Broad-spectrum amidase gene aphA for degrading amide alcohol antibiotics and amide herbicides as well as encoding protein and application of broad-spectrum amidase gene aphA

Abstract

The invention discloses a broad-spectrum amidase gene aphA for degrading amidol antibiotics and amide herbicides as well as an encoded protein and application thereof. The nucleotide sequence of the amidase gene is SEQ ID NO.1, the full length is 912bp, 303 amino acids are encoded, and the amino acid sequence is SEQ ID NO.2. The invention further discloses a preparation method of the broad-spectrum amidase gene aphA for degrading the amidol antibiotics and the amide herbicides. The gene aphA disclosed by the invention is the first disclosed amido alcohol antibiotic amidase gene cloned from a single isolated strain genome, amidase coded by the gene can hydrolyze amido bonds of amido alcohol antibiotics, and can also degrade various amide chemical herbicides such as propanil, bromuron, chlorpyriuron and chlorbrouron, so that the amido alcohol antibiotic amidase gene has a wide application prospect. The broad-spectrum amidase coded by the gene and the genetically engineered bacteria containing the amidase gene have very important theoretical and application values in the aspects of degradation of amido alcohol antibiotic pollutants in water environments such as sewage treatment plants, livestock and poultry breeding wastewater and the like, remediation of herbicide-polluted soil and the like.

Description

Technical Field

[0001] The invention belongs to the field of applied environmental microorganisms and agriculture, and specifically relates to a broad-spectrum amidase gene aphA for degrading amide alcohol antibiotics and amide herbicides, a coded protein and an application thereof. Background Art

[0002] Amide alcohol antibiotics include chloramphenicol (CHL), thiamphenicol (TAP) and florfenicol (Ff). Chloramphenicol, as the first generation of broad-spectrum amide alcohol antibiotics, was first isolated from Streptomyces venezuelae in the 1950s. Because it has good antibacterial effects on chlamydia, mycoplasma, Gram-negative bacteria, etc., it is widely used in the clinical treatment of human diseases. During long-term use, it was found that chloramphenicol would bring a series of serious side effects, including aplastic anemia, adverse reactions of the nervous system, bone marrow suppression, and potential carcinogenicity and genotoxicity. Due to its low price, availability and broad spectrum, it is widely used in the control of infectious diseases in my country's aquatic products, livestock and poultry, bee breeding industries, etc., resulting in chloramphenicol residues in meat, eggs and milk of farmed animals to varying degrees. Thiamphenicol is a second-generation synthetic broad-spectrum antibacterial drug that still has a strong immunosuppressive effect. Florfenicol is widely used as a veterinary drug in livestock and poultry farming to prevent and treat bacterial diseases. Antibiotics in the environment mainly come from industrial wastewater discharge, wastewater generated by animal husbandry and aquaculture, animal feces and the use of medical antibiotics. At present, three amide antibiotics can be detected simultaneously in the water environment of different countries and regions. This combined toxicity undoubtedly poses a potential threat to human health. The residues of multiple antibiotics in the environment will also accelerate the emergence of multidrug-resistant bacteria and the spread of resistance genes, destroy the balance of microecology, affect aquatic organisms and terrestrial plants, and have many negative effects.

[0003] At present, there are very limited reports on strains that efficiently degrade amide antibiotics, and there is an even greater lack of strains that can simultaneously degrade multiple amide antibiotics. Therefore, screening for efficient amide antibiotic-degrading strains and tapping into degradation gene resources have the following effects and guiding significance in the research and development of bioremediation technologies for amide antibiotic-polluted environments: (i) they can be used to detoxify residual amide antibiotics in small-scale environmental water bodies such as sewage treatment plants and livestock and poultry farm wastewater; (ii) they can be used to assess the risk and fate of amide antibiotics in the environment. Therefore, the degradation gene has very important theoretical and application value in the elimination of amide antibiotic residues and the research and development of diagnostic gene resources for risk assessment of amide antibiotics in the environment. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the present invention provides a broad-spectrum amidase gene aphA, which is an amidase gene for hydrolyzing amide alcohol antibiotics and amide chemical herbicides. It is a brand-new amidase gene. The amidase AphA encoded by the gene can both break the amide bond in the structural formula of amide alcohol antibiotics and hydrolyze the amide bond in the structural formula of amide chemical herbicides, and has a very good effect on the degradation and conversion of amide alcohol antibiotics and amide herbicides.

