JPH0120875B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for obtaining 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its ester in which microorganisms accumulate from a medium containing cholic acid and/or its ester. 3α using the ester as a substrate,
Bacteria belonging to the genus Arthrobacter, Brevibacterium, Corynebacterium, or Nocardia that produce 7α-dihydroxy-12-keto-5β-cholanic acid and/or its esters were treated with cholic acid and/or its esters. 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its ester are produced by culturing in a nutrient medium containing 3α,7α-dihydroxy-12-keto-5β-cholanic acid and collected. -Regarding a method for producing colanic acid and/or its ester. Conventionally, various methods have been known for obtaining 3α,7α-dihydroxy-12-keto-5β-cholanic acid as a metabolite of microorganisms using cholic acid as a substrate. For example, the method using Streptomyces gelaticus 1164 strain by Hayakawa et al. [Journal of Biochemistry (Japan), Vol. 44, No. 2, No. 109-
113 (1957) and Proceedings of the Japan Academy, Vol. 32, pp. 519-522 (1956)]; Method using Aspergillus cinnamomeus HUT2026 strain by Hasegawa et al. [Hiroshima Journal of Medical・Science, Vol. 8, No. 3, pp. 277-283 (1959)]; and Kikuchi et al.'s method using Staphylococcus epidermidis H-1 strain [Journal of Biochemistry, Vol. 72, No. 1 Issue No. 165~
172 pages (1972)]. The present inventors have discovered that 3α and 7α are metabolites of microorganisms.
- In order to advantageously obtain dihydroxy-12-keto-5β-cholanic acid and/or its ester, as a result of long-term screening of microorganisms that utilize cholic acid and/or its ester as a substrate, Arthrobacter Certain bacteria belonging to the genus Brevibacterium, Corynebacterium, or Nocardia use cholic acid and/or its ester as a substrate to produce 3α,7α-dihydroxy-12-
Producing keto-5β-cholanic acid and/or its ester, and converting cholic acid and/or its ester in suspension or crystalline form into the above-mentioned colanic acid and/or its ester without solubilizing the cholic acid and/or its ester. They have discovered that it is possible to do so, and have completed the present invention. 3α,7α-dihydroxy-12- using cholic acid and/or its ester obtained by the present inventors as a substrate.
Bacteria belonging to the genus Arthrobacter, Brevibacterium, Corynebacterium, or Nocardia that have the ability to produce keto-5β-cholanic acid and/or its ester include Arthrobacter CA-35 (Arthrobacter CA-35). 35) Bacterial strain (Feikoken Bacteria No. 5145), Arthrobacter CA-
35−A589−29−32 (Arthrobacter CA−35−
A589-29-32) strain (Feikoken Bacterial Serial No. 5522), Brevibacterium CA-6 (Brevibacterium CA-6)
-6) Bacterial strain (FEI Bacterium No. 5144), Corynebacterium CA-53 strain (FEI BAC No. 5532), and Nocardia CA
-173 (Nocardia CA-173) strain
No. 5531), and the mycological properties of each are listed in the table below.

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[Table] Based on the mycological properties shown in the table above, Arthrobacter CA-35 strain, Arthrobacter CA-
35-A589-29-32 strain, Brevibacterium CA
Identification of Corynebacterium CA-6 strain, Corynebacterium CA-53 strain, and Nocardia CA-173 strain was carried out using Verges Manual of Determination.
