Method for preparing product with controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid, product with controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid and application of method

Abstract

The invention discloses a method for preparing a product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid, the product with the controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid and application of the method, and belongs to the technical field of biology. According to the method, the alpha-type enzyme, the lactic dehydrogenase and the beta-type enzyme are adopted to convert a reaction substrate containing taurochenodeoxycholic acid into a product with a controllable ratio of TUDCA to TCDCA through a multi-path enzymatic reaction, and the method has a relatively high application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing a product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid by three-enzyme enzymatic bioconversion, the product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid and application of the method, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Tauroursodeoxycholic acid is the main effective component of bear bile, and bear bile powder obtained by drying bear bile is a precious Chinese medicinal material, which has the effects of protecting liver and gallbladder, resisting allergy, resisting fatigue, resisting tumor, resisting inflammation, resisting virus, relieving fever and relieving pain, and is mainly used for treating diseases related to abnormal bile acid metabolism, and is also used for treating cardiovascular diseases and hepatobiliary diseases. The effective component of the drug Tarolith is tauroursodeoxycholic acid.

[0003] Taurocholenodeoxycholic acid is the main component of avian and livestock bile, has the effects of immune regulation and anti-inflammation, and has obvious antitussive and expectorant effects, can be used for promoting small intestinal stem cell proliferation, reducing damage to digestive tract tissue structure, protecting the digestive tract, and has the functions of enhancing mucosal immune function and protecting the liver. Meanwhile, taurocholenodeoxycholic acid has different effects on the apoptosis of different immune cells.

[0004] Tauroursodeoxycholic acid is a characteristic component of bear bile, while taurocholenodeoxycholic acid widely exists in avian and livestock bile acids, and the difference between the two lies in the configuration of the 7-hydroxyl group. Both α-type hydroxysteroid dehydrogenase and β-type hydroxysteroid dehydrogenase have bidirectional oxidation-reduction activity, and the epimerization of the 7-hydroxyl group can be realized through enzymatic reaction.

[0005] Different proportions of tauroursodeoxycholic acid and taurocholenodeoxycholic acid represent different pharmacological and pharmacodynamic effects. The current patents and literature reports on artificial bear bile powder prepared by bioconversion technology show that the ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid in the artificial bear bile powder is usually between 1.1 and 1.5, and it is difficult to break through 1.5. SUMMARY

[0006] [TECHNICAL PROBLEM]

[0007] The first technical problem to be solved by the present application is to provide a preparation method capable of preparing a characteristic product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid.

[0008] The second technical problem to be solved by the present application is to provide a characteristic product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid.

[0009] ​The third technical problem to be solved by the present application is to provide application of the preparation method in the mixture of tauroursodeoxycholic acid and taurocholenodeoxycholic acid or the tauroursodeoxycholic acid compound.

[0010] [Technical scheme]

[0011] The present application provides a method for preparing a product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid by biological transformation, which comprises the following steps: a method for converting a reaction substrate containing taurocholenodeoxycholic acid into a product with controllable ratio of tauroursodeoxycholic acid and taurocholenodeoxycholic acid by enzymatic reaction in the presence of coenzyme and cofactor, using α-type enzyme, lactate dehydrogenase and β-type enzyme. The tauroursodeoxycholic acid is TUDCA, and the taurocholenodeoxycholic acid is TCDCA. The α-type enzyme, lactate dehydrogenase and β-type enzyme are in the form of intracellular enzyme or free enzyme.

[0012] Intracellular enzyme refers to intracellular enzyme, which exists inside the cell and the cell structure remains intact. Free enzyme refers to enzyme existing in the form of monomer or complex after the expression cell is broken.

[0013] In some embodiments of the present application, the coenzyme is coenzyme I or coenzyme II, and the cofactor is sodium pyruvate; the reaction substrate can be poultry gall powder or other mixture with taurocholenodeoxycholic acid content of more than 40%.

