Bile acid converting microorganism and process for preparing bile acid

A microorganism having a capability of introducing a 7.beta.-hydroxyl group into a 7-unsubstituted bile acid, and a process for preparing a bile acid having a 7.beta.-hydroxyl group characterized by bringing the above microorganism into contact with a 7-unsubstituted bile acid to convert the acid to a bile acid having a 7.beta.-hydroxyl group. The process permits ursodeoxycholic acid useful as a cholagogue and intermediates therefor to be efficiently prepared.

TECHNICAL FIELD 
The present invention relates to a microorganism producing a bile acid 
having a 7.beta.-hydroxyl group such as 3.alpha., 
7.beta.-dihydroxy-5.beta.-cholanic acid (hereinafter, referred to as 
ursodeoxycholic acid) or the like, which is useful as a cholagogue, and to 
a process for preparing ursodeoxycholic acid or a bile acid having a 
7.beta.-hydroxyl group, which is an intermediate for preparing the former, 
utilizing the microorganism. 
BACKGROUND ART 
For introducing a 7.beta.-hydroxyl group into a 7-unsubstituted bile acid 
utilizing the conversion by a microorganism, the following methods have 
been known: 
1) A process for preparing 3.alpha., 7.beta., 
12.alpha.-trihydroxy-5.beta.-cholanic acid (hereinafter, referred to as 
ursocholic acid) from 3.alpha., 12.alpha.-dihydroxy-5.beta.-cholanic acid 
(hereinafter, referred to as deoxycholic acid) using one or more 
microorganisms belonging to the genus Pleurotus, the genus Coriolus, the 
genus Daedaleopsis, the genus Panaeolus, the genus Marasmius, the genus 
Crinipellis, the genus Pholiota or the genus Fusarium. (Japanese Patent 
Publication No. 34038 of 1989) 
2) A process for preparing ursodeoxycholic acid from 
3.alpha.-hydroxy-5.beta.-cholanic acid (hereinafter, referred to as 
lithocholic acid) using a microorganism belonging to the genus Fusarium. 
(Japanese Patent Publication No. 29397 of 1991) 
3) A process for preparing a conjugated form of ursodeoxycholic acid from a 
conjugated form of lithocholic acid using a microorganism belonging to the 
genus Mortierella. (Japanese Patent Publication No. 29438 of 1993) 
When a 7.beta.-hydroxyl group was introduced using these microorganisms, 
however, it was necessary to adjust the concentration of a substrate at a 
level as low as approximately 0.02-0.1% v/w. Therefore, a great technical 
problem arose that a colossal apparatus for production was required when 
production at an industrial scale was carried out. 
On the other hand, microorganisms of the genus Penicillium have been known 
having a bile acid-converting capacity by introducing a 1.beta.-hydroxyl 
group and a 15.beta.-hydroxyl group (Journal of Lipid Research, Vol. 22, 
p. 1225 (1981) ); no microorganism of the genus Penicillium, however, has 
been known to have a bile acid-converting capacity by introducing a 
7.beta.-hydroxyl group. 
DISCLOSURE OF INVENTION 
The purpose of the present invention is to provide an industrially useful 
process for preparing a bile acid having a 7.beta.-hydroxyl group by 
obviating the defect in the conventional art where it requires a low 
concentration for a substrate. After conducting extensive studies for 
attaining this purpose, the present inventors have found a novel 
microorganism having a bile acid-converting capacity by introducing a 
7.beta.-hydroxyl group, having confirmed that this microorganism 
introduces a hydroxyl group to the 7.beta.-position of a bile acid at a 
substrate concentration higher than the conventional one, that is, even at 
a substrate concentration as high as 0.5% v/w or above, and thus have 
completed the present invention. 
The present invention provides Penicillium sp. TTUR 422 belonging to the 
genus Penicillium and having a capability of introducing a 
7.beta.-hydroxyl group into a 7-unsubstituted bile acid (hereinafter, 
referred to as TTUR 422), and a process for the preparation of a bile acid 
having a 7.beta.-hydroxyl group which comprises bringing TTUR 422 into 
contact with a 7-unsubstituted bile acid to convert the acid to a bile 
acid having a 7.beta.-hydroxyl group. As shown by the Chemical Reaction 1, 
deoxycholic acid is converted into ursocholic acid and 
12-keto-ursodeoxycholic acid, intermediates for preparing ursodeoxycholic 
acid, and lithocholic acid is converted into ursodeoxycholic acid, by 
bringing TTUR 422 into contact with deoxycholic acid or lithocholic acid. 
