Porphyromonas gingivalis derived single atom nanoszyme, and preparation method and application thereof

Abstract

This invention provides a *Porphyromonas gingivalis*-derived single-atom nanozyme, its preparation method, and its application, belonging to the field of nanosynthesis technology. The preparation method of the *Porphyromonas gingivalis*-derived single-atom nanozyme provided by this invention includes the following steps: enriching and culturing *Porphyromonas gingivalis* in a heme-containing BHI medium to the logarithmic growth phase, centrifuging and drying to obtain *Porphyromonas gingivalis*; carbonizing the *Porphyromonas gingivalis* to obtain the *Porphyromonas gingivalis*-derived single-atom nanozyme; the carbonization temperature is 500-1000℃, and the time is 1-5 hours. The carbon structure formed by *Porphyromonas gingivalis* during the carbonization process provides an anchoring site for the iron element in heme, and the prepared iron-based single-atom nanozyme exhibits excellent peroxidase-like catalytic activity and good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of nanosynthesis technology, specifically relating to a single-atom nanozyme derived from Porphyromonas gingivalis, its preparation method, and its application. Background Technology

[0002] Single-atom nanozymes, as a class of artificial enzymes combining the properties of nanomaterials with the catalytic activity of natural enzymes, achieve 100% atomic utilization by anchoring metal atoms in an isolated form on the surface of a support, fundamentally solving the core problems of insufficient atomic utilization and low catalytic efficiency in traditional nanozymes. Despite the enormous potential of single-atom nanozymes in terms of catalytic activity, their development still faces key challenges. Due to the extremely high surface free energy of single atoms, metal species are prone to migration and aggregation to form nanoparticles, leading to a sharp drop in the density of single-atom active sites. However, the complexity and high cost of current preparation processes make large-scale preparation difficult. Therefore, developing a simple and cost-effective synthetic strategy to achieve atomically dispersed active sites remains a significant challenge. Summary of the Invention

[0003] The purpose of this invention is to provide a single-atom nanozyme derived from *Porphyromonas gingivalis*, its preparation method, and its application. This invention uses *Porphyromonas gingivalis* as a template, and the carbon structure formed by the natural macromolecules within it during high-temperature carbonization provides anchoring sites for the iron element in heme. Finally, an atomically dispersed iron-based single-atom nanozyme is prepared, which has excellent peroxidase-like catalytic activity and good biosafety.

[0004] To achieve the objectives of this invention, the following technical solutions are provided: A method for preparing a single-atom nanozyme derived from *Porphyromonas gingivalis* includes the following steps: Porphyromonas gingivalis was enriched and cultured in BHI medium containing heme to the logarithmic phase, then separated by centrifugation and dried to obtain Porphyromonas gingivalis. The *Porphyromonas gingivalis* was carbonized to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes; the carbonization temperature was 500-1000℃ and the time was 1-5 hours.

[0005] Preferably, the BHI culture medium contains 5-25 μg / mL of heme.

[0006] Preferably, the *Porphyromonas gingivalis* includes one or more of strains BNCC 353909, BNCC 236547, BNCC 337441, and BNCC 336950.

[0007] Preferably, the centrifugation speed is 10,000 rpm and the time is 10 min.

[0008] Preferably, the freeze-drying temperature is -40 to -80°C and the time is 24 to 48 hours.

[0009] The present invention also provides a single-atom nanozyme derived from *Porphyromonas gingivalis* prepared by the preparation method described above, wherein the single-atom nanozyme is an iron-based single-atom nanozyme.

[0010] Preferably, the single-atom nanozyme has a micron-scale morphology.

[0011] Preferably, the single-atom nanozyme has an Fe-N4 active site.

[0012] The present invention also provides the application of the *Porphyromonas gingivalis*-derived single-atom nanozyme described in the above-described technical solution in the preparation of enzyme-catalyzed products.

[0013] Preferably, the enzyme catalytic activity includes peroxidase-like POD catalytic activity, catalase-like CAT catalytic activity, or superoxide dismutase-like SOD catalytic activity.

