JPH0120873B2 - - 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 salts as a metabolite of microorganisms from a medium containing cholate in high yield and in a short period of cultivation. is the Arthrobacter strain CA-35
3α,7α-dihydroxy-12-
A mutant strain that produces keto-5β-cholanic acid and/or a salt thereof is cultured in a nutrient medium containing cholate to produce the above-mentioned colanic acid and/or a salt thereof, and the resulting product is collected. 3α,
The present invention relates to a method for producing 7α-dihydroxy-12-keto-5β-cholanic acid and/or a salt thereof. 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 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 obtain dihydroxy-12-keto-5β-cholanic acid industrially, we have carried out long-term screening for microorganisms that require high concentrations of cholate as a substrate. We discovered that a specific type of bacteria can metabolically produce 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its salts in high yields and in short-term culture using high-concentration cholate as a substrate. The bacterium was transformed into Arthrobacter CA-35 (Arthrobacter CA-35).
35) The strain was named ``Feikoken Bibori No. 5145'' (see Japanese Patent Application No. 106768, 1983). Furthermore, the present inventors have discovered that the above-mentioned Arthrobacter
The CA-35 strain was subjected to conventional mutation treatments, such as irradiation with ultraviolet rays, X-rays, γ-rays, etc., N-methyl-
Mutant strains obtained by contacting with mutagenic agents such as N'-nitro-N-nitrosoguanidine, 4-nitroquinoline-N-oxide, acriflavine, and ethyl methanesulfonate are 3α, 7α using salt as a substrate
The inventors have discovered that -dihydroxy-12-keto-5β-cholanic acid and/or its salts can be metabolically produced extremely selectively, and have completed the present invention. Examples of such mutant strains include the following strains. (1) Arthrobacter CA−35−A589−29−32
(Arthrobacter CA-35-A589-29-32) Strain (Feikoken Bacteria No. 5522) (2) Arthrobacter CA-35-A589-47
(Arthrobacter CA-35-A589-47) Strain (Feikoken Bacteria Collection No. 5523) (3) Arthrobacter CA-35-A849
(Arthrobacter CA-35-A849) Strain (Feikoken Bacteria No. 5524) (4) Arthrobacter CA-35-A-1071-15
(Arthrobacter CA-35-A-1071-15) Strain (Feikoken Bacteria No. 5525) (5) Arthrobacter CA-35-A-1448
(Arthrobacter CA-35-A-1448) Strain (Feikoken Bacteria Collection No. 5526) (6) Arthrobacter CA-35-A-1475
(Arthrobacter CA-35-A-1475) Strain (Feikoken Bacteria Collection No. 5527) (7) Arthrobacter CA-35-A-1766-15
(Arthrobacter CA-35-A-1766-15) Strain (Feikoken Bacteria No. 5528) (8) Arthrobacter CA-35-M-965-3
(Arthrobacter CA-35-M-965-3) Strain (Feikoken Bacteria No. 5529) (9) Arthrobacter CA-35-Y-37-12
(Arthrobacter CA-35-Y-37-12) strain (Feikoken Bacterial Serial No. 5530) Among these mutant strains, especially Arthrobacter CA-35-A589-29-32 strain and Arthrobacter CA- The strain 35-A589-47 has the ability to produce 3α,7α-dihydroxy-12-keto-5β-cholanic acid and/or its salts from cholate with high conversion and high selectivity. Among these mutant strains, Arthrobacter CA-35-A589-29-32 strain, Arthrobacter CA-35-A589-47 strain, Arthrobacter CA-35-A849 strain, and Arthrobacter CA
-35-A-1071-15 strain, Arthrobacter CA
-35-A-1448 strain, Arthrobacter CA-35
-A-1475 strain, Arthrobacter CA-35-A
-1766-15 strain and Arthrobacter CA-35-
M-965-3 strain was obtained by contacting Arthrobacter CA-35 strain with N-methyl-N'-nitro-N-nitrosoguanidine, and Arthrobacter CA-35-Y-37-12 strain was Each strain was obtained by irradiating Slobacter CA-35 strain with ultraviolet light. Specific methods for obtaining these mutant strains are shown in Reference Example 1 below. The following table lists the mycological properties of the parent Arthrobacter CA-35 strain and its mutant strains in comparison with the mycological properties of Arthrobacter simplex IAM1660 strain.

