aphA gene, its encoded protein, and applications of amidases that degrade amide alcohol antibiotics and amide herbicides.

Abstract

This invention discloses an amidase gene for degrading amide alcohol antibiotics and amide herbicides. right here The amidase gene, along with its encoded protein and applications, has the nucleotide sequence SEQ ID NO.1, a full length of 912 bp, encoding 303 amino acids, and its amino acid sequence is SEQ ID NO.2. This invention relates to the gene... right here This is the first publicly disclosed amidase gene for amide alcohol antibiotics cloned from the genome of a single isolated strain. The amidase encoded by this gene can not only hydrolyze the amide bonds of amide alcohol antibiotics, but also degrade various amide chemical herbicides such as propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate. The amidase encoded by this gene, as well as genetically engineered bacteria containing the amidase gene, have very important theoretical and applied value in the degradation of amide alcohol antibiotic pollutants in aquatic environments such as sewage treatment plants and livestock and poultry breeding wastewater, and in the remediation of herbicide-contaminated soil.

Description

Technical Field

[0001] This invention belongs to the fields of applied environmental microbiology and agriculture, specifically relating to a broad-spectrum amidase gene aphA that degrades amyl alcohol antibiotics and amide herbicides, its encoded protein, and its applications. Background Technology

[0002] Amide alcohol antibiotics include chloramphenicol (CHL), thiamphenicol (TAP), and florfenicol (Ff). Chloramphenicol, a first-generation broad-spectrum amide alcohol antibiotic, was first isolated from *Streptomyces venezuelae* in the 1950s. Due to its good antibacterial effects against chlamydia, mycoplasma, and Gram-negative bacteria, it has been widely used in the clinical treatment of human diseases. However, long-term use has revealed a series of serious side effects, including aplastic anemia, adverse neurological reactions, bone marrow suppression, and potential carcinogenicity and genotoxicity. Due to its low cost, availability, and broad spectrum, it is widely used in my country's aquaculture, livestock, and beekeeping industries for the control of infectious diseases, resulting in varying degrees of chloramphenicol residues being detected in the meat, eggs, and milk of farmed animals. Thiamphenicol is a second-generation synthetic broad-spectrum antibacterial drug with strong immunosuppressive effects. Florfenicol is widely used as a veterinary drug in livestock and poultry farming to prevent bacterial diseases. Antibiotics in the environment mainly originate from industrial wastewater discharge, wastewater from livestock and aquaculture, animal feces, and the use of medical antibiotics. Currently, three types of amide alcohol antibiotics can be detected simultaneously in the water environments of different countries and regions; this combined toxicity undoubtedly poses a potential threat to human health. The presence of multiple antibiotics in the environment can also accelerate the emergence of multidrug-resistant bacteria and the spread of resistance genes, disrupt the microecological balance, and affect aquatic organisms and terrestrial plants, among other negative effects.

[0003] Currently, reports on highly efficient amide alcohol antibiotic degrading strains are very limited, and strains capable of simultaneously degrading multiple amide alcohol antibiotics are even more scarce. Therefore, screening highly efficient amide alcohol antibiotic degrading strains and mining degradation gene resources have the following roles and guiding significance for the research and development of biological remediation technologies for environmental pollution caused by amide alcohol antibiotics: (i) It can be used for the detoxification of residual amide alcohol antibiotics in small-scale environmental water bodies such as sewage treatment plants and livestock and poultry farm wastewater; (ii) It can be used to assess the risk and fate of amide alcohol antibiotics in the environment. Therefore, this degradation gene has very important theoretical and applied value in the research and development of gene resources for eliminating amide alcohol antibiotic residues and assessing the risk of amide alcohol antibiotics in the environment. Summary of the Invention

[0004] Objective of the invention: To address the shortcomings of existing technologies, this invention provides a broad-spectrum amidase gene aphA. This gene is a novel amidase gene that hydrolyzes amide alcohol antibiotics and amide herbicides. The amidase AphA encoded by this gene can both break the amide bonds in the structural formulas of amide alcohol antibiotics and hydrolyze the amide bonds in the structural formulas of amide herbicides, thus exhibiting excellent effects in degrading and converting amide alcohol antibiotics and amide herbicides.

