CN117070405A - Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparation of (R) -2-chloropropionic acid - Google Patents

Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparation of (R) -2-chloropropionic acid Download PDF

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CN117070405A
CN117070405A CN202310995706.5A CN202310995706A CN117070405A CN 117070405 A CN117070405 A CN 117070405A CN 202310995706 A CN202310995706 A CN 202310995706A CN 117070405 A CN117070405 A CN 117070405A
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chloropropionic acid
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王普
宁盼
刘汉宇
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses a Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparing (R) -2-chloropropionic acid, wherein the strain can split an environment-friendly halide 2-chloropropionic acid racemate to obtain a thiolactam intermediate (R) -2-chloropropionic acid, and has the advantages of high concentration of a catalytic substrate, high optical purity of the obtained product and the like. The invention uses Pseudomonas taiwan ZJPH2023032 whole cell as a catalyst, and uses a biocatalysis method to split 2-chloropropionic acid raceme to prepare (R) -2-chloropropionic acid, when the substrate concentration is 80mM, the conversion rate of 2-chloropropionic acid reaches 49.9%, ee s The value is more than 99.9%, the complete resolution of the substrate can be realized, and when the concentration of the substrate is 90mM, the conversion rate of the 2-chloropropionic acid is 45.1%，ee s The value was 86.0%.

Description

Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparation of (R) -2-chloropropionic acid
Field of the art
The invention relates to a new microorganism strain, namely Pseudomonas taiwan ZJPH2023032 and application thereof in preparing a thiolamycin intermediate (R) -2-chloropropionic acid by biocatalytically splitting racemate 2-chloropropionic acid (2-CPA).
(II) background art
(R) -2-chloropropionic acid is used as an important medical intermediate and can be used for synthesizing the medicine thiolactam and the nutritional medicine L-alanyl-L-glutamine. Thiolactamycin (TLM) is a thiolactone antibiotic that can be activated by inhibition of β -ketoacyl-Acyl Carrier Protein (ACP) synthases (Kas), and has been reported to possess antipathogenic, antitubercular mycobacterial and antimalarial activities. The L-alanyl-L-glutamine has medicinal value of improving human immunity and improving intestinal health. Therefore, the preparation of the (R) -2-chloropropionic acid with a single configuration has important application value.
(R) -2-chloropropionic acid with molecular formula of C 3 H 5 ClO 2 The chemical structural formula is as follows:
the method for synthesizing (R) -2-chloropropionic acid mainly comprises a chemical method and a biological method, ethyl chloropropionate, formic acid and strong acid cation exchange resin are utilized, and the (R) -2-chloropropionic acid can be prepared and obtained through a transesterification method, wherein toxic and harmful organic reagents such as formic acid, pyridine and the like are needed to be used in the method. The biological process for preparing (R) -2-chloropropionic acid has the advantages of environment friendliness, mild reaction condition, high enantioselectivity and the like. Compared with enzyme catalysis, the whole cell catalysis method can omit the separation and purification process of the enzyme and is easy to operate. However, in the prior art, the report of preparing single-configuration (R) -2-CPA by utilizing whole-cell catalytic resolution of 2-CPA racemization is less, the catalytic efficiency is low, and the catalytic substrate concentration is only 30mM. The invention provides a novel microorganism strain with 2-halogenated acid dehalogenase activity, which can be used for preparing (R) -2-CPA with high selectivity by whole cell catalysis.
(III) summary of the invention
The invention aims to provide a novel microorganism strain, namely Pseudomonas taiwanensis (Pseudomonas taiwanensis) ZJPH2023032, and application of the strain in preparation of a thiolactam intermediate (R) -2-chloropropionic acid by biologically splitting 2-chloropropionic acid racemate, so that the catalytic efficiency is remarkably improved, and the problem of low substrate concentration of the 2-chloropropionic acid racemate split by the existing microorganism is solved.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain with 2-halogenated acid dehalogenase activity, namely Pseudomonas taiwanensis (Pseudomonas taiwanensis) ZJPH2023032, which is preserved in China center for type culture Collection, with a preservation date: 2023, 6, 14, deposit number: cctccc NO: m20231020, address: chinese, university of armed chinese, postal code, 430072.
