An amidease mutant and its use in preparing (s)-pyracthiobane

Abstract

This invention provides an amidase mutant and its application in the preparation of ( S The application of pyrimethanil 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 contains one or more mutations at the following sites: alanine at position 132 is mutated to leucine; serine at position 205 is mutated to glycine; alanine at position 211 is mutated to glycine. Compared with wild-type amidase, the enzyme activity and stereoselectivity of the amidase mutant of this invention are significantly improved; based on the mild and simple preparation method of this mutant, the obtained ( S )-Pyrimethanil possesses excellent chiral selectivity and product optical purity, which aligns with the development trend of green pesticide creation and has significant industrial application value.

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 pyrimethanil. Background Technology

[0002] Penthiopyrad is a representative member of the succinate dehydrogenase inhibitor (SDHI) class of fungicides. Compared with earlier developed SDHI fungicides, penthiopyrad has significant advantages, exhibiting a unique fungicidal spectrum and excellent bioactivity. Penthiopyrad is used on fruit trees, vegetables, and ornamental crops. This compound has broad-spectrum fungicidal activity against a variety of plant pathogenic fungi, showing particularly excellent control effects against diseases such as rust, sclerotinia rot, gray mold, powdery mildew, and apple and pear scab.

[0003] From a stereochemical perspective, the pyrimethanil molecule contains a chiral center, and thus exhibits (…). R )-(-)-pyrimethanil and ( S Two enantiomers of pyrimethanil (+)-pyrimethanil. Studies have shown that these two enantiomers exhibit significant stereoselective biological activity. S The bactericidal activity of the enantiomer was significantly higher than that of the ( R ()-enantiomers. This stereoselective activity may be related to the difference in binding between the enantiomers and the target enzyme (succinate dehydrogenase). In addition to biological activity, pyrimethanil enantiomers also exhibit stereoselectivity in environmental behavior and ecotoxicity. Studies have also found that in soil environments ( S The degradation rate of enantiomers is generally faster than that of ( R -Enantiomer.

[0004] Currently, all industrially produced pyrimethanil products circulating in the global pesticide market are racemic mixtures. R )-and( S (The enantiomer ratio is approximately 1:1), and there has been no commercial application of a single optically pure enantiomer. Given ( S Enantiomers have dual advantages in terms of biological activity and environmental behavior. Currently, the development of synthetic processes with high enantioselectivity has important application value: theoretically, it can reduce the dosage of pesticides by 30-50% to achieve the same control effect, while significantly reducing the risk of exposure to non-target organisms and reducing the accumulation of pesticide residues in environmental media, which is in line with the current development trend of green pesticide creation. 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 Application of pyrimethanil to solve the problem of existing wild-type amidase-catalyzed preparation of (S This paper addresses the technical problems of poor enantioselectivity and low target product content in the production of pyrimethanil, and also provides a method for preparing this mutant and its application in (…). S Application of 1-pyrimethanil in synthesis, establishing an efficient and green chemical-enzymatic preparation process.

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

[0007] An amidase mutant, compared to the amidase with the amino acid sequence shown in SEQ ID No. 1, contains mutations at one or more of the following sites:

[0008] The alanine at position 132 is mutated to leucine;

[0009] The serine at position 205 is mutated to glycine;

[0010] The alanine at position 211 is mutated to glycine.

[0011] 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. 3-6.

[0012] The present invention also provides a recombinant plasmid carrying the above-mentioned encoding gene.

[0013] The present invention also provides a host cell carrying the above-mentioned coding gene or the above-mentioned recombinant plasmid.

[0014] This invention also provides a method for preparing the above-mentioned amidase mutant, comprising the following steps: seed culture is prepared by inoculating the host cells into YPD medium; the seed culture is transferred to BMGY liquid medium at an inoculation rate of 5%-15%; the culture is carried out at 28-30°C, and 0.5%-1.5% methanol is added every 24 hours to induce expression for 96-120 h; the supernatant is collected by centrifugation to obtain crude enzyme solution. Alternatively, seed culture is prepared by inoculating the host cells into YPD medium; the seed culture is inoculated into a fermenter containing BMGY liquid medium to prepare secondary seed culture; the secondary seed culture is inoculated again into BMGY liquid medium; and methanol is added to induce expression for 96-120 h under dissolved oxygen conditions controlled at 20%-40% to obtain crude enzyme solution.