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

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

[0007] The broad-spectrum amidase gene aphA of the present invention encodes the amidase protein AphA, and the amino acid sequence of the amidase protein AphA is shown in SEQ ID NO.2.

[0008] The invention discloses a recombinant expression vector containing 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 invention discloses a genetically engineered bacterium containing the broad-spectrum amidase gene aphA or the recombinant expression vector.

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

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

[0013] The amidase gene aphA described in the present invention is used as a genetic diagnostic resource for evaluating the fate of amide antibiotics in the environment.

[0014] Among them, the broad-spectrum amidase gene aphA or the amidase protein AphA is used in degrading or removing residual pollutants of amide antibiotics and amide herbicides in environmental water and soil.

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

[0016] Furthermore, the broad-spectrum amidase gene AphA described in the present invention is used in constructing antibiotic-detoxifying genetically engineered strains and amide chemical herbicide transgenic crops.

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

[0018] Among them, the amide antibiotics include chloramphenicol, thiamphenicol and florfenicol; the amide herbicides include propanil, bromothiocarb, chlorpyrifos and chlorbromide.

[0019] The invention discloses an application of the broad-spectrum amidase gene aphA in constructing transgenic crops capable of degrading amide herbicides.

[0020] The present invention obtains positive clones from the genome of strain Sphingobium yanoikuyae B1 by shotgun library construction, and finds an ORF with a length of 912 bp annotated as α / β hydrolase through sequencing and amino acid sequence alignment analysis, which is named aphA. The gene aphA fragment containing BamHI and XhoI restriction sites at the end and removing the signal peptide is amplified by PCR technology, connected to the BamHI and XhoI restriction sites of the Escherichia coli high-efficiency expression vector pET-32a (+), transformed into the expression host bacteria BL21 (DE3), IPTG induced expression is performed, and then the protein expression amount is verified. Pure enzyme AphA is obtained by Ni-NTA affinity chromatography purification, and functional verification finds that AphA can not only break the amide bond in the structure of amide alcohol antibiotics chloramphenicol, thiamphenicol and florfenicol, but also can act on amide herbicides propanil, bromothiocarb, chlorothiocarb and chlorbrom.

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

[0022] 1. The present invention successfully cloned an amidase gene aphA. Comparison between Swiss-Prot and NCBI's PDB database revealed that the gene was a new amidase gene with an amino acid sequence similarity of less than 30%.

[0023] 2. The present invention uses PCR technology to amplify a complete amidase gene fragment containing BamHI and XhoI sites at the end, connects it to the Escherichia coli high-efficiency expression vector pET-32a(+), transforms the expression host bacteria BL21(DE3), and can express efficiently after IPTG induction.

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

[0025] 4. The amidase AphA encoded by the amidase gene aphA of the present invention has a wide substrate spectrum. In addition to using amide antibiotics as substrates, it can also break the amide bonds in the amide chemical herbicides propanil, bromothiocarb, chlorpyrifos and chlorbromide. The amidase AphA and the genetically engineered bacteria containing the amidase gene aphA can be used for the degradation and biotransformation of amide antibiotics and / or amide chemical herbicides residues in water bodies and farmland soils, and the effect is remarkable, and it has very important theoretical and application value.

[0026] 5. The genetically engineered strain constructed by the present invention can efficiently express amidase, and the produced enzyme preparation can be used for the degradation or transformation of amide antibiotics and herbicides residual in soil, water and crops. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 Schematic diagram of the construction of the amidase gene aphA vector.

[0029] Figure 3 This is the protein electrophoresis pattern of the Ni-NTA purified and concentrated pET32a-aphA protein;

[0030] Figure 4 HPLC graph of chloramphenicol degradation by amidase AphA and identification of degradation products; A: HPLC graph of chloramphenicol degradation by AphA; B: primary and secondary mass spectra of chloramphenicol degradation products.

[0031] Figure 5 HPLC graph of thiamphenicol degradation by amidase AphA and identification of degradation products; A: HPLC graph of thiamphenicol degradation by AphA; B: primary and secondary mass spectra of thiamphenicol degradation products.

[0032] Figure 6HPLC graph of florfenicol degradation by amidase AphA and identification of degradation products; A: HPLC graph of florfenicol degradation by AphA; B: primary and secondary mass spectra of florfenicol degradation products.

