KR101247290B1 - Enzyme-immobilized magnetic nanoparticles for biodegradation of refractory aromatic compounds and method for producing thereof - Google Patents

Abstract

The present invention relates to an enzyme-immobilized magnetic nanoparticles for biodegrading a biodegradable aromatic compound, which is a water pollutant with high efficiency, and a method for preparing the same, and more specifically, phenol-monooxygenase and catechol, which are enzymes derived from microorganisms. Disclosed are an enzyme-immobilized magnetic nanoparticle in which catechol 1 (2-dioxygenase) is bound to the surface of silica-magnetic nanoparticles and a method for producing the same.

Description

Enzyme-immobilized magnetic nanoparticles for biodegradation of refractory aromatic compounds and method for producing etc

The present invention relates to magnetic nanoparticles to which biodegradable enzymes of hardly decomposable aromatic compounds are biodegradable, which are water contaminants, and methods for preparing the same, and more particularly, phenol-monooxidase which is an enzyme derived from microorganisms. magnetic nanoparticles immobilized with biodegradable enzymes of hardly decomposable aromatic compounds in which (phenol-monooxygenase) and catechol 1 (2-dioxygenase) are bound to the surfaces of silica-magnetic nanoparticles; It relates to a manufacturing method.

The phenol spill in the Nakdong River and the phenol pollution in the Mississippi River in the United States have increased awareness of harmful substances both at home and abroad.In particular, aromatic compounds such as phenol, which are produced in large quantities in the effluent from the paper and plastics business, are released into the environment. It is a toxic substance that enters the human body through the food chain and becomes a carcinogenic substance or acts as an environmental hormone, which seriously affects living organisms.

As a method for removing the hardly decomposable aromatic compounds, methods such as activated carbon, extraction, and chemical oxidation have been known, but high maintenance and treatment costs, limited coverage, and incomplete treatment It has the disadvantage of generating harmful and harmful secondary pollutants. In addition, there is an example using a biological method by a specific microorganism treatment to decompose hardly decomposable aromatic compounds, such as phenol, but due to the very low microbial metabolic efficiency, it is not possible to quickly decompose compounds such as phenols contained in wastewater, It is difficult to cultivate, maintain and recover the microorganisms, which increases the process cost. In order to solve these drawbacks, a research using an enzyme capable of oxidizing and decomposing hardly decomposable aromatic compounds containing phenol has been conducted.

U.S. Patent No. 7214507 discloses a method of applying cytochrome P450 of Asteraceae to decompose xenobiotics, which is a heterologous expression of plant-derived genes. There is a disadvantage in that mass production is difficult.

U.S. Patent No. 06858417 discloses Ralstonia eutropha A method of degrading various aromatic compounds such as phenol, toluene, and benzene using E. coli introduced with new toluene monooxygenase isolated from strain TB64) is disclosed. In order to maintain a constant culture medium and culture environment, it is difficult to reuse microbial strains.

In addition, Korean Patent Registration No. 10-2006-0135353 discloses a method for decomposing phenol by fixing horseradish peroxidase (HRP.EC 1.11.1.7) derived from a flora and fauna on the surface of a porous carbon electrode. However, the enzymes used have disadvantages involving activation by hydrogen peroxide and electrochemical water treatment steps.

Due to the problems of the prior art, by producing a large amount of oxidase that can break the ring-structure of the aromatic compound, it is possible to maintain the enzyme activity more stably than the prior art, and economical by reusing the enzyme There is a need for an enzyme immobilization technology capable of increasing the amount of.

The present invention has been made to solve the above problems of the prior art,

The present invention can effectively use enzymes to decompose difficult-decomposable aromatic compounds by expressing large amounts of oxidases in Escherichia coli, which can break down the ring-structure of phenolic aromatic compounds, and immobilize the oxidases on magnetic nanoparticles. The present invention provides a biodegradable enzyme-immobilized magnetic nanoparticle and a method for producing the same.

In addition, the present invention, the existing aromatic compound oxidase has a large molecular weight and has a complex three-dimensional structure is difficult to mass expression in E. coli and difficult to maintain the stability of the enzyme. However, in the present invention, a large amount of oxidase that does not form a complex structure in E. coli and immobilized on magnetic nanoparticles provides stability to enzymes and at the same time provides a method for effectively decomposing aromatic compounds. do.