[0015] This invention also provides the preparation of the above-mentioned amidase mutant ( S Application of pyrimethanil.

[0016] This invention also provides a chemical-enzymatic method for preparing ( S The method for using pyrimethanil includes the following steps:

[0017] (1) Prepare amidase mutants using the above preparation method;

[0018] (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-1 is a compound having the structure shown in Formula I, where R in Formula I is C2-C2. 10 alkyl groups; the ( S )-M-2 is a compound having the structure shown in Formula II;

[0019]

[0020] Formula I

[0021]

[0022] Formula II

[0023] (3) The key chiral intermediate is separated from the reaction solution of step (2). S )-M-2;

[0024] (4) The key chiral intermediate ( S )-M-2 reacts with substrate M-3 to obtain ( S M-3 is a compound having the structure shown in Formula III.

[0025]

[0026] Formula III

[0027] Preferably, in step (2), the concentration of racemic substrate M-1 is 0.1-80 g / L, and the concentration of amidase mutant is 0.01-5 g / L.

[0028] Preferably, in step (2), the conditions for the enzymatic reaction are controlled as follows: reaction temperature 30-50℃, reaction pH 7.0-11.0, and reaction time 1-36 h.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The amidase mutant of this invention can specifically catalyze the generation of racemic substrate M-1 ( S )-M-2, ( S The proportion of )-type products is as high as 99.9%, which completely overcomes the problem of poor enantioselectivity of wild-type amidases. High-optical-purity key intermediates can be obtained without complicated chiral resolution steps, which significantly simplifies the process.

[0031] The chemical-enzymatic preparation process of the amidase mutant based on this invention has mild enzymatic reaction conditions, does not require harsh conditions such as high temperature and high pressure, and has low energy consumption; the reaction process does not generate any toxic or harmful substances, and can reduce the dosage of pesticides and reduce the risk of environmental residues, which is in line with the concept of green chemical industry and sustainable development.

[0032] Prepared by the method of the present invention ( S )-Pyrimethanil, with a purity of up to 99.9%, an ee value of 99.9%, and a yield of 98%, is far superior in quality to existing racemic mixture products. It has stronger biological activity, better environmental compatibility, and outstanding market competitiveness. Attached Figure Description

[0033] Figure 1 This is an HPLC chromatogram of the wild-type amidase catalyzed product in Example 2;

[0034] Figure 2 This is a three-dimensional structural diagram of the wild-type amidase in Example 3;

[0035] Figure 3 The amidase mutant in Example 5 catalyzed different reaction times. S )-M-2 production. Detailed Implementation

[0036] An amidase mutant, compared with the amidase shown in SEQ ID No. 1, contains one or more of the following site mutations: alanine at position 132 is mutated to leucine; serine at position 205 is mutated to glycine; alanine at position 211 is mutated to glycine.

[0037] 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. 3-6.

[0038] The present invention also provides a recombinant plasmid carrying the above-mentioned coding gene; the preferred preparation method is to clone any one of the DNA sequences shown in SEQ ID No. 3-6 into the EcoRI and NotI sites of the pPIC9K plasmid to obtain the recombinant plasmid pWT.

[0039] The present invention also provides a host cell carrying the above-mentioned coding gene or the above-mentioned recombinant plasmid; a preferred preparation method is: the recombinant plasmid pWT is transformed into a host cell by chemical transformation. Komagataella phaffii Recombinant K was obtained from GS115 competent cells. phaffii WT.

[0040] The present invention also provides a method for preparing the above-mentioned amidase mutant, comprising the following steps: inoculating the above-mentioned host cells into YPD medium (10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose) to prepare a seed culture; transferring the seed culture to BMGY liquid medium (10 g / L yeast extract, 20 g / L tryptone, 13.4 g / L amino-free yeast nitrogen source, 10 g / L glycerol, 3 g / L dipotassium hydrogen phosphate, 12 g / L potassium dihydrogen phosphate), preferably 10%, at an inoculation rate of 5%-15%; culturing at 28-30°C; and inducing expression by adding 0.5%-1.5% methanol every 24 hours for 96-120 h, preferably by adding 1% methanol every 24 hours for 120 h; and collecting the supernatant by centrifugation to obtain a crude enzyme solution.

