Method for preparing nano metal iron oxide material from metallothionein mutant strain

Abstract

The invention discloses a method for preparing a nano metal iron oxide material from a metallothionein mutant strain, and belongs to the technical field of gene engineering. In order to overcome the technical problem of low binding amount of metallothionein and iron ions in nano # imgabs0 # prepared by a biological method in the prior art, the invention provides a method for preparing a nano metal iron oxide material from a metallothionein mutant strain, the metallothionein mutant strain is prepared by mutating metallothionein, and the metallothionein mutant strain is fermented to obtain the nano metal iron oxide material. A # imgabs 1 # nano material precursor is prepared, and the # imgabs 2 # nano material which is uniform in nano particle size and smaller in particle size is prepared through calcination. The biological preparation method of the # imgabs3 nano material provided by the invention is mild in reaction condition, environment-friendly in preparation process, green and environment-friendly, and simple and convenient to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for preparing nano-metal iron oxide materials using a metallothionein mutant strain. Background Art

[0002] Iron (Fe) is one of the most abundant metal elements on earth. The study of the properties and applications of its compounds is a hot topic in the field of chemistry and materials science. ) is an important iron oxide, It has the advantages of being non-toxic, stable, green and environmentally friendly, and low-consumption, and is widely used in catalysts, gas and humidity sensors, inorganic dyes, anodes of photoelectrochemical cells, photocatalytic reactions, water treatment, and lithium-ion batteries.

[0003] It is also a commonly used catalyst for high burning rate propellants, with the characteristics of high catalytic activity. After nano-sizing, compared with micron-sized particles, the specific surface area is increased, the number of surface atoms is increased, the pore structure is rich and there are more unsaturated sites. Studies have shown that The smaller the particle size, the higher the propellant burning rate; therefore, the nano-catalyst With high catalytic activity, catalyst nano-sizing is considered to be an important method to improve the combustion performance of propellants.

[0004] at present, The most commonly used preparation method is the hydrothermal method. This method produces particles with high purity, good crystallinity, and good dispersibility. However, it also suffers from low sample concentrations, and the preparation process involves high pressure and stringent equipment requirements (such as a polytetrafluoroethylene-lined reactor). The biological preparation of nanometals has emerged as a new preparation technology in recent years. The process is safe, non-toxic, and environmentally friendly, requiring no complex equipment. It typically occurs at room temperature and neutral pH, making it easily tolerated by the environment. Summary of the Invention

[0005] The present invention aims to overcome the problems in the prior art of preparing nanoparticles by biological methods. The invention solves the technical problem that the metallothionein has a low binding capacity with iron ions, and provides a method for preparing nano-metal iron oxide materials by using a metallothionein mutant strain.

[0006] One of the purposes of the present invention is to provide a method for preparing nano-metal iron oxide materials using a metallothionein mutant strain, the method comprising the following steps: S1: Mutating the R position of metallothionein to obtain a mutant metallothionein; ligating the mutant metallothionein to a plasmid to obtain a recombinant vector; and transforming the recombinant vector into Escherichia coli to obtain a metallothionein mutant strain; S2: fermenting the metallothionein mutant strain obtained in S1 to obtain a fermentation broth of the metallothionein mutant 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.

[0007] In a preferred embodiment of the present invention, the mutation in S1 refers to amino acid mutation at one or more sites in the metallothionein sequence, and the metallothionein amino acid sequence after mutation contains an active side chain group; 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 mutant metallothionein in S1 further contains a tag, which 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 mutant metallothionein pure protein or a mutant metallothionein crude protein; When system C is pure protein, the protein concentration in system C is 10 mg / L-1000 mg / L; When system C is crude protein, 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, aeration conditions of air or oxygen, and a ventilation volume of 0.5 vvm-30 vvm.

[0014] In a preferred embodiment of the present invention, the step S4 The precursor cleaning solvent is water or water / ethanol alternating washing.

[0015] 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.

[0016] Beneficial effects of the present invention: The present invention provides a metallothionein mutant strain, which is prepared by mutating metallothionein, fermenting the metallothionein mutant strain, extracting crude protein, pure protein and iron ion precursor containing the mutant metallothionein as raw materials, and preparing Nanomaterial precursors, finally prepared by calcination Nanomaterials.

[0017] The metallothionein mutant strain provided by the present invention has increased the binding capacity of metallothionein to iron ions compared with the original metallothionein strain, thereby preparing nanoparticles with uniform size and smaller particle size. , which improves The catalytic activity of nanomaterials. The biological preparation method of nanomaterials is applicable to a variety of metallothioneins, and the reaction conditions are mild, the preparation process is environmentally friendly, green and environmentally friendly, and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the binding equivalents of different metallothioneins and iron ions in Example 1; Figure 2 For the synthesis of GST-MT in Example 2 SEM images of nanomaterials; Figure 3 Synthesis of GST-MT1 in Example 2 SEM images of nanomaterials; Figure 4 For the synthesis of GST-MT crude protein in Example 3 SEM images of nanomaterials; Figure 5 For the synthesis of GST-MT2 crude protein in Example 3 SEM images of nanomaterials; Figure 6 For the synthesis of GST-MT crude protein in Example 3 XRD patterns of nanomaterials; Figure 7 In Example 4 Crude protein synthesis SEM images of nanomaterials; Figure 8 In Example 4 Crude protein synthesis SEM images of nanomaterials. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] In the following examples, metallothionein is abbreviated as MT.

