Method for preparing artificial bear gall powder through double enzymes, artificial bear gall powder and application of method

Abstract

The invention discloses a method for preparing artificial bear gall powder through double enzymes, the artificial bear gall powder and application of the method, and belongs to the technical field of biology. The method comprises the following steps: respectively immobilizing two enzymes, and filling the two immobilized enzymes into a column or a reactor according to a ratio. The method can reasonably and effectively control the proportion of the amount of two enzymes participating in the reaction, so that the enzymatic reaction is effectively controlled, and the stability and consistency of the batch-to-batch conversion reaction are ensured. And after the enzymatic reaction is finished, the immobilized enzyme can be recycled in manners of centrifugation, filtration, magnetic separation and the like and is fixed according to the enzymatic reaction requirement, so that the immobilized enzyme can be recycled for multiple times, the use amount of the enzyme is saved, and the use efficiency of the enzyme is improved. The method provided by the invention is strong in operability, good in repeatability and easy in linear amplification, the ratio of TUDCA to TCDCA in the obtained product can reach 1.33 or above, and the method is suitable for industrial production of artificial bear gall powder.

Description

Technical Field

[0001] The invention relates to a method for preparing artificial bear bile powder with double enzymes, the artificial bear bile powder and the application of the method, and belongs to the field of biotechnology. Background Art

[0002] Bear bile powder has the effects of clearing away heat and detoxifying, calming the liver and improving eyesight, eliminating inflammation, and dissolving gallstones. It is usually obtained by drying the bile of bears. Natural bear bile powder usually contains a high content of TUDCA, whose scientific name is tauroursodeoxycholic acid. It is a combination of ursodeoxycholic acid (UDCA) and taurine. It has good solubility in aqueous solution and is easy for human body to absorb. In recent years, with the vigorous development of the bio-industry, some companies have combined immobilized enzyme technology with enzyme catalysis technology and developed a method of converting artificial bear bile powder or bile conversion products with high TUDCA content through immobilized enzyme method. However, these methods generally have problems such as poor stability between batches, low reuse rate of immobilized enzymes, and inability to linearly amplify, which limits the industrial development of artificial bear bile powder. Chinese invention patent CN201410588581.5 uses chitosan as the main substrate for dual enzyme fixation. This method simultaneously immobilizes α-hydroxysteroid dehydrogenase and β-hydroxysteroid dehydrogenase as dual enzymes, and then uses the immobilized dual enzymes to convert the substrate of livestock bile powder. This method cannot accurately control the ratio between the two enzymes, the stability of the conversion between batches is poor, and the content of the intermediate is also uncontrollable. If the enzyme activity of the two types of chitosan immobilized enzymes after mixing is attenuated, the two types of immobilized enzymes cannot be supplemented and fixed, so it is difficult to reuse them, which limits their application in the industrial production of artificial transformation of bear bile powder. Chinese invention patent CN 202210387293.8 also uses immobilized enzyme technology, but the difference is that it replaces the chitosan column with an ion exchange agarose gel column. Using ion exchange agarose gel as the immobilization substrate, its application scenario is restricted by many factors such as ambient temperature and pH. The interaction between ion exchange agarose gel and the target protease is the mutual attraction between the charge carried by the gel and the opposite charge carried by the protease molecule to produce the "immobilization" effect, and this adsorption force is usually weak. In the process of bear bile powder conversion, the substrate livestock bile powder is acidic. After participating in the reaction, the enzymatic reaction liquid will gradually develop in the direction of acidification. A slight change in the pH in the reaction system will lead to desorption. In addition, this method also fixes the two enzymes at the same time, and it is impossible to accurately control the ratio between the two enzymes. Therefore, the immobilized enzyme method prepared by ion exchange agarose gel has no practical significance in the production of artificial bear bile powder conversion. Summary of the invention

[0003] [Technical issues]

[0004] In order to overcome the shortcomings of the existing artificial bear bile powder conversion technology, the first technical problem to be solved by the present invention is to provide a method for preparing artificial bear bile powder with dual enzymes, which can prepare artificial bear bile powder with characteristic components consistent with or close to those of natural bear bile powder on demand.

