CN106916872B - Method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by efficient biotransformation - Google Patents
Method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by efficient biotransformation Download PDFInfo
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Abstract
The invention provides a method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by high-efficiency biotransformation. The method takes 17 alpha-hydroxyprogesterone as a raw material, and supplements a carbon source in a biotransformation process, so that an adequate carbon source is provided for the growth of thalli, hydroxylase in the thalli keeps high activity, the high-efficiency biotransformation stabilization period of the thalli is prolonged, the feeding amount of the 17 alpha-hydroxyprogesterone in a fermentation culture medium can be higher than 2% (w/v), and the transformation yield of the prepared 11 alpha, 17 alpha-dihydroxyl progesterone reaches over 90%. The optimal range of the feed ratio of the 17 alpha-hydroxyprogesterone in the fermentation medium is 2-20%, and the highest conversion yield of the 11 alpha, 17 alpha-dihydroxyprogesterone can reach 98%. The problems of low equipment output rate, large pollutant discharge amount and the like caused by low reactant feed ratio are effectively solved, the output efficiency is improved, and the production cost is reduced.
Description
Technical Field
The invention belongs to the field of preparation of steroids, and relates to a method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone through biotransformation, in particular to a method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone through efficient biotransformation.
Background
11 alpha, 17 alpha-dihydroxyprogesterone (abbreviated as dihydroxyluteone) is an important corticoid drug intermediate, is used for synthesizing corticoid drugs, and is an important intermediate for producing steroid hormone drugs such as hydrocortisone, dexamethasone, prednisone, betamethasone and the like.
17 alpha-hydroxyprogesterone, a full name of 17 alpha-hydroxy-pregn-4-ene-3, 20-dione, also known as hydroxyprogesterone, is an odorless, white or off-white crystalline powder, is insoluble in water, and is soluble in some organic solvents such as methanol, acetonitrile, chloroform, acetone, etc.
The molecular structure of the conversion of 17 α -hydroxyprogesterone to 11 α,17 α -dihydroxyprogesterone is:
steroid C11 hydroxylation is an indispensable step in the synthesis of various corticosteroids. In 1952, Peterson and Murray firstly use Rhizopus nigricans (Rhizopus nigericans) to hydroxylate the C11 site of progesterone and successfully convert progesterone into 11 alpha-hydroxyprogesterone in one step, so that the process from progesterone to corticosterone is reduced to only 3 steps, and the key problem in the synthesis process of corticosterone is solved. The hydroxyl group on the steroid is very difficult for chemical synthesis, and the hydroxyl group is difficult to be chemically introduced at other positions except the C17 position of the steroid. Steroid C11 hydroxylation is a transformation reaction peculiar to microorganisms, and in recent years, the industrialization of preparing 11 α,17 α -dihydroxyprogesterone from 17 α -hydroxyprogesterone by a biotransformation method has been realized. The method not only solves the problem that 11 alpha, 17 alpha-dihydroxyprogesterone is difficult to chemically synthesize, but also introduces hydroxyl at the 11 position to increase the drug effect of the corticoid compound, thereby providing a new way for hydroxylation of the steroid compound.
However, the steroid compounds have poor water solubility, generally 10-6～10-5mol/L, low mass transfer efficiency in the biotransformation process, and limited reactant charge ratio, wherein in the process of preparing 11 α and 17 α -dyhydroxy progesterone by biotransformation of 17 α -hydroxy progesterone as a raw material, the mass ratio of the reactant 17 α -hydroxy progesterone is lower than 2%, and the mass ratio of the reactant 17 α -hydroxy progesterone is low, so that the yield efficiency is low, the utilization rate of a culture medium is low, and the emission of pollutants is large.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by biotransformation, and particularly provides a method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by high-efficiency biotransformation.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for preparing 11 α,17 α -dihydroxyprogesterone by bioconversion, the method comprising the steps of:
(1) preparing an inclined plane: inoculating ochratoxin strains to a slant culture medium, and culturing at constant temperature to generate yellow spore slant for later use;
(2) seed culture: inoculating strains from the yellow spore slant obtained in the step (1) into a seed culture medium for seed culture to obtain a seed solution;
(3) and (3) biotransformation: inoculating the seed solution obtained in the step (2) into a fermentation medium, adding a reactant 17 alpha-hydroxyprogesterone, and performing shaking culture, wherein in the process of shaking culture, a carbon source and the reactant 17 alpha-hydroxyprogesterone are supplemented, the concentration of the carbon source in a fermentation liquid is kept to be not lower than 0.8mg/mL, the total feeding amount of the 17 alpha-hydroxyprogesterone is 2-20% of the volume of the fermentation medium, and thus, a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone is obtained.