[0014] In some embodiments of the present application, the intracellular enzyme of the α-type enzyme, lactate dehydrogenase and β-type enzyme is in the form of whole cell enzyme bacteria containing α-type enzyme, lactate dehydrogenase and β-type enzyme, respectively.

[0015] In some embodiments of the present application, the intracellular enzyme of the α-type enzyme, lactate dehydrogenase and β-type enzyme is in the form of resuspension enzyme liquid obtained by resuspending whole cell enzyme bacteria of the three enzymes in buffer.

[0016] In some embodiments of the present application, the free enzyme of the α-type enzyme, lactate dehydrogenase and β-type enzyme is in the form of broken liquid enzyme, supernatant enzyme liquid and purified enzyme liquid obtained by breaking, clarifying and purifying the resuspension enzyme liquid of the three enzymes, respectively.

[0017] In some embodiments of the present application, the enzymatic reaction can be carried out in a reaction container reaction system in one step or multiple steps.

[0018] In some embodiments of the present application, the poultry gall powder is refined poultry gall powder with mass percentage of taurocholenodeoxycholic acid ≥63%.

[0019] In some embodiments of the present application, the enzymatic reaction mainly comprises the following steps:

[0020] (1) Take a certain amount of refined poultry gall powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and sodium pyruvate, and adjust the pH to 7.0-8.5 to obtain a reaction substrate solution;

[0021] (2) Mix the reaction substrate solution prepared in step (1) with a certain proportion of α-type enzyme, lactate dehydrogenase, and β-type enzyme uniformly, make up the volume, and fully contact and react under the condition of 20-30°C. After the reaction is completed, the supernatant is obtained by clarification, concentrated, alcohol precipitated, clarified to obtain the supernatant, concentrated, dried to obtain the characteristic product.

[0022] In some embodiments of the present application, the enzymatic reaction mainly includes the following steps:

[0023] (1) Take a certain amount of refined poultry gall powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II, and sodium pyruvate, and adjust the pH to 7.0-8.5 to obtain a reaction substrate solution;

[0024] (2) Mix the reaction substrate solution prepared in step (1) with a certain proportion of α-type enzyme and lactate dehydrogenase uniformly, make up the volume, and fully contact and react under the condition of 20-30°C. After the reaction is completed, the supernatant is obtained by clarification, and the pH is adjusted to 7.0-8.5. A certain amount of β-type enzyme is mixed uniformly, the volume is made up, and fully contacted and reacted under the condition of 20-30°C. After the reaction is completed, the supernatant is obtained by clarification, concentrated, alcohol precipitated, clarified to obtain the supernatant, concentrated, dried to obtain the characteristic product.

[0025] In some embodiments of the present application, the characteristic product can be artificial bear gall powder, or a compound mixture of tauroursodeoxycholic acid and taurocholenodeoxycholic acid in different proportions, or a single compound of tauroursodeoxycholic acid.

[0026] In some embodiments of the present application, the buffer solution is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II is NAD + or NADP + , and the final concentration of coenzyme I or coenzyme II is 1 mM based on the volume of the conversion system after making up the volume; and the final concentration of the sodium pyruvate solution is 0.2 M based on the volume of the conversion system after making up the volume.

[0027] In some embodiments of the present application, the mass ratio between the α-type enzyme enzyme body and the lactate dehydrogenase enzyme body is 1:0.25-1:1, the mass ratio between the α-type enzyme enzyme body and the β-type enzyme enzyme body is 1:5-1:40, and the mass ratio between the α-type enzyme enzyme body and the reaction substrate is 1:10-1:80.

[0028] The present application also provides a product with controllable proportion of tauroursodeoxycholic acid and taurocholenodeoxycholic acid prepared by the above method.

[0029] The present application also provides the use of the method in the preparation of a mixture of tauroursodeoxycholic acid and taurocholenodeoxycholic acid or a single compound of tauroursodeoxycholic acid.