[0005] 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.

[0006] The third technical problem to be solved by the present invention is to provide a method for applying artificial bear bile powder.

[0007] [Technical solution]

[0008] The present invention provides a method for preparing artificial bear bile powder by using double enzymes, the method comprising the following steps: in the presence of a coenzyme, a reaction substrate containing taurochenodeoxycholic acid is subjected to an enzymatic reaction with α-type hydroxysteroid dehydrogenase and β-type hydroxysteroid dehydrogenase, part of the taurochenodeoxycholic acid is converted into tauroursodeoxycholic acid, and artificial bear bile powder is obtained. The taurochenodeoxycholic acid is TCDCA.

[0009] In certain embodiments of the present invention, the α-type enzyme and the β-type enzyme are in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II; and the reaction substrate is livestock bile powder or livestock bile concentrate.

[0010] In certain embodiments of the present invention, the immobilized enzymes of the α-type enzyme and the β-type enzyme are in the form of immobilized α-type enzyme and immobilized β-type enzyme formed by combining the α-type enzyme and the β-type enzyme with immobilization resins, respectively.

[0011] In certain embodiments of the present invention, the immobilized enzyme of the α-type enzyme is in the form of an α-type enzyme immobilized enzyme formed by mixing the α-type enzyme with a certain amount of coenzyme I or coenzyme II and then combining the mixture with an immobilization resin.

[0012] 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.

[0013] 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.

[0014] In certain embodiments of the present invention, the enzymatic reaction mainly comprises the following steps:

[0015] (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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution;

[0016] (2) The reaction substrate solution is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme immobilized enzyme, 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.

[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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution;

[0019] (2) The reaction substrate solution is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 7.0 and 8.5, and the solution is mixed evenly with the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, the supernatant is concentrated, and dried to obtain artificial bear bile powder.

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

[0021] (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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution;

[0022] (2) The reaction substrate solution is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 7.0 and 8.5, and the solution is mixed evenly with the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, the supernatant is concentrated, and dried to obtain artificial bear bile powder.

[0023] In certain embodiments of the present invention, the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme are in the form of enzyme cell weight suspension enzyme liquid containing α-type enzyme, β-type enzyme, and isoenzymes thereof expressed by Escherichia coli, Bacillus subtilis, and yeast, respectively, and the crushed liquid enzyme liquid, supernatant enzyme liquid, and purified enzyme liquid obtained by crushing, clarifying, and purifying are combined with immobilized resin to form the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme.

[0024] In certain embodiments of the present invention, the buffer is purified water, or glycine buffer, Tris buffer, or phosphate buffer; the coenzyme I or coenzyme II refers to NAD + or NADP + , calculated on the volume of the conversion system after constant volume, the final concentration of coenzyme I or coenzyme II is 0.15-0.65 g / L; the final concentration of the refined poultry and livestock bile powder is 170 g / L on the volume of the conversion system after constant volume.

[0025] In certain embodiments of the present invention, the mass ratio of the immobilized α-type enzyme to the immobilized β-type enzyme is 1:2 to 1:10; the mass ratio of the immobilized α-type enzyme to the refined poultry and livestock bile powder is 1:20 to 1:50.

[0026] In certain embodiments of the present invention, the mass ratio of TUDCA to TCDCA in the artificial bear bile powder is 1.33:1 to 1.80:1, the sum of the percentages of TUDCA and TCDCA is ≥58%, and the mass percentage of TUDCA is ≥32%.

[0027] 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.

[0028] The invention also provides application of the method in artificial bear bile powder.

[0029] [Beneficial Effects]

[0030] (1) With the help of resin, enzyme protein molecules can be physically stuck on the micropores of the resin, with a large protein loading capacity and relatively firm fixation. Even if there is slight vibration or pressure, the enzyme protein molecules stuck on the micropores are not easy to fall off. The physical and chemical properties of the resin are relatively stable, and it can adapt to the medium and low pressure environment of 0.4Mpa, and has good stability under the conditions of pH 5.5-8.5.