[0005] The present invention also provides the amidase encoded by the amidase gene aphA and its applications.

[0006] Technical solution: In order to achieve the above objective, the present invention provides a broad-spectrum amidase gene aphA, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the broad-spectrum amidase gene aphA encoded by the present invention is shown in SEQ ID NO.2.

[0008] The present invention contains a recombinant expression vector of the broad-spectrum amidase gene aphA.

[0009] The recombinant expression vector is obtained by inserting the broad-spectrum amidase gene aphA between the BamHI and XhoI sites of the vector pET-32a(+), and the recombinant expression vector is pET-32a(+)-aphA.

[0010] The present invention relates to genetically engineered bacteria containing the broad-spectrum amidase gene aphA or the recombinant expression vector.

[0011] The genetically engineered bacteria are obtained by introducing the broad-spectrum amidase gene aphA or the recombinant expression vector into Escherichia coli BL21(DE3).

[0012] The application of the broad-spectrum amidase gene aphA or the amidase protein AphA described in this invention in the degradation and conversion of amide alcohol antibiotics and amide herbicides.

[0013] The amidase gene aphA described in this invention serves as a genetic diagnostic resource for assessing the fate of amyl alcohol antibiotics in the environment.

[0014] The application of the broad-spectrum amidase gene aphA or the amidase protein AphA in the degradation or removal of residual pollutants such as amide alcohol antibiotics and amide herbicides in environmental water and soil.

[0015] Preferably, the broad-spectrum amidase gene aphA or the amidase protein AphA is used in the degradation or removal of residual pollutants such as amide alcohol antibiotics and amide herbicides from environmental water bodies, such as sewage treatment plants, livestock and poultry breeding wastewater, and pesticide-contaminated surface water and soil.

[0016] Furthermore, the application of the broad-spectrum amidase gene AphA described in this invention in the construction of antibiotic-free genetically engineered strains and transgenic crops containing amide-based chemical herbicides.

[0017] The genetically engineered bacteria of this invention efficiently express broad-spectrum amidases, and the enzyme preparations produced can be used to reduce or remove residual amide alcohol antibiotics in water, soil and other environments, as well as to degrade or transform residual amide chemical herbicides in crops and other environments.

[0018] The amide alcohol antibiotics include chloramphenicol, thiamphenicol, and florfenicol; the amide herbicides include propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate.

[0019] The application of the broad-spectrum amidase gene aphA described in this invention in the construction of transgenic crops that degrade amide herbicides.

[0020] This invention utilizes shotgun library construction to screen positive clones from the genome of *Sphingobium yanoikuyae* B1. Sequencing and amino acid sequence alignment analysis revealed a 912 bp ORF annotated as an α / β hydrolase, named aphA. The aphA gene fragment, containing BamHI and XhoI restriction sites at the ends and with the signal peptide removed, was amplified by PCR and ligated into the BamHI and XhoI restriction sites of the *E. coli* high-efficiency expression vector pET-32a(+). The fragment was then transformed into the host strain BL21(DE3), induced by IPTG, and the protein expression level was verified. The pure enzyme AphA was purified by Ni-NTA affinity chromatography. Functional verification revealed that AphA can cleave the amide bonds in the structures of amide alcohol antibiotics chloramphenicol, thiamphenicol, and florfenicol, and can also act on amide herbicides such as propargite, bromoxynil, chlorfluazuron, and chlorobromopropylate.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] 1. This invention successfully cloned an amidase gene aphA. Comparison with Swiss-Prot and NCBI's PDB databases revealed that this gene is a novel amidase gene with an amino acid sequence similarity of less than 30%.

[0023] 2. This invention amplifies a complete amidase gene fragment containing BamHI and XhoI sites at the ends using PCR technology, ligates it into the E. coli high-efficiency expression vector pET-32a(+), transforms it into the expression host bacterium BL21(DE3), and can achieve high-efficiency expression after IPTG induction.

