JPH0147156B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the production of 12α-hydroxyandroster by microorganisms from deoxycholic acid and/or its salts.
Regarding the method for producing 1,4-diene-3,17-dione, please refer to Pseudomonas putida D4014 for details.
−A1099 (Pseudomonas putida D4014−A1099)
12α-
hydroxyandrosta-1,4-diene-3,
12α-hydroxyandroster-1, which is characterized by producing 17-dione and collecting it;
The present invention relates to a method for producing 4-diene-3,17-dione. Conventionally, deoxycholic acid was treated with microorganisms.
12α-hydroxyandrosta-1,4-diene-3,17-dione (hereinafter referred to as 12α-hydroxyADD)
The following reports can be found on how to obtain First, Pseudomonas sp. NCIB 10590 by PJBarnes et al.
(Pseudomonas sp. NCIB 10590) strain has been reported [J.Chem.Soc.Chem.
Commun, (1974), pp. 115-116 and
Tetrahedron, vol. 32, pp. 89-93 (1976)].
This method was developed for Pseudomonas sp. NCIB in mineral salts medium 10 containing 0.1% deoxycholic acid.
10590 strain was cultured aerobically for 14 hours and 12β-hydroxyandroster was extracted from 10 g of deoxycholic acid.
1,4-diene-3,17-dione 960mg and 12α
-Hydroxypregna-1,4-dien-3-one-20α-carboxylic acid (657 mg) is the main product, and 12α-hydroxy ADD is only obtained as a minor component. Regarding the Pseudomonas SP-NCIB 10590 strain used in this method, nothing has been reported other than that this strain is a bacterium belonging to the genus Pseudomonas collected from animal feces. Also, Owen (RW
Bacteroides fragilis by Owen et al.
XF23 (Bacteroides fragilis XF23) strain or Bacteroides fragilis subsp.
Thetaiotaomicron E59 (Bacteroides fragilis)
12α-hydroxy ADD, 12β from deoxycholic acid using the subsp.
-Hydroxyandrosta-1,4-diene-
Method for obtaining 3,17-dione, 12β-hydroxy-4-androstene-3,17-dione and 12α-hydroxypregna-1,4-dien-3-one-20α-carboxylic acid [Biochem.Soc.Trans ,5
Vol., pp. 1711-1713 (1977)], but all of these methods use anaerobic bacteria, the substrate used is at a low concentration, and culture requires a long period of time. There are various problems in industrial fermentation production such as low yield of the target product. The present inventors conducted a long-term search for microorganisms that produce 12α-hydroxy ADD using deoxycholic acid and/or its salts as substrates, and as a result, specific bacteria belonging to Pseudomonas putida were found to produce 12α-hydroxy ADD using deoxycholic acid and/or its salts. The present inventors have discovered that 12α-hydroxy ADD can be produced with high selectivity and yield using salt as a substrate, and have completed the present invention. The bacteria belonging to Pseudomonas putida obtained by the present inventors include Pseudomonas putida D4014-A1099.
-A1099) strain (Feikoken Joyori No. 207).
This strain is a Pseudomonas strain collected from soil.
This is a mutant strain obtained by subjecting the Pseudomonas putida D4014 strain (Feikoken Joyori No. 205) to mutation treatment. Pseudomonas putida obtained by the present inventors
D4014 strain and Pseudomonas putida D4014−
The following table lists the mycological properties of the A1099 strain.