The invention also provides an application of the pseudomonas taiwanensis ZJPH2023032 in preparing (R) -2-chloropropionic acid by resolving 2-chloropropionic acid raceme.
Further, the application method comprises the following steps: the preparation method comprises the steps of taking wet thalli obtained by fermenting and culturing Pseudomonas taiwanensis ZJPH2023032 as an enzyme source, taking a 2-chloropropionic acid racemate as a resolution substrate, taking phosphate buffer with the pH value of 6.0-8.0 as a reaction medium to form a conversion system, reacting at the temperature of 25-45 ℃ and the speed of 180-200rpm, obtaining a conversion solution containing (R) -2-chloropropionic acid after the reaction is finished, and separating and purifying to obtain a (R) -2-chloropropionic acid product.
Further, the separation and purification method of the (R) -2-chloropropionic acid-containing conversion solution comprises the following steps: extracting the conversion solution containing (R) -2-chloropropionic acid with ethyl acetate with equal volume, centrifuging, collecting ethyl acetate extract phase, and removing ethyl acetate solvent by rotary evaporation and concentration to obtain (R) -2-chloropropionic acid product.
Further, the amount of the wet thalli is 50-600g/L, preferably 200g/L, based on the volume of the buffer solution; the substrate is initially added at a concentration of 50 to 120mM (preferably 80 to 90 mM).
Further, the buffer 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), and most preferably dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with pH 7.5,0.1M.
Further, the reaction time is 6 to 48 hours, more preferably 35 ℃, and the reaction is carried out at 200rpm for 12 to 24 hours.
Further, the reaction medium is dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with pH of 7.5,0.1M, and the reaction condition is 35 ℃ and the reaction is carried out for 12 hours under the condition of 200 rpm; the amount of the wet bacterial cells is 200g/L in terms of buffer volume, and the initial concentration of the substrate is 80-90mM.
Further, the enzyme source is obtained as follows:
(1) Plate culture: inoculating Pseudomonas taiwanensis ZJPH2023032 on a plate culture medium, and culturing at 30 ℃ for 48-72h to obtain a plate culture strain; the composition of the plate culture medium is as follows: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 20g/L of agar and 6.5 pH of water as a solvent.
(2) Seed culture: and (3) picking a loop of the strain after the plate culture, inoculating the loop of the strain to a seed culture medium, and culturing for 12 hours at the temperature of 30 ℃ at 200rpm to obtain a seed culture solution. The seed culture medium consists of: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, water and pH 6.5 as solvent.
(3) Fermentation culture: inoculating the seed culture solution in the step (2) into a fermentation culture medium with an inoculum size of 10% of volume concentration, culturing for 24 hours at 200rpm and 30 ℃ to obtain a fermentation liquid, centrifuging the fermentation liquid at 9000rpm and 4 ℃ for 10 minutes, washing the obtained precipitate with 0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with pH of 7.0, and centrifuging again to obtain the wet thalli, namely enzyme source cells containing (R) -2-chloropropionic acid which can be used for resolving 2-chloropropionic acid raceme. The fermentation medium consists of: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, water and pH 6.5 as solvent.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a new strain with 2-halogenated acid dehalogenase activity, namely Pseudomonas taiwanensis ZJPH2023032, which is easy to culture and simple to prepare; the strain of the invention can split the halide 2-chloropropionic acid racemate polluting the environment to obtain the thiolactamycin intermediate (R) -2-chloropropionic acid, and has high catalytic substrate concentrationThe obtained product has the advantages of high optical purity and the like. The invention uses Pseudomonas taiwan ZJPH2023032 whole cell as a catalyst, and uses a biocatalysis method to split 2-chloropropionic acid raceme to prepare (R) -2-chloropropionic acid, when the substrate concentration is 80mM, the conversion rate of 2-chloropropionic acid reaches 49.9%, ee s The value is more than 99.9%, the complete resolution of the substrate can be realized, and when the concentration of the substrate is 90mM, the conversion rate of the 2-chloropropionic acid is 45.1%, ee s The value was 86.0%. Compared with the prior report level, the invention has the characteristics of high enantiomeric excess value of the product obtained by utilizing the catalysis of the wild strain, low cost, green conversion process, environmental friendliness and the like.