[0041] Alternatively, the host cells described above can be inoculated into YPD medium to prepare a seed culture. This seed culture can then be inoculated into a fermenter containing BMGY liquid medium to prepare a secondary seed culture. Preferably, the fermentation temperature is 28°C, the fermentation speed is 700 rpm, and the fermentation time is 16 h. The secondary seed culture is then inoculated again into BMGY liquid medium, and methanol is added to induce expression for 96-120 h under conditions where dissolved oxygen is controlled at 20%-40%. Preferably, the temperature is controlled at 30°C, the aeration rate is 2 L / min, the pH is controlled at 5.5 by automatic ammonia addition, and the dissolved oxygen (DO) is controlled at 30% by automatic fermentation speed adjustment. After the dissolved oxygen rebounds, glycerol is added, and the culture is continued until the cell OD reaches a certain level. 600 Stop adding glycerol at 220°C. After stopping glycerol addition for 1 hour, cool down to 22°C and add methanol at a rate of 2.5 mL / L / h to induce expression for 96 hours to obtain crude enzyme solution.

[0042] This invention also provides the above-mentioned amidase mutant, or recombinant plasmid, or host cell in the preparation ( S Application of pyrimethanil.

[0043] This invention also provides a chemical-enzymatic method for preparing ( S The method for using pyrimethanil includes the following steps:

[0044] (1) Prepare amidase mutants using the above preparation method;

[0045] (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-1 is a compound having the structure shown in Formula I, where R in Formula I is C2-C2. 10 alkyl groups; the ( S )-M-2 is a compound having the structure shown in Formula II;

[0046]

[0047] (Formula I)

[0048]

[0049] (Formula II)

[0050] (3) The key chiral intermediate is separated from the reaction solution of step (2). S )-M-2;

[0051] (4) The key chiral intermediate ( S )-M-2 reacts with substrate M-3 to obtain ( S M-3 is a compound having the structure shown in Formula III.

[0052]

[0053] (Formula III)

[0054] In this invention, preferably in step (2), the concentration of racemic substrate M-1 is 0.1-80 g / L and the concentration of amidase mutant is 0.01-5 g / L.

[0055] In this invention, the conditions for the enzymatic reaction in step (2) are preferably controlled as follows: reaction temperature 30-50℃, more preferably 35℃; reaction pH 7.0-11.0, more preferably pH 8.5; reaction time 1-36 h.

[0056] 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.

[0057] Example 1

[0058] Construction and fermentation of amidase expression strains

[0059] First, codon optimization was performed based on the amino acid sequence of the wild-type amidase (as shown in SEQ ID No. 1), and a His6 tag was added to the C-terminus to obtain the DNA sequence shown in SEQ ID No. 2. Then, Beijing Qingke Biotechnology Co., Ltd. completed gene synthesis and cloned the DNA into the EcoRI and NotI sites of the pPIC9K plasmid to obtain the recombinant plasmid pWT. The recombinant plasmid pWT was then transformed into... Komagataella phaffii Recombinant K was obtained from GS115 competent cells. phaffii WT.

[0060] Recombination K. phaffiiYPD medium, consisting of 10 g / L yeast extract, 20 g / L tryptone, and 20 g / L p-glucose, was used for culturing. Recombinant K was selected. phaffii Single colonies were cultured overnight at 28°C and 200 rpm in test tubes containing 5 mL of YPD medium to obtain the seed culture. Shake-flask fermentation was performed using BMGY liquid medium containing 10 g / L yeast extract, 20 g / L tryptone, 13.4 g / L amino-free yeast nitrogen source, 10 g / L glycerol, 3 g / L dipotassium hydrogen phosphate, and 12 g / L potassium dihydrogen phosphate. The seed culture was transferred to BMGY liquid medium at a 10% inoculum and cultured at 28°C and 200 rpm. Expression was induced for 120 h by adding 1% methanol every 24 h, and the supernatant was collected by centrifugation to obtain the crude enzyme solution.

