Cyclobutylamine acidified protein as well as preparation and application thereof

Abstract

The invention relates to the technical field of pesticides, in particular to a cyclobutylamine acidified protein as well as preparation and application thereof. The invention provides a cyclobutylamine acidified protein. The protein is acidified by cyclobutylamine acid; amino groups of the protein and carboxyl groups of the cyclobutyric acid are subjected to dehydration condensation to form chemical bond connection. The cyclobutylamine acidified protein provided by the invention can be used for preparing a bactericide, the bactericide is prepared from the following components in parts by mass: 1-20 parts of cyclobutylamine acidified protein, 1-20 parts of a reducing agent and 2-20 parts of a pH regulator, the reducing agent can enable the cyclobutylamine acidified protein to form a protein film on the surface of hyphae, and the pH regulator can be used for regulating the pH value of the hyphae. Therefore, the phenomenon of pesticide loss caused by splashing and rain wash is avoided; and the like. The bactericide provided by the invention can be used for killing cucurbit powdery mildew bacteria, a protein film formed by the bactericide is fixed on the surface of hydrophobic powdery mildew hyphae, the utilization rate of the bactericide can be improved, and the bactericide has a wide prospect in practical application.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticides, and particularly to a cyclobutylamine-acidified protein and its preparation and application. Background Art

[0002] Powdery mildew is a world-wide plant disease that is difficult to control. It occurs on many crops and seriously endangers the crops. Powdery mildew of cucurbits is a relatively common type among crop powdery mildews. It is a leaf disease caused by powdery mildew fungi. When it is severe, the stems and petioles will also be infected to varying degrees, resulting in leaf wilt or pulling out of the seedlings, thus causing a reduction in the yield of cucurbits.

[0003] At present, the control of powdery mildew of cucurbits largely depends on agricultural chemicals, mainly including organosulfur fungicides, benzimidazole fungicides, triazole fungicides and bio-mimetic botanical fungicides. However, synthetic fungicides seriously threaten ecological safety and cucurbit powdery mildew has developed resistance to various fungicides. Among them, L-azetidine-2-carboxylic acid, that is, cyclobutylamine acid, has a good effect on the control of cucurbit powdery mildew. It has a direct contact effect with hyphae and destroys the infected hyphae, thus achieving a therapeutic effect. However, due to the hydrophobic or superhydrophobic characteristics of plant leaves and the surfaces of pathogens, the problem of pesticide droplet splash is serious. When pesticide droplets hit the leaves of plants, they will bounce or splash, resulting in the loss of more than 50% of the pesticides. And a small part of the pesticides that reach the leaf surface may enter groundwater, air and soil due to rain erosion, evaporation and washing, which not only leads to the overuse of pesticides, but also causes environmental pollution and has harmful effects on human health and the ecosystem.

[0004] A common method to improve the splash and rebound of pesticide droplets is to add polymer solutions, surfactants to the liquid or make the droplets charged to improve the deposition of pesticides on the leaf surface of plants. However, synthetic polymers and surfactants are not environmentally friendly and will cause secondary pollution to the environment. In addition, such methods do not consider other natural conditions, such as the impact of rain washing on pesticides, and do not demonstrate the universality in the plant kingdom and the excellent ability to fix pesticides. Summary of the Invention

[0005] Cyclobutylamine acid in the present invention has a good effect on the control of cucurbit powdery mildew. It has a direct contact effect with hyphae and destroys the infected hyphae, thus achieving a therapeutic effect. However, it cannot effectively improve the phenomenon of pesticide droplet splash and rebound. Therefore, the present invention provides a cyclobutylamine-acidified protein, which can be used to prepare a fungicide that can form a protein film at the affected area. The cyclobutylamine-acidified protein is a protein acidified by cyclobutylamine acid;.

[0006] The acidification is that the amino group of the protein and the carboxyl group of cyclobutylamine acid are connected by dehydration condensation to form a chemical bond.