[0031] (2) The preparation method adopts the method of first immobilizing the two enzymes separately, and then loading the immobilized enzymes into a column or a reactor according to the proportion. This method can reasonably and effectively control the ratio of the amount of the two enzymes involved in the reaction, thereby effectively controlling the generation of intermediates and ensuring the stability and consistency of the conversion reaction between batches.

[0032] (3) After an enzymatic reaction is completed, the immobilized enzyme can be recovered by centrifugation, filtration, magnetic separation, etc. and re-immobilized according to the requirements of the enzymatic reaction, so that it can be repeatedly recycled many times, saving the amount of enzyme, improving the efficiency of enzyme use, and reducing the production cost and time cost caused by bacterial transformation.

[0033] (4) The preparation method is highly operable, reproducible, and easy to scale up linearly. The amount and ratio of TUDCA and TCDCA in the obtained product are stable and controllable, and is suitable for industrial production of artificial bear bile powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0035] The two enzymes used in the following examples are: α-type enzyme and β-type enzyme.

[0036] The enzymes used in the following examples mainly include: α-type immobilized enzymes and β-type immobilized enzymes.

[0037] 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.

[0038] In the following embodiments, artificial bear bile powder refers to artificial bear bile powder whose controllable components are consistent with or close to the characteristic components of bear bile powder. In the following embodiments, the buffer is purified water, or glycine buffer, Tris buffer, phosphate buffer, preferably purified water.

[0039] In the following embodiments, the coenzyme is coenzyme I or coenzyme II, preferably coenzyme I.

[0040] In the following embodiments, the bacteria used to express α-hydroxysteroid dehydrogenase and β-hydroxysteroid dehydrogenase, respectively, can be constructed using conventional recombinant Escherichia coli or recombinant Bacillus subtilis construction methods, for example, a gene encoding an enzyme is selected from the prior art and expressed using Escherichia coli or Bacillus subtilis as a host and a conventional expression strategy.

[0041] In the following examples, the recombinant Escherichia coli used is: refer to the Chinese application document with publication number CN109402212A.

[0042] In the following embodiments, the recombinant Bacillus subtilis used: refer to the Chinese application document with application number 2022109651623.

[0043] In the following examples, TUDCA, TCDCA, the intermediate tauro-7-ketolithocholic acid, TCA, and tauro-ursocholic acid were all detected by HPLC-ELSD in accordance with the Chinese Pharmacopoeia Volume IV (2020 Edition) (General Rules for High Performance Liquid Chromatography). The tauro-7-ketolithocholic acid is T-7-KLCA, and the tauro-ursocholic acid is TUA.

[0044] In the following examples, the main methods for preparing the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme are as follows:

[0045] (1) Resin pretreatment: Weigh a certain amount of epoxy resin LXTE-1000, wash it three times with 100 mM pH 7.5 phosphate (PB) buffer at a mass volume ratio of 1:20 (g / L), and then filter it to dryness;

[0046] (2) Preparation of enzyme solution: Weigh the corresponding amount of enzyme bacteria expressing intracellular α-type enzyme and intracellular β-type enzyme, resuspend them in 100 mM pH 7.5 PB buffer at a mass volume (g / L) ratio of 1:20, crush and centrifuge to obtain the supernatant enzyme solution, and make up to volume at a mass volume (g / L) ratio of 1:5.

[0047] (3) Immobilization of α-type enzyme and β-type enzyme: The α-type enzyme supernatant enzyme solution and the β-type enzyme supernatant enzyme solution prepared in step (2) are respectively mixed evenly with the epoxy resin pretreated in step (1), and placed in a shaking incubator at 20° C. to be fully fixed. After the fixation is completed, the α-type enzyme and the β-type enzyme are immobilized by centrifugation to remove the liquid.

