CA1169795A - Microbial process for producing cholanic acid derivatives and microbes used therein - Google Patents

Abstract

Abstract of the disclosure:
The invention relates to a microbial process for producing a cholanic acid derivative of the formula:
wherein X is

Description

9 ~
MICROBIAL PROCESS FOR PRODUCING CHOLANIC
ACID DE~IVA'rIV~S AND MICROB~ USED THEREIN
. . ~

", The present invention relates to a microbial process for p~oducing a cholanic acid derivative of the formula:

(I~
~,~ ~ COOR

x ~ ~OH
H

wherein X is ~-OH or =O; and R is hydrogen, an alkali -metal or an alkaline earth metal; and to microbes used in the process.
The cholanic acid derivative of the formula (I) wherein X is ~-OH, namely, 3~,7~-dihydroxy-12-keto-H

5~-cholanic acid or a salt thereof is a useful intermediate ` for producing a gallstones solubilizer, chenodeoxycholic acid (CDCA). The cholanic acid derivative of the formula (I) wherein X is =O, namely, 7~-hydroxy-3,12-diketo-5~-cholanic acid or a salt thereof is a useful intermediate for producing deoxycholic acid, which is a useful starting material in the production of progesterone and adrenal corticosteroid derivatives.
It has hitherto been known that the cholanic acid derivatives of the formula (I) can be obtained by a mi-crobial process using cholic acid or a salt thereof as a substrate. For example, Hayakawa et al. disclose a - process for producing 3~,7~-dihydroxy-12-keto-5~-cholanic acid by using 5treptomy-c-e-s gelaticus 1164 strain [The Journal of Biochemistry (Japan), Vol. 44, No. 2, pages 109 to 113 (1957); and Proceedings of Japan ~cademy, Vol. 32, : pages 519 to 522 (1956)]. Hasegawa et al. disclose a process for producing 3~,7~-dihydroxy-12-keto-5 ~cholanic acid by using Aspergillis cinnamomeus HUT 2026 strain [Hiroshima Journal of Medical Science, Vol. 8, No. 3, pages 277 to 283 (1959)]. Kikuchi et al. also disclose a process for producing 3~,7 ~dihydroxy-12-keto-5 ~cholanic acid by using Staphylococcus epidermidis H-l strain [Journal of Biochemistry, Vol. 72, No. 1, pages 165 to 172 (1972)]. Further, Hayakawa et al. disclose a process for producing 7~-hydroxy-3,12-diketo~5~-cholanic acid by using Streptomyces gelaticus 1164 s-train [Proceedings of Japan Academy,. Vol. 32, pages 519 to 522 (1956)].
However, in these processes, the concentration of cholic acid as a substrate in a culture medium is low, e.g. not more than 10 g/l. According to the present inventors' experiments, it is necessary to use cholic acid in a low concentration in the production of the cholanic acid derivatives by the known microbial processes since the microbes to be used grow poorly when the concentration of cholic acid in the culture medium is as high as 20 g/l. Moreover, these known processes require too much time for the cultivation. Therefore, it has been desired to develop a process for producing the cholanic acid derivatives in a high yield within a short period of time.

`~ 795 It has now been found that certain microbes belong-ing to the genera Arthrobacter, Brevibacterium and Corynebacterium can grow in a medium containing cholic acid or its salt-as a substrate in a concentration varying widely and can produce the cholanic acid derivatives in a high yield within a short period of time.
One object of the present invention is to provide a microbial process for producing the cholanic acid deriva-tives of the formula (I) in a high yield within a short period of time. Another object of the present invention is to provide novel microbes which are capable of growing in a medium containing cholic acid or a salt thereof as a substrate.
According to the present invention, there is provided a microbial process for producing a cholanic acid deri-vative of'the formula (I) which comprises cultivating a microbe which is capable of growing in a medium containing cholic acid or a salt thereof as a substrate to produce the cholanic acid derivative, in a culture medium con-taining the substrate and collecting the resultingderivative, said microbe being selected from the genera Arthrobacter, Brevibacterium and Corynebacterium.
The cholanic acid derivatives of the formula (I) can be produced by the method of the invention, at least in the preferred forms, in a high yield withln a short period of time by cultivating a specific microbe in a culture medium containing cholic acid or a salt thereof as a substrate.

' `"` 11697g5 The microbes to be used in the present inv~ntion are those isolated ~rom soil and mutants thereof obtained by natural mutation, or produced by, for example, X-ray irradiation, ultraviolet irradiation, a mutating agent e.g. N-methyl~N'-nitro-N-nitroso guanidine, 4-nitro-quinoline-N-oxide, acriflavine or ethylmethane sulfonate or combinations thereof and the like.
Among the microbes being capable of producing the cholanic acid derivative of the formula (I) in a culture medium containing cholic acid or its salt obtained by the present inventor, the representatives are deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan (hereinafter, referred to as FERM) and with American Type Culture Collection, U.S.A.
(hereinafter, referred to as ATCC). They are Arthrobacter CA-35 strain (FERM-P No. 5145; ATCC No. 31651), Arthrobacter CA-35-A589-29-32 strain (FERM-P No. 5522;
. ATCC No. 31652), Arthrobacter CA-35-A589-47 strain (FERM-P
No. 5523; ATCC No. 31653), Arthrobacter CA-35-A849 strain 20 (FERM-P No. 5524; ATCC No. 31654), Arthrobacter CA-35-A-1071-15 strain (FERM-P No. 5525; ATCC No. 31655), Arthrobacter CA-35-A-1448 strain (FERM-P No. 5526; ATCC
No. 31656), Arthrobacter CA-35-A-1475 strain (FERM-P No.
5527; ATCC No. 31657), Arthrobacter CA-35-A-1766-15 strain (FERM-P No. 5528; ATCC No. 31658), Arthrobacter CA-35-M-965-3 strain (FERM-P No. 5529; ATCC No. 31659), Arthrobacter CA-35-Y-37-12 strain (FERM-P No. 5530; ATCC
No. 31660), 1 ~9~9~

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Brevibacterium CA-6 strain (~M-P No. 5144; ATCC No. 31661) and Corynebacterium CA-53 strain (F~RM-P No. 5532;
ATCC No. 31662). Arthrobacter CA-35, Brevibacterium CA-6 and Corynebacterium CA-53 stralns are wild t~pe strains and the other nine Arthrobacter strains are mutants of Arthrobacter CA-35 strain. Arthrobacter CA-35-Y-37-12 strain is produced by ultraviolet irradiation.
The other eight mutants of Arthrobacter CA-35 strain are obtained by treatment of the parent strain with N-( i~ 10 methyl-N'-nitro-N-nitrosoguanidine.
The morphological, cultural and physiological characteristics of these strains are set forth in Table 1.
For comparison, these characteristics of Arthrobacter simplex IAM 1660 strain which has a relation to Arthrobacter CA-35 strain are also shown in Table 1.

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Remarks~
1) The symbols used in Physiological character mean as follows:
The strain has the corresponding eharacter or produees the eorresponding product.
+ : It is difficult to determine whether the strain has the corresponding charaeter or produces the corresponding product or not.
- : The strain does not have the correspondins character or does not produce the correspond-ing product.

