Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparing (r)-2-chloropropionic acid

Abstract

The application discloses a Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparation of (R)-2-chloropropionic acid. The strain can resolve the racemic 2-chloropropionic acid in a polluted environment to obtain an intermediate (R)-2-chloropropionic acid in preparation of thiolactomycin, and has the advantages of high catalytic substrate concentration and high optical purity of the obtained product. The application utilizes the whole cell of the Pseudomonas taiwanensis ZJPH2023032 as a catalyst, and utilizes a biological catalysis method to resolve the racemic 2-chloropropionic acid to prepare (R)-2-chloropropionic acid. When the substrate concentration is 80 mM, the conversion rate of the 2-chloropropionic acid reaches 49.9%, the ee s value is greater than 99.9%, and complete resolution of the substrate can be realized. When the substrate concentration is 90 mM, the conversion rate of the 2-chloropropionic acid is 45.1%, and the ee s value is 86.0%.

Description

(I) Technical Field

[0001] This invention relates to a novel microbial strain—Pseudomonas taiwanensis ZJPH2023032—and its application in the biocatalytic resolution of racemic 2-chloropropionic acid (2-CPA) to prepare the intermediate (R)-2-chloropropionic acid for thiolactamase. (II) Background Technology

[0002] (R)-2-chloropropionic acid, as an important pharmaceutical intermediate, can be used to synthesize the drug thiolactomycin and the nutritional supplement L-alanyl-L-glutamine. Thiolactomycin (TLM) is a thiolactone antibiotic that exerts its activity by inhibiting β-ketoacyl-acylcarrier protein (ACP) synthase (Kas). Reports have shown that thiolactomycin analogs possess antibacterial, anti-tuberculosis, and antimalarial activities. L-alanyl-L-glutamine has medicinal value in enhancing human immunity and improving intestinal health. Therefore, the preparation of a single configuration of (R)-2-chloropropionic acid has significant application value.

[0003] The molecular formula of (R)-2-chloropropionic acid is C3H5ClO2, and its chemical structural formula is:

[0004]

[0005] Currently reported methods for synthesizing (R)-2-chloropropionic acid mainly include chemical and biological methods. The chemical method utilizes ethyl chloropropionate, formic acid, and a strongly acidic cation exchange resin to prepare (R)-2-chloropropionic acid via transesterification, but this method requires the use of toxic and harmful organic reagents such as formic acid and pyridine. The biological method for preparing (R)-2-chloropropionic acid has advantages such as environmental friendliness, mild reaction conditions, and high enantioselectivity. The biological method includes enzymatic catalysis and whole-cell catalysis. Compared with enzymatic catalysis, whole-cell catalysis eliminates the enzyme separation and purification process and is easier to operate. However, in existing technologies, there are few reports on the use of whole-cell catalysis to resolve the racemic mixture of 2-CPA to prepare a single configuration of (R)-2-CPA, and the catalytic efficiency is low, with a substrate concentration of only 30 mM. This invention provides a novel microbial strain with 2-haloacid dehalogenase activity, which can be used for the highly selective whole-cell catalytic preparation of (R)-2-CPA. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a novel microbial strain—Pseudomonas taiwanensis ZJPH2023032—and its application in the biological resolution of racemic 2-chloropropionic acid to prepare the intermediate (R)-2-chloropropionic acid for thiolactamase, which significantly improves catalytic efficiency and solves the problem of low substrate concentration in existing microbial resolution of racemic 2-chloropropionic acid.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a novel strain of Pseudomonas taiwanensis with 2-haloacid dehalogenase activity, ZJPH2023032, which is deposited at the China Center for Type Culture Collection (CCTCC) on June 14, 2023, with accession number CCTCC NO: M 20231020, located at Wuhan University, Wuhan, China, 430072, China.

[0009] The present invention also provides the application of the aforementioned Pseudomonas taiwanensis ZJPH2023032 in the preparation of (R)-2-chloropropionic acid by resolving the racemic mixture of 2-chloropropionic acid.