[0048] In the following examples, when the α-type enzyme and the β-type enzyme are present in the form of immobilized enzymes, the method for determining the enzyme activity values ​​is as follows:

[0049] The enzyme activity value (U / mg immobilized enzyme) of the α-type enzyme was measured: 5mM taurochenodeoxycholic acid was used as the substrate, and NADP with a final concentration of 0.2mM was added to a 250ml pH 8.0 100mM phosphate buffer reaction system. + and 50 mg of α-type immobilized enzyme, react at 25°C for 5 minutes. Take samples, filter, and measure the change of absorbance of the clear solution at 340 nm, and compare it with the blank control to calculate the enzyme activity value of α-type immobilized enzyme.

[0050] The enzyme activity value (U / mg immobilized enzyme) of the β-type enzyme was measured: 5mM tauroursodeoxycholic acid was used as the substrate, and NADP with a final concentration of 0.2mM was added to a 250ml pH 8.0 100mM phosphate buffer reaction system. + and 50 mg of β-type immobilized enzyme, react at 25°C for 5 minutes. Take samples, filter, measure the change of absorbance of the clear solution at 340 nm, and compare with the blank control to calculate the enzyme activity value of β-type immobilized enzyme.

[0051] Example 1 Preparation of artificial bear bile powder by immobilized enzyme catalysis

[0052] Resin pretreatment: Weigh 20 g and 40 g of epoxy resin LXTE-1000 respectively, wash them three times with 40 ml and 80 ml of 100 mM phosphate (PB) buffer with a pH of 7.5 respectively, and then filter and dry.

[0053] Preparation of enzyme solution: Weigh 50g of recombinant E. coli expressing α-type enzyme and 100g of recombinant E. coli expressing β-type enzyme, respectively, stir and resuspend with 200ml and 400ml of 100mM PB buffer with pH 7.5, respectively, and then repeatedly break for 3 times, centrifuge the broken liquid at 20000rpm for 20min, discard the precipitate, collect the supernatant enzyme solution, and make the volume to 250ml and 500ml respectively.

[0054] Immobilization of α-type enzyme: In a 3000 mL shake flask, mix 250 ml of the supernatant of the α-type enzyme with 20 g of the resin treated in the pretreatment of the resin, and then place the shake flask in a shaker at 20°C and 100 rpm for more than 3 hours. Centrifuge the immobilized α-type enzyme at 1000 rpm, discard the supernatant, and collect the immobilized enzyme.

[0055] The enzyme activity value of the α-type enzyme immobilized enzyme was measured. With 5mM taurochenodeoxycholic acid as the substrate, NADP+ with a final concentration of 0.2mM and 50mg of α-type enzyme 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 increase in absorbance of the clear liquid was measured at 340nm. Compare and calculate with the blank control. The enzyme activity value of the α-type enzyme immobilized enzyme is: 1.13U / mg.

[0056] Immobilization of β-type enzyme: In a 3000mL shake flask, 500ml of the supernatant of the β-type enzyme was mixed evenly with 40g of the resin treated in the pretreatment of the resin, and then the shake flask was placed in a shaker at 20°C and 100rpm for more than 3 hours. The immobilized β-type enzyme was centrifuged at 1000rpm, the supernatant was discarded, and the immobilized β-type enzyme was collected.

[0057] The enzyme activity of the immobilized β-type enzyme was measured. Using 5 mM tauroursodeoxycholic acid as the substrate, NADP with a final concentration of 0.2 mM was added to a 250 ml pH 8.0 100 mM phosphate buffer reaction system. + and 50 mg of β-type enzyme immobilized enzyme, react at 25℃ for 5 minutes. Take samples, filter, and measure the increase of absorbance value of the clear liquid at 340nm. Compare and calculate with the blank control. The enzyme activity value of β-type enzyme immobilized enzyme is: 2.62U / mg.

