A method for preparing a steroid drug intermediate

Abstract

The present invention belongs to the field of microbial transformation technology, specifically a method for preparing a steroid drug intermediate, comprising the following steps: strain activation; seed liquid culture; fermentation conversion in a fermenter; extraction of conversion products; purification of conversion products; structural confirmation of conversion products. By using androstenedione as the starting material, a strain is selected to ferment and prepare 1,4-androstenedione, and the culture medium selected by strain activation is LB solid culture medium, which is cultured in a constant temperature incubator at 28°C for 24 hours after inoculation; LB liquid culture medium is selected for seed liquid culture, and a single colony on a plate is picked up with an inoculation loop and inoculated and then cultured in a constant temperature shaker at 28°C for 16 hours; then the substrate 4-androstenedione is fermented; an equal volume of ethyl acetate is added to the culture medium for extraction and separation, and it is in a white powder after decolorization and purification, and the selected strain has a short conversion time, and the conversion rate can reach 70% in only 24 hours, realizing the function of rapid conversion and achieving the effect of improving the conversion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial transformation, in particular to a method for preparing a steroid drug intermediate. Background Art

[0002] Steroid drugs generally refer to steroid drugs, mainly including glucocorticoid drugs, adrenocortical hormone drugs, sex hormone drugs, etc.; steroid drug intermediates are key raw materials for the production of steroid drugs. 4-androstenedione (AD) and 1,4-androstenedione (ADD) are both important steroid drug intermediates. 4-androstenedione (AD) can be used as a precursor of hormone substances such as androgens and protein synthesis-stimulating hormones, and can also be used to synthesize drugs such as spironolactone, hydrocortisone, prednisone oxide, and dexamethasone; while 1,4-androstenedione (ADD) can be used for the synthesis of drugs such as estrogens and oral contraceptives.

[0003] The structures of 4-androstenedione (AD) and 1,4-androstenedione (ADD) differ only at the C1,2 positions. After dehydrogenation at the C1,2 positions, 4-androstenedione (AD) becomes 1,4-androstenedione (AD). Studies have shown that when double bonds are introduced into steroid compounds, such as at the C1,2 positions of the parent nucleus of anti-inflammatory steroid hormone drugs, their anti-inflammatory effects can be multiplied. However, it is difficult to introduce double bonds at specific sites using traditional chemical methods, and microbial methods can effectively solve this problem.

[0004] Among the currently available microbial transformation methods, the most widely used strain is Arthrobacter, but the transformation time of this strain is relatively long and the strain is not easy to separate and purify, resulting in a low conversion rate of the transformation product and thus a low efficiency in the preparation of steroid drug intermediates.

[0005] To this end, the present invention provides a method for preparing a steroid drug intermediate. Summary of the invention

[0006] In order to make up for the shortcomings of the prior art, the technical problems proposed in the background technology that the strain transformation time is long and the strain is not easy to separate and purify, resulting in low conversion rate of the transformation product and thus low efficiency in the preparation of steroid drug intermediates are solved.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a steroid drug intermediate according to the present invention comprises the following steps:

[0008] S1: Activation of bacterial strains: Take the strain Comamonas thiooxydans CSUSB4 and inoculate it into the LB plate with an inoculation loop, and culture it in a constant temperature incubator;

[0009] S2: Seed liquid culture: Take the strain Comamonas thiooxydans CSUSB4 plate, pick a single colony with an inoculation loop and inoculate it into LB liquid medium, and then shake and culture it on a constant temperature shaker;

[0010] S3: fermentation conversion in a fermentation tank: add the pre-sterilized substrate oil solution into the fermentation tank according to aseptic operation specifications, and then aseptically pour the cultured bacteria Comamonas thiooxydans CSUSB4 seed solution into the fermentation tank for constant temperature fermentation;

[0011] S4: Extraction of conversion product: extract the oil layer of the fermentation liquid and let it stand for separation, collect the organic phase; slowly spin dry the extract on a rotary evaporator, wash it with water and dry it in an oven at 50°C to obtain the extraction product;

[0012] S5: Purification of conversion product: The crude product was purified by semi-preparative liquid phase separation to obtain a white powder;

[0013] S6: Structural confirmation of the transformation product: The structure was identified as 1,4-androstenedione.

