CN109536562B - Method for preparing steroid drug intermediate by fermenting and converting phytosterol through mixed bacteria - Google Patents
Method for preparing steroid drug intermediate by fermenting and converting phytosterol through mixed bacteria Download PDFInfo
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Abstract
The invention belongs to the technical field of sterol biotransformation, and particularly relates to a method for preparing a steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by biotransformation of phytosterol through mixed fermentation. The invention inoculates mycobacterium TCCC 11028M3 into a fermentation medium containing phytosterol and transformation medium cyclodextrin, and inoculates aspergillus ochraceus CICC 41473 spore liquid for continuous transformation after culturing for 24 h. The invention realizes one-step conversion from phytosterol to 11 alpha-hydroxy-androst-4-ene-3, 17-dione by using mixed fermentation. The method improves the product yield, shortens the conversion period, simplifies the operation, saves the cost, and is suitable for the production of the steroid compound by biotransformation.
Description
The technical field is as follows:
the invention belongs to the technical field of sterol biotransformation, and particularly relates to a method for preparing a steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by biotransformation of phytosterol through mixed fermentation.
Background art:
the steroid compound is a terpenoid compound containing four cyclopentane polyhydrophenanthrene, and is widely present in animal and plant tissues and some microbial cells. Steroid drugs produced from steroid compounds have a very important regulatory effect on the body, and are widely used for disease treatment and health care. At present, the market demand of steroid drugs is second to that of antibiotics, such as aldosterone antagonist and eplerenone, which have been clinically used for treating various diseases such as heart failure, hypertension, edema, hepatic ascites, respiratory failure and the like. 11 α -hydroxy-androst-4-ene-3, 17-dione (11 α -hydroxyyandrost-4-ene-3, 17-dione, 11 α -OH AD) is the major adrenal steroid in mammals and is a key precursor for the synthesis of halogenated corticosteroids, a pharmacologically valuable analogue of natural corticosteroids. It has been found that various microorganisms can be hydroxylated into 11 alpha-OH AD by androst-4-ene-3,17-dione (AD), mainly some fungal species including Aspergillus ochraceus, Cunninghamella elegans, Rhizopus nigricans, Beauveria bassiana, Metarhizium anisopliae, etc.
For a long time, the main raw material in the production of steroid compounds is natural diosgenin. However, the chemical conversion of natural diosgenin into valuable steroids has many disadvantages, such as high cost, complicated steps, low yield, waste of land and wild plant resources, etc. In recent years, steroid production has focused primarily on the microbial transformation of phytosterols (sitosterol, stigmasterol, brassicasterol, campesterol), which are alternatives to natural diosgenin. Waste phytosterols from agriculture, food, cellulose manufacturing industries can be used to produce valuable steroids without purification. Mycobacteria, rhodococcus, aspergillus oryzae, fusarium moniliforme and the like can transform phytosterol to produce AD, wherein the mycobacteria and the rhodococcus are the microorganisms which produce AD most effectively.
In industrial production, two steps of microbial transformation are needed to produce 11 alpha-OH AD by using phytosterol as a raw material. Firstly, degrading phytosterol by a mycobacteria side chain to generate AD; in the second step, the strain having 11. alpha. hydroxylation ability is used to hydroxylate AD to obtain 11. alpha. -OH AD. In practical operation, for example, extraction and separation of products of each reaction step result in a great waste of manpower, material resources and time. If the above-mentioned two-step microbial conversion reaction is completed in one step, the whole process is simplified, and a large amount of organic solvent for extraction and separation operations is saved, thereby reducing the production cost. The mixed fermentation technology has certain difficulty, mainly because the growth and fermentation conditions of different strains are greatly different, such as temperature, culture medium, pH and the like, the two strains are difficult to respectively exert the maximum enzyme activity under the same fermentation condition. Therefore, it is important to find a suitable set of microbial combinations and fermentation processes that can achieve a one-step conversion of phytosterols to 11 α -OH AD.
The invention content is as follows:
the invention aims to provide a method for preparing a steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by fermenting and converting phytosterol through mixed bacteria, which shortens the fermentation period, saves the cost and improves the product generation rate.
The production strains employed in the present invention are Aspergillus ochraceus (Aspergillus ochracea) and Mycobacterium (Mycobacterium neoaurum).