Chemical Reaction 1 
##STR1## 
Ursocholic acid obtained by the present invention is converted into 
12-keto-ursodeoxycholic acid by oxidizing the hydroxyl group at the 
12-position according to a chemical process using an oxidizing agent or a 
process using a microorganism (Japanese Laid-open Patent Publication No. 
111391 of 1993). 
Then, ursodeoxycholic acid is obtained at a satisfactory yield by 
subjecting 12-keto-ursodeoxycholic acid to Wolff-Kishner reduction. 
Chemical Reaction 2 
##STR2## 
Specific examples of the invention are illustrated below but it should be 
appreciated that the preparation process of the present invention is not 
limited to these examples only. 
(1) A process in which TTUR 422 is cultured in a medium containing a 
7-unsubstituted bile acid to convert the acid to a bile acid having a 
7.beta.-hydroxyl group, which is then obtained. 
(2) A process in which TTUR 422 is cultured in a nutrition medium, and 
after separating TTUR 422 from the medium by centrifugation, filtering by 
a filter paper, etc.; cells are brought into contact with a 
7-unsubstituted bile acid in an appropriate reaction solution to convert 
the acid to a bile acid having a 7.beta.-hydroxyl group, which is then 
obtained. 
(3) A process in which, after immobilizing TTUR 422 separated in the 
process (2) on a carrier such as polyacrylamide, calcium alginate or the 
like, the carrier is brought into contact with a reaction solution 
containing a 7-unsubstituted bile acid to convert the acid to a bile acid 
having a 7.beta.-hydroxyl group, which is then obtained. 
(4) A process in which, after culturing TTUR 422 in a nutrition medium 
until spores are formed and then collected, the spores are placed in a 
reaction solution in which a 7-unsubstituted bile acid is dissolved or 
suspended to convert the acid to a bile acid having a 7.beta.-hydroxyl 
group, which is then obtained. 
(5) A process in which, after immobilizing spores collected in process (4) 
in a manner similar to cells in (3), the spores are brought into contact 
with a reaction solution containing a 7-unsubstituted bile acid to convert 
the acid to a bile acid having a 7.beta.-hydroxyl group, which is then 
obtained. 
In process (1), 7-unsubstituted bile acid can be added appropriately during 
the culturing step. 
In addition, in the above-described processes (2), (3), (4) and (5), it is 
desirable to add organic substances as an energy source, for example, 
glucose, other carbohydrates, casein hydrolysate or yeast extract, at a 
low concentration to the reaction solution. 
The 7-unsubstituted bile acid includes, for example, lithocholic acid, 
deoxycholic acid, 3.alpha.-hydroxy-12-keto-5.beta.-cholanic acid 
(hereinafter, referred to as 12-keto-lithocholic acid) and the like. The 
microorganism according to the present invention has the capability of 
converting these substrate bile acids into bile acids having a 
7.beta.-hydroxyl group but does not show a property of assimilating or 
decomposing these substrate bile acids. 
The concentration of the substrate is more than 0% v/w and 5% v/w or less 
(0-5% v/w), preferably 0.5% v/w or more and 3% v/w or less (0.5-3% v/w), 
and more preferably 0.5% v/w or more and 1% v/w or less (0.5-1% v/w). 
The growth morphology of TTUR 422, the microorganism of the present 
invention, in media is illustrated as follows: 
(1) Growth Morphology in Various Media 
When TTUR 422 was cultured in the oatmeal agar medium (ISP medium, No. 3) 
at 25.degree. C. for 7 days, white wooly colonies having a diameter of 
2.5-3.5 cm were formed with synnema-like structure (synnema (Synnema: 
spores are formed covering a stem) or coremia (Coremia: characterized by a 
stem without spore and a compact oval-shaped top part)) of concentric 
circles having a height of 1-5 mm. Conidia were yellowish gray (No. 68 in 
Today's color/300 by Nippon Shikisai Sha). Yellowish red (No. 15 in 
Today's color/300 by Nippon Shikisai Sha) secretions were observed in 
places. At the reverse side, only the color of the medium was confirmed. 
