Indoxacarb degrading enzyme and its encoding gene, production bacteria and application

Abstract

The present invention provides an indoxacarb degrading enzyme and its encoding gene, production bacteria and application. The amino acid sequence of the indoxacarb degrading enzyme is shown in SEQ ID NO.1, and the encoding gene sequence is shown in SEQ ID NO.2. The production bacteria is a recombinant engineering bacterium including the above encoding gene. The application is the application of the above indoxacarb degrading enzyme in degrading indoxacarb and repairing indoxacarb-contaminated environments, and a corresponding method for degrading indoxacarb or repairing indoxacarb-contaminated environments. The advantages of the present invention are that the indoxacarb degrading enzyme screened by the present invention can efficiently and quickly degrade indoxacarb, and can also be used to repair water bodies, soil and other environments contaminated by indoxacarb, and has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of biodegradation, and in particular to an oxadiazine insecticide indoxacarb degrading enzyme and its encoding gene, production bacteria and application. Background Art

[0002] Indoxacarb is an oxadiazine insecticide that blocks sodium ion channels. It is widely used to control lepidopteran, homoptera and coleoptera pests in the cultivation of fruits, vegetables and grain crops. However, it is also toxic to non-target organisms. For example, indoxacarb residues have strong lethal and sublethal effects on aquatic animals such as fish and crustaceans, and are harmful to some beneficial insects in agriculture and forestry. At the same time, it produces methemoglobin symptoms in mammals such as humans and is accompanied by chronic toxicity risks. The residues of indoxacarb in the environment not only pose a serious threat to human health, but also bring risks to the environment.

[0003] Microbial degradation is one of the important ways to eliminate indoxacarb in the environment. However, due to the limited resources of indoxacarb-degrading strains obtained through screening, there are few reports on related indoxacarb-degrading enzymes and their catalytic mechanisms. Bacillus aryabhattai DPX-1, and applied to the remediation of polluted environment (patent application number CN202411296737.2). In the present invention, a new indoxacarb degrading enzyme gene was screened by whole genome sequencing, and the genome of strain DPX-1 was used as a template to clone and prepare the corresponding genetically engineered bacteria. The degrading enzyme and its genetically engineered bacteria can be used to quickly and efficiently degrade indoxacarb residues and repair water and soil environments contaminated by indoxacarb residues. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an indoxacarb degrading enzyme and its encoding gene, production bacteria and application.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] An indoxacarb degrading enzyme, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] A gene encoding the above-mentioned indoxacarb degrading enzyme, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] A recombinant engineering bacterium comprises the coding gene of the above-mentioned indoxacarb degrading enzyme.

[0009] As one of the preferred embodiments of the present invention, the specific construction method of the recombinant engineered bacteria is as follows: inserting the gene sequence fragment of the indoxacarb degrading enzyme into the pET-30a expression vector through the EcoRI and NotI restriction sites to obtain a recombinant expression vector; transforming the recombinant expression vector into BL21 competent cells, and using a plate containing kanamycin resistance to screen positive clones to obtain the desired recombinant engineered bacteria.

[0010] A method for preparing the above-mentioned indoxacarb degrading enzyme comprises the following steps:

[0011] (1) synthesizing the nucleotide sequence encoding indoxacarb degrading enzyme shown in SEQ ID NO.2;

[0012] (2) Designing PCR primers to amplify the DNA fragment encoding indoxacarb degrading enzyme, the primers are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0013] (3) constructing a recombinant expression vector containing a DNA fragment encoding an indoxacarb-degrading enzyme, wherein the expression vector is pET-30a;

[0014] (4) Transform the recombinant expression vector into BL21 competent cells, and use kanamycin resistance plates to screen positive clones to obtain recombinant engineered bacteria;

[0015] (5) Cultivating the above-mentioned recombinant engineered bacteria to express indoxacarb-degrading enzyme.

[0016] As one of the preferred embodiments of the present invention, the method further comprises step (6): separating and purifying the degradation enzyme by Ni column affinity chromatography.

[0017] An application of the indoxacarb degrading enzyme in degrading indoxacarb and repairing an indoxacarb-contaminated environment.

[0018] As one of the preferred embodiments of the present invention, the polluted environment includes water and soil.

[0019] A method for degrading indoxacarb or repairing an environment polluted by indoxacarb, utilizing the indoxacarb degrading enzyme YvaK.

[0020] As one of the preferred embodiments of the present invention, a crude enzyme solution or a pure enzyme solution is directly used.