The method for producing 11 alpha-hydroxy-androst-4-ene-3, 17-dione by using the microbial transformation phytosterol comprises the following steps:
(1) inoculating 3-14% of inoculum size into mycobacterium seed culture solution in a fermentation culture medium containing 2-30g/L phytosterol, culturing at 25-32 ℃ at 150-;
(2) suspending Aspergillus ochraceus spore in a final spore concentration of 0.5-2 × 106Inoculating the inoculum size of each/mL into the culture solution obtained in the step (1), and converting for 4-7d at the culture temperature of 25-32 ℃ and the rotation speed of 150-220 r/min;
preferably, when the concentration of the phytosterol is 2-7g/L, the product is converted for 4-5d, and the product yield reaches 79-87.3%;
further, the fermentation medium consists of: 25-45g/L of glucose, 2-10g/L of citric acid, 0.05-0.5g/L of ferric ammonium citrate, 0.5-2g/L of magnesium sulfate, 0.5-2g/L of dipotassium phosphate, 3.5-10g/L of diammonium phosphate, 30-60g/L of corn steep liquor, 2-10g/L of silkworm chrysalis powder, 10-100g/L of cyclodextrin and the balance of water, wherein the pH value is 6.5-7.0;
further, the culture method of the mycobacterium seed culture solution is as follows: sterile use of 0.5% Tween 80Washing the slant seeds with aqueous solution to make the thallus OD600The value is controlled to be 1.0 +/-0.2, 3 percent of inoculation amount is inoculated into a seed culture solution, and the seed culture solution is cultured at the temperature of 28 ℃ and the speed of 200r/min for 36-48h to the logarithmic phase;
preferably, the seed culture medium consists of 0.5g/L dipotassium phosphate, 0.5g/L magnesium sulfate, 0.05g/L ferric ammonium citrate, 2g/L citric acid, 2g/L ammonium nitrate, 20g/L glycerol, 10g/L glucose, 10g/L calcium carbonate and the balance of water, and the pH is natural;
further, the aspergillus ochraceus spore suspension is prepared by the following method: the thalli on the inclined plane is washed off by sterile water, and the suspension containing the spores is poured into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and is fully vibrated to prepare the spore suspension with a certain concentration.
Has the advantages that:
(1) the invention provides a high-efficiency method for preparing a steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by fermenting and converting phytosterol through mixed bacteria, which optimizes the optimal fermentation conditions to meet the requirements of high substrate utilization rate and high product yield, shortens the conversion period, simplifies the production process, reduces the production cost, ensures that the substrate concentration is 2-30g/L, the conversion is 4-7d, and the product yield reaches 75.5-87.3%.
(2) The method for generating 11 alpha-hydroxy-androst-4-ene-3, 17-dione by converting phytosterol through mixed culture of two bacteria reduces the inhibition effect of a substrate to a certain extent, and is beneficial to the generation of AD.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the scope of the invention.
The production strains used in the present invention are Aspergillus ochraceus (A. ochracea) and Mycobacterium (M. neoaurum), and the following examples will be described in the present invention with Aspergillus ochraceus (A. ochraceus) CICC 41473 (available to the public by CICC) and Mycobacterium (Mycobacterium neoaurum) TCCC 11028M3(MNR M3) deposited at the institute of Microbiologics and medicine of the university of Tianjin (the strains are disclosed in the scientific literature: Rili Xie, Yanbing Shen, Ning Qin, Yibo Wang, Liqiu Su, Min Wang. genetic engineering in ksdD influe on the ADD/AD ratio of Mycobacterium neoaurum [ J ]. Socientation for industry biology and Biotechnology (DOC) and the methods described in the present invention are described in the university of Microbiologics and technology [ J ]. 10252-1572, the above selected species are to be understood as exemplary and not limiting, and the method of the present invention is applicable to all species of strains known to date.
The one-step conversion from phytosterol to 11 alpha-hydroxyl-androstane-4-alkene-3, 17-diketone is realized through mixed fermentation, and the optimal conditions of fermentation are optimized to meet the requirements of high substrate utilization rate and high product generation rate. Because the fermentation conditions of fungi and bacteria are completely different, the mycobacteria are bacteria, the suitable temperature for growth and transformation is 30 ℃, and the suitable pH is 7.0-7.2; the growth and transformation of the fungus is suitably carried out at a temperature of 28 ℃ and a pH of 4.5 to 5.0. Therefore, one of the key problems of mixed fermentation is to find a suitable fermentation medium, so that in the fermentation medium, the mycobacteria can degrade the phytosterol by side chains to accumulate androst-4-ene-3,17-dione, meanwhile, the fungi can carry out hydroxylation on the C11 alpha position of androst-4-ene-3,17-dione to generate 11 alpha-hydroxy-androst-4-ene-3, 17-dione, and the generation rate of the product is not inferior to that of a single conversion process, thereby realizing the biotransformation reaction for generating 11 alpha-hydroxy-androst-4-ene-3, 17-dione from the phytosterol by mixed fermentation in one step; the fermentation medium provided by the invention can meet the requirements and realize one-step biotransformation of mixed fermentation.
