CN113735175A

CN113735175A - Nano-mimetic enzyme, preparation method and seed soaking agent containing nano-mimetic enzyme

Info

Publication number: CN113735175A
Application number: CN202110946069.3A
Authority: CN
Inventors: 吴洪洪; 李召虎; 刘家浩; 顾江江; 胡金; 李广静; 曹菲菲
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-12-03
Anticipated expiration: 2041-08-17
Also published as: CN113735175B

Abstract

The invention relates to a nano enzyme mimic, a preparation method and application thereof, wherein the nano enzyme mimic is a spherical manganese-based oxide nano material or colloid containing spherical manganese-based oxide nano particles, and the spherical manganese-based oxide particles are modified with a surfactant. Compared with the prior art, the invention has the beneficial effects that: the spherical manganese-based oxide nano material modified by the surfactant has good water solubility, can stably exist in a high-concentration colloid form, and can improve the germination rate and the root growth under the condition of high salt and enhance the salt tolerance when being subsequently applied to seedling raising, thereby further improving the stress resistance of plant seedlings.

Description

Nano-mimetic enzyme, preparation method and seed soaking agent containing nano-mimetic enzyme

Technical Field

The invention belongs to the field of nano materials, and particularly relates to a nano mimic enzyme, a preparation method and a seed soaking agent containing the nano mimic enzyme.

Background

On salinized soil, crop seed germination and seedling establishment are a major concern. The damage of salt to cells and the water stress caused by the reduction of osmotic potential reduce the rate of emergence, the seedling growth is blocked, and the yield is directly influenced. Therefore, it is very important to research the improvement of the salt resistance of the crops in the seedling stage.

Nanopologue is a generic term for nanomaterials that possess antioxidant enzyme activity similar to that of cells. In recent years, researchers have discovered a nanmimic enzyme that efficiently scavenges active oxygen: surface-modified cerium oxide nanomaterials (PNC). PNC can eliminate excessive and accumulated active oxygen in arabidopsis mesophyll cells under salt stress, and enhance plant cell potassium ion (K)⁺) Retention of (D) and sodium ion (Na)⁺) Thereby improving the salt tolerance of the plants. However, since cerium, a rare earth element contained in PNC, is a heavy metal, there is a certain biological safety concern. Manganese (Mn) is an essential micronutrient for plants, and the current research shows that the salt tolerance of cucumbers can be improved by using the nano-mimic enzyme of the manganese-based nano-material, but the existing manganese-based nano-material has poor dispersibility in water, so that the use of the manganese-based nano-material is limited, the salt tolerance is increased to a limited extent, the pretreatment procedure is complicated, the cost is high, and the popularization and the use are difficult.

Disclosure of Invention

In order to solve at least one technical problem, the invention provides a nano-mimetic enzyme, a preparation method and a seed soaking agent containing the nano-mimetic enzyme.

The specific technical scheme is as follows:

the nano-mimic enzyme is characterized in that the nano-mimic enzyme is a spherical manganese-based oxide nano material or colloid containing spherical manganese-based oxide nano particles, and the spherical manganese-based oxide particles are modified with a high-molecular surfactant and have the particle size of 5.0-6.0 nm.

Further, the surfactant is one or two of polyacrylic acid or polyethyleneimine.

Further, if the nano-mimic enzyme contains colloid of spherical manganese-based oxide nano-materials, the concentration of the spherical manganese-based oxide nano-materials in the colloid is 5 g/L-7 g/L, and the dispersion solvent is water.

The preparation method of the nano-mimetic enzyme is characterized by comprising the following steps:

step S1, mixing the divalent manganese salt solution with the high molecular surfactant solution to obtain a system A solution;

and step S2, mixing the solution of the system A with the solution of the precipitator, carrying out hydrothermal reaction under the condition of oxygen, and then purifying to obtain the nano-mimetic enzyme.

Further, in the step S2, the system a solution is added dropwise to the precipitant solution.

Further, in the step S2, the precipitant is ammonia water, and is purified by a dialysis method to obtain a colloid containing spherical manganese-based oxide particles.