The biotransformation preparation process of 11 alpha, 17 alpha-dihydroxyprogesterone is a hydroxylase catalysis process in fermentation thalli, the catalysis activity of the hydroxylase is high when the thalli grow vigorously, and the key for improving the transformation efficiency is to maintain the catalysis activity of the thalli. According to the invention, the carbon source is supplemented in the biotransformation process, and the concentration of the carbon source in the fermentation liquor is kept to be not less than 0.8mg/mL, so that an adequate carbon source is provided for the growth of the thalli, the hydroxylase in the thalli keeps high activity, and the high-efficiency biotransformation stabilization period of the thalli is prolonged, therefore, the high-efficiency biotransformation of 17 alpha-hydroxyprogesterone can be realized under the condition of high reactant feeding amount, the output efficiency is improved, and the production cost and the total pollutant emission are reduced.
In the method of the present invention, the slant culture medium of step (1) comprises the following components in mass-to-volume ratio (w/v): 2.0-5.0% of glucose, 1.0-5.0% of malt extract, 0.5-3.0% of peptone and 2.0-2.5% of agar, wherein the pH value of the slant culture medium is 5.0-6.0, and the slant culture medium is used after steam sterilization.
The mass-to-volume ratio in step (1) means a mass (g) relative to the amount of the components added to 100mL of the medium, for example, 2.0 to 5.0% of glucose is contained in the slant medium, that is, 2.0 to 5.0g of glucose is contained in 100mL of the slant medium. The meaning of the mass-to-volume ratios of the components in the seed medium and in the fermentation medium is the same here below.
In a slant medium, the glucose content may be 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, or 4.8%; the content of malt extract may be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5% or 4.8%; the content of peptones may be 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, or 2.8%; the content of agar may be 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%. The slant culture medium of the present invention has a pH of 5.0-6.0, e.g., 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9, and can be used after steam sterilization at 121 ℃ for 20 min.
Preferably, the temperature of said isothermal cultivation in step (1) is 25-28 ℃, such as 25.5 ℃, 26 ℃, 26.5 ℃, 27 ℃ or 27.5 ℃.
Preferably, the incubation period in step (1) is 4-7 days, such as 4.3 days, 4.5 days, 4.8 days, 5 days, 5.3 days, 5.5 days, 5.8 days, 6 days, 6.3 days, 6.5 days or 6.8 days.
The yellow spore slant obtained in the step (1) can be stored at constant temperature of 4 ℃ in a refrigerator for standby and can be stored for 2-3 months.
In the method of the present invention, the seed culture medium in step (2) comprises the following components in mass-to-volume ratio (w/v): 2.0-5.0% of glucose, 1.0-5.0% of corn steep liquor, 1.0-5.0% of peptone and 0.5-2.0% of yeast extract, wherein the pH value of the seed culture medium is 5.5-6.5, and the seed culture medium is used after steam sterilization.
The content of glucose in the seed medium may be 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.4%, 4.6% or 4.8%; the content of corn steep liquor may be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5% or 4.8%; the content of peptones may be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5%, or 4.8%; the content of yeast extract may be 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%. The pH of the seed medium is 5.5-6.5, e.g., 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, or 6.4. The seed culture medium can be used after being sterilized by steam at 121 ℃ for 20 min.
Preferably, the seed culture in step (2) is performed by shaking culture at 160-220r/min (e.g., 160r/min, 165r/min, 170r/min, 175r/min, 180r/min, 185r/min, 190r/min, 195r/min, 200r/min or 220 r/min).
Preferably, the temperature of the seed culture in step (2) is 25-32 deg.C, such as 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C or 31 deg.C.
Preferably, the seed culture time in step (2) is 20-30h, such as 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h or 29 h.
In the method of the present invention, the fermentation medium in step (3) comprises the following components in mass-to-volume ratio (w/v): 1.0-4.0% of glucose, 0.5-4.0% of corn steep liquor, 0.5-4.0% of peptone and 0.5-3.0% of yeast extract, wherein the pH value of the fermentation medium is 5.5-6.5, and the fermentation medium is used after steam sterilization.
