Acetyl esterase selectively degrading r-indoxacarb enantiomer, gene, producing strain and application thereof

Abstract

The present application relates to the technical field of pesticide residue microbial degradation and contaminated environment remediation, and provides an acetyl esterase capable of selectively degrading R-indoxacarb enantiomer, and the amino acid sequence is shown as SEQ ID NO. 1. The present application also provides a coding gene of the acetyl esterase and a recombinant engineering bacterium comprising the gene. Meanwhile, the present application also provides a preparation method and application of the acetyl esterase. The present application has the advantages that: an acetyl esterase with significant enantiomeric selectivity is found for the first time, the enzyme can preferentially degrade the inactive body R-indoxacarb, has low degrading activity to S-indoxacarb, and can realize selective removal of R-indoxacarb residues in soil, water and agricultural products, and solve the problem of lack of chiral selective degradation in the prior art.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue microbial degradation and environmental remediation technology, and particularly to an acetylesterase that selectively degrades R-indoxacarb enantiomers, its gene, producing strain, and applications. Background Technology

[0002] Indoxacarb is a commonly used carbamate insecticide in my country, primarily used to control lepidopteran pests on crops such as apples, pears, cabbages, kale, and tea. Indoxacarb is a chiral pesticide with two chiral enantiomers: R-indoxacarb and S-indoxacarb. Previous studies have shown significant differences between the two enantiomers in terms of insecticidal activity, non-target biotoxicity, and environmental residual degradation behavior: S-indoxacarb is the effective insecticide but has high acute toxicity to mammals; R-indoxacarb is ineffective and lacks insecticidal activity; S-indoxacarb degrades more readily in alkaline soils, while R-indoxacarb degrades more readily in acidic soils.

[0003] Due to limitations in synthetic pathways and production costs, commercially available indoxacarb in my country is currently mostly a racemic mixture (S-indoxacarb:R-indoxacarb = 1:1) or a mixture of DPX-MP062 (S-indoxacarb:R-indoxacarb = 3:1). After field application, the ineffective R-indoxacarb can easily remain in soil, water, and other environments for a long time. Although its acute toxicity is low, its long-term presence still poses ecological and environmental risks and potential harm to human health.

[0004] Microbial degradation is an important pathway for the degradation of indoxacarb in the environment and a key technical means for the remediation of indoxacarb residue pollution. In recent years, many indoxacarb-degrading strains have been screened, isolated, and identified both domestically and internationally, such as Bacillus cereus from the gut of the diamondback moth. Bacillus cereus It can achieve a 20% degradation rate of 100 mg / L indoxacarb within 6 days; Pseudomonas stearothermia derived from activated sludge ( Pseudomonas stutzeri It can degrade 85% of 20 mg / L indoxacarb within 7 days. However, none of the above-mentioned degrading strains have the ability to selectively degrade specific chiral enantiomers of indoxacarb.

[0005] Previously, our team screened a strain of Bacillus argentis that has the ability to selectively degrade indoxacarb enantiomeric progeny from activated sludge. Bacillus aryabhattai The strain DPX-1 (patent number ZL 202411296737.2) exhibits significantly higher degradation activity against S-indoxacarb than against R-indoxacarb. Based on this, this invention, through whole-genome sequencing, microbial functional verification, and enzymatic experiments, for the first time discovered an acetylesterase from this strain capable of selectively degrading the ineffective form of R-indoxacarb, and obtained pure enzymes through prokaryotic expression and purification. This acetylesterase can selectively and efficiently degrade R-indoxacarb residues in soil, water bodies, and agricultural products, and has significant application value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an acetylesterase that selectively degrades the enantiomer of R-indoxacarb, its gene, producing strain, and application. This acetylesterase has significant enantiomer selectivity and can preferentially degrade the ineffective indoxacarb, R-indoxacarb, while exhibiting low activity in degrading S-indoxacarb. It can achieve selective removal of R-indoxacarb residues in soil, water bodies, and agricultural products, thus solving the problem of the lack of chiral selective degradation in the prior art.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: An acetylesterase that selectively degrades the R-indoxacarb enantiomer, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] A gene encoding the aforementioned acetylesterase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] A recombinant engineered bacterium containing the gene encoding the aforementioned acetylesterase.

