Biological source manganese oxide as well as preparation system and application thereof

Abstract

The invention discloses a biological source manganese oxide material as well as a preparation system and application thereof. The biological source manganese oxide is obtained by oxidizing Mn (II) in vitro by Mn (II) oxidase PomA. The biogenic manganese oxide has a relatively strong degradation effect on five antibiotics such as streptomycin, vancomycin, ciprofloxacin, sulfamethoxazole and oxytetracycline.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology and environmental remediation, and particularly relates to a biogenic manganese oxide as well as a preparation system and application thereof. BACKGROUND

[0002] Manganese oxide refers to a mineral composition formed by oxidation of Mn(II) and having high reactivity, which determines the form, migration and transformation of many substances in the environment and plays an important role in the process of biogeochemical cycle. The manganese oxide produced by biological oxidation of Mn(II) is defined as biogenic manganese oxide (BMO). They are generally formed by bacterial oxidation of Mn(II), such as Leptothrix discophora (SS-1 and SP-6), Pseudomonas putida (MnB1 and GB-1) and Bacillus sp. SG-1, etc. Biogenic manganese oxide has the characteristics of strong adsorption and strong oxidation, and is regarded as a natural, non-secondary pollution, economic and efficient environmental pollutant remediation agent, which has a broader application prospect in treating heavy metal and organic pollutant wastewater than chemically synthesized manganese oxide. Leptothrix discophora Pseudomonas putida The oxidation of Mn(II) by bacteria can be realized through "indirect" and "direct" ways: the indirect action is to adjust the pH value and oxidation-reduction potential of the microenvironment around the cell through metabolic activity, and to release metabolic end products to oxidize Mn(II); the direct action is that the bacteria secrete specific Mn(II) oxidase to the outside of the cell to directly catalyze the oxidation of Mn(II) to higher valence manganese compounds. Among them, some multicopper oxidases (MCOs) combined with Cu(II) coenzyme belong to Mn(II) oxidase, which can catalyze the oxidation of Mn(II) to Mn(III / IV) oxide. However, these multicopper oxidases often only have Mn(II) oxidation ability in the case of living cells, and cannot directly oxidize Mn(II) to form manganese oxide in vitro. Bacillus Previous studies have shown that the crystal structure of biogenic manganese oxide mainly belongs to δ-MnO2, α-Mn2O3, γ-Mn3O4 or amorphous, and no other types of biogenic manganese oxide different from these crystal structures have been found.

[0003] SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art and provides a biogenic manganese oxide as well as a preparation system and application thereof.

[0005] The present application aims to overcome the deficiencies of the prior art and provides a biogenic manganese oxide as well as a preparation system and application thereof.

[0006] ​​In order to achieve the above object, the technical scheme provided by the present application is: The biological manganese oxide is obtained by oxidizing Mn(II) in vitro by a Mn(II) oxidase PomA.

[0007] Preferably, the SEM morphology of the biological manganese oxide is layered.

[0008] More preferably, the XRD main characteristic peaks of the biological manganese oxide are located at 2θ angles of 12.64°, 14.86°, 18.88°, 21.58°, 26.82°, 29.66°, 34.7°, 42.44°, 44.68°, and 60.2°, respectively.

[0009] More preferably, the main component elements of the biological manganese oxide are Mn and O, and the biological manganese oxide mainly contains O-H, Mn-O chemical bonds or functional groups.

[0010] Preferably, the Mn(II) oxidase PomA is derived from a Mn(II) oxidizing bacterium Providencia manganoxydans LLDRA6 (a manganese-oxidizing bacterium strain of Providencia) belongs to the multicopper oxidase family. The manganese-oxidizing bacterium strain of Providencia has been preserved in the China Center for Type Culture Collection in Wuhan, China on December 10, 2018, with a preservation number of CCTCC NO: M 2018876, named as Providencia sp. LLDRA6, and has been protected by a patent.

[0011] Preferably, the biological manganese oxide is prepared by a reaction system of Mn(II) oxidase PomA, MnCl2, CuCl2, and HEPES buffer. In the reaction system, the final concentration of Mn(II) oxidase PomA is 0.03±0.005 mg / mL, the final concentration of MnCl2 is 10±0.5 mmol / L, the final concentration of CuCl2 is 1±0.05 mmol / L, the final concentration of HEPES is 40±5 mmol / L, and the reaction condition is 37℃, 180 rpm shaking culture for 48±5 h.

