CN112391419A - Biological catalysis preparation method of tauroursodeoxycholic acid - Google Patents
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Abstract
The invention discloses a biological catalytic preparation method of tauroursodeoxycholic acid (TUDCA), which comprises the first step of utilizing TCDCA oxidase to dehydrogenate TCDCA to be oxidized into tauro-7-ketolithocholic acid (T-7-KLCA), and utilizing ketoreductase to catalyze acetone to generate isopropanol so as to provide coenzyme regeneration of NADP +/NAD +; in the second step, T-7-KLCA is reduced to TUDCA by T-7-KLCA reductase, and isopropanol dehydrogenase is used to catalyze the generation of acetone from isopropanol to provide coenzyme regeneration of NADPH/NADH. The double enzyme-linked reaction can carry out the second step reaction by simple treatment after the first step reaction is finished without intermediate purification; the addition amount of NADP +/NAD + in the reaction is small, and acetone and isopropanol are mutual substrates and products in the two-step reaction, and can be recycled, so that the production cost and the environmental protection pressure are greatly reduced. Provides a new economic, efficient and environment-friendly large-scale industrial production process for generating TUDCA by the taurochenodeoxycholic acid (TCDCA) double-enzyme-linked reaction.
Description
Technical Field
The invention relates to the field of biocatalysis, in particular to a biocatalysis preparation method of tauroursodeoxycholic acid and a coenzyme regeneration system.
Background
Tauroursodeoxycholic acid (TUDCA), having the chemical name of 3 alpha, 7 beta dihydroxycholanyl-N-taurine, is a conjugated bile acid formed by the shrinkage between the carboxyl group of ursodeoxycholic acid (UDCA) and the amino group of taurine. It was found in 1902 that bear's gall is the main bile acid in bear's gall, and has spasmolytic, anticonvulsive, anti-inflammatory and cholelithiasis-dissolving effects. The traditional Chinese medicine composition is mainly used for treating cholecystolithiasis, primary sclerosing cholangitis, primary biliary cirrhosis, chronic viral hepatitis C and the like in clinic.
The current industrial production of TUDCA is generally a chemical process, and although the process is continuously optimized and improved in recent years, the chemical process is generally complex, low in yield, high in cost, environment-friendly and poor in safety, which is also a direct reason for the high price of TUDCA. In the years, with the popularization of the domestic enzyme catalysis market, a plurality of manufacturers and scientific research units begin to try to produce TUDCA through enzyme catalysis, but most of the currently published documents and patents are only primary attempts of enzyme catalysis processes, and have a great distance from industrial production. The patent CN107287272A adopts escherichia coli which simultaneously expresses 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH) genes to carry out liquid fermentation, and taurochenodeoxycholic acid is converted into tauroursodeoxycholic acid in the fermentation process, although the method can obtain a final product, the time is too long, the patent expression is 1-3 days (preferably 2-3 days), autolysis is easy to occur in the industrial large-scale recombinant escherichia coli fermentation process, and once the autolytic substrate is recycled, the method is a problem, so that the method is only one discussion of an enzyme catalysis process, and the economy and the stability are difficult to meet the requirements of industrial production. Patent CN102994604B uses recombinase 7 alpha-HSDH and 7 beta-HSDH to perform in-situ catalysis on conjugated state CDCA (including TCDCA) in bile/powder to generate conjugated state UDCA (including TUDCA), but it does not use coenzyme regeneration system, adds a large amount of NADP + and NADPH in two-step enzyme catalysis reaction, only this cost exceeds the value of product, and its conversion rate is between 55% -60%, the conversion rate is too low, the post-extraction difficulty is large, the yield is low, and it has no production condition for amplification. According to a coenzyme regeneration system in the patent CN109402212A two-step enzymatic reaction, lactic acid is generated by catalyzing lactic acid dehydrogenase used in the first step and alpha-keto acid, NADPH is converted into NADP +, glucose is converted into sodium gluconate by using glucose dehydrogenase in the second step, and NADP + is converted into NADPH.
Disclosure of Invention
Aiming at the defects in the prior art, the invention realizes the acetone-isopropanol circulation auxiliary tauroursodeoxycholic acid biocatalytic preparation method by adopting double-enzyme-linked catalytic reaction, and the method has the advantages of simple process, low cost, high conversion rate, easier post-treatment and more environmental protection. And simultaneously, the acetone and the isopropanol are easy to distill and recycle, so that the cost is reduced and the subsequent treatment procedures are reduced.