[0010] Furthermore, the application method is as follows: using wet cells of *Pseudomonas taiwanensis* ZJPH2023032 obtained by fermentation culture as the enzyme source, racemic 2-chloropropionic acid as the resolution substrate, and phosphate buffer solution with pH 6.0-8.0 as the reaction medium to form a transformation system, the reaction is carried out at 25-45℃ and 180-200 rpm, after the reaction is completed, a transformation solution containing (R)-2-chloropropionic acid is obtained, and the (R)-2-chloropropionic acid product is obtained after separation and purification.

[0011] Furthermore, the separation and purification method of the (R)-2-chloropropionic acid conversion solution is as follows: the (R)-2-chloropropionic acid conversion solution is extracted with an equal volume of ethyl acetate, centrifuged and the ethyl acetate extract phase is collected, and then concentrated by rotary evaporation to remove the ethyl acetate solvent, thereby obtaining the (R)-2-chloropropionic acid product.

[0012] Furthermore, the amount of wet bacterial cells used is 50-600 g / L based on the buffer volume, preferably 200 g / L; the initial concentration of the substrate is 50-120 mM (preferably 80-90 mM).

[0013] Furthermore, the buffer solution is selected from disodium hydrogen phosphate-citric acid buffer (pH 3.0-6.0), dipotassium hydrogen phosphate-potassium dihydrogen phosphate (pH 6.0-8.0), Tris-HCl buffer (pH 8.0-9.0), glycine-sodium hydroxide buffer (pH 9.0-10.0), with the most preferred being a 0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 7.5.

[0014] Furthermore, the reaction time is 6-48 hours, more preferably 12-24 hours at 35°C and 200 rpm.

[0015] Furthermore, the reaction medium was a 0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a pH of 7.5, and the reaction conditions were 35°C and 200 rpm for 12 h; the wet cell volume was 200 g / L based on the buffer volume, and the initial concentration of the substrate was 80-90 mM.

[0016] Furthermore, the enzyme source is obtained as follows:

[0017] (1) Plate culture: Pseudomonas taiwanensis ZJPH2023032 was inoculated onto a plate culture medium and cultured at 30℃ for 48-72h to obtain the plate culture strain; the plate culture medium composition was: glucose 15g / L, fish meal peptone 20g / L, yeast extract 10g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, sodium chloride 1g / L, magnesium sulfate heptahydrate 0.5g / L, agar 20g / L, water as solvent, pH 6.5.

[0018] (2) Seed culture: One loopful of the strain after plate culture was inoculated into seed culture medium and cultured at 200 rpm and 30℃ for 12 h to obtain seed culture solution. The seed culture medium consisted of: glucose 15 g / L, fish meal peptone 20 g / L, yeast extract 10 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, water as solvent, pH 6.5.

[0019] (3) Fermentation culture: The seed culture solution from step (2) was inoculated into the fermentation medium at a volume concentration of 10%, and cultured at 200 rpm and 30℃ for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 9000 rpm and 4℃ for 10 min. The resulting precipitate was washed with 0.1 M, pH 7.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, and centrifuged again to obtain the wet cells, which are the enzyme source cells containing the enzyme that can be used to resolve the racemic mixture of 2-chloropropionic acid to prepare (R)-2-chloropropionic acid. The fermentation medium consisted of: glucose 15 g / L, fish meal peptone 20 g / L, yeast extract 10 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and water as the solvent, pH 6.5.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] This invention provides a novel strain of *Pseudomonas taiwanensis* ZJPH2023032 with 2-halogenated acid dehalogenase activity. This microbial strain is easy to culture and simple to prepare. This strain can resolve the racemic mixture of 2-chloropropionic acid, a halide compound in the contaminated environment, to obtain the thiolactam intermediate (R)-2-chloropropionic acid, exhibiting advantages such as high substrate concentration and high optical purity of the obtained product. This invention utilizes the whole cell of *Pseudomonas taiwanensis* ZJPH2023032 as a catalyst to prepare (R)-2-chloropropionic acid by resolving the racemic mixture of 2-chloropropionic acid using a biocatalytic method. When the substrate concentration is 80 mM, the conversion rate of 2-chloropropionic acid reaches 49.9%. s With a value greater than 99.9%, complete resolution of the substrate can be achieved. At a substrate concentration of 90 mM, the conversion rate of 2-chloropropionic acid is 45.1%. s The value was 86.0%. Compared with the levels reported in the prior art, the product obtained by the present invention using wild-type strain catalysis has a high enantiomeric excess value, and features low cost, green and environmentally friendly transformation process. (iv) Description of the attached drawings

