阅读说明：本技术 一种高效率生产12-酮基胆酸的方法 (Method for efficiently producing 12-ketocholic acid ) 是由 程玲利 王婷婷 张翔 彭捷 黄清东 于 2021-06-30 设计创作，主要内容包括：本发明公开一种高效率生产12-酮基胆酸的方法,包括以下步骤,(1)种子培养；(2)扩大培养：将步骤(1)所得摇瓶种子接种到发酵罐的液体培养基中进行发酵罐发酵,此过程包括第一阶段和第二阶段,第一阶段进行增殖发酵；第一阶段完成后进行第二阶段发酵,流加诱导物质溶液,同时流加吡格列酮甲醇溶液和氨水,并设置通气比为0.8-1.2,保持溶氧为15-25%,温度为25℃；(3)催化转化。本发明步骤(2)中,添加了吡格列酮作为氧化酶活化激动剂,显著提高12α-羟基类固醇脱氢酶的酶活水平,最高可达1500～1700U/mL,前体物质的投入量从20g/L直接提高到300g/L,大幅提高前体物质的投料量,提高了生产效率。(The invention discloses a method for efficiently producing 12-ketocholic acid, which comprises the following steps of (1) seed culture; (2) and (3) amplification culture: inoculating the shake flask seeds obtained in the step (1) into a liquid culture medium of a fermentation tank for fermentation in the fermentation tank, wherein the process comprises a first stage and a second stage, and the first stage is used for propagation fermentation; after the first stage is finished, performing second stage fermentation, feeding an inducing substance solution, and feeding a pioglitazone methanol solution and ammonia water at the same time, wherein the aeration ratio is set to be 0.8-1.2, the dissolved oxygen is kept at 15-25%, and the temperature is 25 ℃; (3) and (4) performing catalytic conversion. In the step (2), pioglitazone is added as an oxidase activation agonist, so that the enzyme activity level of 12 alpha-hydroxysteroid dehydrogenase is remarkably improved to 1500-1700U/mL at most, the input amount of the precursor is directly increased to 300g/L from 20g/L, the feeding amount of the precursor is greatly increased, and the production efficiency is improved.)

1. A preparation method for efficiently producing 12-ketocholic acid is characterized by comprising the following steps:

(1) seed culture: inoculating the recombinant escherichia coli strain of the 12 alpha-hydroxysteroid dehydrogenase expression gene into a shake flask of a liquid culture medium of the shake flask, maintaining the temperature at 37 ℃, and culturing for 8-12h in a 220rpm shaking table to obtain shake flask seeds;

(2) and (3) amplification culture: inoculating the shake flask seeds obtained in the step (1) into a liquid culture medium of a fermentation tank for fermentation in the fermentation tank, wherein the process comprises a first stage and a second stage, the first stage is used for propagation fermentation, the aeration ratio is set to be 0.8, the dissolved oxygen of the fermentation liquor in the first stage is kept to be more than 50%, the fermentation temperature is kept to be 37 ℃, and the propagation fermentation time is kept within four hours; after the first stage is finished, performing second-stage fermentation, feeding an inducing substance solution, feeding a pioglitazone methanol solution and ammonia water simultaneously, setting the aeration ratio to be 0.8-1.2, keeping the dissolved oxygen to be 15-25%, and keeping the temperature to be 25 ℃, wherein the inducing substance solution comprises 0.1% of lactic acid, 5% of lactose, 2% of glycerol, 0.05% of an antifoaming agent and 0.01mmol/L of IPTG (isopropyl thiogalactoside);

(3) and (3) catalytic conversion: when the density OD600 of the cultured bacteria is more than 25 and the enzyme activity of 12 alpha-hydroxysteroid dehydrogenase is more than 25U/mL, adding an auxiliary substance solution into the fermentation broth, starting to add a precursor substance solution after a period of time, setting the aeration ratio to be 0.8-1.2, keeping the dissolved oxygen to be 30-50%, keeping the fermentation temperature to be 25 ℃, and keeping the pH to be 8.2-8.5; the precursor solution is 40% cholic acid, sodium hydroxide with equal mole equivalent of cholic acid, and the balance of water; the auxiliary substance solution comprises riboflavin with the concentration of 0.01 time of the molar equivalent of cholic acid, a sodium hydroxide solution with the pH value of 11 and the balance of water; and after the precursor substance solution and the auxiliary substance solution are fed for 1h, starting to intermittently detect the concentration of the cholic acid, and finishing the culture when the concentration of the cholic acid is less than or equal to 0.3%.

