Prothioconazole methyltransferase gene pmt76 from mycobacteria and use thereof

Abstract

The present application belongs to the field of environmental biotechnology, and particularly relates to prothioconazole methyltransferase gene pmt76 from mycobacterium and application thereof. ‑3 min ‑1 μM ‑1 The catalytic efficiency of the enzyme pmt76 to (R)-PTC reaches 6.2 x 10 ‑3 min ‑1 μM ‑1 The optimal temperature of the enzyme pmt76 to catalyze (R)-PTC is 30 DEG C, and the reaction condition is mild; meanwhile, the pmt76 of the present application can only catalyze the degradation of PTC, and shows strong specificity. In summary, the prothioconazole methyltransferase found in the present application has good industrial application potential in environmental pollution control.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, and in particular, it relates to the pmt76 gene of prothioconazole methyltransferase from mycobacteria and its applications. Background Technology

[0002] Prothioconazole (PTC) is a chiral triazole fungicide with CAS Registry Number 178928-70-6 and molecular formula C. 14 H 15Cl2N3OS has a relative molecular mass of 344.26. Prothioconazole belongs to the sterol demethylation inhibitor (DMI) class and is classified as subgroup G1 by the International Fungicide Resistance Action Committee (FRAC). Its mechanism of action is to inhibit the demethylation of lanosterol or 2,4-methylenedihydrolanosterol, precursors of sterols in fungi, at the 14-position, thereby blocking ergosterol synthesis and disrupting the integrity of the fungal cell membrane. After application to plants, prothioconazole is metabolized into its bioactive form, dethioconazole-desthio, which inhibits the C14-demethylation process with greater activity. This agent not only has excellent systemic activity but also possesses superior protective, curative, and eradicative activities, with a long residual effect. Prothioconazole has a broad fungicidal spectrum, showing excellent control effects against almost all fungal diseases on cereals, including powdery mildew, Fusarium head blight, sheath blight, and rust. It can also effectively control soil-borne diseases and major foliar diseases of rapeseed and peanuts, such as gray mold, brown spot, black spot, and black shank. Its application crops include wheat, soybeans, rapeseed, rice, peanuts, sugar beets, corn, and cotton. In terms of formulation, prothioconazole can be made into various forms such as suspension concentrates, oil-dispersible suspensions, water-dispersible granules, and emulsifiable concentrates, which can be used for foliar spraying or seed treatment. However, with its widespread use in agricultural production, it has been found in recent years that its residues can easily cause environmental pollution and exhibit chiral selective toxicity. The European Food Safety Authority (EFSA) has confirmed the potential acute risks of prothioconazole and has set maximum residue limits (MRLs) of 0.02-0.1 mg / kg for crops such as corn, sugar beets, potatoes, and peanuts. Canada has set an MRL of 0.2 mg / kg for prothioconazole in sunflowers. Therefore, the degradation treatment of prothioconazole is an important research direction. Jia Ye et al. disclosed (“Enantioselective Metabolism of Chiral Fungicide Prothioconazole by Mycobacterium sp. Y-3 and Its Bioaugmentation”, Journal of Agricultural and Food Chemistry, DOI: 10.1021 / acs.jafc.5c02382) a Mycobacterium Y-3 strain capable of degrading prothioconazole. This strain preferentially metabolizes (R)-prothioconazole via methylation, with a degradation rate 2.5 times that of (S)-prothioconazole. At 37°C and pH 5.0, the half-lives (T1 / 2) of strain Y-3 for 20–80 μM (Rac)-, (S)-, and (R)-prothioconazole were 4.1–7.1 hours, respectively.However, in order to further improve enzymatic methods and obtain strains with stronger degradation capabilities, it is necessary to conduct further research and exploration on methylation-related genes and enzymes in the process of strains degrading prothioconazole. Summary of the Invention

[0003] To address the related problems in the prior art, this invention, through further in-depth research, discovered a prothioconazole methyltransferase gene pmt76 from Mycobacterium sp. Y-3. This lays the foundation for enzymatic degradation of prothioconazole, construction of recombinant prothioconazole-degrading bacteria, and mutation screening to obtain stronger prothioconazole-degrading bacteria, thus completing this invention.

