CN108707618A - A kind of nano enzyme and preparation method thereof based on people's ferritin - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and specifically discloses a nanozyme based on human ferritin and a preparation method thereof. The preparation method uses human ferritin as a fusion partner, places the target enzyme at its N-terminal for fusion expression, and inserts a connecting peptide between the target enzyme and human ferritin. The preparation method of the invention can significantly improve the catalytic activity of the target enzyme to the substrate, and make the enzyme have stronger tolerance to temperature and pH. This preparation method is not limited to the fusion expression of human ferritin, methyl parathion hydrolase and Escherichia coli alkaline phosphatase, and is also applicable to the fusion expression of human ferritin and various enzymes to improve the characteristics of the target enzyme. It has high research value and scientific significance in scientific research and applied basic research.

Description

A nanozyme based on human ferritin and its preparation method

technical field

The invention relates to the technical field of genetic engineering, in particular to a nanozyme based on human ferritin and a preparation method thereof.

Background technique

Recombinant heavy chain of human ferritin (rHF, hereinafter referred to as human ferritin) is a relatively well-researched protein with nanostructure characteristics. It has the ability to self-assemble in vitro to form 24-mer cage-shaped nanoparticles. characteristic. Human ferritin is relatively simpler than other biomacromolecular nanocomponents, and its modification and assembly technology is relatively mature. It can be connected with functional proteins by genetic engineering, especially fusion with the N-terminal of human ferritin, without affecting the integrity of nanoparticles. properties, and functional molecules are displayed on the surface of the cage structure after assembly, these characteristics endow human ferritin with unique advantages in the field of nanomaterials, making human ferritin a perfect module for building self-assembled nanoparticles.

As a product widely used in agricultural production, parathion-type organophosphorus pesticides have been listed as one of the most toxic substances. Its neurotoxicity is due to inhibition of acetylcholinesterase, an enzyme in the nervous system that normally degrades the neurotransmitter acetylcholine, thereby disrupting nervous system function. In agricultural production, the abuse of organophosphorus pesticides caused by pests and diseases makes pesticides remain in soil, water resources, agricultural products, and even food chains, destroying agricultural ecology and seriously threatening human health. Therefore, it is urgent to develop an efficient and pollution-free A parathion-type pesticide degradation method and a high-sensitivity detection method. Methyl parathion hydrolase (MPH, EC 3.1.8.1) is a methyl parathion degrading bacterium ( Pseudomonas sp.strain WBC- 3) Important metabolizing enzymes of parathion isolated and purified in Studies have found that MPH can degrade methyl parathion, ethyl parathion, chlorpyrifos, etc. Although some researchers have successfully constructed the E. coli expression system of MPH, and successfully achieved a large amount of expression of MPH in heterologous hosts (Chu Xiaona, 2003; Liu Lulu et al., 2004), but this is urgent to improve the degradation rate and However, the study of detection sensitivity methodology is far from enough.

Alkaline phosphatase (Alkaline phosphatase, AP, EC 3.1.3.1) is a non-specific phosphomonoesterase, which has the function of catalyzing the hydrolysis of phosphate monoesters to generate inorganic phosphate and corresponding alcohols, phenols or sugar compounds. Alkaline phosphatase has a wide range of sources, and the molecular size and coding sequence of different sources are very different, and the structure, function and catalytic mechanism are also slightly different. Among them, the research on Escherichia coli alkaline phosphatase (EAP) is the most thorough. EAP is a homodimeric metalloenzyme. It is widely used in medicine because of its clear catalytic mechanism and wide range of substrates. , immunology and other fields.

Human ferritin nanoparticles are widely used in the research and application of biosensors, vaccine development, and targeted drug delivery systems, but methyl parathion hydrolase and Escherichia coli alkaline phosphate based on human ferritin nanostructures The recombinant protein of the enzyme (nanozyme), and the research on the catalytic system of these two nanozymes have not been reported yet.

Contents of the invention

The object of the present invention is to provide a nanozyme based on human ferritin and a preparation method thereof in order to overcome the above-mentioned deficiencies of the prior art. In the present invention, methyl parathion hydrolase or Escherichia coli alkaline phosphatase is placed at the N-terminus of human ferritin for fusion expression, and these two enzymes can self-assemble in Escherichia coli to form nanoparticles, and the enzyme activity and stability Both are improved, and the preparation method of the invention can solve the problems of low enzyme activity, too narrow optimum pH and optimum temperature, etc., and expand the application range of the enzyme.

Another object of the present invention is to provide the nanozyme prepared by the above preparation method.

Another object of the present invention is to provide a fusion gene encoding the aforementioned nanozyme.