The glucose content in the fermentation medium may be 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5% or 3.8%; the content of corn steep liquor can be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, or 3.8%; the content of peptones may be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, or 3.8%; the content of yeast extract may be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5% or 2.8%. The pH of the fermentation medium is 5.5-6.5, e.g., 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, or 6.4. The seed culture medium can be used after being sterilized by steam at 121 ℃ for 20 min.
Preferably, the seed liquid of step (3) is inoculated in an amount of 2-10% by volume of the fermentation medium, e.g. 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 9.5%, preferably 2-5%.
Preferably, the mass of the added reactant 17 α -hydroxyprogesterone of step (3) is 2-20% of the volume of the fermentation medium, e.g. 3%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16% or 18%.
The sum of the charged amounts of the first 17 alpha-hydroxyprogesterone and the then optionally supplemented 17 alpha-hydroxyprogesterone is 2-20%.
Preferably, the shaking culture of step (3) is performed at a shaking rate of 160-220r/min (e.g., 160r/min, 165r/min, 170r/min, 175r/min, 180r/min, 185r/min, 190r/min, 195r/min, 200r/min or 220 r/min).
Preferably, the temperature of the shaking culture in step (3) is 25-32 deg.C, such as 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C or 31 deg.C.
Preferably, the carbon source in step (3) is an organic carbon source and/or an inorganic carbon source.
Preferably, the carbon source in step (3) is selected from any one or a combination of at least two of glucose, fructose, galactose, maltose, lactose, sucrose, molasses, starch, pectin, cellulose, fats and oils, amino acids, alcohols, aldehydes, phenols, carbonates or bicarbonates, preferably any one or a combination of at least two of glucose, fructose, molasses, fats and oils, alcohols or carbonates.
Preferably, the carbon source is supplemented in step (3) in a continuous feeding manner or in batches.
In the invention, the carbon source is supplemented to provide a necessary carbon source for the growth of the thalli, so that the hydroxylase in the thalli keeps high activity, the high-efficiency biotransformation stabilization period of the thalli is prolonged, and the high-efficiency biotransformation of 17 alpha-hydroxyprogesterone under the condition of high reactant feeding amount can be realized.
Preferably, the carbon source is supplemented in step (3) in a timely and appropriate amount, and the concentration of the supplemented carbon source is not less than 50g/L (e.g., the concentration of the supplemented carbon source can be 50g/L, 80g/L, 100g/L, 150g/L, 200g/L, 300g/L, 500g/L, 700g/L, etc.) to maintain the concentration of the carbon source in the fermentation broth at not less than 0.8mg/mL, e.g., the concentration of the carbon source in the fermentation broth is not less than 1.0mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, 5mg/mL, 5.5mg/mL, 6mg/mL, 6.5mg/mL, 7mg/mL, or 8mg/mL, etc.
Preferably, the reactant 17 α -hydroxyprogesterone in step (3) is fed in a one-time feeding manner or a fed-batch manner, or is fed in a mixed flow with a carbon source or fed in a fed-batch manner.
The reactant 17 alpha-hydroxyprogesterone in the step (3) can be fed once, namely, the 17 alpha-hydroxyprogesterone is fed once before inoculation, and the subsequent feeding is not needed; or fed-batch, or fed-batch with a mixed flow of carbon source, the total charge of the reactant 17 α -hydroxyprogesterone being 2-20% by volume of the fermentation medium, such as 3%, 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20%, whichever is fed. Here, the total amount of reactants to be fed is 2 to 20g, for example, 3g, 5g, 8g, 10g, 12g, 14g, 16g, 18g or 20g, based on 2 to 20% by volume of the fermentation medium in 100mL of the medium.
