Sphingosine-producing bacterium zjph2023031 and application thereof in preparation of (r)-2-chloropropionic acid

Abstract

The application discloses a bacterium of Sphingosinicella mucoica ZJPH2023031 and application thereof in preparation of (R)-2-chloropropionic acid. The bacterium of Sphingosinicella mucoica ZJPH2023031 is easy to culture and simple to prepare. The bacterium can resolve the racemic 2-chloropropionic acid in a polluted environment to obtain a sulfur lactone mycosporine intermediate (R)-2-chloropropionic acid, and has the advantages of high catalytic substrate concentration, high optical purity of the obtained product and the like. The application utilizes the whole cell of the bacterium of Sphingosinicella mucoica ZJPH2023031 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 2-chloropropionic acid reaches 49.9%, and the ee s value is greater than 99.9%, and complete resolution of the substrate can be achieved. When the substrate concentration is 90 mM, the conversion rate of 2-chloropropionic acid reaches 46.7%, and the ee s value is 92.6%.

Description

(I) Technical Field

[0001] This invention relates to a novel microbial strain—Sphingomonas ZJPH2023031—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-chloropropionic acid, and the catalytic efficiency is relatively 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—Sphingobacterium mucilaginosum ZJPH2023031—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 biological methods for preparing (R)-2-chloropropionic acid.

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

[0008] This invention provides a novel strain of *Sphingobacterium mucilaginosum* ZJPH2023031 with 2-haloacid dehalogenase activity, deposited at the China Center for Type Culture Collection (CCTCC) on June 14, 2023, accession number: CCTCC NO: M 20231019, address: Wuhan University, Wuhan, China, 430072, China.

[0009] The present invention also provides the application of the aforementioned *Sphingomonas sphingolipidae* ZJPH2023031 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 *Sphingomyelin-Bacillus myxobin* ZJPH2023031 obtained by fermentation culture as the enzyme source, racemic 2-chloropropionic acid as the resolution substrate, and a buffer solution with a pH of 3.0-10.0 as the reaction medium to form a transformation system, the reaction is carried out at 20-40℃ and 180-200 rpm, after the reaction is completed, a transformation solution containing (R)-2-chloropropionic acid is obtained, and after separation and purification, the (R)-2-chloropropionic acid product is obtained.

[0011] Furthermore, the method for separating and purifying the conversion solution containing (R)-2-chloropropionic acid is as follows: the conversion solution containing (R)-2-chloropropionic acid is extracted with an equal volume of ethyl acetate, the ethyl acetate extract phase is collected after centrifugation, and then the ethyl acetate solvent is removed by rotary evaporation to obtain the (R)-2-chloropropionic acid product.

[0012] Furthermore, the amount of wet bacterial cells used is 50-600 g / L, preferably 50-150 g / L, based on the buffer volume; the initial concentration of the substrate added is 50-120 mM (preferably 80 mM), based on the buffer volume.

[0013] Furthermore, the buffer solutions selected are 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), and glycine-sodium hydroxide buffer (pH 9.0-10.0). The ionic strength of all buffer solutions is 0.1M, with the most preferred being a 0.1M glycine-sodium hydroxide buffer at pH 9.0.

[0014] Furthermore, the reaction time is 6-48 hours, more preferably 24-36 hours at 30°C and 200 rpm.

[0015] Furthermore, the reaction medium is a 0.1M glycine-sodium hydroxide buffer solution with a pH of 9.0, the amount of wet bacterial cells is 70 g / L based on the buffer volume, the initial concentration of the substrate is 80-90 mM based on the buffer volume, and the reaction conditions are 30°C and 200 rpm for 24 h.

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

[0017] (1) Plate culture: Sphingomyelin Bacillus ZJPH2023031 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 20 g / L, yeast extract 8 g / L, ammonium sulfate 20 g / L, sodium chloride 10 mM, 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 *Sphingosphate Bacillus* ZJPH2023031 with 2-haloacid 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 contaminated in the 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. Compared with existing reports, this invention utilizes the whole cell of *Sphingosphate Bacillus* ZJPH2023031 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 substrate resolution can be achieved. At a substrate concentration of 90 mM, the conversion rate of 2-chloropropionic acid reaches 46.7%. s The value was 92.6%. 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 Photographs of plate rescreening of *Sphingomyelin-1* ZJPH2023031.