[0012] More preferably, in the reaction system, the final concentration of Mn(II) oxidase PomA is 0.03 mg / mL, the final concentration of MnCl2 is 10 mmol / L, the final concentration of CuCl2 is 1 mmol / L, the final concentration of HEPES is 40 mmol / L, and the reaction condition is 37℃, 180 rpm shaking culture for 48 h.

[0013] The biological manganese oxide provided by the present application can be used for preparing an antibiotic degrading agent.

[0014] Preferably, the antibiotic is streptomycin, vancomycin, ciprofloxacin, sulfamethoxazole or terramycin. The initial concentration of the antibiotic is 5 mg / L.

[0015] The application will be further described below: The synthesis system of the biological manganese oxide in the application is composed of Mn(II) oxidase PomA, MnCl2, CuCl2 and HEPES buffer. The specific preparation steps are as follows: 20 mL of PomA solution with an initial concentration of 0.075 mg / mL, 20 mL of HEPES buffer with an initial concentration of 100 mmol / L, 5 mL of MnCl2 solution with an initial concentration of 100 mmol / L, and 5 mL of CuCl2 solution with an initial concentration of 10 mmol / L are added to the system respectively, so that the total volume of the reaction system is 50 mL, the final concentration of Mn(II) oxidase PomA is 0.03 mg / mL, the final concentration of MnCl2 is 10 mmol / L, the final concentration of CuCl2 is 1 mmol / L, and the final concentration of HEPES is 40 mmol / L. The culture is incubated at 37℃ and 180 rpm for 48 h.

[0016] The protein and manganese oxide mixture is centrifuged and washed at a speed of 4000 rpm; phenol, chloroform and methanol are added and ultrasonicated at a power of 200 W; then the mixture is centrifuged at 4000 rpm to remove the supernatant and obtain the sediment; the sediment is centrifuged and washed at a speed of 4000 rpm; the pH of the washed sediment is adjusted to 3.0, and the sediment is shaken on a shaking table at 35℃ and 180 rpm for 0.5 h; then the sediment is centrifuged and washed at a speed of 4000 rpm until the pH of the supernatant is neutral; sodium hypochlorite is added to the centrifuged sediment, and the mixture is shaken on a shaking table at 35℃ and 180 rpm for 4 h; the sediment treated with sodium hypochlorite is centrifuged and washed at a speed of 4000 rpm, and then dried at 60℃ to obtain pure biological manganese oxide.

[0017] The SEM morphology of the biological manganese oxide is layered, and the main characteristic peaks of the XRD are located at 2θ angles of 12.64°, 14.86°, 18.88°, 21.58°, 26.82°, 29.66°, 34.7°, 42.44°, 44.68° and 60.2°. After comparison with the PDF-4+2020 database, it is found that the XRD peak type is completely different from the XRD peak type structure of all known types of manganese oxides.

[0018] The Mn(II) oxidase PomA in the application is derived from the Mn(II) oxidizing bacteria Providencia sp.LLDRA6, and the expression and purification steps are as follows: Firstly, total DNA of the strain LLDRA6 was extracted, and a primer was designed to clone the target gene pomA (GenBank accession: PQ362382), and the recombinant vector and the recombinant bacteria were obtained by using the E. coli expression strain BL21 and the expression vector pET32a (+). The crude enzyme solution was obtained by ultrasonic disruption, and the Mn(II) oxidase PomA capable of synthesizing manganese oxide in vitro was separated and purified by using Ni-NTA agarose protein purification resin.

[0019] The biological manganese oxide can be used for preparing an antibiotic degrading agent. When the biological manganese oxide is used as an oxidizing agent, the degradation efficiency of the biological manganese oxide on streptomycin, vancomycin, ciprofloxacin, sulfamethoxazole and oxytetracycline is 52.86%, 57.87%, 56.87%, 64.77% and 57.88%, respectively; and when the biological manganese oxide is used as a catalyst of PMS, the degradation efficiency of the biological manganese oxide on streptomycin, vancomycin, ciprofloxacin, sulfamethoxazole and oxytetracycline is 89.65%, 77.85%, 96.67%, 93.77% and 89.87%, respectively.

