KR101151792B1 - Coated magnetic silica nano complexes and preparation method thereof - Google Patents

Abstract

The present invention relates to a magnetic silica nanoparticles for enzyme immobilization and a ligand containing a metal such as copper, and a method for preparing the same. The magnetic silica nanoparticles according to the present invention have a uniform particle size distribution and have a size of 10 emu / g. It is characterized by having the above magnetic properties.

Description

Coated magnetic silica nanocomposites and preparation method

The present invention relates to a ligand containing a magnetic / silica nanocomposite and a metal such as copper for enzyme immobilization, and a method for preparing the same. The magnetic silica nanocomposite according to the present invention has a uniform particle size distribution and magnetic materials are mutually different. By combining the core to form a magnetic feature of 10 emu / g or more, by attaching the copper complex to the core / shell nanocomposite surface can be fixed histidine-tagged (His-tagged) enzyme.

Magnetic nanoparticles are well known for their unique magnetic properties, data storage possibilities, bioenvironment separation technologies, magnetic resonance imaging devices, drug carriers, and enzyme immobilization. Silica has been used as a coating for magnetic nanoparticles because it protects the magnetic nanoparticles from dipolar interaction. At the end of the surface of the silica there is also a group of silanol capable of reacting a specific ligand with the organosilane coupling agent (Organosilane Coupling Agent). Due to this property of silica, silica coated magnetic nanoparticles are used for nucleic acid separation, enzyme immobilization, high temperature reaction and the like. However, the difficulty of magnetic separation due to the marked reduction of the magnetic properties of the core / shell structure is a major disadvantage, and therefore the synthesis of strong magnetic magnetic / silica nanoparticles has been criticized.

On the other hand, conventionally known techniques related to particles and methods for preparing the enzyme that can stably immobilize are as follows.

The degree to which the specificity approximates the enzyme specificity of the free state by attaching the functional group to the inorganic carrier covalently, and having the largest number of pore diameters to produce the most active one, regardless of the amount of the enzyme continuously linked thereto. A technique for contacting with an enzyme solution having an amount of is known. (Method for preparing immobilized enzyme, 1983-0002697, December 08, 1983) In the case of a carrier made from the silica gel described above, The specificity is 10-15 units / mg, and the specificity with glucose isomerase is 50-70 units / mg.

Also known is a technique of silica-immobilized enzyme and its preparation method using a cationic surfactant. (Silica-immobilized enzyme and its preparation method, 10-2007-0118805, December 18, 2007) Hydrophobic silicate precursors such as TEOS and the like are fixed by the network structure due to the combination of the cationic surfactant and the silicate, thereby increasing the supporting ratio and maintaining the activity of the enzyme.

In addition, a method of immobilizing an enzyme by making a hydrophobic base on silica by a sol-gel method is known (Lipases immobilized in sol-gel processed hydrophobic materials, EU, 0676414A1, 1995, 11, 10). Magnetite was used as one of the magnetic nanoparticles as in the present patent, but it was not coated with nanoparticles or coated with nanoparticles, but merely mixed with magnetite and solidified.

As described above, in the case of the carrier disclosed in the above document, there is a need for the development of a carrier having a higher activity persistence due to lack of enzyme activity.

The present invention is to overcome the above problems, the present invention was largely aimed at the application of the synthesis and enzymatic immobilization of two nanoparticles having a magnetic cluster as a nucleus. Among the various methods of immobilization, covalent binding minimized the loss of enzymes, but due to the essential amino acids on the active side, often reduced enzyme activity significantly. Metal ions that bind immediately with protein histidine residues to improve enzyme immobilization are known as silica surface materials. Six or seven histidine residue oligonucleotide water peptide (Oligopeptide) when connected to a nitrogen or carbon of the enzyme ends is made histidine-tagged de (His-tagged) enzyme histidine co (Collaborative) bonded to the metal ion, such as Cu ^{2 +} Due to this, it can be strongly immobilized to the metal chelate. A number of reports reported on the application of metal ion-immobilized silica and magnetic silica support protein separation techniques due to the stability of metal chelates and high protein loading performance. In addition, the magnetic / silica compounds immobilized with metal ions and the core / shell magnetic / silica exhibit inherent simple magnetic separation and selective metal affinity purification.

The present invention is a magnetic core; And a nanocomposite comprising a silica shell comprising a metal complex functional group covering the core, thereby providing a nanocomposite of the present invention comprising a magnetic core / silica shell structure capable of strongly fixing an enzyme while maintaining enzymatic activity. It was completed.

