An amidase mutant and its application in the preparation of (S)-fluopyram aniline

Abstract

This invention provides an amidase mutant and its application in the preparation of ( S The application of )-fluopyrazopyr in aniline belongs to the fields of genetic engineering and enzyme catalysis technology. Compared with the amidase shown in SEQ ID No. 1, the amidase mutant of this invention has a mutation at position 109 (alanine) to valine and / or position 232 (leucine) to arginine. Compared with the wild-type amidase, the amidase mutant of this invention has a higher optimum temperature and thermal stability, and can catalyze the enzymatic reaction of racemic substrate M-1 (Formula I) in an aqueous phase to generate the key chiral intermediate (…). S )-M-2 (Formula II), further reacted with compound M-3 (Formula III), to efficiently prepare ( S The method for preparing 1-fluopyrazoline is characterized by mild reaction conditions, simple operation, and promising prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme catalysis technology, and particularly relates to an amidase mutant and its preparation. S Application of )-Fluorazole aniline. Background Technology

[0002] Amidases (EC 3.5.1.X) are a class of important hydrolases that catalyze the hydrolysis of amide bonds to produce the corresponding carboxylic acids and ammonia. Due to their high stereoselectivity, broad substrate spectrum, and mild reaction conditions, amidases show great potential for application in organic synthesis, particularly in the preparation of chiral pharmaceuticals and pesticide intermediates.

[0003] Penflufen, a fungicide developed by Bayer AG, is a succinate dehydrogenase inhibitor primarily used for seed treatment. It effectively controls crop diseases caused by fungi such as Rhizoctonia. Its molecular structure contains a chiral center, which... S )-and( R - Two enantiomers. The commercial product of fluopyram aniline is its racemic form, i.e., an equal mixture of ( S )-type and ( R Type )-. But the main bactericidal agent is ( S )-type,( R The )-type is almost ineffective. Therefore, it is necessary to develop efficient and green methods to synthesize optically pure ( ) S Fluopyram aniline can reduce the total amount of chemicals released into the environment, thus reducing residues and ecological risks.

[0004] Chemical methods for preparing chiral molecules typically require expensive chiral catalysts or chiral resolving agents and may generate environmental pollution. Biochemical enzyme catalysis offers a greener and more efficient approach. However, natural amidases often have limitations in practical industrial applications, such as low enzyme activity and poor thermal stability. Protein engineering techniques, particularly site-directed mutagenesis, are effective means of improving enzyme properties. By rationally designing or directing evolution to replace key amino acid residues in enzyme molecules, catalytic efficiency, stability, and stereoselectivity can be significantly improved. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an amidase mutant and its preparation ( S The application of 1-fluopyram in aniline aims to solve the problems of poor thermal stability and low optimal reaction temperature of natural amidases in existing technologies, which lead to insufficient catalytic efficiency.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides an amidase mutant, which, compared with the amidase shown in SEQ ID No. 1, has alanine at position 109 mutated to valine and / or leucine at position 232 mutated to arginine.

[0008] The present invention also provides the encoding gene of the above-mentioned amidase mutant, the nucleotide sequence of which is shown in SEQ ID No. 3-5.

[0009] The present invention also provides a recombinant plasmid carrying the above-mentioned encoding gene.

[0010] The present invention also provides a host cell carrying the above-mentioned coding gene or the above-mentioned recombinant plasmid.

[0011] This invention also provides a method for preparing the above-mentioned amidase mutant, comprising the following steps: inoculating the above-mentioned host cells into LB medium and culturing at 37±2℃ and 150-250 rpm to prepare a seed culture; transferring the seed culture to a new LB medium and continuing to culture until OD. 600 Once the enzyme concentration reaches 0.6, cool the temperature to 25±2℃ and add IPTG to induce expression, obtaining the crude enzyme solution. Alternatively, the above host cells can be seeded into LB medium and cultured at 37±2℃ and 150-250 rpm to prepare a seed culture; the seed culture can be transferred to a fermenter for fermentation to prepare a secondary seed culture; the secondary seed culture can then be transferred to a new fermenter and cultured until the OD reaches 0.6. 600 Once the enzyme concentration reaches 1.0, the temperature is lowered to 25±2℃ and IPTG is added to induce expression, thus obtaining the crude enzyme solution.