Growth on Czapek's agar medium was somewhat slow, and white wooly colonies 
having a diameter of 2.0-2.5 cm were formed upon the culture at 25.degree. 
C. for 7 days. The reverse side was white. 
(2) Morphological Characteristics 
FIG. 1 shows a micrograph (magnification: 400 times) of the growth 
morphology of TTUR 422 when cultured at 27.degree. C. for a week with 
Miura's medium (1 g of glucose, 1 g of potassium dihydrogen phosphate, 0.2 
g of magnesium sulfate (heptahydrate), 0.2 g of potassium chloride, 2.0 g 
of sodium nitrate, 0.2 g of yeast extract, 13 g of agar, and 1 liter of 
tap water; pH 6.5-7.0). Conidiophores were formed on the surface of the 
mycelium and synnemata, when formed, were present at the upper part of the 
mycelium. The conidiophores were usually compound-verticillate but 
sometimes irregularly branched. The number of metula was 2 or 3 and their 
size was 5-7.times.1.5-2.5 .mu.m. The number of phialide was 3 per metula 
and their size was 8-11.times.1.0-2.0 .mu.m, with the shape being long and 
slender at the top, slender at the foot and warped ampoule-shaped at the 
middle. The spores were spindle-shaped and their size was 
3.0-4.0.times.1.0-2.0 .mu.m. No sexual generation was observed upon a 
long-term culture. From the above-described morphological characteristics, 
the isolated strain was identified as a strain belonging to the genus 
Penicillium. 
Attempts have been made to identify TTUR 422 referring to the 
classification of the genus Penicillium by Pitt (John I. Pitt, The genus 
Penicillium and its telemorphic states Eupenicillium and Talaromyces, 
Academic Press, London, New York, Toronto, Sydney, San Francisco (1979)). 
Upon a culture of the isolated strain on various media including PDA, ISP 
No.3, CYA (Czapek yeast autolysate agar: Pitt, 1973), MEA (Malt extract 
agar) and others, synnemata were formed within 7 days. Species from genus 
Penicillium displaying such morphology include 3 species: Penicillium 
claviforme, Penicillium isariiforme and Penicillium duclauxii. Comparison 
of the species gave the following results: 
1) The synnema of TTUR 422 formed was similar to synnema of isariiforme or 
duclauxii rather than coremia of claviforme. 
2) The comparison of the size of colonies from 3 Penicillium species 
(Penicillium claviforme, Penicillium isariiforme and Penicillium 
duclauxii) was as follows: 
I. CYA medium 
When cultured at 25.degree. C., the size of colonies from TTUR 422 was 
smaller than those from isariiforme and claviforme, and larger than that 
from duclauxii. At 5.degree. C., claviforme grew to the size of several 
millimeters but TTUR 422, isariiforme and duclauxii showed almost no 
growth. At 30.degree. C., no species grew. 
II. G25N medium (25% glycerol nitrate agar, Pitt, 1973) 
When cultured at 25 .degree. C., claviforme grew to a size exceeding 10 mm 
in diameter but TTUR 422, isariiforme and duclauxii only grew to several 
millimeters. 
III. MEA medium 
Growth of isariiforme was larger than the others. 
The size of colony was similar to that of duclauxii in general. 
3) The formed colonies showed various morphologies depending on the kind of 
media, and the color of the conidia was different as shown in Table 1. 
TABLE 1 
______________________________________ 
Color of conidia in various media 
ISP No.3 CYA MEA 
______________________________________ 
TTUR 422 pink-white pink-white pink-white 
(No. 50) (No. 50), (No. 104) 
yellowish gray 
(No. 68) 
claviforme 
olive green Pale yellow- 
grayish yellow- 
ATCC48945 
(No. 128) brown green 
(No. 94) (No. 130) 
isariiforme 
dull yellow- 
yellowish deep yellow 
ATCC48951 
green white (No. 108) 
(No. 135) (No. 106) 
duclauxii 
yellow-brown 
olive green yellowish red 
ATCC9121 (No. 101) (No. 128) (No. 15), 
olive green 
(No. 128) 
______________________________________ 
The color of conidia from TTUR 422 was light in color such as pink-white 
(No. 50) and yellowish gray in above three media. The color of conidia 
from claviforme, isariiforme and duclauxii had a tendency towards deep 
color such as olive green (No. 128), grayish yellow-green (No. 130), dull 
yellow-green (No. 135) and yellow-brown (No. 101). 