[0010] As one of the preferred embodiments of the present invention, the recombinant engineered bacteria uses Escherichia coli as the host, inserts the encoding gene of the acetylesterase into the pET-30a prokaryotic expression vector, transforms the host bacteria, and is then screened to obtain the recombinant engineered bacteria.

[0011] A method for preparing the above-mentioned acetylesterase includes the following steps: (1) Using SEQ ID NO.3 and SEQ ID NO.4 as primers, the acetylesterase encoding gene shown in SEQ ID NO.2 was amplified by PCR; (2) The amplified acetylesterase gene fragment was constructed into the pET-30a prokaryotic expression vector to obtain the recombinant expression vector; (3) Transform the recombinant expression vector into Escherichia coli host bacteria and screen to obtain positive recombinant engineered bacteria; (4) Cultivate the recombinant engineered bacteria and add IPTG to induce acetylesterase expression; (5) After induction, collect the bacterial cells and break them up. Centrifuge to obtain the supernatant, which is acetylesterase.

[0012] As one of the preferred embodiments of the present invention, in step (4), the expression is induced at 16°C for 12 hours.

[0013] As one of the preferred embodiments of the present invention, the method further includes step (6): purifying the obtained acetylesterase by Ni column affinity chromatography to obtain purified acetylesterase.

[0014] Application of the aforementioned acetylesterase in the degradation of R-indoxacarb enantiomers and in the remediation of indoxacarb residue contamination in the environment.

[0015] As one of the preferred embodiments of the present invention, the indoxacarb residue pollutes the environment, including water bodies, soil, and agricultural products.

[0016] A method for degrading R-indoxacarb or remediating environmental pollution caused by indoxacarb residues, utilizing the aforementioned acetylesterase.

[0017] The advantages of this invention compared to the prior art are: (1) This invention utilizes functionally degraded strain Bacillus argentisi. Bacillus aryabhattai A gene for indoxacarb degrading enzyme was cloned from DPX-1. The novel acetylesterase (named Aes) encoded by Aes has high enantioselectivity and can specifically degrade the indoxacarb ineffective form R-indoxacarb. It has low activity in degrading the insecticidal effective form S-indoxacarb, and can achieve precise and selective removal of chiral pesticide ineffective forms, thus making up for the lack of chiral selectivity in existing degradation materials. (2) The present invention successfully constructed a genetically engineered bacterium containing an acetylesterase encoding gene. After induction expression, the crude enzyme obtained by cleavage has excellent selective degradation ability, stable degradation activity, and is easy to prepare and apply. (3) The present invention establishes a simple and efficient purification process. High-purity acetylesterase Aes can be obtained rapidly by Ni column affinity chromatography and imidazole gradient elution. The obtained pure enzyme maintains excellent enantioselective degradation function, which is convenient for enzymatic property research and industrial application. (4) The acetylesterase of the present invention degrades indoxacarb via a detoxicating metabolic pathway. The final degradation product M455 has significantly lower acute and chronic toxicity to non-target aquatic organisms than the parent indoxacarb and intermediate IN-JT333. The degradation process is safe and has no secondary risk. (5) This invention enriches the enantiomeric degradative enzyme resource library of chiral pesticides, and provides a new enzyme preparation and technical solution for the efficient and green remediation of indoxacarb ineffective residues in agricultural products, soil, water and other scenarios. It has important application value in the field of chiral pesticide residue pollution control. Attached Figure Description