[0021] The advantages of the present invention compared to the prior art are:

[0022] (1) The present invention is based on the indoxacarb degradation strain Bacillus aryabhattaiA new indoxacarb-degrading enzyme was screened from the genome of DPX-1, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.2; the enzyme belongs to the carboxylesterase class (EC 3.1.1.1), and metabolite analysis revealed that it can degrade the indoxacarb parent through the ester bond hydrolysis (demethylation) pathway, which is different from the reported degradation pathway, indicating that it is a new indoxacarb-degrading enzyme;

[0023] (2) The present invention constructs and screens a recombinant engineered bacterium containing a novel indoxacarb-degrading enzyme gene (SEQ ID NO. 2); the crude enzyme of the engineered bacterium after being crushed can degrade 10 mg / L indoxacarb in water by 70.3% within 24 hours, and the degradation efficiency is higher than that of the original strain DPX-1 (24 hours, 10 mg / L degradation by 61.3%); indicating that the engineered bacterium of the present invention has higher degradation activity;

[0024] (3) The present invention further uses affinity chromatography to obtain the corresponding new type of degradation enzyme pure enzyme based on the crude enzyme of genetically engineered bacteria, and the obtained pure enzyme also has the activity of degrading indoxacarb;

[0025] (4) The present invention enriches the pesticide degrading enzyme resource library and has great application value in the restoration of water bodies, soil and other environments contaminated by indoxacarb residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the agarose gel electrophoresis diagram of the YvaK gene cloned by the present invention (in the figure, M: marker; lane 1: target gene yvaK);

[0027] Figure 2 The present invention is an SDS-PAGE diagram of the indoxacarb degrading enzyme YvaK during expression and purification (M: marker; lane 1: uninduced precipitation; lane 2: uninduced supernatant; lane 3: induced precipitation; lane 4: induced supernatant; lane 5: effluent; lane 6: 20 mM imidazole elution; lane 7: 50 mM imidazole elution; lane 8: 100 mM imidazole elution; lane 9: 300 mM imidazole elution; lane 10: 500 mM imidazole elution);

[0028] Figure 3 The degradation kinetics of indoxacarb by the crude enzyme YvaK of the present invention are as follows;

[0029] Figure 4 The degradation kinetics of indoxacarb by the pure enzyme YvaK of the present invention are as follows;

[0030] Figure 5 It is a chromatogram of different degradation times of indoxacarb under the action of the degradation enzyme YvaK of the present invention (in the figure, Figure a is a chromatogram at 0h; Figure b is a chromatogram at 12h);

[0031] Figure 6 This is a mass spectrum of KT413, a degradation product of indoxacarb under the action of the degradation enzyme YvaK of the present invention;

[0032] Figure 7 It is a diagram of the binding pattern of the degradation enzyme YvaK of the present invention and indoxacarb (in the diagram, Figure a is a two-dimensional binding pattern diagram; Figure b is a three-dimensional binding pattern diagram);

[0033] Figure 8 It is a comparison of the degradation activity difference of the degradation enzyme YvaK of the present invention before and after mutation. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, culture media, methods and equipment used in the present invention are conventional reagents, culture media, methods and equipment in the art.

[0035] Example 1: Screening of indoxacarb-degrading enzyme YvaK

[0036] Determination of indoxacarb-degrading strains Bacillus aryabhattai The metabolites of DPX-1 (CCTCC NO: M 2024482) were found to degrade the parent indoxacarb by hydrolysis (demethylation) of the carboxylic acid ester bond. Based on this, esterases or hydrolases were screened in the genome of strain DPX-1, and finally a new indoxacarb degrading enzyme was screened and named YvaK. The enzyme belongs to the carboxylesterase class (EC 3.1.1.1), the amino acid sequence is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2. Metabolite analysis found that it can degrade the parent indoxacarb by ester bond hydrolysis (demethylation) pathway.

[0037] Example 2: Cloning of the indoxacarb degrading enzyme YvaK gene

[0038] (1) Based on strains Bacillus aryabhattai The genomic DNA of DPX-1 was used as a template, and the upstream and downstream primers yvaK-F (SEQ ID NO.3) and yvaK-R (SEQ ID NO.4) were designed to amplify the indoxacarb degrading enzyme YvaK gene by PCR. The PCR reaction procedure was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 30s, 34 cycles; 72℃ extension for 10min.

[0039] (2) After the reaction is completed, use 1% agarose gel electrophoresis to detect the target band, such as Figure 1As shown, the target fragment was recovered using a gel recovery kit and set aside.

[0040] (3) The target gene fragment was ligated with the T-vector enzyme to obtain the plasmid pMD-yvaK, which was transformed into DH5α competent cells. After incubation, positive clones were selected and cultured in LB liquid medium and sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The sequencing results showed that the yvaK gene fragment was 741 bp in length. The domain analysis results showed that the yvaK gene had an esterase domain and was further classified as a carboxylesterase (EC 3.1.1.1).

