Natural nitrogenase-like FeMo nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a natural nitrogenase-like FeMo nano-enzyme and a preparation method and application thereof, and relates to the technical field of nano-enzymes and agriculture, the method comprises the following steps: respectively dissolving 1, 3, 4-thiadiazole-2, 5-diamine, molybdenum pentachloride, ferric chloride and 2, 5-thiophenedicarboxylic acid in absolute ethyl alcohol; the preparation method comprises the following steps: adding a molybdenum pentachloride solution and a ferric chloride solution into a 1, 3, 4-thiadiazole-2, 5-diamine solution, and stirring until the solution is yellowish-brown to obtain a FeMo nano-enzyme mixed solution; and adding a 2, 5-thiophenedicarboxylic acid solution into the FeMo nano-enzyme mixed solution, stirring, and sequentially centrifuging, drying and calcining to obtain the natural nitrogenase-like FeMo nano-enzyme. The FeMo nano-enzyme has nitrogen fixation activity similar to that of natural nitrogenase, nitrogen and water can generate ammonia at normal temperature and normal pressure, and the technical problem that in-situ generation of ammonia in plants at normal temperature and normal pressure is difficult in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the field of nanozymes and agricultural technology, and in particular to a natural nitrogenase-like FeMo nanozyme and a preparation method and application thereof. Background Art

[0002] Nitrogen is a key nutrient for living organisms. Many important biochemical components such as nucleic acids, proteins, chlorophyll and hemoglobin contain nitrogen. In plants, nitrogen is a macronutrient second only to carbon, hydrogen and oxygen. Although nitrogen is abundant in the atmosphere, nitrogen cannot be directly absorbed and utilized by organisms due to its highly stable nitrogen-nitrogen triple covalent bond. It needs to be converted into ammonia or nitrate before being utilized. This nitrogen fixation process can be achieved through natural nitrogen fixation or artificial nitrogen fixation. However, in most natural and agricultural ecosystems, the nitrogen content is insufficient and inorganic nitrogen fertilizers need to be applied to increase yields. Ammonia is the main basic component of nitrogen fertilizers and plays a vital role in modern agriculture.

[0003] At present, the industry mainly synthesizes ammonia through the traditional Haber-Bosch process, which requires H2 and N2 to react under high temperature and pressure. This process is energy-intensive and emits a large amount of carbon dioxide. It is a typical "high energy consumption + high carbon emission" industry, and its energy consumption accounts for 1.3-1.5% of the world. Excessive nitrogen fertilizer causes surface and groundwater pollution and soil acidification, which endangers human health and agricultural sustainable development. Microorganisms can fix nitrogen at room temperature and pressure, avoiding the high temperature and high pressure conditions commonly used in industry, thereby significantly reducing energy consumption and greenhouse gas emissions; at the same time, the nitrogen of nitrogen fixation can be efficiently used by plants, which is a clean nitrogen fertilizer with the advantages of environmental protection and sustainable development. However, the efficiency of natural nitrogenase is low and its application range is relatively limited. Therefore, there is an urgent need to develop sustainable alternatives and seek a green, efficient, sustainable and alternative technology to produce NH3 under milder conditions.

[0004] Therefore, using nanozymes with high catalytic activity to simulate the structure and function of natural nitrogenase, and using nitrogen and water to synthesize ammonia at room temperature and pressure has huge advantages over natural enzymes and the current industrial Haber-Bosch process: first, the reaction conditions are mild and the energy consumption is low; second, the reaction substrates are water and nitrogen, and no hydrogen, a compound currently produced by chemical fuels, is required, which effectively reduces carbon emissions. Therefore, exploring low-energy, stable artificial nitrogenase to replace industrial nitrogen fixation and assist natural nitrogenase to expand the range of nitrogen-fixing plants is a scientific problem that needs to be solved urgently. Summary of the invention

[0005] To solve the above technical problems, the object of the present invention is to provide a kind of natural nitrogenase-like FeMo nanozyme and its preparation method and application. This FeMo nanozyme has the nitrogen fixation activity of natural nitrogenase-like, and can generate ammonia from nitrogen and water under normal temperature and pressure, solving the technical problem that it is very difficult to generate ammonia in situ in plants under normal temperature and pressure in the prior art.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: Provide a preparation method of a natural nitrogenase-like FeMo nanozyme, including the following steps:

[0007] (1) Add 1,3,4-thiadiazole-2,5-diamine into absolute ethanol, stir at room temperature to obtain a 1,3,4-thiadiazole-2,5-diamine solution;

[0008] (2) Dissolve molybdenum pentachloride in absolute ethanol to obtain a molybdenum pentachloride solution;

[0009] (3) Dissolve ferric chloride in absolute ethanol to obtain a ferric chloride solution;

[0010] (4) Dissolve 2,5-thiophenedicarboxylic acid in absolute ethanol to obtain a 2,5-thiophenedicarboxylic acid solution;

[0011] (5) Add the molybdenum pentachloride solution obtained in step (2) and the ferric chloride solution obtained in step (3) into the 1,3,4-thiadiazole-2,5-diamine solution obtained in step (1), stir at room temperature, and the solution changes from dark brown to yellowish brown to obtain a FeMo nanozyme mixed solution;

[0012] (6) Add the 2,5-thiophenedicarboxylic acid solution obtained in step (4) into the FeMo nanozyme mixed solution obtained in step (5), stir at room temperature, the solution becomes turbid from yellowish brown, and is successively centrifuged, dried and calcined to obtain a natural nitrogenase-like FeMo nanozyme.

[0013] Further, in step (1), the molar volume ratio of 1,3,4-thiadiazole-2,5-diamine to absolute ethanol is 1-3 mmol: 40-60 mL.

[0014] Further, in step (1), stir at 500-700 r / min for 1-2 h.

[0015] Further, in step (2), the molar volume ratio of molybdenum pentachloride to absolute ethanol is 0.4-0.6 mmol: 2-3 mL.

[0016] Further, in step (3), the molar volume ratio of ferric chloride to absolute ethanol is 0.4-0.6 mmol: 2-3 mL.

[0017] Further, in step (4), the molar volume ratio of 2,5-thiophenedicarboxylic acid to absolute ethanol is 1-3 mmol: 4-6 mL.

[0018] Further, in step (5), the volume ratio of the molybdenum pentachloride solution, ferric chloride solution and 1,3,4-thiadiazole-2,5-diamine solution is 2-3: 2-3: 20-40.

[0019] Further, in step (5), the molar ratio of molybdenum pentachloride to ferric chloride is 1:1.

[0020] Further, in step (6), the volume ratio of the 2,5-thiophenedicarboxylic acid solution to the FeMo nanozyme mixed solution is 4-6: 24-46.

[0021] Further, in step (6), it is calcined at 300-700 °C for 1-3 h.

[0022] Still further, in step (6), it is calcined at 500 °C for 2 h.

[0023] Further, in step (6), it is stirred at room temperature for 1-3 h.

[0024] Further, in step (6), it is centrifuged at 7000-9000 r / min for 2-4 min.

[0025] The present invention also provides a natural-like nitrogenase FeMo nanozyme prepared by the preparation method of the above natural-like nitrogenase FeMo nanozyme.

[0026] The present invention also provides the application of the above natural-like nitrogenase FeMo nanozyme in plant nitrogen fixation.

[0027] The present invention has the following beneficial effects:

[0028] 1. The present invention designs a FeMo nanozyme to simulate natural nitrogenase by introducing FeCl3 and MoCl5 on the basis of 1,3,4-thiadiazole-2,5-diamine and calcining at high temperature under N2. This FeMo nanozyme has the nitrogen fixation activity of natural-like nitrogenase and can generate ammonia from nitrogen and water under normal temperature and pressure. At the same time, through the exploration of preparation conditions, it is found that when the molar ratio of MoCl5 to FeCl3 is 1:1, the nitrogen fixation activity of the FeMo nanozyme is the best, and the ammonium concentration generated in vitro can reach 5.32 ± 1.00 mM.