[0014] Preferably, the strain in S1 is Comamonas thiooxydans CSUSB4, the Comamonas thiooxydans CSUSB4 strain uses 4-androstenedione as a starting material, the concentration of the Comamonas thiooxydans CSUSB4 strain is 5 g / L; the inoculation amount of the Comamonas thiooxydans CSUSB4 strain is 5%; the culture medium selected for activation of the strain is LB solid culture medium, and after inoculation, the strain is cultured at a constant temperature of 28° C. in an incubator for 24 hours.

[0015] Preferably, the LB liquid culture medium in S2 is used to pick a single colony on the plate with an inoculation loop and then cultured in a constant temperature shaker at 28°C at 180r for 16h.

[0016] Preferably, the fermentation conversion in S3 uses LB liquid culture medium, the substrate is dissolved in vegetable soybean oil, sterilized separately and added to the fermenter, the seed liquid is inoculated into the fermenter, and the fermentation is carried out at a constant temperature of 28° C. for 24 hours and a rotation speed of 200 r.

[0017] Preferably, the substrate is 4-androstenedione, and the concentration of the 4-androstenedione is 10 g / L; the ratio of the 4-androstenedione to the vegetable soybean oil is set to 1:10.

[0018] Preferably, the components of the LB liquid culture medium include: 10 g of 4-androstenedione, 100 g of vegetable soybean oil, 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride and 1 L of water.

[0019] Preferably, the pH in the LB liquid culture medium is controlled to be 7.2.

[0020] Preferably, the content of the conversion product in S4 is detected by high performance liquid chromatography, and the product is reconstituted with a mobile phase and then filtered through a 0.22 μm organic membrane to remove impurities.

[0021] Preferably, the conversion product in S4 is extracted using an ethyl acetate oil solution, and the ratio of ethyl acetate to oil is 1:1; the extraction of the conversion product also includes: extracting the product in the oil layer three times, controlling the temperature to 50°C and stirring the extraction for 5-10 minutes, placing it in a separatory funnel and letting it stand for separation, and collecting the organic phase; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 50°C to obtain the extracted product.

[0022] Preferably, the conversion product in S5 is purified by semi-preparative liquid phase separation technology.

[0023] The beneficial effects of the present invention are as follows:

[0024] 1. A method for preparing a steroid drug intermediate according to the present invention comprises the following steps: using androstenedione (AD) as a starting material and fermenting 1,4-androstenedione (AD) using the strain Comamonas thiooxydans CSUSB4; the culture medium selected for the activation of the strain Comamonas thiooxydans CSUSB4 is LB solid culture medium, which is inoculated and cultured in a constant temperature incubator at 28°C for 24 hours; then the seed liquid of the strain Comamonas thiooxydans CSUSB4 is cultured in LB liquid culture medium, a single colony on a plate is picked up with an inoculation loop and inoculated, and then cultured in a constant temperature shaker at 28°C for 16 hours; then the substrate 4-androstenedione is dissolved in vegetable soybean oil at a ratio of 10%, sterilized separately and added to a fermenter, the seed liquid is inoculated in the fermenter, and fermented at 28°C for 24 hours; an equal volume of ethyl acetate is added to the culture medium after culture for extraction and separation, and the product is in a white powder after decolorization and purification, which is identified as 1,4-androstenedione, and the selected strain Comamonas The conversion time of thiooxydansCSUSB4 is short, and the conversion rate can reach 70% in just 24 hours, realizing the function of rapid conversion and achieving the effect of improving the conversion rate.

[0025] 2. The preparation method of a steroid drug intermediate described in the present invention adopts a combination of two-phase culture of organic phase and aqueous phase, and grows in a culture medium containing an organic solvent and an aqueous phase respectively. Since water and some organic matter have different polarities, they may form two immiscible phases when mixed. By adjusting the ratio of the organic phase to the aqueous phase, the yield of the secondary product can be significantly improved; wherein in the two-phase culture, the effect of the organic solvent on cell growth and product metabolism, as well as the oxygen transfer situation, needs to be considered; at the same time, the density difference between the organic phase and the aqueous phase will lead to solution stratification, and the culture conditions need to be reasonably controlled to obtain the best effect.