Magnetic nanoparticles to which the biodegradable enzyme of the hardly decomposable aromatic compound of the present invention is immobilized to solve the above problems are hardly decomposable aromatics on the surface of silica-magnetic nanoparticles coated on the surface of one-cycle transition metal or ions of the transition metal. Compound phenol-monooxidase or catechol-dioxidase immobilized by heterologous expression in Escherichia coli, respectively, in the form of combining histidine, an amino acid containing an imidazole functional group, as a biodegradable enzyme.

Here, the one-cycle transition metal is characterized in that any one selected from the group consisting of nickel, copper, cobalt, zinc.

Here, the ion of the transition metal is characterized in that the ion of the transition metal is any one selected from the group consisting of nickel, copper, cobalt, zinc.

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Here, the oxidase is monophenol-monooxygenase (monophenol-monooxygenase), chlorophenol-monooxygenase (pentachlorophenol-monooxygenase), nitrophenol-monooxidase ( nitrophenol-monooxygenase) is characterized in that the phenol-monooxidase selected from the group consisting of.

Here, the oxidase is catechol 1, 2-dioxygenase, catechol 2, 3-dioxygenase, protocatechuate-dioxide (protocatechuate- dioxygenase, gentisate-dioxygenase, benzene-dioxygenase, benzoate-dioxygenase, toluene-dioxygenase, naphthalene-dioxidase -dioxygenase) is characterized in that the catechol-dioxide enzyme selected from the group consisting of.

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In addition, the present invention is mixed with a FeCl ₂ solution and FeCl ₃ solution, dropping the NH ₄ OH solution drop by drop while stirring and reacted with 0.5 ~ 2M TEOS (tetraethyl orthosilicate) in the reaction product for 20 to 24 hours 1 cycle transition metal or its transition obtained by preparing silica-magnetic nanoparticles having a diameter of 1 to 100 nm and immersing and reacting the silica-magnetic nanoparticles in an aqueous solution containing one cycle transition metal or ions of the transition metal After preparing and preparing silica-magnetic nanoparticles containing metal ions coated on the surface thereof, the silica-magnetic nanoparticles are biodegradable enzymes that biodegradable hardly decomposable aromatic compounds and combine histidine, an amino acid containing an imidazole functional group. Phosphate buffers containing phenol-monooxygenase or catechol-dioxygenase e buffered saline (pH 7.4), and then stirred under the conditions of agitation temperature of 1 ℃ to 10 ℃, stirring time 30 minutes to 2 hours, and then the magnetic nanoparticles bound to the enzyme were separated from the solution using a magnet. Provided is a method for producing magnetic nanoparticles in which a biodegradable enzyme of a hardly decomposable aromatic compound is immobilized by washing with a phosphate buffer solution.

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In addition, in the present invention, the enzyme-immobilized magnetic nanoparticles described above or the enzyme-immobilized magnetic nanoparticles prepared by the above method are contained so as to contain 0.01 to 0.1% by weight relative to the total weight of contaminated water contaminated with an aromatic compound or a phenolic aromatic compound. A method of biodegrading these by input is disclosed.

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The present invention provides a method of increasing the decomposition efficiency by biodegrading the hardly decomposable phenolic aromatic compounds using oxidase and immobilizing the enzymes on the magnetic nanoparticles, and compared with the physical, chemical and biological methods for treating aromatic compounds. It is possible to improve the polluted water environment in an easy, fast and economic way.

1 is a silica-picture depicting the manufacturing process in steps of magnetic nanoparticles - nickel nitrate (nickel nitrate) The reaction was divalent nickel ion having a nickel / silica combination of (Ni ²⁺⁾ on the surfaces of the magnetic nanoparticles.
Figure 2 is a schematic diagram showing the phenol biodegradation pathway of Corynebacterium glutamicum.
3 is a transmission electron microscopy (TEM) photograph showing the outer skin / internal structure of silica-magnetic nanoparticles.
4 is a FT-IR spectroscopic graph of magnetic nanoparticles, silica-magnetic nanoparticles, and nickel / silica-magnetic nanoparticles prepared according to the present invention.
5 is a schematic diagram of a vector DNA cloned (cloning) phenol-monooxidase and catechol-dioxidase gene.
FIG. 6 is a SDS-polyacrylamide gel electrophoresis diagram of phenol-monooxidase and catechol-dioxidase heterologous expression in E. coli, followed by protein purification purified by affinity chromatography.
7 is a fluorochemical and fluorocarbon (CLC) biodegradation process using phenol-monooxidase and catechol-dioxidase immobilized on nickel / silica-magnetic nanoparticles (HPLC, high performance liquid chromatography). Figures analyzed by).