As a preferred technical scheme, the method for preparing 11 alpha, 17 alpha-dihydroxyprogesterone by biotransformation comprises the following steps:
(1) preparing an inclined plane: inoculating Aspergillus ochraceus strain to slant culture medium, and culturing at 25-28 deg.C for 4-7 days to obtain yellow spore slant;
(2) seed culture: inoculating strains from the yellow spore slant obtained in the step (1) into a seed culture medium, and performing shake culture at 25-32 ℃ and 220r/min for 20-30h to obtain a seed solution;
(3) and (3) biotransformation: inoculating the seed liquid obtained in the step (2) into a fermentation medium by an inoculation amount which is 2-10% of the volume of the fermentation medium, simultaneously adding 17 alpha-hydroxyprogesterone as a reactant, performing shaking culture at the temperature of 25-32 ℃ for 220r/min, supplementing a carbon source and 17 alpha-hydroxyprogesterone as the reactant in the shaking culture process, keeping the concentration of the carbon source in the fermentation liquid to be not lower than 0.8mg/mL, and keeping the total feeding amount of the 17 alpha-hydroxyprogesterone to be 2-20% of the volume of the fermentation medium to obtain the fermentation liquid of the 11 alpha, 17 alpha-dihydroxyprogesterone.
In another aspect, the invention provides 11 α,17 α -dihydroxyprogesterone prepared by the method of the first aspect.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the carbon source is supplemented in the biotransformation process, and the concentration of the carbon source in the fermentation liquor is kept to be not less than 0.8mg/mL, so that a necessary carbon source is provided for the growth of thalli, the hydroxylase in the thalli keeps high activity, the high-efficiency biotransformation stabilization period of the thalli is prolonged, the high-efficiency biotransformation of 17 alpha-hydroxyprogesterone can be realized under the condition of high reactant feeding amount of 2-20%, the transformation yield of the 17 alpha-hydroxyprogesterone reaches more than 90% and can reach 98% to the maximum, the problems of low equipment yield, large pollutant discharge amount and the like caused by low reactant feeding ratio are effectively solved, the output efficiency is improved, and the production cost is reduced.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 2.0%, malt extract 1.0%, peptone 0.5%, agar 2.0%, pH 5.0, steam sterilizing at 121 deg.C for 20min), and culturing at 25 deg.C for 4 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 2.0%, corn steep liquor 1.0%, peptone 1.0%, yeast extract 0.5%, pH 5.5, and 121 deg.C steam sterilizing for 20min), and performing shake culture at 25 deg.C and 160r/min for 20 hr to obtain seed liquid required by fermentation;
(3) inoculating the seed solution into a sterilized fermentation medium (1.0% of glucose, 0.5% of corn steep liquor, 0.5% of peptone, 0.5% of yeast extract, 5.5 of pH value, steam sterilization at 121 ℃ for 20min) according to the inoculation amount of 2%, simultaneously adding 4% of 17 alpha-hydroxyprogesterone at one time, carrying out shaking culture at 25 ℃ and 160r/min, and supplementing a sugar solution with the concentration of 50g/L for multiple times in the shaking culture process to keep the sugar concentration in the fermentation medium not lower than 0.8mg/mL so as to obtain the fermentation liquor of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion of 11 α,17 α -dihydroxyprogesterone in the fermentation broth was 94% by High Performance Liquid Chromatography (HPLC) analysis.
Example 2
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 3.0%, malt extract 2.0%, peptone 1.0%, agar 2.0%, pH 5.3, steam sterilizing at 121 deg.C for 20min), and culturing at 26 deg.C for 7 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 3.0%, corn steep liquor 2.0%, peptone 2.0%, yeast extract 1.0%, pH 5.5, and 121 deg.C steam sterilizing for 20min), and performing shaking culture at 28 deg.C and 180r/min for 22h to obtain seed liquid required by fermentation;
(3) inoculating the seed solution into a sterilized fermentation medium (1.5% of glucose, 1.0% of corn steep liquor, 1.0% of peptone, 0.5% of yeast extract, 6.0 of pH value and 20min of steam sterilization at 121 ℃), adding 17 alpha-hydroxyprogesterone of which the total amount is 4% in batches, performing shaking culture at 28 ℃ and 180r/min, adding a 200g/L sugar solution in a flowing manner in the shaking culture process, and keeping the sugar concentration in the fermentation medium not lower than 2mg/mL to obtain a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion rate of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth was 96% by High Performance Liquid Chromatography (HPLC) analysis.