The test was conducted based on Bacteriology, 7th and 8th editions. Arthrobacter CA-35 strain has its morphology,
Based on microscopic findings such as Gram staining and physiological properties, it was identified as a bacterium belonging to the genus Arthrobacter. Arthrobacter strain CA-35 produces a yellow to cream-colored pigment, and it belongs to the genus Arthrobacter, and the pigment-producing bacteria include Arthrobacter oxydans, Arthrobacter
auretuscens and Arthrobacter ureafaciens are present. However, Arthrobacter oxydans and Arthrobacter auretuscens are different from the Arthrobacter CA-35 strain because they are usually negative in Gram staining and have the ability to hydrolyze starch. Also,
Since Arthrobacter ureafaciens is usually negative in Gram staining and does not have nitrate reducing properties, Arthrobacter CA-
Different from 35 strains. Furthermore Arthrobacter CA
-35 strain is identified as being closely related to Arthrobacter simplex, but since Arthrobacter simplex IAM1660 strain does not grow in medium containing 10 g/concentration of cholate, Arthrobacter CA -35 strain clearly differs from Arthrobacter simplex IAM1660 strain in its attitude towards cholate. Therefore, since the Arthrobacter strain CA-35 is different from any standard species belonging to the genus Arthrobacter, it was determined that it constitutes a new species belonging to the genus Arthrobacter. Arthrobacter
CA-35-A589-29-32 strain is Arthrobacter
This is a mutant strain obtained by contacting strain CA-35 with N-methyl-N'-nitro-N-nitrosoguanidine. Since mutant strains are generally considered to belong to the same species as their parent strains, Arthrobacter CA-35-A589-29-
The 32 strains were determined to belong to the same new bacterial species as their parent strain. Brevibacterium strain CA-6 was identified as a bacterium belonging to the genus Brevibacterium based on its microscopic findings such as Gram staining and physiological properties. However, whereas bacteria belonging to the genus Brevibacterium have periflagella, the Brevibacterium CA-6 strain has a polar monoflagellum. It has slightly different properties from bacteria. Corynebacterium strain CA-53 was identified as a bacterium closely related to Corynebacterium equi based on its morphology, microscopic findings such as Gram staining, and physiological properties. nocaldeia
Strain CA-173 seems to belong to the genus Corynebacterium or Nocardia based on microscopic findings and physiological properties, but since the O-F test is oxidative, it belongs to the genus Corynebacterium. It is clearly different.
Therefore, the Nocardia strain CA-173 was determined to be a bacterium belonging to the genus Nocardia. 3α,7α-dihydroxy- by the method of the present invention
The production of 12-keto-5β-cholanic acid and/or its ester is carried out by using the above-mentioned cholic acid and/or its ester as a substrate and producing 3α,7α-dihydroxy-12-
This is carried out by culturing bacteria that produce keto-5β-cholanic acid and/or its ester in a medium containing cholic acid and/or its ester.
The cholic acid ester is preferably a lower alkyl ester of cholic acid such as methyl ester, ethyl ester, propyl ester, butyl ester. The concentration of cholic acid and/or its ester may normally range from about 1 to 500 g
From an economic point of view, such as the yield of 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its ester, culture conditions and operability, the amount is preferably in the range of about 10 to 500 g/, and more preferably in the range of 10 to 300 g/. is more preferable. The culture method is basically the same as that used for culturing general microorganisms, but usually a shaking culture method or an aerated agitation culture method using a liquid medium is used. As a medium, the above cholic acid and/or
Alternatively, it may contain a nutrient source that can be assimilated and utilized by bacteria that produce 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its ester using its ester as a substrate. In addition to cholic acid and/or its esters, carbon sources include pentoses such as arabinose; hexoses such as glucose, mannose, fructose, and galactose; disaccharides such as maltose; starch decomposition products, sugar alcohols, glycerin, etc. Polyhydric alcohols; polypeptone, peptone, meat extract, malt extract, cornstarch liquor, yeast extract, various amino acids, etc. are used. Further, as the nitrogen source, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, etc. are used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Culture conditions are not specific, but usually 25
Shaking culture or aerated agitation culture is performed at ~35°C for 6 hours to 8 days. 3α, 7α accumulated in the culture medium in this way
In order to separate and collect -dihydroxy-12-keto-5β-cholanic acid and/or its ester, first, the bacterial cells in the culture solution, 3α,7α-dihydroxy- Oxidation products of cholic acid and/or its esters, including 12-keto-5β-cholanic acid and/or its esters, and unconverted cholic acid and/or its esters are separated and obtained by filtration or centrifugation. For example, a chloroform-ethanol mixture is added to this to dissolve the oxidized product of cholic acid and/or its ester, as well as unconverted cholic acid and/or its ester, and then the bacterial cells are separated and removed by filtration or centrifugation. , the resulting filtrate or supernatant is evaporated to dryness. To the obtained oxidation product of cholic acid and/or its ester and the mixture of unconverted cholic acid and/or its ester, for example, ethyl acetate is added to the previous culture filtrate or supernatant, and the remaining cholic acid and/or ester is added. Almost completely 3α
Collecting oxidation products of cholic acid and/or its esters, including 7α-dihydroxy-12-keto-5β-cholanic acid and/or its esters, and recovering unconverted cholic acid and/or its esters. Can be done. The thus obtained oxidation product of cholic acid and/or its ester and the mixture of unconverted cholic acid and/or its ester are adsorbed onto a silica gel column, for example, and sequentially eluted with a chloroform-ethanol mixture.