[0041] Example 3: Construction of recombinant engineering bacteria

[0042] (1) The expression vector pET-30a and the plasmid pMD-yvaK (containing the target gene) of Example 2 were double-digested with EcoR I and Xho I restriction endonucleases, respectively. The expression vector pET-30a and the target gene digestion products were recovered by gel excision, and then the target gene fragments after double digestion were ligated with the expression vector pET-30a using T4 ligase to obtain the recombinant plasmid pET-yvaK.

[0043] (2) Transform the recombinant plasmid into E. coli BL21 (DE3) competent cells were cultured and positive clones were screened using resistance plates containing 50 ng / μL kanamycin.

[0044] (3) Select positive clones and culture them in LB medium for 6 hours. Send the bacterial solution to General Biotechnology (Anhui) Co., Ltd. for sequencing to verify the target gene sequence. After verification, the engineered bacteria containing the degradation enzyme YvaK gene were determined to be the target recombinant engineered bacteria.

[0045] Example 4: Expression of indoxacarb-degrading enzyme YvaK and preparation of crude enzyme

[0046] (1) Take 500 μL of the frozen recombinant engineered bacteria (Example 3) and add it to 50 mL of "LB medium containing 50 ng / μL kanamycin" and culture it at 37°C and 150 rpm for 12 h for activation.

[0047] (2) Transfer 500 μL of bacterial solution to 50 mL of LB medium containing 50 ng / μL kanamycin and culture at 37°C and 150 rpm until OD 600 The value reaches 0.6.

[0048] (3) Add 100 mmol / L IPTG solution to the bacterial solution to a final concentration of 0.5 mmol / L.

[0049] (4) Continue to culture the bacterial solution at 16°C and 150 rpm with shaking, and take 1 mL of the induced bacterial solution after 6 hours.

[0050] (5) Wash the bacterial solution with 50 mmoL PBS buffer and resuspend it three times. Centrifuge at 4°C and 10,000 rpm for 10 min to collect the bacteria. The collected cells are disrupted using an ultrasonic disruptor in an ice bath. The instrument operating procedure is as follows: 200 W power, work for 2 s, stop for 2 s, for a total of 100 times, until the solution becomes clear.

[0051] (6) After the crushing is completed, centrifuge and collect the supernatant to obtain the crude enzyme solution.

[0052] The supernatant and precipitate after the disruption in this example were subjected to SDS-PAGE detection, and the supernatant and precipitate after the disruption without IPTG induction were used as a control to verify the expression of the degradation enzyme.

[0053] The results are as follows Figure 2 As shown in “Lanes 1~4” (in the figure, M: marker; Lane 1: uninduced precipitation; Lane 2: uninduced supernatant; Lane 3: induced precipitation; Lane 4: induced supernatant).

[0054] Depend on Figure 2 The results showed that: (1) compared with the control, after IPTG induction, the Escherichia coli containing the yvaK gene had obvious protein bands, among which the supernatant (lane 4) was clearer than the precipitate (lane 3), indicating that the expressed degradation enzyme YvaK protein was soluble; (2) compared with the protein marker, the molecular weight of the carboxylesterase yvaK protein was about 28 kDa, which was basically consistent with the predicted results, indicating that the carboxylesterase yvaK gene was successfully expressed.

[0055] Example 5: Degradation of indoxacarb by crude YvaK enzyme

[0056] 1.25 mL of YvaK crude enzyme solution (Example 4), 10 mg / L indoxacarb and 1.25 mL of PBS buffer were added to the 2.5 mL reaction system, incubated at 37°C, and samples were taken at 0 h, 3 h, 12 h, 24 h, and 28 h. When sampling, 2.5 mL of chromatographic acetonitrile was added to terminate the reaction, mixed evenly and centrifuged, the supernatant was filtered through a 0.22 µm organic phase filter, and the residual concentration of indoxacarb was determined using an ultra-high performance liquid chromatograph. The group without crude enzyme was set as the control group.

[0057] The results are as follows Figure 3 As shown. Figure 3 The results showed that compared with the control group, the crude enzyme obtained by crushing the same amount of bacteria could degrade 10 mg / L indoxacarb by 70.3% in 24 hours; the degradation curve conformed to the first-order kinetics, and the degradation rate constant was 0.0568 h -1, the degradation half-life is 12h; under the conditions of an initial indoxacarb concentration of 10mg / L and a 20% inoculation amount, compared with the degradation of indoxacarb by the degradation bacteria DPX-1 (degrading 61.3% of indoxacarb in 24h), the crude enzyme in this example has a higher degradation efficiency. At the same time, this is also the first time that the degradation enzyme YvaK has been found to have the activity of degrading indoxacarb.

