CN113462602A

CN113462602A - Corksaiella radicata ZJPH202011 and application thereof

Info

Publication number: CN113462602A
Application number: CN202110776859.1A
Authority: CN
Inventors: 王普; 孔祥鑫; 桂铅; 于双嵘
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2021-10-01
Anticipated expiration: 2041-07-09
Also published as: CN113462602B

Abstract

The invention provides a new strain for catalyzing asymmetric reduction of 4' -bromo-2, 2, 2-trifluoroacetophenone to prepare (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol for rhizogenic coxsackiella ZJPH202011 and application thereof, and the strain is adopted to catalyze and prepare the (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol and has the advantages of high concentration of catalytic substrate, high optical purity of product and the like. Compared with the existing report level, the method has the advantages that the enantiomeric excess value of the product obtained by catalysis of the wild type strain is high, the e.e. value is larger than 90.66%, the microbial strain is easy to culture and rich in source, no expensive coenzyme is needed to be added in the conversion process, and the method has the characteristics of low cost, green conversion process, environment-friendliness and the like. The invention reduces 4' -bromo-2, 2, 2-trifluoro acetophenone by adopting Corksaiella radicata (Kosakonia radicitans) ZJPH202011 cells as a catalyst, and when the concentration of a substrate is 30mM, the e.e. value of the target product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol is more than 93.82 percent, and the yield reaches 75.54 percent.

Description

Corksaiella radicata ZJPH202011 and application thereof

(I) technical field

The invention relates to a new microbial strain, namely rhizogenic Kosakonia radicinacis ZJPH202011, and application thereof in catalyzing asymmetric reduction of 4' -bromo-2, 2, 2-trifluoro acetophenone to prepare (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol.

(II) background of the invention

The chemical structural formula of the (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol is as follows:

(R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol is an important intermediate for synthesizing tryptophan hydroxylase (TPH) inhibitors and JANUS kinase (JAK) inhibitors. Among them, TPH inhibitors regulate peripheral serotonin levels by blocking the synthesis of 5-hydroxytryptamine, and are useful for alleviating nausea and vomiting caused by chemotherapy and treating irritable bowel syndrome, which represents LX1031 as a drug. As an oral drug, LX1031 is a TPH inhibitor acting locally, which can improve symptoms of abdominal pain, spasm, constipation, diarrhea, etc. in patients, and is an effective drug for treating irritable bowel syndrome and other gastrointestinal diseases.

The JAK inhibitor can block a JAK/STAT passage, further influence the division, proliferation and differentiation and immune regulation of cells, can treat rheumatoid arthritis, partial cancers and blood system diseases, and represents Odanacatib. Odanacatinb is a selective and reversible inhibitor of cathepsin k (catk), a cysteine protease abundantly expressed in osteoclasts, and is mainly used for the treatment of osteoporosis. Although the drug is stopped being developed because of the increased risk of stroke found in clinical studies, the search for new use of the drug is still ongoing, and Jon J. Vermeire et al found that Odanacatib can reduce the number of internal helminthes by 73% and simultaneously reduce the activity of cysteine protease in parasites by 51% in the process of searching for a drug for treating hookworm disease, which provides new possibility for developing low-dose drugs and substitutes for treating hookworm disease. In addition, in the OVAA patent, the Odanacatib structure is used as a mother nucleus for structural modification, and a plurality of CatK inhibitors are developed, wherein CatK4 and CatK5 show stronger physiological activities.

Disclosure of the invention

The invention aims to provide a new microbial strain, namely rhizogenic Kosakonia radicinacis ZJPH202011, and application of the strain in catalyzing asymmetric reduction of 4' -bromo-2, 2, 2-trifluoroacetophenone to prepare (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol. Compared with the traditional chemical method, the novel method for preparing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol by utilizing the complete cell catalysis of the rhizogenic coxsackiella ZJPH202011 has the characteristics of mild reaction conditions, environmental friendliness, low cost and the like, and compared with enzyme catalysis, the novel method does not need to add expensive auxiliary factors and has higher catalytic yield.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a new strain, namely rhizogenic Kosakonia radicinacitans ZJPH202011, which is preserved in China center for type culture Collection with the preservation date of 2021, 6 and 11 days, and the preservation number is as follows: CCTCC NO: M2021714, address: china, wuhan university, 430072.