[0033] Figure 7 This is the determination of the degradation rate of three amide antibiotics by amidase AphA.

[0034] Figure 8 The pathways of amidase AphA degrading amide antibiotics; A: the pathway of amidase AphA degrading chloramphenicol; B: the pathway of amidase AphA degrading thiamphenicol; C: the pathway of amidase AphA degrading florfenicol.

[0035] Fig. 9 HPLC graph of propanil degradation by amidase AphA and identification of degradation products; A: HPLC graph of propanil degradation by AphA; B: primary and secondary mass spectra of the products of propanil degradation by AphA.

[0036] Fig.10 HPLC graph of bromothiocarb degradation by amidase AphA and identification of degradation products; A: HPLC graph of bromothiocarb degradation by AphA; B: Primary and secondary mass spectra of the products of bromothiocarb degradation by AphA.

[0037] Fig.11 HPLC diagram of chloranil degradation by amidase AphA and identification of degradation products; A: HPLC diagram of chloranil degradation by AphA; B: primary and secondary mass spectra of the products of chloranil degradation by AphA.

[0038] Fig.12 HPLC graph of chlorbromidone degradation by amidase AphA and identification of degradation products; A: HPLC graph of chlorbromidone degradation by AphA; B: primary and secondary mass spectra of the products of chlorbromidone degradation by AphA.

[0039] Fig.13 Determination of the degradation rate of four amide herbicides by amidase AphA.

[0040] Fig.14 These are the pathways for degradation of amide chemical herbicides by amidase AphA; A: the pathway for degradation of propanil by amidase AphA; B: the pathway for degradation of bromosulfuron by amidase AphA; C: the pathway for degradation of chlorosulfuron by amidase AphA; D: the pathway for degradation of chlorsulfuron by amidase AphA. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

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

[0043] Strain Sphingobium yanoikuyae B1, 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 Novozyme Biotechnology (Nanjing) Co., Ltd.;

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

[0047] Example 1

[0048] Extraction of total genomic DNA from strain B1

[0049] After the strain Sphingobium yanoikuyae B1 was cultured in LB medium to the logarithmic growth phase, the genomic DNA of B1 was extracted using the genome extraction kit of Novozymes according to the instructions, dissolved in sterile water (pH 7.0), and the DNA concentration was accurately quantified by Qubit. Among them, the 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 amidase gene aphA

[0052] 1. PCR amplification of sequences

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

[0054] Forward primer SEQ ID NO.3:

[0055] GCCATGGCTGATATCGGATCC CACCATCATCATCATCATATGCAGACGCC

[0056] GTCGGAT

[0057] Reverse primer SEQ ID NO.4:

[0058] GTGGTGGTGGTGGTGCTCGAG ATGATGATGATGATGGTGTTGCTCTGCT

[0059] TTCGCCGC

[0060] The aphA fragment of the amide antibiotic amidase gene of the strain Sphingobium yanoikuyae B1 was amplified from the total genomic DNA. 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, respectively.

[0061] Target gene amplification system (50μL):

[0062]

[0063] Target gene PCR amplification procedure:

[0064]

[0065] The agarose gel electrophoresis of the PCR amplification product of the amidase gene aphA of amide antibiotics is shown in Figure 1 shown.

[0066] 2. PCR product recovery

[0067] The target fragment was obtained by purifying the PCR amplified fragment by gel recovery kit in step 1. The pET-32a(+) vector was linearized by double restriction enzyme digestion with BamHI and XhoI and then recovered by gel to obtain the linearized vector fragment.

[0068] Vector pET-32a(+) double enzyme digestion reaction system (50μL):

[0069]

[0070]

[0071] The purified aphA gene fragment and the linearized vector fragment were homologously recombined and connected at 37°C for 30 minutes according to the following reaction system to obtain the recombinant expression vector pET-32a(+)-aphA. The transformation was carried out by heat shock method at 42°C. The competent strain was DH5α. 10 μL of the enzyme-linked product was used to transform 500 μL of competent cells and spread on a plate containing 100 mg L -1 Place the plate containing ampicillin on a LB plate and incubate it inverted overnight at 37°C.

[0072] Homologous recombination system (20 μL):

[0073]

[0074] A single colony from the overnight culture was selected and cultured in a 5 mL LB test tube to the logarithmic phase, and the plasmid was extracted and sent to the company for sequencing.

