Doped holoferritin nanoszyme and biosynthesis method and application thereof

Abstract

The present application relates to a kind of doped Holoferritin nanoenzymes and its biosynthesis method and application, belong to the cross technical field of nanobiology, biosynthetic enzyme, the doped Holoferritin nanoenzymes except with the protein basic characteristic of Holoferritin nanoenzymes, also have class oxidase activity and class peroxidase activity, can improve the problem of current ferritin cavity doping scheme difficulty, catalytic activity single, in complex or specific catalytic scene with application potential.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of nanobiology and biomimetic synthetic enzymes, and relates to a doped Holoferritin nanozyme, its biosynthesis method, and its application. Background Technology

[0002] Natural enzymes, with their highly efficient and specific catalytic capabilities, have shown great potential in fields such as biomedicine and industrial catalysis. However, most natural enzymes are proteins, which have inherent limitations such as poor stability, sensitivity to the environment, high extraction costs, and difficulty in long-term storage, severely restricting their practical applications. To overcome these bottlenecks, nanozyme technology has emerged. Among them, nanozymes, as a novel type of enzyme composed of nanomaterials, combine the unique physicochemical properties of nanomaterials with the catalytic function of enzymes, exhibiting significant advantages such as high stability, scalable preparation, and tunable catalytic activity, and have become one of the ideal alternatives to natural enzymes.

[0003] Ferritin is a complex composed of 24 similar subunits that self-assemble into hollow ferritin nanocage structures, exhibiting excellent biocompatibility and structural stability. Its unique cavity structure allows it to serve as a biological template in confined nanomaterial synthesis and a nanocarrier in drug encapsulation and delivery. Natural iron storage proteins (Holoferritin) possess ferrooxidase activity, transferring products into protein cavities for storage, thereby participating in the regulation of iron metabolism and oxidative stress in vivo. This inherent catalytic ability combined with its structural characteristics makes ferritin not only a stable carrier for enzymes but also a potential ideal substitute for natural enzymes, thus demonstrating unique application advantages in the fields of ferritin nanomaterials and nanozyme catalysis. However, current research on ferritin-based nanozymes still faces significant bottlenecks. First, the iron core chemical composition of natural ferritin is relatively simple, limiting the regulatory dimensions of its catalytic behavior. Second, the precise synthesis of doped iron cores with specific chemical compositions within the ferritin cavity using biomimetic mineralization strategies remains challenging and lacks systematic research; most studies focus on the construction of single-metal systems, with insufficient understanding of the mechanisms by which multi-element synergistic regulation of ferritin catalytic performance. Therefore, the purpose of this invention is to provide a novel method for preparing doped Holoferritin nanozymes. Summary of the Invention

[0004] In view of this, the present invention provides a doped Holoferritin nanozyme and its biosynthesis method and application, in order to solve the problems of the difficulty of current ferritin cavity doping schemes and the single catalytic activity, which limit its application potential in complex or specific catalytic scenarios.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A doped Holoferritin nanozyme, wherein the doped Holoferritin nanozyme has peroxidase-like activity and oxidase-like activity.

[0007] This invention also discloses a method for the biosynthesis of doped Holoferritin nanozymes, the biosynthesis method comprising the following steps:

[0008] (1) The gene encoding the amino acid sequence of ferritin was cloned into a prokaryotic expression vector to construct a prokaryotic expression vector, and the plasmid vector was transformed into prokaryotic expression cells to obtain a single clone strain;

[0009] (2) After picking single colonies for expansion culture, IPTG and phosphate solution were added to the culture medium at the same time to induce expression in prokaryotic cells. After induced expression for ≥4 hours, ferrous salt solution was added to continue inducing expression.

[0010] (3) After a total of 8 to 12 hours of induction expression in prokaryotic cells, the cells were purified to obtain the doped Holoferritin nanozyme.

[0011] Furthermore, the phosphate in the phosphate solution is either sodium dihydrogen phosphate or disodium hydrogen phosphate, and the ferrous salt in the ferrous salt solution is either ferrous sulfate or ferrous chloride.

[0012] Furthermore, the concentration of the phosphate solution is 1-20 mM, and the concentration of the ferrous salt solution is 1-40 mM; wherein the ferrous salt solution can be added in batches during the induction of expression. The preferred concentration of the ferrous salt solution is 5 mM.

[0013] Furthermore, the amino acid sequence of the ferritin is selected from gene sequences encoding ferritin in animals, plants, and microorganisms.