[0012] This invention also provides the above-mentioned amidase mutant, or recombinant plasmid, or host cell in the preparation ( S Application of )-Fluorazole aniline.

[0013] This invention also provides a method for preparing ( S The method for using 1-fluopyrazopyr aniline includes the following steps:

[0014] (1) Prepare amidase mutants using the above preparation method;

[0015] (2) Using racemic substrate M-1 as reactant, an enzymatic reaction is carried out under the catalysis of the amidase mutant in step (1) to generate the key chiral intermediate ( S )-M-2; wherein, M-1 is a compound having the structure shown in Formula I, and the ( S )-M-2 is a compound having the structure shown in Formula II;

[0016]

[0017] Formula I

[0018]

[0019] Formula II

[0020] (3) The key chiral intermediate is separated from the reaction solution of step (2). S )-M-2;

[0021] (4) The key chiral intermediate ( S )-M-2 reacts with substrate M-3 to obtain ( S )-Fluorazole aniline, wherein M-3 is a compound having the structure shown in Formula III.

[0022]

[0023] Formula III

[0024] Preferably, in step (2), the concentration of racemic substrate M-1 is 0.1-100 g / L, and the concentration of amidase mutant is 0.01-5 g / L.

[0025] Preferably, in step (2), the conditions for the enzymatic reaction are controlled as follows: reaction temperature 35-65℃, reaction pH 8.0-10.0, and reaction time 1-12 h.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention improves the conformational stability of amidases. Compared with wild-type amidases (optimal temperature 40℃, residual enzyme activity of only 20% after incubation at 50℃ for 1 hour), the optimal temperatures of single-point mutants A109V and L232R are increased to 45℃ and 50℃, respectively. The optimal temperature of double mutant A109V-L232R is stable at 50℃, and the residual enzyme activity after incubation at 50℃ for 1 hour reaches 61%, which is more than 3 times that of wild-type amidases. This effectively solves the problem of easy inactivation of natural amidases under medium and high temperature reaction conditions and broadens the temperature range applicable to enzymatic reactions.

[0028] The conformational optimization of the amidase mutant of this invention simultaneously enhances enzyme-substrate binding and catalytic activity. In the same reaction system, the double mutant A109V-L232R catalyzes the formation of (… S The rate of )-M-2 was significantly improved, reaching a yield of 41.2 g / L in just 8 hours, while the yield of wild-type amidase was only 12.5 g / L after 12 hours. The product generation efficiency was increased by more than 3 times, and the reaction time was shortened by 33%, which greatly improved production efficiency and reduced time costs.

[0029] The amidase mutant of the present invention has extremely high stereoselectivity, catalyzing the generation of ( SM-2 has excellent optical purity and is subsequently reacted with M-3 to prepare ( S When processing )-fluopyram aniline, the product yield reaches 95%, and the purity and ee value are both as high as 99.9%, which is far superior to existing racemic products. Moreover, it eliminates the need for complex chiral separation steps, simplifying the process and avoiding product loss during the separation process.

[0030] This invention employs a route combining bio-enzyme catalysis and chemical synthesis. The enzymatic reaction is carried out in an aqueous phase under mild conditions and without the addition of any toxic or harmful reagents. Compared with traditional chemical methods, it significantly reduces environmental pollution and ecological risks, aligns with the development trend of green pesticide creation, and has broad application prospects. Attached Figure Description

[0031] Figure 1 The results of RMSF analysis of wild-type amidase in Example 2 at 350 K;

[0032] Figure 2 The results of the optimal reaction temperature analysis for amidase and amidase mutant in Example 4;

[0033] Figure 3 The results of thermostability analysis of amidase and amidase mutant in Example 4;

[0034] Figure 4 For the different reaction times catalyzed by amidase and amidase mutant in Example 5 ( S The production of )-M-2. Detailed Implementation

[0035] The present invention provides an amidase mutant, which, compared with the amidase shown in SEQ ID No. 1, has alanine at position 109 mutated to valine and / or leucine at position 232 mutated to arginine.