In addition, the invention also provides application of the rhizogenic coxsackiella ZJPH202011 in preparing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol by catalyzing asymmetric reduction of 4' -bromo-2, 2, 2-trifluoroacetophenone.

Further, the application is as follows: taking wet thalli obtained by fermenting and culturing rhizogenic Coxsackia ZJPH202011 as an enzyme source cell, taking 4' -bromo-2, 2, 2-trifluoroacetophenone as a substrate, adding an auxiliary substrate, taking a phosphate buffer solution with the pH of 6.0-8.0 as a reaction medium to form a conversion system, reacting at the temperature of 25-37 ℃ and the speed of 150 plus 250rpm (preferably at the temperature of 30 ℃ and the speed of 200rpm), and obtaining a conversion solution containing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol after the reaction is finished; the cosubstrate is one or more of the following: glucose, sucrose, maltose, glycerol, isopropanol, methanol, lysine, tyrosine, glutamine.

Further, the conversion solution containing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was isolated and purified as follows: extracting the conversion solution containing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol with ethyl acetate of the same volume, centrifuging, collecting ethyl acetate extract phase, and concentrating by a rotary evaporator to remove the ethyl acetate solvent to obtain a crude extract of the product; silica gel soaked by petroleum ether is filled in a chromatographic column to prepare the silica gel chromatographic column, and then the petroleum ether: the silica gel column was equilibrated with an eluent of ethyl acetate 8:1(v/v), and the crude extract of the obtained product was applied to a silica gel column, followed by washing with petroleum ether: and (2) eluting with ethyl acetate (8: 1 (v/v)) as an eluent, collecting and combining eluents containing the target product, and evaporating to dryness by using a rotary evaporator to obtain the (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol product.

Further, the dosage of the substrate is 2-10g/L (preferably 2.5-7.6 g/L) based on the volume of the phosphate buffer solution, and the dosage of the wet cells is 100-600g/L (preferably 100-500 g/L) based on the volume of the phosphate buffer solution; the dosage of the auxiliary substrate is 30-200g/L (preferably 50-150 g/L) based on the volume of the phosphate buffer solution.

Preferably, the co-substrate is glycerol, most preferably in an amount of 600g/L based on the volume of the phosphate buffer.

Further, the phosphate buffer is preferably a potassium phosphate buffer or a sodium phosphate buffer, and most preferably a potassium phosphate buffer.

Further, the wet cells of the present invention are obtained by the following method:

1) slant culture: selecting single colony of the root-promoting Corksaiella ZJPH202011 to inoculate into a first slant culture medium, culturing for 24h at 30-37 ℃, selecting colony growing on the first slant culture medium to inoculate into a second slant culture medium again, culturing for 24h at 30-37 ℃ to obtain slant strain after slant culture twice (storing the slant strain in a refrigerator at 4 ℃); the final concentration composition of each component in the first slant culture medium and the second slant culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 2g/L， NaCl 1g/L，MgSO₄0.244g/L of agar, 20g/L of agar, water as a solvent and pH 6.5;

2) seed culture: slant culture twice from the slant culture obtained in step 1)Taking a full ring of the surface strain, inoculating the surface strain into a seed culture medium, culturing at 25-37 ℃ and 150-250rpm for 10-15 h (preferably culturing at 30 ℃ and 200rpm for 12h) to obtain a seed solution; the final concentration composition of each component in the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄·7H₂O is 0.5g/L, the solvent is water, and the pH value is 6.5;

3) fermentation culture: inoculating the seed liquid into a fermentation culture medium at an inoculation amount of 6-15% (preferably 10%) by volume concentration, and performing fermentation culture at 150-250rpm at 25-37 ℃ for 24-32h (preferably at 30 ℃ and 200rpm for 24 h); after the fermentation culture is finished, the obtained fermentation liquor is centrifuged, and the obtained precipitate is treated with K with the pH of 7.0 and the M of 0.1₂HPO₄-KH₂PO₄Washing with buffer solution, and centrifuging again to obtain the wet thallus, namely the enzyme source cell containing the substrate 4' -bromo-2, 2, 2-trifluoro acetophenone bioreduction capable of being catalyzed; the final concentration of each component in the fermentation medium is as follows: sucrose 25g/L, (NH)₄)₂SO₄ 5g/L，K₂HPO₄2g/L, solvent is water, and pH is 6.5.