In addition, the method is different from the prior art, the aspergillus ochraceus is added in a spore suspension mode instead of culturing mature aspergillus ochraceus thalli, the method improves the product generation rate, simplifies the fermentation process and saves the time cost and the economic cost required by culture of the aspergillus ochraceus thalli. In order to compare the advantages and the disadvantages of the two, the invention respectively optimizes the double-bacterium fermentation process (hereinafter referred to as sequential transformation) added with mature thalli of aspergillus ochraceus and the double-bacterium fermentation process (hereinafter referred to as mixed culture) added with spores of aspergillus ochraceus. Example 1 analyzes the influence of the inoculation amount of aspergillus ochraceus on the yield, example 2 analyzes the influence of the inoculation time of aspergillus ochraceus on the yield, and the optimized sequential transformation process conditions obtained in the examples 1 and 2 are combined; examples 3 and 4 analyzed the effect of Aspergillus ochraceus spore and substrate addition sequence on transformation and finally determined the transformation process conditions for mixed culture.
The cyclodextrin of the present invention may be natural cyclodextrin, such as α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, or derivatives of the above cyclodextrins, such as hydroxypropyl- β -cyclodextrin, methyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin, carboxymethyl- β -cyclodextrin, hydroxyethyl- β -cyclodextrin, sulfo- β -cyclodextrin, hydroxypropyl- γ -cyclodextrin, or methyl- γ -cyclodextrin. The following examples are all described by way of example of HP-beta-CD.
The present invention will be further described with reference to specific examples.
Example 1: effect of Aspergillus ochraceus inoculum size on the sequential conversion of phytosterols to 11 alpha-OH AD
(1) Adopting Mycobacterium (M.neoaurum) TCCC 11028M3(MNR M3) as a production strain, streaking the strain taken from glycerinum tube on a slant culture medium, culturing at 29 ℃ for 3-4 days, and activating the slant twice;
(2) seed culture of Mycobacterium TCCC 11028M3(MNR M3): pouring a certain amount of 0.5 percent Tween 80 sterile aqueous solution onto the solid culture medium in the step (1) under the aseptic operation to wash down the inclined plane seeds so as to ensure the OD of thalli600The value is controlled to be 1.0 +/-0.2, the mixture is inoculated into 30mL of seed culture medium by 3 percent of inoculation amount, and the mixture is cultured at the temperature of 28 ℃ and at the speed of 200r/min until logarithmic phase growth (36-48 h);
(3) mycobacterium TCCC 11028M3(MNR M3) fermentation culture: accurately weighing a proper amount of phytosterol to enable the final concentration of the phytosterol in a fermentation culture medium to be 3g/L, inoculating the seed culture solution in the step (2) into 50mL of fermentation culture medium containing the phytosterol according to the inoculation amount of 8%, and culturing for 5d at the temperature of 28 ℃ and at the speed of 200 r/min;
(4) adopting Aspergillus ochraceus (A. ochracea) CICC 41473 as a production strain, growing on a PDA slant for 5-7 days, and storing at 4 ℃;
(5) preparation of aspergillus ochraceus CICC 41473 spore suspension: pouring a certain amount of sterile water on the PDA solid culture medium in the step (4) under the aseptic operation condition, scraping off spores by using an inoculating loop, pouring the suspension containing the spores into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and fully vibrating to prepare spore suspension with a certain concentration (counting by a blood counting cell counting plate);
(6) fermenting and culturing aspergillus ochraceus CICC 41473: the spore suspension prepared in the step (5) is used for controlling the final concentration of the spores to be 106Inoculating the strain per mL into Aspergillus ochraceus CICC 41473 fermentation medium, filling 50mL of fermentation medium into a conical flask with the liquid filling amount of 250mL, culturing at 28 ℃, and culturing at the rotating speed of 200r/min for 24-28h to a logarithmic phase;
