Bacillus subtilis carbon nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses bacillus subtilis carbon nano enzyme as well as a preparation method and application thereof. The bacillus subtilis carbon nano-enzyme is prepared by adopting a solvothermal method, and the enzyme is obtained by carrying out high-temperature reaction, dialysis, rotary evaporation, centrifugal concentration, repeated freeze thawing, centrifugation, filtration and the like on bacillus subtilis, reduced glutathione and formamide, and finally carrying out vacuum freeze drying. The bacillus subtilis carbon nano-enzyme prepared by adopting a green and simple method can realize efficient and green degradation of residual pesticides in the environment, can improve the photosynthetic efficiency of plants and promote growth, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of nanoenzyme technology, and in particular to a Bacillus subtilis carbon nanoenzyme, its preparation method, and its application. Background Technology

[0002] Neonicotinoid insecticides (NEOs) are a new class of insecticides that act on the central nervous system of insects, effectively controlling piercing-sucking pests such as aphids, whiteflies, leafhoppers, and thrips, as well as some microscopic lepidopteran and coleopteran pests. After application, only a small portion of NEOs can be absorbed and utilized by crops; the majority enters various environmental media such as soil, water, and sediment. NEOs have a long half-life in soil, high water solubility, and a high detection rate in the environment. The main types of NEOs detected include imidacloprid, thiamethoxam, and thiamethoxam, with imidacloprid being detected at a much higher level than other NEOs, due to its higher market share and frequency of use. Because of their long half-lives in microbial degradation and hydrolysis, photodegradation is the primary pathway for NEOs to degrade in the environment.

[0003] Previous research on carbon nanozymes has primarily focused on carbon dots (CDs) as a substrate, constructing composite nanomaterials by doping them with various heavy metals. These composite materials exhibit spherical nanoparticle morphology, possessing both excellent dispersibility and hydrophilicity. Their surfaces are rich in active groups such as carboxyl, amino, and hydroxyl groups, enabling the formation of stable coordination structures. Based on these characteristics, existing studies have explored the antioxidant and antibacterial properties of nanomaterials synthesized using this method, as well as their application in the degradation of organic dyes. However, none of these studies have addressed the two major directions of pesticide residue degradation and plant growth promotion. Furthermore, the strategy of using heavy metals as dopant not only significantly increases the synthesis cost of the materials but also poses potential environmental risks and negative impacts on plant growth.

[0004] In existing technologies, the degradation methods for imidacloprid pesticides mainly rely on metal doping and modification, often using hydrothermal or solvothermal methods to synthesize degradable materials, which then degrade imidacloprid under light conditions. Most studies report that these materials can achieve degradation rates of over 95%–99% for imidacloprid; however, some techniques have stringent light requirements, demanding specific ultraviolet (UV) light environments to achieve the desired degradation effect. Furthermore, current research has not explored the regulatory effects of the synthesized materials on plant growth, making it difficult to meet the requirements for environmentally friendly agricultural applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a method for preparing Bacillus subtilis carbon nanozymes, characterized by comprising the following steps: S1: Add reduced glutathione and Bacillus subtilis to the reaction vessel, then add formamide, stir evenly and then react to obtain the reaction product; S2: Dialyze the reaction product obtained in S1, purify it by dialysis, evaporate it by rotary evaporation, remove the precipitate by centrifugation, and collect the supernatant. S3: Repeatedly freeze-thaw and centrifuge the supernatant, then filter the solution with a filter membrane, and then freeze-dry it under vacuum to obtain Bacillus subtilis carbon nanozyme.

[0006] Furthermore, the reaction temperature is 160-180°C, and the time is 8-10 hours; the dialysis time is 8-10 days.

[0007] Furthermore, the mass ratio of Bacillus subtilis to reduced glutathione is 1:90-1:100.

[0008] The present invention also provides a Bacillus subtilis carbon nanozyme, which is prepared according to the preparation method described above.

[0009] The present invention also provides an application of Bacillus subtilis carbon nanozyme in the degradation of neonicotinoid insecticides. By doping the carbon nanozyme with Bacillus subtilis, the enzyme activity is improved, and it has the properties of photooxidase under light, which can efficiently degrade imidacloprid.

[0010] Furthermore, the Bacillus subtilis carbon nanozyme degrades neonicotinoid insecticides under light; the light is visible light, and the illumination time is 80-90 minutes.

[0011] Furthermore, the reaction concentration of the Bacillus subtilis carbon nanozyme is 10-30 μg / ml, preferably 25 μg / ml; the pH is 3-11, preferably 7; and the temperature is 25-45°C, preferably 25°C.