The nanocomposite of the present invention maintains high magnetic force as a core-shell composite by allowing magnetic bodies to form a core together, and a metal complex is fixed on a silica surface, and is strongly resistant to metal chelate through a combination of metal ions and enzymes. By fixing the enzyme, there is an advantage that the enzyme activity is high and the activity can be maintained for a long time.

1 is a TEM image (a) of a magnetic nanoparticles and a TEM image (b) of a magnetic / silica nanocomposite.
2 is a magnetic curve (a) of the magnetic nanoparticles and the magnetic curve (b) of the magnetic core / shell.
3 is an XRD analysis of magnetic / silica nanocomposites.
FIG. 4 shows magnetic / silica nanocomposites (a) with copper attached without ligands in the FT-IR spectrum, magnetic / silica nanocomposites (b) with ligands II present, and magnetic / silica with copper attached and ligands II simultaneously. Nanocomposite (c) spectrum. Small inset ⅰ is the FT-IR spectrum before and after the addition of copper to the magnetic / silica nanocomposite with Ligand I, and inset ii is the FT-IR spectrum before and after the addition of copper to the magnetic / silica nanoparticles with Ligand III. to be.
Figure 5 shows the change in enzyme activity when repeated use at 37 ℃.
6 shows ligands I-Cu (a), ligands II-Cu (b), and ligands III-Cu (c) in a schematic diagram of the core / shell of the copper complex.

The present invention is a magnetic core having a silica coating layer; And a metal complex covalently bonded to the coating layer and having three nitrogen atoms bonded to one metal atom.

In the present invention, the nanocomposite is characterized in having a magnetism of 10 emu / g or more.

In the present invention, the average diameter of the magnetic core may be 1 to 200nm, more specifically 50 to 150nm.

The average size of the nanoparticles may be 100 to 500nm, more specifically 150 to 200nm. When the average size of the particles exceeds 500 nm, since the thickness of the silica tends to increase in proportion to the size of the entire composite after a certain size (200 nm), the magnetic force may decrease, and the size of the particles If too small, the amount of the magnetic material is small compared to the thickness of the silica, there is a fear that the magnetic force is reduced. However, the particle size is not very important if the magnetic body is present in the shell, and the nanocomposite of the present invention is characterized by having a magnetism of 10 emu / g or more, so if the magnetic force has a large particle size is not important. not.

In the present invention, the magnetic core may be a cluster of one or more magnetic materials or magnetic alloy nanoparticles.

In the present invention, the magnetic material is Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And M _p O _q (M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and p and q satisfy the expressions "0 <p <3 and 0 <q <5", respectively.) At least one selected from the group consisting of, the magnetic alloy may be CoCu, CoPt, FePt, CoSm, NiFe or NiFeCo, but is not limited thereto.

In the present invention, the thickness of the silica coating layer may be 10 to 100 nm, more specifically 10 to 30 nm. When the thickness of the silica coating layer is thicker than 100 nm, the magnetic force of the composite is very low due to the silica coating layer, there is a fear that a problem that can not play the role of the magnetic composite.

In the present invention, the metal complex may be a nanocomposite represented by the following formula (1):

[Formula 1]

In the formula, M represents a metal atom,

n represents 0, 1 or 2.

In the present invention, M represents a metal exhibiting 2 ⁺ value by coordinating three nitrogen atoms, more specifically Cu, Zn, Fe, Co, Mg, Ba, St, Mn, n is 1, It is not limited to this.

In the present invention, the metal complex of Formula 1 is bonded to the silica coating layer through an organic linker.

In the present invention, the organic linker may be represented by the following formula (2).

[Formula 2]

In the formula, R ₁ represents an alkyl group having 1 to 4 carbon atoms,

R ₂ represents an alkylene group having 1 to 6 carbon atoms,

n represents the integer of 0-3.

In the present invention, the enzyme immobilized on the nanoparticle may be a histidine-tagged enzyme, but is not limited thereto.

In the present invention, the nanocomposite may be used for enzyme immobilization, but is not limited thereto.

The present invention also comprises the steps of a) mixing the magnetic body and the silica precursor in the presence of a solvent to obtain a magnetic body having a silica coating layer; And b) combining the metal composite having three nitrogen atoms coordinated with one metal atom with the silica coating layer.

In the present invention, the magnetic body may be prepared by pyrolyzing the magnetic precursor by heating a mixture of the magnetic precursor and a solvent or a solvent containing a surfactant.