[0020] The biological manganese oxide is synthesized in vitro by using the Mn(II) oxidase PomA, and the synthesized manganese oxide has a wide application prospect in catalyzing antibiotic degradation and other environmental remediation. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The preparation system diagram of the biological manganese oxide is shown in (a) reaction 0h, (b) reaction 48h and (c) LBB staining. Figure 2 The actual object diagram of the biological manganese oxide is shown in (a) reaction 0h, (b) reaction 48h and (c) LBB staining. Figure 3 The SEM characterization diagram of the biological manganese oxide is shown in (a) reaction 0h, (b) reaction 48h and (c) LBB staining. Figure 4 The characterization diagram of the biological manganese oxide is shown in (a) XRD, (b) XPS and (c) FTIR. Figure 5 (a-e): the effect of the biological manganese oxide (BMO) on degradation of different antibiotics: (a) streptomycin (ST), (b) vancomycin (VH), (c) ciprofloxacin (CIP), (d) sulfamethoxazole (SMX) and (e) oxytetracycline (OTC); (f-j): the effect of the biological manganese oxide (BMO) / PMS on degradation of different antibiotics: (f) streptomycin (ST), (g) vancomycin (VH), (h) ciprofloxacin (CIP), (i) sulfamethoxazole (SMX) and (j) oxytetracycline (OTC). DETAILED DESCRIPTION

[0022] I. Preparation method of Mn(II) oxidase.

[0023] 1. Inducible expression of Mn(II) oxidase: Recombinant Escherichia coli BL21-pET- pomA Inoculated into LB liquid medium and incubated at 37°C and 170 rpm on a shaker until OD. 600 When the pH reached approximately 0.6, 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce a large expression of the PomA protein. After adding IPTG, the cells were cultured at 16°C and 150 rpm for 16 h. The cells were collected by centrifugation at 4°C and 10,000 rpm for 10 min, resuspended in PBS buffer (pH=7), and sonicated using a cell disruptor (2 s on, 3 s off, sonication for 10 min) to obtain cell lysates. Cell debris was removed by centrifugation at 4°C and 10,000 rpm for 20 min. The resulting supernatant was the crude enzyme solution containing a large amount of the target protein.

[0024] 2. Purification and preparation of Mn(II) oxidase: The protein was purified using a Ni-NTA agarose resin purification kit. The crude enzyme solution was filtered through a 0.4 μm filter and set aside for later use. The specific purification steps are as follows: ① Allow the storage buffer in the Ni-NTA resin to flow out by gravity. Equilibrate the Ni-NTA resin with 5-6 column volumes of ultrapure water, and then equilibrate it with 5-6 column volumes of binding buffer (PBS buffer containing 20 mM imidazole). Use a flow rate of 0.5-1 mL / min to slowly drain the buffer from the resin.

[0025] ② Prepare the sample solution by mixing the filtered crude enzyme solution with the binding buffer at a 1:1 ratio, ensuring the total volume of the sample solution is twice the column volume. Add the sample solution to the column and collect the flow-through into a centrifuge tube. If there is excess sample, it can be loaded again; reflowing can improve the binding force between the sample and the packing material.

[0026] ③ Wash the column with two column volumes of binding buffer (PBS buffer containing 20 mM imidazole) and elution buffer (PBS buffer containing 100 mM imidazole), and collect the flow-through. Test the absorbance of the flow-through at 280 nm. Repeat the steps with a new collection tube each time the buffer is changed, until the absorbance of the flow-through at 280 nm is close to the baseline, indicating that contaminating proteins have been thoroughly removed.

[0027] IV. Elution of the target protein with the elution buffer (PBS buffer containing 125 mM imidazole) until the absorbance at 280 nm of the eluate approaches the baseline.

[0028] V. Post-column treatment: the column material is washed with 5 volumes of elution buffer (PBS buffer containing 200 mM imidazole), then equilibrated with 5 volumes of binding buffer (PBS buffer containing 20 mM imidazole), and finally washed with 5 volumes of deionized water, and then 20% ethanol is added as a protective solution, and the column material is stored at 2-8°C.

[0029] VI. The purity of the target protein is detected by SDS-PAGE, and the desired PomA protein solution is obtained.

[0030] 3. Concentration determination of Mn(II) oxidase: The concentration of the obtained enzyme solution is determined by using a Bradford protein concentration determination kit, and the specific determination steps are as follows: I. Take 80 μL of BSA (bovine serum albumin) standard protein solution (5 mg / mL) and add it to 1920 μL of PBS buffer, so that the final concentration of BSA is 0.2 mg / mL.