[0022] Example 1: In this example, the R position of the metallothionein (MT) enzyme whose amino acid sequence is shown in SEQ ID NO. 1 is mutated. The specific mutation positions are shown in Table 1 to obtain mutant metallothioneins. The amino acid sequences of the mutant metallothioneins are shown in SEQ ID NOs. 2-5.

[0023] In this example, the GST tag with the sequence shown in SEQ ID NO.6 was linked to the non-mutated metallothionein with the amino acid sequence shown in SEQ ID NO.1 and the mutant metallothionein with the amino acid sequences shown in SEQ ID NOs.2-5, respectively, to obtain GST-MT (shown in SEQ ID NO.7), GST-MT1 (shown in SEQ ID NO.8), GST-MT2 (shown in SEQ ID NO.9), GST-MT3 (shown in SEQ ID NO.10), and GST-MT4 (shown in SEQ ID NO.11) proteins, respectively. The GST-MT, GST-MT1, GST-MT2, GST-MT3, and GST-MT4 proteins were respectively linked to the pETDuet-1 plasmid to obtain recombinant vectors. The recombinant vectors were respectively transformed into BL21 (DE3) Escherichia coli to obtain recombinant strains. After fermentation, the fermentation broth of the recombinant strains was obtained, which was centrifuged at 8000 rpm and 4°C for 5 min, and the precipitate was collected. The cells in the precipitate were broken by PIS crushing pressure, and the supernatant crude enzyme solution was collected by centrifugation. After purification using a GST column, ultrafiltration was performed and the enzyme protein solutions of GST-MT, GST-MT1, GST-MT2, GST-MT3 and GST-MT4 were obtained respectively.

[0024] Different equivalent ratios of ferrous chloride solution (3, 6, 9, 11, 12, 13, 14, 15, 17, 19, 21, 23, 25) were added to the GST-MT, GST-MT1, GST-MT2, GST-MT3, and GST-MT4 enzyme protein solutions obtained above, resulting in a final concentration of metallothionein in the mixed solution of The reaction mixture was allowed to react at room temperature for 10 minutes. The reaction mixture was then analyzed using a UV spectrophotometer at wavelengths between 220 nm and 400 nm. UV absorption increased with increasing iron ion concentration. At a wavelength of 280 nm, UV absorption no longer varied with increasing iron ion concentration, indicating the maximum absorption equivalent of metallothionein for iron ions.

[0025] The results are as follows Figure 1 As shown, compared with the non-mutated metallothionein, the mutated metallothionein has an increased binding capacity for iron ions, from 7 equivalents to 8-11 equivalents.

[0026] Table 1

[0027] Note: The interpretation of the mutation position in the table is as follows, for example: R22C means that the 22nd amino acid in the original metallothionein mutated from R (arginine, Arg) to C (cysteine, Cys).

[0028] Example 2: S1: The GST tag represented by SEQ ID NO.6 was linked to the unmutated metallothionein (MT) represented by the amino acid sequence of SEQ ID NO.1 and the mutant metallothionein represented by the amino acid sequence of SEQ ID NO.2, respectively, to obtain GST-MT (represented by SEQ ID NO.7) and GST-MT1 (represented by SEQ ID NO.8), respectively; the GST-MT and GST-MT1 proteins were respectively linked to the pETDuet-1 plasmid to obtain recombinant vectors; the recombinant vectors were respectively transformed into BL21 (DE3) Escherichia coli to obtain recombinant strains; 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: The precipitate collected in S2 was resuspended in 250 mL of 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 34 pis, centrifuged, and the supernatant was collected to obtain system C (composed of GST-MT and GST-MT1 proteins purified and diluted on a GST purification column, at a concentration of 20 mg / L). 150 mL of system C was added to a 250 mL reactor, and 298.1 mg of FeCl2·4H2O was accurately weighed and dissolved 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] In this example, the above-mentioned GST-MT and GST-MT1 proteins were prepared to obtain The nanomaterials were tested by SEM, and the results were as follows Figure 2 and Figure 3 As shown in the figure, compared with the non-mutated metallothionein (GST-MT), the mutant metallothionein (GST-MT1) increased the binding capacity with iron ions. The particle size of the nanomaterial is significantly reduced from 124 nm to 86.8 nm. It can be seen that the mutant metallothionein provided by the present invention has the effect of increasing the binding capacity of metallothionein to iron ions and reducing The ability of nanomaterials to increase particle size.