[0004] The technical solution of the present invention is as follows:

[0005] In one aspect, the present invention discloses a prothioconazole methyltransferase gene pmt76, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] ATGACTGCTCCAGAAGACACCACGCGCCATTTCGAAGAGATGTACCGCGACGAGCGGACATCGCACGGTCTGCCTGCCGCCACCCCGTGGGACATCGGAGGGCCCCAACCGGTCGTCCAGCAACTCGTCGCGGTCGGCGCCGTCAAAGGGGAGGTACTCGACCCGGGCACCGGCCCCGGCCACCATGCCATCCACTACGCGTCAAAGGGCCTGTCGGCCACGGGCATCGACGCATCGCCCACGGCGATCGAGCGGGCCAAGGAGAACGCGGCGAAAGCCGGGGTGACGGTGGACTTCCAGGTGGCCGATGCCACCAAACTCGAAGGCCTGGAAGGGCGTTTCGACACCGTCGTCGACTCCGCGTTCTTCCATGTGTTCACCGGCGAGCAGGAACTGCAGAAGTCCTACGTGCGCGCATTGCACCGGGCCACCAAACCCGGGGCGCGGTTGTTCATGTTCGAATTCGCCGATCACAACGTCAACGGCTTCCGGATGCCGCGCTACCTCACCGCCGACGATTTCCGTGCGGTGCTGCCCGACGCCGGCTGGGACATCACCTACCTCGGGCCCACCACCTATCAGGGCAACGTCAGCGTCGAGACGTTCGAGATGATGGCCGCTCGCAACCCCGACATGGCCGAGGGGATGCAACCGCTGCTGGAGCGCTTGCGGGTGATCGAGCCGTGGCTGATCGACGGCCGCGTGCACATGCCGTTCTGGGAGGTGCACGCCACCCGGATGGGCTGA

[0007] In one aspect of the present invention, the present invention discloses a prothioconazole methyltransferase pmt76, the amino acid sequence of which is set forth in SEQ ID NO.2.

[0008] MTAPEDTTRHFEEMYRDERTSHGLPAATPWDIGGPQPVVQQLVAVGAVKGEVLDPGTGPGHHAIHYASKGLSATGIDASPTAIERAKENAAKAGVTVDFQVADATKLEGLEGRFDTVVDSAFFH VFTGEQELQKSYVRALHRATKPGARLFMFEFADHNVNGFRMPRYLTADDFRAVLPDAGWDITYLGPTTYQGNVSVETFEMMAARNPDMAEGMQPLLERLRVIEPWLIDGRVHMPFWEVHATRMG*

[0009] In one aspect, the present invention discloses an expression vector containing the prothioconazole methyltransferase gene pmt76. The expression vector can be a prokaryotic expression vector or a eukaryotic expression vector; preferably, the expression vector is a prokaryotic expression vector.

[0010] In one aspect, the present invention discloses a recombinant strain containing an expression vector containing the prothioconazole methyltransferase gene pmt76.

[0011] In one embodiment, the strain is Escherichia coli.

[0012] In one aspect, the present invention discloses the application of strains containing the prothioconazole methyltransferase pmt76 gene expression vector in the degradation of prothioconazole.

[0013] In one aspect, the present invention discloses the prothioconazole methyltransferase pmt76 in the degradation of prothioconazole.

[0014] In one embodiment, the prothioconazole is R-prothioconazole or S-prothioconazole.

[0015] In one aspect, the present invention also discloses a formulation for degrading prothioconazole, wherein the formulation contains a strain expressing the prothioconazole methyltransferase gene pmt76 gene; or the formulation contains prothioconazole methyltransferase pmt76.

[0016] In one aspect, the present invention also discloses an immobilized enzyme preparation, wherein the immobilized enzyme is prothioconazole methyltransferase PMT76. Beneficial effects

[0017] This invention identified the methyltransferase gene pmt76, which is effective in degrading prothioconazole, and its recombinant expression, resulting in the enzyme pmt76 with a catalytic efficiency of 6.2 × 10⁻⁶ for (R)-PTC. -3 min -1 μM -1 The optimal temperature for this enzyme to catalyze (R)-PTC is 30 °C, and the reaction conditions are mild. At the same time, the pmt76 of this invention can only catalyze the degradation of PTC, exhibiting extremely strong specificity. Attached Figure Description

[0018] Figure 1 Electrophoresis image of PCR amplification of the target gene pmt76, where M is the marker and 3 is the pmt76 gene.