[0023] Figure 2 Colony morphology of *Sphingomyelin-1* ZJPH2023031.

[0024] Figure 3 Gram staining results of *Sphingomyelin-1* ZJPH2023031.

[0025] Figure 4 Phylogenetic tree of *Sphingospholipidobacterium* ZJPH2023031.

[0026] Figure 5 Liquid chromatography spectra of (R)-2-chloropropionic acid and (S)-2-chloropropionic acid standards.

[0027] Figure 6 Liquid chromatography-mass spectra of the (R)-2-chloropropionic acid conversion extract prepared by biocatalytic resolution of the racemic 2-chloropropionic acid by strain Sphingomyelin Bacillus ZJPH2023031. (V) Detailed Implementation

[0028] 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:

[0029] The culture medium used in this invention is as follows:

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The fermentation medium consisted of: 20 g / L glucose, 8 g / L yeast extract, 20 g / L ammonium sulfate, 10 mM sodium chloride, and water as the solvent, with a pH of 6.5.

[0035] Example 1: Screening of strains with 2-haloacid dehalogenase activity

[0036] Strain source: *Sphingomyelin-1* ZJPH2023031 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:

[0037] In the initial screening of the strain, sodium dichloropropionate (2-CPANa) was used as the sole carbon source. Enrichment culture of 2-CPA-convertible strains was carried out by gradually increasing the concentration of 2-CPANa in the liquid culture medium. A 500mM 2-CPANa stock solution was prepared 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 incubated 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, incubated at 30℃ with shaking for 24h. Finally, 20mL of the second enrichment culture was added to another shake flask containing liquid culture medium, and 13.3mL of 2-CPANa stock solution was added for further enrichment culture, incubated at 30℃ with shaking for 24h. The enrichment culture was repeated three times.

[0038] Secondary screening of bacterial strains: In a clean bench, add 100 μL of enriched bacterial culture to an EP tube, 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 Spread 100 μL onto an indicator plate and incubate statically in a 30°C biochemical incubator for 24-48 hours. You will then observe that the color of some areas on the plate changes from green to yellow (see...). Figure 1 This indicates that the strain possesses 2-haloacid dehalogenase activity. Single yellow colonies were picked from the plate and streaked onto agar plates to obtain pure cultures. The isolated strains were Gram-stained and examined under a microscope. The pure cultures with different morphologies after microscopic examination were then transferred to seed culture medium for glycerol preservation.

[0039] 1 mL of the glycerol-preserved bacterial strain 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 wet bacterial cells. 500 mg of wet bacterial cells were added to 5 mL of 0.1 M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0). After mixing the bacterial 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 Value. The optimal strain for preparing (R)-2-chloropropionic acid by resolving the racemic mixture of 2-chloropropionic acid was obtained through screening and is denoted as strain ZJPH2023031.

[0040] 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.

[0041] The substrate conversion rate is calculated as follows:

[0042] 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 was obtained as: 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)

[0043] The formula for calculating the conversion rate of 2-chloropropionic acid is:

[0044] Conversion rate (%) = [1 - (R)] t +St ) / (R0+S0)]×100

[0045] R t S represents the concentration of (R)-2-chloropropionic acid at the end of the reaction. t R0 represents the initial concentration of (S)-2-chloropropionic acid at the end of the reaction, and S0 represents the initial concentration of (R)-2-chloropropionic acid.

[0046] The optical purity of the substrate is determined by the enantiomeric excess value (ee). s The formula is as follows:

[0047]

[0048] S + and S - The peak areas of (R)-2-chloropropionic acid and (S)-2-chloropropionic acid in the substrate, respectively, are represented by the peak areas in the chiral liquid chromatography detection results.

[0049] Example 2: Identification of strain ZJPH2023031

[0050] Morphological observation: Strain ZJPH2023031 was inoculated onto agar plates and incubated at 30℃ for 48-72 hours. 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 in the attached table. Figure 2 The bacteria are short rod-shaped and Gram-negative. Results are shown in the attached table. Figure 3 .

