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Preparation method of tauroursodeoxycholic acid

A technology of tauroursodeoxycholic acid and conversion method, which is applied in the field of preparation of tauroursodeoxycholic acid

Pending Publication Date: 2017-10-24
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] TCDCA is a bile acid compound widely present in the bile of chickens, ducks, geese and other poultry and livestock. Using TCDCA as a raw material and directly using genetically modified Escherichia coli as a transformed strain, the method for preparing TUDCA has not been reported so far. and can achieve rational utilization of resources

Method used

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  • Preparation method of tauroursodeoxycholic acid
  • Preparation method of tauroursodeoxycholic acid
  • Preparation method of tauroursodeoxycholic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Whole-gene synthesis of codon-optimized 7α-HSDH and 7β-HSDH genes

[0065] 7α-HSDH and 7β-HSDH are 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, respectively;

[0066] The two 7α-HSDHs were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Escherichia coli strain TW14359, GenBank accession number: CU928163.2),

[0067] Two 7β-HSDH genes were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Ruminococcus gnavus strain N53, GenBank accession number: KF052988.1),

[0068] The above sequence was entrusted to Yingwei Jieji (Shanghai) Trading Co., Ltd. to use GeneArt software to optimize the codon of the gene sequence according to the codon preference of Escherichia coli, and to carry out the whole gene synthesis, which are respectively recorded as α1, α2, β1, and β2.

[0069] The codon-optimized 7α-HSDH derived from Clostridium sardiniense (denoted as α 1 ) gene...

Embodiment 2

[0071] Construction of engineered bacteria

[0072] 1) Expanded culture using pMD as a carrier with a synthetic target gene α 1 、α 2 , β 1 , β 2 coli, and DH5α-pETM6 strain, take 10μl sample, add 10ml of LB (Amp + ) medium, cultivated on a shaker at 37° C. for 12 to 16 hours, and the shaker speed was 225 rpm.

[0073] 2) Use the plasmid extraction kit purchased from Shanghai Chuangying Biotechnology Co., Ltd. to extract the plasmids of the above bacteria, and operate according to the operation instructions of the kit.

[0074] 3) pMD-α obtained by double enzyme digestion and extraction 1 , pMD-α 2 , pMD-β 1 , pMD-β 2 Plasmid, and expression vector pETM6, the enzyme digestion system is as follows:

[0075]

[0076] Digest at 37°C for 2 to 4 hours, and purify the target fragment with a gel recovery kit.

[0077] 4) The recovered 850bp target gene fragment α 1 、α 2 , β 1 , β 2 , respectively, and the 5.6kb pETM6 carrier fragment, the connection system is as follo...

Embodiment 3

[0101] Cultivation of engineering bacteria and transformation of substrates

[0102] 1) Inoculate the engineered bacteria into LB solid plate medium, activate and cultivate at 37°C for 12 hours;

[0103] 2) Pick a single colony from the LB solid plate medium and inoculate it in LB (Amp + ) liquid culture medium in the Erlenmeyer flask, at 225rpm / min, 37 ℃ shaking culture for 14 hours;

[0104] 3) The above culture was inoculated into liquid M9 medium at a ratio of 1:50, cultured with shaking at 225rpm / min and 37°C for 3.5 hours, and 1mM isopropyl-β-D-thiogalactopyranoside ( IPTG), continue to cultivate for 3 hours;

[0105] 4) Centrifuge the above culture at 5000g / min, collect the bacteria, and concentrate 15 times into a new M9 medium supplemented with 100mg / L Amp+, 1mM IPTG, and 8mM TCDCA. Shake culture at 225 rpm and 37° C. for 2 days to carry out fermentative transformation of the substrate.

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Abstract

The invention relates to a preparation method of tauroursodeoxycholic acid and belongs to the technical field of biology. The preparation method comprises the following steps: firstly, carrying out liquid fermentation on escherichia coli which simultaneously expresses 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) and 7beta-hydroxy steroid dehydrogenase (7beta-HSDH) genes; secondly, directly converting taurochenodeoxycholic acid into the tauroursodeoxycholic acid. The preparation method has the advantages of stability, controllability, high conversion rate, simplicity, quickness, mild conditions and zero pollution; the taurochenodeoxycholic acid is derived from chicken bile, goose bile or duck bile, is easy to obtain and is of great significance to theoretical research and extensive utilization of medicinal value of the tauroursodeoxycholic acid.

Description

technical field [0001] The present invention relates to a kind of preparation method of tauroursodeoxycholic acid, specifically, it relates to a kind of in situ conversion of tauroursodeoxycholic acid (TCDCA) into taurine by genetically modified non-pathogenic microorganism. A method for sulfursodeoxycholic acid (TUDCA) belongs to the field of biotechnology. Background technique [0002] Use exogenous natural or synthetic organic compounds as substrates, add them to the growing biological system or enzyme system, and cultivate them under suitable conditions, so that the substrate interacts with the enzymes in the biological system to produce Structural changes, a process called biotransformation. Biotransformation uses a variety of enzyme systems with different catalytic functions to transform the chemical components of traditional Chinese medicines, which can produce new natural compounds, or improve the water solubility of active ingredients in traditional Chinese medicin...

Claims

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Application Information

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IPC IPC(8): C12P41/00C12P33/00C12N15/70C12R1/19
CPCC12N9/0006C12P33/00C12P41/00C12Y101/01159C12Y101/01201
Inventor 赵淑娟王峥涛杨莉史杰周吉燕胡之璧张金家
Owner SHANGHAI UNIV OF T C M
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