[0058] Example 6: Purification of indoxacarb-degrading enzyme YvaK

[0059] In order to ensure the remediation efficiency in the actual indoxacarb-contaminated environment, Ni column affinity chromatography was further used on the basis of Example 5 to obtain pure enzyme of the degradation enzyme YvaK.

[0060] (1) The crude enzyme solution was loaded onto a Ni column. After washing with a washing solution, the degradation enzyme protein was eluted with different concentrations of imidazole (20 mM, 50 mM, 100 mM, 300 mM, 500 mM).

[0061] The effluent after washing with washing solution and the elution solution with different concentrations of imidazole were subjected to SDS-PAGE detection. The results are as follows: Figure 2 As shown in lanes 5 to 10 (in the figure, M: marker; lane 5: effluent; lane 6: 20mM imidazole elution; lane 7: 50mM imidazole elution; lane 8: 100mM imidazole elution; lane 9: 300mM imidazole elution; lane 10: 500mM imidazole elution). Among them, 50mM (lane 7) and 100mM (lane 8) imidazole elution effects are the best, and the pure enzyme obtained has fewer impurities and higher concentration.

[0062] (2) Combine and mix the 50mM imidazole and 100mM imidazole eluates, desalt them by ultrafiltration and dialysis, and replace the imidazole buffer in the purification process with PBS buffer: add the eluate to a 30kDa ultrafiltration tube, centrifuge at 4℃ and 4000rpm for 25min, discard the lower layer of waste liquid, add 10mL 10mmol / L PBS buffer, centrifuge at 4℃ and 4000rpm for 25min, discard the lower layer of waste liquid, and repeat this step 3 times. Move the purified protein in the ultrafiltration tube into a dialysis bag, dialyze with 10mmol / L PBS buffer for 12h, and obtain the pure enzyme of the degradation enzyme YvaK.

[0063] Example 7: Degradation of indoxacarb by pure indoxacarb-degrading enzyme YvaK

[0064] 0.1 mg / mL YvaK pure enzyme and 5 mg / L indoxacarb were added to 1 mL PBS buffer, incubated at 37°C, and samples were taken at 0 h, 3 h, 6 h, 15 h, and 24 h. When sampling, 1 mL of chromatographic acetonitrile was added to terminate the reaction, mixed evenly, and centrifuged. The supernatant was filtered through a 0.22 µm organic phase filter membrane, and the residual concentration of indoxacarb was determined using an ultra-high performance liquid chromatograph. The group without pure enzyme was set as the control group.

[0065] The results are as follows Figure 4 As shown. Figure 4 The results showed that the degradation of 5 mg / L indoxacarb by Yvak pure enzyme followed the first-order kinetics (R 2 >0.9), the degradation rate constant k is 0.1072h -1 The half-life was 6.46 h, indicating that the pure enzyme Yvak had the activity of degrading indoxacarb, and the degradation activity was better than that of the crude enzyme solution.

[0066] Example 8: Degradation products of indoxacarb degrading enzyme YvaK

[0067] The residues of indoxacarb parent and its degradation products at different reaction times (0h, 12h) under the action of pure enzyme YvaK were determined by ultra-high performance liquid chromatography. Figure 5 As shown in the figure, the retention time of indoxacarb is 4.339 min, and the retention time of the degradation product is 2.991 min; as the reaction proceeds, the residual amount of indoxacarb parent gradually decreases, and the content of the degradation product gradually increases. The results show that the purified degradation enzyme YvaK can degrade indoxacarb.

[0068] Next, high-resolution mass spectrometry was used to determine the structure of the degradation product under the action of pure enzyme. Figure 6 As shown in the figure: By analyzing the structure of the degradation product, compared with the parent indoxacarb, the product has lost the methyl group on the carboxylic acid methyl ester connected to the indene ring (carboxylic acid ester hydrolysis), and the metabolite is determined to be KT413. This is significantly different from the structure of the main hydrolysis product of indoxacarb JT333 (DCJW) reported previously, indicating that the degradation pathway is different, indicating that the degradation enzyme YvaK is a new indoxacarb degradation enzyme.

[0069] Example 9: Modeling and molecular docking of indoxacarb-degrading enzyme YvaK

[0070] (1) The AlphaFold2 model was used to model the structure of the degradation enzyme YvaK, ​​and the protein structure was used as the initial structure for subsequent molecular docking. The initial structure was processed using AutoDock Tools 1.5.6, the original charge of the protein was preserved, and a pdbqt file was generated for docking.

