Method for preparing product with controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid by using four enzymes, 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 by using four enzymes, 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 immobilized enzymes of the alpha-type enzyme, the lactic dehydrogenase, the beta-type enzyme and the glucose dehydrogenase 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 invention relates to a method for preparing a product with a controllable ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid by using four enzymes, the product with a controllable ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid and the application of the method, and belongs to the field of biotechnology. Background Art

[0002] Tauroursodeoxycholic acid is the main active ingredient of bear bile. Bear bile powder obtained by drying bear bile is a precious Chinese medicinal material. It has the effects of protecting the liver and promoting bile secretion, anti-allergy, anti-fatigue, and anti-tumor. It also has the effects of sterilization, anti-inflammatory, antiviral, antipyretic and analgesic. It is mainly used clinically to treat diseases related to abnormal bile acid metabolism, and is also used as an auxiliary treatment for cardiovascular diseases and hepatobiliary diseases. The active ingredient is tauroursodeoxycholic acid.

[0003] Taurochenodeoxycholic acid is the main component of livestock bile, and has immunomodulatory, anti-inflammatory, and significant antitussive and expectorant effects. It can be used to promote the proliferation of small intestinal stem cells, reduce damage to the digestive tract tissue structure, protect the digestive tract, enhance mucosal immune function, and protect the liver. At the same time, taurochenodeoxycholic acid has different effects on the apoptosis of different immune cells.

[0004] Tauroursodeoxycholic acid is a characteristic component of bear bile, while tauroursodeoxycholic acid is widely present in the bile acid of poultry and livestock. The difference between the two is the different configuration of the hydroxyl group at position 7. Both α-hydroxysteroid dehydrogenase and β-hydroxysteroid dehydrogenase have bidirectional redox activity and can achieve the diastereoisomerization of the hydroxyl group at position 7 through enzymatic reactions.

[0005] Different ratios of tauroursodeoxycholic acid and taurochenodeoxycholic acid show different pharmacological effects. At present, patents and literature reports on artificial bear bile powder show that the ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid in artificial bear bile powder prepared by bioconversion technology is usually between 1.1 and 1.5, and it is difficult to exceed 1.5. Summary of the invention

[0006] [Technical issues]

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

[0008] The second technical problem to be solved by the present invention is to provide a characteristic product with a controllable ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid, wherein the product is prepared by the above-mentioned preparation method.

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

[0010] [Technical solution]

[0011] The present invention provides a method for preparing a product with a controllable ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid by bioconversion, the method comprising the following steps: in the presence of coenzymes and cofactors, a reaction substrate containing tauroursodeoxycholic acid is subjected to an enzymatic reaction with an α-type enzyme, lactate dehydrogenase, a β-type enzyme and glucose dehydrogenase, so that the reaction substrate containing tauroursodeoxycholic acid is converted into a product with a controllable ratio of tauroursodeoxycholic acid and tauroursodeoxycholic acid. The tauroursodeoxycholic acid is TUDCA, and the tauroursodeoxycholic acid is TCDCA.

[0012] In certain embodiments of the present invention,

[0013] The four enzymes, namely, α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase, are present in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II, and the cofactors are sodium pyruvate and glucose; the reaction substrate is poultry and livestock bile powder, or other mixtures with a taurochenodeoxycholic acid content of more than 40%; the product with a controllable ratio of tauroursodeoxycholic acid to tauroursodeoxycholic acid refers to a characteristic product with a controllable ratio of 1.1:1 to 1.5:1, or more than 1.5, or all of which are tauroursodeoxycholic acid.

[0014] In certain embodiments of the present invention, the immobilized enzymes of the four enzymes, namely, α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase, exist in the form of α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase, which are respectively combined with immobilization resins to form α-type enzyme immobilized enzyme, lactate dehydrogenase immobilized enzyme, β-type enzyme immobilized enzyme, and glucose dehydrogenase immobilized enzyme.