[0012] Furthermore, the neonicotinoid insecticide is imidacloprid; the initial concentration of imidacloprid is 1-3 ppm, preferably 1 ppm. As the concentration of imidacloprid increases, the degradation effect of Bacillus subtilis carbon nanoenzyme gradually weakens, showing a negative correlation. A higher degradation rate is observed at a concentration of 1-3 ppm.

[0013] This invention also provides the application of Bacillus subtilis carbon nanozyme in promoting plant photosynthesis.

[0014] Furthermore, the plant is lettuce; the concentration of the Bacillus subtilis carbon nanozyme promoting photosynthesis is 0-30 mg / L. In some embodiments, spraying BS-CD on lettuce can effectively promote the plant's photosynthesis and growth.

[0015] In summary, compared with the prior art, the Bacillus subtilis carbon nanozyme prepared by the present invention using a green and simple method can efficiently degrade imidacloprid and promote the photosynthesis of lettuce, thus having broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of the synthesis of carbon nanozymes in Example 1 of the present invention.

[0018] Figure 2 The image shows the UV-absorbing spectra of TMB, BS-CD+TMB+Light, and BS-CD+TMB at 300-800 nm as detected by a spectrophotometer in Example 2 of this invention.

[0019] Figure 3 This is a kinetic curve of BS-CD nanozyme oxidation of TMB at different substrate concentrations in Example 2 of the present invention.

[0020] Figure 4 This is the Michaelis-Menten fitting curve of BS-CD to TMB concentration in Example 2 of the present invention.

[0021] Figure 5 This is a comparison of the absorbance of BS-CD and GSH-CD at 652 nm before and after the addition of substrate TMB in Example 2 of the present invention.

[0022] Figure 6 This is a graph showing the decrease in the concentration of different BS-CD nanozymes and the dissolution of imidacloprid in Example 3 of the present invention.

[0023] Figure 7 This is a first-order kinetic analysis diagram of the decrease in imidacloprid concentration at different BS-CD nanozyme concentrations in Example 3 of the present invention.

[0024] Figure 8 The graph shows the rate and degradation rate of imidacloprid at different BS-CD nanozyme concentrations in Example 3 of this invention.

[0025] Figure 9 This is a graph showing the degradation curves of imidacloprid by BS-CD nanozyme at different initial concentrations of imidacloprid in Example 4 of the present invention.

[0026] Figure 10This is a first-order kinetic analysis diagram of the degradation of imidacloprid by BS-CD nanozyme at different initial concentrations of imidacloprid in Example 4 of the present invention.

[0027] Figure 11 The graph shows the rate and degradation rate of BS-CD nanozyme degrading imidacloprid at different initial concentrations of imidacloprid in Example 4 of this invention.

[0028] Figure 12 This is a graph showing the degradation curves of imidacloprid by BS-CD nanozymes at different pH levels in Example 5 of the present invention.

[0029] Figure 13 This is a primary kinetic analysis diagram of the degradation of imidacloprid by BS-CD nanozyme at different pH levels in Example 5 of the present invention.

[0030] Figure 14 The graph shows the rate and degradation rate of BS-CD nanozyme degrading imidacloprid at different pH levels in Example 5 of this invention.

[0031] Figure 15 This is a graph showing the degradation curves of imidacloprid by BS-CD nanozymes at different temperatures in Example 6 of the present invention.

[0032] Figure 16 This is a first-order kinetic analysis diagram of the degradation of imidacloprid by BS-CD nanozymes at different temperatures in Example 6 of the present invention.

[0033] Figure 17 The graph shows the rate and degradation rate of BS-CD nanozyme degrading imidacloprid at different temperatures in Example 6 of this invention.

[0034] Figure 18 This is a graph showing the repeated degradation efficiency of BS-CD nanozyme on imidacloprid under optimal conditions in Example 7 of the present invention.

[0035] Figure 19 The diagram shows the degradation pathway of imidacloprid by the BS-CD nanozyme obtained in Example 8 of this invention.

[0036] Figure 20 This is a phenotypic comparison of lettuce at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0037] Figure 21 This is a comparison diagram of lettuce stem length at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0038] Figure 22 This is a comparison diagram of the root length of lettuce under different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0039] Figure 23 This is a comparison chart of the fresh weight of lettuce at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0040] Figure 24 This is a comparison chart of the dry weight of lettuce at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0041] Figure 25 This is a comparison chart of carotenoid content in lettuce at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0042] Figure 26 This is a comparison chart of chlorophyll content in lettuce at different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0043] Figure 27 This is a comparison chart of the soluble sugar content of lettuce under different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0044] Figure 28 This is a comparison chart of the soluble protein content of lettuce under different BS-CD nanozyme concentrations in Example 10 of the present invention.