The magnetic precursor may be a metal precursor, a metal nitrate compound, a metal sulfate compound, a metal acetylacetonate compound, a metal fluoroacetoacetate compound, a metal halide (MXa, M = Ti, Zr, Ta, Nb, Mn, Sr, Ba, W, Mo, Sn, Pb, X = F, Cl, Br, I, compounds of 0 <a≤5) series, metal perchlorolate compounds, metal chaphamate series It may be selected from the group consisting of a compound of, a metal styretic compound and an organometallic compound.

The surfactants include organic acids (C _n COOH, C _n : hydrocarbons, 7 ≦ _n ≦ 30), organic amines (C _n NH ₂ , Cn: hydrocarbons, 7 ≦ _n ≦ 30), and alkane thiols (C _n SH). , C _n : hydrocarbon, 7 ≦ _n ≦ 30), phosphonic acid (C _n PO (OH) ₂ , C _n : hydrocarbon, 7 ≦ _n ≦ 30), alkyl phosphate, trioctyl phosphine oxide, tributyl phosphine , Alkyl sulfates and tetraalkyl ammonium halides.

The organic solvent is an ether compound (C _{n 2} O, C _n : hydrocarbon, 5 ≦ _n ≦ 30), hydrocarbons (C _n Hm, 7 ≦ _n ≦ 30), organic acid (C _n COOH, C _n : hydrocarbon, 7 ≦ _n ≦ 30), organic amine (C _n NH ₂ , C _n : hydrocarbon, 7 ≦ _n ≦ 30) and alkane thiol (C _n SH, C _n : hydrocarbon, 7 ≦ _n ≦ 30) Can be.

In the preparation method of the present invention, the silica precursor may be tetraethyl orthosilicate, tetramethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane or mixtures thereof. However, it is not limited thereto.

In the process of the invention, step a) can be carried out in the presence of a catalyst.

The catalyst may be any basic compound such as NH ₄ OH or NaOH, and an acidic compound such as nitric acid, hydrochloric acid, or sulfuric acid without limitation, but it is preferable to use NH ₄ OH which is a weak basic catalyst.

In the present invention, a metal complex in which three nitrogen atoms are coordinated with one metal atom provides a method for preparing an enzyme-fixed nanocomposite according to Formula 3 below:

(3)

In the formula, R ₁ represents an alkyl group having 1 to 4 carbon atoms,

R ₂ represents an alkylene group having 1 to 6 carbon atoms,

M represents a metal atom,

n represents 0, 1 or 2.

The present invention also provides a method for immobilizing an enzyme comprising the step of mixing the nanocomposite of claim 1 in a solution containing the enzyme.

In the present invention, the enzyme may be a histidine-tagged enzyme.

The present invention also comprises the steps of immobilizing an enzyme in the nanocomposite of the present invention; And separating the nanocomposites to which the enzyme is immobilized by applying a magnetic field.

[ Example ]

Manufacturing Process Overview

New complexes of surface-modified magnetic / silica core / shell nanospheres were prepared using copper for enzyme immobilization. First, magnetic / silica core / shell nanospheres were prepared through hydrolysis and condensation of TEOS (tetraethyl orthoSilicate). (Iii) 3-chloropropyltriethoxy silane [3-Chloropropyltriethoxy Silane (CPS)] and di- (2-picolyl) amine [di- (2-picolyl) amine (DPA)], (ii) 3-amino Propyltriethoxy silane [3-Aminopropyltriethoxy Silane (APS)] and salicylaldehyde [Salicylaldehyde (SAL)], or (iii) 3-glycidoxypropyltrimethoxy silane [3-Glycidoxypropyltrimethoxy Silane (GLYMO)] Iodidiacetic Acid (IDA), the condensation products of these three materials, is close to the ligand for the copper complex on the surface of the nanospheres of magnetic silica. These materials were charged with copper ions and used as a model of His-tagged enzyme for immobilization of His-tagged Bacillus stearothermopilus L1 lipase.

Substance preparation

Benzyl Ether (99%), Dodecanediol (90%), Dodecanediol (90%), Iron Tri (acetylacetonate) (97%) [Iron Tri (Acetylacetonate) (97% )], Oleic acid (90%), oleyl amine (70%), tetraethylorthosilicate, 3-aminopropyltriethoxy Silane (APS), 3-chloropropyltriethoxy Silane (3-Chloropropyltriethoxy Silane (CPS), 3-Glycidoxypropyltrimethoxy Silane (GLYMO), Salicylaldehyde (Salicylaldehyde, SAL), Di- (2-Picolyl) amine [Di- (2-picolyl) Amine, DPA], Iminodiacetic Acid (IDA), and Copper (II) Nitrate Hydrate (98%). The above items were used by Sigma Aldrich (Seoul, Korea), and analytical grade toluene and ethanol were purchased from Dae Jung (Seoul, Korea).