Example 3
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 2.5%, malt extract 1.5%, peptone 2.0%, agar 2.5%, pH 6.0, steam sterilizing at 121 deg.C for 20min), and culturing at 27 deg.C for 6 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 3.0%, corn steep liquor 1.5%, peptone 1.5%, yeast extract 1.0%, pH 6.0, steam sterilizing at 121 deg.C for 20min), and performing shake culture at 28 deg.C and 220r/min for 30h to obtain seed liquid required for fermentation. Inoculating the seed liquid into a sterilized fermentation culture medium (4.0% of glucose, 4.0% of corn steep liquor, 4.0% of peptone, 3.0% of yeast extract, 5.5 of pH value, steam sterilization at 121 ℃ for 20min), performing shaking culture at 28 ℃ and 220r/min, and adding a mixed suspension of a reactant 17 alpha-hydroxyprogesterone and a sugar solution (200g/L) in the shaking culture process, wherein the total feed ratio of the reactants is 4%, and the sugar concentration in the fermentation culture medium is kept to be not lower than 3mg/mL, so as to obtain a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion rate of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth was 98% by High Performance Liquid Chromatography (HPLC) analysis.
Example 4
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 5.0%, malt extract 5.0%, peptone 3.0%, agar 2.5%, pH 6.0, steam sterilizing at 121 deg.C for 20min), and culturing at 28 deg.C for 7 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 5.0%, corn steep liquor 5.0%, peptone 5.0%, yeast extract 2.0%, pH 6.5, and 121 deg.C steam sterilizing for 20min), and performing shake culture at 32 deg.C and 200r/min for 24h to obtain seed liquid required by fermentation;
(3) inoculating the seed solution into sterilized fermentation medium (glucose 3.0%, corn steep liquor 2.5%, peptone 2.5%, yeast extract 1.0%, pH 6.5, 121 deg.C steam sterilizing for 20min) at 5%, and performing shake culture at 26 deg.C and 200 r/min. Adding 17 alpha-hydroxyprogesterone with total amount of 6% in batches, performing shaking culture at 26 ℃ at 200r/min, and adding 200g/L sugar solution during the shaking culture to keep the sugar concentration in the fermentation medium not lower than 1.5mg/mL to obtain a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion yield of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth is 94% by High Performance Liquid Chromatography (HPLC) analysis.
Example 5
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 5.0%, malt extract 5.0%, peptone 3.0%, agar 2.5%, pH 6.0, steam sterilizing at 121 deg.C for 20min), and culturing at 26 deg.C for 6 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 2.5%, corn steep liquor 2.0%, peptone 2.0%, yeast extract 1.0%, pH 6.0, steam sterilizing at 121 deg.C for 20min), and shake culturing at 30 deg.C and 200r/min for 24 hr to obtain seed liquid required by fermentation;
(3) inoculating the seed solution into a sterilized fermentation medium (3.0% of glucose, 2.5% of corn steep liquor, 2.5% of peptone, 1.0% of yeast extract, 6.5 of pH value, steam sterilization at 121 ℃ for 20min), performing shaking culture at 30 ℃ and 220r/min, and supplementing a mixed suspension of a reactant 17 alpha-hydroxyprogesterone and a sugar solution (100g/L) in batches during the shaking culture process, wherein the total feeding ratio of the reactants is 6%, and the sugar concentration in the fermentation medium is kept to be not lower than 2mg/mL, so as to obtain a fermentation broth of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion yield of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth is 95% by High Performance Liquid Chromatography (HPLC) analysis.
Example 6
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 3.0%, malt extract 2.0%, peptone 1.5%, agar 2.0%, pH 5.5, steam sterilizing at 121 deg.C for 20min), and culturing at 26 deg.C for 7 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 2.0%, corn steep liquor 2.0%, peptone 2.0%, yeast extract 1.5%, pH 6.5, and 121 deg.C steam sterilizing for 20min), and performing shake culture at 28 deg.C and 180r/min for 30h to obtain seed liquid required by fermentation;
(3) inoculating the seed solution into a sterilized fermentation medium (3.0% of glucose, 1.5% of corn steep liquor, 1.5% of peptone, 1.5% of yeast extract, 6.5 of pH value and 20min of steam sterilization at 121 ℃), adding 20% of 17 alpha-hydroxyprogesterone at one time, carrying out shaking culture at 28 ℃ and 180r/min, supplementing 100g/L sugar solution for a certain time, and keeping the sugar concentration in the fermentation medium to be not lower than 2.5mg/mL to obtain a fermentation solution of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion yield of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth is 97% by High Performance Liquid Chromatography (HPLC) analysis.