[0022] Figure 1 Colony morphology of Pseudomonas taiwanensis ZJPH2023032.

[0023] Figure 2 Gram staining results of Pseudomonas taiwanensis ZJPH2023032.

[0024] Figure 3 Phylogenetic tree of Pseudomonas taiwanensis ZJPH2023032.

[0025] Figure 4 Liquid chromatography spectra of (R)-2-chloropropionic acid and (S)-2-chloropropionic acid standards.

[0026] Figure 5 Liquid chromatography-mass spectra of (R)-2-chloropropionic acid conversion extract prepared by resolving the racemic mixture of 2-chloropropionic acid from strain ZJPH2023032 of Pseudomonas taiwanensis. (V) Specific Implementation Methods:

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0028] The culture medium used in this invention is as follows:

[0029] The liquid culture medium consisted of: 3.2 g / L disodium hydrogen phosphate dodecahydrate, 0.5 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.098 g / L magnesium sulfate heptahydrate, 0.5 g / L yeast extract, pH 7.0, and water as the solvent.

[0030] The plate culture medium consisted of: 15 g / L glucose, 20 g / L fish meal peptone, 10 g / L yeast extract, 2 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 20 g / L agar, and water as the solvent, with a pH of 6.5.

[0031] The indicator plate culture medium consisted of: 2 g / L ammonium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 0.098 g / L anhydrous magnesium sulfate, 5 g / L sodium chloride, 0.08 g / L bromothymol blue, 20 g / L agar powder, and water as the solvent, with a pH of 7.0.

[0032] The seed culture medium consisted of: 15 g / L glucose, 20 g / L fish meal peptone, 10 g / L yeast extract, 2 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, with water as the solvent and a pH of 6.5.

[0033] The fermentation medium consisted of: 15 g / L glucose, 20 g / L fish meal peptone, 10 g / L yeast extract, 2 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, with water as the solvent and a pH of 6.5.

[0034] Example 1: Screening of bacterial strains with 2-haloacid dehalogenase activity

[0035] Strain source: *Pseudomonas fortatum* ZJPH2023032 was isolated and screened from soil samples containing chemical reagent contaminants from the vicinity of the laboratory on the Moganshan campus of Zhejiang University of Technology. The specific screening method is as follows:

[0036] During the initial screening of the strain, sodium dichloropropionate (2-CPANa) was used as the sole carbon source. The concentration of 2-CPANa added to the liquid culture medium was gradually increased to enrich the strains that could transform into 2-CPA. 2-CPANa was prepared into a 500mM stock solution using a pH 7.0, 0.1M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer and added to the enrichment medium as needed: 10g of soil sample was weighed into 100mL of liquid culture medium, and 2mL of 2-CPANa stock solution was added as the sole carbon source. The mixture was then cultured at 30℃ with shaking for 24h. Then, 20mL of the enriched culture was inoculated into another shake flask containing liquid culture medium, and 7.7mL of 2-CPANa stock solution was added for a second enrichment culture. The mixture was cultured at 30℃ with shaking for 24h. 20mL of the second enrichment culture was then added into another shake flask containing liquid culture medium, and 13.3mL of 2-CPANa stock solution was added for further enrichment culture. The mixture was cultured at 30℃ with shaking for 24h. The enrichment culture was repeated three times.