(IV) description of the drawings
FIG. 1, colony morphology of Pseudomonas taiwanensis ZJPH2023032.
FIG. 2, gram stain of Pseudomonas taiwan ZJPH2023032.
FIG. 3, phylogenetic tree of Pseudomonas taiwanensis ZJPH2023032.
FIG. 4, liquid phase assay of (R) -2-chloropropionic acid, (S) -2-chloropropionic acid standard.
FIG. 5, liquid phase detection spectra of (R) -2-chloropropionic acid conversion extract prepared by resolution of Pseudomonas taiwan ZJPH2023032 strain on 2-chloropropionic acid raceme.
(V) the specific embodiment:
the invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the culture medium used in the practice of the invention is as follows:
the liquid culture medium comprises the following components: disodium hydrogen phosphate dodecahydrate 3.2g/L, ammonium sulfate 0.5g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate heptahydrate 0.098g/L, yeast extract 0.5g/L, pH 7.0, and water as solvent.
The composition of the plate culture medium is as follows: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 20g/L of agar and 6.5 pH of water as a solvent.
The indicated plate medium composition was: 2g/L of monoammonium phosphate, 1g/L of dipotassium phosphate, 0.098g/L of anhydrous magnesium sulfate, 5g/L of sodium chloride, 0.08g/L of bromothymol blue, 20g/L of agar powder, water and pH 7.0 as solvent.
The seed culture medium consists of: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, water and pH 6.5 as solvent.
The fermentation medium consists of: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, water and pH 6.5 as solvent.
Example 1: bacterial screening with 2-haloacid dehalogenase activity
Strain sources: pseudomonas taiwanensis ZJPH2023032 is obtained by separating and screening soil samples containing chemical reagent pollutants around laboratories in the mountain school area of Zhejiang university industry Mo Gan, wherein the specific screening method is as follows:
in the process of strain primary screening, sodium dichloropropionate (2-CPANa) is used as the sole carbon source, enrichment culture of the convertible 2-CPA strain is carried out by gradually increasing the concentration of the 2-CPANa added into a liquid culture medium, and the 2-CPANa is prepared into 500mM 2-CPANa mother solution by using a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with pH of 7.0,0.1M and then added into the enrichment culture medium as required: weighing 10g of soil sample in 100mL of liquid culture medium, adding 2mL of 2-CPANa mother solution as a unique carbon source, and carrying out shake culture at 30 ℃ for 24h; then 20mL of the enriched culture solution is inoculated into another shake flask filled with the liquid culture medium, 7.7mL of 2-CPANa mother solution is added for secondary enrichment culture, shaking culture is carried out for 24 hours at 30 ℃,20 mL of the secondary enrichment culture solution is taken out, the secondary enrichment culture solution is added into the shake flask filled with the liquid culture medium, 13.3mL of 2-CPANa mother solution is added for continuous enrichment culture, and shaking culture is carried out for 24 hours at 30 ℃. Enrichment culture was repeated 3 times in total.
And (3) strain re-screening: adding 100 μl of enriched culture bacteria solution into EP tube on ultra-clean workbench, adding 900 μl of sterile water, and mixing thoroughly to obtain 10 -1 Diluting the bacterial liquid. Then take 100. Mu.L of 10 -1 Adding the diluted bacterial solution into another EP tube, adding 900 μl of sterile water, and mixing thoroughly to obtain 10 -2 Diluting the bacterial liquid; and so on, finallyDiluted to 10 by gradient -6 . The above dilutions (10) -2 、10 -3 、10 -4 、10 -5 、10 -6 ) 100 mu L of the strain is coated on an indication flat plate culture medium, and after the strain is cultured in a biochemical incubator at 30 ℃ for 24-48 hours, the color of a partial area on the flat plate is observed to change from green to yellow, which indicates that the strain is a strain with 2-haloacid dehalogenase activity. And (3) picking a single colony which is yellow from a flat plate, streaking and inoculating the single colony onto a flat plate culture medium to obtain a pure culture, gram staining and microscopic examination are carried out on the separated strain, and then the pure culture with different forms after microscopic examination is transferred into a seed culture medium and then subjected to glycerol pipe preservation.