(7) and (3) biotransformation: inoculating 25%, 50%, 75% and 100% of the Aspergillus ochraceus CICC 41473 fermentation culture solution in the step (6) into the mycobacterium TCCC 11028M3(MNR M3) fermentation culture medium in the step (3) respectively to perform a transformation experiment, wherein the culture temperature is 28 ℃, and the transformation is carried out for 2-3d at the rotating speed of 200 r/min;
wherein the slant culture medium in the step (1) comprises 0.5g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 0.05g/L ferric ammonium citrate, 2g/L citric acid, 2g/L ammonium nitrate, 20g/L glycerol, 10g/L glucose, 10g/L calcium carbonate, 20g/L agar and the balance of water, and is sterilized under high-pressure steam at 115 ℃ for 20 min;
the seed culture medium in the step (2) comprises 0.5g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric ammonium citrate, 2g/L of citric acid, 2g/L of ammonium nitrate, 20g/L of glycerol, 10g/L of glucose, 10g/L of calcium carbonate and the balance of water, the pH is natural, and the seed culture medium is sterilized under high-pressure steam at 115 ℃ for 20 min;
the fermentation medium in the step (3) comprises 10g/L of glucose, 2g/L of citric acid, 0.05g/L of ferric ammonium citrate, 0.5g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 3.5g/L of diammonium phosphate, 38g/L of cyclodextrin and the balance of water, the pH value is 7.0-7.2, and the fermentation medium is sterilized for 20min under high-pressure steam at the temperature of 121 ℃;
the PDA culture medium in the step (4) comprises 200g/L of potato, 20g/L of glucose, 20g/L of agar and the balance of water, the pH is natural, and the potato is sterilized for 20min under high-pressure steam at 115 ℃;
the fermentation medium in the step (6) comprises 15g/L glucose, 40g/L corn steep liquor, 2g/L silkworm chrysalis meal, 1.5g/L ammonium sulfate and the balance of water, wherein the pH value is 4.5, and the fermentation medium is sterilized for 20min under high-pressure steam at 121 ℃;
(8) and (3) product detection: sampling the conversion solution obtained in the step (7) every 12h until the conversion is finished. Each sample was taken by adding 1ml of the fermentation broth to a 10ml centrifuge tube containing 2ml of ethyl acetate and further extracting with ultrasound using an ultrasonic cleaner. 1mL of the extracted supernatant was centrifuged at 13000r/min for 10min in a 1.5mL centrifuge tube. Accurately taking 0.1ml of centrifuged supernatant into a 1.5ml small centrifuge tube, placing the centrifuge tube in a fume hood for natural evaporation, accurately taking 1ml of a redissolving agent for redissolving in the volatilized centrifuge tube, performing ultrasonic treatment in an ultrasonic cleaning instrument for 10min, performing centrifugal treatment at 13000r/min for 10min, and analyzing the content of androstane-4-ene-3, 17-dione (AD) and 11 alpha-hydroxy-androstane-4-ene-3, 17-dione (11 alpha-OH AD) by using a high performance liquid chromatography.
(9) And (3) conversion result: as shown in Table 1, after the side chain degradation of mycobacteria and the conversion of phytosterol for 5d, when the inoculum size of Aspergillus ochraceus added is more than 50%, the substrate is basically completely transferred within 42 h. When the inoculation amount of aspergillus ochraceus is 50%, the generation rate of 11 alpha-hydroxy-androst-4-ene-3, 17-dione is 83%.
TABLE 1
| Inoculum size (%) | 25 | 50 | 75 | 100 |
| Production Rate (%) | 69.3 | 83 | 79.4 | 78.2 |
Example 2: effect of Aspergillus ochraceus inoculation time on sequential phytosterol production (11 alpha-OH AD)
The procedure was as in example 1 except for the following.
In the sequential transformation system, when the mycobacteria are allowed to grow to a certain stage and the side chain degrades phytosterol to generate androst-4-ene-3,17-dione (AD) with a certain concentration, it is advantageous to inoculate Aspergillus ochraceus to the whole flora system at a proper time, considering that the mold has a distinct advantage over the growth and propagation of mycobacteria.