2) The symbols used in Productlon of acids and gases from earbohydrates mean as follows:
i) growth, acids and gases:
The strain was cultivated in Hugh and Leifson medium provided that eaeh of the earbohydrates 1 to 15 is substituted for the earbon source and the growth thereof and acids and gases produetion were observed.
~ : The strain grows or a acid or a gas is produced.
+ : It is diffieult to determine whether the strain grows or a aeid or a gas is produced or not.
- : The strain does not grow or a acid or gas is not produced.
`` ~ ii~ assimilation:
The culture medium comprising NH4N03 (2 g/l), r 1 ~ B 9 7 9 ~

KH2PO~ (2 g/1), X2HPO4 (5 g/1), MgSO4-7H2t.2 g/1), yeast extract (0.1 g/1) and one of the carbohydrates 1 to 15 (5 g/l) was put into a tube (diameter 21 mm), the strain was cultivated with shaking therein and its assimilation (growtn) was observed.
- . The strain does not gro~
+ : The strain grows slightly.
+ : The strain grows.
++ : The strain grows wel].
,.
+++ : The strain grows very well.
On tne basis of these morphological, cultural and physiological characteristics, the classification of the strains has been determined according to sergey's Mannual of Determinative Bacteriology 7th and 8th Editions.
It is determined that Arthrobacter CA-35 strain has a relation to Arthrobacter simplex in view of its microscopic observation such as form, gram stain and the like as well as its physiological character. However, ,6 20 Arthrobacter CA-35 strain is different from Arthrobacter simplex IAM 1660 strain in view of their pigment production, assimilation of carbohydrates and growth in a medium containing cholic acid. Arthrobacter CA-35 strain can grow in a medium containing sodium cholate as the sole carbon source in a high concentration such as about 20 to 500 g/1 to produce as main metabolic products 7C~-hydroxy-3,12-diketo-5~cholanic acid, 3~,7r~-dihydro~y-12-keto-5~-cholanic acid, 7~,12~ dihydro~y-3-keto-5~' . .

.

3 1~979 r c cholanic acid and/or sodium salts thereof whereas Arthrobacter simple~ IAM ].660 strain can hardly grow _ in a medium containing sodium cholate as the sole carbon source in a concentration of 10 g/l.
. Arthrobacter CA-35 strain produces yellow to - cream-colored pigments. On the other hand, as a microbe which belongs to the genus Arthrob~cter and has a pigment productivity, there e~ist Arthrobacter oxydans, Arthrobacter aurescens and Arthrobacter ureafaciens. However, .. ..
~ 10 Arthrobacter CA-35 strain is also different from these .~
mlcrobes since Arthrobacter oxYdans and Arthrobacter aurescens are usually gram-negative and hydrolyze starch and Arthrobacte~
ureafaclens is usually gram-negative and does not reduce a nitrate. Thus, it is believed that Arthrobacter CA-35 . strain constitutes a new species belonging to the genus Arthrobacter since it is different from strains of standard species belonging to the genus Arthrobacter.
Although some of the mutants of Arthrobacter ~ CA-35 strain are quite different from the parent strain ` 20 in view of flagella, it is determined that these mutants belong to Arthrobacter since, generally, a mutant is classified into the same species of its parent strain.
- It is determined that Brevibacterium CA-6 strain belongs to the genus Brevibacterium in view of its microscopic observation such as yram stain-and the like and its physiological character. However, srevibacterium CA~6 strain is somewhat different from other microbes belonging to the genus Brevibacterium since the other microbes 7 ~ ~

.` ;
have peritrichous flagella whereas Brevibacterium CA-6 strain has polar flagellum and so on.
Further, it is determined that Corynebacterium CA~53 strain has a close relation to Corvnebacterium equi.
The process of the presen-t invention is carried out by cultivating a microbe which is capable of growing in a medium containing cholic acid or its salt as a substrate, selected from the genera Arthrobacter, Brevibacterium and Corynehacterium, like the above in 10 a culture medium containing cholic acid or its salt as c a substrate.
In the present invention, cholic acid per se can be used as a substrate. There can be also used an alkali metal salt of cholic acid such as sodium cholate, potassium cholate or the like or an alkaline earth metal salt of cholic acid such as calcium cholate, magnesium cholate or the like, preferably an alkali metal salt.
When a cholate lS used, it is dissolved in a water to prepare an aqueous solution containing the cholate in a 20 predetermined concentrarion. Alternatively, a certain amount of an alkali metal compound or an alkaline earth metal compound which forms a salt with cholic acid may previously be dissolved in water and added thereto cholic acid to obtain an aqueous solution containing a cholate in a pre-determined concentration. Concentration of cholic acid or lts salt may be varied widely in a range of from about 1 to 500 g/l as cholic acid. In view of a yield of the desired cholanic acid der-ivatives of the formula (I), conditions for cultivation 979~
, and economic efficiency such as operability,wo~kability and the like, it is recommended that cholic acid or its salt ls used in a concentration of about 5 to 300 y/l, preEerably, about 10 to 200 g/l as cholic acid.
Cultiva-tion can be carried out according to a known method and, usually, a shaking or submerged culture using a liquid medium is employed.
As a medium, there can be used one containing nutrients which can be assimilated by a microbe to be 10 used. A medium can contain cholic acid or its salt as the sole carbon source or an additional carbon source such as a pentose (e.g. arabinose etc.), a hexose (e.g.
glucose, mannose, fructose, galactose etc.), a disaccharide (e.g. maltose etc.), a starch decomposition product (e.g. dextrin etc.), a sugar alcohol (e.g. sorbitol etc.), a polyvalent alcohol (e.g. glycerol etc.), a mixture thereof or the like and/or another nutrient such as a polypeptone, a peptone, meat extract, malt e~tract, corn steep liquor, yeast e~tract, an amino acid, a 20 mixture thereof or the like. Usually, an additional carbon source and/or ano-ther nutrient can be added to a medium in a concentration of about 0.1 to 10 g/l.
As a nitrogen source, there can be used, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, a mixture thereo~
and the like. Usually, a nitrogen source can be added to a medium in a concentration of about 0.5 to 5 g/l.
Further, as inorganic salts, dipotassium hydrogen phosphate, -~ 1~979~

po-tassium dihydrogen phosphate, magnesium sulfate and the like can be added to a medium, usually, in a concentraiton of about 0.1 to 10 g/l. Cultivation can be carried out .in a shaking or submerged culture at 25 to 35~C for 6 hours to 5 days.
According to the process of the present invention, cholic acid or its salt used as a substrate is converted into the cholanic acid derivatives of the formula (I) and a small amounL of 7c,r,12C'-dihydroxy-3-keto-5r'-cholanic acid or its salt when Arthrobacter CA-35 strain is used.
It has been found that the cholanic acid derivative of the ; formula (I) wherein X is ~-OH, namely, 3~,7c~-dihydroxy-H

