Use of an amidase gene and the protein encoded thereby in degrading propanil

Abstract

The present application belongs to the technical field of biological remediation and enzyme catalytic reaction. More particularly, it relates to the application of an amidase gene and the protein coded by the gene in degrading propanil. The present application first clones and identifies a novel amidase gene arfP from Pandora, and the amidase ArfP coded by the gene has excellent catalytic degradation activity on the amide herbicide propanil, and can efficiently remove the propanil residues in the agricultural environment. The amidase ArfP of the present application can be widely applied in the biological remediation of propanil contaminated environment, and is suitable for various habitats such as farmland soil, irrigation water and underground water contaminated by propanil, and provides a safe and efficient new tool for biological remediation for green agricultural product production and ecological safety protection, and has good application prospect and popularization value.

Description

Technical Field

[0001] This invention belongs to the field of bioremediation and enzyme catalysis technology. More specifically, it relates to the application of an amidase gene and its encoded protein in the degradation of barnyardgrass. Background Technology

[0002] Propanil is a widely used herbicide for grassy weeds worldwide. It works by inhibiting electron transport in the photosystem II of weeds, causing chlorosis and yellowing of leaves, ultimately leading to weed death. Due to its advantages such as broad spectrum of weed control, rapid effect, and relatively high safety for rice, propanil remains one of the most widely used herbicides in rice-growing areas.

[0003] However, the excessive and inappropriate application of propanil has led to serious environmental and ecological safety problems. After application in paddy fields, propanil is highly susceptible to migration via surface runoff and seepage, causing complex pollution of surface water, groundwater, and soil, and exhibiting severe acute toxicity to various aquatic organisms. Studies have detected propanil concentrations in irrigation water as high as 3600 μg / L, far exceeding the maximum permissible concentration (1 mg / L) in aquatic environments. A safety assessment report from the European Food Safety Authority indicates that propanil poses a high acute risk to herbivorous birds, can have toxic effects on mammals and fish within certain concentration ranges, and can adversely affect the human immune system; acute poisoning can cause methemoglobinemia, which can be fatal in severe cases. Therefore, how to efficiently and safely remove propanil residues from the agricultural environment has become a crucial issue urgently needing to be addressed in the fields of pesticide residue management and ecological environmental protection.

[0004] Currently, among the various strategies for pesticide residue remediation, microbial degradation is widely recognized as the most promising technological approach due to its advantages such as environmental friendliness, low cost, and strong sustainability. The degradation of pesticides by microorganisms essentially relies on the catalytic hydrolysis reaction catalyzed by specific enzymes within the microorganisms. Therefore, discovering novel amidases capable of efficiently hydrolyzing the amide bonds of propargite is of great significance for developing green bioremediation technologies for propargite residues. Summary of the Invention

[0005] The present invention aims to provide a novel amidase gene arfP and its encoded amidase ArfP, which can specifically degrade phoxim and efficiently remove phoxim residues in the agricultural environment, providing a new enzymatic tool for the bioremediation of phoxim-contaminated environments.

[0006] The first objective of this invention is to provide an amidase ArfP.

[0007] A second objective of this invention is to provide a polynucleotide.

[0008] A third objective of this invention is to provide an expression vector.

[0009] The fourth objective of this invention is to provide a strain expressing the amidase ArfP protein.

[0010] A fifth object of the present invention is to provide the application of the above-mentioned amidase ArfP, the above-mentioned polynucleotide, the above-mentioned expression vector, or the above-mentioned amidase ArfP protein expression strain.

[0011] The sixth object of the present invention is a product for degrading barnyardgrass.

[0012] The seventh object of the present invention is a method for degrading barnyardgrass.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides an amidase ArfP, the amino acid sequence of which is shown in SEQ ID NO.2.

[0014] As an alternative implementation, the preparation method of the above-mentioned amidase ArfP is as follows: cloning the nucleotide sequence encoding the above-mentioned amidase ArfP into a prokaryotic expression vector to construct a recombinant prokaryotic expression plasmid, and then introducing the recombinant prokaryotic expression plasmid into a prokaryotic expression system for expression to obtain the amidase ArfP.