1mL of the strain preserved by the glycerol pipe is inoculated into 100mL of seed culture medium, activated for 24 hours at 30 ℃ and 200rpm, and then the seed culture solution is inoculated into a fermentation culture medium with the inoculum size of 10% of the volume concentration, and is cultured for 24 hours at 200rpm and 30 ℃ to obtain the fermentation solution. The fermentation broth was centrifuged at 9000rpm at 4℃for 10min, and the resulting precipitate was washed with 0.1M potassium dihydrogen phosphate-potassium dihydrogen phosphate buffer having pH 7.0, and centrifuged again to obtain the wet cell. 500mg of wet cells were taken, 5mL of a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.0) was added, the cells were mixed with the buffer, 300. Mu.L of a 2-CPANa mother solution was added as a substrate for the conversion reaction, and the mixture was converted for 24 hours at 200rpm in a shaker at 30 ℃. After the reaction is finished, 150 mu L of concentrated hydrochloric acid with the mass concentration of 36% is added into a reaction system, acidized for 10min at 25 ℃, extracted for 30min by 5mL of ethyl acetate, supernatant containing (R) -2-chloropropionic acid is obtained after centrifugation, the supernatant is filtered by a 0.45 mu m filter head, the peak areas of unconverted substrate and product are detected by liquid chromatography, and the conversion rate and ee of the substrate are calculated s And (3) screening to obtain a dominant strain for preparing (R) -2-chloropropionic acid by resolving the raceme of the 2-chloropropionic acid, and marking the dominant strain as strain ZJPH2023032.
Liquid chromatography detection conditions: the chromatographic column isID (4.6 mM. Times.250 mM,5 μm) with n-hexane (V) as mobile phase: ethanol (V) =98:2, detection wavelength 254nm, flow rate 1mL/min, and sample injection amount 5 μl.
The substrate conversion rate calculation method comprises the following steps:
drawing a standard curve: a standard solution with the final concentration of 10 mM, 50mM, 100 mM, 150 mM and 200mM is prepared by preparing a 2-CPANa mother solution with the final concentration of 500mM by using a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution with the pH of 7.0,0.1M, liquid chromatography detection is carried out, and standard curves of (S) -2-chloropropionic acid ((S) -2-CPA) and (R) -2-chloropropionic acid ((R) -2-CPA) are respectively drawn. The standard curve equation of the (S) -2-chloropropionic acid is as follows: y= 1993.1x-3024.8 (R 2 =0.999), (R) -2-chloropropionic acid standard curve is: y=1679.3x+313.97 (R 2 ＝0.999)
The calculation formula of the conversion rate of the 2-chloropropionic acid is as follows:
conversion (%) = [1- (R) t +S t )/(R 0 +S 0 )]×100
R t Concentration of (R) -2-chloropropionic acid at the end of the reaction, S t At the end of the reaction, the concentration of (S) -2-chloropropionic acid, R 0 Is the initial concentration of (R) -2-chloropropionic acid, S 0 The optical purity of the substrate for the initial concentration of (S) -2-chloropropionic acid is determined by the enantiomeric excess value (ee s ) To characterize. The calculation formula is as follows:
S + and S-represents peak areas of (R) -2-chloropropionic acid and (S) -2-chloropropionic acid in the chiral liquid phase detection result substrate, respectively.
Example 2: identification of Strain ZJPH2023032
Morphological observation: inoculating the strain ZJPH2023032 to a plate culture medium, culturing at 30 ℃ for 48-72 hours, observing bacterial colonies and bacterial forms, wherein the bacterial colonies of the strain are yellow, round, neat in edge, smooth in surface and slightly convex in glossy center; the texture is soft and easy to pick. The results are shown in FIG. 1. The cell morphology was short bar-like and gram staining was negative, and the results are shown in FIG. 2.
Physiological and biochemical test: the strain ZJPH2023032 was subjected to VITEK physiological and biochemical property identification (see Table 1), and the physiological and biochemical characteristics thereof were consistent with those of Pseudomonas taiwan.
