Use of a bacillus aryabhattai in the preparation of (r)-dmpm

Abstract

The application mainly provides application of Bacillus aryabhattai in preparation of (R)-DMPM, solves the racemization problem of chiral acid in chemical methyl esterification and the high cost problem of commercial enzyme esterification by using esterification performance, and realizes low-cost green production of an important intermediate of precise mefenoxam. The stereoselectivity of the enzyme-producing microbial strain Bacillus aryabhattai WZZ10 used in the application is high, preparation is simple and convenient, stability is good, and the enzyme-producing microbial strain can be repeatedly used, so that production cost and environmental pollution are reduced. The esterification rate and e.e.p of (R)-DMPM prepared by the application are 56.41% and 99.12% respectively.

Description

(1) Technical field

[0001] The present invention relates to the esterification of (R)-N-(2,6-dimethylphenyl)aminopropionic acid, and in particular to a method for obtaining optically pure (R)-N-(2,6-dimethylphenyl)aminopropionic acid methyl ester by methyl esterification of (R)-N-(2,6-dimethylphenyl)aminopropionic acid using Bacillus arylium WZZ10. (2) Background technology

[0002] Amide pesticides, such as metalaxyl, are low-toxic, highly effective, and broad-spectrum systemic insecticides. They are highly effective against plant diseases caused by downy mildew, phytophthora, and pythium, and possess strong efficacy, making them one of the most widely used fungicides today. Their fungicidal mechanism is to inhibit protein synthesis within the pathogen, resulting in nutrient depletion and a lack of growth. Metalaxyl is a racemic compound consisting of a pair of optical isomers. (R,S)-N-(2,6-dimethylphenyl)aminopropionic acid methyl ester (R,S)-DMPM is a key chiral intermediate in the preparation of metalaxyl. The R-form of metalaxyl (metalaxyl-S) is many times more active than the S-form. Therefore, the preparation of high-purity (R)-DMPM is crucial for the production of high-purity metalaxyl in the pesticide industry.

[0003] Currently, (R)-DMPM is primarily prepared by chemical and biological methods. The chemical method, which primarily uses L-lactic acid and 2,6-dimethylaniline as raw materials, is still the most mature and fast and efficient production process. However, the reaction process is complex and requires the addition of toxic and hazardous substances such as organic solvents and sulfuric acid, which pollute the environment. Furthermore, the optical purity of the (R)-DMPM produced is only around 92%, requiring secondary purification. Another method involves the selective enzymatic resolution of racemic metalaxyl to produce high-purity (R)-2,6-dimethylphenylaminopropionic acid ((R)-MAP-acid), which is then further enzymatically methylated to yield high-purity metalaxyl-M. (3) Summary of the invention

[0004] The present invention mainly provides a new esterification scheme for (R)-N-(2,6-dimethylphenyl)aminopropionic acid, which utilizes Bacillus arylboroyi to esterify (R)-N-(2,6-dimethylphenyl)aminopropionic acid to prepare optically pure (R)-N-(2,6-dimethylphenyl)aminopropionic acid. The esterification performance of the new scheme is utilized to solve the racemization problem of chiral acids in the chemical methylation process and the high cost problem of esterification using commercial enzymes, thereby realizing low-cost green production of an important intermediate of metalaxyl-M.

[0005] The technical route adopted by the present invention is:

[0006] The application provides application of Bacillus aryabhattai in preparation of (R)-N-(2,6-dimethylphenyl) aminopropanamide methyl ester ((R)-DMPM) from methyl esterification (R)-2,6-dimethylphenyl aminopropanoic acid ((R)-MAP-acid), and the Bacillus aryabhattai is Bacillus aryabhattai CCTCC NO: M20191099.

[0007] The Bacillus aryabhattai WZZ10 is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M20191099, a preservation date of December 25, 2019, and an address of Wuhan, China, Wuhan University, and a postcode of 430072, and has been disclosed in a patent application (CN111534472A, filed on June 11, 2020).

[0008] Preferably, the application is as follows: the wet mycelium obtained by fermentation culture of the Bacillus aryabhattai is freeze-dried to obtain dry mycelium as a catalyst, high-purity (R)-MAP-acid and anhydrous methanol are used as substrates, t-butyl alcohol and water are used as solvents, reaction is carried out at 25-40 DEG C, 100-200 rpm for 10-20 h (preferably 35 DEG C, 200 rpm for 12 h), the reaction solution is separated and purified, and (R)-N-(2,6-dimethylphenyl) aminopropanamide methyl ester is obtained.

