CN114134067A - Escherichia coli and application thereof - Google Patents
Escherichia coli and application thereof Download PDFInfo
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- CN114134067A CN114134067A CN202111216464.2A CN202111216464A CN114134067A CN 114134067 A CN114134067 A CN 114134067A CN 202111216464 A CN202111216464 A CN 202111216464A CN 114134067 A CN114134067 A CN 114134067A
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- 241000588724 Escherichia coli Species 0.000 title claims abstract description 22
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000004380 Cholic acid Substances 0.000 claims abstract description 28
- 229960002471 cholic acid Drugs 0.000 claims abstract description 28
- 235000019416 cholic acid Nutrition 0.000 claims abstract description 28
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims abstract description 28
- MIHNUBCEFJLAGN-UHFFFAOYSA-N 12-oxo-chenodeoxycholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(=O)C2 MIHNUBCEFJLAGN-UHFFFAOYSA-N 0.000 claims abstract description 11
- MIHNUBCEFJLAGN-DMMBONCOSA-N 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholanic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)C(=O)C1 MIHNUBCEFJLAGN-DMMBONCOSA-N 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 238000011081 inoculation Methods 0.000 claims abstract description 4
- 238000011068 loading method Methods 0.000 claims abstract description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 25
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 4
- 125000003716 cholic acid group Chemical group 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 13
- 239000000047 product Substances 0.000 description 11
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 10
- 229960001661 ursodiol Drugs 0.000 description 10
- 239000007795 chemical reaction product Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 3
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003613 bile acid Substances 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 108010073927 12-alpha-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- OHXPGWPVLFPUSM-KLRNGDHRSA-N 3,7,12-trioxo-5beta-cholanic acid Chemical compound C1CC(=O)C[C@H]2CC(=O)[C@H]3[C@@H]4CC[C@H]([C@@H](CCC(O)=O)C)[C@@]4(C)C(=O)C[C@@H]3[C@]21C OHXPGWPVLFPUSM-KLRNGDHRSA-N 0.000 description 1
- 102100039358 3-hydroxyacyl-CoA dehydrogenase type-2 Human genes 0.000 description 1
- 108010032887 7 beta-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 108010014831 7-alpha-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000006856 Wolf-Kishner-Huang Minlon reduction reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 229960001701 chloroform Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229960002997 dehydrocholic acid Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006345 epimerization reaction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
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Abstract
The invention relates to the field of microorganism application, and particularly discloses escherichia coli and application thereof. The Escherichia coli is characterized in that: escherichia coli DH5 alpha purchased from Biotechnology engineering (Shanghai) Co., Ltd is used as an original strain, and is inoculated into LB culture medium according to one thousandth of inoculation amount, the bottle loading amount is 100mL/500mL, the strain is placed into a shaking table, and the strain is cultured for 24h at 37 ℃ and 200rpm, so that Escherichia coli is obtained. The invention provides Escherichia coli DH5 alpha, which can oxidize hydroxyl at 12-position of cholic acid to synthesize 12-oxo-chenodeoxycholic acid, and the conversion rate can reach 100%.
Description
Technical Field
The invention relates to the field of microorganism application, in particular to escherichia coli and application thereof.
Background
Ursodeoxycholic Acid (Ursoxycholic Acid), chemical name 3 alpha, 7 beta-dihydroxy-5 beta-cholestane-24-Acid. Ursodeoxycholic acid is a rare medicinal material, and can promote secretion of endogenous bile acid and reduce reabsorption; antagonize the cytotoxic action of hydrophobic bile acid and protect the liver cell membrane; it can dissolve cholesterol calculus, has immunoregulation effect, and is a common medicine for protecting liver and resisting inflammation. The market of ursodeoxycholic acid in China is about 8.5 hundred million in 2015, but 95% of hospital markets are imported products, the market share of Germany Fuke large pharmaceutical factory is the largest, and the sale amount of ursodeoxycholic acid (the trade name is YouSefu) in China reaches 6.6 hundred million RMB in 2015. Therefore, it is very necessary to provide a method for preparing ursodeoxycholic acid efficiently and simply.