Manufacturing example  One

Magnetite nanoparticles coated with oleic acid / oleylamine were prepared by a high temperature decomposition method. Preparation of monodisperse magnetic nanoparticles is already well known and has not been specifically described.

Iron tri (acetylacetonate, 2 mmol) [Iron tri (acetylacetonate, 2 mmol)], dodecanediol (10 mmol) [Dodecanediol (10 mmol)], oleic acid (6 mmol) [Oleic Acid (6 mmol)], oleylamine (6 mmol) [Oleylamine (6mmol)] and benzyl ether (20mmol) [Benzyl Ether (20 ml)] were mixed well under stirring under nitrogen composition.

The above mixture was heated gradually to 200 ° C. over 2 hours. Upon reaching 200 ° C., it was immediately heated to 270 ° C. and maintained for 1 hour. After lowering the temperature to room temperature, ethanol (40 ml) was added to the magnetite nanoparticle precipitate. The particles were separated for 1 hour using a centrifuge (at 1450 RPM per gram) and then washed with hexane. The obtained magnetite nanoparticles were stored in hexane.

Manufacturing example  2

150 g of magnetic nanoparticles were mixed with 10 ml of toluene and then mixed with 160 ml of ethanol, 40 ml of distilled water, and 5 ml of ammonium hydroxide (30 wt%) [Ammonium Hydroxide (30 wt%)]. After mixing well at room temperature for 20 minutes, 2 ml of tetraethylorthoSilicate was added over a day. The resulting magnetic / silica core / shell nanoparticles were collected using a magnet.

Example  1 and Comparative example  One

Ligand I (3-Chloropropyltriethoxy Silane-di- (2-picolyl) Amine, CPS-DPA) was obtained by reacting CPS and DPA in toluene. Ligand II (3-AminoPropyltriethoxy Silane-SalicylAldehyde, APS-SAL) was obtained by reacting APS and SAL in toluene.

To completely convert CPS and APS to ligands I and II, excess DPA and SAL were added. All of the above reactions were carried out at 90 ° C. over 6 hours.

Comparative example  2

Ligand III (3-Glycidoxypropyltriethoxy Silane-IminoDiacetic Acid, GLYMO-IDA) is already known and therefore its preparation has not been described herein.

The resulting organosilanes (1 mmol each) were implanted on the surface of the magnetic / silica nanoparticles (100 mg) incubated at 90 ° C. for 3 hours. The particles were washed with toluene to remove other unbound ligands. After centrifugation and drying, 30 mg of particles were added to 50 ml of an ethanol solution of copper (II) nitrate hydrate (0.2 wt%). Thereafter, the mixture was incubated with stirring at 200 rpm for 3 hours at room temperature.

Experimental Example  One

To immobilize the lipase, 7.5 mg of copper-filled magnetic / silica nanoparticles were added to 7.5 ml of NaCl (0.85% w / v) solution containing His-tagged B. Stearothermopilus L1 lipase (0.8465 mg protein / ml). After 4 hours of incubation, the cells were washed twice with NaCl solution (0.85% w / v). And the upper solution was separated using a magnet and stored at 4 ° C. The total protein in the particles is determined by measuring the protein concentration in the upper layer using a lipase solution and the Lowry method.

Experimental Example  2

Lipase activity is determined by measuring the degree of hydrolysis of the oil. The oil is emulsified by mixing 1% (v / v) olive oil (Sigma Chemical Co.) with NaCl (20 mM), CaCl ₂ (1 mM) and rubbery arabic (1% w / v, Sigma Chemical Co.) Made. The reaction was started by adding immobilized lipase. The pH and temperature of the reaction were maintained at 7.7 and 37 ° C, respectively. Fatty acid extracted from oil hydration was titrated using a pH-stat titrator (Stat Titrator (718 Stat Titorino, Metrohm, Switzerland)) and 10 mM NaOH.

One unit of lipase activity is defined as the amount of enzyme needed to liberate the amount of fatty acid that can titrate 1 μmol of NaOH for one minute. After the reaction, the particles were collected by magnetic separation, washed twice with NaCl solution (0.85% w / v) and reused. The specific activity of lipase was defined as the enzyme activity divided by the protein content and expressed in U / mg protein.