The analytical detection method of the transformation liquid comprises the following steps: after the reaction is finished, terminating the reaction of the conversion solution by using ethyl acetate with the same volume (containing the internal standard tetradecane), extracting for 30min by oscillation, standing, taking the upper extraction liquid, analyzing and detecting by adopting a gas chromatography, and finally calculating to obtain the yield of the target product and the enantiomeric excess value (e.e. value).

Compared with the prior art, the invention has the following beneficial effects: the invention provides a new strain for catalyzing asymmetric reduction of 4' -bromo-2, 2, 2-trifluoro acetophenone to prepare (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol, and the strain is adopted to catalyze and prepare the (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol, and has the advantages of high concentration of catalytic substrate, high optical purity of product and the like. Compared with the existing reported level, the method has the advantages that the enantiomeric excess value of the product obtained by catalysis of the wild type strain is high, the e.e. value is greater than 90.66%, the microbial strain is easy to culture and rich in source, no expensive coenzyme is required to be added in the conversion process, and the method has the characteristics of low cost, green conversion process, environment-friendliness and the like. The invention reduces 4' -bromo-2, 2, 2-trifluoro acetophenone by using Kosakonia radicitans ZJPH202011 cells as a catalyst, and when the concentration of a substrate is 30mM, the e.e. value of the target product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol is more than 93.82 percent, and the yield reaches 75.54 percent.

(IV) description of the drawings

FIG. 1 shows a gas chromatography chromatogram of a substrate, 4' -bromo-2, 2, 2-trifluoroacetophenone, and products, namely (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol and (S) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol (with an internal standard, tetradecane).

FIG. 2 is a gas phase detection chromatogram (containing internal standard tetradecane) of asymmetric reduction reaction extract of a rhizogenic Kosakholderia ZJPH202011 strain.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in the examples of the present invention, the substrate and the co-substrate are measured in buffer volumes.

Example 1: strain screening and identification

1. The strain source is as follows: the rhizogenic Kosakechu (Kosakonia radicinatins) ZJPH202011 is obtained by separating and screening corn field soil samples from Fuwu town of Fenyang city of Shanxi province, and the specific screening method is as follows:

primary screening: 2g of the soil sample was weighed and added to a 250mL Erlenmeyer flask containing 100mL of physiological saline with a mass concentration of 0.9%, shaken at 30 ℃ and 200rpm for 30min, and then 2mL of the soil sample suspension was added to a 250mL Erlenmeyer flask containing 100mL of seed medium, and cultured at 30 ℃ and 200rpm for 24 h. After the culture solution becomes turbid, transferring 2mL of the bacterial solution to a 250mL conical flask filled with 100mL of enrichment medium for culture for 3-5 days. The enrichment medium takes 4' -bromo-2, 2, 2-trifluoro acetophenone as a unique carbon source, and the final concentration composition is as follows: 10mM of 4' -bromo-2, 2, 2-trifluoro acetophenone, (NH)₄)₂SO₄ 2.0g/L，KH₂PO₄ 2.0g/L，NaCl 1.0g/L，MgSO₄0.244g/L, solvent is water, pH 6.5.

Re-screening: the enrichment culture solution obtained by the steps is diluted to 10 degrees by physiological saline in a gradient way^-5、10^-6Then, absorbing 100-; after the colonies grow out, selecting colonies with different forms, streaking and inoculating the colonies onto a separation plate, and standing and culturing at 30 ℃ for 1-3 d; selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 30 ℃ and 200rpm for 12 hours to obtain a seed solution; then, the seed solution was inoculated into a fermentation medium at a volume concentration of 10%, and cultured at 30 ℃ and 200rpm for 24 hours. And after the fermentation is finished, centrifuging the fermentation liquor at 8000rpm for 10min to obtain wet thalli, namely the enzyme-containing source cells.

And (3) carrying out a biotransformation reaction process: 100g/L of cells containing the enzyme source, 10mM of 4' -bromo-2, 2, 2-trifluoroacetophenone (by volume of buffer), 100g/L of glucose (co-substrate) were added to a ground flask containing 10mL of phosphate buffer (0.1M, pH 7.0) and the conversion product was obtained after bioconversion at 30 ℃ and 200rpm for 24 h.