[0018] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification product of the acetylesterase gene fragment of this invention (in the image, M: marker; lane 1: amplified). aes (gene fragments) Figure 2This is an SDS-PAGE electrophoresis image of the induced expression, solubility analysis, and affinity purification of acetylesterase Aes of the present invention (in the figure, M: marker; lane 1: bacterial culture before IPTG induction; lane 2: bacterial culture after IPTG induction; lane 3: supernatant after lysis of the bacterial culture after induction; lane 4: precipitate after lysis of the bacterial culture after induction; lane 5: effluent from the supernatant obtained in lane 3 after loading onto a Ni column; lane 6: 20 mM imidazole elution buffer; lane 7: 50 mM imidazole elution buffer; lane 8: 100 mM imidazole elution buffer). Figure 3 This is a kinetic curve of the degradation of indoxacarb by the crude acetylesterase Aes of the present invention; Figure 4 The results of the degradation kinetics determination of R-indoxacarb and S-indoxacarb by the pure acetylesterase Aes enzyme of the present invention are shown in the figure (the red curve is the degradation curve of R-indoxacarb by the pure acetylesterase Aes enzyme, and the blue curve is the degradation curve of S-indoxacarb by the pure acetylesterase Aes enzyme). Figure 5 This invention presents the mass spectra of the degradation products of indoxacarb by pure acetylesterase Aes enzyme, determined by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (Figure A is the mass spectrum of degradation intermediate IN-JT333; Figure B is the mass spectrum of the final degradation product M455). Figure 6 This is a schematic diagram of the two-step hydrolysis pathway of indoxacarb by the acetylesterase Aes of this invention; Figure 7 This invention utilizes the ECOSAR model to assess the acute toxicity (LC50) of indoxacarb and its degradation products (IN-JT333, M455) to non-target aquatic organisms (fish, water fleas, green algae). 50 / EC 50 Heatmap of toxicity and chronic toxicity (ChV) (in the graph, the redder the color, the higher the toxicity, and the greener the color, the lower the toxicity). Figure 8 This is a graph showing the binding curves of pure acetylesterase Aes and the chiral enantiomer of indoxacarb, determined by surface plasmon resonance technology in this invention (Figure A shows the binding curve of acetylesterase Aes with S-indoxacarb; Figure B shows the binding curve of acetylesterase Aes with R-indoxacarb; different colored curves correspond to different substrate concentrations). Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents, methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in this technical field.

[0020] Example 1: Source and Acquisition of Acetylesterase Aes: The acetylesterase Aes of this invention is derived from an indoxacarb-degrading strain obtained in a previous screening. Bacillus aryabhattai DPX-1 (CCTCC NO:M 2024482).

[0021] Our team identified the indoxacarb-degrading enzyme gene from this strain through whole-genome sequencing and functional gene annotation, combined with gene cloning, prokaryotic expression, and enzymatic verification. aes This gene encodes an acetylesterase, named Aes.

[0022] The amino acid sequence of acetylesterase Aes is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. Functional assays have confirmed that this enzyme has selective degradation activity against the R-indoxacarb enantiomer, a discovery made for the first time in this invention.

[0023] Example 2: Cloning of the acetylesterase Aes gene: (1) with Bacillus aryabhattai Using DPX-1 genomic DNA as a template, upstream and downstream primers aes-F and aes-R were designed to target the acetylesterase-encoding gene. aes PCR amplification was performed using primer sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The PCR reaction program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 30s, for 34 cycles; and 72℃ extension for 10min.

[0024] (2) The amplification products were detected by 1% agarose gel electrophoresis. Figure 1 The target fragment was recovered using a gel recovery kit.

[0025] (3) The recovered target fragment was sent to General Biotech (Anhui) Co., Ltd. for sequencing. The sequencing results showed that the target gene was 933 bp in length and the nucleotide sequence was as shown in SEQ ID NO.2. The gene annotation showed that the gene encodes an acetylesterase (EC 3.1.1), named Aes.

[0026] Example 3: Construction of recombinant engineered bacteria: (1) The target gene fragment recovered in Example 2 and the pET-30a prokaryotic expression vector were double-digested using EcoRI and XhoI restriction endonucleases, respectively. The double-digestion system was as follows: 2.5 μL each of EcoRI and XhoI, 5 μL of H-buffer, 25 μL of target gene fragment, and 15 μL of ddH2O; incubated at 37℃ for 1 h; after gel recovery of the digestion products, they were ligated using T4 ligase to obtain the recombinant expression plasmid pET- aes .