[0036] The present invention also provides the encoding gene of the above-mentioned amidase mutant, the nucleotide sequence of which is shown in SEQ ID No. 3-5.

[0037] The present invention also provides a recombinant plasmid carrying the above-mentioned encoding gene.

[0038] The present invention also provides a host cell carrying the above-mentioned coding gene or the above-mentioned recombinant plasmid.

[0039] This invention also provides a method for preparing the above-mentioned amidase mutant, using shake-flask fermentation, comprising the following steps: inoculating the above-mentioned host cells into LB medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride), and culturing at 37±2℃ and 150-250 rpm to prepare a seed culture, preferably at 37℃ and 200 rpm; transferring the seed culture to a new LB medium and continuing to culture until OD. 600 The concentration reached 0.6, and the temperature was lowered to 25±2℃ and IPTG was added to induce expression. Preferably, the temperature was lowered to 25℃ and 0.2 mM IPTG was added to induce expression for 14 h to obtain crude enzyme solution. Further preferred method was to centrifuge the crude enzyme solution to collect the bacterial cells, resuspend them in an equal volume of deionized water, and collect the supernatant by sonication and centrifugation.

[0040] Alternatively, fermentation can be carried out in a fermenter, comprising the following steps: The host cells are inoculated into LB medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride) and cultured at 37±2℃ and 150-250 rpm to prepare a seed culture, preferably at 37℃ and 200 rpm; the seed culture is transferred to a fermenter (12.0 g / L yeast extract, 15.0 g / L tryptone, 8.9 g / L Na2HPO4·12H2O, 3.4 g / L KH2PO4, 2.67 g / L NH4Cl, 0.71 g / L Na2SO4, 0.49 g / L MgSO4·7H2O) for fermentation to prepare a secondary seed culture, preferably fermented for 8 hours; the secondary seed culture is then transferred to a new fermenter, preferably with an initial fermentation temperature of 37℃, a rotation speed of 700 rpm, and an aeration rate of 1.5 L·min. -1 After the dissolved oxygen rebounded, it began to rise at 8 g·L⁻¹. -1 ·h -1 Glycerol was added to the culture medium at a constant rate, and the culture was incubated until OD500 reached. 600 Reach 1.0; cool to 25±2℃ and add IPTG to induce expression, preferably cool to 25℃ and add 0.2 mM IPTG for induction, until OD... 600 After ceasing growth, a crude enzyme solution was obtained. Further optimization involved centrifuging the crude enzyme solution to collect the bacterial cells, then resuspending the cells in deionized water to control OD. 600 The bacterial cells were broken up using a high-pressure homogenizer at a concentration of 80, and the supernatant was collected by centrifugation.

[0041] This invention also provides the above-mentioned amidase mutant, or recombinant plasmid, or host cell in the preparation ( S Application of )-Fluorazole aniline.

[0042] This invention also provides a method for preparing ( S The method for using 1-fluopyrazopyr aniline includes the following steps:

[0043] (1) Prepare amidase mutants using the above preparation method;

[0044] (2) Using racemic substrate M-1 as reactant, an enzymatic reaction is carried out under the catalysis of the amidase mutant in step (1) to generate the key chiral intermediate ( S )-M-2; wherein, M-1 is a compound having the structure shown in Formula I, and the ( S )-M-2 is a compound having the structure shown in Formula II;

[0045]

[0046] Formula I

[0047]

[0048] Formula II

[0049] (3) The key chiral intermediate is separated from the reaction solution of step (2). S )-M-2;

[0050] (4) The key chiral intermediate ( S )-M-2 reacts with substrate M-3 to obtain ( S )-Fluorazole aniline, wherein M-3 is a compound having the structure shown in Formula III.

