Method for preparing nano metal iron oxide material from iron ion binding protein with specific binding capacity

Abstract

The invention relates to a method for preparing a nano metal iron oxide material from iron ion binding protein with specific binding capacity, and belongs to the technical field of genetic engineering. In order to overcome the technical problems of non-specific binding of metallothionein and iron ions and relatively low production efficiency in a traditional nano # imgabs0 # biological preparation method, the invention provides an iron ion binding protein with specific binding capacity, and a specific binding iron ion polypeptide sequence is connected on the basis of metallothionein, so that the iron ion binding protein with specific binding capacity is obtained. The specific binding amount of iron ions is increased, and the specific binding iron ion protein is used as a template to prepare the # imgabs 1 # nano material. According to the method for preparing the nano-metal iron oxide material through specific binding of the iron ion protein, reaction conditions are mild, # imgabs2 # is prepared and synthesized in an oriented mode through high selectivity of the specific binding iron ion protein, the preparation process is easy to control, and the particle size range of the prepared # imgabs3 # is 20-1000 nm.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing nano-metal iron oxide materials from an iron ion binding protein with specific binding ability. Background Art

[0002] Iron(III) oxide ) is an important transition metal oxide, widely used in catalysis, pigments, biomedicine and other fields. Among them, It is also a commonly used catalyst for high burning rate propellants and has the characteristics of high catalytic activity. Nano-sizing can reduce particle size, increase specific surface area, and promote the increase of surface atoms. Nano-sized crystals have complex microstructures and various lattice defects, and have high catalytic activity. Therefore, catalyst nano-sizing is considered an important method to improve the combustion performance of propellants. Related research shows that The smaller the particle size, the higher the propellant burning rate.

[0003] at present, Traditional preparation methods include chemical precipitation, sol-gel, hydrothermal, template, hydrothermal, etc. Chemical reaction preparation methods are often accompanied by the appearance of some toxic by-products, which pollute the environment. The use of biological methods to prepare nanometals is a new preparation technology that has emerged in recent years. The preparation process is safe, non-toxic, green, and does not require complex equipment. It generally occurs at room temperature and neutral pH conditions and is easily accepted by the environment. At present, there are literature reports on the biological preparation of nanometals by metallothionein. However, metallothionein can interact with most metal ions, which seriously affects the nanostructure of the nanostructured production efficiency. Summary of the Invention

[0004] The present invention is to overcome the traditional nano The biological preparation method has the technical problems of non-specific binding of metallothionein to iron ions and low production efficiency. A method for preparing nano-metal iron oxide materials using iron ion binding proteins with specific binding ability is provided.

[0005] One of the purposes of the present invention is to provide a method for preparing nano-metal iron oxide materials using an iron ion binding protein with specific binding ability, the method comprising the following steps: S1: linking metallothionein to an iron-binding protein with specific binding ability to obtain a recombinant sequence; linking the recombinant sequence to a plasmid to obtain a recombinant vector; and transforming the recombinant vector into Escherichia coli to obtain a recombinant strain; S2: fermenting the recombinant strain obtained in S1 to obtain a fermentation broth of the recombinant strain, centrifuging it, and collecting the precipitate; S3: Resuspend the precipitate collected in S2 in system A to obtain system B; disrupt the cells in system B with crushing pressure, centrifuge, and collect the supernatant to obtain system C; weigh the iron ion solution and dissolve it in system C to obtain system D; S4: The system D obtained in S3 is stirred and aerated at a certain temperature to obtain Nano precursors; The nano precursor was added to the solvent, washed and centrifuged 2-3 times, and calcined to obtain Nanomaterials.

[0006] In a preferred embodiment of the present invention, the iron ion binding protein with specific binding ability described in S1 includes but is not limited to the amino acid sequences shown in SEQ ID NO.1-13.

[0007] In a preferred embodiment of the present invention, the iron ion binding protein with specific binding ability described in S1 contains active side chain groups; The active side chain groups are amino acids containing carboxyl, amino, and guanidine groups, and the amino acids include but are not limited to Lys, Asp, and Glu; The active side chain group is a cyclic amino acid, including but not limited to Glu and Asp; The active side chain group contains amino acids that can interact with ferrous iron through coordination bonds or ionic bonds, and the amino acids include but are not limited to Cys, Arg, and Ser.