12-keto-5,~-cholanic acid or its salt is predominantly produced when the above mutants oflArthrobacter CA-35 are used. Therefore, when 3r~,7r~-dihydroxy-12-keto-5a-cholanic acid or its salt is desired, it is preferable to use these mutants, especially, Arthrobacter CA-35-A589-29-32 strain (FERM-P No. 5522; ATCC No. 31652), Arthrobacter CA-35-A589-47 strain (FERM-P No. 5523;
20 ATCC No. 31653), and Arthrobacter CA-35-A-1766-15 strain (FERM-P No. 5528; ATCC No. 31658). Particulary, Arthrobacter CA-35-A589-29-32 strain and Arthrobacter CA-35-A589-47 strain are more preferable. When Brevibacterium CA-6 strain is used, cholic acid or its salt is converted into the cholanic acid derivatives of the formula (I) and a small amount of 7cc-hydroxy-3~l2-di~eto-~ -cholenic acid or its salt. When Corynebacterium CA-53 strain is used, cholic 7 9 ~
r -acid or its salt is converted into the cholanic acid derivatives of the formula (I) and 7~-hydroxy-3,12-diketo-~4-cholenic acid or its salt.
After completion of cul-tivation, products accumulated in the medium are separated from the medium and purified. Firstly, insoluble materials in the medium such as microbial cells and the like are removed from the medium by a known method such as filtration, centrifu-gation and the like. Then, a filtrate or supernatant is acidified by addition of an acid such as hydrochloric acid to precipitate products therein. At the same time, cholic acid or its salt used as a substrate which remains without being converted is also precipitated as cholic acid.
After separation of the resulting precipitate, the filtrate or supernatant is further extracted with an inert organic solvent such as ethyl acetate, or the like and the solvent is distilled off from the extract to obtain a resldue.
Thereby, remainlng products and the substrate are almost completely recovered. The resulting residue is combined with the above obtained precipitate.
A mixture of products and cholic acid thus obtained is subjected to chromatography to isolate the desired product. For example, products obtained by using Arthrobacter CA-35 strain or a mutant thereof can be isolated as follows: Products and cholic acid are converted into methyl esters thereof and the obtained methyl esters are subjected to chromatography on a silica gel column by successively eluting with chloroform, chloroform-7 ~ 5 ethanol (99 : 1, v/v)-and then chloroform-ethanol (97 : 3, v/v). The aesired 7~-hydroxy-3,12-diketo-51~-cholanic aci.d methyl ester is eluted with chloroform. By elution with chloroform-ethanol (99 : 1, v/v), firstly, one of the by-products, 7~,12~-dihydroxy-3-keto-5/~-cholanic acid methyl ester and then, the desired 3~,7~~dihydroxy-12-keto-5j~-cholanic acid methyl ester are eluted. Methyl cholate is eluted with chloroform-ethanol (97 : 3, v/v).
These methyl esters can be converted into the corresponding - 10 acids by hydrolysis according to a standard method.
Alternatively, when srevibacterium CA-6 strain or CorYnebacterium CA-53 strain is used, products can be -isolated, for example,as follows: A mixture of products and cholic acid are directly subjected to chromatography on a silica gel by succesively eluting with chloroform-ethanol (98 : 2, v/v) and then chloroform-e-thanol (97 : 3, v/v). The desired 7~-hydroxy-3,12-diketo-5r-cholanic acid is eluted with chloroform-ethanol (98 : 2, v~v).
By elution with chloroform-ethanol (97 : 3, v/v), firstly, a by-product, 7~-hydroxy-3,12-diketo-~4-cholenic acid is eluted and then, the desired 3~,7C~-dihydroxy-12-keto-5~-cholanic acid is eluted.
3C~,7~-dihydroxy-12-keto-5~-cholanic acid or its salt obtained by the present invention can be readily reduced according to Huang Minlon reduction me-thod to give a useful gallstones solubilizer, chenodeoxycholic acid (CDCA).
7~-hydroxy-3,12-diketo-5r-cholanic acid or its salt . .

- . :l16~37 : '

4~
can be readily converted into deoxycholic acid, which is a useful starting material in the production of progesterone and adrenal corticosteroid derivatives, by dehydration, followed by hydrogenation according to a standard method.
The following examples and reference e~amples ~urther illustrate the present invention in detail but are not to be construed to limit the scope thereof.
reparation of mutants One loopful of Arthrobacter CA-35 strain c 10 (FERM-P No. 5145; ATCC No. 31651) cultivated on a slant medium (medium ~; NaOH 0.5 %, cholic acid 5.0 %, peptone 0.5 %r yeast extract 0.5 %, NaC1 0.5 % and agar 1.5 %) was inoculated into a medium (10 ml, medium B: NaOH 1 ~, cholic acid 10 %, NH4NO3 0.2 %, KH2PO4 0-2 %, K2HPO4 0.5 %, ~gSO4 7H2O 0.02 % and yeast extract 0.01 %) in a test tube (200 mm x 21 mm in diameter), and incubated with shaking at 30C for 24 hours. The resulting culture (0.3 ml) was added to a medium (10 ml, medium C: NaOH
0.05 %, cholic acid 0.5 %, glucose 0.5 %, NH4NO3 0.2 %, 2 4 , 2HPO4 0.5 %r MgSO4 7H2O 0.02 % 2nd yeast extract 0.01 %) in a test tube (200 mm x 21 mm in diameter) and incubated with shaking at 30C for 15 to 16 hours.
The microbial cells in a log phase were harvested by a membrane filter (pore size 0.45/~) under aseptic conditions, washed with 0.1 M phosphate bu~fer (pH 7.0, 20 ml) and suspended in the same buffer (25 ml).
A treatment of mutation was carried out by adding N-methyl-N'-nitro-N-nitrosoguanidine in a final .. . .

concentration of 50 ~g/ml to the above cell suspension . ~
(4 ml) in test tube (200 mm x 21 mm in diameter) and incubating the mixture with shaking at 30C for 45 minutes. Under this condition, a lethal rate of Arthrobacter CA-35 strain was about 80 %.
The cells thus treated were harvested by a membrane filter (pore size, 0.45J~) under aseptic conditions, washed with 0.1 M phosphate buffer (pH 7.0, 20 ml) and suspended in the same buffer (25 ml). The resulting cell c 10 suspension was diluted with a sterilized normal saline solution and spreaded on agar plates (medium D: NaOH 0.1 %, cholic acid 1.0 %, NH4NO3 0.2 %, XH2PO4 0.2 %, K2HPO4 0-5 g6 MgSO4~7H2O 0.02 %, yeast extract 0.01 % and agar 1.5 %) so as to form 300 to 500 colonies per one plate. The plates were incubated at 30C for 4 days. Each pin point colony thus formed was isolated and incubated on a plate (medium E: peptone 0.5 %~ yeast extract 0.5 %, NaCl 0.5 %
and agar 1.5 %) at 30C for 24 hours. Each colony formed on the plate of medium E was replicated on a plate of 20 medium D and incubated at 30~C for 20 hours.
Each colony on the above plate of medium E, of which replicated colony did not grow on the plate of medium D, was incubated on a slant medium (medium F: NaOH
0.1 %, cholic acid 1.0 %, peptone 0.5 %, yeast extract 0.5 %, NaCl 0.5 % and agar 1.5 %) at 30C for 24 hours.
One loopful of this slant culture was inoculated into a medium (10 ml, medium G: NaOH 0.5 %, cholic acid 5.0 %, glucose 0-5 %, NH4NO3 0.2 %, KH2PO4 0-2 ~, K2HPO4 0-5 %, ' ' - .