Another object of the present invention is to provide an expression vector containing the above nucleic acid.

Another object of the present invention is to provide a host bacterium containing the above expression vector.

Another object of the present invention is to provide a preparation for degrading organophosphorus pesticides or catalyzing the hydrolysis of phosphate monoesters.

In order to achieve the above object, the present invention is achieved through the following schemes:

A method for preparing a nanozyme based on human ferritin, using human ferritin as a fusion partner, placing a target enzyme at its N-terminus for fusion expression, and inserting a connecting peptide between the target enzyme and human ferritin. The connecting peptide is a flexible connecting peptide whose sequence is GGGGS.

The preparation method of the invention can significantly improve the catalytic activity of the target enzyme to the substrate, and make the enzyme have stronger tolerance to temperature and pH, and has the characteristics of high efficiency, simplicity, and wide application range, and is suitable for genetic engineering, biological Research in chemistry and molecular biology.

Preferably, the preparation method includes the following steps: cloning human ferritin and the target enzyme gene, splicing to obtain the human ferritin-linking peptide-target enzyme fusion protein gene, constructing a recombinant plasmid, transforming E. coli host bacteria, screening to obtain engineering strains, Cultivate engineering bacteria, induce the expression of human ferritin-connecting peptide-target enzyme fusion protein, purify by nickel ion affinity chromatography, and obtain human ferritin-connecting peptide-target enzyme fusion protein.

Preferably, the target enzyme is a hydrolase.

More preferably, the hydrolase is methyl parathion hydrolase or Escherichia coli alkaline phosphatase.

The following takes methyl parathion hydrolase and Escherichia coli alkaline phosphatase as examples to illustrate the characteristics of the present invention: human ferritin (rHF) is used as a fusion partner, methyl parathion hydrolase (MPH) or Escherichia coli alkali phosphatase (EAP) was fused to the N-terminal of rHF to construct the fusion expression vector pET28a-MPH-rHF or pET28a-EAP-rHF. Both fusion proteins MPH-rHF and EAP-rHF can be highly expressed in Escherichia coli, and high-purity MPH-rHF and EAP-rHF can be obtained by nickel ion affinity chromatography. The aggregation form of the fusion protein was detected by transmission electron microscopy, and it was found that the fusion proteins MPH-rHF and EAP-rHF successfully self-assembled to form nanostructures in vitro, indicating that the fusion of MPH or EAP at the N-terminus of rHF did not affect the self-assembly of rHF to form a cage Nano-structure. By comparing the hydrolysis activity of nanozyme MPH-rHF and free enzyme MPH on the substrate (parathion), it was found that nanozyme MPH-rHF had higher catalytic activity than free enzyme MPH; compared with free enzyme MPH, The optimum temperature of nanozyme MPH-rHF changed from 35°C to 40°C, and the optimum pH changed from 9.5 to 10.5. By comparing the catalytic activity of the nanozyme EAP-rHF and the free enzyme EAP on the substrate (p-nitrophenyl phosphate, p-nitrophenyl phosphate, pNPP), it was found that the nanozyme EAP-rHF has higher catalytic activity; compared with the free enzyme EAP, nanozyme EAP-rHF has higher tolerance to temperature and pH.

Therefore, the present invention claims the use of human ferritin in improving the catalytic activity and/or stability of methyl parathion hydrolase or Escherichia coli alkaline phosphatase; tolerance.

The present invention also claims to protect the nanozyme prepared by the above preparation method. The nanozyme is a fusion protein, and the sequence from the N-terminal to the C-terminal is the target enzyme, connecting peptide and human ferritin.

Preferably, the amino acid sequence of the fusion protein is any one of SEQ ID NO:1-SEQ ID NO:2.

The amino acid sequence of the fusion protein MPH-rHF is shown in SEQ ID NO:1.

The amino acid sequence of the fusion protein EAP-rHF is shown in SEQ ID NO:2.

The present invention also claims a fusion gene encoding the aforementioned nanozyme, the nucleotide sequence of the fusion gene being any one of SEQ ID NO:3-SEQ ID NO:4.

The nucleotide sequence of the fusion gene MPH - rHF is shown in SEQ ID NO:3.

The nucleotide sequence of the fusion gene EAP - rHF is shown in SEQ ID NO:4.

The present invention also claims an expression vector containing the above nucleic acid, and the expression vector is pET28a-MPH-rHF or pET28a-EAP-rHF.

The present invention also claims a host bacterium containing the above expression vector, the host bacterium is E. coli BL21 (DE3) or E. coli BL21 (DE3) pLysS competent cell.