[0031] II. Standard group preparation: take 16 1.5 mL centrifuge tubes, and add each solution according to the proportions in the following table, and set 1 replicate for each standard (Table 1); Table 1

[0032] III. Sample group preparation: dilute the enzyme solution 10 times with PBS buffer for standby. Take 4 1.5 mL centrifuge tubes, and divide them into 2 replicate groups, and the replicate centrifuge tubes have the same number, and the two tubes with different numbers are added with 100 μL of the original protein solution and the protein diluent, respectively.

[0033] IV. Add 1 mL of Bradford working solution to each tube and mix quickly. After reaction for 5 min at room temperature 25-30°C, take the A595 value of each tube on the spectrophotometer with No. 0 tube as the blank control. Draw the standard curve on the coordinate paper or in the Microsoft Excel software, with the average A595 value of each tube in the standard group as the ordinate and the corresponding protein concentration as the abscissa. According to the average A595 value of the two same sample diluents, the protein concentration of the sample after dilution is calculated on the standard curve. Select the appropriate dilution of the sample to calculate the final sample protein concentration, and then calculate the original sample protein concentration from the dilution factor.

[0034] V. Dilute the protein solution to the desired concentration for the experiment with PBS buffer.

[0035] II. Preparation and identification of biological manganese oxides.

[0036] 1. Preparation of biological manganese oxides: The reaction system was set to 50 mL, and 20 mL of PomA solution with an initial concentration of 0.075 mg / mL, 20 mL of HEPES buffer with an initial concentration of 100 mmol / L, 5 mL of MnCl2 solution with an initial concentration of 100 mmol / L, and 5 mL of CuCl2 solution with an initial concentration of 10 mmol / L were added to the system, respectively, so that the total volume of the reaction system was 50 mL, the final concentration of Mn(II) oxidase PomA was 0.03 mg / mL, the final concentration of MnCl2 was 10 mmol / L, the final concentration of CuCl2 was 1 mmol / L, and the final concentration of HEPES was 40 mmol / L. The culture was incubated at 37°C and 180 rpm for 48 h.

[0037] 2. Extraction of biological manganese oxides: ① The protein / manganese oxide mixture was collected in a 50 mL centrifuge tube and centrifuged at 4000 rpm and room temperature for 10 min. After discarding the supernatant, the sediment was washed and centrifuged with deionized water. This step was repeated until the supernatant was clear, and the sediment was retained.

[0038] ② 5 mL of phenol was added to the sediment, and after ultrasonic treatment for 60 min, the sediment was centrifuged according to the conditions of step ①, and the supernatant was discarded.

[0039] ③ 5 mL of a mixture of phenol and chloroform with a volume ratio of 1:1 was added to the sediment, and after ultrasonic treatment for 30 min, the sediment was centrifuged according to the conditions of step ①, and the supernatant was discarded.

[0040] ④ 20 mL of a mixed solution of methanol, chloroform, and deionized water (volume ratio of methanol, chloroform, and deionized water is 12:3:5) was added, and ultrasonic treatment was performed again for 30 min. The supernatant was removed by centrifugation, and the sediment was washed with deionized water as in step ①.

[0041] ⑤ The sediment was resuspended by adding a previously prepared HCl solution, and the suspension in the 50 mL centrifuge tube was acidified to pH 3.0. The suspension was shaken for 30 min on a shaker. The supernatant was removed by centrifugation, and the sediment was washed with deionized water as in step ① until the supernatant was neutral.

[0042] ⑥ NaClO was added, and the suspension was shaken on a shaker for 4 h. The supernatant was removed by centrifugation, and the sediment was washed with deionized water as in step ①.

[0043] ⑦ The sediment was dried at 60°C to obtain biological manganese oxides, which were stored at room temperature for future use.

[0044] 3. Characterization and identification of biological manganese oxides: A small amount of purified and dried bio-manganese oxide particle powder was weighed and directly adhered to conductive glue. After vacuum spraying of gold powder, the surface morphology structure of the manganese oxide was observed by scanning electron microscopy.