[0015] In certain embodiments of the present invention, the immobilized enzymes of the four enzymes, α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase, exist in the form of a community immobilized enzyme formed by combining at least two of the four enzymes, α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase, with an immobilized resin.

[0016] In certain embodiments of the present invention, the immobilized enzymes of the four enzymes, namely, α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase, exist in the form of α-type enzyme-lactate dehydrogenase immobilized enzyme formed by combining α-type enzyme and lactate dehydrogenase with immobilized resin, and β-type enzyme-glucose dehydrogenase immobilized enzyme formed by combining β-type enzyme and glucose dehydrogenase with immobilized resin.

[0017] In certain embodiments of the present invention, the poultry and livestock bile powder is refined poultry and livestock bile powder with a mass percentage of taurochenodeoxycholic acid ≥ 63%.

[0018] In certain embodiments of the present invention, the enzymatic reaction can be carried out in one or more steps in a reaction vessel reaction system or in a multi-combination immobilized enzyme column reaction system.

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

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

[0021] (2) The reaction substrate solution I is mixed evenly with the α-type enzyme immobilized enzyme, the lactate dehydrogenase immobilized enzyme, the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain the characteristic product.

[0022] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

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

[0024] (2) The reaction substrate solution I is mixed evenly with a certain proportion of α-type enzyme-lactate dehydrogenase immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated after clarification, precipitated with alcohol, the supernatant is concentrated after clarification, and dried to obtain the characteristic product.

[0025] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0026] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II and sodium pyruvate, adjust the pH to between 7.0 and 8.5, and make up the volume to obtain a reaction substrate solution II;

[0027] (2) The reaction substrate solution II is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and lactate dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20-30°C for a sufficient reaction. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 7.0 and 8.5. The mixture is mixed evenly with β-type enzyme immobilized enzyme and glucose dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20-30°C for a sufficient reaction. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain the characteristic product.

[0028] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0029] (1) Weigh a certain amount of refined poultry and livestock bile powder, dissolve it in a buffer solution, add coenzyme I or coenzyme II and sodium pyruvate, and adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution II;

[0030] (2) The reaction substrate solution II is mixed evenly with a certain amount of α-type enzyme-lactate dehydrogenase immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 7.0 and 8.5, and then mixed evenly with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, and the supernatant is concentrated and dried to obtain the characteristic product.

[0031] In certain embodiments of the present invention, the buffer is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II is NAD + or NADP + , based on the volume of the conversion system after constant volume, the final concentration of coenzyme I or coenzyme II is 1 mM; the final concentration of sodium pyruvate is 0.2 M based on the volume of the conversion system after constant volume; the final concentration of glucose is 0.1 M based on the volume of the conversion system after constant volume.

[0032] In certain embodiments of the present invention, the mass ratio between the α-type enzyme immobilized enzyme and the lactate dehydrogenase immobilized enzyme is 1:0.25 to 1:1, the mass ratio between the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme is 1:5 to 1:40, the mass ratio between the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme is 1:0.5 to 1:1, and the mass ratio between the α-type enzyme immobilized enzyme and the reaction substrate is 1:10 to 1:80.

[0033] In certain embodiments of the present invention, the mass ratio between the α-type enzyme and the lactate dehydrogenase in the α-type enzyme-lactate dehydrogenase immobilized enzyme is 1:0.25-1:1, the mass ratio between the β-type enzyme and the glucose dehydrogenase in the β-type enzyme-glucose dehydrogenase immobilized enzyme is 1:0.5-1:1, the mass ratio between the α-type enzyme-lactate dehydrogenase immobilized enzyme and the β-type enzyme-glucose dehydrogenase immobilized enzyme is 1:5-1:20, and the mass ratio between the α-type enzyme-lactate dehydrogenase immobilized enzyme and the reaction substrate is 1:5-1:40.

[0034] The present invention also provides a product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid prepared by the above method, wherein the product is a mixture in which the ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid is 1.1:1 to 1.5:1 (i.e. artificial bear bile powder), or a ratio of 1.5 or more, or a single tauroursodeoxycholic acid compound.

