Method for preparing artificial bear gall powder by biotransformation through three-enzyme enzymatic method, artificial bear gall powder and application of method

Abstract

The invention discloses a method for preparing artificial bear gall powder by biotransformation through a three-enzyme enzymatic method, the artificial bear gall powder and application of the method, and belongs to the technical field of biology. According to the method, alpha-type enzyme, lactic dehydrogenase, beta-type enzyme and glucose dehydrogenase are adopted to convert a reaction substrate containing taurochenodeoxycholic acid into artificial bear gall powder through a multi-path enzymatic reaction, and the method has high application value.

Description

Technical Field

[0001] The invention relates to a method for preparing artificial bear bile powder by three-enzyme enzymatic biotransformation, the artificial bear bile powder and the application of the method, and belongs to the field of biotechnology. Background Art

[0002] Bear bile powder is the irregular fragments, granules or powder of bile from the gallbladder of black bears or brown bears after surgery. It is a precious Chinese herbal medicine and ranks first among the four precious animal herbal medicines in traditional Chinese medicine. It has the effects of clearing away heat and detoxification, protecting the liver and promoting bile secretion, relieving pain and stopping spasms, anticonvulsant, antibacterial and anti-inflammatory, and litholysis. It can also promote the synthesis of glycogen from sugar and lactose, enhance the action and secretion of insulin, and thus has the effect of lowering blood sugar. The current annual output of bear bile powder is low, which is difficult to meet the people's growing health needs.

[0003] Therefore, in order to solve the shortage of bear bile powder in the market, it is increasingly important to accelerate the research and development of artificial bear bile powder and promote the sustainable development of the bear bile powder industry. Taurochenodeoxycholic acid is the main component of poultry bile. At present, poultry bile is mainly used as raw material, and biotechnology is used to catalyze and convert the main bile acid components in it in vitro to synthesize tauroursodeoxycholic acid, and prepare artificial bear bile powder similar to natural bear bile powder. For example, Chinese invention patent CN201610852312.4 discloses a method for producing artificial bear bile powder by fermentation with engineered bacteria. It constructs a double gene co-expression vector and a multi-gene co-expression vector containing 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase respectively, uses poultry bile products as raw materials, adds substrates to the culture medium, adds to the fermentation broth of co-expressing 7α-steroid dehydrogenase and 7-β-steroid dehydrogenase, and converts part of taurochenodeoxycholic acid into tauroursodeoxycholic acid while fermenting. In this method, the composition of poultry and livestock bile powder is complex, and adding it during the fermentation process will have a certain impact on the normal growth of cells, thereby affecting the activity of the enzyme. Moreover, the conversion substrate concentration of this method is low, the conversion efficiency is unstable, and the content of the reaction intermediate tauro7-ketolithocholic acid is high. And the conversion cycle of this method is long, which is not suitable for industrial scale-up production. Chinese invention patent CN201210473483.8 converts the bound chenodeoxycholic acid in poultry and livestock bile into bound ursodeoxycholic acid by a two-step enzymatic method. The content of the reaction intermediate tauro7-ketolithocholic acid in this method is high and uncontrollable, affecting the quality of the product. The taurochenodeoxycholic acid is TCDCA, the tauroursodeoxycholic acid is TUDCA, and the tauro7-ketolithocholic acid is T7-KLCA.

[0004] Although the artificial bear bile powder prepared by the current α-type enzyme and β-type enzyme dual enzyme enzymatic conversion technology is highly similar to the natural bear bile powder and the technology is stable, it still cannot solve the problem of high content of the reaction intermediate T7-KLCA. This is determined by the dual enzyme enzymatic reaction mechanism and is difficult to solve simply by optimizing the enzymatic reaction conditions. Summary of the invention

[0005] [Technical issues]

[0006] Since the existing artificial bear bile powder preparation technology has the problem of high content of intermediate T7-KLCA, the present invention aims to provide a method for preparing artificial bear bile powder, so as to realize the preparation of artificial bear bile powder with the same or close characteristic components as natural bear bile powder on demand.