TABLE 1 physiological and biochemical characterization results
16S rRNA sequence analysis: the 16S rRNA gene sequence of the strain ZJPH2023032 is completed by Beijing qing department Biotechnology Co., ltd, the length of the 16S rRNA sequence of the strain ZJPH2023032 is 1456bp, and the specific nucleotide sequence is shown as SEQ ID NO. 1. The resulting strain 16S rRNA sequence (SEQ ID NO. 1) was subjected to homology alignment (BLAST) at NCBI (http:// www.ncbi.nlm.nih.gov), and a phylogenetic tree was established using MEGA11, which revealed that: the sequence homology of strain ZJPH2023032 to Pseudomonas taiwanensis DSW 21245 reaches 94%, and the result is shown in FIG. 3.
The strain ZJPH2023032 was identified as Pseudomonas taiwan (Pseudomonas taiwanensis) by combining morphological characteristics, physiological and biochemical characteristics and 16S rRNA sequence, and the date of preservation: 2023, 6, 14, deposit number: cctccc: m20231020, address: chinese, university of armed chinese, postal code, 430072.
SEQ ID NO.1
CACCCCAGTCATGAATCACACCGTGGTAACCGTCCTCCCGAAGGTTAGACTAGC
TACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCG
GGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCAC
GCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTGTGAGATTAGCT
CCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCC
CAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTC
ACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGG
GTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAG
CCATGCAGCACCTGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGT
TCTCTGCATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACA
TGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGC
CGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAAATCTCAAG
GATTCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATC
CTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGC
CTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATT
CCACCACCCTCTACCGTACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTG
AGCCCGGGGCTTTCACATCCAACTTAACGAACCACCTACGCGCGCTTTACGCCC
AGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAG
TTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACAGCAAGGTATTAGCTTACTG
CCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCG
GCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCC
GTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACC
AGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACC
TAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGAC
GTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTC
CTAGGCATTACTCACCCGTCCGCCGCTGAATCAAGGAGCAAGCTCCCGTCATCCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTC。
Example 3: preparation of Pseudomonas taiwanensis ZJPH2023032 wet cell
Plate culture: the Pseudomonas taiwanensis ZJPH2023032 is inoculated on a plate culture medium and cultured for 48-72 hours at the temperature of 30 ℃ to obtain a plate culture strain.
Seed culture: the strain after the plate culture is picked up and inoculated into a shake flask filled with a seed culture medium, and is cultured for 12 hours at the speed of 200rpm and the temperature of 30 ℃ to obtain a seed culture solution.
Fermentation culture: inoculating the seed culture solution into a fermentation culture medium with an inoculum size of 10% of volume concentration, culturing at 200rpm and 30 ℃ for 24 hours to obtain a fermentation broth, centrifuging the fermentation broth at 9000rpm and 4 ℃ for 10 minutes to obtain wet thalli, and obtaining the enzyme source cells for preparing (R) -2-chloropropionic acid by splitting 2-chloropropionic acid.
Example 4:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.0), the amount of the wet cells added was 100g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 30℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 34.0%, ee, as measured by the liquid phase detection method of example 1 s The value was 54.4%.
Example 5:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 100g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 30℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 38.0%, ee, as measured by the liquid phase detection method of example 1 s The value was 60.8%.
Example 6:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 8.0), the amount of the wet cells added was 100g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 30℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 36.1% and ee was measured by the liquid phase measurement method of example 1 s The value was 55.1%.
Example 7:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 200g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 30℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 40.1%, ee, as measured by the liquid phase detection method of example 1 s The value was 67.3%.
Example 8:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 300g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 30℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 37.4% and ee was measured by the liquid phase measurement method of example 1 s The value was 59.2%.
Example 9:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 200g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 35℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 45.1% and ee was measured by the liquid phase measurement method of example 1 s The value was 86.0%, and the liquid chromatogram was shown in FIG. 5.
Under the same conditions, when the substrate concentration was changed to 80mM, the reaction was carried out at 35℃and 200rpm for 24 hours, the conversion rate of 2-chloropropionic acid was 49.9%, and ee was measured s The value was 99.9%.