[0037] Secondary screening of bacterial strains: Add 100 μL of enriched bacterial culture to an EP tube in a clean bench, then add 900 μL of sterile water and mix thoroughly to obtain 10 strains. -1 Dilute the bacterial suspension. Then take 100 μL of 10 -1 The diluted bacterial solution was added to another EP tube, along with 900 μL of sterile water. The mixture was thoroughly mixed to obtain 10... -2 Dilute the bacterial culture; and so on, finally using a serial dilution to 10⁻⁶. -6 Take 10g of the above diluted solution. -2 10 -3 10 -4 10 -5 10 -6 100 μL of the culture was spread onto an indicator plate and incubated at 30°C for 24-48 hours. The color of some areas on the plate changed from green to yellow, indicating that the strain possessed 2-haloacid dehalogenase activity. Single yellow colonies were picked from the plate and streaked onto another plate to obtain pure cultures. The isolated strains were Gram-stained and examined under a microscope. Pure cultures showing different morphologies after microscopic examination were then transferred to seed culture media and preserved in glycerol tubes.

[0038] The strain preserved in 1 mL of glycerol was inoculated into 100 mL of seed culture medium and activated at 30 °C and 200 rpm for 24 h. The seed culture was then inoculated into fermentation medium at a volume concentration of 10% and cultured at 200 rpm and 30 °C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 9000 rpm and 4 °C for 10 min. The resulting precipitate was washed with 0.1 M pH 7.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer and centrifuged again to obtain the wet cells. 500 mg of the wet cells were added to 5 mL of 0.1 M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0). After mixing the cells and buffer, 300 μL of 2-CPANa stock solution was added as the substrate for the transformation reaction. The mixture was transformed at 30 °C and 200 rpm for 24 h in a shaker. After the reaction was complete, 150 μL of 36% concentrated hydrochloric acid was added to the reaction system. The mixture was acidified at 25 °C for 10 min, then extracted with 5 mL of ethyl acetate for 30 min. After centrifugation, a supernatant containing (R)-2-chloropropionic acid was obtained. This supernatant was filtered through a 0.45 μm filter, and the peak areas of the unconverted substrate and product were detected by liquid chromatography. The substrate conversion rate and ee were calculated. s The dominant strain capable of resolving the racemic mixture of 2-chloropropionic acid to prepare (R)-2-chloropropionic acid was obtained by screening and designated as strain ZJPH2023032.

[0039] Liquid chromatography detection conditions: The chromatographic column used is ID (4.6mM×250mM, 5μm), mobile phase was n-hexane (V): ethanol (V) = 98:2, detection wavelength was 254nm, flow rate was 1mL / min, and injection volume was 5μL.

[0040] The substrate conversion rate is calculated as follows:

[0041] Standard curves were plotted: A 500 mM stock solution of 2-CPANa was prepared using a 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution (pH 7.0). This stock solution was then diluted to obtain standard solutions with concentrations of 10, 50, 100, 150, and 200 mM. These solutions were then analyzed by liquid chromatography (LC) to plot standard curves for (S)-2-chloropropionic acid ((S)-2-CPA) and (R)-2-chloropropionic acid ((R)-2-CPA). The equation for the (S)-2-chloropropionic acid standard curve is: y = 1993.1x - 3024.8(R) 2 =0.999), the standard curve for (R)-2-chloropropionic acid is: y = 1679.3x + 313.97(R) 2 =0.999)

[0042] The formula for calculating the conversion rate of 2-chloropropionic acid is:

[0043] Conversion rate (%) = [1 - (R)] t +S t ) / (R0+S0)]×100

[0044] R t S represents the concentration of (R)-2-chloropropionic acid at the end of the reaction. t R0 is the initial concentration of (R)-2-chloropropionic acid at the end of the reaction, and S0 is the initial concentration of (S)-2-chloropropionic acid. The optical purity of the substrate is determined by the enantiomeric excess value (ee). s It is characterized by ) . The calculation formula is:

[0045]

[0046] S + S and S- represent the peak areas of (R)-2-chloropropionic acid and (S)-2-chloropropionic acid in the substrate, respectively, in the chiral liquid chromatography detection results.