In this way, 3α,7α-dihydroxy-12-keto-5β-cholanic acid and its ester can be obtained. The obtained 3α,7α-dihydroxy-12-keto-
The ester of 5β-cholanic acid can be converted into 3α,7α-dihydroxy-12-keto-5β-cholanic acid by hydrolysis using a conventional method. The 3α,7α-dihydroxy-12-keto-5β-cholanic acid obtained by the method of the present invention can be easily converted to chenodeoxycholic acid (CDCA), which is useful as a gallstone dissolving agent, by subjecting it to the Huang-Minlong reduction reaction. The present invention will be explained in more detail below using Examples. Reference example 1 Acquisition of Arthrobacter CA-35-A589-29-32 strain (Feikoken Bibori No. 5522) Medium 1 (composition: sodium hydroxide 0.5%, cholic acid 5.0%, peptone 0.5%, yeast extract One platinum loop of Arthrobacter CA-35 strain grown on a slant of 0.5% sodium chloride, 0.5% sodium chloride, and 1.5% agar) was placed in medium 2 (composition) prepared in advance in a test tube (inner diameter 21 mm, length 200 mm). : Sodium hydroxide 1%,
Cholic acid 10%, ammonium nitrate 0.2%, phosphoric acid 2
Potassium hydrogen 0.2%, potassium monohydrogen phosphate 0.5%,
The cells were inoculated into 10 ml of 0.02% magnesium sulfate heptahydrate and 0.01% yeast extract, and cultured with shaking at 30°C for 24 hours. Pour 0.3 ml of this culture solution into a test tube (inner diameter
21 mm, length 200 mm) prepared in medium 3 (composition: sodium hydroxide 0.05%, cholic acid 0.5%, glucose 0.5%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.2%, potassium monohydrogen phosphate 0.5%, magnesium sulfate. Heptahydrate 0.02% and yeast extract 0.01
%) and cultured with shaking at 30°C for 15-16 hours. Next, the bacterial cells in the logarithmic growth phase were
Aseptically filter and collect bacteria using a membrane filter.
After washing with 20 ml of 0.1M phosphate buffer (PH7.0), it was suspended in 25 ml of the same buffer. Put 4 ml of this bacterial suspension into a test tube (inner diameter 21 mm, length 200 mm) and add N-methyl-N'- to a final concentration of 50 μg/ml.
Add nitro-N-nitrosoguanidine and heat to 30°C.
Mutation treatments were performed by shaking for 45 minutes at . Note that the mortality rate of Arthrobacter CA-35 strain under this treatment condition was about 80%. The mutant-treated bacterial cells were collected by aseptic filtration using a 0.45μ membrane filter, washed with 20ml of 0.1M phosphate buffer (PH7.0), and then suspended in 25ml of the same buffer. The obtained bacterial suspension was diluted with sterilized physiological saline and mixed with medium 4 (composition: sodium hydroxide 0.1%, cholic acid 1.0%, ammonium nitrate).
0.2%, potassium dihydrogen phosphate 0.2%, potassium monohydrogen phosphate 0.5%, magnesium sulfate heptahydrate 0.02
%, yeast extract 0.01%, and agar 1.5%) so that 300 to 500 colonies appeared on an agar plate, and then cultured at 30°C for 4 days. Isolate the smallest colonies among the colonies that appeared and incubate them on an agar plate in medium 5 (composition: peptone 0.5%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) at 30°C.
The cells were cultured for 24 hours. The colonies that appeared here were replicated onto an agar plate in medium 4 and cultured at 30°C for 20 hours. Colonies in medium 5, which correspond to colonies that do not grow in medium 4, were transferred to medium 6 (composition: sodium hydroxide 0.1%, cholic acid 1.0%, peptone).