4) Morphology under microscope 
Penicilli (Penicilli:Penicillus) of TTUR 422 were formed like a blooming 
flower as shown in FIG. 1 and showed a characteristic morphology having 
many curved ampoule-shaped radial phialides. On the other hand, penicilli 
of claviforme, isariiforme and duclauxii were formed directed towards the 
top and no shape of phialide characteristic in TTUR 422 was observed. In 
addition, the conidium of TTUR 422 had a shape of an oval narrower than 
those of claviforme, isariiforme and duclauxii. 
Since TTUR 422 had one characteristic partly different from all other 
species as described above, it was confirmed to be a novel species 
belonging to the genus Penicillium and forming synnema. 
Therefore, this strain was deposited in Name: National Institute of 
Bioscience and Human-Technology Agency of Industrial Science and 
Technology, Ministry of Industrial Trade and Industry, Address: 1-3, 
Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan (Postal Zip-code: 305) on 
Feb. 22, 1996, as Penicillium sp. TTUR 422 (Deposition Number: FERM 
BP-5410). 
The physiological properties of the strain TTUR 422 are as follows: 
(1) Optimum growth condition (pH and temperature) 
The optimum growth pH is 5-6, and the optimum growth temperature is 
25-27.degree. C. 
(2) Viable range (pH and temperature) 
The range of pH is 3-9, and the range of temperature is 5-29.degree. C. 
(3) Other remarkable characteristics 
1) Synnemata were formed within 7 days of culture. 
2) Liquid culture shows a yeast-like growth. 
Now, picking up of TTUR 422 is described below. From soils at various 
places in the Kanto area were isolated molds growing on PDA plate medium 
supplemented with 5% v/w sodium cholate. They were inoculated 
independently in middle-sized test tubes (18.phi..times.180 mm) containing 
5 ml of a medium for testing capacity of conversion, and cultured at 
28.degree. C. for 7 days with shaking. At the end of culturing, the 
strains were screened for capacity of converting deoxycholic acid into 
ursocholic acid by qualitatively analyzing the liquid media with thin 
layer chromatography in order to compare them with an authentic sample of 
ursocholic acid. 
The medium for testing capacity of conversion contained 24 g of potato 
dextrose broth, 5 g of yeast extract and 5 g of sodium deoxycholic acid as 
a substrate for conversion reaction in 1 liter of de-ionized water, and 
adjusted to pH 7.5. 
As the result, TTUR 422 was obtained as a strain isolated from soil 
collected at Kitamoto-shi, Saitama-ken, on Aug. 9, 1993, and having the 
best capacity for converting deoxycholic acid into ursocholic acid.

MODE FOR CARRYING OUT THE INVENTION 
The examples of the present invention will now be described by embodiments, 
which should not be construed as a limitation upon the scope of the 
present invention. 
&lt;Embodiment 1&gt; 
Into a 5 liter fermenter was placed 2.5 liter of a liquid medium 
(containing 10 g of glucose, 20 g of special grade Esusan meat (soybean 
protein, manufactured by Ajinomoto), 10 g of yeast extract and 10 g of 
deoxycholic acid (substrate concentration; 1% v/w) in 1 liter of water) 
adjusted to pH 7.5 with 4N-NaOH. The liquid medium in the abovementioned 
fermenter was inoculated with 25 ml of a culture solution of TTUR 422 
which was previously cultured with shaking in a medium composed of 0.4% 
potato extract, 2% glucose and 0.5% yeast extract at 27.degree. C. for 2 
days. The inoculation was aerobically cultured with a stirring velocity of 
300 rpm and an aeration rate of 2.5 1/minutes for 7 days, adjusting at a 
culturing temperature of 27.degree. C. and pH of 7.5. 