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

The present invention is a method for increasing the stability of the enzyme by environmentally friendly biodegradation method of phenolic aromatic compounds using oxidase selected from microorganisms, animals and plants, enzyme immobilized magnetic nano-immobilized on the surface of silica-magnetic nanoparticles By providing particles.

According to the present invention, the magnetic nanoparticles to which the biodegradable enzyme of the hardly decomposable aromatic compound is immobilized are hardly decomposable aromatic compounds whose histidine is labeled on the surface of the silica-magnetic nanoparticles containing one cycle transition metal or ions of the transition metal on the surface thereof. The biodegradable enzyme is immobilized and completed.

The one-cycle transition metal is preferably used any one selected from the group consisting of nickel, copper, cobalt, zinc.

The ion of the transition metal is preferably an ion of the transition metal, which is any one selected from the group consisting of nickel, copper, cobalt and zinc.

According to the present invention, the biodegradable enzyme of the hardly degradable aromatic compound is preferably immobilized phenol-monooxygenase or catechol-dioxygenase on the magnetic nanoparticles.

More preferably, the biodegradable enzyme of the hardly degradable aromatic compound is monophenol-monooxygenase, chlorophenol-monooxygenase, pentachlorophenol-monooxygenase, Phenol-monooxidase selected from the group consisting of nitrophenol-monooxygenase, or catechol 1, 2-dioxygenase, catechol 2, 3-dioxidase (catechol 2, 3-dioxygenase), protocatechuate-dioxygenase, gentisate-dioxygenase benzene-dioxygenase, benzoate-dioxide Dioxygenase), toluene-dioxygenase (toluene-dioxygenase), naphthalene-dioxygenase (naphthalene-dioxygenase) is more preferably a catechol-dioxide enzyme selected from the group consisting of.

According to the present invention, it is preferable to use the biodegradable enzyme of the hardly degradable aromatic compound including 4 to 12 consecutive histidine sequences at the terminal of the amino acid sequence.

According to the present invention, the phenol-monooxidase or catechol-dioxidase is used in combination with histidine, an amino acid containing an imidazole functional group, in the form of heterologous expression in Escherichia coli, respectively, for mass production.

According to the present invention, a method for preparing magnetic nanoparticles to which a biodegradable enzyme of a hardly decomposable aromatic compound is immobilized prepares silica-magnetic nanoparticles containing one cycle transition metal or ions of these transition metals on a surface thereof, The particles were added to histidine-labeled oxidase dissolved in phosphate buffered saline (pH 7.4), and then stirred. Then, magnetic nanoparticles bound to the enzyme were separated from the solution using a magnet and washed with phosphate buffer. Is completed.

Here, the stirring is preferably stirred under the conditions of 1 ℃ ~ 10 ℃, stirring time 30 minutes ~ 2 hours. More preferably, it is best to stir at 4 ° C. for 1 hour.

The one-cycle transition metal is preferably any one selected from the group consisting of nickel, copper, cobalt, zinc.

The ion of the transition metal is preferably an ion of a transition metal which is any one selected from the group consisting of nickel, copper, cobalt and zinc.

The biodegradable enzyme of the hardly degradable aromatic compound is phenol-monooxygenase or catechol-dioxygenase, and the phenol-monooxidase or catechol-dioxidase contains an imidazole functional group. The heterologous expression in the form of combining histidine, an amino acid, is used.

As the biodegradable enzyme of the hardly degradable aromatic compound, it is preferable to use an enzyme including 4 to 12 consecutive histidine sequences at the terminal of the amino acid sequence.

Enzymatic immobilized magnetic nanoparticles provided according to the present invention as described above biodegraded the aromatic compound or phenolic aromatic compound by the immobilized enzyme if left for a predetermined time in a contaminated water contaminated with aromatic compounds or phenolic aromatic compounds Or breaks their ring-structure and at the same time eliminates toxicity.

In this case, the enzyme-immobilized magnetic nanoparticles are preferably added in an amount of 0.01 to 0.1% by weight relative to the total weight of the contaminated water.