[0030] [Advantages]

[0031] 1) The present application is suitable for the preparation of artificial bear bile powder, or a mixture of tauroursodeoxycholic acid and taurocholenodeoxycholic acid in different proportions, or a single compound of tauroursodeoxycholic acid.

[0032] 2) The present application adopts three-enzyme enzymatic conversion, which can control the full conversion of intermediates to products.

[0033] 3) The present application uses a three-enzyme reaction system of α-type enzyme, lactate dehydrogenase, and β-type enzyme, which can recycle coenzymes and reduce the use amount of α-type enzyme.

[0034] 4) The present application uses whole-cell enzyme bacteria, resuspended enzyme liquid, broken enzyme liquid, supernatant enzyme liquid, and purified enzyme enzyme liquid of the three enzymes of α-type enzyme, lactate dehydrogenase, and β-type enzyme for the preparation of products with controllable proportions of tauroursodeoxycholic acid and taurocholenodeoxycholic acid. This technology can make any combination of the above enzymes for enzymatic reaction, and all can achieve the same conversion effect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The present application is a schematic diagram. DETAILED DESCRIPTION

[0036] The three enzymes used in the following examples are: α-type enzyme, lactate dehydrogenase, and β-type enzyme.

[0037] In the following examples, the use forms of each enzyme mainly include: enzyme bacteria, resuspended enzyme liquid, broken enzyme liquid, supernatant enzyme liquid, and purified enzyme enzyme liquid of the three enzymes of α-type enzyme, lactate dehydrogenase, and β-type enzyme.

[0038] In the following examples, the enzymatic reaction forms mainly include: the enzymatic reaction can be carried out in one step or multiple steps in the reaction system of the reaction container.

[0039] In the following examples, the characteristic product is artificial bear bile powder, or a mixture of tauroursodeoxycholic acid and taurocholenodeoxycholic acid in different proportions, or a single compound of tauroursodeoxycholic acid.

[0040] In the following examples, the buffer is purified water or glycine buffer, preferably purified water.

[0041] In the following examples, the coenzyme is coenzyme I or coenzyme II, preferably coenzyme I

[0042] In the following examples, the coenzyme is sodium pyruvate and glucose.

[0043] In the following examples, the recombinant Escherichia coli used is the Chinese application document with reference number CN109402212A.

[0044] In the following examples, when the use form of each enzyme is enzyme cell resuspension enzyme liquid, or broken liquid enzyme liquid, or supernatant enzyme liquid, or purified enzyme enzyme liquid, the determination method of enzyme activity value refers to the Chinese application document with reference number CN109402212A.

[0045] In the following examples, the detection of TUDCA and TCDCA both uses HPLC-ELSD, and is detected in accordance with the Chinese Pharmacopoeia (2020 Edition) (General Test for High Performance Liquid Chromatography).

[0046] Example 1: Enzymatic conversion of TUDCA and TCDCA to prepare characteristic products with a ratio of 1.1-1.5

[0047] (1) Take 1000g of refined poultry and livestock gall powder, dissolve in 2000ml of pure water, stir uniformly, add coenzyme NAD + solution to a final concentration of 1mM and pyruvic acid sodium solution to a final concentration of 0.2M, adjust the pH to 8.5 with 5M sodium hydroxide, add 37.5ml of mixed resuspension enzyme liquid containing 6.25g of α-type enzyme whole cell enzyme bacteria and 6.25g of lactate dehydrogenase whole cell enzyme bacteria, supplement with pure water to make the reaction liquid to 3500ml, the α-type enzyme activity value reaches 0.51U / ml, and the reaction liquid is placed in a 25℃ constant temperature water bath for 6h;

[0048] (2) After the reaction liquid obtained in step (1) is placed in a 60℃ constant temperature water bath for 20min, centrifugation is performed, the supernatant is taken, glucose is added to a final concentration of 0.1M, the pH is adjusted to 8.5 with 5M sodium hydroxide, 750ml of resuspension enzyme liquid containing 250g of β-type enzyme whole cell enzyme bacteria is added, the reaction liquid is made to 4500ml with pure water, and it is placed in a 25℃ constant temperature water bath for 8h to obtain a conversion liquid;