[0019] Figure 2 E. coil BL21 (DE3) (pET-pmt76) PTC liquid phase diagram.

[0020] Figure 3 SDS-PAGE gel image of the fusion protein His-pmt76, where M is the protein marker; 1 is the crude pmt76 protein, and 2 is the purified pmt76 protein.

[0021] Figure 4 : pmt76's ability to convert PTC.

[0022] Figure 5 The products generated by pmt76 catalyzing PTC were detected by HPLC and UPLC-Q-TOF-MS. A is the HPLC chromatogram of pmt76 converting (Rac)-PTC; B is the mass spectrum.

[0023] Figure 6 Enzyme kinetic curves of pmt76 catalyzing the conversion of (R)-PTC and (S)-PTC.

[0024] Figure 7 The effect of temperature on the activity of pmt76 enzyme.

[0025] Figure 8 Effect of pH on the activity of pmt76 enzyme.

[0026] Figure 9 The effects of metal ions and chemical reagents on the activity of pmt76 enzyme. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0028] Example 1: Cloning, expression, and purification of the prothioconazole methyltransferase gene

[0029] 1.1 PCR amplification of methyltransferase candidate genes

[0030] Using Mycobacterium sp. Y-3 as a template, the target gene pmt76 was amplified by PCR using pre-designed primers. After separation of the amplification products by agarose gel electrophoresis, a single gel band containing the target DNA fragment was rapidly excised under UV light. The target fragment was purified using the FastPure® Gel DNA Extraction Mini Kit. The gene sequence of pmt76 is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2.

[0031] 1.2 Construction of heterologous expression vector for methyltransferase

[0032] The homologous recombination seamless cloning kit ligated the amplified pmt76 gene with the linearized expression vector pET28a (+) to obtain the exogenous recombinant expression vector pET28a-pmt76. The recombinant vector was transformed into E coli BL21(DE3). After the reaction, plasmids were extracted, and PCR detection was performed. The bacterial culture corresponding to a single PCR product with the same size as the target band was sent to Anhui General Biotechnology Co., Ltd. for sequencing. The successfully constructed and correctly expressed strain E. coli BL21(DE3) was mixed with sterilized 30% glycerol at a 1:1 ratio and stored at -80 ℃ for later use.

[0033] 1.3 Test of the transformation ability of recombinant strains to prothioconazole (PTC)

[0034] The successfully constructed recombinant plasmids were inoculated into 30 mL LB medium containing 50 mg / L Km and cultured at 37℃ with shaking at 150 r / min until the OD600 reached 0.6. Then, 0.1 mM IPTG and 40 mg / L (Rac)-PTC were added to the medium, and the culture was further induced for 24 h at 150 r / min. One mL of culture medium was taken, and an equal volume of methanol was added. The mixture was thoroughly mixed by inversion and centrifuged at 12000 r / min for 5 min. The supernatant was filtered through a 0.22 μm organic filter membrane, and the concentration change of (Rac)-PTC in the culture medium was detected by HPLC.

[0035] 1.4 Preparation and purification of crude enzyme solution of recombinant protein

[0036] Single colonies of recombinant E coli BL21 (DE3) containing pET28a-pmt76 were picked and inoculated into 300 mL LB liquid medium containing 50 mg / L Km. The cultures were incubated at 37 °C with shaking until the OD600 reached 0.6. IPTG was then added to the E coli BL21 (pET-pmt76) culture to a final concentration of 0.5 mM, and the cultures were induced at 25 °C for 12 h with shaking. After induction, the cultures were centrifuged at 4 °C, 10000 r / min for 8 min, and the precipitated bacterial cells were collected. The bacterial cells were resuspended in pre-chilled 20 mM Tris-HCl (pH=7.4) buffer, washed twice by centrifugation at 5000 r / min for 10 min, and finally resuspended in 25 mL buffer. The resuspended bacterial solution was then sonicated and centrifuged at 10000 r / min for 30 min at 4 ℃. The collected supernatant was the pmt76 crude enzyme solution. The sample was placed on ice or stored at 4 ℃ for later use. Recombinant protein purification was performed using a Ni-TED.6EF pre-packed gravity column. After purification, the molecular size of the recombinant protein was determined by SDS-PAGE, and the protein concentration was determined using the Bradford Protein Assay Kit (Takara).