Silica-magnetic nanoparticles used as the enzyme support disclosed in the present invention can be synthesized by various methods, but can be synthesized by wet method among them. First, a solution of FeCl ₂ and FeCl ₃ are mixed, and then 0.5 to 2 M, preferably 1 M of tetraethyl orthosilicate (TEOS) is added to the reacted product by dropping the NH ₄ OH solution dropwise while stirring for 20 to 24 hours. Preferably, the reaction can be carried out for 24 hours to have a diameter of 1 to 100 nm, more preferably 2 to 50 nm.

In the present invention, silica-magnetic nanoparticles are bonded to a covalent bond type such as Si-O-Si formed on a silica surface by forming a chelate of one-cycle transition metal ions such as nickel, copper, cobalt, and zinc. do.

Proteins capable of binding to silica-magnetic nanoparticles containing transition metals or ions of transition metals, which are enzyme binders of the present invention, include enzymes comprising 4-12 consecutive histidine sequences at the ends of the amino acid sequences of the enzymes; Protein structure.

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited to the contents of the following examples.

Example  One : Nickel ion  Synthesis of Coated Silica-Magnetic Nanoparticles

1.0 ml of ₂ M FeCl ₂ solution and 4.0 ml of 1 M FeCl ₃ solution were mixed together, and 50 ml of 0.7 M NH ₄ OH solution was added dropwise while stirring. The product produced after the reaction for about 1 hour was magnetically separated and washed with ethanol 2-3 times and dried to prepare magnetite nanoparticles.

100 mg of magnetite was added to 100 ml of cyclohexane and 3.3 ml of oleic acid was dispersed for 3 hours. Next, 100 ml of the magnetite dispersion solution was added to a solution of 44 g of IGEPALCO-520 dispersed in 900 ml of cyclohexane, followed by stirring for 15 minutes. 8 ml of NH ₄ OH was added under stirring, followed by stirring for 15 minutes. Then, 1 M of TEOS (tetraethyl orthosilicate) was added thereto and stirred at room temperature for 24 hours. After the reaction, methanol (methanol) was added and the precipitate obtained by centrifugation was washed with ethanol and dried for 24 hours using a vacuum desiccator to prepare silica-coated magnetic nanoparticles. (See FIGS. 1-3).

1 g of the prepared silica-magnetic nanoparticles were mixed with 0.1 M Ni (NO ₃ ) ₂ .6H ₂ O solution at room temperature for 1 hour. After the reaction, the precipitate separated by the magnet was washed 2-3 times with distilled water, and air-dried to prepare nickel / silica-magnetic nanoparticles coated with nickel ions on the surface. The product confirmed the binding of nickel ions by fourier transform-infrared (FT-IR) spectroscopy (FIG. 4).

Example  2 : Monooxidase  Expression of oxidase and dioxide

Corynebacterium glutamicum ATCC through nucleotide search (BLASTN, http://blast.ncbi.nlm.nih.gov/Blast.cgi) at the National Institute of Biological Information (NCBI, www.ncbi.nih.gov) 13032 ( Corynebacterium Identify the sequence of the gene encoding phenol-monooxygenase of glutamicum ATCC 13032 (cg 2966) and the gene encoding catate 3 (catechol 1, 2-dioxygenase) ( catA 3) A nucleotide primer having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 capable of specifically amplifying each gene was prepared by a polymerase chain reaction (hereinafter, abbreviated as "PCR"). (See Table 1 below).

Table 1 below is the nucleotide sequence of the nucleotide primer having SEQ ID NO: 1, 2, 3, 4.

SEQ ID NO: Base number Remarks Sequence (5 ′ → 3 ′) One 28 cg2966-P1 (forward direction) cac catatg cagtttcattatgaaggat ( Nde I) 2 29 cg2966-P2 (reverse direction) tat gcggccgc g ttcgcgttgattagctg ( Not I) 3 27 catA3-P3 (forward direction) cag catatg acaaccaccaccgcagac ( Nde I) 4 27 catA3-P4 (reverse) aat aagctt cgcgcccggcgcgagcac ( Hind III)

(In Table 1, the underlined portions are Nde I (SEQ ID NO: 1 and SEQ ID NO: 3), Not I (SEQ ID NO: 2) and Hind III (SEQ ID NO: 4) restriction enzyme sites, respectively.)