[0014] Furthermore, the amino acid sequence of the ferritin is derived from Japanese shrimp.

[0015] Furthermore, the prokaryotic expression plasmid vector used can be a common prokaryotic expression plasmid vector such as the PET series; the prokaryotic expression strain can be a common expression-type bacterial strain such as Escherichia coli or Bacillus subtilis;

[0016] Furthermore, the specific operation of the purification process is as follows:

[0017] After induction of expression, the cells were centrifuged to obtain a cell pellet. The cells were then lysed and the supernatant was obtained. Nuclease was added and incubated at 37°C for 30-60 minutes. The volume of nuclease added was 0.005-0.015% of the supernatant. The solution was then heated at 65°C for 20 minutes and the supernatant was obtained again. The protein was purified using a 45% saturated ammonium sulfate solution for 6-8 hours to obtain a high-purity doped Holoferritin nanozyme.

[0018] Magnetic stirring or ultrasonic methods can be used for incubation.

[0019] Furthermore, the expression was induced by the ferrous salt solution for 4–8 hours.

[0020] The application of the doped Holoferritin nanozyme or the prepared doped Holoferritin nanozyme disclosed in this invention in the field of catalytic detection.

[0021] The application of the doped Holoferritin nanozyme or the prepared doped Holoferritin nanozyme disclosed in this invention in the preparation of reagents or pharmaceuticals for scavenging peroxide-like and / or oxide-like substances.

[0022] The beneficial effects of this invention are as follows:

[0023] The prepared phosphorus-doped Holoferritin nanozyme, while possessing ferrooxidase activity, further endows the nanozyme with both peroxidase-like (POD) and oxooxidase-like (OXD) activities through precise phosphorus doping. It exhibits a particularly strong affinity for hydrogen peroxide, demonstrating intense POD enzyme activity, thus solving the problem of single catalytic activity and achieving effective regulation of multiple enzyme activities. Therefore, the phosphorus-doped Holoferritin nanozyme not only combines the excellent biocompatibility, targeting ability, and nanocage structure of ferritin itself, but also possesses multiple enzyme active centers, making this material highly valuable and promising for research in catalytic detection and in agents or reagents for scavenging peroxides or oxides. Attached Figure Description

[0024] Figure 1 Electrophoresis results of Holoferritin nanozymes prepared for the examples, comparative examples, and blank control;

[0025] Figure 2 Transmission electron microscopy and FTIR images of the Holoferritin nanozyme prepared as a blank control and in Example 1;

[0026] Figure 3The chemical state and overall electronic structure of the Holoferritin nanozyme prepared as a blank control and in Example 1 are shown in the figure.

[0027] Figure 4 The particle size distribution of Holoferritin nanozymes prepared for the examples, comparative examples, and blank control is shown in the figure.

[0028] Figure 5 Potential characterization of Holoferritin nanozymes prepared for examples, comparative examples, and blank controls;

[0029] Figure 6 The graph shows the iron and phosphorus content of Holoferritin nanozymes prepared for the examples, comparative examples, and blank control.

[0030] Figure 7 Holoferritin nanozyme-like oxidase activity prepared for examples, comparative examples, and blank controls;

[0031] Figure 8 The peroxidase-like activity of Holoferritin nanozymes prepared as examples, comparative examples, and blank controls. Detailed Implementation

[0032] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0033] Example 1: Preparation of doped Holoferritin nanozymes

[0034] (1) The cDNA encoding ferritin from Japanese shrimp (Marsupenaeus japonicus, MjFer) (LOC122256452) was synthesized and subcloned into the pET-3a plasmid of Escherichia coli, thus transforming it into the DH5α recombinant plasmid of Escherichia coli. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) recipient cells to obtain a single-clone strain.

[0035] (2) Single clones of the strain were picked and placed in 5 mL of Luria-Bertani (LB) medium containing ampicillin. Activation culture was performed at 37°C for 5 hours. Then, the culture was transferred to 500 mL of LB medium and cultured at 37°C until the optical density reached 0.6, completing the expansion culture. IPTG solution (isopropyl-β-D-thiogalactoside) and sodium dihydrogen phosphate solution were then added to the LB medium simultaneously. The IPTG solution concentration was 1 M, and the added volume was 200 µL. The sodium dihydrogen phosphate solution concentration was 5 mM, and the added volume was 20 mL. After culturing at 37°C for 4 hours, ferrous sulfate solution was added and the culture was continued at 37°C for another 8 hours of induction. During the induction expression period, ferrous sulfate solution needed to be added slowly in multiple batches over half an hour. The concentration of the added ferrous sulfate solution was 20 mM, and the total volume after multiple additions was 20 mL. Holoferritin was induced to be expressed in *E. coli* cells.

