CN105219829A - A kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione - Google Patents

Abstract

The invention discloses a method for preparing 9α-hydroxy-androst-1,4-diene-3,17-dione, which uses phytosterol as a substrate and utilizes mycobacterium <i>Mycobacterium</i> ( CICC? 21097) and Rhodococcus <i> Rhodococcus? 9α-hydroxy-androst-1,4-diene-3,17-dione was prepared by mixed fermentation of two kinds of microorganisms in rhodochrous</i> (CGMCC? 4.1480). In the present invention, the highest molar conversion rate of phytosterols to 9α-OH-ADD reaches 84.3% by adding complex solubilizers in the fermentation medium. At the same time, the purification steps of the two-step method for preparing 9α-hydroxy-androst-1,4-diene-3,17-dione are reduced, and the amount of solvent used is reduced.

Description

A method for preparing 9α-hydroxyl-androst-1,4-diene-3,17-dione

technical field

The invention belongs to the field of microbial pharmacy, and relates to a 9α-hydroxy-androsta-1,4-diene-3,17-dione, specifically a method for preparing 9α-hydroxy-androsta-1,4-diene-3,17-dione by using mixed bacteria to biotransform phytosterols. The method of ster-1,4-diene-3,17-dione.

Background technique

Steroids, also known as steroids, are a class of compounds containing cyclopentane polyhydrophenanthrene nuclei. Its basic structure is shown below. It consists of three six-membered rings and one five-membered ring, called A, B, C, and D rings respectively. Generally, there are angular methyl groups on the 10th and 13th positions of the steroid nucleus ( -CH ₃ ), there may be a hydroxyl group (-OH) or a keto group (-C=O) at the 3rd, 9th, and 11th positions, some double bonds exist in the A ring or B ring, and there are side chains of different lengths at the 17th position. A series of compounds with unique physiological functions are formed due to the differences in substituents, double bond positions or stereo configurations on the steroidal core.

General structure of steroids

Steroid drugs are the second largest class of drugs in the pharmaceutical industry after antibiotics. Steroid drugs have various functions such as anti-allergic, anti-shock, anti-inflammation, and anti-allergic reactions. They are widely used in the treatment of bronchial asthma, Rheumatoid arthritis, eczema, anemia, and the promotion of fracture and wound healing are also used to treat endocrine diseases such as Addison's, and play an important role in body development, immune regulation, skin disease treatment, and birth control. Steroids are effective in the treatment of hormone-dependent tumors. With the rapid development of steroid chemistry, steroid hormone drugs have become an important category in the field of medicine, and are widely used in clinical practice.

At present, the research and development of new strains and new technologies for the transformation of steroidal drugs by microbial methods is in good shape. 9α-OH-ADD and 9α-OH-ADD are important raw materials and key intermediates for the synthesis of steroid drugs, and are favored by many researchers.

Research work at home and abroad mainly focuses on the preparation of androst-4-ene-3,17-dione (4-AD) or androst-1,4-diene-3,17-dione ( ADD), and the use of bacteria such as Nocardia or Rhodococcus to carry out C _9α hydroxylation of 4-AD or ADD to prepare 9α-hydroxyl-androst-4-ene-3,17-dione (9α-OH-AD ) or 9α-hydroxy-androst-1,4-diene-3,17-dione (9α-OH-ADD).

(1) Preparation of 4-AD or ADD from sterols

The microorganisms used for the degradation of sterol side chains mainly include Mycobacterium, Nocardia, Arthrobacterium, Brevibacterium and the like. Numerous literatures and patents have shown that 4-AD and ADD will be produced simultaneously during the side chain degradation process using phytosterols, and the production mass ratio of the two will vary depending on the strain and production process. For example, in patent CN104403974A, Bacillus amyloliquefaciens is used to degrade phytosterols by side chains, and the mass ratio of 4-AD to ADD is 18:1; in patent CN1544618A, Mycobacteriumsp. The mass ratio to ADD is 1:10. The conversion time is generally 100-120h.