2. The method for preparing 12-ketocholic acid with high efficiency according to claim 1, wherein disodium hydrogen phosphate with a concentration of 2% by mass is added into the inducing substance solution.

3. The method for preparing 12-ketocholic acid with high efficiency according to claim 1, wherein the liquid medium is prepared by preparing a solution of corn steep liquor dry powder, yeast extract, sodium chloride and water, wherein the concentration of the corn steep liquor dry powder is 10g/L, the concentration of the yeast extract is 5g/L and the concentration of NaCl is 10g/L, firstly preparing a 1g/L potassium bicarbonate solution, heating 500mL of the potassium bicarbonate solution to 40-45 ℃, adding the corn steep liquor dry powder into the heated potassium bicarbonate solution, stirring for dissolution, cooling to room temperature, then adding the yeast extract and sodium chloride, and adding water to a constant volume of 1000mL to obtain the liquid medium.

4. The preparation method for efficiently producing 12-ketocholic acid according to claim 1, wherein the concentration of the ammonia water is 1% by mass, and the concentration of the pioglitazone methanol solution is 0.3 g/L.

5. The preparation method for efficiently producing 12-ketocholic acid according to claim 1, wherein in the step (2), the feeding time of the inducing substance solution is 3-6h, and the feeding time of the pioglitazone methanol solution is 3 h; the feeding flow rate of the inducing substance solution is 20ml/(L x h), and the feeding flow rate of the pioglitazone methanol solution is 0.1ml/(L x h); the flow rate of the ammonia water was 5ml/(L × h).

6. The method according to claim 1, wherein in step (3), the feeding time of the auxiliary substance solution is 3 hours, the feeding time of the precursor substance solution is 1 hour, the feeding flow rate of the precursor substance solution is 750ml/(L x h), and the feeding flow rate of the auxiliary substance solution is 0.2ml/(L x h).

Technical Field

The invention belongs to the technical field of biology and medicine, and particularly relates to a method for efficiently producing 12-ketocholic acid.

Background

The invention patent application with the application number of 202010458182.2 discloses a preparation method of 12-ketocholic acid, which specifically comprises the following steps: (1) inoculating the recombinant Escherichia coli strain of the 12 alpha-hydroxysteroid dehydrogenase expression gene into a biological culture container containing an LB liquid culture medium for culture according to the inoculum size with the volume fraction of 2-6%; (2) when the dissolved oxygen value of the culture solution is lower than 60%, feeding an inducing substance solution; (3) after the feeding of the inducing substance solution is finished, when the density OD600 of the cultured bacteria reaches 15 and the enzyme content of 12 alpha-hydroxysteroid dehydrogenase reaches 12U/mL, feeding the precursor substance and the auxiliary substance simultaneously, and feeding for 6 hours in total; (4) detecting the concentration of cholic acid 2h after the precursor and auxiliary substances are fed, and finishing the culture when the concentration is 0.3%. The method of the present invention is described in [0033] to improve the production efficiency of 12-ketocholic acid by increasing the amount of a precursor by ensuring the dynamic stability of 12 α -hydroxysteroid dehydrogenase, but it was found that the method of the present invention can ensure the dynamic stability of 12 α -hydroxysteroid dehydrogenase in the production process, but the overall enzyme activity level is low, and therefore the production efficiency is still low due to the limitation of the enzyme activity level although the amount of the precursor of the present invention is increased.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method of 12-ketocholic acid, which improves the enzyme activity level by feeding pioglitazone methanol solution in the process of induced proliferation culture, thereby greatly improving the addition of precursor substances and improving the production efficiency.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method for efficiently producing 12-ketocholic acid comprises the following steps:

(1) seed culture: inoculating the recombinant escherichia coli strain of the 12 alpha-hydroxysteroid dehydrogenase expression gene into a shake flask of a liquid culture medium of the shake flask, maintaining the temperature at 37 ℃, and culturing for 8-12h in a 220rpm shaking table to obtain shake flask seeds;

(2) and (3) amplification culture: inoculating the shake flask seeds obtained in the step (1) into a liquid culture medium of a fermentation tank for fermentation in the fermentation tank, wherein the process comprises a first stage and a second stage, the first stage is used for propagation fermentation, the aeration ratio is set to be 0.8, the dissolved oxygen of the fermentation liquor in the first stage is kept to be more than 50%, the fermentation temperature is kept to be 37 ℃, and the propagation fermentation time is kept within four hours; after the first stage is finished, performing second-stage fermentation, feeding an inducing substance solution, feeding a pioglitazone methanol solution and ammonia water simultaneously, setting the aeration ratio to be 0.8-1.2, keeping the dissolved oxygen to be 15-25%, and keeping the temperature to be 25 ℃, wherein the inducing substance solution comprises 0.1% of lactic acid, 5% of lactose, 2% of glycerol, 0.05% of an antifoaming agent and 0.01mmol/L of IPTG (isopropyl thiogalactoside);

(3) and (3) catalytic conversion: when the density OD600 of the cultured bacteria is more than 25 and the enzyme activity of 12 alpha-hydroxysteroid dehydrogenase is more than 25U/mL, adding an auxiliary substance solution into the fermentation broth, starting to add a precursor substance solution after a period of time, setting the aeration ratio to be 0.8-1.2, keeping the dissolved oxygen to be 30-50%, keeping the fermentation temperature to be 25 ℃, and keeping the pH to be 8.2-8.5; the precursor solution is 40% cholic acid, sodium hydroxide with equal mole equivalent of cholic acid, and the balance of water; the auxiliary substance solution comprises riboflavin with the concentration of 0.01 time of the molar equivalent of cholic acid, a sodium hydroxide solution with the pH value of 11 and the balance of water; and after the precursor substance solution and the auxiliary substance solution are fed for 1h, starting to intermittently detect the concentration of the cholic acid, and finishing the culture when the concentration of the cholic acid is less than or equal to 0.3%.

In the scheme, the propagation fermentation is carried out in the step (2) in the first stage to obtain a large amount of recombinant escherichia coli of 12 alpha-hydroxysteroid dehydrogenase expression genes, the induced propagation culture is carried out in the second stage to obtain 12 alpha-hydroxysteroid dehydrogenase, and in the second stage, a pioglitazone methanol solution and ammonia water are fed while an inducing substance solution is fed, wherein in the invention, pioglitazone which is usually used as an antidiabetic drug is used as an oxidase activation agonist; in the first stage, the fermentation time of the first stage is shortened, namely the propagation fermentation time is shortened, and the first stage is controlled within four hours, because the situation that the fermentation time of the first stage exceeds four hours in the production process is found, in the fermentation liquid obtained in the second stage, the enzyme activity level is lower than that of the first stage within four hours, the probable reason is suspected to be that the time of the first stage is too long, and the accumulated fermentation byproducts in the process are too much to influence the activation effect of pioglitazone, therefore, in the invention, the synergistic effect between material addition and fermentation processes is combined to determine that the fermentation time of the first stage is within four hours, and the optimal time is between three and four hours; in the second stage, ammonia fed back supplements a nitrogen source, the fed-back pioglitazone methanol solution supplements a carbon source, so that the carbon source is sufficient in the fermentation process, and pioglitazone is added as an oxidase activation agonist, namely pioglitazone is an activation agonist of 12 alpha-hydroxysteroid dehydrogenase, so that the enzyme activity level of the 12 alpha-hydroxysteroid dehydrogenase is remarkably improved to 1500-1700U/mL at most, and as the enzyme activity is greatly improved, more precursor substances can be added during catalytic conversion, more precursor substances can be catalytically converted at the same time, and the production efficiency is improved.