The final concentration of each component in the seeds and the fermentation medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄0.244g/L, solvent is water, pH 6.5. The plate medium was isolated to constitute the enrichment medium, and 20g/L agar was added. The nutrient agar plate culture medium comprises the same seed culture medium, and 20g/L agar is added.

And after the conversion reaction is finished, extracting the conversion solution by using ethyl acetate containing the internal standard tetradecane in the same volume, centrifuging, taking the upper extraction liquid for analysis and detection, and measuring the yield and the enantiomeric excess value (e.e. value) of the target product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol in the conversion solution by adopting chiral gas chromatography.

The gas chromatogram of the substrate 4' -bromo-2, 2, 2-trifluoroacetophenone, the product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol and the (S) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol standard (containing the internal standard tetradecane) is shown in FIG. 1. The microbial strain ZJPH202011 with higher enantiomeric excess value (e.e. value > 90.66%) and higher yield is obtained by strain screening, and a gas chromatogram (containing an internal standard tetradecane) of a reaction extract obtained by utilizing the strain after biological reduction is shown in figure 2.

Gas chromatography conditions: agilent 7820A gas chromatograph, Varian CP-Chirasil-Dex CB capillary gas chromatography column (25 m. times.0.25 mm. times.0.25 μm), FID detector. The carrier gas is high-purity nitrogen, the flow is 2mL/min, the injection port temperature and the detector temperature are both 250 ℃, and the temperature rise program is as follows: keeping the temperature at 142 ℃ for 0min, increasing the temperature to 152 ℃ at 1 ℃/min, keeping the temperature for 0min, and carrying out sample injection at 1 mu L with the split ratio of 15: 1.

Gas chromatography analysis and detection: and detecting the concentration of the product after the conversion reaction and the concentration of the residual substrate, and calculating the conversion rate of the substrate, the yield of the product and the e.e. value.

The yield calculation method comprises the following steps:

drawing a standard curve: preparing substrate and product standard solutions with final concentration of 50mM, diluting to obtain solutions with concentrations of 10, 15, 20, 25, 30 and 40mM respectively, and detecting by gas chromatography respectively. And integrating the obtained chromatogram to obtain a peak area, drawing by taking the ratio of the peak area of the substrate or the product to the peak area of the tetradecane as a horizontal coordinate, and taking the ratio of the concentration of the substrate or the product to the concentration of the tetradecane as a vertical coordinate, performing linear regression fitting, and drawing a standard curve. Obtaining a substrate standard curve equation: 2.1833x +0.0395, R²0.9983; product standard curve equation: 1.767x +0.1912, R²＝0.9991。

The product yield calculation formula is as follows:

yield (%) ═ C_p/C₀X 100% of C in the formula 1_pThe concentration of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol after the reaction was completed, C₀Is the initial concentration of 4' -bromo-2, 2, 2-trifluoroacetophenone.

The optical purity of the product is characterized by enantiomeric excess (e.e.). The calculation formula is as follows:

in the formula C_RAnd C_SAre respectively R-configuration and S-Configurational 1- (4-bromophenyl) -2,2, 2-trifluoroethanol molar concentration.

2. The culture characteristics of the strain ZJPH202011 are as follows:

the morphological characteristics of the screened strain ZJPH202011 are as follows: the basic characteristics of the bacterial colony appearance, color, growth speed and the like of the strain ZJPH202011 are observed on a nutrient agar plate. The bacterial colony on the nutrient agar plate is yellow and round, grows fast, has a raised center, is neat in the edge and smooth in the surface, and is turbid in liquid culture; and observing the cell morphology of the dyed microorganisms by using an optical microscope, wherein a single thallus is in a short rod shape.