【table】

[Table] Based on the mycological properties shown in the table above, Pseudomonas putida strain D4014 and Pseudomonas putida D4014-A1099 strain were identified. Pseudomonas putida strain D4014 is a bacillus, has polar flagella, has negative Gram staining, positive oxidase and catalase reactions, and is aerobic. Based on the physiological properties such as the O-F test result being oxidative, bacteria belonging to the genus Pseudomonas are classified according to the Verges Manual of Determinative Bacteriology, 7th and 8th editions. It was identified that Furthermore, Pseudomonas putida strain D4014 is believed to belong to the Pseudomonas putida species because the culture solution has a fluorescent color, it does not liquefy gelatin, it grows at 37°C, and it produces arginine dihydrase. Identified. Furthermore, it is generally believed that a mutant strain belongs to the same species as its parent strain, and it was determined that the Pseudomonas putida strain D4014-A1099 belongs to the species Putida of the genus Pseudomonas. Production of 12α-hydroxy ADD by the method of the present invention involves culturing bacteria belonging to Pseudomonas putida that produces 12α-hydroxy ADD using deoxycholic acid and/or its salt as a substrate in a medium containing deoxycholic acid and/or its salt. This is done by culturing the cells. Specific examples of the salts of deoxycholic acid include salts of deoxycholic acid with alkali metals such as sodium and potassium. The concentration of deoxycholic acid and/or its salt is usually about 1
The amount of 12α- produced may be in the range of ~100g
From economical viewpoints such as the yield of hydroxy ADD, culture conditions, and operability, the amount is preferably in the range of about 2 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 using a liquid medium or an aerated agitation culture method is used. The medium may contain a nutrient source that can be assimilated and utilized by bacteria belonging to Pseudomonas putida that produce 12α-hydroxy ADD using the above deoxycholic acid and/or its salt as a substrate. As a carbon source, deoxycholic acid and/or its salt may be used as a single carbon source, but preferably deoxycholic acid and/or its salt is combined with glucose,
Glycerin, peptone, meat extract, malt extract,
It is best to use yeast extract etc. Examples of nitrogen sources include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate,
Inorganic nitrogen sources such as sodium nitrate, potassium nitrate, or organic nitrogen sources such as polypeptone, peptone, meat extract, etc. are used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually carried out at 25 to 35°C for 10 hours to 7 days. In order to separate and collect the 12α-hydroxy ADD accumulated in the culture solution in this way, the culture solution is made alkaline and then an organic solvent that dissolves the 12α-hydroxy ADD and phase separates from water, such as ethyl acetate, is used. , chloroform, a mixture of chloroform and methanol, etc. are used for extraction. This extraction with an organic solvent may be carried out on the culture fluid containing the bacterial cells, or the bacterial cells and other insoluble components in the culture fluid may be separated and removed by filtration or centrifugation in advance, and the resulting culture The test may be carried out on the filtrate or supernatant. When using the latter extraction method, the insoluble components that are separated and removed by filtration or centrifugation, if necessary, can be extracted with the organic solvent described above, so that they can be precipitated or precipitated in the culture solution. 12α-hydroxy
ADD can be recovered. By collecting the extracts thus obtained and distilling off the solvent, almost the entire amount of the target 12α-hydroxy ADD can be recovered. In addition to 12α-hydroxy ADD, the obtained extract contains almost no residual substrate deoxycol and/or its salts and by-products, and can be extracted by recrystallization from ethyl acetate, dichloromethane/methanol, etc. High purity 12α-hydroxy ADD can be obtained. 12α-hydroxy obtained by the method of the present invention
ADD is a 4-androstene-3,17-dione that is a raw material for the synthesis of steroids such as spironolactone, which is useful as a diuretic, by partially hydrogenating its A ring and chemically removing the hydroxyl group at the 12th position. can be induced to For example, 12α-hydroxy ADD with tris(triphenylphosphine)
By partial hydrogenation using chlororhodium
12α-hydroxy-4-androstene-3,17
- obtained dione [C.Djerassi and J.Gutzwiller, J.
Am.Chem.Soc., vol. 88. pp. 4538-4539 (1966)
], 12α with the keto groups at positions 3 and 17 protected by reacting this 12α-hydroxy-4-androstene-3,17-dione with 2-methyl-2-ethyl-1,3-dioxolane. -hydroxy-5-androstene-3,17-dione-3,
Obtain 17-bis(ethylene acetal) [G.
Rosenkranz et al., J.Am.Chem.Soc., vol. 76.
See pages 5024-5026 (1954)]. This acetal was tosylated with p-toluenesulfonyl chloride and reduced with lithium triethylborohydride.
After removing the hydroxyl group at the 12th position, 4-androstene-3,17-dione can be obtained by treating with mineral acid/acetone to remove the protecting groups at the 3rd and 17th positions. The present invention will be explained in more detail below using Examples and Reference Examples. Reference example Method for obtaining Pseudomonas putida D4014-A1099 strain Medium 1 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, peptone 0.5%, yeast extract
Pseudomonas putida grown on slants of 0.5% sodium chloride, 0.5% sodium chloride and 1.5% agar).
A loopful of D4014 strain was prepared in advance in a test tube in medium 2 (composition: 2% deoxycholic acid, 0.2% sodium hydroxide, 0.2% ammonium nitrate, 0.1% potassium dihydrogen phosphate, 0.6 potassium dihydrogen phosphate).