697g5 ,: ' MgSO4-7H2O 0.02 % and yeast ex-tract 0.01 %) in a test tube (200 mm x 21 mm in diameter) and incuba-ted with shaking at 30C for 3 days~
Upon examining products accumulated ln each medium G by thin layer chromatography, a microbe which selectively produced 3~,7C~-dihydroxy-12 keto-5~-cholanic acid or its salt was found and named Arthrobacter CA-35-M-965-3 strain.
The above mentioned procedure is hereafter { 10 referred to as "procedure M". According to the procedure M, a treatment of mutation was earried out by using Arthrobacter CA-35-M-965-3 strain as a parent strain. As the result, three strains which seleetively produeed 3cc, 7~-dihydroxy-12-keto-5~-cholanic acid or its salt were found and named Arthrobacter CA-35-A589, Arthrobacter CA-35-A849 and Arthrobacter CA-35-A-1071 strains, respectively.
From the colony of Arthrobacter CA-35-A589 strain, two superior strains were isolated and these strains were named Arthrobacter CA-35-A589-29-32 strain and Arthrobacter CA-35-( 20 A589-47 strain, respectively. One superior strain was also isolated from the colony of Arthrobaeter CA-35-A-1071 strain and named Arthrobacter CA-35-A-1071-15 strain.
Further one superior strain was produced by using Arthrobacter CA-35-A849 strain as a parent strain according to the procedure M and named Arthrobacter CA-35-A-1448 strain. Arthrobacter CA-35-A-1475 strain was produced by using Arthrobacter CA-35-A-1448 strain as a parent strain according to the procedure M.

~ 169795 , t , .
Arthrobacter CA-35-A-1766 strain was produeed by using r' Arthrobaeter CA-35-A-1071 strain as a parent strain according to the procedure ~, and Arthrobaeter CA-35-A-1766-15 strain was isolated from the colony of the resulting Arthrobacter CA-35-A-1766 strain.
Arthrobacter CA-35-Y-37-12 strain was produced according to the following procedure.
One loopful of Arthrobacter CA-35 strain (FE~-P
No. 5145; ATCC No. 31651) eultivated on a slant of medium c 10 A was inoculated into medium B (10 ml) in a test tube (200 mm Y 21 mm in diameter) and incubated with shaking at 30C for 20 hours. Microbial cells were harvested by eentrifuging (10,000 r.p.m.) at 5~C for 5 minutes under aseptic conditions. The cells were suspended in a sterili2ed water (10 ml). The suspension was put lnto a Petri dish (diameter 7.5 cm) placed at about 27 cm distant under a 15 W ultraviolet lamp (2537 A) in a germ-free box and irradiated with ultraviolet rays for 10 minutes. Under this condition, a lethal rate of 20 Arthrobacter CA-35 strain was about 99.9 %.
~fter irradiation, the microbial cells were harvested by a membrane filter, washed with 0.1 M phosphate buffer and suspended in the same buffer. The resulting suspension was diluted with a normal saline solution and the above eultivation using media D to G was repeated to obtain a superior strain which selectively produeed 3~,7~-dihydroxy-12-keto-5~-cholanic acid or its salt.

.

t Example 1 ~
~rthrobacter CA-35 strain (F'ERM-p No. 5145;
ATCC No. 31651) was cultivated as follows:
Composition of culture medium Cholic acid 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 10 Yeast extract 0.1 g Sodium hydroxide 10 g Distilled water to 1 liter The above ingredients were admixed to obtain a culture m,edium (1 liter). Each 100 ml portion of the culture medium was distributed to ten Sakaguchi flasks (volume 500 ml) and autoclaved at 120C for 15 minutes.
To each flask, there was added 10 ml portion of seed ~ culture which was obtained by previously cultivating ; the strain in the same medium with shaking at 30C for 2 days. Each flask was incubated on a shaker at 30C
for 2 days.
After completion of cultivation, the culture broth was combined and centrifuged to remove microbial cells. The resulting supernatant was acidified by addition of lN aqueous hydrochloric acid (600 ml) to form a precipitate. The precipitate was separated and the remaining solution was extrac-ted with ethyl acetate (1 liter). Ethyl acetate was distilled off from the '; .

1 16979~
. .

r extract by a rotary evaporator and the residue was combined - with the above precipitate to obtain a mixture of cholanic acid derivatives and cholic acid (85 g).
A small portion of the mixture was dissolved in methanol in a concentration of 1 %. The solution (10 ~1) was injected to a high-speed liquid chromatography apparatus equipped with a ~sondapak C-18 column (HLC-GPC-244 type produced by Waters in U.S.A.).
The column was eluted with water-methanol (30 :
70, v/v, pH 2.5) at the rate of 1 ml/min. and refractive index of the eluate was measured.
The accompanying Figure 1 shows the resulting chromatogram. The peaks A, B, C and D in Figure 1 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5k~cholanic acid, 3~,7~-dihydroxy-12-keto-5,~-cholanic acid, 7~,12~-dihydroxy 3-keto-5~-cholanic acid and cholic acid, respectively.
~urther, when the compounds in the fractions corresponding to the peaks A, B and C were isolated and C 20 their chemical constitutions were determined based on the mass spectra, IR spectra and NMR spectra thereof, these spectra showed that these compounds were 7~-hydroxy-: 3,12-diketo-5~ cholanic acid, 3~,7,~-dihydroxy-12-keto-

5,~,-cholanic acid and 7~,12~'-dihydroxy-3-keto-5~-cholanic `~ acid, respectively. The accompanying Figures 2, 3 and 4 show the IR spectra (A, B and C) of methyl esters of the compounds corresponding to the peaks A, B and C in comparison with those of the reference standards (A', B' and C'), , , ~ 79~

respectively.
The melt.ing points of the compounds and methyl esters thereof corresponding to the peaks A, B and C
are shown in 'rable 2.
Table 2 Compounds Melting points of Melting poins of the methyl esters the free acids ( o C ) ( o C ) Found Literature Found Literature ( 10 Peak A 150 - 152 152 - 154 Peak B 154 - 156 156 - 157 219 - 220 221 - 222 Peak C 168 - 169 169 - 172 185 - 186 185 - 188 The yield of the products and -the amount of cholic acid which remained without being converted were calculated based on the area ratio of the chromatogram of Figure 1. The results are shown in Table 3.
` Table 3 ( Compounds Area ratio Conversion Yield or rate (%) amount (g) .
7~-hydroxy-3,12-diketo-5~-cholanic 28.54 28.54 24.3 acid (peak A) 3~,7~-dihydroxy-12-keto-5~-cholanic 63.06 63.06 53.6 acid (peak B) 7~,12~-dihydroxy-3-keto-5~'-cholanic 1.54 1.54 1.30 acid (peak C) cholic acid 6.86 6.86 5.80 (peak D) ; - - ------- .. _ _ .

'.
.

.