(1) And (3) biotransformation: degrading phytosterol at a mycobacteria side chain to generate androst-4-ene-3,17-dione with a certain concentration, namely adding aspergillus ochraceus CICC 41473 thallus into the transformed 1d, 2d, 3d, 4d and 5d of the mycobacteria according to the inoculation amount of 50% to perform subsequent transformation experiments, wherein the culture temperature is 28 ℃, and the transformation is carried out for 2d at the rotating speed of 200 r/min;
(2) and (3) conversion result: the conversion rate is fastest in 48 hours of fermentation and the product generation rate tends to be stable from 84 hours to the end of fermentation in the process of degrading phytosterol to generate androst-4-ene-3,17-dione by mycobacteria side chains. As shown in Table 2, the yield of Aspergillus ochraceus products obtained by adding the optimal inoculation amount to the 4 th day of mycobacterial fermentation is 84.5%, which is higher than the 3 rd day by 3.2% and shortens the fermentation period compared with the 5 th day (83.2%).
TABLE 2
| Inoculation time (d) | 1 | 2 | 3 | 4 | 5 |
| Production Rate (%) | 74.2 | 77.6 | 81.3 | 84.5 | 83.2 |
By discussing the influence of the inoculation amount and the inoculation time of the second strain in the sequential transformation system on the whole fermentation system, the sequential transformation system is established by screening out the aspergillus ochraceus strain and the mycobacteria at the early stage, a scheme for respectively culturing sequential transformation is established, and the optimal transformation process for preliminarily determining the product transformation is as follows: the optimal inoculation amount of aspergillus ochraceus is 50%, the effect of adding aspergillus ochraceus is the best when mycobacteria are fermented for 4d, and the product generation rate after 2d of transformation is 84.5%.
TABLE 3
Example 3: study on hybrid culture for converting phytosterol to generate 11 alpha-OH AD
The procedure was as in example 1 except for the following.
(1) And (3) biotransformation: inoculating Mycobacterium TCCC 11028M3 seed culture solution into 50mL fermentation culture medium according to 8% inoculation amount, and inoculating the prepared aspergillus ochraceus CICC 41473 spore suspension with final spore concentration of 106Inoculating the inoculum size of each/mL into a fermentation culture medium, and then adding phytosterol with final concentration of 3g/L at 24h, 36h and 48h respectively, wherein the culture temperature is 28 ℃, and the phytosterol is converted for 5d at the rotating speed of 200 r/min;
the fermentation medium comprises 30g/L of glucose, 2g/L of citric acid, 0.05g/L of ferric ammonium citrate, 0.5g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 3.5g/L of diammonium phosphate, 40g/L of corn steep liquor, 2g/L of silkworm chrysalis meal, 38g/L of cyclodextrin and the balance of water, the pH value is 6.5-7.0, and the fermentation medium is sterilized under high-pressure steam at the temperature of 121 ℃ for 20 min;
(2) and (3) conversion result: as shown in Table 4, the conversion rates of the added substrate after 24 hours of cell culture were 72.6% and 71.1% respectively at pH 6.5 and 7.2, and the fermentation period was shortened to some extent.
TABLE 4
Example 4: influence of Aspergillus ochraceus and substrate addition sequence on mixed culture conversion of phytosterol to 11 alpha-OH AD
The procedure was as in example 3 except for the following.
(1) The mycobacterium TCCC 11028M3 is fermented and cultured: inoculating the seed culture solution into 50mL of fermentation medium containing phytosterol according to the inoculation amount of 8%, wherein the culture temperature is 28 ℃, and the seed culture solution is converted for 24 hours at the rotating speed of 200 r/min;
(2) and (3) biotransformation: the second bacteria and substrate are added in sequence, namely, the phytosterol substrate with the concentration of 3g/L is added into the fermentation medium, and the final concentration of 10 is inoculated in 24 hours, 48 hours, 72 hours and 96 hours of conversion respectively6Culturing Aspergillus ochraceus spore liquid at a spore amount of one per mL at 28 deg.C, and converting at 200r/min for 5 d; (3) and (3) conversion result: as shown in Table 5, the yield of the 5d transformed product after 24h addition of the second strain was 87.3%, which is twice as high as the other inoculation times and greatly shortens the fermentation period.
TABLE 5
| Inoculation time (h) | 24 | 48 | 72 | 96 |
| Production Rate (%) | 87.3 | 39.2 | 48.4 | 43.6 |
Through discussing the influence of the adding sequence of the second bacterium and the substrate in the mixed bacterium culture system on the whole fermentation system, a scheme for mixed culture and transformation of the two bacteria is constructed, and as shown in table 6, the optimal transformation process for primarily determining product transformation is as follows: firstly adding a substrate into a mixed fermentation culture medium, degrading side chains for 24h, then adding aspergillus ochraceus with a certain amount of spores for hydroxylation conversion for 4-5d, wherein the effect is optimal, and the product generation rate reaches 78.5-87.3%.
