CN110760488A - Fermentation method of high-content 12 α -hydroxysteroid dehydrogenase fermentation broth - Google Patents
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Abstract
The invention provides a fermentation method of a high-content 12 α -hydroxysteroid dehydrogenase fermentation broth, which belongs to the technical field of biology and medicine, and is characterized in that standby recombinant escherichia coli of a 12 α -hydroxysteroid dehydrogenase expression gene is taken as a strain, the strain is inoculated into a novel sterile fermentation culture medium according to the inoculation amount of 2-6% in parts by volume for culture, an induction culture medium is fed-batch after 2 hours, and the total fed-batch time is 8 hours.
Description
Technical Field
The invention relates to the technical field of biology and medicine, in particular to a fermentation method of high-content 12 α -hydroxysteroid dehydrogenase fermentation liquor.
Background
Enzymes are proteins or RNAs produced by living cells, which have a high specificity for their substrates and a high catalytic efficiency. Enzymes are extremely important biocatalysts, which dominate many catalytic processes such as metabolism, nutrition and energy conversion of organisms, and most of the reactions closely related to life processes are enzyme-catalyzed reactions.
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.
Grant publication No. CN102007210B discloses a 12 α -hydroxysteroid dehydrogenase, a nucleic acid sequence for encoding the same, an expression cassette and a vector, a recombinant microorganism comprising an appropriate encoding nucleic acid sequence, a method for producing the 12 α -hydroxysteroid dehydrogenase, a method for enzymatic oxidation of 12 α -hydroxysteroids using the enzyme, a method for enzymatic reduction of 12-steroids using the enzyme, a method for qualitative or quantitative determination of 12-steroids or 12 α -hydroxysteroids using the 12 α -hydroxysteroid dehydrogenase, and a method for preparation of ursodeoxycholic acid comprising catalyzing bile acid oxidation using the 12 α -hydroxysteroid dehydrogenase, the activity of 12 α -hydroxysteroid dehydrogenase is only 20-100U/mg.
In the prior art, the enzyme activity of 12 α -hydroxysteroid dehydrogenase (12 α -HSDH) is only about 50U/mL, and the activity is low, so that the high cholic acid conversion efficiency is low, the large-scale production of chenodeoxycholic acid cannot be realized, and the production cost of the chenodeoxycholic acid is high.
Disclosure of Invention
Aiming at the defects that the enzyme activity of the prior 12 α -hydroxysteroid dehydrogenase is only 50U/mL and the activity is lower, the invention provides a fermentation method of fermentation liquor with high content of 12 α -hydroxysteroid dehydrogenase.
A fermentation method of fermentation liquor with high content of 12 α -hydroxysteroid dehydrogenase comprises taking recombinant Escherichia coli of 12 α -hydroxysteroid dehydrogenase expression gene as a spare strain, inoculating the strain into a novel sterile fermentation culture medium according to the inoculum size of 2-6% in parts by volume, culturing, feeding an induction culture medium after 2h, and feeding the induction culture medium for 8h in total.
In the technical scheme of the application, in the fermentation method, in the process of producing 12 α -hydroxysteroid dehydrogenase (12 α -HSDH) by fermenting recombinant escherichia coli containing 12 α -hydroxysteroid dehydrogenase expression genes as strains, a traditional LB culture medium is replaced by a novel culture medium with high nutrition and low cost, a process fed-batch induction culture medium is adopted, and through culture, the density OD (600) of a culture strain body reaches 50-60, and meanwhile the enzyme activity of the 12 α -hydroxysteroid dehydrogenase reaches 700-900U/L.
Preferably, the recombinant Escherichia coli of the 12 α -hydroxysteroid dehydrogenase expression gene is a secondary fermentation strain, is stored in a glycerol tube, is inoculated into a triangular shake flask containing the LB liquid sterile culture medium according to the inoculation amount with the volume fraction of 1-5%, and is cultured in a shaking table at 37 ℃, 40-50 RH% and 240rpm for 12h for later use.
Preferably, the novel sterile fermentation medium comprises, by weight, 0.1-0.5% of corn flour, 2-6% of corn steep liquor, 0.05-0.2% of nitrate, 0.05-0.2% of ammonium salt, 0.1-1% of ammonia water, 0.1-0.5% of phosphate, 0.2-0.5% of sodium chloride, 0.02-0.05% of magnesium sulfate, 0.02-0.05% of zinc sulfate, 0.05% of an antifoaming agent, and the balance of water.