[0049] (3) The conversion liquid obtained in step (2) is centrifuged, and the obtained supernatant is concentrated to 1L, 4L of 95% ethanol is added, slowly stirred for 30min, placed in a 16℃ constant temperature water bath for 4h, and then centrifuged, the obtained supernatant is concentrated by rotary evaporation, vacuum dried to a water content of less than 3% to obtain 959.43g of product with a TUDCA and TCDCA ratio of 1.1-1.5.

[0050] The TUDCA and TCDCA ratio of the characteristic product prepared in step (3) was detected by HPLC-ELSD according to the Chinese Pharmacopoeia (2020 edition) (General method for high performance liquid chromatography), the percentage content of TUDCA was 38.66%, the percentage content of TCDCA was 28.64%, the percentage content of TUDCA+TCDCA was 67.30%, and the TUDCA / TCDCA ratio was 1.35.

[0051] Example 2: Preparation of a characteristic product with a TUDCA and TCDCA ratio of more than 1.5 by a three-enzyme enzymatic method

[0052] (1) 1000 g of refined poultry and livestock gall powder was dissolved in 2000 ml of pure water, stirred uniformly, and coenzyme NAD + solution to a final concentration of 1 mM and pyruvic acid sodium solution to a final concentration of 0.2 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, 78 ml of mixed resuspended enzyme solution containing 13 g of α-type enzyme whole cell enzyme bacteria and 13 g of lactate dehydrogenase whole cell enzyme bacteria was added, the reaction solution was made up to 3500 ml with pure water, the α-type enzyme activity value reached 1.13 U / ml, and the reaction solution was placed in a 25°C constant temperature water bath for 6 h;

[0053] (2) The reaction solution obtained in step (1) was placed in a 60°C constant temperature water bath for termination reaction for 20 min, then centrifuged, and the supernatant was taken, glucose was added to a final concentration of 0.1 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, 750 ml of resuspended enzyme solution containing 250 g of β-type enzyme whole cell enzyme bacteria was added, the reaction solution was made up to 4500 ml with pure water, and was placed in a 25°C constant temperature water bath for 8 h to obtain a conversion solution;

[0054] (3) The conversion solution obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, slowly stirred for 30 min, placed in a 16°C constant temperature water bath for 4 h of alcohol precipitation, then centrifuged, and the obtained supernatant was concentrated by rotary evaporation, vacuum dried to a water content of less than 3% to obtain 963.27 g of a product with a TUDCA and TCDCA ratio of more than 1.5.

[0055] The TUDCA and TCDCA ratio of the characteristic product prepared in step (3) was detected by HPLC-ELSD according to the Chinese Pharmacopoeia (2020 edition) (General method for high performance liquid chromatography), the percentage content of TUDCA was 38.66%, the percentage content of TCDCA was 28.64%, the percentage content of TUDCA+TCDCA was 67.30%, and the TUDCA / TCDCA ratio was 1.35.

[0056] Example 3: Preparation of TUDCA single compound by three-enzyme enzymatic conversion

[0057] (1) Take 1000g of refined poultry and livestock gall powder, dissolve in 2000ml of pure water, stir evenly, add coenzyme NAD + solution to a final concentration of 1mM and sodium pyruvate solution to a final concentration of 0.2M, adjust the pH to 8.5 with 5M sodium hydroxide, add 156ml of mixed resuspended enzyme solution containing 26g of whole cell enzyme bacteria of α-type enzyme and 26g of whole cell enzyme bacteria of lactate dehydrogenase, make up the reaction solution to 3500ml with pure water, the α-type enzyme activity value reaches 2.21U / ml, and the reaction solution is placed in a 25℃ constant temperature water bath for 6h;

