Method for jointly degrading dicarboximide bactericide by bacterium-enzyme medium system

Abstract

The invention discloses a method for jointly degrading a dicarboximide bactericide through a bacterium-enzyme medium system. According to the method, Pseudomonas sp. F13, laccase and a laccase medium are adopted for jointly degrading the dicarboximide bactericide and metabolites of the dicarboximide bactericide. The preservation number of the pseudoarthrobacter sp. F13 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20241866, and the preservation number of the pseudoarthrobacter sp. F13 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20241866. Experimental results show that under the condition that the pH is 4.8 and 6.0, 100% degradation of the dicarboximide fungicide can be achieved through the method, accumulation of 3, 5-DCA is not detected, the method provides a new way for degradation of procymidone and toxic intermediate metabolite 3, 5-DCA, and the method is simple, easy to operate and suitable for market popularization and utilization.

Description

Technical Field

[0001] The invention belongs to the field of environmental treatment and microbial technology, and particularly relates to a method for degrading dicarboximide fungicides by combining microorganisms with a laccase-mediator system. Background Art

[0002] Dicarboximide fungicides are widely used for field fungal disease control and are low-toxic systemic fungicides. Dicarboximide fungicides enter the human body through food, and their parent and toxic metabolites remain seriously in the environment, posing a threat to the safety of humans and ecosystems. Moreover, 3,5-dichloroaniline (3,5-DCA), the main metabolite of dicarboximide fungicides, is a substance with carcinogenic risk in dietary assessments, which is more toxic and persistent than the parent compound. Therefore, the biodegradation of dicarboximide fungicides and their metabolites has attracted attention.

[0003] Laccase is a Cu-containing 2+ Polyphenol oxidase has a wide substrate specificity and can efficiently degrade a variety of aromatic compound-containing environmental pollutants such as phenols and their derivatives, aromatic amines and their derivatives, carboxylic acids and their derivatives, and steroid hormones. It is considered to be an intelligent, environmentally friendly and potential biocatalytic tool. The laccase and small molecule compounds constitute the laccase mediator system (LMS), which further expands the application range of laccase and shows more efficient degradation ability. However, the combination of laccase-mediator system and functional microorganisms in the biodegradation and biodetoxification of dicarboximide fungicides has not been reported. Summary of the invention

[0004] The present invention provides a method for degrading dicarboximide fungicides by a bacteria-enzyme system. Pseudarthrobacter sp.) F13, laccase, and laccase mediator are used in combination to degrade dicarboximide fungicides and their metabolites, among which Pseudoarthrobacter ( Pseudarthrobacter sp.) F13 was deposited in the China Center for Type Culture Collection on August 28, 2024, with the deposit number CCTCC NO: M 20241866, and the deposit address is Wuhan University, Wuhan, China.

[0005] The laccase addition amount is 2 U / mL, the laccase mediator is a phenolic small molecule compound such as catechol, and the laccase mediator addition amount is 0.5-2 mM.

[0006] The laccase in the present invention is derived from a crude enzyme solution produced by a laccase-producing strain or a conventional commercially available laccase.

[0007] The specific steps of the method of the present invention are as follows: (1) After activation, the slant-stored Pseudomonas aeruginosa F13 was inoculated on LB solid medium, inoculated into LB liquid medium, cultured at 37°C and 150 rpm, centrifuged to collect the cells, washed, and resuspended to obtain a cell suspension; (2) Pseudomonas aeruginosa F13, laccase, and phenolic small molecule compounds were added to a solution containing a dicarboximide fungicide, and the solution was incubated at 30°C and 150 rpm with shaking to degrade the dicarboximide fungicide. The content of the dicarboximide fungicide and 3,5-dichloroaniline in the reaction solution was detected by HPLC.

[0008] Advantages and technical effects of the method of the present invention: The method of the present invention is to treat Pseudoarthrobacter ( Pseudarthrobacter sp.) F13 and laccase, and used to degrade dicarboximide fungicides and their metabolites in the presence of laccase mediators. The experimental results showed that under pH 4.8 and 6.0, the method could achieve 100% degradation of dicarboximide fungicides, and no accumulation of 3, 5-DCA was detected. The method of the invention provides a new way for the degradation of carbendazim and its toxic intermediate metabolite 3, 5-DCA, and the method of the invention is simple and easy to operate, and is suitable for market promotion and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The results of laccase activity detection during the preparation of crude laccase solution; Figure 2 The relative activity of laccase and the growth of cells under different pH reaction conditions; Figure 3 is the degradation rate of 3,5-DCA by the method of the present invention; Figure 4 It is the degradation rate of procymidone by the method of the present invention under different pH conditions. DETAILED DESCRIPTION

[0010] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; GYP liquid medium: 2 g glucose, 5 g yeast extract powder, 5 g tryptone, 1 g MgSO4·7H2O, 1 L distilled water, natural pH, sterilized at 121℃ for 30 minutes; solid medium with 1.5% agar added; MSM liquid culture medium: K2HPO4 0.2g, KH2PO4 0.8g, MgSO4⋅7H2O 0.2g, FeSO4⋅7H2O 0.005g, Na2MoO4⋅2H2O 0.003g, CaCl2 0.1g, (NH4)2SO4 0.5g, distilled water 1L, pH 7.0, solid culture medium plus 2% agar powder.