[0075] 3. Analysis of nucleotide sequence of gene aphA

[0076] The positive clone obtained in step 2 was entrusted to Shanghai Shenggong Biotechnology Co., Ltd. for sequence determination, and the nucleotide sequence of the amidase gene aphA was measured to be SEQ ID NO.1, with a total length of 912 bp. According to the nucleotide sequence of the amidase gene aphA, it was inferred that it was 303 amino acids (excluding the stop codon), with a theoretical size of 31.8 KDa. The amino acid sequence of the amidase AphA encoded by the amidase gene aphA had a signal peptide at the N-terminus, and the predicted signal peptide cleavage site was located between amino acids Ala20 and Gln21. After removing the signal peptide, the size of the amidase AphA was 283 amino acids.

[0077] The AphA protein sequence was subjected to an online BLAST homology comparison in the Swiss-Prot database. Only four similar sequences were found in the comparison results, of which two sequences had verified biological functions. They were the esterase EstD from Thermotoga maritima (accession number is Q9WYH1.1) that hydrolyzes short acyl ester bonds and the isopropylcysteine ​​methylesterase encoded by At5g15860 in the plant Arabidopsisthaliana (accession number is Q8VYP9.1). The amino acid sequence similarities were 26.09% and 22.98%, respectively, proving that the present invention is a completely new gene.

[0078] SEQ ID NO.1 (aphA nucleic acid sequence):

[0079] ATGCGATACCGTTCATTAGGCCTGGCTACAGTCTTGATTGTCACGAGCACCGCTTGGGCGCAGACGCCGTCGGATAACCAACATGCGCTGATTGGACCGCCCCAACTCCACGCCCTTCCCGTCACCGCTCCGACGCTCGTCGAGGCCTATGGTCCTGATCCTCTGCAGATCGGTGAACTGCGCTTGCCTGCAGGTCCTGGCCCGTTTCCCGTCGTCATGGTCATTCACGGTGGCTGCTGGACCAAAGGCTATGAGACACTGGCAGGCACGGCGCCGTTGGCGAGCGCCTTGACGGACAAGGGCGTGGCGACATGGAATATAGAGTATCGACAGGTTGGTGACACCCGCGGCGGGTGGCCTGGGACCTTCCAGGATTGGGGAGCGGCGCTCGATCACTTGAGGGTGCTGGCACGAACCCAGCCTCTGGACCTGAAGCATGTGGTGACGGTGGGACATTCGGCCGGCGCCCACGCTGCCCTGTGGCTGGCGGCCCGTCCCAGATTGCCTGCCGACAGCGAGGTCCGCGGCGCTGATCCACTGAAGGTTTCCGCCGCGGTCGCCATCGACGGCCCAGGCGACCTGCGAACCCTGTACGGGTTCGACAAGGATATTTGCGGCCGCCCGATCTTCGTCAACCTGTTCGGGGGCGCTCCTGACGCACAGGCGGGCCGCTACCGTCAGGCAAATCCGATCGAATGGCTGCCTCTGGCCGTGCCTCAATTCATGGTCGCTTCCGTCGTGCTGGAGCCTTCAGCCGCGCAAGCCTATGCAGCAGCCGCACAGTCGGCCGGCGATAAGGCGACGGTGATCACCCTGGAGAATGCGGGCCACTTCAACATGCTTTCGCCTGCGGACCCCACCTGGGCGCCGGTAGAGGCCGCGATCCTCGCGGCGGCGAAAGCAGAGCAATAG

[0080] SEQ ID NO.2 (AphA amino acid sequence (including signal peptide), MRYRSLGLATVLIVTSTAWA is the signal peptide):

[0081] MRYRSLGLATVLIVTSTAWAQTPSDNQHALIGPPQLHALPVTAPTLVEAYGPDPLQIGELRLPAGPGPFPVVMVIHGGCWTKGYETLAGTAPLASALTDKGVATWNIEYRQVGDTRGGWPGTFQDWGAALDHLRVLARTQPLDLKHVVTVG HSAGAHAALWLAARPRLPADSEVRGADPLKVSAAVAIDGPGDLRTLYGFDKDICGRPIFVNLFGGAPDAQAGRYRQANPIEWLPLAVPQFMVASVVLEPSAAQAYAAAAQSAGDKATVITLENAGHFNMLSPADPTWAPVEAAILAAAKAEQ