TABLE 6
Through the examples 1-4, the product formation rate, the substrate utilization rate, the by-product rate, the fermentation period and the like of the two schemes are comprehensively compared, the by-product rate of the sequential conversion is lower by 1.7% compared with that of the mixed culture, the fermentation period is about 6d, but the time-space yield STY (the target product amount generated in different volumes in unit time and used for representing the process efficiency) is larger and the product formation rate is higher because the mixed culture reduces the fermentation culture process of aspergillus ochraceus. In conclusion, the mixed bacteria fermentation of the mixed culture of the two bacteria is adopted to realize the one-step preparation of the steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione from the phytosterol.
Example 5: mixed culture and transformation of phytosterol into 11 alpha-OH AD
(1) The strain obtained from Glycine max (L.) Merr is streaked on slant culture medium by using Mycobacterium (M. neoaurum) TCCC 11028M3(MNR M3) as production strain, and cultured at 29 deg.C for 3-4 days. Activating the inclined plane twice;
(2) seed culture: pouring a certain amount of 0.5 percent Tween 80 sterile aqueous solution onto the solid culture medium in the step (1) under the aseptic operation to wash down the inclined plane seeds so as to ensure the OD of thalli600The value is controlled to be 1.0 +/-0.2. Inoculating 3% of the strain into 30mL of seed culture solution, and culturing at 28 ℃ at 200r/min until logarithmic phase (36-48 h);
(3) fermentation culture: accurately weighing a proper amount of phytosterol to ensure that the final concentration of the phytosterol in a fermentation culture medium is 5 g/L. Inoculating the seed culture solution in the step (2) into 50mL of fermentation medium containing phytosterol according to the inoculation amount of 10%, and culturing at 30 ℃ for 24h at 180 r/min;
(4) aspergillus ochraceus CICC 41473 is adopted as a production strain, and is stored at 4 ℃ after 5-7 days of growth on a PDA inclined plane;
(5) spore suspension: pouring a certain amount of sterile water on the PDA solid culture medium in the step (4) under the aseptic operation condition, scraping off spores by using an inoculating loop, pouring the suspension containing the spores into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and fully vibrating to prepare spore suspension with a certain concentration (counting by a blood counting cell counting plate);
(6) and (3) biotransformation: then the spore suspension prepared in the step (5) is used for controlling the final concentration of the spores to be 1.5 multiplied by 106Inoculating the inoculum size of each/mL into the fermentation culture medium in the step (3), wherein the culture temperature is 30 ℃, and the strain is converted for 4d at the rotating speed of 180 r/min; after 4d conversion, the product formation was 79.1%.
Wherein the slant culture medium in the step (1) comprises 0.5g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 0.05g/L ferric ammonium citrate, 2g/L citric acid, 2g/L ammonium nitrate, 20g/L glycerol, 10g/L glucose, 10g/L calcium carbonate, 20g/L agar and 20min sterilization under high-pressure steam at 115 ℃; the seed culture medium in the step (2) comprises 0.5g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric ammonium citrate, 2g/L of citric acid, 2g/L of ammonium nitrate, 20g/L of glycerol, 10g/L of glucose and 10g/L of calcium carbonate, has natural pH and is sterilized under high-pressure steam at 115 ℃ for 20 min; the fermentation medium in the step (3) comprises 25g/L of glucose, 2g/L of citric acid, 0.05g/L of ferric ammonium citrate, 0.5g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 3.5g/L of diammonium phosphate, 30g/L of corn steep liquor, 2g/L of silkworm chrysalis meal, 40g/L of cyclodextrin and the balance of water, the pH value is 6.5-7.0, and the fermentation medium is sterilized under high-pressure steam at 121 ℃ for 20 min; the PDA culture medium in the step (4) comprises 200g/L of potato, 20g/L of glucose, 20g/L of agar, natural pH, and sterilizing with 115 deg.C high pressure steam for 20 min.