Example 7
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 2.0%, malt extract 1.0%, peptone 1.5%, agar 2.5%, pH 5.5, steam sterilizing at 121 deg.C for 20min), and culturing at 24 deg.C for 7 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 1.5%, corn steep liquor 2.0%, peptone 2.0%, yeast extract 0.5%, pH 6.0, and 121 deg.C steam sterilizing for 20min), and performing shake culture at 28 deg.C and 200r/min for 24 hr to obtain seed liquid required by fermentation;
(3) inoculating the seed liquid into a sterilized fermentation culture medium (glucose 3.0%, corn steep liquor 1.5%, peptone 1.5%, yeast extract 1.5%, pH 6.5, steam sterilization at 121 ℃ for 20min) according to an inoculation amount of 5%, adding 17 alpha-hydroxyprogesterone in batches, and performing shaking culture at 28 ℃ and 200r/min, wherein the total feed ratio is 20%. Adding sugar solution with the concentration of 100g/L for a certain time, and keeping the sugar concentration in the fermentation medium not lower than 3mg/mL to obtain the fermentation liquid of the 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion yield of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth is 98% by High Performance Liquid Chromatography (HPLC) analysis.
Example 8
In this example, 11 α,17 α -dihydroxyprogesterone was prepared by the following method, specifically including the steps of:
(1) inoculating Aspergillus ochraceus test tube strain to slant culture medium (glucose 3.0%, malt extract 2.5%, peptone 1.5%, agar 2.0%, pH 5.5, steam sterilizing at 121 deg.C for 20min), and culturing at 26 deg.C for 7 days to obtain spore slant;
(2) taking out the prepared spore slant, inoculating with an inoculating loop, inoculating in seed culture medium (glucose 1.5%, corn steep liquor 2.5%, peptone 2.5%, yeast extract 1.5%, pH 6.5, and 121 deg.C steam sterilizing for 20min), and performing shake culture at 28 deg.C and 180r/min for 30h to obtain seed liquid required by fermentation;
(3) inoculating the seed liquid into a sterilized fermentation culture medium (2.0% of glucose, 2.5% of corn steep liquor, 1.5% of peptone, 2.0% of yeast extract, 6.5 of pH value, steam sterilization at 121 ℃ for 20min), performing shaking culture at 28 ℃ and 220r/min, and adding a mixed suspension of a reactant 17 alpha-hydroxyprogesterone and a sugar solution (200g/L) in the shaking culture process, wherein the total feed ratio of the reactants is 20%, and the sugar concentration in the fermentation culture medium is kept to be not lower than 5mg/mL, so as to obtain a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone.
The conversion yield of 11 alpha, 17 alpha-dihydroxyprogesterone in the fermentation broth is 98% by High Performance Liquid Chromatography (HPLC) analysis.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (19)
1. A method for preparing 11 α,17 α -dihydroxyprogesterone by bioconversion, comprising the steps of:
(1) preparing an inclined plane: inoculating ochratoxin strains to a slant culture medium, and culturing at constant temperature to generate yellow spore slant for later use;
(2) seed culture: inoculating strains from the yellow spore slant obtained in the step (1) into a seed culture medium for seed culture to obtain a seed solution;
(3) and (3) biotransformation: inoculating the seed solution obtained in the step (2) into a fermentation medium, adding a reactant 17 alpha-hydroxyprogesterone, performing shaking culture, supplementing a carbon source and the reactant 17 alpha-hydroxyprogesterone in the shaking culture process, keeping the concentration of the carbon source in a fermentation liquid to be not lower than 0.8mg/mL, and keeping the total feeding amount of the 17 alpha-hydroxyprogesterone to be 2-20% of the volume of the fermentation medium to obtain the fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone, wherein the total feeding amount of 2-20% of the volume of the fermentation medium means that the total reactant fed into 100mL of the culture medium is 2-20 g;
and (3) the concentration of the supplemented carbon source is not lower than 50 g/L.
2. The method according to claim 1, wherein the slant medium of step (1) comprises the following components in mass-to-volume ratio (w/v): 2.0-5.0% of glucose, 1.0-5.0% of malt extract, 0.5-3.0% of peptone and 2.0-2.5% of agar, wherein the pH value of the slant culture medium is 5.0-6.0, and the slant culture medium is used after steam sterilization, and the mass-to-volume ratio refers to the mass g of the components added in 100mL of the culture medium.