[0008] In a preferred embodiment of the present invention, the iron ion binding protein with specific binding ability described in S1 further contains a tag, and the tag includes but is not limited to a GST tag.

[0009] In a preferred embodiment of the present invention, the plasmid in S1 includes but is not limited to pETDuet-1, and the Escherichia coli includes but is not limited to Escherichia coli BL21 (DE3).

[0010] In a preferred embodiment of the present invention, the composition of the system A in S3 is: a Tris-HCl solution with a concentration of 0.01-0.2 M and a pH value of 6.0-7.5, and the crushing pressure is 34 PIS.

[0011] In a preferred embodiment of the present invention, the system C in S3 is a pure protein or crude protein solution; When system C is a pure protein that binds to iron ions with specific binding ability, the protein concentration in system C is 10 mg / L-1000 mg / L; When system C is an iron ion-binding crude protein with specific binding ability, the protein concentration in system C is 50 mg / L-5000 mg / L.

[0012] In a preferred embodiment of the present invention, the iron ion solution in S3 is a divalent or trivalent iron ion solution, and the final concentration of iron ions in the system D is 7 mM-1000 mM.

[0013] In a preferred embodiment of the present invention, the stirring and aeration reaction conditions in S4 are: stirring speed of 50-800 rpm, reaction temperature of 25-40 ° C, reaction time of 1-24 h, reaction pH of 6.0-8.0, ventilation conditions of air or oxygen, ventilation volume of 0.5 vvm-30 vvm; The precursor cleaning solvent is water or water / ethanol alternating washing.

[0014] In a preferred embodiment of the present invention, the temperature of the calcination treatment in S4 is 500-900° C., and the calcination treatment time is 1-3 h.

[0015] Beneficial effects of the present invention: The present invention provides an iron ion binding protein with specific binding ability, which is prepared by connecting a specific iron ion binding polypeptide sequence on the basis of metallothionein to increase the specific binding capacity of iron ions. Nanomaterials; Fermenting a recombinant strain containing metallothionein and a protein specifically binding to iron ions, extracting crude protein, pure protein and iron ion precursor of the iron ion binding protein with specific binding ability as raw materials, and preparing Nanomaterial precursors, finally prepared by calcination Nanomaterials.

[0016] The iron ion binding protein with specific binding ability provided by the present invention has high selectivity and can be prepared and synthesized in a targeted manner. , the preparation process is easy to control; the metallothionein that specifically binds to iron ion protein has a higher iron ion specificity than the original metallothionein, which can effectively promote the biological synthesis of small-particle nano-iron oxide. The method for preparing nano-metal iron oxide materials by specifically binding to iron ion protein provided by the present invention is applicable to a variety of metallothioneins, has mild reaction conditions, is environmentally friendly in preparation process, and is easy to operate. The particle size of nanomaterials ranges from 20 to 1000 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison of the retention rates of GST-MT, GST-MT-SEQ ID NO.1, GST-SEQ ID NO.8-MT, and GST-MT-SEQ ID NO.12 recombinant proteins in Example 1 when binding to iron ions under interference from foreign ions; Figure 2 This is a comparison of the retention rates of GST-MT', GST-MT'-SEQ ID NO.1 and GST-MT'-SEQ ID NO.7 recombinant proteins binding to iron ions under the interference of foreign ions in Example 1; Figure 3 For the synthesis of GST-SEQ ID NO.8-MT crude protein in Example 3 SEM images of nanomaterials; Figure 4 For the synthesis of GST-SEQ ID NO.8-MT crude protein in Example 3 XRD patterns of nanomaterials; Figure 5 Synthesis of crude protein of GST-MT'-SEQ ID NO.7 in Example 4 XRD patterns of nanomaterials; Figure 6 Synthesis of crude protein of MT"-SEQ ID NO.4 in Example 5 XRD patterns of nanomaterials; Figure 7 Synthesis of GST-MT-SEQ ID NO.12 protein in Example 2 SEM images of nanomaterials; Figure 8 For the GST-MT synthesis in Comparative Example 1 SEM images of nanomaterials. DETAILED DESCRIPTION

[0018] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0020] In the following examples, metallothionein is abbreviated as MT.