[0007] The second technical problem to be solved by the present invention is to provide an artificial bear bile powder whose characteristic components are consistent with or close to those of natural bear bile powder.

[0008] The third technical problem to be solved by the present invention is to provide a method for applying artificial bear bile powder.

[0009] [Technical solution]

[0010] The invention provides a method for preparing artificial bear bile powder by three-enzyme enzymatic biotransformation. The method comprises the following main steps: in the presence of coenzymes and cofactors, a reaction substrate containing taurochenodeoxycholic acid is subjected to enzymatic reaction with α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase and glucose dehydrogenase to prepare artificial bear bile powder; the α-type enzyme, β-type enzyme and glucose dehydrogenase are present in the form of intracellular enzymes or free enzymes.

[0011] Intracellular enzymes refer to enzymes that exist inside cells and their cell structures remain intact. Free enzymes refer to enzymes that exist as monomers or complexes after their expressing cells are broken.

[0012] In certain embodiments of the present invention, the coenzyme is coenzyme I or coenzyme II, and the cofactor is glucose; the reaction substrate may be poultry and livestock bile powder or poultry and livestock bile concentrate.

[0013] In certain embodiments of the present invention, the intracellular enzymes of the α-type enzyme, β-type enzyme, and glucose dehydrogenase exist in the form of whole-cell enzyme bacteria expressing the α-type enzyme, β-type enzyme, and glucose dehydrogenase, respectively.

[0014] In certain embodiments of the present invention, the intracellular enzymes of the α-type enzyme, β-type enzyme and glucose dehydrogenase exist in the form of resuspended enzyme solutions obtained by resuspending the whole-cell enzyme bacteria of the three enzymes in a buffer solution.

[0015] In certain embodiments of the present invention, the free enzyme forms of the α-type enzyme, β-type enzyme and glucose dehydrogenase are obtained by resuspending the whole-cell enzyme bacteria of the three enzymes in a buffer solution to obtain a resuspended enzyme solution, and then crushing, clarifying and purifying the obtained crushed enzyme solution, supernatant enzyme solution and purified enzyme solution.

[0016] In certain embodiments of the present invention, the enzymatic reaction can be carried out in a reaction vessel reaction system in one step or multiple steps.

[0017] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0018] (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 glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution;

[0019] (2) The reaction substrate solution prepared in step (1) is uniformly mixed with a certain proportion of α-type enzyme, β-type enzyme and glucose dehydrogenase, the volume is fixed, and the mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, alcohol precipitation is performed, the supernatant is concentrated by clarification, and drying is performed to obtain artificial bear bile powder.

[0020] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0021] (1) Alternatively, a certain amount of refined poultry and livestock bile powder is weighed and dissolved in a buffer solution, coenzyme I or coenzyme II and glucose are added, and the pH is adjusted to between 6.5 and 8.5 to obtain a reaction substrate solution;

[0022] (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain proportion of α-type enzyme and β-type enzyme, fixed to volume, and fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain proportion of β-type enzyme and glucose dehydrogenase, fixed to volume, and fully contacted and reacted at 20-30° C. 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 artificial bear bile powder.

[0023] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0024] (1) Alternatively, a certain amount of refined poultry and livestock bile powder is weighed and dissolved in a buffer solution, coenzyme I or coenzyme II and glucose are added, and the pH is adjusted to between 6.5 and 8.5 to obtain a reaction substrate solution;

[0025] (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain proportion of α-type enzyme and β-type enzyme, fixed to volume, and fully contacted with each other at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain amount of glucose dehydrogenase, fixed to volume, and fully contacted with each other at 20-30° C. 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 artificial bear bile powder.

[0026] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0027] (1) Alternatively, a certain amount of refined poultry and livestock bile powder is weighed and dissolved in a buffer solution, coenzyme I or coenzyme II and glucose are added, and the pH is adjusted to between 6.5 and 8.5 to obtain a reaction substrate solution;

[0028] (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain amount of α-type enzyme, fixed to volume, and fully contacted with the reaction at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain proportion of β-type enzyme and glucose dehydrogenase, 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, concentrated, and dried to obtain artificial bear bile powder.