:~ 1 6979~

....
E~ample 2 The same procedure as described in Example 1 was repeated except that the glucose (5.0 g/l) was further added to the medium to obtain a mixture of products and cholic acid which remained without beiny conver-ted (91.0 g).
The mixture (91.0 g) was dissolved in methanol ~270 ml) and added thereto conc. hydrochloric acid (9 ml). The resulting solution was heated under reflux for 20 minutes to convert the products and cholic acid into methyl esters thereof.
Silica gel C-200 (1500 g) was packed in a column (1200 mm x 70 mm in diameter) and the above methyl esters were adsorbed thereto. The column was eluted with chloroform to obtain~,7~-hydroxy-3,12-diketo-5~-cholanic acid methyl ester (25.1 g). Then, the column was eluted with chloro-form-ethanol (99 : 1, v/v) to obtain, firstly, 7~,12~Y-dihydroxy-3-keto-5~-cholanic acid methyl ester (1.40 g) and then 3~,7.C-dihydroxy-12-keto-5~-cholanic acid methyl ester (56.4 g).
2Q These methyl esters were hydrolized to obtain 7~-hydroxy-3,12-diketo-5~-cholanic acid (24. 8 g), 7~,12~-dihydroxy-3-keto-5,~-cholanic acid (1.38 g) and 3~X,7~-dihydroxy-12-keto-5~-cholanic acid (56.0 g).
Example 3 The same procedure as described in Example 1 was repeated except that a concentration of cholic acid was varied and sodium hydroxide was added to a medium in an amount of 10 % by weigh-t based on cholic acid 979~

.

used. The yield of products and the amount oE cholic acid which remained without being converted at each concent~ation of the substrate (cholic acid) are shown in Table 4.
Table 4 Compounds Yields Substrate concentration (g/l) 3~,7~-dihydroxy-12-keto-5~-cholanic 27.1 g 108.3 g 47.4 g acid 7~-hvdroxy-3,12-diketo-5~-cholanic 12.3 g 49.0 g 21.4 g acid 7~,12~-dihydroxy-3-keto-5~cholanic0.7 g 2.65 g 1.20 g acid cholic acidamount 20.1 g 230 g Example 4 The same procedure as described in Example C 2 was repeated except that each flask was incubated on a shaker at 30C for 24 hours to obtain a mixture of products and cholic acid which remained without being converted (98.0 g). The mix-ture was subjected to high-speed liquid chromatography under the same conditions as in Example 1 to determine the composition thereof.
The results are shown in Table 5.

.

7 g ~

Table 5 , Compounds ratio 3C~,7~'-dihydroxy-12-keto-5~-cholanic acid 67.7 7~'h~droxy-3,12-diketo-S,~-cholanic acid 8.3 7^c, 12~X-dihydroxy-3-keto-5~ cholanic acid 4.0 cholic acid 20.0 Example 5 The same procedure as described in Example 2 was repeated except that 20 ml portion of seed culture obtained by cultivating at 30C for 24 hours was added to each flask to ob~ain a mixture of products and cholic acid which remained without being converted (117 .0 g).
~ liquid - The mixture was subjected to high-speed/chroma-tography under the same conditions as in Example 1 to determin the composition thereof. The results are shown in Table

6.
Table 6 C ~ .
Compounds ratio 3~,7~-dihydroxy-12-keto-5,~-cholanic acid 63.3

7~-hydroxy-3,12-diketo-5 ~cholanic acid 23.5 7~,12~~dihydroxy-3-keto-5k~cholanic aci.d 2.0 cholic acid 11.2 -Example 6 Arthrobacter CA-35-A589-29-32 strain (FER~I-P

No. 5521; ATCC No. 31652) was cultivated as follows:

.

Composition of culture medium Cholic acid 100 g Ammonium nitrate 2.0 g Potassium dihydrogen phosphate 2.0 g Dipotassium hydrogen phosphate 5.0 g Magnesium sulEate heptahydrate 0.2 g Yeast extract 0.1 g Sodium hydroxide 10 g Glucose 5.0 g 10 Tap water to 1 liter C The above ingredients other than glucose were admixed in tap water to adjust the volume to 800 ml and autoclaved at 120C for 15 minutes. Glucose was dissolved in tap water (200 ml) and autoclaved at 110C for 30 minutes. After cooling, both solutions were combined to obtain a culture medium (1 liter). Each 100 ml portion of the culture medium was distributed to ten Sakaguchi flasks (volume 500 ml) under aseptic conditions.
To each flask, there was added 2 ml portion of seed ( 20 culture which was obtained by previously cultivating the strain in the same medium with shaking at 30C for 14 hours. Each flask was incubated on a shaker at 30C
for 3 days.
~ftèr completion of cultivation~ the culture broth was combined and centrifuged to remove microbial cells. The resulting superna-tant was acidified by addition of lN aqueous hydrochloric acid (600 ml) to form a precipitate. The precipitate was separated and the t residual solution was extracted with ethyl ace~ate (1 liter). Ethyl acetate was distilled off from the extract by a rotary evaporat~r and the residue was combined with the above precipitate to obtain a mix-ture of cholanic acid derivatives and cholic acid (99.3 g).
A small portion o~ the mixture was dissolved in methanol in a concentration of 2 ~. The solution (10 /bl) was injected to a high-speed liquid chromatography apparatus equipped with a/~Bondapak C-18 column (HLC-10 GPC-244 type).
C The column was eluted with water~methanol (30 : 70, v/v, pH 4.0) at the rate of 1 ml/min. and refractive index of the elute was measured.
The accompanying Figure S shows the resulting chromatogram. The peaks B and D in Figure 5 are correspond-ing to those of the reference standards of 3~,7c~-dihydroxy-; 12-keto-5~-cholanic acid and cholic acid, respectively.
When the compound in the fraction corresponding to the peak B was isolated and its chemical constitution C 20 was determined based on the mass spectrum, IR spectrum and NMR spectrum, these spectra showed that the compound was 3~,7~-dihydroxy-12-keto-5~-cholanic acid.
The yield of the products and the amount of cholic acid which remained without being converted were calculated based on the area ratio of the chromatogram of `\ Figure 5. The results are shown in Ta~le 7.

~ .

11~;97~5 ,. . .

.
Table 7 - *
Compounds Conversion ~ield or rate (~) amount (y) 3~,7Q'-dihydroxy-12-keto-5~~ 98.85 98.16 eholanie aeid (peak B) Other derivatives produeed from cholie acid 0.59 0.59 eholie aeid (peak D) 0.56 0.55 .. _ _ _ _ _ _, _ _ _ _ _ . . . . ..
- * : conversion rate was ealculated based on the area ratio.

C 10 Example 7 The same proeedure as deseribed in Example 6 was repeated exee~ that a mierobial eells suspension ` whieh was prepared by separating mierobial cells from the seed culture ~10 ml) by eentrifugation and suspending them in sterilized water (10 ml) was added to each flask in stead of 2 ml of seed culture to obtain a mixture of produets and remaining eholic acid (99.5 g). The mixture (g9.5 g) was dissolved in methanol (270 ml) and added thereto c eone. hydrochlorie acid (9 ml). The resulting solution was heated under reflex for 20 minutes to convert the products and eholie aeid into methyl esters thereof.
Silica gel C-200 (1500 g) was pac~ed in a eolumn (1200 mm x 70 mm in diameter) and the above methyl esters were adsorbed thereto. The column was eluted with ehloroform-ethanol (99 : 1, v/v) to obtain 3~,7~-dihydro~y-12-keto-5p~cholanie acid methyl ester (102.0 g) which was hydrolyzed to obtain 3~,7~-dihydro~y-12-keto-5.~-eholanie acid (98.5 g).

~ ,.. .

~ 16~7~5 . .
~xample 8 The same procedure as described in ~ample 6 was repeated except that the concen-tration of cholic acid was varied, sodium hydroxide ~as added to a medium in an : amount of 10 % by weic3ht based on cholic acid used and 1~ aqueous hydrochloric acid was added in an amount of 60 ml per lg of sodium hydroxide used to form a precipitate.
The conversion rate or yeild of the product and the amount of remaining cholic acid at each concentration of the c 10 substrate (cholie aeid) are shown in Table 8.
. . .