Molecular biological identification: 16S rDNA sequencing of the strain ZJPH202011 was performed by Venezo Biotechnology engineering (Shanghai) Ltd. Using the extracted total DNA of the cells as a template, 16S rDNA of the strain was amplified using universal primers NL1 and NL4, and the PCR product was subjected to 1% agarose gel electrophoresis. The sequence of the 16S rDNA gene of the strain ZJPH202011 is confirmed to be shown in SEQ ID NO.1 by sequencing. SEQ ID NO. 1: CTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCAC AGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAA ACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACG TCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAG ATGGGATTAGCTAGTAGGCGGGGTAATGGCCCACCTAGGCGACGATCCCTAGCT GGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTA CGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCA TGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGG AAGGCGGTACGGTTAATAACCGTGCTGATTGACGTTACCCGCAGAAGAAGCACC GGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGG AATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCC CCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTCGTAGA GGGAGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATAC CGGTGGCGAAGGCGGCCTCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGT GGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGAT TTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACC GCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCG CACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTG GTCTTGACATCCACAGAACCTGGCAGAGATGCCGGGGTGCCTTCGGGAACTGT GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAG TCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACT CAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCA TCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAG AAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATT GGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGAATCAGA ATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGG GAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCAC TTTGTGATTCATGACTGGGGTGAAGTCGT

The 16S rDNA sequence (SEQ ID NO: 1) of strain ZJPH202011 was subjected to homology alignment (BLAST) at NCBI website (http:// www.ncbi.nlm.nih.gov), and the results showed that: the sequence homology of strain ZJPH202011 with Kosakonia radiancinnans strain DSM 107547 (GenBank accession No. CP040392.1) was 93%.

The strain ZJPH202011 is identified as kojikucheng kosaxiella (Kosakonia radicinatins) named as kojikucheng kojikuchen kojijun (Kosakonia radicitans) ZJPH202011 according to the morphological characteristics of the strain ZJPH202011 and combined with molecular biology identification, and is preserved in China center for type culture collection with the preservation date of 2021, 6 and 11 days, and the preservation number is: CCTCC NO: m2021714, deposit address: china, wuhan university, 430072.

Example 2: obtaining of Wet cells

The slant culture medium comprises: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L， KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄0.244g/L of agar, 20g/L of agar, water as a solvent and pH 6.5;

the seed culture medium comprises the following components: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L， KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄·7H₂O is 0.5g/L, the solvent is water, and the pH value is 6.5;

the fermentation medium comprises the following components: sucrose 25g/L, (NH)₄)₂SO₄ 5g/L，KH₂PO₄2g/L, solvent is water, and pH is 6.5.

1) Slant culture: selecting single colony of the rhizogenic Kosakholderia ZJPH202011, inoculating the single colony into a slant culture medium, culturing at 30 ℃ for 24h, selecting single colony on the slant culture medium, inoculating and culturing for 24h, repeating the steps twice, and storing the obtained slant in a refrigerator at 4 ℃;

2) seed culture: picking a loop of slant strain, inoculating the slant strain into 100mL of seed culture medium, and culturing at 30 ℃ and 200rpm for 12h to obtain a seed solution;

3) fermentation culture: 10mL of the seed solution was inoculated into 100mL of a fermentation medium at an inoculum size of 10% by volume, and the medium was incubated at 30 ℃ and 200rpm in a 100mL/250mL Erlenmeyer flask for 24 hours. After the fermentation is finished, the fermentation liquor is centrifuged, and the obtained precipitate is treated with K of 0.1M and pH 7.0₂HPO₄-KH₂PO₄Washing with buffer solution, centrifuging to obtain wet thallus, i.e. enzyme source cell containing 4' -bromo-2, 2, 2-trifluoro acetophenone for catalyzing biological reduction of substrate.

Example 3:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 100g/L buffer; adding 20mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 100g/L glucose by volume of buffer solution as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The analytical test was carried out by the test method of example 1, and the results were as follows: the concentration of the product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was 9.23mM, the concentration of the (S) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was 0.37mM, the residual concentration of the substrate 4 '-bromo-2, 2, 2-trifluoroacetophenone was 9.26mM, and the above-mentioned detection results were calculated to obtain an e.e. value of the product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol of 92.26%, a yield of 46.14%, and a conversion rate of the substrate 4' -bromo-2, 2, 2-trifluoroacetophenone of 53.70%.

Example 4:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 100g/L buffer; adding 20mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 10% (v/v) isopropanol by volume of buffer solution as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 93.55% and a yield of 43.36% by analytical detection using the detection method of example 1.