[0014] This invention provides a method for preparing *Porphyromonas gingivalis*-derived single-atom nanozymes, comprising the following steps: enriching and culturing *Porphyromonas gingivalis* in heme-containing BHI medium to the logarithmic growth phase, centrifuging and drying to obtain *Porphyromonas gingivalis*; carbonizing the *Porphyromonas gingivalis* to obtain the *Porphyromonas gingivalis*-derived single-atom nanozymes; the carbonization temperature is 500-1000℃, and the time is 1-5 h. This invention uses *Porphyromonas gingivalis* as a raw material to synthesize single-atom nanozymes (PFMs) with Fe-N4 active sites through high-temperature carbonization. Microorganisms, as a renewable and high-quality carbon source, can provide sufficient precursors for the synthesis of carbon-based carriers of single-atom nanozymes through simple pretreatment, thereby achieving efficient and scalable gram-scale synthesis. Furthermore, the topological structure of natural macromolecules within microorganisms provides an ideal framework for anchoring metal single-atom sites, preventing the migration and aggregation of metal species during synthesis. Compared to other bacteria, *Porphyromonas gingivalis* lacks an endogenous pathway for iron porphyrin synthesis and can only meet its iron and protoporphyrin IX requirements by taking up exogenous heme. This provides a basis for the metal sites of its derived single-atom nanozymes. Therefore, *Porphyromonas gingivalis* exhibits the advantage of cost-effective large-scale synthesis of iron-based single-atom nanozymes. This invention not only provides new ideas for the rational design and large-scale synthesis of microbially derived single-atom nanozymes but also offers a new strategy for the preparation of biomaterials with good biosafety. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a SEM image of the single-atom nanozyme prepared in this invention; Figure 2 TEM image of the single-atom nanozyme prepared in this invention; Figure 3 The XRD pattern of the single-atom nanozyme prepared in this invention; Figure 4 XPS image of the single-atom nanozyme prepared in this invention; Figure 5 XAFS image of the single-atom nanozyme prepared in this invention; Figure 6 This is a diagram showing the POD-like catalytic activity of the single-atom nanozyme prepared in this invention; Figure 7 This is a comparison of the POD catalytic activity of single-atom nanozymes prepared at different temperatures according to the present invention; Figure 8 This is a cell MTT assay diagram of the single-atom nanozyme prepared in this invention. Figure 9 This is a cell live / dead staining experiment diagram of the single-atom nanozyme prepared in this invention. Detailed Implementation

[0017] This invention provides a method for preparing a single-atom nanozyme derived from *Porphyromonas gingivalis*, comprising the following steps: Porphyromonas gingivalis was enriched and cultured in BHI medium containing heme to the logarithmic phase, then separated by centrifugation and dried to obtain Porphyromonas gingivalis. The *Porphyromonas gingivalis* was carbonized to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes; the carbonization temperature was 500-1000℃ and the time was 1-5 hours.

[0018] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0019] In this invention, the BHI culture medium contains 5-25 μg / mL of heme, and in specific embodiments it can be 10 or 15 μg / mL.

[0020] In this invention, the *Porphyromonas gingivalis* includes one or more of strains BNCC 353909, BNCC 236547, BNCC 337441, and BNCC 336950.

[0021] In this invention, the centrifugation speed is 10,000 rpm and the time is 10 min.

[0022] In this invention, the freeze-drying temperature is -40 to -80°C, and in a specific embodiment it can be -60 or -50°C; the time is 24 to 48 hours, and in a specific embodiment it can be 36 hours.

[0023] In this invention, the carbonization is carried out in an inert argon atmosphere; the carbonization process further includes water washing and freeze-drying. This invention does not impose any particular limitations on the conditions for water washing and freeze-drying.

[0024] The present invention also provides a single-atom nanozyme derived from *Porphyromonas gingivalis* prepared by the preparation method described above, wherein the single-atom nanozyme is an iron-based single-atom nanozyme.

[0025] In this invention, the single-atom nanozyme has a micron-scale morphology.

[0026] In this invention, the single-atom nanozyme has an Fe-N4 active site.

[0027] The present invention also provides the application of the *Porphyromonas gingivalis*-derived single-atom nanozyme described in the above-described technical solution in the preparation of products with multi-enzyme catalytic activity.

[0028] In this invention, the enzyme catalytic activity includes peroxidase-like POD catalytic activity, catalase-like CAT catalytic activity, or superoxide dismutase-like SOD catalytic activity.

[0029] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the *Porphyromonas gingivalis*-derived single-atom nanozyme, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Under anaerobic conditions at 37°C, *Porphyromonas gingivalis* (BNCC 353909) was enriched and cultured in brain heart broth (BHI) containing 25 μg / mL heme. After the bacteria reached the logarithmic growth phase, well-grown bacteria were collected from the BHI medium by centrifugation at 10,000 rpm and then freeze-dried in a vacuum freeze dryer. Subsequently, the freeze-dried bacterial powder was transferred to a magnetic boat and placed in a tube furnace, where it was carbonized at 900°C for 2 hours under an argon atmosphere (Ar). After natural cooling, the resulting black powder was collected, washed three times repeatedly with deionized water, and then freeze-dried to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes (PFMs).