Example 10:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 200g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 40℃and 200rpm for 24 hours, and the conversion of 2-chloropropionic acid was 36.6% and ee was measured by the liquid phase measurement method of example 1 s The value was 56.7%.
Example 11:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogenphosphate-potassium dihydrogenphosphate buffer (0.1M, pH 7.5), the amount of the wet cells added was 200g/L based on the wet weight, 2-CPA as a substrate was added at a final concentration of 90mM, and the mixture was placed in a shaker at 35℃and 200rpm to be converted for 12 hours, and the conversion of 2-chloropropionic acid was 43.4% and ee was measured by the liquid phase measurement method of example 1 s The value was 79.0%.
Example 12:
the wet cells obtained in accordance with the method of example 3 were resuspended in 5mL of dipotassium hydrogen phosphate-phosphorusAdding wet thallus with wet weight of 200g/L into acid potassium dihydrogen buffer (0.1M, pH 7.5), adding 2-CPA with final concentration of 90mM as substrate, placing into shaking table at 25deg.C and 200rpm for conversion for 36 hr, detecting by liquid phase detection method in example 1, and converting 2-chloropropionic acid with conversion rate of 43.9%, ee s The value was 81.3%.
Example 13: investigation of the conversion Capacity of Pseudomonas putida ZJPH1412 to biocatalyse 2-CPA raceme preparation (R) -2-chloropropionic acid
(1) Pseudomonas putida (Pseudomonas putida) ZJPH1412, deposited with China center for type culture Collection, date of deposit: 2016, 4 months, 11 days, deposit number: CCTCC NO: M201687. This strain has been disclosed in applicant's prior patent application (publication number: CN106497843A publication date: 2019, 5, 31). The culture method of the strain and the preparation process of the enzyme source cells are according to the prior patent application (publication number: CN106497843A publication date: 2019, 5, 31).
(2) Biocatalytic preparation of (R) -2-chloropropionic acid from 2-CPA racemate
0.5g of Pseudomonas putida ZJPH1412 wet thallus obtained by fermentation is resuspended in 5mL of potassium phosphate buffer (0.1M, pH 7.0), the added amount of the wet thallus is 100g/L based on wet weight, 2-CPA with the final concentration of 90mM is added as a substrate, and the substrate is placed in a shaking table at 30 ℃ and 200rpm for conversion for 24 hours. Detection was performed using the liquid phase detection method in example 1.
Conclusion: pseudomonas putida ZJPH1412 cannot biocatalyse the preparation of (R) -2-chloropropionic acid from 2-chloropropionic acid racemate.
Example 14: investigation of the conversion Capacity of Acinetobacter ZJPH1806 biocatalysis for preparing (R) -2-chloropropionic acid from 2-CPA raceme
(1) Acinetobacter (Acinetobacter sp.) ZJPH1806, deposited in China center for type culture Collection, date of deposit: 3.29 days 2019, deposit number: cctccc NO:2019214. this strain has been disclosed in applicant's prior patent application (publication number: CN110016444B publication date: 2019, 7, 16).
(2) Biocatalytic preparation of (R) -2-chloropropionic acid from 2-CPA racemate
0.5g of the Acinetobacter ZJPH1806 wet thalli prepared by fermentation is resuspended in 5mL of potassium phosphate buffer solution (0.1M, pH 7.0), the addition amount of the wet thalli is 100g/L based on the wet weight, 2-CPA with the final concentration of 90mM is added as a substrate, and the substrate is placed in a shaking table at 30 ℃ and 200rpm for conversion for 24 hours. Detection was performed using the liquid phase detection method in example 1.
Conclusion: acinetobacter ZJPH1806 cannot biocatalytically prepare (R) -2-chloropropionic acid from 2-chloropropionic acid racemate.
Example 15: transformation capability investigation of 2-CPA raceme preparation (R) -2-chloropropionic acid by promoting root growth of Coxsackie bacteria ZJPH202011 biocatalysis
(1) Root growth promoting Cola's bacteria (Kosakonia radicincitans) ZJPH202011, deposited with China center for type culture Collection, date of deposit: 2021, 6, 11, deposit number: cctccc NO: m2021714. This strain has been disclosed in applicant's prior patent application (publication number: CN113462602A publication date: 2022, 5, 24 days).