Example 6: mixed culture and transformation of phytosterol into 11 alpha-OH AD
(1) The strain obtained from Glycine max (L.) Merr is streaked on a slant culture medium using Mycobacterium neoaurum TCCC 11028M3(MNR M3) as a production strain, and cultured at 29 deg.C for 3-4 days. Activating the inclined plane twice;
(2) seed culture: pouring a certain amount of 0.5 percent Tween 80 sterile aqueous solution onto the solid culture medium in the step (1) under the aseptic operation to wash down the inclined plane seeds so as to ensure the OD of thalli600The value is controlled to be 1.0 +/-0.2. Inoculating 3% of the strain into 30mL of seed culture solution, and culturing at 28 ℃ at 200r/min until logarithmic phase (36-48 h);
(3) fermentation culture: accurately weighing a proper amount of phytosterol to ensure that the final concentration of the phytosterol in a fermentation culture medium is 7 g/L. Inoculating the seed culture solution in the step (2) into 50mL of fermentation medium containing phytosterol according to the inoculation amount of 14%, and culturing at 32 ℃ for 24h at 220 r/min;
(4) aspergillus ochraceus CICC 41473 is adopted as a production strain, and is stored at 4 ℃ after 5-7 days of growth on a PDA inclined plane;
(5) spore suspension: pouring a certain amount of sterile water on the PDA solid culture medium in the step (4) under the aseptic operation condition, scraping off spores by using an inoculating loop, pouring the suspension containing the spores into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and fully vibrating to prepare spore suspension with a certain concentration (counting by a blood counting cell counting plate);
(6) and (3) biotransformation: then the spore suspension prepared in the step (5) is used for controlling the final concentration of the spores to be 2 multiplied by 106Inoculating the inoculum size of each/mL into the fermentation culture medium in the step (3), wherein the culture temperature is 32 ℃, and the strain is converted for 5d at the rotating speed of 220 r/min; after 5d of conversion, the product formation was 81.0%.
Wherein the fermentation medium in the step (3) comprises 45g/L of glucose, 10g/L of citric acid, 0.5g/L of ferric ammonium citrate, 2g/L of magnesium sulfate, 2g/L of dipotassium phosphate, 10g/L of diammonium phosphate, 60g/L of corn steep liquor, 10g/L of silkworm chrysalis meal, 50g/L of cyclodextrin and the balance of water, the pH value is 6.5-7.0, and the fermentation medium is sterilized under high-pressure steam at 121 ℃ for 20 min; the rest of the medium was the same as in example 5.
Example 7: mixed culture and transformation of phytosterol into 11 alpha-OH AD
(1) The strain obtained from Glycine max (L.) Merr is streaked on slant culture medium by using Mycobacterium (M. neoaurum) TCCC 11028M3(MNR M3) as production strain, and cultured at 29 deg.C for 3-4 days. Activating the inclined plane twice;
(2) seed culture: pouring a certain amount of 0.5 percent Tween 80 sterile aqueous solution onto the solid culture medium in the step (1) under the aseptic operation to wash down the inclined plane seeds so as to ensure the OD of thalli600The value is controlled to be 1.0 +/-0.2. Inoculating 3% of the strain into 30mL of seed culture solution, and culturing at 28 ℃ at 200r/min until logarithmic phase (36-48 h);
(3) fermentation culture: accurately weighing a proper amount of phytosterol to ensure that the final concentration of the phytosterol in a fermentation culture medium is 20 g/L. Inoculating the seed culture solution in the step (2) into 50mL of fermentation medium containing phytosterol according to the inoculation amount of 14%, and culturing at 32 ℃ for 24h at 220 r/min;
(4) aspergillus ochraceus CICC 41473 is adopted as a production strain, and is stored at 4 ℃ after 5-7 days of growth on a PDA inclined plane;
(5) spore suspension: pouring a certain amount of sterile water on the PDA solid culture medium in the step (4) under the aseptic operation condition, scraping off spores by using an inoculating loop, pouring the suspension containing the spores into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and fully vibrating to prepare spore suspension with a certain concentration (counting by a blood counting cell counting plate);
(6) and (3) biotransformation: then the spore suspension prepared in the step (5) is used for controlling the final concentration of the spores to be 2 multiplied by 106Inoculating the inoculum size of each/mL into the fermentation culture medium in the step (3), wherein the culture temperature is 32 ℃, and the strain is converted for 7d at the rotating speed of 220 r/min; after 7d conversion, the product formation was 79.2%.
Wherein the fermentation medium in the step (3) comprises 45g/L of glucose, 10g/L of citric acid, 0.5g/L of ferric ammonium citrate, 2g/L of magnesium sulfate, 2g/L of dipotassium phosphate, 10g/L of diammonium phosphate, 60g/L of corn steep liquor, 10g/L of silkworm chrysalis powder, 70g/L of cyclodextrin and the balance of water, the pH value is 6.5-7.0, and the fermentation medium is sterilized under high-pressure steam at 121 ℃ for 20 min; the rest of the medium was the same as in example 5.