Further, the concentration of the divalent manganese salt solution is 0.8-1.2 mol/L, the concentration of the surfactant solution is 800-1000 g/L, and the volume ratio of the divalent manganese salt solution to the surfactant solution is 1: (1.5-2.5); the precipitator is ammonia water, the purification is carried out by adopting a dialysis method, and the volume ratio of the solution of the system A to the ammonia water is 1: (1.5-2.5).

Further, in the reaction system, the weight ratio of the divalent manganese salt to the surfactant substance is 1: (0.8 to 1.5).

An application of the above nano-mimetic enzyme in improving the salt tolerance of seedlings.

Further, the application of the nano-mimetic enzyme in preparing a seed soaking agent or a culture medium.

Further, when the nano-mimetic enzyme is colloid, the concentration of the nano-mimetic enzyme is 1.5 mg/L-2.5 mg/L when the culture medium is prepared.

Compared with the prior art, the invention has the beneficial effects that:

(1) the spherical manganese-based oxide nano material modified by the macromolecular surfactant has good water solubility, can stably exist in a high-concentration colloid form, and can improve the germination rate and the root growth under the condition of high salt and enhance the salt tolerance when being subsequently applied to seedling raising, thereby further improving the stress resistance of the plant seedlings.

(2) The invention adopts the steps of firstly mixing divalent manganese salt with polyacrylic acid or polyethyleneimine high molecular surfactant, so that manganese raw material is firstly dispersed in the surfactant, the form and particle size of the manganese raw material can be controlled, and then the manganese raw material is mixed with a precipitator in the follow-up process, and then the hydrothermal reaction is carried out, so that the water solubility of the nano material is improved.

(3) The invention adds the mixed system of the divalent manganese salt and the surfactant into the precipitator solution drop by drop, and can improve the yield of the manganese-based oxide nano material.

(4) According to the invention, ammonia water is used as a precipitator, so that the degree of hydrothermal reaction can be controlled, and the shape and particle size can be controlled.

Drawings

FIG. 1 shows the results of TEM appearance test of example 1;

FIG. 2 shows the valence state detection result of X-ray photoelectron spectroscopy;

FIG. 3 is the TEM appearance test result of comparative example 2;

FIG. 4 shows the results of the particle size test of comparative example 2;

FIG. 5 shows that the salt stress of example 5 shows that the germination rate of the rape seeds treated by the nannociceptive seed soaking is higher;

FIG. 6 shows the root growth of Arabidopsis seedlings treated with Nanopolyase under salt stress in example 6.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Polyacrylic acid: sigma, MW 1800.

The ammonia water was 32% of commercial ammonia water.

Example 1

The embodiment provides a colloidal nano-mimetic enzyme, which comprises the following specific preparation steps:

step S1: mixing MnSO₄.H₂O0.425 g in 2.5mL of purified water, 4.5g of polyacrylic acid (weight average molecular weight 1800) in 5mL of purified water; mixing the manganese sulfate solution and the polyacrylic acid solution, and mixing for 15 minutes at 2500rpm on a vortex instrument to obtain a system A solution;

step S2: the mixed solution was added dropwise to 15mL of aqueous ammonia, and stirred on a magnetic stirrer at 500rpm for 24 hours; putting the stirred solution into a 50mL polytetrafluoroethylene reaction kettle, and carrying out hydrothermal reaction at 120 ℃ for 24 hours; and (3) subpackaging the reacted solution in 2mL centrifuge tubes, centrifuging at 4000g normal temperature for 1 hour, taking the supernatant, dialyzing for 24 hours by using a dialysis bag (MW 3500), and changing water every 8 hours to obtain the colloid containing the nano manganese-based oxide. FIG. 1 shows that the crystal lattice is consistent with the published crystal lattice of trimanganese tetroxide (Sun Y, Yu Z, Wang W, et al. Selective gas detection using Mn 3O 4/WO 3 compositions a sending layer [ J ]. Beilstein Journal of Nanotechnology,2019,10(1): 1423) -1433.), and the concentration of the nano-manganese-based oxide is tested to be 6g/L after freeze-drying, and the yield is calculated by solid: 10.2 percent

The phenotype was observed, and the result is shown in FIG. 1, which is apparent as a spherical form.