In recent years, biological methods have become a new synthetic method for ursodeoxycholic acid in view of the low efficiency of chemical synthesis and the serious environmental pollution, and cholic acid, chenodeoxycholic acid, and dehydrocholic acid have also attracted attention as substrates for synthesizing ursodeoxycholic acid. Among them, the synthesis of ursodeoxycholic acid using cholic acid as a substrate is the most inexpensive method. The synthetic scheme of using cholic acid as a substrate to synthesize ursodeoxycholic acid is that hydroxyl at 12 position of cholic acid is oxidized, then carbonyl oxygen at 12 position is removed through Huang Minlon reaction to generate chenodeoxycholic acid, and then 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase are used to perform epimerization on hydroxyl at 7 position, and finally the epimer of the chenodeoxycholic acid, namely the ursodeoxycholic acid, is obtained.
The current biological method for synthesizing 12-oxo-chenodeoxycholic acid (12-oxo-CDCA) by oxidizing 12-hydroxy of cholic acid is to utilize 12 alpha-hydroxysteroid dehydrogenase to oxidize 12-hydroxy of cholic acid so as to synthesize 12-oxo-chenodeoxycholic acid.
Coli is one of the most widely used exogenous gene expression systems. Coli DH5 α is one of the most commonly used host bacteria in genetic engineering due to its high transformation rate. The Escherichia coli used in the invention can oxidize cholic acid to synthesize 12-oxo-chenodeoxycholic acid.
Disclosure of Invention
The invention provides escherichia coli and application thereof in order to make up for the defects of the prior art.
The invention is realized by the following technical scheme:
an escherichia coli characterized by: escherichia coli DH5 alpha purchased from Biotechnology engineering (Shanghai) Co., Ltd is used as an original strain, and is inoculated into LB culture medium according to one thousandth of inoculation amount, the bottle loading amount is 100mL/500mL, the strain is placed into a shaking table, and the strain is cultured for 24h at 37 ℃ and 200rpm, so that Escherichia coli is obtained.
The invention also discloses application of the escherichia coli in conversion of cholic acid into 12-oxo-chenodeoxycholic acid.
Preferably, the Escherichia coli converts cholic acid into 12-oxo-chenodeoxycholic acid at pH6.0-8.0 and temperature 30-37 deg.C.
More preferably, when the amount of the cholic acid is 10g/L, it takes 2.5 hours to complete the cholic acid conversion reaction in 3g of Escherichia coli DH 5. alpha. cells.
The invention provides Escherichia coli DH5 alpha, which can oxidize hydroxyl at 12-position of cholic acid to synthesize 12-oxo-chenodeoxycholic acid, and the conversion rate can reach 100%.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a thin layer chromatography assay of the reaction product of the present invention;
FIG. 2 is an evaporative light detection profile of the reaction product of the present invention;
FIG. 3 is a mass spectrometry detection profile of a reaction product of the present invention;
FIG. 4 is a NMR spectroscopy analysis chart of the reaction product of the present invention.
In FIG. 1, CA is cholic acid control, and 1-6 are samples at different reaction times.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to specific examples to facilitate understanding of the present invention, but the present invention is not limited thereto.
The culture medium of the invention is as follows:
LB culture medium: tryptone 1%, yeast powder 0.5%, sodium chloride 1%, pH is natural.
The material sources in the following examples are:
coli DH5 α was purchased from Biotechnology engineering (Shanghai) Ltd.
Example 1: preparation of crude enzyme solution of Escherichia coli DH5 alpha
Inoculating Escherichia coli DH5 alpha into LB culture medium in one thousandth of inoculation amount, bottling in 100ml/500ml, placing into a shaking table, culturing at 37 ℃ and 200rpm for 24 hours, centrifuging the culture solution at 4000rpm, collecting cell precipitate, and washing with normal saline to obtain resting cells.
1g of the resting cells obtained as described above were suspended in 40ml of a phosphate buffer (100 mM, pH 8.0), and disrupted by ultrasonication in an ice-water bath to give a crude enzyme solution of Escherichia coli DH 5. alpha.
Example 2: escherichia coli DH5 alpha for catalyzing conversion reaction with cholic acid as substrate
In a 500ml Erlenmeyer flask, 20ml of phosphate buffer (100 mM, pH 8.0), 1mM cholic acid, 1mM NAD +, and crude enzyme solution of Escherichia coli DH 5. alpha. obtained in example 1 were sequentially added. The reaction was carried out at 37 ℃ and 150rpm, and the reaction was terminated after sampling and examining intermittently for 1.5 hours.