[0007] The above-mentioned cyclobutylamine-acidified protein, wherein the protein is one or a combination of more than one of plant-derived protein, lysozyme, bovine serum albumin, lactalbumin, insulin protein, lactoferrin, human serum albumin, hemoglobin, myoglobin, collagen, chymotrypsin, catalase, horseradish peroxidase, cytochrome C, α-lactalbumin, and β-lactoglobulin.

[0008] The above-mentioned cyclobutylamine-acidified protein, wherein the molar ratio of cyclobutylamine acid to the protein is 1:1 to 50:1.

[0009] Meanwhile, the present invention also provides a preparation method for the above-mentioned cyclobutylamine-acidified protein, including: by mole fraction, mixing 1 to 50 parts of cyclobutylamine acid, 1 part of protein, 1 to 200 parts of dehydrating agent, and 1 to 100 parts of activating agent as solutes in a solvent for reaction to obtain the cyclobutylamine-acidified protein described in any one of claims 1 to 3.

[0010] The above-mentioned preparation method for the cyclobutylamine-acidified protein, wherein the dehydrating agent is EDCI, the activating agent is NHS, and the solvent is 10×PBS solution.

[0011] Secondly, the present invention also provides a bactericide, which is composed of the following raw materials by mass fraction: 1 to 20 parts of the cyclobutylamine-acidified protein described in claims 1 to 3, 1 to 20 parts of reducing agent, and 2 to 20 parts of pH regulator; The pH regulator controls the pH of the bactericide after being dissolved in water to be 7 to 10.

[0012] For the above-mentioned bactericide, the reducing agent is one or a combination of several of dithiothreitol, 2-mercaptoethanol, TCEP, L-cysteine, reduced glutathione, β-mercaptoethanol, dimercaptosuccinic acid, sodium sulfite, and thioglycerol; the pH regulator is one or a combination of several of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate.

[0013] Furthermore, the present invention provides an application of the bactericide in preventing and controlling powdery mildew of cucurbits, and the use concentration of the bactericide is 0.01 mg / mL to 10 mg / mL.

[0014] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: 1. The cyclobutylamine-acidified protein provided by the present invention has a simple composition. Cyclobutylamine acid itself has a bactericidal function, and the protein can further form a protein film, providing a brand-new raw material for plant agronomy and laying a solid foundation for the bactericide provided by the present invention.

[0015] 2. The fungicide provided by the present invention has simple components and easily available raw materials. Under the action of a reducing agent, proteins undergo amyloid transformation to form amyloid aggregates. Since amyloid protein aggregates tend to accumulate at the gas-liquid interface, the surface tension of the solution is reduced, making the solution easier to wet the superhydrophobic substrate. At the same time, the amyloid-like protein aggregates will further assemble at the solid-liquid interface to form an adhesive film, i.e., a protein film, which can assist in stably adhering to the superhydrophobic hyphae, thereby achieving the sterilization effect while avoiding the phenomenon of pesticide droplets splashing and rebounding. And through in vitro experiments and in vivo experiments, it is proved that the fungicide of the present invention can eradicate powdery mildew, and the eradication rate reaches 90%.

[0016] 3. The fungicide prepared by the present invention has strong stability in organic solvents: after being soaked in various organic solvents such as ultrasonic, ethanol, n-hexane, petroleum ether, DMF, and chloroform for 2 hours, it can still exist stably. This characteristic not only broadens its application scenarios but also enhances its adaptability in complex environments.