The size and shape of the produced materials were observed by operating 300kV of high resolution TEM (HR-TEM) called JEOL JEM-300. Samples for HR-TEM analysis were obtained by dropping a drop of solution on a copper grid and evaporating sufficiently at room temperature. In addition, X-ray diffractometry (XRD) was used to observe the crystals of nanoparticles, and Rigaku's D / max-3A model was used. Varian Excalibur Series was used for FT-IR analysis.

In addition, a VSM (Vibration Sample Magnetometer; Lake Shore Model 7300) was used to evaluate the magnetic properties. ICP-AES (Inductively Coupled Plasma-Atomic Emission Spectrometer, OPTIMA 4300 DV, Perkin Elmer) was used to measure the iron and copper contents.

Experimental Example  3

Figure 1a shows the TEM image of the magnetic nanoparticles, it can be seen that the sphere is less than 7nm. Wrapped in oleic acid / olileyamine and immediately dispersed in toluene. Adding ethanol to the particles dispersed in toluene produces emulsified particles. Growth of the silica shell around the magnetic nanoparticles includes the basic catalytic hydration of TEOS and the secondary condensation of silica occurring on the surface of the magnetic core.

Experimental Example  4

1B shows a TEM image of magnetic / silica core / shell particles. It is also spherical and has an average size of 100 nm and an average thickness of the silica shell is 20 nm. Magnetic nanoparticle fragments in the core are clearly visible in FIG. Even when molded into this magnetite silica shell, it shows a saturated magnetic value as high as 21 emu / g (see FIG. 2). This suggests that the magnetic separation can be easily separated.

Experimental Example  5

XRD analysis showed that the magnetic / silica nanoparticles having the crystal structure were made by the sol-gel method. Checking the XRD pattern, it can be seen that the pattern made of Crystalline Magnetite and the core / shell have the same similarity (see FIG. 3).

Experimental Example  6

The process of making the copper-filled core / shell nanoparticles is shown in FIG. 6. Organosilane Ligand was implanted with the dehydration of Si-OH group through the silica surface. The copper complex was formed when the addition reaction of copper (II) ions occurred. The copper composite formed was confirmed by IR-Spectroscopy.

When implanting Ligand II, several CH oscillation zones were found in the region 1000-1250 cm ⁻¹ (FIG. 4B), which was not found in the magnetic / silica (FIG. 4A) prior to implantation.

The characteristic section of the free azomethine group (-N = CH-) of ligand II was found to be 1645 cm ^-1 or less (Fig. 4b). However, this zone is at a lower 1628cm nanoparticles with a Cu ^{2 + charge-and} also indicate the coordinated movement of the ^first and below (Fig. 4c) and a copper azomethine nitrogen. Similar shifts in nitrogen were observed due to the coordination bonds of copper and nitrogen (Figs. 4 and 4ii).

Analysis of copper-filled nanoparticles (Table 1) using an ICP analyzer showed similar ratios of copper and iron in nanoparticles implanted with ligand I and nanoparticles implanted with ligand III. The lowest copper-iron ratio was nanoparticles implanted with Ligand II. If the coordination ratio of copper and His-tagged lipase is the same as for all tested ligands, the specific activity of the immobilized His-tagged lipase may be lowest for the ligand-implanted nanoparticles.

Table 1 ICP Analysis of Magnetic / Silica Nanoparticles

Experimental Example  7

When His-tagged enzyme is added to a suspension of copper-filled nanoparticles, the enzyme is immobilized via coordination of copper with histidine residues. Table 2 summarizes the enzyme immobilization efficiency and specific activity of the enzyme immobilized on various magnetic / silica nanoparticles filled with copper and the protein content as an index of immobilization.

[Table 2] Comparison of protein content and specific activity of His-tagged lipase

The protein content and specific activity of the enzyme solution were 0.8462ml / mL and 730U / mg protein, respectively. The highest immobilization efficiency and the most specific binding of His-tagged enzyme was obtained from Ligand I. Ligand II, on the other hand, had the lowest efficiency for protein immobilization and coordinated histidine. These results showed a good agreement between the data shown in Table 1 and the expected values. The shape of the copper complex with three ligands is shown in Concept 1. Interestingly, two arrangements have been proposed for the copper complex with ligand II (concept 1 (b)). In the left arrangement, two molecules of Ligand II are attached simultaneously and do not coordinate with His-tagged lipase with copper.