Further, disodium hydrogen phosphate with the mass percentage concentration of 2% is added into the inducing substance solution. Although 12 α -hydroxysteroid dehydrogenase is continuously produced during the proliferation induction, 12 α -hydroxysteroid dehydrogenase is unstable and easily inactivated, and when disodium hydrogen phosphate is not added, a large amount of 12 α -hydroxysteroid dehydrogenase is produced, which macroscopically shows that the enzyme activity level is continuously increased within a certain period of time, and the level of the added precursor substance is not reached, so that the precursor substance cannot be added for catalytic conversion, and thus the inactivated 12 α -hydroxysteroid dehydrogenase is wasted. In order to reduce the inactivation rate of the 12 alpha-hydroxysteroid dehydrogenase, disodium hydrogen phosphate is added into an inducing substance solution and mixed in a system, so that the effect on the 12 alpha-hydroxysteroid dehydrogenase is realized, the enzyme activity stability is improved, and the time for the enzyme activity of the 12 alpha-hydroxysteroid dehydrogenase to be more than 25U/mL is shortened. Simple detection on the enzyme activity stability of the 12 alpha-hydroxysteroid dehydrogenase shows that the 12 alpha-hydroxysteroid dehydrogenase without disodium hydrogen phosphate needs to be stored at 2-8 ℃ in a cold storage mode, and is inevitably inactivated by more than 80% after 4-6 h, while the 12 alpha-hydroxysteroid dehydrogenase with disodium hydrogen phosphate can be stored at room temperature for 14-16 h, and the enzyme activity is kept at more than 95%. There is provided a concept that when the 12 α -hydroxysteroid dehydrogenase is to be preserved, disodium hydrogen phosphate can be added thereto to extend the preservation time.

Further, the preparation method of the liquid culture medium comprises the steps of preparing a solution from corn steep liquor dry powder, a yeast extract, sodium chloride and water, wherein the concentration of the corn steep liquor dry powder is 10g/L, the concentration of the yeast extract is 5g/L, and the concentration of NaCl is 10g/L, firstly, preparing a potassium bicarbonate solution with the concentration of 1g/L, heating 500mL of the potassium bicarbonate solution to 40-45 ℃, adding the corn steep liquor dry powder into the heated potassium bicarbonate solution, stirring for dissolving, cooling to room temperature, then adding the yeast extract and the sodium chloride, and fixing the volume of water to 1000mL to obtain the liquid culture medium. The corn steep liquor dry powder is a nutrient substance which is prepared by taking fresh corn steep liquor as a raw material and performing low-temperature instant heating spray drying, the corn steep liquor dry powder is used as a main nitrogen source in a conventional method, the corn steep liquor dry powder in the market is called to be completely dissolved, but the corn steep liquor dry powder cannot be completely dissolved in the using process, and great difference exists among different manufacturers, if the liquid culture medium is filtered, partial nutrition is lost, if the precipitation liquid culture medium is directly used, the obtained shake flask seeds are not free from precipitation impurities, when the shake flask seeds mixed with the precipitation impurities are inoculated to a fermentation tank culture medium during enlarged culture, the inoculation concentration is low, and the dissolution condition of each batch of production along with the corn steep liquor dry powder is different, the inoculation concentration among batches is deviated, and in addition, if the precipitation impurities exist in the liquid culture medium, the actual OD600 value of the indicator "the thallus density OD600 is more than 25" for catalytic conversion may be less than 25, so that the incomplete dissolution of the dry corn steep liquor powder in water may cause various unstable factors, and finally the process may be unstable. In order to solve the problem, in the technical scheme, firstly, the corn steep liquor dry powder is dissolved in a heated potassium bicarbonate solution, the potassium bicarbonate solution is alkalescent and is heated to 40-45 ℃, under the condition, the corn steep liquor dry powder is effectively dissolved, the influence of a liquid culture medium on the fermentation process is avoided, and the process is stabilized; and because potassium bicarbonate is added into the culture medium, potassium ions are provided for the escherichia coli, and in the process of induced proliferation culture, the potassium ions have an activating effect on enzymes synthesized by the escherichia coli and metabolized by lactose, so that the utilization rate of the lactose in the induced substance solution is improved, and the catalytic conversion time is shorter compared with that of sodium bicarbonate.