[0017] 4. The fungicide of the present invention has good weather resistance. After being placed in various extreme environments for 30 days, such as at a high temperature of 70°C, a low temperature of -24°C, and a simulated light environment (14400 LX), the protein pesticide fungicide shows excellent stability. Whether under high temperature, low temperature or light conditions, it can achieve a long-term fixation effect, ensuring the effectiveness of the product under different climatic conditions and environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1a It is a scanning electron microscope image of the surface of blank powdery mildew hyphae; Figure 1b It is a scanning electron microscope image of the surface of cucumber powdery mildew hyphae after spraying the protein pesticide fungicide prepared in Example 3; Figure 2a It is a scanning electron microscope image of the surface of cucumber powdery mildew hyphae before the protein film formed in Example 9 is washed by simulated rain; Figure 2b It is a scanning electron microscope image of the surface of cucumber powdery mildew hyphae after the protein film formed in Example 9 is washed by simulated rain; Figure 3 is a scanning electron microscope image of the weather resistance experiment of the protein film formed by the protein pesticide fungicide obtained in Example 4, where, Figure 3aIt is a scanning electron microscope image of a protein film formed on a superhydrophobic surface placed in a low-temperature environment (-24°C, 30 days); Figure 3b It is a scanning electron microscope image placed in a high-temperature environment (70°C, 30 days); Figure 3c It is a scanning electron microscope image placed in an environment simulating light (144000 LX, 30 days); Figure 4 is a scanning electron microscope image of the protein film formed by the protein pesticide bactericide in Example 3 soaked in different organic solvents for 2 hours. Among them, Figure 4a It is a scanning electron microscope image of the protein film in an ultrasonic environment; Figure 4b It is a scanning electron microscope image of the protein film in a dichlorine environment; Figure 4c It is a scanning electron microscope image of the protein film in a DMF environment; Figure 4d It is a scanning electron microscope image of the protein film in a petroleum ether environment; Figure 4e It is a scanning electron microscope image of the protein film in an ethanol environment; Figure 4f It is a scanning electron microscope image of the protein film in a n-hexane environment; Figure 5 It is a data comparison chart of the eradication rate of the bactericidal eradication experiment. Detailed implementation manners

[0020] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The % in the following embodiments is the mass percentage unless otherwise specified. The ratios in the following embodiments are all mass ratios unless otherwise specified.

[0021] Examples 1 to 3 illustrate a cyclobutylamine-acidified protein and its preparation provided by the present invention.

[0022] Example 1 A cyclobutylamine-acidified lysozyme provided in this example is composed of cyclobutylamine acid and lysozyme. The cyclobutylamine acid is L-azetidine-2-carboxylic acid, and the molar ratio of the cyclobutylamine acid to lysozyme is 50:1.

[0023] A preparation method of cyclobutylamine-acidified lysozyme provided by this embodiment includes the following steps: Dissolve the solute in the solvent, stir at room temperature for 4 h, and then dialyze and freeze-dry to obtain cyclobutylamine-acidified lysozyme; By mole fraction, the solute is 50 parts of cyclobutylamine acid, 1 part of lysozyme, 200 parts of EDCI, and 100 parts of NHS, and the solvent is 10×PBS, and the mass ratio of the solvent to the solute is 20:1.

[0024] Example 2 A cyclobutylamine-acidified bovine serum albumin provided by this embodiment is composed of cyclobutylamine acid and bovine serum albumin. The cyclobutylamine acid is L-azetidine-2-carboxylic acid, and the molar ratio of the cyclobutylamine acid to bovine serum albumin is 1:1.

[0025] A preparation method of cyclobutylamine-acidified bovine serum albumin provided by this embodiment includes the following steps: Dissolve the solute in the solvent, stir at room temperature for 4 h, and then dialyze and freeze-dry to obtain cyclobutylamine-acidified bovine serum albumin; By mole fraction, the solute is 1 part of cyclobutylamine acid, 1 part of bovine serum albumin, 1 part of EDCI, and 1 part of NHS, and the solvent is 10×PBS, and the mass ratio of the solvent to the solute is 10:1.

[0026] Example 3 A cyclobutylamine-acidified soybean protein provided by this embodiment is composed of cyclobutylamine acid and soybean protein. The cyclobutylamine acid is L-azetidine-2-carboxylic acid, and the molar ratio of the cyclobutylamine acid to soybean protein is 25:1.