0.5%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) and incubate at 30°C.
The cells were cultured for 24 hours. Culture medium 7 (composition: sodium hydroxide 0.5%,
Cholic acid 5.0%, glucose 0.5%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.2%, potassium monohydrogen phosphate 0.5%, magnesium sulfate heptahydrate
Inoculate 10ml of yeast extract (0.02% and yeast extract 0.01%),
The cells were cultured with shaking at 30°C for 3 days. The products in each culture solution obtained were assayed by thin layer chromatography, and the target 3α,7α-dihydroxy-12-
One strain that selectively accumulates keto-5β-cholanic acid and/or its salts was found and named Arthrobacter strain CA-35-M-965-3. From now on, I will summarize the method for obtaining this mutant strain and simply explain.
This is called NTG treatment. By using Arthrobacter CA-35-M-965-3 strain as the parent strain and subjecting it to NTG treatment,
We found three strains that selectively accumulate 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its salts, and one of these strains was isolated from Arthrobacter.
The strain was named CA-35-A589. Two excellent strains were isolated from the colony of Arthrobacter CA-35-A589 strain, and one strain among them was named Arthrobacter CA-35-A589-29-32 strain. Example 1 Arthrobacter strain CA-35-A589-29-32 (Feikoken Bibori No. 5522) was cultured by the following method. Add tap water to 5.0 g of cholic acid, 0.5 g of glucose, 0.2 g of ammonium nitrate, 0.2 g of potassium dihydrogen phosphate, 0.5 g of dipotassium hydrogen phosphate, 0.02 g of magnesium sulfate heptahydrate, and 0.05 g of yeast extract to bring the volume to 100 ml. This was used as a culture medium. Pour this medium into a 500ml Sakaguchi flask and heat at 120℃ for 15 minutes.
Steam sterilization was performed for 1 minute. Inoculum grown in advance in the same medium as above in a test tube shaker for 2 days
10 ml was inoculated into the medium in the above 500 ml Sakaguchi flask, and cultured with shaking for 5 days. Cholic acid was in an undissolved state in the medium, and as the culture progressed, the crystal form changed to clearly different crystals of 3α,7α-dihydroxy-12-keto-5β-cholanic acid. After culturing, the obtained culture solution was centrifuged to precipitate the bacterial cells, oxidation products of cholic acid, and unconverted cholic acid, and separate them from the supernatant. 100 ml of a mixture of chloroform/ethanol at a volume ratio of 1/1 was added to the precipitate consisting of the bacterial cells, the oxidation product of cholic acid, and unconverted cholic acid, and the oxidation product of cholic acid and unconverted cholic acid were added. It was dissolved in the mixture. The bacterial cells were removed by filtration, and the filtrate was concentrated to dryness under reduced pressure to obtain a mixture of cholic acid oxidation products and unconverted cholic acid. To this, 100 ml of ethyl acetate was added to the culture supernatant. extract,
The oxidation product of cholic acid obtained by concentrating this extract to dryness and the mixture of unconverted cholic acid were combined to obtain 5.2 g of a mixture. Take a very small portion of this mixture, add methanol to it to make a 2% solution, and apply 10μ of this solution to a high performance liquid chromatography system (manufactured by Waters, USA, HLC-GPC-
244 type). As a mobile phase, a mixed solution of water/methanol in a volume ratio of 30/70 adjusted to pH 4.0 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method, and the chromatogram shown in FIG. 1 was obtained. Peak A and peak B in this chromatogram were determined to be 3α and 7α, respectively, by comparing them with the peaks of the standard product.
-dihydroxy-12-keto-5β-cholanic acid and cholic acid. In addition, a compound corresponding to the peak A portion was fractionated, and this compound was converted into 3α,7α-dihydroxy-12-keto-5β by the following method.