[0036] (3) The cells were purified by centrifugation at 8000 r / min, washed and resuspended with Tris-HCl buffer (25 mM, pH 8.0), and the collected cells were sonicated for 20 min under ice bath conditions (power 325W, sonication for 2 seconds, interval 3 seconds). Then, the cells were centrifuged (10000 r / min, 4℃, 10 min) to remove cell debris and obtain supernatant. 0.005% (volume percentage) of nuclease was added to the supernatant and incubated at 37℃ with stirring for 1 hour.

[0037] After incubation, the solution was heated at 65°C for 20 min to inactivate nucleases and denature heat-sensitive proteins. Then, it was centrifuged (10,000 r / min, 4°C, 10 min) to collect the supernatant. The protein was precipitated with 45% saturated ammonium sulfate solution and allowed to stand at 4°C for 6 hours. Then, it was centrifuged again (10,000 r / min, 4°C, 20 min) to collect the precipitated target protein. The protein was reconstituted with Tris-HCl buffer (25 mM, pH 8.0) and dialyzed three times (8 hours each time) to obtain high-purity doped Holoferritin nanozyme.

[0038] Example 2:

[0039] The only difference between this embodiment and Example 1 is that the concentration of the added sodium dihydrogen phosphate solution is 2.5 mM and the amount added is 20 mL.

[0040] Example 3:

[0041] The only difference between this embodiment and Example 1 is that the concentration of the added sodium dihydrogen phosphate solution is 10mM and the amount added is 20mL.

[0042] Comparative Example 1:

[0043] The monoclonal strain prepared in Example 1 was placed in 5 mL of Luria-Bertani (LB) medium containing ampicillin and activated at 37°C for 5 hours. Then, it was inoculated onto 500 mL of LB medium and cultured at 37°C until the optical density reached 0.6, completing the expansion culture. Next, 200 µL of 1 M IPTG solution was added to the LB medium, followed by ferrous sulfate solution, and the culture was continued at 37°C for 12 hours of induction. During the induction expression period, ferrous sulfate solution was slowly added in multiple batches over half an hour, with a concentration of 5 mM, and the total volume after multiple additions was 20 mL. This induced the synthesis and expression of Holoferritin in *E. coli* cells. The resulting cells were then purified using the same procedure as in Example 1 to obtain unadulterated Holoferritin nanozymes.

[0044] Comparative Example 2:

[0045] The only difference between this comparative example and Comparative Example 1 is that the concentration of the added ferrous sulfate solution is 10 mM, 20 mL.

[0046] Comparative Example 3:

[0047] The monoclonal strain prepared in Example 1 was placed in 5 mL of Luria-Bertani (LB) medium containing ampicillin and activated at 37°C for 5 hours. Then, it was inoculated onto 500 mL of LB medium and cultured at 37°C until the optical density reached 0.6 to complete the expansion culture. Then, IPTG solution with a concentration of 1M and an addition volume of 200 µL was added to the LB medium and cultured at 37°C for 12 hours to induce E. coli cells to synthesize and express ferritin. Then, the obtained cells were purified using the same procedure as in Example 1 to finally obtain unadulterated ferritin nanozyme.

[0048] Blank comparison:

[0049] The only difference between the blank control and Comparative Example 1 is that the concentration of the added ferrous sulfate solution is 20 mM and 20 mL.

[0050] Experiments were conducted on the nanozymes prepared in the above examples, comparative examples, and blank controls.

[0051] Among them, Example 1 is referred to as HP 5 group, Example 2 as HP 2.5 group, Example 3 as HP 10 group, Comparative Example 1 as Holo 5 group, Comparative Example 2 as Holo 10 group, Comparative Example 3 as ferritin group, and blank control as HP 0 / Holo 20 group.

[0052] Experiment 1: Electrophoretic analysis of Holoferritin nanozymes prepared by doped Holoferritin nanozymes, comparative examples, and blank controls.