[0015] Specifically, the prokaryotic expression vector is pET-28b.

[0016] Specifically, the prokaryotic expression system is Escherichia coli.

[0017] The present invention provides a polynucleotide comprising a nucleotide sequence encoding the above-mentioned amidase ArfP.

[0018] As an alternative implementation, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO.1.

[0019] The present invention provides an expression vector comprising the above-mentioned polynucleotides.

[0020] The present invention provides an ArfP protein expression strain containing the above-mentioned polynucleotide or the above-mentioned expression vector.

[0021] This invention clones an amidase from Pandora bacteria, the amino acid sequence of which is shown in SEQ ID NO.2. This enzyme has a significant ability to degrade propanil and can be used to remove propanil residues from the environment. Therefore, this invention claims protection for the following applications: Application of the above-mentioned amidase ArfP or the above-mentioned amidase ArfP protein-expressing strains in the degradation of phoxim or the remediation of phoxim contamination environments.

[0022] The application of the above-mentioned amidase ArfP, the above-mentioned polynucleotide, the above-mentioned expression vector, or the above-mentioned amidase ArfP protein expression strain in the preparation of products that degrade tardigrade.

[0023] The application of the above-mentioned amidase ArfP, the above-mentioned polynucleotide, the above-mentioned expression vector, or the above-mentioned amidase ArfP protein expression strain in the preparation of products for the remediation of environments contaminated by tarantula.

[0024] The present invention provides a product for degrading barnyardgrass, comprising the above-mentioned amidase ArfP or the above-mentioned amidase ArfP protein-expressing strain.

[0025] This invention provides a method for degrading barnyardgrass, using the above-mentioned amidase ArfP or the above-mentioned product for treatment.

[0026] The present invention has the following beneficial effects: This invention is derived from Pandora bacteria ( Pandoraea pnomenusa A novel amidase gene, arfP, was cloned and identified in pET-28b / E. coli The BL21(DE3) prokaryotic expression system successfully yielded a biologically active recombinant amidase ArfP protein. The amidase ArfP of this invention exhibits excellent catalytic degradation activity against the amide herbicide propargite. Experimental results show that after 16 h of reaction between amidase ArfP and propargite, the propargite content in the reaction system significantly decreased, with a degradation rate as high as 83.78%.

[0027] The amidase ArfP and its expression strain provided by this invention can be widely used in the bioremediation of environments contaminated with pampas grass, including farmland soil, irrigation water, and groundwater contaminated with pampas grass. Treatment with this enzyme or its expression strain can effectively reduce pampas grass residue levels in the environment, providing a safe and efficient new bioremediation tool for green agricultural production and ecological security protection, with promising application prospects and widespread application value. Attached Figure Description

[0028] Figure 1 amidase gene arfP A diagram illustrating the cloning expression strategy.

[0029] Figure 2 Genetically engineered bacteria E. coli GC-MS chromatogram of BL21(DE3)-ArfP degrading *Pteris vittata*; Figure A is the GC-MS chromatogram of the sample with added *Pteris vittata* stock solution but without culture; Figure B is the GC-MS chromatogram of the sample after 36 h of culture.

[0030] Figure 3 Genetically engineered bacteria E. coli Mass spectrometry results of the parent compound, *Pteris vittata*, in the degradation of *Pteris vittata* catalyzed by BL21(DE3)-ArfP.

[0031] Figure 4 Genetically engineered bacteria E. coli 3,4-DCA mass spectrometry results of the metabolites of BL21(DE3)-ArfP catalyzed degradation of phoxim.

[0032] Figure 5 SDS-PAGE image of unpurified amidase ArfP; Lane M: Marker; Lane 1: E. coli Electrophoresis results of the supernatant of BL21(DE3)-empty vector strain; Lane 2: E. coli Precipitation electrophoresis results of BL21(DE3)-empty vector strain; Lane 3: engineered strain E. coli Electrophoresis results of BL21(DE3)-ArfP supernatant; Lane 4: engineered bacteria E. coli Precipitation electrophoresis results of BL21(DE3)-ArfP.

[0033] Figure 6 The results of HPLC determination of the degradation effect of ArfP enzyme on barnyardgrass.