[0061] Fermentation in a 50 L fermenter: The seed culture was transferred to a 5 L fermenter with an initial inoculum of 10% BMGY medium (2 L BMGY). The fermentation temperature was 28°C, the fermentation speed was 700 rpm, and fermentation lasted 16 h to prepare a secondary seed culture. The secondary seed culture was then transferred to the 50 L fermenter with an inoculum of 10% using BMGY liquid medium. The temperature was controlled at 30°C, the aeration rate at 2 L / min, and the pH was maintained at 5.5 by automatic ammonia replenishment. Dissolved oxygen (DO) was maintained at 30% by automatic rotation speed adjustment. Glycerol was added after dissolved oxygen rebounded. The culture was continued until the cell OD reached the target level. 600 Stop adding glycerol when the temperature reaches approximately 220°C. One hour after stopping glycerol addition, cool the temperature to 22°C and add methanol at a rate of 2.5 mL / L / h to induce expression for 96 hours. After the induction process is complete, centrifuge and collect the supernatant, which is the crude enzyme solution.

[0062] Example 2

[0063] Amidease activity analysis

[0064] Crude amidase was prepared according to the shake-flask fermentation method of Example 1. The His6-tagged recombinant protein in the crude enzyme was purified using Ni-NTA resin. Protein concentration was determined 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 (R being a C2 alkyl group) was added to the reaction sample, resulting in a final substrate concentration of 50 mM. An equal volume of buffer was used in place of the enzyme in the control group. The sample was shaken (100 rpm) at 35°C for 4 h. 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.

[0065] The liquid chromatography conditions were as follows: Mobile phase ratio: 0.1% phosphoric acid water: acetonitrile = 80:20. Chromatographic column: LuxCellulose-3 chiral column (4.6 × 250 mm), detector: UV detector, flow rate 0.4 mL / min, 230 nm, column temperature 28℃, time 30 min, injection volume 10 µL.

[0066] HPLC analysis results of wild-type amidase are as follows Figure 1 As shown, this enzyme can hydrolyze racemic M-1 to generate ( S )-M-2 and ( R )-M-2, but generate ( R M-2 is more common.

[0067] Example 3

[0068] To improve the specificity of wild-type amidase for M-1, the three-dimensional structure of wild-type amidase was predicted using the AlphaFold tool. The structure with the highest score was selected for analysis and compared with the amidase in the PDB database (PDB ID: 4YJI). The amino acids within a 5A distance of the ligand were analyzed. The three-dimensional structure of wild-type amidase is shown below. Figure 2 As shown in Table 1, amino groups other than the active site were selected as candidate mutation sites.

[0069] Table 1 Candidate mutation sites for wild-type amidases

[0070]

[0071] In order to eliminate the wild-type amidase catalyzing the generation of ( R To enhance the activity of )-M-2, the candidate mutation sites were selected to mutate amino acid residues with smaller side chain space to amino acid residues with larger side chain space steric hindrance. Therefore, sites 132, 205, and 211 were further selected as mutation directions.

[0072] Single-point mutations were introduced into the amidase 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 pWT as a template. The PCR product was recovered from the gel and digested with DpnI restriction enzyme before seamless cloning and ligation. The ligation product was transformed into *E. coli* DH5α competent cells, and the transformants were verified by sequencing. The correctly transformed plasmid was then transferred into K. phaffii Recombinant strains A132L, S205L, and A211L were obtained from GS115 competent cells, among which the amidase encoding gene of A132L is shown in SEQ ID No. 3.

[0073] Table 2 Primers used in Example 3

[0074]

[0075] The recombinant strain was subjected to shake-flask fermentation and activity analysis according to the methods of Examples 1 and 2. The results are shown in Table 3. The A132L site mutation resulted in amidase catalysis producing only (…). S )-M-2, produces almost no ( R )-M-2. Mutations in S205L and A211L lead to ( S The yield of )-M-2 dropped sharply, to only 1.6% and 3.5%, indicating that the amino acid at this position is not conducive to the production of () by amidase catalysis. S The activity of )-M-2 is greatly affected.

[0076] Table 3. Activity analysis of amidases and amidase mutants

[0077]

[0078] Example 4

[0079] Site-directed mutagenesis analysis of amidase mutant A132L

[0080] To improve the activity of the amidase mutant A132L, site-directed mutagenesis was performed on the amidase mutant A132L using the primers shown in Table 4, following the method in Example 3, to generate mutants A132L-S205G, A132L-A211G, and A132L-S205G-A211G, whose amidase coding sequences are shown in SEQ ID No. 4-6, respectively.

[0081] Table 4 Primers used in Example 4

[0082]

[0083] The recombinant strains were subjected to shake-flask fermentation and activity analysis according to the methods of Examples 1 and 2. The results are shown in Table 5. Mutations at both S205G and A211G sites significantly improved the substrate conversion rate. Simultaneous mutations of both S205G and A211G resulted in even higher conversion rates, and no (…) were generated. R )-M-2.