[0009] Preferably, the mass ratio of (R)-2,6-dimethylphenyl aminopropanoic acid to the catalyst is 1:0.05-1, preferably 1:0.02; the water is added in an amount of 0.1-1.0 mL / g, preferably 0.24 mL / g, based on the mass of (R)-2,6-dimethylphenyl aminopropanoic acid; the t-butyl alcohol is added in an amount of 4-50 mL / g, preferably 10 mL / g, based on the mass of (R)-2,6-dimethylphenyl aminopropanoic acid; and the anhydrous methanol is added in an amount of 1-5 mL / g, preferably 2 mL / g, based on the mass of (R)-2,6-dimethylphenyl aminopropanoic acid.

[0010] Preferably, the freeze-drying is that the wet mycelium is frozen at-20 DEG C, and then freeze-dried by a freeze dryer at a cold trap of-80 DEG C and a vacuum degree of 20 Pa for 12 h to obtain the dry mycelium.

[0011] Preferably, the wet mycelium is prepared according to the following method.

[0012] (1) Slant culture: Bacillus aye-hadou was inoculated into LB solid medium and cultured at 28°C for 1 day to obtain slant cells. The LB solid medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar. The solvent was deionized water, and the pH was 7.0.

[0013] (2) Seed culture: Take the slant bacteria and inoculate them into seed culture medium. Cultivate them at 28°C and 180 rpm for 12-24 h to obtain seed solution. The seed culture medium composition is as follows: peptone 25 g / L, glucose 20 g / L, K2HPO4 1.0 g / L, (NH4)2SO4 5 g / L, MgSO4 0.25 g / L, olive oil 10 mL / L, solvent is water, pH 7.0, sterilize at 115°C for 30 min.

[0014] (3) Fermentation culture: Under sterile conditions, the seed liquid was inoculated into the fermentation medium at an inoculum concentration of 1-2% by volume, and cultured at 28°C and 180 r / min for 24 h to obtain the fermentation broth, which was centrifuged at 12000 rpm for 3-10 min, the supernatant was discarded, and the wet cells were collected; the fermentation medium composition was: glucose 5 g / L, peptone 20 g / L, K2HPO4 1.0 g / L, (NH4)2SO4 1 g / L, MgSO4 0.25 g / L, the solvent was deionized water, pH 7.0, and sterilized at 115°C for 30 min.

[0015] The reaction model of the present invention in which (R)-MAP-acid is catalyzed by Bacillus arylphylloides and anhydrous methanol to prepare (R)-DMPM is:

[0016]

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] The enzyme-producing microbial strain Bacillus aryeboldii used in the present invention has strong stereoselectivity, is simple and convenient to prepare, has good stability, can be reused, and reduces production costs and environmental pollution.

[0019] The esterification rate and eep of the (R)-DMPM prepared by the present invention are 56.41% and 99.12% respectively, and the purity is improved from 92% of the chemical method to more than 99% today, which simplifies the complex reaction process of the chemical method and reduces the pollution caused by the chemical method. (IV) Description of the accompanying drawings

[0020] Figure 1 This is the liquid chromatogram of (R)-MAP-acid.

[0021] Figure 2The liquid chromatograms of (R)-MAP-acid and (S)-MAP-acid.

[0022] Figure 3 Liquid chromatograms of (R)-DMPM, (S)-DMPM and (R)-MAP-acid.

[0023] Figure 4 This is a liquid chromatography analysis chart of the reaction solution for preparing (R)-DMPM by enzymatic esterification using the strain numbered CZG-18 in Example 2. (V) Specific implementation methods

[0024] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] The (R)-MAP-acid used in the present invention has a purity of 98%), and other reagents used in the experiments, such as anhydrous methanol, tert-butyl alcohol, concentrated hydrochloric acid, and ethyl acetate, are all commercially available analytical grade. Tert-butyl alcohol and isopropanol are both chromatographic grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0026] Example 1: Preparation of dry bacterial powders of different lipase-producing bacteria:

[0027] Soil samples from the experimental field of Moganshan Campus of Zhejiang University of Technology were collected for enrichment culture. 1 g of soil was added to a shake flask containing 50 mL of sterile water and a small amount of glass beads, and cultured at 30°C and 180 rpm for 5 h. 1 mL of the suspension was taken and gradiently diluted to 10 -6 , take 200 μL and spread it on LB solid medium and culture it at 30℃ for 24h.