[0034] Figure 7 SDS-PAGE images of purified amidase ArfP: Lane 1: Electrophoresis results of purified and concentrated ArfP protein; Lane 2: Electrophoresis results of loading buffer; Lane 3: Electrophoresis results of flow-through buffer; Lane 4: Electrophoresis results of 60 mmol / L imidazole elution buffer; Lane 5: Electrophoresis results of 100 mmol / L imidazole elution buffer; Lanes 6-7: Electrophoresis results of 200 mmol / L imidazole elution buffer; Lane 8: Electrophoresis results of 300 mmol / L imidazole elution buffer. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] The plasmid miniprep kit is manufactured by Kangwei Century Biotechnology Co., Ltd., and its catalog number is CW0500M.

[0038] The ClonExpress® Ultra One Step Cloning Kit is manufactured by Nanjing Novizan Biotechnology Co., Ltd., and its product number is C115-01.

[0039] Bacterial genomic DNA extraction kit, manufactured by Tiangen Biotech Co., Ltd., catalog number DP302.

[0040] Luria-Bertani medium (LB, g / L): yeast extract 5.0 g, peptone 10.0 g, sodium chloride 10.0 g. Luria-Bertani solid medium: 15 g agar powder per 1 L of medium.

[0041] The buffer formulations described in the following examples are as follows: PBS (phosphate buffered saline solution, pH 7.4): 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4.

[0042] Pandora bacteria ( Pandoraea pnomenusa ZQ05 has been disclosed in Chinese patent application CN115261254A.

[0043] BCA protein concentration assay kit, manufactured by Novizan, catalog number E112.

[0044] Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0045] Enemy barnyard grass, CAS No.: 709-98-8, structural formula:

[0046] 3,4-Dichloroaniline (3,4-DCA), CAS No.: 95-76-1, has the following structural formula:

[0047] Example 1: Nucleotide and amino acid sequences of amidase ArfP In this embodiment, the amidase ArfP was derived from Pandora (Bacteria spp.) Pandoraea pnomenusa ZQ05.

[0048] The nucleotide sequence of the amidase ArfP is shown in SEQ ID NO.1. SEQ ID NO.1:

[0049] The amino acid sequence of the amidase ArfP is shown in SEQ ID NO.2. SEQ ID NO.2: MGKSHSPVHWKSAAEIVELVKSKQISPREVVESTIDLIEQRDPGLNAVVYKAYDEAREKAAALERRIMQGEPVGMLAGVPTLIKDMFVPKPGWPYTLGGIRALKDARGAAGVWSTYPLKMSGED SLLLGQTNSPVYGFRGTTDNTFFGPTRNPFNLDFNAGGSSGGAAALVADGIVPVAGGTDGGGSIRNPAAWTNTYGFQPSIGRVPFKSRPNAFHPGPYIYEGPITRTVRDAALAMNVLHGFDRRD PASLRVKLDFTSALAQGVRGKKIGTLLNYGVFPVQQEIQDLIGKAARVFTELGAHVEFVDLGIPYSQKQMSDAWCRMVAIPTVASMQALRKEGIDLYGEHRADIPDALMKWIDAVADISVQQIS ADQLLRTTVFDCMNSVFDHFDLLLAPTLACMPVRNATDGCTEGPSQINGEEIDPLIGWCMTYLTNFSGHPSASVPAGLIDGLPAGMLIIGDRQADLDVIAASAAFEQASPWSQYYDIPAGRPL*.

[0050] Example 2 Construction of an expression vector for amidase ArfP protein The flowchart for constructing the ArfP protein expression vector for amidase is as follows: Figure 1 As shown.

[0051] 1. Plasmid extraction Genomic DNA of Pandora bacillus ZQ05 was extracted according to the instructions of the bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.) and used as a template for subsequent PCR amplification.

[0052] pET-28b plasmid was extracted according to the instructions of the plasmid miniprep kit (Kangwei Century Biotechnology Co., Ltd.). The extracted pET-28b plasmid DNA was dissolved in the kit's TE buffer, and the purity and concentration of the plasmid DNA were detected using a Nannodrop spectrophotometer. The extracted pET-28b plasmid was subjected to double enzyme digestion to obtain a linearized plasmid. The digestion temperature was 37℃, and the digestion time was 2 h. The specific digestion reaction system is shown in Table 1. The linearized pET-28b plasmid was obtained.