[0045] After tabletting of the manganese oxide powder, the purified manganese oxide sample was tested using a Bruker D8 advance X-ray diffractometer (Cu target, Kα ray source, λ = 0.154 nm, Ni filter). The test conditions were as follows: 2θ scanning range of 10°-80°, scanning speed of 2° / min, electron generation current of 40 mA, and acceleration voltage of 40 kV. ° ° °

[0046] III. Degradation of antibiotics by bio-manganese oxide 1. Degradation of typical antibiotics by bio-manganese oxide itself: The prepared bio-manganese oxide was added to a 100 mL conical flask containing 50 mL of 10 mmol / L NaCl solution, and streptomycin (ST), vancomycin (VH), ciprofloxacin (CIP), sulfamethoxazole (SMX), and oxytetracycline (OTC) were added, respectively, so that the final concentration of the antibiotics was 5 mg / L and the final concentration of the bio-manganese oxide was 0.5 g / L. The reaction flask was placed in a constant-temperature shaker in the dark, and incubated at a temperature of 35°C and a rotation speed of 180 r / min. Samples were taken every 5 min on a clean bench, and treated by membrane filtration, i.e., the sample was filtered with a 0.22 μm organic filter membrane, and the remaining antibiotic concentration was determined by HPLC. Three replicates were set for each reaction.

[0047] 2. Degradation of typical antibiotics by bio-manganese oxide advanced oxidation system: The prepared mother liquor solution with a concentration of 0.5 g / L of each antibiotic was added to a 100 mL conical flask containing 50 mL of 10 mmol / L NaCl solution, so that the final concentration of the antibiotic was 5 mg / L. 0.025 g of purified and dried bio-manganese oxide was added to each antibiotic solution, so that the final concentration of the bio-manganese oxide was 0.5 g / L. Then, 0.1 mol / L of an oxidizing agent (persulfate, PMS) was added, so that the final concentration of the oxidizing agent was 1 mmol / L, and the degradation reaction was carried out. PMS oxidizing agent solution alone was used as a blank control, and three replicates were set for each experimental group. The degradation reaction was carried out in the dark at 35°C and a rotation speed of 180 rpm, and samples were taken every 5 min on a clean bench and treated by membrane filtration, i.e., the sample was filtered with a 0.22 μm organic filter membrane, and the remaining antibiotic concentration was determined by HPLC. Three replicates were set for each reaction. ​​​

[0048] IV. Results and analysis As shown in Figure 1 (a), the freshly prepared PomA / Mn(II) reaction solution was light blue. After 48 h of reaction at 37 °C with 180 rpm shaking, the PomA / Mn(II) reaction solution was brown-black (b), indicating the possible formation of manganese oxides. Further LBB staining of the reaction solution showed that it was blue (c), indicating that Mn(II) oxidation indeed occurred. As can be seen from Figure 1 , the appearance of the biogenic manganese oxide was brown and powdery, and the hardness was low. Through SEM electron scanning (d), it was found that, in terms of microstructure, the biogenic manganese oxide was stacked by irregular flaky structures, and the surface was smooth, with a flaky structure size of about 500 nm x 500 nm. Figure 1 Figure 2 Figure 3 From the XRD (X-ray diffraction) results of (a), it can be seen that the biogenic manganese oxide has high crystallinity, and there are many characteristic diffraction peaks, with 2θ angles of 12.64°, 14.86°, 18.88°, 21.58°, 26.82°, 29.66°, 34.7°, 42.44°, 44.68°, and 60.2°. However, after comparison with the PDF-4+2020 database, it was found that these characteristic peaks could not be matched with any manganese oxide material in the standard card library, indicating that the biogenic manganese oxide is a new type of manganese oxide. XPS full spectrum (b) showed that the material contains elements such as Mn, O, P, C, and N, but the main elements are Mn and O. From the FTIR spectrum (Fig. (4c)), it can be seen that the material mainly contains O-H, Mn-O, and other chemical bonds or functional groups. In summary, the material is a new type of biogenic manganese oxide with good crystallinity.

[0049] Figure 4 Figure 4 The results of the biogenic manganese oxide (BMO) itself degrading typical antibiotics are shown in

[0050] Figure 5 ​​​​(a-e) are shown. The biogenic manganese oxide (BMO) has degradation effect on various antibiotics. The biogenic manganese oxide (BMO) has a faster degradation rate of antibiotics within 15 min, and the degradation rate gradually decreases with the extension of reaction time. At 25 min, the degradation reaction of antibiotics gradually tends to equilibrium. Finally, at 30 min, the degradation efficiency of the biogenic manganese oxide (BMO) on streptomycin (ST) is 52.86%, vancomycin (VH) is 57.87%, ciprofloxacin (CIP) is 56.87%, sulfamethoxazole (SMX) is 64.77%, and oxytetracycline (OTC) is 57.88%. In comparison, the commercial manganese oxides (chemical sources), including MnO, Mn2O3, MnO2and Mn3O4, have little degradation effect on these antibiotics.