3. The method according to claim 1, wherein the temperature of the isothermal culture of step (1) is 25-28 ℃.
4. The method according to claim 1, wherein the incubation period in step (1) is 4 to 7 days.
5. The method of claim 1, wherein the seed medium of step (2) comprises the following components in a mass to volume ratio (w/v): 2.0-5.0% of glucose, 1.0-5.0% of corn steep liquor, 1.0-5.0% of peptone and 0.5-2.0% of yeast extract, wherein the pH value of the seed culture medium is 5.5-6.5, and the seed culture medium is used after steam sterilization, and the mass-to-volume ratio refers to the mass g of the components added in 100mL of the culture medium.
6. The method as claimed in claim 1, wherein the seed culture of step (2) is shake culture at 220r/min 160-.
7. The method according to claim 1, wherein the temperature of the seed culture in step (2) is 25-32 ℃.
8. The method according to claim 1, wherein the seed culture time in step (2) is 20-30 h.
9. The method according to claim 1, wherein the fermentation medium of step (3) comprises the following components in a mass to volume ratio (w/v): 1.0-4.0% of glucose, 0.5-4.0% of corn steep liquor, 0.5-4.0% of peptone and 0.5-3.0% of yeast extract, wherein the pH value of the fermentation medium is 5.5-6.5, and the fermentation medium is used after steam sterilization, and the mass-to-volume ratio refers to the mass g of the components added in 100mL of the medium.
10. The method of claim 1, wherein the seed liquid of step (3) is inoculated in an amount of 2-10% by volume of the fermentation medium.
11. The method of claim 1, wherein the seed liquid of step (3) is inoculated in an amount of 2-5% by volume of the fermentation medium.
12. The method as claimed in claim 1, wherein the shaking culture of step (3) is performed at a shaking rate of 160-220 r/min.
13. The method according to claim 1, wherein the temperature of the shaking culture in the step (3) is 25 to 32 ℃.
14. The method according to claim 1, wherein the carbon source in step (3) is an organic carbon source and/or an inorganic carbon source.
15. The method according to claim 1, wherein the carbon source in step (3) is selected from any one or a combination of at least two of glucose, fructose, galactose, maltose, lactose, sucrose, molasses, starch, pectin, cellulose, fats and oils, amino acids, alcohols, aldehydes, phenols, carbonates or bicarbonates.
16. The method according to claim 1, wherein the carbon source in step (3) is selected from any one of glucose, fructose, molasses, fats and oils, alcohol and carbonate or a combination of at least two of the foregoing.
17. The method of claim 1, wherein the carbon source is added in step (3) in a continuous feeding manner or in batches.
18. The method of claim 1, wherein the reactant 17 α -hydroxyprogesterone in step (3) is fed in a single batch or fed-batch manner, or fed-batch manner with a carbon source mixed stream.
19. Method according to claim 1, characterized in that it comprises the following steps:
(1) preparing an inclined plane: inoculating Aspergillus ochraceus strain to slant culture medium, and culturing at 25-28 deg.C for 4-7 days to obtain yellow spore slant;
(2) seed culture: inoculating strains from the yellow spore slant obtained in the step (1) into a seed culture medium, and performing shake culture at 25-32 ℃ and 220r/min for 20-30h to obtain a seed solution;
(3) and (3) biotransformation: inoculating the seed solution obtained in the step (2) into a fermentation medium by an inoculation amount of 2-10% of the volume of the fermentation medium, simultaneously adding 17 alpha-hydroxyprogesterone as a reactant, performing shaking culture at the temperature of 25-32 ℃ for 220r/min, supplementing a carbon source and 17 alpha-hydroxyprogesterone as the reactant in the shaking culture process, wherein the concentration of the supplemented carbon source is not lower than 50g/L, the concentration of the carbon source in the fermentation liquid is not lower than 0.8mg/mL, the total feeding amount of the 17 alpha-hydroxyprogesterone is 2-20% of the volume of the fermentation medium, and thus obtaining a fermentation liquid of 11 alpha, 17 alpha-dihydroxyprogesterone, wherein the total feeding amount of 2-20% of the volume of the fermentation medium means that the total feeding amount of the reactants in 100mL of the fermentation medium is 2-20 g.
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