[0026] 3. A method for preparing a steroid drug intermediate of the present invention, which is analyzed by HPLC and high performance liquid chromatography, and the method for detecting the content of the conversion product is high performance liquid chromatography, after re-dissolving with a mobile phase, filtering with a 0.22 μm organic membrane to remove impurities, after HPLC analysis of the substrate conversion is complete, the fermentation tank is inactivated at 121° C., the fermentation liquid oil layer is collected, and the product in the oil layer is extracted three times according to the ratio of ethyl acetate: oil = 1:1, the temperature is controlled at 50° C. and stirred for extraction for 5-10 minutes, and the mixture is placed in a separating funnel and allowed to stand for separation, and the organic phase is collected; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 50° C. to obtain an extracted product, and after extraction, the obtained crude product is separated and purified by semi-preparative liquid phase separation, and the product is white powder after decolorization and purification to improve its purity and quality, and the chemical and physical properties of the extracted and purified product are identified, such as by mass spectrometry, nuclear magnetic resonance and other means to determine its structure, and the molecular structure is identified as: 1,4-androstenedione. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings.

[0028] Figure 1 It is a flow chart of the preparation method of the steroid drug intermediate in the present invention;

[0029] Figure 2 is a molecular structure diagram of 4-androstenedione in the present invention;

[0030] Figure 3 It is the molecular structure diagram of 1,4-androstenedione in the present invention;

[0031] Figure 4 It is a schematic diagram of the preparation process of the steroid drug intermediate in the present invention. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0033] Embodiment 1:

[0034] like Figures 1 to 4 As shown, a method for preparing a steroid drug intermediate according to an embodiment of the present invention comprises the following steps:

[0035] S1: Activation of bacterial strains: Take the strain Comamonas thiooxydans CSUSB4 and inoculate it into the LB plate with an inoculation loop, and culture it in a constant temperature incubator;

[0036] S2: Seed liquid culture: Take the strain Comamonas thiooxydans CSUSB4 plate, pick a single colony with an inoculation loop and inoculate it into LB liquid medium, and then shake and culture it on a constant temperature shaker;

[0037] S3: fermentation conversion in a fermentation tank: add the pre-sterilized substrate oil solution into the fermentation tank according to aseptic operation specifications, and then aseptically pour the cultured bacteria Comamonas thiooxydans CSUSB4 seed solution into the fermentation tank for constant temperature fermentation;

[0038] S4: Extraction of conversion product: extract the oil layer of the fermentation liquid and let it stand for separation, collect the organic phase; slowly spin dry the extract on a rotary evaporator, wash it with water and dry it in an oven at 50°C to obtain the extraction product;

[0039] S5: Purification of conversion product: The crude product was purified by semi-preparative liquid phase separation to obtain a white powder;

[0040] S6: Structural confirmation of the transformation product: The structure was identified as 1,4-androstenedione.

[0041] Since the transformation time of Arthrobacter species is long and the species is not easy to separate and purify, the conversion rate of the transformation product is low, and thus the efficiency of preparing the steroid drug intermediate is low. The preparation method provided by the present invention uses 4-androstenedione (AD) as a starting material and uses the species Comamonas thiooxydans CSUSB4;

[0042] Take the frozen glycerol tube of the strain Comamonas thiooxydans CSUSB4, put it in an ice box to thaw, inoculate it into the LB plate with an inoculation loop on an ultra-clear workbench, and culture it in a constant temperature incubator at 28°C for 24 hours. Then pick a single colony with an inoculation loop and inoculate it again into the LB plate, and culture it in a constant temperature incubator at 28°C for 24 hours.