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7 ~ ~
, ~ ~ , ,, o a~ o . ~ ~ ~

O ~ 3 . ,_1 CO O' . b~ ~
O ~D ~
~n a~ o . ~ ~ O

~-1 ~ O ~r-l ~t 0 ~1 h ~ G~ o ~J o ~ ~
h U o~Oo\
s:~ O CO o ,0 * )' ~ ~ o\ . o~O
O U~ ~ . .
Q ~ o . . ~ ~ $
d . ~ ~:
~ ,1 03 X ~: h Q O ~
~ ~S ~0 ~ 0 E~ ~ ~ ,1 Ul ,~ U U
~ ' Q~ ..
~ I U) O
O
O
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7 ~ 5 .

Example 9 The same procedure as described in Example 6 was repeated excep-t that Arthrobacter CA-35-A589-47 strain (FE~l-P No. 5523; ATCC Mo. 31653) was used in stead of Arthrobacter CA-35-AS89-29-32 strain, the concentrations of cholic acid and sodium hydroxide in the medium were changed to 50 g/l and 5 g/l, respectively and each flask was incubated at 30C for 35 hours to obtain a mixture of products and remaining cholic acid (49.31 g).
The mixture was subjected to same high-speed li~uid chromatography under the same conditions as in ~xample 6.
The accompanying Figure 6 shows the resulting chromatogram. The peaks A, B, C and D in Figure 6 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5,~-cholanic acid, 3~,7~-dihydroxy-12-keto-5~-cholanic acid, 7~,12~-dihydroxv-3-keto-~-c cholanic acid and cholic acid, respectively.
When the fractions corresponding to the peaks A, B, C and D were separated and subjected to thin layer chromatography, each fraction corresponding to the peak B, C or D contained a single compound. The fraction corresponding to -the peak A was a mixture of 7~-hydroxy-3,12-diketo-5~-cholanic acid and another unidentified material.
The yield of the products and the amount of cholic acid which remained without being converted ~' .

; ~ ~69'~
... .
. .
were calculated based on the area ratio of the chromatogram of Figure 6. The results are shown in Table 9.
Table 9 ;.
Compounds ConversionYield or rate (%)amount (g) _ 7~-hydroxy-3,12-diketo-5~-cholanic acid and unidentified 0.80 0.39 material (peak A) 3C~,7~'-dihydroxy-12-keto-5p~cholanic acid 98.01 48.33 (peak B) 7~,12~-dihydroxy-3-keto-5/--cholanic acid 0.25 0.13 (peak C) other 0.24 0.12 ` derivatives cholic acid (peak D) 0.70 0.35 Example 10 The same procedure as described in Example 9 was repeated except that Arthrobacter CA-35-A8g9 strain (FERM-P No. 5524; ATCC No. 31654) was used in stead of C ~rthrobacter CA-35-A589-47 strain to obtain a mixture of products and remaining cholic acid (49.34 g).
The mixture was subjected to high-speed liquid chromatography under the same conditions as in Example 6.
The accompanying Figure 7 shows the resulting chromatogram. The peaks A, B, C and D in Figure 7 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5~-cholanic acid, 3~,7~-dihydroxy-30 12-keto-5~-cholanic acid, 7C~,12C~-dihydroxy-3-keto-5~-cholanic acid and cholic acid, respectively.
.

t 16~79~

, When the fractions corresponding to the peaks A, ~3, C and D were separated and subjected to thin layer chromatography~ each ~raction contained a single compound.
The ~ield of the products and the amount of cholic acid which remained without being converted were calculated based on the area ratio of the chromatogram of Figure 7. The results are shown in Table 10.
Table 10 Compounds ConversionYield of C 10 rate (%)amount (g) 7C~-hydroxy-3~l2-diketo-5k~cholanic acid 3.2 1.58 (peak A) 3~,7~-dihydroxy-12-keto-5 -cholanic acid 83.5 41.20 (peak B) 7~,12~dihydroxy-3-keto-5~-cholanic acid 7.9 3.90 (peak C) derivatives 2.8 1.38 cholic acid (peak D) 2.6 1.28 C
Example 11 The same procedure as described in Example 9 was repeated except that Arthrobacter CA-35-A-1071-15 strain (FERM-~ No. 5525; ATCC No. 31655) was used in stead of Arthrobacter CA-35-A589-47 strain to obtain a mixture of products and remaining cholic acid (49.31 g).

The mixture was subjected to high-speed liquid 3Q chromatography under the same conditions as in Example 6.
The accompanying Figure 8 shows the resulting .
, 9 ~ 9 5 .~ .

chromatogram. The peaks A, B, C and D in Figure 8 correspond to -those of the reference standards ol 7~-hydroxy-3,12-diketo-S/"-cholanic acid, 3~,7~-dih~droxy-12-keto-Sj~-cholanic acid, 7c~,12c~-dihydroxy-3-keto~S/~-cholanic acid and cholic acid, respectively.
When the fractions corresponding to the peaks A, B, C and D were separated and subjected to -thin layer chromatography, each fraction correspondiny to the peak B, C or D contained a single compound. The fraction C 10 corresponding to the peak A was a mixture of 7~-hydroxy~
3,12-diketo-S~-cholanic acid and another unidentified material.
The yield of the products and -the amount of remaining eholic acid were calculated based on the area ratio of the chromatogram of Figure 8. The results are shown in Table 11.
Table 11 : CompoundsConversion Yield or C rate (O) amount (g) 7C~-hydroxy-3,12-diketo-5~-cholanic acid and un-5 2 2 56 identified material (peak A) 3C~,7~-dihydroxy-12-keto-5~-cholanic acid 93.9 46.30 (peak B) 7C~,12C~-dihydroxy-3-keto-5,¢cholanic acid 0.2 0.10 (peak C) other derivatives 0.2 0.10 cholic acid 0.5 0.25 1 ~B979~
, Example 12 The same procedure as described in Example 9 was repeated e~cept that ~rthrobacter C~-35-A-1448 strain (FE~l-P No. 5526; ATCC No. 31656) was used in stead of Arthrobacter CA-35-A589-47 strain to obtain a mixture of products and remaining cholic acid - (49.30 g).
The mixture was subjected to high-speed li~uld chromatography under the same conditions as in Example 6.
c 10 The accompanying Figure 9 shows the resulting chromatogram. The peaks A, B, C and D in Figure 9 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-S~-cholanic acid, 3~,7~-dihydroxy-12-keto-5~ cholanic acid, 7~,12~-dihydroxy-3-keto-5,~-cholanic acid and cholic acid, respectively.
When the fractions corresponding to the peaks A, B, C and D were separated and subjected to thin layer chromatography, each fraction corresponding to the peak B, C or D contained a single compound. The fraction corresponding to the peak A was a mixture of 7~-hydroxy-3,12-diketo-5,~-cholanic acid and another unidentified material.
The yields of the products and ~he amount of cholic acid which remained without being converted were calculated based on the area ratio of the chromatogram of Figure 9. The results are shwon in Table 12.