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[Table] Attitude towards sodium cholate The Arthrobacter strain CA-35 consumes about 20-500 g of sodium cholate as the sole carbon source.
It can grow in a medium containing a high concentration of 7α-hydroxy-3,12-diketo-5β-cholanic acid and/or its sodium salt, 3α,7α-dihydroxy-12 using sodium cholate as a substrate.
-keto-5β-cholanic acid and/or its sodium salt and 7α,12α-dihydroxy-3-keto-
5β-cholanic acid and/or its sodium salt can be produced as the main metabolite. On the other hand, the mutant strain of Arthrobacter CA-35 described above grows poorly or does not grow at all in a medium containing sodium cholate as the sole carbon source. Also, Arthrobacter simplex
The IAM1660 strain has difficulty growing in a medium containing 10 g/sodium cholate as the sole carbon source. Arthrobacter with the above mycological properties
The taxonomic position of the CA-35 strain was searched using Virgie's Manual of Determinative Bacteriology, 7th and 8th editions. The Arthrobacter strain CA-35 clearly differs from the Arthrobacter simplex IAM1660 strain, particularly in pigment production, sugar assimilation, and attitude toward sodium cholate. Arthrobacter
The CA-35 strain produces a yellow to cream-colored pigment, and pigment-producing bacteria belonging to the genus Arthrobacter include Arthrobacter oxydans, Arthrobacter auretuscens, and Arthrobacter ureafaciens. exist. 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. Furthermore, Arthrobacter ureafaciens is different from the Arthrobacter CA-35 strain because Gram staining is usually negative and it does not have nitrate reducing properties. Therefore,
Since the Arthrobacter strain CA-35 is different from any standard species belonging to the genus Arthrobacter, it was determined that it is a microorganism constituting a new species belonging to the genus Arthrobacter. Additionally, mutant strains are generally considered to belong to the same species as their parent strains, and the above-mentioned Arthrobacter CA
-35 mutant strains were determined to belong to the same new bacterial species as their parent strains. 3α,7α-dihydroxy- by the method of the present invention
Production of 12-keto-5β-cholanic acid and/or its salts is carried out by culturing the aforementioned mutant strain of Arthrobacter CA-35 in a medium containing cholate. Specifically, the cholate is a salt of an alkali metal such as sodium or potassium, or a salt of an alkaline earth metal such as calcium or magnesium, and preferably an alkali metal salt of cholic acid. Cholate may be prepared as is into an aqueous solution of a predetermined concentration and used as a substrate, or a predetermined amount of an alkali metal compound or alkaline earth metal compound that can form a salt with cholic acid is dissolved in water in advance. Thereafter, cholic acid may be added to the aqueous solution to adjust the concentration to a predetermined concentration, and the cholate salt may be used as a substrate. The concentration of cholate usually ranges from about 1 to 500 g/m, but the metabolically produced 3α,7α-dihydroxy-12-keto-5β-
From economic viewpoints such as the yield of colanic acid and/or its salt, culture conditions, and operability, the amount is preferably in the range of about 10 to 500 g/, and more preferably in the range of 20 to 300 g/. 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. The medium may be any medium as long as it contains a nutrient source that can be assimilated and utilized by the mutant strain of Arthrobacter CA-35. Carbon sources include bentoses such as arabinose; hexoses such as glucose, mannose, fructose, and galactose; disaccharides such as maltose; starch decomposition products, sugar alcohols, and polyhydric alcohols such as glycerin; or voribeptone, 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. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually performed at 25 to 35°C for 6 hours to 5 days. 3α and 7α accumulated in the culture medium in this way
- To separate and collect dihydroxy-12-keto-5β-cholanic acid and/or its salts, first remove the bacterial cells and other insoluble components in the culture solution by filtration or centrifugation, and then For example, add hydrochloric acid to the filtrate or supernatant. By acidifying the culture filtrate or supernatant in this manner, converted products of cholate, including 3α,7α-dihydroxy-12-keto-5β-cholanic acid, are precipitated. here,
3α,7α-dihydroxy-12-keto-5β precipitates
-7α as a converted product of cholate other than colanic acid
-hydroxy-3,12-diketo-5β-cholanic acid,
Examples include 7α,12α-dihydroxy-3-keto-5β-cholanic acid. In addition, at this time, unconverted cholate is precipitated as cholic acid. For example, by adding ethyl acetate to the solution from which the precipitate has been removed, the remaining converted product of the above-mentioned cholate and unconverted cholic acid and/or
or its salt, and then distilling off the ethyl acetate to almost completely collect the converted products of cholate, including 3α,7α-dihydroxy-12-keto-5β-cholanic acid, and remove the unconverted cholate. of cholic acid and/or its salts can be recovered. The thus obtained converted product of cholate and unconverted cholic acid and/or its salts are converted into, for example, methyl ester, and then adsorbed onto a silica gel column, and then chloroform, chloroform/ethanol, etc.
It is sequentially eluted with a 99/1 volume ratio mixture and a 97/3 volume ratio mixture of chloroform/ethanol. That is, 7α-hydroxy-3,12-diketo-5β-cholanic acid methyl ester was eluted with chloroform, and 7α-hydroxy-3,12-diketo-5β-cholanic acid methyl ester was eluted with chloroform/ethanol.
First, 7α,12α-dihydroxy-3-keto-5β-cholanic acid methyl ester is eluted using a mixed solution with a volume ratio of 99/1, and then the target methyl 3α,7α-dihydroxy-12-keto-5β-cholanic acid is eluted. Elute the ester. Furthermore, chloroform/
Cholic acid methyl ester is eluted with a mixture of ethanol in a 97/3 volume ratio. Obtained 3α,
7α-dihydroxy-12-keto-5β-cholanic acid methyl ester 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 Obtaining a mutant strain of Arthrobacter CA-35 Medium 1 (composition: sodium hydroxide 0.5%, cholic acid 5.0%, peptone 0.5%, yeast extract 0.5%, sodium chloride 0.5% and agar 1.5%) One platinum loop of Arthrobacter CA-35 strain grown on a slant was placed in medium 2 (composition: 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 solution (PH7.0), it was suspended in 25 ml of the same buffer solution. 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 sterile 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.
