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Method for synthesizing ursodeoxycholic acid through enzymatic method

A technology of ursodeoxycholic acid and chenodeoxycholic acid, which is applied in the field of enzymatically catalyzing chenodeoxycholic acid to synthesize ursodeoxycholic acid, can solve the problem of low conversion rate of chenodeoxycholic acid, complicated post-treatment, Environmental pollution and other problems, to achieve the effect of easy control of industrial scale-up production, simple extraction and refining, and high selectivity

Inactive Publication Date: 2018-07-06
ZHONGSHAN BAILING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, cholic acid and chenodeoxycholic acid are mainly used as raw materials to prepare ursodeoxycholic acid through multi-step chemical reactions at home and abroad. A large amount of solvents, oxidants and reducing agents are used in the reaction process, which causes serious environmental pollution; The conversion rate of oxycholic acid is low, and the post-treatment is complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Add 10 g of chenodeoxycholic acid and 100 ml of 50 mMPH9.0 phosphate buffer into the reaction flask, slowly add 10% sodium hydroxide to adjust the pH of the solution to between 7.5 and 8.0, and the chenodeoxycholic acid is completely dissolved. Add 10 mg of NAD, 0.15 g of NOX, and 30 mg of 7-α steroid dehydrogenase, feed oxygen, control the flow rate of oxygen to 5 ml / min, and react at room temperature until the residual CDCA is less than 1.0%. After the reaction is complete, stop feeding oxygen, raise the temperature to 50-55 degrees and maintain the temperature and stir for 20 minutes.

[0024] Cool the material to room temperature, add 4.5 g of L-sodium malate, 0.18 g of malate dehydrogenase, and 15 mg of 7-β steroid dehydrogenase to the reaction solution, and control the reaction between pH 7.0-7.5 at 25-30 degrees. Until the residual 7-ketolithocholic acid is lower than 0.1%. After the reaction is completed, add 10% sodium hydroxide solution to adjust the pH of th...

Embodiment 2

[0026] Add 10 g of chenodeoxycholic acid and 100 ml of 50 mMPH9.0 phosphate buffer into the reaction flask, slowly add 10% sodium hydroxide to adjust the pH of the solution to between 7.5 and 8.0, and the chenodeoxycholic acid is completely dissolved. Add 15 mg of NAD, 0.20 g of NOX, and 33 mg of 7-α steroid dehydrogenase, feed oxygen, control the flow rate of oxygen to 5.5 ml / min, and react at room temperature until the residual CDCA is less than 1.0%. After the reaction is complete, stop feeding oxygen, raise the temperature to 50-55 degrees and maintain the temperature and stir for 30 minutes.

[0027] Cool the material to room temperature, add 5.0 g of L-sodium malate, 0.18 g of malate dehydrogenase, and 20 mg of 7-β steroid dehydrogenase to the reaction solution, and control the reaction between pH 7.0-7.5 at 25-30 degrees. Until the residual 7-ketolithocholic acid is lower than 0.1%. After the reaction is completed, add 10% sodium hydroxide solution to adjust the pH of ...

Embodiment 3

[0029] Add 10 g of chenodeoxycholic acid and 100 ml of 50 mMPH9.0 phosphate buffer into the reaction flask, slowly add 10% sodium hydroxide to adjust the pH of the solution to between 7.5 and 8.0, and the chenodeoxycholic acid is completely dissolved. Add NAD18mg, NOX0.25g, 7-alpha steroid dehydrogenase 35mg, feed oxygen, control the oxygen flow rate to be 6.0ml / min, and react at room temperature until CDCA residual is less than 1.0%. After the reaction is complete, stop feeding oxygen, raise the temperature to 50-55 degrees and maintain the temperature and stir for 25 minutes.

[0030] Cool the material to room temperature, add 5.5 g of L-sodium malate, 0.25 g of malate dehydrogenase, and 25 mg of 7-β steroid dehydrogenase to the reaction solution, and control the reaction between pH 7.0-7.5 at 25-30 degrees. Until the residual 7-ketolithocholic acid is lower than 0.1%. After the reaction is completed, add 10% sodium hydroxide solution to adjust the pH of the solution above ...

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Abstract

The invention discloses a method for synthesizing ursodeoxycholic acid through an enzymatic method and a method for synthesizing the ursodeoxycholic acid by taking chenodeoxycholic acid as a raw material. The method takes the chenodeoxycholic acid as a base material and comprises the following steps: dissolving the chenodeoxycholic acid into a 50mM phosphate buffer solution; firstly, catalyticallyoxidizing the chenodeoxycholic acid by utilizing 7-alpha hydroxysteroid dehydrogenase in the presence of NAD, NOX2 and under the condition that oxygen is introduced, so as to obtain 7-ketolithocholicacid; then under the condition that the NAD, L-malic acid and malic dehydrogenase exist, catalytically reducing the 7-ketolithocholic acid by utilizing the 7-alpha hydroxysteroid dehydrogenase to obtain the ursodeoxycholic acid. According to the method disclosed by the invention, an organic solvent is not used and operation is simple; reaction conditions are moderate and easy to control and the utilization rate of the raw materials is high; the conversion rate reaches 99 percent or more.

Description

technical field [0001] The invention provides an enzymatic method for catalyzing chenodeoxycholic acid to synthesize ursodeoxycholic acid. The invention belongs to the technical field of bioengineering. Background technique [0002] The chemical name of ursodeoxycholic acid is: 3α, 7β-dihydroxy-5β-cholanic acid, which has the same molecular formula as chenodeoxycholic acid, but different three-dimensional structures. Chemically, the structural relationship between these two compounds is called isomerism Construct. Ursodeoxycholic acid is a white crystalline powder, odorless and bitter. It is easily soluble in ethanol, chloroform, glacial acetic acid, dilute lye, slightly soluble in ether, hardly soluble in water and dilute mineral acid, with a melting point of 203°C. Ursodeoxycholic acid UDCA is a non-toxic hydrophilic cholic acid, which can competitively inhibit the absorption of toxic endogenous cholic acid in the ileum, by activating the signaling network composed of c...

Claims

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Application Information

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IPC IPC(8): C12P33/02
CPCC12P33/02
Inventor 秦和平张和平钟义华祝国祥刘良伏
Owner ZHONGSHAN BAILING BIOTECHNOLOGY CO LTD
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