The present invention also claims a preparation for degrading organophosphorus pesticides or catalyzing the hydrolysis of phosphate monoesters, said preparation including the above-mentioned nanozyme, fusion gene, expression vector or host bacteria.

Compared with the prior art, the present invention has the following beneficial effects:

The nanozyme (fusion protein) is formed by the preparation method provided by the present invention, which respectively improves the catalytic activity of methyl parathion hydrolase or Escherichia coli alkaline phosphatase on the substrate, and enhances the tolerance of the enzyme to temperature and pH sex. This preparation method is not limited to the fusion expression of human ferritin, methyl parathion hydrolase and Escherichia coli alkaline phosphatase, but is also applicable to the fusion expression of human ferritin and various enzymes to improve the characteristics of the target enzyme. It has high research value and scientific significance in research and applied basic research.

Description of drawings

Figure 1 is a schematic diagram of the recombinant enzyme construction strategy of the present invention; wherein the target enzyme gene can be methyl parathion hydrolase gene ( MPH ) or Escherichia coli alkaline phosphatase gene ( EAP ); linker is the nucleotide sequence of GGGGS; rHF is human ferritin heavy chain gene.

Fig. 2 is the SDS-PAGE electrophoresis figure of the recombinant enzyme purified in the embodiment; a is the electrophoresis figure of purified MPH-rHF and MPH, wherein M is protein Marker, swimming lane 1 is the MPH of purification, and swimming lane 2 is the MPH-rHF of purification rHF; b is the electropherogram of purified EAP-rHF and EAP, where M is protein marker, lane 1 is purified EAP, and lane 2 is purified EAP-rHF.

Figure 3 is a transmission electron microscope (TEM) image of the recombinant enzyme in the example; a is the aggregated form of the recombinant enzyme MPH-rHF; b is the aggregated form of the recombinant enzyme EAP-rHF.

Fig. 4 is the result of the influence of temperature on enzyme activity in the embodiment; a is the influence of temperature on MPH-rHF and MPH; b is the influence of temperature on EAP-rHF and EAP.

Figure 5 is the result of the effect of pH on enzyme activity in the examples; a is the effect of pH on MPH-rHF and MPH; b is the effect of pH on EAP-rHF and EAP.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

Example 1 Construction of fusion expression vector of methyl parathion hydrolase and human ferritin

Fusion protein design strategy: using human ferritin (rHF) as a fusion partner, placing methyl parathion hydrolase (MPH) at the N-terminus of rHF for expression, and a linker between MPH and rHF connect. The schematic diagram of the gene sequence of the fusion protein MPH-rHF is shown in Fig. 1 .

Construction of expression vectors: According to the vectors pET20-MPH and pET28a-rHF provided by Wuhan Institute of Virology, Chinese Academy of Sciences, specific amplification primers were designed, and Nde I and Bam H I restriction sites were introduced at both ends of the MPH gene ; The nucleotide sequence of the linker (GGGGS) is introduced at the 5' end, and Bam H I and Hind III restriction sites are introduced at both ends. The MPH and pET28a vectors were recovered by double enzyme digestion with Nde I and Bam H I, respectively, and the digested products were ligated, and transformed into E. coli DH5α, and the positive clones were screened and sequenced. The sequencing results showed that the vector pET28a-MPH was successfully constructed, and the correct positive clones Expand the culture, extract the plasmid and save it for later use. Then pET28a-MPH and the amplified rHF fragment were digested with Bam H I and Hin d III, the digested products were ligated, and transformed into E. coli DH5α, positive clones were screened and sequenced. The sequencing results showed that the expression vector pET28a-MPH was successfully constructed -rHF, the correct positive clones are expanded and cultivated, and the plasmids are extracted and stored for later use.

Example 2 Induced expression and purification of fusion protein MPH-rHF

The expression vectors pET28a-MPH-rHF and pET28a-MPH successfully constructed in Example 1 were transferred into E. coli BL21 (DE3) competent cells by the heat shock method, and a single colony was picked in a medium containing 50 μg/mL kana In the LB medium containing kanamycin, shake culture at 37°C and 200 rpm for 5 h, then transfer to 500 mL LB medium containing 50 μg/mL kanamycin at a ratio of 1:100, and at 37 Under the conditions of ℃ and 200 rpm, shake until OD ₆₀₀ =0.6 or so, add the inducer IPTG to make the final concentration 0.5mM, and induce for 8 h at 30℃ and 150 rpm. Centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant and collect the cells. Each gram of bacteria was resuspended with 20 mL of binding buffer (20 mM Tris, 0.5 M sodium chloride and 5 mM imidazole, pH7.9), ultrasonically disrupted, and centrifuged at 4°C and 6000 rpm for 15 min. Collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, then perform nickel ion affinity chromatography (Ni-NTA) purification, gradient elution, collect the eluate in separate tubes and perform SDS-PAGE electrophoresis, the electrophoresis results are shown in the figure 2a.