[0024] 3. The amidase AphA encoded by the amidase gene aphA of this invention is the first amidase of amide alcohol antibiotics cloned from the genome of a pure culture strain. This amidase can simultaneously hydrolyze the amide bonds of three antibiotics: chloramphenicol, thiamphenicol, and florfenicol, and has very good broad-spectrum activity.

[0025] 4. The amidase AphA encoded by the amidase gene aphA of this invention has a broad substrate spectrum. In addition to using amide alcohol antibiotics as substrates, it can also break the amide bonds in amide chemical herbicides such as propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate. This amidase AphA and the genetically engineered bacteria containing the amidase gene aphA can be used for the degradation and biotransformation of residues of amide alcohol antibiotics and / or amide chemical herbicides in water bodies and farmland soils, with significant effects and very important theoretical and applied value.

[0026] 5. The genetically engineered strain constructed in this invention can efficiently express amidase, and the enzyme preparations produced can be used for the degradation or transformation of amide antibiotics and herbicides remaining in soil, water bodies and crops. Attached Figure Description

[0027] Figure 1 shows the agarose gel electrophoresis diagram of PCR amplification of the aphA gene.

[0028] Figure 2 is a schematic diagram of the construction of the aphA vector for the amidase gene.

[0029] Figure 3 shows the protein electrophoresis pattern of pET32a-aphA protein after Ni-NTA purification and concentration;

[0030] Figure 4 shows the HPLC chromatogram of chloramphenicol degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of chloramphenicol degradation by AphA; B: Primary and secondary mass spectra of chloramphenicol degradation products.

[0031] Figure 5 shows the HPLC chromatogram of thiamphenicol degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of thiamphenicol degradation by AphA; B: Primary and secondary mass spectra of thiamphenicol degradation products.

[0032] Figure 6 shows the HPLC chromatogram of florfenicol degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of florfenicol degradation by AphA; B: Primary and secondary mass spectra of florfenicol degradation products.

[0033] Figure 7 shows the degradation rate of three amino alcohol antibiotics by amidase AphA.

[0034] Figure 8 shows the pathways of amidase AphA degradation of amyl alcohol antibiotics; A: AphA degradation of chloramphenicol pathway; B: AphA degradation of thiamphenicol pathway; C: AphA degradation of florfenicol pathway.

[0035] Figure 9 shows the HPLC chromatogram of the degradation of barnyardgrass by amidase AphA and the identification of the degradation products; A: HPLC chromatogram of barnyardgrass degradation by AphA; B: Primary and secondary mass spectra of the products of barnyardgrass degradation by AphA.

[0036] Figure 10 shows the HPLC chromatogram of bromosulphuron degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of bromosulphuron degradation by AphA; B: Primary and secondary mass spectra of the products of bromosulphuron degradation by AphA.

[0037] Figure 11 shows the HPLC chromatogram of chlorotoluene degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of chlorotoluene degradation by AphA; B: Primary and secondary mass spectra of chlorotoluene degradation products by AphA.

[0038] Figure 12 shows the HPLC chromatogram of chlorobromodiphenyl ether (CBOE) degradation by amidase AphA and the identification of degradation products; A: HPLC chromatogram of CBOE degradation by AphA; B: Primary and secondary mass spectra of CBOE degradation products by AphA.

[0039] Figure 13 shows the degradation rate of four amide herbicides by amidase AphA.

[0040] Figure 14 shows the pathways of amidase AphA in degrading amide-based chemical herbicides; A: AphA degradation pathway of propargite; B: AphA degradation pathway of bromoxynil; C: AphA degradation pathway of chlorantraniliprole; D: AphA degradation pathway of chlorobromopropylate. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1. Journal of Industrial Microbiology & Biotechnology, 23(4-5), 294-302. Provided by Nanjing Agricultural University.