[0051] Physiological and biochemical tests: The physiological and biochemical characteristics of strain ZJPH2023031 were identified by VITEK (see Table 1), and its physiological and biochemical characteristics were consistent with those of *Sphingosphate Bacillus*.

[0052] Table 1. Results of Physiological and Biochemical Characteristic Identification

[0053]

[0054]

[0055] 16S rRNA sequence analysis: The 16S rRNA gene sequence of strain ZJPH2023031 was obtained by Sangon Biotech (Shanghai) Co., Ltd., and its 16S rRNA sequence length was determined to be 1311 bp. The nucleotide sequence is shown in SEQ ID NO.1. The obtained 16S rRNA sequence 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 ZJPH2023031 shared 91% sequence homology with Sphingobacterium mucilaginosum strain THG-SQA8. The results are shown in [Figure number missing]. Figure 4 .

[0056] Based on the morphological characteristics, physiological and biochemical properties, and 16S rRNA sequence of the strain, strain ZJPH2023031 was identified as *Sphingobacterium mucilaginosum*, deposited on June 14, 2023, with accession number CCTCC NO: M 20231019, located at Wuhan University, Wuhan, China, 430072, China.

[0057] SEQ ID NO.1

[0058]

[0059] Example 3: Preparation of wet cells of *Sphingomyelin-Bacillus myxobin* ZJPH2023031

[0060] Plate culture: Sphingomyelin Bacillus ZJPH2023031 was inoculated onto a plate culture medium and cultured at 30℃ for 48-72 h to obtain the plate culture strain.

[0061] 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.

[0062] 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 that can be biologically separated from 2-chloropropionic acid to prepare (R)-2-chloropropionic acid.

[0063] Example 4:

[0064] 0.5 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (0.1 M, pH 7.0). The wet bacterial cell addition was 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 the conversion rate was 20.4%. s The value is 20.5%.

[0065] Example 5:

[0066] 0.5 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0). The wet bacterial cell addition was 100 g / L on a wet weight basis. 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 34.2%. s The value is 50.2%.

[0067] Example 6:

[0068] 0.5 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 10.0). The wet bacterial cell addition was 100 g / L on a wet weight basis. 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 31.1%. s The value is 42.1%.

[0069] Example 7:

[0070] One g of wet bacterial cells obtained according to the method in Example 3 was resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0). The wet bacterial cell addition was 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 28.4%. s The value is 36.2%.

[0071] Example 8:

[0072] 0.25 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0). The wet bacterial cell addition was 50 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 42.8%. s The value is 77.1%.

[0073] Example 9:

[0074] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 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 detection method described in Example 1, and the conversion rate was 46.7%. s The value was 92.6%, and the liquid chromatogram is shown below. Figure 6 .

[0075] Under the same conditions, when 2-CPA at a final concentration of 80 mM was added as a substrate, the conversion rate of 2-chloropropionic acid was 49.9% at 30°C and 200 rpm for 24 h. s The value is 99.9%.

[0076] Example 10:

[0077] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 2-CPA was added as a substrate to a final concentration of 100 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 detection method described in Example 1, and the conversion rate was 37.5%. s The value is 58.8%.

[0078] Example 11:

[0079] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 2-CPA was added as a substrate to a final concentration of 100 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 detection method described in Example 1, and the conversion rate was 26.1%. s The value is 30.7%.

[0080] Example 12:

[0081] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 2-CPA was added as a substrate to a final concentration of 100 mM, and the mixture was incubated in a shaker at 25°C and 200 rpm for 24 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography detection method described in Example 1, and the conversion rate was 40.8%. s The value is 69.5%.

[0082] Example 13:

[0083] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 2-CPA was added as a substrate to a final concentration of 100 mM, and the mixture was incubated in a shaker at 25°C and 200 rpm for 12 h. The conversion rate of 2-chloropropionic acid was detected using the liquid chromatography detection method described in Example 1, and was 31.3%. s The value is 40.3%.

[0084] Example 14:

[0085] 0.35 g of wet bacterial cells obtained according to the method in Example 3 were resuspended in 5 mL of glycine-sodium hydroxide (0.1 M, pH 9.0), with the wet bacterial cell addition amount being 70 g / L on a wet weight basis. 2-CPA was added as a substrate to a final concentration of 100 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 41.9%. s The value is 73.2%.