(2) 4-AD or ADD to prepare 9α-OH-AD or 9α-OH-ADD

The microorganisms used for the steroid 9α-hydroxylation reaction mainly include Rhodococcus, Corynebacterium, Nocardia and the like. For example, patent CN103667126A introduces a method for preparing 9α-OH-AD by using Mycobacterium smegmatis; patent CN103343155A discloses a method for preparing 9α-hydroxyandrostenedione by using Mycobacterium fortuitousus. The conversion time is generally around 96-100h.

In summary, based on the current literature and patents, the preparation of 9α-OH-ADD by using phytosterol biological method needs to be completed by two-step fermentation (i.e. two-step method). OH-ADD reported in the literature. The two-step method generally uses mycobacteria to convert phytosterols into ADD, and then extracts and refines ADD to obtain ADD, and then uses Nocardia or Rhodococcus strains to perform 9α-hydroxylation of ADD to obtain 9α-OH-ADD. However, the above-mentioned two-step method has the following disadvantages: ① When phytosterols are converted to ADD direction, the utilization efficiency of phytosterols is low. ② The products generated during the side chain degradation fermentation process all contain 4-AD and ADD at the same time. Since 4-AD and ADD have similar structures, it is difficult to separate and purify. ③ The later extraction and purification of the product ADD requires the use of a large amount of suitable organic solvents for extraction and purification to obtain ADD with higher purity, which can be used as a substrate for subsequent 9α-hydroxylation. Therefore, the development of a more efficient and time-saving industrial biological method for preparing 9α-OH-ADD has high application value and prospects.

Contents of the invention

Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for preparing 9α-hydroxy-androst-1,4-diene-3,17-dione, the preparation of 9α-hydroxy- The method of androst-1,4-diene-3,17-dione solves the problem of using two-step fermentation in the prior art to prepare 9α-hydroxy-androst-1,4-diene-3,17-dione The method has the technical problems of low conversion rate and difficult separation and purification.

The invention provides a method for preparing 9α-hydroxy-androst-1,4-diene-3,17-dione, which uses phytosterol as a substrate and uses two kinds of mycobacterium Mycobacterium and Rhodococcus rhodochrous Preparation of 9α-hydroxy-androst-1,4-diene-3,17-dione by mixed microbial fermentation.

Further, the method includes the following steps:

1) a step of preparing a fermentation medium containing phytosterols;

2) Adding a composite solubilizer in the fermentation medium, the composite solubilizer is composed of ethanol and polyethylene glycol, in the fermentation medium, the concentration of the ethanol is 10.0-50.0g /L, the concentration of the polyethylene glycol is 5.0-20.0g/L;

3) A step of inoculating Mycobacterium and Rhodococcus rhodochrous respectively, the inoculum amount of Mycobacterium is 10.0-15.0%, and after 0-48 hours of transformation culture, 10.0-20.0% rose For Rhodococcus rhodochrous, the fermentation temperature is 28-32°C, the fermentation time is 72-120h, and the pH of the fermentation medium is between 6.5-7.5;

4) A step of extracting and refining the product. After the fermentation is completed, inactivate the fermentation broth at 70-90°C, cool it to 20-30°C, press filter with plate and frame, take the filter cake for extraction, and then filter, decolorize, distill, Refined to obtain 9α-hydroxy-androst-1,4-diene-3,17-dione. Further, the formula of the fermentation medium is as follows: glucose 10.0-30.0g/L, ammonium dihydrogen phosphate 2.0-4.0g/L, ferric ammonium citrate 0.03-0.08g/L, magnesium sulfate heptahydrate 1.5-3.5g /L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium dihydrogen phosphate 0.5-1.5g/L, beet molasses 5.0-9.0g/L, phytosterol 30.0-40.0g/L.

Further, the preservation number of the Mycobacterium Mycobacterium is CICC21097 from the China Industrial Microorganism Culture Collection Management Center; the preservation number of the Rhodococcus rhodochrous is CGMCC4.1480 from the China General Microbiology Culture Collection Management Center. center.