In order to achieve the above object, the present invention adopts the following aspects.
A biological catalysis preparation method of tauroursodeoxycholic acid is characterized by comprising the following steps:
(1) the first step of enzyme-catalyzed reaction: catalyzing acetone to generate isopropanol by using ketoreductase in the presence of NADP + or NAD +, dehydrogenating TCDCA to be oxidized into taurocholic 7-ketolithocholic acid T-7-KLCA by using TCDCA oxidase, and obtaining first-step enzyme reaction liquid;
(2) the second step of enzyme catalytic reaction: after the residual enzyme is removed from the first step of enzyme reaction liquid, a certain amount of isopropanol is added, isopropanol dehydrogenase is used for catalyzing the isopropanol to generate acetone so as to provide coenzyme regeneration of NADPH/NADH, and T-7-KLCA reductase is used for reducing the T-7-KLCA into TUDCA.
Preferably, in the above biological catalytic preparation method of tauroursodeoxycholic acid of the present invention, the step (1) comprises: dissolving acetone in water, adding TCDCA, continuously stirring, fully dissolving, then adding NADP +/NAD +, then adding ketoreductase and TCDCA oxidase, and reacting; the ratio of the addition amount of acetone to NADP + or NAD + to TCDCA oxidase to TCDCA is 0.1-0.3 g to 1.2-2.5 ml to 0.0001-0.0008 g to 0.2-0.5 g to 1 g; the reaction concentration of the TCDCA is 10-25%.
In the above-mentioned method for preparing tauroursodeoxycholic acid by biocatalysis according to the present invention, the reaction conditions in step (1) are more preferably: the temperature of the reaction system is 25-37 ℃, and the pH is adjusted to 6.0-8.5; detecting the content of TCDCA and T-7-KLCA in a liquid phase in the reaction process, and finishing the reaction when the substrate reacts for more than 99.0 percent.
In the above method for preparing tauroursodeoxycholic acid by biocatalysis of the present invention, preferably, in the step (2), the ratio of the addition amounts of the isopropanol dehydrogenase, isopropanol, T-7-KLCA reductase and T-7-KLCA is: 0.2 to 0.5g, 0.5 to 1.5ml, 0.3 to 0.5g, 1 g.
In the above-mentioned method for preparing tauroursodeoxycholic acid by biocatalysis of tauroursodeoxycholic acid of the present invention, the reaction conditions in the step (2) are more preferably: the system temperature is 25-37 ℃, and the pH is adjusted to 6.0-8.5; detecting the content of T-7-KLCA and TUDCA by using a liquid phase in the reaction process, and finishing the reaction when the substrate reacts for more than 99.0 percent.
In the above-mentioned method for preparing tauroursodeoxycholic acid by biocatalysis of the present invention, preferably, the ketoreductase, TCDCA oxidase, isopropanol dehydrogenase, and T-7-KLCA reductase are immobilized enzymes, enzyme solutions, or lyophilized enzyme powders, wherein the enzyme solutions or immobilized enzymes are directly metered during metering, and the lyophilized enzyme powders are first dissolved in pure water 15 times the mass of the enzyme solutions and then metered.
When the enzyme is enzyme liquid or enzyme freeze-dried powder, after the reactions in the steps (1) and (2) are finished, adjusting the pH to 3.0-4.0 or 9.0-10.0, stirring for 3-5 h to denature and precipitate the enzyme, and centrifuging to remove the precipitate and leave supernatant.
Preferably, in the above biological catalytic preparation method of tauroursodeoxycholic acid of the invention, after the enzyme is removed from the reaction solution in the step (2), the reaction solution is filtered by a 0.1 μm microporous filter membrane, then acetone is recovered by vacuum condensation at 56 ℃, isopropanol is recovered by vacuum extraction at 82 ℃, and then the water is removed by vacuum evaporation or spray drying continuously, so that the TUDCA dry powder is finally obtained.
The invention realizes the conversion of acetone and isopropanol through ketone reductase and isopropanol dehydrogenase, realizes the conversion of NADP +/NAD + and NADPH/NADH, combines the catalytic action of TCDCA oxidase and T-7-KLCA reductase, generates tauroursodeoxycholic acid (T-7-KLCA) from TCDCA first, and finally obtains tauroursodeoxycholic acid TUDCA. The isopropanol which is the reaction product of the first step can be directly used as a reaction substrate of the second step, only a small amount of isopropanol needs to be added, and in addition, the acetone and the isopropanol solvent have low boiling points, are easy to recover and treat, are easy to realize the reaction circulation, and reduce the overall cost.