[0051]

[0052] Formula III

[0053] In this invention, preferably in step (2), the concentration of racemic substrate M-1 is 0.1-100 g / L, and the concentration of amidase mutant is 0.01-5 g / L.

[0054] In this invention, in step (2), the conditions for the enzymatic reaction are preferably controlled as follows: reaction temperature 35-65℃, more preferably 50℃; reaction pH 8.0-10.0, more preferably pH 8.5; reaction time 1-12 h.

[0055] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Construction and fermentation of amidase expression strains

[0058] The encoding gene for wild-type amidase was artificially synthesized. First, codon optimization was performed based on the amino acid sequence of the amidase (as shown in SEQ ID No. 1) to obtain the DNA sequence shown in SEQ ID No. 2. Then, Beijing Qingke Biotechnology Co., Ltd. completed the gene synthesis and cloned the DNA into the Nde I and Xho I sites of the pET-28a plasmid to obtain the recombinant plasmid pET-WT. The recombinant plasmid pET-WT was transformed into E. coli BL21(DE3) competent cells using the heat shock method to obtain recombinant E. coli. E. coli WT.

[0059] Recombinant Escherichia coli was cultured and fermented in shake flasks using LB medium, which consists of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride. Single colonies of recombinant Escherichia coli were picked and placed in test tubes containing 5 mL of LB medium, and incubated overnight at 37°C and 200 rpm to obtain the seed culture.

[0060] Shake-flask fermentation: Transfer the seed culture to fresh LB medium at a 3% inoculum and incubate at 37°C and 200 rpm until OD500. 600 When the concentration reaches approximately 0.6, the temperature is lowered to 25°C, and 0.2 mM IPTG is added for induction of expression. After 14 h of induction, the bacterial cells are collected by centrifugation, resuspended in an equal volume of deionized water, and the supernatant is collected by sonication and centrifugation to obtain crude enzyme solution.

[0061] Fermentation in a 50 L fermenter: The seed culture was transferred to a 5 L fermenter containing 2 L LB medium at a 3% inoculum level. Fermentation was carried out for 8 hours to prepare the secondary seed culture. The 50 L fermenter contained an initial 20 L fermentation medium, consisting of 12.0 g / L yeast extract, 15.0 g / L peptone, 8.9 g / L Na₂HPO₄·12H₂O, 3.4 g / L KH₂PO₄, 2.67 g / L NH₄Cl, 0.71 g / L Na₂SO₄, and 0.49 g / L MgSO₄·7H₂O. All the prepared secondary seed culture was transferred to the 50 L fermenter. The initial fermentation temperature was 37℃, the rotation speed was 700 rpm, and the aeration rate was 1.5 L / min. -1 After the dissolved oxygen rebound, it began to rise at 8 g·L⁻¹. -1 ·h -1 Glycerol was added to the culture medium at a uniform rate. The culture was continued until the bacterial cell OD reached a certain level. 600 After reaching 1, the temperature was lowered to 25°C and 0.2 mM IPTG was added to induce protein expression. The expression was then induced until the bacterial cell OD... 600 Once growth ceases, fermentation stops. The cells are collected by centrifugation and then resuspended in deionized water to control OD (occurrence depth). 600 The concentration was 80. The bacterial cells were broken up using a high-pressure homogenizer, and the supernatant was collected by centrifugation to obtain the crude enzyme solution.