The present invention uses phytosterol as a substrate, utilizes Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) to prepare 9α-OH-ADD through mixed fermentation of two microorganisms, and also adds compound Solubilizer, biotransforms phytosterols, completes the degradation of phytosterol side chains and C _9α -position hydroxylation in one step, and makes the highest molar conversion rate of phytosterols to 9α-OH-ADD reach 84.3%.

The method of the present invention has following advantage compared with two-step method (promptly make phytosterol biotransformation into ADD earlier, then use ADD as substrate, obtain 9α-OH-ADD at its C _9α position hydroxylation): (1) one-step 9α-OH-ADD is prepared by using mixed bacteria to transform phytosterols. The hydroxylation process is more conducive to the conversion of phytosterols to ADD and improves the utilization efficiency of phytosterols. (2) The purification steps are reduced, the extraction and purification process of ADD in the two-step method is removed, the two-step purification is changed into one step, and the solvent usage is reduced. (3) Reduce the production of by-products 4-AD and 9α-OH-AD, increase the biotransformation rate of phytosterols to 9α-OH-ADD, and shorten the biological fermentation cycle.

The present invention utilizes the advantages of the synergistic effect between the two bacterial strains to promote the conversion of phytosterols to 9α-OH-ADD, reduce the production of by-products 4-AD and 9α-OH-AD, and increase the conversion of phytosterols to 9α-OH-ADD. The biological efficiency of ADD shortens the biological fermentation cycle and greatly reduces the production cost.

Compared with the prior art, the technical progress of the present invention is remarkable. The present invention adopts a one-step conversion method, reduces the purification steps, that is, eliminates the extraction and purification process of ADD in the two-step method, makes the two-step purification into one step, greatly reduces the amount of solvent used, and reduces the production cost.

Description of drawings

Figure 1 shows the process of preparing 9α-OH-ADD by biotransforming phytosterols with mixed bacteria.

detailed description

The present invention is further illustrated below by specific examples, but the present invention is not limited.

The Mycobacterium Mycobacterium (CICC21097) that the present invention adopts is the microorganism of public sale, comes from, China Industrial Microorganism Strain Preservation Management Center, the address of this institution is: No. 6 Building (100015), No. 24 Courtyard, Jiuxianqiao Middle Road, Chaoyang District, Beijing , can be purchased by phone, or through the website (http://www.china-cicc.org), anyone can purchase as long as they pay the relevant fees.

The rhodococcus rhodochrous (CGMCC4.1480) that the present invention adopts is the microorganism of public sale, comes from, China common microbial culture preservation and management center, the address of this organization is: No. 3, courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, China The Institute of Microbiology, Academy of Sciences, can be purchased by phone or through the website (http://www.cgmcc.net). Anyone can purchase it as long as they pay the relevant fees.

Example 1

Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) were cultured on slant and seeds to obtain the strains required for fermentation. 1. Preparation of mycobacterium Mycobacterium (CICC21097) slant strain and seed solution

(1) Incline cultivation

Slant medium: peptone 5.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, glucose 10.0g/L, agar 20.0g/L, cultured at 29°C for 5-7 days.

(2) Seed cultivation

Seed medium: glucose 15.0g/L, peptone 5.0g/L, beef extract 3.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.5g/L, ammonium dihydrogen phosphate 3.5g/L, phosphoric acid Dipotassium hydrogen 1.0g/L, potassium dihydrogen phosphate 1.0g/L, pH 7.0, sterilized by moist heat at 121°C for 30min.

The bacterial strain cultivated in step (1) was inoculated under sterile conditions with an inoculation loop into a 250mL shake flask containing 50mL seed medium, and cultivated on a rotary shaker at 200r/min for 48h at 30°C to prepare Get the seed liquid.

2. Preparation of Rhodococcus rhodochrous (CGMCC4.1480) slant strain and seed solution

(1) Incline cultivation

Slant medium: glucose 10.0g/L, tryptone 15.0g/L, soybean peptone 5.0g/L, sodium chloride 5.0g/L, agar 20.0g/L, cultured at 30°C for 5-6 days.