[0045] Figure 29 The absorption spectra of chloroplasts isolated from lettuce at different BS-CD nanozyme concentrations obtained in Example 10 of this invention are shown.

[0046] Figure 30 The fluorescence emission spectra of chloroplasts isolated from lettuce at different BS-CD nanozyme concentrations obtained in Example 10 of this invention are shown at an excitation wavelength of 438 nm.

[0047] Figure 31 The fluorescence emission spectra of chloroplasts isolated from lettuce at different BS-CD nanozyme concentrations obtained in Example 10 of this invention are shown at an excitation wavelength of 475 nm. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0049] Example 1: Preparation of BS-CD Bacillus subtilis carbon nanozyme (BS-CD) was synthesized using a solvothermal method. The synthetic route is as follows: Figure 1As shown, 21 mg of Bacillus subtilis, 2.1 g of reduced glutathione, and 70 mL of formamide were mixed thoroughly and added to a reaction vessel, then reacted at 160 °C for 8 h. The resulting solution was dialyzed against a 3500 Da dialysis bag for 7 days. After dialysis and purification, most of the solvent was removed by rotary evaporation, and then the solution was concentrated by centrifugation (10000 rpm, 30 min) to remove the precipitate. The freeze-thaw and centrifugation steps were repeated, and the solution was then filtered through a 0.22 μm filter membrane. Finally, the solution was freeze-dried under vacuum for 72 h to obtain a green powder, namely BS-CD.

[0050] Example 2: Detection of BS-CD-like oxidase activity 100 μL of BS-CD (100 μg / mL) and 100 μL of TMB (1 mM) were mixed and brought to a final volume of 1 mL. The mixture was incubated under illumination for 3 min. The absorbance of the reaction system in the wavelength range of 300–800 nm was measured using a UV-Vis spectrophotometer. Under the same experimental conditions, different concentrations of TMB chromogenic substrate (100 μM, 200 μM, 300 μM, 400 μM, 500 μM) were added, and the absorbance at 652 nm was measured every 3 s using a UV-Vis spectrophotometer to characterize the peroxidase activity of BS-CD. Based on the detection results at different substrate concentrations, the kinetics of the BS-CD nanozyme were analyzed. Its classical kinetic parameters, including the maximum reaction rate (…), were calculated using the Lineweaver-Burk double reciprocal method. v max ) and Michaelis constant (K m ).

[0051]

[0052] in ν max This represents the maximum initial velocity, which is determined by... ν Definition. [S] is the concentration of the substrate, and the Michaelis constant is given by K. m K indicates m Used as a measure of enzyme affinity for substrate. K m The lower the value, the greater the affinity between the substrate and the enzyme.

[0053] like Figure 2 As shown, the mixture turns blue and the ultraviolet absorption is significantly enhanced only when TMB+BS-CD+light is present. Figure 3 The results showed that as the substrate concentration increased, more light-absorbing material was produced per unit time, indicating that increasing the substrate concentration promoted the activity of BS-CD nanozymes. Through... Figure 4 Analysis yields its K mThe concentration was 169.03 μM, smaller than the 181.9 μM of the prior art, indicating that BS-CD exhibits good oxidase-like properties under light irradiation. (The text abruptly ends here.) Figure 5 Analysis shows that the oxidase-like activity of nanozyme materials is enhanced after the addition of Bacillus subtilis. The oxidase-like activity of BS-CD is about 207% of that of GSH-CD, which is a carbon nanozyme modified with reduced glutathione without the addition of Bacillus subtilis.

[0054] Example 3: Degradation effect of imidacloprid at different BS-CD concentrations In this application, a xenon lamp was used as the light source, placed 50 cm above the reaction vessel. A constant-temperature magnetic stirrer was used for stirring (300 rpm). BS-CD was prepared at five concentration gradients: 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, and 30 μg / ml. The initial concentration of imidacloprid was 1 ppm. The temperature was controlled at 25℃, pH=7, and the total volume of the reaction solution was 10 ml. The absorbance at 270 nm was measured every 10 minutes using a UV spectrophotometer. The incubation period was 80 minutes in total, with three parallel experimental groups per group. The degradation efficiency (%) was calculated using the following formula:

[0055] The kinetics of the degradation process were evaluated using the following equations:

[0056] Where C0 and C t t represents the concentration of imidacloprid at times t=0 and t=t, and k is the first-order rate constant.