[0043] LB liquid medium was used for seed liquid culture. 100 ml LB liquid medium was prepared in a 250 ml conical flask at 121 °C and sterilized for 30 min. A single colony was picked from a Comamonas thiooxydans CSUSB4 plate with an inoculation loop and inoculated into the LB liquid medium. The culture was then placed on a constant temperature shaker at 28 °C and shaken at 180 r for 16 h.

[0044] Prepare 10L LB liquid culture medium, adjust the pH to 7.2, put it into a fermenter, sterilize it at 121°C for 30 minutes, wait for the fermentation liquid to cool to room temperature, first add the pre-sterilized substrate oil solution into the fermenter according to the aseptic operation specification, and then aseptically pour the cultured strain Comamonas thiooxydans CSUSB4 seed liquid into the fermenter, adjust the speed to 200r, and ferment it at a constant temperature of 28°C for 24 hours;

[0045] After the substrate conversion is complete according to HPLC analysis, the fermentation tank is inactivated at 121°C, the fermentation liquid oil layer is collected, and the product in the oil layer is extracted three times according to the ratio of ethyl acetate: oil = 1:1, the temperature is controlled at 50°C and stirred for extraction for 5-10 minutes, and the mixture is placed in a separatory funnel for standing and liquid separation, and the organic phase is collected; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water, and dried in an oven at 50°C to obtain the extracted product, and a white crystalline product is obtained;

[0046] The crude product was purified by semi-preparative liquid phase separation to obtain a white powder. The sample was sent to Jiangsu Yongjian Inspection and Testing Co., Ltd. for structural confirmation, and the structure was identified as 1,4-androstenedione.

[0047] like Figure 1 and Figure 4 As shown, the strain in S1 is Comamonas thiooxydans CSUSB4, the Comamonas thiooxydans CSUSB4 strain uses 4-androstenedione as a starting material, and the concentration of the Comamonas thiooxydans CSUSB4 strain is 5 g / L; the inoculation amount of the Comamonas thiooxydans CSUSB4 strain is 5%; the culture medium selected for the activation of the strain is LB solid culture medium, and after inoculation, it is cultured at a constant temperature of 28° C. in an incubator for 24 hours.

[0048] When the strain activation provided by the present invention is used, 5 g / L of the strain Comamonas thiooxydans CSUSB4 is added to a bioconversion system with a substrate 4-androstenedione (AD) concentration of 10 g / L. The strain is a bacterial strain with unique biochemical characteristics and research value, has good medical research value in terms of drug resistance and genome, and has a specific enzyme system that can catalyze the conversion of androstenedione. The product 1,4-androstenedione (AD) is prepared by fermentation. The culture medium selected for the Comamonas thiooxydans CSUSB4 strain activation is an LB solid culture medium, and after inoculation, the culture is cultured at a constant temperature of 28°C in an incubator for 24 hours.

[0049] like Figure 1 and Figure 4 As shown, the LB liquid culture medium in S2 is used to pick a single colony on the plate with an inoculation loop and then cultured in a constant temperature shaker at 28° C. at 180 r for 16 h.

[0050] The seed liquid culture provided by the present invention maintains the concentration of nutrient-promoting substances in the culture liquid, reduces costs, shortens the culture cycle, and increases yields when used. During the seed liquid culture process, it is necessary to control appropriate culture conditions, such as temperature, pH value, and ventilation volume, to obtain the best bacterial strain growth effect, and obtain bacterial strains with strong vitality and sufficient quantity for subsequent experiments or production use; the seed liquid culture uses LB liquid culture medium for pre-culture of bacterial strains and culture of genetically engineered receptor bacteria, prepares 100 ml of LB liquid culture medium in a 250 ml conical flask, sterilizes at 121° C. for 30 min, takes a bacterial strain Comamonas thiooxydans CSUSB4 plate, picks a single colony with an inoculation loop and inoculates it in the LB liquid culture medium, and then cultures it in a constant temperature shaker at 28° C. at 180 r for 16 h;

[0051] When using a constant temperature shaker, please note: the samples should be placed symmetrically to keep the equipment running in balance; the temperature and oscillation speed should be accurately set according to the experimental requirements.