[0028] Single colonies were selected and inoculated onto Rhodamine B colorimetric plates and incubated at 30°C for 72 hours. Based on the order of color change zones, the ratio of color change zone diameter to colony diameter, strains with high lipase activity and short enzyme production cycles were selected. These strains were inoculated onto LB slant medium and cultured at 30°C for 24 hours. The strains were then refrigerated at 4°C and numbered CZG-1 to CZG-24.

[0029] Rhodamine B colorimetric plate: Mix component A and component B in a 10:1 volume ratio to form a mixed solution. Add 0.1% rhodamine B solution (filter sterilized) to the mixed solution in a 1:100 volume ratio, mix thoroughly, and pour onto a plate as a color indicator. Component A consists of: 1.0 g / L (NH4)2SO4, 1.0 g / L K2HPO4, 5.0 g / L KC1, 0.25 g / LMgSO4, 0.1 g / L FeSO4, 5.0 g / L peptone, 20 g / L agar, dissolved in water, natural pH, placed in a conical flask, sterilized at 115°C for 30 min, and cooled to 60°C; Component B consists of: olive oil and 2% polyvinyl alcohol (PVA) aqueous solution at a volume ratio of 1:3, emulsified at 10,000 rpm for 3 min using a cell disruptor, rest for 5 min, emulsified again for 3 min, transferred to a conical flask, sterilized at 115°C for 30 min, and cooled to 60°C.

[0030] Different strains with lipase activity were inoculated into LB solid medium and cultured at 30°C for 24 hours. LB solid medium formula: 10g / L peptone, 5g / L yeast powder, 10g / L NaCl, 20g / L agar, dissolved in water, adjusted to pH 7.0, and sterilized at 115°C for 30 minutes.

[0031] A single colony was selected and inoculated into the seed culture medium, and cultured in a shaking incubator at 30°C and 180 rpm for 12 h. The inoculum was transferred to the fermentation medium at a volume concentration of 2%, and cultured in a shaking incubator at 30°C and 180 rpm for 24 h.

[0032] The fermentation broth was divided into 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 10 min. The bacterial pellet was retained and frozen in a -20°C refrigerator for 24 h. The tube was placed in a vacuum freeze dryer and freeze-dried for 12 h in a cold trap of -80°C and a vacuum degree of 20 Pa to obtain dry bacterial powder. The dry bacterial powder was aseptically divided and numbered for subsequent experiments.

[0033] Seed culture medium formula: peptone 20g / L, glucose 30g / L, K2HPO41.0g / L, (NH4)2SO45g / L, MgSO40.25g / L, olive oil 10mL / L, dissolve in water and adjust pH to 7.0.

[0034] The fermentation medium composition is: glucose 5g / L, peptone 20g / L, K2HPO41.0g / L, (NH4)2SO41g / L, MgSO40.25g / L, the solvent is deionized water, pH7.0, sterilized at 115℃ for 30min.

[0035] Example 2: Esterification reaction of (R)-MAP-acid with bacterial powders of different numbers:

[0036] To a 10 mL stoppered glass tube, add 0.025 g of dried bacterial powder of different numbers prepared by the method of Example 1, 0.5 g of (R)-MAP-acid, 0.5 mL of anhydrous methanol, 0.12 mL of water, and 5 mL of tert-butanol. The mass ratio of dried bacterial powder to (R)-MAP-acid was 0.05:1. The volume of anhydrous methanol added was 1 mL / g (based on the mass of (R)-MAP-acid), the volume of water added was 0.24 mL / g (based on the mass of (R)-MAP-acid), and the volume of tert-butanol added was 10 mL / g (based on the mass of (R)-MAP-acid). The glass reaction tube was placed in a shaking water bath at 35°C and 200 rpm for 12 h. After the reaction, 20 μL of the reaction solution was added to 1 mL of liquid mobile phase to dissolve the sample and the content of (R)-DMPM, (S)-DMPM and (R)-MAP-acid was detected by high performance liquid chromatography. The liquid chromatography analysis of the reaction solution of (R)-DMPM prepared by enzymatic esterification of strain CZG-18 was performed. Figure 4 As shown, the liquid chromatogram of (R)-MAP-acid standard is as follows Figure 1 As shown, the liquid chromatograms of (R)-MAP-acid and (S)-MAP-acid standards are as follows Figure 2 As shown, the liquid chromatograms of (R)-DMPM, (S)-DMPM and (R)-MAP-acid are as follows Figure 3 The enzymatic esterification rate and eep of (R)-DMPM were calculated, and the results are shown in Table 1.

