JPH0573394B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to 7α-hydroxyandrosta-4-
Regarding the method for producing ene-3,17-dione, please refer to 3α,7α-dihydroxy-5β-cholanic acid (hereinafter referred to as
The present invention relates to a method for producing 7α-hydroxyandrost-4-ene-3,17-dione (hereinafter referred to as 7α-hydroxy 4AD) by allowing microorganisms to act on CDCA (referred to as CDCA) and/or its salt. 7α-hydroxy 4AD produced by the method of the present invention is 17β-hydroxy-7α-mercapto-4AD, which is known as an anti-aldosterone diuretic.
17β-hydroxy-17α-(3-hydroxypropyl)andro, a synthetic intermediate of 3-oxo-17α-pregna-4-ene-21-carboxylic acid γ-lactone 7α-acetate (hereinafter referred to as spironolactone) It is useful as a raw material compound for producing star-4,6-dien-3-one. [Prior Art] Several cases have been reported in which 7α-hydroxy 4AD was obtained by allowing microorganisms to act on CDCA. For example, METenneson et al.
NCIB10590 (Pseudomonas sp.
NCIB10590) strain was cultured aerobically to produce 7α-hydroxyandrosta-1,4-diene-3,17-
dione (hereinafter referred to as 7α-hydroxy ADD),
7α-Hydroxypregna-1,4-diene-3
-one-20-carboxylic acid, 7α-hydroxypregna-4-en-3-one-20-carboxylic acid, androsta-1,4,6-triene-3,17-dione, etc. of its products
7α-hydroxy ADD and androsta-1,4,
It has been reported that 6-triene-3,17-dione is expected to be generated via 7α-hydroxy 4AD as a metabolic intermediate [Journal of Steroid Biochemistry].
of Steroid Biochemistry), Volume 10, No. 311~
See page 316 (1979)]. Owen (RW
Owen et al., the above-mentioned Pseudomonas sp.
The NCIB10590 strain was cultured anaerobically in an inorganic salt medium containing CDCA, and the bacteria were cultured with cola-4,6-dien-3-one-24-acid, androsta-4,6-dien-3,
reported that 17-dione was obtained as the main product, and various by-products such as 7α-hydroxy 4AD were obtained [Journal of Lipid Research, Vol. 24] ,
See pages 1109-1118 (1983)]. Regarding the Pseudomonas sp. NCIB10590 strain used in the methods of Tenneson et al. and Owen et al., nothing has been reported other than that this strain belongs to the genus Pseudomonas and was collected from animal feces. Not [Journal of the Chemical Society,
Chemical Communications (Journal of
the Chemical Society,Chemical
Communications), 1974, pp. 115-116]. Also, in Japanese Patent Publication No. 57-500226, Pseudomonas sp. ATCC31752 (Pseudomonas sp.
ATCC31752) strain was cultured aerobically in a medium containing bile concentrate, and 7α-hydroxy
It has been described that a number of steroid products such as 4AD have been obtained. [Problems to be solved by the invention] The above conventional methods all involve 7α-hydroxy 4AD.
It cannot be said that this method is suitable for industrially advantageous production. Therefore, an object of the present invention is to provide an industrially advantageous method for producing 7α-hydroxy 4AD with high selectivity and good yield by allowing microorganisms to act on CDCA. [Means for Solving the Problems] According to the present invention, the above object is achieved by using CDCA and/or
or by culturing bacteria belonging to Pseudomonas putida species or Pseudomonas stuttieri species that have the ability to produce 7α-hydroxy4AD using its salt as a substrate in a medium containing CDCA and/or its salt.
This is achieved by providing a method for producing 7α-hydroxy 4AD, which is characterized by producing and collecting 7α-hydroxy 4AD. Examples of bacteria belonging to the genus Pseudomonas putida that have the ability to produce 7α-hydroxy 4AD using CDCA and/or its salt as a substrate used in the method of the present invention include Pseudomonas putida D4014-A1615 (Pseudomonas putida D4014-A1615).
D4014-A1615) strain (Feikoken Bibori No. 6572), and uses CDCA and/or its salts as a substrate.
Examples of bacteria belonging to the genus Pseudomonas species having the ability to produce 7α-hydroxy 4AD include Pseudomonas stuttieri M-1492.
(Pseudomonas stutzeri M-1492) strain (Feikoken Bacteria No. 8771). Pseudomonas putida D4014-A1615 strain was collected from soil.
D4014) is a mutant strain obtained by performing mutation treatment on the bacterial strain (Feikoken Jokyo No. 205), and the mycological properties of these strains are disclosed in JP-A-58-149698 or JP-A-59. As stated in Publication No.-39298. Also, Pseudomonas stuttieri M-
The 1492 strain was a Pseudomonas strain collected from soil.
Pseudomonas stutzeri C-37-2 (Pseudomonas stutzeri
These are mutant strains obtained by subjecting C-37-2) strain (Feikoken Bibori No. 8770) to mutation treatment, and the mycological properties of these strains are shown in the following table.