The nano particle size detection is carried out on the nano particle size, and the particle size is as follows: 5.60 nm.

The valence state is detected, and the result is shown in fig. 2, which shows that the nanomaterial contains bivalent, trivalent and tetravalent manganese.

Example 2

The embodiment provides a solid type nano-mimetic enzyme, which is prepared by the following specific steps:

step S1: mixing MnSO₄·H₂O0.425 g in 2.5mL of purified water, 4.5g of polyacrylic acid (weight average molecular weight 1800) in 5mL of purified water; mixing the manganese sulfate solution and the polyacrylic acid solution, and mixing for 15 minutes at 2500rpm on a vortex instrument to obtain a system A solution;

step S2: the mixed solution was added dropwise to 15mL of aqueous ammonia, and stirred on a magnetic stirrer at 500rpm for 24 hours; putting the stirred solution into a 50mL polytetrafluoroethylene reaction kettle, and carrying out hydrothermal reaction at 120 ℃ for 24 hours; and (3) subpackaging the reacted solution in 2mL centrifuge tubes, centrifuging at 4000g normal temperature for 1 hour, taking the supernatant, dialyzing for 24 hours by using a dialysis bag (MW 3500), and changing water every 8 hours to obtain the colloid containing the nano manganese-based oxide.

Step S3: and freeze-drying the colloid containing the nano manganese-based oxide to obtain the solid nano manganese-based oxide.

Example 3

step S2: adding 15mL of ammonia water dropwise into the mixed solution, and stirring the solution on a magnetic stirrer at 500rpm for 24 hours; putting the stirred solution into a 50mL polytetrafluoroethylene reaction kettle, and carrying out hydrothermal reaction at 120 ℃ for 24 hours; and (3) subpackaging the reacted solution in 2mL centrifuge tubes, centrifuging at 4000g normal temperature for 1 hour, taking the supernatant, dialyzing for 24 hours by using a dialysis bag (MW 3500), and changing water every 8 hours to obtain the colloid containing the nano manganese-based oxide. HRTEM results showed that the lattice is associated with the published manganomanganic oxide (Sun Y, Yu Z, Wang W, et al₃O₄/WO₃composites as a sensing layer[J]The crystal lattice of Beilstein Journal of Nanotechnology,2019,10(1):1423-1433.) was consistent, and subsequently tested after lyophilization to determine the concentration of the nano-manganese-based oxide to be 3.4g/L, as calculated as solids, with the yield: 6 percent of

The phenotype of the strain is observed, and the appearance shows a spherical shape.

The nano particle size detection is carried out on the nano particle size, and the particle size is as follows: 6.0nm

The valence state detection is carried out on the nanometer material, and the result shows that the nanometer material simultaneously contains bivalent, trivalent and quadrivalent manganese.

Comparative example 1

The comparative example provides a method for preparing manganese-based oxide rice particles, which comprises the following specific operations:

and (3) magnetically stirring 2g of manganese acetate and 100mL of ethanol uniformly, reacting in a hydrothermal kettle at 120 ℃ for 24h, centrifuging at 10000rpm for 5min, washing for 3 times, and performing vacuum drying for 24h to obtain a brown solid.

Performing apparent detection on the manganese oxide nanoparticles, wherein the phenotype of the manganese oxide nanoparticles is spherical manganese oxide nanoparticles;

the particle size of the product was measured and found to be 6.0 nm.

Comparative example 2

The comparative example provides a preparation method of a nano-particle colloid containing manganese-based oxide, which comprises the following specific operations:

and step A1, performing magnetic stirring on 2g of manganese acetate, 50mL of ethanol and 50mL of ethylene glycol uniformly, performing reaction in a hydrothermal kettle at 120 ℃ for 24h, centrifuging at 10000rpm for 5min, washing for 3 times, and performing vacuum drying for 24h to obtain a brown solid, thus obtaining the nano manganese-based oxide matrix.