[0071] (2) Chem3D was used to draw the structure, and then the MOPAC program was used to optimize the molecular structure and calculate the PM3 atomic charges for subsequent molecular docking. AutoDock Tools 1.5.6 was used to process the ligand structure and generate a pdbqt file for docking.

[0072] (3) Molecular docking was performed using the AutoDock 4.2.6 software package. The center coordinates of the docking box were set to the center of the protein to enclose the entire protein structure. The number of grid points in the X, Y, and Z directions was set to 100 × 100 × 100, the number of dockings was set to 50, and the remaining parameters used the default values.

[0073] (4) The energy optimization of the docking results was performed using the Amber14 force field. The optimization process was carried out in two steps: first, 1000 steps of the steepest descent method were used for optimization, and then 5000 steps of the conjugate gradient method were used to further optimize the structure. The final result was used as the model for subsequent analysis.

[0074] The docking results are as follows Figure 7 As shown. Figure 7 It can be seen that there is a strong binding energy between indoxacarb and the degradation enzyme YvaK (-7.712 kcal / mol); indoxacarb can bind to the active pocket composed of Tyr104, Gly103, Val106, Pro107, Ala182, Asp245, Pro183, Tyr181, Ile108 and Thr211 amino acid residues in the degradation enzyme YvaK; further analysis of the interaction between the two showed that indoxacarb binds to the amino acid residues in the active pocket mainly through hydrophobic interaction and hydrogen bonding ( Figure 7 a).

[0075] Example 10: Point mutation and activity determination of indoxacarb degrading enzyme YvaK

[0076] In order to verify the catalytic active site obtained in Example 9, the Gly103 residue that forms a hydrogen bond with indoxacarb in the active pocket of the degradation enzyme YvaK was subjected to point mutation by point mutation method.

[0077] The expression vector pET-YvaK obtained in Example 3 was used as a template, and the Gly103 residue was used as a mutation point. PCR amplification was performed using overlapping primers Gly103Ile-F and Gly103Ile-R (the sequences of primers Gly103Ile-F and Gly103Ile-R are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively). 1µL of DMT enzyme was added to the PCR product to digest the methylated plasmid (non-mutant plasmid template). After reacting at 37°C for 1h, 5µL of DMT enzyme digestion product was taken into 50µL DMT competent cells, and after ice bathing for 25min, heat shock was performed in a 42°C water bath for 45s, and immediately placed on ice for 2min. Then 250µL of LB medium cooled to room temperature was added, and the culture was shaken at 37°C and 150r / min for 1h. Positive mutants were screened using kanamycin resistance plates, and the degradation enzyme activity after mutation was determined after sequencing verification.

[0078] 1.25 mL of crude enzyme solution before and after mutation, 10 mg / L indoxacarb and 1.25 mL of PBS buffer were added to the 2.5 mL reaction system, and the mixture was incubated at 37 °C. Samples were taken at 0 h and 24 h. When sampling, 2.5 mL of chromatographic acetonitrile was added to terminate the reaction, and the mixture was mixed evenly and centrifuged. The supernatant was filtered through a 0.22 µm organic phase filter membrane, and the residual concentration of indoxacarb was determined by ultra-high performance liquid chromatography. The group without degradative enzyme was set as the control group.

[0079] Point mutation results are as follows Figure 8 As shown. Figure 8 The results showed that compared with the control group, the wild-type degradation enzyme YvaK could degrade 10 mg / L indoxacarb by 59.59% in 24 hours, while the mutant degradation enzyme could not degrade indoxacarb (with no significant difference compared with the control), indicating that the Gly103 residue is the key site for the degradation enzyme YvaK to catalyze the degradation of indoxacarb.

[0080] In summary, the degradation enzyme YvaK of the present invention, whether it is a crude enzyme or a pure enzyme, can efficiently and quickly degrade indoxacarb, and can also be used to repair water bodies, soil and other environments contaminated by indoxacarb, and has important application value.

[0081] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of an indoxacarb degrading enzyme in degrading indoxacarb and repairing an indoxacarb-contaminated environment, characterized in that: The amino acid sequence of the indoxacarb degrading enzyme is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The polluted environment includes water and soil.

3. A method for degrading indoxacarb or repairing an environment polluted by indoxacarb, characterized in that: The indoxacarb degrading enzyme having an amino acid sequence as shown in SEQ ID NO.1 is used.

4. The method for degrading indoxacarb or repairing an environment polluted by indoxacarb according to claim 3, characterized in that: Directly use crude enzyme solution or pure enzyme solution.

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