[0035] The present invention also provides application of the method in a mixture of tauroursodeoxycholic acid and taurochenodeoxycholic acid or a tauroursodeoxycholic acid compound.

[0036] [Beneficial Effects]

[0037] 1) The present invention is applicable to the preparation of artificial bear bile powder, or the preparation of a compound mixture of tauroursodeoxycholic acid and taurochenodeoxycholic acid in different proportions, or the preparation of a single tauroursodeoxycholic acid compound.

[0038] 2) The present invention adopts four-enzyme enzymatic conversion, which can control the intermediate to be fully converted into the product.

[0039] 3) The present invention uses a four-enzyme reaction system of α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase. This reaction system can recycle the coenzyme and reduce the usage of α-type enzyme.

[0040] 4) The present invention uses the immobilized enzymes prepared by combining the four enzymes of α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase with immobilized resins, or co-immobilized immobilized enzymes, to prepare products with a controllable ratio of tauroursodeoxycholic acid to tauroursodeoxycholic acid. This technology can make any combination of the above enzymes perform enzymatic reactions and achieve the same conversion effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0042] The four enzymes used in the following examples are: α-type enzyme, lactate dehydrogenase, β-type enzyme, and glucose dehydrogenase.

[0043] The enzymes used in the following examples mainly include: α-type enzyme immobilized enzyme, lactate dehydrogenase immobilized enzyme, β-type enzyme immobilized enzyme, glucose dehydrogenase immobilized enzyme, α-type enzyme-lactate dehydrogenase immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme.

[0044] The enzymatic reaction forms in the following embodiments mainly include: the enzymatic reaction can be carried out in one step or multiple steps in a reaction container reaction system or in a multi-combination immobilized enzyme column reaction system.

[0045] In the following examples, the characteristic product is artificial bear bile powder, or a compound mixture of tauroursodeoxycholic acid and taurochenodeoxycholic acid in different proportions, or a single compound of tauroursodeoxycholic acid.

[0046] In the following examples, the buffer is purified water or glycine buffer, preferably purified water.

[0047] In the following embodiments, the coenzyme is coenzyme I or coenzyme II, preferably coenzyme I

[0048] In the following examples, the cofactors are sodium pyruvate and glucose.

[0049] In the following examples, the recombinant Escherichia coli used is: refer to the Chinese application document with publication number CN109402212A.

[0050] In the following examples, TUDCA and TCDCA were detected by HPLC-ELSD in accordance with the Chinese Pharmacopoeia Volume 4 (2020 Edition) (General Rules for High Performance Liquid Chromatography).

[0051] In the following examples, when each enzyme is used in the form of an immobilized enzyme, the method for determining the enzyme activity value is as follows:

[0052] The enzyme activity value (U / mg immobilized enzyme) of α-type immobilized enzyme was measured: 5mM taurochenodeoxycholic acid was used as substrate, NADP+ with a final concentration of 0.2mM and 50mg α-type immobilized enzyme were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. Samples were taken, filtered, and the change in absorbance of the clear solution was measured at 340nm, and the enzyme activity value of α-type immobilized enzyme was calculated by comparison with the blank control.

[0053] The enzyme activity value (U / mg immobilized enzyme) of the β-type immobilized enzyme was measured: 5mM tauroursodeoxycholic acid was used as the substrate, NADP+ with a final concentration of 0.2mM and 50mg of the β-type immobilized enzyme were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. The sample was taken and filtered, and the change in the absorbance value of the clear liquid was measured at 340nm, and the enzyme activity value of the β-type immobilized enzyme was calculated by comparing it with the blank control.

[0054] The enzyme activity value (U / mg immobilized enzyme) of the immobilized lactate dehydrogenase was measured: 5mM sodium pyruvate was used as the substrate, NADH with a final concentration of 0.2mM and 50mg of immobilized lactate dehydrogenase were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. The sample was taken and filtered, and the change of the absorbance value of the clear liquid was measured at 340nm, and the enzyme activity value of the immobilized lactate dehydrogenase was calculated by comparing with the blank control.