As shown in Table 1, it can be seen that the copper content of ligand II is less than half of the content seen in ligands I and III. Through this, it can be seen that the left array of FIG. 6 (b) is more dominant than the right array. Therefore, the lowest efficiency of enzyme immobilization due to ligand II is inevitable. Based on the data of Ligand I and Ligand II shown in Table 2, it can be seen that Pyridine Nitrogen of Ligand I is more stable in covalent bond between His-tagged lipase and copper than carboxylate oxygen of Ligand III.

Immobilized lipase was regenerated through magnetic separation and reused three times. 5 shows how the activity of the enzyme changes as the number of reuse increases. After the first use, all three nanoparticles showed a marked decrease in enzyme activation. However, it remained almost constant in future use. The lipase immobilized on the nanoparticles implanted with Ligand I, which showed the highest relative activity (about 70%), had the highest three ligands and copper bonds. On the other hand, magnetic / silica particles without any ligand showed only a small degree of relative activity when reused.

Claims (19)

A magnetic core having a silica coating layer; And
A nanocomposite covalently bonded to the coating layer, three nitrogen atoms coordinated with one metal atom, and comprising a metal composite represented by the following Chemical Formula 1;
[Formula 1]

In Formula 1, M is a metal having 2 ⁺ valence by coordinating three nitrogen atoms, and n is 1 or 2.
The method of claim 1,
The nanocomposite is characterized in that the nanocomposite has a magnetic force of 10 emu / g or more.
The method of claim 1,
Nanocomposite, characterized in that the average diameter of the magnetic core is 1 to 200nm.
The method of claim 1,
The magnetic core is a nanocomposite, characterized in that the cluster (cluster) of one or more magnetic material or magnetic alloy nanoparticles.
The method of claim 4, wherein
The magnetic material is Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And M _p O _q (M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and p and q satisfy the expressions "0 <p ≤ 3 and 0 <q ≤ 5", respectively.) At least one selected from the group consisting of, wherein the magnetic alloy is at least one selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo nanocomposite.
The method of claim 1,
Nanocomposite, characterized in that the thickness of the silica coating layer is 10 to 100 nm
delete delete The method of claim 1,
The metal composite represented by Chemical Formula 1 is bonded to the silica coating layer through an organic linker.
10. The method of claim 9,
The organic linker is a nanocomposite, characterized in that:
(2)

In Formula 2, R ₁ represents an alkyl group having 1 to 4 carbon atoms,
R ₂ represents an alkylene group having 1 to 6 carbon atoms,
n represents the integer of 0-3.
a) mixing a magnetic body and a silica precursor in the presence of a solvent to obtain a magnetic body having a silica coating layer; And
b) a method of manufacturing a nanocomposite comprising combining three nitrogen atoms with one metal atom and coordinating the metal composite represented by Formula 1 with the silica coating layer;
[Formula 1]

In Formula 1, M is a metal having 2 ⁺ valence by coordinating three nitrogen atoms, and n is 1 or 2.
The method of claim 11,
Magnetic material is a method of producing a nanocomposite, characterized in that the magnetic precursor is prepared by heating a mixture of a surfactant or a solvent containing a surfactant by thermal decomposition of the magnetic precursor.
The method of claim 11,
The silica precursor is at least one selected from the group consisting of tetraethyl orthosilicate, tetramethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane and mixtures thereof Method for producing a nanocomposite.
The method of claim 11,
Step a) is carried out in the presence of a catalyst.
The method of claim 11,
Method for producing a nanocomposite characterized in that the metal complex in which three nitrogen atoms are coordinated with one metal atom is represented by the following formula (3):
(3)

In Formula 3, R ₁ represents an alkyl group having 1 to 4 carbon atoms,
R ₂ represents an alkylene group having 1 to 6 carbon atoms,
M represents a metal which exhibits ²⁺ valence by coordinating with three nitrogen atoms,
n represents 1 or 2.
Method of immobilizing the enzyme comprising the step of mixing the nanocomposite of claim 1 in a solution containing the enzyme.
The method of claim 16,
An enzyme immobilization method, characterized in that the histidine-tagged enzyme (His-tagged enzyme).
Immobilizing the enzyme on the nanocomposite of claim 1; And
Separation method of the enzyme comprising the step of separating the nanocomposite to which the enzyme is fixed by applying a magnetic field.
The method of claim 18,
An isolation method for an enzyme, characterized in that the enzyme is a histidine-tagged enzyme.

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