[0029] 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 0.15-0.8 g / L; the final concentration of glucose is 0.2 M based on the volume of the conversion system after constant volume.

[0030] In certain embodiments of the present invention, the mass ratio of the α-type enzyme enzyme bacteria and the β-type enzyme enzyme bacteria to the refined poultry and livestock bile powder is 1:2:20 to 1:10:50; the mass ratio of the glucose dehydrogenase enzyme bacteria to the refined poultry and livestock bile powder is 1:10 to 1:25.

[0031] In certain embodiments of the present invention, the poultry and livestock bile powder is a refined poultry and livestock bile powder with a mass percentage of taurochenodeoxycholic acid ≥ 63% and a mass percentage of taurochenodeoxycholic acid in the poultry and livestock bile powder ≤ 3%. The taurochenodeoxycholic acid is TCA.

[0032] The present invention also provides artificial bear bile powder prepared by the above method. The artificial bear bile powder refers to artificial bear bile powder whose characteristic components are consistent with or close to those of natural bear bile powder.

[0033] The invention also provides application of the method in artificial bear bile powder.

[0034] [Beneficial Effects]

[0035] (1) In the process of preparing artificial bear bile powder by the dual enzyme enzymatic method of α-type enzyme and β-type enzyme, T-7-KLCA is an inevitable intermediate product in the process of converting TCDCA to TUDCA, and its content is usually 1.7-3%. The difference between the present invention and the present invention lies in that the third enzyme glucose dehydrogenase is introduced into the reaction system, so that the conversion reaction is further promoted in the direction of the product, and the content of the reaction intermediate T-7-KLCA is reduced to a lower level, so that the intermediate T-7-KLCA is more thoroughly converted into the reaction product. The final percentage content of T-7-KLCA can be reduced to within 0.35%, which is 6 to 11 times different, and meets the limit requirements of the intermediate T-7-KLCA of natural bear bile powder, reflecting that the three-enzyme multi-path enzymatic method composed of glucose dehydrogenase, α-type enzyme and β-type enzyme can play a key role in reducing the percentage content of the intermediate 7-ketolithocholic acid.

[0036] (2) The present invention uses refined poultry and livestock bile powder as a substrate and a multi-path enzymatic method using three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, to prepare artificial bear bile powder with a TUDCA content of more than 35%, a TCDCA content of more than 25%, a TUA content of less than 0.8%, a TCA content of less than 1.9%, a T-7-KLCA content of less than 0.35%, a TUDCA+TCDCA content of more than 60%, and a TUDCA / TCDCA content of more than 1.35. The artificial bear bile powder has better stability and consistency between batches and can be scaled up.

[0037] (3) The present invention uses a three-enzyme reaction system of α-type enzyme, β-type enzyme and glucose dehydrogenase. This reaction system can recycle the coenzyme and reduce the usage of β-type enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0039] The three enzymes used in the following examples are: α-type enzyme, β-type enzyme, and glucose dehydrogenase.

[0040] The enzymes used in the following examples mainly include: enzyme cells, resuspended enzyme solutions, broken enzyme solutions, supernatant enzyme solutions, and purified enzyme solutions of α-type enzymes, β-type enzymes, and glucose dehydrogenase.

[0041] The enzymatic reaction forms in the following embodiments mainly include: the enzymatic reaction can be carried out in a reaction vessel reaction system in one step or multiple steps.

[0042] In the following embodiments, artificial bear bile powder refers to a product whose characteristic components are consistent with or close to those of natural bear bile powder.

[0043] In the following examples, the buffer is purified water or glycine buffer, preferably purified water.

[0044] In the following embodiments, the coenzyme is coenzyme I or coenzyme II, preferably coenzyme I

[0045] In the following examples, the cofactor is glucose.

[0046] In the following examples, the recombinant Escherichia coli used is: refer to the Chinese application document with publication number CN109402212A.

[0047] In the following examples, when each enzyme is used in the form of an enzyme cell resuspension solution; or a broken solution solution; or a supernatant solution; or a purified enzyme solution, the method for determining the enzyme activity value is referred to the Chinese application document with publication number CN109402212A.