(2) Seed cultivation

Seed medium: glucose 10.0g/L, yeast extract 20.0g/L, fish peptone 5.0g/L, corn starch 9.0g/L, dipotassium hydrogen phosphate 2.5g/L, pH 7.0, 121°C damp heat sterilization for 30min .

The bacterial strain cultivated in step (1) was inoculated with an inoculation loop under sterile conditions in a 250mL shake flask containing 50mL seed medium, and cultivated for 30h on a rotary shaker at 200r/min at 30°C to prepare Get the seed solution.

3. Fermentation

Fermentation medium: phytosterol 35.0g/L, glucose 21.0g/L, ammonium dihydrogen phosphate 3.5g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate heptahydrate 2.0g/L, dipotassium hydrogen phosphate 1.2g /L, potassium dihydrogen phosphate 1.0g/L, beet molasses 6.0g/L, pH7.0.

The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a complex solubilizing agent was added to the medium: ethanol 30.0g/L, polyethylene glycol 10.0g/L. Carry out inoculation again, the inoculation amount of mycobacterium MycobacteriumCICC (21097) is 12.0%, inoculate Rhodococcus rhodochrous when mycobacterium inoculates, the inoculum amount of Rhodococcus rhodochrous (CGMCC4.1480) is 17.0%, uses 5L The fermenter was used for biotransformation, with a liquid filling capacity of 70%, an aeration rate of 1.0 v/v min, a temperature controlled at 30° C., and a rotational speed of 300 r/min. During the conversion period, samples were taken every 12 hours for TLC monitoring. Fermented and cultured for 96 hours, sampled for TLC analysis, there were no phytosterol spots, and the transformation was basically completed. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high performance liquid chromatography. The content of 9α-OH-ADD in the above-mentioned obtained fermentation broth is 17.5g/L, and the content of 9α-OH-AD is 0.25g/L , the molar conversion rate of phytosterol was 72.4%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. Refined by beating with toluene to finally obtain the fine product of 9α-OH-ADD. The analytical purity by high performance liquid chromatography was 98.7%.

Example 2

Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) strains were prepared into seed liquid (the method is the same as in Example 1), and then fermented.

Fermentation medium: phytosterol 35.0g/L, ethanol 31.0g/L, polyethylene glycol 9.0g/L, glucose 20.0g/L, ammonium dihydrogen phosphate 3.0g/L, ferric ammonium citrate 0.05g/L, Magnesium sulfate heptahydrate 2.5g/L, dipotassium hydrogen phosphate 1.0g/L, potassium dihydrogen phosphate 1.0g/L, beet molasses 6.0g/L, pH7.0.

The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the medium: ethanol 31.0 g/L, polyethylene glycol 9.0 g/L. Carry out inoculation again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 12.0%, inoculate Rhodococcus rhodochrous after mycobacterium transformation culture 24h, the inoculum size of Rhodococcus rhodochrous (CGMCC4.1480) is 18.0%, use A 5L fermenter was used for biotransformation, with a liquid volume of 70%, an aeration rate of 1.0v/v·min, a temperature controlled at 30°C, and a rotational speed of 300r/min. During the transformation period, samples were taken every 12 hours for TLC monitoring. Fermented and cultured for 96 hours, sampled for TLC analysis, there were no phytosterol spots, and the transformation was basically completed. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high performance liquid chromatography. The content of 9α-OH-ADD in the above-mentioned obtained fermentation broth is 20.4g/L, and the content of 9α-OH-AD is 0.19g/L , the molar conversion rate of phytosterols was 84.3%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. It was refined by beating with toluene to finally obtain the refined product of 9α-OH-ADD, the purity of which was analyzed by high performance liquid chromatography was 99.1%.

Example 3

Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) strains were used to prepare seed liquid (the method is the same as in Example 1), and then fermented.

Fermentation medium: phytosterol 35.0g/L, glucose 22.0g/L, ammonium dihydrogen phosphate 3.0g/L, ferric ammonium citrate 0.05g/L, magnesium sulfate heptahydrate 2.0g/L, dipotassium hydrogen phosphate 1.0g /L, potassium dihydrogen phosphate 1.0g/L, beet molasses 6.0g/L, pH7.0.