[0037] Results: See Figure 1 The amplified pmt76 target gene is shown, with a molecular weight of approximately 747 bp; see also Figure 2High-performance liquid chromatography (HPLC) results showed that the characteristic peaks of PTC (retention time RT = 5.6 min) in the culture medium of recombinant E. coli BL21 (pET-pmt76) all exhibited significant attenuation. Simultaneously, a novel product peak appeared at a retention time of 10.2 min, whose elution time perfectly matched the characteristic peak of the PTM standard (RT = 10.2 min). These results indicate that recombinant E. coli can catalyze the conversion of PTC to PTM, and pmt76 is the functional gene responsible for catalyzing the methylation reaction of PTC. See also... Figure 3 SDS-PAGE analysis showed that after purification, the size of the pmt76 enzyme was approximately 30 kDa, consistent with the theoretical value of 29.1 kDa.

[0038] Example 2: Comparison of the ability of pmt76 to convert different configurations of PTC

[0039] A final concentration of 40 μM (Rac)- / (R)- / (S)-PTC and 1 mM SAM were added to 500 μL of purified enzyme PMT76 in a 30 °C water bath. Samples were taken at different time points, and an equal volume of acetonitrile was added to terminate the enzyme reaction. The terminated enzyme reaction solution was vortexed, centrifuged at 12000 r / min for 5 min, and filtered through a 0.22 μm organic phase filter. The concentrations of substrate and product were determined by HPLC using a chiral column. All treatments were performed in triplicate.

[0040] Results: pmt76 achieved a 54% conversion rate of (Rac)-PTC within 95 min, with the EF value decreasing from 0.50 to 0.49. Figure 4 ).

[0041] Example 3: Metabolic pathway analysis of pmt76 to PTC conversion

[0042] Add 40 μM (Rac)-PTC and 1 mM SAM to 500 μL of enzyme-catalyzed reaction solution containing an appropriate amount of purified PMT76 enzyme. Incubate in a 30 °C water bath for 30 min, then terminate the reaction with an equal volume of acetonitrile. Vortex the terminated enzyme reaction solution, centrifuge at 12000 r / min for 5 min, filter through a 0.22 μm organic phase filter, and analyze by HPLC. After confirming the presence of a distinct product peak, dilute the sample (1:1 methanol:water) to a concentration less than 1 ppm, vortex, centrifuge at 13500 r / min for 3 min, filter through a 0.22 μm organic phase filter, and detect the peak using UPLC-Q-TOF-MS. Identify the product based on the primary and secondary mass spectra.

[0043] Results: See Figure 5The product generated after the pmt76-catalyzed (Rac)-PTC conversion was identified by UPLC-QTOF-MS. The results showed that the characteristic peak at m / z = 358.0559 in the primary mass spectrum was consistent with that of prothioconazole [M+CH2+H]. + The theoretical value of 358.0543 is consistent; the characteristic diagnostic ions of the secondary mass spectrometry (m / z = 98.9632 and 125.0158) match the fragmentation pattern of the structural azole of methylthiobacillus (triazole methylthio and o-chlorobenzyl structural units), and their mass-to-charge ratio is consistent with the relative molecular mass of PTM (see [link]). Figure 5 (B) This study confirmed that Pmt76 can catalyze the methylation reaction of PTC to produce the product PTM.

