-X MS / MS analysis, and the immobilized trypsin used was no longer hydrolyzed. After washing with a buffer until no residual protein remained, it was stored at 4 ^° C until the next use. In proteomic analysis using LOMS / MS, the peptides from which enolase is hydrolyzed through conventional enolase hydrolysis are organized into a database, and LC-MS / The peak area of the peptide detected by MS analysis was compared with the existing database to confirm the efficiency of hydrolysis. In this experiment, the enolase was determined from the peak area weighting database of the peptide peptide detected by LC-MS / MS analysis.

Five representative peptides that can be considered to be hydrolyzed peptides of (enolase) were selected and their area summed to determine their hydrolysis efficiency.

<ιιο> Enolase was hydrolyzed using 4 types of immobilized trypsin repeated for 59 days, followed by enolase hydrolyzed peptides via LC-MS / MS. The analysis results are as follows. First, in the case of covalent enzyme immobilization methods (CA / EtOH-NF and CA / NF), the initial hydrolysis efficiency is lower than that of enzyme coating, and the efficiency decreases rapidly over time when reused. It could be confirmed (see FIGS. 6 and 7). This results in the low stability of the covalent enzyme immobilization method, demonstrating that enolase was not fully digested under in-solution digestion experimental conditions. On the other hand, comparing the covalent enzyme immobilization method (CA / EtOH-NF and CA / NF) in nanofibers and dispersed nanofibers, the relative stability of CA / EtOH-NF is higher. This is due to the spread of nanofibers through alcohol treatment, which leads to an increase in the surface area and thus to the attachment of more enzymes, so that the enzymes lose their activity over time but remain active.

: The remaining enzyme is relatively large compared to the enzyme attached to the existing nanofibers. When the <ιπ> enzyme coating method is used, it can be seen that high hydrolysis efficiency is maintained even when reused for 59 days (see FIGS. 8 and 9). In addition, it can be seen that the enzyme coating method (EC / EtOH-NF) using dispersed nanofibers has about three times higher hydrolysis efficiency than the conventional enzyme coating method (EC / NF) using nanofibers. (See FIG. 10).

Through this, it can be seen that the enzyme coating method maintains high stability and activity compared to the covalent enzyme fixation method. When using dispersed nanofibers, the amount of the enzyme immobilized increases the existing nanofibers. Compared with the results used, it was confirmed that the high activity. In addition, this enzyme immobilization method was confirmed that can be successfully used in the trypsin hydrolysis process in the proteomic analysis, which is the actual field of practice.

Example 4 Fabrication of Specific Trypsin Hydrolysis Column

<ιΐ4> Liquid phase hydrolysis using biocatalytic nanofiber (in-

"Solution digestion" showed excellent hydrolysis efficiency during protein hydrolysis.

Has the problem of reducing the time for hydrolysis. Many efforts have been made to solve this problem, and enzyme stabilization is still a problem. In this study, an enzymatic column using enzymatic coating method was proposed to provide a solution to reduce enzyme stabilization and hydrolysis time. It was fabricated and applied to the hydrolysis process.

FIG. 5 shows the entire picture of the enzyme column system for protein hydrolysis and the dispersed nanofibers through the enzyme treatment (EC-TR / NF column) and alcohol treatment using the enzyme coating method in the conventional nanofibers. Column using enzyme coating method

-TR / EtOH-NF column).

<! 16> The enzyme column system using the enzyme coating method has the advantage of significantly reducing the hydrolysis time (within 5 minutes) compared to the conventional in-solution digestion method. In addition, since the enzyme coating stabilizes the enzyme, it has the advantage of maintaining the hydrolysis efficiency even when reused.

<ιΐ7> However, as EC-TR / NF columns continue to be used, the nanofibers in the column are compressed by fluid pressure, resulting in an empty space, and thus the fluid flowing in the column. Bypassing into the voids reduces protein hydrolysis efficiency. EC-TR / EtOH-NF columns, on the other hand, are nano-islets in the column. ^; ᅳ Oil is completely dispersed and nanofibers are compressed to prevent empty spaces, preventing bypass and having high protein hydrolysis efficiency even when reused.

<ιΐ8> First to determine the rate of flow of enolase solution into the column

The flow rate of the EC-TR / NF column was changed (0.1-5 ml / h), and the degree of hydrolysis by hydrolysis of the enolase was analyzed by LC / MS-MS. As a result, the enolase was more hydrolyzed as the flow rate was enjoyed (see FIG. 11). This can be seen that as the ow rate decreases, the time for which the enolase solution stays in the column increases, so that the time for enolase contact with trypsin increases and more hydrolysis occurs. From these results, the final flow rate (flow rate) was determined to be 2 ^ / min.