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, and 1.5% agar) and incubate at 30 °C.
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%),
Culture was carried out with shaking at 30°C for 3 days. The metabolites in each culture solution obtained were assayed by thin layer chromatography, and the target 3α,7α-dihydroxy-12
We found a strain that selectively capsulate -keto-5β-cholanic acid and/or its salts, and named it 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 these strains were isolated from Arthrobacter CA.
−35−A589 strain, Arthrobacter CA−35−
A849 strain and Arthrobacter CA-35-A-
It was named strain 1071. Arthrobacter obtained
Two superior bacterial strains were isolated from the colony of CA-35-A589 strain, and each strain was
They were named strain CA-35-A589-29-32 and Arthrobacter strain CA-35-A589-47. Furthermore, one excellent strain was isolated from the colony of Arthrobacter CA-35-A-1071 strain, and this strain was named Arthrobacter CA-35-A-1071-15 strain. Arthrobacter CA-35-A849 strain was used as the parent strain, and by treating it with NTG, 3α, 7α-
dihydroxy-12-keto-5β-cholanic acid and/or
We found several strains that selectively accumulate the salts thereof, and named the best strain among them the Arthrobacter CA-35-A-1448 strain. Arthrobacter CA-35-A-1448 strain was used as the parent strain, and by treating it with NTG,
We found multiple strains that selectively accumulate 7α-dihydroxy-12-keto-5β-cholanic acid and/or its salts, and selected the best strain among them as Arthrobacter CA-35-A-1475 strain. It was named. By using Arthrobacter CA-35-A-1071 strain as the parent strain and treating it with NTG, 3α,
We found multiple strains that selectively accumulate 7α-dihydroxy-12-keto-5β-cholanic acid and/or its salts, and selected the best strain among them as Arthrobacter CA-35-A-1766 strain. It was named.
One excellent strain was isolated from the resulting colony of Arthrobacter CA-35-A-1766 strain and named Arthrobacter CA-35-A-1766-15 strain. Arthrobacter CA-35-Y-37-12 strain was obtained by the following method. One platinum loop of Arthrobacter CA-35 strain grown on the slant of medium 1 was inoculated into 10 ml of medium 2 prepared in advance in a test tube (inner diameter 21 mm, length 200 mm), and incubated at 30°C for 20 hours. Cultured with shaking. This culture solution is aseptically separated into salt centers (5
℃, 10,000 rpm, 5 minutes) to collect the bacteria, and suspend them in 10 ml of sterile water. The obtained bacterial suspension was placed in a 7.5 cm inner diameter shear tray placed approximately 27 cm below the sterile box with a 15 W ultraviolet lamp (wavelength 2537 Å) suspended, and irradiated with ultraviolet light for 10 minutes. Mutation processing was performed using Furthermore, under this treatment condition, Arthrobacter
The mortality rate of the CA-35 strain was approximately 99.9%. 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 cultured using medium 4 to 7 in the same manner as in the NTG treatment described above to obtain 3α,7α-dihydroxy-12-keto-5β. - We have discovered a strain that selectively accumulates colanic acid and/or its salts, and we have identified this strain as Arthrobacter CA-35-Y.
The strain was named -37-12. Reference example 2 Arthrobacter strain CA-35
No. 5145) was cultured using the following method. cholic acid
Add distilled water to 100 g, ammonium nitrate 2.0 g, potassium dihydrogen phosphate 2.0 g, dipotassium hydrogen phosphate 5.0 g, magnesium sulfate heptahydrate 0.2 g, yeast extract 0.1 g, and sodium hydroxide 10 g to reduce the volume to 1.
This was used as a culture medium. 500% of this medium
Dispense into 10 bottles of 100ml each into Sakaguchi flasks and heat at 120°C.
Steam sterilization was performed for 15 minutes. Inoculum, which had been previously grown in the same medium as the above medium for 2 days in a test tube shaker, was added to 10 ml per 500 ml Sakaguchi flask, and cultured with shaking for 2 days. After culturing, these culture fluids are collected, and after removing the bacterial cells with a centrifuge, hydrochloric acid is added to the obtained culture supernatant to make the supernatant acidic with hydrochloric acid, resulting in converted and unconverted cholate. Acid precipitated. This precipitate was collected, the remaining solution was extracted with 1 part of ethyl acetate, the ethyl acetate was distilled off using a rotary evaporator, and the resulting residue was combined with the previous precipitate to convert 85 g of cholate. A mixture of cholic acid and unconverted cholic acid was obtained.
Take a small portion of this mixture, add methanol to it to make a 1% solution, and apply 10μ of this solution to a high-performance liquid chromatography system equipped with a Microbondapak C-18 column (manufactured by Waters, USA).
HLC-GPC-244 type). as mobile phase
A mixed solution of water/methanol in a 30/70 volume ratio adjusted to pH 2.5 was flowed at a sulfur rate of 1 ml/min, and detection was performed using the refractive index method, and the chromatogram shown in FIG. 1 was obtained. Peak A in this chromatogram,
Peak B, peak C, and peak D were each identified as 7α-hydroxy- by comparing them with the peaks of standard products.
3,12-diketo-5β-cholanic acid, 3α,7α-dihydroxy-12-keto-5β-cholanic acid, 7α,12α-
It was consistent with that of dihydroxy-3-keto-5β-cholanic acid and cholic acid. In addition, compounds corresponding to each portion of peak A, peak B, and peak C were fractionated and the structures of the compounds were confirmed by mass spectra, infrared absorption spectra, and NMR spectra.
-hydroxy-3,12-diketo-5β-cholanic acid,
3α,7α-dihydroxy-12-keto-5β-cholanic acid and 7α,12α-dihydroxy-3-keto-5β-
It was colanic acid. In addition, the infrared absorption spectra (A, B, and C) of the compound corresponding to each part of peak A, peak B, and peak C are for the compound after methyl esterification, and they are compared with the infrared absorption spectrum of the standard product. (A′, B′ and
Figures 2, 3 and 4 in contrast to C') respectively.
Show in diagram. Further, the compounds corresponding to peak A, peak B, and peak C in the fractionated chromatogram were methyl esterified, and then the melting points were measured, and the results were as shown in the following table.