[0044] Example 4: Measurement of kinetic parameters of pmt76

[0045] In a 500 μL Tris-HCl enzymatic reaction system containing an appropriate amount of pmt76, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, and 180 μM of (S)-PTC and (R)-PTC were added, respectively, and the reaction was carried out in a 30 °C water bath for 30 min. The enzymatic reaction system consisted of 1 mL of 20 mM Tris-HCl (pH = 7.4) buffer, with a final concentration of 40 μM (Rac)-PTC, an appropriate amount of SAM, and an appropriate amount of pmt76 added. Specific enzyme activity was defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate per 1 min. An equal volume of acetonitrile was added to the above reaction solution, and the mixture was thoroughly inverted to terminate the enzymatic reaction. The mixture was centrifuged at 12000 r / min for 3 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The concentrations of PTC and the product PTM were determined by HPLC. The substrate concentration and reaction rate in the reaction were nonlinearly fitted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) to obtain K. m Vmax and k cat Values. All processes are set up with three parallel groups.

[0046] Results: Enzyme kinetic analysis showed that the catalytic efficiency k of Pmt76 for (R)-PTC was [missing value]. cat / K m = 6.2×10 -3 min -1 μM -1 ( Figure 6 (A), its catalytic efficiency k for (S)-PTC cat / K m = 6.2×10 -3 min-1 μM -1 ( Figure 6 (B), which indicates that Pmt76 has the same catalytic efficiency for (R)- / (S)-PTC, i.e., no obvious stereoselectivity.

[0047] Example 5: Effect of temperature on pmt76 enzyme activity

[0048] Add appropriate amounts of PMT76 purified enzyme, 0.5 mM SAM, and 40 μM (S)- / (R)-PTC to the buffer solution. React at temperatures of 4, 16, 25, 30, 37, 42, 50, 60, and 70 °C for 20 min, then take samples and add an equal volume of acetonitrile to terminate the enzyme reaction. HPLC was used to determine the substrate and product concentrations, and the relative enzyme activity at different temperatures was calculated with the highest enzyme activity defined as 100%. No enzyme was added as a blank control, and both the control and treatment groups were set up in triplicate.

[0049] Results: The optimal reaction temperature for Pmt76 with both substrates was 30 ℃. Figure 7 When the temperature is within the range of 4–30 °C, the enzyme activity of Pmt76 gradually increases with increasing temperature; when the temperature is above 30 °C, the relative enzyme activity of Pmt76 catalyzing the conversion of (R)- / (S)-PTC begins to gradually decrease; when the temperature is above 60 °C or below 4 °C, the relative enzyme activity of Pmt76 catalyzing (R)- / (S)-PTC is below 20%. Therefore, the enzyme activity of Pmt76 catalyzing (R)- / (S)-PTC is quite sensitive to temperature.

[0050] Example 6: Effect of pH on pmt76 enzyme activity

[0051] Different pH buffer systems were prepared: 50 mM acetate-sodium acetate (pH 3.5-6.0), 50 mM citric acid-sodium citrate (pH 5.5-8.5), 50 mM Tris-HCl buffer (pH 7.0-9.0), and 50 mM glycine-NaOH buffer (pH 9.0-11.0). Appropriate amounts of PMT76 purified enzyme, 0.5 mM MSAM, and 40 μM (S)- / (R)-PTC were added to each, and the reactions were carried out in a water bath at the optimal temperature for 20 min. The reaction was terminated by adding an equal volume of acetonitrile. The concentrations of substrate and product were determined by HPLC, and the relative enzyme activities under other pH conditions were calculated with the highest enzyme activity defined as 100%.

[0052] Results: Different pH values ​​had different effects on the relative enzyme activity of Pmt76 catalyzing prothioconazole. The optimal catalytic pH for the conversion of (R)- / (S)-prothioconazole by Pmt76 was 9.0. Figure 8Pmt76 exhibits the ability to convert (R)- / (S)-PTC within a pH range of 5.5–11.0. However, at pH < 5.0, the relative enzyme activity of Pmt76 in catalyzing the conversion of (R)- / (S)-prothioconazole is less than 5%. Furthermore, Pmt76 shows poor conversion efficiency for (R)- / (S)-PTC in an acetate-sodium acetate (HAc-NaAc) buffer system.