[0053] Table 1. Double enzyme digestion reaction system and conditions

[0054] 2. Amplification and recovery of the target fragment Using the genomic DNA of Pandoraella ZQ05 as a template, specific primers were designed and synthesized for PCR amplification of the amidase ArfP gene fragment. The specific primer sequences are shown in Table 2. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4. After the amplification reaction, 1% agarose gel electrophoresis was used to verify whether the target band met the expected size. Then, depending on the specificity, either routine product recovery or gel excision was selected to obtain the target fragment.

[0055] Table 2. Specific primers for PCR amplification of the ArfP gene fragment of amidase.

[0056] Table 3 PCR reaction system

[0057] Table 4 PCR reaction procedure

[0058] 3. Ligation of plasmid vector and target fragment The linearized pET-28b plasmid and the target fragment were subjected to seamless cloning based on the Gibson recombination principle. The plasmid vector and the target fragment were ligated according to the instructions of the ClonExpress® Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to obtain the expression vector.

[0059] 4. Construction and validation of expression strains The expression vector was transformed into *E. coli* expression strain E. coli BL21(DE3) to obtain transformants. Single transformants were picked and cultured in LB broth containing 50 mg / L kanamycin at 37°C and 200 rpm using a shaker. PCR was used to verify the correctness of the obtained strain. The successfully transformed positive clones were named as follows: E. coli BL21(DE3)-ArfP.

[0060] Example 3 Identification of degradation products I. Preparation of the degradation reaction system The engineered bacteria from Example 2 E. coliBL21(DE3)-ArfP was inoculated into LB medium and cultured overnight by shaking to obtain a seed culture. The seed culture was transferred at a ratio of 1:20 (v:v) to 20 mL of LB medium containing 100 mg / L Kan, and cultured until OD600 = 0.6 ± 0.1. IPTG was added to a final concentration of 0.5 mM, and induction was performed at 16℃ and 120 rpm for 16 h. Then, propanil stock solution (10000 mg / L, methanol as solvent) was added to a final concentration of 25 mg / L, and culture was continued for 36 h to obtain the culture medium. The culture medium was centrifuged at 12000 rpm for 5 min, and the supernatant was collected as the sample to be tested.

[0061] II. Sample Extraction and GC-MS Detection Conditions Accurately pipette 1.0 mL of the supernatant (sample to be tested), add 2.0 mL of dichloromethane, and vortex extract for 2 min. After standing and separating the layers, collect the lower organic phase and dry it overnight in a fume hood. Redissolve the residue in 1.0 mL of chromatographic grade methanol, vortex to mix, filter through a 0.22 μm organic microporous membrane, and transfer to a chromatographic vial for analysis.

[0062] The samples were analyzed using gas chromatography-mass spectrometry (GC-MS). The specific method is as follows: The injection port temperature was set to 220℃; the injection volume was 1 μL. High-purity helium was used as the mobile phase carrier gas at a flow rate of 1.2 mL / min. The gas chromatography column used was an HP-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm), and the detector was an Agilent 6890N / 5977B.

[0063] The column temperature program for sample analysis was designed as follows: 50℃ for 1.5 min; increase to 150℃ at 40℃ / min and hold for 1 min; then increase to 200℃ at 10℃ / min and hold for 6 min; finally, heat to 260℃ at 50℃ / min and hold for 6 min. The injection port temperature was set to 220℃, and the ion source temperature and inner surface temperature of the mass spectrometer were both set to 250℃, with a solvent delay of 3 min.

[0064] The target compound was identified by comparing its mass spectrum with that of standard compounds in the National Institute of Standards and Technology (NIST) database and combining the degree of matching with the molecular weight of the ion peaks.