[0011] Example 1: Pseudoarthrobacter Pseudarthrobacter Preparation of sp. F13 bacterial suspension 1. Soil from a vegetable greenhouse in a farmland in Yuxi County, Yunnan Province, where the pesticide procymidone was used all year round, was collected and crushed through an 80-mesh sieve. 5 g of the sieved soil was added to a 250 mL conical flask containing 50 mg / L procymidone and 50 mL MSM liquid culture medium. The soil was cultured at 37 °C and 150 rpm for 8 hours. d, let the conical flask stand, take the supernatant culture solution, transfer it to the sterilized MSM liquid medium containing 50 mg / L procymidone at a 10% inoculation rate, and continue to acclimate and enrich the culture at 37°C and 150 rpm for 8 days. After repeated enrichment and subculture for 6 times, take the last enrichment culture solution for 10-fold gradient dilution and spread it on the MSM solid medium containing 50 mg / L procymidone. When the single colony with regular colonies grows at 37°C, pick the single colony and streak it on the MSM solid medium containing 50 mg / L procymidone to purify it, and obtain 9 purified strains; the purified strains were inoculated in the MSM liquid medium containing 50 mg / L procymidone, respectively, and cultured at 37°C and 150 rpm for 6 days. The degradation effect of each strain on procymidone was determined by high performance liquid chromatography, and the strain F13 with the highest degradation rate on procymidone was selected. The strain F13 was determined to be Pseudomonas aeruginosa through morphological and molecular identification results. Pseudarthrobacter sp.), named Pseudarthrobacter sp. F13; 2. Store Pseudoarthrobacter at -80℃ ( Pseudarthrobacter sp.) F13 was streaked on LB solid medium for activation and cultured at 37°C. When a large single colony grew on the LB solid medium, a single colony was inoculated into 30 mL of sterilized LB liquid medium and cultured at 37°C with constant temperature and shaking overnight. 600 When the pH value is 1.2-1.4, the culture solution is centrifuged at 8000 rpm for 5 min, the supernatant is discarded, the bacteria are washed twice with 0.9% NaCl solution, and then the bacteria are resuspended with 3 mL of sterile saline to prepare a cell suspension for use.

[0012] Example 2: Preparation of crude laccase solution and activity determination 1. Place the laccase-producing thermotolerant Trametes fungi stored on the slant surface ( Trametes trogii) S0301 picked the bacterial block and inoculated it on GYP solid medium. After culturing at 28℃ for 5 days, the strain was expanded and then the mycelium was transferred to enzyme production medium (glucose 2.8g, tryptone 1g, FeSO4·7H2O 0.0112g, CuSO4·5H2O 0.042g, natural pH, distilled water to 100mL, sterilized at 115℃ for 20min; after sterilization, vitamin B1 with a final concentration of 2mg / L, phenol 0.3054mg / L, Tween-80 60mg / L, and 0.3054mg / L PEG4000, wherein vitamin B1, phenol and Tween-80 are sterilized by filtration with a sterile 0.22 μm filter membrane), cultured at 28°C and 150 rpm constant temperature shaking, and sampling was started to measure the laccase activity in the culture solution on the second day of culture. When the activity of the crude laccase enzyme solution reached the highest, the culture solution was collected, and after centrifugation at 8000 rpm for 5 minutes, the supernatant was collected as the crude laccase enzyme solution; 2. Mix 0.1 mL of crude laccase solution, 0.65 mL of 100 mM phosphate buffer (pH 4.8), and 0.75 mL of 2 mM ABTS reagent, and detect the change in absorbance at 420 nm within 3 minutes; One unit of enzyme activity is defined as the amount of enzyme that oxidizes 1 μmol of ABTS per minute. The laccase activity calculation formula is U (U / L) = N×V1×(A3-A0) / (ε×L) / V2 / t = N×1.5mL×(A3-A0) / 36000 / 0.1×10 6 / 3min, where: N is the dilution multiple; V1 is the total volume of the reaction system; V2 is the total volume of the enzyme in the reaction system; ε is the extinction coefficient, and the ε of ABTS at 420nm is 36000; A0: initial absorbance value; A3: final absorbance value; L: optical path length.

[0013] Results Figure 1 The activity of laccase in the crude laccase solution increased with the increase of culture time and reached the highest level on the 8th day, with a laccase activity of 511.11 U / mL.