The invention has the advantages of
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
1) the enzyme reaction related to the technology can adopt immobilized enzyme, enzyme liquid or enzyme powder, the immobilized enzyme can be recycled, the enzyme liquid or the enzyme powder is simple to manufacture, and the enzymes in the three states can be flexibly selected according to the production scale, so that the cost optimization is realized;
2) a coenzyme regeneration system is adopted, the addition amount of the coenzyme is low, and the production cost is greatly reduced;
3) the isopropanol and NADP +/NAD + products of the first step reaction can be directly used as substrates in the second step reaction, so that the production cost is saved;
4) acetone and isopropanol can be easily subjected to fractional distillation and recycled, so that the production cost is saved, and the environmental protection pressure is reduced.
Drawings
FIG. 1 is a diagram showing a process of a double enzyme-linked reaction.
Detailed Description
Example 1: adding 125ml of acetone and 300ml of pure water into a 1L three-hole reaction bottle, stirring and mixing, placing the reaction bottle in a 25 ℃ constant-temperature water bath kettle, adding 50.16g of 98% purity TCDCA, continuously stirring for half an hour to fully dissolve the TCDCA into a mixed solution, adding 0.05g of NADP +, adjusting the pH to 6.0-6.2, adding 5.35g of ketoreductase (immobilized enzyme) and 10.52g of TCDCA oxidase (immobilized enzyme), complementing the volume to 500ml with pure water, reacting for 15-20 hours at 150rpm, controlling the temperature to 25-26 ℃ and the pH to 6.0-6.2 during the reaction, detecting the contents of TCDCA and T-7-KLCA by using a liquid phase, stopping the reaction when the substrate reaction is over 99.0%, passing through a 60-mesh reaction liquid, recovering the immobilized enzyme, and waiting for the rest of the system to enter a second-step of enzyme reaction.
Example 2: about 600ml of reaction system, firstly adding the reaction solution obtained after enzyme removal in the example 1 into a 1L three-hole reaction bottle, placing the reaction bottle in a constant-temperature water bath kettle at 25 ℃, starting and stirring 150rpm, adding 75ml of isopropanol into the reaction solution, adjusting the pH value to 6.0-6.2, then adding 10.13g of isopropanol dehydrogenase enzyme solution and 15.62g T-7-KLCA reductase enzyme solution, reacting for 15-20 h at 150rpm, controlling the temperature and the pH value within the process requirement range during the reaction, detecting the contents of T-7-KLCA and TUDCA by using a liquid phase, stopping the reaction when the substrate reaction exceeds 99.0%, adjusting the pH value of the reaction solution to 4.0 by using concentrated hydrochloric acid, continuously stirring for 3h, centrifuging for 10min at 10000g to remove precipitates, collecting supernatant, filtering by using a 0.1 mu m microporous filter membrane, vacuumizing and condensing and recovering acetone at 56 ℃ to obtain 113.6ml of acetone, vacuumizing and recovering isopropanol at 82 ℃ to obtain 62.9ml of isopropanol, and then, continuously utilizing a rotary evaporator to vacuumize, heat and evaporate in a rotary manner, and finally obtaining 46.18g of TUDCA dry powder with the purity of more than 98%, wherein the yield is 92.1%.
Example 3: adding 375ml of acetone and 500ml of pure water into a 2L three-hole reaction bottle, stirring and mixing, placing the reaction bottle in a 37 ℃ constant-temperature water bath kettle, adding 250.11g of 98% TCDCA, continuously stirring for half an hour to fully dissolve the TCDCA into a mixed solution, adding 0.20g of NAD +, adjusting the pH to 8.3-8.5, adding 75.59g of ketoreductase (immobilized enzyme) and 124.64g of TCDCA oxidase enzyme solution (immobilized enzyme), complementing the volume to 1000ml with pure water, reacting at 300rpm for 10-15 hours, controlling the temperature to 36-37 ℃ and the pH to 8.3-8.5 during the reaction, detecting the contents of TCDCA and T-7-KLCA with a liquid phase, stopping the reaction when the substrate reaction exceeds 99.0%, recycling the immobilized enzyme from a reaction liquid with a 60-mesh screen, and waiting for the rest of the system to enter a second enzyme reaction.