[0021] Example 1: (1) Metallothionein (MT) containing a GST tag (shown as SEQ ID NO.15) and having an amino acid sequence shown as SEQ ID NO.14 was linked to different specific iron ion binding proteins to obtain recombinant proteins (GST-MT-SEQ ID NO.1, GST-SEQ ID NO.8-MT and GST-MT-SEQ ID NO.12); the amino acid sequences of the different specific iron ion binding proteins were shown as SEQ ID NO.1, SEQ ID NO.8 and SEQ ID NO.12, respectively.

[0022] (2) Metallothionein (MT') containing a GST tag (shown as SEQ ID NO.15) and having an amino acid sequence shown as SEQ ID NO.16 was linked to different specific iron ion binding proteins to obtain recombinant proteins (GST-MT'-SEQ ID NO.1 and GST-MT'-SEQ ID NO.7); the amino acid sequences of the different specific iron ion binding proteins were shown as SEQ ID NO.1 and SEQ ID NO.7, respectively.

[0023] In this example, the Fe 2+ Ion binding specificity, the detection steps are as follows: Add the final concentration of 0.02 M sodium acetate solution at pH 5.0. The above-mentioned recombinant proteins with different binding (GST-MT-SEQ ID NO.1, GST-SEQ ID NO.8-MT and GST-MT-SEQ ID NO.12; GST-MT'-SEQID NO.1 and GST-MT'-SEQ ID NO.7) and the recombinant proteins not linked to the iron ion-specific binding protein (GST-MT and GST-MT') were added. Fe 2+ Ionic solution was placed at 60℃ and incubated for 90 min to obtain a mixed solution; The mixture was added with 6 times the volume of ethanol, allowed to stand at room temperature for 60 min, centrifuged at 12000 rpm for 30 min, the supernatant was removed, and the mixture was dried in an oven for 24 h. Diluent (from the Iron Ion Colorimetric Detection Kit), Fe 2+ Detect the concentration and record it as C0; Add the final concentration of 0.02 M sodium acetate solution at pH 5.0. The above-mentioned recombinant proteins with different binding were added Metal ion mixture (the metal ion mixture contains Fe 2+、Zn 2+ Mg 2+ ), incubate at 60℃ for 90 min to obtain a mixed solution; take the above The mixture was added with 6 times the volume of ethanol, allowed to stand at room temperature for 60 min, centrifuged at 12000 rpm for 30 min, the supernatant was removed, and dried in an oven for 24 h. Diluent (purchased from the Iron Ion Colorimetric Detection Kit) was used to detect Fe 2+ The concentration was detected and recorded as C1.

[0024]

[0025] The results are as follows Figure 1 As shown in the figure, the metallothionein MT-linked recombinant proteins specifically targeting iron ions (GST-MT-SEQ ID NO.1, GST-SEQ ID NO.8-MT, and GST-MT-SEQ ID NO.12) showed significantly higher iron ion binding retention rates under the interference of foreign ions than the recombinant sequence protein (GST-MT) not linked to the specific iron ion binding protein. Among them, the recombinant protein linked to the specific iron ion binding protein shown in SEQ ID NO.10 still had a 98.9% retention rate under the interference of foreign ions.

[0026] The results are as follows Figure 2 As shown, the recombinant proteins obtained by linking metallothionein MT' to a protein specifically targeting iron ions (GST-MT'-SEQ ID NO. 1 and GST-MT'-SEQ ID NO. 7) showed significantly higher iron ion binding retention rates in the presence of foreign ions than the recombinant protein not linked to the protein specifically targeting iron ions (GST-MT'). Specifically, the recombinant protein linked to the protein specifically targeting iron ions with the amino acid sequence of SEQ ID NO. 7 maintained a 92.7% retention rate in the presence of foreign ions.

[0027] In summary, the specific iron ion targeting protein provided by the present invention can bind to different metallothioneins and increase the iron ion specific binding capacity of metallothioneins.