[0058] (2) The reaction solution obtained in step (1) is placed in a 60℃ constant temperature water bath for 20 minutes to terminate the reaction, then centrifuged, and the supernatant is taken, glucose is added to a final concentration of 0.1M, the pH is adjusted to 8.5 with 5M sodium hydroxide, 750ml of resuspended enzyme solution containing 250g of whole cell enzyme bacteria of β-type enzyme is added, the reaction solution is made up to 4500ml with pure water, and is placed in a 25℃ constant temperature water bath for 8h to obtain a conversion solution;

[0059] (3) The conversion solution obtained in step (2) is centrifuged, and the obtained supernatant is concentrated to 1L, 4L of 95% ethanol is added, slowly stirred for 30 minutes, placed in a 16℃ constant temperature water bath for 4 hours for alcohol precipitation, then centrifuged, and the obtained supernatant is concentrated by rotary evaporation, vacuum dried to a water content of less than 3% to obtain 949.89g of product with TCDCA completely converted to TUDCA.

[0060] The HPLC-ELSD is used to detect the characteristic product of TCDCA completely converted to TUDCA prepared in step (3) according to the "Chinese Pharmacopoeia Four Parts (2020 Edition)" (General Method for High Performance Liquid Chromatography), and the percentage content of TUDCA is more than 99%.

[0061] Example 4: Preparation of TUDCA and TCDCA ratio between 1.1 and 1.5 by three-enzyme enzymatic conversion

[0062] (1) Take 1000g of refined poultry and livestock gall powder, dissolve in 2000ml of pure water, stir evenly, add coenzyme NAD +The solution was adjusted to a final concentration of 1 mM and a sodium pyruvate solution was adjusted to a final concentration of 0.2 M, glucose was added to a final concentration of 0.1 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, 1185 ml of a mixed resuspended enzyme solution containing 10 g of whole-cell enzyme bacteria of the α-type enzyme, 10 g of whole-cell enzyme bacteria of lactate dehydrogenase, and 250 g of whole-cell enzyme bacteria of the β-type enzyme was added, the reaction solution was brought to a volume of 4500 ml with purified water, the α-type enzyme enzyme activity value reached 0.80 U / ml, and the reaction solution was placed in a 25°C constant-temperature water bath for 12 h to obtain a conversion solution.

[0063] (2) The conversion solution obtained in step (1) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the solution was slowly stirred for 30 min, then placed in a 16°C constant-temperature water bath for 4 h of alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation, vacuum dried to a water content of less than 3%, and dried to obtain 959.51 g of characteristic product with a TUDCA to TCDCA ratio of 1.1-1.5.

[0064] The characteristic product with a TUDCA to TCDCA ratio of 1.1-1.5 prepared in step (3) was detected using HPLC-ELSD in accordance with the “Chinese Pharmacopoeia, Volume 4 (2020 Edition)” (General Rules for High Performance Liquid Chromatography), the TUDCA content was 39.06%, the TCDCA content was 27.13%, the TUDCA+TCDCA content was 66.19%, and the TUDCA / TCDCA ratio was 1.44.

[0065] Example 5: Preparation of 10 Kg of TUDCA single compound by three-enzyme enzymatic conversion

[0066] The specific implementation of Example 5 is the same as that of Example 3, and the main difference is that the buffer solution is adjusted to 10 mM glycine from pure water; the amount of refined poultry gall powder is 10 Kg; the amount of whole-cell enzyme bacteria of the α-type enzyme used is 260 g, the amount of whole-cell enzyme bacteria of lactate dehydrogenase used is 260 g, and the amount of whole-cell enzyme bacteria of the β-type enzyme used is 2600 g; the volume of the reaction solution in step (1) is 35 L, and the enzyme activity value of the α-type enzyme reaches 4.37 U / ml; the volume of the reaction solution in step (2) is 45 L; and the amount of 95% ethanol used in step (3) is 16 L, i.e., the entire enzymatic reaction system is scaled up by 10 times. 9.73 Kg of TUDCA single compound characteristic product was prepared.