[0055] The enzyme activity value (U / mg immobilized enzyme) of glucose dehydrogenase immobilized enzyme was measured: 5mM glucose was used as substrate, NAD+ with a final concentration of 0.2mM and 50mg glucose dehydrogenase were added to a 250ml pH8.0 100mM phosphate buffer reaction system, and the reaction was carried out at 25°C for 5 minutes. Samples were taken and filtered, and the change of absorbance value of the clear liquid was measured at 340nm, and the enzyme activity value of glucose dehydrogenase immobilized enzyme was calculated by comparison with the blank control.

[0056] Example 1: Preparation of a characteristic product having a ratio of TUDCA to TCDCA between 1.1 and 1.5 by enzymatic conversion using four immobilized enzymes

[0057] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was added to a final concentration of 1 mM and the sodium pyruvate solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide. 60 ml of a mixed immobilized enzyme resuspension containing 13 g of α-type enzyme immobilized enzyme and 6.5 g of lactate dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 3500 ml with pure water and stirred evenly. The activity value of the α-type enzyme reached 0.58 U / ml. The reaction solution was placed in a 25°C constant temperature water bath and slowly stirred for 8 h.

[0058] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, 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, 900 ml of a mixed immobilized enzyme resuspension containing 200 g of β-type enzyme immobilized enzyme and 100 g of glucose dehydrogenase immobilized enzyme was added, and the reaction solution was fixed to 4500 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25° C. and slowly stirred for reaction for 12 h to obtain a conversion solution;

[0059] (3) The conversion solution obtained in step (2) was centrifuged, and the resulting supernatant was concentrated to 1 L. 4 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%. 970.01 g of a characteristic product having a TUDCA to TCDCA ratio of 1.1 to 1.5 was obtained.

[0060] Using HPLC-ELSD, the characteristic product with a ratio of TUDCA to TCDCA between 1.1 and 1.5 prepared in step (3) was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was 38.49%, the percentage of TCDCA was 28.73%, the percentage of TUDCA + TCDCA was 67.22%, and TUDCA / TCDCA = 1.34.

[0061] Example 2: Preparation of a characteristic product having a ratio of TUDCA to TCDCA of more than 1.5 by enzymatic conversion using four immobilized enzymes

[0062] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was added to a final concentration of 1 mM and the sodium pyruvate solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide. 135 ml of a mixed immobilized enzyme resuspension containing 30 g of α-type enzyme immobilized enzyme and 15 g of lactate dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 3500 ml with pure water and stirred evenly. The activity value of the α-type enzyme reached 1.27 U / ml. The reaction solution was placed in a 25°C constant temperature water bath and slowly stirred for 8 h.

[0063] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, 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, 1200 ml of a mixed immobilized enzyme resuspension containing 300 g of β-type enzyme immobilized enzyme and 100 g of glucose dehydrogenase immobilized enzyme was added, and the reaction solution was fixed to 8000 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25° C. and slowly stirred for reaction for 12 h to obtain a conversion solution;

[0064] (3) The conversion solution obtained in step (2) was centrifuged, and the resulting supernatant was concentrated to 2 L. 8 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%. 965.41 g of a characteristic product having a TUDCA to TCDCA ratio of more than 1.5 was obtained.

[0065] Using HPLC-ELSD, the characteristic product with a ratio of TUDCA to TCDCA above 1.5 prepared in step (3) was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was 39.44%, the percentage of TCDCA was 24.51%, the percentage of TUDCA+TCDCA was 63.95%, and TUDCA / TCDCA=1.61.