Further, the mass percentage concentration of the ammonia water is 1%, and the concentration of the pioglitazone methanol solution is 0.3 g/L.

Further, in the step (2), the feeding time of the induction substance solution is 3-6h, and the feeding time of the pioglitazone methanol solution is 3 h; the feeding flow rate of the inducing substance solution is 20ml/(L x h), and the feeding flow rate of the pioglitazone methanol solution is 0.1ml/(L x h); the flow rate of the ammonia water was 5ml/(L × h).

Further, in the step (3), the feeding time of the auxiliary substance solution is 3 hours, the feeding time of the precursor substance solution is 1 hour, the feeding flow rate of the precursor substance solution is 750ml/(L × h), and the feeding flow rate of the auxiliary substance solution is 0.2ml/(L × h).

Compared with the prior art, the invention has the beneficial effects that:

(1) pioglitazone is added as an oxidase activation agonist, namely pioglitazone is an activation agonist of 12 alpha-hydroxysteroid dehydrogenase, so that the enzyme activity level of the 12 alpha-hydroxysteroid dehydrogenase is remarkably improved and can reach 1500-1700U/mL at most, the input amount of a precursor substance is directly increased to 300g/L from 20g/L (the volume of 300g/L is the volume of original fermentation liquor, and the total volume of a solvent containing the precursor substance is not added), the feeding amount of the precursor substance is greatly improved, and the production efficiency is improved;

(2) the fermentation time of the first stage is limited within four hours, so that the activation effect of pioglitazone is prevented from being influenced by excessive fermentation byproducts accumulated due to overlong time of the first stage, the propagation fermentation time is shortened to a certain extent, the process period is shortened, and the production efficiency is improved;

(3) disodium hydrogen phosphate with the mass percentage concentration of 2% is added into the inducing substance solution to reduce the inactivation rate of the 12 alpha-hydroxysteroid dehydrogenase, so that the 12 alpha-hydroxysteroid dehydrogenase can be stored at normal temperature for a longer time;

(4) the liquid culture medium has no sediment, so that the influence of the liquid culture medium on the fermentation process is avoided; and the potassium bicarbonate in the liquid culture medium has an activating effect on enzymes metabolized with lactose, so that the utilization rate of the lactose in the inducing substance solution is improved.

Drawings

FIG. 1 is a production system;

in the figure: 1-fermentation tank, 2-stirring rod, 3-exhaust pipe, 4-balance pipe, 5-feeding pipe, 6-feeding measuring cup, 7-flow regulator, 8-air-supplementing device, 9-feeding pipe, 10-air pipe and 11-liquid sterile filter.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate only some, but not all, of the embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art without any inventive step are within the scope of the present invention.

The present invention uses the following production system:

as shown in fig. 1, this production system includes fermentation cylinder 1, the inside puddler 2 that is equipped with of fermentation cylinder 1, 1 top of fermentation cylinder is equipped with blast pipe 3, 3 tops of blast pipe are the gas vent, gas vent department is equipped with the air discharge valve, 3 middle parts of blast pipe and balance pipe 4 intercommunication, balance pipe 4's the other end and the 6 top of reinforced graduated flask intercommunication, 6 tops of reinforced graduated flask are equipped with filling tube 5, be equipped with liquid aseptic filter 11 on the filling tube 5, reinforced graduated flask 6's exit end is equipped with inlet pipe 9, be equipped with flow regulator 7 on this inlet pipe 9, the inlet pipe 9 other end communicates with air hose 10, air hose 10 one end links to each other with air supplement unit 8, the other end of air hose 10.