[0058] Enzymatic reaction:

[0059] (1) Weigh 1000g of refined poultry and livestock bile powder and dissolve it in 2000ml of 0.1M glycine buffer, stir evenly, and add coenzyme NADP +The solution was adjusted to a final concentration of 0.15 g / L, the pH was adjusted to 8.5, 180 ml of a mixed immobilized enzyme resuspension containing 20 g of α-type enzyme immobilized enzyme and 40 g of β-type enzyme immobilized enzyme was added, 0.1 M glycine buffer was added to make the reaction solution 4000 ml, and the reaction solution was stirred evenly. The reaction solution was placed in a 25°C constant temperature water bath and slowly stirred for reaction;

[0060] (2) Sampling the conversion solution at 2h, 4h, 6h, 12h, and 18h ​​of conversion, respectively, and clarifying the supernatant to dry the dried product to a water content of less than 3%, and then testing the content;

[0061] Using HPLC-ELSD, the components in the dried product obtained by sampling the transformation product at different transformation time periods in step (2) were detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The test results were calculated and organized as follows, see Table 1.

[0062] Table 1 Conversion results corresponding to different conversion times

[0063]

[0064] Examples 2 to 8 and Comparative Examples 1 to 4: Effects of different conditions on the preparation of artificial bear bile powder by immobilized enzyme catalysis

[0065] The specific implementation methods of Examples 2 to 8 and Comparative Examples 1 to 4 are the same as those of Example 1, with the main differences being the type of enzyme expression strain, the ratio of the volume of the enzyme solution used for immobilization to the mass of the resin (L / Kg), the mass ratio of the immobilized enzyme of the α-type enzyme to the immobilized enzyme of the β-type enzyme under the premise that the mass ratio of the immobilized enzyme of the α-type enzyme to the substrate is 1:50, the buffer type of the conversion system, the pH value of the conversion system, the concentration of the coenzyme in the conversion system, the conversion temperature and time. For specific conditions, see Table 2.

[0066] Using HPLC-ELSD, the components in the dried products obtained by sampling the conversion products of Examples 2 to 8 and Comparative Examples 1 to 4 were detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The test results were calculated and organized as follows, see Table 3.

[0067] Table 2 Specific parameters of different conditions in Examples 2 to 8 and Comparative Examples 1 to 7

[0068]

[0069]

[0070] Table 3 Content and ratio of components in the dried products obtained from the conversion products prepared in Examples 2 to 8 and Comparative Examples 1 to 7

[0071]

[0072]

[0073] Example 10 Preparation of 10 kg artificial bear bile powder by immobilized enzyme catalysis

[0074] The specific implementation method of Example 10 is the same as that of Example 1, with the main difference being that 10,000 g of refined poultry and livestock bile powder, 200 g of α-type immobilized enzyme, and 400 g of β-type immobilized enzyme are used, so the entire reaction system is correspondingly enlarged 10 times, but the reaction time is 6 hours, and 9,600.05 g of artificial bear bile powder is obtained.

[0075] Using HPLC-ELSD, the components of the artificial bear bile powder obtained in Example 12 were detected in accordance with the "Chinese Pharmacopoeia Volume IV (2020 Edition)" (General Rules for High Performance Liquid Chromatography). The test results were calculated and collated: the content of TUDCA+TCDCA was 56.99%, of which the content of TUDCA was 34.46%, and TUDCA / TCDCA=1.53.

[0076] Example 11 Immobilized enzyme mixed column conversion

[0077] (1) Weigh 1000g of refined poultry and livestock bile powder and dissolve it in 5000ml of pure water, stir evenly, and add coenzyme NADP + The solution was stirred to a final concentration of 0.15 g / L, the pH value of the solution was adjusted to 7.5, the volume was made up to 6000 ml with pure water, and the mixture was stirred evenly. The mixture was introduced into a mixed immobilized enzyme column containing 50 g of α-type enzyme immobilized enzyme and 500 g of β-type enzyme immobilized enzyme in a constant temperature water bath at 25°C at a constant flow rate. The enzymatic reaction was fully contacted and reacted in the column for 12 hours in a manner of circulating the column at a constant flow rate to obtain a conversion solution.