[0066] Example 3: Preparation of TUDCA single compound characteristic product by enzymatic conversion using four immobilized enzymes

[0067] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 2000ml of pure water, stir evenly, and add coenzyme NAD + The solution was added to a final concentration of 1 mM and the sodium pyruvate solution was added to a final concentration of 0.2 M. The pH was adjusted to 8.5 with 5 M sodium hydroxide. 600 ml of a mixed immobilized enzyme resuspension containing 100 g of α-type enzyme immobilized enzyme and 100 g of lactate dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 4000 ml with pure water and stirred evenly. The activity value of the α-type enzyme reached 5.02 U / ml. The reaction solution was placed in a 25°C constant temperature water bath and slowly stirred for 8 h.

[0068] (2) The reaction solution obtained in step (1) was centrifuged to terminate the reaction, and the supernatant was taken. Glucose was added to a final concentration of 0.1 M, and the pH was adjusted to 8.5 with 5 M sodium hydroxide. 4500 ml of a mixed immobilized enzyme resuspension containing 1000 g of β-type enzyme immobilized enzyme and 500 g of glucose dehydrogenase immobilized enzyme was added. The reaction solution was fixed to 9000 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25° C. and slowly stirred for reaction for 12 h to obtain a conversion solution.

[0069] (3) The conversion solution obtained in step (2) was centrifuged, and the resulting supernatant was concentrated to 3.5 L. 14 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 967.20 g of a single compound characteristic product of TUDCA.

[0070] Using HPLC-ELSD, the characteristic product of the conversion of TCDCA prepared in step (3) into TUDCA was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was more than 99%.

[0071] Example 4: Preparation of a characteristic product with a ratio of TUDCA to TCDCA between 1.1 and 1.5 by enzymatic conversion using four immobilized enzymes

[0072] (1) Weigh 1000 g of refined poultry and livestock bile powder, dissolve it in 5000 ml of 10 mM glycine buffer, stir evenly, and add coenzyme NAD + solution to a final concentration of 1 mM and sodium pyruvate solution to a final concentration of 0.2 M, glucose to a final concentration of 0.1 M, 5 M sodium hydroxide was used to adjust the pH to 8.5, 10 mM glycine buffer was added to a constant volume of 7000 ml, stirred evenly, and introduced at a constant flow rate into a mixed immobilized enzyme column placed in a 25° C. constant temperature water bath containing 13 g of α-type enzyme immobilized enzyme, 7.5 g of lactate dehydrogenase immobilized enzyme, 130 g of β-type enzyme immobilized enzyme and 65 g of glucose dehydrogenase immobilized enzyme, wherein the α-type enzyme activity value reached 0.60 U / ml, and the enzymatic reaction was contacted and reacted in the column for 16 hours in a constant flow rate cyclic loading manner to obtain a conversion intermediate solution;

[0073] (2) The conversion solution obtained in step (1) was centrifuged, and the resulting supernatant was concentrated to 2 L. 8 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%. 961.24 g of a characteristic product having a TUDCA to TCDCA ratio of 1.1 to 1.5 was obtained.

[0074] Using HPLC-ELSD, the characteristic product with a ratio of TUDCA to TCDCA between 1.1 and 1.5 prepared in step (2) was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was 38.67%, the percentage of TCDCA was 28.43%, the percentage of TUDCA + TCDCA was 67.10%, and TUDCA / TCDCA = 1.36.

[0075] Example 5: Preparation of a characteristic product having a ratio of TUDCA to TCDCA of more than 1.5 by enzymatic conversion using four immobilized enzymes

[0076] (1) Weigh 1000 g of refined poultry and livestock bile powder, dissolve it in 5000 ml of 10 mM glycine buffer, stir evenly, and add coenzyme NAD +solution to a final concentration of 1 mM and sodium pyruvate solution to a final concentration of 0.2 M, glucose to a final concentration of 0.1 M, 5 M sodium hydroxide was used to adjust the pH to 8.5, 10 mM glycine buffer was added to a constant volume of 7000 ml, stirred evenly, and introduced at a constant flow rate into a mixed immobilized enzyme column placed in a 25° C. constant temperature water bath containing 30 g of α-type enzyme-lactate dehydrogenase immobilized enzyme and 300 g of β-type enzyme-glucose dehydrogenase immobilized enzyme, wherein the α-type enzyme activity value reached 1.19 U / ml, and the enzymatic reaction was contacted and reacted in the column for 16 hours in a constant flow rate cyclic loading manner to obtain a conversion intermediate solution;

[0077] (2) The conversion solution obtained in step (1) was centrifuged, and the resulting supernatant was concentrated to 2 L. 8 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%. 958.39 g of a characteristic product having a TUDCA to TCDCA ratio of more than 1.5 was obtained.