[0044] BamHI and XhoI were purchased from TAKARA Bioengineering (Nanjing) Co., Ltd.;

[0045] Escherichia coli DH5α and BL21(DE3) were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0046] The E. coli high expression vector pET-32a(+) was purchased from Novogen.

[0047] Example 1

[0048] Total genomic DNA extraction from strain B1

[0049] After the *Sphingobium yanoikuyae* B1 strain was cultured in LB medium to the logarithmic growth phase, genomic DNA was extracted from B1 using the Novizan Genomic DNA Extraction Kit according to the manufacturer's instructions. The DNA was dissolved in sterile water (pH 7.0), and the concentration was precisely quantified using a Qubit assay. High-concentration DNA samples with OD values ​​between 1.8 and 2.0 were used for library construction.

[0050] Example 2

[0051] Cloning and sequence analysis of the amidase gene aphA

[0052] 1. PCR amplification of the sequence

[0053] Design primers to amplify the target gene aphA:

[0054] Forward primer SEQ ID NO.3:

[0055] GCCATGGCTGATATCGGATCC CACCATCATCATCATCATATGCAGACGCCGTCGGAT

[0056] Reverse primer SEQ ID NO.4:

[0057] GTGGTGGTGGTGGTGCTCGAG ATGATGATGATGATGGTGTTGCTCTGCTTTCGCCGC

[0058] The aphA fragment of the amidotropic antibiotic amidase gene was amplified from the total genomic DNA of strain Sphingobium yanoikuyae B1. The signal peptide and the stop codon were removed from the fragment, and six histidine tags were added to the N-terminus and C-terminus of the protein AphA.

[0059] Target gene amplification system (50 μL):

[0060]

[0061] PCR amplification procedure for the target gene:

[0062]

[0063] Agarose gel electrophoresis image of the PCR amplification product of the amidotropic antibiotic amidase gene aphA is shown below. Figure 1 As shown.

[0064] 2. PCR product recovery

[0065] In step 1, the PCR amplified fragment was purified using a gel extraction kit to obtain the target fragment. The pET-32a(+) vector was linearized by double digestion with BamHI and XhoI, and the linearized vector fragment was obtained by gel extraction.

[0066] pET-32a(+) double enzyme digestion reaction system (50 μL):

[0067]

[0068] The purified aphA gene fragment and the linearized vector fragment were homologously recombinated. Ligation was performed at 37°C for 30 minutes to obtain the recombinant expression vector pET-32a(+)-aphA. Transformation was carried out using a 42°C heat shock method with DH5α as the competent cell strain. 10 μL of the enzyme ligation product was used to transform 500 μL of competent cells, which were then plated onto a medium containing 100 mg L... -1 Ampicillin was incubated overnight in an inverted incubator at 37°C on LB agar plates.

[0069] Homologous recombination system (20 μL):

[0070]

[0071] Single colonies cultured overnight were selected and cultured in 5 mL LB tubes until the logarithmic growth phase. Plasmids were then extracted and sent to the company for sequencing.

[0072] 3. Nucleotide sequence analysis of gene aphA

[0073] The positive clones obtained in step 2 were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The nucleotide sequence of the amidase gene aphA was determined to be SEQ ID NO.1, with a full length of 912 bp. Based on the nucleotide sequence of the amidase gene aphA, it is estimated to be 303 amino acids (excluding the stop codon), with a theoretical size of 31.8 kDa. The amidase AphA encoded by the amidase gene aphA has a signal peptide at the N-terminus. The predicted signal peptide cleavage site is located between amino acids Ala20 and Gln21. After removing the signal peptide, the size of amidase AphA is 283 amino acids.

[0074] Online BLAST homology comparison of the AphA protein sequence was performed in the Swiss-Prot database. Only four similar sequences were found, two of which have been verified for biological function: EstD, an esterase that hydrolyzes short acyl ester bonds from Thermotoga maritima (accession number Q9WYH1.1), and isopropylcysteine ​​methyl esterase encoded by At5g15860 from the plant Arabidopsis thaliana (accession number Q8VYP9.1). The amino acid sequence similarity was 26.09% and 22.98%, respectively, proving that this invention is a novel gene.