[0028] Example 2: S1: A metallothionein (MT) containing a GST tag (shown in SEQ ID NO.15) and having an amino acid sequence shown in SEQ ID NO.14 was linked to a specific iron-binding protein having an amino acid sequence shown in SEQ ID NO.12 to obtain a recombinant protein (GST-MT-SEQ ID NO.12); the recombinant protein was ligated into a pETDuet-1 plasmid to obtain a recombinant vector; the recombinant vector was transformed into BL21 (DE3) Escherichia coli to obtain a recombinant strain; S2: Ferment the recombinant strain obtained in S1 to obtain the recombinant strain fermentation liquid, centrifuge at 8000 rpm and 4°C for 5 min, and collect the precipitate; S3: Resuspend the precipitate collected in S2 in 250 mL of system A (composed of 0.01 M Tris-HCl solution, pH 6.5) to obtain system B; disrupt the cells in system B with a crushing pressure of 34 PIS, centrifuge, and collect the supernatant to obtain system C (composed of pure iron ion-binding protein with specific binding ability purified by GST purification column, concentration of 25 mg / L); add 150 mL of system C to a 250 mL reactor, accurately weigh 298.1 mg of FeCl2·4H2O and dissolve it in system C to a final iron ion concentration of 10 mM to obtain system D; S4: The system D obtained in S3 was subjected to a stirring and aeration reaction at a certain temperature. The stirring and aeration reaction conditions were: a stirring speed of 200 rpm, a reaction temperature of 25°C, a reaction time of 1.5 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 0.5 vvm; and nanoparticles were obtained. Precursor; nano The precursor was added to 300 mL of water, washed and centrifuged twice, centrifuged at 10,000 rpm for 10 min, and calcined at 800 ° C for 2 h to obtain Nanomaterials.

[0029] Example 3: S1: A metallothionein (MT) containing a GST tag (shown in SEQ ID NO.15) and having an amino acid sequence shown in SEQ ID NO.14 was linked to a specific iron-binding protein having an amino acid sequence shown in SEQ ID NO.8 to obtain a recombinant protein (GST-SEQ ID NO.8-MT); the recombinant protein was ligated into a pETDuet-1 plasmid to obtain a recombinant vector; the recombinant vector was transformed into BL21 (DE3) Escherichia coli to obtain a recombinant strain; S2: Ferment the recombinant strain obtained in S1 to obtain the recombinant strain fermentation liquid, centrifuge at 8000 rpm and 4°C for 5 min, and collect the precipitate; S3: The precipitate collected in S2 was resuspended in system A (composed of 0.01 M Tris-HCl solution, pH 6.5) to obtain system B; the cells in system B were disrupted at a crushing pressure of 34 psi, centrifuged, and the supernatant was collected to obtain system C (composed of 300 mg / L iron-binding crude protein with specific binding capacity); 2.3 L of system C was added to a 5 L reactor, and FeCl2·4H2O was weighed and dissolved in system C until the final iron ion concentration in the system was 300 mM to obtain system D; S4: The system D obtained in S3 was subjected to a stirring and aeration reaction at a certain temperature. The stirring and aeration reaction conditions were: a stirring speed of 800 rpm, a reaction temperature of 25°C, a reaction time of 1.5 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 3 vvm; nanoparticles were obtained. Precursor; nano The precursor was added to 300 mL of water, washed and centrifuged twice, centrifuged at 10000 rpm for 10 min, and calcined at 800 °C for 2 h to obtain Nanomaterials.

[0030] In this example, the above-mentioned GST-SEQ ID NO.8-MT crude protein was prepared to obtain The nanomaterials were tested by SEM and XRD, and the results were as follows Figure 3 and Figure 4 As shown in the figure, the particle size of 135.4 nm was successfully prepared in this example. Nanomaterials, indicating that the iron ion binding crude protein with specific binding ability provided by the present invention can be successfully prepared Nanomaterials.

[0031] Example 4: S1: A metallothionein (MT') containing a GST tag (shown in SEQ ID NO.15) and having an amino acid sequence shown in SEQ ID NO.16 was linked to a specific iron-binding protein (shown in SEQ ID NO.7) to obtain a recombinant protein (GST-MT'-SEQ ID NO.7); the recombinant protein was ligated into a pETDuet-1 plasmid to obtain a recombinant vector; the recombinant vector was transformed into BL21 (DE3) Escherichia coli to obtain a recombinant strain; S2: Ferment the recombinant strain obtained in S1 to obtain the recombinant strain fermentation liquid, centrifuge at 8000 rpm and 4°C for 5 min, and collect the precipitate; S3: The precipitate collected in S2 was resuspended in system A (composed of 0.01 M Tris-HCl solution, pH 6.5) to obtain system B; the cells in system B were disrupted at a crushing pressure of 34 psi, centrifuged, and the supernatant was collected to obtain system C (composed of crude protein with specific iron binding ability, concentration of 250 mg / L); 2.3 L of system C was added to a 5 L reactor, and FeCl2·4H2O was weighed and dissolved in system C until the final iron ion concentration in the system was 300 mM, to obtain system D; S4: The system D obtained in S3 was subjected to a stirring and aeration reaction at a certain temperature. The stirring and aeration reaction conditions were: a stirring speed of 800 rpm, a reaction temperature of 25°C, a reaction time of 2 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 3 vvm; nanoparticles were obtained. Precursor; nano The precursor was added to 300 mL of water, washed and centrifuged twice, centrifuged at 10000 rpm for 10 min, and calcined at 800 °C for 2 h to obtain Nanomaterials.