[0052] like Figure 1 and Figure 4 As shown, the fermentation conversion in S3 uses LB liquid culture medium, the substrate is dissolved in vegetable soybean oil, sterilized separately and added to the fermentation tank, the seed liquid is inoculated into the fermentation tank, and the fermentation is carried out at a constant temperature of 28° C. for 24 hours and a rotation speed of 200 r.

[0053] The substrate is 4-androstenedione, and the concentration of the 4-androstenedione is 10 g / L; the ratio of the 4-androstenedione to the vegetable soybean oil is set to 1:10.

[0054] When the LB liquid culture medium provided by the present invention is used, 10 L LB liquid culture medium is prepared, the pH value of which is adjusted to 7.2, and the medium is placed in a fermenter, sterilized at 121° C. for 30 minutes, and after the fermented liquid is cooled to room temperature, a pre-sterilized substrate oil solution is first added to the fermenter according to a sterile operation specification, and then the cultured strain Comamonas thiooxydans CSUSB4 seed liquid is aseptically poured into the fermenter, the rotation speed is adjusted to 200 r, and the fermentation is carried out at a constant temperature for 24 hours at 28° C.

[0055] like Figure 1 and Figure 2 As shown, the components of the LB liquid culture medium include: 10 g of 4-androstenedione, 100 g of vegetable soybean oil, 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride and 1 L of water; the pH in the LB liquid culture medium is controlled to be 7.2.

[0056] When used, the LB liquid culture medium provided by the present invention contains 10g 4-androstenedione (AD), 100g vegetable soybean oil, 10g peptone, 5g yeast powder and 5g sodium chloride in 1L water, and the pH of the fermentation medium is adjusted to 7.2 to provide suitable fermentation conditions.

[0057] like Figure 1 and Figure 3 As shown, the content of the conversion product in S4 is detected by high performance liquid chromatography, and after re-dissolving with mobile phase, it is filtered and removed with a 0.22 μm organic membrane;

[0058] The conversion product in S4 is extracted using an ethyl acetate oil solution, and the ratio of ethyl acetate to oil is 1:1; the extraction of the conversion product also includes: extracting the product in the oil layer three times, controlling the temperature to 50°C and stirring the extraction for 5-10 minutes, placing it in a separatory funnel and standing for separation, and collecting the organic phase; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 50°C to obtain the extracted product.

[0059] When the conversion product provided by the present invention is used, after the substrate conversion is completely analyzed by high performance liquid chromatography (HPLC), the fermentation tank is inactivated at 121°C, the fermentation liquid oil layer is collected, and the product in the oil layer is extracted three times according to the ratio of ethyl acetate: oil = 1:1, the temperature is controlled at 50°C and stirred for extraction for 5-10 minutes, and the mixture is placed in a separating funnel for standing and liquid separation, and the organic phase is collected; after the extraction is repeated three times, the extracts are combined and slowly dried on a rotary evaporator, washed with water, and dried in an oven at 50°C to obtain the extracted product. A white crystalline product is obtained, wherein the content of 1,4-androstenedione is 70%.

[0060] The method for detecting the content of the conversion product is high performance liquid chromatography, using a mobile phase (chromatographically pure methanol: chromatographically pure acetonitrile: 0.1% phosphoric acid solution = 108:81:100) to re-dissolve and then filter with a 0.22 μm organic membrane to remove impurities. The HPLC analysis conditions are shown in Table 1:

[0061] Table 1 HPLC analysis conditions

[0062]

[0063] like Figures 1 to 4 As shown, the conversion product in S5 is purified by semi-preparative liquid phase separation technology.

[0064] When the conversion product provided by the present invention is used, after the fermentation is completed, the target product is extracted from the fermentation broth. This generally involves steps such as solvent extraction, chromatographic separation, and crystallization. After extraction, the obtained crude product is separated and purified by semi-preparative liquid phase, and the product is in the form of a white powder after decolorization and purification to improve its purity and quality. The chemical and physical properties of the extracted and purified product are identified, such as by mass spectrometry, nuclear magnetic resonance, etc. to determine its structure. At the same time, it is also necessary to evaluate the biological activity or application value of the product, and the final identified structure is: 1,4-androstenedione.