(2) Biocatalytic preparation of (R) -2-chloropropionic acid from 2-CPA racemate
0.5g of the wet thalli of the root growth promoting Cola-type bacteria ZJPH202011 prepared by fermentation is resuspended in 5mL of potassium phosphate buffer (0.1M, pH 7.0), the addition of the wet thalli is 100g/L by wet weight, 2-CPA with the final concentration of 90mM is added as a substrate, and the substrate is placed in a shaking table at 30 ℃ and is transformed for 24 hours at 200 rpm. Detection was performed using the liquid phase detection method in example 1.
Conclusion: the root growth promoting Cola's bacteria ZJPH202011 cannot biocatalytically prepare the thiolactamycin intermediate (R) -2-chloropropionic acid from the 2-chloropropionic acid racemate.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (8)

1. Pseudomonas taiwanensis (Pseudomonas taiwanensis) ZJPH2023032, deposited with China center for type culture Collection, date of deposit: 2023, 6, 14, deposit number: cctccc NO: m20231020, address: chinese, university of armed chinese, postal code, 430072.
2. Use of pseudomonas taiwanensis ZJPH2023032 according to claim 1 for the resolution of 2-chloropropionic acid racemate for the preparation of (R) -2-chloropropionic acid.
3. The application according to claim 2, wherein the method of application is: the preparation method comprises the steps of taking wet thalli obtained by fermenting and culturing Pseudomonas taiwanensis ZJPH2023032 as an enzyme source, taking a 2-chloropropionic acid racemate as a resolution substrate, taking phosphate buffer with the pH value of 6.0-8.0 as a reaction medium to form a conversion system, reacting at the temperature of 25-45 ℃ and the speed of 180-200rpm, obtaining a conversion solution containing (R) -2-chloropropionic acid after the reaction is finished, and separating and purifying to obtain a (R) -2-chloropropionic acid product.
4. The use according to claim 3, wherein the amount of wet cells is 50-600g/L by volume of buffer; the initial addition concentration of the substrate is 50-120 mM.
5. The use according to claim 3, wherein the reaction medium is a dipotassium hydrogenphosphate-potassium dihydrogenphosphate buffer having a pH of 7.5 and 0.1M.
6. The use according to claim 3, wherein the reaction is carried out at 35℃and 200rpm for 12-24 hours.
7. Use according to one of claims 3 to 6, characterized in that the reaction medium is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer having a pH of 7.5 and 0.1M, the reaction conditions being 35 ℃,200rpm for 12 hours; the amount of the wet bacterial cells is 200g/L in terms of buffer volume, and the initial concentration of the substrate is 80-90mM.
8. The use according to claim 3, wherein the enzyme source is obtained by:
(1) Plate culture: inoculating Pseudomonas taiwanensis ZJPH2023032 on a plate culture medium, and culturing at 30 ℃ for 48-72h to obtain a plate culture strain; the composition of the plate culture medium is as follows: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 20g/L of agar, water as a solvent and pH 6.5;
(2) Seed culture: selecting a loop of strain after plate culture, inoculating the loop of strain to a seed culture medium, and culturing for 12 hours at 200rpm and 30 ℃ to obtain a seed culture solution; the seed culture medium consists of: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride and 0.5g/L of magnesium sulfate heptahydrate, wherein the solvent is water, and the pH value is 6.5;
(3) Fermentation culture: inoculating the seed culture solution in the step (2) into a fermentation culture medium with an inoculum size of 10% of volume concentration, culturing for 24 hours at 200rpm and 30 ℃ to obtain a fermentation liquid, centrifuging the fermentation liquid at 9000rpm and 4 ℃ for 10min, washing the obtained precipitate with 0.1M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with pH of 7.0, and centrifuging again to obtain the wet thalli, wherein the fermentation culture medium comprises the following components: 15g/L of glucose, 20g/L of fish meal peptone, 10g/L of yeast extract, 2g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 1g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, water and pH 6.5 as solvent.
CN202310995706.5A 2023-08-08 2023-08-08 Pseudomonas taiwanensis ZJPH2023032 and application thereof in preparation of (R) -2-chloropropionic acid Pending CN117070405A (en)

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