More preferably, the novel sterile fermentation medium comprises, by weight, 0.3% of corn flour, 4% of corn steep liquor, 0.075% of nitrate, 0.075% of ammonium salt, 0.45% of ammonia water, 0.3% of phosphate, 0.15% of sodium chloride, 0.015% of magnesium sulfate, 0.015% of zinc sulfate, 0.05% of an antifoaming agent, and the balance of water.
Preferably, the induction culture medium comprises, by weight, 0.1-0.5% of lactic acid, 5-10% of lactose, 2-10% of glycerol, 0.5-2% of glucose, 0.05-0.2% of ammonium salt, 0.2-0.5% of sodium chloride, 0.02-0.05% of magnesium sulfate, 0.02-0.05% of zinc sulfate, 0.05% of an antifoaming agent, and the balance of water.
More preferably, the induction medium comprises, by weight, 0.3% of lactic acid, 7.5% of lactose, 6% of glycerol, 1.2% of glucose, 0.12% of ammonium salt, 0.35% of sodium chloride, 0.03% of magnesium sulfate, 0.04% of zinc sulfate, 0.05% of an antifoaming agent, and the balance of water.
Preferably, the culture conditions when inoculating into a novel sterile fermentation culture medium for culture are that the temperature is 37 ℃ in 0-3 hours, the temperature is 25-33 ℃ after 3 hours, the pH is 6-7 in 0-8 hours, the pH is 7-8 after 8 hours, sterile air is introduced while stirring reaction is started, the dissolved oxygen content is not less than 15% in 0-6 hours by adjusting the aeration ratio of 0.3-2.0 and the stirring power of 20-50 HZ, and the dissolved oxygen content is kept at 15-30% after 6 hours; the induction culture medium is fed according to the first 2 hours with the flow rate of 5ml/(L H), the second 2 hours with the flow rate of 15ml/(L H), the third 2 hours with the flow rate of 35ml/(L H), the fourth 2 hours with the flow rate of 20ml/(L H), and the total of 8 hours.
More preferably, the culture conditions when inoculating into the novel sterile fermentation medium for culture are that the temperature is 37 ℃ in 0-3 hours, the temperature is 29 ℃ after 3 hours, the pH is 6.5 in 0-8 hours, the pH is 7.5 after 8 hours, sterile air is introduced while stirring reaction is started, the dissolved oxygen content is not less than 15% after 0-6 hours by adjusting the aeration ratio of 0.3-2.0 and the stirring power of 20-50 HZ, and the dissolved oxygen content is kept at 22% after 6 hours; the induction culture medium is fed according to the first 2 hours with the flow rate of 5ml/(L H), the second 2 hours with the flow rate of 15ml/(L H), the third 2 hours with the flow rate of 35ml/(L H), the fourth 2 hours with the flow rate of 20ml/(L H), and the total of 8 hours.
Preferably, fermenting for 15.5-16.5 hours and putting in a tank.
More preferably, the fermentation is carried out for 16 hours.
In the technical scheme of the application, the recombinant Escherichia coli of the 12 α -hydroxysteroid dehydrogenase expression gene is commercially available as a strain.
Compared with the prior art, the invention has the beneficial effects that:
(1) the fermentation method is characterized in that in the process of producing 12 α -hydroxysteroid dehydrogenase (12 α -HSDH) by fermenting recombinant escherichia coli containing 12 α -hydroxysteroid dehydrogenase expression genes as strains, a novel culture medium with high nutrition and low cost replaces a traditional LB culture medium and a process fed-batch induction culture medium is adopted, and through culture, the density OD (600) of a culture strain body reaches 50-60, and meanwhile, the enzyme activity of the 12 α -hydroxysteroid dehydrogenase reaches 700-900U/L.
(2) The secondary fermentation strain has strong activity, rapid propagation, reasonable density of prepared thallus, and high activity of the produced 12 α -hydroxysteroid dehydrogenase.
(3) The organic nitrogen source of the novel sterile fermentation medium comes from corns, the organic carbon source comes from corn steep liquor, the corn steep liquor contains higher organic phosphorus and inorganic phosphorus, the nitrogen source, the carbon source and the phosphorus source are sufficient, the cost is low, in addition, the nutrition collocation of all the components is reasonable, and all the nutritional components are sufficient, so that the growth of recombinant escherichia coli and the production of secondary metabolites are facilitated.