[0075] SEQ ID NO.1 (aphA nucleic acid sequence):

[0076] ATGCGATACCGTTCATTAGGCCTGGCTACAGTCTTGATTGTCACGAGCACCGCTTGGGCGCAGACGCCGTCGGATAACCAACATGCGCTGATTGGACCGCCCCAACTCCACGCCCTTCCCGTCACCGCTCCGACGCTCGTCGAGGCCTATGGTCCTGATCCTCTGCAGATCGGTGAACTGCGCTTGCCTGCAGGTCCTGGCCCGTTTCCCGTCGTCATGGTCATTCACGGTGGCTGCTGGACCAAAGGCTATGAGACACTGGCAGGCACGGCGCCGTTGGCGAGCGCCTTGACGGACAAGGGCGTGGCGACATGGAATATAGAGTATCGACAGGTTGGTGACACCCGCGGCGGGTGGCCTGGGACCTTCCAGGATTGGGGAGCGGCGCTCGATCACTTGAGGGTGCTGGCACGAACCCAGCCTCTGGACCTGAAGCATGTGGTGACGGTGGGACATTCGGCCGGCGCCCACGCTGCCCTGTGGCTGGCGGCCCGTCCCAGATTGCCTGCCGACAGCGAGGTCCGCGGCGCTGATCCACTGAAGGTTTCCGCCGCGGTCGCCATCGACGGCCCAGGCGACCTGCGAACCCTGTACGGGTTCGACAAGGATATTTGCGGCCGCCCGATCTTCGTCAACCTGTTCGGGGGCGCTCCTGACGCACAGGCGGGCCGCTACCGTCAGGCAAATCCGATCGAATGGCTGCCTCTGGCCGTGCCTCAATTCATGGTCGCTTCCGTCGTGCTGGAGCCTTCAGCCGCGCAAGCCTATGCAGCAGCCGCACAGTCGGCCGGCGATAAGGCGACGGTGATCACCCTGGAGAATGCGGGCCACTTCAACATGCTTTCGCCTGCGGACCCCACCTGGGCGCCGGTAGAGGCCGCGATCCTCGCGGCGGCGAAAGCAGAGCAATAG

[0077] SEQ ID NO. 2 (AphA amino acid sequence (including signal peptide), MRYRSLGLATVLIVTSTAWA is the signal peptide):

[0078] MRYRSLGLATVLIVTSTAWAQTPSDNQHALIGPPQLHALPVTAPTLVEAYGPDPLQIGELRLPAGPGPFPVVMVIHGGCWTKGYETLAGTAPLASALTDKGVATWNIEYRQVGDTRGGWPGTFQDWGAALDHLRVLARTQPLDLKHVVTVG HSAGAHAALWLAARPRLPADSEVRGADPLKVSAAVAIDGPGDLRTLYGFDKDICGRPIFVNLFGGAPDAQAGRYRQANPIEWLPLAVPQFMVASVVLEPSAAQAYAAAAQSAGDKATVITLENAGHFNMLSPADPTWAPVEAAILAAAKAEQ

[0079] Example 3

[0080] High-efficiency expression of the amidase gene aphA in Escherichia coli BL21(DE3)(pET-32a(+))

[0081] Plasmids extracted from positive clones in Example 2 were transformed into the expression host bacterium BL21(DE3) (transformation method as described in Example 2). The recombinant vector pET-32a(+)-aphA was introduced into Escherichia coli BL21(DE3) to obtain a recombinant genetically engineered strain, which was then plated on a substrate containing 100 mg L... -1 Ampicillin plates were incubated overnight at 37°C inverted. Positive transformants (i.e., single colonies of BL21(DE3) / pET32a-aphA from overnight culture) were selected for expression. The expression process of the amidase gene aphA in BL21(DE3) is as follows: Figure 2 As shown.