[0078] Using HPLC-ELSD, the characteristic product with a ratio of TUDCA to TCDCA of more than 1.5 prepared in step (2) was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was 39.37%, the percentage of TCDCA was 24.30%, the percentage of TUDCA+TCDCA was 63.67%, and TUDCA / TCDCA=1.62.

[0079] Example 6: Preparation of TUDCA single compound characteristic product by enzymatic conversion using four immobilized enzymes

[0080] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 5000ml of pure water, stir evenly, and add coenzyme NAD + solution to a final concentration of 1 mM and a sodium pyruvate solution to a final concentration of 0.2 M, adjust the pH to 8.5 with 5 M sodium hydroxide, make up to 7000 ml with pure water, stir evenly, and enter at a constant flow rate into a mixed immobilized enzyme column containing 200 g of a uniformly mixed α-type enzyme-lactate dehydrogenase immobilized enzyme in a 25° C. constant temperature water bath, wherein the α-type enzyme activity value reaches 4.89 U / ml, and the enzymatic reaction is contacted and reacted in the column for 8 hours in a constant flow rate cyclic loading manner to obtain a conversion intermediate solution;

[0081] (2) Glucose was added to the conversion intermediate solution prepared in step (1) to a final concentration of 0.1 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, the volume was made up to 9000 ml with purified water, and the mixture was stirred evenly. The mixture was introduced into a mixed immobilized enzyme column containing 1500 g of a uniformly mixed β-type enzyme-glucose dehydrogenase immobilized enzyme in a constant temperature water bath at 25° C. at a constant flow rate, and reacted in the column for 12 h in a cyclic loading manner at a constant flow rate to obtain a conversion solution;

[0082] (3) The conversion solution obtained in step (2) was centrifuged, and the resulting supernatant was concentrated to 3.5 L. 14 L of 95% ethanol was added and slowly stirred for 30 minutes. The solution was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The resulting supernatant was then centrifuged. The resulting supernatant was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 957.18 g of a characteristic product in which TCDCA was completely converted to TUDCA.

[0083] Using HPLC-ELSD, the characteristic product of the conversion of TCDCA prepared in step (3) into TUDCA was detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the percentage of TUDCA was more than 99%.

[0084] Comparative Example 1: Effect of lactate dehydrogenase on the conversion of TCDCA to TUDCA

[0085] The specific implementation of Comparative Example 1 is the same as that of Example 4, except that in step (1), the amount of immobilized lactate dehydrogenase used is 26 g, which is twice the amount of immobilized α-type enzyme used. When other conditions remain unchanged, the reaction is completed to obtain a conversion solution.

[0086] Using HPLC-ELSD, the conversion liquid was sampled and tested in accordance with the "Chinese Pharmacopoeia Part IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The percentage of TUDCA was 35.02%, the percentage of TCDCA was 33.35%, the percentage of TUDCA+TCDCA was 68.37%, TUDCA / TCDCA=1.05, the conversion effect was poor, and TUDCA / TCDCA failed to reach 1.1-1.5.

[0087] Comparative Example 2: Effect of Glucose Dehydrogenase on the Conversion of TCDCA to TUDCA

[0088] The specific implementation of Comparative Example 2 is the same as that of Example 3, except that in step (1), the amount of immobilized glucose dehydrogenase used is 100 g, which is 10% of the amount of immobilized β-type enzyme used. When other conditions remain unchanged, the reaction is completed to obtain a conversion solution.