The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the medium: ethanol 30.0g/L, polyethylene glycol 9.0g/L. Carry out inoculation again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 13.0%, inoculate Rhodococcus rhodochrous after mycobacterium transformation culture 48h, the inoculum size of Rhodococcus rhodochrous (CGMCC4.1480) is 17.0%, use A 5L fermenter was used for biotransformation, with a liquid volume of 70%, an aeration rate of 1.0v/v·min, a temperature controlled at 30°C, and a rotational speed of 300r/min. During the transformation period, samples were taken every 12 hours for TLC monitoring. Fermented and cultured for 96 hours, sampled for TLC analysis, there were no phytosterol spots, and the transformation was basically completed. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high performance liquid chromatography. The content of 9α-OH-ADD in the above-mentioned obtained fermentation broth is 18.3g/L, and the content of 9α-OH-AD is 0.31g/L , the molar conversion rate of phytosterol was 75.7%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. It was refined by beating with toluene to finally obtain the refined product of 9α-OH-ADD, the purity of which was analyzed by high performance liquid chromatography was 98.6%.

Example 4

Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) strains were used to prepare seed liquid (the method is the same as in Example 1), and then fermented.

Fermentation medium: phytosterol 30.0g/L, glucose 10.0g/L, ammonium dihydrogen phosphate 2.0g/L, ferric ammonium citrate 0.08g/L, magnesium sulfate heptahydrate 3.5g/L, dipotassium hydrogen phosphate 1.5g /L, potassium dihydrogen phosphate 1.5g/L, beet molasses 9.0g/L, pH7.5.

The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the medium: ethanol 10.0g/L, polyethylene glycol 5.0g/L. Carry out inoculation again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 10.0%, inoculate Rhodococcus rhodochrous after mycobacterium transformation culture 24h, the inoculum size of Rhodococcus rhodochrous (CGMCC4.1480) is 10.0%, use A 5L fermenter was used for biotransformation, with a liquid volume of 70%, an aeration rate of 1.0v/v·min, a temperature controlled at 28°C, and a rotational speed of 300r/min. During the transformation period, samples were taken every 12 hours for TLC monitoring. After 72 hours of fermentation, sampling and TLC analysis showed that there were no phytosterol spots, and the transformation was basically completed. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high-performance liquid chromatography. The content of 9α-OH-ADD in the fermentation broth obtained above is 17.1g/L, and the content of 9α-OH-AD is 0.20g/L , the molar conversion rate of phytosterol was 82.5%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. It was refined by beating with toluene to finally obtain the refined product of 9α-OH-ADD, the purity of which was analyzed by high performance liquid chromatography was 98.8%.

Example 5

Mycobacterium Mycobacterium (CICC21097) and Rhodococcus rhodochrous (CGMCC4.1480) strains were used to prepare seed liquid (the method is the same as in Example 1), and then fermented.

Fermentation medium: phytosterol 40.0g/L, glucose 30.0g/L, ammonium dihydrogen phosphate 4.0g/L, ferric ammonium citrate 0.03g/L, magnesium sulfate heptahydrate 1.5g/L, dipotassium hydrogen phosphate 0.5g /L, potassium dihydrogen phosphate 0.5g/L, beet molasses 5.0g/L, pH6.5.

The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the culture medium: ethanol 50.0g/L, polyethylene glycol 20.0g/L. Carry out inoculation again, the inoculum size of mycobacterium MycobacteriumCICC (21097) is 15.0%, inoculate Rhodococcus rhodochrous after mycobacterium transformation culture 24h, the inoculum size of Rhodococcus rhodochrous (CGMCC4.1480) is 20.0%, use A 5L fermenter was used for biotransformation, with a liquid volume of 70%, an aeration rate of 1.0v/v·min, a temperature controlled at 32°C, and a rotational speed of 300r/min. During the transformation period, samples were taken every 12h for TLC monitoring. After 120 hours of fermentation, sampling and TLC analysis showed that there were no phytosterol spots, and the transformation was basically completed. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high performance liquid chromatography. The content of 9α-OH-ADD in the above-mentioned obtained fermentation broth is 21.5g/L, and the content of 9α-OH-AD is 0.24g/L , the molar conversion rate of phytosterol was 77.8%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. It was refined by beating with toluene to finally obtain the refined product of 9α-OH-ADD, the purity of which was analyzed by high performance liquid chromatography was 98.1%.