[0053] The doped Holoferritin nanozymes prepared in Examples 1-3 and those prepared in Comparative Examples 1-3 and the blank control were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (Native-PAGE) to determine their protein purity and subunit molecular weight. For SDS-PAGE, a 5% stacking gel and a 12% separating gel were used; for Native-PAGE, an 8% polyacrylamide gradient gel was used. Both were run at 80 V for 30 min and then switched to a constant voltage of 120 V for electrophoresis. After electrophoresis, the samples were further stained with Coomassie Brilliant Blue R-250 for observation.

[0054] The results obtained are as follows Figure 1 As shown, the results are analyzed as follows:

[0055] Holoferritin nanozymes prepared in Comparative Examples 1-3 and blank control ( Figure 1 -A, B) and the doped Holoferritin nanozymes prepared in Examples 1-3 ( Figure 1 Samples C and D both showed a single, uniform band at 19 kDa in SDS-PAGE, consistent with the theoretical molecular weight of the Holoferritin subunit. Native-PAGE showed a clear band at approximately 440 kDa, considered to be the intact oligomeric structure of Holoferritin. Neither electrophoresis result showed diffuse bands of other proteins or self-degradation bands, indicating the successful preparation of a high-purity, doped Holoferritin nanozyme with an intact structure. This preliminarily demonstrates that the phosphorus doping process of this invention disrupts the basic structural framework of the ferritin cage.

[0056] Experiment 2: Electron microscopy and particle size and potential characterization analysis of doped Holoferritin nanozymes

[0057] 1. Electron microscopy and FTIR analyses were performed on the doped Holoferritin nanozyme prepared in Example 1 and the Holoferritin nanozyme prepared as a blank control. The specific experimental procedures are as follows:

[0058] Electron microscopy analysis: The doped Holoferritin nanozyme prepared in Example 1 and the Holoferritin nanozyme prepared as a blank control were analyzed using a Jem-1400 plus transmission electron microscope (Electronics Corporation, Japan). The sample preparation process was as follows: 5 µL of sample was cast onto a copper grid coated with a thin layer of amorphous carbon for 1 min to allow the target particles to be absorbed on its surface. Excess protein solution was removed with filter paper, and then the sample was dried at room temperature for several minutes. The copper grid was negatively stained with a 2% phosphotungstic acid solution for 30 s, and excess solution was removed with filter paper. Electron microscopy was performed, and images were acquired and analyzed.

[0059] FTIR analysis: The purified sample was freeze-dried into powder and analyzed using a Spectrum 100 Fourier transform infrared spectrometer (PerkinElmer, USA). The absorption characteristics in the 600–4000 cm⁻¹ region were determined through 32 scans. Results were obtained. Figure 2 As shown.

[0060] The results obtained are as follows Figure 2 As shown, according to Figure 2 The results show that:

[0061] Example 1 ( Figure 2 -C) produced a significant color change and the appearance of a phosphate peak at ~1050 cm⁻¹. -1 This can be considered a successful doping of phosphate. Figure 2 In both Example 1 (HP 5) and the blank control (HP 0) samples of -A and B, regular, nearly spherical nanoparticles were present, indicating that the prepared doped Holoferritin nanozyme has a complete structure, and the phosphorus doping process of this invention did not destroy the basic structural framework of the ferritin cage. Furthermore, the images show that the doped Holoferritin nanozyme of Example 1 is uniform in size and dispersed, indicating that the doped Holoferritin nanozyme prepared by this invention has good dispersibility, which provides a good foundation for subsequent chemical applications.

[0062] 2. The specific XPS steps for detecting the chemical state and overall electronic structure of the elements in the nanozymes prepared in Example 1 and the blank control are as follows:

[0063] After pressing an appropriate amount of sample into a pellet, attach it to the sample tray and place the sample into the sample chamber of the Thermo Scientific K-Alpha XPS instrument. When the pressure in the sample chamber is less than 2.0 × 10⁻⁷ mbar, send the sample into the analysis chamber. The spot size is 400 μm, the operating voltage is 12 kV, and the filament current is 6 mA. The full spectrum scanning pass energy is 150 eV with a step size of 1 eV; the narrow spectrum scanning pass energy is 50 eV with a step size of 0.1 eV.