[0014] Example 3: Effect of laccase mediators on the stability of crude laccase solution and the growth of F13 cells 1. Add 50 mg / L procymidone, 0.2 U / mL laccase crude enzyme solution, 2 mM laccase mediator, and 0.2 mL of 10 mM citrate buffer to MSM medium (pH 4.8) containing 1% yeast extract. Mix well and add 0.1 mL of Pseudomonas aeruginosa F13 (OD 600=1.2), 0.42mL sterile water to prepare 2mL enzyme-bacteria reaction system, wherein the laccase mediators are 1-hydroxybenzotriazole (HBT), 2,2'-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS), acetosyringone (AS), catechol, and pyrogallic acid; the enzyme-bacteria reaction system is cultured at 30°C and 150rpm for 8 days under aerobic shaking, and samples are taken every 24 hours to detect the laccase activity, and a blank control with only MSM culture medium and buffer added is set at the same time; Laccase relative activity = (A0 / A t )×100%, where A0 is the initial activity of laccase, A t is the laccase activity after incubation; The test results showed that catechol had the least inhibitory effect on the crude laccase enzyme solution, with the residual laccase activity being 58.0% and 37.8% after 1d and 5d, respectively. HBT had a stronger inhibitory effect on the laccase activity, with the residual activity being 15.8% after 5d. Therefore, catechol was selected in this study for the construction of the laccase-F13 bacterial degradation system.

[0015] 2. Add 50 mg / L procymidone, 2 U / mL laccase crude enzyme solution, 2 mM catechol, and 0.2 mL of 10 mM citrate buffer (pH 4.8 or pH 6.0) to MSM medium (pH 4.8 or pH 6.0) containing 1% yeast extract. Mix well and add 0.1 mL of Pseudomonas aeruginosa F13 (OD 600 =1.2), 0.42 mL of sterile water to prepare 2 mL of enzyme-bacteria reaction system; Results Figure 2 , when the system pH was 4.8 and 6.0, the maximum absorbance value of strain F13 was close to 3.0 on the 4th to 5th day and remained stable in the next 3 days; When the system pH was 4.8 and 6.0, the relative activities of laccase on the second day were 98.3% and 89.6%, respectively. In the following 6 days, the laccase activity remained stable, with relative activities exceeding 50.0%.

[0016] Example 4: Degradation experiment of procymidone and its metabolite 3,5-DCA by laccase crude enzyme solution, catechol and Pseudomonas aeruginosa F13 In MSM medium (pH 4.8 or pH 6.0) containing 1% yeast extract, add 50 mg / L procymidone, 2 U / mL laccase crude enzyme solution, 2 mM catechol, and 0.2 mL of 10 mM citrate buffer (pH 4.8 or pH 6.0), mix well, and add 0.1 mL of Pseudomonas aeruginosa F13 resting cells (OD 600 = 1.2), 0.42 mL of sterile water to prepare 2 mL of enzyme-bacteria reaction system; The mixture was cultured at 30°C and 150 rpm, and samples were taken every 24 hours. The contents of procymidone and the intermediate product 3,5-DCA in the reaction system were determined by HPLC, and the degradation rates of procymidone and 3,5-DCA were calculated. The experimental groups were set up as follows: a control group (C0) with the same amount of fungicide added but without the addition of Pseudomonas aeruginosa F13 resting cells, catechol and laccase; a treatment group (C1) with the same amount of fungicide added, the addition of Pseudomonas aeruginosa F13 resting cells and the laccase mediator system t ); A complete blank control group containing an equal amount of MSM culture medium and buffer; Degradation rate D% = (C0-C t ) / C0×100% Results Figure 3 In the pH 4.8 system, catechol and laccase crude enzyme solution can effectively degrade 3,5-DCA, with a degradation rate of 95.0% at 3 h and almost complete degradation (about 99.6%) after 24 h.

[0017] Figure 4 The results showed that in the pH 4.8 system, the Pseudomonas aeruginosa F13 strain combined with catechol to degrade procymidone, with degradation rates of 26.0%, 67.3%, 87.5%, 87.5%, and 100% on the 2nd, 4th, 6th, and 8th days, respectively. In the pH 6.0 system, the degradation rates on the 2nd, 4th, 6th, and 8th days were 70.1%, 89.2%, 100%, and 100%, respectively. In addition, no accumulation of 3, 5-DCA was detected.

[0018] Example 5: Experiment on the degradation of procymidone by commercial laccase and catechol The reaction system of this example is the same as the pH 6 reaction system of Example 4, except that commercially available laccase is used instead of T. trogii The crude laccase solution prepared by S0301 was cultured at 30°C and 150 rpm under aerobic shaking for 8 days. Samples were taken every 24 hours to measure the degradation rate of procymidone. On the 6th day, the degradation rate of procymidone reached 100%.

Claims

1. A method for degrading dicarboximide fungicides by a bacteria-enzyme system, characterized in that: Using Pseudomonas aeruginosa ( Pseudarthrobacter sp.) F13, laccase, and laccase mediators jointly degrade dicarboximide fungicides and their metabolites; among them, Pseudomonas aeruginosa ( Pseudarthrobacter sp.) F13 is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20241866.

2. The method for degrading dicarboximide fungicides by a bacteria-enzyme system according to claim 1, characterized in that: The laccase mediator is catechol.

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