[0048] In the following examples, TUDCA, TCDCA, T-7-KLCA, TCA, and TUA were all detected by HPLC-ELSD in accordance with the Chinese Pharmacopoeia Volume IV (2020 Edition) (General Rules for High Performance Liquid Chromatography). The taurocholic acid is TUA.

[0049] Examples 1-4: Preparation of artificial bear bile powder by enzymatic conversion of heavy suspension enzyme solution

[0050] Embodiment 1:

[0051] (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 stirred to a final concentration of 0.15 g / L, glucose was added to a final concentration of 0.2 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, 1950 ml of a mixed heavy suspension enzyme solution containing 50 g of α-type enzyme whole-cell enzyme bacteria, 500 g of β-type enzyme whole-cell enzyme bacteria and 100 g of glucose dehydrogenase whole-cell enzyme bacteria was added, and the reaction solution was fixed to 6000 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25 ° C for 5 hours to obtain a conversion solution;

[0052] (2) The conversion solution obtained in step (1) 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 mixture was placed in a constant temperature water bath at 16° C. and allowed to stand for 4 hours for alcohol precipitation. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%. 967.32 g of a product with a TUDCA to TCDCA ratio between 1.1 and 1.5 was obtained.

[0053] The artificial bear bile powder prepared in step (2) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.04%, the content of TCDCA was 27.69%, the content of TUA was 0.61%, and the content of TCA was 1.82%. The content of T-7-KLCA was 0.28%, the percentage of TUDCA+TCDCA was 66.73%, and TUDCA / TCDCA=1.41.

[0054] Embodiment 2:

[0055] (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 stirred to a final concentration of 0.8 g / L, glucose was added to a final concentration of 0.2 M, the pH was adjusted to 8.5 with 5 M sodium hydroxide, 1650 ml of a mixed resuspension enzyme solution containing 50 g of α-type enzyme whole-cell enzyme bacteria and 500 g of β-type enzyme whole-cell enzyme bacteria was added, and the reaction solution was fixed to 5000 ml with pure water, and the reaction solution was placed in a constant temperature water bath at 25 ° C for 4 hours;

[0056] (2) The reaction solution obtained in step (1) was placed in a constant temperature water bath at 60° C. for termination reaction for 20 minutes, and then centrifuged. The supernatant was taken, and the pH was adjusted to 8.5 with 5M sodium hydroxide. 1125 ml of a mixed resuspension enzyme solution containing 250 g of β-type enzyme whole-cell enzyme bacteria and 100 g of glucose dehydrogenase whole-cell enzyme bacteria was added. The reaction solution was fixed to 7000 ml with pure water, and placed in a constant temperature water bath at 25° C. for reaction for 5 h to obtain a conversion solution.

[0057] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 952.93 g of artificial bear bile powder.

[0058] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.03%, the content of TCDCA was 26.73%, the content of TUA was 0.65%, and the content of TCA was 1.75%. The content of T-7-KLCA was 0.26%, the percentage of TUDCA+TCDCA was 65.76%, and TUDCA / TCDCA=1.46.

[0059] Embodiment 3:

[0060] (1) Weigh 1000 g of refined poultry and livestock bile powder, dissolve it in 2000 ml of 10 mM glycine solution, stir evenly, and add coenzyme NAD + The solution was stirred to a final concentration of 0.5 g / L, glucose was added to a final concentration of 0.2 M, the pH was adjusted to 6.5 with 5 M sodium hydroxide, 450 ml of a mixed resuspension enzyme solution containing 50 g of α-type enzyme whole-cell enzyme bacteria and 100 g of β-type enzyme whole-cell enzyme bacteria was added, 10 mM glycine solution was added to make the reaction solution volume 4000 ml, and the reaction solution was placed in a constant temperature water bath at 25 ° C for 4 hours;

[0061] (2) The reaction solution obtained in step (1) was placed in a constant temperature water bath at 60° C. to terminate the reaction for 20 minutes, then centrifuged, the supernatant was taken, the pH was adjusted to 6.5 with 5M sodium hydroxide, 120 ml of a mixed resuspension enzyme solution containing 40 g of whole cell enzyme bacteria of glucose dehydrogenase was added, 10 mM glycine solution was added to make the reaction solution volume 5000 ml, and the solution was placed in a constant temperature water bath at 25° C. to react for 5 h to obtain a conversion solution;

[0062] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 952.87 g of artificial bear bile powder.