The working process of the production system is as follows: fermenting the fermentation liquor in a fermentation tank 1, and stirring the fermentation liquor by a stirring rod 2; the fed-batch liquid material passes through liquid aseptic filter 11 and gets into reinforced graduated flask 6, reinforced graduated flask 6 passes through balance pipe 4 with fermentation cylinder 1 and keeps pressure balance, flow regulator 7 flow regulation, liquid material gets into air pipe 10, partial solvent volatilizees, material and gas get into fermentation cylinder 1 together, mix with the zymotic fluid furthest in fermentation cylinder 1, and simultaneously, when the discharge valve door of blast pipe 3 was closed, the solvent that volatilizees among the fermentation cylinder 1 was through balance pipe 4, reinforced graduated flask 7, inlet pipe 9, air pipe 10, get back to in the fermentation cylinder 1 again, the organic raw materials who volatilizees converts into the gaseous state to mend promptly, the utilization ratio of organic raw materials has been improved.

The recombinant Escherichia coli strain expressing the 12 alpha-hydroxysteroid dehydrogenase gene used in the present invention is commercially available as in the prior art invention patent 202010458182.2 in the background art.

Example 1

Preparing a liquid culture medium: preparing a solution from corn steep liquor dry powder, a yeast extract, sodium chloride and water, wherein the concentration of the corn steep liquor dry powder is 10g/L, the concentration of the yeast extract is 5g/L, and the concentration of NaCl is 10 g/L; the preparation method comprises the steps of firstly, preparing a potassium bicarbonate solution with the concentration of 1g/L, heating 500mL of the potassium bicarbonate solution to 40-45 ℃, adding the corn steep liquor dry powder into the heated potassium bicarbonate solution, stirring for dissolving, cooling to room temperature, then adding the yeast extract and sodium chloride, and fixing the volume to 1000mL with water to obtain the liquid culture medium.

Example 2

(1) Seed culture: inoculating the recombinant escherichia coli strain of the 12 alpha-hydroxysteroid dehydrogenase expression gene into a shake flask of a liquid culture medium of the shake flask, and culturing for 8 hours in a 220rpm shaking table at the temperature of 37 ℃ under 40-60RH to obtain shake flask seeds;

(2) and (3) amplification culture: inoculating the shake flask seeds obtained in the step (1) into a liquid culture medium of a fermentation tank for fermentation in the fermentation tank, wherein the process comprises a first stage and a second stage, the first stage is used for propagation fermentation, the aeration ratio is set to be 0.8, the dissolved oxygen of the fermentation liquor in the first stage is kept to be more than 50%, the fermentation temperature is kept to be 37 ℃, and the propagation fermentation time is four hours; after the first stage is finished, performing second-stage fermentation, feeding an inducing substance solution, feeding a pioglitazone methanol solution and ammonia water, setting the aeration ratio to be 1.2, keeping the dissolved oxygen to be 15-25%, and setting the temperature to be 25 ℃, wherein the inducing substance solution contains 0.1% of lactic acid, 5% of lactose, 2% of glycerol, 0.05% of an antifoaming agent and 0.01mmol/L of IPTG (isopropyl thiogalactoside); the mass percentage concentration of the ammonia water is 1%, and the concentration of the pioglitazone methanol solution is 0.3 g/L; the feeding time of the inducing substance solution is 3-6h, and the feeding time of the pioglitazone methanol solution is 3 h; the feeding flow rate of the inducing substance solution is 20ml/(L x h), and the feeding flow rate of the pioglitazone methanol solution is 0.1ml/(L x h); the flow rate of the ammonia water is 5ml/(L x h);