[0082] Example 3

[0083] High-level expression of amidase gene aphA in Escherichia coli BL21(DE3)(pET-32a(+))

[0084] The plasmid extracted from the positive clone in Example 2 was selected and transformed into the expression host bacteria BL21 (DE3) (the transformation method is referred to Example 2), and the recombinant vector pET-32a (+) -aphA was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant genetic engineering strain, which was spread on a plate containing 100 mg L -1 The plate containing ampicillin was incubated at 37°C and inverted overnight. The positive transformants (i.e., single colonies BL21(DE3) / pET32a-aphA grown overnight) were selected for expression. The expression process of the amidase gene aphA in BL21(DE3) is as follows: Figure 2 shown.

[0085] The positive transformants were selected and inoculated into fresh LB liquid medium (containing 100 mg L -1 Ampicillin) was cultured at 37℃ for about 4h, OD 600 The p-value was about 0.6, and IPTG with a final concentration of 0.2 mM was added to induce the expression of pET-32a(+)-aphA. After induction at 16°C for 16 hours, the bacteria were collected and resuspended in 15 mL PBS (20 mM, pH 7.4) buffer, ultrasonically disrupted for 10 minutes, and centrifuged at 12000 rpm and 4°C for 10 minutes. The supernatant was the crude enzyme solution containing AphA; in addition, the bacterial solution after induction at 16°C for 16 hours was directly used to verify the degradation effect of three amide antibiotics.

[0086] The induced bacterial solution (transformant BL21(DE3) / pET32a-aphA) prepared above was inoculated at a volume ratio of 5% to a final concentration of 25 mg L -1 The culture medium was added with chloramphenicol, thiamphenicol and florfenicol in an inorganic salt medium and placed in a shaker at 30°C and 180 rpm. Three replicates were set for each treatment. 1 mL of sample was taken out after 48 hours and centrifuged at 12000 rpm for 10 minutes. The supernatant was taken out and filtered through a water filter membrane. The residual amount of the three amide antibiotics in the detection system was detected by high performance liquid chromatography. The degradation rates of the three amide antibiotics by the transformant BL21(DE3) / pET32a-aphA were calculated based on the substrate detection amount of the control group.

[0087] The degradation effects of the positive transformants on the three amide antibiotics are shown in Table 1. The degradation efficiency of the 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%.

[0088] Table 1 Degradation effect of transformant BL21(DE3) / pET32a-aphA on amide antibiotics

[0089] Types of antibiotics Chloramphenicol Thiamphenicol Florfenicol Degradation rate 77.42％ 51.32％ 74.90％

[0090] Specific steps for AphA protein purification: The AphA crude enzyme solution prepared above was filtered through a 0.22μM filter and added to a Ni-NTA affinity chromatography column. The Ni-NTA column was washed with 30mM, 50mM, 100mM, 150mM, 200mM, 250mM and 300mM imidazole in a gradient from low concentration to high concentration, and the eluate was collected respectively. The purification status was identified by SDS-PAGE pattern. Figure 3 , Figure 3 The results showed that the purified sample had a single band at 48 KDa, which was consistent with the theoretical size of the fusion protein AphA, 48.21 KDa.

[0091] Example 4

[0092] Degradation of chloramphenicol, thiamphenicol and florfenicol by AphA and identification of their metabolites

[0093] 20 mM PBS buffer (pH 7.4), 25 mg L -1The substrates chloramphenicol, thiamphenicol and florfenicol were added with an appropriate amount of the pure enzyme AphA eluted with 150 mM imidazole in Example 3 above, and reacted 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 taken out, extracted twice, combined, dried, dissolved with 250 μL methanol, and filtered through a 0.22 μm organic phase filter to remove impurities to obtain a liquid sample. The metabolites in the reaction solution were determined by LC / MS / MS.