[0065] III. Experimental Results Genetically engineered bacteria E. coli The GC-MS chromatogram of BL21(DE3)-ArfP degrading phoxim is shown below. Figure 2 As shown, genetically engineered bacteria E. coliThe mass spectrometry results of the parent compound *Pterostilbene* catalyzed by BL21(DE3)-ArfP for degradation are as follows: Figure 3 As shown, genetically engineered bacteria E. coli The 3,4-DCA mass spectrometry results of the metabolite catalyzed by BL21(DE3)-ArfP for the degradation of phellandrene are as follows: Figure 4 As shown. The results indicate that the genetically engineered bacteria E. coli BL21(DE3)-ArfP can degrade barnyardgrass to generate 3,4-DCA. GC-MS total ion chromatogram showed that barnyardgrass eluted at 7.9 min (m / z 217.0), while 3,4-DCA eluted at 5.5 min (m / z 161.0). The NIST database clearly supports the view that 3,4-DCA is the major metabolite of barnyardgrass.

[0066] Example 4: Quantitative Analysis of Dianthus parasiticus and 3,4-DCA I. Verification of the Inducible Expression and Degradation Capacity of Engineered Bacteria 1. Preparation of the degradation reaction system Experimental group: The engineered bacteria from Example 2 E. coli BL21(DE3)-ArfP was inoculated into LB medium and cultured overnight by shaking to obtain a seed culture. The seed culture was then transferred at a ratio of 1:20 (v:v) to 20 mL of LB medium containing 100 mg / L Kan and cultured until OD500. 600 =0.6±0.1, add IPTG to a final concentration of 0.5 mM, and induce for 16 h at 16℃ and 120 rpm. Then add propargite stock solution (10000 mg / L, methanol as solvent) to a final concentration of 50 mg / L and continue culturing for 16 h to obtain the culture medium. Centrifuge the culture medium at 12000 rpm for 5 min and collect the supernatant as the sample to be tested.

[0067] Control group: Empty vector pET-28b was transformed into Escherichia coli expression strain E. coli In BL21(DE3), we obtain E .coli BL21(DE3) - empty vector strain. The difference between the control group and the experimental group is that the engineered strain... E. coli BL21(DE3)-ArfP replaced with E. coli BL21(DE3)-empty vector strain.

[0068] 2. Sample extraction and volume adjustment Liquid-liquid extraction of the reaction supernatant was performed using dichloromethane. The specific method was as follows: 1 mL of supernatant (sample to be tested) was taken, and twice the volume of dichloromethane was added. The mixture was vortexed for 2 min to mix thoroughly. The mixture was allowed to stand to separate the aqueous phase (upper layer) and the organic phase (lower layer). The aqueous phase solution was discarded, and the lower organic phase was evaporated to dryness. 1 mL of chromatographic grade methanol was added, and the mixture was vortexed for 1 min to dissolve thoroughly. The supernatant was then drawn through a 0.22 μm microporous membrane using a syringe to prepare the sample to be tested. The sample was placed in a chromatographic vial for testing and was used to determine the residues of propanil and 3,4-DCA.

[0069] 3. Determination by high performance liquid chromatography (HPLC) The residue of argentamycin in the sample was determined using high performance liquid chromatography (HPLC). Specific detection conditions were as follows: The stationary phase was a Kinetex® 5µm PFP 100Å LC column (250×4.60 mm, Phenomenex), and the mobile phase was methanol:water (7:3, v:v); the flow rate was 1 mL / min; the injection volume was 10 μL; the column temperature was 25℃, and the detection wavelength was 250 nm.

[0070] 4. SDS-PAGE electrophoresis engineered bacteria E. coli BL21(DE3)-ArfP and E. coli The BL21(DE3)- empty vector strain was inoculated into LB medium and cultured overnight by shaking to obtain seed culture. The seed culture was then transferred at a ratio of 1:20 (v:v) to 20 mL of LB medium containing 100 mg / L Kan and cultured until OD500. 600 =0.6±0.1, add IPTG to a final concentration of 0.5 mM, and induce for 16 h at 16℃ and 120 rpm to obtain bacterial culture. Pipette 1000 μL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 3 min at 4℃, discard the supernatant, and collect the bacterial cells in a new clean centrifuge tube. Add 100 μL of RIPA protein lysis buffer to the bacterial cells, mix well, and incubate on ice or at 4℃ for 30 min; centrifuge the resulting cell lysis buffer at 12000 rpm for 3 min at 4℃, and collect the supernatant in a new centrifuge tube; resuspend the precipitate in 100 μL of sterile water. The supernatant and precipitate are used for SDS-PAGE analysis.

