阅读说明：本技术 一种12-酮基胆酸的制备方法 (Preparation method of 12-ketocholic acid ) 是由 程玲利 黄清东 张翔 彭捷 邓治荣 周彩娥 王婷婷 向世明 龙柯利 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种12-酮基胆酸的制备方法,属于生物与医药技术领域,用于生产12-酮基胆酸,以解决现有的12α-羟基类固醇脱氢酶(12α-HSDH)高胆酸转化效率低下,且12α-羟基类固醇脱氢酶分离纯化成本较高的问题,含12α-羟基类固醇脱氢酶表达基因的重组大肠杆菌为菌种发酵生产12α-羟基类固醇脱氢酶(12α-HSDH)的过程中,当培养液溶解氧值低于60％时,流加诱导物质溶液,诱导物质溶液流加完毕,培养菌菌体密度OD(600)达到15且12α-羟基类固醇脱氢酶的酶含量达到12U/ml时,流加前体物质,同时流加辅助物质,共计流加6h,最终得到含量高达80～150g/L的12-酮基胆酸溶液。(The invention discloses a preparation method of 12-ketocholic acid, belonging to the technical field of biology and medicine, which is used for producing 12-ketocholic acid and solving the problems that the prior 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH) has low conversion efficiency of high cholic acid and the separation and purification cost of the 12 alpha-hydroxysteroid dehydrogenase is higher, in the process of producing the 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH) by fermenting recombinant escherichia coli containing 12 alpha-hydroxysteroid dehydrogenase expression genes as a strain, when the dissolved oxygen value of a culture solution is lower than 60 percent, an inducing substance solution is fed in, the density OD (600) of the cultured bacteria reaches 15, and the enzyme content of the 12 alpha-hydroxysteroid dehydrogenase reaches 12U/ml, a precursor substance is fed in, and auxiliary substances are simultaneously fed in, adding for 6h in total to finally obtain the 12-ketocholic acid solution with the content of 80-150 g/L.)

1. A preparation method of 12-ketocholic acid is characterized by comprising the following steps: 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 OD 600 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%.

2. The method for preparing 12-ketocholic acid according to claim 1, wherein: the fermentation production period of the escherichia coli strain in the step (1) is 17-20 h.

3. The method for preparing 12-ketocholic acid according to claim 1, wherein: in the step (1), the recombinant escherichia coli of the 12 alpha-hydroxysteroid dehydrogenase expression gene is a secondary fermentation strain.

4. The method of claim 3, wherein the preparation method of 12-ketocholic acid comprises the following steps: the second-stage fermentation strain is obtained by inoculating strain liquid stored in glycerol tube into LB-containing liquid culture medium triangular shake flask according to inoculum size of 2% by volume fraction, and culturing in shaking table at 37 deg.C, 40-50 RH% and 240rpm for 12 h.

5. The method for preparing 12-ketocholic acid according to claim 1, wherein: the culture in the step (1) comprises bacteria proliferation culture, the dissolved oxygen value of the culture solution is more than 60%, the culture temperature is 37 ℃, the pH value is 6-7, the aeration ratio of sterile air is 0.6, the stirring power is started to be 20HZ, the pressure of the exhaust gas in the culture container is 0.02Mpa, and then the step (2) is carried out.

6. The method for preparing 12-ketocholic acid according to claim 1, wherein: and (3) when the dissolved oxygen value of the culture solution in the step (2) is kept at 15-30%, the culture temperature is 25 ℃, the pH value is 6.5-7.5, and the pressure of the exhaust gas in the culture container is 0.02 Mpa.

7. The method for preparing 12-ketocholic acid according to claim 1, wherein: and (3) taking the inducing substance in the step (2) as an inducing culture medium, feeding the inducing culture medium with a flow rate of 20ml/(L H), wherein the inducing culture medium comprises 0.1 percent of lactic acid, 5 percent of lactose, 2 percent of glycerol, 0.05 percent of antifoaming agent, 0.01mmol/L of IPTG and the balance of water by weight percentage, and feeding the inducing culture medium for 8 hours in total.

8. The method of claim 7, wherein the preparation method of 12-ketocholic acid comprises the following steps: 1 hour before the induction culture medium is fed completely, 2ml of culture solution is taken every half hour for detecting the density OD 600 of the cultured bacteria and the enzyme content of 12 alpha-hydroxysteroid dehydrogenase.