Example 4: about 1300ml of reaction system, firstly adding the reaction solution of example 1 after enzyme removal into a 2L three-hole reaction bottle, placing the reaction bottle in a constant temperature water bath kettle at 37 ℃, starting and stirring at 300rpm, adding 250ml of isopropanol into the reaction solution, adjusting the pH to 8.3-8.5, then adding 124.32g of isopropanol dehydrogenase freeze-dried powder aqueous solution (15 times of mass pure water for dissolution) and 123.97g T-7-KLCA reductase freeze-dried powder aqueous solution (15 times of mass pure water for dissolution), reacting at 300rpm for 10-15 h, controlling the temperature and the pH within the process requirement range during the reaction, detecting the contents of T-7-KLCA and TUDCA by using a liquid phase, stopping the reaction when the substrate reaction exceeds 99.0%, adjusting the pH of the reaction solution to 10.0 by using NaOH solution, continuously stirring for 3h, centrifuging for 10min at 10000g, removing precipitates, collecting supernatant, filtering by using a 0.1 mu m microporous filter membrane, vacuumizing and condensing the rest reaction solution at 56 ℃ for recovering acetone, 329.4ml of acetone is obtained, isopropanol is recovered by vacuumizing at 82 ℃ to obtain 232.6ml of isopropanol, and then a rotary evaporator is used for vacuumizing, heating and rotary steaming to finally obtain 225.35g of TUDCA dry powder with the purity of more than 98%, and the yield is 90.1%.
Example 5: adding 130L of acetone and 250L of pure water into a 1T stainless steel reaction tank, starting to stir at 200rpm, controlling the temperature of a jacket to be 29-30 ℃, then 75.08kg of TCDCA with 98 percent purity is added and the stirring is continued for half an hour to ensure that the TCDCA is fully dissolved into the mixed solution, then adding 0.03kg of NADP +, adjusting the pH value to 7.8-8.0, adding 7.96kg of ketoreductase lyophilized powder aqueous solution (dissolved by 15 times of pure water by mass) and 30.12kg of TCDCA oxidase lyophilized powder aqueous solution (dissolved by 15 times of pure water by mass), complementing the volume to 500L by pure water, reacting at constant temperature for 12-15 h, controlling the temperature to be 29-30 ℃ and the pH value to be 7.8-8.0 during the reaction, detecting the contents of TCDCA and T-7-KLCA by liquid phase, stopping the reaction when the substrate reaction is over 99.0 percent, adjusting the pH of the reaction solution to 9.8-10.0 by using 2M NaOH solution, stirring for 4 hours, removing precipitates by using a disc centrifuge, and pumping the supernatant into a reaction tank for waiting for entering a second step of enzyme reaction.
Example 6: about 600L of reaction system, firstly adding the reaction solution after removing the precipitate in the embodiment 5 into a stainless steel reaction tank of 1T, opening and stirring 200rpm, controlling the temperature of the reaction solution by a jacket to 29-30 ℃, adding 75L of isopropanol into the reaction solution, adjusting the pH to 7.5-7.8, then adding 25.37kg of isopropanol dehydrogenase enzyme solution and 30.22kg of T-7-KLCA reductase enzyme solution, reacting at constant temperature for 12-15 h, controlling the temperature and the pH within the process requirement range during the reaction, detecting the content of T-7-KLCA and TUDCA by using a liquid phase, stopping the reaction when the substrate reaction exceeds 99.0%, adjusting the pH to 3.5-3.7 by using concentrated hydrochloric acid, removing the precipitate by a disc centrifuge after stirring for 5h, further filtering the supernatant by a 0.1 mu m membrane filter to remove the precipitate, vacuumizing and condensing and recycling acetone at 56 ℃ of the remaining reaction solution to obtain 109.3L of acetone, vacuumizing and recycling the isopropanol at 82 ℃, 63.1L of isopropanol is obtained, and then the reaction solution is concentrated by a double-effect evaporator and then is spray-dried to obtain 65.51kg of TUDCA dry powder, and the recovery rate is 87.3%.
The above-mentioned embodiments are merely illustrative of the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.