Comparative Example 1

The mycobacterium Mycobacterium (CICC21097) strain is prepared into a seed solution (the method is the same as in Example 1), and then fermented.

The fermentation medium was as described in Example 2, and the fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the medium: ethanol 31.0 g/L, polyethylene glycol 9.0 g/L. Carry out inoculation again, the inoculum amount of mycobacterium Mycobacterium (CICC21097) is 12.0%, uses 5L fermenter to carry out biotransformation, and liquid filling capacity is 70%, and ventilation rate is 1.0v/v·min, and temperature is controlled at 30 ℃, rotating speed The temperature was 300r/min, and samples were taken every 12h for TLC monitoring during the transformation. Fermented and cultured for 96 hours, sampling was analyzed by TLC, and the conversion stagnated. The sample is extracted with ethyl acetate, and the supernatant collected by centrifugation is analyzed by high performance liquid chromatography. The content of ADD in the fermented liquid obtained above is 16.7g/L, the content of 4-AD is 0.87g/L, and the molar conversion rate of phytosterols was 72.9%.

After fermentation, inactivate the fermentation broth at 85°C, cool it to 25°C, press filter with plate and frame, extract the filter cake with dichloromethane, and then obtain the crude ADD product through filtration, decolorization and distillation, and refine the crude product with toluene , and finally obtain the ADD fine product, the purity of which is 98.5% by high performance liquid chromatography.

Comparative Example 2

The bacterial strain of Rhodococcus rhodochrous (CGMCC4.1480) is prepared into seed liquid (the method is the same as in Example 1), and then fermented.

The fermentation medium was as described in Example 2, and the substrate was changed from 35.0 g/L phytosterol to 24.0 g/L ADD. The fermenter containing the fermentation medium was sterilized by high-pressure steam at 121° C. for 15 minutes.

At the beginning of the culture, a compound solubilizing agent was added to the medium: ethanol 31.0 g/L, polyethylene glycol 9.0 g/L. Carry out inoculation again, the inoculum size of Rhodococcus rhodochrous (CGMCC4.1480) is 18.0%, uses 5L fermenter to carry out biotransformation, and liquid filling capacity is 70%, and ventilation rate is 1.0v/v min, and temperature is controlled at 30 °C, the rotational speed was 300r/min, and samples were taken every 12h for TLC monitoring during the conversion period. Fermented and cultured for 72 hours, sampled for TLC analysis, there was no ADD spot, and the transformation was basically completed. The sample was extracted with ethyl acetate, and the supernatant collected by centrifugation was analyzed by high performance liquid chromatography. The content of 9α-OH-ADD in the fermentation broth obtained above was 22.6g/L, and the molar conversion rate of ADD was 93.8%.

After the fermentation, the fermentation liquid was inactivated at 85°C, cooled to 25°C, plate and frame filter press, the filter cake was extracted with dichloromethane, and the crude product of 9α-OH-ADD was obtained through filtration, decolorization and distillation. It was refined by beating with toluene to finally obtain the refined product of 9α-OH-ADD, the purity of which was analyzed by high performance liquid chromatography was 99.1%.

As can be seen from the examples, the one-step method has the following advantages compared with the two-step method:

①It helps to improve the conversion efficiency of phytosterols. As described in Comparative Examples 1 and 2, the two-step method biotransforms phytosterols to prepare 9α-OH-ADD, and the final molar conversion rate of phytosterols is 72.9%×93.8%=68.4%; and as described in Example 2, the one-step method The biotransformation of phytosterols to prepare 9α-OH-ADD has a molar conversion rate of phytosterols as high as 84.3%.