[0082] Pick the above positive transformants and inoculate them into fresh LB liquid medium (containing 100 mg L). -1 Ampicillin) cultured at 37°C for approximately 4 hours, OD 600 The concentration was approximately 0.6. IPTG was added to a final concentration of 0.2 mM to induce the expression of pET-32a(+)-aphA. After induction at 16°C for 16 h, the bacterial cells were collected, resuspended in 15 mL of PBS (20 mM, pH 7.4) buffer, sonicated for 10 min, and centrifuged at 12000 rpm and 4°C for 10 min. The supernatant contained the crude enzyme solution of AphA. Additionally, the bacterial solution induced at 16°C for 16 h was directly used to verify the degradation effect on three amino alcohol antibiotics.

[0083] The induced bacterial culture (transformant BL21(DE3) / pET32a-aphA) prepared above was inoculated at a volume ratio of 5% into a solution containing a final concentration of 25 mg / L. -1 The samples were placed in inorganic salt culture media containing chloramphenicol, thiamphenicol, and florfenicol at 30°C and 180 rpm in a shaker. Each treatment was repeated three times. After 48 hours, 1 mL of each sample was collected, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The samples were filtered through an aqueous filter membrane, and the residual amounts of the three amide alcohol antibiotics in the system were detected by high performance liquid chromatography. The degradation rate of the three amide alcohol antibiotics by the transformant BL21(DE3) / pET32a-aphA was calculated based on the substrate detection amount of the control group.

[0084] The degradation effects of the positive transformants on the three amyl alcohol antibiotics are shown in Table 1. The degradation efficiency of transformant BL21(DE3) / pET32a-aphA on chloramphenicol reached 77.42%, the degradation efficiency on thiamphenicol was 51.32%, and the degradation efficiency on florfenicol was 74.90%.

[0085] Table 1. Degradation effect of transformant BL21(DE3) / pET32a-aphA on amide alcohol antibiotics

[0086]

[0087] The specific steps for purifying AphA protein were as follows: The crude AphA enzyme solution prepared above was filtered through a 0.22 μM filter and then added to a Ni-NTA affinity chromatography column. The Ni-NTA column was washed sequentially with imidazole at concentrations ranging from low to high (30 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM), and the eluents were collected after each wash. The purification results were identified by SDS-PAGE chromatography. Figure 3 , Figure 3 This indicates that the purified sample has a single band at 48 kDa, which is consistent with the theoretical size of the fusion protein AphA, which is 48.21 kDa.

[0088] Example 4

[0089] AphA degradation of amyl alcohol antibiotics chloramphenicol, thiamphenicol, and florfenicol, and identification of their metabolites.

[0090] 20 mM PBS buffer (pH 7.4), 25 mg L -1Substrate chloramphenicol, thiamphenicol, and florfenicol were added to an appropriate amount of the purified enzyme AphA eluted with 150 mM imidazole from Example 3 above, and the reaction was carried out at 35°C for 2 h. The reaction was terminated with an equal volume of ethyl acetate, and the mixture was extracted by shaking for 10 min. After separation, the organic phase was collected, extracted twice, combined, dried, dissolved in 250 μL of methanol, and filtered through a 0.22 μm organic phase filter to remove impurities, obtaining a liquid sample. The metabolites in the reaction solution were determined by LC / MS / MS.

[0091] Validation method for degradation effect by high performance liquid chromatography (HPLC): The contents of amide alcohol antibiotics chloramphenicol, thiamphenicol, and florfenicol in the extract were determined by HPLC. HPLC conditions: mobile phase acetonitrile:water (40:60, V / V), C18 reversed-phase column (4.6 × 250 mm, 5 µm, Agilent Technologies), column temperature 40°C, UV detector, detection wavelengths 272 nm (chloramphenicol), 235 nm (thiamphenicol), and 233 nm (florfenicol), injection volume 20 μL, flow rate 1.0 mL·min⁻¹ -1 The degradation products were identified using liquid chromatography-mass spectrometry (LC-MS) as follows: First, the enzyme reaction solution was extracted with three volumes of ethyl acetate. The sample was dried and then dissolved in 250 μL of chromatographic grade methanol. The solution was then filtered through a 0.22 µm pore size membrane. LC conditions: injection volume 2 µL, flow rate 0.2 mL / min. -1 Temperature: 40°C. Sampling rate: 5.0 μs. -1 The program was gradient elution (0-1 min 5% methanol, 1-14 min 95% methanol, 14-17 min 95% methanol, 17-17.10 min 5% methanol, 17.10 min-20 min Stop).