Upon termination of the culturing, cells were removed by centrifugation, 
and the culture solution was extracted thrice with twice the amount of 
ethyl acetate after adjusting pH to 3 with hydrochloric acid. The ethyl 
acetate layer was washed several times with water and dried over anhydrous 
sodium sulfate. Ethyl acetate was evaporated under reduced pressure to 
give 24.5 g of crystals. To a column (2.2 cm in diameter and 70 cm in 
height) packed with 95 g of silica gel (FL60D, manufactured by Fuji 
Silicia Chemicals) was applied 1 g of the crystals, and the column was 
then fractionally eluted using eluents consisting of the mixed solvents 
described below in exact order. Fractions showing each spot of 
12-keto-ursodeoxycholic acid and ursocholic acid on TLC analysis were 
collected and evaporated to give 367 mg of 12-keto-ursodeoxycholic acid 
and 88 mg of ursocholic acid in crystals respectively. 
(Eluents) 
1) chloroform:acetone:acetic acid=8:2:0.3, 500 ml 
2) chloroform:acetone:acetic acid=7:2:0.5, 600 ml 
3) chloroform:acetone:acetic acid=7:2:1, 600 ml 
4) chloroform:acetone:acetic acid=3:1:1, 600 ml 
5) chloroform:acetone:acetic acid=1:1:1, 600 ml 
The fact that the conversion products were ursocholic acid and 
12-keto-ursodeoxycholic acid was confirmed by agreement of Rf values in 
TLC analysis, retention times in HPLC analysis, NMR spectra and mass 
analysis values for authentic samples of both ursocholic acid and 
12-keto-ursodeoxycholic acid and values for the conversion products. 
Conditions for TLC Analysis and Rf Values Thereof 
Carrier; Kieselgel 60 (0.25 mm in thickness, manufactured by Merck) 
Developing solvent 1; chloroform:acetone:acetic acid=7:2:1 (volume ratio) 
Developing solvent 2; benzene:isopropanol:acetic acid=40:10:1 (volume 
ratio) 
Coloring; Coloring is effected by spraying the phosphorus molybdic 
acid-sulfuric acid reagent (a reagent prepared by dissolving 1 g of 
phosphorus molybdic acid in 20 ml of methanol and adding 1 ml of 
concentrated sulfuric acid) and heating until the spot of the bile acid 
becomes deep blue. 
Rf Values 
Developing solvent 1: ursocholic acid: authentic sample/Embodiment 
1=0.14/0.14 
Developing solvent 1: 12-keto-ursodeoxycholic acid: authentic 
sample/Embodiment 1=0.25/0.25 
Developing solvent 2: ursocholic acid: authentic sample/Embodiment 
1=0.24/0.24 
Developing solvent 2: 12-keto-ursodeoxycholic acid: authentic 
sample/Embodiment 1=0.38/0.38 
Conditions for HPLC Analysis and Retention Times Thereof 
Column; Inertsil ODS column (column size: 4.6.phi..times.250 mm) 
Detection; differential refraction 
Mobile phase; 0.03 M Na.sub.2 HPO.sub.4 buffer (adjusted to pH 3 with 
H.sub.3 PO.sub.4): 
acetonitrile: methanol=37:30:40 
Flow rate of mobile phase; 1 ml/minute 
Retention time; ursocholic acid: authentic sample/Embodiment 1=8.0 
minutes/8.0 minutes 
12-keto-ursodeoxycholic acid: authentic sample/Embodiment 1=6.1 minutes/6.1 
minutes 
.sup.1 H-NMR spectrum (DMSO-d6, ppm) 
12-keto-ursodeoxycholic acid 
0.77 (3H, d, J=6.1 Hz), 0.99 (6H, s), 3.32 (2H, s), 4.13 (1H, d, J=6.7 Hz), 
4.44 (1H, d, J=4.9 Hz), 11.93 (1H, s) 
Ursocholic Acid 
0.60 (3H, d, J=6.1 Hz), 0.85 (3H, s), 0.92 (3H, s), 3.33 (2H, br), 3.76 
(1H, s), 3.82 (1H, d, J=6.7 Hz), 4.18 (1H, d, J=3.7 Hz), 4.46 (1H, d, 
J=4.3 Hz), 11.94 (1H, s) 
Mass Analysis Value (LC/MS, Mode: APCI, MH.sup.+) 
APCI (m/z); ursocholic acid: authentic sample/Embodiment 1=409/409 
12-keto-ursodeoxycholic acid: authentic sample/Embodiment 1=407/407 
Molecular weight; ursocholic acid: authentic sample/Embodiment 1=408/408 
12-keto-ursodeoxycholic acid: authentic sample/Embodiment 1=406/406 
&lt;Embodiment 2&gt; 
Conversion was carried out according to Embodiment 1 with the same strain 
and the same medium but using 12-keto-lithocholic acid (substrate 
concentration; 1% v/w) in place of the substrate deoxycholic acid to give 
24.6 g of the conversion product. HPLC analysis conducted under the 
conditions described in Embodiment 1 showed that the composition ratio of 
12-keto-ursodeoxycholic acid in the conversion product was 43.9%. 