[0071] II. Experimental Results SDS-PAGE electrophoresis results are as follows: Figure 5 As shown, the results indicate that engineered bacteria E. coli The molecular weight of the recombinant protein ArfP expressed by BL21(DE3)-ArfP is consistent with the previously predicted value (approximately 58.2 kDa).

[0072] The HPLC results of the effect of amidase ArfP on the degradation of phoxim are as follows: Figure 6 As shown, the results indicate that the content of *Pyrrosia lingua* was significantly reduced in the reaction system treated with the amidase ArfP, decreasing by 83.78%. Example 5 Purification and Enzymatic Characterization of Amidease ArfP I. Large-scale expression and purification of amidase ArfP 1. Expand training The engineered bacteria from Example 2 E. coli BL21(DE3)-ArfP was inoculated into LB medium and cultured overnight by shaking to obtain a seed culture. The seed culture was then inoculated at a ratio of 1:100 (v / v) into two 2 L Erlenmeyer flasks containing 500 mL of LB liquid medium (containing 100 μg / mL Kan), and cultured at 37℃ and 200 r / min for 3–4 h, with OD values ​​monitored periodically until the OD value reached the target level. 600 =0.6±0.1. Then, IPTG with a final concentration of 0.5 mM was added, and the mixture was induced at 16℃ and 120 rpm for 16 h to obtain the bacterial culture.

[0073] 2. Protein breakage Transfer the bacterial culture to a centrifuge bottle; centrifuge at 6000 r / min, 4℃ for 15 min, and discard the supernatant. Resuspend the bacterial cells in 80 mL PBS. Disrupt the cells using an ultrasonic disruptor (Wiggens HD2000, Germany) under ice bath conditions for a total disruption time of 30 min (3 s working, 3 s interval), for a total of 2 disruptions. After ultrasonic disruption, centrifuge the cell disruption buffer at 8000 r / min, 4℃ for 30 min, separating the supernatant from the precipitate. Transfer the disrupted sample to a 50 mL centrifuge tube, then filter through a 0.45 μm low protein binding filter membrane, collecting the supernatant, i.e., the crude ArfP protein enzyme solution, for affinity purification.

[0074] 3. Affinity chromatography purification of ArfP protein ArfP protein was purified using gravity column purification. The purification column was a HisTrap™ HP 5 mL column (Changzhou Tiandi Renhe Biotechnology Co., Ltd.), and the column packing material was Ni NTA Beads (Changzhou Tiandi Renhe Biotechnology Co., Ltd.; catalog number SA004025). The specific purification procedure was performed according to the Ni NTA Beads product instructions. The buffer formulation is shown in Table 5. The specific steps are as follows: (1) Use Lysis Buffer, which is 5 times the volume of Ni NTA Beads gravity column, to equilibrate the column multiple times to ensure that the packing material and the target protein are in the same buffer environment. This step needs to be repeated 3 times. (2) Slowly add the crude protein enzyme solution to the equilibrated column and ensure that the sample stays in the column for no less than 2 minutes to promote full contact between the crude enzyme solution and the medium. Then collect the effluent. To improve the binding efficiency, this loading process should be repeated 3 times. (3) Use 10 column volumes of Wash Buffer (60 mM imidazole) to wash away non-specifically bound proteins and collect the wash buffer. (4) Use 10 column volumes of Elution Buffer (imidazolium concentrations include 100 mM, 200 mM and 300 mM) for elution and collect the eluent. During this process, the eluent should be collected in segments, with each column volume of eluent collected in one tube and tested separately.

[0075] Table 5. Buffers and formulations required for purification of soluble histidine-tagged proteins

[0076] 4. Protein ArfP purified and concentrated by ultrafiltration The collected effluent, washing buffer, and elution buffer were analyzed by SDS-PAGE, using the same method as in Example 4. After verifying the purity of the protein purification solution by SDS-PAGE, the protein purification sample was concentrated using an ultrafiltration tube (Merck, 30 kDa). To avoid the adverse effects of imidazole on protein activity, the original buffer was replaced with PBS buffer. Finally, the target protein was quantitatively analyzed using a BCA protein assay kit (Novizan, Kit-E112), with the protein concentration adjusted to 100 µg / mL, yielding the purified and concentrated ArfP protein.