[0092] Mass spectrometry detection was performed using an AB Sciex high-resolution tandem mass spectrometer, a Triple TOF 5600+LC / MS / MS system, with a DuoSpray ion source, positive ion detection mode, and a mass scan range (m / z) of 50-800.

[0093] HPLC results of the degradation of chloramphenicol by the amidase AphA encoded by the gene aphA are as follows: Figure 4 As shown in A, Figure 4 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade chloramphenicol. The product was identified by mass spectrometry as p-nitrophenylserine alcohol. Figure 4 B, by Figure 4Data analysis revealed that chloramphenicol, after being hydrolyzed by amidase AphA, cleaves the amide bond to generate p-nitrobenzenesilyl alcohol. The HPLC results of amidase AphA degradation of thiamphenicol are shown below. Figure 5 As shown in A, Figure 5 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade thiamphenicol. The product was identified by mass spectrometry as thiamphenicol amine. Figure 5 B, by Figure 5 Data analysis revealed that thiamphenicol is converted to thiamphenicol amine after hydrolysis by the amidase AphA, which breaks the amide bond. The HPLC results of florfenicol degradation by amidase AphA are shown below. Figure 6 As shown in A, Figure 6 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade florfenicol. The product was identified by mass spectrometry as florfenicolamine. Figure 6 B, by Figure 6 Data analysis revealed that florfenicol is converted to florfenicolamine by hydrolysis of the amide bond by the amidase AphA. Figure 7 As shown, the degradation rates of three amide alcohol antibiotics by amidase AphA were 75.17% (chloramphenicol), 58.11% (thiamphenicol), and 71.08% (florfenicol), respectively. The reaction pathway of amidase AphA hydrolyzing amide alcohol antibiotics is summarized in... Figure 8 A (Chloramphenicol conversion equation). Figure 8 B (Conversion equation of thiamphenicol) Figure 8 C (Fluorfenicol conversion equation).

[0094] Example 5

[0095] Degradation of amide-based chemical herbicides propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate by AphA and identification of their metabolites.

[0096] 20 mM PBS buffer (pH 7.4), 30 mg L -1 Substrates such as propargyl, bromoxynil, chlorobromopropylate, and chlorobromopropylate were added, along with an appropriate amount of the purified enzyme AphA eluted with 150 mM imidazole from Example 3 above. The reaction was carried out at 35°C for 1 h. An equal volume of methanol was added to terminate the enzyme reaction, and the mixture was shaken thoroughly for 10 min, centrifuged at 12000 rpm for 3 min, and filtered through a 0.22 μm organic phase filter to remove impurities. High-performance liquid chromatography (HPLC) was used to detect the degradation of amide herbicides, and LC / MS / MS was used to identify the metabolites in the reaction solution.

[0097] Validation method for degradation effect by high performance liquid chromatography (HPLC): The degradation of amide herbicides propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate in the reaction solution was determined by HPLC. HPLC conditions: mobile phase: propargite:acetonitrile:water (70:30, V / V); bromoxynil, chlorpyrifos, and chlorobromopropylate:acetonitrile:water (65:35, V / V); C18 reversed-phase column (4.6 × 250 mm, 5 µm, Agilent Technologies); column temperature: 40°C; UV detector; measurement wavelength: 245 nm (propargite), 250 nm (bromopropylate, chlorpyrifos, and chlorobromopropylate); injection volume: 20 μL; flow rate: 1.0 mL·min⁻¹. -1 The degradation products were determined using liquid chromatography-mass spectrometry (LC-MS) as follows: First, the enzyme reaction solution was extracted with three volumes of dichloromethane. The sample was dried and then dissolved in 250 μL of chromatographically pure methanol. The solution was then filtered through a 0.22 µm filter membrane. LC conditions: injection volume 2 µL, flow rate 0.2 mL / min. -1 Temperature: 40°C. Sampling rate: 5.0 μs. -1 .