Claims (8)
1. A biological catalysis preparation method of tauroursodeoxycholic acid is characterized by comprising the following steps:
(1) the first step of enzyme-catalyzed reaction: catalyzing acetone to generate isopropanol by using ketoreductase in the presence of NADP + or NAD +, dehydrogenating TCDCA to be oxidized into taurocholic 7-ketolithocholic acid T-7-KLCA by using TCDCA oxidase, and obtaining first-step enzyme reaction liquid;
(2) the second step of enzyme catalytic reaction: after the residual enzyme is removed from the first step of enzyme reaction liquid, a certain amount of isopropanol is added, isopropanol dehydrogenase is used for catalyzing the isopropanol to generate acetone so as to provide coenzyme regeneration of NADPH/NADH, and T-7-KLCA reductase is used for reducing the T-7-KLCA into TUDCA.
2. The method for preparing tauroursodeoxycholic acid in a biocatalytic manner according to claim 1, wherein the process of the step (1) is: dissolving acetone in water, adding TCDCA, continuously stirring, fully dissolving, then adding NADP +/NAD +, then adding ketoreductase and TCDCA oxidase, and reacting; the ratio of the addition amount of acetone to NADP + or NAD + to TCDCA oxidase to TCDCA is 0.1-0.3 g to 1.2-2.5 ml to 0.0001-0.0008 g to 0.2-0.5 g to 1 g; the reaction concentration of the TCDCA is 10-25%.
3. The method for preparing tauroursodeoxycholic acid by biocatalysis according to claim 2, wherein the reaction conditions of the step (1) are as follows: the temperature of the reaction system is 25-37 ℃, and the pH is adjusted to 6.0-8.5; detecting the content of TCDCA and T-7-KLCA in a liquid phase in the reaction process, and finishing the reaction when the substrate reacts for more than 99.0 percent.
4. The method for preparing tauroursodeoxycholic acid by biocatalysis according to claim 1, wherein in the step (2), the ratio of the addition amount of isopropanol dehydrogenase to T-7-KLCA reductase to T-7-KLCA is as follows: 0.2 to 0.5g, 0.5 to 1.5ml, 0.3 to 0.5g, 1 g.
5. The process for the biocatalytic preparation of tauroursodeoxycholic acid according to claim 4, wherein the reaction conditions in the step (2) are: the system temperature is 25-37 ℃, and the pH is adjusted to 6.0-8.5; detecting the content of T-7-KLCA and TUDCA by using a liquid phase in the reaction process, and finishing the reaction when the substrate reacts for more than 99.0 percent.
6. The method for preparing tauroursodeoxycholic acid according to claim 1, 2 or 4, wherein the ketoreductase, TCDCA oxidase, isopropanol dehydrogenase and T-7-KLCA reductase are immobilized enzymes, enzyme liquids or enzyme lyophilized powders, the enzyme liquids or immobilized enzymes are directly metered during metering, and the enzyme lyophilized powders are metered after being dissolved by pure water 15 times the mass of the enzyme lyophilized powders.
7. The biocatalytic preparation method of tauroursodeoxycholic acid according to claim 1, 2 or 4, characterized in that, when the enzyme is enzyme liquid or enzyme freeze-dried powder, after the reaction of the steps (1) and (2) is finished, the pH is adjusted to 3.0-4.0 or 9.0-10.0, the enzyme is stirred for 3-5 hours to denature and precipitate the enzyme, and the supernatant liquid is left after the precipitate is removed by centrifugation.
8. The method for preparing tauroursodeoxycholic acid according to claim 7, wherein the reaction solution obtained in step (2) is subjected to enzyme removal, then filtered by a 0.1 μm microporous membrane, then subjected to vacuum-pumping at 56 ℃ for condensation and recovery of acetone, subjected to vacuum-pumping at 82 ℃ for recovery of isopropanol, and then subjected to vacuum evaporation or spray drying for moisture removal, thereby obtaining TUDCA dry powder.
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|---|---|---|---|---|
| CN113736842A (en) * | 2021-09-02 | 2021-12-03 | 四川澄华生物科技有限公司 | Method for efficiently preparing tauroursodeoxycholic acid by multiple cells |
| CN113736842B (en) * | 2021-09-02 | 2024-04-19 | 四川澄华生物科技有限公司 | Method for efficiently preparing tauroursodeoxycholic acid by multiple cells |
| CN114592027A (en) * | 2022-03-03 | 2022-06-07 | 北京岳达生物科技有限公司 | Method for preparing tauroursodeoxycholic acid by two-step method |
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