Adding anhydrous ethanol with the volume twice that of the reaction solution for extraction, and removing precipitates; and carrying out suction filtration on the supernatant for three times to obtain a clear ethanol solution. And (4) carrying out rotary evaporation at 65 ℃ by using a rotary evaporator to obtain a concentrated solution. Adding three times of ethyl acetate into the ethanol concentrated solution for extraction, and carrying out rotary evaporation and concentration at 65 ℃ to obtain a product which is a white crystal, namely a reaction product.
Example 3: identification of products of E.coli DH5 alpha catalytic conversion reaction
(1) Detection of product by thin layer chromatography
A small amount of the product of example 2 was dissolved in ethyl acetate and spotted. Performing qualitative analysis by thin layer chromatographyThe components of the starter are as follows: trichloromethane: acetone: glacial acetic acid =1.2ml:0.6ml:30 μ L. The color developing agent is prepared by Mncl2 Concentrated H2SO4Methanol =0.2g, 17ml, 60 ml.
Preparing cholic acid standard substances for qualitative reference, spotting 2 μ (diameter not more than 4 mm) at a distance of 1cm from the bottom edge of the chromatography plate, spreading in a chromatography tank, taking out when the developing agent is 1cm away from the upper edge of the chromatography plate, developing in a developer, and blow-drying. Then placing the mixture at 105 ℃ for baking. The results are shown in FIG. 1.
(2) Evaporative light detection of the product
A small sample of the product of example 2 was taken and dissolved in ethyl acetate and the time of the reaction product peak was determined by evaporation of light. The results are shown in FIG. 2.
(3) Mass spectrometry detection of products
A small amount of the product sample obtained in example 2 was dissolved in ethyl acetate, and the reaction product was detected by mass spectrometry for peak time. The results are shown in FIG. 3.
(4) Detection of products by nuclear magnetic resonance spectroscopy
A small amount of the product sample obtained in example 2 was dissolved in ethyl acetate, and the molecular weight of the reaction product was determined by NMR spectroscopy. The results are shown in FIG. 4.
Example 4: the conditions for synthesizing 12-oxo-chenodeoxycholic acid by catalyzing cholic acid with Escherichia coli DH5 alpha are optimized
1g of the resting cells obtained in example 1 were suspended in 40ml of a phosphate buffer (100 mM, pH 8.0) and disrupted by sonication in an ice-water bath to give a crude enzyme solution of Escherichia coli DH 5. alpha. 20ml of phosphate buffer (100 mM, pH 8.0), 1-8g/L cholic acid, 1mM NAD +, and crude enzyme solution of Escherichia coli DH 5. alpha. were sequentially added to a 500ml Erlenmeyer flask. Reacting at 37 ℃ and 150rpm, intermittently sampling and detecting, reacting for 2 hours, and completely reacting cholic acid, namely, the conversion rate is 100%.
When the cholic acid in the reaction system is 10g/L, the cholic acid reaction needs 3 hours completely.
When the cholic acid amount is 10g/L, the bacterial amount is increased to 3g of bacterial cells, and the cholic acid reaction takes 2.5 hours completely.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (4)
1. An escherichia coli characterized by: escherichia coli DH5 alpha purchased from Biotechnology engineering (Shanghai) Co., Ltd is used as an original strain, and is inoculated into LB culture medium according to one thousandth of inoculation amount, the bottle loading amount is 100mL/500mL, the strain is placed into a shaking table, and the strain is cultured for 24h at 37 ℃ and 200rpm, so that Escherichia coli is obtained.
2. Use of E.coli as claimed in claim 1 for the conversion of cholic acid to 12-oxo-chenodeoxycholic acid.
3. Use according to claim 2, characterized in that: the Escherichia coli converts cholic acid into 12-oxo-chenodeoxycholic acid at pH6.0-8.0 and temperature 30-37 deg.C.
4. Use according to claim 3, characterized in that: when the amount of the cholic acid is 10g/L, the cholic acid conversion reaction takes 2.5 hours for 3g of Escherichia coli DH5 alpha cells.
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