[0094] HPLC degradation effect verification method: HPLC was used to determine the contents of chloramphenicol, thiamphenicol and florfenicol in the extract. The liquid chromatography conditions were as follows: mobile phase acetonitrile: water (40:60, V / V), C18 reverse phase column (4.6×250 mm, 5 μm, Agilent Technologies), column temperature 40 °C, UV detector, measurement wavelength 272 nm (chloramphenicol), 235 nm (thiamphenicol), and 233 nm (florfenicol), injection volume 20 μL, flow rate 1.0 mL min -1 . The degradation products were identified by liquid chromatography-mass spectrometry as follows: first, the above enzyme reaction solution was extracted with 3 volumes of ethyl acetate, the sample was blown dry, and then 250 μL of methanol (chromatographic grade) was added to dissolve, and filtered with a filter membrane (pore size 0.22 μm). Liquid chromatography conditions: injection volume 2 μL, flow rate 0.2 mL·min -1 , temperature 40℃. Sampling speed: 5.0ul s -1 The elution program was gradient (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).

[0095] Mass spectrometry detection was performed using an AB Sciex high-resolution tandem mass spectrometer, Triple TOF 5600+LC / MS / MS system, DuoSpray ion source, positive ion detection mode, and mass scan range (m / z): 50-800.

[0096] The 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 AphA encoded by the amidase gene aphA can degrade chloramphenicol, and the resulting product is identified as p-nitrophenylserinol by mass spectrometry. Figure 4 B, by Figure 4 The data analysis showed that chloramphenicol was hydrolyzed by amidase AphA to break the amide bond and generate p-nitrophenylserinol. The HPLC results of amidase AphA degradation of thiamphenicol are shown in Figure 5 As shown in A, Figure 5A indicates that the amidase AphA encoded by the amidase gene aphA can degrade thiamphenicol, and the generated product is identified as thiamphenicol amine by mass spectrometry. Figure 5 B, by Figure 5 The data analysis showed that thiamphenicol was hydrolyzed by amidase AphA to break the amide bond and generate thiamphenicol amine. The HPLC results of florfenicol degradation by amidase AphA are shown in Figure 6 As shown in A, Figure 6 A indicates that the amidase AphA encoded by the amidase gene aphA can degrade florfenicol, and the resulting product is identified as florfenicol amide by mass spectrometry. Figure 6 B, by Figure 6 The data analysis showed that florfenicol was hydrolyzed by amidase AphA to break the amide bond and generate florfenicol amide. Figure 7 As shown in Figure 2, the degradation rates of the three amide antibiotics by amidase AphA were 75.17% (chloramphenicol), 58.11% (thiamphenicol) and 71.08% (florfenicol). The reaction pathways of the amidase AphA hydrolyzing amide antibiotics are summarized in Figure 8 A (Chloramphenicol conversion equation), Figure 8 B (Thiamethoxam conversion equation), Figure 8 C (florfenicol conversion equation).

[0097] Example 5

[0098] Degradation of amide herbicides propanil, bromothiocarb, chlorpyrifos and chlorbromide by AphA and identification of their metabolites

[0099] 20 mM PBS buffer (pH 7.4), 30 mg L -1 Substrates of propanil, bromothiocarb, chlorpyrifos and chlorbrom were added with an appropriate amount of the pure enzyme AphA eluted with 150 mM imidazole in Example 3 above, and reacted at 35°C for 1 hour. An equal volume of methanol was added to terminate the enzyme reaction, and the mixture was fully shaken for 10 minutes, centrifuged at 12000 rpm for 3 minutes, filtered through a 0.22 μm organic phase filter to remove impurities, and the degradation of amide herbicides was detected by high performance liquid chromatography, and the metabolites in the reaction solution were identified by LC / MS / MS.

[0100] HPLC degradation effect verification method: HPLC was used to determine the degradation of amide herbicides propanil, bromothiocarb, chlorpyrifos and chlorbrom in the reaction solution. Liquid chromatography conditions: mobile phase, propanil: acetonitrile: water (70:30, V / V); bromothiocarb, chlorpyrifos and chlorbrom: acetonitrile: water (65:35, V / V), C18 reverse phase column (4.6×250mm, 5μm, Agilent Technologies), column temperature of 40℃, UV detector, measurement wavelength of 245nm (propanil), 250nm (bromothiocarb, chlorpyrifos and chlorbrom), injection volume of 20μL, flow rate of 1.0mL·min -1 The degradation products were determined by liquid chromatography-mass spectrometry as follows: first, the above enzyme reaction solution was extracted with 3 volumes of dichloromethane, the sample was blown dry, and then 250 μL of methanol (chromatographic grade) was added to dissolve it and filtered with a filter membrane (pore size 0.22 μm). Liquid chromatography conditions: injection volume 2 μL, flow rate 0.2 mL·min -1 , temperature 40℃. Sampling speed: 5.0ul s -1 .