【table】

[Table] Based on the mycological properties shown in the table above, Pseudomonas stuttieri strain C-37-2 and Pseudomonas stuttieri strain M-1492 were identified. Pseudomonas stuttieri strain C-37-2 is a bacillus, has a polar monoflagellate, has negative Gram staining, and has positive oxidase and catalase reactions. , being aerobic;
Bergey's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bacteriology)
Based on Determinative Bacteriology) 8th edition,
It was identified as a bacterium belonging to the genus Pseudomonas. Furthermore, Pseudomonas stuttieri C-37-
The two strains were identified as belonging to the Pseudomonas sp. stuttieri species because they do not produce soluble pigments, perform denitrification reactions, do not decompose gelatin, and decompose starch. Additionally, mutant strains are generally considered to belong to the same species as their parent strain, and Pseudomonas stuttieri M
The -1492 strain was determined to be a bacterium belonging to the Pseudomonas sp. The production of 7α-hydroxy 4AD by the method of the present invention is carried out using CDCA and/or its salt as a substrate.
- It is carried out by culturing bacteria belonging to Pseudomonas putida species or Pseudomonas stuttieri species that have the ability to produce hydroxy 4AD in a medium containing CDCA and/or its salt.
Specific examples of the salt of CDCA include salts of alkali metals such as sodium and potassium, or salts of alkaline earth metals such as calcium and magnesium. The concentration of CDCA and/or its salt in the medium may usually range from about 1 to 100 g/
From the viewpoint of easily obtaining 7α-hydroxy 4AD in a high yield under mild conditions, the amount is preferably in the range of about 10 to 50 g/. The culture method is basically the same as that used for aerobic culture of general microorganisms, but usually a shaking culture method or an aerated agitation culture method using a liquid medium is used. As a medium, use the above CDCA and/or its salt as a substrate and 7α-hydroxy.
It is sufficient that it contains a nutrient source that can be assimilated and utilized by bacteria belonging to Pseudomonas putida species or Pseudomonas stuttieri species that have the ability to produce 4AD. As a carbon source, CDCA and/or its salt may be used as a single carbon source, or CDCA
and/or pentoses such as arabinose; glucose, mannose, fructose,
Hexoses such as galactose; Disaccharides such as sucrose and maltose; Starch decomposition products;
Sugar alcohols, polyhydric alcohols such as glycerin; or polypeptone, peptone, meat extract,
Malt extract, cornstarch liquor, yeast extract, various amino acids, organic acids, etc. may be used in combination. As the nitrogen source, for example, inorganic nitrogen sources such as ammonium nitrate, sodium nitrate, potassium nitrate, etc., or organic nitrogen sources such as voripeptone, peptone, meat extract, etc. are used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. There is no particular culture condition, but it is usually cultured at 25-35℃ for 10 hours to 7 days.
Perform daily shaking culture or aerated agitation culture. Most of the 7α-hydroxy 4AD accumulated in the culture solution in this way is usually obtained as a precipitate. To separate and collect the accumulated 7α-hydroxy 4AD, it is extracted using an organic solvent that dissolves 7α-hydroxy 4AD and phase separates from water, such as ethyl acetate, chloroform, or a mixture of chloroform and methanol. is taken. Extraction with an organic solvent may be performed on a culture containing precipitates and bacterial cells, or may be performed on insoluble components separated from the culture by filtration or centrifugation. When the former extraction method is used, it is preferable to make the culture solution alkaline by adding an aqueous alkaline solution such as an aqueous solution of sodium hydroxide, and then perform the extraction operation using the organic solvent described above. When the latter extraction method is adopted, an alkaline aqueous solution such as an aqueous solution of sodium hydroxide is preferably added to the culture filtrate or supernatant, which is separated and removed by filtration or centrifugation as necessary. After making the supernatant alkaline, the 7α-hydroxy dissolved in the culture solution is extracted by using the organic solvent described above.
4AD can be collected. By collecting the extracts thus obtained and distilling off the solvent, almost the entire amount of the target 7α-hydroxy 4AD can be recovered. Recovered 7α−
Hydroxy-4AD is purified, for example, by recrystallization from methanol, an aqueous methanol solution, or the like. 7α-Hydroxy 4AD produced by the method of the present invention can be produced by, for example, spironolactone via 17β-hydroxy-17α-(3-hydroxypropyl)androsta-4,6-dien-3-one by the following method. be guided by.