<ιΐ9> Figure 12 shows the enzyme column using the enzyme coating method in the conventional nanofibers (EC-

TR / NF column) and enolase hydrolysis using enzymatic column (EC-TR / EtOH-NF column) using enzyme coating method on nanofibers dispersed through alcohol treatment. When 100 ng / enolase solution was flowed into the enzyme column at a rate of 2 / min, the residence time could be reduced to within 5 minutes. Pass through column

One effluent was analyzed by LTQ-FT mass spectrometer to identify hydrolyzed peptide fragments. In addition, we validate IDed peptides from the results of a search search for sequence coverage analysis.

For the validation, a peptide prophet tool was used in a pipeline called TPP provided by ISB.

In the case of i20> EC-TR / NF columns, the chromatographic analysis showed relatively clear peaks that can be confirmed as enolase. Specific peptides cut from enolase) were identified, and amino acid sequence coverage of 68.4) was confirmed. On the other hand, in the case of the EC-TR / EtOH-NF column, the chromatographic analysis showed a very clear peak that could be confirmed as enlase. specific peptides cut from the enolase were identified and the amino acid sequence coverage of 80.5% was determined.

Confirmed (see Table 2). Through these results, the enzymatic column using the enzyme coating method was able to significantly reduce the hydrolysis time and at the same time obtain efficient protein hydrolysis results.In the case of the EC-TR / EtOH-NF column, the nanofibers were compressed by the fluid pressure. Higher hydrolysis by preventing voids from occurring and preventing bypass It could prove to show efficiency.

<121> [Table 2]

<Example 5> Measurement of the persistence of the enzyme column

<i23> To compare the hydrolysis performance of EC-TR / EtOH-NF with conventional in-solution digestion method and trypsin bead which is currently commercially available. Each sample was stirred at 250 rpm for 6 hours at a high temperature of 50, followed by hydrolysis of the model protein enolase. At the same time, liquid phase hydrolysis (in-solution digestion method, free trypsin) was used. Free trypsin used in trypsin: protein = 1:50 ratio and hydrolyzed for 12 hours) was used to collect a certain amount of commercial trypsin, and commercialized trypsin beads (Promega, V9012) and EC—TR / Et0H-NF were sampled. After washing, the same sample was reused.

<i24> Figure 13 is a graph showing the vertical axis of the protein hydrolytic ability after stirring the EC-TR / EtOH-NF, commercially available trypsin beads, free enzyme at a high temperature of 50 ° C to confirm thermal stability The amount of the hydrolyzed peptide was calculated by area using a machine called LC-MS / MS. The initial hydrolysis capacity of EC-TR / EtOH-NF was slightly higher than that of in-solution digestion and commercialized trypsin bead. In addition, after reaction at 250 rpm and 50 ^° C for 60 hours, the hydrolysis was carried out again. As a result, the hydrolytic ability of EC-TR / EtOH-NF was maintained. However, free trypsin and commercialized trypsin bead were It can be seen that the degradation capacity was significantly reduced. This result implies that EC-TR / EtOH-NF can be reused at room temperature as well as 50 ^° C without loss of hydrolysis performance.

<Example 6> Measurement of the dispersion degree of the nanofiber according to the alcohol concentration

In order to measure the dispersion of nanofibers according to alcohol treatment, 1 mg of nanofibers were treated with various concentrations of ethanol solution (0-100% v / v), and then the dispersion degree was checked. FIG. 14 is a photograph taken after alcohol treatment with nanofibers, washing with water and removing alcohol according to the concentration. As a result, when the vial containing 1 mg of nanofibers and alcohol solution was shaken for 5 minutes and vortexed for 10 minutes, the structure of nanofibers after mixing was not significantly different after 10% (v / v) alcohol concentration. . On the other hand, as the concentration of alcohol increases, the nanofibers were easily dispersed, and it was confirmed that the nanofibers were dispersed at an alcohol concentration of more than 20% ( _V / _V ). These dispersed nanofibers are used to remove alcohol Even after washing with water for harm, the degree of dispersion did not change and was found to remain dispersed (see FIG. 14). Maintaining the dispersed shape of the nanofibers dispersed by alcohol treatment even after removing the alcohol maintains the shape of the nanofibers even though the reaction proceeds in a buffer for enzyme fixation, thereby increasing the amount of enzyme immobilized.