[0047] Example 2: Identification of strain ZJPH2023032

[0048] Morphological observation: Strain ZJPH2023032 was inoculated onto agar plates and incubated at 30℃ for 48-72 h. Colony and cell morphology were then observed. The colonies were yellow, round, with neat edges, a smooth and glossy surface, and a slightly convex center; they were soft and easily picked up. Results are shown below. Figure 1The bacteria are short rod-shaped and Gram-negative. Results are shown in the attached table. Figure 2 .

[0049] Physiological and biochemical tests: VITEK physiological and biochemical performance of strain ZJPH2023032 was identified (see Table 1), and its physiological and biochemical characteristics were consistent with those of Pseudomonas taiwanensis.

[0050] Table 1. Results of Physiological and Biochemical Characteristic Identification

[0051]

[0052] 16S rRNA sequence analysis: The 16S rRNA gene sequence of strain ZJPH2023032 was obtained by Beijing Qingke Biotechnology Co., Ltd., and the length of the 16S rRNA sequence of strain ZJPH2023032 was determined to be 1456 bp. The specific nucleotide sequence is shown in SEQ ID NO.1. The obtained 16S rRNA sequence (SEQ ID NO.1) of the strain was subjected to homology alignment (BLAST) on NCBI (http: / / www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA11. The results showed that strain ZJPH2023032 and Pseudomonas taiwanensis DSW 21245 had 94% sequence homology. The results are shown in [see figure]. Figure 3 .

[0053] Based on the morphological characteristics, physiological and biochemical properties and 16S rRNA sequence of the strain, strain ZJPH2023032 was identified as *Pseudomonas taiwanensis*, deposited on June 14, 2023, with accession number CCTCC: M20231020, address: Wuhan University, Wuhan, China, 430072, China.

[0054] SEQ ID NO.1

[0055] CACCCCAGTCATGAATCACACCGTGGTAACCGTCCTCCCGAAGGTTAGACTAGC

[0056] TACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCG

[0057] GGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCAC

[0058] GCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTGTGAGATTAGCT

[0059] CCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCC

[0060] CAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTC

[0061] ACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGG

[0062] GTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAG

[0063] CCATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGT

[0064] TCTCTGCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACA

[0065] TGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGC

[0066] CGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAAATCTCAAG

[0067] GATTCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATC

[0068] CTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGC

[0069] CTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATT

[0070] CCACCACCCTCTACCGTACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTG

[0071] AGCCCGGGGCTTTCACATCCAACTTAACGAACCACCTACGCGCGCTTTACGCCC

[0072] AGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAG

[0073] TTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATTAGCTTACTG

[0074] CCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCG

[0075] GCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCC

[0076] GTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACC

[0077] AGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACC

[0078] TAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGAC

[0079] GTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTC

[0080] CTAGGCATTACTCACCCGTCCGCCGCTGAATCAAGGAGCAAGCTCCCGTCATCCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTC。

[0081] Example 3: Preparation of wet cells of *Pseudomonas taiwanensis* ZJPH2023032

[0082] Plate culture: Pseudomonas taiwanensis ZJPH2023032 was inoculated onto agar plates and cultured at 30°C for 48-72 hours to obtain the plate cultured strain.

[0083] Seed culture: Pick a loopful of the strain after plate culture and inoculate it into a shake flask containing seed culture medium. Incubate at 200 rpm and 30°C for 12 h to obtain seed culture solution.

[0084] Fermentation culture: The seed culture solution was inoculated into the fermentation medium at a volume concentration of 10% and cultured at 200 rpm and 30℃ for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 9000 rpm and 4℃ for 10 min to obtain wet cells, which are the enzyme source cells for resolving 2-chloropropionic acid to prepare (R)-2-chloropropionic acid.