(3) and (3) catalytic conversion: when the density OD600 of the cultured bacteria is more than 25 and the enzyme activity of 12 alpha-hydroxysteroid dehydrogenase is more than 25U/mL, feeding an auxiliary substance solution into 1L of fermentation liquor, and feeding a precursor substance solution after 20-30 min;

setting aeration ratio at 1.2, maintaining dissolved oxygen at 30-50%, and maintaining fermentation temperature at 25 deg.C and pH at 8.2-8.5; after the precursor substance solution and the auxiliary substance solution are fed in uniformly for 1h, starting to intermittently detect the concentration of the cholic acid, detecting every twenty minutes, and finishing the culture when the concentration of the cholic acid is less than or equal to 0.3%; the precursor solution is 40% cholic acid, sodium hydroxide with equal mole equivalent of cholic acid, and the balance of water; the auxiliary substance solution comprises riboflavin with the concentration of 0.01 time of the molar equivalent of cholic acid, a sodium hydroxide solution with the pH value of 11 and the balance of water; the feeding time of the auxiliary substance solution is 3 hours, and the feeding time of the precursor substance solution is 1 hour; the feed rate of the precursor solution was 750 ml/(L.multidot.h), and the feed rate of the auxiliary solution was 0.2 ml/(L.multidot.h).

In this example, the liquid medium used was the liquid medium prepared in example 1.

And when the density OD600 of the cultured bacteria is more than 25 and the enzyme activity of the 12 alpha-hydroxysteroid dehydrogenase is more than 25U/mL, taking out 20mL12 alpha-hydroxysteroid dehydrogenase, filtering the alpha-hydroxysteroid dehydrogenase by a ceramic membrane for coarse purification, refrigerating and hermetically storing the coarse purified enzyme solution at 2-8 ℃, wherein the enzyme is inactivated by more than 50% after four hours, and the enzyme is inactivated by more than 80% after six hours.

In this example, the amount of the precursor added to 1L of the fermentation broth was 300g, whereas the amount of the precursor added in the prior art mentioned in the background art was 20 g.

Example 3

The present example is different from example 2 in that disodium hydrogen phosphate was added to the inducer solution at a concentration of 2% by mass in addition to example 2.

In the catalytic conversion of step (3), the time for starting the feeding of the precursor substance solution and the auxiliary substance solution is about half an hour earlier than that of example 2.

In addition, when the density OD600 of the cultured bacteria is more than 25 and the enzyme activity of the 12 alpha-hydroxysteroid dehydrogenase is more than 25U/mL, two 20mL12 alpha-hydroxysteroid dehydrogenases are taken out, ceramic membrane filtration and coarse purification are respectively carried out on the two parts, the enzyme solution after coarse purification is refrigerated and hermetically stored at the temperature of 2-8 ℃, one part is stored for six hours, the other part is stored for sixteen hours, the enzyme activity of the enzyme solution after six hours of storage is detected to be kept at more than 98%, and the enzyme activity of the enzyme solution after sixteen hours of storage is kept at more than 80%.

Comparative example 1

The comparative example is different from example 2 in that no methanol solution of pioglitazone was fed during the second stage fermentation in step (2).

In the course of measuring cholic acid concentration, it was found that when the cholic acid concentration of example 2 was less than 0.3%, the cholic acid concentration of the present comparative example was 9.3%, and when the cholic acid concentration of the present comparative example was less than 0.3%, it took 260 minutes more than that of example 2.

Comparative example 2

The comparative example is different from example 2 in that the liquid medium used in the comparative example is prepared by preparing a solution of corn steep liquor dry powder having a concentration of 10g/L, yeast extract having a concentration of 5g/L and NaCl into 900mL of water, sufficiently dissolving, then fixing the volume to 1000mL, and then filtering to obtain a liquid medium.