(4) In the fed-batch induction culture medium, because glycerol and lactose are the best carbon source for inducing the engineering bacteria of the escherichia coli, the invention is characterized in that the induction is carried out in four sections, the density of the recombinant escherichia coli is reasonable, and the activity of the produced 12 α -hydroxysteroid dehydrogenase is high.
(5) The culture conditions when the strain is inoculated into a novel sterile fermentation culture medium for culture are that the temperature is 37 ℃ in 0-3 hours, the temperature is 25-33 ℃ after 3 hours, the pH is 6-7 in 0-8 hours, the pH is 7-8 after 8 hours, sterile air is introduced while stirring reaction is started, the dissolved oxygen content is not lower than 15% in 0-6 hours by adjusting the aeration ratio of 0.3-2.0 and the stirring power of 20-50 HZ, and the strain is kept at 15-30% after 6 hours, the culture medium is induced to flow 5mL/(L H) in the first 2 hours, 15mL/(L H) in the second 2 hours, 35mL/(L H) in the third 2 hours, 20mL/(L H) in the fourth 2 hours, and a fermentation system with 8 hours is fed-batch, and the system meets the growth nutrition requirement of S-type logarithm of the microorganism in a growth period, so that the strain density OD (600) reaches 50-12 α, and the enzyme activity of 12-hydroxysteroid dehydrogenase reaches 700U/mL-900 mL.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1
A fermentation method of a high-content 12 α -hydroxysteroid dehydrogenase fermentation broth includes the steps of inoculating a spare recombinant escherichia coli strain of a 12 α -hydroxysteroid dehydrogenase expression gene into a novel sterile fermentation culture medium according to an inoculation amount of 2% in parts by volume for culture, feeding an induction culture medium after 2 hours, feeding the induction culture medium in a feeding mode for 8 hours in total, placing the recombinant escherichia coli strain of the 12 α -hydroxysteroid dehydrogenase expression gene into a triangular shaking flask containing LB liquid according to an inoculation amount of 1-5% in a glycerol tube for culture for 12 hours in a shaking table at 37 ℃, 40 RH% and 240rpm for later use, placing the novel sterile fermentation culture medium into a fermentation tank for culture for 12 hours in a feeding mode, wherein the novel sterile fermentation culture medium comprises 0.1% of corn flour, 2% of corn steep liquor, 0.05% of nitrate, 0.05% of ammonium salt, 0.1% of ammonia water, 0.1% of phosphate, 0.2% of sodium chloride, 0.02% of magnesium sulfate, 0.02% of zinc sulfate, 0.05% of defoaming agent and the balance of water according to the weight percentage, the fermentation medium is prepared by adding 0.1%, stirring after fermentation of lactic acid, 0.1%, 2% of lactic acid, 5% of lactose, 0.1%, 2% of glucose, stirring, 0.2% of 2% of sodium chloride, 2.2% of 2H, stirring, 2% of 2H, stirring, 2H, stirring, the flow rate of fermentation medium, the flow rate of the fermentation medium is 0.7.7H, the fermentation medium, the flow rate of 10.7H of the flow rate of the fermentation medium is 0.15H, the flow rate of the fermentation medium, the flow rate of the flow rate is 0.7H of the fermentation medium, the flow rate of the fermentation medium is adjusted to 20H, the fermentation medium is adjusted to 20H flow rate of the fermentation medium, the fermentation medium is adjusted to 20H, the fermentation medium is adjusted to 20H, the.
Example 2
A fermentation method of a high-content 12 α -hydroxysteroid dehydrogenase fermentation broth includes the steps of inoculating a spare recombinant escherichia coli strain of a 12 α -hydroxysteroid dehydrogenase expression gene into a novel sterile fermentation culture medium according to an inoculation amount of 4% in parts by volume for culture, feeding an induction culture medium after 2 hours, feeding the induction culture medium in a feeding mode for 8 hours in total, placing the recombinant escherichia coli strain of the 12 α -hydroxysteroid dehydrogenase expression gene into a triangular shaking flask containing LB liquid according to an inoculation amount of 1-5% in a glycerol tube for culture for 12 hours in a shaking table at 37 ℃, 45 RH% and 240rpm for later use, placing the novel sterile fermentation culture medium in a shaking table at 37 ℃, 45 RH% for 12 hours for later use, adding 0.015% of zinc sulfate, 0.05% of an antifoaming agent and the balance of water according to weight percentage, adding 0.3% of corn flour, 4% of corn steep liquor, 0.075% of nitrate, 0.075% of ammonium salt, 0.45% of ammonia water, 0.3% of phosphate, 0.15% of sodium chloride, 0.015% of magnesium sulfate, 0.05% of an antifoaming agent and 0.05% of a balance of water, adding 0.35-35% of a fermentation medium, stirring the fermentation medium, adding a fermentation medium for fermentation time, adding 0.35H/(0.35H) for fermentation, adding 0.35-35H of a fermentation medium, stirring, adding 0.35H of a fermentation medium for fermentation medium, stirring medium for fermentation time, adding 0.6H/(0.35H) for fermentation time, adding 0.35H of a fermentation medium for fermentation medium, adding 0.35H of 2H/(0.6H/(0.35H) for fermentation time, adding 0.6H) for fermentation medium for fermentation time, adding 0.6H/(0.6H) for fermentation time, stirring, adding 0.2H/(0.35H/(0.2H) for fermentation time, and 2H) for fermentation time, adding 0.2H), adding 0.2H/(0.2H) for fermentation, stirring, adding 0.2H for fermentation, and stirring, adding 0.2H) for fermentation medium for fermentation time, adding.