[0051] The results of the degradation of typical antibiotics by the advanced oxidation system composed of the biogenic manganese oxide (BMO) and PMS are shown in Figure 5 (f-j) are shown. When the biogenic manganese oxide (BMO) is used as a catalyst and PMS is used as an oxidant, the advanced oxidation system has a faster degradation rate of antibiotics within 10 min, and the degradation rate gradually decreases with the extension of reaction time. At 15 min, the degradation reaction of antibiotics gradually tends to equilibrium. Finally, at 30 min, the degradation efficiency of the system on streptomycin is 89.65%, vancomycin is 77.85%, ciprofloxacin is 96.67%, sulfamethoxazole is 93.77%, and oxytetracycline is 89.87%. In comparison, the advanced oxidation system composed of the commercial manganese oxides (chemical sources), including MnO, Mn2O3, MnO2and Mn3O4, and PMS has much lower degradation ability on these antibiotics than the advanced oxidation system composed of the biogenic manganese oxide (BMO) and PMS.

Claims

1. A biogenic manganese oxide characterized in that, The biological manganese oxide is obtained by oxidizing Mn(II) in vitro by Mn(II) oxidase PomA.

2. The biogenic manganese oxide of claim 1, wherein, The SEM morphology of the biological manganese oxide is layered.

3. The biogenic manganese oxide of claim 2, wherein, The XRD main characteristic peaks of the biological manganese oxide are located at 2θ angles of 12.64°, 14.86°, 18.88°, 21.58°, 26.82°, 29.66°, 34.7°, 42.44°, 44.68°, and 60.2°.

4. The biogenic manganese oxide of claim 3, wherein, The main component elements of the biological manganese oxide are Mn and O, and the biological manganese oxide mainly contains O-H, Mn-O chemical bonds or functional groups.

5. The biogenic manganese oxide of claim 1, wherein, The Mn(II) oxidizing enzyme PomA is derived from a Mn(II) oxidizing bacterium Providencia LLDRA6, belonging to the multicopper oxidase family.

6. The biogenic manganese oxide according to any one of claims 1 to 5, characterized in that The biological manganese oxide is prepared by the following reaction system: Mn(II) oxidase PomA, MnCl2, CuCl2, and HEPES buffer; the final concentration of Mn(II) oxidase PomA in the reaction system is 0.03±0.005 mg / mL, the final concentration of MnCl2 is 10±0.5 mmol / L, the final concentration of CuCl2 is 1±0.05 mmol / L, the final concentration of HEPES is 40±5 mmol / L, and the reaction condition is 37℃, 180 rpm shaking culture for 48±5 h.

7. The biogenic manganese oxide of claim 6, wherein, The final concentration of Mn(II) oxidase PomA in the reaction system is 0.03 mg / mL, the final concentration of MnCl2 is 10 mmol / L, the final concentration of CuCl2 is 1 mmol / L, the final concentration of HEPES is 40 mmol / L, and the reaction condition is 37℃, 180 rpm shaking culture for 48 h.

8. Use of the biological manganese oxide of claim 6 in the preparation of an antibiotic degradation agent.

9. Use according to claim 8, wherein the compound is ###0002### The antibiotic is streptomycin, vancomycin, ciprofloxacin, sulfamethoxazole, or terramycin.

10. A reaction system for the preparation of a biogenic manganese oxide according to any one of claims 1 to 5, characterized in that The reaction system is composed of Mn(II) oxidase PomA, MnCl2, CuCl2, and HEPES buffer; the final concentration of Mn(II) oxidase PomA in the reaction system is 0.03±0.005 mg / mL, the final concentration of MnCl2 is 10±0.5 mmol / L, the final concentration of CuCl2 is 1±0.05 mmol / L, the final concentration of HEPES is 40±5 mmol / L, and the reaction condition is 37℃, 180 rpm shaking culture for 48±5 h.

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