[0037] Table 1: Esterification rate and selectivity of (R)-MAP-acid by freeze-dried bacterial powder with different numbers (NA means not present)

[0038]

[0039] The calculation formulas for the esterification rate and product selectivity eep of freeze-dried bacterial powders with different numbers are as follows:

[0040]

[0041] In formula 2-1, S0 represents the liquid phase peak area of ​​(R)-MAP-acid when the methyl esterification reaction time is 0; St is the liquid phase peak area of ​​(R)-MAP-acid in the reaction solution after a period of reaction (t minutes).

[0042]

[0043] In formula 2-2, AP1 represents the liquid phase peak area of ​​(R)-DMPM; AP2 represents the liquid phase peak area of ​​(S)-DMPM.

[0044] High performance liquid chromatography: Waters 1525 (USA), chromatographic column: Cellud-Y (5 μm, 4.6 × 250 mm, Yuexu, China), mobile phase: chromatographic grade tert-butyl alcohol / isopropanol / trifluoroacetic acid = 98 / 2 / 0.1 (v / v / v), flow rate: 0.5 mL / min, column temperature: 30°C, injection volume: 10 μL.

[0045] According to Table 1, the strain numbered CZG-18 was selected for 16s rDNA identification:

[0046] The 16s rDNA sequence of CZG-18 was identified by sequencing as shown in SEQ ID NO.1:

[0047]

[0048] Comparison of the sequence with the NCBI database revealed that the strain numbered CZG-18 is consistent with the previously published sequence of Bacillus aryabhattai WZZ10. This strain was used as a strain for preparing (R)-DMPM. The strain was deposited with the China Center for Type Culture Collection, with the deposit number CCTCCNO: M20191099, on December 25, 2019, at Wuhan University, Wuhan, China, zip code 430072, and has been disclosed in a patent application (CN111534472A, filed June 11, 2020).

[0049] Example 3: Freeze-dried powder of Bacillus arylpolytii WZZ10

[0050] The Bacillus aryabendii WZZ10 strain was inoculated into LB solid medium (composition as in Example 1) and cultured at 28°C for 1 day to obtain a slant cell. The slant cell was inoculated into 50 mL of seed medium (composition as in Example 1) and cultured at 28°C and 180 rpm for 12 h to obtain a seed solution. 1 mL of the seed solution was inoculated into 100 mL of fermentation medium (composition as in Example 1) and cultured at 28°C and 180 rpm for 24 h to obtain a fermentation broth. The fermentation broth was centrifuged at 12000 rpm for 3 min, the supernatant was discarded, and the wet cell was frozen at -20°C and then freeze-dried in a vacuum freeze dryer at -80°C and a vacuum of 20 Pa for 12 h to obtain 0.06 g of freeze-dried bacterial powder of Bacillus aryabendii WZZ10.

[0051] Example 4: Application 1 of Bacillus arylsi WZZ10 in the preparation of (R)-N-(2,6-dimethylphenyl)aminopropionic acid:

[0052] 0.01 g of lyophilized Bacillus aryetii WZZ10 powder prepared by the method of Example 3, 0.5 g of (R)-MAP-acid, 1.0 mL of anhydrous methanol, 0.12 mL of water, and 6 mL of tert-butanol were added to a glass reaction tube. The mass ratio of lyophilized Bacillus aryetii WZZ10 powder to (R)-MAP-acid was 0.02:1. The volume of anhydrous methanol added was 2 mL / g based on the mass of (R)-MAP-acid, the volume of water added was 0.24 mL / g based on the mass of (R)-MAP-acid, and the volume of tert-butanol added was 12 mL / g based on the mass of (R)-MAP-acid. The glass reaction tube was placed in a shaking water bath at 35°C and 200 rpm for 14 h. The reaction was assayed using the method of Example 2. The esterification yield of the prepared (R)-DMPM was 50.73%, and the eep value was 99.12%.

[0053] Example 5: Application 2 of Bacillus aygulii WZZ10 for preparing (R)-N-(2,6-dimethylphenyl)aminopropanoic acid

[0054] Into a glass reaction tube, 50 mg of Bacillus aygulii WZZ10 freeze-dried bacterial powder prepared by the method of Example 3, 0.1 g of (R)-MAP-acid, 200 μL of anhydrous methanol, 20 μL of water, and 1 mL of t-butanol were added, wherein the mass ratio of Bacillus aygulii WZZ10 freeze-dried bacterial powder to (R)-MAP-acid was 0.02:1, the volume of anhydrous methanol added was 2 mL / g based on the mass of (R)-MAP-acid, the amount of water added was 0.24 mL / g based on the mass of (R)-MAP-acid, and the volume of t-butanol added was 10 mL / g based on the mass of (R)-MAP-acid. The glass reaction tube was placed in a water bath shaker at 35°C and 200 rpm for 24 h, and the esterification rate of (R)-DMPM prepared was detected by the method of Example 2, which was 55.64%, and the e.e.p was 99.05%.