Example 3
A fermentation method of fermentation liquor of 12 α -hydroxysteroid dehydrogenase with high content includes inoculating a spare recombinant Escherichia coli strain of 12 α -hydroxysteroid dehydrogenase expression genes into a novel sterile fermentation culture medium according to an inoculation amount of 6% in parts by volume for culture, feeding an induction culture medium after 2 hours, feeding the induction culture medium in a feeding mode for 8 hours in total, placing the recombinant Escherichia coli strain of the 12 α -hydroxysteroid dehydrogenase expression genes into a triangular shaking flask containing LB liquid according to an inoculation amount of 1-5% in parts by volume in a glycerol tube for culture for 12 hours in a shaking table at 37 ℃, 50 RH% and 240rpm, placing the novel sterile fermentation culture medium into 7818% and 7818% in percentage by weight, wherein the novel sterile fermentation culture medium comprises 0.5% of corn flour, 6% of corn steep liquor, 0.2% of nitrate, 0.2% of ammonium salt, 1% of ammonia water, 0.5% of phosphate, 0.5% of sodium chloride, 0.05% of magnesium sulfate, 0.05% of zinc sulfate, 0.05% of defoaming agent and the balance of water, the induction culture medium is prepared by adding 0.05% of glucose dehydrogenase, stirring the fermentation medium, the fermentation medium is added with stirring for fermentation at a pH ratio of 0.35-7 hours, the fermentation time of 10%, the fermentation medium is 0.5%, the fermentation medium is added with the fermentation time of 10%, the fermentation time of 0.5% of 0.35-7 hours, the fermentation medium is added with the fermentation time of 10% of 2% of glucose, the fermentation medium, the flow rate of 2H/(0.7.7H/(0.7H), the flow rate of 10.7H/(0.7H) of 10.7H), the flow rate of 10.7H) of 10H, the flow rate of 10.7H, the fermentation medium, the flow rate of the flow.
The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.
Claims (10)
1. A fermentation method of fermentation liquor with high content of 12 α -hydroxysteroid dehydrogenase is characterized in that standby recombinant escherichia coli with 12 α -hydroxysteroid dehydrogenase expression genes is taken as a strain, the strain is inoculated into a novel sterile fermentation culture medium according to the inoculum size of 2-6% in parts by volume for culture, an induction culture medium is fed after 2 hours, and the feeding is carried out for 8 hours in total.
2. The fermentation method of fermentation broth with high content of 12 α -hydroxysteroid dehydrogenase as claimed in claim 1, wherein the recombinant Escherichia coli with 12 α -hydroxysteroid dehydrogenase expressed gene is a second-level fermentation strain, stored in a glycerin tube, inoculated into a triangular flask containing LB liquid sterile medium according to the inoculum size of 1-5% by volume fraction, and cultured in a shaker at 37 ℃, 40-50 RH% and 240rpm for 12h for later use.
3. The fermentation method of fermentation broth of 12 α -hydroxysteroid dehydrogenase in high content according to claim 1, wherein the novel sterile fermentation medium comprises corn flour 0.1-0.5 wt%, corn steep liquor 2-6 wt%, nitrate 0.05-0.2 wt%, ammonium salt 0.05-0.2 wt%, ammonia 0.1-1 wt%, phosphate 0.1-0.5 wt%, sodium chloride 0.2-0.5 wt%, magnesium sulfate 0.02-0.05 wt%, zinc sulfate 0.02-0.05 wt%, defoaming agent 0.05 wt%, and water in balance.