[0085] Example 4:

[0086] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.0), with a wet bacterial cell addition of 100 g / L. 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 30°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and the conversion rate was 34.0%. s The value is 54.4%.

[0087] Example 5:

[0088] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 100 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 30°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and was 38.0%. s The value is 60.8%.

[0089] Example 6:

[0090] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 8.0), with a wet bacterial cell addition of 100 g / L. 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 30°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and was 36.1%. s The value is 55.1%.

[0091] Example 7:

[0092] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 200 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 30°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and the conversion rate was 40.1%. s The value is 67.3%.

[0093] Example 8:

[0094] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 300 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 30°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and was 37.4%. s The value is 59.2%.

[0095] Example 9:

[0096] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 200 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 35°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and the conversion rate was 45.1%. s The value was 86.0%, and the liquid chromatogram is shown below. Figure 5 As shown.

[0097] Under the same conditions, when the substrate concentration was changed to 80 mM, the conversion rate of 2-chloropropionic acid was 49.9% at 35℃ and 200 rpm for 24 h. s The value is 99.9%.

[0098] Example 10:

[0099] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 200 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 40 °C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and was 36.6%. s The value is 56.7%.

[0100] Example 11:

[0101] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 200 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 35°C and 200 rpm for 12 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and was 43.4%. s The value is 79.0%.

[0102] Example 12:

[0103] The wet bacterial cells obtained according to the method in Example 3 were suspended in 5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1 M, pH 7.5), with a wet bacterial cell addition of 200 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 90 mM, and the mixture was incubated in a shaker at 25°C and 200 rpm for 36 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography method described in Example 1, and the conversion rate was 43.9%. s The value is 81.3%.

[0104] Example 13: Investigation of the biocatalytic conversion ability of *Pseudomonas putida* ZJPH1412 to prepare (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0105] (1) *Pseudomonas putida* ZJPH1412, deposited at the China Center for Type Culture Collection (CCTCC), date of deposit: April 11, 2016, accession number: CCTCC NO: M 2016187. This strain has been disclosed in the applicant's previous patent application (publication number: CN106497843A, publication date: May 31, 2019). The culture method and enzyme source cell preparation process of the strain are based on the previous patent application (publication number: CN106497843A, publication date: May 31, 2019).

[0106] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from racemic 2-CPA mixture

[0107] 0.5 g of wet cells of *Pseudomonas putida* ZJPH1412, prepared by fermentation, were resuspended in 5 mL of potassium phosphate buffer (0.1 M, pH 7.0). The wet cell concentration was 100 g / L (wet weight). 2-CPA was added to a final concentration of 90 mM as substrate, and the mixture was incubated in a shaker at 30 °C and 200 rpm for 24 h. The results were detected using the liquid chromatography method described in Example 1.

[0108] Conclusion: Pseudomonas putida ZJPH1412 cannot biocatalyze the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0109] Example 14: Investigation of the biocatalytic conversion ability of Acinetobacter ZJPH1806 to prepare (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0110] (1) Acinetobacter sp. ZJPH1806, deposited at the China Center for Type Culture Collection, deposit date: March 29, 2019, accession number: CCTCC NO: 2019214. This strain has been disclosed in the applicant's previous patent application (publication number: CN110016444B, publication date: July 16, 2019).

[0111] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from racemic 2-CPA mixture

[0112] 0.5 g of wet Acinetobacter ZJPH1806 cells obtained through fermentation were resuspended in 5 mL of potassium phosphate buffer (0.1 M, pH 7.0). The wet cell addition was 100 g / L (wet weight). 2-CPA was added to a final concentration of 90 mM as substrate, and the mixture was incubated in a shaker at 30 °C and 200 rpm for 24 h. The results were detected using the liquid chromatography method described in Example 1.