7 ~ 5 , Table 12 -Compounds Conversion ~ield or rate (%) amount (y) - 7~-hydroxy-3,12-diketo-5~-- cholanic acid and unidentified 6.5 3.20 material (peak A) 3c~,7~-dihydroxy-12-keto-5k~cholanic acid 88.0 43.39 (peak B) 7~,12c~-dihydroxy-3-keto-5~-cholanic acid 0.3 0.15 c (peak C) other derivatives 0.1 0.05 cholic acid (peak D) 5.1 2.51 Example 13 'The same procedure as described in Example 9 was repeated except that Arthrobacter CA-35-A-1475 strain (FE~-P No. 5527; ATCC No. 31657) was used in stead of Arthrobacter CA-35-AS89-47 strain to obtain a mixture of products and cholic acid which remained without being c converted (49 35 g) The mixture was subjected to hi~h-speed liquid chromatography under the same conditions as in Example 6.
The accompanying Figure 10 shows the resulting chromatogram. The peaks A, B, C and D in Figure 10 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5~'-cholanic acid, 3~,7Q~dihydroxy-12-keto-5~-cholanic acid, 7C~,12C~-dihydroxy-3-keto-5~-cholanic acid and cholic acid, respectively.
When the fractions corresponding to the peaks ~' .

:` :

~ ~69795 .

- A, B, C and D were separated and subjec-ted to thin layer chromatography, each fraction contained a single compound.
The yield of the produc-ts and the amount of cholic acid were calculated based on the area ratio o the chromatogram of Figure 10. The results are shown in Table 13.
Table 13 Compounds ConversionYield or rate (~)amount (g) 7~-hydroxy-3,12-diketo-5~-cholanic acid 3.26 1.61 (peak A) 3~,7~-dihydroxy-12-keto-5~-cholanic acid 92.0 45.40 (peak B) 7~,12~-dihydroxy-3-keto-5l~-cholanic acid 0.20 0.10 (peak C) other derivatives small amount cholic acid (peak D) 4.54 2.24 .
Example 14 ( The same procedure as described in Example 9 was repeated except that Arthrobacter CA-35-A-1766-15 strain (PERM-P No. 5528; ATCC No. 31658) was used in stead of Arthrobacter CA-35-A589-47 strain to obtain a mixture of the products and cholic acid (49.27 g).
The mixture was subjected to high-speed liquid chromatography under the same condi-tions as in Example 6.
The accompanying Figure 11 shows the resulting chromatogram. The peaks A, B and D in Figure ll correspond-979~
, .` <
to those of the reference standards of 7~-hydroxy-3,12-diketo-5~-cholanic acid, 3~,7~-dihydroxy-12-keto-5 cholanic acid and cholic acid, respectively.
When the fractions corresponding to the peaks A, B and D were separated and subjected to thin layer chromatography, each fraction contained a single compound.
The yield of the products and the amount of remaining cholic acid were calculated based on the area ratio of the chromatogram of Figure 11. The results c 10 are shown ln Table 14.
Table 14 Compounds Conversion Yield or rate (%) amount (g) 7C`~-hydroxy-3~12-diketO-5~p- 0 23 0.11 cholanic acid (peak A) ` 3~,7~-dihydroxy-12-keto- 97 02 47 80 5~-cholanic acid (peak B) other derivatives 0.75 0.37 cholic acid (peak D) 2.0 0.99 Example 15 The same procedure as described in Example 9 was repeated except tha-t Arthrobacter CA-35-M-965-3 strain (FERM-P No. 5529; ATCC No. 31659) was used irl stead of Arthrobacter CA-35-A589-47 strain to obtain a mixture of products and cholic acid (49.36 g).
The mixture was subjected to high-speed liquid chromatography under the same conditions as in Example 6.
The accompanying Figure 12 shows the resulting - ' r 1 1 ~ 9 7 9 5~

chromatogram. The peaks A, B and D in Figure 12 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5/~-cholanic acid, 3~,7~-dihydrox~-12-keto-5~-cholanlc acid and cholic acid, respectively.
When the fractions corresponding to the peaks A, B and D were separated and subjected to thin layer chromatography, each fraction corresponding to the peak B or D contained a single compound. The fraction corresponding to the peak A was a mixture of 7c~-hydroxy-3,12-diketo-5~-cholanic acid and another unidentified material.
The yield of the products and the amount of remaining cholic acid were calculated based on the area ratio of ~he chromatogram of Figure 12. The results are shown in Table 15.
Table 15 . . ~
Compounds Conversion Yiel~ or rate ~) amount (g) 7C~-hydroxy-3,12-diketo-5~-cholanic acid and unidentified 8.6 4.24 material (peak A) 3~,7~-dihydroxy-12~keto-85 3 42.11 5~-cholanic acid (peak B) other derivatives 2.0 0.99 cholic acld (peak D) 4.1 2.02 _xample 16 The same procedure as described in E~ample 9 was repeated except that ~rthrobacter CA-35-~-37-12 strain (FE~-P No. 5530; ATCC No. 31660) was used in `\
r 1 ~ ~; 9 7 9 ~;
c .
stead of Ar-throbacter CA-35-AS89-47 strain to obtain a mixture of prod~cts and cholic acid (49.33 y).
The mixture was subjected to high-speed liquid chromatography under the same conditions as in Example 6.
The accompanyiny Figure 13 shows the resulting chromatogram. The peaks A, B, C and D in Figure 13 correspond to those of the reference standards of 7~-hydroxy-3,12-diketo-5~-cholanic acid, 3~,7~-dihydroxy-12-keto-5~-cholanic acid, 7c~,12~-dihydroxy-3-keto-5~ cholanic C 10 acid and cholic acid, respectively.
When the fractions corresponding to the peaks , B, C and D were separated and subjected to thin layer chromatography, each fraction contained a single compound.
~ The yeild of the products and the amount of remaining cholic acid were calculated based on the area ratio of the chromatogram of Figure 13. The results are shown in Table 16.
Table 16 C .. .. ~
Compounds Conversion Yield or rate (%) amount (g) 7~-hydroxy-3,12-diketo-5~-cholanic acid (peak A)7-7 3.80 3~,7~-dihydroxy-12-keto- 9O 0 44 40 5~-cholanic acid (peak B) 7~,12~-dihydroxy-3-keto- 0 3 0.15 5~-cholanlc acid (peak C) cholic acid (peak D) 2.0 0.98 7 ~ 5 ., Example 17 ` The same procedure as described in Example 1 was repeated except that Brevibacterium CA-6 strain tFERM-P No. 5144; ATCC No 31661) was used ln stead of Arthrobacter CA-35 strain , The resulting mixture of products and remaining cholic acid was subjected to high-speed liquid chromato-graphy as in Example 1.
The accompanying Figure 14 shows the resulting chromatogram.
When the compounds in the fractions corresponding to the peaks E, A, B and D were isolated and their chemical constitutlons were determined based on the mass spectra, IR
spectra a~d NMR spectra thereof, these spectra showed that these compounds were 7x-hydroxy-3,12-diketo-~4-cholenic acid, 7~-hydroxy-3,12-diketo-5,~-cholanic acid, ~,7~-dihydroxy-12-keto-S,~-cholanic acid and cholic acid, respectively. The compounds in the fractions correspond-c ing to the peaks X and Y were unidentified.
In the chromatogram of Figure 14, the area ratio of the peaks other than peaks X and Y (i.e. the area ratio E : A : B : D) is 12.2 : 22.2 : 44.9 : 20.7.
Example 18 The same procedure as described in Example 1 was repeated except that Corynebacterium CA-53 strain (FERM-P No. 5532; ATCC No. 31662) was used in stead of Arthrobacter CA-35 strain to obtain a mixture of products and cholic acid (82 g).