[0055] Example 6: Application 3 of Bacillus aygulii WZZ10 for preparing (R)-N-(2,6-dimethylphenyl)aminopropanoic acid

[0056] Into a glass reaction tube, 10 mg of Bacillus aygulii WZZ10 freeze-dried bacterial powder prepared by the method of Example 3, 0.02 g of (R)-MAP-acid, 40 μL of anhydrous methanol, 10 μL of water, and 1 mL of t-butanol were added, wherein the mass ratio of Bacillus aygulii WZZ10 freeze-dried bacterial powder to (R)-MAP-acid was 0.02:1, the volume of anhydrous methanol added was 2 mL / g based on the mass of (R)-MAP-acid, the amount of water added was 1 mL / g based on the mass of (R)-MAP-acid, and the volume of t-butanol added was 50 mL / g based on the mass of (R)-MAP-acid. The glass reaction tube was placed in a water bath shaker at 35°C and 200 rpm for 24 h, and the esterification rate of (R)-DMPM prepared was detected by the method of Example 2, which was 48.38%, and the e.e.p was 99.43%.

[0057] The above merely describes preferred embodiments of the present application and is not intended to limit the technical content of the present application in any form. Any simple modification, equivalent change, and modification made to the above embodiments according to the technical essence of the present application all fall within the protection scope of the present application.

Claims

1. An application of Bacillus arylpolytriae in the methylation of (R)-2,6-dimethylphenylaminopropionic acid to prepare (R)-N-(2,6-dimethylphenyl)aminopropionic acid methyl ester, characterized in that: The application comprises the following steps: using wet cells obtained by fermentation and culturing Bacillus aryabhattai and freeze-drying the dry cells as a catalyst, using (R)-2,6-dimethylphenylaminopropionic acid and anhydrous methanol as substrates, and using tert-butanol and water as solvents; reacting at 25-40° C. and 100-200 rpm for 10-24 hours; separating and purifying the reaction solution to obtain (R)-N-(2,6-dimethylphenyl)aminopropionic acid methyl ester; and the Bacillus aryabhattai is Bacillus aryabhattai (Bacillus aryabhattai) CCTCC NO: M20191099.

2. The use according to claim 1, characterized in that The mass ratio of the (R)-2,6-dimethylphenylaminopropionic acid to the catalyst is 1:0.05-1; the volume usage of the anhydrous methanol is 1-5 mL / g based on the mass of the (R)-2,6-dimethylphenylaminopropionic acid.

3. The use according to claim 1, characterized in that The amount of water added is 0.1-1.0 mL / g based on the mass of (R)-2,6-dimethylphenylaminopropionic acid; the amount of tert-butanol added is 4-50 mL / g based on the mass of (R)-2,6-dimethylphenylaminopropionic acid.

4. The use according to claim 1, wherein The freeze drying is to freeze the wet cells at -20°C, and then freeze dry them in a freeze dryer at -80°C and a vacuum degree of 20Pa for 12 hours to obtain dry cells.

5. The use according to claim 1, characterized in that The wet cells are prepared as follows: (1) Slant culture: Bacillus arylpolyticum was inoculated into LB solid medium and cultured at 28°C for 1 day to obtain slant cells. The LB solid medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar. The solvent was deionized water, and the pH was 7.

0. (2) Seed culture: Take the slant bacteria and inoculate them into seed culture medium. Culture them at 28°C and 180 rpm for 12-24 h to obtain seed solution. The seed culture medium composition is as follows: peptone 25 g / L, glucose 20 g / L, K2HPO4 1.0 g / L, (NH4)2SO4 5 g / L, MgSO4 0.25 g / L, olive oil 10 mL / L, solvent is water, pH 7.

0. (3) Fermentation culture: Under sterile conditions, the seed liquid was inoculated into the fermentation medium at an inoculum concentration of 1-2% by volume, and cultured at 28°C and 180 r / min for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 12000 rpm for 3-10 min, the supernatant was discarded, and the wet cells were collected; the fermentation medium composition was: glucose 5 g / L, peptone 20 g / L, K2HPO4 1.0 g / L, (NH4)2SO4 1 g / L, MgSO4 0.25 g / L, the solvent was deionized water, pH 7.0.

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