Finally, the enzyme stabilization method using the polymer nanofibers, which was previously studied, was further extended to disperse the polymer nanofibers through alcohol treatment to increase the surface area, thereby increasing the amount of immobilized enzyme compared to the conventional method. The efficiency could be increased and stabilized using an enzyme coating method. The created an immobilized enzyme liquid phase hydrolysis using a result of over _(n i _so lution digestion) method using a 59 days when enzyme coating method was able to confirm that maintains a high proteolytic efficiency.

In addition, the enzyme column using the enzyme coating method to shorten the hydrolysis time

"As a result of the hydrolysis of the protein, the hydrolysis time can be greatly enjoyed compared to the existing time (4-12 hours) and the high hydrolysis efficiency (retention time: within 5 minutes) was confirmed. In other words, the enzyme column system using enzyme coating can reduce the hydrolysis time while maintaining high stability.The development of such a system enables the use of various experiments, that is, the actual protein quality, for future practical use. ， Development of optimized system through research on the identification of hydrolysis efficiency in low concentration protein.

<129>

Industrial Applicability

The enzyme column including the immobilized enzyme of the present invention can reduce the total proteomic analysis time by hydrolyzing the target protein in a short time. In addition, since the enzyme activity is stabilized using the enzyme coating method, the lifespan of the column is greatly increased, and thus it may be usefully used in the proteomic analysis industry.

<i3l>

<132>

Claims

[Range of request]

[Claim 11

1) packing polymer fibers into a column;

2) treating the polymer fibers packed in the column with an alcohol solution to disperse the high molecular fibers in the column to increase the inter-fiber space;

3) removing the treated alcohol solution in the column; And

4) adding an enzyme to the polymer fiber packed in the column to immobilize the enzyme on the surface of the polymer fiber.

[Claim 2]

The method of claim 1, wherein the polymer fibers are polymer nanofibers or polymer micron fibers and a method for producing an enzyme column, characterized in that it has a three-dimensional network structure.

[Claim 3]

In U,

The polymer fibers are polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid, A method for producing an enzyme column, characterized in that at least one selected from the group consisting of polyglycolic acid polycaprolactone, collagen, polyaniline and poly (styrene-co-maleic anhydride). '

[Claim 4]

The method of claim 1,

The alcohol solution is a method of producing an enzyme column, characterized in that it comprises any one or more selected from the group consisting of methanol, ethanol, propanol and butanol.

[Claim 5]

The method of claim 1,

The concentration of the alcohol solution is 15 to 100% (ν / ν) method for producing an enzyme column, characterized in that. ^'

[Claim 6]

In U,

Step 3) is a method for producing an enzyme column, characterized in that washing the inside of the column without including a drying step.

[Claim 7]

The method of claim 1,

Method for producing an enzyme column, characterized in that to add a cross-linking agent in the column in order to immobilize the enzyme on the surface of the polymer fiber in the step 4).

[Claim 8]

The method of claim 7,

The crosslinking agent includes any one or more selected from the group consisting of diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, and glutaraldehyde Method for producing an enzyme column characterized by.

[Claim 9]

The method of claim 1,

The enzyme is trypsin, chymotrypsin, subtilisin, papain, thermolysine, lipase, peroxidase, tyrosinase, laccase, celase, xylanase, lactase, organic phosphohydrolase, cholinesterase ， Glycolytic enzyme, alcohol dehydrogenase, glucose dehydrogenase, and glucose isomerization enzyme production method, characterized in that any one or more selected from the group consisting of isomerase.

[Claim 10]

column; And an enzyme column packed inside the column, the polymer fiber being treated with an alcohol solution to be dispersed in the column and having an increased inter-fiber space.

[Claim 11]

The enzyme column according to claim 10, wherein the polymer fibers are polymer nanofibers or polymer micron fibers and have a three-dimensional network structure.

[Claim 12]

The method of claim 10,

The polymer fibers may be polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid, Enzyme column, characterized in that at least one selected from the group consisting of polyglycolic acid polycaprolactone, collagen, polyaniline and poly (styrene-co-maleic anhydride).

[Claim 13]

The method of claim 10, wherein the enzyme is fixed on the surface of the polymer fiber Enzyme column.

[Claim 14]

The method of claim 13,

The enzyme is trypsin, chymotrypsin, subtilisin, papain, thermolysine, lipase, peroxidase, tyrosinase, laccase, celase, xylanase, lactase, organic phosphohydrase, choline An enzyme column, characterized in that at least one selected from the group consisting of esterases, glycosylase, alcohol dehydrogenase, glucose dehydrogenase, and glucose isomerase.

[Claim 15]

The method of claim 10,

Enzyme column, characterized in that the enzyme column is filled with a polymer island flow path is increased inter-fiber space (inter-fiber space).