Silica gel column chromatography was carried out in a manner similar to 
that in Embodiment 1 using 1 g sample of the conversion product. As a 
result, 395 mg of 12-keto-ursodeoxycholic acid was obtained showing a 
purity of 97.6% by HPLC analysis. 
The fact that the conversion product was 12-keto-ursodeoxycholic acid was 
confirmed, because Rf value in TLC analysis, retention time in HPLC 
analysis, NMR spectrum and mass analysis value for an authentic sample of 
12-keto-ursodeoxycholic acid and the conversion product agreed with the 
values described in Embodiment 1. 
&lt;Embodiment 3&gt; 
Conversion was carried out according to Embodiment 1 with the same strain 
and the same medium but using lithocholic acid, in an amount of 5 g per 1 
liter (substrate concentration; 0.5% v/w), in place of the substrate 
deoxycholic acid to give 24.7 g of the conversion product. HPLC analysis 
conducted under the conditions described below showed that the composition 
ratio of ursodeoxycholic acid in the conversion product was 5.1%. 
Silica gel column chromatography was carried out in a manner similar to 
that in Embodiment 1 using 1 g sample of the conversion product. As the 
result, 46 mg of ursodeoxycholic acid was obtained showing a purity of 
97.6% by HPLC analysis. 
The fact that the conversion product was ursodeoxycholic acid was confirmed 
by agreement of Rf value in TLC analysis, retention time in HPLC analysis, 
NMR spectrum and mass analysis value for authentic sample of 
ursodeoxycholic acid and values for the conversion product. 
Conditions for TLC Analysis and Rf Values Thereof 
Conditions for analysis; the same as in Embodiment 1 
Rf Values 
Developing solvent 1; ursodeoxycholic acid: authentic sample/Embodiment 
3=0.38/0.38 
Developing solvent 2; ursodeoxycholic acid: authentic sample/Embodiment 
3=0.42/0.42 
Conditions for HPLC Analysis and Observed Retention Times 
Conditions for analysis; the same as in Embodiment 1 
Retention time; ursodeoxycholic acid: authentic sample/Embodiment 3=18.5 
minutes/18.5 minutes 
.sup.1 H-NMR spectrum (DMSO-d6, ppm) 
0.62 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 3.33 (2H, s), 3.87 (1H, d, J=6.7 
Hz), 4.43 (1H, d, J=4.3 Hz), 11.93 (1H, s) 
Mass Analysis Value (LC/MS, Mode: APCI, MH.sup.+) 
APCI (m/z); ursodeoxycholic acid: authentic sample/Embodiment 4=393/393 
Molecular weight; ursodeoxycholic acid: authentic sample/Embodiment 
3=392/392 
INDUSTRIAL APPLICABILITY 
As described above, the microorganism according to the present invention 
enables introduction of a hydroxyl group into the 7.beta.-position of a 
bile acid at a higher substrate concentration as compared to the 
conventional microorganism, and by utilizing the microorganism, 3.alpha., 
7.beta.-dihydroxy-5.beta.-cholanic acid, which is useful as a cholagogue, 
or preparing intermediates thereof can be produced with good efficiency.