[0067] The TUDCA single compound characteristic product prepared in step (3) was detected using HPLC-ELSD in accordance with the “Chinese Pharmacopoeia, Volume 4 (2020 Edition)” (General Rules for High Performance Liquid Chromatography), and the TUDCA content was more than 99%.

[0068] Example 1: Effect of lactate dehydrogenase on conversion of TCDCA to TUDCA

[0069] The specific implementation of Comparative Example 1 is the same as that of Example 1, with the main difference being that the amount of lactate dehydrogenase whole-cell enzyme bacteria used in step (1) is 12.5 g, which is twice the amount of α-type enzyme whole-cell enzyme bacteria used, and under the condition that other conditions remain unchanged, the reaction is completed to obtain a conversion liquid.

[0070] The conversion liquid sample was detected using HPLC-ELSD in accordance with the Chinese Pharmacopoeia (2020 edition) (General Rules for High Performance Liquid Chromatography), and the percentage content of TUDCA was 34.13%, the percentage content of TCDCA was 34.47%, the percentage content of TUDCA+TCDCA was 68.60%, and TUDCA / TCDCA = 0.99. The conversion effect was poor, and TUDCA / TCDCA failed to reach 1.1-1.5.

[0071] Comparative Example 2: Effect of α-type enzyme enzyme activity value less than 0.5 U / ml on conversion of TCDCA to TUDCA

[0072] The specific implementation of Comparative Example 2 is the same as that of Example 1, with the main difference being that the amount of α-type enzyme whole-cell enzyme bacteria used in step (1) is reduced to 4.5 g and the mixed resuspension enzyme solution of lactate dehydrogenase whole-cell enzyme bacteria 4.5 g, so the α-type enzyme enzyme activity value is only 0.45 U / ml, which is less than 0.5 U / ml, and under the condition that other conditions remain unchanged, the reaction is completed to obtain a conversion liquid.

[0073] The conversion liquid sample was detected using HPLC-ELSD in accordance with the Chinese Pharmacopoeia (2020 edition) (General Rules for High Performance Liquid Chromatography), and the percentage content of TUDCA was 35.15%, the percentage content of TCDCA was 33.16%, the percentage content of TUDCA+TCDCA was 68.31%, and TUDCA / TCDCA = 1.06. The conversion effect was poor, and TUDCA / TCDCA failed to reach 1.1-1.5.

[0074] The various enzyme conversion techniques of the present application can be used to prepare conversion products of tauroursodeoxycholic acid and taurochenodeoxycholic acid in any controllable ratio as needed, or to completely convert the substrate taurochenodeoxycholic acid into tauroursodeoxycholic acid, thereby providing a material basis and conditions for further expanding its application in drug development and new drug research.

[0075] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid, characterized in that, Under the participation of coenzyme and cofactor, the reaction substrate containing tauroursodeoxycholic acid is converted into the product with controllable ratio of tauroursodeoxycholic acid and taurine chenodeoxycholic acid by the enzymatic reaction of three enzymes of α-type hydroxysteroid dehydrogenase, lactate dehydrogenase and β-type hydroxysteroid dehydrogenase; The α-type hydroxysteroid dehydrogenase is referred to as α-type enzyme, the β-type hydroxysteroid dehydrogenase is referred to as β-type enzyme, and the product with controllable ratio of tauroursodeoxycholic acid and taurine chenodeoxycholic acid is referred to as characteristic product or product; The three enzymes of α-type enzyme, lactate dehydrogenase and β-type enzyme exist in the form of intracellular enzyme or free enzyme.

2. The method of claim 1, wherein, The coenzyme is coenzyme I or coenzyme II, the cofactor is sodium pyruvate, and the reaction substrate is poultry gall powder or other mixture with tauroursodeoxycholic acid content of more than 40%.