~1~97~5 ., The resultlng mixture was subject to high-speed liquid chromatograpy under the same conditions as in Example 1 except that water-methanol (30 : 70, v/v, pH 4.0) was used, in stead of water-methanol (30 : 70, v/v, pH 2.5) as an eluate.
The accompanying Figure 15 shows the resulting chromatogram. The peaks ~, B, C and D in Figure 15 correspond to those oE the reference standards of 7~-hydroxy-3,12-diketo-5~-cholanic acid, 3~,7~-dihydroxy-c 10 12-keto-5p-cholanic acid, 7CC-hydrolxy-3,12-diketo-~ -cholenic acid and cholic acid, respectively.
When the compounds in the fractions corresponding to the peaks A, B and C were isolated and their chemical constitutions were determined based on the mass spectra, IR spectra and NMR spectra thereof, these spectra showed that these compounds were 7~-hydroxy-3,12-diketo-5~-cholanic acid, 3~,7~-dihydroxy-12-keto-5~-cholanic acid and 7~-hydroxy-3,12-diketo-~`~4-cholenic acid, respectively.
Reference example 1 3~,7~-dihydroxy-12-keto-51~-cholanic acid (10 g) thus obtained was placed in a three-necked (round bottom) flask and slowly added thereto ethylene glycol (100 ml) and aqueous potassium hydroxide solution (potassium hydroxide . -10 g ~ water 20 ml). Further, 85 % hydrazine hydrate (10 ml) was added and then, the resulting mixture was refluxed at 100C for 2 hours. The temperature was gradually elevated and the reflux was continued at 185 to 190C
for 4 hours -to distill of~ hydrazine hydrate. After -~ 1~9795 ., cooling, the reaction mixture was diluted with excess water and adjusted to pH 3 to form a precipitate. The precipitate was collec-ted by filtration, washed wi-th water and air-dried to obtain 3c~,7~-dihydroxy-5~cholanic acid (CDCA), yield 3.5 g.
Reference example 2 In a three-necXed (round bottom) flask, 7c~-hydroxy-3,12-diketo-5k~cholanic acid t3 g) obtained in the above examples was dissolved in pyridine (70 ml) and added thereto c 10 tosyl chloride (1 mol). The resulting mixture was reacted at 0C for 1 hour and then, refluxed for 5 hours. After reflux, 3N aqueous hydrochloric acid was added and the reaction mix-ture was extracted with ether (100 ml). Ether was distilled off from the extract under a reduced pressure to give ~6 _ and/or ~7 -3,12-diketo-cholenic acid (2.5 g). The resultant (2 g) was dissolved in lN aqueous sodium hydroxide (10 ml) and added thereto Raney nickel (0.1 g). After addition of hydrogen (0.01 mol), the mixture was reacted overnight to obtain 3~,12~-dihydroxy-5~-cholanic acid (deoxycholic acid) and 3~,12C~dihydroxy-5~cholanic acid (yield : 90 %).

Claims (17)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A microbial process for producing a cholanic acid derivative of the formula:
wherein X is or =O; and R is hydrogen, an alkali metal or an alkaline earth metal, which process comprises cultivating a microbe which is capable of growing in a medium containing cholic acid or a salt thereof as a substrate to produce the cholanic acid derivative in a culture medium containing the substrate, and collecting the resulting derivative, said microbe being selected from the genera Arthrobacter, Brevibacterium and Corynebacterium.

2. A microbial process according to Claim 1, wherein the microbe is a member selected from the group consisting of Arthrobacter CA -35 strain (FERM-P NO. 5145; ATCC No.
31651), Arthrobacter CA-35-A589-29-32 strain (FERM-P NO.
5522; ATCC No. 31652), Arthrobacter CA-35-A589-47 strain (FERM-P NO, 5523; ATCC NO. 31653), Arthrobacter CA-35-A849 s-train (FERM-P NO. 5524; ATCC NO. 31654), Arthrobacter CA-35-A-1071-15 strain (FERM-P No. 5525; ATCC No. 31655), Arthrobacter CA-35-A-1448 strain (FERM-P NO. 5526; ATCC
No. 31656), Arthrobacter CA-35-A-1475 strain (FERM-P No.
5527; ATCC No. 31657), Arthrobacter CA-35-A-1766-15 strain (FERM-P No. 5288; ATCC No. 31658), Arthrobacter CA-35-M-965-3 strain (FERM-P No. 5529;
ATCC No. 31659), Arthrobacter CA-35-Y-37-12 strain (FERM-P No. 5530; ATCC No. 31660), Brevibacterium CA-6 strain (FERM-P No. 5144; ATCC No. 31661), and Corynebacterium CA-53 strain (FERM-P No. 5532; ATCC No. 31662).

3. A microbial process according to Claim 1, wherein the cholanic acid derivatives produced is 3.alpha.,7.alpha.-dihydroxy-12-keto-5.beta.-cholanic acid.

4. A microbial process according to Claim 3, wherein the microbe is a member selected from the group consisting of Arthrobacter CA-35-A589-29-32 strain (FERM-P No. 5522; ATCC No. 31652), Arthrobacter CA-35-A589-47 strain (FERM-P No. 5523; ATCC No. 31653), Arthrobacter CA-35-A849 strain (FERM-P No. 5524; ATCC
No. 31654), Arthrobacter CA-35-A-1071-15 strain (FERM-P
No. 5525; ATCC No. 31655), Arthrobacter CA-35-A-1448 strain (FERM-P No. 5526; ATCC No. 31656), Arthrobacter CA-35-A-1475 strain (FERM-P No. 5527; ATCC No. 31657), Arthrobacter CA-35-A-1766-15 strain (FERM-P No. 5528;
ATCC No. 31658), Arthrobacter CA-35-M-965-3 strain (FERM-P
No. 5529; ATCC No. 31659), Arthrobacter CA-35-Y-37-12 strain (FERM-P No. 5530; ATCC No. 31660).

5. A microbial process according to Claim 1, wherein the substrate is used in a concentration of 1 to 500 g/l as cholic acid.

6. A microbial process according to Claim 5,, wherein the substrate is used in a concentration of 5 to 300 g/l as cholic acid.

7. A microbial process according to Claim 6, wherein the substrate is used in a concentration of 10 to 200 g/l as cholic acid.

8. Arthrobacter sp. which is capable of growing in a medium containing cholic acid or a salt thereof as a substrate to produce cholanic acid derivatives of the formula:
wherein X is or =O; and R is hydrogen, an alkali metal or an alkaline earth metal; said Arthrobacter sp.
being in substantially pure form.

9. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A589-29-32 strain (FERM-P No. 5522; ATCC No. 31652).

10. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A589-47 strain (FERM-P No. 5523; ATCC No. 31653).

11. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A849 strain (FERM-P No. 5524; ATCC No. 31654).

12. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A-1071-15 strain (FERM-P No. 5525; ATCC No. 31655).

13. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A-1448 strain (FERM-P No. 5526; ATCC No. 31656).

14. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A-1475 strain (FERM-P No. 5527; ATCC No. 31657).

15. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-A-1766-15 strain (FERM-P No. 5528; ATCC No. 31658).

16. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-M-965-3 strain (FERM-P No. 5529; ATCC No. 31659).

17. Arthrobacter sp. of Claim 8 being Arthrobacter CA-35-Y-37-12 strain (FERM-P No. 5530; ATCC No. 31660).