Example 5:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 100g/L buffer; adding 20mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 50g/L glycerol by volume of buffer solution as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 95.03% and a yield of 50.64% as determined by analysis using the assay method of example 1.

Example 6:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.1M, pH 7.0) at a wet cell loading of 100g/L buffer; adding 20mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 600g/L glycerol by volume of buffer solution as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 94.47% and a yield of 74.53% by analytical examination using the test method of example 1.

Example 7:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.1M, pH 6.5) at a wet cell loading of 100g/L buffer; adding 20mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 600g/L glycerol by volume of buffer solution as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 95.46% and a yield of 49.42% by analytical detection using the detection method of example 1.

Example 8:

the wet cells obtained according to the method of example 2 were resuspended in 10mL of potassium phosphate buffer (0.05M, pH 7.0) at a wet cell loading of 100g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoroacetophenone in an amount of 20mM based on the volume of the buffer, adding glycerol in an amount of 600g/L based on the volume of the buffer as an auxiliary substrate, and converting in a shaker at 30 ℃ and 200rpm for 24 hours. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 93.77% and a yield of 68.98% by analytical detection using the detection method of example 1.

Example 9:

the wet cells obtained in example 2 were resuspended in 100mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 100g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoroacetophenone in an amount of 20mM based on the volume of the buffer, adding glycerol in an amount of 600g/L based on the volume of the buffer as an auxiliary substrate, and converting in a shaker at 30 ℃ and 200rpm for 24 hours. The product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to have an e.e. value of 94.81% and a yield of 74.89% using the assay procedure of example 1.

Example 10:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 100g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoro acetophenone in 20mM buffer volume, adding glycerol in 600g/L buffer volume as auxiliary substrate, and reacting in a shaker at 37 deg.C and 200rpm for 24 h. The e.e. value of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to be 94.95% and the yield was 78.52% by analytical examination using the examination method of example 1.

Example 11:

the wet cells obtained in example 2 were resuspended in 10mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 50g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoro acetophenone in 20mM buffer volume, adding glycerol in 600g/L buffer volume as auxiliary substrate, and reacting in a shaker at 37 deg.C and 200rpm for 24 h. The e.e. value of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was 96.63% by analytical examination using the examination method of example 1, and the yield was 52.42%.

Example 12:

the wet cells obtained according to the method of example 2 were resuspended in 10mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 150g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoro acetophenone in 20mM buffer volume, adding glycerol in 600g/L buffer volume as auxiliary substrate, and reacting in a shaker at 37 deg.C and 200rpm for 24 h. The e.e. value of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to be 94.12% and the yield was 76.81% by analytical examination using the examination method of example 1.

Example 13:

the wet cells obtained according to the method of example 2 were resuspended in 10mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 150g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoro acetophenone in 30mM buffer volume, adding glycerol in 600g/L buffer volume as auxiliary substrate, and reacting in a shaker at 37 deg.C and 200rpm for 19 h. The e.e. value of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to be 93.91% and the yield was 68.71% by analytical detection using the detection method of example 1.

Example 14:

the wet cells obtained according to the method of example 2 were resuspended in 10mL of potassium phosphate buffer (0.2M, pH 7.0) at a wet cell loading of 150g/L buffer; adding substrate 4' -bromo-2, 2, 2-trifluoro acetophenone in 30mM buffer volume, adding glycerol in 600g/L buffer volume as auxiliary substrate, and reacting in a shaker at 37 deg.C and 200rpm for 24 h. The e.e. value of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol was calculated to be 93.82% and the yield was 75.54% by analytical examination using the examination method of example 1.

Example 15:

1) selecting Enterobacter cloacae (Enterobacter cloacae) ZJPH1903, and storing in China center for type culture Collection with the address: china, wuhan university, 430072, accession number: CCTCC NO: m2019821, preservation date of 2019, 10 months and 14 days. This strain has been disclosed in a prior patent application (publication No. CN110982757A, published: 2020, 4/10). The culture method of the strain and the preparation process of the enzyme-containing cells are in accordance with the prior patent application (publication No. CN110982757A, published as: 2020, 4/10).