3. The method of claim 1, wherein, The three enzymes of α-type enzyme, lactate dehydrogenase and β-type enzyme exist in the form of intracellular enzyme, which are respectively expressed as whole-cell enzyme bacteria containing α-type enzyme, lactate dehydrogenase and β-type enzyme.

4. The method of claim 1, wherein, The three enzymes of α-type enzyme, lactate dehydrogenase and β-type enzyme exist in the form of intracellular enzyme, which are respectively obtained by resuspending whole-cell enzyme bacteria of the three enzymes in buffer solution.

5. The method of claim 1, wherein, The three enzymes of α-type enzyme, lactate dehydrogenase and β-type enzyme exist in the form of free enzyme, which are respectively obtained by resuspending whole-cell enzyme bacteria of the three enzymes in buffer solution, and then crushing, clarifying and purifying the resuspended enzyme solution to obtain a broken enzyme solution, supernatant enzyme solution and purified enzyme solution.

6. The method of claim 1, wherein, The enzymatic reaction can be carried out in one step or multiple steps in a reaction container reaction system.

7. The method according to any one of claims 1 to 6, characterized in that, The enzymatic reaction mainly includes the following steps: (1) a certain amount of reaction substrate is weighed and dissolved in buffer solution, coenzyme I or coenzyme II and sodium pyruvate are added, and the pH is adjusted to 7.0-8.5 to obtain a reaction substrate solution; (2) the reaction substrate solution prepared in step (1) is mixed with a certain proportion of α-type enzyme, lactate dehydrogenase and β-type enzyme, and the volume is adjusted, and the reaction is fully contacted at 20-30℃, after the reaction is completed, the supernatant is obtained by clarification, concentration, alcohol precipitation, clarification, concentration and drying to obtain the characteristic product.

8. The method according to any one of claims 1 to 6, characterized in that, The enzymatic reaction mainly includes the following steps: (1) a certain amount of reaction substrate is weighed and dissolved in buffer solution, coenzyme I or coenzyme II and sodium pyruvate are added, and the pH is adjusted to 7.0-8.5 to obtain a reaction substrate solution; (2) the reaction substrate solution prepared in step (1) is mixed with a certain proportion of α-type enzyme and lactate dehydrogenase, and the volume is adjusted, and the reaction is fully contacted at 20-30℃, after the reaction is completed, the supernatant is obtained by clarification, the pH is adjusted to 7.0-8.5, and a certain amount of β-type enzyme is mixed and the volume is adjusted, and the reaction is fully contacted at 20-30℃, after the reaction is completed, the supernatant is obtained by clarification, concentration, alcohol precipitation, clarification, concentration and drying to obtain the characteristic product.

9. The method of any one of claims 4, 5, 7, 8, wherein, The buffer is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II refers to NAD + or NADP + , the final concentration of coenzyme I or coenzyme II is 1 mM according to the volume of the conversion system after constant volume; the final concentration of sodium pyruvate solution is 0.2 M according to the volume of the conversion system after constant volume.

10. The method according to any one of claims 2 to 9, characterized in that, The mass ratio between the α-type enzyme enzyme body and the lactate dehydrogenase enzyme body is 1:0.25-1:1, the mass ratio between the α-type enzyme enzyme body and the β-type enzyme enzyme body is 1:5-1:40, and the mass ratio between the α-type enzyme enzyme body and the reaction substrate is 1:10-1:

80.

11. The method of claim 2, wherein, The poultry gall powder is refined poultry gall powder with the mass percentage of taurocholic acid being greater than or equal to 63%.

12. A product of controllable ratio of taurocholic acid to taurochenodeoxycholic acid, characterized in that, The product is a mixture of taurocholic acid and taurochenodeoxycholic acid with a ratio of 1.1:1-1.5:1 or more than 1.5, or a single compound of taurocholic acid, prepared by the method of any one of claims 1-11.

13. The use of the method of any one of claims 1-11 in a mixture of taurocholic acid and taurochenodeoxycholic acid or a compound of taurocholic acid.

Images