[0030] Example 3: S1: The GST tag of SEQ ID NO.6 was linked to the unmutated metallothionein (MT) of SEQ ID NO.1 and the mutant metallothionein of SEQ ID NO.3, respectively, to obtain GST-MT (SEQ ID NO.7) and GST-MT2 (SEQ ID NO.9) proteins, respectively; the GST-MT and GST-MT2 proteins were ligated into the pETDuet-1 plasmid to obtain recombinant vectors; the recombinant vectors were transformed into BL21 (DE3) Escherichia coli to obtain recombinant strains; 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: 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 34 pis, centrifuged, and the supernatant was collected to obtain system C (composed of crude GST-MT and GST-MT2 proteins that had not been purified using a GST purification column, at a concentration of 390 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 to a final iron ion concentration of 350 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 3.62 vvm; 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.

[0031] In this example, the above-mentioned GST-MT crude protein and GST-MT2 crude protein were prepared respectively. The nanomaterials were tested by SEM, and the results were as follows Figure 4 and Figure 5As shown in the figure, compared with the non-mutated metallothionein (GST-MT), the mutated metallothionein (GST-MT2) increased the binding capacity with iron ions, and the prepared The particle size of the nanomaterial was significantly reduced from 159 nm to 132.8 nm.

[0032] In this example, GST-MT crude protein was prepared to obtain Nanomaterials were tested by XRD, and the results were as follows Figure 6 As shown, it is found that the X-ray diffraction peaks are The results were consistent, indicating that crude metallothionein can be successfully prepared Nanomaterials.

[0033] It can be seen that the crude mutant metallothionein provided by the present invention can be successfully prepared Nanomaterials can increase the binding capacity of metallothionein and iron ions, reduce The ability of nanomaterials to increase particle size.

[0034] Example 4: S1: Mutate the R position of the metallothionein protein with the amino acid sequence of SEQ ID NO.12. The specific mutation positions are shown in Table 2 to obtain a mutant metallothionein protein (amino acid sequence of SEQ ID NO.13); ), the amino acid sequence of the mutant metallothionein shown in SEQ ID NO.13 ( ) were respectively connected to the pETDuet-1 plasmid to obtain recombinant vectors; the above recombinant vectors were respectively transformed into BL21 (DE3) Escherichia coli to obtain recombinant strains; 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: The precipitate collected in S2 was resuspended in system A (composed of 0.01 M Tris-HCl solution with a pH of 6.5) to obtain system B; the cells in system B were disrupted with a crushing pressure of 34 PIS, centrifuged, and the supernatant was collected to obtain system C (composed of the GST purification column-unpurified and crude protein, concentration was 200 mg / L); 2 L of system C was added to a 5 L reactor, and FeCl2·4H2O was weighed and dissolved in system C to a final iron ion concentration of 70 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 h, a reaction pH value of 6.4-6.6, an aeration condition of air, and a ventilation volume of 3.62 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.

[0035] This embodiment is for the above and Crude protein was prepared The nanomaterials were tested by SEM, and the results were as follows Figure 7 and Figure 8 As shown, compared with the non-mutated metallothionein ( ), mutant metallothionein ( ) increases the binding capacity with iron ions, which is more conducive to the preparation of smaller particle size Nanomaterials will The particle size of the nanomaterial was reduced from 128 nm to 85 nm.

[0036] It can be seen that in the preparation method provided by the present invention, the mutant metallothionein increases the binding capacity with iron ions and reduces The particle size of the nanomaterial is small, and the preparation method is applicable to a variety of metallothioneins.

[0037] Table 2

[0038] Note: The interpretation of the mutation position in the table is as follows, for example: the 6th amino acid in the original metallothionein mutated from V (valine, Val) to C (cysteine, Cys).

[0039] 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 a metallothionein mutant strain, characterized in that: The method comprises the following steps: S1: Mutating the R position of metallothionein to obtain a mutant metallothionein; ligating the mutant metallothionein to a plasmid to obtain a recombinant vector; and transforming the recombinant vector into Escherichia coli to obtain a metallothionein mutant strain; S2: fermenting the metallothionein mutant strain obtained in S1 to obtain a fermentation broth of the metallothionein mutant 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 mutation in S1 refers to an amino acid mutation at one or more sites in the metallothionein sequence, whereby the metallothionein amino acid sequence contains an active side chain group after the mutation; 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.

3. The method according to claim 1, wherein The mutant metallothionein in S1 further comprises a tag, which includes but is not limited to a GST tag.

4. 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).

5. 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.

6. The method according to claim 1, wherein The system C described in S3 is a mutant metallothionein pure protein or a mutant metallothionein crude protein; When system C is pure protein, the protein concentration in system C is 10 mg / L-1000 mg / L; When system C is crude protein, the protein concentration in system C is 50 mg / L-5000 mg / L.

7. 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.

8. 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.

9. The method according to claim 1, wherein As described in S4 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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