And A2, mixing the nano manganese-based oxide matrix with sodium oleate for 10min, and performing ultrasonic modification for 10 min.

The surface appearance of the sample was measured, and the sample was fibrous and had a nanoparticle concentration of 5.6g/L, as shown in FIG. 3.

The particle size was measured and, as shown in FIG. 4, the length was 400nm and the width was 6.0 nm.

Comparative example 3

and step A1, performing magnetic stirring on 2g of manganese acetate, 100mL of ethanol and 100mL of glycol uniformly, performing reaction in a hydrothermal kettle at 120 ℃ for 24h, centrifuging at 10000rpm for min, washing for 3 times, and performing vacuum drying for 24h to obtain a brown solid to obtain the nano manganese-based oxide matrix.

And A2, mixing the nano manganese-based oxide matrix and sodium citrate in ethylene glycol, and performing ultrasonic modification for 1 h.

The appearance of the strain is detected, and the phenotype of the strain is fibrous;

the particle size was measured to be 400nm in length and 6.0nm in width, similarly to comparative example 2.

Comparative example 4

And A2, mixing the nano manganese-based oxide matrix and hexadecyl trimethyl ammonium bromide in ethylene glycol, and performing ultrasonic modification for 1 h.

the particle size was measured and found to be 400nm in length and 6.0nm in width, similar to comparative example 2.

Example 4

In this example, the water solubility test of the nanomaterials prepared in example 1 and comparative examples 1 to 4 was carried out by the following specific operations:

after the nano-mimetic enzyme of example 1 was allowed to stand for 1 hour, it was observed whether or not it precipitated.

10mg of the nanomaterial of comparative example 1 was dispersed in 3mL of pure water, and then left to stand for 1 hour, and then it was observed whether or not precipitation occurred.

1mL of the colloids of comparative examples 2 to 4 was dispersed in 3mL of pure water and then allowed to stand for 1 hour, and then it was observed whether or not precipitation occurred. The test results are shown in table 1.

TABLE 1 Water solubility test of the nanomaterials of the examples and comparative examples

Nano material	Dispersibility	Precipitation conditions
			Example 1	Dispersible	Whether or not
Comparative example 1	Can not be dispersed	Is that
			Comparative example 2	Dispersible	Is that
Comparative example 3	Dispersible	Is that
			Comparative example 4	Dispersible	Is that

The embodiment of the invention is different from the comparative example 1 in that the active polymer surfactant is adopted to modify the nano material, the comparative example 1 adopts hydroxyl groups modified on the surface, and in the preparation process, the polymer surfactant and the divalent manganese salt are mixed firstly, and then the alkaline precipitator is dripped to carry out hydrothermal reaction. As can be seen from the results in Table 1, it was not only dispersed but also stably present in water.

The embodiment of the invention is different from the comparative example 2 in that the nano material is spherical and modifies the macromolecular surfactant, while the comparative example 2 is fibrous, compared with the comparative example 2, the water solubility of the invention is more excellent, and stable colloid can be formed.

The difference between the embodiment of the invention and the comparative examples 3 to 4 is that the surfactant is introduced into the hydrothermal reaction system through the macromolecule, the appearance is spherical, the particle size is controlled to be 5.60nm, and from the results in table 1, compared with the comparative examples 2 to 4, the fibrous surface modified active surfactant has improved dispersibility and stability.

Example 5

The embodiment provides an application experiment for improving the salt tolerance of plants by using pseudo-nano enzyme as a seed soaking agent component, which comprises the following specific operation steps:

after rape seeds of the species of Zhongshuang 11 are disinfected by sodium hypochlorite, the nano pseudoenzyme in the example 1 and the nano pseudoenzyme in the proportion of 2 are diluted by clear water into a seed soaking agent containing 300mg/L nano particles, clear water is adopted for soaking seeds for comparison, the rape seeds are placed in square culture dishes uniformly paved with quartz sand, 30 seeds (5 multiplied by 6) are placed in each culture dish, 200mM NaCl solution is added to ensure that the seeds can be contacted, the number of the germinated seeds is counted every day, and the germination rate is calculated. The germination rates 7 days after seed soaking are shown in table 2, wherein the results of the seed soaking experiments are shown in fig. 5, and it can be seen from the results of table 2 and fig. 5 that the germination rates are further improved by soaking seeds with the pseudonanoenzyme compared with those without the nanoenzyme.