[0062] Example 2

[0063] Analysis of the thermal stability of amidases and amidase mutants

[0064] The three-dimensional structure of wild-type amidases was predicted using the AlphaFold tool, and the structure with the highest score was selected for subsequent analysis. GROMACS was used for protein thermal stability analysis, selecting the OPLS-AA / L all-atomforce field and the TIP3P water model, and generating the corresponding topology file. The protein was placed at the center of a cubic water tank, with the tank boundary at least 1.0 nm away from the protein to ensure no non-physical interactions between the protein and its periodic mirror image. Subsequently, the protein was subjected to electrolyte separation with Na+. + The system was neutralized with Cl, simulating a physiological ion concentration (150 mM). System energy minimization was performed using the steepest descent method until the maximum force was less than 1000 kJ·mol⁻¹. -1 ·nm -1 Next, equilibration was performed for 100 ps in the NVT ensemble (equal particle number, equal volume, isothermal), and the system and protein were coupled separately to 350 K using the V-rescale temperature control method. Finally, equilibration was performed for 100 ps in the NPT ensemble (equal particle number, isobaric, isothermal), using Berendsen pressure coupling (target pressure of 1 bar) to stabilize the system density. After the system was fully equilibrated, a long-term unconstrained molecular dynamics simulation production run was conducted. The simulation time for each temperature point should be at least 100 ns to ensure sufficient sampling of protein conformation. The simulation was performed in the NPT ensemble using Parrinello-Rahman pressure coupling and V-rescale temperature coupling. All bond lengths were constrained using the LINCS algorithm with an integration step size of 2 fs. Trajectory data was saved every 10 ps for subsequent analysis. The RMSF of the α-carbon atom of each amino acid residue was calculated using the gmx rmsf command to identify the flexibility of local protein regions. The analysis results are as follows: Figure 1 As shown, amino acid residues 103 and 236 of the wild-type amidase exhibit large RMSF values, indicating that the region is highly flexible. Therefore, the region containing these two amino acid residues was selected as a candidate region for subsequent mutation.

[0065] Mutation analysis was performed on 10 amino acids near amino acid residues 103 and 236 using ThermoNet software to predict changes in protein free energy after mutation. Table 1 shows the results of the largest decreases in free energy after mutation.

[0066] Table 1. Analysis of free energy changes after single-point mutation of amidase.

[0067]

[0068] Example 3

[0069] Construction of amidase mutant expression strains

[0070] Single-point mutations of amidase were introduced using primers shown in Table 2. The mutation site and homologous arm were designed at the 5' end of the primers, and then PCR amplification was performed using plasmid pET-WT as a template. The PCR products were recovered from the gel and digested with Dpn I restriction enzyme before seamless cloning and ligation. The ligation products were transformed into *E. coli* DH5α competent cells, and the transformants were verified by sequencing. The verified transformants were then transferred into *E. coli* BL21(DE3) competent cells to obtain recombinant strains D99T, D105S, A109V, L232R, and S240A. The coding gene for the A109V mutant is shown in SEQ ID No. 3, and the coding gene for the L232R mutant is shown in SEQ ID No. 4. A double-point mutant, A109V-L232R, was also constructed using the A109V plasmid as a template; its coding gene is shown in SEQ ID No. 5.

[0071] Table 2 Primers used for mutant construction.

[0072]

[0073] Example 4

[0074] Activity analysis of amidases and amidase mutants

[0075] Crude enzyme solutions of wild-type and mutant from Example 3 were prepared according to the shake-flask fermentation method of Example 1. The His6-tagged recombinant protein in the crude enzyme solution was purified using Ni-NTA resin. Protein concentration was detected using the Coomassie Brilliant Blue assay, with bovine serum albumin as the standard curve. 10 μL of purified enzyme (pre-diluted to 1 mg / mL) was placed in 890 μL of 100 mM glycine-sodium hydroxide buffer, with an initial pH of 8.5. 100 μL of 500 mM substrate M-1 was added to the reaction sample, resulting in a final substrate concentration of 50 mM. The control group used an equal volume of buffer instead of the enzyme solution. The sample was shaken (100 rpm) at 30°C for 30 min. Protein inactivation was achieved by boiling for 10 min. The reaction solution was extracted with ethyl acetate, the solvent was removed, and the precipitate was redissolved in methanol for liquid chromatography analysis to detect the product. S The amount of )-M-2 generated.

