Method for preparing ursodeoxycholic acid through cyclic enzyme catalysis

A technology for ursodeoxycholic acid and catalytic preparation is applied in the field of catalyzed preparation of ursodeoxycholic acid by circulating enzymes, which can solve the problems of high cost of sewage treatment, non-compliance with environmental protection policies, unfavorable large-scale stable production, etc., and achieves easy recovery and treatment. , The effect of reducing subsequent processing procedures and low cost

Pending Publication Date: 2021-01-29
UNIV OF JINAN
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  • Abstract
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  • Application Information

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Problems solved by technology

Patent CN106086149A discloses a method for preparing UDCA by chemical-enzymatic method. CDCA is oxidized to 7-KLCA with sodium hypochlorite, and 7-KLCA is catalyzed by enzymatic method to UDCA. The problem with chemical oxidation is that the residual oxidant will affect the next step of enzymatic catalysis, although Treated with sodium sulfite, but there are still unstable factors, which is not conducive to large-scale stable production. The glucose used for the regeneration of the coenzyme catalyzed by the second step generates a large amount of sodium gluconate after the reaction. Although it has no effect on the reaction itself, the resulting sewage However, the processing cost is very high, which does not meet the current increasingly stringent environmental protection policies. Patents such as CN107995929A, CN109055473A, and CN110643584A all use glucose coenzyme regeneration systems, while some patents use coenzyme regeneration systems such as lactic acid and sodium malate. There is a certain pressure of sewage treatment

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  • Method for preparing ursodeoxycholic acid through cyclic enzyme catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Reaction system 500ml, first add 120ml of acetone and 250ml of pure water into a 1L three-hole reaction bottle and stir to mix. The reaction bottle is placed in a 25°C constant temperature water bath, then add 50.16g of CDCA and continue to stir for half an hour, let Dissolve it fully into the mixed solution, then add 0.02g NADP+, adjust the pH to 6.0-6.2, then add 5.42g ketoreductase and 15.28g 7-αHSDH, make up the volume with pure water, react at 300rpm for 12-15h, control during the reaction The temperature and pH are within the range of process requirements, and the content of CDCA and 7-KLCA is detected by liquid phase. When the substrate reaction exceeds 98%, the reaction is terminated. The reaction liquid is passed through a 60-mesh filter to recover the immobilized enzyme, and the rest of the system is waiting to enter the second step enzyme reaction.

Embodiment 2

[0027] Example 2: The reaction system is about 550ml. First, add the first step enzyme reaction solution prepared in Example 1 into a 1L three-hole reaction bottle, place the reaction bottle in a 30°C constant temperature water bath, turn on the stirring at 300rpm and stir for half an hour. Add 50ml of isopropanol to the reaction solution, adjust the pH to 7.0-7.2, then add 15.31g of isopropanol dehydrogenase and 25.22g of 7-βHSDH, react at 300rpm, control the temperature and pH during the reaction within the range of process requirements, and use The content of 7-KLCA and UDCA was detected by liquid phase. When the substrate reaction exceeded 98%, the reaction was terminated. The reaction liquid passed through a 60-mesh filter to recover the immobilized enzyme. ml, 82°C vacuum recovery isopropanol, to obtain isopropanol 45.5ml.

[0028] After the organic phase was removed from the reaction solution, it was extracted three times with 380ml ethyl acetate, the organic phases wer...

Embodiment 3

[0029] Example 3: The reaction system is 1000L, first add 240L acetone and 500L pure water into a 1.5T stainless steel reaction tank, turn on the stirring at 200rpm, and control the temperature of the jacket at 30°C, then add 100.09kg CDCA and continue stirring for half an hour to allow it to fully dissolve To the mixed solution, then add 0.05kg NADP+, adjust the pH to 7.0-7.2, then add 15.35kg ketoreductase and 50.07kg 7-αHSDH, make up the volume to 1000L with pure water, and react at constant temperature. During the reaction, control the temperature and pH at Within the range of process requirements, the content of CDCA and 7-KLCA is detected by liquid phase. When the substrate reaction exceeds 98%, the reaction is terminated. The reaction solution passes through a 60-mesh filter to recover the immobilized enzyme, and the rest of the system waits for the second step of enzyme reaction.

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Abstract

The invention discloses an application of acetone/isopropanol circulation in preparation of ursodeoxycholic acid (UDCA) by an enzyme method, and provides an environment-friendly, economic and efficient new idea for preparation of UDCA by a two-step enzyme method. The method comprises the following steps of 1, reducing acetone into isopropanol by using ketoreductase, and converting NADPH/NADH intoNADP+/NAD+ to provide NADP+/NAD+ for catalyzing chenodeoxycholic acid (CDCA) to generate 7KLCA by using 7alpha steroid dehydrogenase (7alpha HSDH); in the second step, isopropanol being oxidized intoacetone through isopropanol dehydrogenase, NADP+/NAD+ being converted into NADPH/NADH, and NADPH/NADH being provided for catalyzing 7KLCA to generate UDCA through 7beta steroid dehydrogenase (7beta HSDH). According to the two-step enzyme method, immobilized enzyme is utilized, the process is simple, second-step reaction can be carried out through simple treatment after first-step reaction is finished, and intermediate purification is not needed; the addition amount of NADP+/NAD+ in the reaction is small, and acetone and isopropanol can be recycled, so production cost and the environmental protection pressure are greatly reduced.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a method for preparing ursodeoxycholic acid catalyzed by circulating enzymes. Background technique [0002] Ursodeoxycholic acid (UDCA), whose chemical name is 3α,7β-dihydroxy-5β-cholestane-24-acid, is an organic compound with an odorless, bitter taste. In medicine, it is used to increase the secretion of bile acid, change the composition of bile, reduce cholesterol and cholesterol lipid in bile, and help the cholesterol in gallstones dissolve gradually. It is used for the treatment of gallstones, cholestatic liver disease, fatty liver, and various types of hepatitis. , toxic liver disorders, cholecystitis, cholangitis and biliary dyspepsia, bile reflux gastritis, eye diseases, etc. [0003] In recent years, the domestic UDCA market has experienced explosive growth. The current production process is generally a chemical process, which extracts CDCA from animal bile and then uses chem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/06C12P33/02
CPCC12P33/06C12P33/02
Inventor 高娟张欣玉郁桂聪郑豪蕾
Owner UNIV OF JINAN
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