[0078] (2) The conversion liquid obtained in step (1) 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 and dried to a dry product with a water content of less than 3%, thereby obtaining 958.37 g of artificial bear bile powder.

[0079] 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). The test results were calculated and collated to obtain: the content of TUDCA+TCDCA was 58.31%, of which the content of TUDCA was 33.60%, and TUDCA / TCDCA=1.36.

[0080] Example 12 Immobilized enzymes were loaded into columns and converted

[0081] (1) Weigh 1000g of refined poultry and livestock bile powder, dissolve it in 5000ml of pure water, 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, the volume was made up to 6000 ml with pure water, and the mixture was stirred evenly. The mixture was introduced into an immobilized enzyme column containing 50 g of α-type immobilized enzyme in a constant temperature water bath at 25°C at a constant flow rate. The enzymatic reaction was circulated on the column at a constant flow rate for 8 hours to obtain a conversion intermediate solution.

[0082] (2) Add 5M sodium hydroxide to the conversion intermediate solution prepared in step (1) to adjust the pH to 7.5, add pure water to make the volume 7000 ml, stir evenly, and introduce into an immobilized enzyme column containing 500 g of β-type enzyme immobilized enzyme placed in a constant temperature water bath at 25°C at a constant flow rate, and react in the column for 12 hours in a manner of circulating on the column at a constant flow rate to obtain a conversion solution;

[0083] (3) The conversion liquid obtained in step (2) was centrifuged, and the obtained supernatant was concentrated to 1L, 4L 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 956.91g of artificial bear bile powder.

[0084] 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). The test results were calculated and collated to obtain: the content of TUDCA+TCDCA was 58.13%, of which the content of TUDCA was 33.71%, and TUDCA / TCDCA=1.38.

[0085] Examples 13-21 Effect of repeated use of immobilized enzymes on the preparation of artificial bear bile

[0086] The specific implementation methods of Examples 13 to 21 are the same as those of Example 12, with the main difference being that the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme used in Example 13 are the immobilized enzymes re-cleaned and balanced after the completion of Example 12; the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme used in Examples 14 to 21 are the immobilized enzymes re-cleaned and balanced after the completion of the previous example. Using HPLC-ELSD, the components in the artificial bear bile powder samples obtained in Examples 13 to 21 were detected in accordance with the "Chinese Pharmacopoeia Volume 4 (2020 Edition)" (General Rules for High Performance Liquid Chromatography), and the test results were calculated and sorted as follows, see Table 4.

[0087] Table 4 Test results of characteristic components of artificial bear bile powder prepared in Examples 13 to 21

[0088] Example TUDCA content (%) Content of TUDCA+TCDCA (%) TUDCA / TCDCA Example 13 35.00 59.66 1.42 Embodiment 14 35.10 59.82 1.42 Embodiment 15 35.01 60.01 1.40 Example 16 34.91 60.20 1.38 Embodiment 17 35.10 60.17 1.40 Embodiment 18 34.81 60.22 1.37 Embodiment 19 34.55 60.33 1.34 Embodiment 20 34.35 60.57 1.31 Embodiment 21 34.19 60.69 1.29

[0089] The above repeated transformation experiments were completed within 1 month, and each transformation took 12 hours. After the transformation was completed, the immobilized enzyme was collected by centrifugation and stored at 4°C.

[0090] The data of Examples 12 to 19 show that the immobilized enzyme prepared by this method can be reused at least 8 times under similar conditions, and TUDCA / TCDCA can be maintained above 1.34.

[0091] The present invention can provide a novel preparation method of artificial bioconversion bear bile powder. The method uses resin substances as carriers for immobilizing hydroxysteroid dehydrogenase, and fixes hydroxysteroid dehydrogenase protein molecules on the carriers through unique functional groups on the carriers, which is stable and reliable, has a large loading capacity and is not easy to fall off.

[0092] The preparation method adopts the method of first immobilizing the two enzymes separately, which can reasonably and effectively control the ratio of the amounts of the two enzymes involved in the reaction, thereby effectively controlling the generation of intermediates and achieving the stability of the conversion reaction between batches.