[0029] 2. The present invention realizes the preparation of nitrogen-fixing nanozymes through a high-temperature calcination method, and verifies the effectiveness of FeMo nanozymes in the detection of free ammonia content in vitro and the ammonia production and ammonia assimilation in plants. The FeMo nanozymes of the present invention can be used to achieve low-energy-consuming and stable artificial nitrogenase to replace industrial nitrogen fixation, assist natural nitrogenase to expand the range of nitrogen-fixing plants, can be applied to the increase of crop yields and incomes, have the advantages of environmental protection and sustainable development, and have a very high transformation prospect. Description of the Drawings

[0030] Figure 1 Schematic diagram of the preparation process of FeMo nanozymes in Example 1;

[0031] Figure 2 Morphology characterization result diagram of FeMo nanozymes;

[0032] Figure 3 In vitro nitrogen fixation activity test result diagram of FeMo nanozymes prepared under different conditions;

[0033] Figure 4 Nitrogen fixation activity test result diagram of FeMo nanozymes under different usage conditions;

[0034] Figure 5 Nitrogen fixation activity test result diagram of FeMo nanozymes on Nicotiana benthamiana plants. Detailed Embodiments

[0035] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0036] Example 1

[0037] A kind of natural nitrogenase-like FeMo nanozyme, the preparation schematic diagram is as Figure 1 shown, and its preparation method includes the following steps:

[0038] (1) Add 2 mmol of 1,3,4-thiadiazole-2,5-diamine to 30 mL of absolute ethanol, stir at 600 r / min at room temperature for 1.5 h to obtain a 1,3,4-thiadiazole-2,5-diamine solution;

[0039] (2) Dissolve 0.5 mmol of molybdenum pentachloride in 2.5 mL of absolute ethanol to obtain a molybdenum pentachloride solution;

[0040] (3) Dissolve 0.5 mmol of ferric chloride in 2.5 mL of absolute ethanol to obtain a ferric chloride solution;

[0041] (4) Dissolve 2 mmol of 2,5-thiophenedicarboxylic acid in 5 mL of absolute ethanol to obtain a 2,5-thiophenedicarboxylic acid solution;

[0042] (5) Add 2.5 mL of the molybdenum pentachloride solution obtained in step (2) and 2.5 mL of the iron(III) chloride solution obtained in step (3) to 30 mL of the 1,3,4-thiadiazole-2,5-diamine solution obtained in step (1), stir at room temperature, and the solution changes from dark brown to yellowish brown to obtain a FeMo nanozyme mixed solution;

[0043] (6) Add 5 mL of the 2,5-thiophenedicarboxylic acid solution obtained in step (4) to 35 mL of the FeMo nanozyme mixed solution obtained in step (5), stir at room temperature for 2 h, the solution becomes turbid from yellowish brown, centrifuge at 8000 r / min for 3 min and then dry, and then calcine at 500 °C for 2 h to obtain a natural-like nitrogenase FeMo nanozyme.

[0044] Example 2

[0045] A natural-like nitrogenase FeMo nanozyme, and its preparation method includes the following steps:

[0046] (1) Add 1 mmol of 1,3,4-thiadiazole-2,5-diamine to 20 mL of absolute ethanol, stir at 500 r / min at room temperature for 1 h to obtain a 1,3,4-thiadiazole-2,5-diamine solution;

[0047] (2) Dissolve 0.4 mmol of molybdenum pentachloride in 2 mL of absolute ethanol to obtain a molybdenum pentachloride solution;

[0048] (3) Dissolve 0.4 mmol of iron(III) chloride in 2 mL of absolute ethanol to obtain an iron(III) chloride solution;

[0049] (4) Dissolve 1 mmol of 2,5-thiophenedicarboxylic acid in 4 mL of absolute ethanol to obtain a 2,5-thiophenedicarboxylic acid solution;

[0050] (5) Add 2 mL of the molybdenum pentachloride solution obtained in step (2) and 2 mL of the iron(III) chloride solution obtained in step (3) to 20 mL of the 1,3,4-thiadiazole-2,5-diamine solution obtained in step (1), stir at room temperature, and the solution changes from dark brown to yellowish brown to obtain a FeMo nanozyme mixed solution;

[0051] (6) Add 4 mL of the 2,5-thiophenedicarboxylic acid solution obtained in step (4) to 24 mL of the FeMo nanozyme mixed solution obtained in step (5), stir at room temperature for 1 - 3 h, the solution becomes turbid from yellowish brown, centrifuge at 7000 r / min for 2 - 4 min and then dry, and then calcine at 300 °C for 1 h to obtain a natural-like nitrogenase FeMo nanozyme.