Next, genomic DNA was extracted from the Corynebacterium glutamicum ATCC 13032 strain, and the cg 2966 gene was amplified by PCR using primers having the nucleotide sequences of SEQ ID NOs: 1 and 2 as templates. The catA 3 gene was also amplified by PCR using primers having the nucleotide sequences of SEQ ID NOs: 3 and 4. PCR amplification products were identified by agarose gel electrophoresis and separated and purified.

The PCR amplified cg 2966 and catA 3 genes and the pET-21a (Novegen, Madison, WI, USA) vectors were treated with Nde I, Not I and Hind III restriction enzymes, respectively, isolated and purified to T ₄ DNA ligase. (ligase) to make a recombinant vector DNA was named "pSY-PMO" and "pSY-Cat", respectively (see Figure 5). Since, Escherichia coli BL21 ( Escherichia coli BL21) (Novagen, Madison, WI, USA) was transformed.

E. coli transformed by introducing the prepared pSY-PMO and pSY-Cat vectors was abbreviated as LB (Luria-Bertani, hereinafter “LB”) to which 100 μg / ml ampicillin was added, and the composition was bactotripton 10 g / l, bacterium yeast extract 5 g / l, sodium chloride 5 g / l, and the same below) After incubation in an early exponential phase in a liquid medium, 0.5 mM IPTG (Isopropyl β-D-1- The addition of thiogalactopyranoside) induced protein overexpression of the introduced genes cg 2966 and catA 3. Thereafter, the cells were disrupted and purified by affinity chromatography using a histidine label (His-tag) bound to the target protein. Overexpression and purification efficiency of phenol-monooxidase (628aa, 69.7kDa) and catechol-dioxide (301aa, 33.1kDa), the target proteins, were confirmed by SDS-polyacrylamide gel electrophoresis (FIG. 6). .

Example 3 Combination of Nickel / Silica-Magnetic Nanoparticles with Enzymes

Histidine-labeled phenol-monoxidase and catechol-dioxidase (1 mg) dissolved in nickel / silica-magnetic nanoparticles (5 mg) prepared in Example 1 in 10 mM phosphate buffered saline (pH 7.4). / Ml) and stirred at 4 ° C. for about 1 hour. Magnetic nanoparticles bound to the enzyme were separated from the solution using a magnet and washed twice with phosphate buffer. In the present invention, the immobilized enzyme produced by the above process was named as PMO @ Ni / Si-MNPs and CatA @ Ni / Si-MNPs, respectively. The amount of enzyme immobilized on the nickel / silica-magnetic nanoparticles of Ni / Si-MNPs @ PMO and Ni / Si-MNPs @ CatA was measured by quantitative analysis by a bicinchoninic acid (BCA) test. The results are shown in Table 2 (enzyme immobilization efficiency).

PMO @ Ni / Si-MNPs
(Phenol-monooxidase) CatA @ Ni / Si-MNPs
(Catechol-dioxide) Amount of fixed enzyme
(Μg enzyme / mg nanoparticle) 6.2 ± 1.5 3.6 ± 0.2 Enzyme Immobilization Efficiency (%) 39.8 ± 7.3 25.5 ± 6.4

Example  4: Biodegradation Experiment of Aromatic Compound Using Enzyme Immobilized on Magnetic Nanoparticles

PMO @ Ni / Si-MNPs and CatA @ Ni / Si-MNPs, the immobilized enzymes prepared above, were used to biodegrade phenol and catechol, which are hardly decomposable aromatic compounds. 30 mg of PMO @ Ni / Si-MNPs was added to 0.1 mM phenol solution and left at 37 ° C. for 12 hours with stirring. The decomposition efficiency was 90.03%. In addition, 5 mg of CatA @ Ni / Si-MNPs was added to a 0.1 mM catechol solution and left at room temperature for 2 hours with stirring to show a decomposition efficiency of 88.75% (FIG. 7). In each step of the process, it was found that phenol-monooxidase and catechol-dioxidase immobilized on magnetic nanoparticles effectively biodegrade phenol and catechol, which are hardly decomposable aromatic compounds.