[0089] Using HPLC-ELSD, the conversion liquid was sampled and tested in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The TUDCA percentage was 95.61%, and the conversion effect was poor. The TUDCA percentage did not reach more than 99%.

[0090] Comparative Example 3: Effect of α-type enzyme activity value lower than 0.5 U / ml on the conversion of TCDCA to TUDCA

[0091] The specific implementation of Comparative Example 3 is the same as that of Example 1, with the main difference being that in step (1), the amount of the α-type enzyme immobilized enzyme is reduced to a mixed immobilized enzyme resuspension of 10 g and 10 g of the lactate dehydrogenase immobilized enzyme, so the α-type enzyme activity value is only 0.40 U / ml, less than 0.5 U / ml. When other conditions remain unchanged, the reaction is completed to obtain a conversion solution.

[0092] Using HPLC-ELSD, the conversion liquid was sampled and tested in accordance with the "Chinese Pharmacopoeia Part IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The percentage of TUDCA was 34.68%, the percentage of TCDCA was 33.64%, the percentage of TUDCA+TCDCA was 68.32%, TUDCA / TCDCA=1.02, and the conversion effect was poor, and TUDCA / TCDCA failed to reach 1.1-1.5.

[0093] By utilizing the various enzyme conversion technologies of the present invention, conversion products of tauroursodeoxycholic acid and taurochenodeoxycholic acid in any controllable ratio can be prepared as needed, or the substrate tauroursodeoxycholic acid can be completely converted into tauroursodeoxycholic acid, thereby providing a material basis and conditions for further expanding its application in medicine and new drug research and development.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid using four enzymes, characterized in that: In the presence of coenzymes and cofactors, a reaction substrate containing taurochenodeoxycholic acid is subjected to an enzymatic reaction with α-hydroxysteroid dehydrogenase, lactate dehydrogenase, β-hydroxysteroid dehydrogenase and glucose dehydrogenase to convert the reaction substrate containing taurochenodeoxycholic acid into a product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid; 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 a controllable ratio of tauroursodeoxycholic acid and taurochenodeoxycholic acid is referred to as characteristic product or product.

2. The method according to claim 1, characterized in that The four enzymes, α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase, are in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II, and the cofactors are sodium pyruvate and glucose; the reaction substrate is poultry bile powder, or other mixtures with a taurochenodeoxycholic acid content of more than 40%.

3. The method according to claim 2, characterized in that The immobilized enzyme forms of the four enzymes, namely, α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase, are formed by combining α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase with immobilized resins, respectively.

4. The method according to claim 2, characterized in that: The immobilized enzymes of the α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase are in the form of a community immobilized enzyme formed by combining at least two of the four enzymes with an immobilization resin.

5. The method according to claim 2 or 4, characterized in that: The immobilized enzyme forms of the four enzymes, namely, α-type enzyme, lactate dehydrogenase, β-type enzyme and glucose dehydrogenase, are α-type enzyme-lactate dehydrogenase immobilized enzyme formed by combining α-type enzyme and lactate dehydrogenase with immobilized resin, and β-type enzyme-glucose dehydrogenase immobilized enzyme formed by combining β-type enzyme and glucose dehydrogenase with immobilized resin.

6. The method according to claim 2, characterized in that The poultry and livestock bile powder is refined poultry and livestock bile powder with a mass percentage of taurochenodeoxycholic acid of ≥63%.

7. The method according to claim 1, characterized in that The enzymatic reaction can be carried out in one step or multiple steps in a reaction container reaction system or in a multi-combination immobilized enzyme column reaction system.

8. The method according to any one of claims 1 to 3 and 6 to 7, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, sodium pyruvate, and glucose, and adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution I; (2) The reaction substrate solution I is mixed evenly with the α-type enzyme immobilized enzyme, the lactate dehydrogenase immobilized enzyme, the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, precipitated with alcohol, the supernatant is concentrated by clarification, and dried to obtain the characteristic product.