Example 3 Determination of Recombinase MPH-rHF and Free Enzyme MPH Kinetic Parameters

The relative steady-state kinetics of recombinant enzyme MPH-rHF and free enzyme MPH to the model substrate methyl parathion was determined by using a Shimadzu UV-2550 spectrophotometer to detect the absorbance of the product p-nitrophenol at 405 nm parameter. The total volume of the standard reaction system is 800 μL, which contains 50 nM MPH-rHF or MPH and different concentrations of methyl parathion (dissolved in methanol, when the concentration of methanol is less than 4%, the enzyme activity will not be affected), buffer The solution was 20 mM Tris-HCl at pH 8.0. The absorbance value was adjusted to zero before the addition of the substrate, and the reaction was initiated after adding methyl parathion and mixing well, and the change of the absorbance value at a wavelength of 405 nm within 4 min was recorded at 37°C. GraphPad Prism was used to fit the data of the three experiments to obtain the enzymatic kinetic parameters, as shown in Table 1 (the results are the average of the three experiments). It can be seen from Table 1 that compared with the free enzyme MPH, the catalytic efficiency ( k _cat / K _m ) of the recombinant enzyme MPH-rHF was increased by more than 1 times.

Table 1 Comparison of kinetic parameters between recombinant enzyme MPH-rHF and free enzyme MPH

parameter MPH MPH-rHF Km ( _μM ) 36.98±7.65 65.80±29.59 k _cat (min ^-1 ) 177.8±12.75 687.1±141.2 k _cat / K _m (min ^-1 μM ^-1 ) 4.81 10.45

Example 4 Transmission Electron Microscopy (TEM) Detection of the Aggregated Form of the Recombinase MPH-rHF

Add 20 μL MPH-rHF solution and 20 μL negative staining solution dropwise to the smooth surface of the parafilm to form raised droplets; gently clamp the edge of the copper grid and cover the carbon film surface on the sample solution to allow the sample to adsorb to the surface of the carbon film for 3 to 5 minutes; carefully clamp the copper mesh, use filter paper to absorb the liquid from the edge of the copper mesh, and cover the carbon film surface of the copper mesh to the negative Dye the surface of the solution for 5-7 minutes. Also use filter paper to absorb the liquid from the edge, place it on a plate covered with a layer of filter paper, and mark it; after standing for 2 to 4 hours, TEM characterization can be performed. Electron microscope observation was completed by South China Agricultural University Test Center (FEI, Netherlands, analytical transmission electron microscope).

The self-assembly of MPH-rHF to form a cage-shaped nanoparticle structure was characterized by electron microscopy, as shown in Figure 3a: there is a granular regular structure with a diameter of about 12 nm [12.86±2.17 nm (n=300)], indicating that the N in rHF End-fused MPH did not affect the self-assembly of human ferritin to form cage-like nanostructures, and the isolated and purified MPH-rHF successfully self-assembled into cage-like nanoparticle structures in vitro.

Example 5 Effect of temperature on the activity of nanozyme MPH-rHF

Place the reaction system containing 50 nM of the enzyme to be tested at various temperatures (4°C, 15°C, 30°C, 37°C, 40°C, 50°C, 60°C, and 65°C), and after incubation for 30 min, the activity proceeds rapidly. For detection, after 10 min of reaction, 50 μL of 1 M NaOH was added to terminate the reaction. Taking each temperature as the horizontal axis, the absorption value of the product at 405 nm at each temperature is plotted on the vertical axis. It can be seen from Figure 4a that the nanozyme MPH-rHF has better tolerance to high temperature, and exhibits the best activity at 40°C, which is different from the optimum temperature of the free enzyme MPH at about 35°C; The enzyme MPH-rHF has good activity up to 60°C, while the enzyme activity of the free enzyme MPH drops sharply after exceeding 40°C, which indicates that the fusion with human ferritin enhances the temperature tolerance of MPH.

Example 6 The Effect of pH on the Activity of Nanozyme MPH-rHF

20 mM Tris-HCl buffer solution with pH values of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0 was selected as the enzyme reaction buffer at an enzyme concentration of 50 nM and a substrate concentration of 0.0625 The enzyme activity was measured under the mM reaction system, and 50 μL of 1 M NaOH was added to terminate the reaction after 10 min of reaction. With the pH value on the horizontal axis, the absorbance value of the product at each pH value at 405 nm is plotted on the vertical axis, as shown in Figure 5a, it can be seen that the nanozyme MPH-rHF has better enzymatic activity at pH 10.5, The free enzyme MPH is expressed at about pH 9.5; and it can be seen from the figure that the nanozyme also has good adaptability to pH, which indicates that the fusion with human ferritin enhances the tolerance of MPH to pH.

