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Method for preparing tauroursodeoxycholic acid by two-step method

A technology for tauroursodeoxycholic acid and tauroursodeoxycholic acid is applied in the field of preparing tauroursodeoxycholic acid by a two-step method, which can solve the problems of many reaction steps, high price, complicated preparation and the like, and achieves the The effect of short reaction time, saving raw material cost and simplifying process

Pending Publication Date: 2022-06-07
北京岳达生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0006] ③ After ursodeoxycholic acid and ethyl chloroformate form mixed anhydride, they condense with p-hydroxypropiophenone to obtain the active phenolic ester of ursodeoxycholic acid, which is then reacted with taurine to prepare TUDCA with a total yield of about 64%. , this method has more reaction steps
[0007] Comparing the three synthetic methods, the latter two require EEDQ and DEPC, which are expensive and cumbersome to prepare
[0008] Taurochenodeoxycholic acid (TCDCA) is widely present in the bile of poultry such as chickens, ducks, geese, etc. People use more to prepare chenodeoxycholic acid by hydrolyzing these bile, and prepare TUDCA by using bound TCDCA There are not many reports on the method

Method used

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  • Method for preparing tauroursodeoxycholic acid by two-step method
  • Method for preparing tauroursodeoxycholic acid by two-step method
  • Method for preparing tauroursodeoxycholic acid by two-step method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Chemical oxidation of sodium hypochlorite by TCDCA to generate taurine 7-ketolithocholic acid (T-7K)

[0065] Take 300g of TCDCA (98%) and add 300mL of methanol, after dissolving, add 100g of phosphoric acid, dropwise add 300mL of sodium hypochlorite aqueous solution (available chlorine 12%), and conduct the reaction under temperature control, and the reaction process temperature does not exceed 5 °C. After the dropwise addition, the TCDCA residue was detected by liquid phase, and the TCDCA residue was determined by liquid chromatography. The TCDCA residue was 0.43%, less than 1.0%. The reaction was completed. After adding 6.0 g of anhydrous sodium sulfite and stirring for 30 min, slowly add 300 mL of water dropwise, filter and place it in. Dry in a vacuum drying oven at 75°C for 10h to obtain the product T-7K, weigh 292g, and determine the purity of 95.76%.

[0066] figure 2 is the MS spectrum of taurine 7-ketolithocholic acid (T-7K); image 3 is the 1H-NM...

Embodiment 2

[0067] Example 2 Preparation and expression of tauroursodeoxycholidase-reducing engineered bacteria.

[0068] 1. Whole-gene synthesis of codon-optimized 7β-HSDH gene and GDH gene

[0069] 1.7β-HSDH and GDH genes are 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase, respectively

[0070] 2.7β-HSDH is derived from: Clostridium absonum (GenBank accession number: JN191345.1)

[0071] GDH source: Bacillus subtilis (GenBank accession number NC-000964)

[0072] 3. The above sequence was entrusted to Sangon Bio (Shanghai) Co., Ltd. to optimize the gene sequence according to the codon preference of Escherichia coli, and to add EcoRⅤ and XhoI enzymes to the 5' and 3' ends of the 7β-HSDH gene sequence, respectively. The cleavage site was added to the 5' and 3' ends of the GDH gene sequence, respectively, with EcoRI and HindIII restriction sites, and the whole gene was synthesized. The gene sequence of the obtained codon-optimized 7β-HSDH is shown in SEQ ID NO.1, and the gene ...

Embodiment 3

[0136] Example 3 Whole cell enzyme-catalyzed preparation of tauroursodeoxycholic acid.

[0137] Take 250 g of T-7k obtained in Example 1, add 1150 mL of purified water, add 137.5 g of dextrose monohydrate, stir well, and after the temperature reaches 25 ° C, add 50 g of the bacterial cells prepared in Example 2 (mixed with 100 mL of purified water). After suspending evenly), add NADP + After 0.25g, the reaction was started, the rotation speed was 400rpm, and the reaction pH was adjusted between 6.5 and 8.0 with 1M sodium hydroxide during the reaction. The reaction process was monitored by TLC plate, and samples were taken every 2 hours for monitoring. After 8 hours of reaction, liquid chromatography was used to determine that the residual T-7k was 0.43%, less than 1.0%. The reaction was completed, and the reaction solution of tauroursodeoxycholic acid was obtained.

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Abstract

The invention discloses a method for preparing tauroursodeoxycholic acid by a two-step method. Comprising the following steps: 1, carrying out chemical oxidation reaction on taurochenodeoxycholic acid (TCDCA) to obtain tauro7-ketolithocholic acid (T-7K); and 2, carrying out whole-cell catalytic reduction on the tauroursodeoxycholic acid (TUDCA) prepared from the tauro7-ketolithocholic acid (T-7K) obtained in the step 1 through coexpression of 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) and glucose dehydrogenase (GDH), so as to obtain the tauroursodeoxycholic acid (TUDCA). According to the method, TCDCA with a wide source is mainly utilized, tauro7-ketolithocholic acid (T-7K) is firstly produced through chemical oxidation, then the tauro7-ketolithocholic acid is converted into tauroursodeoxycholic acid by utilizing 7beta-steroid dehydrogenase (7beta-HSDH), and meanwhile, NADP < + > is cyclically regenerated by co-expressed glucose dehydrogenase in the presence of glucose. According to the method, the two zymoproteins are co-expressed, whole-cell catalysis is carried out in the catalysis process, high activity is guaranteed, meanwhile, the zymoproteins are better in stability, and high-yield and high-purity preparation of tauroursodeoxycholic acid can be achieved.

Description

technical field [0001] The present invention relates to a method for preparing tauroursodeoxycholic acid, in particular to a method for preparing tauroursodeoxycholic acid (TUDCA) by a two-step method, namely tauroursodeoxycholic acid (TCDCA) from natural sources. First through chemical oxidation, tauro 7-ketolithocholic acid (T7K) is generated, and then tauro 7-ketolithocholic acid is converted to tauroursodeoxycholic acid (TUDCA) by co-expression in Escherichia coli. The method belongs to the technical field of chemical synthesis. Background technique [0002] Tauroursodeoxycholic acid (TUDCA), chemical name 2-[[(3α,5β,7β)-3,7-dihydroxy-24-oxocholestan-24-yl]amino]ethanesulfonic acid Acid dihydrate is a conjugated bile acid formed by shrinking between the carboxyl group of ursodeoxycholic acid (UDCA) and the amino group of taurine. In 1902, TUDCA was discovered from bear bile, which is the main bile acid in bear bile, and has the functions of antispasmodic, anticonvulsan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/06C12P33/00C12N15/70C12N15/53C12R1/19
CPCC12P33/06C12P33/00C12N9/0006C12N15/70C12Y101/01119C12Y101/01201C12N2800/22Y02A50/30
Inventor 张天王力边朝阳薛童朱丽惠
Owner 北京岳达生物科技有限公司
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