[0113] Conclusion: Acinetobacter ZJPH1806 cannot biocatalyze the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0114] Example 15: Investigation of the biocatalytic conversion ability of *Sacchariformis* ZJPH202011 in the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0115] (1) Kosakonia radicincitans ZJPH202011, deposited at the China Center for Type Culture Collection (CCTCC), deposit date: June 11, 2021, accession number: CCTCC NO: M 2021714. This strain has been disclosed in the applicant's previous patent application (publication number: CN113462602A, publication date: May 24, 2022).

[0116] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from racemic 2-CPA mixture

[0117] 0.5 g of wet cells of *Sacchariformis* ZJPH202011, prepared by fermentation, were resuspended in 5 mL of potassium phosphate buffer (0.1 M, pH 7.0). The wet cell concentration was 100 g / L (wet weight). 2-CPA was added to a final concentration of 90 mM as substrate, and the mixture was incubated in a shaker at 30 °C and 200 rpm for 24 h. The results were detected using the liquid chromatography method described in Example 1.

[0118] Conclusion: The root-promoting bacteria ZJPH202011 cannot biocatalyze the preparation of the intermediate (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0119] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. *Pseudomonas taiwanensis* ( Pseudomonas taiwanensis ZJPH2023032, deposited at the China Center for Type Culture Collection, deposited on June 14, 2023, accession number: CCTCC NO: M 20231020, address: Wuhan University, Wuhan, China, 430072, China.

2. A method for preparing the *Pseudomonas taiwanensis* ZJPH2023032 according to claim 1 by resolving the racemic 2-chloropropionic acid mixture (… R Applications of 2-chloropropionic acid.

3. The application as described in claim 2, characterized in that, The application method is as follows: Using wet cells of *Pseudomonas taiwanensis* ZJPH2023032 obtained through fermentation as the enzyme source, 2-chloropropionic acid racemic mixture as the resolution substrate, and phosphate buffer solution with a pH of 6.0-8.0 as the reaction medium, a transformation system is constructed. The reaction is carried out at 25-40℃ and 180-200 rpm. After the reaction, a solution containing (…) is obtained. R The conversion solution of 2-chloropropionic acid was purified to obtain ( R )-2-chloropropionic acid products.

4. The application as described in claim 3, characterized in that, The amount of wet bacterial cells used is 50-600 g / L based on the volume of the buffer solution; the initial concentration of the substrate is 50-120 mM.

5. The application as described in claim 3, characterized in that, The reaction medium is a 0.1 M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a pH of 7.

5.

6. The application as described in claim 3, characterized in that, The reaction was carried out at 35°C and 200 rpm for 12-24 hours.

7. The application as described in any one of claims 3 to 6, characterized in that, The reaction medium was a 0.1 M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a pH of 7.

5. The reaction conditions were 35°C and 200 rpm for 12 h. The wet cell volume was 200 g / L based on the buffer volume, and the initial concentration of the substrate was 80-90 mM.

8. The application as described in claim 3, characterized in that, The enzyme source was obtained as follows: (1) Plate culture: Pseudomonas taiwanensis ZJPH2023032 was inoculated onto plate culture medium and cultured at 30℃ for 48-72 h to obtain plate culture strain; the plate culture medium composition was: glucose 15 g / L, fish meal peptone 20 g / L, yeast extract 10 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, agar 20 g / L, solvent was water, pH 6.5; (2) Seed culture: One loopful of the strain after plate culture was inoculated into seed culture medium and cultured at 200 rpm and 30℃ for 12 h to obtain seed culture solution; The seed culture medium consisted of: glucose 15 g / L, fish meal peptone 20 g / L, yeast extract 10 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, water as solvent, pH 6.5; (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into the fermentation medium at an inoculation amount of 10% by volume. The culture was carried out at 200 rpm and 30℃ for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 9000 rpm and 4℃ for 10 min. The precipitate was washed with 0.1 M, pH 7.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer. After centrifugation again, the wet cells were obtained. The fermentation medium consisted of: glucose 15 g / L, fish meal peptone 20 g / L, yeast extract 10 g / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 2 g / L, sodium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and water as the solvent, pH 6.5.

Images