Example 7 Construction of fusion expression vector of Escherichia coli alkaline phosphatase and human ferritin

Fusion protein design strategy: Human ferritin (rHF) was used as a fusion partner, and Escherichia coli alkaline phosphatase (EAP) was expressed at the N-terminus of rHF, and EAP was connected to rHF through a linker . The schematic diagram of the gene sequence of the fusion protein EAP-rHF is shown in Fig. 1 .

Construction of the expression vector: the genome was extracted from the ATCC 25922 strain already in the laboratory. Primers were designed according to the sequence of EAP in NCBI, and the nucleotide sequences of EAP containing stop codon and without stop codon were respectively amplified (both ends contained Nde I and Bam H I restriction sites); then Nde I and Bam H I enzyme double-digests the amplified nucleotide sequence of EAP containing stop codon and without stop codon and pET28a vector, ligates the digested products, transforms them into E.coli DH5α competent cells, and screens Positive clones were sequenced, and the sequencing results showed that the expression vector pET28a-EAP (with or without stop codon) was successfully constructed. According to the sequencing results, the correct positive clones were expanded and cultivated, and the plasmids were extracted and stored for later use.

From the existing pET28a-rHF vector in the laboratory, design primers (the nucleotide sequence of GGGGS introduced at the 5' end) to amplify the nucleotide sequence of rHF containing the linker (including Bam H I and Hind III restriction sites); The amplified rHF nucleotide sequence containing linker and the pET28a-EAP vector without stop codon were double digested with Bam H I and Hind III enzymes, and the digested products were ligated and transformed into E.coli DH5α sensory The positive clones were screened and sequenced. The sequencing results showed that the expression vector pET28a-EAP-rHF was successfully constructed. According to the sequencing results, the correct positive clones were expanded and cultivated, and the plasmids were extracted and stored for later use.

Example 8 Induced expression and purification of fusion protein EAP-rHF

Transform the reserved expression vectors pET28a-EAP and pET28a-EAP-rHF into E. coli BL21(DE3) pLysS competent cells by heat shock method, and pick a single clone in 5 mL of medium containing kanamycin 50 mg/mL After shaking in LB medium at 37°C and 200 rpm for 5 h, transfer to 500 mL of fresh LB medium containing kanamycin 50 μg/mL at a ratio of 1:100, and shake at 37°C and 200 rpm Shake the bacteria until OD ₆₀₀ = 0.6, add the inducer IPTG to make the final concentration 0.5 mM, and harvest the bacteria after induction at 25°C and 150 rpm for 24 h. Centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant, and resuspend the cells with pre-cooled ddH ₂ O. Centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant and keep the cells.

Resuspend each gram of bacteria with 20 mL buffer (20 mM Tris-HCl, 1 mM EDTA, pH8.0), add PMSF with a final concentration of 1 mM, place the sample on ice for ultrasonic disruption, and store the broken sample at 4 °C , centrifuged at 5000 rpm for 30 min, passed the supernatant through a 0.22 μm filter membrane, performed nickel ion affinity chromatography (Ni-NTA) purification, gradient elution, collected the eluate in separate tubes, and performed SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 2b .

Example 9 Determination of Kinetic Parameters of Recombinase EAP-rHF and Free Enzyme EAP

With p-nitrophenylphosphate (pNPP) as the catalytic substrate, EAP reacts with pNPP to produce p-nitrophenol, which has a specific absorption value at 405 nm. The relevant kinetic parameters were determined by analyzing the catalytic ability of the free enzyme EAP and the nanozyme EAP-RHF to the substrate pNPP. The enzyme activity assay was performed on a 96-well plate with a total reaction volume of 250 μL and an enzyme concentration of 5 nM. Before the reaction, incubate the DEA buffer solution (pH9.8, 1 mM Mg ²⁺ ) containing different concentrations of pNPP and the enzyme solution at 37°C for 5 min, and put them into the instrument immediately after mixing. The sample well without enzyme solution was used as the sample well. Adjust the sample to zero, and monitor the change of the absorbance at 405 nm within 5 minutes at 37°C. GraphPad Prism was used to fit the data of the three experiments, and the enzymatic kinetic parameters were plotted, as shown in Table 2 (the results are the average of the three experiments). It can be seen from Table 2 that the catalytic efficiency ( k _cat / K _m ) of the nanozyme EAP-rHF is 6.7 times higher than that of the free enzyme EAP.