[Table] The yield of the converted cholate and the remaining amount of unconverted cholic acid obtained from the area ratio of the chromatogram shown in FIG. 1 are calculated as shown in the following table.

[Table] Example 1 Arthrobacter strain CA-35-A589-29-32 (Feikoken Bacteria No. 5522) was cultured by the following method. Cholic acid 100g, ammonium nitrate 2.0g, potassium dihydrogen phosphate 2.0g, dipotassium hydrogen phosphate 5.0
g, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of yeast extract, and 10 g of sodium hydroxide were added with tap water to adjust the volume to 800 ml. This solution was heated to 120℃.
Steam sterilized for 15 minutes. Separately, dissolve 5g of glucose in 200ml of tap water, and add this solution to 110ml of tap water.
The mixture was steam sterilized at ℃ for 30 minutes, and after cooling, it was added to the above solution to adjust the volume to 1, and this was used as a culture medium.
Next, add 100 ml of this medium to a 500 ml Sakaguchi flask.
The mixture was aseptically dispensed into 10 tubes. Add 2 cultures of inoculum grown in the same medium as above in a test tube shaker for 14 hours per 500 ml Sakaguchi flask.
ml and cultured with shaking for 3 days. After culturing, collect these culture fluids and remove the bacterial cells using a centrifuge.
When hydrochloric acid was added to the obtained culture supernatant to make the supernatant acidic with hydrochloric acid, converted cholate and unconverted cholic acid were precipitated. This precipitate was collected, the remaining solution was extracted with 1 part of ethyl acetate, the ethyl acetate was distilled off using a rotary evaporator, and the resulting residue was combined with the previous precipitate to obtain 99.3 g of cholate. A mixture of converted and unconverted cholic acid was obtained. 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 equipped with a Microbondapak C-18 column (manufactured by Waters, USA).
HLC-GPC-244 type). as 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. 5 was obtained. By comparing peaks B and D in this chromatogram with the peaks of the standard product, 3α, 7α-dihydroxy-12-keto-5β-
It was consistent with that of colanic acid and cholic acid. Also,
A compound corresponding to the peak B portion was separated, and the structure of the compound was confirmed by its mass spectrum, infrared absorption spectrum, and NMR spectrum in the same manner as in the reference example, and it was found that the above 3α,7α-dihydroxy-12- It was keto-5β-cholanic acid. The yield of the obtained 3α,7α-dihydroxy-12-keto-5β-cholanic acid and the remaining amount of unconverted cholic acid were calculated from the area ratio of the chromatogram shown in FIG. 5 as shown in the following table.