[0101] Mass spectrometry detection was performed using an AB Sciex high-resolution tandem mass spectrometer, Triple TOF 5600+LC / MS / MS system, with DuoSpray as the ion source and a mass scanning range (m / z): 50-800.

[0102] The HPLC results of the amidase AphA encoded by the gene aphA degrading the amide chemical herbicide propanil are as follows Fig. 9 As shown in A, Fig. 9 A indicates that the amidase AphA encoded by the amidase gene aphA can degrade propanil, and the generated product is identified as 3,4-dichloroaniline by mass spectrometry. Fig. 9 B, by Fig. 9 The data analysis showed that propanil was hydrolyzed by AphA to break the amide bond to generate 3,4-dichloroaniline. The HPLC results of AphA degradation of bromothiocarb were as follows: Fig.10 As shown in A, Fig.10 A indicates that the amidase AphA encoded by the amidase gene aphA can degrade bromoaniline, and the generated product is identified as 4-bromoaniline by mass spectrometry. Fig.10 B, by Fig.10 The data analysis showed that bromobenzene was hydrolyzed by amidase AphA to break the amide bond and generate 4-bromoaniline. The HPLC results of amidase AphA degradation of bromobenzene are as follows Fig.11 As shown in A, Fig.11 A indicates that the amidase AphA encoded by the amidase gene aphA can degrade chloranil, and the generated product is identified as 4-chloroaniline by mass spectrometry. Fig.11 B, by Fig.11The data analysis showed that chlorbromidone was hydrolyzed by amidase AphA to break the amide bond and generate 4-chloroaniline. The HPLC results of amidase AphA degradation of chlorbromidone are as follows Fig.12 As shown in A, Fig.12 A indicates that the amidase AphA encoded by the amidase gene aphA can degrade chlorbromid, and the generated product is identified as 4-bromo-3-chloroaniline by mass spectrometry. Fig.12 B, by Fig.12 The data analysis showed that chlorbromidrone was hydrolyzed by amidase AphA to break the amide bond and generate 4-bromo-3-chloroaniline. Fig.13 As shown in Figure 2, the degradation rates of the three amide herbicides by amidase were 98.47% (propanil), 55.77% (bromothiocarb), 92.17% (chlorothiocarb) and 93.70% (chlorbrom). The reaction pathways of amidase AphA in degrading amide chemical herbicides are summarized in Fig.14 A(propanil conversion equation), Fig.14 B (Bromsulfuron conversion equation), Fig.14 C (Chloralkonium chloride conversion formula), Fig.14 D (Chlorbromomethyl conversion equation).

Claims

1. A broad-spectrum amidase gene aphA, characterized in that: The nucleotide sequence of the amidase gene aphA is shown in SEQ ID NO.

1.

2. An amidase protein AphA encoded by the broad-spectrum amidase gene aphA according to claim 1, characterized in that: The amino acid sequence of the amidase protein AphA is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the broad-spectrum amidase gene aphA according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that 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(+).

5. A genetically engineered bacterium containing the broad-spectrum amidase gene aphA according to claim 1 or the recombinant expression vector according to claim 3.

6. The genetically engineered bacterium according to claim 5, characterized in that: The genetically engineered bacteria are obtained by introducing the broad-spectrum amidase gene aphA described in claim 1 or the recombinant expression vector described in claim 3 into Escherichia coli BL21 (DE3).

7. Use of the broad-spectrum amidase gene aphA according to claim 1 or the amidase protein AphA according to claim 2 in the degradation and conversion of amide antibiotics and amide herbicides.

8. The use according to claim 7, characterized in that: The broad-spectrum amidase gene aphA or the amidase protein AphA is preferably used in the degradation or removal of residual pollutants of amide alcohol antibiotics and amide herbicides in environmental water and soil.

9. The use according to claim 7, characterized in that: The amide antibiotics include chloramphenicol, thiamphenicol and florfenicol; the amide herbicides include propanil, bromothiocarb, chlorpyrifos and chlorbromide.

10. Use of the broad-spectrum amidase gene aphA according to claim 1 in constructing transgenic crops that degrade amide herbicides.

Images