[0086] Example 15: Investigation of the biocatalytic conversion ability of *Pseudomonas putida* ZJPH1412 to prepare (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0087] (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).

[0088] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from racemic 2-CPA mixture

[0089] 0.5 g of wet *Pseudomonas putida* ZJPH1412 cells obtained through fermentation was resuspended in 5 mL of potassium phosphate buffer (pH 7.0, 0.1 M), with the wet cell addition amount being 100 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 65 mM, 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.

[0090] Conclusion: Pseudomonas putida ZJPH1412 cannot biocatalyze the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0091] Example 16: Investigation of the biocatalytic conversion ability of Acinetobacter ZJPH1806 to prepare (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0092] (1) Acinetobacter sp. ZJPH1806, deposited at the China Center for Type Culture Collection, deposit date: March 29, 2019, accession number: CCTCC NO: M 2019214. This strain has been disclosed in the applicant's previous patent application (publication number: CN110016444B, publication date: July 16, 2019).

[0093] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from 2-CPA

[0094] 0.5 g of wet Acinetobacter ZJPH1806 cells obtained through fermentation were resuspended in 5 mL of potassium phosphate buffer (pH 7.0, 0.1 M), with the wet cell addition amount being 100 g / L (wet weight). 2-CPA was added as a substrate to a final concentration of 65 mM, 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 detection method described in Example 1.

[0095] Conclusion: Acinetobacter ZJPH1806 cannot biocatalyze the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0096] Example 17: Investigation of the biocatalytic conversion ability of *Sacchariformis* ZJPH202011 in the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-CPA.

[0097] (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).

[0098] (2) Biocatalytic preparation of (R)-2-chloropropionic acid from racemic 2-CPA mixture

[0099] 0.5 g of wet cells of *Sacchariformis* ZJPH202011, prepared by fermentation, were resuspended in 5 mL of potassium phosphate buffer (pH 7.0, 0.1 M). The wet cell concentration was 100 g / L (wet weight). 2-CPA was added to a final concentration of 65 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.

[0100] Conclusion: The root-promoting bacteria ZJPH202011 cannot biocatalyze the preparation of (R)-2-chloropropionic acid from the racemic mixture of 2-chloropropionic acid.

[0101] 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. Sphingosine monoclonal antibodies (SMPs) Sphingobacterium mucilaginosum ZJPH2023031, deposited at the China Center for Type Culture Collection, deposited on June 14, 2023, accession number: CCTCC NO: M 20231019, address: Wuhan University, Wuhan, China, 430072, China.

2. A method for preparing the *Sphingomyelin-Bacillus* ZJPH2023031 according to claim 1 by resolving the racemic mixture of 2-chloropropionic acid (… 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 *Sphingomyelin-Bacillus* ZJPH2023031 obtained through fermentation as the enzyme source, 2-chloropropionic acid racemic mixture as the resolution substrate, and a buffer solution of pH 7.0-10.0 as the reaction medium, a transformation system is constructed. The reaction is carried out at 20-40℃ 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 buffer solution; the initial concentration of the racemic 2-chloropropionic acid is 50-120 mM based on the volume of buffer solution.

5. The application as described in claim 3, characterized in that, The reaction medium is a 0.1 M glycine-sodium hydroxide buffer solution with a pH of 9.

0.

6. The application as described in claim 3, characterized in that, The reaction was carried out at 30°C and 200 rpm for 24-36 h.

7. The application as described in any one of claims 3-6, characterized in that, The reaction medium is a 0.1 M glycine-sodium hydroxide buffer solution with a pH of 9.

0. The amount of wet bacterial cells used is 70 g / L based on the buffer volume. The initial concentration of the racemic 2-chloropropionic acid is 80-90 mM based on the buffer volume. The reaction conditions are 30°C and 200 rpm for 24 h.

8. The application as described in claim 3, characterized in that, The enzyme source is obtained as follows: (1) Plate culture: Sphingomyelin Bacillus ZJPH2023031 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 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 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 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. The fermentation medium consisted of: glucose 20 g / L, yeast extract 8 g / L, ammonium sulfate 20 g / L, sodium chloride 10 mM, and water as the solvent, pH 6.5.

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