[0098] Mass spectrometry detection was performed using an AB Sciex high-resolution tandem mass spectrometer, a Triple TOF 5600+LC / MS / MS system, with a DuoSpray ion source and a mass scan range (m / z) of 50-800.

[0099] HPLC results of the amidase AphA, encoded by the gene aphA, degrading the amide herbicide propargite, are as follows: Figure 9 As shown in A, Figure 9 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade phlebsiella pneumoniae. The product was identified by mass spectrometry as 3,4-dichloroaniline. See [link to article]. Figure 9 B, by Figure 9 Data analysis revealed that after hydrolysis by the amidase AphA, amide bonds of phoxim were broken, yielding 3,4-dichloroaniline. The HPLC results of the degradation of bromoxynil by amidase AphA are shown below. Figure 10 As shown in A, Figure 10 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade bromosulone. The product was identified by mass spectrometry as 4-bromoaniline. Figure 10 B, by Figure 10 Data analysis revealed that bromosulfoformis, after being hydrolyzed by the amidase AphA, cleaves the amide bond to generate 4-bromoaniline. The HPLC results of bromosulfoformis degradation by the amidase AphA are shown below. Figure 11 As shown in A, Figure 11 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade chloroquine. The product was identified by mass spectrometry as 4-chloroaniline. Figure 11B, by Figure 11 Data analysis revealed that chlorobromopropylate is converted to 4-chloroaniline by hydrolysis with amidase AphA, which breaks the amide bond. The HPLC results of chlorobromopropylate degradation by amidase AphA are shown below. Figure 12 As shown in A, Figure 12 A indicates that the amidase gene aphA encodes amidase AphA, which can degrade chlorobromopropylate. The product was identified by mass spectrometry as 4-bromo-3-chloroaniline. Figure 12 B, by Figure 12 Data analysis revealed that chlorobromopropylate, after being hydrolyzed by the amidase AphA, cleaves the amide bond to generate 4-bromo-3-chloroaniline. For example... Figure 13 As shown, the degradation rates of the three amide herbicides by amidase were 98.47% (propargyl), 55.77% (bromosulfuron), 92.17% (chlorfluazuron), and 93.70% (chlorobromosulfuron). The reaction pathway of amidase AphA in degrading amide chemical herbicides is summarized in... Figure 14 A (Equation for the transformation of barnyard grass). Figure 14 B (Bromisoproline conversion equation) Figure 14 C (Green Valley Long Transformation Equation) Figure 14 D (Chlorobromide conversion equation).

Claims

1. An amidase gene aphA Alternatively, the application of the amidase protein AphA in the degradation and conversion of amide alcohol antibiotics and herbicides, wherein the amide alcohol antibiotics are chloramphenicol, thiamphenicol, and florfenicol; the herbicides are propargite, bromoxynil, chlorpyrifos, and chlorobromopropylate; and the amidase gene... aphA The nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence of the amidase protein AphA is shown in SEQ ID NO.

2.

2. A device containing the amidase gene of claim 1 aphA The application of a recombinant expression vector or genetically engineered bacteria containing the recombinant expression vector in the degradation and transformation of amide alcohol antibiotics and herbicides, wherein the amide alcohol antibiotics are chloramphenicol, thiamphenicol, and florfenicol; the herbicides are propargyl, bromoxynil, chlorantraniliprole, and chlorobromopropylate; and the amidase gene... aphA The nucleotide sequence is shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that, The genetically engineered bacteria are those containing the amidase gene. aphA The recombinant expression vector was introduced into Escherichia coli BL21(DE3) to obtain the desired result.

4. The application according to claim 1 or 2, characterized in that, The application is to degrade or remove residual pollutants such as amide alcohol antibiotics and herbicides from environmental water bodies and soil.

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