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〔Example〕

EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples. Reference example 1 Obtaining Pseudomonas stuttieri strain M-1492 Medium 1 (composition: peptone 0.5%, meat extract 0.5%,
Pseudomonas stuttieri C-37- grown on a slant with 0.5% salt and 1.5% agar; PH: 7.8)
Place a loopful of the two strains in a test tube (inner diameter: 21 mm;
Medium 2 (composition:
CDCA 3%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02
%, glucose 0.05%, peptone 0.05% and sodium hydroxide 0.3%; PH: 7.5).
Culture was carried out with shaking at 30°C for 24 hours. 0.4 of this culture
ml of culture medium 3 (composition: CDCA 0.5%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate) prepared in advance in a test tube (inner diameter: 21 mm; length: 200 mm). Heptahydrate 0.02%, yeast extract 0.02% and glucose 0.1
%; PH: 8.5) and cultured with shaking at 30°C for 6 hours. Next, the cells in the logarithmic growth phase were collected by aseptic filtration using a membrane filter with a pore size of 0.45μ, and added to 20ml of 0.1M phosphate buffer (PH: 7.0).
After washing with water, the cells were suspended in 25 ml of the same buffer. Pour 4ml of this bacterial suspension into a test tube (inner diameter: 21mm: length: 200mm)
N-methyl-N'-nitro-N-nitrosoguanidine was added to this to a final concentration of 50 μg/ml, and after 10 minutes, 0.1 ml of it was added to medium 4 [composition: 7α-hydroxy 4AD0.1 %, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, hydrogen phosphate 2
Potassium 0.6%, magnesium sulfate heptahydrate
0.02%, yeast extract 0.05% and polyoxyethylene (20) sorbitan monooleate (Tween
80) 0.05%; PH: 7.5], and then added penicillin G to a concentration of 100 U/ml.
The culture was incubated with shaking at 30°C for 18 hours. The obtained culture solution was diluted and applied to a plate of medium 1 so that about 100 to 500 colonies appeared on the plate, and then cultured overnight at 30°C. After the colonies that appeared were isolated on a slant of medium 1, each isolate was cultured with shaking in 10 ml of medium 2 for 2 days. The products in each culture solution obtained were assayed by thin layer chromatography, and the target 7α-
We found one strain that selectively accumulates hydroxy4AD and identified it as Pseudomonas stuttieri M.
The strain was named −1492. Example 1 Pseudomonas putida strain D4014-A1615 was cultured by the following method. CDCA 10g, ammonium nitrate 2.0g, potassium dihydrogen phosphate 1.0g, dipotassium hydrogen phosphate 6.0g, magnesium sulfate heptahydrate 0.2g, yeast extract 0.2g, glucose 0.5g,
Add tap water to a mixture of 0.5 g of peptone and 1.0 g of sodium hydroxide to adjust the volume to 1 (PH:
8.5) This was used as a culture medium. This medium was placed in a 2 volume fermenter and steam sterilized at 120°C for 15 minutes.
In advance, use the same medium as above in a test tube shaker.
50 ml of the inoculum grown for 20 hours was added to the above medium, and culture with aeration was performed for 48 hours under conditions of a stirring speed of 300 rpm, an aeration rate of 0.5 vvm, and 30°C. After culturing, the culture solution was thoroughly mixed and a portion thereof was analyzed by thin layer chromatography, and it was confirmed that no CDCA remained. By adding 1N aqueous sodium hydroxide solution to the culture solution