Claims (18)

Heterogeneously expressed in Escherichia coli in the form of a single-cycle transition metal or ion of the transition metal bonded to the surface of silica-magnetic nanoparticles coated on the surface by binding histidine, an amino acid containing an imidazole functional group, as a biodegradable aromatic compound biodegradable enzyme. A non-degradable aromatic compound biodegradable enzyme is immobilized magnetic nanoparticles, characterized in that the mass-produced phenol-monooxidase or catechol-dioxidase is immobilized. The method of claim 1,
The one-cycle transition metal is any one selected from the group consisting of nickel, copper, cobalt, zinc, biodegradable enzyme immobilized magnetic nanoparticles of a non-degradable aromatic compound. The method of claim 1,
The ion of the transition metal is a biodegradable enzyme immobilized magnetic nanoparticles of a hardly decomposable aromatic compound, characterized in that the ion of the transition metal is any one selected from the group consisting of nickel, copper, cobalt, zinc. delete The method of claim 1,
The phenol-monooxidase is monophenol-monooxygenase, chlorophenol-monooxygenase, pentachlorophenol-monooxygenase, nitrophenol-monooxidase (nitrophenol-monooxygenase) biodegradable enzyme immobilized magnetic nanoparticles of a non-degradable aromatic compound, characterized in that any one selected from the group consisting of. The method of claim 1,
The catechol-dioxidase is catechol 1, 2-dioxygenase, catechol 2, 3-dioxygenase, protocatechuate-dioxide (protocatechuate) -dioxygenase, gentisate-dioxygenase, benzene-dioxygenase, benzoate-dioxygenase, toluene-dioxygenase, naphthalene-dioxide Naphthalene-dioxygenase) is a biodegradable enzyme immobilized magnetic nanoparticles of a hardly decomposable aromatic compound, characterized in that any one selected from the group consisting of. delete delete After mixing FeCl ₂ solution and FeCl ₃ solution, drop the NH ₄ OH solution drop by drop while stirring, and add 0.5-2M of tetraethyl orthosilicate (TEOS) to the reaction product for 20-24 hours to react. 1-phase transition metal or a transition metal ion obtained by preparing a silica-magnetic nanoparticle having a surface and immersing the silica-magnetic nanoparticle in an aqueous solution containing one-cycle transition metal or an ion of the transition metal surface Prepared by preparing silica-magnetic nanoparticles coated on the substrate, and then heterogeneously expressing the silica-magnetic nanoparticles in the form of combining histidine, an amino acid containing an imidazole functional group, with an enzyme for biodegrading a hardly decomposable aromatic compound. After adding phenol-monooxygenase or catechol-dioxygenase to dissolved phosphate buffer solution, stirring temperature 1 After stirring under the conditions of ℃ ~ 10 ℃, stirring time 30 minutes ~ 2 hours, the magnetic nanoparticles bound to the enzyme using a magnet is separated from the solution, washed with phosphate buffer solution, characterized in that the Method for producing biodegradable enzyme-immobilized magnetic nanoparticles. delete The method of claim 9,
The one-cycle transition metal is any one selected from the group consisting of nickel, copper, cobalt, zinc, a method for producing a biodegradable enzyme-immobilized magnetic nanoparticles of a non-degradable aromatic compound. The method of claim 9,
The ion of the transition metal is a method for producing a biodegradable enzyme-immobilized magnetic nanoparticles of a hardly decomposable aromatic compound, characterized in that the ion of the transition metal is any one selected from the group consisting of nickel, copper, cobalt, zinc. delete The method of claim 9,
The phenol-monooxygenase or catechol-dioxygenase is an hardly degradable aromatic compound, characterized in that the enzyme comprises 4 to 12 consecutive histidine sequences at the end of the amino acid sequence. Method for producing biodegradable enzyme-immobilized magnetic nanoparticles. delete 10. Contamination of magnetic nanoparticles to which biodegradable enzymes of the hardly decomposable aromatic compounds according to any one of claims 1 to 2, 3 and 5 to 6 are immobilized with an aromatic compound or a phenolic aromatic compound. Method of biodegrading them by adding 0.01 to 0.1% by weight based on the total weight of water. delete Claim 10, claim 11 to claim 12 of the magnetic nanoparticles of which the biodegradation effect of the hardly decomposable aromatic compound prepared by the method according to any one of the manufacturing method is immobilized with an aromatic compound or a phenolic aromatic compound Biodegradation by adding 0.01 to 0.1% by weight relative to the total weight of the contaminated water.

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