Since the mass percentage of lactose is decreased by diluting the inducer solution after the inducer solution is added to the system, the lactose concentration of the system before the precursor is fed to the system in example 2 and comparative example 2 is measured as the conventional high performance liquid chromatography method in the lactose utilization ratio of comparative example 2 and comparative example 2, and the difference between example 2 and comparative example 2 is expressed as a relative value, and the lactose concentration of example 2 is 23% lower than that of comparative example 2.

Comparative example 3

The present comparative example is different from example 2 in that the liquid medium of the present comparative example is prepared by the following method: preparing a solution from corn steep liquor dry powder, a yeast extract, sodium chloride and water, wherein the concentration of the corn steep liquor dry powder is 10g/L, the concentration of the yeast extract is 5g/L, and the concentration of NaCl is 10 g/L; the preparation method comprises the steps of firstly, preparing a sodium bicarbonate solution with the concentration of 1g/L, heating 500mL of the sodium bicarbonate solution to 40-45 ℃, adding the corn steep liquor dry powder into the heated sodium bicarbonate solution, stirring for dissolving, cooling to room temperature, then adding the yeast extract and sodium chloride, and adding water to a constant volume of 1000mL to obtain the liquid culture medium.

The lactose concentration of the system before the precursor material is fed in example 2, comparative example 2 and comparative example 3 is measured by the existing high performance liquid chromatography method, and the difference between example 2, comparative example 2 and comparative example 3 is expressed by relative values, and the lactose concentration of example 2 is 23% lower than that of comparative example 2 and the lactose concentration of comparative example 3 is 1% lower than that of comparative example 2.

The time for example 2, comparative example 2 and comparative example 3 cholic acid concentrations of less than 0.3% was recorded, with the time for example 2 being the shortest and the time for comparative example 2 and comparative example 3 being comparable.

Combining example 2, comparative example 2 and comparative example 3, it can be seen that the utilization of lactose is affected to a greater extent by potassium ions, rather than the weak alkalinity of the liquid medium; since example 2 is shorter than comparative example 2 and comparative example 3 in reaching cholic acid concentration of less than 0.3%, it is suspected that the possible reason is that potassium bicarbonate causes other effects while improving the utilization rate of lactic acid, and the catalytic effect of 12 α -hydroxysteroid dehydrogenase is improved in combination with pioglitazone.

Comparative example 4

(1) Seed culture: inoculating the recombinant escherichia coli strain of the 12 alpha-hydroxysteroid dehydrogenase expression gene into a shake flask of a liquid culture medium of the shake flask, and culturing for 8 hours in a 220rpm shaking table at the temperature of 37 ℃ under 40-60RH to obtain shake flask seeds;

(2) and (3) amplification culture: dividing the shake flask seeds obtained in the step (1) into a group A and a group B, respectively inoculating the shake flask seeds of the group A and the group B into liquid culture mediums in different fermentation tanks for fermentation, wherein the first-stage fermentation of the group A is carried out for six hours, the first-stage fermentation of the group B is carried out for four hours, and other conditions are the same: setting the aeration ratio to be 0.8, keeping the dissolved oxygen of the fermentation liquor at the first stage to be more than 50 percent, and keeping the fermentation temperature to be 37 ℃; the conditions in the second stage are the same: fermenting for 10 hours in the second stage, feeding an inducing substance solution, feeding a pioglitazone methanol solution and ammonia water simultaneously, setting the aeration ratio to be 1.2, keeping the dissolved oxygen to be 15-25%, and the temperature to be 25 ℃, wherein the inducing substance solution comprises 0.1% of lactic acid, 5% of lactose, 2% of glycerol, 0.05% of an antifoaming agent and 0.01mmol/L of IPTG (isopropyl thiogalactoside); the mass percentage concentration of the ammonia water is 1%, and the concentration of the pioglitazone methanol solution is 0.3 g/L; (3) detecting enzyme activity: after fermentation, respectively taking system liquid of the group A and the group B to detect the enzyme activity.

The detection result shows that the enzyme activity of the group A is 1521U/mL, and the enzyme activity of the group B is 1683U/mL.