[0027] (2) The recombinant expression plasmid pET- aes Transform to E. coli Positive clones were screened using kanamycin-resistant plates in BL21(DE3) competent cells, and the positive clones were sent to General Biotech (Anhui) Co., Ltd. for sequencing. aes Gene sequence. Once verified, the saved positive clone contains the gene encoding the target acetylesterase. aes Recombinant engineered bacteria.

[0028] Example 4: Induced expression of acetylesterase Aes: (1) Pick a single colony of the recombinant engineered bacteria obtained in Example 3 and inoculate it into 50 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C and 150 r / min to obtain seed culture.

[0029] (2) Take 500 μL of seed culture and put it into 50 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37 °C and shake at 150 r / min until OD. 600 The value reached 0.6.

[0030] (3) Add 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to the above bacterial culture to induce acetylesterase Aes expression, and adjust the culture conditions to 16℃, 150 r / min, and induce expression by constant temperature shaking for 12 h.

[0031] (4) Centrifuge the bacterial culture at 4℃ and 8000r / min for 5min and collect the bacterial precipitate; wash and resuspend the bacterial cells three times with 10mM PBS buffer (pH 7.0), and finally resuspend the bacterial cells with 25mL PBS buffer.

[0032] (5) The resuspended bacterial culture was disrupted using an ultrasonic disruptor in an ice bath. The procedure was as follows: 40W power, 2s operation, 2s stop, until the solution became clear. After disruption, the supernatant was collected by centrifugation and filtered through a 0.22µm filter membrane to obtain the crude enzyme solution.

[0033] (6) The expression of acetylesterase Aes was detected by SDS-PAGE (with the pre-induction bacterial culture without IPTG as the control group).

[0034] The results are as follows Figure 2 As shown in the figure. The results showed that, compared with the control group without IPTG, the addition of IPTG significantly induced the expression of the target protein; the expressed target protein was mainly in the supernatant, indicating that the expressed acetylesterase Aes was soluble; the theoretical molecular weight of the acetylesterase Aes protein sequence was approximately 46 kDa, and the displayed target protein band was consistent with the predicted result, indicating that acetylesterase Aes was successfully expressed.

[0035] Example 5: Verification of the degradation of indoxacarb by crude acetylesterase Aes: In this embodiment, the crude acetylesterase Aes prepared in Example 4 was used to verify the in vitro degradation activity and degradation kinetics of indoxacarb. The specific steps are as follows: (1) Prepare multiple identical enzymatic reaction systems (2.5 mL per system), with the same components in each system, including the following: 1.25 mL crude acetylesterase (Aes) solution (Example 4), 1.25 mL PBS buffer (pH 7.0), and commercially available indomethacin racemic mixture at a final concentration of 10 mg / L; after thorough mixing, place in a constant temperature environment of 37°C for reaction. Set up 3 parallel reaction systems, and at the same time set up a negative control group (without adding crude enzyme solution, but replacing it with an equal amount of PBS buffer) to ensure experimental repeatability.

[0036] (2) For each reaction system, samples were taken at five time points: 0h, 3h, 12h, 24h and 28h after the start of the reaction. When sampling, one complete reaction system (2.5mL) was taken at each time point as the test sample. An equal volume of chromatographic acetonitrile (2.5mL) was added to terminate the reaction and extract indoxacarb residues from the sample. After vortexing for 30s, the sample was centrifuged. The supernatant was then filtered through a 0.22µm organic phase filter membrane for instrumental analysis.

[0037] (3) The indoxacarb residue concentration of the samples at each time point was determined by ultra-high performance liquid chromatography, and the degradation rate was calculated; the degradation kinetic curve was fitted with the reaction time as the abscissa and the indoxacarb residue as the ordinate.