After adjusting the pH to 10, the culture solution was diluted with chloroform/
Extraction was performed with 5 parts of an ethanol mixed solvent (volume ratio: 4/1). Insoluble substances such as denatured bacterial cells were filtered from the extract, and the solvent was distilled off from the filtrate under reduced pressure to obtain 7.0 g of a dry product. Analysis of this dried product by high performance liquid chromatography revealed that the dried product was 7α-hydroxy 4AD with a purity of 86%. This 7α-hydroxy 4AD was recrystallized using an 80% by volume methanol aqueous solution to obtain 4.0 g of purified product. The analysis results of the purified product of 7α-hydroxy 4AD are shown below. Melting point: 245-247°C Mass spectrum m/e: 302 [M] ⁺ 284 [M- _H2O ] ⁺ Also, the presence of 3-keto-4-ene was confirmed from m/e = 124. NMR ( _CDCl3 , 90MHz) δ: 0.92 (s, 3H, 18- _CH3 ), 1.22 (s, 3H, 19
-CH ₃ ), 4.09 (m, 1H, 7β-H), 5.79 (s,
1H, 4-H) ppm Example 2 Culture was carried out in the same manner as in Example 1 to obtain a culture solution. As a result of analyzing a portion of the culture solution by thin layer chromatography, it was confirmed that no CDCA remained in this culture solution.
The culture solution was centrifuged, and the obtained precipitate and bacterial cells were mixed with a chloroform/ethanol mixed solvent (volume ratio:
4/1) and filtered the mixture. 5.0 by concentrating this filtrate under reduced pressure.
g of dried solid product was obtained. As a result of analyzing this dried product by high performance liquid chromatography, it was found that the dried product was 7α-hydroxy 4AD with a purity of 92%. Example 3 In Example 1, Pseudomonas putida
Pseudomonas stuttieri M-1492 strain was used instead of D4014-A1615 strain, and
The amount of CDCA used was changed from 10g to 30g, and the amount of sodium hydroxide used in the medium was changed from 1.0g to 3.0g.
By performing the same operation except that the amount was changed to 21.9 g of a dried product, 21.9 g of the dried product was obtained. In addition, as a result of analyzing a part of the obtained culture solution by thin layer chromatography, it was confirmed that no CDCA remained in the culture solution. As a result of analyzing the dry solid product by high performance liquid chromatography, the purity of the dry solid product was 85%.
It turned out to be 7α-hydroxy 4AD.
By recrystallizing this 7α-hydroxy 4AD using methanol, 10.5 g of purified product was obtained. Reference example 2

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〔Effect of the invention〕

According to the present invention, as is clear from the above examples, 7α-hydroxy 4AD can be produced with high selectivity and good yield.

Claims (1)

[Claims] 1 Pseudomonas having the ability to produce 7α-hydroxyandrost-4-ene-3,17-dione using 3α,7α-dihydroxy-5β-cholanic acid and/or its salt as a substrate
Bacteria belonging to the genus Putida species or the genus Pseudomonas stutzeri species are cultured in a medium containing 3α,7α-dihydroxy-5β-cholanic acid and/or a salt thereof.
7α-hydroxyandrost-4-ene-3,
7α-Hydroxyandrosta-4-, which is characterized by producing 17-dione and collecting it.
A method for producing ene-3,17-dione.