%, magnesium sulfate heptahydrate 0.02%, and yeast extract 0.02%) and cultured with shaking at 30°C for 14 to 15 hours. Culture medium 3 (composition: deoxycholic acid 0.5
%, sodium hydroxide 0.05%, glucose 0.1%,
Ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1
%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02% and yeast extract 0.02%).
ml and cultured at 30°C for 8 to 9 hours. Then,
The cells in the logarithmic growth phase were collected by aseptic filtration using a 0.45 μ membrane filter, washed with 20 ml of 0.1 M phosphate buffer (PH: 7.0), and washed with 25 ml of the same buffer.
ml. Mutation treatment was carried out by adding N-methyl-N'-nitro-N-nitrosoguanidine to the mixture at a final concentration of 50 μg/ml and allowing it to stand for 3 to 4 minutes. The mutant-treated bacterial cells were collected by filtration using a 0.45μ membrane filter, washed with 20ml of 0.1M phosphate buffer (PH: 7.0), and then suspended in 20ml of the same buffer. The obtained bacterial suspension was diluted with sterile physiological saline and added to medium 4.
(Composition: Deoxycholic acid 0.5%, sodium hydroxide 0.05%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02
% and agar 1.5%) on an agar plate so that 500 to 1000 colonies appear, and then incubated at 30°C.
The cells were cultured for 3 to 4 days. After isolating the tiny colonies among the colonies that appeared on the slant of medium 1,
Culture medium 5 (composition: deoxycholic acid 0.2%, sodium hydroxide
0.02%, glucose 0.1%, ammonium nitrate 0.2
%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%
and yeast extract 0.02%) and inoculated at 30℃.
It was cultured with shaking for 24 hours. The products in each of the obtained culture solutions were assayed by thin layer chromatography, and one strain was found that selectively accumulated the target 12α-hydroxy ADD, and this was named Pseudomonas putida D4014-A1099. I named it. Example 1 Pseudomonas putida strain D4014-A1099 (Feikoken Joyori No. 207) was cultured by the following method. Deoxycholic acid 0.5g, glucose 0.1g,
Ammonium nitrate 0.2g, potassium dihydrogen phosphate 0.1
g, dipotassium hydrogen phosphate 0.6 g, magnesium sulfate heptahydrate 0.02 g, yeast extract 0.02 g, and sodium hydroxide 0.05 g, add tap water to make the volume 100.
ml (PH: 8.4) and used as a medium. Pour this medium into a 500ml Sakaguchi flask and heat at 120℃ for 15 minutes.
Steam sterilization was performed for 1 minute. Add 10 ml of the inoculum grown in advance in the same medium as the above medium for 15 hours in a test tube shaker to the above 500 ml Sakaguchi flask,
Shaking culture was performed at 30°C for 28 hours. After culturing, collect the culture fluid and remove the bacterial cells using a centrifuge. Add a 1N aqueous solution of sodium hydroxide to the resulting culture supernatant to bring the pH of the culture supernatant to around 12. It was adjusted. Then, the culture supernatant was diluted with 300% ethyl acetate.
ml, and 12α-
360mg of hydroxyADD was obtained. Take a part of the obtained 12α-hydroxy ADD,
Methanol was added to this to make a 2% solution, and 25μ of this solution was injected into a high performance liquid chromatography system (HLC-GPC-244 model, manufactured by Waters, USA) equipped with a Micro Bondapak C-18 column. Water/methanol adjusted to pH 4.0 as the mobile phase.
A mixed solution with a volume ratio of 30/70 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method. The area ratio of each peak in the obtained liquid chromatogram was determined using an integrator (Shimadzu Chromato Pack C-R1A, manufactured by Shimadzu Corporation), and from this area ratio, the above 12α-hydroxy
When the purity of ADD was determined, it was 99.5%. Furthermore, the 12α-hydroxy ADD obtained above was confirmed by the following method. Mass spectrum m/z: 300 [M] ₊

Claims (1)

[Claims] 1 Pseudomonas putida D4014−A1099
(Pseudomonas putida D4014-A1099) strain (Feikoken Jokyo No. 207) was cultured in a medium containing deoxycholic acid and/or its salts. −
12α-Hydroxyandrosta-1,4- which is characterized by producing dione and collecting it.
Method for producing diene-3,17-dione.