[0076] The liquid chromatography conditions were as follows: Mobile phase ratio: 0.1% phosphoric acid water: acetonitrile = 7:3. Chromatographic column: Lux Cellulose-3 chiral column (4.6 × 250 mm), detector: UV detector, flow rate 0.4 mL / min, 230 nm, column temperature 25℃, time 30 min, injection volume 10 µL.

[0077] To determine the optimal temperature for amidase, the samples were reacted at different temperatures while keeping other conditions constant, following the conditions described above. Catalytic activity analysis was performed on the wild-type and the mutant prepared in Example 3, and the results are as follows: Figure 2 As shown, the optimal temperature for wild-type amidase is 40℃. The enzyme activities of mutants D99T, D105S, and S240A at different temperatures are similar to or slightly lower than those of the wild type. The optimal temperature for mutant A109V is increased to 45℃, while the optimal temperatures for mutants L232R and A109V-L232R are increased to 50℃, with mutant A109V-L232R exhibiting the highest activity at this temperature.

[0078] To further investigate the thermostability changes of deamidase, the wild-type enzyme and the mutant enzyme with an increased optimum temperature were incubated at 50°C and pH 8.5 in glycine-sodium hydroxide buffer for 1 h, and then their residual enzyme activity was measured. The results are as follows: Figure 3 As shown, the wild-type enzyme had only 20% residual enzyme activity after incubation, while the residual enzyme activities of mutants A109V, L232R, and A109V-L232R were 28%, 43%, and 61%, respectively.

[0079] Example 5

[0080] Preparation by amidase hydrolysis ( S )-M-2

[0081] Crude enzyme solutions of wild-type amidase and amidase mutant A109V-L232R were prepared using the 50 L fermenter fermentation method described in Example 1. 5 L of deionized water, 1 kg of racemic M-1, and 5 L of crude enzyme solution (containing 10 g / L amidase mutant A109V-L232R) were added to a 20 L reactor. During the reaction, the pH was controlled at 8.5, the reaction temperature for wild-type amidase was 40°C, and the reaction temperature for mutant amidase was 50°C. Samples were taken every 2 hours to monitor product formation. The reaction was stopped after 14 hours of catalysis. Results are as follows: Figure 4 As shown, wild-type amidase can only catalyze the production of 12.5 g / L of ( ) after 12 h. S The amidase mutant can generate 41.2 g / L of (M-2) in just 8 hours. S )-M-2.

[0082] Example 6

[0083] ( S Preparation of 1-Fluoropyram aniline

[0084] The reaction solution catalyzed by the amidase mutant A109V-L232R in Example 5 was heated to 100°C and boiled for 10 min. After cooling, the denatured protein was removed by centrifugation. The product in the reaction solution was extracted three times with an equal volume of ethyl acetate. Excess ethyl acetate was removed by rotary evaporation under reduced pressure. The remaining liquid was dried over anhydrous sodium sulfate to obtain the product. S )-M-2 (430 g).

[0085] Will( S M-2 (50 g) and pyridine (46 mL) were dissolved in dichloromethane (100 mL), and then M-3 (54.8 g) was added. The reaction was carried out at room temperature for 9 h. After the reaction was complete, water (50 mL) was added for dilution. The aqueous phase was extracted with ethyl acetate (50 mL × 3) after separation. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The residue was crystallized by ethanol-water precipitation, and 85.9 g of a grayish-white solid was obtained by filtration (yield 95%, purity 99.9%, ee value 99.9%). NMR and mass spectrometry showed that the structure was correct, and the absolute configuration was confirmed as ( S )-Fluorazole aniline.