Table 2 Kinetic parameters of recombinant enzyme EAP-rHF and free enzyme EAP

parameter EAP EAP-rHF Km ( _μM ) 35.10±18.31 87.68±15.81 k _cat (min ^-1 ) 186.2±14.63 3592±140.3 k _cat / K _m (min ⁻¹ μM ⁻¹ ) 5.31 40.97

Example 10 Transmission Electron Microscopy (TEM) Detection of the Aggregated Form of the Recombinase EAP-rHF

The purified recombinant enzyme EAP-rHF was analyzed by TEM electron microscope: take 10 μL of the protein sample solution on a smooth parafilm, clamp the edge of the copper omentum with tweezers, cover it on the protein solution, and infiltrate it for 10 min; take 10 μL of 2 %PTA solution (pH 8.0) on another smooth sealing film, erect the copper mesh soaked in the protein solution, blot the excess protein solution on the membrane with filter paper, and then gently place the copper mesh on the 2% PTA solution The excess liquid on the membrane was soaked for 10 minutes; the excess liquid on the membrane was blotted dry with filter paper, and placed at room temperature. After the copper mesh was dry, the morphology was observed on the machine (the electron microscope observation was completed by the Wuhan Institute of Virology, Chinese Academy of Sciences), as shown in Figure 3b It shows that there is a granular regular structure with a diameter of about 12 nm [12.54±1.35 nm (n=20)].

Example 11 Effect of temperature on the activity of nanozyme EAP-rHF and free enzyme EAP

Place the enzyme to be tested and the substrate buffer solution containing 0.0125 mM at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C and 80°C. After warming for 5 min, take 50 μL of the enzyme solution and 200 μL of substrate buffer, quickly detect the activity, and add 50 μL of 1 M NaOH to terminate the reaction after 10 min of reaction. Taking temperature as the horizontal axis, the absorption value of the product at 405 nm at each temperature is plotted on the vertical axis. As shown in Figure 4b, similar to the free enzyme EAP, the nanozyme EAP-rHF still has higher activity under high temperature conditions; however, the activity of the nanozyme EAP-rHF is stronger than that of the free enzyme EAP at all temperature conditions, which indicates that Fusion to human ferritin enhances the temperature tolerance of EAP.

Example 12 Effect of pH on Nanozyme EAP-rHF and Free Enzyme EAP Activity

DEA buffer solutions with pH values of 6.5, 7.0, 8.0, 9.0, 10.0 and 10.5 were selected as the enzyme reaction buffer, and the enzyme activity was measured in a 250 μL reaction system with an enzyme concentration of 5 nM and a substrate concentration of 0.0125 mM for 10 min. Then add 50 μL of 1 M NaOH to stop the reaction. Take the pH value on the horizontal axis, and plot the absorbance value of the product at 405 nm at each pH value on the vertical axis. It can be seen from Figure 5b that the optimum pH of both nanozyme EAP-rHF and free enzyme EAP is around 8.0, but the activity of nanozyme EAP-rHF is higher than that of free enzyme EAP under each pH condition, which shows that Fusion of human ferritin enhances the pH tolerance of EAP.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

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Gly Leu Phe Ala Asp Gly Asn Met Pro Val Arg Trp Gln Gly Pro Lys