[0038] The results are as follows Figure 3As shown in the figure. The results showed that the residual concentration of indoxacarb in the negative control did not decrease significantly within 28 hours, indicating that indoxacarb could be stably maintained under these reaction conditions; while the residual concentration of indoxacarb in the Aes crude enzyme treatment group of this invention showed a significant decreasing trend with the extension of reaction time, and 64.31±0.60% of 10 mg / L indoxacarb could be degraded within 28 hours; further fitting analysis showed that the degradation process of indoxacarb by the Aes crude enzyme conformed to a first-order kinetic model, and the degradation rate constant obtained by fitting was 0.039±0.001 h. -1 The corresponding degradation half-life was 17.48 ± 0.62 h. These results fully demonstrate that the crude acetylesterase Aes obtained in this invention possesses highly efficient indoxacarb degradation activity and can effectively remove indoxacarb residues from the system.

[0039] Example 6: Purification and degradation activity assay of acetylesterase Aes: This embodiment purifies the crude acetylesterase Aes solution prepared in Example 4 and determines its degradation kinetic parameters. The specific steps are as follows: (1) The crude acetylesterase Aes was purified by affinity chromatography column. The crude enzyme solution obtained in Example 4 was loaded onto a Ni NTA Beads 6FF gravity column, washed with washing buffer, and then eluted with different concentrations of imidazole (20mM, 50mM, 100mM). The eluents from each step were measured by SDS-PAGE.

[0040] The SDS-PAGE results are shown below. Figure 2 The results showed that the effluent after loading the crude enzyme solution onto the column no longer contained the target protein, indicating that the acetylesterase Aes had been successfully attached to the Ni column. Adding different concentrations of imidazole could successfully elute the target protein, and the target protein obtained after elution with 100 mM imidazole had a higher concentration and purity.

[0041] The target protein eluted with 100mM imidazole was desalted by ultrafiltration and then dialyzed with 10mM PBS buffer for 12 hours to obtain pure acetylesterase Aes enzyme.

[0042] (2) Degradation kinetics of indoxacarb by purified acetylesterase (Aes) on two chiral enantiomers. In 5 mL glass tubes, indoxacarb enantiomers at a final concentration of 1 mg / L and purified acetylesterase solution at 100 ng / mL were added respectively. The total solution volume was brought up to 1 mL with PBS. All treatments were performed in triplicate. Samples were incubated at 37°C with shaking at 150 r / min, and samples were taken at 0, 6, 12, 18, and 24 h. The reaction was terminated by adding 1 mL of acetonitrile at each sampling. After mixing, the supernatant was centrifuged and filtered through a 0.22 μm organic phase filter membrane. The concentration of indoxacarb was determined by high-performance liquid chromatography (HPLC).

[0043] The results are as follows Figure 4As shown. Compared with the negative control without the addition of degrading enzymes, the purified acetylesterase Aes enzyme exhibited degradative activities for both R-indoxacarb and S-indoxacarb. Its degradation rate for 1 mg / L R-indoxacarb was significantly faster than that for S-indoxacarb, with degradation rate constants of 0.046 h⁻¹. -1 With 0.017h -1 .

[0044] Example 7: Verification of the degradation products of indoxacarb by acetylesterase Aes: In this embodiment, the degradation products of indoxacarb by purified acetylesterase Aes (Example 6) were determined using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q TOF / MS).

[0045] The detection method is as follows: chromatographic column ACQUITY UPLC BEH C 18 (2.1 mm × 100 mm); mobile phase: acetonitrile aqueous solution; flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 5 μL; detection wavelength: 310 nm; gradient elution: 0–1 min 5% acetonitrile aqueous solution, 1–10 min 5% acetonitrile aqueous solution–100% acetonitrile aqueous solution, 10–12 min 100% acetonitrile aqueous solution, 12–15 min 100% acetonitrile aqueous solution–5% acetonitrile aqueous solution.

[0046] Test results as follows Figure 5 As shown: Two degradation products were detected. One is the N-demethoxycarbonyl metabolite IN-JT333 of indoxacarb, which is also the main metabolite and insecticidal active ingredient of indoxacarb in the target insect. The other is product M455, which is a further ester bond hydrolysis product of IN-JT333. This product, namely acetylesterase Aes, can hydrolyze the parent indoxacarb in two steps. The specific degradation pathway is as follows: Figure 6 As shown. This is also the first discovery of an indoxacarb-degrading enzyme with a two-step hydrolysis function.