[0052] Example 3

[0053] A kind of natural-like nitrogenase FeMo nanozyme, and its preparation method includes the following steps:

[0054] (1) Add 3 mmol of 1,3,4-thiadiazole-2,5-diamine to 40 mL of absolute ethanol, stir at 700 r / min at room temperature for 2 h to obtain a 1,3,4-thiadiazole-2,5-diamine solution;

[0055] (2) Dissolve 0.6 mmol of molybdenum pentachloride in 3 mL of absolute ethanol to obtain a molybdenum pentachloride solution;

[0056] (3) Dissolve 0.6 mmol of ferric chloride in 3 mL of absolute ethanol to obtain a ferric chloride solution;

[0057] (4) Dissolve 3 mmol of 2,5-thiophenedicarboxylic acid in 6 mL of absolute ethanol to obtain a 2,5-thiophenedicarboxylic acid solution;

[0058] (5) Add 3 mL of the molybdenum pentachloride solution obtained in step (2) and 3 mL of the ferric chloride solution obtained in step (3) to 40 mL of the 1,3,4-thiadiazole-2,5-diamine solution obtained in step (1), stir at room temperature, and the solution changes from dark brown to yellowish brown to obtain a FeMo nanozyme mixed solution;

[0059] (6) Add 6 mL of the 2,5-thiophenedicarboxylic acid solution obtained in step (4) to 46 mL of the FeMo nanozyme mixed solution obtained in step (5), stir at room temperature for 3 h, the solution becomes turbid from yellowish brown, centrifuge at 9000 r / min for 4 min and then dry, and then calcine at 600 °C for 3 h to obtain the natural-like nitrogenase FeMo nanozyme.

[0060] Example 1

[0061] The FeMo nanozyme of Example 1 was characterized by SEM, TEM and particle size tester, and the results are as Figure 2 shown. Among them, A is the scanning electron microscope image of the FeMo nanozyme (scale = 200 nm), B is the particle size diagram of the FeMo nanozyme, C is the transmission electron microscope image of the FeMo nanozyme (scale = 200, 100, 50, 20 nm), and D is the element mapping diagram of the FeMo nanozyme. The results prove the successful synthesis of the FeMo nanozyme.

[0062] Example 2

[0063] A series of FeMo nanozymes were prepared by adjusting the preparation conditions, and the nitrogen fixation activities of the FeMo nanozymes were compared based on the concentration of ammonium generated in vitro. A blank group (ddH2O), a standard curve group (ammonium chloride), and an experimental group (FeMo nanozyme, 1 mg / mL) were set up. In the standard curve group, a standard solution was prepared from an ammonium chloride standard, and in the experimental group, the FeMo nanozyme was dispersed in ddH2O to prepare a 1 mg / mL stock solution for standby (ice bath probe ultrasound: 150 W for 3 s, interval 2 s, total time 5 minutes). 100 μL of the supernatant solution of each group was taken and placed in a 1.5 mL EP tube, and then 200 μL of 0.33 M sodium phenolate, 10 μL of 0.2% nitroprusside, and 200 μL of 2%-3% sodium hypochlorite were added in sequence. After thorough mixing, the absorbance at 630 nm was measured, a standard curve was plotted, and the concentration of ammonium generated in vitro by a series of FeMo nanozymes was calculated according to the standard curve to compare their nitrogen fixation activities. The results are as Figure 3 shown, where A is a schematic diagram of free ammonia generated in vitro by an ammonium chloride standard solution, B is a standard curve, C is a schematic diagram of a solution of free ammonia generated in vitro by FeMo nanozyme, and D is a statistical result of free ammonia generated in vitro by FeMo nanozyme.

[0064] It can be Figure 3 seen that a series of FeMo nanozymes synthesized under different optimized conditions have different nitrogen fixation activities in vitro. Among them, the FeMo nanozyme with a molar ratio of MoCl5 to FeCl3 of 1:1, a calcination temperature of 500 °C, and a calcination time of 2 h has the strongest nitrogen fixation activity.

[0065] Example 3

[0066] Referring to the test method of Example 2, the nitrogen fixation activities of FeMo nanozymes under different usage conditions were tested respectively. The results are as Figure 4 shown, where A is a schematic diagram of free ammonia generated in vitro by an ammonium chloride standard solution, B is a standard curve, C is a schematic diagram of a solution of free ammonia generated in vitro by FeMo nanozyme under different conditions, and D is a statistical result of free ammonia generated in vitro by FeMo nanozyme under different conditions.

[0067] It can be Figure 4 seen that the nitrogen fixation activity of FeMo nanozyme increases under light-shielded conditions, revealing that the nitrogen fixation activity can be improved under light-shielded conditions; the nitrogen fixation activity of FeMo nanozyme increases under acidic conditions, revealing that under acidic conditions, it may be due to the solution providing more H + thus increasing the nitrogen fixation activity.