[Table] Example 2 Using the Arthrobacter CA-35-A589-29-32 strain (same as above), instead of adding 2 ml of the inoculum per Sakaguchi flask to the culture medium in Example 1, 10 ml of the inoculum culture solution was added. Culture was carried out in the same manner as in Example 1, except for adding a bacterial suspension obtained by centrifuging the inoculum to 10 ml of sterile water. 99.5 g of a mixture of cholic acid was obtained. 99.5g of this mixture
was dissolved in 270 ml of methanol, 9 ml of concentrated hydrochloric acid was added thereto, and the mixture was boiled under reflux for 20 minutes to methyl esterify the converted cholate and unconverted cholic acid contained in the mixture. Column (diameter 70mm x 1500g of silica gel C-200
After adsorbing the above methyl ester, 3α, 7α-
102.0 g of dihydroxy-12-keto-5β-cholanic acid methyl ester was obtained. By hydrolyzing this, 98.5 g of 3α,7α-dihydroxy-12-keto-5β-cholanic acid was obtained. Example 3 Using Arthrobacter CA-35-A589-29-32 strain (same as above), changing the concentration of cholic acid in Example 1, and using sodium hydroxide 1/10 weight of cholic acid Example 1 except for adding
Culture was carried out in the same manner as above, and 3α,7α-dihydroxy-12-keto-
The yield of 5β-cholanic acid and the remaining amount of unconverted cholic acid were determined and shown in the following table.

[Table] Example 4 Arthrobacter CA-35- in Example 1
Arthrobacter CA instead of A589-29-32 strain
-35-A589-47 strain (Feikoken Bibori No. 5523) was used, the concentrations of cholic acid and sodium hydroxide were changed to 50 g/, and 5 g/, respectively, and the culture time was
By culturing and treating in the same manner as in Example 1 except that the time was shortened to 35 hours, 49.31 g of a mixture of cholate converted product and unconverted cholic acid was obtained.
The chromatogram shown in FIG. 6 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of standard products to identify 7α-hydroxy-3,12-diketo-5β-cholanic acid, 3α,7α-dihydroxy-12-keto, respectively. −5β
-Colanic acid, 7α,12α-dihydroxy-3-keto-5β-cholanic acid and cholic acid.
In addition, when compounds corresponding to each portion of peak A, peak B, peak C, and peak D were fractionated and examined using thin layer chromatography, peak B, peak C, and peak D were detected. The compound corresponding to the part was a single compound, but
The compound corresponding to the peak A portion was a mixture of 7α-hydroxy-3,12-diketo-5β-cholanic acid and other unidentified substances. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area ratio of the chromatogram shown in FIG. 6 as shown in the following table.