9. The method for preparing 12-ketocholic acid according to claim 1, wherein: in the step (3), the precursor is fed-batch according to the feeding amount of 100ml/(L H), the precursor is cholic acid alkali solution, the concentration of the cholic acid alkali solution is 20 percent cholic acid, sodium hydroxide with equal cholic acid molar equivalent, and the balance is water; the auxiliary substance is fed according to the feeding flow of 0.2ml/(L H), the auxiliary substance comprises riboflavin alkali solution, riboflavin with the riboflavin alkali solution concentration of 0.01 times of cholic acid molar equivalent, sodium hydroxide solution with pH11, and the balance of water.

10. The method for preparing 12-ketocholic acid according to claim 1, wherein: and (4) detecting the concentration of the cholic acid by adopting a high performance liquid chromatography.

Technical Field

A preparation method of 12-ketocholic acid belongs to the technical field of biology and medicine, is used for producing 12-ketocholic acid, and particularly relates to a preparation method of 12-ketocholic acid.

Background

The biocatalysis technology is a technology for carrying out catalytic reaction by using enzyme or microbial cells or animal and plant cells as a biocatalyst. Enzymes have many advantages as biocatalysts over chemical catalysts: the enzyme catalysis reaction is generally carried out under the conditions of normal temperature, normal pressure and nearly neutral, so the investment is less, the energy consumption is less and the operation safety is high; the biological catalyst has extremely high catalytic efficiency and reaction speed, and the catalytic efficiency can be 107-1013 times higher than that of the chemical catalytic reaction.

Chenodeoxycholic Acid (CDCA) is colorless needle crystal, and has no odor and bitter taste. The medicine is hardly dissolved in water, is easily dissolved in ethanol, glacial acetic acid and is slightly dissolved in chloroform, and is one of the most used medicines for treating gallstones in the world at present. The main effect is to reduce the saturation of cholesterol in the bile, and after most patients take CDCA (when CDCA accounts for 70% of bile salt in the bile), the lipid recovers micelle state, and the cholesterol is in unsaturated state, so that the cholesterol in the calculus is dissolved and dropped off. The large dose of CDCA (10-15 mg/kg per day) can inhibit cholesterol synthesis and increase bile secretion of cholelithiasis patients, but the secretion of bile salt and phospholipid is kept unchanged, and the CDCA is a raw material for synthesizing ursodeoxycholic acid (UDCA) and other steroid compounds.

The 12-position hydroxyl of cholic acid is converted into keto by an enzyme method, and chenodeoxycholic acid is obtained by a Huang Minglong reaction, and the cholic acid is a main component of animal bile acid such as chicken, duck, pig, cattle, sheep, rabbit and the like, so that the chenodeoxycholic acid obtained by a semi-synthesis method can greatly improve the production advantages of the chenodeoxycholic acid.

Authorization notice number: CN102007210B, discloses 12 alpha-hydroxysteroid dehydrogenase, a nucleic acid sequence for encoding the same, an expression cassette and a vector; a recombinant microorganism comprising an appropriate coding nucleic acid sequence; a method for producing the 12 α -hydroxysteroid dehydrogenase; a method for enzymatically oxidizing 12 alpha-hydroxysteroids using said enzyme, a method for enzymatically reducing 12-steroids using said enzyme, a method for qualitatively or quantitatively determining 12-steroids or 12 alpha-hydroxysteroids using said 12 alpha-hydroxysteroid dehydrogenase; and a method for preparing ursodeoxycholic acid, comprising catalyzing oxidation of cholic acid using the 12 α -hydroxysteroid dehydrogenase enzyme.

Disclosure of Invention

The invention aims to: provides a preparation method of 12-ketocholic acid, which aims to solve the defects that the prior 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH) has low transformation efficiency of high cholic acid and the separation and purification cost of the 12 alpha-hydroxysteroid dehydrogenase is high.

The technical scheme adopted by the invention is as follows:

a preparation method of 12-ketocholic acid 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 OD (600) 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%.

In the technical scheme of the application, in the process of producing 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH) by fermenting the recombinant escherichia coli containing the 12 alpha-hydroxysteroid dehydrogenase expression gene for strains, when the dissolved oxygen value of a culture solution is lower than 60%, feeding an inducing substance solution, finishing feeding the inducing substance solution, feeding a precursor substance when the density OD (600) of a cultured strain reaches 15 and the enzyme content of the 12 alpha-hydroxysteroid dehydrogenase reaches 12U/ml, simultaneously feeding an auxiliary substance, and feeding for 6 hours in total, thereby finally obtaining a 12 ketostone cholesterol solution with the content of 80-150 g/L.