[0086] Should( S The NMR and mass spectrometry results for )-fluopyrazoline are as follows: 1 H NMR (500 MHz, CDCl3) δ 7.80 (dd, J =7.6, 1.8 Hz, 1H), 7.41 – 7.33 (m, 1H), 7.29 – 7.25 (m, 1H), 7.24 – 7.16 (m,2H), 3.75 (d, J = 1.3 Hz, 3H), 3.05 – 2.93 (m, 1H), 2.50 (s, 3H), 1.60 – 1.49(m, 2H), 1.48 – 1.39 (m, 1H), 1.23 (d, J = 6.8 Hz, 3H), 0.91 – 0.81 (m, 6H). 13 CNMR (126 MHz, CDCl3) δ 159.58 (d, J CF = 4.7 Hz), 151.42 (d, J CF = 279.2 Hz), 150.22(d, JCF = 8.1 Hz), 139.68, 134.09, 126.44, 126.32, 126.02, 125.02, 96.16 (d, J CF =9.8 Hz), 46.83, 34.05, 31.03, 25.65, 22.92, 22.61, 21.64, 14.87. HRMS (ESI) C 18 H 25 FN3O + [M + H] + Calculated value: 318.1976, measured value: 318.1970.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amidase mutant, characterized in that, Compared with the amidase shown in SEQ ID No. 1, the amidase mutant has alanine at position 109 mutated to valine and / or leucine at position 232 mutated to arginine.

2. The encoding gene of the amidase mutant according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No. 3-5.

3. A recombinant plasmid carrying the encoding gene of claim 2.

4. A host cell carrying the encoding gene of claim 2 or the recombinant plasmid of claim 3.

5. The method for preparing the amidase mutant according to claim 1, characterized in that, The process includes the following steps: inoculating the host cells described in claim 4 into LB medium and culturing at 37±2℃ and 150-250 rpm to prepare a seed culture; transferring the seed culture to fresh LB medium and continuing to culture until OD. 600 Once the concentration reaches 0.6, the temperature is lowered to 25±2℃ and IPTG is added to induce expression, thus obtaining the crude enzyme solution.

6. The method for preparing the amidase mutant according to claim 1, characterized in that, The process includes the following steps: inoculating the host cells described in claim 4 into LB medium and culturing them at 37±2℃ and 150-250 rpm to prepare a seed culture; transferring the seed culture to a fermenter and fermenting it to prepare a secondary seed culture; The secondary seed culture was transferred to a new fermenter and cultured until OD reached. 600 Once the enzyme concentration reaches 1.0, the temperature is lowered to 25±2℃ and IPTG is added to induce expression, thus obtaining the crude enzyme solution.

7. The amidase mutant of claim 1, or the recombinant plasmid of claim 3, or the host cell of claim 4 in the preparation ( S Application of )-Fluorazole aniline.

8. A method for preparing ( S The method for using 1-fluopyrazopyr aniline is characterized by, Includes the following steps: (1) An amidase mutant is prepared by the preparation method described in claim 5 or 6; (2) Using racemic substrate M-1 as reactant, an enzymatic reaction is carried out under the catalysis of the amidase mutant in step (1) to generate the key chiral intermediate ( S )-M-2; wherein, M-1 is a compound having the structure shown in Formula I, and the ( S )-M-2 is a compound having the structure shown in Formula II; Formula I; Formula II; (3) The key chiral intermediate is separated from the reaction solution of step (2). S )-M-2; (4) The key chiral intermediate ( S )-M-2 reacts with substrate M-3 to obtain ( S )-Fluorazole aniline, wherein M-3 is a compound having the structure shown in Formula III; Formula III.

9. The method according to claim 8, characterized in that, In step (2), the concentration of racemic substrate M-1 added is 0.1-100 g / L, and the concentration of amidase mutant added is 0.01-5 g / L.

10. The method according to claim 8, characterized in that, In step (2), the conditions for the enzymatic reaction are controlled as follows: reaction temperature 35-65℃, reaction pH 8.0-10.0, and reaction time 1-12 h.

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