[0064] The results obtained are as follows Figure 3 As shown in the analysis results, the following can be concluded:

[0065] XPS results for Fe 2p showed that Holoferritin, the blank control, contained Fe 2p at 710.95 eV, 713.53 eV and 724.45 eV, 727.33 eV, respectively. 3 / 2 and Fe 2p 1 / 2 The main binding energy peak, the generation of which is attributed to Fe 3+ The presence of ions. Therefore, it can be considered that the iron core structure in the Holoferritin prepared in the blank control mainly exists in the form of FeOOH and Fe2O3. The Holoferritin prepared in Example 1 has Fe 2p ions at 711.00 eV and 714.46 eV. 3 / 2 The main binding energy peak is Fe. 3+ Multiple fission occurs. Furthermore, Fe exhibits activity at 724.07 eV and 728.26 eV. 3+ Fe 2p generated by spin-orbit coupling 1 / 2 The main binding peak is observed. Additionally, Fe is observed at 719.06 eV. 3+ Satellite peaks. Most importantly, clear Fe peaks can be observed at 708.05 eV and 720.85 eV. 2+ (Fe 2p) 3 / 2 ) and (Fe 2p 1 / 2 The main binding energy peak of the sample confirms the presence of a small amount of divalent iron. Therefore, it can be concluded that the core structure of Holoferritin prepared in Example 1 is doped with a small amount of divalent iron.

[0066] 3. The particle size and potential of the doped Holoferritin nanozymes prepared in Examples 1-3, Comparative Examples 1-3, and the blank control were characterized and analyzed. The specific experimental procedures are as follows:

[0067] Holoferritin nanozymes prepared in the examples and comparative examples, along with a blank control, were characterized for particle size and potential. Holoferritin was diluted to a concentration of 0.5 µM with Tris-HCl buffer (25 mM, pH 8.0), and its particle size distribution and zeta potential were measured using a Malvern particle size analyzer (Zetasizer Nano ZS90, UK). The detector angle was 90°, the laser wavelength was 633 nm, and the equilibration time was set to 2 min. A total of 11 runs were performed, with 3 measurements taken. All samples were measured and analyzed in cuvettes with a path length of 10 mm. The analytical model was the Malvern software's general model (standard resolution), and the zeta potential analysis model was set to the automatic mode of the Malvern software. All values ​​were measured three times at 25 °C.

[0068] The results obtained are as follows Figure 4 , Figure 5 As shown in the results, we can conclude that:

[0069] The particle size distribution of the samples in Examples 1 (HP 5), 2 (HP 2.5), 3 (HP 10), 1 (Holo 5), 2 (Holo 10), 3 (ferritin), and the blank control (HP 0 / Holo 20) all showed a single-peak distribution characteristic, with an average hydrodynamic diameter of about 12 nm, indicating that the protein particles maintained excellent monodispersity in solution.

[0070] Further measurements of the zeta potential (ζ-potential) of the particles revealed that the ζ-potential of the comparative and blank control samples decreased with increasing iron content, while the ζ-potential of the sample in the example group remained essentially unchanged. This indicates that iron ions affect the surface charge environment, while phosphorus doping essentially does not alter the basic structure and charge environment of the protein surface, thus exhibiting ζ-potential stability. Phosphorus doping may lead to the formation of defective structures in the iron nucleus, resulting in disorder and the creation of vacancy structures. This structural transformation reconstructs the active site, significantly lowering the energy barrier of the reaction. Simultaneously, this change in active structure promotes the adsorption and accumulation of oxidase / peroxidase substrates at the enzyme's active site, facilitating enzymatic reactions. Therefore, compared to simply increasing the iron content in the comparative example, phosphorus doping alters the activity regulation strategy.

[0071] Experiment 3: Elemental Content Analysis of Doped Holoferritin Nanozymes

[0072] Elemental analysis was performed on the doped Holoferritin nanozymes prepared in Examples 1-3, and the Holoferritin nanozymes prepared in Comparative Examples 1-3 and the blank control. The specific experimental procedures are as follows:

[0073] Iron and phosphorus contents of Holo (undoped) and Holo-P (phosphorus-doped) samples were determined using an inductively coupled plasma mass spectrometer (ICP-OES / MS; Agilent 5110, USA). Samples (0.5 mL) from Examples 1 (HP 5), 2 (H-P2.5), 3 (HP 10), Comparative Examples 1 (Holo 5), 2 (Holo 10), 3 (ferritin), and the blank control (HP 0 / Holo 20) were mixed with HNO3 (2 mL, 100%) and H2O2 (0.5 mL, 30%), and then digested using a microwave digester under the following conditions: 1800 W, 120 °C, held for 5 min; increased from 120 °C to 150 °C at a rate of 6 °C / min, held for 10 min; then increased to 180 °C at a rate of 6 °C / min, and held for 30 min. The sample was then diluted to 20 mL with ultrapure water. ICP-OES operating conditions were as follows: RF power, 1250 W; pump rate, 60 r / min; auxiliary gas, 12.0 L / min; nebulizer gas, 0.7 L / min. Protein concentration was determined according to the kit specifications. The iron content value (mg / L) obtained from ICP-OES / MS was converted to a molar ratio of protein content (Holo / HP) to iron.