260 265 270

Ala Thr Tyr His Gly Asn Ile Asp Lys Pro Ala Val Thr Cys Thr Pro

275 280 285

Asn Pro Gln Arg Asn Asp Ser Val Pro Thr Leu Ala Gln Met Thr Asp

290 295 300

Lys Ala Ile Glu Leu Leu Ser Arg Asn Glu Lys Gly Phe Phe Leu Gln

305 310 315 320

Val Glu Gly Ala Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro Cys

325 330 335

Gly Gln Ile Gly Glu Thr Val Asp Leu Asp Glu Ala Val Gln Arg Ala

340 345 350

Leu Glu Phe Ala Lys Lys Asp Gly Asn Thr Leu Val Ile Val Thr Ala

355 360 365

Asp His Ala His Ala Ser Gln Ile Val Ala Pro Asp Thr Lys Ala Pro

370 375 380

Gly Leu Thr Gln Ala Leu Asn Thr Lys Asp Gly Ala Val Met Val Met

385 390 395 400

Ser Tyr Gly Asn Ser Glu Glu Asp Ser Gln Glu His Thr Gly Ser Gln

405 410 415

Leu Arg Ile Ala Ala Tyr Gly Pro His Ala Ala Asn Val Val Gly Leu

420 425 430

Thr Asp Gln Thr Asp Leu Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu

435 440 445

Lys Gly Gly Gly Gly Gly Ser Met Thr Thr Ala Ser Thr Ser Gln Val Arg

450 455 460

Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn

465 470 475 480

Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe

485 490 495

Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His

500 505 510

Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln

515 520 525

Asn Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp

530 535 540

Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His

545 550 555 560

Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala

565 570 575

Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr

580 585 590

Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val Thr

595 600 605

Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu

610 615 620

Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser

625 630 635

<210> 3

<211> 1452

<212>DNA

<213> Escherichia coli

<400> 3

gccgcaccgc aggtgcgcac ctcggccccc ggctactacc ggatgctgct gggcgacttc 60

gaaatcaccg cgctgtcgga cggcacggtg gcgctgccgg tcgacaagcg gctgaaccag 120

ccggccccga agacgcagag cgcgctggcc aagtccttcc agaaagcgcc gctcgaaacc 180

tcggtcaccg gttacctcgt caacaccggc tccaagctgg tgctggtgga caccggcgcg 240

gccggcctgt tcggccccac cctgggccgg ctggcggcca acctcaaggc cgcaggctat 300

cagcccgagc aggtcgacga gatctacatc accccacatgc accccgacca cgtgggcggc 360

ttgatggtgg gtgagcaact ggcgttcccg aacgcggtgg tgcgtgcgga ccagaaagaa 420

gccgatttct ggctcagcca gaccaacctc gacaaggccc cggacgacga gagcaaaggc 480

ttcttcaaag gcgccatggc ctcgctgaac ccctatgtga aggccggcaa gttcaagcct 540

ttctcgggga acaccgacct ggtgcccggc atcaaagcgc tggccagcca cggccacacc 600

ccgggccaca ccacctacgt ggtcgaaagc caggggcaaa agctcgccct gctcggcgac 660

ctgatactcg tcgccgcggt gcagttcgac gaccccagcg tcacgaccca gctcgacagc 720

gacagcaagt ccgtcgcggt ggagcgcaag aaggccttcg cggatgccgc caagggcggc 780

tacctgatcg cggcgtccca cctgtcgttc cccggcatcg gccacatccg cgccgaaggc 840

aagggctacc gtttcgtgcc ggtgaactac tcggtcgtca accccaaggg tggaggtgga 900

tcgatgacga ccgcgtccac ctcgcaggtg cgccagaact accaccagga ctcagaggcc 960

gccatcaacc gccagatcaa cctggagctc tacgcctcct acgtttacct gtccatgtct 1020

tactactttg accgcgatga tgtggccttg aagaactttg ccaaatactt tcttcaccaa 1080

tctcatgagg agagggaaca tgctgagaaa ctgatgaagc tgcagaacca acgaggtggc 1140

cgaatcttcc ttcaggatat caagaaacca gactgtgatg actgggagag cgggctgaat 1200

gcgatggagt gtgcattaca tttggaaaaa aatgtgaatc agtcactact ggaactgcac 1260

aaactggcca ctgacaaaaa tgacccccat ttgtgtgact tcattgagac aattacctg 1320

aatgagcagg tgaaagccat caaagaattg ggtgaccacg tgaccaactt gcgcaagatg 1380

ggagcgcccg aatccggctt ggcggaatat ctctttgaca agcacaccct gggagacagt 1440

gataatgaaa gc 1452

<210> 4

<211> 1911

<212>DNA

<213> Escherichia coli

<400> 4

acaccagaaa tgcctgttct ggaaaaccgg gctgctcagg gcgatattac tgcacccggc 60

ggtgctcgcc gcttaacggg tgatcagacc gccgctctgc gtgattctct tagcgataaa 120

cctgcaaaaa atattatttt gctgattggc gatgggatgg gggactcgga aattactgcc 180

gcacgcaatt atgccgaagg tgcgggcggc ttttttaaag gtatcgatgc cttaccgctt 240

accgggcaat acactcacta tgcgctgaat aaaaaaaccg gcaaaccgga ctacgtcacc 300

gactcggctg catcagcaac cgcctggtca actggtgtca aaacctataa cggcgcgctg 360

ggcgtcgata ttcacgaaaa agatcaccca acgattctgg aaatggcaaa agccgcaggt 420

ctggcgaccg gtaacgtttc taccgcagag ttgcaggatg ccacgcccgc tgcgctggtg 480

gcgcatgtga cctcgcgcaa atgctacggt ccgagtgcga ccagtgaaaa atgttcgggt 540