[0032] This example is to prepare the crude protein of GST-MT'-SEQ ID NO.7 The nanomaterials were subjected to XRD analysis, and the results were as follows Figure 5 As shown, the X-ray diffraction peaks The nanomaterials are consistent, proving that this example was successfully prepared Nanomaterials.

[0033] Example 5: S1: The metallothionein (MT'') with the amino acid sequence of SEQ ID NO. 17 was linked to the iron-binding protein with the amino acid sequence of SEQ ID NO. 4 to obtain a recombinant protein (MT"-SEQ ID NO. 4); the recombinant protein was ligated into the pETDuet-1 plasmid to obtain a recombinant vector; the recombinant vector was transformed into BL21 (DE3) Escherichia coli to obtain a recombinant strain; S2: Ferment the recombinant strain obtained in S1 to obtain the recombinant strain fermentation liquid, centrifuge at 8000 rpm and 4°C for 5 min, and collect the precipitate; S3: The precipitate collected in S2 was resuspended in system A (composed of 0.01 M Tris-HCl solution, pH 6.5) to obtain system B; the cells in system B were disrupted at a crushing pressure of 34 psi, centrifuged, and the supernatant was collected to obtain system C (composed of crude protein with specific binding capacity for iron ions, at a concentration of 150 mg / L); 3 L of system C was added to a 5 L reactor, and FeCl2·4H2O was weighed and dissolved in system C until the final iron ion concentration in the system was 100 mM to obtain system D; S4: The system D obtained in S3 was subjected to a stirring and aeration reaction at a certain temperature. The stirring and aeration reaction conditions were: a stirring speed of 800 rpm, a reaction temperature of 25°C, a reaction time of 2 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 3 vvm; nanoparticles were obtained. Precursor; nano The precursor was added to 300 mL of water, washed and centrifuged twice, centrifuged at 10000 rpm for 10 min, and calcined at 800 °C for 2 h to obtain Nanomaterials.

[0034] In this example, the crude protein of MT"-SEQ ID NO.4 was prepared. The nanomaterials were subjected to XRD analysis, and the results were as follows Figure 6 As shown, the X-ray diffraction peaks The nanomaterials are consistent, proving that this example was successfully prepared Nanomaterials.

[0035] Comparative Example 1: S1: A metallothionein (MT) containing a GST tag (amino acid sequence shown in SEQ ID NO. 15) and an amino acid sequence shown in SEQ ID NO. 14 was ligated into the pETDuet-1 plasmid to obtain a recombinant vector (GST-MT); the recombinant vector was transformed into BL21 (DE3) Escherichia coli to obtain a recombinant strain; S2: Ferment the recombinant strain obtained in S1 to obtain the fermentation broth of the recombinant strain, centrifuge at 8000 rpm and 4°C for 5 min, and collect the precipitate; S3: Resuspend the precipitate collected in S2 in 250 mL of system A (composed of 0.01 M Tris-HCl solution, pH 6.5) to obtain system B; disrupt the cells in system B with a crushing pressure of 34 psi, centrifuge, and collect the supernatant to obtain system C (composed of recombinant protein purified using a GST purification column, concentration 25 mg / L); add 150 mL of system C to a 250 mL reactor, accurately weigh 298.1 mg of FeCl2·4H2O and dissolve it in system C to a final iron ion concentration of 10 mM to obtain system D; S4: The system D obtained in S3 was subjected to a stirring and aeration reaction at a certain temperature. The stirring and aeration reaction conditions were: a stirring speed of 200 rpm, a reaction temperature of 25°C, a reaction time of 1.5 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 0.5 vvm; and nanoparticles were obtained. Precursor; nano The precursor was added to 300 mL of water, washed and centrifuged twice, centrifuged at 10,000 rpm for 10 min, and calcined at 800 °C for 2 h to obtain Nanomaterials.