[0077] II. Enzyme kinetic analysis of amidase ArfP Enzyme kinetics of amidase ArfP: 1 mL of substrate propargyl was added to 5 mL centrifuge tubes at concentrations of 10, 20, 30, 40, and 50 μmol / L, respectively. Purified and concentrated ArfP protein was then added to the centrifuge tubes to achieve a final ArfP protein concentration of 100 µg / mL. After mixing, the mixture was incubated at 30°C and 200 rpm for 30 min in a shaker. Then, 2 mL of dichloromethane was added for direct extraction. The degradation rate of propargyl was detected by HPLC (HPLC detection method was the same as in Example 4). Subsequently, the enzyme kinetics of ArfP degradation of propargyl were calculated using the Michaelis-Menten equation, as shown below: V = V max [ S ] / ( K m +[ S ]) Formula 1; k cat = V max / [ E ] Formula 2.

[0078] In the formula, K m Representing the Michaelis constant, it is an indicator of the affinity between an enzyme and its substrate. V max This represents the maximum reaction rate (µM / min) that the enzyme can achieve at saturated substrate concentration. S Substrate concentration (µM); k cat This is a specificity constant, also representing the transformation number, which is the maximum number of substrate molecules that the enzyme can transform per minute; E [] indicates the concentration of the enzyme.

[0079] III. Experimental Results SDS-PAGE experimental results are as follows: Figure 7 As shown, the molecular weight of the purified and concentrated ArfP protein was consistent with the previously observed crude enzyme value (approximately 58.2 kDa), and it exhibited a single band in SDS-PAGE. This result indicates that the ArfP protein has been successfully purified, and the protein concentration was subsequently adjusted to 100 µg / mL.

[0080] The kinetic parameters of the degradation of taraxeribrus catalyzed by the amidase ArfP are shown in Table 6, among which the Michaelis constant ( K m The value was 6.500335 µM, and the maximum reaction rate was ( V max The conversion rate was 60.35415 µmol / L / min. Based on Formula 2, the conversion number was further calculated as follows ( k cat The duration was 27.31 min. -1 . k cat / K m This represents the affinity of the amidase ArfP for the substrate, with a value of 4.20 min. -1 µM -1 This indicates that the amidase ArfP has good substrate affinity and catalytic efficiency for phoxim.

[0081] Table 6 Enzyme kinetic parameters of amidase ArfP

[0082] Note: K m It is the Michaelis constant; V max The maximum reaction rate; k cat Turnover; k cat / K m This represents the enzyme's catalytic efficiency and affinity for its substrate.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An amidase ArfP, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A polynucleotide, characterized in that, The polynucleotide includes a nucleotide sequence encoding the amidase ArfP described in claim 1.

3. The polynucleotide according to claim 2, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

1.

4. An expression carrier, characterized in that, It contains the polynucleotide described in claim 2 or 3.

5. A strain expressing the amidase ArfP protein, characterized in that, It comprises the polynucleotide of claim 2 or 3 or the expression vector of claim 4.

6. The application of the amidase ArfP of claim 1 or the amidase ArfP protein-expressing strain of claim 5 in the degradation of propargite or the remediation of propargite contamination in an environment.

7. The use of the amidase ArfP of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4, or the amidase ArfP protein expression strain of claim 5 in the preparation of products that degrade tardigrade.

8. The application of the amidase ArfP of claim 1, the polynucleotide of claim 2 or 3, the expression vector of claim 4, or the amidase ArfP protein expression strain of claim 5 in the preparation of products for the remediation of environments contaminated by tarantula.

9. A product for degrading phoxim, characterized in that, The strain containing the amidase ArfP protein expression strain of claim 1 or claim 5.

10. A method for degrading barnyardgrass, characterized in that, The product is processed using the amidase ArfP described in claim 1 or the product described in claim 9.

Images