4. The fermentation method of a fermentation broth with high content of 12 α -hydroxysteroid dehydrogenase as claimed in claim 3, wherein the novel sterile fermentation medium comprises corn flour 0.3%, corn steep liquor 4%, nitrate 0.075%, ammonium salt 0.075%, ammonia 0.45%, phosphate 0.3%, sodium chloride 0.15%, magnesium sulfate 0.015%, zinc sulfate 0.015%, defoaming agent 0.05%, and water in balance.
5. The fermentation method of 12 α -hydroxysteroid dehydrogenase fermentation broth according to claim 1, wherein the induction medium comprises, by weight, 0.1-0.5% lactic acid, 5-10% lactose, 2-10% glycerol, 0.5-2% glucose, 0.05-0.2% ammonium salt, 0.2-0.5% sodium chloride, 0.02-0.05% magnesium sulfate, 0.02-0.05% zinc sulfate, 0.05% antifoaming agent, and the balance water.
6. The fermentation method of 12 α -hydroxysteroid dehydrogenase highly fermented liquid according to claim 5, wherein the induction medium comprises 0.3 wt% of lactic acid, 7.5 wt% of lactose, 6 wt% of glycerol, 1.2 wt% of glucose, 0.12 wt% of ammonium salt, 0.35 wt% of sodium chloride, 0.03 wt% of magnesium sulfate, 0.04 wt% of zinc sulfate, 0.05 wt% of antifoaming agent, and the balance water.
7. The fermentation method of a fermentation broth with high 12 α -hydroxysteroid dehydrogenase content according to claim 1, wherein the culture conditions for inoculation into a novel sterile fermentation medium are 0-3 hours at 37 ℃, 3 hours later at 25-33 ℃, 0-8 hours at 6-7, 8 hours later at 7-8, sterile air is introduced while stirring is started, the dissolved oxygen content is not less than 15% in 0-6 hours by adjusting the aeration ratio to 0.3-2.0 and the stirring power to 20-50 HZ, the culture medium is maintained at 15-30% after 6 hours, the culture medium is induced to flow at 5ml/(L H) in the first 2 hours, 15ml/(L H) in the second 2 hours, 35ml/(L H) in the third 2 hours, and 20ml/(L H) in the fourth 2 hours, and the total flow is 8H.
8. The fermentation method of a fermentation broth containing 12 α -hydroxysteroid dehydrogenase as claimed in claim 7, wherein the culture conditions for inoculation into a novel sterile fermentation medium are 0-3 hr at 37 ℃, 3 hr at 29 ℃, 0-8 hr at 6.5, 8 hr at 7.5, introducing sterile air while starting stirring, adjusting the aeration ratio to 0.3-2.0 and the stirring power to 20-50 HZ to make the dissolved oxygen content to be not less than 15% in 0-6 hr, and keeping the dissolved oxygen content at 22% after 6 hr, and the culture medium is induced to flow at 5ml/(L H) for the first 2 hr, 35ml/(L H) for the second 2 hr, and 20ml/(L H) for the fourth 2 hr for 8 hr.
9. The fermentation method of fermentation broth with high content of 12 α -hydroxysteroid dehydrogenase as claimed in claim 1, wherein the fermentation is carried out for 15.5-16.5 hours.
10. The fermentation method of fermentation broth with high content of 12 α -hydroxysteroid dehydrogenase as claimed in claim 1, wherein the fermentation is carried out for 16 hours and the fermentation broth is discharged.
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| CN111676202B (en) * | 2020-08-05 | 2023-04-25 | 重庆极泽生物科技有限公司 | Fermentation process for coexpression of hydroxysteroid dehydrogenase |
| CN113416684A (en) * | 2021-06-30 | 2021-09-21 | 四川澄华生物科技有限公司 | Method for efficiently producing 12-ketocholic acid |
| CN113416684B (en) * | 2021-06-30 | 2022-03-29 | 四川澄华生物科技有限公司 | Method for efficiently producing 12-ketocholic acid |
| CN114107237A (en) * | 2021-09-07 | 2022-03-01 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
| CN114107237B (en) * | 2021-09-07 | 2024-06-04 | 伊犁川宁生物技术股份有限公司 | Method for producing chenodeoxycholic acid oxidase by fermentation |
| CN114134067A (en) * | 2021-10-19 | 2022-03-04 | 山东睿智医药科技有限公司 | Escherichia coli and application thereof |
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