[0031] Example 2 Under anaerobic conditions at 37°C, *Porphyromonas gingivalis* (BNCC 236547) was enriched and cultured in brain heart broth (BHI) containing 25 μg / mL heme. After the bacteria reached the logarithmic growth phase, well-grown bacteria were collected from the BHI medium by centrifugation at 10,000 rpm and then freeze-dried in a vacuum freeze dryer. Subsequently, the freeze-dried bacterial powder was transferred to a magnetic boat and placed in a tube furnace for carbonization at 800°C for 2 hours under an argon atmosphere (Ar). After natural cooling, the resulting black powder was collected, washed three times repeatedly with deionized water, and then freeze-dried to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes (PFMs).

[0032] Example 3 Under anaerobic conditions at 37°C, *Porphyromonas gingivalis* (BNCC 337441) was enriched and cultured in brain heart broth (BHI) containing 25 μg / mL heme. After the bacteria reached the logarithmic growth phase, well-grown bacteria were collected from the BHI medium by centrifugation at 10,000 rpm and then freeze-dried in a vacuum freeze dryer. Subsequently, the freeze-dried bacterial powder was transferred to a magnetic boat and placed in a tube furnace for carbonization at 700°C for 2 hours under an argon atmosphere (Ar). After natural cooling, the resulting black powder was collected, washed three times repeatedly with deionized water, and then freeze-dried to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes (PFMs).

[0033] Example 4 Under anaerobic conditions at 37°C, *Porphyromonas gingivalis* (BNCC 336950) was enriched and cultured in brain heart broth (BHI) containing 25 μg / mL heme. After the bacteria reached the logarithmic growth phase, well-grown bacteria were collected from the BHI medium by centrifugation at 10,000 rpm and freeze-dried in a vacuum freeze dryer. Subsequently, the freeze-dried bacterial powder was transferred to a magnetic boat and placed in a tube furnace for carbonization at 1000°C for 2 hours under an argon atmosphere (Ar). After natural cooling, the resulting black powder was collected, washed three times repeatedly with deionized water, and freeze-dried to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes (PFMs).

[0034] Test Example 1 The single-atom nanozymes PFMs prepared in Example 1 were observed using scanning electron microscopy, and the results are as follows: Figure 1 As shown. From Figure 1 As can be seen, single-atom nanozymes PFMs exhibit a micron-scale morphology.

[0035] The single-atom nanozymes PFMs prepared in Example 1 were observed using transmission electron microscopy, and the results are as follows: Figure 2 As shown, single-atom nanozymes PFMs contain elements such as C, N, O, S, P, and Fe.

[0036] The single-atom nanozymes PFMs prepared in Example 1 were analyzed using X-ray diffraction, and the results are as follows: Figure 3 As shown. From Figure 3 The results show that the single-atom nanozymes PFMs exhibit broad diffraction at 24.5° and 44.5°, but no diffraction peaks related to elemental iron and iron oxides are observed, indicating that the material has a high degree of graphitization and that no iron aggregation or oxidation has occurred.

[0037] The single-atom nanozymes PFMs prepared in Example 1 were analyzed using X-ray photoelectron spectroscopy, and the results are as follows: Figure 4 As shown. From Figure 4 It can be seen from the data that the synthesized single-atom nanozymes PFMs are mainly composed of elements such as C, N, O, S, P, and Fe. The fine spectrum of Fe further proves the successful anchoring of Fe.

[0038] The single-atom nanozymes PFMs prepared in Example 1 were analyzed using X-ray absorption fine structure spectroscopy, and the results are as follows: Figure 5 As shown. From Figure 5 It can be seen that the single-atom nanozymes PFMs have Fe-N4 active sites similar to phthalocyanine iron FePc, but no Fe-Fe active sites similar to iron foil were found, which proves the successful construction of single-atom active sites.

[0039] Test Example 2 The catalytic activity of PFMs-like single-atom nanozymes prepared in Example 1: POD enzymes catalyze the production of reactive oxygen species (ROS) from H₂O₂. These ROS oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue ox-TMB, with a maximum UV-Vis absorption spectrum at 652 nm. This test example uses a TMB-assisted colorimetric reaction to evaluate the POD-like catalytic activity of PDMs.