[0074] The results obtained are as follows Figure 6 As shown in the results, we can conclude that:

[0075] Comparative group ( Figure 6 The iron content in samples A and B increased with increasing ferrous sulfate gradient, and the phosphorus content also increased with increasing ferrous sulfate gradient. This result may be due to the co-precipitation of phosphorus and iron during iron core biomineralization. For the example group samples, the phosphorus content gradually increased with increasing phosphate gradient, while the total iron content was suppressed and showed a decreasing trend, and the phosphorus-iron ratio gradually increased. Figure 6 -C, D, E). External phosphorus regulation may lead to phosphorus replacing some iron sites during biomineralization, resulting in a decrease in iron content compared to the control group. This suggests that phosphorus may embed itself within the iron core and coordinate with iron, causing the iron core to form a complex structure with lattice defects. This reconstructs the coordination environment within the iron core, thereby regulating the redox potential of iron ions, lowering the reaction energy barrier, and facilitating substrate adhesion, thus enhancing enzyme activity. Experiment 4: Enzyme Activity Determination of Oxidase-like and Peroxidase-like Activities of Doped Holoferritin Nanozymes

[0076] Holoferritin nanozymes prepared in the examples, comparative examples, and blank controls were analyzed for their oxidase-like and peroxidase-like activities using the classic 3,3′,5,5′-tetramethylbenzidine colorimetric method. The specific experimental procedures are as follows:

[0077] Oxidase activity assay: In the absence of H2O2, catalytic oxidation was performed using 3,3′,5,5′-tetramethylbenzidine (TMB, final concentration 0.75 mM) substrate in 0.2 M acetate-sodium acetate buffer (pH 4.0), with a Holoferritin protein solution concentration of 0.25 μM. Absorbance was measured at 652 nm using a multi-plate reader.

[0078] Peroxidase-like activity assay: In the presence of H₂O₂, catalytic oxidation was performed using 3,3′,5,5′-tetramethylbenzidine (TMB, final concentration 0.75 mM) substrate in 0.2 M acetate-sodium acetate buffer (pH 4.0), with a Holoferritin protein solution concentration of 0.25 μM. Absorbance was measured at 652 nm using a multi-plate reader.

[0079] The results obtained are as follows Figure 7 and Figure 8 As shown in the analysis results, the following can be concluded:

[0080] 1. Analysis of peroxidase activity results:

[0081] The oxidase-like activities of the samples in Comparative Example 1 (Holo 5 group) and Comparative Example 2 (Holo 10 group) were significantly higher than those of the ferritin in Comparative Example 3 (ferritin group) without an added ferrous sulfate mineralized iron core structure. This is mainly attributed to the intact iron core and high iron loading of Holo, which provides more active sites for catalyzing oxide substrates. Furthermore, with the addition of phosphorus, the oxidase-like activities of the sample groups in the Example groups gradually increased, showing a dose-dependent improvement. The enhancement mechanism may mainly stem from the fact that phosphorus doping modulates the iron core structure, optimizes the surface adsorption characteristics of the iron core, reduces the enzyme's affinity for the substrate, and thus enhances the catalytic performance.

[0082] 2. Analysis of peroxidase-like activity results:

[0083] In the comparative group, the peroxidase-like activity of the samples increased with increasing iron content, exhibiting a clear dose-dependent relationship. This suggests that the iron core itself provides the necessary active site for peroxidase activity. In contrast, the peroxidase-like activity of the sample examples was generally higher than that of the comparative group, confirming that phosphorus doping effectively optimizes the microenvironment of the iron core catalytic center. Furthermore, the peroxidase-like activity initially increased and then decreased with increasing phosphorus content, showing a non-linear relationship where the enzyme activity did not increase with increasing phosphorus-iron ratio.