-Identified as colanic acid. Identification by thin layer chromatography 10 μ of a 2% methanol solution of the above compound was developed for 10 cm using a developing solvent with a composition of benzene: ethyl acetate: acetic acid (30:10:1), and after drying, sulfuric acid: ethanol ( When a coloring reagent solution with a composition of 1:1) is sprayed and heated at 115℃ for 15 minutes, the standard product 3α,
A spot of the same color was observed at the same position (Rf = 0.48) as 7α-dihydroxy-12-keto-5β-cholanic acid. Melting point: 219-220℃ Mass spectrum m/e: 406 [M] ⁺ 388 [M] ⁺ -H ₂ O 370 [M] ⁺ -2H ₂ O NMR spectrum (90MHz) δ ^DMSO-d6 _HMS : 0.70 (3H, d) 21- _CH3 ; 0.88, 0.90 (6H, s) 18- _CH3 , 19- _CH3 ; 3.24 (1H, broad) 3β-H; 3.67 (1H, broad) 7β-H; 4.87, 4.91 (2H ) 3α-OH, 7α-OH From the area ratio of the chromatograms shown in Figure 1, the yield of the obtained 3α,7α-dihydroxy-12-keto-5β-cholanic acid and the remaining amount of unconverted cholic acid are calculated. As shown in the table below.

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[Table] Example 2 Using Arthrobacter CA-35-A589-29-32 strain (same as above), changing the concentration of cholic acid in Example 1, mixing chloroform/ethanol at a volume ratio of 1/1. By culturing and treating in the same manner as in Example 1 except for dissolving the oxidized product of cholic acid obtained by using acidic ethyl acetate instead of the solution and unconverted cholic acid, each substrate concentration was determined. 3α,7α-dihydroxy-12 in
The yield of -keto-5β-cholanic acid and the remaining amount of unconverted cholic acid were determined and shown in the following table.

[Table] Example 3 Arthrobacter CA-35- in Example 1
Arthrobacter CA instead of A589-29-32 strain
4.7 g of a mixture of cholic acid oxidation products and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 1, except that the -35 strain (Feikoken Bibori No. 5145) was used. Ta. The resulting mixture was subjected to thin layer chromatography and liquid chromatography in the same manner as in Example 1 to confirm the presence of 3α,7α-dihydroxy-12-keto-5β-cholanic acid. Example 4 In Example 1, Arthrobacter CA-35-
Brevibacterium instead of A589-29-32 strain
By culturing and treating in the same manner as in Example 1 except for using strain CA-6 (Feikoken Bibori No. 5144), 4.9 g of a mixture of cholic acid oxidation products and unconverted cholic acid was obtained. Obtained. The resulting mixture was subjected to thin layer chromatography and liquid chromatography in the same manner as in Example 1 to confirm the presence of 3α,7α-dihydroxy-12-keto-5β-cholanic acid. Example 5 In Example 1, Arthrobacter CA-35-
Corynebacterium instead of A589-29-32 strain
By culturing and treating in the same manner as in Example 1 except using strain CA-53 (Feikoken Bibori No. 5532), 4.8 g of a mixture of the oxidation product of cholic acid and unconverted cholic acid was obtained. Obtained. The resulting mixture was subjected to thin layer chromatography and liquid chromatography in the same manner as in Example 1 to confirm the presence of 3α,7α-dihydroxy-12-keto-5β-cholanic acid. Example 6 In Example 1, Arthrobacter CA-35-
Nocardia CA- instead of A589-29-32 strain
By culturing and treating in the same manner as in Example 1 except for using strain 173 (Feikoken Bibori No. 5531), 4.9 g of a mixture of cholic acid oxidation products and unconverted cholic acid was obtained. . The resulting mixture was subjected to thin layer chromatography and liquid chromatography in the same manner as in Example 1 to confirm the presence of 3α,7α-dihydroxy-12-keto-5β-cholanic acid. Example 7 Arthrobacter CA-35-A589-29-32 strain (same as above) was cultured by the following method. Add tap water to 5.0 g of methyl cholate, 0.5 g of glucose, 0.2 g of ammonium nitrate, 0.2 g of potassium dihydrogen phosphate, 0.5 g of dipotassium hydrogen phosphate, 0.02 g of magnesium sulfate heptahydrate, and 0.05 g of yeast extract to make up the volume. The volume was adjusted to 100 ml and used as a culture medium. This medium was placed in a 500 ml Sakaguchi flask and steam sterilized at 120°C for 15 minutes. Inoculum 10 grown in advance in the same medium as above in a test tube shaker for 2 days