2) The wet cells cultured as described above were resuspended in 10mL of phosphate buffer (0.1M, pH 7.0) at a wet weight concentration of 100g/L buffer; adding 10mM 4' -bromo-2, 2, 2-trifluoro acetophenone by volume of buffer solution as substrate, adding 100g/L glucose as auxiliary substrate, and reacting in a shaker at 30 deg.C and 200rpm for 24 h. The assay of example 1 was used to determine and calculate the e.e. value and yield of the product (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol.

And (4) conclusion: enterobacter cloacae (Enterobacter cloacae) ZJPH1903 transformed 4' -bromo-2, 2, 2-trifluoroacetophenone into (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol with an e.e. value of 13.77% in 55.44% yield.

Example 16:

1) rhizopus microsporum fibrous variant (Rhizopus microsporus var. Rhizopus) ZJPH1308 was selected and deposited in the chinese type culture collection with the deposit number: CCTCC NO: m2014645, date of deposit 2014, 12 months and 14 days, address: wuhan, Wuhan university, 430072, China. This strain has been disclosed in a prior patent application (publication No.: CN104893989A, published: 2015, 9/9). The culture method of the strain and the preparation process of the cells containing the enzyme source are in accordance with the prior patent application (publication No. CN104893989A, published: 2015, 9/9).

And (4) conclusion: rhizopus microsporus var. rhizomorpha ZJPH1308 could not transform 4' -bromo-2, 2, 2-trifluoroacetophenone to produce (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol.

Example 17:

1) rhodococcus erythropolis XS1012 is selected and preserved in China center for type culture Collection, the preservation date is 2013, 12 and 11 days, and the preservation address is as follows: china, wuhan university, 430072, accession number: CCTCC NO: m2013650. This species has been disclosed in a prior patent application (publication No. CN103773724A, published: 5/7/2014). The culture method of the bacterial strain and the preparation process of the enzyme-containing source cell were carried out according to the prior patent application (publication No. CN103773724A, published: 2014, 5 and 7).

And (4) conclusion: transformation of 4' -bromo-2, 2, 2-trifluoroacetophenone to (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol by Rhodococcus erythropolis (XS) 1012 gave an e.e. value of 41.36% with a yield of 63.30%.

Sequence listing

<110> Zhejiang industrial university

<120> rhizogenic Coxsackia ZJPH202011 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1474

<212> DNA

<213> Gekkaibachia radicicola (Kosakonia radicinatins)