TABLE 2 statistical results of seed germination after seed soaking treatment

Seed soaking agent	Percentage of germination (%)
		Example 1	53.33±2.45
Comparative example 2	22.92±4.52
		Control group	23.89±2.84

Example 6

The embodiment provides an application of improving salt tolerance of a plant as a culture medium component by using a pseudo-nano enzyme, and the specific operation is as follows:

the seeds of Arabidopsis thaliana of Columbia 0 are sown on MS culture medium, and transplanted to new culture medium after 5 days of germination. NaCl is added into a culture medium, the final concentration is 100mM, sterilization is carried out, different contents of the nano enzyme mimic of the embodiment 1 are mixed into different culture media before solidification, solidification is carried out on a flat plate, the culture medium containing 0.5mg/L of the nano enzyme mimic, 2mg/L of the nano enzyme mimic culture medium and 10mg/L of the nano enzyme mimic culture medium are finally obtained, Arabidopsis thaliana is transplanted on the flat plate, the flat plate is sealed by a sealing film and is cultured under light, the root growth condition of the Arabidopsis thaliana is observed after 10 days, and the result is shown in figure 6, wherein 5A is the root growth condition of the Arabidopsis thaliana without adding the nano enzyme mimic, 5B is the root growth condition of the Arabidopsis thaliana of the culture medium containing 0.5mg/L of the nano enzyme mimic, 5C is the root growth condition of the Arabidopsis thaliana of the culture medium containing 2.0mg/L of the nano enzyme mimic, and 5D is the root growth condition of the Arabidopsis thaliana of the nano enzyme culture medium containing 10.0mg/L of the nano enzyme, the result shows that the Arabidopsis seedlings provided with the nano-mimetic enzyme treatment have more lateral roots, the content of the nano-mimetic enzyme in the culture medium is 2.0mg/L, and the root growth condition is more excellent.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The nano-enzyme is characterized in that the nano-enzyme is a spherical manganese-based oxide nano material or colloid containing the spherical manganese-based oxide nano material, and the spherical manganese-based oxide particles are modified with a high-molecular surfactant and have the particle size of 5.0-6.0 nm.

2. The nanophase mimetic enzyme according to claim 1, wherein said polymeric surfactant is one or both of polyacrylic acid and polyethyleneimine.

3. The NanoTase according to claim 1, wherein if the NanoTase is a colloid containing spherical manganese-based oxide nanomaterials, the concentration of the spherical manganese-based oxide nanomaterials in the colloid is 5g/L to 7g/L, and the dispersion solvent is water.

4. The method for preparing a Nanomctosidase according to any one of claims 1 to 3, comprising the steps of:

5. The method of claim 4, wherein the system A solution is added dropwise to the precipitant solution in step S2.

6. The method for preparing nano-mimetic enzyme according to claim 4, wherein in step S2, the precipitant solution is ammonia water, and a colloid containing the spherical manganese-based oxide nanomaterial is obtained after purification by dialysis.

7. The method for preparing nano-mimetic enzyme according to claim 4, wherein the concentration of the divalent manganese salt solution is 0.8mol/L to 1.2mol/L, the concentration of the surfactant solution is 800g/L to 1000g/L, and the volume ratio of the divalent manganese salt solution to the surfactant solution is 1: (1.5-2.5); and purifying the precipitant ammonia water by a dialysis method, wherein the volume ratio of the solution of the system A to the ammonia water is 1: (1.5-2.5).

8. Use of the nanophase quasiplase according to any one of claims 1 to 3 for improving the salt tolerance of seedlings.

9. Use according to claim 8, wherein the nanophase mimicry is used for the preparation of a seed soaking agent or for the preparation of a culture medium.