Preferably, the fermentation production period of the Escherichia coli strain in the step (1) is 17-20 h.

Preferably, the recombinant Escherichia coli expressing the 12 a-hydroxysteroid dehydrogenase gene in step (1) is a secondary fermentation strain.

More preferably, the second-stage fermentation strain is obtained by inoculating strain liquid stored in glycerol tube into LB-containing liquid culture medium triangular shake flask according to the inoculum size of 2% by volume fraction, and culturing in a shaking table at 37 deg.C, 40-50 RH% and 240rpm for 12 h.

Preferably, the step (1) comprises bacterial proliferation culture, wherein the dissolved oxygen value of the culture solution is more than 60%, the culture temperature is 37 ℃, the pH value is 6-7, the aeration ratio of sterile air is 0.6, the stirring power is 20HZ, the pressure of the exhaust gas in the culture container is 0.02Mpa, and then the step (2) is carried out.

Preferably, in the step (2), when the dissolved oxygen value of the culture solution is maintained at 15-30%, the culture temperature is 25 ℃, the pH value is 6.5-7.5, and the pressure of the exhaust gas in the culture container is 0.02 MPa.

Preferably, the inducing substance in the step (2) is an inducing medium, the feeding flow rate is 20ml/(L x H), the inducing medium comprises 0.1% of lactic acid, 5% of lactose, 2% of glycerol, 0.05% of antifoaming agent, 0.01mmol/L of IPTG and the balance of water by weight percentage, and the total feeding time is 8H.

More preferably, 1 hour before the completion of the feeding of the induction medium, 2ml of the culture medium is taken every half hour to measure the density OD (600) of the cultured microorganism and the enzyme content of 12 alpha-hydroxysteroid dehydrogenase.

Preferably, in the step (3), the precursor is fed-batch at a feeding amount of 100ml/(L H), the precursor is cholic acid alkali solution, the concentration of the cholic acid alkali solution is 20% cholic acid and sodium hydroxide with equal cholic acid molar equivalent, and the balance is water; the auxiliary substance is fed according to the feeding flow of 0.2ml/(L H), the auxiliary substance comprises riboflavin alkali solution, riboflavin with the riboflavin alkali solution concentration of 0.01 times of cholic acid molar equivalent, sodium hydroxide solution with pH11, and the balance of water.

Preferably, in the step (4), the cholic acid concentration is detected by high performance liquid chromatography.

In the technical scheme of the application: recombinant E.coli expressing the 12 alpha-hydroxysteroid dehydrogenase gene is commercially available.

In the technical scheme of the application: IPTG: isopropyl-beta-D-thiogalactoside, a commonly used molecular biology agent, is an inducer of beta-galactosidase and beta-galactose permease.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the invention discloses a preparation method of 12-ketocholic acid, belongs to the technical field of biology and medicine, and is used for producing 12-ketocholic acid so as to solve the problems that the existing 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH) is low in high cholic acid conversion efficiency and the separation and purification cost of the 12 alpha-hydroxysteroid dehydrogenase is high.

2. Compared with the prior special culture process aiming at improving the content of 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH), the invention uses the conventional LB culture medium to culture the recombinant escherichia coli containing the 12 alpha-hydroxysteroid dehydrogenase expression gene, and reduces the complex configurations of raw materials, equipment, process control and the like of the special culture process (a fermentation method of fermentation liquor with high content of 12 alpha-hydroxysteroid dehydrogenase).

3. The invention is different from the existing pure enzyme catalysis process, is a whole-cell catalysis process, and reduces the rigor degree of the pure enzyme catalysis environmental conditions (the control requirements of temperature, pH, mixing, biological pollution, impurities and the like are strict).

4. The invention is different from the existing pure enzyme catalysis, is a whole-cell catalysis process, removes a series of process operations such as enzyme separation, purification, preservation, use and the like, not only improves the production efficiency, but also greatly reduces the environmental pollution.

5. The method is different from the existing pure enzyme catalysis, is a whole-cell catalysis process, ensures the dynamic stability of 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH), ensures that the inventory of precursor cholic acid is far greater than that of the existing pure enzyme catalysis precursor cholic acid, and greatly improves the production efficiency of 12-ketocholic acid on the whole because the reaction time is far shorter than that of the existing pure enzyme catalysis.

Drawings

FIG. 1 liquid chromatogram of cholic acid in example 2;

FIG. 2 liquid chromatogram of 12-ketocholic acid in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.