[Table] Example 5 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
-49.34 g of a mixture of cholate converted product and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 4, except that strain A849 (Feikoken Bibori No. 5524) was used. Example 1 from this mixture
The chromatogram shown in FIG. 7 was obtained by the same method. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of the standard product, and 7α-hydroxy-3, 7α-hydroxy-3, and
It was consistent with that of 12-diketo-5β-cholanic acid, 3α,7α-dihydroxy-12-keto-5β-cholanic acid, 7α,12α-dihydroxy-3-keto-5β-cholanic acid, and cholic acid. In addition, when compounds corresponding to each portion of peak A, peak B, peak C, and peak D were separated and examined using thin layer chromatography, peaks A,
The compounds corresponding to peak B, peak C, and peak D were all single compounds. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area of the chromatogram shown in FIG. 7 as shown in the following table.

[Table] Example 6 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
-A-1071-15 strain (Feikoken Bibori No. 5525) was cultured and treated in the same manner as in Example 4 to produce 49.31 g of a mixture of cholate converted product and unconverted cholic acid. Obtained. The chromatogram shown in FIG. 8 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of the standard product to identify 7α-hydroxy-3,12-diketo-5β-cholanic acid, 3α, and 7α, respectively.
-dihydroxy-12-keto-5β-cholanic acid, 7α,
It was consistent with that of 12α-dihydroxy-3-keto-5β-cholanic acid and cholic acid. Note that peak A,
Compounds corresponding to each portion of peak B, peak C, and peak D were separated and examined using thin layer chromatography, and it was found that the compounds corresponded to portions of peak B, peak C, and peak D. Although the compound was a single compound, the compound corresponding to the peak A portion was a mixture of 7α-hydroxy-3,12-diketo-5β-cholanic acid and other unidentified substances. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area ratio of the chromatogram shown in FIG. 8 as shown in the following table.

[Table] Example 7 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
49.30 g of a mixture of converted cholic acid and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 4, except for using the -A-1448 strain (Feikoken Bibori No. 5526). . The chromatogram shown in FIG. 9 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of standard products to identify 7α-hydroxy-3,12-diketo-5β-cholanic acid, 3α, 7α-
Dihydroxy-12-keto-5β-cholanic acid, 7α,
It was consistent with that of 12α-dihydroxy-3-keto-5β-cholanic acid and cholic acid. Note that peak A,
Compounds corresponding to each portion of peak B, peak C, and peak D were separated and examined using thin layer chromatography, and it was found that the compounds corresponded to portions of peak B, peak C, and peak D. Although the compound was a single compound, the compound corresponding to the peak A portion was a mixture of 7α-hydroxy-3,12-diketo-5β-cholanic acid and other unidentified substances. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area ratio of the chromatogram shown in FIG. 9 as shown in the following table.

[Table] Example 8 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
49.35 g of a mixture of cholate converted product and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 4 except for using the -A-1475 strain (Feikoken Bibori No. 5527). . The chromatogram shown in FIG. 10 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of standard products to identify 7α-hydroxy-3,12-diketo-5β-cholanic acid, 3α, 7α-
Dihydroxy-12-keto-5β-cholanic acid, 7α,
It was consistent with that of 12α-dihydroxy-3-keto-5β-cholanic acid and cholic acid. Note that peak A,
Compounds corresponding to each portion of peak B, peak C, and peak D were separated and examined using thin layer chromatography, and it was found that the portions of peak A, peak B, peak C, and peak D All the compounds corresponding to were single compounds. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area ratio of the chromatogram shown in FIG. 10 as shown in the following table.

[Table] Example 9 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
-A-1766-15 strain (Feikoken Bibori No. 5528) was cultured and treated in the same manner as in Example 4 to produce 49.27 g of a mixture of cholate converted product and unconverted cholic acid. Obtained. The chromatogram shown in FIG. 11 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, and peak D in this chromatogram were compared with the peak of the standard product, and each 7α-hydroxy-
It was consistent with that of 3,12-diketo-5β-cholanic acid, 3α,7α-dihydroxy-12-keto-5β-cholanic acid, and cholic acid. In addition, peak A, peak B
Compounds corresponding to each part of peak A, peak B, and peak D were fractionated and examined using thin layer chromatography. As a result, all of the compounds corresponding to parts of peak A, peak B, and peak D were isolated. It was one compound. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid are calculated from the area ratio of the chromatogram shown in FIG. 11 as shown in the following table.