[0057] Figure 6 and Figure 7 The degradation curves and degradation kinetics of imidacloprid at different BS-CD concentrations were shown, and it was found that the degradation effect of imidacloprid was best at a concentration of 25 μg / ml. Figure 8 At a concentration of 25 μg / ml, imidacloprid exhibited the highest degradation rate and degradation efficiency, at 95.42% and 0.0408 min, respectively. -1 .

[0058] Example 4: Degradation effect of imidacloprid at different initial concentrations The difference from Example 3 is that the BS-CD concentration is 25 μg / ml, and the imidacloprid concentrations are 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, and 3 ppm, respectively.

[0059] Depend on Figure 9-10 The data shows that as the concentration of imidacloprid increases, the degradation effect gradually weakens, showing a negative correlation. Figure 11It can be found that when the concentration of imidacloprid is 1.5 ppm, the degradation rate decreases to 79.53%, and the degradation rate is 0.01984 min. -1 When the concentration of imidacloprid was 3 ppm, the degradation rate decreased to 58.5%, and the degradation rate was 0.0087 min. -1 .

[0060] Example 5: Degradation effect of imidacloprid at different pH levels The difference from Example 3 is that the BS-CD concentration is 25 μg / ml, and the pH values ​​of the buffer solutions are set to 3, 5, 7, 9, and 11, respectively.

[0061] Depend on Figure 12-13 It can be seen that imidacloprid achieves optimal degradation at pH 7, and also maintains relatively good degradation performance at pH 5, 9, and 11. Combined with... Figure 14 Further analysis of the data showed that, although the degradation rate of imidacloprid was relatively slow at pH 5, 9, and 11, the final degradation rate still exceeded 90%. These results indicate that BS-CD is only ineffective in degrading imidacloprid under extremely acidic conditions, while exhibiting good degradation performance in other acidic and alkaline environments.

[0062] Example 6: Degradation effect of imidacloprid at different temperatures The difference from Example 3 is that the BS-CD concentration is 25 μg / ml, and the temperatures are 25℃, 30℃, 35℃, 40℃, and 45℃.

[0063] Figure 15-17 The results show that BS-CD has good degradation ability at 25℃ and 30℃, but 25℃ is better than 30℃. As the temperature increases, the degradation rate and degradation percentage decrease continuously, indicating that BS-CD has a better degradation effect at 25℃ and 30℃.

[0064] Example 7: Repeated Cycle Degradation Rate of Imidacloprid by BS-CD The above examples show that the optimal conditions for BS-CD degradation of imidacloprid are: BS-CD concentration of 25 μg / mL, initial imidacloprid concentration of 1 ppm, pH = 7.0, and temperature of 25℃. Under these optimal conditions, the used BS-CD was recovered and reused in degradation experiments with 1 ppm imidacloprid solution, for a total of 5 cycles.

[0065] Figure 18Data shows that the degradation efficiencies of BS-CD in the second to fifth cycles were 88.64%, 81.39%, 69.71%, and 51.34%, respectively. After five cycles, its degradation efficiency remained above 50%, indicating that BS-CD possesses excellent structural stability and catalytic durability, meeting the application requirements for multiple cycles.

[0066] Example 8: Speculation on the degradation of imidacloprid by BS-CD The solution with BS-CD concentration of 25 μg / ml, initial imidacloprid concentration of 1 ppm, pH=7.0, and reaction temperature of 25℃ was collected and ultrafiltered (7000 rpm, 20 min) in a light-protected environment. The ultrafiltered solution was then filtered through a 0.22 μm filter membrane, and finally the degradation products were measured using a triple quadrupole time-of-mass spectrometer.

[0067] Figure 19 The degradation products obtained by BS-CD degradation of imidacloprid were clarified, mainly compound III-(6-chloronicotinic acid (m / z=157)), as well as compound II and some undegraded imidacloprid and other small molecules. The degradation pathway is consistent with other data.

[0068] Example 9: Comparison of BS-CD degradation of imidacloprid with other studies. The degradation performance of BS-Cd on imidacloprid was compared with that of six other known carbon nanomaterial photocatalysts through photodegradation experiments. The specific experimental conditions and results are shown in Table 1.

[0069] Table 1 Comparison of BS-Cd with existing carbon nanomaterials for degrading imidacloprid

[0070] The results in Table 1 demonstrate that the BS-Cd obtained by this invention can efficiently degrade imidacloprid without harsh acid or alkaline environments and under visible light illumination, showcasing its excellent practicality and application potential.