[0053] Example 7 Effects of metal ions and chemical reagents on the activity of pmt76 enzyme

[0054] In a buffer system at the optimal pH, appropriate amounts of pmt76 purified enzyme, 0.5 mM SAM, and 40 μM (S)- / (R)-PTC were added, followed by the addition of various metal ions (K+, K+, and PTC) to a final concentration of 1.0 mM. + Na + Ag + Zn 2+ Mg 2+ Cu 2+ Ca 2+ Mn 2+ Ni 2+ Fe 2+ Fe 3+ and Cr 3+ Various inhibitors, including EDTA (1 mM), 1,10-phenanthroline, and detergents CTAB and SDS (0.5% m / v), were reacted in a water bath at the optimal temperature. The enzyme activity under the reaction system without any added metal ions or other chemical reagents was taken as 100%, and the relative enzyme activities under other reagent conditions were calculated.

[0055] Results: See Figure 9 Adding different metal ions to the enzyme-catalyzed reaction system will inhibit the relative enzyme activity of Pmt76 to varying degrees, among which 1 mM Ni 2+ Cr 3+ Fe 2+ Fe 3+ Cu 2+ Zn 2+ Ag +The enzyme activity of Pmt76 catalyzing (R)- / (S)-PTC was significantly inhibited, with inhibition rates ranging from 65.0% to 99.9%. The addition of different chemical reagents showed varying degrees of inhibitory or promoting effects on Pmt76 enzyme activity. Specifically, 1 mM CTAB and 0.5% (m / v) detergent SDS significantly inhibited Pmt76 catalyzing (R)- / (S)-PTC activity, while 1 mM PMSF showed moderate inhibition, with inhibition rates of approximately 55%–70%. 1 mM 1,10-phenanthroline showed moderate inhibition of Pmt76 catalyzing (S)-PTC activity, but had no significant effect on its catalyzing (R)-PTC activity.

[0056] Example 8 Substrate Spectral Analysis

[0057] A suitable amount of purified pmt76 enzyme and 1 mM SAM were added to a 500 μL Tris-HCl enzymatic reaction system, followed by the addition of 20 mg / L of each of the following five PTC analogs: 4-amino-5-(2-furan)-4H-1,2,4-triazol-3-thione, 4-amino-5-(4-chlorophenyl)-1,3-dithiacyclopentadien-2-thione, 5-(4-fluorophenyl)-4-methyl-4H-1,2,4-triazol-3-thiol, 4-phenyl-4H-1,2,4-triazol-3-mercaptoethanol, and 4-ethyl-5-phenyl-4H-[1,2,4]triazol-3-thiol. The reaction was carried out at 30 °C for 24 h, and the enzyme reaction was terminated by adding an equal volume of acetonitrile. The conversion of each analog was detected by HPLC, and peaks were detected by UPLC-Q-TOF-MS. The products were identified based on the primary and secondary mass spectra.

[0058] Results: As shown in Table 1, pmt76 is only active against R-prothioconazole and S-prothioconazole, while other species have similar catalytic activity.

[0059] Table 1: Substrate degradation profile of pmt76 enzyme

[0060]

[0061] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A prothioconazole methyltransferase gene pmt76, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A prothioconazole methyltransferase pmt76, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The expression vector contains the pmt76 gene of prothioconazole methyltransferase, and the expression vector is selected from prokaryotic expression vectors or eukaryotic expression vectors; preferably, the expression vector is a prokaryotic expression vector.

4. A recombinant bacterial strain, characterized in that, The recombinant strain contains an expression vector containing the prothioconazole methyltransferase gene pmt76.

5. The recombinant strain according to claim 4, characterized in that, The strain in question is Escherichia coli.

6. Application of strains containing the prothioconazole methyltransferase pmt76 gene expression vector in the degradation of prothioconazole.

7. The application of prothioconazole methyltransferase PMT76 in the degradation of prothioconazole, characterized in that, The prothioconazole is R-prothioconazole or S-prothioconazole.

8. A formulation for degrading prothioconazole, characterized in that, The preparation contains a strain expressing the pmt76 gene of prothioconazole methyltransferase; or the preparation contains prothioconazole methyltransferase pmt76.

9. An immobilized enzyme preparation, characterized in that, The immobilized enzyme in the immobilized enzyme preparation is prothioconazole methyltransferase PMT76.

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