②It helps to shorten the fermentation cycle. As described in Comparative Examples 1 and 2, the two-step microbial transformation time is about 168h, and as described in Example 2, the one-step mixed fermentation produces 9α-OH-ADD biotransformation time is about 96h, and the biotransformation time of the one-step method The time is shortened by about 72h compared with the two-step method.

③Reduce the amount of solvent used. The one-step method removes the extraction and purification process of ADD in the two-step method, makes the two-step purification into one step, reduces the amount of solvent used, and reduces the production cost.

④Reduce the production of main by-products 4-AD and 9α-OH-AD. By the comparison of Example 2 and Comparative Example 1, it can be seen that when utilizing mycobacteria to carry out single bacterium biotransformation to phytosterols, there will be the generation of ADD and 4-AD simultaneously, and the mass ratio of the two is about 19: 1. However, when 9α-OH-ADD is produced by mixed fermentation of two kinds of bacteria, 9α-OH-ADD and 9α-OH-AD will be produced at the same time, and the mass ratio of the two is about 107:1. That is, the one-step method of mixed bacteria transformation of phytosterols to produce 9α-OH-ADD can effectively reduce the production of by-products 4-AD and 9α-OH-AD, and its hydroxylation process is more conducive to the conversion of phytosterols to ADD, improving the production of phytosterols. usage efficiency.

The above content is only a basic description of the concept of the present invention, and any equivalent transformation made according to the technical solution of the present invention shall belong to the protection scope of the present invention.

Claims (4)

1. prepare a method for 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione, it is characterized in that: be substrate with plant sterol, utilize mycobacterium mycobacteriumand rhodococcus rhodochrous rhodococcusrhodochroustwo kinds of microorganism mixed fungus fermentations prepare 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.

2. a kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione according to claim 1, is characterized in that: comprise the steps:

1) step of the fermention medium of a preparation containing plant sterol;

2) in described fermention medium, compound chaotropic agent is added, described compound chaotropic agent is made up of ethanol and polyoxyethylene glycol, in described fermention medium, the concentration of described ethanol is 10.0-50.0g/L, and the concentration of described polyoxyethylene glycol is 5.0-20.0g/L;

3) mycobacterium is inoculated respectively for one mycobacteriumand rhodococcus rhodochrous rhodococcusrhodochrousstep, mycobacterium mycobacteriuminoculum size be 10.0-15.0%, transform and cultivate after 0-48h, then access the rhodococcus rhodochrous of 10.0-20.0% rhodococcusrhodochrous, leavening temperature is 28-32 DEG C, and fermentation time is 72-120h, and fermention medium pH is between 6.5-7.5;

4) step of a product Hydrolysis kinetics, after fermentation ends, by fermented liquid 70-90 DEG C deactivation, after being cooled to 20-30 DEG C, filter press, gets filter cake and extracts, more after filtration, decolouring, distillation, refining, obtain 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione.

3. one according to claim 1 prepares 9 Alpha-hydroxies-androstane-1,4-diene-3, the method of 17-diketone, is characterized in that the formula of described fermention medium is as follows: glucose 10.0-30.0g/L, primary ammonium phosphate 2.0-4.0g/L, ferric ammonium citrate 0.03-0.08g/L, magnesium sulfate heptahydrate 1.5-3.5g/L, dipotassium hydrogen phosphate 0.5-1.5g/L, potassium primary phosphate 0.5-1.5g/L, beet sirup 5.0-9.0g/L, plant sterol 30.0-40.0g/L.

4. a kind of method preparing 9 Alpha-hydroxies-Androsta-1,4-diene-3,17-dione according to claim 1, is characterized in that: described mycobacterium mycobacteriumpreserving number be CICC21097, from Chinese industrial Microbiological Culture Collection administrative center; Described rhodococcus rhodochrous rhodococcusrhodochrouspreserving number be CGMCC4.1480, from China General Microbiological culture presevation administrative center.