Although the present invention has been described in detail above with reference to the general description, the specific embodiments and experiments, it can be modified or improved based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. A method for preparing a steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by fermenting and converting phytosterol through mixed bacteria is characterized by comprising the following steps:
(1) inoculating 3-14% of inoculum size into mycobacterium seed culture solution in a fermentation culture medium containing 2-30g/L phytosterol, culturing at 25-32 ℃ at 150-;
(2) suspending Aspergillus ochraceus spore in a final spore concentration of 0.5 × 106-2×106Inoculating the inoculum size of each/mL into the culture solution obtained in the step (1), and converting for 4-7d at the culture temperature of 25-32 ℃ and the rotation speed of 150-220 r/min;
the Aspergillus ochraceus is Aspergillus ochraceus (A), (B)A.ochraceus）CICC 41473；
The mycobacterium is mycobacterium (A), (B), (C)Mycobacterium neoaurum）TCCC 11028 M3（MNR M3）；
The fermentation medium comprises the following components: 25-45g/L of glucose, 2-10g/L of citric acid, 0.05-0.5g/L of ferric ammonium citrate, 0.5-2g/L of magnesium sulfate, 0.5-2g/L of dipotassium phosphate, 3.5-10g/L of diammonium phosphate, 30-60g/L of corn steep liquor, 2-10g/L of silkworm chrysalis powder, 10-100g/L of cyclodextrin, the balance of water and the pH value of 6.5-7.0.
2. The method for preparing the steroid drug intermediate 11 α -hydroxy-androst-4-ene-3, 17-dione by fermenting phytosterol with mixed bacteria as claimed in claim 1, wherein the culture method of the mycobacteria seed culture solution is as follows: washing the slant seeds with 0.5% Tween 80 sterile water solution to make the thallus OD600The value is controlled to be 1.0 +/-0.2, 3-15% of inoculation amount is inoculated into a seed culture medium, and the seed culture medium is cultured at 28 ℃, 200r/min and 36-48h to logarithmic phase.
3. The method for preparing steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by mixed fermentation and conversion of phytosterol as claimed in claim 2, wherein the seed culture medium comprises dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferric ammonium citrate 0.05g/L, citric acid 2g/L, ammonium nitrate 2g/L, glycerol 20g/L, glucose 10g/L, calcium carbonate 10g/L, and the balance of water, has a natural pH, and is sterilized under high pressure steam at 115 ℃ for 20 min.
4. The method for preparing the steroid drug intermediate 11 alpha-hydroxy-androst-4-ene-3, 17-dione by fermenting and converting phytosterol with mixed bacteria as claimed in claim 1, wherein the aspergillus ochraceus spore suspension is prepared by the following method: the thalli on the inclined plane is washed off by sterile water, and the suspension containing the spores is poured into a sterilized triangular flask which is attached with 3 layers of gauze and is filled with glass beads, and is fully vibrated to prepare the spore suspension with a certain concentration.
5. The method for preparing steroid drug intermediate 11 α -hydroxy-androst-4-ene-3, 17-dione by mixed fermentation of phytosterols as claimed in claim 1, wherein the conversion time is 4-5 days at a phytosterol concentration of 2-7 g/L.
6. The method for preparing steroid drug intermediate 11 α -hydroxy-androst-4-ene-3, 17-dione by fermenting phytosterol with mixed bacteria as claimed in claim 1, comprising the steps of:
(1) inoculating mycobacterium seed culture solution into a fermentation culture medium containing 3g/L phytosterol according to the inoculation amount of 8%, and culturing at 28 ℃ at 200r/min for 24 h;
(2) suspending Aspergillus ochraceus spore in a spore suspension with a final concentration of 106Inoculating the inoculum size of each/mL into the culture solution obtained in the step (1), and converting for 5d at the culture temperature of 28 ℃ and the rotating speed of 200 r/min;
the fermentation medium comprises 30g/L of glucose, 2g/L of citric acid, 0.05g/L of ferric ammonium citrate, 0.5g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 3.5g/L of diammonium phosphate, 40g/L of corn steep liquor, 2g/L of silkworm chrysalis powder, 38g/L of cyclodextrin and the balance of water, the pH value is 6.5-7.0, and the fermentation medium is sterilized under high-pressure steam at the temperature of 121 ℃ for 20 min.
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