9. The method according to any one of claims 1 to 2 and 4 to 7, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II, sodium pyruvate, and glucose, and adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution I; (2) The reaction substrate solution I is mixed evenly with a certain proportion of α-type enzyme-lactate dehydrogenase immobilized enzyme and β-type enzyme-glucose dehydrogenase immobilized enzyme, and the volume is fixed. The mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated after clarification, precipitated with alcohol, the supernatant is concentrated after clarification, and dried to obtain the characteristic product.

10. The method according to any one of claims 1 to 3 and 6 to 7, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II and sodium pyruvate, adjust the pH to between 7.0 and 8.5, and make up the volume to obtain reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and lactate dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20-30°C for a sufficient reaction. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 7.0 and 8.

5. The mixture is mixed evenly with β-type enzyme immobilized enzyme and glucose dehydrogenase immobilized enzyme, and the mixture is brought into contact with each other at 20-30°C for a sufficient reaction. After the reaction is completed, the supernatant is clarified and concentrated, precipitated with alcohol, and the supernatant is clarified and concentrated, and dried to obtain the characteristic product.

11. The method according to any one of claims 1 to 2 and 4 to 7, characterized in that: The enzymatic reaction mainly comprises the following steps: (1) Weigh a certain amount of reaction substrate, dissolve it in a buffer solution, add coenzyme I or coenzyme II and sodium pyruvate, and adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution II; (2) The reaction substrate solution II is mixed evenly with a certain amount of α-type enzyme-lactate dehydrogenase immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is clarified and glucose is added to adjust the pH to between 7.0 and 8.5, and then mixed evenly with a certain amount of β-type enzyme-glucose dehydrogenase immobilized enzyme, fixed to volume, and fully contacted with the reaction at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, and the supernatant is concentrated and dried to obtain the characteristic product.

12. The method according to any one of claims 8 to 11, characterized in that: The buffer is purified water or 10-100 mM glycine buffer; the coenzyme I or coenzyme II is NAD + or NADP + , based on the volume of the conversion system after constant volume, the final concentration of coenzyme I or coenzyme II is 1 mM; the final concentration of sodium pyruvate is 0.2 M based on the volume of the conversion system after constant volume; the final concentration of glucose is 0.1 M based on the volume of the conversion system after constant volume.

13. The method according to any one of claims 2 to 3, 8 and 10, characterized in that: The mass ratio between the α-type enzyme immobilized enzyme and the lactate dehydrogenase immobilized enzyme is 1:0.25-1:1, the mass ratio between the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme is 1:5-1:40, the mass ratio between the β-type enzyme immobilized enzyme and the glucose dehydrogenase immobilized enzyme is 1:0.5-1:1, and the mass ratio between the α-type enzyme immobilized enzyme and the reaction substrate is 1:10-1:

80.

14. The method according to any one of claims 2 and 4, 5, 9, and 11, characterized in that: The mass ratio between the α-type enzyme and the lactate dehydrogenase in the α-type enzyme-lactate dehydrogenase immobilized enzyme is 1:0.25-1:1, the mass ratio between the β-type enzyme and the glucose dehydrogenase in the β-type enzyme-glucose dehydrogenase immobilized enzyme is 1:0.5-1:1, the mass ratio between the α-type enzyme-lactate dehydrogenase immobilized enzyme and the β-type enzyme-glucose dehydrogenase immobilized enzyme is 1:5-1:20, and the mass ratio between the α-type enzyme-lactate dehydrogenase immobilized enzyme and the reaction substrate is 1:5-1:

40.

15. A product with a controllable ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid, characterized in that: Prepared by the method of any one of claims 1 to 14, the product is a mixture of tauroursodeoxycholic acid and taurochenodeoxycholic acid in a ratio of 1.1:1 to 1.5:1, or above 1.5, or a single tauroursodeoxycholic acid compound.

16. Use of the method according to any one of claims 1 to 14 in a mixture of tauroursodeoxycholic acid and taurochenodeoxycholic acid or a tauroursodeoxycholic acid compound.

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