[0063] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 38.95%, the content of TCDCA was 28.22%, the content of TUA was 0.53%, and the content of TCA was 1.85%. The content of T-7-KLCA was 0.31%, the percentage of TUDCA+TCDCA was 67.17%, and TUDCA / TCDCA=1.38.

[0064] Embodiment 4:

[0065] (1) Weigh 1000 g of refined poultry and livestock bile powder, dissolve it in 2000 ml of 10 mM glycine solution, stir evenly, and add coenzyme NAD + The solution was adjusted to a final concentration of 0.15 g / L, the pH was adjusted to 7.5 with 5 M sodium hydroxide, 60 ml of a resuspended enzyme solution containing 20 g of α-type enzyme whole-cell enzyme bacteria was added, 10 mM glycine solution was added to make the reaction solution 4000 ml, and the reaction solution was placed in a 25 ° C constant temperature water bath for 2 h;

[0066] (2) The reaction solution obtained in step (1) was placed in a 60°C constant temperature water bath for termination reaction for 20 minutes, then centrifuged, the supernatant was taken, glucose was added to a final concentration of 0.2 M, the pH was adjusted to 7.5 with 5 M sodium hydroxide, 240 ml of a mixed resuspension enzyme solution containing 40 g of β-type enzyme whole-cell enzyme bacteria and 40 g of glucose dehydrogenase whole-cell enzyme bacteria was added, 10 mM glycine solution was added to make the reaction solution volume 5000 ml, and the solution was placed in a 25°C constant temperature water bath for reaction for 4 h to obtain a conversion solution;

[0067] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1 L, 4 L of 95% ethanol was added, and the mixture was slowly stirred for 30 minutes. The mixture was placed in a constant temperature water bath at 16°C and allowed to stand for 4 hours for alcohol precipitation, and then centrifuged. The supernatant obtained by centrifugation was concentrated by rotary evaporation and vacuum dried to a dry product with a water content of less than 3%, thereby obtaining 958.51 g of artificial bear bile powder.

[0068] The artificial bear bile powder prepared in step (3) was tested using HPLC-ELSD in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 39.23%, the content of TCDCA was 25.64%, the content of TUA was 0.78%, and the content of TCA was 1.65%. The content of T-7-KLCA was 0.24%, the percentage of TUDCA+TCDCA was 64.87%, and TUDCA / TCDCA=1.53.

[0069] Comparative Example 1: Effect of Glucose Dehydrogenase on the Conversion of Artificial Bear Bile Powder

[0070] The specific implementation of Comparative Example 1 is the same as that of Example 3, except that glucose dehydrogenase is not used in step (2), and the reaction itself becomes a double enzyme reaction of only α-type enzyme and β-type enzyme. When other corresponding conditions remain unchanged, the reaction is completed to obtain 963.12g of artificial bear bile powder.

[0071] The artificial bear bile powder prepared in Comparative Example 1 was tested using HPLC-ELSD in accordance with the Chinese Pharmacopoeia Part IV (2020 Edition) (General Rules for High Performance Liquid Chromatography), wherein the content of TUDCA was 38.57%, the content of TCDCA was 29.45%, the content of TUA was 0.59%, and the content of TCA was 1.82%. The content of T-7-KLCA was 1.91%, the percentage of TUDCA+TCDCA was 68.02%, TUDCA / TCDCA=1.31, wherein the percentage of 1.91% T-7-KLCA was more than 6 times higher than the percentage of T-7-KLCA in Implementation 3 and other embodiments.