ml was inoculated into the medium in the above test tube, and cultured with shaking for 7 days. After culturing, the obtained culture solution is centrifuged to precipitate the bacterial cells, oxidation products of methyl cholate, and unconverted methyl cholate,
It was separated from the supernatant. Add 50 ml of a mixture of chloroform/ethanol at a volume ratio of 1/1 to the precipitate (hydrated material) consisting of bacterial cells, oxidation products of methyl cholate, and unconverted methyl cholate to obtain the oxidation products of methyl cholate. and unconverted methyl cholate were dissolved in the mixture. The bacterial cells and water were removed by liquid separation, and the resulting filtrate was concentrated to dryness under reduced pressure to obtain 4.4 g of a mixture of the oxidation product of methyl cholate and unconverted methyl cholate. Take a small portion of this mixture, add methanol to it to make a 2% solution, and apply 10μ of this solution to a high performance liquid chromatography system (manufactured by Waters Corporation, USA, HLC-
GPC-244). As a mobile phase, a mixed solution of water/methanol in a 20/80 volume ratio adjusted to pH 4.0 was flowed at a flow rate of 1 ml/min, and detection was performed using a refractive index method, and the chromatogram shown in FIG. 2 was obtained.
Peak C and peak D in this chromatogram were determined by comparing them with the peaks of the standard product.
It was consistent with that of methyl 3α,7α-dihydroxy-12-keto-5β-cholanate and methyl cholate.
In addition, a compound corresponding to the portion of peak C was fractionated, and this compound was identified as methyl 3α,7α-dihydroxy-12-keto-5β-cholanate by the method described below. Identification by thin layer chromatography 10μ of a 2% methanol solution of the above compound was mixed in isooctane:ethyl acetate:acetic acid (10:10:2).
Developed for 10 cm using a developing solvent with the composition, and after drying,
When a coloring reagent solution with a composition of sulfuric acid and ethanol (1:1) is sprayed and heated at 115°C for 15 minutes, the standard product
A spot with the same color was observed at the same position as methyl 3α,7α-dihydroxy-12-keto-5β-cholanate (Rf=0.37). Melting point: 154-156℃ Mass spectrum m/e: 420 [M] ⁺ 402 [M] ⁺ -H ₂ O 384 [M] ⁺ -2H ₂ O NMR spectrum (90MHz) δ ^CDCl3 _HMS : 0.8 (3H, d) 21- _CH3 ; 0.9 (6H, d) 18- _CH3 , 19- _CH3 ; 3.4 (1H, broad) 3β-H; 3.6 (3H, s)-COOCH3 _3.8-3.9 (1H, broad) 7β- H From the area ratio of the chromatogram in Figure 2, the yield of the obtained methyl 3α,7α-dihydroxy-12-keto-5β-cholate and the remaining amount of unconverted methyl cholate are calculated as shown in the following table. It is.

[Table] Examples 8 to 10 In Example 7, Arthrobacter CA-35-
Arthrobacter CA instead of A589-29-32 strain
−35 strain (same as above), Brevibacterium CA
-6 strains (same as above) or Corynebacterium
By culturing and treating in the same manner as in Example 7 except for using the CA-53 strain (same as above), 3α,7α-dihydroxy-12-keto-5β-cholane was obtained using each strain. The yield of methyl cholate and the remaining amount of unconverted methyl cholate were determined and shown in the following table.

【table】 [Brief explanation of drawings]

Figure 1 shows a liquid chromatogram of the oxidation product of cholic acid obtained in Example 1 and unconverted cholic acid, and Figure 2 shows the oxidation product of methyl cholate and unconverted cholic acid obtained in Example 7. Figure 3 depicts the liquid chromatogram of methyl cholate of the transformation.

Claims (1)

[Claims] 1 Arthrobacter, Brevibacterium, Corynebacterium, or Nocardia that produce 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its ester using cholic acid and/or its ester as a substrate Bacteria belonging to
3α,7α-dihydroxy-12-keto-
3α, 7α characterized by producing and collecting 5β-cholanic acid and/or its ester
- A method for producing dihydroxy-12-keto-5β-cholanic acid and/or its ester.