[0065] Embodiment 2:

[0066] The strain in S1 is Comamonas thiooxydans CSUSB4, which uses 4-androstenedione as a starting material, and the concentration of the Comamonas thiooxydans CSUSB4 is 6 g / L; the inoculation amount of the Comamonas thiooxydans CSUSB4 is 6%; the culture medium selected for activation of the strain is LB solid culture medium, and after inoculation, the strain is cultured at a constant temperature of 25° C. in an incubator for 28 hours.

[0067] The LB liquid culture medium in S2 is used to pick a single colony on the plate with an inoculation loop and then cultured in a constant temperature shaker at 25° C. at 200 r for 18 h.

[0068] The fermentation conversion in S3 adopts LB liquid culture medium, the substrate is dissolved in vegetable soybean oil, sterilized separately and added to the fermentation tank, the seed liquid is inoculated into the fermentation tank, and the fermentation is carried out at a constant temperature of 25° C. for 28 hours at a rotation speed of 180 r.

[0069] The substrate is 4-androstenedione, and the concentration of the 4-androstenedione is 9 g / L; the ratio of the 4-androstenedione to the vegetable soybean oil is set to 1:8.

[0070] The components of the LB liquid culture medium include: 8 g of 4-androstenedione, 80 g of vegetable soybean oil, 8 g of peptone, 4 g of yeast powder, 4 g of sodium chloride and 0.8 L of water.

[0071] The pH in the LB liquid culture medium was controlled to be 7.1.

[0072] The content of the conversion product in S4 is detected by high performance liquid chromatography, and the product is reconstituted with a mobile phase and then filtered through a 0.2 μm organic membrane to remove impurities.

[0073] The conversion product in S4 is extracted using an ethyl acetate oil solution, and the ratio of ethyl acetate to oil is 1:1; the extraction of the conversion product also includes: extracting the product in the oil layer three times, controlling the temperature to 60°C and stirring the extraction for 5 minutes, placing it in a separatory funnel and standing for separation, and collecting the organic phase; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 60°C to obtain the extracted product.

[0074] Embodiment three:

[0075] The strain in S1 is Comamonas thiooxydans CSUSB4, which uses 4-androstenedione as a starting material, and the concentration of the Comamonas thiooxydans CSUSB4 is 7 g / L; the inoculation amount of the Comamonas thiooxydans CSUSB4 is 7%; the culture medium selected for activation of the strain is LB solid culture medium, and after inoculation, the strain is cultured at a constant temperature of 30° C. in an incubator for 20 hours.

[0076] The LB liquid culture medium in S2 is used to pick a single colony on the plate with an inoculation loop and then cultured in a constant temperature shaker at 30°C and 220r for 15h.

[0077] The fermentation conversion in S3 adopts LB liquid culture medium, the substrate is dissolved in vegetable soybean oil, sterilized separately and added to the fermentation tank, the seed liquid is inoculated into the fermentation tank, and the fermentation is carried out at a constant temperature of 30° C. for 20 hours at a rotation speed of 220 r.

[0078] The substrate is 4-androstenedione, and the concentration of the 4-androstenedione is 12 g / L; the ratio of the 4-androstenedione to the vegetable soybean oil is set to 1:11.

[0079] The components of the LB liquid culture medium include: 12 g of 4-androstenedione, 120 g of vegetable soybean oil, 12 g of peptone, 6 g of yeast powder, 6 g of sodium chloride and 1.2 L of water.

[0080] The pH in the LB liquid culture medium was controlled to be 7.3.

[0081] The content of the conversion product in S4 is detected by high performance liquid chromatography, and the product is reconstituted with a mobile phase and then filtered through a 0.25 μm organic membrane to remove impurities.

[0082] The conversion product in S4 is extracted using an ethyl acetate oil solution, and the ratio of ethyl acetate to oil is 1:1; the extraction of the conversion product also includes: extracting the product in the oil layer three times, controlling the temperature to 40°C and stirring the extraction for 10 minutes, placing it in a separatory funnel and standing for separation, and collecting the organic phase; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 40°C to obtain the extracted product.