aacgctctgg aaaaaggcgg aaaaggatcg attaccgaac agctgcttaa cgcccgtgcc 600

gatgttacgc ttggcggcgg cgcaaaaacc tttgctgaaa cggcaaccgc cggtgaatgg 660

cagggaaaaa cgctgcgtga acaggcacag gcgcgtggtt atcagttggt gagcgatgct 720

gcctcactga attcggtgac ggaagcgaat cagcaaaaac ccctattagg actgtttgct 780

gacggcaata tgccagtgcg ctggcaagga ccgaaagcaa cgtaccacgg taatatagat 840

aagcccgcag tcacctgtac gcctaatccg caacgtaatg acagtgtacc gaccctggcg 900

cagatgaccg acaaagccat tgaattgttg agtagaaatg agaaaggctt tttcctgcaa 960

gttgaaggtg cgtcaatcga taaacaggat cacgctgcga atccttgtgg gcaaattggc 1020

gagacggtcg atctcgatga agccgtacaa cgggcgctgg aattcgctaa aaaggatggt 1080

aacacgttgg tcatagtcac cgctgatcac gcccatgcca gccagatagt tgcgccagat 1140

accaaagctc cgggcctcac ccaggcgcta aataccaaag atggcgcagt gatggtgatg 1200

agttacggga actccgaaga ggattcacaa gaacataccg gcagtcagtt gcgtattgcg 1260

gcgtatggcc cgcatgccgc caatgttgtt ggactgaccg accagaccga tctcttctac 1320

accatgaaag ccgctctggg gctgaaaggt ggaggtggat cgatgacgac cgcgtccacc 1380

tcgcaggtgc gccagaacta ccaccaggac tcagaggccg ccatcaaccg ccagatcaac 1440

ctggagctct acgcctccta cgtttacctg tccatgtctt actactttga ccgcgatgat 1500

gtggccttga agaactttgc caaatacttt cttcaccaat ctcatgagga gagggaacat 1560

gctgagaaac tgatgaagct gcagaaccaa cgaggtggcc gaatcttcct tcaggatatc 1620

aagaaaccag actgtgatga ctgggagagc gggctgaatg cgatggagtg tgcattacat 1680

ttggaaaaaa atgtgaatca gtcactactg gaactgcaca aactggccac tgacaaaaat 1740

gacccccatt tgtgtgactt cattgagaca cattacctga atgagcaggt gaaagccatc 1800

aaagaattgg gtgaccacgt gaccaacttg cgcaagatgg gagcgcccga atccggcttg 1860

gcggaatatc tctttgacaa gcacaccctg ggagacagtg ataatgaaag c 1911

Claims (10)

1. a kind of preparation method of the nano enzyme based on people's ferritin, which is characterized in that using people's ferritin as fusion partner, by mesh Mark enzyme is placed in its N-terminal and carries out amalgamation and expression, and connection peptide is inserted between target enzyme and people's ferritin.

2. preparation method according to claim 1, which is characterized in that include the following steps：Human cloning ferritin and target enzyme Gene, splicing obtain people's ferritin-connection peptide-target enzyme antigen-4 fusion protein gene, and construction recombination plasmid converts escherichia coli host Bacterium, screening obtain engineered strain, culturing engineering bacterium, the expression of induction people ferritin-connection peptide-target enzyme fusion proteins, through nickel Ion affinity chromatography purifies, and separation obtains people's ferritin-connection peptide-target enzyme fusion proteins.

3. according to any preparation method of claims 1 or 2, which is characterized in that the target enzyme is hydrolase.

4. preparation method according to claim 3, which is characterized in that the hydrolase is Methyl Parathion Hydrolase or large intestine Bacillus alkaline phosphatase.

5. the nano enzyme being prepared by any one of Claims 1 to 4 preparation method, which is characterized in that the nano enzyme For fusion protein, target enzyme, connection peptide and people's ferritin are followed successively by from N-terminal to C-terminal.

6. nano enzyme according to claim 5, which is characterized in that the amino acid sequence of the fusion protein is SEQ ID NO:1~SEQ ID NO:Any bar in 2.

7. the fusion of nano enzyme described in a kind of coding claim 6, which is characterized in that the nucleotide of the fusion Sequence is SEQ ID NO:3~SEQ ID NO:Any bar in 4.

8. a kind of expression vector containing nucleic acid described in claim 7, which is characterized in that the expression vector is pET28a- MPH-rHF or pET28a-EAP-rHF.

9. a kind of host strain containing expression vector described in claim 8, which is characterized in that the host strain isE. coli BL21（DE3）OrE. coli BL21（DE3）PLysS competent cells.

10. a kind of preparation hydrolyzed for degrading organic phosphor pesticides or catalytic phosphatase monoesters, which is characterized in that the preparation includes The fusion described in nano enzyme, claim 7, expression vector according to any one of claims 8 or right described in claim 6 are wanted Seek the host strain described in 9.