[0027] A preparation method of cyclobutylamine-acidified soybean protein provided by this embodiment includes the following steps: Dissolve the solute in the solvent, stir at room temperature for 4 h, and then dialyze and freeze-dry to obtain cyclobutylamine-acidified soybean protein; By mole fraction, the solute is 25 parts of cyclobutylamine acid, 1 part of soybean protein, 100 parts of EDCI, and 50 parts of NHS, and the solvent is 10×PBS, and the mass ratio of the solvent to the solute is 15:1.

[0028] A bactericide of the present invention will be described below through Examples 4 to 6.

[0029] Example 4 The cyclobutylamine-acidified protein in a bactericide in this embodiment selects the cyclobutylamine-acidified bovine serum albumin in Example 2. A bactericide in this embodiment is composed of the following components by mass: 1 part of cyclobutylamine-acidified bovine serum albumin, 1 part of reducing agent, and 2 parts of pH regulator. The reducing agent is dithiothreitol, and the pH regulator is sodium citrate. The pH regulator controls the pH of the bactericide dissolved in water to be 7.

[0030] Example 5 For the cyclobutylamine acidified protein of the fungicide in this example, the cyclobutylamine acidified lysozyme in Example 1 is selected. The fungicide in this example is composed of the following components by mass: 10 parts of cyclobutylamine acidified lysozyme, 5 parts of reducing agent, and 5 parts of pH regulator. The reducing agent is L-cysteine, and the pH regulator is sodium carbonate. The pH regulator controls the pH of the fungicide dissolved in water to be 9.

[0031] Example 6. For the cyclobutylamine acidified protein of the fungicide in this example, the cyclobutylamine acidified soy protein in Example 3 is selected. The fungicide in this example is composed of the following components by mass: 20 parts of cyclobutylamine acidified soy protein, 20 parts of reducing agent, and 20 parts of pH regulator. The reducing agent is a composite reducing agent of 2-mercaptoethanol and TCEP, and the pH regulator is potassium carbonate. The pH regulator controls the pH of the fungicide dissolved in water to be 10.

[0032] To prove the beneficial effects of the present invention, the inventor added 50 mg of the fungicide in Example 4 to 10 mL of deionized water and gently shook it until the fungicide was fully dissolved to obtain a 5 mg / mL fungicide solution with a pH of 9. Then, various performance tests were carried out on this fungicide solution. The specific experiments are shown in Examples 7 to 11.

[0033] Example 7 In this example, the fixation ability of the bactericidal effect in Example 5 was verified. The specific operation is as follows: Taking Figure 1a the powdery mildew hyphae with superhydrophobic properties shown as an example, a 5 mg / mL fungicide solution was evenly sprayed on the surface of the powdery mildew hyphae cut into a size of 1.8 cm × 1.8 cm, and the surface of the powdery mildew hyphae was washed with deionized water. After natural drying, it was observed with a scanning electron microscope. The results are as Figure 1b shown, which can prove that the protein film formed by the fungicide of the present invention can be effectively fixed on the powdery mildew hyphae.

[0034] Example 8 In this example, the rain resistance ability of the protein film formed by the fungicide of the present invention in Example 7 was verified. The specific operation was to simulate the rain washing process of the powdery mildew hyphae with the protein film formed in Example 7 as Figure 2a shown. The simulated precipitation intensity was 6 mm / h, and the simulated time was 3 h. The results are as Figure 2b shown, indicating that the protein film formed by the fungicide of the present invention in Example 7 has good rain resistance ability.

[0035] Example 9 In this example, the weather resistance of the bactericidal effect in Example 7 was verified. The specific operation is as follows; The powdery mildew hyphae after sterilization in Example 7 were divided into five parts. Four of them were placed in an environment of 70 °C for 30 days, -24 °C for 30 days, a simulated light environment (14400 LX) for 30 days, and a microbial environment for 30 days respectively. And the samples placed in the normal room temperature environment were used as the control test. Observation was carried out with a scanning electron microscope, and the results proved that the protein product of the present invention showed excellent stability in the above various environments and could achieve a long-term fixation effect at low temperature (see Figure 3a ), high temperature (see Figure 3b ), and light (see Figure 3c ).