[Table] Example 10 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
49.36 g of a mixture of cholate converted product and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 4, except that -M-965-3 strain (Feikoken Bibori No. 5529) was used. Obtained. The chromatogram shown in FIG. 12 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, and peak D in this chromatogram were compared with the peak of the standard product, and each 7α-hydroxy-
It was consistent with that of 3,12-diketo-5β-cholanic acid, 3α,7α-dihydroxy-12-keto-5β-cholanic acid, and cholic acid. In addition, peak A, peak B
Compounds corresponding to each part of peak B and peak D were fractionated and examined using thin layer chromatography, and it was found that the compounds corresponding to parts of peak B and peak D were a single compound. but,
The compound corresponding to the peak A portion was a mixture of 7α-hydroxy-3,12-diketo-5β-cholanic acid and other unidentified substances. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid were calculated from the area ratio of the chromatogram shown in FIG. 12 as shown in the following table.

[Table] Example 11 Arthrobacter CA-35- in Example 4
Arthrobacter CA-35 instead of A589-47 strain
49.33 g of a mixture of cholate-converted product and unconverted cholic acid was obtained by culturing and treating in the same manner as in Example 4, except that the -Y-37-12 strain (Feikoken Bibori No. 5530) was used. Obtained. The chromatogram shown in FIG. 13 was obtained from this mixture by the same method as in Example 1. Peak A, peak B, peak C, and peak D in this chromatogram were compared with the peaks of the standard product to identify 7α-hydroxy-3,12-diketo-5β-cholanic acid, 3α, and 7α, respectively.
-dihydroxy-12-keto-5β-cholanic acid, 7α,
It was consistent with that of 12α-dihydroxy-3-keto-5β-cholanic acid and cholic acid. Note that peak A,
Compounds corresponding to each portion of peak B, peak C, and peak D were separated and examined using thin layer chromatography, and it was found that the portions of peak A, peak B, peak C, and peak D All the compounds corresponding to were single compounds. The yield of the obtained cholate converted product and the remaining amount of unconverted cholic acid were calculated from the area ratio of the chromatogram shown in FIG. 13 as shown in the following table.

【table】 [Brief explanation of drawings]

Figure 1 shows liquid chromatograms of the converted cholic acid and unconverted cholic acid obtained in Reference Example 2, and Figures 2 to 4 show peak A, peak B, and peak C of the chromatogram in Figure 1, respectively. The infrared absorption spectra (A, B, and C) of methyl esterified compounds corresponding to each part of are shown in comparison with the infrared absorption spectra (A', B', and C') of standard products. . Figures 5-13 represent liquid chromatograms of cholate converted products and unconverted cholic acid obtained in Examples 1 and 4-11, respectively.

Claims (1)

[Claims] 1. 3α,7α-dihydroxy-12 using cholate obtained by subjecting Arthrobacter CA-35 strain (Feikoken Bibori No. 5145) to mutation treatment as a substrate.
- A mutant strain that produces keto-5β-cholanic acid and/or its salts is cultivated in a nutrient medium containing cholate to produce the above-mentioned colanic acid and/or its salts, and then collected. Characterized by 3α,
A method for producing 7α-dihydroxy-12-keto-5β-cholanic acid and/or a salt thereof. 2 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is A589-29-32 strain (Feikoken Bibori No. 5522). 3 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the A589-47 strain (Feikoken Bibori No. 5523). 4 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is A849 strain (Feikoken Bibori No. 5524). 5 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the A-1071-15 strain (Feikoken Bibori No. 5525). 6 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the A-1448 strain (Feikoken Bibori No. 5526). 7 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the A-1475 strain (Feikoken Bibori No. 5527). 8 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the A-1766-15 strain (Feikoken Bibori No. 5528). 9 The mutant strain is Arthrobacter CA-35-
The manufacturing method according to claim 1, which is the M-965-3 strain (Feikoken Bibori No. 5529). 10 The mutant strain is Arthrobacter CA-35
-Y-37-12 strain (Feikoken Bibori No. 5530), the manufacturing method according to claim 1.