[0084] Table 5. Activity analysis of amidase mutants

[0085]

[0086] Example 5

[0087] Preparation of amidase mutant by hydrolysis ( S )-M-2

[0088] Crude enzyme solutions of amidase mutants A132L and A132L-S205G-A211G were prepared using the 50 L fermenter fermentation method described in Example 1. 5 L of deionized water, 0.8 kg of racemic M-1 (R being a C2 alkyl group), and 5 L of crude enzyme solution (containing 8 g / L amidase mutants) were added to a 20 L reactor. During the reaction, the pH was controlled at 8.5, and the reaction temperature was 35°C. Samples were taken every 2 hours to monitor product formation. The reaction was stopped after 18 hours of catalysis. Results are as follows: Figure 3 As shown, after 16 h of catalysis by the amidase mutant A132L ( S The yield of )-M-2 reached 34.6 g / L, while the amidase mutant A132L-S205G-A211 catalyzed for 12 h ( S The yield of )-M-2 reached 35.46 g / .

[0089] Example 6

[0090] ( S Preparation of pyraclostrobin

[0091] The reaction solution catalyzed by the amidase mutant A132L-S205G-A211 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 (342g).

[0092] Will( S M-2 (50 g) and pyridine (44 mL) were dissolved in dichloromethane (100 mL), and M-3 (63.8 g) was added. The reaction was carried out at room temperature for 9 h. After the reaction was complete, the mixture was diluted with water (50 mL), and the aqueous phase was extracted with ethyl acetate (50 mL × 3). 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 with ethanol-water, and filtered to obtain 97.7 g of a grayish-white solid (yield 98%, 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 )-Pyrimethanil.

[0093] Should( S The NMR and mass spectrometry results for pyraclostrobin are as follows: 1 H NMR (500 MHz, CDCl3) δ 8.03 (s, 1H), 7.57 (s, 1H), 7.40 (d, J = 5.4 Hz, 1H), 7.12 (d, J= 5.4 Hz, 1H), 3.96 (s, 3H), 3.14 – 3.01 (m, 1H), 1.63 – 1.50 (m, 2H), 1.50 – 1.41 (m, 1H), 1.24 (d, J = 6.8Hz, 3H), 0.85 (dd, J = 6.4, 1.9 Hz, 6H). 13 C NMR (126 MHz, CDCl3) δ 158.36,139.72, 137.82 (q, J = 37.8 Hz), 136.61, 129.71, 124.37, 121.27 (q, J = 268.9Hz), 121.22, 117.37, 48.13, 39.97, 30.33, 25.76, 23.14, 22.60, 22.52. HRMS (ESI) C 16 H 21 F3N3OS + [M + H] + Calculated value: 360.1352, measured value: 360.1348.

[0094] 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, The amidase mutant has the following site mutations compared to the amidase shown in SEQ ID No. 1: The alanine at position 132 is mutated to leucine; Or, the alanine at position 132 may be mutated to leucine and the serine at position 205 to glycine. Or the alanine at position 132 may be mutated to leucine and the alanine at position 211 may be mutated to glycine; Or, alanine at position 132 may be mutated to leucine, serine at position 205 to glycine, and alanine at position 211 to glycine.

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. 3-6.

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 YPD medium to prepare a seed culture, transferring the seed culture to BMGY liquid medium at an inoculation rate of 5%-15%, culturing at 28-30°C, and adding 0.5%-1.5% methanol every 24 hours to induce expression for 96-120 hours, and centrifuging to collect the supernatant to obtain a 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 YPD medium to prepare a seed culture; inoculating the seed culture into a fermenter containing BMGY liquid medium to prepare a secondary seed culture; inoculating the secondary seed culture into BMGY liquid medium again; and, under the condition of controlling dissolved oxygen at 20%-40%, adding methanol to induce expression for 96-120 h to obtain a 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 of ( S Application of pyrimethanil.

8. A chemical-enzymatic method for preparing ( S The method for pyrimethanil is characterized in that, 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-1 is a compound having the structure shown in Formula I, where R in Formula I is C2-C2. 10 alkyl groups; 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 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 0.1-80 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 30-50℃, reaction pH 7.0-11.0, and reaction time 1-36 h.

Images