<400> 1

ctcagattga acgctggcgg caggcctaac acatgcaagt cgaacggtag cacagagagc 60

ttgctctcgg gtgacgagtg gcggacgggt gagtaatgtc tgggaaactg cctgatggag 120

ggggataact actggaaacg gtagctaata ccgcataacg tcgcaagacc aaagaggggg 180

accttcgggc ctcttgccat cagatgtgcc cagatgggat tagctagtag gcggggtaat 240

ggcccaccta ggcgacgatc cctagctggt ctgagaggat gaccagccac actggaactg 300

agacacggtc cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag 360

cctgatgcag ccatgccgcg tgtgtgaaga aggccttcgg gttgtaaagc actttcagcg 420

gggaggaagg cggtacggtt aataaccgtg ctgattgacg ttacccgcag aagaagcacc 480

ggctaactcc gtgccagcag ccgcggtaat acggagggtg caagcgttaa tcggaattac 540

tgggcgtaaa gcgcacgcag gcggtctgtc aagtcggatg tgaaatcccc gggctcaacc 600

tgggaactgc attcgaaact ggcaggctgg agtctcgtag agggaggtag aattccaggt 660

gtagcggtga aatgcgtaga gatctggagg aataccggtg gcgaaggcgg cctcctggac 720

gaagactgac gctcaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780

ccacgccgta aacgatgtcg atttggaggt tgtgcccttg aggcgtggct tccggagcta 840

acgcgttaaa tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaattgac 900

gggggcccgc acaagcggtg gagcatgtgg tttaattcga tgcaacgcga agaaccttac 960

ctggtcttga catccacaga acctggcaga gatgccgggg tgccttcggg aactgtgaga 1020

caggtgctgc atggctgtcg tcagctcgtg ttgtgaaatg ttgggttaag tcccgcaacg 1080

agcgcaaccc ttatcctttg ttgccagcgg tccggccggg aactcaaagg agactgccag 1140

tgataaactg gaggaaggtg gggatgacgt caagtcatca tggcccttac gaccagggct 1200

acacacgtgc tacaatggcg catacaaaga gaagcgacct cgcgagagca agcggacctc 1260

ataaagtgcg tcgtagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc 1320

tagtaatcgt gaatcagaat gtcacggtga atacgttccc gggccttgta cacaccgccc 1380

gtcacaccat gggagtgggt tgcaaaagaa gtaggtagct taaccttcgg gagggcgctt 1440

accactttgt gattcatgac tggggtgaag tcgt 1474

Claims

1. The rhizogenic Coxsackia ZJPH202011 is preserved in China center for type culture Collection with the preservation date of 2021, 6 months and 11 days, and the preservation numbers are as follows: CCTCC NO: M2021714, address: china, wuhan university, 430072.

2. The use of the koxsackia rhizogenes ZJPH202011 as claimed in claim 1 for the preparation of (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol by asymmetric reduction of 4' -bromo-2, 2, 2-trifluoroacetophenone.

3. The use according to claim 2, characterized in that said use is: taking wet thalli obtained by fermenting and culturing rhizogenic Kosakholderia ZJPH202011 as enzyme source cells, taking 4' -bromo-2, 2, 2-trifluoroacetophenone as a substrate, adding an auxiliary substrate, taking a phosphate buffer solution with the pH of 6.0-8.0 as a reaction medium to form a conversion system, reacting at the temperature of 25-37 ℃ and the speed of 150 plus 250rpm, and obtaining a conversion solution containing (R) -1- (4-bromophenyl) -2,2, 2-trifluoroethanol after the reaction is finished; the cosubstrate is one or more of the following: glucose, sucrose, maltose, glycerol, isopropanol, methanol, lysine, tyrosine, glutamine.

4. Use according to claim 3, characterized in that: the dosage of the substrate is 2-10g/L based on the volume of the phosphate buffer solution.

5. Use according to claim 3, characterized in that: the dosage of the wet thallus is 600g/L based on the volume of the phosphate buffer solution.

6. Use according to claim 3, characterized in that: the dosage of the auxiliary substrate is 30-200g/L based on the volume of the phosphate buffer solution.

7. Use according to claim 3, characterized in that: the auxiliary substrate is glycerol, and the dosage of the glycerol is 600g/L based on the volume of the phosphate buffer solution.

8. Use according to claim 3, characterized in that: the phosphate buffer is potassium phosphate buffer or sodium phosphate buffer.

9. The use of claim 8, wherein: the phosphate buffer is potassium phosphate buffer.

10. The use according to claim 2, wherein the wet biomass is obtained by:

1) slant culture: selecting a single colony of the rhizogenic Kosakholderia ZJPH202011 to inoculate into a first slant culture medium, culturing for 24h at 30-37 ℃, selecting a colony growing on the first slant culture medium to inoculate into a second slant culture medium again, and culturing for 24h at 30-37 ℃ to obtain slant strains after slant culture twice; the final concentration composition of each component in the first slant culture medium and the second slant culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄0.244g/L of agar, 20g/L of agar, water as a solvent and pH 6.5;

2) seed culture: selecting a full ring from the slant strains obtained in the step 1) after slant culture twice, inoculating the full ring into a seed culture medium, and culturing at 25-37 ℃ and 250rpm for 10-15 h to obtain a seed solution; the final concentration composition of each component in the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)₄)₂SO₄ 2g/L，KH₂PO₄ 2g/L，NaCl 1g/L，MgSO₄·7H₂O is 0.5g/L, the solvent is water, and the pH value is 6.5;

3) fermentation culture: inoculating the seed liquid into a fermentation culture medium by an inoculation amount with the volume concentration of 6-15%, and carrying out fermentation culture at the temperature of 25-37 ℃ and the rotation speed of 150-250rpm for 24-32 h; after the fermentation culture is finished, the obtained fermentation liquor is centrifuged, and the obtained precipitate is treated with K with the pH of 7.0 and the M of 0.1₂HPO₄-KH₂PO₄Washing with a buffer solution, and centrifuging again to obtain the wet thalli; the final concentration of each component in the fermentation medium is as follows: sucrose 25g/L, ammonium sulfate 5g/L, K₂HPO₄2g/L, solvent is water, and pH is 6.5.