[0071] Example 10: BS-CD promotes photosynthesis in lettuce Lettuce seeds were thoroughly cleaned with ultrapure water and placed in petri dishes lined with moistened filter paper, which was changed every 3 days. After 12 days of cultivation, lettuce seedlings with uniform growth were selected and transplanted into a standard 1 / 2 concentration Hoagland nutrient solution for another 8 days. Seedlings with consistent growth were then selected again and evenly divided into 6 groups. One group served as the control group (H2O), while the other five groups were treated with BS-CD solutions at concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / L, respectively. Each group of plants was sprayed daily with the corresponding solution, with 5 mL sprayed per plant. Plant material was harvested 10 days after treatment. The stem and root lengths of the plants were measured using a ruler, the fresh weight was measured using an electronic balance, the chlorophyll and carotenoid contents were measured after extraction with 95% ethanol, the soluble sugars were measured using the sulfuric acid-anthrone method, the soluble proteins were measured using the Coomassie brilliant blue staining method, and the chloroplasts were isolated and purified using differential centrifugation. All experiments were performed in triplicate, and the experimental data are expressed as mean ± standard deviation.

[0072] like Figure 20-28 As shown, treatment with BS-CD solutions of 5 mg / L, 10 mg / L, 20 mg / L, and 30 mg / L all promoted the growth and photosynthesis of lettuce seedlings. The best growth was observed with BS-CD solution at 20 mg / L, with increases in stem length, root length, fresh weight, dry weight, carotenoid content, chlorophyll content, soluble sugar content, and soluble protein content compared to the WT group (24%, 39%, 40%, 63%, 55%, 29%, 58%, and 33%, respectively). However, with BS-CD solution at 40 mg / L, these indicators decreased by 19%, 8%, 3%, 16%, 1%, 16%, 20%, and 16%, respectively, compared to the WT group. These results demonstrate that BS-CD solution has a dual effect on lettuce seedling growth: at low concentrations (20 mg / L), it significantly promotes growth and physiological activity, while at high concentrations (40 mg / L), it has a certain inhibitory effect.

[0073] Figure 29 The results showed that the chloroplast absorption spectrum pattern after BS-CD treatment was the same as that of the WT group. Compared with the WT group, the chloroplast absorption spectrum after BS-CD treatment at a concentration of 20 mg / L had a stronger absorption intensity in the wavelength range of 300-750 nm. Figures 30-31 The results showed that when chloroplasts isolated from lettuce were excited at wavelengths of 438 or 475 nm, the concentration of 20 mg / L increased by 42% and 28% compared to WT, respectively. This indicates that BS-CD modification more strongly enhances the sensitivity of chlorophyll to light absorption and light energy capture.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing Bacillus subtilis carbon nanozyme, characterized in that, Includes the following steps: S1: Add reduced glutathione and Bacillus subtilis to the reaction vessel, then add formamide, stir evenly and then react to obtain the reaction product; S2: Dialyze the reaction product obtained in S1, purify it by dialysis, evaporate it by rotary evaporation, remove the precipitate by centrifugation, and collect the supernatant. S3: Repeatedly freeze-thaw and centrifuge the supernatant, then filter the solution with a filter membrane, and then freeze-dry it under vacuum to obtain Bacillus subtilis carbon nanozyme.

2. The method for preparing Bacillus subtilis carbon nanozyme according to claim 1, characterized in that, The reaction temperature is 160-180°C, and the time is 8-10 hours; the dialysis time is 8-10 days.

3. The method for preparing Bacillus subtilis carbon nanozyme according to claim 1, characterized in that, The mass ratio of Bacillus subtilis to reduced glutathione is 1:90-1:

100.

4. A Bacillus subtilis carbon nanozyme, characterized in that, It is prepared according to any one of claims 1-3.

5. The application of Bacillus subtilis carbon nanozyme according to claim 4 in the degradation of neonicotinoid insecticides.

6. The application according to claim 5, characterized in that, The Bacillus subtilis carbon nanozyme degrades neonicotinoid insecticides under light; the light is visible light, and the illumination time is 80-90 minutes.

7. The application according to claim 5, characterized in that, The reaction concentration of the Bacillus subtilis carbon nanozyme is 10-30 μg / ml, preferably 25 μg / ml; the pH is 3-11, preferably 7; and the temperature is 25-45°C, preferably 25°C.

8. The application according to claim 5, characterized in that, The neonicotinoid insecticide is imidacloprid; the initial concentration of the imidacloprid is 1-3 ppm, preferably 1 ppm.

9. The application of Bacillus subtilis carbon nanozyme as described in claim 4 in promoting plant photosynthesis.

10. The application according to claim 9, characterized in that, The plant is lettuce; the concentration of the Bacillus subtilis carbon nanoenzyme that promotes photosynthesis is 0-30 mg / L.

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