[0084] 3. Phosphorus doping exhibits differentiated regulation of oxidase-like and peroxidase-like activities. For oxidase-like activities, phosphorus doping modulates the iron core structure, and activity increases with increasing phosphorus content. However, for peroxidase-like activities, the catalytic process involves larger molecular substrates. Appropriate phosphorus doping may replace specific sites in the iron core structure, forming phosphorus-iron coordination, thereby regulating the distribution and coordination environment of iron ions and enhancing peroxidase-like activity. However, excessive phosphorus doping may shield certain catalytically active sites, thus inhibiting catalytic activity. Therefore, phosphorus doping forms a specific iron core structure of ferrous iron, which both regulates the iron core and reconstructs the coordination environment within the iron core, thus creating a dual enzyme active center with both oxidase-like and peroxidase-like activities.

[0085] In summary, the above experiments demonstrate that this invention uses Japanese shrimp ferritin as the target gene. A recombinant plasmid was constructed, transformed into *E. coli* host bacteria, and the resulting engineered strain was cultured. Phosphate was added for doping while inducing shrimp ferritin synthesis, inducing phosphorus-regulated iron core structure-ferritin bioexpression in *E. coli*. This resulted in highly dispersed, non-single-activity, nanoscale phosphorus-doped ferritin nanoparticles with both oxidase-like and peroxidase-like activities. Unlike simple iron loading strategies, phosphorus doping essentially does not alter the protein surface structure but forms a specific coordination structure with iron ions, reconstructing the electronic structure and coordination environment of the iron core active center. This significantly lowers the energy barrier of redox reactions, thereby fundamentally enhancing its intrinsic catalytic activity as an oxidase / peroxidase-like enzyme, forming a differentiated regulatory mechanism. This preparation method is mild and convenient, providing a new technical path for developing efficient and stable biomimetic nanozymes.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A doped Holoferritin nanoszyme, characterized in that, The doped Holoferritin nanozyme exhibits peroxidase-like and peroxidase-like activities.

2. The method for biosynthesizing a doped Holoferritin nanozyme according to claim 1, characterized in that, The biosynthesis method includes the following steps: (1) The gene encoding the amino acid sequence of ferritin was cloned into a prokaryotic expression vector to construct a prokaryotic expression vector, and the plasmid vector was transformed into prokaryotic expression cells to obtain a single clone strain; (2) After picking single colonies for expansion culture, IPTG and phosphate solution were added to the culture medium at the same time to induce expression in prokaryotic cells. After induced expression for ≥4 hours, ferrous salt solution was added to continue inducing expression. (3) After a total of 8 to 12 hours of induction expression in prokaryotic cells, the cells were purified to obtain doped Holoferritin nanozymes.

3. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 2, characterized in that, The phosphate in the phosphate solution is either sodium dihydrogen phosphate or disodium hydrogen phosphate, and the ferrous salt in the ferrous salt solution is either ferrous sulfate or ferrous chloride.

4. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 3, characterized in that, The concentration of the phosphate solution is 1-20 mM, and the concentration of the ferrous salt solution is 1-40 mM.

5. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 1, characterized in that, The amino acid sequence of the ferritin is selected from gene sequences encoding ferritin in animals, plants, and microorganisms.

6. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 5, characterized in that, The amino acid sequence of the ferritin is derived from Japanese shrimp.

7. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 1, characterized in that, The specific operation of the purification process is as follows: After induction of expression, the cells were centrifuged to obtain a cell pellet. The cells were then lysed and the supernatant was obtained. Nuclease was added and incubated at 37°C for 30-60 minutes. The volume of nuclease added was 0.005-0.015% of the supernatant. The solution was then heated at 65°C for 20 minutes and the supernatant was obtained again. The protein was purified using a 45% saturated ammonium sulfate solution for 6-8 hours to obtain a high-purity doped Holoferritin nanozyme.

8. The method for biosynthesizing a doped Holoferritin nanozyme as described in claim 1, characterized in that, The expression was induced by the ferrous salt solution for 4 to 8 hours.

9. The application of the Holoferritin nanozyme as described in claim 1 or the doped Holoferritin nanozyme prepared according to any one of claims 2-8 in the field of catalytic detection.

10. The use of the doped Holoferritin nanozyme as described in claim 1 or the doped Holoferritin nanozyme prepared according to any one of claims 2-8 in the preparation of reagents or pharmaceuticals for scavenging peroxide-like substances and / or oxide-like substances.

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