[0040] First, an acidic phosphate buffer solution (20 mM, pH=3) containing TMB (0.5 mM) was prepared as control group 1. Then, H2O2 (5 mM) and PFMs (25 μg / mL) were added to the above solution to create the H2O2 group (control group 2) and PFMs group (control group 3), respectively. The experimental group solutions consisted of acidic phosphate buffer solutions containing the above concentrations of TMB, H2O2, and PFMs. After thorough mixing, the solutions were incubated for 10 min, and the color change of the mixture was observed visually, with the UV-Vis absorption spectrum (500–800 nm) recorded.

[0041] The results are as follows Figure 6 As shown, PFMs exhibit good POD-like catalytic activity under acidic conditions. Furthermore, to compare the POD-like catalytic activity of single-atom nanozymes PFMs prepared at different temperatures, the enzyme activity of PFMs synthesized at 500℃, 600℃, 700℃, 800℃, 900℃, and 1000℃ was verified using the above-mentioned reagent concentrations. The results are as follows... Figure 7 As shown, single-atom nanozymes PFMs synthesized at different temperatures all exhibit good POD-like catalytic activity, with the best activity observed at 900℃.

[0042] Test Example 3 Example 1: MTT assay of single-atom nanozymes PFMs prepared in cells: Mouse fibroblasts (L929 cells) were seeded in 96-well plates and incubated overnight at a density of 2 × 10⁶ cells per well. 4 Cells were cultured overnight until adherence. Different concentrations of PFMs (0–100 μg / mL) were prepared and co-cultured with the cells for 12 h. Then, 10 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 h. After incubation, the culture medium was aspirated from all wells, and the precipitated formazan crystals were dissolved in 100 μL of DMSO. After complete dissolution, the absorbance of each well at 490 nm was measured using a multi-functional microplate reader. The results are shown below. Figure 8 As shown in the figure. The results indicate that even at a material concentration as high as 100 μg / mL, there was no significant effect on cell viability.

[0043] Test Example 4 Single-atom nanozymes (PFMs) prepared in Example 1: Cell live / dead staining: L929 cells were seeded in 6-well plates and incubated overnight at a density of 1 × 10⁶ cells per well. 6 Cells were cultured overnight until adherence. Different concentrations of PFMs (0–100 μg / mL) were prepared and co-cultured with the cells for 12 h. Then, the prepared Calcein-AM / propidium iodide PI working solution was added to each well and incubated in the dark for 30 min. After incubation, the cells were washed three times with physiological saline, and the morphology of the stained cells was photographed using a fluorescence microscope. Cell viability and mortality staining results are shown below. Figure 9 As shown, even at a material concentration as high as 100 μg / mL, no significant changes in cell morphology were observed.

[0044] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a single-atom nanozyme derived from *Porphyromonas gingivalis*, characterized in that, Includes the following steps: Porphyromonas gingivalis was enriched and cultured in BHI medium containing heme to the logarithmic phase, then separated by centrifugation and dried to obtain Porphyromonas gingivalis. The *Porphyromonas gingivalis* was carbonized to obtain *Porphyromonas gingivalis*-derived single-atom nanozymes; the carbonization temperature was 500-1000℃ and the time was 1-5 hours.

2. The preparation method according to claim 1, characterized in that, The BHI culture medium contains 5-25 μg / mL of heme.

3. The preparation method according to claim 1, characterized in that, The *Porphyromonas gingivalis* includes one or more of the following strains: BNCC353909, BNCC 236547, BNCC 337441, and BNCC 336950.

4. The preparation method according to claim 1, characterized in that, The centrifugation speed was 10,000 rpm and the time was 10 min.

5. The preparation method according to claim 1, characterized in that, The freeze-drying temperature is -40~-80℃, and the time is 24~48h.

6. The *Porphyromonas gingivalis*-derived single-atom nanozyme prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The single-atom nanozyme is an iron-based single-atom nanozyme.

7. The *Porphyromonas gingivalis*-derived single-atom nanozyme according to claim 6, characterized in that, The single-atom nanozyme has a micron-scale morphology.

8. The *Porphyromonas gingivalis*-derived single-atom nanozyme according to claim 6, characterized in that, The single-atom nanozyme has an Fe-N4 active site.

9. The use of the *Porphyromonas gingivalis*-derived single-atom nanozyme according to any one of claims 6 to 8 in the preparation of enzyme-catalyzed products.

10. The application according to claim 9, characterized in that, The enzyme catalytic activity includes peroxidase-like POD catalytic activity, catalase-like CAT catalytic activity, or superoxide dismutase-like SOD catalytic activity.

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