[0036] Effect experiment: The present invention prepares the obtained The nanomaterials were tested for particle size by SEM. Figure 7 and Figure 8 As shown in Example 2, the amino acid sequence introduced is the specific iron binding protein shown in SEQ ID NO.12 to prepare The particle size of the nanomaterial is 31.6 nm, compared with the nanomaterial prepared in Comparative Example 1. Nanomaterials (119.1 nm) have a smaller particle size.

[0037] It can be seen that the specific iron ion binding protein provided by the present invention can be prepared and synthesized in a directional manner. , which can improve the binding force between metallothionein and iron ions, and is more conducive to the preparation of small particle size Nanomaterials; and, applicable to a variety of metallothioneins.

[0038] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing nano-metal iron oxide materials using an iron ion binding protein with specific binding ability, characterized in that: The method comprises the following steps: S1: linking metallothionein to an iron-binding protein with specific binding ability to obtain a recombinant sequence; linking the recombinant sequence to a plasmid to obtain a recombinant vector; and transforming the recombinant vector into Escherichia coli to obtain a recombinant strain; S2: fermenting the recombinant strain obtained in S1 to obtain a fermentation broth of the recombinant strain, centrifuging it, and collecting the precipitate; S3: Resuspend the precipitate collected in S2 in system A to obtain system B; disrupt the cells in system B with crushing pressure, centrifuge, and collect the supernatant to obtain system C; weigh the iron ion solution and dissolve it in system C to obtain system D; S4: The system D obtained in S3 is stirred and aerated at a certain temperature to obtain Nano precursors; The nano precursor was added to the solvent, washed and centrifuged 2-3 times, and calcined to obtain Nanomaterials.

2. The method according to claim 1, wherein The iron ion binding protein with specific binding ability described in S1 includes but is not limited to the amino acid sequences shown in SEQ ID NO. 1-13.

3. The method according to claim 1, wherein The iron ion binding protein with specific binding ability described in S1 contains active side chain groups; The active side chain groups are amino acids containing carboxyl, amino, and guanidine groups, and the amino acids include but are not limited to Lys, Asp, and Glu; The active side chain group is a cyclic amino acid, including but not limited to Glu and Asp; The active side chain group contains amino acids that can interact with ferrous iron through coordination bonds or ionic bonds, and the amino acids include but are not limited to Cys, Arg, and Ser.

4. The method according to claim 1, wherein The iron ion binding protein with specific binding ability described in S1 further contains a tag, which includes but is not limited to a GST tag.

5. The method according to claim 1, wherein The plasmid in S1 includes but is not limited to pETDuet-1, and the Escherichia coli includes but is not limited to Escherichia coli BL21 (DE3).

6. The method according to claim 1, wherein The composition of the system A in S3 is: a Tris-HCl solution with a concentration of 0.01-0.2 M and a pH value of 6.0-7.5, and the crushing pressure is 34 PIS.

7. The method according to claim 1, wherein The system C described in S3 is a pure protein or crude protein solution; When system C is a pure protein that binds to iron ions with specific binding ability, the protein concentration in system C is 10 mg / L-1000 mg / L; When system C is an iron ion-binding crude protein with specific binding ability, the protein concentration in system C is 50 mg / L-5000 mg / L.

8. The method according to claim 1, wherein The iron ion solution in S3 is a divalent or trivalent iron ion solution, and the final concentration of iron ions in the system D is 7 mM-1000 mM.

9. The method according to claim 1, wherein The stirring and aeration reaction conditions in S4 are: stirring speed of 50-800 rpm, reaction temperature of 25-40°C, reaction time of 1-24 h, reaction pH of 6.0-8.0, aeration conditions of air or oxygen, and aeration volume of 0.5 vvm-30 vvm; The precursor cleaning solvent is water or water / ethanol alternating washing.

10. The method according to claim 1, wherein The calcination temperature in S4 is 500-900° C., and the calcination time is 1-3 h.

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