[0093] After the immobilized α-type enzyme and β-type enzyme are completed in one substrate conversion reaction, the immobilized enzymes can be recovered by centrifugation, filtration, magnetic adsorption, etc., and supplemented and fixed according to the degree of enzyme activity attenuation of the two enzymes and the requirements of the enzymatic reaction, so as to achieve the effect of multiple recycling, saving enzyme dosage and ensuring the stability of enzymatic reaction between batches. The artificial bear bile powder obtained by this conversion method has high consistency and stability between batches, and the cost is controllable. The content and ratio of TUDCA and TCDCA in the artificial bear bile powder are also ideally controllable.

[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 artificial bear bile powder using double enzymes, characterized in that: In the presence of coenzyme, a reaction substrate containing taurochenodeoxycholic acid is subjected to enzymatic reaction with α-hydroxysteroid dehydrogenase and β-hydroxysteroid 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.

2. The method according to claim 1, characterized in that The α-type enzyme and the β-type enzyme are in the form of immobilized enzymes; the coenzyme is coenzyme I or coenzyme II; and the reaction substrate is livestock bile powder or livestock bile concentrate.

3. The method according to claim 2, characterized in that The immobilized enzymes of the α-type enzyme and the β-type enzyme exist in the form of α-type enzyme immobilized enzyme and β-type enzyme immobilized enzyme formed by combining the α-type enzyme and the β-type enzyme with immobilization resins respectively.

4. The method according to claim 2 or 3, characterized in that: The immobilized enzyme of the α-type enzyme exists in the form of an α-type enzyme immobilized enzyme formed by mixing the α-type enzyme with a coenzyme and then combining the α-type enzyme with an immobilization resin.

5. 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%.

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 or in a multi-combination immobilized enzyme column 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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution is mixed evenly with a certain proportion of α-type enzyme immobilized enzyme and β-type enzyme immobilized enzyme, 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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 7.0 and 8.5, and the solution is mixed evenly with the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, the supernatant is concentrated, and dried to obtain artificial bear bile powder.

9. The method according to 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 adjust the pH to between 7.0 and 8.5 to obtain a reaction substrate solution; (2) The reaction substrate solution is mixed evenly with a certain proportion of the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is clarified and the pH is adjusted to between 7.0 and 8.5, and the solution is mixed evenly with the β-type enzyme immobilized enzyme, the volume is fixed, and the reaction is fully contacted at 20-30°C. After the reaction is completed, the supernatant is concentrated, precipitated with alcohol, the supernatant is concentrated, and dried to obtain artificial bear bile powder.

10. The method according to any one of claims 7 to 9, characterized in that: The α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme are in the form of enzyme cell weight suspension enzyme liquids respectively expressed by Escherichia coli, Bacillus subtilis and yeast containing α-type enzyme, β-type enzyme and isozymes thereof, and the crushed liquid enzyme liquid, supernatant enzyme liquid and purified enzyme liquid obtained by crushing, clarifying and purifying are combined with immobilized resin to form the α-type enzyme immobilized enzyme and the β-type enzyme immobilized enzyme.

11. The method according to any one of claims 7 to 9, characterized in that: The buffer is purified water, or glycine buffer, Tris buffer, or phosphate 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 0.15-0.65 g / L.

12. The method according to any one of claims 7 to 9, characterized in that: The mass ratio of the α-type enzyme immobilized enzyme to the β-type enzyme immobilized enzyme is 1:2-1:10; the mass ratio of the α-type enzyme immobilized enzyme to the reaction substrate is 1:20-1:

50.

13. The method according to claims 7 to 9, characterized in that: The mass ratio of tauroursodeoxycholic acid to taurochenodeoxycholic acid in the artificial bear bile powder obtained by the reaction is 1.33:1-1.80:1, the sum of the percentages of tauroursodeoxycholic acid and taurochenodeoxycholic acid is ≥58%, and the mass percentage of tauroursodeoxycholic acid is ≥32%.

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

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

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