[0047] Example 8: Prediction of the toxicity of degradation products to non-target aquatic organisms: Building upon Example 7, this embodiment further utilizes the ECOSAR model to evaluate the acute toxicity (LC50) of the two degradation products to non-target aquatic organisms (fish, water fleas, and green algae). 50 or EC 50 ) and chronic toxicity (ChV).

[0048] The results are as follows Figure 7As shown in the figure. The results showed that indoxacarb had high acute and chronic toxicity to aquatic organisms, with acute toxicity to fish, water fleas, and green algae at 0.929 mg / L, 0.811 mg / L, and 0.381 mg / L, respectively. The product IN-JT333 was also highly toxic, but the final product M455 of IN-JT333 under the action of the acetylesterase Aes in this invention showed a significant reduction in toxicity to aquatic organisms, indicating that the degradation process of indoxacarb by acetylesterase is a detoxification process that can reduce the risk of residues.

[0049] Example 9: Determination of the chiral enantiomer affinity of acetylesterase (Aes) to indoxacarb: To verify the enantioselective degradation function of acetylesterase Aes, this example further utilizes surface plasmon resonance to determine the affinity of pure acetylesterase Aes enzyme (Example 6) for two chiral enantiomers of indoxacarb.

[0050] First, the CM5 chip was activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the purified acetylesterase Aes was coupled to the chip and blocked with ethanolamine. After solvent correction, R-indoxacarb and S-indoxacarb were diluted to several concentrations in a 96-well plate and passed through the chip from low to high concentration at a flow rate of 30 μL / min for 60 s. After each concentration point, the chip was regenerated with 10 mM glycine hydrochloride (pH 2.0) solution for 5 min, and this process was repeated until all corresponding concentrations of the compounds were obtained. Finally, the data were fitted to a 1:1 Langmuir binding model using Biacore Insight software (Cytiva, V6.0) to calculate the binding and dissociation constants.

[0051] The results are as follows Figure 8 As shown. The results show that the dissociation constant K of acetylesterase Aes with R-indoxacarb and S-indoxacarb is... D They are 5.43×10 -7 With 3.57×10 -5 This indicates that acetylesterase Aes has a stronger affinity for R-indoxacarb; the binding rate constant K of acetylesterase Aes with R-indoxacarb and S-indoxacarb is... a They are 9.19×10 5 With 2.73×10 2 This indicates that acetylesterase Aes binds to R-indoxacarb more rapidly. This result corroborates the results of the previous examples, fully demonstrating that acetylesterase Aes can selectively degrade the ineffective R-indoxacarb.

[0052] In summary, this invention successfully achieved... Bacillus aryabhattai Acetylesterase gene cloned from DPX-1 aesA recombinant engineered bacterium capable of solublely expressing acetylesterase Aes was constructed. The prepared acetylesterase Aes exhibits highly efficient indoxacarb degradation activity and significant enantiomeric selectivity for the R-indoxacarb ineffective form. This enzyme can degrade indoxacarb to the low-toxicity product M455 through two consecutive hydrolysis steps, achieving attenuation and removal of indoxacarb. Based on this, the acetylesterase Aes of this invention has promising application prospects in the selective degradation and ecological risk control of indoxacarb residues in water, soil, and agricultural products.

[0053] 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 within the protection scope of the present invention.

Claims

1. The application of an acetylesterase in the degradation of R-indoxacarb enantiomers and the remediation of R-indoxacarb residue-contaminated environments, characterized in that, The amino acid sequence of the acetylesterase is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The environmental pollution caused by R-indoxacarb residues includes water bodies, soil, and agricultural products.

3. A method for degrading R-indoxacarb or remediating R-indoxacarb residue pollution in the environment, characterized in that, Acetylesterase with an amino acid sequence as shown in SEQ ID NO.1 was used.

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