[0083] Working principle: First, take the frozen glycerol tube of the strain Comamonas thiooxydans CSUSB4, put it in an ice box to thaw, inoculate it into the LB plate with an inoculation loop on an ultra-clear workbench, and culture it in a constant temperature incubator at 28°C for 24 hours. Then, use an inoculation loop to pick up a single colony and inoculate it again into the LB plate, and culture it in a constant temperature incubator at 28°C for 24 hours. Among them, LB liquid culture medium is used for seed liquid culture. 100ml LB liquid culture medium is prepared in a 250ml conical flask at 121°C and sterilized for 30 minutes. Take the strain Comamonas thiooxydans CSUSB4 plate, pick up a single colony with an inoculation loop and inoculate it into the LB liquid culture medium, and then culture it on a constant temperature shaker at 28°C with a rotation speed of 180r for 16 hours; then prepare 10L LB liquid medium, adjust the pH to 7.2, put it into the fermentation tank, and sterilize it at 121℃ for 30min. After the fermentation liquid cools to room temperature, add the pre-sterilized substrate oil solution into the fermentation tank according to the aseptic operation specification, and then add the cultured bacteria Comamonas thiooxydans The CSUSB4 seed liquid was aseptically poured into the fermentation tank, and the speed was adjusted to 200r. The fermentation was carried out at a constant temperature of 28°C for 24h. After the substrate conversion was completely analyzed by HPLC, the bacteria were inactivated at 121°C in the fermentation tank, and the oil layer of the fermentation liquid was collected. The product in the oil layer was extracted three times according to the ratio of ethyl acetate: oil = 1:1. The temperature was controlled at 50°C and stirred for extraction for 5-10min. The product was placed in a separating funnel and allowed to stand for separation, and the organic phase was collected. After three rounds of extraction, the extracts were combined and slowly dried on a rotary evaporator, washed with water, and dried in an oven at 50°C to obtain the extracted product, and a white crystalline product was obtained. Finally, the obtained crude product was separated and purified by semi-preparative liquid phase separation, and the sample was structurally confirmed, and the chemical and physical properties of the extracted and purified product were identified, such as by mass spectrometry, nuclear magnetic resonance, etc. The structure was determined to be 1,4-androstenedione.

[0084] In the biotransformation system with a substrate 4-androstenedione (AD) concentration of 10 g / L, 5 g / L of the strain Comamonas thiooxydans CSUSB4 was added to ferment and prepare the product 1,4-androstenedione (AD). The selected strain Comamonas thiooxydans CSUSB4 has a short conversion time, and the conversion rate can reach 70% in only 24 hours, achieving the function of rapid conversion and achieving the effect of improving the conversion rate; the ratio of 4-androstenedione to vegetable soybean oil is 1:10; ensure that the purity of 4-androstenedione is high enough to avoid the influence of impurities on the fermentation process; the strain Comamonas thiooxydans CSUSB4 is a bacterial strain with unique biochemical characteristics and research value. It has good medical research value in terms of drug resistance and genome. It has a specific enzyme system that can catalyze the conversion of androstenedione. When using seed liquid culture, the concentration of nutrient-promoting substances in the culture medium is maintained, the cost is reduced, the culture cycle is shortened, and the yield is increased. During the seed liquid culture process, it is necessary to control appropriate culture conditions, such as temperature, pH value, and ventilation volume, to obtain the best bacterial growth effect and obtain bacteria with strong vitality and sufficient quantity for subsequent experiments or production use.

[0085] The organic phase and the aqueous phase are combined to grow in culture medium containing organic solvent and aqueous phase respectively. Due to the different polarities of water and some organic matter, they may form two immiscible phases when mixed. By adjusting the ratio of organic phase to aqueous phase, the yield of secondary products can be significantly improved. In the two-phase culture, the effect of organic solvent on cell growth and product metabolism, as well as oxygen transfer, needs to be considered. At the same time, the density difference between organic phase and aqueous phase will lead to solution stratification, and the culture conditions need to be reasonably controlled to obtain the best effect.