[0072] The present invention proposes a new method for preparing artificial bear bile powder. Through the refining of livestock bile powder and a three-enzyme multi-path enzymatic method, the content of the intermediate T-7-KLCA is reduced, and artificial bear bile powder with characteristic components closer to natural bear bile powder is prepared, thereby solving the problem of high intermediate T-7-KLCA content in the existing artificial bear bile powder preparation technology.

[0073] 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 artificial bear bile powder by three-enzyme enzymatic biotransformation, 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, β-hydroxysteroid dehydrogenase and glucose dehydrogenase to prepare artificial bear bile powder. The α-type hydroxysteroid dehydrogenase is referred to as the α-type enzyme, and the β-type hydroxysteroid dehydrogenase is referred to as the β-type enzyme; The three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, exist in the form of intracellular enzymes or free enzymes.

2. The method according to claim 1, characterized in that The coenzyme is coenzyme I or coenzyme II, and the cofactor is glucose; the reaction substrate is poultry and livestock bile powder or poultry and livestock bile concentrate.

3. The method according to claim 1, characterized in that The intracellular enzymes of the three enzymes, α-type enzyme, β-type enzyme and glucose dehydrogenase, exist in the form of whole-cell enzyme bacteria expressing α-type enzyme, β-type enzyme and glucose dehydrogenase respectively.

4. The method according to claim 1, characterized in that The intracellular enzymes of the α-type enzyme, the β-type enzyme and the glucose dehydrogenase exist in the form of resuspended enzyme solutions obtained by resuspending the whole-cell enzyme bacteria of the three enzymes in a buffer solution.

5. The method according to claim 1, characterized in that The free enzyme forms of the α-type enzyme, β-type enzyme and glucose dehydrogenase are obtained by resuspending the whole-cell enzyme bacteria of the three enzymes in a buffer solution to obtain a resuspended enzyme solution, and then crushing, clarifying and purifying the obtained crushed enzyme solution, supernatant enzyme solution and purified enzyme solution.

6. 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 vessel reaction system.

7. The method according to any one of claims 1 to 6, 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 glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution prepared in step (1) is uniformly mixed with a certain proportion of α-type enzyme, β-type enzyme and glucose dehydrogenase, the volume is fixed, and the mixture is fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is concentrated by clarification, alcohol precipitation is performed, the supernatant is concentrated by clarification, and drying is performed to obtain artificial bear bile powder.

8. The method according to any one of claims 1 to 6, 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 glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain proportion of α-type enzyme and β-type enzyme, fixed to volume, and fully contacted and reacted at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain proportion of β-type enzyme and glucose dehydrogenase, fixed to volume, and fully contacted and reacted at 20-30° C. 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 artificial bear bile powder.

9. The method according to any one of claims 1 to 6, 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 glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain proportion of α-type enzyme and β-type enzyme, fixed to volume, and fully contacted with each other at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain amount of glucose dehydrogenase, fixed to volume, and fully contacted with each other at 20-30° C. 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 artificial bear bile powder.

10. The method according to any one of claims 1 to 6, 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 glucose, and adjust the pH to between 6.5 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution prepared in step (1) is mixed evenly with a certain amount of α-type enzyme, fixed to volume, and fully contacted with the reaction at 20-30° C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 6.5 and 8.5, and mixed evenly with a certain proportion of β-type enzyme and glucose dehydrogenase, 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, concentrated, and dried to obtain artificial bear bile powder.

11. The method according to any one of claims 4 to 10, 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 0.15-0.8 g / L; the final concentration of glucose is 0.2 M based on the volume of the conversion system after constant volume.

12. The method according to claim 1, characterized in that The mass ratio of the α-type enzyme bacteria and the β-type enzyme bacteria to the reaction substrate is 1:2:20 to 1:10:50; the mass ratio of the glucose dehydrogenase enzyme bacteria to the reaction substrate is 1:10 to 1:

25.

13. 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% and a mass percentage of taurochenodeoxycholic acid in the poultry and livestock bile powder of ≤3%.

14. An artificial bear bile powder, characterized in that: Prepared by the method according to any one of claims 1 to 13.

15. Application of the method according to any one of claims 1 to 13 in artificial bear bile powder.

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