[0068] Example 4 Testing the nitrogen fixation activity in plants

[0069] The FeMo nanozyme of Example 1 was used to treat Nicotiana benthamiana (4-week-old leaves) by leaf injection and foliar spraying respectively. After 5 days, the plant phenotypes were observed. Fresh leaves were frozen in liquid nitrogen and ground into powder, and the contents of glutamate (Glu) and glutamine (Gln) were measured using the following kits: Glu (G0427W, Greeson, Suzhou), Gln (G0429W29, Greeson, Suzhou). The results are as Figure 5 shown. Among them, A is the phenotype diagram of Nicotiana benthamiana plants by leaf injection, B is the statistical chart of glutamine content in Nicotiana benthamiana leaves, C is the statistical chart of glutamate content in Nicotiana benthamiana leaves, D is the phenotype diagram of Nicotiana benthamiana plants by foliar spraying, E is the statistical chart of glutamine content in Nicotiana benthamiana leaves, and F is the statistical chart of glutamate content in Nicotiana benthamiana leaves.

[0070] It can be Figure 5 seen that after the application of FeMo nanozyme by both leaf injection and foliar spraying, the plant leaves are larger and the plants grow stronger. When the application concentration of FeMo nanozyme is 0.1 g / L, the content of Glu in tobacco leaves decreases and the content of Gln increases, which proves that FeMo nanozyme has ammonia assimilation effect.

[0071] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of a quasi-natural nitrogenase FeMo nanozyme, characterized in that, It includes the following steps: (1) Add 1,3,4-thiadiazole-2,5-diamine into absolute ethanol and stir at room temperature to obtain a 1,3,4-thiadiazole-2,5-diamine solution; (2) Dissolve molybdenum pentachloride in absolute ethanol to obtain a molybdenum pentachloride solution; (3) Dissolve ferric chloride in absolute ethanol to obtain a ferric chloride solution; (4) Dissolve 2,5-thiophenedicarboxylic acid in absolute ethanol to obtain a 2,5-thiophenedicarboxylic acid solution; (5) Add the molybdenum pentachloride solution obtained in step (2) and the ferric chloride solution obtained in step (3) into the 1,3,4-thiadiazole-2,5-diamine solution obtained in step (1), stir at room temperature, and the solution changes from dark brown to yellowish brown to obtain a FeMo nanozyme mixed solution; (6) Add the 2,5-thiophenedicarboxylic acid solution obtained in step (4) into the FeMo nanozyme mixed solution obtained in step (5), stir at room temperature, the solution becomes turbid from yellowish brown, and after centrifugation, drying and calcination in sequence, a natural nitrogenase-like FeMo nanozyme is obtained.

2. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that, In step (1), the molar volume ratio of 1,3,4-thiadiazole-2,5-diamine to absolute ethanol is 1 - 3 mmol: 20 - 40 mL.

3. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that, In step (2), the molar volume ratio of molybdenum pentachloride to absolute ethanol is 0.4 - 0.6 mmol: 2 - 3 mL.

4. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that In step (3), the molar volume ratio of ferric chloride to absolute ethanol is 0.4 - 0.6 mmol: 2 - 3 mL.

5. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that, In step (4), the molar volume ratio of 2,5-thiophenedicarboxylic acid to absolute ethanol is 1 - 3 mmol: 4 - 6 mL.

6. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that, In step (5), the volume ratio of the molybdenum pentachloride solution, ferric chloride solution and 1,3,4-thiadiazole-2,5-diamine solution is 2 - 3: 2 - 3: 20 - 40.

7. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that In step (6), the volume ratio of the 2,5-thiophenedicarboxylic acid solution and the FeMo nanozyme mixed solution is 4 - 6: 24 - 46.

8. The preparation method of the quasi-natural nitrogenase FeMo nanozyme according to claim 1, characterized in that, In step (6), calcine at 300 - 700 °C for 1 - 3 h.

9. The natural nitrogenase-like FeMo nanozyme prepared by the preparation method of the natural nitrogenase-like FeMo nanozyme according to any one of claims 1 - 8.

10. The application of the natural nitrogenase-like FeMo nanozyme according to claim 9 in plant nitrogen fixation.

Images