[0086] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a steroid drug intermediate, characterized in that: The following steps are involved: S1: Activation of bacterial strains: Take the strain Comamonas thiooxydans CSUSB4 and inoculate it into the LB plate with an inoculation loop, and culture it in a constant temperature incubator; S2: Seed liquid culture: Take the strain Comamonas thiooxydans CSUSB4 plate, pick a single colony with an inoculation loop and inoculate it into LB liquid medium, and then shake and culture it on a constant temperature shaker; S3: fermentation conversion in a fermentation tank: add the pre-sterilized substrate oil solution into the fermentation tank according to aseptic operation specifications, and then aseptically pour the cultured bacteria Comamonas thiooxydans CSUSB4 seed solution into the fermentation tank for constant temperature fermentation; S4: Extraction of conversion product: extract the oil layer of the fermentation liquid and let it stand for separation, collect the organic phase; slowly spin dry the extract on a rotary evaporator, wash it with water and dry it in an oven at 50°C to obtain the extraction product; S5: Purification of conversion product: The crude product was purified by semi-preparative liquid phase separation to obtain a white powder; S6: Structural confirmation of the transformation product: The structure was identified as 1,4-androstenedione.

2. The method for preparing a steroid drug intermediate according to claim 1, characterized in that: The strain in S1 is Comamonas thiooxydans CSUSB4, which uses 4-androstenedione as a starting material, and the concentration of the Comamonas thiooxydans CSUSB4 is 5 g / L; the inoculation amount of the Comamonas thiooxydans CSUSB4 is 5%; the culture medium selected for activation of the strain is LB solid culture medium, and after inoculation, the strain is cultured at a constant temperature of 28° C. in an incubator for 24 hours.

3. The method for preparing a steroid drug intermediate according to claim 2, characterized in that: The LB liquid culture medium in S2 is used to pick a single colony on the plate with an inoculation loop and then cultured in a constant temperature shaker at 28°C at 180r for 16h.

4. The method for preparing a steroid drug intermediate according to claim 3, characterized in that: The fermentation conversion in S3 adopts LB liquid culture medium, the substrate is dissolved in vegetable soybean oil, sterilized separately and added to the fermentation tank, the seed liquid is inoculated into the fermentation tank, and the fermentation is carried out at a constant temperature of 28° C. for 24 hours and a rotation speed of 200 r.

5. The method for preparing a steroid drug intermediate according to claim 4, characterized in that: The substrate is 4-androstenedione, and the concentration of the 4-androstenedione is 10 g / L; the ratio of the 4-androstenedione to the vegetable soybean oil is set to 1:

10.

6. The method for preparing a steroid drug intermediate according to claim 5, characterized in that: The components of the LB liquid culture medium include: 10 g of 4-androstenedione, 100 g of vegetable soybean oil, 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride and 1 L of water.

7. The method for preparing a steroid drug intermediate according to claim 6, characterized in that: The pH in the LB liquid culture medium was controlled to be 7.

2.

8. The method for preparing a steroid drug intermediate according to claim 7, characterized in that: The content of the conversion product in S4 is detected by high performance liquid chromatography, and the product is reconstituted with a mobile phase and then filtered through a 0.22 μm organic membrane to remove impurities.

9. The method for preparing a steroid drug intermediate according to claim 8, characterized in that: The conversion product in S4 is extracted using an ethyl acetate oil solution, and the ratio of ethyl acetate to oil is 1:1; the extraction of the conversion product also includes: extracting the product in the oil layer three times, controlling the temperature to 50°C and stirring the extraction for 5-10 minutes, placing it in a separatory funnel and standing for separation, and collecting the organic phase; after three rounds of extraction, the extracts are combined and slowly dried on a rotary evaporator, washed with water and dried in an oven at 50°C to obtain the extracted product.

10. The method for preparing a steroid drug intermediate according to claim 9, characterized in that: The conversion product in S5 is purified by semi-preparative liquid phase separation technology.

Images