[0036] Example 10 In this example, the sterilization effect in Example 7 was verified for its stability in organic solvents. The specific operation is as follows: The powdery mildew hyphae after sterilization in Example 7 were evenly divided into six parts, and they were respectively placed in ultrasonic, ethanol, n-hexane, petroleum ether, DMF, and chloroform. After 2 hours, they were taken out and the surface was observed with a scanning electron microscope (see Figure 4). The results proved that the fungicide product of the present invention could stably exist in ultrasonic (see Figure 4a ), dichloride (see Figure 4b ), DMF (see Figure 4c ), petroleum ether (see Figure 4d ), ethanol (see Figure 4e ), and n-hexane (see Figure 4f ).

[0037] Example 11 In this example, the fungicidal eradication performance of the fungicide in Example 5 was verified. The specific operation is as follows: The cucumber powdery mildew hyphae potted plants were divided into three groups, with 6 potted plants in each group, and the following treatments were carried out respectively: The first group was the blank group without treatment, the second group was sprayed with 25 mL of 10 mg / mL cyclobutylamine acid solution (prepared with deionized water), and the third group was sprayed with 25 mL of 10 mg / mL fungicide prepared in Example 5. The results proved that a fungicide could effectively eradicate the powdery mildew on the surface of cucumber leaves, had a positive effect on cucumber leaves compared with the untreated blank group, and reduced the usage amount of the fungicide by 60% - 70% compared with the second group (see Figure 5 ).

[0038] As described above, the basic principle, main features, and advantages of the present invention are preferably described. The above examples and descriptions are only for describing the preferred embodiments of the present invention. The present invention is not limited by the above examples. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A cyclobutyric acid-modified protein, characterized in that The cyclobutanamine-acidified protein is a protein that has been acidified with cyclobutanamine; The acidification is to form a chemical bond connection between the amino group of the protein and the carboxyl group of cyclobutane through dehydration condensation.

2. The cyclobutyric acid-modified protein according to claim 1, characterized in that The protein is one or more combinations of plant-derived protein, lysozyme, bovine serum albumin, whey albumin, insulin protein lactoferrin, human serum albumin, hemoglobin, myoglobin, collagen, chymosin, catalase, horseradish peroxidase, cytochrome C, α-lactalbumin and β-lactoglobulin.

3. The cyclobutyric acid-modified protein according to claim 2, characterized in that The molar ratio of cyclobutane to protein is 1:1 to 50:

1.

4. A method for preparing a cyclobutyric acid-containing protein, characterized in that: include: The cyclobutyric acid-modified protein according to any one of claims 1 to 3 is obtained by mixing 1 to 50 parts of cyclobutyric acid, 1 part of protein, 1 to 200 parts of a dehydrating agent and 1 to 100 parts of an activating agent as solutes in a solvent for reaction.

5. The method for preparing cyclobutyric acid-modified protein according to claim 4, characterized in that: The dehydrating agent is EDCI, the activating agent is NHS, and the solvent is 10×PBS solution.

6. The method for preparing cyclobutyric acid-modified protein according to claim 4, characterized in that: The mass ratio of the solvent to the solute is 10:1 to 20:

1.

7. A bactericide, characterized in that: The composition is composed of the following raw materials, by weight: 1 to 20 parts of the cyclobutane-acidified protein according to claims 1 to 3, 1 to 20 parts of a reducing agent, and 2 to 20 parts of a pH adjusting agent; The pH regulator controls the pH of the bactericide to be 7-10 after being dissolved in water.

8. The fungicide according to claim 7, characterized in that The reducing agent is one or a combination of dithiothreitol, 2-mercaptoethanol, TCEP, L-cysteine, reduced glutathione, β-mercaptoethanol, dimercaptosuccinic acid, sodium sulfite, and thioglycerol; the pH adjusting agent is one or a combination of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate.

9. Use of the fungicide according to any one of claims 6 to 8 in preventing and controlling powdery mildew of melons.

10. The use according to claim 9, characterized in that: The fungicide is used at a concentration of 0.01 mg / mL to 10 mg / mL.

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